Comparing cell viability and ethanol fermentation of the thermotolerant yeast Kluyveromyces marxianus and Saccharomyces cerevisiae on steam-exploded biomass treated with laccase.

PubMed

Moreno, Antonio D; Ibarra, David; Ballesteros, Ignacio; González, Alberto; Ballesteros, Mercedes

2013-05-01

In this study, the thermotolerant yeast Kluyveromyces marxianus CECT 10875 was compared to the industrial strain Saccharomyces cerevisiae Ethanol Red for lignocellulosic ethanol production. For it, whole slurry from steam-exploded wheat straw was used as raw material, and two process configurations, simultaneous saccharification and fermentation (SSF) and presaccharification and simultaneous saccharification and fermentation (PSSF), were evaluated. Compared to S. cerevisiae, which was able to produce ethanol in both process configurations, K. marxianus was inhibited, and neither growth nor ethanol production occurred during the processes. However, laccase treatment of the whole slurry removed specifically lignin phenols from the overall inhibitory compounds present in the slurry and triggered the fermentation by K. marxianus, attaining final ethanol concentrations and yields comparable to those obtained by S. cerevisiae. Copyright © 2012 Elsevier Ltd. All rights reserved.

Protein enrichment of an Opuntia ficus-indica cladode hydrolysate by cultivation of Candida utilis and Kluyveromyces marxianus.

PubMed

Akanni, Gabriel B; du Preez, James C; Steyn, Laurinda; Kilian, Stephanus G

2015-03-30

The cladodes of Opuntia ficus-indica (prickly pear cactus) have a low protein content; for use as a balanced feed, supplementation with other protein sources is therefore desirable. We investigated protein enrichment by cultivation of the yeasts Candida utilis and Kluyveromyces marxianus in an enzymatic hydrolysate of the cladode biomass. Dilute acid pretreatment and enzymatic hydrolysis of sun-dried cladodes resulted in a hydrolysate containing (per litre) 45.5 g glucose, 6.3 g xylose, 9.1 g galactose, 10.8 g arabinose and 9.6 g fructose. Even though K. marxianus had a much higher growth rate and utilized l-arabinose and d-galactose more completely than C. utilis, its biomass yield coefficient was lower due to ethanol and ethyl acetate production despite aerobic cultivation. Yeast cultivation more than doubled the protein content of the hydrolysate, with an essential amino acid profile superior to sorghum and millet grains. This K. marxianus strain was weakly Crabtree positive. Despite its low biomass yield, its performance compared well with C. utilis. This is the first report showing that the protein content and quality of O. ficus-indica cladode biomass could substantially be improved by yeast cultivation, including a comparative evaluation of C. utilis and K. marxianus. © 2014 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Fermentation of cacao (Theobroma cacao L.) seeds with a hybrid Kluyveromyces marxianus strain improved product quality attributes.

PubMed

Leal, Gildemberg Amorim; Gomes, Luiz Humberto; Efraim, Priscilla; de Almeida Tavares, Flavio Cesar; Figueira, Antonio

2008-08-01

Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage ('sweatings') from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.

Trichoderma sp. spores and Kluyveromyces marxianus cells magnetic separation: Immobilization on chitosan-coated magnetic nanoparticles.

PubMed

Palacios-Ponce, Sócrates; Ramos-González, Rodolfo; Ruiz, Héctor A; Aguilar, Miguel A; Martínez-Hernández, José L; Segura-Ceniceros, Elda P; Aguilar, Cristóbal N; Michelena, Georgina; Ilyina, Anna

2017-07-03

In the present study, the interactions between chitosan-coated magnetic nanoparticles (C-MNP) and Trichoderma sp. spores as well as Kluyveromyces marxianus cells were studied. By Plackett-Burman design, it was demonstrated that factors which directly influenced on yeast cell immobilization and magnetic separation were inoculum and C-MNP quantity, stirring speed, interaction time, and volume of medium, while in the case of fungal spores, the temperature also was disclosed as an influencing factor. Langmuir and Freundlich models were applied for the mathematical analysis of adsorption isotherms at 30°C. For Trichoderma sp. spore adsorption isotherm, the highest correlation coefficient was observed for lineal function of Langmuir model with a maximum adsorption capacity at 5.00E + 09 spores (C-MNP g -1 ). Adsorption isotherm of K. marxianus cells was better adjusted to Freundlich model with a constant (K f ) estimated as 2.05E + 08 cells (C-MNP g -1 ). Both systems may have a novel application in fermentation processes assisted with magnetic separation of biomass.

Improving xylitol production at elevated temperature with engineered Kluyveromyces marxianus through over-expressing transporters.

PubMed

Zhang, Jia; Zhang, Biao; Wang, Dongmei; Gao, Xiaolian; Hong, Jiong

2015-01-01

Three transporter genes including Kluyveromyces marxianus aquaglyceroporin gene (KmFPS1), Candida intermedia glucose/xylose facilitator gene (CiGXF1) or glucose/xylose symporter gene (CiGXS1) were over-expressed in K. marxianus YZJ017 to improve xylitol production at elevated temperatures. The xylitol production of YZJ074 that harbored CiGXF1 was improved to 147.62g/L in Erlenmeyer flask at 42°C. In fermenter, 99.29 and 149.60g/L xylitol were produced from 99.55 and 151.91g/L xylose with productivity of 4.14 and 3.40g/L/h respectively at 42°C. Even at 45°C, YZJ074 could produce 101.30g/L xylitol from 101.41g/L xylose with productivity of 2.81g/L/h. Using fed-batch fermentation through repeatedly adding non-sterilized substrate directly, YZJ074 could produce 312.05g/L xylitol which is the highest yield reported to date. The engineered strains YZJ074 which can produce xylitol at elevated temperatures is an excellent foundation for xylitol bioconversion. Copyright © 2014 Elsevier Ltd. All rights reserved.

Respiratory capacity of the Kluyveromyces marxianus yeast isolated from the mezcal process during oxidative stress.

PubMed

Arellano-Plaza, Melchor; Gschaedler-Mathis, Anne; Noriega-Cisneros, Ruth; Clemente-Guerrero, Mónica; Manzo-Ávalos, Salvador; González-Hernández, Juan Carlos; Saavedra-Molina, Alfredo

2013-07-01

During the mezcal fermentation process, yeasts are affected by several stresses that can affect their fermentation capability. These stresses, such as thermal shock, ethanol, osmotic and growth inhibitors are common during fermentation. Cells have improved metabolic systems and they express stress response genes in order to decrease the damage caused during the stress, but to the best of our knowledge, there are no published works exploring the effect of oxidants and prooxidants, such as H2O2 and menadione, during growth. In this article, we describe the behavior of Kluyveromyces marxianus isolated from spontaneous mezcal fermentation during oxidative stress, and compared it with that of Saccharomyces cerevisiae strains that were also obtained from mezcal, using the W303-1A strain as a reference. S. cerevisiae strains showed greater viability after oxidative stress compared with K. marxianus strains. However, when the yeast strains were grown in the presence of oxidants in the media, K. marxianus exhibited a greater ability to grow in menadione than it did in H2O2. Moreover, when K. marxianus SLP1 was grown in a minibioreactor, its behavior when exposed to menadione was different from its behavior with H2O2. The yeast maintained the ability to consume dissolved oxygen during the 4 h subsequent to the addition of menadione, and then stopped respiration. When exposed to H2O2, the yeast stopped consuming oxygen for the following 8 h, but began to consume oxygen when stressors were no longer applied. In conclusion, yeast isolated from spontaneous mezcal fermentation was able to resist oxidative stress for a long period of time.

Evaluation of Kluyveromyces marxianus FII 510700 grown on a lactose-based medium as a source of a natural bioemulsifier.

PubMed

Lukondeh, Tredwell; Ashbolt, Nicholas J; Rogers, Peter L

2003-12-01

Mannoprotein with emulsification properties was extracted from the cell walls of Kluyveromyces marxianus grown on a lactose-based medium by autoclaving cells in a citrate buffer at pH 7. The purified product was evaluated for chemical and physical stability to establish its potential use as a natural emulsifier in processed foods. The yield of purified bioemulsifier from this strain of K. marxianus was 4-7% of the original dry cell weight. The purified product, at a concentration of 12 g l(-1), formed emulsions that were stable for 3 months when subjected to a range of pH (3-11) and NaCl concentrations (2-50 g l(-1)). The composition of this mannoprotein was 90% carbohydrate (mannan) and 4-6% protein. These values are similar to mannoprotein extracted from cells of Saccharomyces cerevisiae, which is the traditional source. Consequently K. marxianus cultivated on a low-cost lactose-based medium such as whey, a lactose-rich clean waste of the dairy industry, could be developed as a source of bioemulsifier for use in the food industry.

Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing.

PubMed

Hu, Nan; Yuan, Bo; Sun, Juan; Wang, Shi-An; Li, Fu-Li

2012-09-01

Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40 °C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200 g L(-1)) at 40 °C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2 g L(-1), which corresponded to the theoretical ethanol yield of 90.0 and 79.7 %, respectively. In the range of 30 to 40 °C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP.

Protein enrichment of an Opuntia ficus-indica cladode hydrolysate by cultivation of Candida utilis and Kluyveromyces marxianus

PubMed Central

Akanni, Gabriel B; du Preez, James C; Steyn, Laurinda; Kilian, Stephanus G

2015-01-01

BACKGROUND The cladodes of Opuntia ficus-indica (prickly pear cactus) have a low protein content; for use as a balanced feed, supplementation with other protein sources is therefore desirable. We investigated protein enrichment by cultivation of the yeasts Candida utilis and Kluyveromyces marxianus in an enzymatic hydrolysate of the cladode biomass. RESULTS Dilute acid pretreatment and enzymatic hydrolysis of sun-dried cladodes resulted in a hydrolysate containing (per litre) 45.5 g glucose, 6.3 g xylose, 9.1 g galactose, 10.8 g arabinose and 9.6 g fructose. Even though K. marxianus had a much higher growth rate and utilized l-arabinose and d-galactose more completely than C. utilis, its biomass yield coefficient was lower due to ethanol and ethyl acetate production despite aerobic cultivation. Yeast cultivation more than doubled the protein content of the hydrolysate, with an essential amino acid profile superior to sorghum and millet grains. CONCLUSIONS This K. marxianus strain was weakly Crabtree positive. Despite its low biomass yield, its performance compared well with C. utilis. This is the first report showing that the protein content and quality of O. ficus-indica cladode biomass could substantially be improved by yeast cultivation, including a comparative evaluation of C. utilis and K. marxianus. © 2014 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:25371280

Fermentation of molasses using a thermotolerant yeast, Kluyveromyces marxianus IMB3: simplex optimisation of media supplements.

PubMed

Gough, S; Flynn, O; Hack, C J; Marchant, R

1996-09-01

The use of molasses as a substrate for ethanol production by the thermotolerant yeast Kluyveromyces marxianus var. marxianus was investigated at 45 degrees C. A maximum ethanol concentration of 7.4% (v/v) was produced from unsupplemented molasses at a concentration of 23% (v/v). The effect on ethanol production of increasing the sucrose concentration in 23% (v/v) molasses was determined. Increased sucrose concentration had a similar detrimental effect on the final ethanol produced as the increase in molasses concentration. This indicated that the effect may be due to increased osmotic activity as opposed to other components in the molasses. The optimum concentration of the supplements nitrogen, magnesium, potassium and fatty acid for maximum ethanol production rate was determined using the Nelder and Mead (Computer J 7:308-313, 1965) simplex optimisation method. The optimum concentration of the supplements were 0.576 g1(-1) magnesium sulphate, 0.288 g1(-1) potassium dihydrogen phosphate and 0.36% (v/v) linseed oil. Added nitrogen in the form of ammonium sulphate did not affect the ethanol production rate.

Direct fermentation of Jerusalem artichoke tuber powder for production of l-lactic acid and d-lactic acid by metabolically engineered Kluyveromyces marxianus.

PubMed

Bae, Jung-Hoon; Kim, Hyun-Jin; Kim, Mi-Jin; Sung, Bong Hyun; Jeon, Jae-Heung; Kim, Hyun-Soon; Jin, Yong-Su; Kweon, Dae-Hyuk; Sohn, Jung-Hoon

2018-01-20

An efficient production system for optically pure l- and d-lactic acid (LA) from Jerusalem artichoke tuber powder (JAP) was developed by metabolic engineering of Kluyveromyces marxianus. To construct LA-producing strains, the ethanol fermentation pathway of K. marxianus was redirected to LA production by disruption of KmPDC1 and expression of l- and d-lactate dehydrogenase (LDH) genes derived from Lactobacillus plantarum under the control of the K. marxianus translation elongation factor 1α promoter. To further increase the LA titer, the l-LA and d-LA consumption pathway of host strains was blocked by deletion of the oxidative LDH genes KmCYB2 and KmDLD1. The recombinant strains produced 130g/L l-LA and 122g/L d-LA by direct fermentation from 230g/L JAP containing 140g/L inulin, without pretreatment or nutrient supplementation. The conversion efficiency and optical purity were ≫>95% and ≫>99%, respectively. This system using JAP and the inulin-assimilating yeast K. marxianus could lead to a cost-effective process for the production of LA. Copyright © 2017 Elsevier B.V. All rights reserved.

Saccharomyces cerevisiae and Kluyveromyces marxianus Cocultures Allow Reduction of Fermentable Oligo-, Di-, and Monosaccharides and Polyols Levels in Whole Wheat Bread.

PubMed

Struyf, Nore; Laurent, Jitka; Verspreet, Joran; Verstrepen, Kevin J; Courtin, Christophe M

2017-10-04

Fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) are small molecules that are poorly absorbed in the small intestine and rapidly fermented in the large intestine. There is evidence that a diet low in FODMAPs reduces abdominal symptoms in approximately 70% of the patients suffering from irritable bowel syndrome. Wheat contains relatively high fructan levels and is therefore a major source of FODMAPs in our diet. In this study, a yeast-based strategy was developed to reduce FODMAP levels in (whole wheat) bread. Fermentation of dough with an inulinase-secreting Kluyveromyces marxianus strain allowed to reduce fructan levels in the final product by more than 90%, while only 56%  reduction was achieved when a control Saccharomyces cerevisiae strain was used. To ensure sufficient CO 2 production, cocultures of S. cerevisiae and K. marxianus were prepared. Bread prepared with a coculture of K. marxianus and S. cerevisiae had fructan levels ≤0.2% dm, and a loaf volume comparable with that of control bread. Therefore, this approach is suitable to effectively reduce FODMAP levels in bread.

Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater.

PubMed

Yuan, W J; Zhao, X Q; Ge, X M; Bai, F W

2008-12-01

To study fuel ethanol fermentation with Kluyveromyces marxianus ATCC8554 from Jerusalem artichoke (Helianthus tuberosus) grown in salina and irrigated with a mixture of seawater and freshwater. The growth and ethanol fermentation of K. marxianus ATCC8554 were studied using inulin as substrate. The activity of inulinase, which attributes to the hydrolysis of inulin, the main carbohydrate in Jerusalem artichoke, was monitored. The optimum temperatures were 38 degrees C for growth and inulinase production, and 35 degrees C for ethanol fermentation. Aeration was not necessary for ethanol fermentation with the K. marxianus from inulin. Then, the fresh Jerusalem artichoke tubers grown in salina and irrigated with 25% and 50% seawater were further examined for ethanol fermentation with the K. marxianus, and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25% seawater. Furthermore, the dry meal of the Jerusalem artichoke tubers irrigated with 25% seawater was examined for ethanol fermentation at three solid concentrations of 200, 225 and 250 g l(-1), and the highest ethanol yield of 0.467, or 91.5% of the theoretical value of 0.511, was achieved for the slurry with a solid concentration of 200 g l(-1). Halophilic Jerusalem artichoke can be used for fuel ethanol production. Halophilic Jerusalem artichoke, not competing with grain crops for arable land, is a sustainable feedstock for fuel ethanol production.

Production of bioethanol from effluents of the dairy industry by Kluyveromyces marxianus.

PubMed

Zoppellari, Francesca; Bardi, Laura

2013-09-25

Whey and scotta are effluents coming from cheese and ricotta processing respectively. Whey contains minerals, lipids, lactose and proteins; scotta contains mainly lactose. Whey can be reused in several ways, such as protein extraction or animal feeding, while nowadays scotta is just considered as a waste; moreover, due to very high volumes of whey produced in the world, it poses serious environmental and disposal problems. Alternative destinations of these effluents, such as biotechnological transformations, can be a way to reach both goals of improving the added value of the agroindustrial processes and reducing their environmental impact. In this work we investigated the way to produce bioethanol from lactose of whey and scotta and to optimize the fermentation yields. Kluyveromyces marxianus var. marxianus was chosen as lactose-fermenting yeast. Batch, aerobic and anaerobic, fermentations and semicontinuous fermentations in dispersed phase and in packed bed reactor were carried out of row whey, scotta and mix 1:1 whey:scotta at a laboratory scale. Different temperatures (28-40°C) were also tested to check whether the thermotolerance of the chosen yeast could be useful to improve the ethanol yield. The best performances were reached at low temperatures (28°C); high temperatures are also compatible with good ethanol yields in whey fermentations, but not in scotta fermentations. Semicontinuous fermentations in dispersed phase gave the best fermentation performances, particularly with scotta. Then both effluents can be considered suitable for ethanol production. The good yields obtained from scotta allow us to transform this waste in a source. Copyright © 2012 Elsevier B.V. All rights reserved.

Influence of carbon and nitrogen source on production of volatile fragrance and flavour metabolites by the yeast Kluyveromyces marxianus.

PubMed

Gethins, Loughlin; Guneser, Onur; Demirkol, Aslı; Rea, Mary C; Stanton, Catherine; Ross, R Paul; Yuceer, Yonca; Morrissey, John P

2015-01-01

The yeast Kluyveromyces marxianus produces a range of volatile molecules with applications as fragrances or flavours. The purpose of this study was to establish how nutritional conditions influence the production of these metabolites. Four strains were grown on synthetic media, using a variety of carbon and nitrogen sources and volatile metabolites analysed using gas chromatography-mass spectrometry (GC-MS). The nitrogen source had pronounced effects on metabolite production: levels of the fusel alcohols 2-phenylethanol and isoamyl alcohol were highest when yeast extract was the nitrogen source, and ammonium had a strong repressing effect on production of 2-phenylethyl acetate. In contrast, the nitrogen source did not affect production of isoamyl acetate or ethyl acetate, indicating that more than one alcohol acetyl transferase activity is present in K. marxianus. Production of all acetate esters was low when cells were growing on lactose (as opposed to glucose or fructose), with a lower intracellular pool of acetyl CoA being one explanation for this observation. Bioinformatic and phylogenetic analysis of the known yeast alcohol acetyl transferases ATF1 and ATF2 suggests that the ancestral protein Atf2p may not be involved in synthesis of volatile acetate esters in K. marxianus, and raises interesting questions as to what other genes encode this activity in non-Saccharomyces yeasts. Identification of all the genes involved in ester synthesis will be important for development of the K. marxianus platform for flavour and fragrance production. Copyright © 2014 John Wiley & Sons, Ltd.

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.

PubMed

Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji

2013-12-01

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.

Ethanol production from Jerusalem artichoke tubers (Helianthus tuberosus). Using Kluyveromyces marxcianus and Saccharomyces rosei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Margaritis, A.; Bajpai, P.

1982-04-01

This article examines the potential of Jerusalem artichoke as a source for ethanol and single-cell protein SCP. In addition, experimental results are presented on batch fermentation kinetics employing two strains of Kluyveromyces marxianus and one strain of Saccharomyces rosei grown on the extract derived from the tubers of Jerusalem artichoke. Of the three cultures examined, Kluyveromyces marxianus UCD (FST) 55-82 was found to be the best producer of ethanol grown in a simple medium at 35 degrees C. The ethanol production was found to be growth-associated having a mu max = 0.41/h and the ethanol and biomass yields were determinedmore » to be Y p/s = 0.45 (88% of the theoretical) and Y x/s = 0.04 with 92% of the original sugars utilized. On the basis of carbohydrate yields of Jerusalem artichoke reported in the literature and these batch kinetic studies with Kluyveromyces marxianus, the calculated ethanol yields were found to range from 1400 kg ethanol/acre/yr to a maximum of 2700 kg ethanol/acre/yr. The SCP yields for Kluyveromyces marxianus were calculated to range between 130 to 250 kg dry wt cell/acre/yr. The potential for developing an integrated process to produce ethanol and SCP is also discussed. (Refs. 27).« less

Kluyveromyces marxianus and Saccharomyces boulardii Induce Distinct Levels of Dendritic Cell Cytokine Secretion and Significantly Different T Cell Responses In Vitro

PubMed Central

Smith, Ida M.; Baker, Adam; Christensen, Jeffrey E.; Boekhout, Teun; Frøkiær, Hanne; Arneborg, Nils; Jespersen, Lene

2016-01-01

Interactions between members of the intestinal microbiota and the mucosal immune system can significantly impact human health, and in this context, fungi and food-related yeasts are known to influence intestinal inflammation through direct interactions with specialized immune cells in vivo. The aim of the present study was to characterize the immune modulating properties of the food-related yeast Kluyveromyces marxianus in terms of adaptive immune responses indicating inflammation versus tolerance and to explore the mechanisms behind the observed responses. Benchmarking against a Saccharomyces boulardii strain with probiotic effects documented in clinical trials, we evaluated the ability of K. marxianus to modulate human dendritic cell (DC) function in vitro. Further, we assessed yeast induced DC modulation of naive T cells toward effector responses dominated by secretion of IFNγ and IL-17 versus induction of a Treg response characterized by robust IL-10 secretion. In addition, we blocked relevant DC surface receptors and investigated the stimulating properties of β-glucan containing yeast cell wall extracts. K. marxianus and S. boulardii induced distinct levels of DC cytokine secretion, primarily driven by Dectin-1 recognition of β-glucan components in their cell walls. Upon co-incubation of yeast exposed DCs and naive T cells, S. boulardii induced a potent IFNγ response indicating TH1 mobilization. In contrast, K. marxianus induced a response dominated by Foxp3+ Treg cells, a characteristic that may benefit human health in conditions characterized by excessive inflammation and positions K. marxianus as a strong candidate for further development as a novel yeast probiotic. PMID:27898740

Kluyveromyces marxianus and Saccharomyces boulardii Induce Distinct Levels of Dendritic Cell Cytokine Secretion and Significantly Different T Cell Responses In Vitro.

PubMed

Smith, Ida M; Baker, Adam; Christensen, Jeffrey E; Boekhout, Teun; Frøkiær, Hanne; Arneborg, Nils; Jespersen, Lene

2016-01-01

Interactions between members of the intestinal microbiota and the mucosal immune system can significantly impact human health, and in this context, fungi and food-related yeasts are known to influence intestinal inflammation through direct interactions with specialized immune cells in vivo. The aim of the present study was to characterize the immune modulating properties of the food-related yeast Kluyveromyces marxianus in terms of adaptive immune responses indicating inflammation versus tolerance and to explore the mechanisms behind the observed responses. Benchmarking against a Saccharomyces boulardii strain with probiotic effects documented in clinical trials, we evaluated the ability of K. marxianus to modulate human dendritic cell (DC) function in vitro. Further, we assessed yeast induced DC modulation of naive T cells toward effector responses dominated by secretion of IFNγ and IL-17 versus induction of a Treg response characterized by robust IL-10 secretion. In addition, we blocked relevant DC surface receptors and investigated the stimulating properties of β-glucan containing yeast cell wall extracts. K. marxianus and S. boulardii induced distinct levels of DC cytokine secretion, primarily driven by Dectin-1 recognition of β-glucan components in their cell walls. Upon co-incubation of yeast exposed DCs and naive T cells, S. boulardii induced a potent IFNγ response indicating TH1 mobilization. In contrast, K. marxianus induced a response dominated by Foxp3+ Treg cells, a characteristic that may benefit human health in conditions characterized by excessive inflammation and positions K. marxianus as a strong candidate for further development as a novel yeast probiotic.

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation.

PubMed

Crafack, Michael; Mikkelsen, Morten B; Saerens, Sofie; Knudsen, Morten; Blennow, Andreas; Lowor, Samuel; Takrama, Jemmy; Swiegers, Jan H; Petersen, Gert B; Heimdal, Hanne; Nielsen, Dennis S

2013-10-01

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12-24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)5-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed. © 2013.

Enhanced production of extracellular inulinase by the yeast Kluyveromyces marxianus in xylose catabolic state.

PubMed

Hoshida, Hisashi; Kidera, Kenta; Takishita, Ryuta; Fujioka, Nobuhisa; Fukagawa, Taiki; Akada, Rinji

2018-06-01

The production of extracellular proteins by the thermotolerant yeast Kluyveromyces marxianus, which utilizes various sugars, was investigated using media containing sugars such as glucose, galactose, and xylose. SDS-PAGE analysis of culture supernatants revealed abundant production of an extracellular protein when cells were grown in xylose medium. The N-terminal sequence of the extracellular protein was identical to a part of the inulinase encoded by INU1 in the genome. Inulinase is an enzyme hydrolyzing β-2,1-fructosyl bond in inulin and sucrose and is not required for xylose assimilation. Disruption of INU1 in the strain DMKU 3-1042 lost the production of the extracellular protein and resulted in growth defect in sucrose and inulin media, indicating that the extracellular protein was inulinase (sucrase). In addition, six K. marxianus strains among the 16 strains that were analyzed produced more inulinase in xylose medium than in glucose medium. However, expression analysis indicated that the INU1 promoter activity was lower in the xylose medium than in the glucose medium, suggesting that enhanced production of inulinase is controlled in a post-transcriptional manner. The production of inulinase was also higher in cultures with more agitation, suggesting that oxygen supply affects the production of inulinase. Taken together, these results suggest that both xylose and oxygen supply shift cellular metabolism to enhance the production of extracellular inulinase. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Lactose-induced cell death of beta-galactosidase mutants in Kluyveromyces lactis.

PubMed

Lodi, Tiziana; Donnini, Claudia

2005-05-01

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.

[Production and partial characterization of beta-galactosidase from Kluyveromyces lactis grown in deproteinized whey].

PubMed

Ramírez Matheus, Alejandra O; Rivas, Nilo

2003-06-01

The purpose of this work was to optimize the beta-galactosidase production by Kluyveromyces lactis, applying the Surface Response Methodology (SRM) and using deproteinized whey as fermentation medium. An Orthogonal Central Compound Design (OCCD) was used without repetition, with four factors: temperature, pH, agitation speed and fermentation time. Then, enzyme activity (U/ml) as response variable was used. Thirty trials in twenty-five treatments, with six repetitions at the central point, were carried out, in a New Brunswick Bioflo 2000 fermentor with a volume of 2 liters. The deproteinized whey obtained by thermocoagulation was chemically analyzed. The results were: moisture 93.83%, total solids 6.17%, protein 0.44%, lactose 4.85%, acidity 0.43% and pH 4.58. The best conditions in the enzyme production were: temperature 30.3 degrees C, pH 4.68, agitation speed 191 r.p.m. and fermentation time 18.5 h. with an enzyme production of 8.3 U/ml. The degree of purification obtained was 7.4 times and the yield was 50.8%. The purified enzyme had an optimum temperature of 60 degrees C and a pH of 6.2. This work shows that the yeast Kluyveromyces lactis grown in deproteinized whey is able to produce the enzyme beta-galactosidase and SRM can be used in the fermentology processes, specifically in determining the best suitable operation conditions.

Bioproduction of 2-phenylethanol and 2-phenethyl acetate by Kluyveromyces marxianus through the solid-state fermentation of sugarcane bagasse.

PubMed

Martínez, Oscar; Sánchez, Antoni; Font, Xavier; Barrena, Raquel

2018-06-01

2-Phenylethanol (2-PE) and 2-phenethyl acetate (2-PEA) are important aroma compounds widely used in food and cosmetic industries due to their rose-like odor. Nowadays, due to the growing demand for natural products, the development of bioprocesses for obtaining value-added compounds has become of great significance. 2-PE and 2-PEA can be produced through the biotransformation of L-phenylalanine using the generally recognized as safe strain Kluyveromyces marxianus. L-phenylalanine bioconversion systems have been typically focused on submerged fermentation processes (SmF), but there is no information about other alternative productive approaches. Here, the solid-state fermentation (SSF) of sugarcane bagasse supplemented with L-phenylalanine was investigated as a sustainable alternative for producing 2-PE and 2-PEA in a residue-based system using Kluyveromyces marxianus as inoculum. An initial screening of the operational variables indicated that air supply, temperature, and initial moisture content significantly affect the product yield. Besides, it was found that the feeding strategy also affects the production and the efficiency of the process. While a basic batch system produced 16 mg products per gram of residue (dry basis), by using split feeding strategies (fed-batch) of only sugarcane bagasse, a maximum of 18.4 mg Products  g -1 residue were achieved. Increase in product yield was also accompanied by an increase in the consumption efficiency of nutrients and precursor. The suggested system results as effective as other more complex SmF systems to obtain 2-PE and 2-PEA, showing the feasibility of SSF as an alternative for producing these compounds through the valorization of an agro-industrial residue.

Effect of oxygenation and temperature on glucose-xylose fermentation in Kluyveromyces marxianus CBS712 strain

PubMed Central

2014-01-01

Background The yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized. Results In the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes. Conclusions In the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to

Effect of oxygenation and temperature on glucose-xylose fermentation in Kluyveromyces marxianus CBS712 strain.

PubMed

Signori, Lorenzo; Passolunghi, Simone; Ruohonen, Laura; Porro, Danilo; Branduardi, Paola

2014-04-08

The yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized. In the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes. In the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to the impairment of the

Effect of lignocellulosic degradation compounds from steam explosion pretreatment on ethanol fermentation by thermotolerant yeast Kluyveromyces marxianus.

PubMed

Oliva, Jose Miguel; Sáez, Felicia; Ballesteros, Ignacio; González, Alberto; Negro, Maria José; Manzanares, Paloma; Ballesteros, Mercedes

2003-01-01

The filtrate from steam-pretreated poplar was analyzed to identify degradation compounds. The effect of selected compounds on growth and ethanolic fermentation of the thermotolerant yeast strain Kluyveromyces marxianus CECT 10875 was tested. Several fermentations on glucose medium, containing individual inhibitory compounds found in the hydrolysate, were carried out. The degree of inhibition on yeast strain growth and ethanolic fermentation was determined. At concentrations found in the prehy-drolysate, none of the individual compounds significantly affected the fermentation. For all tested compounds, growth was inhibited to a lesser extent than ethanol production. Lower concentrations of catechol (0.96 g/L) and 4-hydroxybenzaldehyde (1.02 g/L) were required to produce the 50% reduction in cell mass in comparison to other tested compounds.

Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.

PubMed

Yarimizu, Tohru; Nakamura, Mikiko; Hoshida, Hisashi; Akada, Rinji

2015-02-14

Targeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signal sequence has been identified. In this study, simple modeling of signal sequences was attempted using Gaussia princeps secretory luciferase (GLuc) in the yeast Kluyveromyces marxianus, which allowed comprehensive recombinant gene construction to substitute synthetic signal sequences. Mutational analysis of the GLuc signal sequence revealed that the GLuc hydrophobic peptide length was lower limit for effective secretion and that the N-terminal basic residue was indispensable. Deletion of the 16th Glu caused enhanced levels of secreted protein, suggesting that this hydrophilic residue defined the boundary of a hydrophobic peptide stretch. Consequently, we redesigned this domain as a repeat of a single hydrophobic amino acid between the N-terminal Lys and C-terminal Glu. Stretches consisting of Phe, Leu, Ile, or Met were effective for secretion but the number of residues affected secretory activity. A stretch containing sixteen consecutive methionine residues (M16) showed the highest activity; the M16 sequence was therefore utilized for the secretory production of human leukemia inhibitory factor protein in yeast, resulting in enhanced secreted protein yield. We present a new concept for the provision of secretory signal sequence ability in the yeast K. marxianus, determined by the number of residues of a single hydrophobic residue located between N-terminal basic and C-terminal acidic amino acid boundaries.

The major facilitator superfamily transporter Knq1p modulates boron homeostasis in Kluyveromyces lactis.

PubMed

Svrbicka, Alexandra; Toth Hervay, Nora; Gbelska, Yvetta

2016-03-01

Boron is an essential micronutrient for living cells, yet its excess causes toxicity. To date, the mechanisms of boron toxicity are poorly understood. Recently, the ScATR1 gene has been identified encoding the main boron efflux pump in Saccharomyces cerevisiae. In this study, we analyzed the ScATR1 ortholog in Kluyveromyces lactis--the KNQ1 gene, to understand whether it participates in boron stress tolerance. We found that the KNQ1 gene, encoding a permease belonging to the major facilitator superfamily, is required for K. lactis boron tolerance. Deletion of the KNQ1 gene led to boron sensitivity and its overexpression increased K. lactis boron tolerance. The KNQ1 expression was induced by boron and the intracellular boron concentration was controlled by Knq1p. The KNQ1 promoter contains two putative binding motifs for the AP-1-like transcription factor KlYap1p playing a central role in oxidative stress defense. Our results indicate that the induction of the KNQ1 expression requires the presence of KlYap1p and that Knq1p like its ortholog ScAtr1p in S. cerevisiae functions as a boron efflux pump providing boron resistance in K. lactis.

Mathematical modeling of Kluyveromyces marxianus growth in solid-state fermentation using a packed-bed bioreactor.

PubMed

Mazutti, Marcio A; Zabot, Giovani; Boni, Gabriela; Skovronski, Aline; de Oliveira, Débora; Di Luccio, Marco; Rodrigues, Maria Isabel; Maugeri, Francisco; Treichel, Helen

2010-04-01

This work investigated the growth of Kluyveromyces marxianus NRRL Y-7571 in solid-state fermentation in a medium composed of sugarcane bagasse, molasses, corn steep liquor and soybean meal within a packed-bed bioreactor. Seven experimental runs were carried out to evaluate the effects of flow rate and inlet air temperature on the following microbial rates: cell mass production, total reducing sugar and oxygen consumption, carbon dioxide and ethanol production, metabolic heat and water generation. A mathematical model based on an artificial neural network was developed to predict the above-mentioned microbial rates as a function of the fermentation time, initial total reducing sugar concentration, inlet and outlet air temperatures. The results showed that the microbial rates were temperature dependent for the range 27-50 degrees C. The proposed model efficiently predicted the microbial rates, indicating that the neural network approach could be used to simulate the microbial growth in SSF.

Acquisition of the yeast Kluyveromyces marxianus from unpasteurised milk by a kefir grain enhances kefir quality.

PubMed

Gethins, Loughlin; Rea, Mary C; Stanton, Catherine; Ross, R Paul; Kilcawley, Kieran; O'Sullivan, Maurice; Crotty, Suzanne; Morrissey, John P

2016-08-01

Kefir is a fermented milk beverage consumed for nutritional and health tonic benefits in many parts of the world. It is produced by the fermentation of milk with a consortium of bacteria and yeast embedded within a polysaccharide matrix. This consortium is not well defined and can vary substantially between kefir grains. There are little data on the microbial stability of kefir grains, nor on interactions between microbes in the grain and in the milk. To study this, a grain was split, with one half of each stored at -20°C and the other half passaged repeatedly in whole unpasteurised milk. Grains passaged in the unpasteurised milk recovered vigour and acquired the yeast Kluyveromyces marxainus from the milk which was confirmed to be the same strain by molecular typing. Furthermore, these passaged grains produced kefir that was distinguished chemically and organoleptically from the stored grains. Some changes in ultrastructure were also observed by scanning electron microscopy. The study showed that kefir grains can acquire yeast from their environment and the final product can be influenced by these newly acquired yeasts. Kluyveromyces marxianus is considered to be responsible for some of the most important characteristics of kefir so the finding that this yeast is part of the less stable microbiota is significant. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Immobilized Kluyveromyces marxianus cells in carboxymethyl cellulose for production of ethanol from cheese whey: experimental and kinetic studies.

PubMed

Roohina, Fatemeh; Mohammadi, Maedeh; Najafpour, Ghasem D

2016-09-01

Cheese whey fermentation to ethanol using immobilized Kluyveromyces marxianus cells was investigated in batch and continuous operation. In batch fermentation, the yeast cells were immobilized in carboxymethyl cellulose (CMC) polymer and also synthesized graft copolymer of CMC with N-vinyl-2-pyrrolidone, denoted as CMC-g-PVP, and the efficiency of the two developed cell entrapped beads for lactose fermentation to ethanol was examined. The yeast cells immobilized in CMC-g-PVP performed slightly better than CMC with ethanol production yields of 0.52 and 0.49 g ethanol/g lactose, respectively. The effect of supplementation of cheese whey with lactose (42, 70, 100 and 150 g/l) on fermentative performance of K. marxianus immobilized in CMC beads was considered and the results were used for kinetic studies. The first order reaction model was suitable to describe the kinetics of substrate utilization and modified Gompertz model was quite successful to predict the ethanol production. For continuous ethanol fermentation, a packed-bed immobilized cell reactor (ICR) was operated at several hydraulic retention times; HRTs of 11, 15 and 30 h. At the HRT of 30 h, the ethanol production yield using CMC beads was 0.49 g/g which implies that 91.07 % of the theoretical yield was achieved.

Heterologous expression of Aspergillus terreus fructosyltransferase in Kluyveromyces lactis.

PubMed

Spohner, Sebastian C; Czermak, Peter

2016-06-25

Fructo-oligosaccharides are prebiotic and hypocaloric sweeteners that are usually extracted from chicory. They can also be produced from sucrose using fructosyltransferases, but the only commercial enzyme suitable for this purpose is Pectinex Ultra, which is produced with Aspergillus aculeatus. Here we used the yeast Kluyveromyces lactis to express a secreted recombinant fructosyltransferase from the inulin-producing fungus Aspergillus terreus. A synthetic codon-optimised version of the putative β-fructofuranosidase ATEG 04996 (XP 001214174.1) from A. terreus NIH2624 was secreted as a functional protein into the extracellular medium. At 60°C, the purified A. terreus enzyme generated the same pattern of oligosaccharides as Pectinex Ultra, but at lower temperatures it also produced oligomers with up to seven units. We achieved activities of up to 986.4U/mL in high-level expression experiments, which is better than previous reports of optimised Aspergillus spp. fermentations. Copyright © 2016 Elsevier B.V. All rights reserved.

Cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025.

PubMed

Rocha, Maria Valderez Ponte; Rodrigues, Tigressa Helena Soares; Melo, Vania M M; Gonçalves, Luciana R B; de Macedo, Gorete Ribeiro

2011-08-01

The potential of cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025 was evaluated in this work. This strain was preliminarily cultivated in a synthetic medium containing glucose and xylose and was able to produce ethanol and xylitol at pH 4.5. Next, cashew apple bagasse hydrolysate (CABH) was prepared by a diluted sulfuric acid pretreatment and used as fermentation media. This hydrolysate is rich in glucose, xylose, and arabinose and contains traces of formic acid and acetic acid. In batch fermentations of CABH at pH 4.5, the strain produced only ethanol. The effects of temperature on the kinetic parameters of ethanol fermentation by K. marxianus CE025 using CABH were also evaluated. Maximum specific growth rate (μ(max)), overall yields of ethanol based on glucose consumption [Formula: see text] and based on glucose + xylose consumption (Y ( P/S )), overall yield of ethanol based on biomass (Y ( P/X )), and ethanol productivity (P (E)) were determined as a function of temperature. Best results of ethanol production were achieved at 30°C, which is also quite close to the optimum temperature for the formation of biomass. The process yielded 12.36 ± 0.06 g l(-1) of ethanol with a volumetric production rate of 0.257 ± 0.002 g l(-1) h(-1) and an ethanol yield of 0.417 ± 0.003 g g(-1) glucose.

Ethanol production using whole plant biomass of Jerusalem artichoke by Kluyveromyces marxianus CBS1555.

PubMed

Kim, Seonghun; Park, Jang Min; Kim, Chul Ho

2013-03-01

Jerusalem artichoke is a low-requirement sugar crop containing cellulose and hemicellulose in the stalk and a high content of inulin in the tuber. However, the lignocellulosic component in Jerusalem artichoke stalk reduces the fermentability of the whole plant for efficient bioethanol production. In this study, Jerusalem artichoke stalk was pretreated sequentially with dilute acid and alkali, and then hydrolyzed enzymatically. During enzymatic hydrolysis, approximately 88 % of the glucan and xylan were converted to glucose and xylose, respectively. Batch and fed-batch simultaneous saccharification and fermentation of both pretreated stalk and tuber by Kluyveromyces marxianus CBS1555 were effectively performed, yielding 29.1 and 70.2 g/L ethanol, respectively. In fed-batch fermentation, ethanol productivity was 0.255 g ethanol per gram of dry Jerusalem artichoke biomass, or 0.361 g ethanol per gram of glucose, with a 0.924 g/L/h ethanol productivity. These results show that combining the tuber and the stalk hydrolysate is a useful strategy for whole biomass utilization in effective bioethanol fermentation from Jerusalem artichoke.

Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

PubMed

Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

2011-02-01

A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry. Copyright © 2010 Elsevier Ltd. All rights reserved.

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1.

PubMed

Baruffini, Enrico; Goffrini, Paola; Donnini, Claudia; Lodi, Tiziana

2006-12-01

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.

Carboxylic acids permeases in yeast: two genes in Kluyveromyces lactis.

PubMed

Lodi, Tiziana; Fontanesi, Flavia; Ferrero, Iliana; Donnini, Claudia

2004-09-15

Two new genes KlJEN1 and KlJEN2 were identified in Kluyveromyces lactis. The deduced structure of their products is typical of membrane-bound carriers and displays high similarity to Jen1p, the monocarboxylate permease of Saccharomyces cerevisiae. Both KlJEN1 and KlJEN2 are under the control of glucose repression mediated by FOG1 and FOG2, corresponding to S. cerevisiae GAL83 and SNF1 respectively, and KlCAT8, proteins involved in glucose signalling cascade in K. lactis. KlJEN1, but not KlJEN2, is induced by lactate. KlJEN2 in contrast is expressed at high level in ethanol and succinate. The physiological characterization of null mutants showed that KlJEN1 is the functional homologue of ScJEN1, whereas KlJEN2 encodes a dicarboxylic acids transporter. In fact, KlJen1p [transporter classification (TC) number: 2.A.1.12.2.] is required for lactate uptake and therefore for growth on lactate. KlJen2p is required for succinate transport, as demonstrated by succinate uptake experiments and by inability of Kljen2 mutant to grow on succinate. This carrier appears to transport also malate and fumarate because the Kljen2 mutant cannot grow on these substrates and the succinate uptake is competed by these carboxylic acids. We conclude that KlJEN2 is the first yeast gene shown to encode a dicarboxylic acids permease.

Metals sorption from aqueous solutions by Kluyveromyces marxianus: process optimization, equilibrium modeling and chemical characterization.

PubMed

Pal, Rama; Tewari, Saumyata; Rai, Jai P N

2009-10-01

The dead Kluyveromyces marxianus biomass, a fermentation industry waste, was used to explore its sorption potential for lead, mercury, arsenic, cobalt, and cadmium as a function of pH, biosorbent dosage, contact time, agitation speed, and initial metal concentration. The equilibrium data fitted the Langmuir model better for cobalt and cadmium, but Freundlich isotherm for all metals tested. At equilibrium, the maximum uptake capacity (Qmax) was highest for lead followed by mercury, arsenic, cobalt, and cadmium. The RL values ranged between 0-1, indicating favorable sorption of all test metals by the biosorbent. The maximum Kf value of Pb showed its efficient removal from the solution. However, multi-metal analysis depicted that sorption of all metals decreased except Pb. The potentiometric titration of biosorbent revealed the presence of functional groups viz. amines, carboxylic acids, phosphates, and sulfhydryl group involved in heavy metal sorption. The extent of contribution of functional groups and lipids to biosorption was in the order: carboxylic>lipids>amines>phosphates. Blocking of sulfhydryl group did not have any significant effect on metal sorption.

Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis

PubMed Central

Colussi, Paul A.; Specht, Charles A.; Taron, Christopher H.

2005-01-01

Endogenous proteins secreted from Kluyveromyces lactis were screened for their ability to bind to or to hydrolyze chitin. This analysis resulted in identification of a nucleus-encoded extracellular chitinase (KlCts1p) with a chitinolytic activity distinct from that of the plasmid-encoded killer toxin α-subunit. Sequence analysis of cloned KlCTS1 indicated that it encodes a 551-amino-acid chitinase having a secretion signal peptide, an amino-terminal family 18 chitinase catalytic domain, a serine-threonine-rich domain, and a carboxy-terminal type 2 chitin-binding domain. The association of purified KlCts1p with chitin is stable in the presence of high salt concentrations and pH 3 to 10 buffers; however, complete dissociation and release of fully active KlCts1p occur in 20 mM NaOH. Similarly, secreted human serum albumin harboring a carboxy-terminal fusion with the chitin-binding domain derived from KlCts1p also dissociates from chitin in 20 mM NaOH, demonstrating the domain's potential utility as an affinity tag for reversible chitin immobilization or purification of alkaliphilic or alkali-tolerant recombinant fusion proteins. Finally, haploid K. lactis cells harboring a cts1 null mutation are viable but exhibit a cell separation defect, suggesting that KlCts1p is required for normal cytokinesis, probably by facilitating the degradation of septum-localized chitin. PMID:15932978

Characterization of a nucleus-encoded chitinase from the yeast Kluyveromyces lactis.

PubMed

Colussi, Paul A; Specht, Charles A; Taron, Christopher H

2005-06-01

Endogenous proteins secreted from Kluyveromyces lactis were screened for their ability to bind to or to hydrolyze chitin. This analysis resulted in identification of a nucleus-encoded extracellular chitinase (KlCts1p) with a chitinolytic activity distinct from that of the plasmid-encoded killer toxin alpha-subunit. Sequence analysis of cloned KlCTS1 indicated that it encodes a 551-amino-acid chitinase having a secretion signal peptide, an amino-terminal family 18 chitinase catalytic domain, a serine-threonine-rich domain, and a carboxy-terminal type 2 chitin-binding domain. The association of purified KlCts1p with chitin is stable in the presence of high salt concentrations and pH 3 to 10 buffers; however, complete dissociation and release of fully active KlCts1p occur in 20 mM NaOH. Similarly, secreted human serum albumin harboring a carboxy-terminal fusion with the chitin-binding domain derived from KlCts1p also dissociates from chitin in 20 mM NaOH, demonstrating the domain's potential utility as an affinity tag for reversible chitin immobilization or purification of alkaliphilic or alkali-tolerant recombinant fusion proteins. Finally, haploid K. lactis cells harboring a cts1 null mutation are viable but exhibit a cell separation defect, suggesting that KlCts1p is required for normal cytokinesis, probably by facilitating the degradation of septum-localized chitin.

Polygalacturonase and ethanol production in Kluyveromyces marxianus: potential use of polygalacturonase in foodstuffs.

PubMed

Serrat, Manuel; Bermúdez, Rosa C; Villa, Tomás G

2004-04-01

The coproduction of ethanol and polygalacturonase (PG) in a pilot-scale batch fermentor using yeast extract--glucose (YD)--and sugar beet molasses (SBM)-based media was implemented utilizing a new high-PG-producing strain of Kluyveromyces marxianus. A certain growth inhibition was observed in SBM medium, causing ethanol and PG production to be lower. Ethanol productivity and accumulation values of 1.94 g/(L x h) and 40 g/L, respectively, were attained in YD, whereas the best fermentation efficiency (95.1%) was achieved with SBM medium. Maximal PG synthesis occurred at the end of cell growth, with values of 1.08 and 0.46 U/(mg x h) for the YD and SBM media, respectively. When the cultures reached stationary phase, PG production stopped. The highest accumulation level (17 U/mL) occurred in YD medium, in agreement with previous laboratory-scale studies carried out for this strain. The potential applications of the crude enzyme preparations were evaluated with different fruit juices and vegetable slices. The enzyme was able to increase the filtration rate of orange, pear, and apple juices by twofold. Additionally, complete clarification of apple juice was readily accomplished, whereas cucumber, carrot, and banana tissues were macerated to a lesser extent. Copyright 2004 Humana Press Inc.

Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions.

PubMed

Yuan, W J; Chang, B L; Ren, J G; Liu, J P; Bai, F W; Li, Y Y

2012-01-01

Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase-producing yeast Kluyveromyces marxianus Y179 and fed-batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch-based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l(-1) was achieved when 0·15 g l(-1) K(2) HPO(4) was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Jerusalem artichoke tubers are an alternative to grain-based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation.

PubMed

Yamaoka, Chizuru; Kurita, Osamu; Kubo, Tomoko

2014-12-01

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool. Copyright © 2014 Elsevier GmbH. All rights reserved.

Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.

PubMed

Hoshida, Hisashi; Murakami, Nobutada; Suzuki, Ayako; Tamura, Ryoko; Asakawa, Jun; Abdel-Banat, Babiker M A; Nonklang, Sanom; Nakamura, Mikiko; Akada, Rinji

2014-01-01

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright © 2013 John Wiley & Sons, Ltd.

Growth and ethanol fermentation ability on hexose and pentose sugars and glucose effect under various conditions in thermotolerant yeast Kluyveromyces marxianus.

PubMed

Rodrussamee, Nadchanok; Lertwattanasakul, Noppon; Hirata, Katsushi; Suprayogi; Limtong, Savitree; Kosaka, Tomoyuki; Yamada, Mamoru

2011-05-01

Ethanol fermentation ability of the thermotolerant yeast Kluyveromyces marxianus, which is able to utilize various sugars including glucose, mannose, galactose, xylose, and arabinose, was examined under shaking and static conditions at high temperatures. The yeast was found to produce ethanol from all of these sugars except for arabinose under a shaking condition but only from hexose sugars under a static condition. Growth and sugar utilization rate under a static condition were slower than those under a shaking condition, but maximum ethanol yield was slightly higher. Even at 40°C, a level of ethanol production similar to that at 30°C was observed except for galactose under a static condition. Glucose repression on utilization of other sugars was observed, and it was more evident at elevated temperatures. Consistent results were obtained by the addition of 2-deoxyglucose. The glucose effect was further examined at a transcription level, and it was found that KmGAL1 for galactokinase and KmXYL1 for xylose reductase for galactose and xylose/arabinose utilization, respectively, were repressed by glucose at low and high temperatures, but KmHXK2 for hexokinase was not repressed. We discuss the possible mechanism of glucose repression and the potential for utilization of K. marxianus in high-temperature fermentation with mixed sugars containing glucose.

Fermentation and aerobic metabolism of cellodextrins by yeasts. [Candida wickerhamii; C. guiliermondii; C. molischiana; Debaryomyces polymorphus; Pichia guilliermondii; Clavispora lusitaniae; Kluyveromyces lactis; Brettanomyces claussenii; Rhodotorula minuta; Dekkera intermedia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Freer, S.N.

1991-03-01

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wicherhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization {<=} 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettanomycesmore » claussenii, Brettanomyces anomalus, Kluyveromyces dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization >3 produced an enzyme(s) that hydrolyzed cellotretose.« less

Statistical investigation of Kluyveromyces lactis cells permeabilization with ethanol by response surface methodology.

PubMed

de Faria, Janaína T; Rocha, Pollyana F; Converti, Attilio; Passos, Flávia M L; Minim, Luis A; Sampaio, Fábio C

2013-12-01

The aim of our study was to select the optimal operating conditions to permeabilize Kluyveromyces lactis cells using ethanol as a solvent as an alternative to cell disruption and extraction. Cell permeabilization was carried out by a non-mechanical method consisting of chemical treatment with ethanol, and the results were expressed as β-galactosidase activity. Experiments were conducted under different conditions of ethanol concentration, treatment time and temperature according to a central composite rotatable design (CCRD), and the collected results were then worked out by response surface methodology (RSM). Cell permeabilization was improved by an increase in ethanol concentration and simultaneous decreases in the incubation temperature and treatment time. Such an approach allowed us to identify an optimal range of the independent variables within which the β-galactosidase activity was optimized. A maximum permeabilization of 2,816 mmol L(-1) oNP min(-1) g(-1) was obtained by treating cells with 75.0% v/v of ethanol at 20.0 °C for 15.0 min. The proposed methodology resulted to be effective and suited for K. lactis cells permeabilization at a lab-scale and promises to be of possible interest for future applications mainly in the food industry.

Comparison of a pectinolytic extract of Kluyveromyces marxianus and a commercial enzyme preparation in the production of Ives (Vitis labrusca) grape juice.

PubMed

Piemolini-Barreto, Luciani Tatsch; Antônio, Regina Vasconcellos; Echeverrigaray, Sergio

2015-05-01

This study analyses the effect of the crude enzymatic extract produced by Kluyveromyces marxianus (EEB) in the maceration and clarification of juice produced from Ives (Vitis labrusca) grapes compared to the commercial enzyme preparation Pectinex(®)Ultra Color (PEC). Treatments were conducted with a total pectinolytic activity of 1 U/mL of fruit juice, at 40 °C, for 60 min. After the enzymatic treatment, the juices were evaluated with respect to yield, viscosity, and degree of clarification, as well as the effect of the enzymes on polyphenol concentration, anthocyanins, and juice color. The results showed that both EEB and PEC increase yield, reduce viscosity and contribute to the clarification of grape juice. After enzyme treatment with the EEB preparation, the extraction yield increased 28.02 % and decreased 50.70 % in viscosity during the maceration of the pulp. During the juice production process clarification increased 11.91 %. With PEC, higher values for these parameters: 42.36, 63.20, and 26.81 % respectively, were achieved. The addition of EEB resulted in grape juice with better color intensity and extraction of phenolic compounds and anthocyanins. Considering all comparison criteria, the enzymatic extract of K. marxianus NRRL-Y-7571 can potentially be used in the production of juice.

Cloning, production, and functional expression of the bacteriocin enterocin A, produced by Enterococcus faecium T136, by the yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans.

PubMed

Borrero, Juan; Kunze, Gotthard; Jiménez, Juan J; Böer, Erik; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2012-08-01

The bacteriocin enterocin A (EntA) produced by Enterococcus faecium T136 has been successfully cloned and produced by the yeasts Pichia pastoris X-33EA, Kluyveromyces lactis GG799EA, Hansenula polymorpha KL8-1EA, and Arxula adeninivorans G1212EA. Moreover, P. pastoris X-33EA and K. lactis GG799EA produced EntA in larger amounts and with higher antimicrobial and specific antimicrobial activities than the EntA produced by E. faecium T136.

Transformation of Saccharomyces cerevisiae with linear DNA killer plasmids from Kluyveromyces lactis.

PubMed Central

Gunge, N; Murata, K; Sakaguchi, K

1982-01-01

Protoplasts of Saccharomyces cerevisiae were mixed with linear DNA plasmids, pGKl1 and pGKl2, isolated from a Kluyveromyces lactis killer strain and treated with polyethylene glycol. Out of 2,000 colonies regenerated on a nonselective medium, two killer transformants were obtained. The pGKl plasmids and the killer character were stably maintained in one (Pdh-1) of them. Another transformant, Pdl-1, was a weak killer, and the subclones consisted of a mixture of weak and nonkiller cells. The weak killers were characterized by the presence of pGKl1 in a decreased amount, and nonkillers were characterized by the absence of pGKl1. The occurrence of two new plasmids which migrated faster than pGKl1 in an agarose gel was observed in Pdl-1 and its subclones, whether weak or nonkillers. Staining with 4',6-diamidino-2-phenylindole revealed that the pGKl plasmids exist in the cytosol of transformant cells with numerous copy numbers. Images PMID:7045080

Automated UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 and selection for microaerophilic growth and ethanol production at elevated temperature on biomass sugars.

PubMed

Hughes, Stephen R; Bang, Sookie S; Cox, Elby J; Schoepke, Andrew; Ochwat, Kate; Pinkelman, Rebecca; Nelson, Danielle; Qureshi, Nasib; Gibbons, William R; Kurtzman, Cletus P; Bischoff, Kenneth M; Liu, Siqing; Cote, Gregory L; Rich, Joseph O; Jones, Marjorie A; Cedeño, David; Doran-Peterson, Joy; Riaño-Herrera, Nestor M; Rodríguez-Valencia, Nelson; López-Núñez, Juan C

2013-08-01

The yeast Kluyveromyces marxianus is a potential microbial catalyst for fuel ethanol production from a wide range of biomass substrates. To improve its growth and ethanol yield at elevated temperature under microaerophilic conditions, K. marxianus NRRL Y-1109 was irradiated with UV-C using automated protocols on a robotic platform for picking and spreading irradiated cultures and for processing the resulting plates. The plates were incubated under anaerobic conditions on xylose or glucose for 5 mo at 46 °C. Two K. marxianus mutant strains (designated 7-1 and 8-1) survived and were isolated from the glucose plates. Both mutant strains, but not wild type, grew aerobically on glucose at 47 °C. All strains grew anaerobically at 46 °C on glucose, galactose, galacturonic acid, and pectin; however, only 7-1 grew anaerobically on xylose at 46 °C. Saccharomyces cerevisiae NRRL Y-2403 did not grow at 46 °C on any of these substrates. With glucose as a carbon source, ethanol yield after 3 d at 46 °C was higher for 8-1 than for wild type (0.51 and 0.43 g ethanol/g glucose, respectively). With galacturonic acid as a carbon source, the ethanol yield after 7 d at 46 °C was higher for 7-1 than for wild type (0.48 and 0.34 g ethanol/g galacturonic acid, respectively). These mutant strains have potential application in fuel ethanol production at elevated temperature from sugar constituents of starch, sucrose, pectin, and cellulosic biomass.

Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and alpha-amylase to produce ethanol.

PubMed

Wang, Rongliang; Wang, Dongmei; Gao, Xiaolian; Hong, Jiong

2014-01-01

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α-amylase from Aspergillus oryzae as well as α-amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date. © 2014 American Institute of Chemical Engineers.

Simultaneous fermentation of glucose and xylose at elevated temperatures co-produces ethanol and xylitol through overexpression of a xylose-specific transporter in engineered Kluyveromyces marxianus.

PubMed

Zhang, Biao; Zhang, Jia; Wang, Dongmei; Han, Ruixiang; Ding, Rui; Gao, Xiaolian; Sun, Lianhong; Hong, Jiong

2016-09-01

Engineered Kluyveromyces marxianus strains were constructed through over-expression of various transporters for simultaneous co-fermentation of glucose and xylose. The glucose was converted into ethanol, whereas xylose was converted into xylitol which has higher value than ethanol. Over-expressing xylose-specific transporter ScGAL2-N376F mutant enabled yeast to co-ferment glucose and xylose and the co-fermentation ability was obviously improved through increasing ScGAL2-N376F expression. The production of glycerol was blocked and acetate production was reduced by disrupting gene KmGPD1. The obtained K. marxianus YZJ119 utilized 120g/L glucose and 60g/L xylose simultaneously and produced 50.10g/L ethanol and 55.88g/L xylitol at 42°C. The yield of xylitol from consumed xylose was over 98% (0.99g/g). Through simultaneous saccharification and co-fermentation at 42°C, YZJ119 produced a maximal concentration of 44.58g/L ethanol and 32.03g/L xylitol or 29.82g/L ethanol and 31.72g/L xylitol, respectively, from detoxified or non-detoxified diluted acid pretreated corncob. Copyright © 2016 Elsevier Ltd. All rights reserved.

Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis.

PubMed

Zhan, Fei Xiang; Wang, Qin Hong; Jiang, Si Jing; Zhou, Yu Ling; Zhang, Gui Min; Ma, Yan He

2014-12-16

Xylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking. The xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread. Xylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory

Growth kinetics and physiological behavior of co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis, fermenting carob sugars extracted with whey.

PubMed

Rodrigues, B; Lima-Costa, M E; Constantino, A; Raposo, S; Felizardo, C; Gonçalves, D; Fernandes, T; Dionísio, L; Peinado, J M

2016-10-01

Alcoholic fermentation of carob waste sugars (sucrose, glucose and fructose) extracted with cheese whey, by co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis has been analyzed. Growth and fermentation of S. cerevisiae in the carob-whey medium showed an inhibition of about 30% in comparison with water-extracted carob. The inhibition of K. lactis on carob-whey was greater (70%) when compared with the whey medium alone, due to osmolarity problems. Oxygen availability was a very important factor for K. lactis, influencing its fermentation performance. When K. lactis was grown alone on carob-whey medium, lactose was always consumed first, and glucose and fructose were consumed afterwards, only at high aeration conditions. In co-culture with S. cerevisiae, K. lactis was completely inhibited and, at low aeration, died after 3 days; at high aeration this culture could survive but growth and lactose fermentation were only recovered after S. cerevisiae became stationary. To overcome the osmolarity and K. lactis' oxygen problems, the medium had to be diluted and a sequential fermentative process was designed in a STR-3l reactor. K. lactis was inoculated first and, with low aeration (0.13vvm), consumed all the lactose in 48h. Then S. cerevisiae was inoculated, consuming the total of the carob sugars, and producing ethanol in a fed-batch regime. The established co-culture with K. lactis increased S. cerevisiae ethanol tolerance. This fermentation process produced ethanol with good efficiency (80g/l final concentration and a conversion factor of 0.4g ethanol/g sugar), eliminating all the sugars of the mixed waste. These efficient fermentative results pointed to a new joint treatment of agro-industrial wastes which may be implemented successfully, with economic and environmental sustainability for a bioethanol industrial proposal. Copyright © 2016 Elsevier Inc. All rights reserved.

Improving the Organoleptic Properties of a Craft Mezcal Beverage by Increasing Fatty Acid Ethyl Ester Contents through ATF1 Expression in an Engineered Kluyveromyces marxianus UMPe-1 Yeast.

PubMed

Campos-García, Jesús; Vargas, Alejandra; Farías-Rosales, Lorena; Miranda, Ana L; Meza-Carmen, Víctor; Díaz-Pérez, Alma L

2018-05-02

Mezcal, a traditional beverage that originated in Mexico, is produced from species of the Agavaceae family. The esters associated with the yeasts utilized during fermentation are important for improving the organoleptic properties of the beverage. We improved the ester contents in a mezcal beverage by using the yeast Kluyveromyces marxianus, which was engineered with the ATF1 gene. ATF1 expression in the recombinant yeast significantly increased compared with that in the parental yeast, but its fermentative parameters were unchanged. Volatile-organic-compound-content analysis showed that esters had significantly increased in the mezcal produced with the engineered yeast. In a sensory-panel test, 48% of the panelists preferred the mezcal produced from the engineered yeast, 30% preferred the mezcal produced from the wild type, and 15 and 7% preferred the two mezcal types produced following the routine procedure. Correlation analysis showed that the fruitiness/sweetness description of the mezcal produced using the ATF1-engineered K. marxianus yeast correlated with the content of the esters, whose presence improved the organoleptic properties of the craft mezcal beverage.

Ethanol production from sunflower meal biomass by simultaneous saccharification and fermentation (SSF) with Kluyveromyces marxianus ATCC 36907.

PubMed

Camargo, Danielle; Gomes, Simone D; Sene, Luciane

2014-11-01

The lignocellulosic materials are considered promising renewable resources for ethanol production, but improvements in the processes should be studied to reduce operating costs. Thus, the appropriate enzyme loading for cellulose saccharification is critical for process economics. This study aimed at evaluating the concentration of cellulase and β-glucosidase in the production of bioethanol by simultaneous saccharification and fermentation (SSF) of sunflower meal biomass. The sunflower biomass was pretreated with 6% H2SO4 (w/v), at 121 °C, for 20 min, for hemicellulose removal and delignificated with 1% NaOH. SSF was performed with Kluyveromyces marxianus ATCC 36907, at 38 °C, 150 rpm, for 72 h, with different enzyme concentrations (Cellulase Complex NS22086-10, 15 and 20 FPU/gsubstrate and β-Glucosidase NS22118, with a cellulase to β-glucosidase ratio of 1.5:1; 2:1 and 3:1). The best condition for ethanol production was cellulase 20 FPU/gsubstrate and β-glucosidase 13.3 CBU/gsubstrate, resulting in 27.88 g/L ethanol, yield of 0.47 g/g and productivity of 0.38 g/L h. Under this condition the highest enzymatic conversion of cellulose to glucose was attained (87.06%).

Lignocellulosic sugar management for xylitol and ethanol fermentation with multiple cell recycling by Kluyveromyces marxianus IIPE453.

PubMed

Dasgupta, Diptarka; Ghosh, Debashish; Bandhu, Sheetal; Adhikari, Dilip K

2017-07-01

Optimum utilization of fermentable sugars from lignocellulosic biomass to deliver multiple products under biorefinery concept has been reported in this work. Alcohol fermentation has been carried out with multiple cell recycling of Kluyveromyces marxianus IIPE453. The yeast utilized xylose-rich fraction from acid and steam treated biomass for cell generation and xylitol production with an average yield of 0.315±0.01g/g while the entire glucose rich saccharified fraction had been fermented to ethanol with high productivity of 0.9±0.08g/L/h. A detailed insight into its genome illustrated the strain's complete set of genes associated with sugar transport and metabolism for high-temperature fermentation. A set flocculation proteins were identified that aided in high cell recovery in successive fermentation cycles to achieve alcohols with high productivity. We have brought biomass derived sugars, yeast cell biomass generation, and ethanol and xylitol fermentation in one platform and validated the overall material balance. 2kg sugarcane bagasse yielded 193.4g yeast cell, and with multiple times cell recycling generated 125.56g xylitol and 289.2g ethanol (366mL). Copyright © 2017 Elsevier GmbH. All rights reserved.

Partial purification and characterization of exoinulinase from Kluyveromyces marxianus YS-1 for preparation of high-fructose syrup.

PubMed

Singh, Ram Sarup; Dhaliwal, Rajesh; Puri, Munish

2007-05-01

An extracellular exoinulinase (2,1-beta-D fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable (100%) for 3 h at the optimum temperature of 50 degrees C. Mn2+ and Ca2+ produced a 2.4-fold and 1.2-fold enhancement in enzyme activity, whereas Hg2+ and Ag2+ completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6 mg/ml and 41.3 mg/ml, respectively.

Toxicity of nalidixic acid on candida albicans, Saccharomyces cerevisiae, and Kluyveromyces lactis.

PubMed

Sobieski, R J; Brewer, A R

1976-03-01

The antibacterial drug nalidixic acid (Nal) can suppress the growth of Candida albicans at levels of the drug normally found in urine. Growth suppression increases as drug levels are increased, and Nal also causes a similar proportional inhibition of the synthesis of all cellular macromolecules. However, growth temperature (25 versus 37 C) and the divalent cations Mg(2+) and Mn(2+) can increase C. albicans resistance to Nal. Also, nitrogen depletion of Candida shows that Nal-treated and untreated cells exhibit no difference in leucine uptake during readaptation to nitrogen. In Nal-treated, nitrogen-starved cells, ribonucleic acid and deoxyribonucleic acid (DNA) biosynthesis are less affected than in unstarved Nal-treated cells, but of the two nucleic acids DNA synthesis is the most affected. Nal-resistant strains of C. albicans exhibit a slight toxicity for macromolecular synthesis. Nal treatment of a synchronized population of Saccharomyces cerevisiae results in an increase in the culture mean doubling time of, at most, 20%, but Nal causes the loss of synchronous cell division. With a synchronized population of Kluyveromyces lactis, Nal causes an increase in the mean doubling time of upwards of 300%, with synchrony of cell division being maintained. It is known that S. cerevisiae asynchronously synthesizes mitochondrial DNA during the cell cycle, whereas with K. lactis it is synchronous. Thus, with C. albicans Nal toxicity is dependent both on the dose and the physiological state of the cell. Furthermore, Nal inhibits growth of yeast with synchronous mitochondrial DNA synthesis more adversely than yeast with asynchronous mitochondrial DNA synthesis.

Production of antioxidant and ACE-inhibitory peptides from Kluyveromyces marxianus protein hydrolysates: Purification and molecular docking.

PubMed

Mirzaei, Mahta; Mirdamadi, Saeed; Ehsani, Mohamad Reza; Aminlari, Mahmoud

2018-04-01

Kluyveromyces marxianus protein hydrolysates were prepared by two different sonicated-enzymatic (trypsin and chymotrypsin) hydrolysis treatments to obtain antioxidant and ACE-inhibitory peptides. Trypsin and chymotrypsin hydrolysates obtained by 5 h, exhibited the highest antioxidant and ACE-inhibitory activities. After fractionation using ultrafiltration and reverse phase high performance liquid chromatography (RP-HPLC) techniques, two new peptides were identified. One fragment (LL-9, MW = 1180 Da) with the amino acid sequence of Leu-Pro-Glu-Ser-Val-His-Leu-Asp-Lys showed significant ACE inhibitory activity (IC 50  = 22.88 μM) while another peptide fragment (VL-9, MW = 1118 Da) with the amino acid sequence of Val-Leu-Ser-Thr-Ser-Phe-Pro-Pro-Lys showed the highest antioxidant and ACE inhibitory properties (IC 50  = 15.20 μM, 5568 μM TE/mg protein). The molecular docking studies revealed that the ACE inhibitory activities of VL-9 is due to interaction with the S2 (His513, His353, Glu281) and S'1 (Glu162) pockets of ACE and LL-9 can fit perfectly into the S1 (Thr345) and S2 (Tyr520, Lys511, Gln281) pockets of ACE. Copyright © 2017. Published by Elsevier B.V.

Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis.

PubMed

Jo, Hyun-Joo; Noh, Jin-Seok; Kong, Kwang-Hoon

2013-08-01

Brazzein is an intensely sweet-tasting protein with high water solubility, heat stability, and taste properties resembling those of carbohydrate sweeteners. In the present study, we describe the expression of the synthetic gene encoding brazzein, a sweet protein in the yeast Kluyveromyces lactis. The synthetic brazzein gene was designed based on the biased codons of the yeast, so as to optimize its expression, as well as on the extracellular secretion for expression in an active, soluble form. The synthesized brazzein gene was cloned into the secretion vector pKLAC2, which contains the yeast prepropeptide signal from the Saccharomycescerevisiae α-mating factor. The constructed plasmid pKLAC2-des-pE1M-brazzein was introduced into the yeast K. lactis GG799. The yeast transformants were cultured for high-yield secretion of the recombinant des-pE1M-brazzein in YPGal medium for 96 h at 30°C. The expressed recombinant des-pE1M-brazzein was purified by CM-Sepharose column chromatography and approximately 104 mg/L was obtained. The purity and conformational state of the recombinant des-pE1M-brazzein were confirmed using SDS-PAGE, HPLC, and circular dichroism. The identity of the recombinant protein was also confirmed by N-terminal amino acid analysis and taste testing. The purified recombinant des-pE1M-brazzein had an intrinsic sweetness in its minor form, approximately 2130 times sweeter than sucrose on a weight basis. These results demonstrate that the K. lactis expression system is useful for producing the recombinant brazzein in active form at a high yield with attributes useful in the food industry. Copyright © 2013 Elsevier Inc. All rights reserved.

Reaction kinetics and galactooligosaccharide product profiles of the β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae.

PubMed

Yin, Huifang; Bultema, Jelle B; Dijkhuizen, Lubbert; van Leeuwen, Sander S

2017-06-15

β-Galactosidase enzymes are used in the dairy industry to convert lactose into galactooligosaccharides (GOS) that are added to infant formula to mimic the molecular sizes and prebiotic functions of human milk oligosaccharides. Here we report a detailed analysis of the clearly different GOS profiles of the commercial β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae. Also the GOS yields of these enzymes differed, varying from 48.3% (B. circulans) to 34.9% (K. lactis), and 19.5% (A. oryzae). Their incubation with lactose plus the monosaccharides Gal or Glc resulted in altered GOS profiles. Experiments with 13 C 6 labelled Gal and Glc showed that both monosaccharides act as acceptor substrates in the transgalactosylation reactions. The data shows that the lactose isomers β-d-Galp-(1→2)-d-Glcp, β-d-Galp-(1→3)-d-Glcp and β-d-Galp-(1→6)-d-Glcp are formed from acceptor reactions with free Glc and not by rearrangement of Glc in the active site. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

PubMed Central

Saliola, Michele; Bartoccioni, Paola Chiara; De Maria, Ilaria; Lodi, Tiziana; Falcone, Claudio

2004-01-01

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose. PMID:15189981

The modeling of ethanol production by Kluyveromyces marxianus using whey as substrate in continuous A-Stat bioreactors.

PubMed

Gabardo, Sabrina; Pereira, Gabriela Feix; Rech, Rosane; Ayub, Marco Antônio Záchia

2015-09-01

We investigated the kinetics of whey bioconversion into ethanol by Kluyveromyces marxianus in continuous bioreactors using the "accelerostat technique" (A-stat). Cultivations using free and Ca-alginate immobilized cells were evaluated using two different acceleration rates (a). The kinetic profiles of these systems were modeled using four different unstructured models, differing in the expressions for the specific growth (μ) and substrate consumption rates (r s), taking into account substrate limitation and product inhibition. Experimental data showed that the dilution rate (D) directly affected cell physiology and metabolism. The specific growth rate followed the dilution rate (μ≈D) for the lowest acceleration rate (a = 0.0015 h(-2)), condition in which the highest ethanol yield (0.52 g g(-1)) was obtained. The highest acceleration rate (a = 0.00667 h(-2)) led to a lower ethanol yield (0.40 g g(-1)) in the system where free cells were used, whereas with immobilized cells ethanol yields increased by 23 % (0.49 g g(-1)). Among the evaluated models, Monod and Levenspiel combined with Ghose and Tyagi models were found to be more appropriate for describing the kinetics of whey bioconversion into ethanol. These results may be useful in scaling up the process for ethanol production from whey.

Production, purification, and characterization of a polygalacturonase from a new strain of Kluyveromyces marxianus isolated from coffee wet-processing wastewater.

PubMed

Serrat, Manuel; Bermúdez, Rose Catalina; Villa, Tomás Gonzáles

2002-03-01

A new high polygalacturonase (PG)-producing Kluyveromyces marxianus strain was isolated from coffee wet-processing wastewater. PG production in this strain is not repressed in the presence of 100 g/L of glucose and, being growth-associated, reached its maximum accumulation in the culture medium at the beginning of the stationary phase. Oxygen and galacturonic acid negatively regulated enzyme synthesis, and glucose as the carbon source afforded better enzyme yields than lactose. The data reported here show that this strain exhibits the highest index of PG production among the wild-type strains reported so far (18.8 U/mL). PG was readily purified by ion-exchange chromatography on SP-Sepharose FF. The activity corresponded to a single protein with an M(r) of 41.7kDa according to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The enzyme was stable in the pH range of 3.0-5.0 and displayed an optimal temperature of 55 degrees C; it showed a typical endosplitting way of substrate hydrolysis and exhibited a fair degree of activity on pectin with a high degree of esterification.

Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to fermentative metabolism by deletion of NDI1

PubMed Central

González-Siso, María Isabel; Touriño, Alba; Vizoso, Ángel; Pereira-Rodríguez, Ángel; Rodríguez-Belmonte, Esther; Becerra, Manuel; Cerdán, María Esperanza

2015-01-01

In this paper, we report the metabolic engineering of the respiratory yeast Kluyveromyces lactis by construction and characterization of a null mutant (Δklndi1) in the single gene encoding a mitochondrial alternative internal dehydrogenase. Isolated mitochondria of the Δklndi1 mutant show unaffected rate of oxidation of exogenous NADH, but no oxidation of matrix NADH; this confirms that KlNdi1p is the only internal NADH dehydrogenase in K. lactis mitochondria. Permeabilized cells of the Δklndi1 mutant do not show oxidation of matrix NADH, which suggests that shuttle systems to transfer the NADH from mitochondrial matrix to cytosol, for being oxidized by external dehydrogenases, are not functional. The Δklndi1 mutation decreases the chronological life span in absence of nutrients. The expression of KlNDI1 is increased by glutathione reductase depletion. The Δklndi1 mutation shifts the K. lactis metabolism from respiratory to fermentative: the Δklndi1 strain shows reduced respiration rate and increased ethanol production from glucose, while it does not grow in non-fermentable carbon sources such as lactate. The biotechnological benefit of the Δklndi1 mutant for bioethanol production from waste cheese whey lactose was proved. PMID:25186243

Heterologous expression of an α-amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae.

PubMed

Brain-Isasi, Stephanie; Álvarez-Lueje, Alejandro; Higgins, Thomas Joseph V

2017-06-15

Phaseolamin or α-amylase inhibitor 1 (αAI) is a glycoprotein from common beans (Phaseolus vulgaris L.) that inhibits some insect and mammalian α-amylases. Several clinical studies support the beneficial use of bean αAI for control of diabetes and obesity. Commercial extracts of P. vulgaris are available but their efficacy is still under question, mainly because some of these extracts contain antinutritional impurities naturally present in bean seeds and also exhibit a lower specific activity αAI. The production of recombinant αAI allows to overcome these disadvantages and provides a platform for the large-scale production of pure and functional αAI protein for biotechnological and pharmaceutical applications. A synthetic gene encoding αAI from the common bean (Phaseolus vulgaris cv. Pinto) was codon-optimised for expression in yeasts (αAI-OPT) and cloned into the protein expression vectors pKLAC2 and pYES2. The yeasts Kluyveromyces lactis GG799 (and protease deficient derivatives such as YCT390) and Saccharomyces cerevisiae YPH499 were transformed with the optimised genes and transformants were screened for expression by antibody dot blot. Recombinant colonies of K. lactis YCT390 that expressed and secreted functional αAI into the culture supernatants were selected for further analyses. Recombinant αAI from K. lactis YCT390 was purified using anion-exchange and affinity resins leading to the recovery of a functional inhibitor. The identity of the purified αAI was confirmed by mass spectrometry. Recombinant clones of S. cerevisiae YPH499 expressed functional αAI intracellularly, but did not secrete the protein. This is the first report describing the heterologous expression of the α-amylase inhibitor 1 (αAI) from P. vulgaris in yeasts. We demonstrated that recombinant strains of K. lactis and S. cerevisiae expressed and processed the αAI precursor into mature and active protein and also showed that K. lactis secretes functional αAI.

Attraction of Coffee Bean Weevil, Araecerus fasciculatus, to Volatiles from the Industrial Yeast Kluyveromyces lactis.

PubMed

Yang, Shuai; Mei, Xiang-Dong; Zhang, Xiao-Fang; Li, Yao-Fa; She, Dongmei; Zhang, Tao; Ning, Jun

2017-02-01

The coffee bean weevil (CBW), Araecerus fasciculatus (De Geer, 1775) (Coleoptera: Anthribidae) is an important pest of stored products such as grains, coffee beans, cassava, and traditional Chinese medicine materials. In China, CBW causes large losses of Daqu, a traditional Chinese liquor fermentation starter, and, unfortunately, the use of conventional insecticides against CBW is not suitable in Daqu storage. We found CBW to be highly attracted to fermenting yeast cultures, such as Kluyveromyces lactis. Eight volatile compounds, produced by fermenting cultures and not by sterile samples, were identified by gas chromatography coupled with mass spectrometry. Five of these substances elicited significant responses in Y-tube behavioral bioassays. Field trapping experiments revealed 2-phenylethanol and 2-phenylethyl acetate to be crucial for attraction of CBW. Results show that yeast volatiles play an important role in host location, and that 2-phenylethanol and 2-phenylethyl acetate could be utilized as potential attractants in monitoring and control systems against this important pest.

Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production.

PubMed

López-Alvarez, Arnoldo; Díaz-Pérez, Alma Laura; Sosa-Aguirre, Carlos; Macías-Rodríguez, Lourdes; Campos-García, Jesús

2012-05-01

In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Recombination Can Cause Telomere Elongations as Well as Truncations Deep within Telomeres in Wild-Type Kluyveromyces lactis Cells ▿

PubMed Central

Bechard, Laura H.; Jamieson, Nathan; McEachern, Michael J.

2011-01-01

In this study, we examined the role of recombination at the telomeres of the yeast Kluyveromyces lactis. We demonstrated that an abnormally long and mutationally tagged telomere was subject to high rates of telomere rapid deletion (TRD) that preferentially truncated the telomere to near-wild-type size. Unlike the case in Saccharomyces cerevisiae, however, there was not a great increase in TRD in meiosis. About half of mitotic TRD events were associated with deep turnover of telomeric repeats, suggesting that telomeres were often cleaved to well below normal length prior to being reextended by telomerase. Despite its high rate of TRD, the long telomere showed no increase in the rate of subtelomeric gene conversion, a highly sensitive test of telomere dysfunction. We also showed that the long telomere was subject to appreciable rates of becoming elongated substantially further through a recombinational mechanism that added additional tagged repeats. Finally, we showed that the deep turnover that occurs within normal-length telomeres was diminished in the absence of RAD52. Taken together, our results suggest that homologous recombination is a significant process acting on both abnormally long and normally sized telomeres in K. lactis. PMID:21148753

Novel technology development through thermal drying of encapsulated Kluyveromyces marxianus in micro- and nano-tubular cellulose in lactose fermentation and its evaluation for food production.

PubMed

Papapostolou, Harris; Servetas, Yiannis; Bosnea, Loulouda A; Kanellaki, Maria; Koutinas, Athanasios A

2012-12-01

A novel technology development based on the production of a low-cost starter culture for ripening of cheeses and baking is reported in the present study. The starter culture comprises thermally dried cells of Kluyveromyces marxianus encapsulated in micro- and nano-tubular cellulose. For production of a low-cost and effective biocatalyst, whey was used as raw material for biomass production and thermal drying methods (convective, conventional, and vacuum) were applied and evaluated at drying temperatures ranging from 35 to 60 °C. The effect of drying temperature of biocatalysts on fermentability of lactose and whey was evaluated. Storage stability and suitability of biocatalysts as a commercial starter cultures was also assessed and evaluated. All thermally dried biocatalysts were found to be active in lactose and whey fermentation. In all cases, there was sugar conversion ranging from 92 to 100 %, ethanol concentration of up to 1.47 % (v/v), and lactic acid concentrations ranged from 4.1 to 5.5 g/l. However, convective drying of the encapsulated cells of K. marxianus in micro- and nano-tubular cellulose was faster and a more effective drying method while drying at 42 °C appear to be the best drying temperature in terms of cell activity, ethanol, and lactic acid formation. Storage of the biocatalysts for 3 months at 4 °C proved maintenance of its activity even though fermentation times increased by 50-100 % compared with the fresh dried ones.

Evolutionary relationships among pathogenic Candida species and relatives.

PubMed Central

Barns, S M; Lane, D J; Sogin, M L; Bibeau, C; Weisburg, W G

1991-01-01

Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida. PMID:2007550

Use of synthetic genes for cloning, production and functional expression of the bacteriocins enterocin A and bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis.

PubMed

Jiménez, Juan J; Borrero, Juan; Gútiez, Loreto; Arbulu, Sara; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2014-06-01

The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZαA and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni.

Potential application of aqueous two-phase systems and three-phase partitioning for the recovery of superoxide dismutase from a clarified homogenate of Kluyveromyces marxianus.

PubMed

Simental-Martínez, Jesús; Rito-Palomares, Marco; Benavides, Jorge

2014-01-01

Superoxide dismutase (SOD; EC 1.15.1.1) is an antioxidant enzyme that represents the primary cellular defense against superoxide radicals and has interesting applications in the medical and cosmetic industries. In the present work, the partition behavior of SOD in aqueous two-phase systems (ATPS) (using a standard solution and a complex extract from Kluyveromyces marxianus as sample) was characterized on different types of ATPS (polymer-polymer, polymer-salt, alcohol-salt, and ionic liquid (IL)-salt). The systems composed of PEG 3350-potassium phosphate, 45% TLL, 0.5 M NaCl (315 U/mg, 87% recovery, and 15.1-fold purification) and t-butanol-20% ammonium sulfate (205.8 U/mg, 80% recovery and 9.8-fold purification), coupled with a subsequent 100 kDa ultrafiltration stage, allowed the design of a prototype process for the recovery and partial purification of the product of interest. The findings reported herein demonstrate the potential of PEG-salt ATPS for the potential recovery of SOD. © 2014 American Institute of Chemical Engineers.

Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799.

PubMed

Galindo-Leva, Luz Ángela; Hughes, Stephen R; López-Núñez, Juan Carlos; Jarodsky, Joshua M; Erickson, Adam; Lindquist, Mitchell R; Cox, Elby J; Bischoff, Kenneth M; Hoecker, Eric C; Liu, Siqing; Qureshi, Nasib; Jones, Marjorie A

2016-07-01

Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL.

Molecular cloning of the plasma membrane H(+)-ATPase from Kluyveromyces lactis: a single nucleotide substitution in the gene confers ethidium bromide resistance and deficiency in K+ uptake.

PubMed Central

Miranda, M; Ramírez, J; Peña, A; Coria, R

1995-01-01

A Kluyveromyces lactis strain resistant to ethidium bromide and deficient in potassium uptake was isolated. Studies on the proton-pumping activity of the mutant strain showed that a decreased H(+)-ATPase specific activity was responsible for the observed phenotypes. The putative K. lactis PMA1 gene encoding the plasma membrane H(+)-ATPase was cloned by its ability to relieve the potassium transport defect of this mutant and by reversing its resistance to ethidium bromide. Its deduced amino acid sequence predicts a protein 899 residues long that is structurally colinear in its full length to H(+)-ATPases cloned from different yeasts, except for the presence of a variable N-terminal domain. By PCR-mediated amplification, we identified a transition from G to A that rendered the substitution of the fully conserved methionine at position 699 by isoleucine. We attribute to this amino acid change the low capacity of the mutant H(+)-ATPase to pump out protons. PMID:7730265

KNQ1, a Kluyveromyces lactis gene encoding a transmembrane protein, may be involved in iron homeostasis.

PubMed

Marchi, Emmanuela; Lodi, Tiziana; Donnini, Claudia

2007-08-01

The original purpose of the experiments described in this article was to identify, in the biotechnologically important yeast Kluyveromyces lactis, gene(s) that are potentially involved in oxidative protein folding within the endoplasmic reticulum (ER), which often represents a bottleneck for heterologous protein production. Because treatment with the membrane-permeable reducing agent dithiothreitol inhibits disulfide bond formation and mimics the reducing effect that the normal transit of folding proteins has in the ER environment, the strategy was to search for genes that conferred higher levels of resistance to dithiothreitol when present in multiple copies. We identified a gene (KNQ1) encoding a drug efflux permease for several toxic compounds that in multiple copies conferred increased dithiothreitol resistance. However, the KNQ1 product is not involved in the excretion of dithiothreitol or in recombinant protein secretion. We generated a knq1 null mutant, and showed that both overexpression and deletion of the KNQ1 gene resulted in increased resistance to dithiothreitol. KNQ1 amplification and deletion resulted in enhanced transcription of iron transport genes, suggesting, for the membrane-associated protein Knq1p, a new, unexpected role in iron homeostasis on which dithiothreitol tolerance may depend.

Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis.

PubMed

Cardarelli, Silvia; Giorgi, Mauro; Naro, Fabio; Malatesta, Francesco; Biagioni, Stefano; Saliola, Michele

2017-09-22

Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological effect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specific hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms. Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purified A1, A2 and A3 isoforms displaying K m , V max and Sildenafil inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth deficiencies and interferences with the endogenous cAMP/cGMP signal transduction pathways. To our knowledge, this is the first time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specific PDE inhibitors for therapeutic applications in many pathologies.

Development of mutated Kluyveromyces marxianus strains for ethanol production at elevated temperature from biomass hydrolysate

USDA-ARS?s Scientific Manuscript database

The yeast K. marxianus has advantages over the most commonly used industrial ethanologen, Saccharomyces cerevisiae, such as the ability to grow at 47°C, to produce ethanol at temperatures above 40°C, and to grow on a wide variety of substrates, including starch, sucrose, pectins, and cellulosic biom...

Regulation of glycolysis in Kluyveromyces lactis: role of KlGCR1 and KlGCR2 in glucose uptake and catabolism.

PubMed

Neil, H; Lemaire, M; Wésolowski-Louvel, M

2004-03-01

In Kluyveromyces lactis, the casein kinase I (Rag8p) regulates the transcription of glycolytic genes and the expression of the low-affinity glucose transporter gene RAG1. This control involves the transcription factor Sck1p, a homologue of Sgc1p of Saccharomyces cerevisiae. SGC1 is known to interact genetically with ScGCR1 and ScGCR2, which code for regulators of glycolytic gene expression. Therefore, we studied the role of KlGCR1 and KlGCR2 genes in K. lactis. The Klgcr1 null mutant could not grow on glucose when respiration was blocked by antimycin A (Rag(- )phenotype). In contrast, the Klgcr2 null mutant could grow under the same conditions, although at a reduced rate. In both mutants, the transcription of glycolytic genes was affected, while that of ribosomal protein genes was not modified. Furthermore, the transcription of the glucose permease genes was also found to be affected in the two mutants, although dissimilarly. While RAG1 transcription decreased at high glucose concentrations, the expression of the high-affinity glucose permease gene HGT1 was unexpectedly impaired under gluconeogenic conditions, in the absence of glucose. Gel mobility shift assays performed with purified maltose-binding protein-KlGcr1p showed that KlGcr1p could interact directly with the promoters of the glycolytic genes, but not with the promoters of the glucose permease genes. Thus, the control exerted by KlGcr1p and KlGcr2p upon glucose transporter genes is probably indirect.

Immobilization of β-galactosidase from Kluyveromyces lactis onto polymeric membrane surfaces: effect of surface characteristics.

PubMed

Güleç, Hacı Ali

2013-04-01

The aim of this study was to investigate the effects of surface characteristics of plain and plasma modified cellulose acetate (CA) membranes on the immobilization yield of β-galactosidases from Kluyveromyces lactis (KLG) and its galacto-oligosaccharide (GOS) yield, respectively. Low pressure plasma treatments involving oxygen plasma activation, plasma polymerization (PlsP) of ethylenediamine (EDA) and PlsP of 2-mercaptoethanol were used to modify plain CA membrane surfaces. KLG enzyme was immobilized onto plain and oxygen plasma treated membrane surfaces by simple adsorption. Oxygen plasma activation increased the hydrophylicity of CA membrane surfaces and it improved the immobilization yield of the enzyme by 42%. KLG enzyme was also immobilized onto CA membrane surfaces through amino groups created by PlsP of EDA via covalent binding. Plasma action at 60W plasma power and 15 min. exposure time improved the amount of membrane bounded enzyme by 3.5-fold. The enrichment of the amount of amino groups via polyethyleneimine (PEI) addition enhanced this increase from 3.5-fold to 4.5-fold. Although high enzyme loading was achived (65-83%), both of the methods dramatically decreased the enzyme activity (11-12%) and GOS yield due to probably negative effects of active amino groups. KLG enzyme was more effectively immobilized onto thiolated CA membrane surface created by PlsP of 2-mercaptoethanol with high immobilization yield (70%) and especially high enzyme activity (46%). Immobilized enzymes on the CA membranes treated by PlsP were successively reutilized for 5-8 cycles at 25°C and enzymatic derivatives retained approximately 75-80% of their initial activites at the end of the reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Purification and characterization of a novel tannase produced by Kluyveromyces marxianus using olive pomace as solid support, and its promising role in gallic acid production.

PubMed

Mahmoud, Abeer E; Fathy, Shadia A; Rashad, Mona M; Ezz, Magda K; Mohammed, Amira T

2018-02-01

Tannase is considered one of the most important industrial enzymes that find great applications in various sectors. Production of tannases through solid state fermentation (SSF) using agro-industrial wastes is an eco-friendly and cheap technology. Tannase was produced by the yeast Kluyveromyces marxianus using olive pomace as a solid support under SSF. It was purified using ammonium sulfate fractional precipitation followed by Sephadex G-200 gel filtration resulting in 64.6% enzyme yield with 1026.12U/mg specific activity and 24.21 purification fold. Pure tannase had molecular weight of 65 KDa and 66.62 KDa by SDS-PAGE and gel filtration, respectively. It showed a maximal activity at 35°C having two different pH optima, one of which is acidic (4.5) and the other one is alkaline (8.5). The enzyme was stable in the acidic range of pH (4.0-5.5) for 30min, and thermostable within the temperature range 30-70°C. Using tannic acid, the enzyme had a Km value of 0.77mM and Vmax of 263.20μmolemin -1 ml -1 . The effect of different metal ions on enzymatic activity was evaluated. HPLC analysis data indicated that the purified enzyme could carry out 24.65% tannic acid conversion with 5.25 folds increase in gallic acid concentration within 30min only. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on L-arabinose and D-xylose.

PubMed

Knoshaug, Eric P; Vidgren, Virve; Magalhães, Frederico; Jarvis, Eric E; Franden, Mary Ann; Zhang, Min; Singh, Arjun

2015-10-01

Genes encoding L-arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on L-arabinose was dependent on a functioning L-arabinose transporter, or by screening a differential display library, respectively. These transporters also transport D-xylose and were designated KmAXT1 (arabinose-xylose transporter) and PgAXT1, respectively. Transport assays using L-arabinose showed that KmAxt1p has K(m) 263 mM and V(max) 57 nM/mg/min, and PgAxt1p has K(m) 0.13 mM and V(max) 18 nM/mg/min. Glucose, galactose and xylose significantly inhibit L-arabinose transport by both transporters. Transport assays using D-xylose showed that KmAxt1p has K(m) 27 mM and V(max) 3.8 nM/mg/min, and PgAxt1p has K(m) 65 mM and V(max) 8.7 nM/mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K(m) 371 mM and V(max) 341 nM/mg/min for L-arabinose, and K(m) 25 mM and V(max) 76 nM/mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both L-arabinose and D-xylose, one scenario for the complete usage of biomass-derived pentose sugars would require only the low-affinity, high-throughput transporter Gal2p and one additional high-affinity general pentose transporter, rather than dedicated D-xylose or L-arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications

PubMed Central

Rico-Díaz, Agustín; Álvarez-Cao, María-Efigenia; Escuder-Rodríguez, Juan-José; González-Siso, María-Isabel; Cerdán, M. Esperanza; Becerra, Manuel

2017-01-01

Kluyveromyces lactis β-galactosidase (Kl-β-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45 °C compared to Kl-β-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5 °C, respectively, higher than Kl-β-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-β-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-β-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-β-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis. PMID:28361909

Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications.

PubMed

Rico-Díaz, Agustín; Álvarez-Cao, María-Efigenia; Escuder-Rodríguez, Juan-José; González-Siso, María-Isabel; Cerdán, M Esperanza; Becerra, Manuel

2017-03-31

Kluyveromyces lactis β-galactosidase (Kl-β-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45 °C compared to Kl-β-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5 °C, respectively, higher than Kl-β-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-β-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-β-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-β-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis.

Molecular analysis of UAS(E), a cis element containing stress response elements responsible for ethanol induction of the KlADH4 gene of Kluyveromyces lactis.

PubMed

Mazzoni, C; Santori, F; Saliola, M; Falcone, C

2000-01-01

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity, which is specifically induced by ethanol and insensitive to glucose repression. In this work, we report the molecular analysis of UAS(E), an element of the KlADH4 promoter which is essential for the induction of KlADH4 in the presence of ethanol. UAS(E) contains five stress response elements (STREs), which have been found in many genes of Saccharomyces cerevisiae involved in the response of cells to conditions of stress. Whereas KlADH4 is not responsive to stress conditions, the STREs present in UAS(E) seem to play a key role in the induction of the gene by ethanol, a situation that has not been observed in the related yeast S. cerevisiae. Gel retardation experiments showed that STREs in the KlADH4 promoter can bind factor(s) under non-inducing conditions. Moreover, we observed that the RAP1 binding site present in UAS(E) binds KlRap1p.

Sequential application of waste whey as a medium component for Kluyveromyces lactis cultivation and a co-feeder for lipase immobilization by CLEA method.

PubMed

Veteikytė, Aušra; Šiekštelė, Rimantas; Tvaska, Bronius; Matijošytė, Inga

2017-05-01

Currently, much attention is paid to technologies which can be drivers of the circular economy across different sectors, in particular, to develop technologies for utilization or reusability of biocompatible materials from industrial waste. One of such is the milk whey, which is a cheap biobased raw material, the disposal of which is a major problem for the dairy industry. Our proposed and investigated technology is based on a continuous exploitation of the whey combining microbiology and biotechnology. Primarily, whey was used as a nutrition source for the cultivation of Kluyveromyces lactis with the aim to produce the targeted biocatalyst-lipase. During cultivation, the whey was transformed into the hydrolyzed form, which was further successfully applied as a protein feeder (external linker) for immobilization of lipase by cross-linked enzyme aggregate (CLEA) method. The first time use of whey as a co-feeder for immobilization of enzymes by CLEA method has shown promising results and increased the stability of lipases for temperature and organic solvents. Hydrolysis of rapeseed oil catalyzed with immobilized derivatives was obtained with 45-96% efficiency at non-optimized conditions. Additionally, the determined kinetic parameters indicated that the rate of p-nitrophenyl palmitate hydrolysis was not changed drastically after immobilization.

Analysis of the yeast short-term Crabtree effect and its origin

PubMed Central

Hagman, Arne; Säll, Torbjörn; Piškur, Jure

2014-01-01

The short-term Crabtree effect is defined as the immediate occurrence of aerobic alcoholic fermentation in response to provision of a pulse of excess sugar to sugar-limited yeast cultures. Here we have characterized ten yeast species with a clearly defined phylogenetic relationship. Yeast species were cultivated under glucose-limited conditions, and we studied their general carbon metabolism in response to a glucose pulse. We generated an extensive collection of data on glucose and oxygen consumption, and ethanol and carbon dioxide generation. We conclude that the Pichia,Debaryomyces,Eremothecium and Kluyveromyces marxianus yeasts do not exhibit any significant ethanol formation, while Kluyveromyces lactis behaves as an intermediate yeast, and Lachancea,Torulaspora,Vanderwaltozyma and Saccharomyces yeasts exhibit rapid ethanol accumulation. Based on the present data and our previous data relating to the presence of the long-term Crabtree effect in over 40 yeast species, we speculate that the origin of the short-term effect may coincide with the origin of the long-term Crabtree effect in the Saccharomycetales lineage, occurring ∼ 150 million years ago. PMID:25161062

Chemical and microbiological characterisation of kefir grains.

PubMed

Garrote, G L; Abraham, A G; De Antoni, G L

2001-11-01

Chemical and microbiological composition of four Argentinean kefir grains from different sources as well as characteristics of the corresponding fermented milk were studied. Kefir grains CIDCA AGK1, AGK2 and AGK4 did not show significant differences in their chemical and microbiological composition. In contrast, protein and yeast content of AGK3 was higher than in the other grains. Although grain microflora comprised lactobacilli, lactococcus, acetic acid bacteria and yeast, we found an important difference regarding species. Lactococcus lactis subsp. lactis, Lactobacillus kefir, Lactobacillus plantarum, Acetobacter and Saccharomyces were present in all types of kefir grain. While Leuconostoc mesenteroides was only isolated from grains CIDCA AGK1 and Lactococcus lactis subsp. lactis biovar diacetylactis, Lactobacillus parakefir and Kluyveromyces marxianus were only isolated from CIDCA AGK2 grains. All grains produced acid products with pH between 3.5 and 4.0. The apparent viscosity of AGK1 fermented milk was greater than the product obtained with AGK4. All fermented milks had inhibitory power towards Escherichia coli but AGK1 and AGK2 supernatants were able to halt the bacterial growth for at least 25 h. Grain weight increment in AGK1, AGK2 and AGK3 during growth in milk did not show significant differences. Despite their fermenting activity, AGK4 grains did not increase their weight.

Investigation on culturable microflora in Tibetan kefir grains from different areas of China.

PubMed

Gao, Jie; Gu, Fengying; Abdella, Nesredin H; Ruan, Hui; He, Guoqing

2012-08-01

Four samples of Tibetan kefir grains (TK-ZJUJ 01-04) from Tibet and surrounding areas were investigated via phenotypic and genotypic methods to compare and analyze the diversity of culturable microflora among different origins. As a result, 4 genera of microorganisms from TK-ZJUJ01: Bacillus subtilis (2.9 × 10(7) cfu/mL), Lactococcus lactis (8.2 × 10(7) cfu/mL), Kluyveromyces marxianus (3.0 × 10(6) cfu/mL), Saccharomyces cerevisiae (9.0 × 10(6) cfu/mL); 4 genera from TK-ZJUJ02: Lactobacillus kefiri (1.0 × 10(8) cfu/mL), Pichia kudriavzevii (5.0 × 10(6) cfu/mL), K. marxianus (1.9 × 10(7) cfu/mL), Kazachstania unispora (6.2 × 10(7) cfu/mL); 6 genera from TK-ZJUJ03: Leuconostoc lactis (4.6 × 10(7) cfu/mL), L. lactis (3.0 × 10(7) cfu/mL), Lactobacillus plantarum (3.0 × 10(7) cfu/mL), K. unispora (3.0 × 10(6) cfu/mL), K. marxianus (2.0 × 10(6) cfu/mL), (1.7 × 10(7) cfu/mL); and 4 genera from TK-ZJUJ04: L. plantarum (1.8 × 10(7) cfu/mL), Acetobacter fabarum (5.0 × 10(6) cfu/mL), K. unispora (6.2 × 10(7) cfu/mL), Pichia guilliermondii (6.2 × 10(7) cfu/mL) were identified. Yeasts like P. kudriavzevii and P. guilliermondii isolated in this study were the first time reported in Tibetan kefir grains. For TK-ZJUJ 01-03, lactic acid bacteria were the major microorganisms, which accounted for more than 50% of all the microbial population, while for TK-ZJUJ04, the largest microbial group was yeasts which accounted for more than 50%. In a word, study of diversity and composition of microflora provided us theoretical foundation for further investigation and application of Tibetan kefir grains. This is the basic research in order to develop and industrialize a new kind of yogurt starter which is naturally formed microbiota with both lactic acid bacteria and yeasts in it. © 2012 Institute of Food Technologists®

Protein Kinases Involved in Mating and Osmotic Stress in the Yeast Kluyveromyces lactis▿

PubMed Central

Kawasaki, Laura; Castañeda-Bueno, María; Sánchez-Paredes, Edith; Velázquez-Zavala, Nancy; Torres-Quiroz, Francisco; Ongay-Larios, Laura; Coria, Roberto

2008-01-01

Systematic disruption of genes encoding kinases and mitogen-activated protein kinases (MAPKs) was performed in Kluyveromyces lactis haploid cells. The mutated strains were assayed by their capacity to mate and to respond to hyperosmotic stress. The K. lactis Ste11p (KlSte11p) MAPK kinase kinase (MAPKKK) was found to act in both mating and osmoresponse pathways while the scaffold KlSte5p and the MAPK KlFus3p appeared to be specific for mating. The p21-activated kinase KlSte20p and the kinase KlSte50p participated in both pathways. Protein association experiments showed interaction of KlSte50p and KlSte20p with Gα and Gβ, respectively, the G protein subunits involved in the mating pathway. Both KlSte50p and KlSte20p also showed interaction with KlSte11p. Disruption mutants of the K. lactis PBS2 (KlPBS2) and KlHOG1 genes of the canonical osmotic response pathway resulted in mutations sensitive to high salt and high sorbitol but dispensable for mating. Mutations that eliminate the MAPKK KlSte7p activity had a strong effect on mating and also showed sensitivity to osmotic stress. Finally, we found evidence of physical interaction between KlSte7p and KlHog1p, in addition to diminished Hog1p phosphorylation after a hyperosmotic shock in cells lacking KlSte7p. This study reveals novel roles for components of transduction systems in yeast. PMID:18024598

Dietary influence of kefir on microbial activities in the mouse bowel.

PubMed

Marquina, Domingo; Santos, A; Corpas, I; Muñoz, J; Zazo, J; Peinado, J M

2002-01-01

In this work the microflora present in kefir, a fermented milk product, was studied together with the effect of kefir administration on different groups of indigenous bacteria of mouse bowel. Kefir microflora was composed of lactic acid bacteria, acetic acid bacteria and yeasts. Yeast population was composed of Saccharomyces cerevisiae, S. unisporus, Candida kefir, Kluyveromyces marxianus and K. lactis. The streptococci levels in kefir treated mice increased by 10-fold and the levels of sulfite-reducing clostridia decreased by 100-fold. The number of lactic acid bacteria increased significantly. The administration of kefir significantly increased the lactic acid bacteria counts in the mucosa of the bowel. Ingestion of kefir specifically lowered microbial populations of Enterobacteriaceae and clostridia. This is the first long-term study about the effects of the kefir administration on the intestinal microflora of mice.

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

PubMed Central

Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal

2016-01-01

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a

The diversity of eukaryotic microbiota in the traditional Slovak sheep cheese--bryndza.

PubMed

Laurencík, M; Sulo, P; Sláviková, E; Piecková, E; Seman, M; Ebringer, L

2008-09-30

We investigated the occurrence and diversity of yeasts and filamentous fungi in bryndza an artisanal Slovak soft spreadable cheese prepared from raw sheep milk or from a mixture of sheep and cow milk. Samples collected during four months of the summer production period from two locations (northern and southern parts of central Slovakia) contained 10(5)-10(7) (cfu) yeasts and about 10(2) (cfu) of mold per gram of wet weight. Further characterization by conventional taxonomy and sequence comparison of D1/D2 region from 26S rRNA gene revealed Mucor circinelloides v. Tieghem as the predominant filamentous fungus. A novel Geotrichum sp. together with Kluyveromyces (K. lactis/K. marxianus) was identified as the most abundant yeast species. Occasionally other yeasts, such as Candida inconspicua, Candida silvae, Pichia fermentans and Trichosporon domesticum were found. Conventional taxonomy readily identified isolates to the genus level, but DNA sequence comparison was capable of discriminating them at the species level.

Hydrogen-driven asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol by Ralstonia eutropha transformant expressing alcohol dehydrogenase from Kluyveromyces lactis.

PubMed

Oda, Takahiro; Oda, Koji; Yamamoto, Hiroaki; Matsuyama, Akinobu; Ishii, Masaharu; Igarashi, Yasuo; Nishihara, Hirofumi

2013-01-10

Conversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alcohol dehydrogenase from Kluyveromyces lactis in a hydrogen-oxidizing bacterium: Ralstonia eutropha. This is the first report of hydrogen-driven in vivo coupling reaction of the alcohol dehydrogenase and indigenous soluble NAD-reducing hydrogenase. Asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol, which is a commercial building block for antibacterial agents, was performed using the transformant as the microbial cell catalyst. The two enzymes coupled in vitro in vials without a marked decrease of reactivity during the 20 hr reaction because of the hydrogenase reaction, which generates no by-product that affects enzymes. Alcohol dehydrogenase was expressed functionally in R. eutropha in an activity level equivalent to that of indigenous NAD-reducing hydrogenase under the hydrogenase promoter. The hydrogen-driven in vivo coupling reaction proceeded only by the transformant cell without exogenous addition of a cofactor. The decrease of reaction velocity at higher concentration of hydroxyacetone was markedly reduced by application of an in vivo coupling system. Production of (R)-1,2-propanediol (99.8% e.e.) reached 67.7 g/l in 76 hr with almost a constant rate using a jar fermenter. The reaction velocity under 10% PH2 was almost equivalent to that under 100% hydrogen, indicating the availability of crude hydrogen gas from various sources. The in vivo coupling system enabled cell-recycling as catalysts. Asymmetric reduction of hydroxyacetone by a coupling reaction of the two enzymes continued in both in vitro and in vivo systems in the presence of hydrogen. The in vivo reaction system using R. eutropha transformant expressing

Yeasts from autochthonal cheese starters: technological and functional properties.

PubMed

Binetti, A; Carrasco, M; Reinheimer, J; Suárez, V

2013-08-01

The aim of this work was to identify 20 yeasts isolated from autochthonal cheese starters and evaluate their technological and functional properties. The capacities of the yeasts to grow at different temperatures, pH, NaCl and lactic acid concentrations as well as the proteolytic and lipolytic activities were studied. Moreover, survival to simulated gastrointestinal digestion, hydrophobicity, antimicrobial activity against pathogens and auto- and co-aggregation abilities were evaluated. The sequentiation of a fragment from the 26S rDNA gene indicated that Kluyveromyces marxianus was the predominant species, followed by Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces lactis and Galactomyces geotrichum. RAPD with primer M13 allowed a good differentiation among strains from the same species. All strains normally grew at pH 4.7-5.5 and temperatures between 15 and 35°C. Most of them tolerated 10% NaCl and 3% lactic acid. Some strains showed proteolytic (eight isolates) and/or lipolytic (four isolates) capacities. All strains evidenced high gastrointestinal resistance, moderate hydrophobicity, intermediate auto-aggregation and variable co-aggregation abilities. No strains inhibited the growth of the pathogens assayed. Some strains from dairy sources showed interesting functional and technological properties. This study has been the first contribution to the identification and characterization of yeasts isolated from autochthonal cheese starters in Argentina. Many strains could be proposed as potential candidates to be used as probiotics and/or as co-starters in cheese productions. © 2013 The Society for Applied Microbiology.

Microbiota of Minas cheese as influenced by the nisin producer Lactococcus lactis subsp. lactis GLc05.

PubMed

Perin, Luana Martins; Dal Bello, Barbara; Belviso, Simona; Zeppa, Giuseppe; Carvalho, Antônio Fernandes de; Cocolin, Luca; Nero, Luís Augusto

2015-12-02

Minas cheese is a popular dairy product in Brazil that is traditionally produced using raw or pasteurized cow milk. This study proposed an alternative production of Minas cheese using raw goat milk added of a nisin producer Lactococcus lactis subsp. lactis GLc05. An in situ investigation was carried on to evaluate the interactions between the L. lactis subsp. lactis GLc05 and the autochthonous microbiota of a Minas cheese during the ripening; production of biogenic amines (BAs) was assessed as a safety aspect. Minas cheese was produced in two treatments (A, by adding L. lactis subsp. lactis GLc05, and B, without adding this strain), in three independent repetitions (R1, R2, and R3). Culture dependent (direct plating) and independent (rep-PCR and PCR-DGGE) methods were employed to characterize the microbiota and to assess the possible interferences caused by L. lactis subsp. lactis GLc05. BA amounts were measured using HPLC. A significant decrease in coagulase-positive cocci was observed in the cheeses produced by adding L. lactis subsp. lactis GLc05 (cheese A). The rep-PCR and PCR-DGGE highlighted the differences in the microbiota of both cheeses, separating them into two different clusters. Lactococcus sp. was found as the main microorganism in both cheeses, and the microbiota of cheese A presented a higher number of species. High concentrations of tyramine were found in both cheeses and, at specific ripening times, the BA amounts in cheese B were significantly higher than in cheese A (p<0.05). The interaction of nisin producer L. lactis subsp. lactis GLc05 was demonstrated in situ, by demonstration of its influence in the complex microbiota naturally present in a raw goat milk cheese and by controlling the growth of coagulase-positive cocci. L. lactis subsp. lactis GLc05 influenced also the production of BA determining that their amounts in the cheeses were maintained at acceptable levels for human consumption. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification and assessment of kefir yeast potential for sugar/ethanol-resistance

PubMed Central

Miguel, M.G.C.P.; Cardoso, P.G.; Magalhães-Guedes, K.T.; Schwan, R.F.

2013-01-01

Biochemical and molecular analysis was used for identification of different kefir yeasts species from Brazil, Canada and the United States of America. The sugar/ethanol-resistant activity of the yeasts was evaluated. Saccharomyces cerevisiae and Kluyveromyces marxianus had the highest growth rates, suggesting biotechnological applications possible for these strains. PMID:24159292

Dynamics of yeast immobilized-cell fluidized-bed bioreactors systems in ethanol fermentation from lactose-hydrolyzed whey and whey permeate.

PubMed

Gabardo, Sabrina; Pereira, Gabriela Feix; Klein, Manuela P; Rech, Rosane; Hertz, Plinho F; Ayub, Marco Antônio Záchia

2016-01-01

We studied the dynamics of ethanol production on lactose-hydrolyzed whey (LHW) and lactose-hydrolyzed whey permeate (LHWP) in batch fluidized-bed bioreactors using single and co-cultures of immobilized cells of industrial strains of Saccharomyces cerevisiae and non-industrial strains of Kluyveromyces marxianus. Although the co-culture of S. cerevisiae CAT-1 and K. marxianus CCT 4086 produced two- to fourfold the ethanol productivity of single cultures of S. cerevisiae, the single cultures of the K. marxianus CCT 4086 produced the best results in both media (Y EtOH/S = 0.47-0.49 g g(-1) and Q P = 1.39-1.68 g L(-1) h(-1), in LHW and LHWP, respectively). Ethanol production on concentrated LHWP (180 g L(-1)) reached 79.1 g L(-1), with yields of 0.46 g g(-1) for K. marxianus CCT 4086 cultures. Repeated batches of fluidized-bed bioreactor on concentrated LHWP led to increased ethanol productivity, reaching 2.8 g L(-1) h(-1).

Phenotypic variation in Lactococcus lactis subsp. lactis isolates derived from intestinal tracts of marine and freshwater fish.

PubMed

Itoi, S; Yuasa, K; Washio, S; Abe, T; Ikuno, E; Sugita, H

2009-09-01

We compared phenotypic characteristics of Lactococcus lactis subsp. lactis derived from different sources including the intestinal tract of marine fish and freshwater fish, and cheese starter culture. In the phylogenetic analysis based on partial 16S rRNA gene nucleotide sequences (1371 bp), freshwater fish-, marine fish- and cheese starter culture-derived strains were identical to that of L. lactis subsp. lactis previously reported. Fermentation profiles determined using the API 50 CH system were similar except for fermentation of several sugars including l-arabinose, mannitol, amygdalin, saccharose, trehalose, inulin and gluconate. The strains did have distinct levels of halotolerance: marine fish-derived strains > cheese starter-derived strain > freshwater fish-derived isolate. Lactococcus lactis subsp. lactis showed extensive diversity in phenotypic adaptation to various environments. The phenotypic properties of these strains suggested that L. lactis subsp. lactis strains from fish intestine have additional functions compared with the cheese starter-derived strain that has previously described. The unique phenotypic traits of the fish intestinal tract-derived L. lactis subsp. lactis might make them useful as a probiotics in aquaculture, and contribute to the development of functional foods and novel food additives, since the strains derived from fish intestines might have additional functions such as antibacterial activity.

Draft Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a Citrate-Fermenting Strain

PubMed Central

Zuljan, Federico; Espariz, Martín; Blancato, Victor S.; Esteban, Luis; Alarcón, Sergio

2016-01-01

We report the draft genome sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a natural strain isolated from artisanal cheese from northwest Argentina. L. lactis subsp. lactis bv. diacetylactis is one of the most important microorganisms used as starter culture around the world. The CRL264 strain constitutes a model microorganism in the studies on the generation of aroma compounds (diacetyl, acetoin, and 2,3-butanediol) by lactic acid bacteria. Our genome analysis shows similar genetic organization to other available genomes of L. lactis bv. diacetylactis strains. PMID:26847906

Draft Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a Citrate-Fermenting Strain.

PubMed

Zuljan, Federico; Espariz, Martín; Blancato, Victor S; Esteban, Luis; Alarcón, Sergio; Magni, Christian

2016-02-04

We report the draft genome sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a natural strain isolated from artisanal cheese from northwest Argentina. L. lactis subsp. lactis bv. diacetylactis is one of the most important microorganisms used as starter culture around the world. The CRL264 strain constitutes a model microorganism in the studies on the generation of aroma compounds (diacetyl, acetoin, and 2,3-butanediol) by lactic acid bacteria. Our genome analysis shows similar genetic organization to other available genomes of L. lactis bv. diacetylactis strains. Copyright © 2016 Zuljan et al.

Genome and metabolic engineering in non-conventional yeasts: Current advances and applications.

PubMed

Löbs, Ann-Kathrin; Schwartz, Cory; Wheeldon, Ian

2017-09-01

Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisia e is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.

Novel Antibacterial Activity of Lactococcus Lactis Subspecies Lactis Z11 Isolated from Zabady

PubMed Central

Enan, Gamal; Abdel-Shafi, Seham; Ouda, Sahar; Negm, Sally

2013-01-01

The purpose of this study was to select and characterize a probiotic bacterium with distinctive antimicrobial activities. In this respect, Lactococcus lactis subspecies lactis Z11 (L. lactis Z11) isolated from Zabady (Arabian yoghurt) inhibited other strains of lactic acid bacteria and some food-born pathogens including Listeria monocytogenes, Bacillus cereus and staphylococcus aureus. The inhibitory activity of cell free supernatant (CFS) of L. lactis Z11 isolated from zabady was lost by proteolytic enzymes, heat resistant. Consequently, the active substance(s) of CFS was characterized as a bacteriocin. This bacteriocin has been shown to consist of protein but has no lipidic or glucidic moieties in its active molecule. Its activity was stable in the pH range 2.0 to 7.0 and was not affected by organic solvents. The L. lactis Z11 bacteriocin was produced in CFS throughout the mide to the late exponential phase of growth of the producer organism and maximum bacteriocin production was obtained at initial pH 6.5 at incubation temperature of about 30°C. PMID:24151453

Nucleotide sequence of the COX1 gene in Kluyveromyces lactis mitochondrial DNA: evidence for recent horizontal transfer of a group II intron.

PubMed

Hardy, C M; Clark-Walker, G D

1991-07-01

The cytochrome oxidase subunit 1 gene (COX1) in K. lactis K8 mtDNA spans 8,826 bp and contains five exons (termed E1-E5) totalling 1,602 bp that show 88% nucleotide base matching and 91% amino acid homology to the equivalent gene in S. cerevisiae. The four introns (termed K1 cox1.1-1.4) contain open reading frames encoding proteins of 786, 333, 319 and 395 amino acids respectively that potentially encode maturase enzymes. The first intron belongs to group II whereas the remaining three are group I type B. Introns K1 cox1.1, 1.3, and 1.4 are found at identical locations to introns Sc cox1.2, 1.5 a, and 1.5 b respectively from S. cerevisiae. Horizontal transfer of an intron between recent progenitors of K. lactis and S. cerevisiae is suggested by the observation that K1 cox1.1 and Sc cox1.2 show 96% base matching. Sequence comparisons between K1 cox1.3/Sc cox1.5 a and K1 cox1.4/Sc cox1.5 b suggest that these introns are likely to have been present in the ancestral COX1 gene of these yeasts. Intron K1 cox1.2 is not found in S. cerevisiae and appears at an unique location in K. lactis. A feature of the DNA sequences of the group I introns K1 cox1.2, 1.3, and 1.4 is the presence of 11 GC-rich clusters inserted into both coding and noncoding regions. Immediately downstream of the COX1 gene is the ATPase subunit 8 gene (A8) that shows 82.6% base matching to its counterpart in S. cerevisiae mtDNA.

Microbial diversity and dynamics during the production of May bryndza cheese.

PubMed

Pangallo, Domenico; Saková, Nikoleta; Koreňová, Janka; Puškárová, Andrea; Kraková, Lucia; Valík, Lubomír; Kuchta, Tomáš

2014-01-17

paradoxus. The diversity of yeasts and fungi encompassed Alternaria alternata, "Ascomycete sp.", Aspergillus fumigatus, Beauveria brongniartii, Candida xylopsoci, C. inconspicua, Cladosporium cladosporioides, Debaromyces hansenii, Fomes fomentarius, Galactomyces candidus, Gymnoascus reesii, Chaetomium globosum, Kluyveromyces marxianus, Metarhizium anisopliae, Penicillium aurantiogriseum, P. camemberti, P. freii, P. polonicum, P. viridicatum, Pichia kudriavzevii, Sordaria alcina, Trichosporon lactis and Yarrowia lipolytica. © 2013.

Oxygen-Dependent Transcriptional Regulator Hap1p Limits Glucose Uptake by Repressing the Expression of the Major Glucose Transporter Gene RAG1 in Kluyveromyces lactis▿

PubMed Central

Bao, Wei-Guo; Guiard, Bernard; Fang, Zi-An; Donnini, Claudia; Gervais, Michel; Passos, Flavia M. Lopes; Ferrero, Iliana; Fukuhara, Hiroshi; Bolotin-Fukuhara, Monique

2008-01-01

The HAP1 (CYP1) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae, Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1-equivalent gene (KlHAP1) in K. lactis. KlHap1p showed a number of sequence features and some gene targets (such as KlCYC1) in common with its S. cerevisiae counterpart, and KlHAP1 was capable of complementing the hap1 mutation. However, the KlHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28°C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The ΔKlhap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis. PMID:18806211

Genetic and phenotypic features defining industrial relevant Lactococcus lactis, L. cremoris and L. lactis biovar. diacetylactis strains.

PubMed

Manno, Mariano Torres; Zuljan, Federico; Alarcón, Sergio; Esteban, Luis; Blancato, Victor; Espariz, Martín; Magni, Christian

2018-06-23

Lactococcus lactis strains constitute one of the most important starter cultures for cheese production. In this study, a genome-wide analysis was performed including 68 available genomes of L. lactis group strains showing the existence of two species (L. lactis and L. cremoris) and two biovars (L. lactis biovar. diacetylactis and L. cremoris biovar. lactis). The proposed classification scheme revealed coherency among phenotypic (through in silico and in vivo bacterial function profiling), phylogenomic (through maximum likelihood trees) and genomic (using overall genome sequence-based parameters) approaches. Strain biodiversity for the industrial biovar. diacetylactis was also analyzed, finding they are formed by at least three variants with the CC1 clonal complex as the only one distributed worldwide. These findings and methodologies will help improve the selection of L. lactis group strains for industrial use as well as facilitate the interpretation of previous or future research studies on this diverse group of bacteria. Copyright © 2018. Published by Elsevier B.V.

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

PubMed Central

Harris, L J; Fleming, H P; Klaenhammer, T R

1992-01-01

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut. Images PMID:1622214

Characterization of a Wild, Novel Nisin A-Producing Lactococcus Strain with an L. lactis subsp. cremoris Genotype and an L. lactis subsp. lactis Phenotype, Isolated from Greek Raw Milk

PubMed Central

Parapouli, Maria; Delbès-Paus, Céline; Kakouri, Athanasia; Koukkou, Anna-Irini; Montel, Marie-Christine

2013-01-01

Several molecular taxonomic studies have revealed that many natural (wild) Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. lactis subspecies lactis cluster genotypically within subspecies cremoris and vice versa. Recently, we isolated two wild nisin-producing (Nis+) L. lactis strains, M78 and M104, of the lactis phenotype from Greek raw milk (J. Samelis, A. Lianou, A. Kakouri, C. Delbès, I. Rogelj, B. B. Matijašic, and M. C. Montel, J. Food Prot. 72:783–790, 2009); strain M78 possess a novel nisin A sequence (GenBank accession number HM219853). In this study, the actual subspecies identity of M78 and M104 isolates was elucidated, using 16S rRNA and acmA (encoding lactococcal N-acetylmuramidase) gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh (encoding lactate dehydrogenase) gene phylogenies. Except the acmA gene analysis, molecular tools revealed that isolates M78 and M104 clustered with strains of the cremoris genotype, including the LMG 6897T strain, while they were distant from strains of the lactis genotype, including the LMG 6890T strain. The two wild isolates had identical repetitive sequence-based PCR (rep-PCR), randomly amplified polymorphic DNA (RAPD), plasmid, and whole-cell protein profiles and shared high 16S rRNA (99.9%) and ldh (100%) gene sequence homologies. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG 6890T strain. To our knowledge, this is the first complete identification report on a wild L. lactis subsp. cremoris genotype of the lactis phenotype which is capable of nisin A production and, thus, has strong potential for use as a novel dairy starter and/or protective culture. PMID:23542625

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: evaluation of the probiotic potential.

PubMed

Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

2014-01-01

Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its probiotic potential. Lc. lactis DF4Mi was resistant to acidic pH and oxbile, presented co-aggregation with Listeria monocytogenes, and was not affected by several drugs from different generic groups, being sensitive to most tested antibiotics. These properties indicate that this Lc. lactis strain can be used for enhancement of dairy foods safety and quality, in combination with potential probiotic properties.

Purification and partial characterization of bacteriocin produced by Lactococcus lactis ssp. lactis LL171.

PubMed

Kumari, Archana; Akkoç, Nefise; Akçelik, Mustafa

2012-04-01

Lactic acid bacteria (LAB) are possessing ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this regard, novel bacteriocin compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was purified by ammonium sulphate precipitation, dialysis and gel filtration. The molecular weight (≈3.4 kDa) of obtained bacteriocin was confirmed by SDS-PAGE, which revealed single peptide band. Molecular identification of LAB strain isolated from Tulum Cheese was conducted using 16S rDNA gene sequencing as Lactococcus lactis ssp. lactis LL171. The amino acid sequences (KKIDTRTGKTMEKTEKKIELSLKNMKTAT) of the bacteriocin from Lactococcus lactis ssp. lactis LL171 was found unique and novel than reported bacteriocins. Further, the bacteriocin was possessed the thermostable property and active at wide range of pH values from 1 to 11. Thus, bacteriocin reported in this study has the potential applications property as food preservative agent.

Comparison of the acidifying activity of Lactococcus lactis subsp. lactis strains isolated from goat's milk and Valdeteja cheese.

PubMed

Alonso-Calleja, C; Carballo, J; Capita, R; Bernardo, A; García-López, M L

2002-01-01

This work was carried out to study the acid production by Lactococcus lactis subsp. lactis strains isolated from goat's milk and goat cheese (Valdeteja variety) in order to select a suitable starter culture for industrial goat cheese manufacturing. The titrable acidity of 45 Lactococcus lactis subsp. lactis strains isolated from a home-made batch of Valdeteja cheese with excellent sensory characteristics was measured over a period of 18 h. The strains were divided into two groups depending on the acid production rate: 20 fast acid producer (F) strains and 25 slow acid producer (S) strains. The kinetic parameters (lag phase, maximum acid production rate and value of upper asymptote curve) of the acid production curves for F and S strains were significantly (P < 0.001) different. Significant (P < 0.001) differences between titrable acidity of F and S strains were observed after the second hour of incubation. An F strain acetoin producer (Lactococcus lactis subsp. lactis 470Ch2) was selected as autochthonous starter culture for industrial Valdeteja goat cheese manufacturing.

Melanin determination by high performance liquid chromatography (HPLC) for K. marxianus

USDA-ARS?s Scientific Manuscript database

Ultraviolet light (UV) mutated K. marxianus was found to turn dark brown during a growth assay. This brown color was hypothesized to be melanin overproduction influenced by the UV exposure. Cell cultures were oxidized and HPLC analyzed to determine melanin concentrations. The resulting melanin con...

Draft Genome Sequence of the Putrescine-Producing Strain Lactococcus lactis subsp. lactis 1AA59

PubMed Central

del Rio, Beatriz; Linares, Daniel M.; Fernandez, María; Mayo, Baltasar; Martín, M. Cruz

2015-01-01

We report here the 2,576,542-bp genome annotated draft assembly sequence of Lactococcus lactis subsp. lactis 1AA59. This strain—isolated from a traditional cheese—produces putrescine, one of the most frequently biogenic amines found in dairy products. PMID:26089428

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Characterization of the bacteriocin

PubMed Central

Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

2014-01-01

Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality. PMID:25763065

A new search for thermotolerant yeasts, its characterization and optimization using response surface methodology for ethanol production.

PubMed

Arora, Richa; Behera, Shuvashish; Sharma, Nilesh K; Kumar, Sachin

2015-01-01

The progressive rise in energy crisis followed by green house gas (GHG) emissions is serving as the driving force for bioethanol production from renewable resources. Current bioethanol research focuses on lignocellulosic feedstocks as these are abundantly available, renewable, sustainable and exhibit no competition between the crops for food and fuel. However, the technologies in use have some drawbacks including incapability of pentose fermentation, reduced tolerance to products formed, costly processes, etc. Therefore, the present study was carried out with the objective of isolating hexose and pentose fermenting thermophilic/thermotolerant ethanologens with acceptable product yield. Two thermotolerant isolates, NIRE-K1 and NIRE-K3 were screened for fermenting both glucose and xylose and identified as Kluyveromyces marxianus NIRE-K1 and K. marxianus NIRE-K3. After optimization using Face-centered Central Composite Design (FCCD), the growth parameters like temperature and pH were found to be 45.17°C and 5.49, respectively for K. marxianus NIRE-K1 and 45.41°C and 5.24, respectively for K. marxianus NIRE-K3. Further, batch fermentations were carried out under optimized conditions, where K. marxianus NIRE-K3 was found to be superior over K. marxianus NIRE-K1. Ethanol yield (Y x∕s ), sugar to ethanol conversion rate (%), microbial biomass concentration (X) and volumetric product productivity (Q p ) obtained by K. marxianus NIRE-K3 were found to be 9.3, 9.55, 14.63, and 31.94% higher than that of K. marxianus NIRE-K1, respectively. This study revealed the promising potential of both the screened thermotolerant isolates for bioethanol production.

A new search for thermotolerant yeasts, its characterization and optimization using response surface methodology for ethanol production

PubMed Central

Arora, Richa; Behera, Shuvashish; Sharma, Nilesh K.; Kumar, Sachin

2015-01-01

The progressive rise in energy crisis followed by green house gas (GHG) emissions is serving as the driving force for bioethanol production from renewable resources. Current bioethanol research focuses on lignocellulosic feedstocks as these are abundantly available, renewable, sustainable and exhibit no competition between the crops for food and fuel. However, the technologies in use have some drawbacks including incapability of pentose fermentation, reduced tolerance to products formed, costly processes, etc. Therefore, the present study was carried out with the objective of isolating hexose and pentose fermenting thermophilic/thermotolerant ethanologens with acceptable product yield. Two thermotolerant isolates, NIRE-K1 and NIRE-K3 were screened for fermenting both glucose and xylose and identified as Kluyveromyces marxianus NIRE-K1 and K. marxianus NIRE-K3. After optimization using Face-centered Central Composite Design (FCCD), the growth parameters like temperature and pH were found to be 45.17°C and 5.49, respectively for K. marxianus NIRE-K1 and 45.41°C and 5.24, respectively for K. marxianus NIRE-K3. Further, batch fermentations were carried out under optimized conditions, where K. marxianus NIRE-K3 was found to be superior over K. marxianus NIRE-K1. Ethanol yield (Yx∕s), sugar to ethanol conversion rate (%), microbial biomass concentration (X) and volumetric product productivity (Qp) obtained by K. marxianus NIRE-K3 were found to be 9.3, 9.55, 14.63, and 31.94% higher than that of K. marxianus NIRE-K1, respectively. This study revealed the promising potential of both the screened thermotolerant isolates for bioethanol production. PMID:26388844

Saccharomyces cerevisiae Bat1 and Bat2 Aminotransferases Have Functionally Diverged from the Ancestral-Like Kluyveromyces lactis Orthologous Enzyme

PubMed Central

Colón, Maritrini; Hernández, Fabiola; López, Karla; Quezada, Héctor; González, James; López, Geovani; Aranda, Cristina; González, Alicia

2011-01-01

Background Gene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism. Principal Findings Our results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs). This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1), while catabolic substrates are accumulated in the cytosol (Bat2). Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition. Conclusions Our study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the biosynthetic and catabolic

Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis▿

PubMed Central

Saliola, Michele; Scappucci, Gina; De Maria, Ilaria; Lodi, Tiziana; Mancini, Patrizia; Falcone, Claudio

2007-01-01

In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield. PMID:17085636

Transcriptome analysis and related databases of Lactococcus lactis.

PubMed

Kuipers, Oscar P; de Jong, Anne; Baerends, Richard J S; van Hijum, Sacha A F T; Zomer, Aldert L; Karsens, Harma A; den Hengst, Chris D; Kramer, Naomi E; Buist, Girbe; Kok, Jan

2002-08-01

Several complete genome sequences of Lactococcus lactis and their annotations will become available in the near future, next to the already published genome sequence of L. lactis ssp. lactis IL 1403. This will allow intraspecies comparative genomics studies as well as functional genomics studies aimed at a better understanding of physiological processes and regulatory networks operating in lactococci. This paper describes the initial set-up of a DNA-microarray facility in our group, to enable transcriptome analysis of various Gram-positive bacteria, including a ssp. lactis and a ssp. cremoris strain of Lactococcus lactis. Moreover a global description will be given of the hardware and software requirements for such a set-up, highlighting the crucial integration of relevant bioinformatics tools and methods. This includes the development of MolGenIS, an information system for transcriptome data storage and retrieval, and LactococCye, a metabolic pathway/genome database of Lactococcus lactis.

Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol.

PubMed

Ballesteros, I; Ballesteros, M; Cabañas, A; Carrasco, J; Martín, C; Negro, M J; Saez, F; Saez, R

1991-01-01

A total of 27 yeast strains belonging to the groups Candida, Saccharomyces, and Kluyveromyces were screened for their ability to grow and ferment glucose at temperatures ranging 32-45 degrees C. K. marxianus and K. fragilis were found to be the best ethanol producing organisms at the higher temperature tested and, so, were selected for subsequent simultaneous saccharification and fermentation (SSF) studies. SSF experiments were performed at 42 and 45 degrees C, utilizing Solkafloc (10%) as cellulose substrate and a cellulase loading of 15 FPU/g substrate. Best results were achieved at 42 degrees C with K. marxianus L. G. and K. fragilis L. G., both of which produced close to 38 g/L ethanol and 0.5 ethanol yield, in 78 h.

Detection and Viability of Lactococcus lactis throughout Cheese Ripening

PubMed Central

Cocolin, Luca

2014-01-01

Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese. PMID:25503474

A Preliminary Study of Europium Uptake by Yeast Cells. The Case of Kluveromyces Marxianus

NASA Astrophysics Data System (ADS)

Anagnostopoulos, V.; Symeopoulos, B.

2008-08-01

The objective of the present work is an exploration of a cost effective recovery of lanthanides, either for minimizing the industrial processes losses, or for reasons related to Radioactive Waste Management. Specifically, the uptake of europium from aqueous solutions by Kluveromyces marxianus cells was studied. Moreover, this biotechnological approach turns out to be environmental friendly, considering that cells of Kluveromyces marxianus are readily available as wastes from food fermentation industries. Europium [152Eu+154Eu]-labelled solutions were used providing better accuracy and reproducibility of measurements, mainly in low concentration range. The effect of pH, contact time and europium initial concentration were investigated. Adsorption data were fitted to Langmuir and Freundlich sorption models and Scatchard plots were used to reveal the existence of at least two types of binding sites.

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication*

PubMed Central

Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

2016-01-01

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

[A comparison of the properties of bacteriocins formed by Lactococcus lactis subsp. lactis strains of diverse origin].

PubMed

Stoianova, L G; Egorov, N S; Fedorova, G B; Katrukha, G S; Netrusov, A I

2007-01-01

Bacteriocins formed by four strains of Lactococcus lactis subsp. lactis have been studied and compared: 729 (a natural strain isolated from milk), 1605 (a mutant of strain 729), F-116 (a recombinant obtained by fusing of protoplasts of the two related strain 729 and 1605), and a nisin-forming strain obtained by adaptive selection at Moscow State University. Antimicrobial activity studies revealed differences between the strains in the effects on individual groups of microorganisms; the activities of the strains were also distinct from that of Nisaplin (a commercial preparation of the bacteriocin nisin). Methods for isolation and purification of bacteriocins have been developed, making it possible to obtain individual components of antibiotic complexes as chromatographically pure preparations. Bacteriocins formed by the strains of Lactococcus lactis subsp. lactis have been identified and differences in their biological and physicochemical properties, established. A novel potent broad-spectrum antibiotic substance distinct from nisin has been isolated from the recombinant strain F-116.

Evaluation of Galactose Adapted Yeasts for Bioethanol Fermentation from Kappaphycus alvarezii Hydrolyzates.

PubMed

Nguyen, Trung Hau; Ra, Chae Hun; Sunwoo, In Yung; Jeong, Gwi-Taek; Kim, Sung-Koo

2016-07-28

Bioethanol was produced from Kappaphycus alvarezii seaweed biomass using separate hydrolysis and fermentation (SHF). Pretreatment was evaluated for 60 min at 121°C using 12% (w/v) biomass slurry with 364 mM H2SO4. Enzymatic saccharification was then carried out at 45°C for 48 h using Celluclast 1.5 L. Ethanol fermentation with 12% (w/v) K. alvarezii hydrolyzate was performed using the yeasts Saccharomyces cerevisiae KCTC1126, Kluyveromyces marxianus KCTC7150, and Candida lusitaniae ATCC42720 with or without prior adaptation to high concentrations of galactose. When non-adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 11.5 g/l, 6.7 g/l, and 6.0 g/l of ethanol were produced, respectively. When adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 15.8 g/l, 11.6 g/l, and 13.4 g/l of ethanol were obtained, respectively. The highest ethanol concentration was 15.8 g/l, with YEtOH = 0.43 and YT% = 84.3%, which was obtained using adapted S. cerevisiae.

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli.

PubMed Central

Sesma, F; Gardiol, D; de Ruiz Holgado, A P; de Mendoza, D

1990-01-01

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:2117878

A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation†

PubMed Central

Wang, Hua; Yu, Weizhu; Coolbear, Tim; O’Sullivan, Dan; McKay, Larry L.

1998-01-01

A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc−) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis. PMID:9572935

Short communication: Genotypic and phenotypic identification of environmental streptococci and association of Lactococcus lactis ssp. lactis with intramammary infections among different dairy farms.

PubMed

Werner, B; Moroni, P; Gioia, G; Lavín-Alconero, L; Yousaf, A; Charter, M E; Carter, B Moslock; Bennett, J; Nydam, D V; Welcome, F; Schukken, Y H

2014-11-01

Lactococcus species are counted among a large and closely related group of environmental streptococci and streptococci-like bacteria that include bovine mastitis pathogenic Streptococcus, Enterococcus, and Aerococcus species. Phenotypic and biochemical identification methods can be inaccurate and unreliable for species within this group, particularly for Lactococcus spp. As a result, the incidence of Lactococcus spp. on the farm may have been historically underreported and consequently little is known about the clinical importance of this genus as a mastitis pathogen. We used molecular genetic identification methods to accurately differentiate 60 environmental streptococci and streptococci-like bacteria isolated from cows with high somatic cell count and chronic intramammary infection (IMI; >2 somatic cell scores above 4) among 5 geographically distinct farms in New York and Minnesota that exhibited an observed increase in IMI. These isolates were phenotypically identified as Streptococcus uberis and Streptococcus spp. Genetic methods identified 42 isolates (70%) as Lactococcus lactis ssp. lactis, including all 10 isolates originally phenotypically identified as Streptococcus uberis. Antibiotic inhibition testing of all Lc. lactis ssp. lactis showed that 7 isolates were resistant to tetracycline. In the present study, a predominance of Lc. lactis ssp. lactis was identified in association with chronic, clinical bovine IMI among all 5 farms and characterized antimicrobial resistance for treatment therapies. Routine use by mastitis testing labs of molecular identification methods for environmental streptococci and streptococci-like bacteria can further define the role and prevalence of Lc. lactis ssp. lactis in association with bovine IMI and may lead to more targeted therapies. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

PubMed

Smith, I M; Baker, A; Arneborg, N; Jespersen, L

2015-11-01

The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast

Antihypertensive and hypolipidemic effect of milk fermented by specific Lactococcus lactis strains.

PubMed

Rodríguez-Figueroa, J C; González-Córdova, A F; Astiazaran-García, H; Hernández-Mendoza, A; Vallejo-Cordoba, B

2013-07-01

The antihypertensive and hypolipidemic effects of milk fermented by specific Lactococcus lactis strains in spontaneously hypertensive rats (SHR) were investigated. The SHR were fed ad libitum milk fermented by Lc. lactis NRRL B-50571, Lc. lactis NRRL B-50572, Captopril (40mg/kg of body weight, Sigma-Aldrich Co., St. Louis, MO) or purified water for 4 wk. Results suggested that Lc. lactis fermented milks presented a significant blood pressure-lowering effect. No significant difference was noted among milk fermented by Lc. lactis NRRL B-50571 and Captopril by the second and third week of treatment. Additionally, milk fermented by Lc. lactis strains modified SHR lipid profiles. Milk fermented by Lc. lactis NRRL B-50571 and B-50572 were able to reduce plasma low-density lipoprotein cholesterol and triglyceride contents. Thus, milk fermented by Lc. lactis strains may be a coadjuvant in the reduction of hypertension and hyperlipidemia and may be used as a functional food for better cardiovascular health. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication.

PubMed

Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

2016-01-01

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

Identification of yeasts and evaluation of their distribution in Taiwanese Kefir and Viili starters.

PubMed

Wang, S Y; Chen, H C; Liu, J R; Lin, Y C; Chen, M J

2008-10-01

The objective of the present study was to investigate yeast communities in kefir grains and viili starters in Taiwan through conventional microbiological cultivation and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The DNA sequencing was used as a validity technique to ensure that all isolates within each group belonged to just one species, and to confirm the identified results of PCR-DGGE. Results indicated that a combination of conventional microbiological cultivation with PCR-DGGE and sequencing could successfully identify 4 yeast species from both types of cultures in Taiwan. Kluyveromyces marxianus, Saccharomyces turicensis, and Pichia fermentans were found in Taiwanese kefir grains with a distribution of 76, 22, and 2%, respectively, whereas Klu. marxianus, Saccharomyces unisporus and P. fermentans were identified in viili starters corresponding to 58, 11, and 31% of the total cell counts, respectively. Furthermore, the culture-independent method was applied to identify the yeast species using DGGE. Only 2 yeast species, Klu. marxianus and S. turicensis, were found in kefir grains and 2, Klu. marxianus and P. fermentans, in viili starters. These results suggest that in samples containing multiple species, PCR-DGGE may fail to detect some species. Sequences of yeast isolates reported in this study have been deposited in the GenBank database under accession nos. DQ139802, AF398485, DQ377652, and AY007920.

Expression of six peptidases from Lactobacillus helveticus in Lactococcus lactis.

PubMed

Luoma, S; Peltoniemi, K; Joutsjoki, V; Rantanen, T; Tamminen, M; Heikkinen, I; Palva, A

2001-03-01

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.

Expression of Six Peptidases from Lactobacillus helveticus in Lactococcus lactis

PubMed Central

Luoma, Susanna; Peltoniemi, Kirsi; Joutsjoki, Vesa; Rantanen, Terhi; Tamminen, Marja; Heikkinen, Inka; Palva, Airi

2001-01-01

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration. PMID:11229915

Suppression of oral tolerance by Lactococcus lactis in mice.

PubMed

Sakai, Tohru; Hirota, Yuko; Nakamoto, Mariko; Shuto, Emi; Hosaka, Toshio; Makino, Seiya; Ikegami, Shuji

2011-01-01

Although oral ovabumin (OVA) administration suppressed the antibody (Ab) response in OVA-immunized mice, Lactococcus lactis increased OVA-specific IgG2a in these mice. L. lactis increased the casein-specific IgG level in NC/Nga mice fed on a casein diet. The percentage of CD4(+)CD25(+) cells was increased in DO11.10 mice orally given OVA, but this increase of CD4(+)CD25(+) cells were suppressed in L. lactis-fed DO11.10 mice.

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Lactococcus lactis ssp. lactis and Lactococcus garvieae isolated from bovine intramammary infections.

PubMed

Plumed-Ferrer, C; Barberio, A; Franklin-Guild, R; Werner, B; McDonough, P; Bennett, J; Gioia, G; Rota, N; Welcome, F; Nydam, D V; Moroni, P

2015-09-01

In total, 181 streptococci-like bacteria isolated from intramammary infections (IMI) were submitted by a veterinary clinic to Quality Milk Production Services (QMPS, Cornell University, Ithaca, NY). The isolates were characterized by sequence analysis, and 46 Lactococcus lactis ssp. lactis and 47 Lactococcus garvieae were tested for susceptibility to 17 antibiotics. No resistant strains were found for β-lactam antibiotics widely used in clinical practice (penicillin, ampicillin, and amoxicillin), and all minimum inhibitory concentrations (MIC) were far from the resistance breakpoints. Eight strains had MIC intermediate to cefazolin. The random amplification of polymorphic DNA (RAPD)-PCR fingerprint patterns showed a slightly higher heterogeneity for Lc. lactis ssp. lactis isolates than for Lc. garvieae isolates. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Use of waste materials for Lactococcus lactis development.

PubMed

Rodríguez, Noelia; Torrado, Ana; Cortés, Sandra; Domínguez, José Manuel

2010-08-15

Lactococcus lactis is an interesting microorganism with several industrial applications, particularly in the food industry. As well as being a probiotic species, L. lactis produces several metabolites with interesting properties, such as lactic acid (LA) and biosurfactants. Nevertheless, L. lactis is an especially demanding species since it has strong nutritional requirements, implying the use of complex and expensive culture media. The results showed the potential of L. lactis CECT-4434 as a LA and biosurfactant producer. The economical cost of L. lactis cultures can be reduced by replacing the MRS medium by the use of two waste materials: trimming vine shoots as C source, and 20 g L(-1) distilled wine lees (vinasses) as N, P and micronutrient sources. From the hemicellulosic fraction, 14.3 g L(-1) LA and 1.7 mg L(-1) surfactin equivalent were achieved after 74 h (surface tension reduction of 14.4 mN m(-1)); meanwhile, a simultaneous saccharification and fermentation process allowed the generation of 10.8 g L(-1) LA and 1.5 mg L(-1) surfactin equivalent after 72 h, reducing the surface tension by 12.1 units at the end of fermentation. Trimming vine shoots and vinasses can be used as alternative economical media for LA and cell-bound biosurfactant production. Copyright (c) 2010 Society of Chemical Industry.

Thermal inactivation kinetics of Lactococcus lactis subsp. lactis bacteriophage pll98-22.

PubMed

Sanlibaba, Pinar; Buzrul, S; Akkoç, Nefise; Alpas, H; Akçelik, M

2009-03-01

Survival curves of Lactococcus lactis subsp. lactis bacteriophage pll98 inactivated by heat were obtained at seven temperature values (50-80 degrees C) in M17 broth and skim milk. Deviations from first-order kinetics in both media were observed as sigmoidal shapes in the survival curves of pll98. An empirical model with four parameters was used to define the thermal inactivation. Number of parameters of the model was reduced from four to two in order to increase the robustness of the model. The reduced model produced comparable fits to the full model. Both the survival data and the calculations done using the reduced model (time necessary to reduce the number of phage pll98 six- or seven- log10) indicated that skim milk is a more protective medium than M17 broth within the assayed temperature range.

Transformation of Streptococcus lactis Protoplasts by Plasmid DNA †

PubMed Central

Kondo, Jeffery K.; McKay, Larry L.

1982-01-01

Polyethylene glycol-treated protoplasts prepared from Streptococcus lactis LM3302, a lactose-negative (Lac−) derivative of S. lactis ML3, were transformed to lactose-fermenting ability by a transductionally shortened plasmid (pLM2103) coding for lactose utilization. Images PMID:16346019

Feasibility of biohydrogen production from industrial wastes using defined microbial co-culture.

PubMed

Chen, Peng; Wang, Yuxia; Yan, Lei; Wang, Yiqing; Li, Suyue; Yan, Xiaojuan; Wang, Ningbo; Liang, Ning; Li, Hongyu

2015-05-06

The development of clean or novel alternative energy has become a global trend that will shape the future of energy. In the present study, 3 microbial strains with different oxygen requirements, including Clostridium acetobutylicum ATCC 824, Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D, were used to construct a hydrogen production system that was composed of a mixed aerobic-facultative anaerobic-anaerobic consortium. The effects of metal ions, organic acids and carbohydrate substrates on this system were analyzed and compared using electrochemical and kinetic assays. It was then tested using small-scale experiments to evaluate its ability to convert starch in 5 L of organic wastewater into hydrogen. For the one-step biohydrogen production experiment, H1 medium (nutrient broth and potato dextrose broth) was mixed directly with GAM broth to generate H2 medium (H1 medium and GAM broth). Finally, Clostridium acetobutylicum ATCC 824, Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D of three species microbial co-culture to produce hydrogen under anaerobic conditions. For the two-step biohydrogen production experiment, the H1 medium, after cultured the microbial strains Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D, was centrifuged to remove the microbial cells and then mixed with GAM broth (H2 medium). Afterward, the bacterial strain Clostridium acetobutylicum ATCC 824 was inoculated into the H2 medium to produce hydrogen by anaerobic fermentation. The experimental results demonstrated that the optimum conditions for the small-scale fermentative hydrogen production system were at pH 7.0, 35°C, a mixed medium, including H1 medium and H2 medium with 0.50 mol/L ferrous chloride, 0.50 mol/L magnesium sulfate, 0.50 mol/L potassium chloride, 1% w/v citric acid, 5% w/v fructose and 5% w/v glucose. The overall hydrogen production efficiency in the shake flask fermentation group was 33.7 mL/h(-1).L(-1), and those the two

Lactococcus lactis-based vaccines: current status and future perspectives.

PubMed

Bahey-El-Din, Mohammed; Gahan, Cormac G M

2011-01-01

Lactococcus lactis offers significant potential as a platform for the delivery of vaccines especially via mucosal routes of administration. The organism has an established history of safe use in the food industry and is highly amenable to genetic manipulation, with many systems available for efficient production of secreted and surface-expressed proteins. Here we describe the benefits of using this organism as a vaccine delivery platform and outline how L. lactis based antigen delivery may be improved. Finally we discuss the safe use of L. lactis vectors and outline the potential for use of biological containment systems and killed lactococcal preparations.

Lactococcus lactis Metabolism and Gene Expression during Growth on Plant Tissues

PubMed Central

Golomb, Benjamin L.

2014-01-01

Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits of Lactococcus lactis during growth in an Arabidopsis thaliana leaf tissue lysate (ATL). L. lactis KF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strain L. lactis IL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded with L. lactis KF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes for L. lactis growth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-type L. lactis strain KF147 but not an xylA deletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. lactis carbohydrate metabolism on plant tissues. These findings show that certain strains of L. lactis are well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations. PMID:25384484

Comparative and functional genomics of the Lactococcus lactis taxon; insights into evolution and niche adaptation.

PubMed

Kelleher, Philip; Bottacini, Francesca; Mahony, Jennifer; Kilcawley, Kieran N; van Sinderen, Douwe

2017-03-29

Lactococcus lactis is among the most widely studied lactic acid bacterial species due to its long history of safe use and economic importance to the dairy industry, where it is exploited as a starter culture in cheese production. In the current study, we report on the complete sequencing of 16 L. lactis subsp. lactis and L. lactis subsp. cremoris genomes. The chromosomal features of these 16 L. lactis strains in conjunction with 14 completely sequenced, publicly available lactococcal chromosomes were assessed with particular emphasis on discerning the L. lactis subspecies division, evolution and niche adaptation. The deduced pan-genome of L. lactis was found to be closed, indicating that the representative data sets employed for this analysis are sufficient to fully describe the genetic diversity of the taxon. Niche adaptation appears to play a significant role in governing the genetic content of each L. lactis subspecies, while (differential) genome decay and redundancy in the dairy niche is also highlighted.

Fructanase and fructosyltransferase activity of non-Saccharomyces yeasts isolated from fermenting musts of Mezcal.

PubMed

Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

2012-04-01

Fructanase and fructosyltransferase are interesting for the tequila process and prebiotics production (functional food industry). In this study, one hundred thirty non-Saccharomyces yeasts isolated from "Mezcal de Oaxaca" were screened for fructanase and fructosyltransferase activity. On solid medium, fifty isolates grew on Agave tequilana fructans (ATF), inulin or levan. In liquid media, inulin and ATF induced fructanase activities of between 0.02 and 0.27U/ml depending of yeast isolate. High fructanase activity on sucrose was observed for Kluyveromyces marxianus and Torulaspora delbrueckii, while the highest fructanase activity on inulin and ATF was observed for Issatchenkia orientalis, Cryptococcus albidus, and Candida apicola. Zygosaccharomyces bisporus and Candida boidinii had a high hydrolytic activity on levan. Sixteen yeasts belonging to K. marxianus, T. delbrueckii and C. apicola species were positive for fructosyltransferase activity. Mezcal microbiota proved to showed to be a source for new fructanase and fructosyltransferases with potential application in the tequila and food industry. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of yeast and bacteria involved in the mezcal fermentation of Agave salmiana.

PubMed

Escalante-Minakata, P; Blaschek, H P; Barba de la Rosa, A P; Santos, L; De León-Rodríguez, A

2008-06-01

To identify the yeast and bacteria present in the mezcal fermentation from Agave salmiana. The restriction and sequence analysis of the amplified region, between 18S and 28S rDNA and 16S rDNA genes, were used for the identification of yeast and bacteria, respectively. Eleven different micro-organisms were identified in the mezcal fermentation. Three of them were the following yeast: Clavispora lusitaniae, Pichia fermentans and Kluyveromyces marxianus. The bacteria found were Zymomonas mobilis subsp. mobilis and Zymomonas mobilis subsp. pomaceae, Weissella cibaria, Weissella paramesenteroides, Lactobacillus pontis, Lactobacillus kefiri, Lactobacillus plantarum and Lactobacillus farraginis. The phylogenetic analysis of 16S rDNA and ITS sequences showed that microbial diversity present in mezcal is dominated by bacteria, mainly lactic acid bacteria species and Zymomonas mobilis. Pichia fermentans and K. marxianus could be micro-organisms with high potential for the production of some volatile compounds in mezcal. We identified the community of bacteria and yeast present in mezcal fermentation from Agave salmiana.

SYSTEMATICS OF THE GENERA SACCHAROMYCES, SCHIZOSACCHAROMYCES, ENDOMYCOPSIS, KLUYVEROMYCES, SCHWANNIOMYCES AND BRETTANOMYCES: PROTON MAGNETIC RESONANCE SPECTRA OF THE MANNANS AND MANNOSE-CONTAINING POLYSACCHARIDES AS AN AID IN CLASSIFICATION,

DTIC Science & Technology

Endomycopsis, Kluyveromyces, Brettanomyces , Nematospora and Schwanniomyces and of some apparently related species of Torulopsis were determined, grouped...mannans produced by Saccharomyces, Kluyveromyces, Nematospora, Brettanomyces and Torulopsis were placed in 10 groups. The galactomannans formed by the

Lactococcus lactis As a Versatile Vehicle for Tolerogenic Immunotherapy

PubMed Central

Cook, Dana P.; Gysemans, Conny; Mathieu, Chantal

2018-01-01

Genetically modified Lactococcus lactis bacteria have been engineered as a tool to deliver bioactive proteins to mucosal tissues as a means to exert both local and systemic effects. They have an excellent safety profile, the result of years of human consumption in the food industry, as well as a lack of toxicity and immunogenicity. Also, containment strategies have been developed to promote further application as clinical protein-based therapeutics. Here, we review technological advancements made to enhanced the potential of L. lactis as live biofactories and discuss some examples of tolerogenic immunotherapies mediated by mucosal drug delivery via L. lactis. Additionally, we highlight their use to induce mucosal tolerance by targeted autoantigen delivery to the intestine as an approach to reverse autoimmune type 1 diabetes. PMID:29387056

Secretory expression of a heterologous nattokinase in Lactococcus lactis.

PubMed

Liang, Xiaobo; Zhang, Lixin; Zhong, Jin; Huan, Liandong

2007-05-01

Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P( nisZ) and signal peptide SP(Usp) were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

PubMed

Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

2017-05-01

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

Lactolisterin BU, a Novel Class II Broad-Spectrum Bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4.

PubMed

Lozo, Jelena; Mirkovic, Nemanja; O'Connor, Paula M; Malesevic, Milka; Miljkovic, Marija; Polovic, Natalija; Jovcic, Branko; Cotter, Paul D; Kojic, Milan

2017-11-01

Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes , Staphylococcus aureus , Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C 18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti , a bacteriocin with analogy to aureocin A. IMPORTANCE Lactolisterin BU, a broad-spectrum leaderless bacteriocin produced by L. lactis subsp. lactis bv. diacetylactis BGBU1-4, expresses strong antimicrobial activity against food spoilage and foodborne pathogens, such as Listeria monocytogenes , Staphylococcus aureus , Bacillus spp., and streptococci. Lactolisterin BU showed the highest similarity to aureocin-like bacteriocins produced by different bacteria. The operon for synthesis is located on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4, indicating possible horizontal transfer among producers. Copyright © 2017 American Society for Microbiology.

Lactolisterin BU, a Novel Class II Broad-Spectrum Bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4

PubMed Central

Lozo, Jelena; Mirkovic, Nemanja; O'Connor, Paula M.; Malesevic, Milka; Miljkovic, Marija; Polovic, Natalija; Cotter, Paul D.

2017-01-01

ABSTRACT Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti, a bacteriocin with analogy to aureocin A. IMPORTANCE Lactolisterin BU, a broad-spectrum leaderless bacteriocin produced by L. lactis subsp. lactis bv. diacetylactis BGBU1-4, expresses strong antimicrobial activity against food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU showed the highest similarity to aureocin-like bacteriocins produced by different bacteria. The operon for synthesis is located on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4, indicating possible horizontal transfer among producers. PMID:28842543

Introduction of Peptidase Genes from Lactobacillus delbrueckii subsp. lactis into Lactococcus lactis and Controlled Expression

PubMed Central

Wegmann, U.; Klein, J. R.; Drumm, I.; Kuipers, O. P.; Henrich, B.

1999-01-01

Peptidases PepI, PepL, PepW, and PepG from Lactobacillus delbrueckii subsp. lactis, which have no counterparts in Lactococcus lactis, and peptidase PepQ were examined to determine their potential to confer new peptidolytic properties to lactococci. Controllable expression of the corresponding genes (pep genes) was achieved by constructing translational fusions with the promoter of the nisA gene (PnisA). A suitable host strain, UKLc10, was constructed by chromosomal integration of the genes encoding the NisRK two-component system into the fivefold peptidase-deficient mutant IM16 of L. lactis. Recombinants of this strain were used to analyze growth, peptidase activities, peptide utilization, and intracellular protein cleavage products. After nisin induction of PnisA::pep fusions, all of the peptidases were visible as distinct bands in protein gels. Despite the fact that identical transcription and translation signals were used to express the pep genes, the relative amounts of individual peptidases varied considerably. All of the peptidases exhibited activities in extracts of recombinant UKLc10 clones, but only PepL and PepG allowed the clones to utilize specific peptide substrates as sources of essential amino acids. In milk medium, induction of pepG and induction of pepW resulted in growth acceleration. The activities of all five peptidases during growth in milk medium were revealed by high-performance liquid chromatography analyses of intracellular amino acid and peptide pools. PMID:10543778

The effect of nisin from Lactococcus lactis subsp. lactis on refrigerated patin fillet quality

NASA Astrophysics Data System (ADS)

Adilla, S. N.; Utami, R.; Nursiwi, A.; Nurhartadi, E.

2017-04-01

The effect of nisin from Lactococcus lactis subsp. lactis with spraying method application on quality of patin fillet during refrigerated storage (4±1°C) was investigated. The quality of patin fillet based on total plate count (TPC), pH, TVB-N, and TBA values during 16 days at 4±1°C. Completely Randomized Design (CDR) was used in one factor (nisin activity) at 0 IU/ml, 500 IU/ml, 1000 IU/ml, and 2000 IU/ml. The observation was done at 0, 4th, 8th, 12th, and 16th days of storage. The result showed that variation of nisin activity significantly affected the quality of fillet according to TPC, pH, and TVB-N values, however no significant difference on the obtained of TBA value. Nisin in 500 IU/ml, 1000 IU/ml, and 2000 IU/ml could extend the shelf-life of fillet until 4th, 8th, and 12th days respectively based on standard in all parameters.

Diversity Analysis of Dairy and Nondairy Lactococcus lactis Isolates, Using a Novel Multilocus Sequence Analysis Scheme and (GTG)5-PCR Fingerprinting▿

PubMed Central

Rademaker, Jan L. W.; Herbet, Hélène; Starrenburg, Marjo J. C.; Naser, Sabri M.; Gevers, Dirk; Kelly, William J.; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E. T.

2007-01-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)5-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene. PMID:17890345

Diversity analysis of dairy and nondairy Lactococcus lactis isolates, using a novel multilocus sequence analysis scheme and (GTG)5-PCR fingerprinting.

PubMed

Rademaker, Jan L W; Herbet, Hélène; Starrenburg, Marjo J C; Naser, Sabri M; Gevers, Dirk; Kelly, William J; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E T

2007-11-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.

Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

PubMed Central

Mulder, Joyce; Wels, Michiel; Kuipers, Oscar P.; Bron, Peter A.

2017-01-01

ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially

Improvement of the respiration efficiency of Lactococcus lactis by decreasing the culture pH.

PubMed

Shi, Weijia; Li, Yu; Gao, Xueling; Fu, Ruiyan

2016-03-01

The growth characteristics and intracellular hemin concentrations of Lactococcus lactis grown under different culture pH and aeration conditions were examined to investigate the effect of culture pH on the respiration efficiency of L. lactis NZ9000 (pZN8148). Cell biomass and biomass yield of L. lactis grown with 4 μg hemin/ml and O2 were higher than those without aeration when the culture pH was controlled at 5-6.5. The culture pH affected the respiratory efficiency in the following order of pH: 5 > 5.5 > 6 > 6.5; the lag phase increased as the culture pH decreased. Hemin accumulation was sensitive to culture pH. Among the four pH conditions, pH 5.5 was optimal for hemin accumulation in the cells. The highest intracellular hemin level in L. lactis resting cells incubated at different pH saline levels (5-6.5) was at pH 5.5. The respiration efficiency of L. lactis under respiration-permissive conditions increases markedly as the culture pH decreases. These results may help develop high cell-density L. lactis cultures. Thus, this microorganism may be used for industrial applications.

Effect of freeze-drying on viability and in vitro probiotic properties of a mixture of lactic acid bacteria and yeasts isolated from kefir.

PubMed

Bolla, Patricia A; Serradell, María de los Angeles; de Urraza, Patricio J; De Antoni, Graciela L

2011-02-01

The effect of freeze-drying on viability and probiotic properties of a microbial mixture containing selected bacterial and yeast strains isolated from kefir grains (Lactobacillus kefir, Lactobacillus plantarum, Lactococcus lactis, Saccharomyces cerevisiae and Kluyveromyces marxianus) was studied. The microorganisms were selected according to their potentially probiotic properties in vitro already reported. Two types of formulations were performed, a microbial mixture (MM) suspended in milk and a milk product fermented with MM (FMM). To test the effect of storage on viability of microorganisms, MM and FMM were freeze-dried and maintained at 4°C for six months. After 180 days of storage at 4°C, freeze-dried MM showed better survival rates for each strain than freeze-dried FMM. The addition of sugars (trehalose or sucrose) did not improve the survival rates of any of the microorganisms after freeze-drying. Freeze-drying did not affect the capacity of MM to inhibit growth of Shigella sonnei in vitro, since the co-incubation of this pathogen with freeze-dried MM produced a decrease of 2 log in Shigella viability. The safety of freeze-dried MM was tested in mice and non-translocation of microorganisms to liver or spleen was observed in BALB/c mice feed ad libitum during 7 or 20 days. To our knowledge, this is the first report about the effect of freeze-drying on viability, in vitro probiotic properties and microbial translocation of a mixture containing different strains of both bacteria and yeasts isolated from kefir.

Lactococcus lactis subsp. tructae subsp. nov. isolated from the intestinal mucus of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss).

PubMed

Pérez, Tania; Balcázar, José Luis; Peix, Alvaro; Valverde, Angel; Velázquez, Encarna; de Blas, Ignacio; Ruiz-Zarzuela, Imanol

2011-08-01

The species Lactococcus lactis currently includes three subspecies; L. lactis subsp. lactis and L. lactis subsp. cremoris, isolated from milk sources, and L. lactis subsp. hordniae, isolated from the leafhopper Hordnia circellata. In this study, three strains, designated L105(T), I3 and L101, were isolated from the intestinal mucus of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss). These strains were closely related to members of the species Lactococcus lactis. Strain L105(T) showed 99.4 % 16S rRNA gene sequence similarity to that of the type strains L. lactis subsp. lactis NCDO 604(T) and L. lactis subsp. hordniae NCDO 2181(T) and showed 99.9 % similarity to the type strain Lactococcus lactis subsp. cremoris NCDO 607(T). Analysis of two housekeeping genes, rpoB and recA, confirmed the close relationship between the novel strains and L. lactis subsp. cremoris with similarities of 99.3 and 99.7 %, respectively. The three strains could, however, be differentiated from their closest relatives on the basis of several phenotypic characteristics, as was the case for L. lactis subsp. lactis and L. lactis subsp. hordniae, which were also closely related on the basis of 16S rRNA, rpoB and recA gene sequence similarities. The strains isolated in this study represent a new subspecies, for which the name Lactococcus lactis subsp. tructae subsp. nov. is proposed. The type strain is L105(T) ( = LMG 24662(T)  = DSM 21502(T)).

Transcriptome analysis of Lactococcus lactis subsp. lactis during milk acidification as affected by dissolved oxygen and the redox potential.

PubMed

Larsen, Nadja; Moslehi-Jenabian, Saloomeh; Werner, Birgit Brøsted; Jensen, Maiken Lund; Garrigues, Christel; Vogensen, Finn Kvist; Jespersen, Lene

2016-06-02

Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L. lactis subsp. lactis DSM20481(T) during milk acidification as affected by oxygen depletion and the decrease of redox potential. Fermentations were carried out at different initial levels of dissolved oxygen (dO2) obtained by milk sparging with oxygen (high dO2, 63%) or nitrogen (low dO2, 6%). Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species (ROS), oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk. Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed. Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations. The knowledge of molecular responses involved in adaptation of L. lactis to the shifts of redox state and pH during milk fermentations is important for the dairy industry to ensure better control of cheese production. Copyright © 2016 Elsevier B.V. All rights reserved.

Lactococcus lactis subsp. lactis infection in Bester sturgeon, a cultured hybrid of Huso huso × Acipenser ruthenus, in Taiwan.

PubMed

Chen, Ming-Hui; Hung, Shao-Wen; Shyu, Ching-Lin; Lin, Cheng-Chung; Liu, Pan-Chen; Chang, Chen-Hsuan; Shia, Wei-Yau; Cheng, Ching-Fu; Lin, Shiun-Long; Tu, Ching-Yu; Lin, Yu-Hsing; Wang, Way-Shyan

2012-10-01

Approximately 5300 hybrid sturgeons with an average body weight of 600-800 g were farmed in 3 round tankers measuring 3m in diameter each containing 28,000 L of aerated groundwater. According to the owner's description, the diseased fish had anorexia, pale body color, and reddish spots on the abdomen. The morbidity and lethality rates in this outbreak were about 70% (3706/5300) and 100% (3706/3706), respectively. The clinical examination revealed enteritis, enlarged abdomen, and rapid respiration rate. The gross findings revealed a volume of about 4 mL of ascites. The histopathological examination showed multiple massive, hemorrhagic or coagulative necrotic foci in the liver and spleen. Furthermore, there was diffuse infiltration of glycogen in hepatic cells, and a few polymorphonuclear and mononuclear leucocytes were observed surrounding the spleen. Some bacterial clumps were noted around the necrotic foci. We also observed that there was moderate to severe, acute, multifocal, coagulative necrosis in the renal parenchyma, with some necrotic foci present beneath the margin of the kidney. Additionally, multifocal, coagulative necrosis was found in the pancreas. Results of microbiologic examinations, including biochemical characteristics, PCR amplification of 16S rRNA gene, sequencing and comparison, and phylogenetic analysis, revealed the pathogen of this infection was Lactococcus lactis subsp. lactis, and based on the results of an antimicrobial agent sensitivity test the bacterium was only sensitive to ampicillin and florfenicol. Additionally, results of in vivo experimental infections in hybrid tilapia showed that 1×10(8) and 1×10(9) CFU/mL of our isolate caused death in all fish and LD(50) values ranged from 10(2) to 10(5) CFU/mL. To the best of the authors' knowledge, this is the first reported case of Lactococcus lactis subsp. lactis infection in hybrid sturgeon. Copyright © 2011 Elsevier Ltd. All rights reserved.

Portal vein thrombosis and liver abscess due to Lactococcus lactis.

PubMed

Güz, Galip; Yeğin, Zeynep Arzu; Doğan, Ibrahim; Hizel, Kenan; Bali, Musa; Sindel, Sükrü

2006-06-01

A 26-year-old man was admitted with fever and abdominal pain. Abdominal ultrasonography and Doppler ultrasound eventually revealed portal vein thrombosis and a pyogenic liver abscess (17x11x11 cm). Lactococcus lactis was isolated from a culture of the abscess material. This organism is not a common pathogen in humans. This is the first published description of portal vein thrombosis and pyogenic liver abscess due to L. lactis.

[Characteristics and identification of bacteriocins produced by Lactococcus lactis subsp. lactis 194-K].

PubMed

Ustiugova, E A; Timofeeva, A V; Stoianova, L G; Netrusov, A I; Katrukha, G S

2012-01-01

The Lactococcus lactis subsp. lactis 194-K strain has been established to be able to produce two bacteriocins, one of which was identified as the known lantibiotic nisin A, and the other 194-D bacteriocin represents a polypeptide with a 2589-Da molecular mass and comprises 20 amino acid residues. Both bacteriocins were produced in varying proportions in all of the studied nutrient media, which support the growth of the producer. Depending on the cultivation medium, the nisin A content was 380- to 1123-fold lower in the 194-K stain culture fluid than that of the 194-D peptide. In comparision to to nisin A Bacteriocin 194-D possessed a wide range of antibacterial activity and suppressed the growth of both Gram-positive and Gram-negative bacteria. An optimal medium for 194-D bacteriocin synthesis was shown to be a fermentation medium which contained yeast extract, casein hydrolysate, and potassium phosphate. The biosynthesis ofbacteriocin 194-D by the 194-K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture fluid was observed at 14-20 h of the strain's growth.

Transcriptome Analysis of Lactococcus lactis in Coculture with Saccharomyces cerevisiae▿

PubMed Central

Maligoy, Mathieu; Mercade, Myriam; Cocaign-Bousquet, Muriel; Loubiere, Pascal

2008-01-01

The study of microbial interactions in mixed cultures remains an important conceptual and methodological challenge for which transcriptome analysis could prove to be the essential method for improving our understanding. However, the use of whole-genome DNA chips is often restricted to the pure culture of the species for which the chips were designed. In this study, massive cross-hybridization was observed between the foreign cDNA and the specific Lactococcus lactis DNA chip. A very simple method is proposed to considerably decrease this nonspecific hybridization, consisting of adding the microbial partner's DNA. A correlation was established between the resulting cross-hybridization and the phylogenetic distance between the microbial partners. The response of L. lactis to the presence of Saccharomyces cerevisiae was analyzed during the exponential growth phase in fermentors under defined growth conditions. Although no differences between growth kinetics were observed for the pure and the mixed cultures of L. lactis, the mRNA levels of 158 genes were significantly modified. More particularly, a strong reorientation of pyrimidine metabolism was observed when L. lactis was grown in mixed cultures. These changes in transcript abundance were demonstrated to be regulated by the ethanol produced by the yeast and were confirmed by an independent method (quantitative reverse transcription-PCR). PMID:17993564

Analyses of the probiotic property and stress resistance-related genes of Lactococcus lactis subsp. lactis NCDO 2118 through comparative genomics and in vitro assays

PubMed Central

Saraiva, Tessália D. L.; Silva, Wanderson M.; Pereira, Ulisses P.; Campos, Bruno C.; Benevides, Leandro J.; Rocha, Flávia S.; Figueiredo, Henrique C. P.; Azevedo, Vasco; Soares, Siomar C.

2017-01-01

Lactococcus lactis subsp. lactis NCDO 2118 was recently reported to alleviate colitis symptoms via its anti-inflammatory and immunomodulatory activities, which are exerted by exported proteins that are not produced by L. lactis subsp. lactis IL1403. Here, we used in vitro and in silico approaches to characterize the genomic structure, the safety aspects, and the immunomodulatory activity of this strain. Through comparative genomics, we identified genomic islands, phage regions, bile salt and acid stress resistance genes, bacteriocins, adhesion-related and antibiotic resistance genes, and genes encoding proteins that are putatively secreted, expressed in vitro and absent from IL1403. The high degree of similarity between all Lactococcus suggests that the Symbiotic Islands commonly shared by both NCDO 2118 and KF147 may be responsible for their close relationship and their adaptation to plants. The predicted bacteriocins may play an important role against the invasion of competing strains. The genes related to the acid and bile salt stresses may play important roles in gastrointestinal tract survival, whereas the adhesion proteins are important for persistence in the gut, culminating in the competitive exclusion of other bacteria. Finally, the five secreted and expressed proteins may be important targets for studies of new anti-inflammatory and immunomodulatory proteins. Altogether, the analyses performed here highlight the potential use of this strain as a target for the future development of probiotic foods. PMID:28384209

Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

PubMed

Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

2015-12-01

Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese.

The Prophylactic Effect of Probiotic Enterococcus lactis IW5 against Different Human Cancer Cells

PubMed Central

Nami, Yousef; Haghshenas, Babak; Haghshenas, Minoo; Abdullah, Norhafizah; Yari Khosroushahi, Ahmad

2015-01-01

Enterococcus lactis IW5 was obtained from human gut and the potential probiotic characteristics of this organism were then evaluated. Results showed that this strain was highly resistant to low pH and high bile salt and adhered strongly to Caco-2 human epithelial colorectal cell lines. The supernatant of E. lactis IW5 strongly inhibited the growth of several pathogenic bacteria and decreased the viability of different cancer cells, such as HeLa, MCF-7, AGS, HT-29, and Caco-2. Conversely, E. lactis IW5 did not inhibit the viability of normal FHs-74 cells. This strain did not generate toxic enzymes, including β-glucosidase, β-glucuronidase, and N-acetyl-β-glucosaminidase and was highly susceptible to ampicillin, gentamycin, penicillin, vancomycin, clindamycin, sulfamethoxazol, and chloramphenicol but resistant to erythromycin and tetracyclin. This study provided evidence for the effect of E. lactis IW5 on cancer cells. Therefore, E. lactis IW5, as a bioactive therapeutics, should be subjected to other relevant tests to verify the therapeutic suitability of this strain for clinical applications. PMID:26635778

Glycolysis Controls Plasma Membrane Glucose Sensors To Promote Glucose Signaling in Yeasts

PubMed Central

Cairey-Remonnay, Amélie; Deffaud, Julien; Wésolowski-Louvel, Micheline; Lemaire, Marc

2014-01-01

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeast Kluyveromyces lactis, extracellular glucose controls the expression of major glucose permease gene RAG1 through a cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of the RAG1 gene is abolished in glycolytic mutants. Here we show that glycolysis regulates RAG1 expression through the K. lactis Rgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor of K. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved in S. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation. PMID:25512610

Respiration-dependent utilization of sugars in yeasts: a determinant role for sugar transporters.

PubMed

Goffrini, Paola; Ferrero, Iliana; Donnini, Claudia

2002-01-01

In many yeast species, including Kluyveromyces lactis, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions. If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place. This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step. We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth. By introducing additional sugar permease genes, we have obtained K. lactis strains that were capable of growing on galactose and raffinose in the absence of respiration. High dosages of both the permease and maltase genes were indeed necessary for K. lactis cells to grow on maltose in the absence of respiration. These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in K. lactis and probably in many other yeasts.

Respiration-Dependent Utilization of Sugars in Yeasts: a Determinant Role for Sugar Transporters

PubMed Central

Goffrini, Paola; Ferrero, Iliana; Donnini, Claudia

2002-01-01

In many yeast species, including Kluyveromyces lactis, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions. If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place. This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step. We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth. By introducing additional sugar permease genes, we have obtained K. lactis strains that were capable of growing on galactose and raffinose in the absence of respiration. High dosages of both the permease and maltase genes were indeed necessary for K. lactis cells to grow on maltose in the absence of respiration. These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in K. lactis and probably in many other yeasts. PMID:11751819

Optimization of the simultaneous saccharification and fermentation process using thermotolerant yeasts.

PubMed

Ballesteros, I; Oliva, J M; Ballesteros, M; Carrasco, J

1993-01-01

Different treatments to improve the thermotolerance of fermenting yeasts for simultaneous ethanol saccharification and fermentation process of cellulosic materials have been examined. Yeasts of the genera Saccharomyces and Kluyveromyces were tested for growth and fermentation at progressively higher temperatures in the range of 42-47 degrees C. The best results were obtained with K. marxianus LG, which was then submitted to different treatments in order to achieve thermotolerant clones. A total of 35 new clones were obtained that dramatically improved the SSF of 10% Solka-floc substrate at 45 degrees C when compared to the original strain, some with ethanol concentrations as high as 33 g/L.

Structural studies of the cell wall polysaccharide from Lactococcus lactis UC509.9.

PubMed

Vinogradov, Evgeny; Sadovskaya, Irina; Grard, Thierry; Murphy, James; Mahony, Jennifer; Chapot-Chartier, Marie-Pierre; van Sinderen, Douwe

2018-05-22

Lactococcus lactis is the most widely utilised starter bacterial species in dairy fermentations. The L. lactis cell envelope contains polysaccharides, which, among other known functions, serve as bacteriophage receptors. Our previous studies have highlighted the structural diversity of these so-called cell wall polysaccharides (CWPSs) among L. lactis strains that could account for the narrow host range of most lactococcal bacteriophages. In the present work, we studied the CWPS of L. lactis strain UC509.9, an Irish dairy starter strain that is host to the temperate and well-characterized P335-type phage Tuc2009. The UC509.9 CWPS structure was analyzed by methylation, deacetylation/deamination, Smith degradation and 2D NMR spectroscopy. The CWPS consists of a linear backbone composed of a tetrasaccharide repeat unit, partially substituted with a branched phosphorylated oligosaccharide having a common trisaccharide and three non-stoichiometric substitutions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis ▿ †

PubMed Central

Ladero, Victor; Rattray, Fergal P.; Mayo, Baltasar; Martín, María Cruz; Fernández, María; Alvarez, Miguel A.

2011-01-01

Lactococcus lactis is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of L. lactis in dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains of L. lactis subsp. lactis and L. lactis subsp. cremoris that are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster of L. lactis is composed of a putative regulatory gene, aguR, followed by the genes (aguB, aguD, aguA, and aguC) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing L. lactis strains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution. PMID:21803900

Geraniol dehydrogenase, the key enzyme in biosynthesis of the alarm pheromone, from the astigmatid mite Carpoglyphus lactis (Acari: Carpoglyphidae).

PubMed

Noge, Koji; Kato, Makiko; Mori, Naoki; Kataoka, Michihiko; Tanaka, Chihiro; Yamasue, Yuji; Nishida, Ritsuo; Kuwahara, Yasumasa

2008-06-01

Geraniol dehydrogenase (GeDH), which plays an important role in the biosynthesis of neral, an alarm pheromone, was purified from the astigmatid mite Carpoglyphus lactis. The enzyme was obtained in an apparently homogeneous and active form after 1879-fold purification through seven steps of chromatography. Car. lactis GeDH was determined to be a monomer in its active form with a relative molecular mass of 42 800, which is a unique subunit structure in comparison with already established alcohol dehydrogenases. Car. lactis GeDH oxidized geraniol into geranial in the presence of NAD+. NADP+ was ineffective as a cofactor, suggesting that Car. lactis GeDH is an NAD+-dependent alcohol dehydrogenase. The optimal pH and temperature for geraniol oxidation were determined to be pH 9.0 and 25 degrees C, respectively. The Km values for geraniol and NAD+ were 51.0 microm and 59.5 microm, respectively. Car. lactis GeDH was shown to selectively oxidize geraniol, whereas its geometrical isomer, nerol, was inert as a substrate. The high specificity for geraniol suggests that Car. lactis GeDH specializes in the alarm pheromone biosynthesis of Car. lactis. Car. lactis GeDH is composed of 378 amino acids. Structurally, Car. lactis GeDH showed homology with zinc-dependent alcohol dehydrogenases found in mammals and a mosquito (36.6-37.6% identical), and the enzyme was considered to be a member of the medium-chain dehydrogenase/reductase family, in view of the highly conserved sequences of zinc-binding and NAD+-binding sites. Phylogenetic analyses indicate that Car. lactis GeDH could be categorized as a new class, different from other established alcohol dehydrogenases.

Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

PubMed Central

Kelly, William J.; Ward, Lawrence J. H.; Leahy, Sinead C.

2010-01-01

A large collection of Lactococcus lactis strains, including wild-type isolates and dairy starter cultures, were screened on the basis of their phenotype and the macrorestriction patterns produced from pulsed-field gel electrophoresis (PFGE) analysis of SmaI digests of genomic DNA. Three groups of dairy starter cultures, used for different purposes in the dairy industry, and a fourth group made up of strains isolated from the environment were selected for analysis of their chromosomal diversity using the endonuclease I-CeuI. Chromosome architecture was largely conserved with each strain having six copies of the rRNA genes, and the chromosome size of individual strains ranged between 2,240 and 2,688 kb. The origin of L. lactis strains showed the greatest correlation with chromosome size, and dairy strains, particularly those with the cremoris phenotype, had smaller chromosomes than wild-type strains. Overall, this study, coupled with analysis of the sequenced L. lactis genomes, provides evidence that defined strain dairy starter cultures have arisen from plant L. lactis strains. Adaptation of these strains to the dairy environment has involved loss of functions resulting in smaller chromosomes and acquisition of genes (usually plasmid associated) that facilitate growth in milk. We conclude that dairy starter cultures generally and the industrially used cremoris and diacetylactis phenotype strains in particular comprise a specialized group of L. lactis strains that have been selected to become an essential component of industrial processes and have evolved accordingly, so that they are no longer fit to survive outside the dairy environment. PMID:20847124

Evaluation of Lactococcus lactis Isolates from Nondairy Sources with Potential Dairy Applications Reveals Extensive Phenotype-Genotype Disparity and Implications for a Revised Species

PubMed Central

Cavanagh, Daniel; Casey, Aidan; Altermann, Eric; Cotter, Paul D.; Fitzgerald, Gerald F.

2015-01-01

Lactococcus lactis is predominantly associated with dairy fermentations, but evidence suggests that the domesticated organism originated from a plant niche. L. lactis possesses an unusual taxonomic structure whereby strain phenotypes and genotypes often do not correlate, which in turn has led to confusion in L. lactis classification. A bank of L. lactis strains was isolated from various nondairy niches (grass, vegetables, and bovine rumen) and was further characterized on the basis of key technological traits, including growth in milk and key enzyme activities. Phenotypic analysis revealed all strains from nondairy sources to possess an L. lactis subsp. lactis phenotype (lactis phenotype); however, seven of these strains possessed an L. lactis subsp. cremoris genotype (cremoris genotype), determined by two separate PCR assays. Multilocus sequence typing (MLST) showed that strains with lactis and cremoris genotypes clustered together regardless of habitat, but it highlighted the increased diversity that exists among “wild” strains. Calculation of average nucleotide identity (ANI) and tetranucleotide frequency correlation coefficients (TETRA), using the JSpecies software tool, revealed that L. lactis subsp. cremoris and L. lactis subsp. lactis differ in ANI values by ∼14%, below the threshold set for species circumscription. Further analysis of strain TIFN3 and strains from nonindustrial backgrounds revealed TETRA values of <0.99 in addition to ANI values of <95%, implicating that these two groups are separate species. These findings suggest the requirement for a revision of L. lactis taxonomy. PMID:25841018

Production of the small heat shock protein Lo18 from Oenococcus oeni in Lactococcus lactis improves its stress tolerance.

PubMed

Weidmann, Stéphanie; Maitre, Magali; Laurent, Julie; Coucheney, Françoise; Rieu, Aurélie; Guzzo, Jean

2017-04-17

Lactococcus lactis is a lactic acid bacterium widely used in cheese and fermented milk production. During fermentation, L. lactis is subjected to acid stress that impairs its growth. The small heat shock protein (sHsp) Lo18 from the acidophilic species Oenococcus oeni was expressed in L. lactis. This sHsp is known to play an important role in protein protection and membrane stabilization in O. oeni. The role of this sHsp could be studied in L. lactis, since no gene encoding for sHsp has been detected in this species. L. lactis subsp. cremoris strain MG1363 was transformed with the pDLhsp18 plasmid, which is derived from pDL278 and contains the hsp18 gene (encoding Lo18) and its own promoter sequence. The production of Lo18 during stress conditions was checked by immunoblotting and the cellular distribution of Lo18 in L. lactis cells after heat shock was determined. Our results clearly indicated a role for Lo18 in cytoplasmic protein protection and membrane stabilization during stress. The production of sHsp in L. lactis improved tolerance to heat and acid conditions in this species. Finally, the improvement of the L. lactis survival in milk medium thanks to Lo18 was highlighted, suggesting an interesting role of this sHsp. These findings suggest that the expression of a sHsp by a L. lactis strain results in greater resistance to stress, and, can consequently enhance the performances of industrial strains. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese

PubMed Central

Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

2015-01-01

Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products. PMID:26221109

Antilisterial Activity of Nisin-Like Bacteriocin-Producing Lactococcus lactis subsp. lactis Isolated from Traditional Sardinian Dairy Products

PubMed Central

Cosentino, Sofia; Fadda, Maria Elisabetta; Deplano, Maura; Melis, Roberta; Pomata, Rita; Pisano, Maria Barbara

2012-01-01

With the aim of selecting LAB strains with antilisterial activity to be used as protective cultures to enhance the safety of dairy products, the antimicrobial properties of 117 Lactococcus lactis subsp. lactis isolated from artisanal Sardinian dairy products were evaluated, and six strains were found to produce bacteriocin-like substances. The capacity of these strains to antagonize Listeria monocytogenes during cocultivation in skimmed milk was evaluated, showing a reduction of L. monocytogenes counts of approximately 4 log units compared to the positive control after 24 h of incubation. In order for a strain to be used as bioprotective culture, it should be carefully evaluated for the presence of virulence factors, to determine what potential risks might be involved in its use. None of the strains tested was found to produce biogenic amines or to possess haemolytic activity. In addition, all strains were sensitive to clinically important antibiotics such as ampicillin, tetracycline, and vancomycin. Our results suggest that these bac+ strains could be potentially applied in cheese manufacturing to control the growth of L. monocytogenes. PMID:22536018

Enhanced enzymatic hydrolysis and ethanol production from cashew apple bagasse pretreated with alkaline hydrogen peroxide.

PubMed

da Costa, Jessyca Aline; Marques, José Edvan; Gonçalves, Luciana Rocha Barros; Rocha, Maria Valderez Ponte

2015-03-01

The effect of combinations and ratios between different enzymes has been investigated in order to assess the optimal conditions for hydrolysis of cashew apple bagasse pretreated with alkaline hydrogen peroxide (the solids named CAB-AHP). The separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes were evaluated in the ethanol production. The enzymatic hydrolysis conducted with cellulase complex and β-glucosidase in a ratio of 0.61:0.39, enzyme loading of 30FPU/g(CAB-AHP) and 66CBU/g(CAB-AHP), respectively, using 4% cellulose from CAB-AHP, turned out to be the most effective conditions, with glucose and xylose yields of 511.68 mg/g(CAB-AHP) and 237.8 mg/g(CAB-AHP), respectively. Fermentation of the pure hydrolysate by Kluyveromyces marxianus ATCC 36907 led to an ethanol yield of 61.8kg/ton(CAB), corresponding to 15 g/L ethanol and productivity of 3.75 g/( Lh). The ethanol production obtained for SSF process using K. marxianus ATCC 36907 was 18 g/L corresponding to 80% yield and 74.2kg/ton(CAB). Copyright © 2014 Elsevier Ltd. All rights reserved.

Butanol is cytotoxic to Lactococcus lactis while ethanol and hexanol are cytostatic.

PubMed

Hviid, Anne-Mette Meisner; Ruhdal-Jensen, Peter; Kilstrup, Mogens

2017-04-01

Lactic acid bacteria currently used extensively by the dairy industry have a superior tolerance towards short-chain alcohols, which makes them interesting targets for use in future bio-refineries. The mechanism underlying the alcohol tolerance of lactic acid bacteria has so far received little attention. In the present study, the physiological alcohol stress response of Lactococcus lactis subsp. cremoris MG1363 towards the primary, even-chain alcohols ethanol, butanol and hexanol, was characterized. The alcohol tolerance of L. lactis was found to be comparable to those reported for highly alcohol-resistant lactic acid bacteria. Combined results from alcohol survival rate, live/dead staining, and a novel usage of the β-galactosidase assay, revealed that while high concentrations of ethanol and hexanol were cytostatic to L. lactis, high concentrations of butanol were cytotoxic, causing irreparable damages to the cell membrane.

Cloning and sequence analysis of the invertase gene INV 1 from the yeast Pichia anomala.

PubMed

Pérez, J A; Rodríguez, J; Rodríguez, L; Ruiz, T

1996-02-01

A genomic library from the yeast Pichia anomala has been constructed and employed to clone the gene encoding the sucrose-hydrolysing enzyme invertase by complementation of a sucrose non-fermenting mutant of Saccharomyces cerevisiae. The cloned gene, INV1, was sequenced and found to encode a polypeptide of 550 amino acids which contained a 22 amino-acid signal sequence and ten potential glycosylation sites. The amino-acid sequence shows significant identity with other yeast invertases and also with Kluyveromyces marxianus inulinase, a yeast beta-fructofuranosidase which has a different substrate specificity. The nucleotide sequences of the 5' and 3' non-coding regions were found to contain several consensus motifs probably involved in the initiation and termination of gene transcription.

A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain.

PubMed

Camperio, Cristina; Armas, Federica; Biasibetti, Elena; Frassanito, Paolo; Giovannelli, Carlo; Spuria, Liliana; D'Agostino, Claudia; Tait, Sabrina; Capucchio, Maria Teresa; Marianelli, Cinzia

2017-01-01

Lactococcus lactis is one of the most important microorganisms in the dairy industry and has "generally recognized as safe" (GRAS) status. L. lactis belongs to the group of lactic acid bacteria (LAB) and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU) or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU) or S. aureus (≈ 102 CFU/ml) were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1β and TNF-α levels were also found in

A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain

PubMed Central

Biasibetti, Elena; Frassanito, Paolo; Giovannelli, Carlo; Spuria, Liliana; D’Agostino, Claudia; Tait, Sabrina; Capucchio, Maria Teresa

2017-01-01

Lactococcus lactis is one of the most important microorganisms in the dairy industry and has “generally recognized as safe” (GRAS) status. L. lactis belongs to the group of lactic acid bacteria (LAB) and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU) or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU) or S. aureus (≈ 102 CFU/ml) were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1β and TNF-α levels were also found

Endosomal recognition of Lactococcus lactis G121 and its RNA by dendritic cells is key to its allergy-protective effects.

PubMed

Stein, Karina; Brand, Stephanie; Jenckel, André; Sigmund, Anna; Chen, Zhijian James; Kirschning, Carsten J; Kauth, Marion; Heine, Holger

2017-02-01

Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic inflammatory reactions. We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection. L lactis G121-induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor-treated (bafilomycin and cytochalasin D) human monocyte-derived dendritic cells (moDCs), bone marrow-derived mouse dendritic cells (BMDCs), and moDC/naive CD4 + T-cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy. L lactis G121-treated murine BMDCs and human moDCs released T H 1-polarizing cytokines and induced T H 1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of T H 1-polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll-like receptor (Tlr) 13 -/- BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The T H 1-polarizing activity of L lactis G121-treated human DCs was blocked by TLR8-specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide-binding oligomerization domain-containing protein (NOD) 2. Bacterial RNA is the main driver of L lactis G121-mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8. Copyright © 2016 American Academy of Allergy, Asthma & Immunology

Kefir-isolated bacteria and yeasts inhibit Shigella flexneri invasion and modulate pro-inflammatory response on intestinal epithelial cells.

PubMed

Bolla, P A; Abraham, A G; Pérez, P F; de Los Angeles Serradell, M

2016-02-01

The aim of this work was to evaluate the ability of a kefir-isolated microbial mixture containing three bacterial and two yeast strains (MM) to protect intestinal epithelial cells against Shigella flexneri invasion, as well as to analyse the effect on pro-inflammatory response elicited by this pathogen. A significant decrease in S. flexneri strain 72 invasion was observed on both HT-29 and Caco-2 cells pre-incubated with MM. Pre-incubation with the individual strains Saccharomyces cerevisiae CIDCA 8112 or Lactococcus lactis subsp. lactis CIDCA 8221 also reduced the internalisation of S. flexneri into HT-29 cells although in a lesser extent than MM. Interestingly, Lactobacillus plantarum CIDCA 83114 exerted a protective effect on the invasion of Caco-2 and HT-29 cells by S. flexneri. Regarding the pro-inflammatory response on HT-29 cells, S. flexneri infection induced a significant activation of the expression of interleukin 8 (IL-8), chemokine (C-C motif) ligand 20 (CCL20) and tumour necrosis factor alpha (TNF-α) encoding genes (P<0.05), whereas incubation of cells with MM did not induce the expression of any of the mediators assessed. Interestingly, pre-incubation of HT-29 monolayer with MM produced an inhibition of S. flexneri-induced IL-8, CCL20 and TNF-α mRNA expression. In order to gain insight on the effect of MM (or the individual strains) on this pro-inflammatory response, a series of experiments using a HT-29-NF-κB-hrGFP reporter system were performed. Pre-incubation of HT-29-NF-κB-hrGFP cells with MM significantly dampened Shigella-induced activation. Our results showed that the contribution of yeast strain Kluyveromyces marxianus CIDCA 8154 seems to be crucial in the observed effect. In conclusion, results presented in this study demonstrate that pre-treatment with a microbial mixture containing bacteria and yeasts isolated from kefir, resulted in inhibition of S. flexneri internalisation into human intestinal epithelial cells, along with the

Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A can efficiently internalize and deliver DNA in human epithelial cells.

PubMed

Innocentin, Silvia; Guimarães, Valeria; Miyoshi, Anderson; Azevedo, Vasco; Langella, Philippe; Chatel, Jean-Marc; Lefèvre, François

2009-07-01

Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA(+)), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA(+) and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA(+)) or its fibronectin binding domains C and D (L. lactis CD(+)). L. lactis FnBPA(+) and L. lactis InlA(+) showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD(+) was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA(+) or L. lactis InlA(+). Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.

Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis.

PubMed

van der Els, Simon; James, Jennelle K; Kleerebezem, Michiel; Bron, Peter A

2018-04-15

CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN -targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis , while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria. IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the

Recombinant porcine epidermal growth factor-secreting Lactococcus lactis promotes the growth performance of early-weaned piglets

PubMed Central

2014-01-01

Background Epidermal growth factor (EGF) is an important growth factor in regulation of cell proliferation, differentiation, survival and apoptosis. Studies showed that food-grade Lactococcus lactis (L. lactis) and NICE expression system have superior performance in exogenous protein expression. This study aimed to construct and express porcine EGF (pEGF), and use L. lactis as vehicle for producing and delivering pEGF. Furthermore, investigating biological activity of pEGF and exploring applications feasibility of combination effects of L. lactis and pEGF on early weaned piglets’ production. Results A recombinant Lactococcus lactis which produced and secreted pEGF at 1000 ng/ml in culture supernatant was generated. Secreted pEGF was a fully biologically active protein, as demonstrated by its capacity to stimulate L929 mouse fibroblast cell line proliferation in vitro. For in vivo study, forty piglets were randomly allocated to control, antibiotic control, empty vector-expressing L. lactis (LL-EV) and pEGF-secreting L. lactis (LL-pEGF). After 14 d of rearing, final body weight and average daily gain in LL-pEGF were greater (P < 0.05, 8.95 vs. 8.37 kg, 206.1 vs. 157.7 g/day, respectively) than those in control, but no significant differences between LL-pEGF, LL-EV and antibiotic control. Overall period average daily feed intake was higher in LL-pEGF, LL-EV and antibiotic control than in control (P < 0.05, 252.9, 255.6, 250.0, 207.3 g/day, respectively). No significant difference was observed on ADFI/ADG. LL-pEGF increased villous height in the duodenum, jejunum and ileum than in control and LL-EV (P < 0.05). Sucrase in the 3 intestinal segments, aminopeptidase A in the duodenum and Jejunum, aminopeptidase N and dipeptidase IV in the duodenum in LL-pEGF were higher than those in control (P < 0.05). Furthermore, Escherichia coli and Enterococcus counts decreased in the ileum and Lactobacillus increased in the ileum and cecum digesta in LL-pEGF compare with the

Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71

PubMed Central

Varma, Nadimpalli Ravi S.; Toosa, Haryanti; Foo, Hooi Ling; Alitheen, Noorjahan Banu Mohamed; Nor Shamsudin, Mariana; Arbab, Ali S.; Yusoff, Khatijah; Abdul Rahim, Raha

2013-01-01

In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle. PMID:23476790

Use of murine models to detect the allergenicity of genetically modified Lactococcus lactis NZ9000/pNZPNK.

PubMed

Chiang, Shen-Shih; Liu, Chin-Feng; Ku, Ting-Wei; Mau, Jeng-Leun; Lin, Hsin-Tang; Pan, Tzu-Ming

2011-04-27

By introducing aprN into Lactococcus lactis NZ9000, the genetically modified L. lactis NZ9000/pNZPNK successfully expressed the nattokinase. The safety assessment of this novel strain was based on allergenicity of pepsin digestion stability and murine model serologic identity. Subjecting to the GM strain and host to pepsin digestion, the soluble fractions and cell debris were fast degraded completely. Feeding with ovalbumin resulted in significantly higher production of IgG1 and IgE as compared to that of L. lactis NZ9000/pNZPNK or L. lactis NZ9000. Further, the serum IgG2a level increased dose-dependently at week 2 and induced immune reaction toward Th1 pathway. Secretion of cytokines IL-4 and IL-10 fed with lactococci was significantly lower than that of the OVA group. L. lactis NZ9000/pNZPNK did not increase the proliferation of type 2 helper T cells in spleen or induce allergenicity in BALB/c mice. On the basis of the results, the new GM lactic acid bacterium is regarded as safe to use.

Bifidobacterium animalis subsp. lactis ATCC 27673 Is a Genomically Unique Strain within Its Conserved Subspecies

PubMed Central

Loquasto, Joseph R.; Barrangou, Rodolphe; Dudley, Edward G.; Stahl, Buffy; Chen, Chun

2013-01-01

Many strains of Bifidobacterium animalis subsp. lactis are considered health-promoting probiotic microorganisms and are commonly formulated into fermented dairy foods. Analyses of previously sequenced genomes of B. animalis subsp. lactis have revealed little genetic diversity, suggesting that it is a monomorphic subspecies. However, during a multilocus sequence typing survey of Bifidobacterium, it was revealed that B. animalis subsp. lactis ATCC 27673 gave a profile distinct from that of the other strains of the subspecies. As part of an ongoing study designed to understand the genetic diversity of this subspecies, the genome of this strain was sequenced and compared to other sequenced genomes of B. animalis subsp. lactis and B. animalis subsp. animalis. The complete genome of ATCC 27673 was 1,963,012 bp, contained 1,616 genes and 4 rRNA operons, and had a G+C content of 61.55%. Comparative analyses revealed that the genome of ATCC 27673 contained six distinct genomic islands encoding 83 open reading frames not found in other strains of the same subspecies. In four islands, either phage or mobile genetic elements were identified. In island 6, a novel clustered regularly interspaced short palindromic repeat (CRISPR) locus which contained 81 unique spacers was identified. This type I-E CRISPR-cas system differs from the type I-C systems previously identified in this subspecies, representing the first identification of a different system in B. animalis subsp. lactis. This study revealed that ATCC 27673 is a strain of B. animalis subsp. lactis with novel genetic content and suggests that the lack of genetic variability observed is likely due to the repeated sequencing of a limited number of widely distributed commercial strains. PMID:23995933

Genome‐scale diversity and niche adaptation analysis of Lactococcus lactis by comparative genome hybridization using multi‐strain arrays

PubMed Central

Siezen, Roland J.; Bayjanov, Jumamurat R.; Felis, Giovanna E.; van der Sijde, Marijke R.; Starrenburg, Marjo; Molenaar, Douwe; Wels, Michiel; van Hijum, Sacha A. F. T.; van Hylckama Vlieg, Johan E. T.

2011-01-01

Summary Lactococcus lactis produces lactic acid and is widely used in the manufacturing of various fermented dairy products. However, the species is also frequently isolated from non‐dairy niches, such as fermented plant material. Recently, these non‐dairy strains have gained increasing interest, as they have been described to possess flavour‐forming activities that are rarely found in dairy isolates and have diverse metabolic properties. We performed an extensive whole‐genome diversity analysis on 39 L. lactis strains, isolated from dairy and plant sources. Comparative genome hybridization analysis with multi‐strain microarrays was used to assess presence or absence of genes and gene clusters in these strains, relative to all L. lactis sequences in public databases, whereby chromosomal and plasmid‐encoded genes were computationally analysed separately. Nearly 3900 chromosomal orthologous groups (chrOGs) were defined on basis of four sequenced chromosomes of L. lactis strains (IL1403, KF147, SK11, MG1363). Of these, 1268 chrOGs are present in at least 35 strains and represent the presently known core genome of L. lactis, and 72 chrOGs appear to be unique for L. lactis. Nearly 600 and 400 chrOGs were found to be specific for either the subspecies lactis or subspecies cremoris respectively. Strain variability was found in presence or absence of gene clusters related to growth on plant substrates, such as genes involved in the consumption of arabinose, xylan, α‐galactosides and galacturonate. Further niche‐specific differences were found in gene clusters for exopolysaccharides biosynthesis, stress response (iron transport, osmotolerance) and bacterial defence mechanisms (nisin biosynthesis). Strain variability of functions encoded on known plasmids included proteolysis, lactose fermentation, citrate uptake, metal ion resistance and exopolysaccharides biosynthesis. The present study supports the view of L. lactis as a species with a very flexible

Utilization of inulin-containing waste in industrial fermentations to produce biofuels and bio-based chemicals.

PubMed

Hughes, Stephen R; Qureshi, Nasib; López-Núñez, Juan Carlos; Jones, Marjorie A; Jarodsky, Joshua M; Galindo-Leva, Luz Ángela; Lindquist, Mitchell R

2017-04-01

Inulins are polysaccharides that belong to an important class of carbohydrates known as fructans and are used by many plants as a means of storing energy. Inulins contain 20 to several thousand fructose units joined by β-2,1 glycosidic bonds, typically with a terminal glucose unit. Plants with high concentrations of inulin include: agave, asparagus, coffee, chicory, dahlia, dandelion, garlic, globe artichoke, Jerusalem artichoke, jicama, onion, wild yam, and yacón. To utilize inulin as its carbon and energy source directly, a microorganism requires an extracellular inulinase to hydrolyze the glycosidic bonds to release fermentable monosaccharides. Inulinase is produced by many microorganisms, including species of Aspergillus, Kluyveromyces, Penicillium, and Pseudomonas. We review various inulinase-producing microorganisms and inulin feedstocks with potential for industrial application as well as biotechnological efforts underway to develop sustainable practices for the disposal of residues from processing inulin-containing crops. A multi-stage biorefinery concept is proposed to convert cellulosic and inulin-containing waste produced at crop processing operations to valuable biofuels and bioproducts using Kluyveromyces marxianus, Yarrowia lipolytica, Rhodotorula glutinis, and Saccharomyces cerevisiae as well as thermochemical treatments.

Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. lactis impairs the growth during citrate metabolism.

PubMed

Augagneur, Y; Garmyn, D; Guzzo, J

2008-01-01

Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions. A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenance of intracellular pH. Experiments performed on resting cells clearly showed that oxaloacetate accumulated temporarily in the supernatant of the mutant. This accumulation could be involved in the perturbations observed during citrate metabolism, as the addition of oxaloacetate in M17 medium inhibited the growth of L. lactis. The mutation of oxaloacetate decarboxylase perturbed citrate metabolism and reduced the benefits of its utilization during growth under acidic conditions. This study allows a better understanding of citrate metabolism and the role of oxaloacetate decarboxylase in the tolerance of lactic acid bacteria to acidic conditions.

Increasing the Heme-Dependent Respiratory Efficiency of Lactococcus lactis by Inhibition of Lactate Dehydrogenase

PubMed Central

Arioli, Stefania; Zambelli, Daniele; Guglielmetti, Simone; De Noni, Ivano; Pedersen, Martin B.; Pedersen, Per Dedenroth; Dal Bello, Fabio

2013-01-01

The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism. PMID:23064338

Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions

PubMed Central

Goel, Anisha; Santos, Filipe; de Vos, Willem M.; Teusink, Bas

2012-01-01

Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. lactis subsp. cremoris MG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L. lactis, mimicking as closely as practically possible its intracellular environment. Procedures were optimized to be carried out in 96-well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established. Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of Vmax over measured in vivo fluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements for L. lactis. PMID:22020503

Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis

PubMed Central

Zhang, Huimin; Wang, Qingjing; Fisher, Derek J.; Cai, Mingzhu; Chakravartty, Vandana; Ye, Huiyan; Li, Ping; Solbiati, Jose O.; Feng, Youjun

2016-01-01

Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment. The in vivo function of the two L. lactis birA genes was judged by their abilities to complement the conditional lethal E. coli birA mutant. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein (BCCP), through the expected biotinoyl-AMP intermediate. Gel shift assays were used to characterize bioY1_LL and BirA1_LL. We also determined the ability to uptake 3H-biotin by L. lactis. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. lactis. PMID:27161258

Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis.

PubMed

Zhang, Huimin; Wang, Qingjing; Fisher, Derek J; Cai, Mingzhu; Chakravartty, Vandana; Ye, Huiyan; Li, Ping; Solbiati, Jose O; Feng, Youjun

2016-05-10

Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment. The in vivo function of the two L. lactis birA genes was judged by their abilities to complement the conditional lethal E. coli birA mutant. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein (BCCP), through the expected biotinoyl-AMP intermediate. Gel shift assays were used to characterize bioY1_LL and BirA1_LL. We also determined the ability to uptake (3)H-biotin by L. lactis. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. lactis.

Natural DNA transformation is functional in Lactococcus lactis ssp. cremoris KW2.

PubMed

David, Blandine; Radziejwoski, Amandine; Toussaint, Frédéric; Fontaine, Laetitia; Henry de Frahan, Marie; Patout, Cédric; van Dillen, Sabine; Boyaval, Patrick; Horvath, Philippe; Fremaux, Christophe; Hols, Pascal

2017-06-16

Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis ssp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a low amount of ComX, the deletion of mecA , clpC , or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species. IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis ssp. cremoris KW2 by the overproduction of the master

Kluyveromyces aestuarii, a potential environmental quality indicator yeast for mangroves in the State of Rio de Janeiro, Brazil

PubMed Central

Araujo, F.V.; Hagler, A. N.

2011-01-01

Kluyveromyces aestuarii was found in sediments from 7 of 8 mangroves in Rio de Janeiro; and absent only at one site with heavy plastic bag pollution. Its presence suggests influence in other habitats from a mangrove and its absence in a mangrove suggests some non- fecal pollution or other habitat alteration. PMID:24031711

Novel angiotensin I-converting enzyme inhibitory peptides produced in fermented milk by specific wild Lactococcus lactis strains.

PubMed

Rodríguez-Figueroa, J C; González-Córdova, A F; Torres-Llanez, M J; Garcia, H S; Vallejo-Cordoba, B

2012-10-01

The ability of specific wild Lactococcus lactis strains to hydrolyze milk proteins to release angiotensin I-converting enzyme (ACE) inhibitory peptides was evaluated. The peptide profiles were obtained from the <3 kDa water-soluble extract and subsequently fractionated by reversed-phase HPLC. The fractions with the lowest half-maximal inhibitory concentration estimated values (peptide concentration necessary to inhibit ACE activity by 50%) were Lc. lactis NRRL B-50571 fraction (F)1 (0.034 ± 0.002 μg/mL; mean ± SD) and Lc. lactis NRRL B-50572B F 0005 (0.041 ± 0.003 μg/mL; mean ± SD). All peptide fractions were analyzed by reversed-phase HPLC tandem mass spectrometry. Twenty-one novel peptide sequences associated with ACE inhibitory (ACEI) activity were identified. Several novel ACEI peptides presented peptides encrypted with proven hypotensive activity. In conclusion, specific wild Lc. lactis strains were able to hydrolyze milk proteins to generate potent ACEI peptides. However, further studies are necessary to find out the relationship between Lc. lactis strain proteolytic systems and their ability to biogenerate hypotensive peptides. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Microbiology neutralization of zearalenone using Lactococcus lactis and Bifidobacterium sp.

PubMed

Król, A; Pomastowski, P; Rafińska, K; Railean-Plugaru, V; Walczak, J; Buszewski, B

2018-01-01

The aim of the study was to neutralize zearalenone by lactic acid bacteria (LAB) such as Lactococcus lactis and Bifidobacterium sp. and investigate the mechanism of zearalenone (ZEA) binding. Neutralization of ZEA by LAB was confirmed by identification of binding kinetics and spectroscopic studies such as Fourier transform infrared spectroscopy (FT-IR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The obtained results showed that the kinetic process of zearalenone binding to L. lactis is not homogeneous but is expressed with an initial rapid stage with about 90% of ZEA biosorption and with a much slower second step. In case of Bifidobacterium sp., the neutralization process is homogeneous; the main stage can be described with about 88% of ZEA biosorption. MALDI-TOF-MS measurements and FTIR analysis confirmed the uptake of zearalenone molecules by bacterial species. Moreover, the assessment of dead and live lactic acid bacteria cells after zearalenone treatment was performed using fluorescence microscopy. Graphical abstract Microbiology neutralization of zearalenone using Lactococcus lactis and Bifidobacterium sp. was confirmed by identification of binding kinetics and spectroscopic studies such as FT-IR spectroscopy and MALDI-TOF-MS spectrometry. The mechanism of ZEA binding was also investigated.

Carpoglyphus lactis (Acari: Astigmata) from various dried fruits differed in associated micro-organisms.

PubMed

Hubert, J; Nesvorná, M; Kopecký, J; Ságová-Marečková, M; Poltronieri, P

2015-02-01

Carpoglyphus lactis is a stored product mite infesting saccharide-rich stored commodities including dried fruits, wine, beer, milk products, jams and honey. The association with micro-organisms can improve the survival of mites on dried fruits. The microbial communities associated with C. lactis were studied in specimens originating from the packages of dried apricot, plums and figs and compared to the laboratory strain reared on house dust mite diet (HDMd). Clone libraries of bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) region were constructed and analysed by operational taxonomic unit (OTU) approach. The 16S rRNA gene libraries differed among the compared diets. The sequences classified to the genera Leuconostoc, Elizabethkingia, Ewingella, Erwinia, Bacillus and Serratia were prevailing in mites sampled from the dried fruits. The ITS library showed smaller differences between the laboratory strain on HDMd and the isolates from dried fruits packages, with the exception of the mite strain from dried plums. The population growth was used as an indirect indicator of fitness and decreased in the order from yeast diet to HDMd and dried fruits. The treatment and pretreatment of mites by antibiotics did not reveal the presence of antagonistic bacteria which might slow down the C. lactis population growth. The shifts of the microbial community in the gut of C. lactis were induced by the diet changes. The identified yeasts and bacteria are suggested as the main food source of stored product mites on dried fruits. The study describes the adaptation of C. lactis to feeding on dried fruits including the interaction with micro-organisms. We also identified potentially pathogenic bacteria carried by the mites to dried fruits for human consumption. © 2014 The Society for Applied Microbiology.

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus: a chronicle of evolution in action.

PubMed

El Kafsi, Hela; Binesse, Johan; Loux, Valentin; Buratti, Julien; Boudebbouze, Samira; Dervyn, Rozenn; Kennedy, Sean; Galleron, Nathalie; Quinquis, Benoît; Batto, Jean-Michel; Moumen, Bouziane; Maguin, Emmanuelle; van de Guchte, Maarten

2014-05-28

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus are lactic acid producing bacteria that are largely used in dairy industries, notably in cheese-making and yogurt production. An earlier in-depth study of the first completely sequenced ssp. bulgaricus genome revealed the characteristics of a genome in an active phase of rapid evolution, in what appears to be an adaptation to the milk environment. Here we examine for the first time if the same conclusions apply to the ssp. lactis, and discuss intra- and inter-subspecies genomic diversity in the context of evolutionary adaptation. Both L. delbrueckii ssp. show the signs of reductive evolution through the elimination of superfluous genes, thereby limiting their carbohydrate metabolic capacities and amino acid biosynthesis potential. In the ssp. lactis this reductive evolution has gone less far than in the ssp. bulgaricus. Consequently, the ssp. lactis retained more extended carbohydrate metabolizing capabilities than the ssp. bulgaricus but, due to high intra-subspecies diversity, very few carbohydrate substrates, if any, allow a reliable distinction of the two ssp. We further show that one of the most important traits, lactose fermentation, of one of the economically most important dairy bacteria, L. delbruecki ssp. bulgaricus, relies on horizontally acquired rather than deep ancestral genes. In this sense this bacterium may thus be regarded as a natural GMO avant la lettre. The dairy lactic acid producing bacteria L. delbrueckii ssp. lactis and ssp. bulgaricus appear to represent different points on the same evolutionary track of adaptation to the milk environment through the loss of superfluous functions and the acquisition of functions that allow an optimized utilization of milk resources, where the ssp. bulgaricus has progressed further away from the common ancestor.

Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence.

PubMed

Freires, Irlan A; Avilés-Reyes, Alejandro; Kitten, Todd; Simpson-Haidaris, P J; Swartz, Michael; Knight, Peter A; Rosalen, Pedro L; Lemos, José A; Abranches, Jacqueline

2017-01-02

In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.

Proposal for creation of a new genus Neomicrococcus gen. nov. to accommodate Zhihengliuella aestuarii Baik et al. 2011 and Micrococcus lactis Chittpurna et al. 2011 as Neomicrococcus aestuarii comb. nov. and Neomicrococcus lactis comb. nov.

PubMed

Prakash, Om; Sharma, Avinash; Nimonkar, Yogesh; Shouche, Yogesh S

2015-11-01

Micrococcus lactis and Zhihengliuella aestuarii were described independently in 2011. Their type strains showed high levels of 16S rRNA gene sequence similarity (99.3%). Phylogenetic analysis revealed that M. lactis MCC 2278T and Z. aestuarii JCM 16166T formed a monophyletic group and showed distant relationships to other members of closely related genera such as Micrococcus, Zhihengliuella, Arthrobacter and Citricoccus. The presence of large proportions of iso-C14:0 and iso-C16:0 with small amounts of iso-C15:0 distinguished M. lactis MCC 2278T and Z. aestuarii JCM 16166T from other members of the genera Micrococcus and Zhihengliuella. Unlike other members of the genera Zhihengliuella and Micrococcus, M. lactis MCC 2278T and Z. aestuarii JCM 16166T showed growth at low concentrations of NaCl. Thus, based on distinctive phylogenetic, chemotaxonomic and physiological features of these two organisms in comparison with other members of the genera Micrococcus and Zhihengliuella, it is clear that they do not fit within the existing classification and deserve separate status. DNA-DNA hybridization between the two type strains was 63%, indicating that they represent separate species. In this study, we propose the creation of a novel genus, Neomicrococcus gen. nov., to accommodate the two species with Neomicrococcus aestuarii gen. nov., comb. nov. (type strain JCM 16166T = KCTC 19557T) as the type species. Neomicrococcus lactis comb. nov. (type strain MCC 2278T = DSM 23694T) is also proposed.

Biofuel production from Jerusalem artichoke tuber inulins: a review

DOE PAGES

Bhagia, Samarthya; Akinosho, Hannah; Ferreira, Jorge F. S.; ...

2017-06-01

Jerusalem artichoke (JA) has a high productivity of tubers that are rich in inulins, a fructan polymer. These inulins can be easily broken down into fructose and glucose for conversion into ethanol by fermentation. This paper discusses tuber and inulin yields, effect of cultivar and environment on tuber productivity, and approaches to fermentation for ethanol production. Consolidated bioprocessing with Kluyveromyces marxianus has been the most popular approach for fermentation into ethanol. Apart from ethanol, fructose can be dehydrated into into 5-hydrolxymethylfurfural followed by catalytic conversion into hydrocarbons. Finally, findings from several studies indicate that this plant from tubers alone canmore » produce ethanol at yields that rival corn and sugarcane ethanol. JA has tremendous potential for use as a bioenergy feedstock.« less

Biofuel production from Jerusalem artichoke tuber inulins: a review

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhagia, Samarthya; Akinosho, Hannah; Ferreira, Jorge F. S.

Jerusalem artichoke (JA) has a high productivity of tubers that are rich in inulins, a fructan polymer. These inulins can be easily broken down into fructose and glucose for conversion into ethanol by fermentation. This paper discusses tuber and inulin yields, effect of cultivar and environment on tuber productivity, and approaches to fermentation for ethanol production. Consolidated bioprocessing with Kluyveromyces marxianus has been the most popular approach for fermentation into ethanol. Apart from ethanol, fructose can be dehydrated into into 5-hydrolxymethylfurfural followed by catalytic conversion into hydrocarbons. Finally, findings from several studies indicate that this plant from tubers alone canmore » produce ethanol at yields that rival corn and sugarcane ethanol. JA has tremendous potential for use as a bioenergy feedstock.« less

Cell lysis induced by membrane-damaging detergent saponins from Quillaja saponaria.

PubMed

Berlowska, Joanna; Dudkiewicz, Marta; Kregiel, Dorota; Czyzowska, Agata; Witonska, Izabela

2015-01-01

This paper presents the results of a study to determine the effect of Quillaja saponaria saponins on the lysis of industrial yeast strains. Cell lysis induced by saponin from Q. saponaria combined with the plasmolysing effect of 5% NaCl for Saccharomyces cerevisiae, Kluyveromyces marxianus yeasts biomass was conducted at 50 °C for 24-48 h. Membrane permeability and integrity of the yeast cells were monitored using fluorescent techniques and concentrations of proteins, free amino nitrogen (FAN) and free amino acids in resulting lysates were analyzed. Protein release was significantly higher in the case of yeast cell lysis promoted with 0.008% Q. saponaria and 5% NaCl in comparison to plasmolysis triggered by NaCl only. Copyright © 2015 Elsevier Inc. All rights reserved.

Subcutaneous or oral immunization of mice with Lactococcus lactis expressing F4 fimbrial adhesin FaeG.

PubMed

Liu, Shujie; Li, Yongming; Xu, Ziwei; Wang, Yicheng

2013-01-01

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea in neonatal and postweaning piglets. Fimbrial adhesion of ETEC has been considered an important colonization factor with antigenicity. To safely and effectively deliver the F4 (K88) fimbrial adhesin FaeG to the immune system, we have previously constructed the secretory expression vector pNZ8112-faeG, and FaeG was produced in cytoplasmic form in Lactococcus lactis. In this work, BALB/c mice were immunized with recombinant L. lactis to further determine the immunogenicity of recombinant FaeG (rFaeG) via the subcutaneous or oral route. Subcutaneous immunization in mice with recombinant L. lactis induced a significant increase in the F4-specific serum IgG titer and the number of antibody-secreting cells (ASCs) in the spleen. Oral immunization of mice with recombinant L. lactis induced mucosal and systemic F4-specific immune responses and increased the number of ASCs in the spleen, mesenteric lymph nodes and Peyer's patches. High-dose (2.8 × 10(11) CFU) recombinant strains and adjuvant cholera toxin B subunit enhanced specific mucosal immune responses. The results suggest the feasibility of delivering rFaeG expressed in L. lactis to the immune system in order to induce an F4-specific immune response.

Beneficial effect of Lactococcus lactis NCC 2287 in a murine model of eosinophilic esophagitis.

PubMed

Holvoet, S; Doucet-Ladevèze, R; Perrot, M; Barretto, C; Nutten, S; Blanchard, C

2016-12-01

Eosinophilic esophagitis (EoE) is a severe inflammatory disease of the esophagus which is characterized histologically by an eosinophilic infiltration into the esophageal tissue. The efficacy of probiotics in the context of atopic diseases has been well investigated but, to date, there has been no study which has evaluated probiotic effects on EoE inflammation. This study sought to identify a probiotic which improves esophageal inflammation in experimental EoE. Two candidate probiotics, Lactococcus lactis NCC 2287 and Bifidobacterium lactis NCC 2818, were tested in a murine model of EoE elicited by epicutaneous sensitization with Aspergillus fumigatus protein extract. Administration of bacterial strains in drinking water was used, respectively, as a preventive or treatment measure, or continuously throughout the study. Inflammatory parameters were assessed in the esophagus, skin, and lungs after allergen challenge. In this EoE model, supplementation with L. lactis NCC 2287 significantly decreased esophageal and bronchoalveolar eosinophilia but only when given as a therapeutic treatment. No significant effect on eosinophilia was observed when NCC 2287 was given as a preventive or a continuous intervention. NCC 2287 supplementation had no significant effect on immunoglobulin levels, skin symptom scores, or on transepidermal water loss. Supplementation with another probiotic, B. lactis NCC 2818, had no significant effect on esophageal eosinophilia. We identified a L. lactis strain, able to attenuate esophageal eosinophilic inflammation in a preclinical model of EoE. This effect is strain specific and depends on the timing and duration of bacterial supplementation. Confirmation of these observations in human clinical trials is warranted. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

L+-lactic acid production from starch by a novel amylolytic Lactococcus lactis subsp. lactis B84.

PubMed

Petrov, Kaloyan; Urshev, Zoltan; Petrova, Penka

2008-06-01

A new Lactococcus lactis subsp. lactis B84, capable of utilizing starch as a sole carbon source and producing L(+)-lactate, was isolated from spontaneously fermented rye sourdough. Aiming at maximum lactic acid productivity, the components of the media and the cultivation conditions were varied. In MRS-starch medium (with absence of yeast and meat extracts), at 33 degrees C, agitation 200 rpm and pH 6.0 for 6 days complete starch hydrolysis occurred and 5.5 gl(-1) lactic acid were produced from 18 gl(-1) starch. The identification of strain B84 was based on genetic criteria. Amplified ribosomal DNA restriction analysis (ARDRA), PCR with species-specific primers and sequencing of the 16S rDNA proved its species affiliation. Four genes for enzymes, involved in starch degradation were detected in B84 genome: amyL, amyY, glgP and apu, coding cytoplasmic and extracellular alpha-amylases, glycogen phosphorylase and amylopullulanase, respectively. Reverse transcription PCR experiments showed that both genes, encoding alpha-amylases (amyL and amyY) were expressed into mRNAs, whereas apu and glgP were not. Amylase activity assay was performed at different pH and temperatures. The cell-bond amylase proved to be the key enzyme, involved in the starch hydrolysis with maximum activity at 45 degrees C and pH 5.4.

Bifidobacterium animalis subsp. lactis decreases urinary oxalate excretion in a mouse model of primary hyperoxaluria

PubMed Central

Whittamore, Jonathan M.; Hatch, Marguerite

2015-01-01

Hyperoxaluria significantly increases the risk of calcium oxalate kidney stone formation. Since several bacteria have been shown to metabolize oxalate in vitro, including probiotic bifidobacteria, we focused on the efficiency and possible mechanisms by which bifidobacteria can infuence oxalate handling in vivo, especially in the intestines, and compared these results with the reported effects of Oxalobacter formigenes. Bifidobacterium animalis subsp. lactis DSM 10140 and B. adolescentis ATCC 15703 were administered to wild-type (WT) mice and to mice defcient in the hepatic enzyme alanine-glyoxylate aminotransferase (Agxt−/−, a mouse model of Primary Hyperoxaluria) that were fed an oxalate-supplemented diet. The administration of B. animalis subsp. lactis led to a significant decrease in urinary oxalate excretion in WT and Agxt−/− mice when compared to treatment with B. adolescent-is. Detection of B. animalis subsp. lactis in feces revealed that 3 weeks after oral gavage with the bacteria 64 % of WT mice, but only 37 % of Agxt−/− mice were colonized. Examining intestinal oxalate fuxes showed there were no significant changes to net oxalate secretion in colonized animals and were therefore not associated with the changes in urinary oxalate excretion. These results indicate that colonization with B. animalis subsp. lactis decreased urinary oxalate excretion by degrading dietary oxalate thus limiting its absorption across the intestine but it did not promote enteric oxalate excretion as reported for O. formigenes. Preventive or therapeutic administration of B. animalis subsp. lactis appears to have some potential to beneficially infuence dietary hyperoxaluria in mice. PMID:25269440

Characterization and stability of lactobacilli and yeast microbiota in kefir grains.

PubMed

Vardjan, T; Mohar Lorbeg, P; Rogelj, I; Čanžek Majhenič, A

2013-05-01

Characterization and stability of lactobacilli and yeasts from kefir grains using culture-dependent and culture-independent methods were investigated in this study. Culture-dependent analysis, followed by sequencing of 16S ribosomal DNA for bacteria and 26S rRNA gene for yeasts, revealed 3 different species of lactobacilli and yeasts, respectively. The most frequently isolated bacterial species were Lactobacillus kefiranofaciens ssp. kefirgranum, Lb. parakefiri, and Lb. kefiri, whereas yeasts belonged to Kluyveromyces marxianus, Kazachstania exigua, and Rhodosporidium kratochvilovae. This study is the first to report on the presence of R. kratochvilovae in kefir grains. On the other hand, PCR-denaturing gradient gel electrophoresis in the culture-independent method showed that the dominant microorganisms were Lb. kefiranofaciens ssp. kefirgranum, Kl. marxianus and Ka. exigua, but did not reveal bands corresponding to Lb. parakefiri, Lb. kefiri, or R. kratochvilovae. Our results support the necessity of combining more techniques for detailed and reliable study of microbial communities in kefir grains. Another interesting finding confirmed that the detected dominant microbiota of kefir grains is very stable and did not change over experimental time. This finding is important to ensure consistent product quality. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin.

PubMed

Vieira-Dalodé, G; Jespersen, L; Hounhouigan, J; Moller, P L; Nago, C M; Jakobsen, M

2007-08-01

To identify the dominant micro-organisms involved in the production of gowé, a fermented beverage, and to select the most appropriate species for starter culture development. Samples of sorghum gowé produced twice at three different production sites were taken at different fermentation times. DNA amplification by internal transcribed spacer-polymerase chain reaction of 288 lactic acid bacteria (LAB) isolates and 16S rRNA gene sequencing of selected strains revealed that the dominant LAB responsible for gowé fermentation were Lactobacillus fermentum, Weissella confusa, Lactobacillus mucosae, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella kimchii. DNA from 200 strains of yeasts was amplified and the D1/D2 domain of the 26S rRNA gene was sequenced for selected isolates, revealing that the yeasts species were Kluyveromyces marxianus, Pichia anomala, Candida krusei and Candida tropicalis. Gowé processing is characterized by a mixed fermentation dominated by Lact. fermentum, W. confusa and Ped. acidilactici for the LAB and by K. marxianus, P. anomala and C. krusei for the yeasts. The diversity of the LAB and yeasts identified offers new opportunities for technology upgrading and products development in gowé production. The identified species can be used as possible starter for a controlled fermentation of gowé.

Fermentation of protopanaxadiol type ginsenosides (PD) with probiotic Bifidobacterium lactis and Lactobacillus rhamnosus.

PubMed

Tan, Joanne Sh; Yeo, Chia-Rou; Popovich, David G

2017-07-01

Ginsenosides are believed to be the principal components behind the pharmacological actions of ginseng, and their bioactive properties are closely related to the type, position, and number of sugar moieties attached to the aglycone; thus, modification of the sugar chains may markedly change their biological activities. In this study, major protopanaxadiol type ginsenosides (PD) Rb1, Rc, and Rb2 were isolated from Panax ginseng and were transformed using two probiotic strains namely Bifidobacterium lactis Bi-07 and Lactobacillus rhamnosus HN001 to obtain specific deglycosylated ginsenosides. It was demonstrated that B. lactis transformed ginsenosides Rb1, Rc, and Rb2 to Rd within 1 h of fermentation and rare ginsenoside F2 by the conversion of Rd after 12-h fermentation. The maximum Rd concentration was 147.52 ± 1.45 μg/mL after 48-h fermentation as compared to 45.85 ± 0.71 μg/mL before fermentation. In contrast, L. rhamnosus transformed Rb1, Rc, and Rb2 into Rd as the final metabolite after 72-h fermentation. B. lactis displayed significantly (p < 0.05) higher β-glucosidase activity against p-nitrophenyl-β-glucopyranoside than L. rhamnosus and higher bioconversion efficiency during fermentation. The present study suggests that the fermentation of major PD type ginsenosides with B. lactis Bi-07 may serve as an effective means to afford bioactive deglycosylated ginsenosides and to create novel ginsenoside extracts.

Listeria monocytogenes and Salmonella enterica affect the expression of nisin gene and its production by Lactococcus lactis.

PubMed

Abdollahi, Soosan; Ghahremani, Mohammad Hossein; Setayesh, Neda; Samadi, Nasrin

2018-06-13

The Lactococcus lactis is known as a probiotic bacterium and also as a producer of nisin. Nisin has been approved by related legal agencies to be used as an antimicrobial peptide in food preservation. In fact, the L. lactis is present in different food products along with other micro-organisms especially pathogenic bacteria. So, it is important to predict the behavior of nisin-producer strain in contact with other pathogens. In this regard, nisin gene expression and the level of secreted biologically active form of nisin by L. lactis subsp. lactis in modified MRS broth and whey solution in co-culture with Listeria monocytogenes or Salmonella enterica were studied. The nisin concentration was determined by microbiological assay method and the transcription level of nisin gene was assayed through quantitative reverse transcription PCR (RT-qPCR). According to our results, the highest concentration of nisin and its gene transcription level were detected in mono- and co-cultures after 16 h of incubation, concurrent with the end of L. lactis exponential phase of growth. The nisin mRNA copies in co-cultures were higher than mono-cultures only at 16 h of incubation. But, differences between nisin concentrations in mono- and co-cultures were significant at 16, 24 h and at 12, 16, 24 h of incubation in the modified MRS medium and whey solution, respectively. This incompatibility could be related to the low availability of components required for nisin precursor modification, transportation and processing in mono-cultures. Overall, the L. lactis produced more mature and active nisin when it was in contact with pathogenic bacteria. Copyright © 2018. Published by Elsevier Ltd.

Manipulation for plasmid elimination by transforming synthetic competitors diversifies lactococcus lactis starters applicable to food products.

PubMed

Kobayashi, Miho; Nomura, Masaru; Kimoto, Hiromi

2007-11-01

This study was designed selectively to eliminate a theta-plasmid from Lactococcus lactis strains by transforming synthetic competitors. A shuttle vector for Escherichia coli and L. lactis, pDB1, was constructed by ligating a partial replicon of pDR1-1B, which is a 7.3 kb theta-plasmid in L. lactis DRC1, with an erythromycin resistance gene into pBluescript II KS(+). This versatile vector was used to construct competitors to common lactococcal theta-plasmids. pDB1 contains the 5' half of the replication origin and the 3' region of repB of pDR1-1B, but lacks the 1.1-kb region normally found between these two segments. A set of primers, Pv3 and Pv4, was designed to amplify the 1.1-kb middle parts of the general theta-replicons of lactococcal plasmids. When the PCR products were cloned into the Nru I and Xho I sites of pDB1, synthetic replicons were constructed and replication activity was restored. A number of theta-plasmids in L. lactis ssp. lactis and cremoris were eliminated selectively by transforming the synthetic competitors. These competitors were easily eliminated by subculture for a short time in the absence of selection. The resulting variants contained no exogenous DNA and are suitable for food products, since part of the phenotype was altered without altering other plasmids indispensable for fermentation.

Lactococcus lactis NCC 2287 Alleviates Food Allergic Manifestations in Sensitized Mice by Reducing IL-13 Expression Specifically in the Ileum

PubMed Central

Zuercher, Adrian W.; Weiss, Marietta; Holvoet, Sébastien; Moser, Mireille; Moussu, Hélène; van Overtvelt, Laurence; Horiot, Stéphane; Moingeon, Philippe; Nutten, Sophie; Prioult, Guénolée; Singh, Anurag; Mercenier, Annick

2012-01-01

Objective. Utilizing a food allergy murine model, we have investigated the intrinsic antiallergic potential of the Lactococcus lactis NCC 2287 strain. Methods. BALB/c mice were sensitized at weekly intervals with ovalbumin (OVA) plus cholera toxin (CT) by the oral route for 7 weeks. In this model, an oral challenge with a high dose of OVA at the end of the sensitization period leads to clinical symptoms. Lactococcus lactis NCC 2287 was given to mice via the drinking water during sensitization (prevention phase) or after sensitization (management phase). Results. Lactococcus lactis NCC 2287 administration to sensitized mice strikingly reduced allergic manifestations in the management phase upon challenge, when compared to control mice. No preventive effect was observed with the strain. Lactococcus lactis NCC 2287 significantly decreased relative expression levels of the Th-2 cytokine, IL-13, and associated chemokines CCL11 (eotaxin-1) and CCL17 (TARC) in the ileum. No effect was observed in the jejunum. Conclusion/Significance. These results taken together designate Lactococcus lactis NCC 2287 as a candidate probiotic strain appropriate in the management of allergic symptoms. PMID:21961022

Lactococcus lactis NCC 2287 alleviates food allergic manifestations in sensitized mice by reducing IL-13 expression specifically in the ileum.

PubMed

Zuercher, Adrian W; Weiss, Marietta; Holvoet, Sébastien; Moser, Mireille; Moussu, Hélène; van Overtvelt, Laurence; Horiot, Stéphane; Moingeon, Philippe; Nutten, Sophie; Prioult, Guénolée; Singh, Anurag; Mercenier, Annick

2012-01-01

Utilizing a food allergy murine model, we have investigated the intrinsic antiallergic potential of the Lactococcus lactis NCC 2287 strain. BALB/c mice were sensitized at weekly intervals with ovalbumin (OVA) plus cholera toxin (CT) by the oral route for 7 weeks. In this model, an oral challenge with a high dose of OVA at the end of the sensitization period leads to clinical symptoms. Lactococcus lactis NCC 2287 was given to mice via the drinking water during sensitization (prevention phase) or after sensitization (management phase). Lactococcus lactis NCC 2287 administration to sensitized mice strikingly reduced allergic manifestations in the management phase upon challenge, when compared to control mice. No preventive effect was observed with the strain. Lactococcus lactis NCC 2287 significantly decreased relative expression levels of the Th-2 cytokine, IL-13, and associated chemokines CCL11 (eotaxin-1) and CCL17 (TARC) in the ileum. No effect was observed in the jejunum. These results taken together designate Lactococcus lactis NCC 2287 as a candidate probiotic strain appropriate in the management of allergic symptoms.

Safety of Bifidobacterium animalis Subsp. Lactis (B. lactis) Strain BB-12-Supplemented Yogurt in Healthy Children.

PubMed

Tan, Tina P; Ba, Zhaoyong; Sanders, Mary E; D'Amico, Frank J; Roberts, Robert F; Smith, Keisha H; Merenstein, Daniel J

2017-02-01

Probiotics are live microorganisms that may provide health benefits to the individual when consumed in sufficient quantities. For studies conducted on health or disease endpoints on probiotics in the United States, the Food and Administration has required those studies to be conducted as investigational new drugs. This phase I, double-blinded, randomized, controlled safety study represents the first requirement of this pathway. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp. lactis (B lactis) strain BB-12 (BB-12)-supplemented yogurt when consumed by a generally healthy group of children. The secondary aim was to assess the effect of BB-12-supplemented yogurt on the gut microbiota of the children. Sixty children ages 1 to 5 years were randomly assigned to consume 4 ounces of either BB-12-supplemented yogurt or nonsupplemented control yogurt daily for 10 days. The primary outcome was to assess safety and tolerability, as determined by the number of reported adverse events. A total of 186 nonserious adverse events were reported, with no significant differences between the control and BB-12 groups. No significant changes due to probiotic treatment were observed in the gut microbiota of the study cohort. BB-12-supplemented yogurt is safe and well-tolerated when consumed by healthy children. The present study will form the basis for future randomized clinical trials investigating the potential effects of BB-12-supplemented yogurt in different disease states.

Assessing the effects of exposure to environmental stress on some functional properties of Bifidobacterium animalis ssp. lactis.

PubMed

Amund, O D; Ouoba, L I I; Sutherland, J P; Ghoddusi, H B

2014-12-01

This study assessed the effects of exposing a strain of Bifidobacterium animalis ssp. lactis to acid, bile and osmotic stresses on antagonistic properties, biofilm formation and antibiotic susceptibility/resistance profile. Exposure to each stress factor appeared to have no significant effect on the antagonism against Escherichia coli NCTC 12900 and Salmonella enterica serovar Enteritidis PT4. No suppression in biofilm formation due to exposure to stress was observed. Bile and osmotic stresses resulted in significantly higher biofilm formation. Expression of an exopolysaccharide synthesis gene, gtf 01207, was significantly higher when the B. animalis ssp. lactis strain was exposed to osmotic stress. Susceptibility of the B. animalis ssp. lactis strain to chloramphenicol, erythromycin, ampicillin and vancomycin, and resistance to tetracycline remained unchanged when exposed to each stress. The expression of a tetracycline resistance gene, tet(W), was significantly higher when exposed to each stress. These results may suggest that the potential for the B. animalis ssp. lactis strain to provide probiotic benefit, after exposure to the stressful conditions of the gastrointestinal tract, remains intact.

PMAA-stabilized ferrofluid/chitosan/yeast composite for bioapplications

NASA Astrophysics Data System (ADS)

Baldikova, Eva; Prochazkova, Jitka; Stepanek, Miroslav; Hajduova, Jana; Pospiskova, Kristyna; Safarikova, Mirka; Safarik, Ivo

2017-04-01

A simple, one-pot process for the preparation of magnetically responsive yeast-based biocatalysts was developed. Saccharomyces cerevisiae, Candida utilis and Kluyveromyces lactis cells were successfully incorporated into chitosan gel magnetically modified with poly(methacrylic acid)-stabilized magnetic fluid (PMAA-FF) during its formation. Magnetic PMAA-FF/chitosan/yeast composites were efficiently employed for invert sugar production. The dependence of invertase activity on used yeast, amount of magnetic biocatalyst, agitation time and after reuse was studied in detail. The tested magnetic biocatalysts retained at least 69% of their initial activity after 8 reuse cycles.

Multilocus sequence typing of Lactococcus lactis from naturally fermented milk foods in ethnic minority areas of China.

PubMed

Xu, Haiyan; Sun, Zhihong; Liu, Wenjun; Yu, Jie; Song, Yuqin; Lv, Qiang; Zhang, Jiachao; Shao, Yuyu; Menghe, Bilige; Zhang, Heping

2014-05-01

To determine the genetic diversity and phylogenetic relationships among Lactococcus lactis isolates, 197 strains isolated from naturally homemade yogurt in 9 ethnic minority areas of 6 provinces of China were subjected to multilocus sequence typing (MLST). The MLST analysis was performed using internal fragment sequences of 12 housekeeping genes (carB, clpX, dnaA, groEL, murC, murE, pepN, pepX, pyrG, recA, rpoB, and pheS). Six (dnaA) to 8 (murC) different alleles were detected for these genes, which ranged from 33.62 (clpX) to 41.95% (recA) GC (guanine-cytosine) content. The nucleotide diversity (π) ranged from 0.00362 (murE) to 0.08439 (carB). Despite this limited allelic diversity, the allele combinations of each strain revealed 72 different sequence types, which denoted significant genotypic diversity. The dN/dS ratios (where dS is the number of synonymous substitutions per synonymous site, and dN is the number of nonsynonymous substitutions per nonsynonymous site) were lower than 1, suggesting potential negative selection for these genes. The standardized index of association of the alleles IA(S)=0.3038 supported the clonality of Lc. lactis, but the presence of network structure revealed by the split decomposition analysis of the concatenated sequence was strong evidence for intraspecies recombination. Therefore, this suggests that recombination contributed to the evolution of Lc. lactis. A minimum spanning tree analysis of the 197 isolates identified 14 clonal complexes and 23 singletons. Phylogenetic trees were constructed based on the sequence types, using the minimum evolution algorithm, and on the concatenated sequence (6,192 bp), using the unweighted pair-group method with arithmetic mean, and these trees indicated that the evolution of our Lc. lactis population was correlated with geographic origin. Taken together, our results demonstrated that MLST could provide a better understanding of Lc. lactis genome evolution, as well as useful information for

Preferential localization of Lactococcus lactis cells entrapped in a caseinate/alginate phase separated system.

PubMed

Léonard, Lucie; Gharsallaoui, Adem; Ouaali, Fahima; Degraeve, Pascal; Waché, Yves; Saurel, Rémi; Oulahal, Nadia

2013-09-01

This study aimed to entrap bioprotective lactic acid bacteria in a sodium caseinate/sodium alginate aqueous two-phase system. Phase diagram at pH=7 showed that sodium alginate and sodium caseinate were not miscible when their concentrations exceeded 1% (w/w) and 6% (w/w), respectively. The stability of the caseinate/alginate two-phase system was also checked at pH values of 6.0 and 5.5. Lactococcus lactis subsp. lactis LAB3 cells were added in a 4% (w/w) caseinate/1.5% (w/w) alginate two-phase system at pH=7. Fluorescence microscopy allowed to observe that the caseinate-rich phase formed droplets dispersed in a continuous alginate-rich phase. The distribution of bacteria in such a system was observed by epifluorescence microscopy: Lc. lactis LAB3 cells stained with Live/Dead(®) Baclight kit™ were located exclusively in the protein phase. Since zeta-potential measurements indicated that alginate, caseinate and bacterial cells all had an overall negative charge at pH 7, the preferential adhesion of LAB cells was assumed to be driven by hydrophobic effect or by depletion phenomena in such biopolymeric systems. Moreover, LAB cells viability was significantly higher in the ternary mixture obtained in the presence of both caseinate and alginate than in single alginate solution. Caseinate/alginate phase separated systems appeared thus well suited for Lc. lactis LAB3 cells entrapment. Copyright © 2013 Elsevier B.V. All rights reserved.

Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

PubMed

Lu, Yifei; Yan, Hongxiang; Deng, Jiezhong; Huang, Zhigang; Jin, Xurui; Yu, Yanlan; Hu, Qiwen; Hu, Fuquan; Wang, Jing

2017-09-18

Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

Code of Federal Regulations, 2011 CFR

2011-04-01

...-galactoside galactohydrase (CAS Reg. No. CBS 683), which converts lactose to glucose and galactose. It is... in § 170.3(o)(9) of this chapter to convert lactose to glucose and galactose. (2) The ingredient is... practice is to use this ingredient in milk to produce lactase-treated milk, which contains less lactose...

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

Code of Federal Regulations, 2010 CFR

2010-04-01

...-galactoside galactohydrase (CAS Reg. No. CBS 683), which converts lactose to glucose and galactose. It is... in § 170.3(o)(9) of this chapter to convert lactose to glucose and galactose. (2) The ingredient is... practice is to use this ingredient in milk to produce lactase-treated milk, which contains less lactose...

Nisin-Producing Lactococcus lactis Strains Isolated from Human Milk

PubMed Central

Beasley, Shea S.; Saris, Per E. J.

2004-01-01

Characterization by partial 16S rRNA gene sequencing, ribotyping, and green fluorescent protein-based nisin bioassay revealed that 6 of 20 human milk samples contained nisin-producing Lactococcus lactis bacteria. This suggests that the history of humans consuming nisin is older than the tradition of consuming fermented milk products. PMID:15294850

Nisin production of Lactococcus lactis N8 with hemin-stimulated cell respiration in fed-batch fermentation system.

PubMed

Kördikanlıoğlu, Burcu; Şimşek, Ömer; Saris, Per E J

2015-01-01

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed-batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed-batch fermentation system with high fidelity (R(2) 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L(-1) h(-1) , 3 μg mL(-1) and 40%, respectively. While 1711 IU mL(-1) nisin was produced by L. lactis N8 in control fed-batch fermentation, 5410 IU mL(-1) nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed-batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed-batch fermentation. © 2015 American Institute of Chemical Engineers.

Determination of the cell wall polysaccharide and teichoic acid structures from Lactococcus lactis IL1403.

PubMed

Vinogradov, Evgeny; Sadovskaya, Irina; Courtin, Pascal; Kulakauskas, Saulius; Grard, Thierry; Mahony, Jennifer; van Sinderen, Douwe; Chapot-Chartier, Marie-Pierre

2018-06-15

In the lactic acid bacterium Lactococcus lactis, a cell wall polysaccharide (CWPS) is the bacterial receptor of the majority of infecting bacteriophages. The diversity of CWPS structures between strains explains, at least partially, the narrow host range of lactococcal phages. In the present work, we studied the polysaccharide components of the cell wall of the prototype L. lactis subsp. lactis strain IL1403. We identified a rhamnose-rich complex polysaccharide, carrying a glycerophosphate substitution, as the major component. Its structure was analyzed by 2D NMR spectroscopy, methylation analysis and MALDI-TOF MS and shown to be distinctly different from currently known lactococcal CWPS structures. It contains a linear backbone of repeated α-l-Rha disaccharide subunits, which is irregularly substituted with a trisaccharide occasionally bearing a glycerophosphate group. A poly (glycerol phosphate) teichoic acid, another important carbohydrate component of the IL1403 cell wall, was also isolated and structurally characterized. Copyright © 2018 Elsevier Ltd. All rights reserved.

Flavins contained in yeast extract are exploited for anodic electron transfer by Lactococcus lactis.

PubMed

Masuda, Masaki; Freguia, Stefano; Wang, Yung-Fu; Tsujimura, Seiya; Kano, Kenji

2010-06-01

Cyclic voltammograms of yeast extract-containing medium exhibit a clear redox peak around -0.4V vs. Ag|AgCl. Fermentative bacterium Lactococcus lactis was hereby shown to exploit this redox compound for extracellular electron transfer towards a graphite anode using glucose as an electron donor. High performance liquid chromatography revealed that this may be a flavin-type compound. The ability of L. lactis to exploit exogenous flavins for anodic glucose oxidation was confirmed by tests where flavin-type compounds were supplied to the bacterium in well defined media. Based on its mid-point potential, riboflavin can be regarded as a near-optimal mediator for microbially catalyzed anodic electron transfer. Riboflavin derivative flavin mononucleotide (FMN) was also exploited by L. lactis as a redox shuttle, unlike flavin adenine dinucleotide (FAD), possibly due to the absence of a specific transporter for the latter. The use of yeast extract in microbial fuel cell media is herein discouraged based on the related unwanted artificial addition of redox mediators which may distort experimental results. Copyright 2009 Elsevier B.V. All rights reserved.

Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments.

PubMed

Korsak, N; Taminiau, B; Leclercq, M; Nezer, C; Crevecoeur, S; Ferauche, C; Detry, E; Delcenserie, V; Daube, G

2015-06-01

in sample D. In relation to 26S pyrosequencing, our study revealed the presence of 3 main yeast species: Naumovozyma spp., Kluyveromyces marxianus, and Kazachastania khefir. For Naumovozyma, further studies are needed to assess the isolation of new species. In conclusion, this study has proved that it is possible to establish the patterns of bacterial and yeast composition of kefir and kefir grain. This was only achieved with the use of high-throughput sequencing techniques. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12-supplemented yogurt in healthy adults on antibiotics: a phase I safety study

PubMed Central

Merenstein, Daniel J; Tan, Tina P; Molokin, Aleksey; Smith, Keisha Herbin; Roberts, Robert F; Shara, Nawar M; Mete, Mihriye; Sanders, Mary Ellen; Solano-Aguilar, Gloria

2015-01-01

Probiotics are live microorganisms that, when administered in sufficient doses, provide health benefits on the host. The United States Food and Drug Administration (FDA) requires phase I safety studies for probiotics when the intended use of the product is as a drug. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp lactis (B. lactis) strain BB-12 (BB-12)-supplemented yogurt when consumed by a generally healthy group of adults who were prescribed a 10-day course of antibiotics for a respiratory infection. Secondary aims were to assess the ability of BB-12 to affect the expression of whole blood immune markers associated with cell activation and inflammatory response. A phase I, double-blinded, randomized controlled study was conducted in compliance with FDA guidelines for an Investigational New Drug (IND). Forty participants were randomly assigned to consume 4 ounces of either BB-12 -supplemented yogurt or non-supplemented control yogurt daily for 10 d. The primary outcome was to assess safety and tolerability, assessed by the number of reported adverse events. A total of 165 non-serious adverse events were reported, with no differences between the control and BB-12 groups. When compared to the control group, B lactis fecal levels were modestly higher in the BB-12-supplemented group. In a small subset of patients, changes in whole blood expression of genes associated with regulation and activation of immune cells were detected in the BB-12-supplemented group. BB-12-supplemented yogurt is safe and well tolerated when consumed by healthy adults concurrently taking antibiotics. This study will form the basis for future randomized clinical trials investigating the potential immunomodulatory effects of BB-12-supplemented yogurt in a variety of disease states. PMID:25569274

Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12-supplemented yogurt in healthy adults on antibiotics: a phase I safety study.

PubMed

Merenstein, Daniel J; Tan, Tina P; Molokin, Aleksey; Smith, Keisha Herbin; Roberts, Robert F; Shara, Nawar M; Mete, Mihriye; Sanders, Mary Ellen; Solano-Aguilar, Gloria

2015-01-01

Probiotics are live microorganisms that, when administered in sufficient doses, provide health benefits on the host. The United States Food and Drug Administration (FDA) requires phase I safety studies for probiotics when the intended use of the product is as a drug. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp lactis (B. lactis) strain BB-12 (BB-12)-supplemented yogurt when consumed by a generally healthy group of adults who were prescribed a 10-day course of antibiotics for a respiratory infection. Secondary aims were to assess the ability of BB-12 to affect the expression of whole blood immune markers associated with cell activation and inflammatory response. A phase I, double-blinded, randomized controlled study was conducted in compliance with FDA guidelines for an Investigational New Drug (IND). Forty participants were randomly assigned to consume 4 ounces of either BB-12 -supplemented yogurt or non-supplemented control yogurt daily for 10 d. The primary outcome was to assess safety and tolerability, assessed by the number of reported adverse events. A total of 165 non-serious adverse events were reported, with no differences between the control and BB-12 groups. When compared to the control group, B lactis fecal levels were modestly higher in the BB-12-supplemented group. In a small subset of patients, changes in whole blood expression of genes associated with regulation and activation of immune cells were detected in the BB-12-supplemented group. BB-12-supplemented yogurt is safe and well tolerated when consumed by healthy adults concurrently taking antibiotics. This study will form the basis for future randomized clinical trials investigating the potential immunomodulatory effects of BB-12-supplemented yogurt in a variety of disease states.

Bile salt tolerance of Lactococcus lactis is enhanced by expression of bile salt hydrolase thereby producing less bile acid in the cells.

PubMed

Bi, Jie; Liu, Song; Du, Guocheng; Chen, Jian

2016-04-01

Changes of bile salt tolerance, morphology and amount of bile acid within cells were studied to evaluate the exact effects of bile salt hydrolase (BSH) on bile salt tolerance of microorganism. The effect of BSHs on the bile salt tolerance of Lactococcus lactis was examined by expressing two BSHs (BSH1 and BSH2). Growth of L. lactis expressing BSH1 or BSH2 was better under bile salt stress compared to wild-type L. lactis. As indicated by transmission electron microscopy, bile acids released by the action of BSH induced the formation of micelles around the membrane surface of cells subject to conjugated bile salt stress. A similar micelle containing bile acid was observed in the cytoplasm by liquid chromatography-mass spectrometry. BSH1 produced fewer bile acid micelles in the cytoplasm and achieved better cell growth of L. lactis compared to BSH2. Expression of BSH improved bile salt tolerance of L. lactis but excessive production by BSH of bile acid micelles in the cytoplasm inhibited cell growth.

Purification and characterization of two new cell-bound bioactive compounds produced by wild Lactococcus lactis strain.

PubMed

Saraiva, Margarete Alice Fontes; Brede, Dag Anders; Nes, Ingolf Figved; Baracat-Pereira, Maria Cristina; de Queiroz, Marisa Vieira; de Moraes, Célia Alencar

2017-07-03

Novel compounds and innovative methods are required considering that antibiotic resistance has reached a crisis point. In the study, two cell-bound antimicrobial compounds produced by Lactococcus lactis ID1.5 were isolated and partially characterized. Following purification by cationic exchange and a solid-phase C18 column, antimicrobial activity was recovered after three runs of RPC using 60% (v/v) and 100% (v/v) of 2-propanol for elution, suggesting that more than one antimicrobial compound were produced by L. lactis ID1.5, which were in this study called compounds AI and AII. The mass spectrum of AI and AII showed major intensity ions at m/z 1070.05 and 955.9 Da, respectively. The compound AI showed a spectrum of antimicrobial activity mainly against L. lactis species, while the organisms most sensitive to compound AII were Bacillus subtilis, Listeria innocua, Streptococcus pneumoniae and Pseudomonas aeruginosa. The antimicrobial activity of both compounds was suppressed by treatment with Tween 80. Nevertheless, both compounds showed high stability to heat and proteases treatments. The isolated compounds, AI and AII, showed distinct properties from other antimicrobial substances already reported as produced by L. lactis, and have a significant inhibitory effect against two clinically important respiratory pathogens. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Molecular and Metabolic Adaptations of Lactococcus lactis at Near-Zero Growth Rates

PubMed Central

Ercan, Onur; Wels, Michiel; Smid, Eddy J.

2014-01-01

This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes. During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY in L. lactis KF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero-growth cultures. The present integrated transcriptome and metabolite (amino acids and previously reported fermentation end products) study provides molecular understanding of the adaptation of L. lactis to conditions supporting low growth rates and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates toward gene regulation patterns involved in zero-growth adaptation. PMID:25344239

Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

PubMed Central

Ng, Daphne T. W.

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts. PMID:23124224

Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

PubMed

Ng, Daphne T W; Sarkar, Casim A

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

Lactococcus lactis expressing food-grade β-galactosidase alleviates lactose intolerance symptoms in post-weaning Balb/c mice.

PubMed

Li, Jingjie; Zhang, Wen; Wang, Chuan; Yu, Qian; Dai, Ruirui; Pei, Xiaofang

2012-12-01

The endogenous β-galactosidase expressed in intestinal microbes is demonstrated to help humans in lactose usage, and treatment associated with the promotion of beneficial microorganism in the gut is correlated with lactose tolerance. From this point, a kind of recombinant live β-galactosidase delivery system using food-grade protein expression techniques and selected probiotics as vehicle was promoted by us for the purpose of application in lactose intolerance subjects. Previously, a recombinant Lactococcus lactis MG1363 strain expressing food-grade β-galactosidase, the L. lactis MG1363/FGZW, was successfully constructed and evaluated in vitro. This study was conducted to in vivo evaluate its efficacy on alleviating lactose intolerance symptoms in post-weaning Balb/c mice, which were orally administered with 1 × 10⁶ CFU or 1 × 10⁸ CFU of L. lactis MG1363/FGZW daily for 4 weeks before lactose challenge. In comparison with naïve mice, the mice administered with L. lactis MG1363/FGZW showed significant alleviation of diarrhea symptoms in less total feces weight within 6 h post-challenge and suppressed intestinal motility after lactose challenge, although there was no significant increase of β-galactosidase activity in small intestine. The alleviation also correlated with higher species abundance, more Bifidobacterium colonization, and stronger colonization resistance in mice intestinal microflora. Therefore, this recombinant L. lactis strain effectively alleviated diarrhea symptom induced by lactose uptake in lactose intolerance model mice with the probable mechanism of promotion of lactic acid bacteria to differentiate and predominantly colonize in gut microbial community, thus making it a promising probiotic for lactose intolerance subjects.

Influence of Technological Treatments on the Functionality of Bifidobacterium lactis INL1, a Breast Milk-Derived Probiotic.

PubMed

Zacarías, María Florencia; Souza, Tassia Costa; Zaburlín, Natalia; Carmona Cara, Denise; Reinheimer, Jorge; Nicoli, Jacques; Vinderola, Gabriel

2017-10-01

The aim of this study is to evaluate the influence of the technological processing on the functionality of the human breast milk probiotic strain Bifidobacterium lactis INL1. In vitro antagonistic activity of B. lactis INL1 was detected for Gram-positive and Gram-negative pathogens. B. lactis INL1 was administered to mice as fresh (F), frozen (Z), spray-dried (S), or lyophilized (L) culture. Immune parameters (IgA, IL-10, and IFN-γ) were determined and histological analysis was performed to assess functionality and protection capacity against Salmonella. In BALB/c mice, F and S cultures induced an increase in the number of IgA-producing cells in the small intestine and IL-10 levels were increased for L culture in the large intestine. In Swiss mice, B. lactis INL1 increased secretory-IgA levels in the small intestine before and after Salmonella infection, both as F or dehydrated culture. Also, an attenuation of damage in the intestinal epithelium and less inflammatory infiltrates were observed in animals that received F and S cultures, whereas in liver only F showed some effect. The anti-inflammatory effect was confirmed in both tissues by myeloperoxidase activity and by IFN-γ levels in the intestinal content. B. lactis INL1 showed inhibitory activity against pathogens and confirmed its probiotic potential in animal models. Technological processing of the probiotic strain affected its functionality. This work provides evidence about the influence of technology on the functionality of probiotics, which may help probiotics and functional food manufacturers to take processing into consideration when assessing the functionality of new strains. © 2017 Institute of Food Technologists®.

Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products.

PubMed

Zycka-Krzesinska, Joanna; Boguslawska, Joanna; Aleksandrzak-Piekarczyk, Tamara; Jopek, Jakub; Bardowski, Jacek K

2015-10-15

To assess the occurrence of antibiotic-resistant Lactic Acid Bacteria (LAB) in Polish raw milk and fermented artisanal products, a collection comprising 500 isolates from these products was screened. Among these isolates, six strains (IBB28, IBB160, IBB161, IBB224, IBB477 and IBB487) resistant to tetracycline were identified. The strains showing atypical tetracycline resistance were classified as Lactococcus lactis: three of them were identified as L. lactis subsp. cremoris (IBB224, IBB477 and IBB487) and the other three (IBB28, IBB160, IBB161) were identified as L. lactis subsp. lactis. The mechanism involving Ribosomal Protection Proteins (RPP) was identified as responsible for tetracycline resistance. Three of the tested strains (IBB28, IBB160 and IBB224) had genes encoding the TetS protein, whereas the remaining three (IBB161, IBB477 and IBB487) expressed TetM. The results also demonstrated that the genes encoding these proteins were located on genetic mobile elements. The tet(S) gene was found to be located on plasmids, whereas tet(M) was found within the Tn916 transposon. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

PubMed Central

Smit, Bart A.; van Hylckama Vlieg, Johan E. T.; Engels, Wim J. M.; Meijer, Laura; Wouters, Jan T. M.; Smit, Gerrit

2005-01-01

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain α-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3′ terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain α-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions. PMID:15640202

Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12®-supplemented yogurt in healthy children

PubMed Central

Tan, Tina P.; Ba, Zhaoyong; Sanders, Mary Ellen; D’Amico, Frank J.; Roberts, Robert F.; Smith, Keisha Herbin; Merenstein, Daniel J.

2016-01-01

Objectives Probiotics are live microorganisms that may provide health benefits to the individual when consumed in sufficient quantities. For studies conducted on health or disease endpoints on probiotics in the United States, the Food and Administration (FDA) has required those studies to be conducted as investigational new drugs. This phase I, double-blinded, randomized, controlled safety study represents the first requirement of this pathway. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12® (BB-12®)-supplemented yogurt when consumed by a generally healthy group of children. The secondary aim was to assess the effect of BB-12®-supplemented yogurt on the gut microbiota of the children. Methods Sixty children aged 1–5 years were randomly assigned to consume four ounces of either BB-12®-supplemented yogurt or non-supplemented control yogurt daily for 10 days. The primary outcome was to assess safety and tolerability, as determined by the number of reported adverse events. Results A total of 186 non-serious adverse events were reported, with no significant differences between the control and BB-12® groups. No significant changes due to probiotic treatment were observed in the gut microbiota of the study cohort. Conclusions BB-12®-supplemented yogurt is safe and well-tolerated when consumed by healthy children. This study will form the basis for future randomized clinical trials investigating the potential effects of BB-12®-supplemented yogurt in different disease states. PMID:28114246

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and Saccharomyces cerevisiae Cells in Dual Matrices.

PubMed

Kokkiligadda, Anusha; Beniwal, Arun; Saini, Priyanka; Vij, Shilpa

2016-08-01

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.

Microbiological and chemical properties of kefir manufactured by entrapped microorganisms isolated from kefir grains.

PubMed

Chen, T-H; Wang, S-Y; Chen, K-N; Liu, J-R; Chen, M-J

2009-07-01

In this study, various yeasts (Kluyveromyces marxianus, Saccharomyces turicensis, Pichia fermentans) and lactic acid bacteria (Lactobacillus kefiranofaciens, Lactobacillus kefiri, Leuconostoc mesenteroides) were entrapped in 2 different microspheres using an entrapment ratio for the strains that was based on the distribution ratio of these organisms in kefir grains. The purpose of this study was to develop a new technique to produce kefir using immobilized starter cultures isolated from kefir grains. An increase in cell counts with fermentation cycles was observed for both the lactic acid bacteria (LAB) and yeasts, whereas the cell counts of kefir grains were very stable during cultivation. Scanning electron microscopy showed that the short-chain lactobacilli and lactococci occupied the surface of the LAB microspheres, whereas the long-chain lactobacilli were inside the microspheres. When the yeasts were analyzed, cells at a high density were entrapped in cracks on the surface and within the microspheres, where they were surrounded by the short-chain lactobacilli. The distribution of the LAB and yeast species in kefir produced from grains and microspheres showed that there was no significant difference between the kefirs produced by the 2 methods; moreover, Leu. mesenteroides and K. marxianus were the predominating microflora in both types of kefir. There was no significant difference in the ethanol and exopolysaccharide contents between the 2 kefirs, although the acidity was different.

Yeast communities associated with artisanal mezcal fermentations from Agave salmiana.

PubMed

Verdugo Valdez, A; Segura Garcia, L; Kirchmayr, M; Ramírez Rodríguez, P; González Esquinca, A; Coria, R; Gschaedler Mathis, A

2011-11-01

The aims of this work were to characterize the fermentation process of mezcal from San Luis Potosi, México and identify the yeasts present in the fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the first fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were identified, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identified yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identifications were confirmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, confirming the results obtained with classic techniques.

Isolation, Identification and Characterization of Yeasts from Fermented Goat Milk of the Yaghnob Valley in Tajikistan

PubMed Central

Qvirist, Linnea A.; De Filippo, Carlotta; Strati, Francesco; Stefanini, Irene; Sordo, Maddalena; Andlid, Thomas; Felis, Giovanna E.; Mattarelli, Paola; Cavalieri, Duccio

2016-01-01

The geographically isolated region of the Yaghnob Valley, Tajikistan, has allowed its inhabitants to maintain a unique culture and lifestyle. Their fermented goat milk constitutes one of the staple foods for the Yaghnob population, and is produced by backslopping, i.e., using the previous fermentation batch to inoculate the new one. This study addresses the yeast composition of the fermented milk, assessing genotypic, and phenotypic properties. The 52 isolates included in this study revealed small species diversity, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae, and one Kazachstania unispora. The K. marxianus strains showed two different genotypes, one of which never described previously. The two genetically different groups also differed significantly in several phenotypic characteristics, such as tolerance toward high temperatures, low pH, and presence of acid. Microsatellite analysis of the S. cerevisiae strains from this study, compared to 350 previously described strains, attributed the Yaghnobi S. cerevisiae to two different ancestry origins, both distinct from the wine and beer strains, and similar to strains isolated from human and insects feces, suggesting a peculiar origin of these strains, and the existence of a gut reservoir for S. cerevisiae. Our work constitutes a foundation for strain selection for future applications as starter cultures in food fermentations. This work is the first ever on yeast diversity from fermented milk of the previously unexplored area of the Yaghnob Valley. PMID:27857705

Microbiological and biochemical aspects of Camembert-type cheeses depend on atmospheric composition in the ripening chamber.

PubMed

Leclercq-Perlat, M-N; Picque, D; Riahi, H; Corrieu, G

2006-08-01

Camembert-type cheeses were prepared from pasteurized milk seeded with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti, and Brevibacterium aurantiacum. Microorganism growth and biochemical dynamics were studied in relation to ripening chamber CO(2) atmospheric composition using 31 descriptors based on kinetic data. The chamber ripening was carried out under 5 different controlled atmospheres: continuously renewed atmosphere, periodically renewed atmosphere, no renewed atmosphere, and 2 for which CO(2) was either 2% or 6%. All microorganism dynamics depended on CO(2) level. Kluyveromyces lactis was not sensitive to CO(2) during its growth phases, but its death did depend on it. An increase of CO(2) led to a significant improvement in G. candidum. Penicillium camemberti mycelium development was enhanced by 2% CO(2). The equilibrium between P. camemberti and G. candidum populations was disrupted in favor of the yeast when CO(2) was higher than 4%. Growth of B. aurantiacum depended more on O(2) than on CO(2). Two ripening progressions were observed in relation to the presence of CO(2) at the beginning of ripening: in the presence of CO(2), the ripening was fast-slow, and in the absence of CO(2), it was slow-fast. The underrind was too runny if CO(2) was equal to or higher than 6%. The nitrogen substrate progressions were slightly related to ripening chamber CO(2) and O(2) levels. During chamber ripening, the best atmospheric condition to produce an optimum between microorganism growth, biochemical dynamics, and cheese appearance was a constant CO(2) level close to 2%.

Novel high-performance metagenome β-galactosidases for lactose hydrolysis in the dairy industry.

PubMed

Erich, Sarah; Kuschel, Beatrice; Schwarz, Thilo; Ewert, Jacob; Böhmer, Nico; Niehaus, Frank; Eck, Jürgen; Lutz-Wahl, Sabine; Stressler, Timo; Fischer, Lutz

2015-09-20

The industrially utilised β-galactosidases from Kluyveromyces spp. and Aspergillus spp. feature undesirable kinetic properties in praxis, such as an unsatisfactory lactose affinity (KM) and product inhibition (KI) by galactose. In this study, a metagenome library of about 1.3 million clones was investigated with a three-step activity-based screening strategy in order to find new β-galactosidases with more favourable kinetic properties. Six novel metagenome β-galactosidases (M1-M6) were found with an improved lactose hydrolysis performance in original milk when directly compared to the commercial β-galactosidase from Kluyveromyces lactis (GODO-YNL2). The best metagenome candidate, called "M1", was recombinantly produced in Escherichia coli BL21(DE3) in a bioreactor (volume 35 L), resulting in a total β-galactosidase M1 activity of about 1100 μkatoNPGal,37 °C L(-1). Since milk is a sensitive and complex medium, it has to be processed at 5-10 °C in the dairy industry. Therefore, the β-galactosidase M1 was tested at 8 °C in milk and possessed a good stability (t1/2=21.8 d), a desirably low apparent KM,lactose,8 °C value of 3.8±0.7 mM and a high apparent KI,galactose,8 °C value of 196.6±55.5 mM. A lactose hydrolysis process (milk, 40 nkatlactose mLmilk,8 °C(-1)) was conducted at a scale of 0.5L to compare the performance of M1 with the commercial β-galactosidase from K. lactis (GODO-YNL2). Lactose was completely (>99.99%) hydrolysed by M1 and to 99.6% (w/v) by K. lactis β-galactosidase after 25 h process time. Thus, M1 was able to achieve the limit of <100 mg lactose per litre milk, which is recommended for dairy products labelled as "lactose-free". Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Acid or erythromycin stress significantly improves transformation efficiency through regulating expression of DNA binding proteins in Lactococcus lactis F44.

PubMed

Wang, Binbin; Zhang, Huawei; Liang, Dongmei; Hao, Panlong; Li, Yanni; Qiao, Jianjun

2017-12-01

Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation. However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. lactis F44 showed a 56.1-fold increase in acid condition (pH 5.0); meanwhile, erythromycin stress (0.04 μg/mL) promoted the transformation efficiency more significantly (76.9-fold). Notably, the transformation efficiency of F44e (L. lactis F44 harboring empty pLEB124) increased up to 149.1-fold under the synergistic stresses of acid and erythromycin. In addition, the gene expression of some DNA binding proteins (DprA, RadA, RadC, RecA, RecQ, and SsbA) changed correspondingly. Especially for radA, 25.1-fold improvement was detected when F44e was exposed to pH 5.0. Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L. lactis through regulating gene expression of DNA binding proteins. We have proposed a simple but promising strategy for improving the transformation efficiency of L. lactis and other hard-transformed microorganisms. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Intramammary infusion of a live culture of Lactococcus lactis in ewes to treat staphylococcal mastitis.

PubMed

Mignacca, Sebastian Alessandro; Dore, Simone; Spuria, Liliana; Zanghì, Pietro; Amato, Benedetta; Duprè, Ilaria; Armas, Federica; Biasibetti, Elena; Camperio, Cristina; Lollai, Stefano A; Capucchio, Maria Teresa; Cannas, Eugenia Agnese; Di Marco Lo Presti, Vincenzo; Marianelli, Cinzia

2017-12-01

Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an evaluation, in two trials, of the efficacy of an intramammary infusion of a live culture of Lactococcus lactis for the treatment of subclinical and clinical mastitis in ewes. In total, 67 animals were enrolled: 19 lactating ewes (study 1), including healthy (N=6) and coagulase-negative staphylococci (CNS)-infected ewes (N=13); and 48 lactating ewes (study 2) with either CNS mastitis (N=32), or Staphylococcus aureus mastitis (N=16), for a total of 123 mammary glands. Intramammary infusions were performed with either L. lactis or PBS for 3 (study 1) or 7 (study 2) consecutive days. Antibiotic-treated and untreated control glands were included. Milk samples for microbiology, somatic cell analysis and milk production were collected before and after treatment.Results/Key findings.L. lactis rapidly activated the mammary glands' innate immune response and initiated an inflammatory response as evidenced by the recruitment of polymorphonuclear neutrophils and increased somatic cell counts. But while leading to a transient clearance of CNS in the gland, this response caused mild to moderate clinical cases of mastitis characterized by abnormal milk secretions and udder inflammation. Moreover, S. aureus infections did not improve, and CNS infections tended to relapse. Under our experimental conditions, the L. lactis treatment led to a transient clearance of the pathogen in the gland, but also caused mild to moderate clinical cases of mastitis. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.

Beta-galactosidase catalyzed selective galactosylation of aromatic compounds.

PubMed

Bridiau, Nicolas; Taboubi, Selma; Marzouki, Nejib; Legoy, Marie Dominique; Maugard, Thierry

2006-01-01

A new approach to galacto-oligosaccharides and galacto-conjugates synthesis performed by the beta-galactosidase from Kluyveromyces lactis is reported. The enzymatic galactosylation of eight kinds of adsorbed aromatic primary alcohols, in particular the two drugs guaifenesin and chlorphenesin, gave the corresponding beta-D-galacto-pyranosides in yields ranging between approximately 10% and 96%. For the first time, we have showed that the adsorption of acceptor substrates onto solid supports such as silica gel influences the yield and the selectivity of galacto-conjugates synthesis. In particular, we observed that adsorption of acceptor favored the synthesis of digalactosylated compounds.

Functional Expression of an Orchid Fragrance Gene in Lactococcus lactis

PubMed Central

Song, Adelene Ai Lian; Abdullah, Janna O.; Abdullah, Mohd Puad; Shafee, Norazizah; Rahim, Raha A.

2012-01-01

Vanda Mimi Palmer (VMP), an orchid hybrid of Vanda tesselata and Vanda Tan Chay Yan is a highly scented tropical orchid which blooms all year round. Previous studies revealed that VMP produces a variety of isoprenoid volatiles during daylight. Isoprenoids are well known to contribute significantly to the scent of most fragrant plants. They are a large group of secondary metabolites which may possess valuable characteristics such as flavor, fragrance and toxicity and are produced via two pathways, the mevalonate (MVA) pathway or/and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. In this study, a sesquiterpene synthase gene denoted VMPSTS, previously isolated from a floral cDNA library of VMP was cloned and expressed in Lactococcus lactis to characterize the functionality of the protein. L. lactis, a food grade bacterium which utilizes the mevalonate pathway for isoprenoid production was found to be a suitable host for the characterization of plant terpene synthases. Through recombinant expression of VMPSTS, it was revealed that VMPSTS produced multiple sesquiterpenes and germacrene D dominates its profile. PMID:22408409

Effect of autochthonous bacteriocin-producing Lactococcus lactis on bacterial population dynamics and growth of halotolerant bacteria in Brazilian charqui.

PubMed

Biscola, Vanessa; Abriouel, Hikmate; Todorov, Svetoslav Dimitrov; Capuano, Verena Sant'Anna Cabral; Gálvez, Antonio; Franco, Bernadette Dora Gombossy de Melo

2014-12-01

Charqui is a fermented, salted and sun-dried meat product, widely consumed in Brazil and exported to several countries. Growth of microorganisms in this product is unlikely due to reduced Aw, but halophilic and halotolerant bacteria may grow and cause spoilage. Charqui is a good source of lactic acid bacteria able to produce antimicrobial bacteriocins. In this study, an autochthonous bacteriocinogenic strain (Lactococcus lactis subsp. lactis 69), isolated from charqui, was added to the meat used for charqui manufacture and evaluated for its capability to prevent the growth of spoilage bacteria during storage up to 45 days. The influence of L. lactis 69 on the bacterial diversity during the manufacturing of the product was also studied, using denaturing gradient gel electrophoresis (DGGE). L. lactis 69 did not affect the counts and diversity of lactic acid bacteria during manufacturing and storage, but influenced negatively the populations of halotolerant microorganisms, reducing the spoilage potential. The majority of tested virulence genes was absent, evidencing the safety and potential technological application of this strain as an additional hurdle to inhibit undesirable microbial growth in this and similar fermented meat products. Copyright © 2014 Elsevier Ltd. All rights reserved.

Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.

PubMed

Robert, S; Van Huynegem, K; Gysemans, C; Mathieu, C; Rottiers, P; Steidler, L

2015-01-01

Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

Strain-Dependent Transcriptome Signatures for Robustness in Lactococcus lactis

PubMed Central

Dijkstra, Annereinou R.; Alkema, Wynand; Starrenburg, Marjo J. C.; van Hijum, Sacha A. F. T.; Bron, Peter A.

2016-01-01

Recently, we demonstrated that fermentation conditions have a strong impact on subsequent survival of Lactococcus lactis strain MG1363 during heat and oxidative stress, two important parameters during spray drying. Moreover, employment of a transcriptome-phenotype matching approach revealed groups of genes associated with robustness towards heat and/or oxidative stress. To investigate if other strains have similar or distinct transcriptome signatures for robustness, we applied an identical transcriptome-robustness phenotype matching approach on the L. lactis strains IL1403, KF147 and SK11, which have previously been demonstrated to display highly diverse robustness phenotypes. These strains were subjected to an identical fermentation regime as was performed earlier for strain MG1363 and consisted of twelve conditions, varying in the level of salt and/or oxygen, as well as fermentation temperature and pH. In the exponential phase of growth, cells were harvested for transcriptome analysis and assessment of heat and oxidative stress survival phenotypes. The variation in fermentation conditions resulted in differences in heat and oxidative stress survival of up to five 10-log units. Effects of the fermentation conditions on stress survival of the L. lactis strains were typically strain-dependent, although the fermentation conditions had mainly similar effects on the growth characteristics of the different strains. By association of the transcriptomes and robustness phenotypes highly strain-specific transcriptome signatures for robustness towards heat and oxidative stress were identified, indicating that multiple mechanisms exist to increase robustness and, as a consequence, robustness of each strain requires individual optimization. However, a relatively small overlap in the transcriptome responses of the strains was also identified and this generic transcriptome signature included genes previously associated with stress (ctsR and lplL) and novel genes, including nan

Altered Fermentation Performances, Growth, and Metabolic Footprints Reveal Competition for Nutrients between Yeast Species Inoculated in Synthetic Grape Juice-Like Medium

PubMed Central

Rollero, Stephanie; Bloem, Audrey; Ortiz-Julien, Anne; Camarasa, Carole; Divol, Benoit

2018-01-01

The sequential inoculation of non-Saccharomyces yeasts and Saccharomyces cerevisiae in grape juice is becoming an increasingly popular practice to diversify wine styles and/or to obtain more complex wines with a peculiar microbial footprint. One of the main interactions is competition for nutrients, especially nitrogen sources, that directly impacts not only fermentation performance but also the production of aroma compounds. In order to better understand the interactions taking place between non-Saccharomyces yeasts and S. cerevisiae during alcoholic fermentation, sequential inoculations of three yeast species (Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae) with S. cerevisiae were performed individually in a synthetic medium. Different species-dependent interactions were evidenced. Indeed, the three sequential inoculations resulted in three different behaviors in terms of growth. P. burtonii and Z. meyerae declined after the inoculation of S. cerevisiae which promptly outcompeted the other two species. However, while the presence of P. burtonii did not impact the fermentation kinetics of S. cerevisiae, that of Z. meyerae rendered the overall kinetics very slow and with no clear exponential phase. K. marxianus and S. cerevisiae both declined and became undetectable before fermentation completion. The results also demonstrated that yeasts differed in their preference for nitrogen sources. Unlike Z. meyerae and P. burtonii, K. marxianus appeared to be a competitor for S. cerevisiae (as evidenced by the uptake of ammonium and amino acids), thereby explaining the resulting stuck fermentation. Nevertheless, the results suggested that competition for other nutrients (probably vitamins) occurred during the sequential inoculation of Z. meyerae with S. cerevisiae. The metabolic footprint of the non-Saccharomyces yeasts determined after 48 h of fermentation remained until the end of fermentation and combined with that of S. cerevisiae. For instance

Altered Fermentation Performances, Growth, and Metabolic Footprints Reveal Competition for Nutrients between Yeast Species Inoculated in Synthetic Grape Juice-Like Medium.

PubMed

Rollero, Stephanie; Bloem, Audrey; Ortiz-Julien, Anne; Camarasa, Carole; Divol, Benoit

2018-01-01

The sequential inoculation of non- Saccharomyces yeasts and Saccharomyces cerevisiae in grape juice is becoming an increasingly popular practice to diversify wine styles and/or to obtain more complex wines with a peculiar microbial footprint. One of the main interactions is competition for nutrients, especially nitrogen sources, that directly impacts not only fermentation performance but also the production of aroma compounds. In order to better understand the interactions taking place between non-Saccharomyces yeasts and S. cerevisiae during alcoholic fermentation, sequential inoculations of three yeast species ( Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae ) with S. cerevisiae were performed individually in a synthetic medium. Different species-dependent interactions were evidenced. Indeed, the three sequential inoculations resulted in three different behaviors in terms of growth. P. burtonii and Z. meyerae declined after the inoculation of S. cerevisiae which promptly outcompeted the other two species. However, while the presence of P. burtonii did not impact the fermentation kinetics of S. cerevisiae , that of Z. meyerae rendered the overall kinetics very slow and with no clear exponential phase. K. marxianus and S. cerevisiae both declined and became undetectable before fermentation completion. The results also demonstrated that yeasts differed in their preference for nitrogen sources. Unlike Z. meyerae and P. burtonii, K. marxianus appeared to be a competitor for S. cerevisiae (as evidenced by the uptake of ammonium and amino acids), thereby explaining the resulting stuck fermentation. Nevertheless, the results suggested that competition for other nutrients (probably vitamins) occurred during the sequential inoculation of Z. meyerae with S. cerevisiae . The metabolic footprint of the non- Saccharomyces yeasts determined after 48 h of fermentation remained until the end of fermentation and combined with that of S. cerevisiae . For instance

Enhance nisin yield via improving acid-tolerant capability of Lactococcus lactis F44.

PubMed

Zhang, Jian; Caiyin, Qinggele; Feng, Wenjing; Zhao, Xiuli; Qiao, Bin; Zhao, Guangrong; Qiao, Jianjun

2016-06-16

Traditionally, nisin was produced industrially by using Lactococcus lactis in the neutral fermentation process. However, nisin showed higher activity in the acidic environment. How to balance the pH value for bacterial normal growth and nisin activity might be the key problem. In this study, 17 acid-tolerant genes and 6 lactic acid synthetic genes were introduced in L. lactis F44, respectively. Comparing to the 2810 IU/mL nisin yield of the original strain F44, the nisin titer of the engineered strains over-expressing hdeAB, ldh and murG, increased to 3850, 3979 and 4377 IU/mL, respectively. These engineered strains showed more stable intracellular pH value during the fermentation process. Improvement of lactate production could partly provide the extra energy for the expression of acid tolerance genes during growth. Co-overexpression of hdeAB, murG, and ldh(Z) in strain F44 resulted in the nisin titer of 4913 IU/mL. The engineered strain (ABGL) could grow on plates with pH 4.2, comparing to the surviving pH 4.6 of strain F44. The fed-batch fermentation showed nisin titer of the co-expression L. lactis strain could reach 5563 IU/mL with lower pH condition and longer cultivation time. This work provides a novel strategy of constructing robust strains for use in industry process.

Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling.

PubMed

Børsting, M W; Qvist, K B; Brockmann, E; Vindeløv, J; Pedersen, T L; Vogensen, F K; Ardö, Y

2015-01-01

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and β-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin

PubMed Central

Castelain, Mickaël; Duviau, Marie-Pierre; Canette, Alexis; Schmitz, Philippe; Loubière, Pascal; Cocaign-Bousquet, Muriel; Piard, Jean-Christophe; Mercier-Bonin, Muriel

2016-01-01

Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0–200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis. PMID:27010408

A novel small RNA S042 increases acid tolerance in Lactococcus lactis F44.

PubMed

Wu, Hao; Song, Shunyi; Tian, Kairen; Zhou, Dandan; Wang, Binbin; Liu, Jiaheng; Zhu, Hongji; Qiao, Jianjun

2018-06-07

Lactococcus lactis, a gram-positive bacterium, encounters various environmental stresses, especially acid stress, during fermentation. Small RNAs (sRNAs) that serve as regulators at post-transcriptional level play important roles in acid stress response. Here, a novel sRNA S042 was identified by RNA-Seq, RT-PCR and Northern blot. The transcription level of s042 was upregulated 2.29-fold under acid stress by Quantitative RT-PCR (qRT-PCR) analysis. Acid tolerance assay showed that overexpressing s042 increased the survival rate of L. lactis F44 and deleting s042 significantly inhibited the viability under acidic conditions. Moreover, the targets were predicted by online software and four genes were chosen as candidates. Among them, argR (arginine regulator) and accD (acetyl-CoA carboxylase carboxyl transferase subunit beta) were validated to be the direct targets activated by S042 through reporter fusion assay. The regulatory mechanism between S042 and its targets was further investigated through Bioinformatics and qRT-PCR. This study served to highlight the role of the novel sRNA S042 in acid resistance of L. lactis and provided new insights into the response mechanism of acid stress. Copyright © 2018 Elsevier Inc. All rights reserved.

Lactococcus lactis subsp. cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro.

PubMed

Zhang, Justina Su; Guri, Anilda; Corredig, Milena; Morales-Rayas, Rocio; Hassan, Ashraf; Griffiths, Mansel; LaPointe, Gisèle

2016-12-01

Lactococcus lactis subsp. cremoris JFR1 has been studied in reduced fat cheese due to its ability to produce exopolysaccharides (EPS) in situ, contributing to improved textural and organoleptic properties. In this study, the effect of strain JFR1 on virulence gene expression and attachment of Salmonella to HT-29 human colon carcinoma cells was investigated. Overnight cultures of L. lactis subsp. cremoris JFR1 containing EPS, grown in M17 media with 0.5% glucose supplementation, decreased attachment as well as down regulated virulence gene expression in Salmonella enterica subsp. enterica when tested on HT-29 cells. However, EPS isolated from milk fermented with L. lactis subsp. cremoris JFR1 did not affect Salmonella virulence gene expression or attachment to HT-29 cells. These results suggest that EPS does not contribute to the attachment of Salmonella to human intestinal cells. However, the possibility that the isolation process may have affected the structural features of EPS cannot be ruled out. Copyright © 2016 Elsevier Ltd. All rights reserved.

Construction of two Lactococcus lactis expression vectors combining the Gateway and the NIsin Controlled Expression systems.

PubMed

Douillard, François P; Mahony, Jennifer; Campanacci, Valérie; Cambillau, Christian; van Sinderen, Douwe

2011-09-01

Over the last 10 years, the NIsin Controlled Expression (NICE) system has been extensively used in the food-grade bacterium Lactococcus lactis subsp. cremoris to produce homologous and heterologous proteins for academic and biotechnological purposes. Although various L. lactis molecular tools have been developed, no expression vectors harboring the popular Gateway recombination system are currently available for this widely used cloning host. In this study, we constructed two expression vectors that combine the NICE and the Gateway recombination systems and we tested their applicability by recombining and over-expressing genes encoding structural proteins of lactococcal phages Tuc2009 and TP901-1. Over-expressed phage proteins were analyzed by immunoblotting and purified by His-tag affinity chromatography with protein productions yielding 2.8-3.7 mg/l of culture. This therefore is the first description of L. lactis NICE expression vectors which integrate the Gateway cloning technology and which are suitable for the production of sufficient amounts of proteins to facilitate subsequent structural and functional analyses. Copyright © 2011 Elsevier Inc. All rights reserved.

The composition of Camembert cheese-ripening cultures modulates both mycelial growth and appearance.

PubMed

Lessard, Marie-Hélène; Bélanger, Gaétan; St-Gelais, Daniel; Labrie, Steve

2012-03-01

The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds: Penicillium camemberti, Geotrichum candidum, Debaryomyces hansenii, and Kluyveromyces lactis on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing P. camemberti, G. candidum, and K. lactis on the surface of the SCMC during a 31-day ripening period. The results showed that P. camemberti and G. candidum quickly dominated the ecosystem, while K. lactis remained less abundant. When added to this ecosystem, D. hansenii completely inhibited the growth of K. lactis in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese.

Comparative Genomics Reveals Chd1 as a Determinant of Nucleosome Spacing in Vivo.

PubMed

Hughes, Amanda L; Rando, Oliver J

2015-07-14

Packaging of genomic DNA into nucleosomes is nearly universally conserved in eukaryotes, and many features of the nucleosome landscape are quite conserved. Nonetheless, quantitative aspects of nucleosome packaging differ between species because, for example, the average length of linker DNA between nucleosomes can differ significantly even between closely related species. We recently showed that the difference in nucleosome spacing between two Hemiascomycete species-Saccharomyces cerevisiae and Kluyveromyces lactis-is established by trans-acting factors rather than being encoded in cis in the DNA sequence. Here, we generated several S. cerevisiae strains in which endogenous copies of candidate nucleosome spacing factors are deleted and replaced with the orthologous factors from K. lactis. We find no change in nucleosome spacing in such strains in which H1 or Isw1 complexes are swapped. In contrast, the K. lactis gene encoding the ATP-dependent remodeler Chd1 was found to direct longer internucleosomal spacing in S. cerevisiae, establishing that this remodeler is partially responsible for the relatively long internucleosomal spacing observed in K. lactis. By analyzing several chimeric proteins, we find that sequence differences that contribute to the spacing activity of this remodeler are dispersed throughout the coding sequence, but that the strongest spacing effect is linked to the understudied N-terminal end of Chd1. Taken together, our data find a role for sequence evolution of a chromatin remodeler in establishing quantitative aspects of the chromatin landscape in a species-specific manner. Copyright © 2015 Hughes and Rando.

Lactose Hydrolysis in Milk and Dairy Whey Using Microbial β-Galactosidases

PubMed Central

Dutra Rosolen, Michele; Gennari, Adriano; Volpato, Giandra; Volken de Souza, Claucia Fernanda

2015-01-01

This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55°C) and Kluyveromyces lactis (at temperatures of 10 and 37°C) β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C), at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry. PMID:26587283

Lactose Hydrolysis in Milk and Dairy Whey Using Microbial β-Galactosidases.

PubMed

Dutra Rosolen, Michele; Gennari, Adriano; Volpato, Giandra; Volken de Souza, Claucia Fernanda

2015-01-01

This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55°C) and Kluyveromyces lactis (at temperatures of 10 and 37°C) β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C), at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry.

Evaluation of acceptor selectivity of Lactococcus lactis ssp. lactis trehalose 6-phosphate phosphorylase in the reverse phosphorolysis and synthesis of a new sugar phosphate.

PubMed

Taguchi, Yodai; Saburi, Wataru; Imai, Ryozo; Mori, Haruhide

2017-08-01

Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce β-d-glucose 1-phosphate (β-Glc1P) and d-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40°C, and was stable in the pH range 5.0-8.0 and at up to 30°C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, β-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, d-mannose 6-phosphate (Man6P). From β-Glc1P and Man6P, a novel sugar phosphate, α-d-Glcp-(1↔1)-α-d-Manp6P, was synthesized with 51% yield.

21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Streptococcus lactis). The preparation contains the enzyme aminopeptidase (CAS Reg. No. 9031-94-1; EC 3.4.11.1) and other peptidases that hydrolyze milk proteins. The preparation is produced by pure culture... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Aminopeptidase enzyme preparation derived from...

21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Streptococcus lactis). The preparation contains the enzyme aminopeptidase (CAS Reg. No. 9031-94-1; EC 3.4.11.1) and other peptidases that hydrolyze milk proteins. The preparation is produced by pure culture... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Aminopeptidase enzyme preparation derived from...

21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

Code of Federal Regulations, 2012 CFR

2012-04-01

... Streptococcus lactis). The preparation contains the enzyme aminopeptidase (CAS Reg. No. 9031-94-1; EC 3.4.11.1) and other peptidases that hydrolyze milk proteins. The preparation is produced by pure culture... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Aminopeptidase enzyme preparation derived from...

21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

Code of Federal Regulations, 2013 CFR

2013-04-01

... Streptococcus lactis). The preparation contains the enzyme aminopeptidase (CAS Reg. No. 9031-94-1; EC 3.4.11.1) and other peptidases that hydrolyze milk proteins. The preparation is produced by pure culture... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Aminopeptidase enzyme preparation derived from...

Yeast community in traditional Portuguese Serpa cheese by culture-dependent and -independent DNA approaches.

PubMed

Gonçalves Dos Santos, Maria Teresa P; Benito, María José; Córdoba, María de Guía; Alvarenga, Nuno; Ruiz-Moyano Seco de Herrera, Santiago

2017-12-04

This study investigated the yeast community present in the traditional Portuguese cheese, Serpa, by culture-dependent and -independent methods. Sixteen batches of Serpa cheeses from various regional industries registered with the Protected Designation of Origin (PDO) versus non-PDO registered, during spring and winter, were used. Irrespective of the producer, the yeast counts were around 5log CFU/g in winter and, overall, were lower in spring. The yeast species identified at the end of ripening (30days), using PCR-RFLP analysis and sequencing of the 26S rRNA, mainly corresponded to Debaryomyces hansenii and Kluyveromyces marxianus, with Candida spp. and Pichia spp. present to a lesser extent. The culture-independent results, obtained using high-throughput sequencing analysis, confirmed the prevalence of Debaryomyces spp. and Kluyveromyces spp. but, also, that Galactomyces spp. was relevant for three of the five producers, which indicates its importance during the early stages of the cheese ripening process, considering it was not found among the dominant viable yeast species. In addition, differences between the identified yeast isolated from cheeses obtained from PDO and non-PDO registered industries, showed that the lack of regulation of the cheese-making practice, may unfavourably influence the final yeast microbiota. The new knowledge provided by this study of the yeast diversity in Serpa cheese, could be used to modify the cheese ripening conditions, to favour desirable yeast species. Additionally, the prevalent yeast isolates identified, Debaryomyces hansenii and Kluyveromyces spp., may have an important role during cheese ripening and in the final sensorial characteristics. Thus, the study of their technological and functional properties could be relevant, in the development of an autochthonous starter culture, to ensure final quality and safety of the cheese. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.

PubMed

Juergens, Hannes; Varela, Javier A; Gorter de Vries, Arthur R; Perli, Thomas; Gast, Veronica J M; Gyurchev, Nikola Y; Rajkumar, Arun S; Mans, Robert; Pronk, Jack T; Morrissey, John P; Daran, Jean-Marc G

2018-05-01

While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.

Isolation and characterisation of an enterocin P-producing Enterococcus lactis strain from a fresh shrimp (Penaeus vannamei).

PubMed

Ben Braïek, Olfa; Ghomrassi, Hamdi; Cremonesi, Paola; Morandi, Stefano; Fleury, Yannick; Le Chevalier, Patrick; Hani, Khaled; Bel Hadj, Omrane; Ghrairi, Taoufik

2017-06-01

Screening for lactic acid bacteria (LAB) from fresh shrimp samples (Penaeus vannamei) collected from retail seafood markets in the Tunisian's coast, resulted in the isolation of an Enterococcus strain termed Q1. This strain was selected for its antagonistic activity against pathogenic bacteria such as Listeria monocytogenes, Pseudomonas aeruginosa, Lactococcus garvieae and against fungi (Aspergillus niger and Fusarium equiseti). The Q1 strain was characterised using standard morphological and biochemical tests, growth assays at different temperatures, pH and salinity. 16S rRNA, rpoA and pheS gene sequencing, as well as the 16S-23S rRNA intergenic spacer analyses, were combined to identify strain Q1 as a strain of Enterococcus lactis. The bacteriocin produced by E. lactis Q1 is thermostable, active in the pH range from 4.0 to 9.0 and has a bactericidal mode of action. The enterocin P structural gene was detected by specific PCR in strain E. lactis Q1, which is in good agreement with SDS-PAGE data of the purified bacteriocin. A lack of significant antibiotic resistance genes and virulence determinants was confirmed by specific PCRs. This work provides the first description of an enterocin P producer E. lactis strain isolated from a fresh shrimp. Based on its safety properties (absence of haemolytic activity, virulence factors and antibiotic resistance genes), this strain has the potential to be used as a natural additive or adjunct protective culture in food biopreservation and/or probiotic culture.

Effects of the probiotic Bifidobacterium animalis subsp. lactis on the non-surgical treatment of periodontitis. A histomorphometric, microtomographic and immunohistochemical study in rats

PubMed Central

Ricoldi, Milla S. T.; Furlaneto, Flávia A. C.; Oliveira, Luiz F. F.; Teixeira, Gustavo C.; Pischiotini, Jéssica P.; Moreira, André L. G.; Ervolino, Edilson; de Oliveira, Maricê N.; Bogsan, Cristina S. B.; Salvador, Sérgio L.

2017-01-01

Lactobacillus probiotics have been investigated in periodontitis. However, the effects of the genus Bifidobacterium on periodontitis are hardly known. This study evaluated the effects of the probiotic (PROB) Bifidobacterium animalis subsp. lactis (B. lactis) HN019 as an adjunct to scaling and root planing (SRP) in rats with experimental periodontitis (EP). At baseline, 32 rats were assigned to 4 groups: C (control), PROB, EP-SRP and EP-SRP-PROB. In groups EP-SRP and EP-SRP-PROB, the mandibular first molars of the animals received a ligature. At day 14, the ligatures were removed and SRP was performed. Animals of groups PROB and EP-SRP-PROB were orally administered with 10 mL/day of 109 colony forming units of B. lactis HN019 for 15 days, starting at day 14. Animals were euthanized at day 29. Histomorphometric, microtomographic and immunohistochemical analyses were performed. Microbiological effects of B. lactis on biofilm were also evaluated. Data were statistically analyzed (ANOVA, Tukey; Kruskal-Wallis, Dunn’s; Two-tailed t-test; p<0.05). Group EP-SRP-PROB presented reduced alveolar bone resorption and attachment loss when compared with Group EP-SRP (p<0.05). Group EP-SRP-PROB showed significantly fewer osteoclasts, increased expression of anti-inflammatory cytokines and reduced expression of proinflammatory cytokines compared with Group EP-SRP (p<0.05). B. lactis promoted a higher ratio between aerobic and anaerobic bacteria in biofilm samples (p<0.05). B. lactis HN019 may have a role in the treatment of EP in rats, as an adjunct to SRP. PMID:28662142

Differentiation of Streptococcus lactis var. maltigenes from Other Lactic Streptococci1

PubMed Central

Gordon, D. F.; Morgan, M. E.; Tucker, J. S.

1963-01-01

Strains of lactic streptococci isolated from samples of raw milk which had developed a malty aroma were subjected to the cultural, physiological, and serological tests commonly employed in the classification of streptococci. None of the strains could be differentiated from Streptococcus lactis by these tests. Resting cells of strains which produced an organoleptically detectable malty aroma when cultured in milk were usually found to possess an active α-ketoacid decarboxylase, indicating the presence of the mechanism responsible for the characteristic aroma production. This decarboxylase activity was either weak or nonexistent in the nonmalty strains, and no activity was detected in known strains of S. lactis, S. cremoris, or S. diacetilactis. The malty strains usually produced higher acidities in milk than did the nonmalty strains, and, in most instances, they developed a granular type of growth sediment in broth, as opposed to a viscid sediment. Many of them gave weakly positive Voges-Proskauer tests in glucose broth with or without added citrate and appeared to be somewhat more resistant to nisin than the nonmalty strains. PMID:13949187

Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis.

PubMed

Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2011-01-01

Replacement of the signal peptide (SP) of the bacteriocins enterocin P (EntP) and hiracin JM79 (HirJM79), produced by Enterococcus faecium P13 and Enterococcus hirae DCH5, respectively, by the signal peptide of Usp45 (SP(usp45)), the major Sec-dependent protein secreted by Lactococcus lactis, permits the production, secretion, and functional expression of EntP and HirJM79 by L. lactis. Chimeric genes encoding the SP(usp45) fused to either mature EntP (entP), with or without the immunity gene (entiP) or to mature HirJM79 (hirJM79), with or without the immunity gene (hiriJM79), were cloned into the expression vector pMG36c, carrying the P(32) constitutive promoter, and into pNZ8048 under control of the inducible PnisA promoter. The production of EntP and HirJM79 by most of the L. lactis recombinant strains was 1.5- to 3.7-fold higher and up to 3.6-fold higher than by the E. faecium P13 and E. hirae DCH5 control strains, respectively. However, the specific antimicrobial activity of the recombinant EntP was 1.1- to 6.2-fold higher than that produced by E. faecium P13, while that of the HirJM79 was a 40% to an 89% of that produced by E. hirae DCH5. Chimeras of SP(usp45) fused to mature EntP or HirJM79 drive the production and secretion of these bacteriocins in L. lactis in the absence of specific immunity and secretion proteins. The supernatants of the recombinant L. lactis NZ9000 strains, producers of EntP, showed a much higher antimicrobial activity against Listeria spp. than that of the recombinant L. lactis NZ9000 derivatives, producers of HirJM79.

Early adaptation to oxygen is key to the industrially important traits of Lactococcus lactis ssp. cremoris during milk fermentation.

PubMed

Cretenet, Marina; Le Gall, Gwenaëlle; Wegmann, Udo; Even, Sergine; Shearman, Claire; Stentz, Régis; Jeanson, Sophie

2014-12-03

Lactococcus lactis is the most used species in the dairy industry. Its ability to adapt to technological stresses, such as oxidative stress encountered during stirring in the first stages of the cheese-making process, is a key factor to measure its technological performance. This study aimed to understand the response to oxidative stress of Lactococcus lactis subsp. cremoris MG1363 at the transcriptional and metabolic levels in relation to acidification kinetics and growth conditions, especially at an early stage of growth. For those purposes, conditions of hyper-oxygenation were initially fixed for the fermentation. Kinetics of growth and acidification were not affected by the presence of oxygen, indicating a high resistance to oxygen of the L. lactis MG1363 strain. Its resistance was explained by an efficient consumption of oxygen within the first 4 hours of culture, leading to a drop of the redox potential. The efficient consumption of oxygen by the L. lactis MG1363 strain was supported by a coherent and early adaptation to oxygen after 1 hour of culture at both gene expression and metabolic levels. In oxygen metabolism, the over-expression of all the genes of the nrd (ribonucleotide reductases) operon or fhu (ferrichrome ABC transports) genes was particularly significant. In carbon metabolism, the presence of oxygen led to an early shift at the gene level in the pyruvate pathway towards the acetate/2,3-butanediol pathway confirmed by the kinetics of metabolite production. Finally, the MG1363 strain was no longer able to consume oxygen in the stationary growth phase, leading to a drastic loss of culturability as a consequence of cumulative stresses and the absence of gene adaptation at this stage. Combining metabolic and transcriptomic profiling, together with oxygen consumption kinetics, yielded new insights into the whole genome adaptation of L. lactis to initial oxidative stress. An early and transitional adaptation to oxidative stress was revealed for L

From field to fermentation: the origins of Lactococcus lactis and its domestication to the dairy environment.

PubMed

Cavanagh, Daniel; Fitzgerald, Gerald F; McAuliffe, Olivia

2015-05-01

Lactococcus lactis is an organism of substantial economic importance, used extensively in the production of fermented foods and widely held to have evolved from plant strains. The domestication of this organism to the milk environment is associated with genome reduction and gene decay, and the acquisition of specific genes involved in protein and lactose utilisation by horizontal gene transfer. In recent years, numerous studies have focused on uncovering the physiology and molecular biology of lactococcal strains from the wider environment for exploitation in the dairy industry. This in turn has facilitated comparative genome analysis of lactococci from different environments and provided insight into the natural phenotypic and genetic diversity of L. lactis. This diversity may be exploited in dairy fermentations to develop products with improved quality and sensory attributes. In this review, we discuss the classification of L. lactis and the problems that arise with phenotype/genotype designation. We also discuss the adaptation of non-dairy lactococci to milk, the traits associated with this adaptation and the potential application of non-dairy lactococci to dairy fermentations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Expression of lycopene biosynthesis genes fused in line with Shine-Dalgarno sequences improves the stress-tolerance of Lactococcus lactis.

PubMed

Dong, Xiangrong; Wang, Yanping; Yang, Fengyuan; Zhao, Shanshan; Tian, Bing; Li, Tao

2017-01-01

Lycopene biosynthetic genes from Deinococcus radiodurans were co-expressed in Lactococcus lactis to produce lycopene and improve its tolerance to stress. Lycopene-related genes from D. radiodurans, DR1395 (crtE), DR0862 (crtB), and DR0861 (crtI), were fused in line with S hine-Dalgarno (SD) sequences and co-expressed in L. lactis. The recombinant strain produced 0.36 mg lycopene g -1  dry cell wt after 48 h fermentation. The survival rate to UV irradiation of the recombinant strain was higher than that of the non-transformed strain. The L. lactis with co-expressed genes responsible for lycopene biosynthesis from D. radiodurans produced lycopene and exhibited increased resistance to UV stress, suggesting that the recombinant strain has important application potential in food industry.

Continuous ethanol fermentation of lactose by a recombinant flocculating Saccharomyces cerevisiae strain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Domingues, L.; Dantas, M.M.; Lima, N.

1999-09-20

Alcohol fermentation of lactose was investigated using a recombinant flocculating Saccharomyces cetevisiae, expressing the LAC4 (coding the {beta}-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus. Data on yeast fermentation and growth on a medium containing lactose as the sole carbon source are presented. In the range of studied lactose concentrations, total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. For the continuously operating bioreactor, an ethanol productivity of 11 g L{sup {minus}1} h{sup {minus}1} (corresponding to a feed lactose concentration of 50 g L{sup {minus}1} and a dilution ratemore » of 0.55 h{sup {minus}1}) was obtained, which is 7 times larger than the continuous conventional systems. The system stability was confirmed by keeping it in operation for 6 months.« less

Effect of the yeast and bacteria biomass on the microbiota in the rumen.

PubMed

Vamanu, E; Vamanu, A; Popa, O; Vassu, Tatiana; Ghindea, Raluca; Pelinescu, Diana; Nita, Sultana; Babeanu, Narcisa

2008-09-15

This study aims at obtaining a probiotic product based on viable biomass from 6 yeast strains and 2 strains of lactic bacteria used for nutrition of animals. The strains are subjected to some resistance tests, at temperature, pH, pepsin, pancreatin and biliary salts so as to make obvious their viability. Tests were done by comparison to the witness strain and respectively a protective solution based on mucin and casein. Based on the resulted viabilities 2 products are formulated. Their effect is tested by inoculating fresh rumen content and supervising the microbic balance for a period of 12 days. After the final tests, it resulted that the product Fpl (20% Saccharomyces cerevisiae 1-29, 10% Kluyveromyces marxianus R-CS, 20% Issatchenkia orientalis R-BC, 30% Lactobacillus paracasei CMGB16, 20% Enterococcus faecium GM8) was chosen because anaerobic strains were preponderant as a consequence of the tests performed with rumen.

Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis.

PubMed

Sadovskaya, Irina; Vinogradov, Evgeny; Courtin, Pascal; Armalyte, Julija; Meyrand, Mickael; Giaouris, Efstathios; Palussière, Simon; Furlan, Sylviane; Péchoux, Christine; Ainsworth, Stuart; Mahony, Jennifer; van Sinderen, Douwe; Kulakauskas, Saulius; Guérardel, Yann; Chapot-Chartier, Marie-Pierre

2017-09-12

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis , a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA , encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan. IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including

Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

PubMed

van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

2016-01-01

RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

Evaluation of hardboard manufacturing process wastewater as a feedstream for ethanol production.

PubMed

Groves, Stephanie; Liu, Jifei; Shonnard, David; Bagley, Susan

2013-07-01

Waste streams from the wood processing industry can serve as feedstream for ethanol production from biomass residues. Hardboard manufacturing process wastewater (HPW) was evaluated on the basis of monomeric sugar recovery and fermentability as a novel feedstream for ethanol production. Dilute acid hydrolysis, coupled with concentration of the wastewater resulted in a hydrolysate with 66 g/l total fermentable sugars. As xylose accounted for 53 % of the total sugars, native xylose-fermenting yeasts were evaluated for their ability to produce ethanol from the hydrolysate. The strains selected were, in decreasing order by ethanol yields from xylose (Y p/s, based on consumed sugars), Scheffersomyces stipitis ATCC 58785 (CBS 6054), Pachysolen tannophilus ATCC 60393, and Kluyveromyces marxianus ATCC 46537. The yeasts were compared on the basis of substrate utilization and ethanol yield during fermentations of the hydrolysate, measured using an HPLC. S. stipitis, P. tannophilus, and K. marxianus produced 0.34, 0.31, and 0.36 g/g, respectively. The yeasts were able to utilize between 58 and 75 % of the available substrate. S. stipitis outperformed the other yeast during the fermentation of the hydrolysate; consuming the highest concentration of available substrate and producing the highest ethanol concentration in 72 h. Due to its high sugar content and low inhibitor levels after hydrolysis, it was concluded that HPW is a suitable feedstream for ethanol production by S. stipitis.

Engineering Trehalose Synthesis in Lactococcus lactis for Improved Stress Tolerance ▿ †

PubMed Central

Carvalho, Ana Lúcia; Cardoso, Filipa S.; Bohn, Andreas; Neves, Ana Rute; Santos, Helena

2011-01-01

Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery. PMID:21515730

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells

PubMed Central

Rezende, Rafael M.; Oliveira, Rafael P.; Medeiros, Samara R.; Gomes-Santos, Ana C.; Alves, Andrea C.; Loli, Flávia G.; Guimarães, Mauro A.F.; Amaral, Sylvia S.; da Cunha, André P.; Weiner, Howard L.; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M.C.

2013-01-01

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. PMID:22939403

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells.

PubMed

Rezende, Rafael M; Oliveira, Rafael P; Medeiros, Samara R; Gomes-Santos, Ana C; Alves, Andrea C; Loli, Flávia G; Guimarães, Mauro A F; Amaral, Sylvia S; da Cunha, André P; Weiner, Howard L; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M C

2013-02-01

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. Copyright © 2012 Elsevier Ltd. All rights reserved.

Growth of infants fed formula supplemented with Bifidobacterium lactis Bb12 or Lactobacillus GG: a systematic review of randomized controlled trials.

PubMed

Szajewska, Hania; Chmielewska, Anna

2013-11-12

Growth is an essential outcome measure for evaluating the safety of any new ingredients, including probiotics, added to infant formulae. The aim of this systematic review was to determine the effects of supplementation of infant formulae with Bifidobacterium lactis Bb12 (B lactis) and/or Lactobacillus rhamnosus GG (LGG) compared with unsupplemented formula on the growth of healthy infants. The MEDLINE, EMBASE, and Cochrane Library databases were searched in June 2013 for relevant randomized controlled trials (RCTs) conducted in healthy term infants. Unpublished data were obtained from the manufacturer of B lactis-supplemented formula. The primary outcome measures were weight, length, and head circumference. Nine eligible trials were identified. Compared with unsupplemented controls, supplementation of infant formula with B lactis had no effect on weight gain [4 RCTs, n = 266, mean difference (MD) 0.96 g/day, 95% confidence interval (CI) -0.70 to 2.63)], length gain (4 RCTs, n = 261, MD -0.39 mm/month, 95% CI -1.32 to 0.53), or head circumference gain (3 RCTs, n = 207, MD 0.56 mm/month, 95% CI -0.17 to 1.30). Data limited to one small (n = 105) trial suggest that infants who received standard infant formula supplemented with LGG grew significantly better. No such effect was observed in infants fed hydrolyzed formula supplemented with LGG. Supplementation of infant formula with B lactis results in growth similar to what is found in infants fed unsupplemented formula. Limited data do not allow one to reach a conclusion regarding the effect of LGG supplementation on infant growth.

Microbial and chemical transformation studies of the bioactive marine sesquiterpenes (S)-(+)-curcuphenol and -curcudiol isolated from a deep reef collection of the Jamaican sponge Didiscus oxeata.

PubMed

El Sayed, Khalid A; Yousaf, Muhammad; Hamann, Mark T; Avery, Mitchell A; Kelly, Michelle; Wipf, Peter

2002-11-01

Microbial and chemical transformation studies of the marine sesquiterpene phenols (S)-(+)-curcuphenol (1) and (S)-(+)-curcudiol (2), isolated from the Jamaican sponge Didiscus oxeata, were accomplished. Preparative-scale fermentation of 1 with Kluyveromyces marxianus var. lactis (ATCC 2628) has resulted in the isolation of six new metabolites: (S)-(+)-15-hydroxycurcuphenol (3), (S)-(+)-12-hydroxycurcuphenol (4), (S)-(+)-12,15-dihydroxycurcuphenol (5), (S)-(+)-15-hydroxycurcuphenol-12-al (6), (S)-(+)-12-carboxy-10,11-dihydrocurcuphenol (7), and (S)-(+)-12-hydroxy-10,11-dihydrocurcuphenol (8). Fourteen-days incubation of 1 with Aspergillus alliaceus (NRRL 315) afforded the new compounds (S)-(+)-10beta-hydroxycurcudiol (9), (S)-(+)-curcudiol-10-one (10), and (S)-(+)-4-[1-(2-hydroxy-4-methyl)phenyl)]pentanoic acid (11). Rhizopus arrhizus (ATCC 11145) and Rhodotorula glutinus (ATCC 15125) afforded (S)-curcuphenol-1alpha-D-glucopyranoside (12) and (S)-curcudiol-1alpha-D-glucopyranoside (13) when incubated for 6 and 8 days with 1 and 2, respectively. The absolute configuration of C(10) and C(11) of metabolites 7-9 was established by optical rotation computations. Reaction of 1 with NaNO(2) and HCl afforded (S)-(+)-4-nitrocurcuphenol (14) and (S)-(+)-2-nitrocurcuphenol (15) in a 2:1 ratio. Acylation of 1 and 2 with isonicotinoyl chloride afforded the expected esters (S)-(+)-curcuphenol-1-O-isonicotinate (16) and (S)-(+)-curcudiol-1-O-isonicotinate (17), respectively. Curcuphenol (1) shows potent antimicrobial activity against Candida albicans, Cryptococcus neoformans, methicillin-resistant Staphylococcus aureus, and S. aureus with MIC and MFC/MBC ranges of 7.5-25 and 12.5-50 microg/mL, respectively. Compounds 1 and 3 also display in vitro antimalarial activity against Palsmodium falciparium (D6 clone) with MIC values of 3600 and 3800 ng/mL, respectively (selectivity index >1.3). Both compounds were also active against P. falciparium (W2 clone) with MIC values of 1800 (S

Biofortification of riboflavin and folate in idli batter, based on fermented cereal and pulse, by Lactococcus lactis N8 and Saccharomyces boulardii SAA655.

PubMed

Chandrasekar Rajendran, S C; Chamlagain, B; Kariluoto, S; Piironen, V; Saris, P E J

2017-06-01

Lactococcus lactis N8 and Saccharomyces boulardii SAA655 were investigated for their ability to synthesize B-vitamins (riboflavin and folate) and their functional role as microbial starters in idli fermentation. In this study, ultra-high performance liquid chromatography and microbiological assay were used to determine the total riboflavin and folate content respectively. Increased levels of folate were evident in both L. lactis N8 and S. boulardii SAA655 cultivated medium. Enhanced riboflavin levels were found only in S. boulardii SAA655 grown medium, whereas decreased riboflavin level was found in L. lactis N8 cultivated medium. To evaluate the functional role of microbial starter strains, L. lactis N8 and S. boulardii SAA655 were incorporated individually and in combination into idli batter, composed of wet grounded rice and black gram. For the experiments, naturally fermented idli batter was considered as control. The results indicated that natural idli fermentation did not enhance the riboflavin level and depleted folate levels by half. In comparison with control, L. lactis N8 and S. boulardii SAA655 incorporated idli batter (individually and in combination) increased riboflavin and folate levels by 40-90%. Apart from compensating the folate loss caused by natural fermentation, S. boulardii SAA655 fermented idli batter individually and in combination with L. lactis N8 also showed the highest leavening character. Moreover, the microbial starter incorporation did not significantly influence the pH of idli batter. Incorporation of L. lactis N8 and S. boulardii SAA655 can evidently enhance the functional and technological characteristics of idli batter. UN General Assembly declared 2016 the International Year of pulses emphasizing the importance of legumes as staple food. Furthermore, this is the first experimental report of in situ biofortifcation of riboflavin and folate using microbes in pulse based fermented staple food. The current study suggests possible

Construction of a lactose-assimilating strain of baker's yeast.

PubMed

Adam, A C; Prieto, J A; Rubio-Texeira, M; Polaina, J

1999-09-30

A recombinant strain of baker's yeast has been constructed which can assimilate lactose efficiently. This strain has been designed to allow its propagation in whey, the byproduct resulting from cheese-making. The ability to metabolize lactose is conferred by the functional expression of two genes from Kluyveromyces lactis, LAC12 and LAC4, which encode a lactose permease and a beta-galactosidase, respectively. To make the recombinant strain more acceptable for its use in bread-making, the genetic transformation of the host baker's yeast was carried out with linear fragments of DNA of defined sequence, carrying as the only heterologous material the coding regions of the two K. lactis genes. Growth of the new strain on cheese whey affected neither the quality of bread nor the yeast gassing power. The significance of the newly developed strain is two-fold: it affords a cheap alternative to the procedure generally used for the propagation of baker's yeast, and it offers a profitable use for cheese whey. Copyright 1999 John Wiley & Sons, Ltd.

The Composition of Camembert Cheese-Ripening Cultures Modulates both Mycelial Growth and Appearance

PubMed Central

Lessard, Marie-Hélène; Bélanger, Gaétan; St-Gelais, Daniel

2012-01-01

The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds: Penicillium camemberti, Geotrichum candidum, Debaryomyces hansenii, and Kluyveromyces lactis on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing P. camemberti, G. candidum, and K. lactis on the surface of the SCMC during a 31-day ripening period. The results showed that P. camemberti and G. candidum quickly dominated the ecosystem, while K. lactis remained less abundant. When added to this ecosystem, D. hansenii completely inhibited the growth of K. lactis in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese. PMID:22247164

A General Method for Selection of α-Acetolactate Decarboxylase-Deficient Lactococcus lactis Mutants To Improve Diacetyl Formation

PubMed Central

Curic, Mirjana; Stuer-Lauridsen, Birgitte; Renault, Pierre; Nilsson, Dan

1999-01-01

The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the α-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp. lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L. lactis NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis strains. PMID:10049884

Regulation of Acetate Kinase Isozymes and Its Importance for Mixed-Acid Fermentation in Lactococcus lactis

PubMed Central

Puri, Pranav; Goel, Anisha; Bochynska, Agnieszka

2014-01-01

Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The Km values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate. PMID:24464460

Growth phase-dependent proteomes of the Malaysian isolated Lactococcus lactis dairy strain M4 using label-free qualitative shotgun proteomics analysis.

PubMed

Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Rahim, Raha Abdul; Mahadi, Nor Muhammad; Illias, Rosli Md; Murad, Abdul Munir Abdul

2014-01-01

Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

Genome Sequence of the Cheese-Starter Strain Lactobacillus delbrueckii subsp. lactis CRL 581.

PubMed

Hebert, Elvira María; Raya, Raúl R; Brown, Lucía; Font de Valdez, Graciela; Savoy de Giori, Graciela; Taranto, María Pía

2013-08-08

We report the genome sequence of Lactobacillus delbrueckii subsp. lactis CRL 581 (1,911,137 bp, GC 49.7%), a proteolytic strain isolated from a homemade Argentinian hard cheese which has a key role in bacterial nutrition and releases bioactive health-beneficial peptides from milk proteins.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia.

PubMed

Dan, Tong; Liu, Wenjun; Sun, Zhihong; Lv, Qiang; Xu, Haiyan; Song, Yuqin; Zhang, Heping

2014-06-09

Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia

PubMed Central

2014-01-01

Background Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Results Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Conclusions Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics. PMID:24912963

Lactococcus lactis Diversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing of epsD.

PubMed

Frantzen, Cyril A; Kleppen, Hans Petter; Holo, Helge

2018-02-01

Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L. lactis subsp . lactis and 28 L. lactis subsp. cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR , a core gene, and epsD , present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk. IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of

Novel features of ARS selection in budding yeast Lachancea kluyveri

PubMed Central

2011-01-01

Background The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined. Results In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis. Conclusions Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs. PMID:22204614

Novel features of ARS selection in budding yeast Lachancea kluyveri.

PubMed

Liachko, Ivan; Tanaka, Emi; Cox, Katherine; Chung, Shau Chee Claire; Yang, Lu; Seher, Arael; Hallas, Lindsay; Cha, Eugene; Kang, Gina; Pace, Heather; Barrow, Jasmine; Inada, Maki; Tye, Bik-Kwoon; Keich, Uri

2011-12-28

The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined. In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis. Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

Micrococcus lactis sp. nov., isolated from dairy industry waste.

PubMed

Chittpurna; Singh, Pradip K; Verma, Dipti; Pinnaka, Anil Kumar; Mayilraj, Shanmugam; Korpole, Suresh

2011-12-01

A Gram-positive, yellow-pigmented, actinobacterial strain, DW152(T), was isolated from a dairy industry effluent treatment plant. 16S rRNA gene sequence analysis indicated that strain DW152(T) exhibited low similarity with many species with validly published names belonging to the genera Micrococcus and Arthrobacter. However, phenotypic properties including chemotaxonomic markers affiliated strain DW152(T) to the genus Micrococcus. Strain DW152(T) had ai-C(15:0) and i-C(15:0) as major cellular fatty acids, and MK-8(H(2)) as the major menaquinone. The cell-wall peptidoglycan of strain DW152(T) had l-lysine as the diagnostic amino acid and the type was A4α. The DNA G+C content of strain DW152(T) was 68.0 mol%. In 16S rRNA gene sequence analysis, strain DW152(T) exhibited significant similarity with Micrococcus terreus NBRC 104258(T), but the mean value of DNA-DNA relatedness between these strains was only 42.3%. Moreover, strain DW152(T) differed in biochemical and chemotaxonomic characteristics from M. terreus and other species of the genus Micrococcus. Based on the above differences, we conclude that strain DW152(T) should be treated as a novel species of the genus Micrococcus, for which the name Micrococcus lactis sp. nov. is proposed. The type strain of Micrococcus lactis sp. nov. is DW152(T) (=MTCC10523(T) =DSM 23694(T)).

Biodiversity and probiotic potential of yeasts isolated from Fura, a West African spontaneously fermented cereal.

PubMed

Pedersen, Line Lindegaard; Owusu-Kwarteng, James; Thorsen, Line; Jespersen, Lene

2012-10-01

Fura is a spontaneously fermented pearl millet product consumed in West Africa. The yeast species involved in the fermentation were identified by pheno- and genotypic methods to be Candida krusei, Kluyveromyces marxianus, Candida tropicalis, Candida rugosa, Candida fabianii, Candida norvegensis and Trichosporon asahii. C. krusei and K. marxianus were found to be the dominant species. Survival in pH 2.5 or in the presence of bile salts (0.3% (w/v) oxgall) and growth at 37°C were independently determined as indicators of the survival potential of the isolates during passage through the human gastrointestinal tract. Selected yeast species isolates were assessed for their probiotic potential. All of the examined yeast isolates survived and grew at human gastrointestinal conditions in pH 2.5, 0.3% (w/v) oxgall at 37°C. The effect on the transepithelial electrical resistance (TEER) across polarized monolayers of intestinal epithelial cells of human (Caco-2) and porcine (IPEC-J2) origin, were determined. The Caco-2 cells and IPEC-J2 cells displayed clearly different relative TEER results. The strains of C. krusei, K. marxianus, C. rugosa and T. asahii were able to increase the relative TEER of Caco-2 monolayers after 48h. In comparison, the relative TEER of IPEC-J2 monolayers decreased when exposed to the same yeasts, even though T. asahii did not differ significantly from Saccharomyces cerevisiae var. boulardii which is used as a human probiotic. C. tropicalis resulted in the largest relative TEER decrease for both the human and the porcine cell model assays. Hyphal growth was observed for C. albicans and C. tropicalis after 48h of incubation with polarized Caco-2 monolayers, whereas this was not the case for the remaining yeast species. In the present study new yeast strains with potential probiotic properties have been isolated to be used potentially as starter cultures for fura production. Copyright © 2012 Elsevier B.V. All rights reserved.

Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis.

PubMed

Linares, Daniel M; Alvarez-Sieiro, Patricia; del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Martin, Ma Cruz; Fernandez, Maria; Alvarez, Miguel A

2015-12-30

Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes. This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten. The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

Bifidobacterium animalis ssp. lactis CNCM-I2494 Restores Gut Barrier Permeability in Chronically Low-Grade Inflamed Mice.

PubMed

Martín, Rebeca; Laval, Laure; Chain, Florian; Miquel, Sylvie; Natividad, Jane; Cherbuy, Claire; Sokol, Harry; Verdu, Elena F; van Hylckama Vlieg, Johan; Bermudez-Humaran, Luis G; Smokvina, Tamara; Langella, Philippe

2016-01-01

Growing evidence supports the efficacy of many probiotic strains in the management of gastrointestinal disorders associated with deregulated intestinal barrier function and/or structure. In particular, bifidobacteria have been studied for their efficacy to both prevent and treat a broad spectrum of animal and/or human gut disorders. The aim of the current work was thus to evaluate effects on intestinal barrier function of Bifidobacterium animalis ssp. lactis CNCM-I2494, a strain used in fermented dairy products. A chronic dinitrobenzene sulfonic acid (DNBS)-induced low-grade inflammation model causing gut dysfunction in mice was used in order to study markers of inflammation, intestinal permeability, and immune function in the presence of the bacterial strain. In this chronic low-grade inflammation mice model several parameters pointed out the absence of an over active inflammation process. However, gut permeability, lymphocyte populations, and colonic cytokines were found to be altered. B. animalis ssp. lactis CNCM-I2494 was able to protect barrier functions by restoring intestinal permeability, colonic goblet cell populations, and cytokine levels. Furthermore, tight junction (TJ) proteins levels were also measured by qRT-PCR showing the ability of this strain to specifically normalize the level of several TJ proteins, in particular for claudin-4. Finally, B. lactis strain counterbalanced CD4(+) lymphocyte alterations in both spleen and mesenteric lymphoid nodes. It restores the Th1/Th2 ratio altered by the DNBS challenge (which locally augments CD4(+) Th1 cells) by increasing the Th2 response as measured by the increase in the production of major representative Th2 cytokines (IL-4, IL-5, and IL-10). Altogether, these data suggest that B. animalis ssp. lactis CNCM-I2494 may efficiently prevent disorders associated with increased barrier permeability.

Influence of the addition of Lactobacillus acidophilus La-05, Bifidobacterium animalis subsp. lactis Bb-12 and inulin on the technological, physicochemical, microbiological and sensory features of creamy goat cheese.

PubMed

Barbosa, Ilsa C; Oliveira, Maria E G; Madruga, Marta S; Gullón, Beatriz; Pacheco, Maria T B; Gomes, Ana M P; Batista, Ana S M; Pintado, Maria M E; Souza, Evandro L; Queiroga, Rita C R E

2016-10-12

The effects of the addition of Lactobacillus acidophilus LA-05, Bifidobacterium animalis subsp. lactis BB-12 and inulin on the quality characteristics of creamy goat cheese during refrigerated storage were evaluated. The manufactured cheeses included the addition of starter culture (Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris - R-704) (CC); starter culture, L. acidophilus LA-05 and inulin (CLA); starter culture, B. lactis BB-12 and inulin (CBB); or starter culture, L. acidophilus LA-05, B. lactis BB-12 and inulin (CLB). In the synbiotic cheeses (CLA, CBB and CLB), the counts of L. acidophilus LA-05 and B. lactis BB-12 were greater than 6log CFU g -1 , the amount of inulin was greater than 6 g per 100 g, and the firmness was reduced. The cheeses evaluated had high brightness values (L*), with a predominance of yellow (b*). CC had higher contents of proteins, lipids and minerals compared to the other cheeses. There was a decrease in the amount of short-chain fatty acids (SCFAs) and an increase of medium-chain (MCFAs) and long-chain fatty acids (LCFAs) in the synbiotic cheeses compared to CC. The amount of conjugated linoleic acid increased in CLA, CBB and CLB. The highest depth of proteolysis and the greatest changes in the release of free amino acids were found in CLB. The addition of inulin and probiotics, alone or in co-culture, did not affect the cheese acceptance. Inulin and probiotics can be used together for the production of creamy goat cheese without negatively affecting the general quality characteristics of the product, and to add value because of its synbiotic potential.

Fuel ethanol production from Jerusalem artichoke stalks using different yeasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Margaritis, A.; Bajpai, P.; Bajpai, P.K.

1983-01-01

The inulin-type sugars present in the stalks of Jerusalem artichoke (Helianthus tuberosus) were extracted with hot water and were used as a substrate to produce fuel EtOH. Seven different yeasts were used to obtain batch kinetic data. The medium consisted of stalk extract from Jerusalem artichoke containing 7.3% total sugars, supplemented with 0.01% oleic acid, 0.01% corn steep liquor, and 0.05% Tween 80. All batch fermentations were carried out in a 1-L bioreactor at 35 degrees and pH 4.6, and the following parameters were measured as a function of time: total sugars, EtOH and biomass concentration, maximum specific growth rate,more » and biomass and EtOH yields. The best EtOH producer was Kluyveromyces marxianus UCD (FST) 55-82 which gave an EtOH-to-sugar yield 97% of the theoretical maximum value, with almost 100% sugar utilization.« less

Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii strains.

PubMed

Greppi, Anna; Krych, Łukasz; Costantini, Antonella; Rantsiou, Kalliopi; Hounhouigan, D Joseph; Arneborg, Nils; Cocolin, Luca; Jespersen, Lene

2015-07-16

Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninety-three yeast isolates from traditional African fermented food products, belonging to nine species (Pichia kudriavzevii, Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces marxianus, Millerozyma farinosa, Candida glabrata, Wickerhamomyces anomalus, Hanseniaspora guilliermondii and Debaryomyces nepalensis) were screened for phytase production on solid and liquid media. 95% were able to grow in the presence of phytate as sole phosphate source, P. kudriavzevii being the best growing species. A phytase coding gene of P. kudriavzevii (PHYPk) was identified and its expression was studied during growth by RT-qPCR. The expression level of PHYPk was significantly higher in phytate-medium, compared to phosphate-medium. In phytate-medium expression was seen in the lag phase. Significant differences in gene expression were detected among the strains as well as between the media. A correlation was found between the PHYPk expression and phytase extracellular activity. Copyright © 2015 Elsevier B.V. All rights reserved.

Production of fermented cheese whey-based beverage using kefir grains as starter culture: evaluation of morphological and microbial variations.

PubMed

Magalhães, Karina Teixeira; Pereira, Maria Alcina; Nicolau, Ana; Dragone, Giuliano; Domingues, Lucília; Teixeira, José António; de Almeida Silva, João Batista; Schwan, Rosane Freitas

2010-11-01

Whey valorization concerns have led to recent interest on the production of whey beverage simulating kefir. In this study, the structure and microbiota of Brazilian kefir grains and beverages obtained from milk and whole/deproteinised whey was characterized using microscopy and molecular techniques. The aim was to evaluate its stability and possible shift of probiotic bacteria to the beverages. Fluorescence staining in combination with Confocal Laser Scanning Microscopy showed distribution of yeasts in macro-clusters among the grain's matrix essentially composed of polysaccharides (kefiran) and bacteria. Denaturing gradient gel electrophoresis displayed communities included yeast affiliated to Kluyveromyces marxianus, Saccharomyces cerevisiae, Kazachatania unispora, bacteria affiliated to Lactobacillus kefiranofaciens subsp. Kefirgranum, Lactobacillus kefiranofaciens subsp. Kefiranofaciens and an uncultured bacterium also related to the genus Lactobacillus. A steady structure and dominant microbiota, including probiotic bacteria, was detected in the analyzed kefir beverages and grains. This robustness is determinant for future implementation of whey-based kefir beverages.

Native yeasts for alternative utilization of overripe mango pulp for ethanol production.

PubMed

Buenrostro-Figueroa, Juan; Tafolla-Arellano, Julio C; Flores-Gallegos, Adriana C; Rodríguez-Herrera, Raúl; De la Garza-Toledo, Heliodoro; Aguilar, Cristóbal N

2017-11-18

Mango fruits (Mangifera indica L.) are highly perishable, causing postharvest losses and producing agroindustrial waste. In the present work, native yeasts were used to evaluate ethanol production in overripe mango pulp. The two isolated strains showed similar sequences in the 18S rDNA region corresponding to Kluyveromyces marxianus, being different to the data reported in the NCBI database. Values of up to 5% ethanol (w/v) were obtained at the end of fermentation, showing a productivity of 4g/l/day, a yield of up to 49% of ethanol and a process efficiency of 80%. These results represent a viable option for using the surplus production and all the fruits that have suffered mechanical injury that are not marketable and are considered as agroindustrial waste, thus achieving greater income and less postharvest losses. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Steam explosion treatment for ethanol production from branches pruned from pear trees by simultaneous saccharification and fermentation.

PubMed

Sasaki, Chizuru; Okumura, Ryosuke; Asada, Chikako; Nakamura, Yoshitoshi

2014-01-01

This study investigated the production of ethanol from unutilized branches pruned from pear trees by steam explosion pretreatment. Steam pressures of 25, 35, and 45 atm were applied for 5 min, followed by enzymatic saccharification of the extracted residues with cellulase (Cellic CTec2). High glucose recoveries, of 93.3, 99.7, and 87.1%, of the total sugar derived from the cellulose were obtained from water- and methanol-extracted residues after steam explosion at 25, 35, and 45 tm, respectively. These values corresponded to 34.9, 34.3, and 27.1 g of glucose per 100 g of dry steam-exploded branches. Simultaneous saccharification and fermentation experiments were done on water-extracted residues and water- and methanol-extracted residues by Kluyveromyces marxianus NBRC 1777. An overall highest theoretical ethanol yield of 76% of the total sugar derived from cellulose was achieved when 100 g/L of water- and methanol-washed residues from 35 atm-exploded pear branches was used as substrate.

Optimization of nisin production by Lactococcus lactis UQ2 using supplemented whey as alternative culture medium.

PubMed

González-Toledo, S Y; Domínguez-Domínguez, J; García-Almendárez, B E; Prado-Barragán, L A; Regalado-González, C

2010-08-01

Lactococcus lactis UQ2 is a nisin A-producing native strain. In the present study, the production of nisin by L. lactis UQ2 in a bioreactor using supplemented sweet whey (SW) was optimized by a statistical design of experiments and response surface methodology (RSM). In a 1st approach, a fractional factorial design (FFD) of the order 2(5-1) with 3 central points was used. The effect on nisin production of air flow, SW, soybean peptone (SP), MgSO(4)/MnSO(4) mixture, and Tween 80 was evaluated. From FFD, the most significant factors affecting nisin production were SP (P = 0.011), and SW (P = 0.037). To find optimum conditions, a central composite design (CCD) with 2 central points was used. Three factors were considered, SW (7 to 10 g/L), SP (7 to10 g/L), and small amounts of added nisin as self-inducer (NI 34.4 to 74.4 IU/L). Nisin production was expressed as international units (IU). From RSM, an optimum nisin activity of 180 IU/mL was predicted at 74.4 IU/L NI, 13.8 g/L SP, and 14.9 or 5.11 g/L SW, while confirmatory experiments showed a maximum activity of 178 +/- 5.2 IU/mL, verifying the validity of the model. The 2nd-order model showed a coefficient of determination (R(2)) of 0.828. Optimized conditions were used for constant pH fermentations, where a maximum activity of 575 +/- 17 IU/mL was achieved at pH 6.5 after 12 h. The adsorption-desorption technique was used to partially purify nisin, followed by drying. The resulting powder showed an activity of 102150 IU/g. Practical Application: Nisin production was optimized using supplemented whey as alternative culture medium, using a native L. lactis UQ2 strain. Soybean peptone, SW, and subinhibitory amounts of nisin were successfully employed to optimize nisin production by L. lactis UQ2. Dried semipurified nisin showed an activity of 102150 IU/g.

A Food-Grade Cloning System for Industrial Strains of Lactococcus lactis

PubMed Central

Sørensen, Kim I.; Larsen, Rasmus; Kibenich, Annette; Junge, Mette P.; Johansen, Eric

2000-01-01

We have previously reported the construction of a food-grade cloning vector for Lactococcus using the ochre suppressor, supB, as the selective marker. This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested. As supB suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides. Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of Lactococcus lactis. The vector uses an amber suppressor, supD, as selectable marker and consists entirely of Lactococcus DNA, with the exception of a small polylinker region. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant L. lactis strains. The usefulness of this system is demonstrated by overexpressing the pepN gene in a number of industrial backgrounds. PMID:10742196

13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses.

PubMed

Azizan, Kamalrul Azlan; Ressom, Habtom W; Mendoza, Eduardo R; Baharum, Syarul Nataqain

2017-01-01

Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13 C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis ( r ) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis' central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis , in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis . Overall, the

A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp. lactis BGMN1-5

PubMed Central

Uzelac, Gordana; Lozo, Jelena; Aleksandrzak-Piekarczyk, Tamara; Gabrielsen, Christina; Kristensen, Tom; Nes, Ingolf F.; Diep, Dzung B.; Topisirovic, Ljubisa

2013-01-01

Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors. PMID:24123824

Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans

USDA-ARS?s Scientific Manuscript database

We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose in Lactococcus lactis using a nisin-controlled gene expression system. Production of DsrI was optimized using several different background vectors, signal peptides, str...

Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid.

PubMed

Song, Li; Cui, Hongyu; Tang, Lijie; Qiao, Xinyuan; Liu, Min; Jiang, Yanping; Cui, Wen; Li, Yijing

2014-07-01

Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L. lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L. casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L. casei ATCC 393 and L. lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier. Copyright © 2014 Elsevier B.V. All rights reserved.

The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808

PubMed Central

Munsch-Alatossava, Patricia; Alatossava, Tapani

2013-01-01

The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lactobacillus delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimize the risks associated with the appearance and attack of phages in the manufacture of yogurt, and Swiss or Italian hard type cheeses, which typically use thermophilic lactic acid bacteria starter cultures containing L. delbrueckii strains among others. This mini review article summarizes the present data concerning (i) the special features, particle structure, and components of phage LL-H and (ii) the structure and properties of lipoteichoic acids (LTAs), which are the phage LL-H receptor components of L. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of L. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed. PMID:24400001

The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808.

PubMed

Munsch-Alatossava, Patricia; Alatossava, Tapani

2013-12-24

The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lactobacillus delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimize the risks associated with the appearance and attack of phages in the manufacture of yogurt, and Swiss or Italian hard type cheeses, which typically use thermophilic lactic acid bacteria starter cultures containing L. delbrueckii strains among others. This mini review article summarizes the present data concerning (i) the special features, particle structure, and components of phage LL-H and (ii) the structure and properties of lipoteichoic acids (LTAs), which are the phage LL-H receptor components of L. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of L. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed.

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties

PubMed Central

Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

2017-01-01

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates. PMID:28702021

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

PubMed

Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

2017-01-01

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus , lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans , with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis . Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

Immunopathological evaluation of recombinant mycobacterial antigen Hsp65 expressed in Lactococcus lactis as a novel vaccine candidate

PubMed Central

Herrera Ramírez, J. C.; De la Mora, A. Ch.; De la Mora Valle, A.; Lopez-Valencia, G.; Hurtado, R. M. B.; Rentería Evangelista, T. B.; Rodríguez Castillo, J. L.; Rodríguez Gardea, A.; Gómez Gómez, S. D.; Medina-Basulto, G. E.

2017-01-01

Bovine tuberculosis (TBB) is a zoonotic disease distributed worldwide and is of great importance for public health and the livestock industry. Several experimental vaccines against this disease have been evaluated in recent years, yielding varying results. An example is the Bacillus Calmette-Guérin (BCG) vaccine, which has been used extensively in humans and tested in cattle showing mixed results related to protection (0-80%) against Mycobacterium bovis. In this study, we used the food-grade bacterium Lactococcus lactis as an expression system for production of mycobacterial protein Hsp65. For this purpose, the construction of a replicable plasmid in strain NZ9000 L. lactis (pVElepr) was conducted, which expressed the Mycobacterium leprae Hsp65 antigen, and was recognized by traded anti-Hsp65 antibodies. The strain NZ9000-pVElepr was applied to calves that were negative to tuberculin test and the immune response was monitored. The results showed that immune response was not significantly increased in calves with NZ9000-pVElepr with respect to control groups, and no injury was observed in any lung or lymph of the calves. Finally, this study suggest that the recombinant NZ9000 strain of L. lactis may protect against the development of M. bovis infection, although studies with longer exposure to this pathogen are necessary to conclude the matter. PMID:29163649

PBP2b plays a key role in both peripheral growth and septum positioning in Lactococcus lactis.

PubMed

David, Blandine; Duchêne, Marie-Clémence; Haustenne, Gabrielle Laurie; Pérez-Núñez, Daniel; Chapot-Chartier, Marie-Pierre; De Bolle, Xavier; Guédon, Eric; Hols, Pascal; Hallet, Bernard

2018-01-01

Lactococcus lactis is an ovoid bacterium that forms filaments during planktonic and biofilm lifestyles by uncoupling cell division from cell elongation. In this work, we investigate the role of the leading peptidoglycan synthase PBP2b that is dedicated to cell elongation in ovococci. We show that the localization of a fluorescent derivative of PBP2b remains associated to the septal region and superimposed with structural changes of FtsZ during both vegetative growth and filamentation indicating that PBP2b remains intimately associated to the division machinery during the whole cell cycle. In addition, we show that PBP2b-negative cells of L. lactis are not only defective in peripheral growth; they are also affected in septum positioning. This septation defect does not simply result from the absence of the protein in the cell growth machinery since it is also observed when PBP2b-deficient cells are complemented by a catalytically inactive variant of PBP2b. Finally, we show that round cells resulting from β-lactam treatment are not altered in septation, suggesting that shape elongation as such is not a major determinant for selection of the division site. Altogether, we propose that the specific PBP2b transpeptidase activity at the septum plays an important role for tagging future division sites during L. lactis cell cycle.

The recombinant Lactococcus lactis oral vaccine induces protection against C. difficile spore challenge in a mouse model.

PubMed

Guo, Shanguang; Yan, Weiwei; McDonough, Sean P; Lin, Nengfeng; Wu, Katherine J; He, Hongxuan; Xiang, Hua; Yang, Maosheng; Moreira, Maira Aparecida S; Chang, Yung-Fu

2015-03-24

Clostridium difficile infection (CDI) causes nosocomial antibiotic-associated diarrhea and colitis in the developed world. Two potent cytotoxins, toxin A (TcdA) and toxin B (TcdB) are the virulence factors of this disease and can be a good vaccine candidate against CDI. In the present study, we genetically engineered Lactococcus lactis to express the nontoxic, recombinant fragments derived from TcdA and TcdB C-terminal receptor binding domains (Tcd-AC and Tcd-BC) as an oral vaccine candidate. The immunogenicity of the genetically engineered L. lactis oral vaccine delivery system (animal groups LAC and LBC or the combination of both, LACBC) was compared with the recombinant TcdA and TcdB C-terminal receptor binding domain proteins (animal groups PAC and PBC or the combination of both, PACBC), which were expressed and purified from E. coli. After the C. difficile challenge, the control groups received PBS or engineered L. lactis with empty vector, showed severe diarrhea symptoms and died within 2-3 days. However, both the oral vaccine and recombinant protein vaccine groups had significantly lower mortalities, body weight decreases and histopathologic lesions than the control sham-vaccine groups (p<0.05) except group LBC which only had a 31% survival rate after the challenge. The data of post infection survival showed that an average of 86% of animals survived in groups PAC and PACBC, 75% of animals survived in group LACBC, and 65% of animals survived in group LAC. All of the vaccinated animals produced higher titers of both IgG and IgA than the control groups (p<0.05), and the antibodies were able to neutralize the cytopathic effect of toxins in vitro. The results of this study indicate that there is a potential to use L. lactis as a delivery system to develop a cost effective oral vaccine against CDI. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of food-grade phytase in Lactococcus lactis from optimized conditions in milk broth.

PubMed

Miao, Yuzhi; Xu, Hui; Fei, Baojin; Qiao, Dairong; Cao, Yi

2013-07-01

The major objective of this study was to engineer lactic acid bacteria to produce the enzyme phytase from a gene native to Bacillus subtilis GYPB04. The phytase gene (phyC) of B. subtilis GYPB04 was cloned into the plasmid pMG36e for expression in Lactococcus lactis. The enzyme activity in L. lactis cultured in GM17 broth was 20.25 U/mL at 36°C. The expressed phytase was characterized as active in a pH range of 2.0-9.0 at a temperature range of 20-80°C, with an optimum pH of 5.5-6.5 and temperature of 60°C. When cultured in food-grade milk broth, the transformed L. lactis grew to an OD(600 nm) value of 1.05 and had a phytase yield of 13.58 U/mL. In same broth under optimized conditions for cell growth and phytase production, the transformant reached an OD(600 nm) value of 1.68 and a phytase yield of 42.12 U/mL, representing approximately 1.6-fold and 3.1-fold increases, respectively, compared to growth in natural milk broth. Fermentation was scaled to 5 L under optimized conditions, and product analysis revealed a final OD(600 nm) value of 1.89 and an extracellular enzyme activity of 24.23 U/mL. The results of this study may be used in the dairy fermentation industry for the development of functional, healthy yogurts and other fermented dairy foods that provide both active phytase and viable probiotics to the consumer. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Assessment of stress tolerance acquisition in the heat-tolerant derivative strains of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus GG.

PubMed

Aakko, J; Sánchez, B; Gueimonde, M; Salminen, S

2014-07-01

The purpose of this study was to investigate the heat-shock response at molecular level in Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis BB-12 and their heat-tolerant derivatives and to characterize the changes that make the derivatives more robust in terms of heat stress. The study strains were exposed for 2 h to a heat-shock treatment, Bif. animalis subsp. lactis BB-12 and its derivative at 50°C and the Lact. rhamnosus GG and its derivative at 60°C. Protein synthesis before and after heat shock was examined using proteomics and RT-qPCR. The analysis revealed that the regulation of seven proteins in both strain pairs was modified as a response to heat or between the original and the derivative strain. The comparison of wild-type strains and the heat-tolerant derivatives suggests that the acquisition of heat tolerance in the Bif. animalis subsp. lactis BB-12 derivative is due to a slightly increased constitutive level of chaperones, while in Lact. rhamnosus GG derivative, the main reason seems to be a higher ability to induce the production of chaperones. This study revealed possible markers of heat tolerance in B. lactis and Lact. rhamnosus strains. This study increases our knowledge on how Lactobacillus and Bifidobacterium strains may acquire heat tolerance. These findings may be useful for improving the heat tolerance of existing probiotic strains as well as screening new heat-tolerant strains. © 2014 The Society for Applied Microbiology.

Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

PubMed Central

Tsakalidou, E.; Anastasiou, R.; Vandenberghe, I.; van Beeumen, J.; Kalantzopoulos, G.

1999-01-01

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified. PMID:10223997

Modeling of the Competitive Growth of Listeria monocytogenes and Lactococcus lactis in Vegetable Broth

PubMed Central

Breidt, Frederick; Fleming, Henry P.

1998-01-01

Current mathematical models used by food microbiologists do not address the issue of competitive growth in mixed cultures of bacteria. We developed a mathematical model which consists of a system of nonlinear differential equations describing the growth of competing bacterial cell cultures. In this model, bacterial cell growth is limited by the accumulation of protonated lactic acid and decreasing pH. In our experimental system, pure and mixed cultures of Lactococcus lactis and Listeria monocytogenes were grown in a vegetable broth medium. Predictions of the model indicate that pH is the primary factor that limits the growth of L. monocytogenes in competition with a strain of L. lactis which does not produce the bacteriocin nisin. The model also predicts the values of parameters that affect the growth and death of the competing populations. Further development of this model will incorporate the effects of additional inhibitors, such as bacteriocins, and may aid in the selection of lactic acid bacterium cultures for use in competitive inhibition of pathogens in minimally processed foods. PMID:9726854

Safety characterisation and inhibition of fungi and bacteria by a novel multiple enterocin-producing Enterococcus lactis 4CP3 strain.

PubMed

Ben Braïek, Olfa; Cremonesi, Paola; Morandi, Stefano; Smaoui, Slim; Hani, Khaled; Ghrairi, Taoufik

2018-03-07

This study aims to characterise a potential bacteriocinogenic lactic acid bacterial strain isolated from a raw pink shrimp (Palaemon serratus) and evaluate its safety aspect. The strain designated as 4CP3 was noted to display antibacterial activities (P < 0.05) against Gram-positive and Gram-negative foodborne pathogens (Listeria monocytogenes and Pseudomonas aeruginosa) and some filamentous fungi (e.g. Aspergillus niger A79). Phenotypic and molecular techniques as well as phylogenetic analysis identified the isolate 4CP3 as Enterococcus lactis. Its produced antimicrobial substance was determined as a bacteriocin that was stable over a wide range of pH (2-10) and after heating at 100 °C for 15 min. The maximum bacteriocin production was 1400 AU/ml recorded after 12 h of incubation in de Man, Rogosa and Sharpe (MRS) broth medium at 30 °C. The mode of action of the bacteriocin produced by 4CP3 strain was identified as bactericidal against L. monocytogenes EGDe 107776 and P. aeruginosa ATCC 27853. By specific PCR amplifications, E. lactis 4CP3 was shown to produce the enterocins A, B and P. To our knowledge, this feature is newly described for E. lactis strain isolated from raw shrimps. Regarding safety aspect of E. lactis 4CP3, it has been demonstrated that this strain was not haemolytic, gelatinase negative, sensitive to vancomycin, and free of common antibiotic resistance genes and virulence factors. Therefore, it may be useful as a safe natural agent in preservation of foods or as a new probiotic strain in food and feed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Brazilian kefir: structure, microbial communities and chemical composition.

PubMed

Magalhães, Karina Teixeira; de Melo Pereira, Gilberto Vinícius; Campos, Cássia Roberta; Dragone, Giuliano; Schwan, Rosane Freitas

2011-04-01

Microbial ecology and chemical composition of Brazilian kefir beverage was performed. The microorganisms associated with Brazilian kefir were investigated using a combination of phenotypic and genotypic methods. A total of 359 microbial isolates were identified. Lactic acid bacteria (60.5%) were the major isolated group identified, followed by yeasts (30.6%) and acetic acid bacteria (8.9%). Lactobacillus paracasei (89 isolates), Lactobacillus parabuchneri (41 isolates), Lactobacillus casei (32 isolates), Lactobacillus kefiri (31 isolates), Lactococcus lactis (24 isolates), Acetobacter lovaniensis (32 isolates), Kluyveromyces lactis (31 isolates), Kazachstania aerobia (23 isolates), Saccharomyces cerevisiae (41 isolates) and Lachancea meyersii (15 isolates) were the microbial species isolated. Scanning electron microscopy showed that the microbiota was dominated by bacilli (short and curved long) cells growing in close association with lemon-shaped yeasts cells. During the 24 h of fermentation, the protein content increased, while lactose and fat content decreased. The concentration of lactic acid ranged from 1.4 to 17.4 mg/ml, and that of acetic acid increased from 2.1 to 2.73 mg/ml. The production of ethanol was limited, reaching a final mean value of 0.5 mg/ml.

Brazilian kefir: structure, microbial communities and chemical composition

PubMed Central

Magalhães, Karina Teixeira; de Melo Pereira, Gilberto Vinícius; Campos, Cássia Roberta; Dragone, Giuliano; Schwan, Rosane Freitas

2011-01-01

Microbial ecology and chemical composition of Brazilian kefir beverage was performed. The microorganisms associated with Brazilian kefir were investigated using a combination of phenotypic and genotypic methods. A total of 359 microbial isolates were identified. Lactic acid bacteria (60.5%) were the major isolated group identified, followed by yeasts (30.6%) and acetic acid bacteria (8.9%). Lactobacillus paracasei (89 isolates), Lactobacillus parabuchneri (41 isolates), Lactobacillus casei (32 isolates), Lactobacillus kefiri (31 isolates), Lactococcus lactis (24 isolates), Acetobacter lovaniensis (32 isolates), Kluyveromyces lactis (31 isolates), Kazachstania aerobia (23 isolates), Saccharomyces cerevisiae (41 isolates) and Lachancea meyersii (15 isolates) were the microbial species isolated. Scanning electron microscopy showed that the microbiota was dominated by bacilli (short and curved long) cells growing in close association with lemon-shaped yeasts cells. During the 24 h of fermentation, the protein content increased, while lactose and fat content decreased. The concentration of lactic acid ranged from 1.4 to 17.4 mg/ml, and that of acetic acid increased from 2.1 to 2.73 mg/ml. The production of ethanol was limited, reaching a final mean value of 0.5 mg/ml. PMID:24031681

Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

PubMed Central

Tramonti, Angela; Lanini, Claudio; Cialfi, Samantha; De Biase, Daniela; Falcone, Claudio

2012-01-01

In the yeast Kluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH in K. lactis led us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+ bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activity in vivo. The appearance of a new G6PDH-specific activity band, following incubation of Saccharomyces cerevisiae and human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes. PMID:23064253

Mechanism of citrate metabolism by an oxaloacetate decarboxylase-deficient mutant of Lactococcus lactis IL1403.

PubMed

Pudlik, Agata M; Lolkema, Juke S

2011-08-01

Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706-714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of L-lactate, indicating exchange between oxaloacetate and L-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate.

Probiotic Yogurt Culture Bifidobacterium Animalis Subsp. Lactis BB-12 and Lactobacillus Acidophilus LA-5 Modulate the Cytokine Secretion by Peripheral Blood Mononuclear Cells from Patients with Ulcerative Colitis.

PubMed

Sheikhi, A; Shakerian, M; Giti, H; Baghaeifar, M; Jafarzadeh, A; Ghaed, V; Heibor, M R; Baharifar, N; Dadafarin, Z; Bashirpour, G

2016-06-01

There are some evidences for the immunomodulation disorders in the response to intestinal microbiota in inflammatory bowel disease. Yogurt is a fermented milk product made with a starter culture consisting of different probiotics which could be colonized in intestine. However, the role of probiotics in the aetiopathogenesis of ulcerative colitis (UC) has not been clarified. To determine how the immune system responds to these bacteria this study was planned. Bifidobacterium lactis BB-12 (B. lactis) and Lactobacillus acidophilus LA-5 (L. acidophilus) were cultivated on MRS broth. PBMCs of 36 UC patients were separated by Ficoll-Hypaque centrifugation and co-cultured with different concentrations of UV killed bacteria in RPMI-1 640 plus 10% FCS for 48/72 h. IL-10, TGF-β, IFN-γ and TNF-α were measured in supernatant of PBMCs by ELISA. Both bacteria significantly augmented IL-10, TGF-β, IFN-γ and TNF-α compared to control (p<0.001). The secretion levels of IL-10 and TGF-β by B. lactis- compared to L. acidophilus-stimulated PBMCs were significantly higher (p<0.05, p<0.01 respectively). The secretion levels of TNF-α and IFN-γ by PBMCs after 72 h were significantly lower compared to 48 h stimulation by B. lactis (p<0.001, p<0.035 respectively). These data show that both probiotics may trigger the pro- and anti-inflammatory immune response of UC patients. It seems that IL-10/TGF-β uprising by B. lactis could be the reason of TNF-α/IFN-γ reduction. Therefore albeit B. lactis still stimulates the effector Th cells but because of more stimulatory effect on Tregs, it could be a good potential therapeutic candidate for further investigation. © Georg Thieme Verlag KG Stuttgart · New York.

The effects of Bifidobacterium animalis ssp. lactis B94 on gastrointestinal wellness in adults with Prader-Willi syndrome: study protocol for a randomized controlled trial.

PubMed

Alyousif, Zainab; Miller, Jennifer L; Sandoval, Mariana Y; MacPherson, Chad W; Nagulesapillai, Varuni; Dahl, Wendy J

2018-04-27

Constipation is a frequent problem in adults with Prader-Willi syndrome. Certain probiotics have been shown to improve transit and gastrointestinal symptoms of adults with functional constipation. The aim of this study is to determine the effect of daily consumption of Bifidobacterium animalis ssp. lactis B94 (B. lactis B94) on stool frequency, stool form, and gastrointestinal symptoms in adults with Prader-Willi syndrome. Adults with Prader-Willi syndrome (18-75 years old, n = 36) will be recruited and enrolled in a 20-week, randomized, double-blind, placebo-controlled, crossover study. Study subjects will be randomized to B. lactis B94 or placebo each for a 4-week period, preceded by a 4-week baseline and followed by 4-week washouts. Subjects will complete daily records of stool frequency and stool form (a proxy of transit time). Dietary intake data also will be collected. Stools, one in each period, will be collected for exploratory microbiota analyses. To our knowledge, this is the first randomized controlled trial evaluating the effectiveness of B. lactis in adults with Prader-Willi syndrome. The results of this study will provide evidence of efficacy for future clinical trials in patient populations with constipation. ClinicalTrials.gov ( NCT03277157 ). Registered on 08 September 2017.

Functional cream cheese supplemented with Bifidobacterium animalis subsp. lactis DSM 10140 and Lactobacillus reuteri DSM 20016 and prebiotics.

PubMed

Speranza, Barbara; Campaniello, Daniela; Monacis, Noemi; Bevilacqua, Antonio; Sinigaglia, Milena; Corbo, Maria Rosaria

2018-06-01

The aim of this study was to develop a functional fresh cream cheese with Bifidobacterium animalis subsp. lactis DSM 10140 or Lactobacillus reuteri DSM 20016 and prebiotics (inulin, FOS and lactulose). The research was divided into two steps: in vitro evaluation of the effects of prebiotic compounds; validation at laboratory level with production of functional cream mini-cheeses. Prebiotics showed a protective effect: B. animalis subsp. lactis DSM 10140 cultivability on Petri dishes was positively influenced by lactulose, whereas fructooligosaccharides (FOS) were the prebiotic compounds able to prolong Lb. reuteri DSM 20016 cultivability. At 30 °C, a prolongation of the death time (more than 300 days) was observed, while the controls showed death time values about 100 days. At 45 °C, death time values increased from 32.2 (control) to 33, 35, and 38 days in the samples added with FOS, inulin and lactulose, respectively. Lactulose and FOS were chosen to be added to cream mini-cheeses inoculated with B. animalis subsp. lactis DSM 10140 and Lb. reuteri DSM 20016, respectively; the proposed functional cream cheese resulted in a product with favourable conditions for the viability of both probiotics which maintained cultivable cells above the recommended level during 28 days of storage at 4 °C with good sensory characteristics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transcriptome analysis shows activation of the arginine deiminase pathway in Lactococcus lactis as a response to ethanol stress.

PubMed

Díez, Lorena; Solopova, Ana; Fernández-Pérez, Rocío; González, Miriam; Tenorio, Carmen; Kuipers, Oscar P; Ruiz-Larrea, Fernanda

2017-09-18

This paper describes the molecular response of Lactococcus lactis NZ9700 to ethanol. This strain is a well-known nisin producer and a lactic acid bacteria (LAB) model strain. Global transcriptome profiling using DNA microarrays demonstrated a bacterial adaptive response to the presence of 2% ethanol in the culture broth and differential expression of 67 genes. The highest up-regulation was detected for those genes involved in arginine degradation through the arginine deiminase (ADI) pathway (20-40 fold up-regulation). The metabolic responses to ethanol of wild type L. lactis strains were studied and compared to those of regulator-deletion mutants MG∆argR and MG∆ahrC. The results showed that in the presence of 2% ethanol those strains with an active ADI pathway reached higher growth rates when arginine was available in the culture broth than in absence of arginine. In a chemically defined medium strains with an active ADI pathway consumed arginine and produced ornithine in the presence of 2% ethanol, hence corroborating that arginine catabolism is involved in the bacterial response to ethanol. This is the first study of the L. lactis response to ethanol stress to demonstrate the relevance of arginine catabolism for bacterial adaptation and survival in an ethanol containing medium. Copyright © 2017 Elsevier B.V. All rights reserved.

Lactobacillus delbrueckii subsp lactis CIDCA 133 modulates response of human epithelial and dendritic cells infected with Bacillus cereus.

PubMed

Rolny, I S; Tiscornia, I; Racedo, S M; Pérez, P F; Bollati-Fogolín, M

2016-11-30

It is known that probiotic microorganisms are able to modulate pathogen virulence. This ability is strain dependent and involves multiple interactions between microorganisms and relevant host's cell populations. In the present work we focus on the effect of a potentially probiotic lactobacillus strain (Lactobacillus delbrueckii subsp. lactis CIDCA 133) in an in vitro model of Bacillus cereus infection. Our results showed that infection of intestinal epithelial HT-29 cells by B. cereus induces nuclear factor kappa B (NF-κB) pathway. Noteworthy, the presence of strain L. delbrueckii subsp.lactis CIDCA 133 increases stimulation. However, B. cereus-induced interleukin (IL)-8 production by epithelial cells is partially abrogated by L. delbrueckii subsp. lactis CIDCA 133. These findings suggest that signalling pathways other than that of NF-κB are involved. In a co-culture system (HT-29 and monocyte-derived dendritic cells), B. cereus was able to translocate from the epithelial (upper) to the dendritic cell compartment (lower). This translocation was partially abrogated by the presence of lactobacilli in the upper compartment. In addition, infection of epithelial cells in the co-culture model, led to an increase in the expression of CD86 by dendritic cells. This effect could not be modified in the presence of lactobacilli. Interestingly, infection of enterocytes with B. cereus triggers production of proinflammatory cytokines by dendritic cells (IL-8, IL-6 and tumour necrosis factor alpha (TNF-α)). The production of TNF-α (a protective cytokine in B. cereus infections) by dendritic cells was increased in the presence of lactobacilli. The present work demonstrates for the first time the effect of L. delbrueckii subsp. lactis CIDCA 133, a potentially probiotic strain, in an in vitro model of B. cereus infection. The presence of the probiotic strain modulates cell response both in infected epithelial and dendritic cells thus suggesting a possible beneficial effect of

Spray-drying process preserves the protective capacity of a breast milk-derived Bifidobacterium lactis strain on acute and chronic colitis in mice

PubMed Central

Burns, Patricia; Alard, Jeanne; Hrdỳ, Jiri; Boutillier, Denise; Páez, Roxana; Reinheimer, Jorge; Pot, Bruno; Vinderola, Gabriel; Grangette, Corinne

2017-01-01

Gut microbiota dysbiosis plays a central role in the development and perpetuation of chronic inflammation in inflammatory bowel disease (IBD) and therefore is key target for interventions with high quality and functional probiotics. The local production of stable probiotic formulations at limited cost is considered an advantage as it reduces transportation cost and time, thereby increasing the effective period at the consumer side. In the present study, we compared the anti-inflammatory capacities of the Bifidobacterium animalis subsp. lactis (B. lactis) INL1, a probiotic strain isolated in Argentina from human breast milk, with the commercial strain B. animalis subsp. lactis BB12. The impact of spray-drying, a low-cost alternative of bacterial dehydration, on the functionality of both bifidobacteria was also investigated. We showed for both bacteria that the spray-drying process did not impact on bacterial survival nor on their protective capacities against acute and chronic colitis in mice, opening future perspectives for the use of strain INL1 in populations with IBD. PMID:28233848

Casein Hydrolysates by Lactobacillus brevis and Lactococcus lactis Proteases: Peptide Profile Discriminates Strain-Dependent Enzyme Specificity.

PubMed

Bounouala, Fatima Zohra; Roudj, Salima; Karam, Nour-Eddine; Recio, Isidra; Miralles, Beatriz

2017-10-25

Casein from ovine and bovine milk were hydrolyzed with two extracellular protease preparations from Lactobacillus brevis and Lactococcus lactis. The hydrolysates were analyzed by HPLC-MS/MS for peptide identification. A strain-dependent peptide profile could be observed, regardless of the casein origin, and the specificity of these two proteases could be computationally ascribed. The cleavage pattern yielding phenylalanine, leucine, or tyrosine at C-terminal appeared both at L. lactis and Lb. brevis hydrolysates. However, the cleavage C-terminal to lysine was favored with Lb. brevis protease. The hydrolysates showed ACE-inhibitory activity with IC 50 in the 16-70 μg/mL range. Ovine casein hydrolysates yielded greater ACE-inhibitory activity. Previously described antihypertensive and opioid peptides were found in these ovine and bovine casein hydrolysates and prediction of the antihypertensive activity of the sequences based on quantitative structure and activity relationship (QSAR) was performed. This approach might represent a useful classification tool regarding health-related properties prior to further purification.

Cloning and functional expression of the mitochondrial alternative oxidase gene (aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions.

PubMed

Papagianni, Maria; Avramidis, Nicholaos

2012-01-05

Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions. There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study, a cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for alternative respiration, from a citric acid producing Aspergillus niger strain was cloned and expressed in L. lactis as a host strain. Expression of aox1 conferred on this organism cyanide-resistant and salicylhydroxamate-sensitive growth. Bioreactor cultures under fully aerobic conditions of the transformed L. lactis showed that the alternative respiratory pathway operates and improves significantly the microorganism's response to oxidizing stress conditions as it enhances biomass production, suppresses lactate formation, and leads to accumulation of large amounts of nisin. Copyright © 2011 Elsevier Inc. All rights reserved.

Effects in the use of a genetically engineered strain of Lactococcus lactis delivering in situ IL-10 as a therapy to treat low-grade colon inflammation.

PubMed

Martín, Rebeca; Chain, Florian; Miquel, Sylvie; Natividad, Jane M; Sokol, Harry; Verdu, Elena F; Langella, Philippe; Bermúdez-Humarán, Luis G

2014-01-01

Irritable bowel syndrome (IBS) is a gastrointestinal disorder characterized by chronic abdominal pain, discomfort, and bloating. Interestingly, there is now evidence of the presence of a low-grade inflammatory status in many IBS patients, including histopathological and mucosal cytokine levels in the colon, as well as the presence of IBS-like symptoms in quiescent inflammatory bowel disease (IBD). The use of a genetically engineered food-grade bacterium, such as Lactococcus lactis, secreting the anti-inflammatory cytokine IL-10 has been proven by many pre-clinical studies to be a successful therapy to treat colon inflammation. In this study, we first reproduced the recovery-recurrence periods observed in IBS-patients in a new chronic model characterized by 2 episodes of DiNitro-BenzeneSulfonic-acid (DNBS)-challenge and we tested the effects of a recombinant strain of L. lactis secreting IL-10 under a Stress-Inducible Controlled Expression (SICE) system. In vivo gut permeability, colonic serotonin levels, cytokine profiles, and spleen cell populations were then measured as readouts of a low-grade inflammation. In addition, since there is increasing evidence that gut microbiota tightly regulates gut barrier function, tight junction proteins were also measured by qRT-PCR after administration of recombinant L. lactis in DNBS-treated mice. Strikingly, oral administration of L. lactis secreting active IL-10 in mice resulted in significant protective effects in terms of permeability, immune activation, and gut-function parameters. Although genetically engineered bacteria are, for now, used only as a "proof-of-concept," our study validates the interest in the use of the novel SICE system in L. lactis to express therapeutic molecules, such as IL-10, locally at mucosal surfaces.

A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403.

PubMed

Mancini, Stefano; Abicht, Helge K; Gonskikh, Yulia; Solioz, Marc

2015-02-01

Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments. © 2014 John Wiley & Sons Ltd.

A CTG Clade Candida Yeast Genetically Engineered for the Genotype-Phenotype Characterization of Azole Antifungal Resistance in Human-Pathogenic Yeasts.

PubMed

Accoceberry, Isabelle; Rougeron, Amandine; Biteau, Nicolas; Chevrel, Pauline; Fitton-Ouhabi, Valérie; Noël, Thierry

2018-01-01

A strain of the opportunistic pathogenic yeast Candida lusitaniae was genetically modified for use as a cellular model for assessing by allele replacement the impact of lanosterol C14α-demethylase ERG11 mutations on azole resistance. Candida lusitaniae was chosen because it is susceptible to azole antifungals, it belongs to the CTG clade of yeast, which includes most of the Candida species pathogenic for humans, and it is haploid and easily amenable to genetic transformation and molecular modeling. In this work, allelic replacement is targeted at the ERG11 locus by the reconstitution of a functional auxotrophic marker in the 3' intergenic region of ERG11 Homologous and heterologous ERG11 alleles are expressed from the resident ERG11 promoter of C. lusitaniae , allowing accurate comparison of the phenotypic change in azole susceptibility. As a proof of concept, we successfully expressed in C. lusitaniae different ERG11 alleles, either bearing or not bearing mutations retrieved from a clinical context, from two phylogenetically distant yeasts, C. albicans and Kluyveromyces marxianus Candida lusitaniae constitutes a high-fidelity expression system, giving specific Erg11p-dependent fluconazole MICs very close to those observed with the ERG11 donor strain. This work led us to characterize the phenotypic effect of two kinds of mutation: mutation conferring decreased fluconazole susceptibility in a species-specific manner and mutation conferring fluconazole resistance in several yeast species. In particular, a missense mutation affecting amino acid K143 of Erg11p in Candida species, and the equivalent position K151 in K. marxianus , plays a critical role in fluconazole resistance. Copyright © 2017 American Society for Microbiology.

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

PubMed Central

Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

1999-01-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains. PMID:10508059

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

PubMed

Torriani, S; Zapparoli, G; Dellaglio, F

1999-10-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

Ethanol fermentation from molasses at high temperature by thermotolerant yeast Kluyveromyces sp. IIPE453 and energy assessment for recovery.

PubMed

Dasgupta, Diptarka; Ghosh, Prasenjit; Ghosh, Debashish; Suman, Sunil Kumar; Khan, Rashmi; Agrawal, Deepti; Adhikari, Dilip K

2014-10-01

High temperature ethanol fermentation from sugarcane molasses B using thermophilic Crabtree-positive yeast Kluyveromyces sp. IIPE453 was carried out in batch bioreactor system. Strain was found to have a maximum specific ethanol productivity of 0.688 g/g/h with 92 % theoretical ethanol yield. Aeration and initial sugar concentration were tuning parameters to regulate metabolic pathways of the strain for either cell mass or higher ethanol production during growth with an optimum sugar to cell ratio 33:1 requisite for fermentation. An assessment of ethanol recovery from fermentation broth via simulation study illustrated that distillation-based conventional recovery was significantly better in terms of energy efficiency and overall mass recovery in comparison to coupled solvent extraction-azeotropic distillation technique for the same.

Effects in the use of a genetically engineered strain of Lactococcus lactis delivering in situ IL-10 as a therapy to treat low-grade colon inflammation

PubMed Central

Martín, Rebeca; Martín, Rebeca; Chain, Florian; Chain, Florian; Miquel, Sylvie; Miquel, Sylvie; Natividad, Jane M; Natividad, Jane M; Sokol, Harry; Sokol, Harry; Verdu, Elena F; Verdu, Elena F; Langella, Philippe; Langella, Philippe; Bermúdez-Humarán, Luis G; Bermúdez-Humarán, Luis G

2014-01-01

Irritable bowel syndrome (IBS) is a gastrointestinal disorder characterized by chronic abdominal pain, discomfort, and bloating. Interestingly, there is now evidence of the presence of a low-grade inflammatory status in many IBS patients, including histopathological and mucosal cytokine levels in the colon, as well as the presence of IBS-like symptoms in quiescent inflammatory bowel disease (IBD). The use of a genetically engineered food-grade bacterium, such as Lactococcus lactis, secreting the anti-inflammatory cytokine IL-10 has been proven by many pre-clinical studies to be a successful therapy to treat colon inflammation. In this study, we first reproduced the recovery-recurrence periods observed in IBS-patients in a new chronic model characterized by 2 episodes of DiNitro-BenzeneSulfonic-acid (DNBS)-challenge and we tested the effects of a recombinant strain of L. lactis secreting IL-10 under a Stress-Inducible Controlled Expression (SICE) system. In vivo gut permeability, colonic serotonin levels, cytokine profiles, and spleen cell populations were then measured as readouts of a low-grade inflammation. In addition, since there is increasing evidence that gut microbiota tightly regulates gut barrier function, tight junction proteins were also measured by qRT-PCR after administration of recombinant L. lactis in DNBS-treated mice. Strikingly, oral administration of L. lactis secreting active IL-10 in mice resulted in significant protective effects in terms of permeability, immune activation, and gut-function parameters. Although genetically engineered bacteria are, for now, used only as a “proof-of-concept,” our study validates the interest in the use of the novel SICE system in L. lactis to express therapeutic molecules, such as IL-10, locally at mucosal surfaces. PMID:24732667

Draft Genome Sequence of Bifidobacterium animalis subsp. lactis Strain CECT 8145, Able To Improve Metabolic Syndrome In Vivo.

PubMed

Chenoll, E; Codoñer, F M; Silva, A; Martinez-Blanch, J F; Martorell, P; Ramón, D; Genovés, S

2014-03-27

Bifidobacterium animalis subsp. lactis strain CECT 8145 is able to reduce body fat content and improve metabolic syndrome biomarkers. Here, we report the draft genome sequence of this strain, which may provide insights into its safety status and functional role.

Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds.

PubMed

Wisselink, H Wouter; Mars, Astrid E; van der Meer, Pieter; Eggink, Gerrit; Hugenholtz, Jeroen

2004-07-01

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

The occurrence and growth of yeasts in Camembert and blue-veined cheeses.

PubMed

Roostita, R; Fleet, G H

1996-01-01

Yeast populations greater than 10(6) cfu/g were found in approximately 54% and 36%, respectively in surface samples of retail Camembert (85 samples) and Blue-veined (45 samples) cheeses. The most predominant species isolated were Debaryomyces hansenii, Candida catenulata, C. lipolytica, C. kefyr, C. intermedia, Saccharomyces cerevisiae, Cryptococcus albidus and Kluyveromyces marxianus. The salt concentration of the surface samples of the cheeses varied between 2.5-5.5% (w/w) (Camembert) and 7.5-8.3 (Blue-veined), depending upon brand, and influenced the yeast ecology, especially the presence of S. cerevisiae. Yeasts grew to populations of 10(6)-10(8) cfu/g when cheeses were stored at either 25 degrees C or 10 degrees C. These populations decreased on continued storage at 25 degrees C, but such decreases were not so evident on storage at 10 degrees C. The properties of yeasts influencing their occurrence and growth in cheese were: fermentation/assimilation of lactose; production of extracellular lipolytic and proteolytic enzymes, utilisation of lactic and citric acids; and growth at 10 degrees C.

Cellulose Biorefinery Based on a Combined Catalytic and Biotechnological Approach for Production of 5-HMF and Ethanol.

PubMed

Sorokina, Ksenia N; Taran, Oxana P; Medvedeva, Tatiana B; Samoylova, Yuliya V; Piligaev, Alexandr V; Parmon, Valentin N

2017-02-08

In this study, a combination of catalytic and biotechnological processes was proposed for the first time for application in a cellulose biorefinery for the production of 5-hydroxymethylfurfural (5-HMF) and bioethanol. Hydrolytic dehydration of the mechanically activated microcrystalline cellulose over a carbon-based mesoporous Sibunt-4 catalyst resulted in moderate yields of glucose and 5-HMF (21.1-25.1 and 6.6-9.4 %). 5-HMF was extracted from the resulting mixture with isobutanol and subjected to ethanol fermentation. A number of yeast strains were isolated that also revealed high thermotolerance (up to 50 °C) and resistance to inhibitors found in the hydrolysates. The strains Kluyveromyces marxianus C1 and Ogataea polymorpha CBS4732 were capable of producing ethanol from processed catalytic hydrolysates of cellulose at 42 °C, with yields of 72.0±5.7 and 75.2±4.3 % from the maximum theoretical yield of ethanol, respectively. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of hyper thermal acid hydrolysis of Kappaphycus alvarezii for enhanced bioethanol production.

PubMed

Ra, Chae Hun; Nguyen, Trung Hau; Jeong, Gwi-Taek; Kim, Sung-Koo

2016-06-01

Hyper thermal (HT) acid hydrolysis of Kappaphycus alvarezii, a red seaweed, was optimized to 12% (w/v) seaweed slurry content, 180mM H2SO4 at 140°C for 5min. The maximum monosaccharide concentration of 38.3g/L and 66.7% conversion from total fermentable monosaccharides of 57.6g/L with 120gdw/L K. alvarezii slurry were obtained from HT acid hydrolysis and enzymatic saccharification. HT acid hydrolysis at a severity factor of 0.78 efficiently converted the carbohydrates of seaweed to monosaccharides and produced a low concentration of inhibitory compounds. The levels of ethanol production by separate hydrolysis and fermentation with non-adapted and adapted Kluyveromyces marxianus to high concentration of galactose were 6.1g/L with ethanol yield (YEtOH) of 0.19 at 84h and 16.0g/L with YEtOH of 0.42 at 72h, respectively. Development of the HT acid hydrolysis process and adapted yeast could enhance the overall ethanol fermentation yields of K. alvarezii seaweed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bioethanol production from Scenedesmus obliquus sugars: the influence of photobioreactors and culture conditions on biomass production.

PubMed

Miranda, J R; Passarinho, P C; Gouveia, L

2012-10-01

A closed-loop vertical tubular photobioreactor (PBR), specially designed to operate under conditions of scarce flat land availability and irregular solar irradiance conditions, was used to study the potential of Scenedesmus obliquus biomass/sugar production. The results obtained were compared to those from an open-raceway pond and a closed-bubble column. The influence of the type of light source and the regime (natural vs artificial and continuous vs light/dark cycles) on the growth of the microalga and the extent of the sugar accumulation was studied in both PBRs. The best type of reactor studied was a closed-loop PBR illuminated with natural light/dark cycles. In all the cases, the relationship between the nitrate depletion and the sugar accumulation was observed. The microalga Scenedesmus was cultivated for 53 days in a raceway pond (4,500 L) and accumulated a maximum sugar content of 29 % g/g. It was pre-treated for carrying out ethanol fermentation assays, and the highest ethanol concentration obtained in the hydrolysate fermented by Kluyveromyces marxianus was 11.7 g/L.

Continuous production of pectinase by immobilized yeast cells on spent grains.

PubMed

Almeida, Catarina; Brányik, Tomás; Moradas-Ferreira, Pedro; Teixeira, José

2003-01-01

A yeast strain secreting endopolygalacturonase was used in this work to study the possibility of continuous production of this enzyme. It is a feasible and interesting alternative to fungal batch production essentially due to the specificity of the type of pectinase excreted by Kluyveromyces marxianus CCT 3172, to the lower broth viscosity and to the easier downstream operations. In order to increase the reactors' productivity, a cellulosic carrier obtained from barley spent grains was tested as an immobilization support. Two types of reactors were studied for pectinase production using glucose as a carbon and energy source--a continuous stirred tank reactor (CSTR) and a packed bed reactor (PBR) with recycled flow. The highest value for pectinase volumetric productivity (P(V)=0.98 U ml(-1) h(-1)) was achieved in the PBR for D=0.40 h(-1), a glucose concentration on the inlet of S(in)=20 g l(-1), and a biomass load in the support of X(i)=0.225 g g(-1). The results demonstrate the attractiveness of the packed bed system for pectinase production.

Selection of Streptococcus lactis Mutants Defective in Malolactic Fermentation

PubMed Central

Renault, Pierre P.; Heslot, Henri

1987-01-01

An enrichment medium and a new sensitive medium were developed to detect malolactic variants in different strains of lactic bacteria. Factors such as the concentration of glucose and l-malate, pH level, and the type of indicator dye used are discussed with regard to the kinetics of malic acid conversion to lactic acid. Use of these media allowed a rapid and easier screening of mutagenized streptococcal cells unable to ferment l-malate. A collection of malolactic-negative mutants of Streptococcus lactis induced by UV, nitrosoguanidine, or transposonal mutagenesis were characterized. The results showed that several mutants were apparently defective in the structural gene of malolactic enzyme, whereas others contained mutations which may either inactivate a putative permease or affect a regulatory sequence. PMID:16347282

Subtilisin QK-2: secretory expression in Lactococcus lactis and surface display onto gram-positive enhancer matrix (GEM) particles.

PubMed

Mao, Ruifeng; Zhou, Kangping; Han, Zhenwei; Wang, Yefu

2016-05-12

Purified from the supernatant of Bacillus subtilis QK02 culture broth, Subtilisin QK-2 is a type of effective thrombolytic reagent that has great exploitable potential. However, the unbearable flavor that occurs with fermentation and the complicated methods that are required to obtain pure products limit the application of this enzyme. Lactic acid bacteria (LAB)-based delivery vehicles are promising as cheap and safe options for medicinal compounds. The secretory expression and surface display using LAB may popularize Subtilisin QK-2 more easily and conveniently with minimal adverse effects. Subtilisin QK-2 was expressed successfully in two forms using lactic acid bacteria. For the secretory expression in Lactococcus lactis, Subtilisin QK-2 was efficiently secreted into the culture using the promoter P nisA and signal peptide SPUsp. The expression levels were not different in L. lactis NZ9000 and NZ3900 without the effect of different selection markers. However, leaky expression was only detected in L. lactis NZ3900. The biological activity of this secreted Subtilisin QK-2 was enhanced by modulating the pH of medium to slightly alkaline during induction and by codon optimization of either the entire gene sequence (qk') or only the propeptide gene sequence (qkpro'). For surface display onto gram-positive enhancer matrix (GEM) particles, n LysM repeats from the C-terminal region of the major autolysin AcmA of L. lactis were fused to either the C-terminus (n = 1, 3, 5) or the N-terminus (n = 1) of the Subtilisin QK-2. These fusion proteins were secreted into the culture medium, and the QK-3LysM was able to bind to the surface of various LAB GEM particles without a loss of fibrinolytic activity. Furthermore, the binding capacity significantly increased with a higher concentration of QK-3LysM. Compared to the free-form Subtilisin QK-2, the QK-3LysM displayed on the surface of GEM particles was more stable in the simulated gastric juice. Combined with the safety and

Mechanism of Citrate Metabolism by an Oxaloacetate Decarboxylase-Deficient Mutant of Lactococcus lactis IL1403 ▿

PubMed Central

Pudlik, Agata M.; Lolkema, Juke S.

2011-01-01

Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706–714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of l-lactate, indicating exchange between oxaloacetate and l-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate. PMID:21665973

Does the scientific evidence support the advertising claims made for products containing Lactobacillus casei and Bifidobacterium lactis? A systematic review.

PubMed

Meléndez-Illanes, Lorena; González-Díaz, Cristina; Chilet-Rosell, Elisa; Álvarez-Dardet, Carlos

2016-09-01

To analyse the scientific evidence that exists for the advertising claims made for two products containing Lactobacillus casei and Bifidobacterium lactis and to conduct a comparison between the published literature and what is presented in the corporate website. Systematic review, using Medline through Pubmed and Embase. We included human clinical trials that exclusively measured the effect of Lactobacillus casei or Bifidobacterium lactis on a healthy population, and where the objective was related to the health claims made for certain products in advertising. We assessed the levels of evidence and the strength of the recommendation according to the classification criteria established by the Oxford Centre for Evidence Based Medicine (CEBM). We also assessed the outcomes of the studies published on the website that did not appear in the search. Of the 440 articles identified, 16 met the inclusion criteria. Only four (25%) of these presented a level of evidence of 1b and a recommendation grade of A, all corresponding to studies on product containing Bifidobacterium lactis, and only 12 of the 16 studies were published on the corporate website (47). There is insufficient scientific evidence to support the health claims made for these products, especially in the case of product containing Lactobacillus casei. © The Author 2015. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase

PubMed Central

Flambard, Benedicte; Helinck, Sandra; Richard, Jean; Juillard, Vincent

1998-01-01

The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing αs1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase. PMID:9603805

Consumption of Dairy Yogurt Containing Lactobacillus paracasei ssp. paracasei, Bifidobacterium animalis ssp. lactis and Heat-Treated Lactobacillus plantarum Improves Immune Function Including Natural Killer Cell Activity.

PubMed

Lee, Ayoung; Lee, Young Ju; Yoo, Hye Jin; Kim, Minkyung; Chang, Yeeun; Lee, Dong Seog; Lee, Jong Ho

2017-05-31

The aim of this study was to investigate the impact of consuming dairy yogurt containing Lactobacillus paracasei ssp. paracasei ( L. paracasei ), Bifidobacterium animalis ssp. lactis ( B. lactis ) and heat-treated Lactobacillus plantarum ( L. plantarum ) on immune function. A randomized, open-label, placebo-controlled study was conducted on 200 nondiabetic subjects. Over a twelve-week period, the test group consumed dairy yogurt containing probiotics each day, whereas the placebo group consumed milk. Natural killer (NK) cell activity, interleukin (IL)-12 and immunoglobulin (Ig) G1 levels were significantly increased in the test group at twelve weeks compared to baseline. Additionally, the test group had significantly greater increases in serum NK cell activity and interferon (IFN)-γ and IgG1 than placebo group. Daily consumption of dairy yogurt containing L. paracasei , B. lactis and heat-treated L. plantarum could be an effective option to improve immune function by enhancing NK cell function and IFN-γ concentration (ClinicalTrials.gov: NCT03051425).

Consumption of Dairy Yogurt Containing Lactobacillus paracasei ssp. paracasei, Bifidobacterium animalis ssp. lactis and Heat-Treated Lactobacillus plantarum Improves Immune Function Including Natural Killer Cell Activity

PubMed Central

Lee, Ayoung; Lee, Young Ju; Yoo, Hye Jin; Kim, Minkyung; Chang, Yeeun; Lee, Dong Seog; Lee, Jong Ho

2017-01-01

The aim of this study was to investigate the impact of consuming dairy yogurt containing Lactobacillus paracasei ssp. paracasei (L. paracasei), Bifidobacterium animalis ssp. lactis (B. lactis) and heat-treated Lactobacillus plantarum (L. plantarum) on immune function. A randomized, open-label, placebo-controlled study was conducted on 200 nondiabetic subjects. Over a twelve-week period, the test group consumed dairy yogurt containing probiotics each day, whereas the placebo group consumed milk. Natural killer (NK) cell activity, interleukin (IL)-12 and immunoglobulin (Ig) G1 levels were significantly increased in the test group at twelve weeks compared to baseline. Additionally, the test group had significantly greater increases in serum NK cell activity and interferon (IFN)-γ and IgG1 than placebo group. Daily consumption of dairy yogurt containing L. paracasei, B. lactis and heat-treated L. plantarum could be an effective option to improve immune function by enhancing NK cell function and IFN-γ concentration (ClinicalTrials.gov: NCT03051425). PMID:28561762

Expression of genes associated with stress conditions by Listeria monocytogenes in interaction with nisin producer Lactococcus lactis.

PubMed

Miranda, Rodrigo Otávio; Campos-Galvão, Maria Emilene Martino; Nero, Luís Augusto

2018-03-01

The use of nisin producers in foods is considered a mitigation strategy to control foodborne pathogens growth, such as Listeria monocytogenes, due to the production of this bacteriocin in situ. However, when the bacteriocin does not reach an adequate concentration, the target bacteria can develop a cross-response to different stress conditions in food, such as acid, thermal and osmotic. This study aimed to evaluate the interaction of a nisin-producing strain of Lactococcus lactis DY-13 and L. monocytogenes in BHI and skim milk, and its influence on general (sigB), acid (gadD2), thermal (groEL) and osmotic (gbu) stress-related genes of the pathogen. L. monocytogenes populations decreased approximately 2log in BHI and 1log in milk after 24h in co-culture with the nisin producer L. lactis, coherent with the increasing expression of nisK. Expression of stress-related genes by L. monocytogenes presented lower oscillation in BHI than in milk, indicating its better ability to survive in milk, despite the higher nisin production. Stress-related genes presented a varied expression by L. monocytogenes in the tested conditions: sigB expression remained stable or reduced over time; gadD2 presented high expression in milk; groEL presented low expression in BHI when compared to milk, trending to decrease overtime; gbu expression in milk after 24h was lower than in BHI. The presented study demonstrated the growth of a nisin producer L. lactis can affect the expression of stress-related genes by L. monocytogenes, and understating these mechanisms is crucial to enhance the conservation methods employed in foods. Copyright © 2017 Elsevier Ltd. All rights reserved.

Long-term administration of pDC-Stimulative Lactococcus lactis strain decelerates senescence and prolongs the lifespan of mice.

PubMed

Sugimura, Tetsu; Jounai, Kenta; Ohshio, Konomi; Suzuki, Hiroaki; Kirisako, Takayoshi; Sugihara, Yoshihiko; Fujiwara, Daisuke

2018-05-01

The decline in immune function caused by aging increases the risk of infectious diseases, tumorigeneses and chronic inflammation, resulting in accelerating senescence. We previously reported a lactic acid bacteria, Lactococcus lactis strain Plasma (synonym of Lactococcus lactis subsp. lactis JCM 5805, Lc-Plasma), that stimulates plasmacytoid dendritic cells (pDCs), which play a crucial role in phylaxis from viral infection. In this study, we investigated the anti-aging effects of long-term oral administration of Lc-Plasma in a senescence-accelerated mouse strain, SAMP6. Mice given Lc-Plasma showed a significant improvement in survival rate at 82 weeks and a decreased senescence score as compared with control mice throughout this study. Anatomic analysis at 82 weeks revealed that the frequency of altered hepatocellular foci was significantly lower, and the incidence of other pathological findings in the liver and lungs tended to be lower in Lc-Plasma mice than in control mice. Transcription level of the IL-1β gene in lungs also tended to be lower in Lc-Plasma mice. Furthermore, the thinning of skin and age-related decrease in muscle mass were also significantly suppressed in the Lc-Plasma group as compared with the control group. Consistent with these phenotypic features, pDCs activity was significantly higher in Lc-Plasma mice than in control mice. In conclusion, long-term administration of Lc-Plasma can decelerate senescence and prolong lifespan via maintenance of the immune system due to activation of pDCs. Copyright © 2018 Elsevier B.V. All rights reserved.

Genome-scale metabolic model for Lactococcus lactis MG1363 and its application to the analysis of flavor formation.

PubMed

Flahaut, Nicolas A L; Wiersma, Anne; van de Bunt, Bert; Martens, Dirk E; Schaap, Peter J; Sijtsma, Lolke; Dos Santos, Vitor A Martins; de Vos, Willem M

2013-10-01

Lactococcus lactis subsp. cremoris MG1363 is a paradigm strain for lactococci used in industrial dairy fermentations. However, despite of its importance for process development, no genome-scale metabolic model has been reported thus far. Moreover, current models for other lactococci only focus on growth and sugar degradation. A metabolic model that includes nitrogen metabolism and flavor-forming pathways is instrumental for the understanding and designing new industrial applications of these lactic acid bacteria. A genome-scale, constraint-based model of the metabolism and transport in L. lactis MG1363, accounting for 518 genes, 754 reactions, and 650 metabolites, was developed and experimentally validated. Fifty-nine reactions are directly or indirectly involved in flavor formation. Flux Balance Analysis and Flux Variability Analysis were used to investigate flux distributions within the whole metabolic network. Anaerobic carbon-limited continuous cultures were used for estimating the energetic parameters. A thorough model-driven analysis showing a highly flexible nitrogen metabolism, e.g., branched-chain amino acid catabolism which coupled with the redox balance, is pivotal for the prediction of the formation of different flavor compounds. Furthermore, the model predicted the formation of volatile sulfur compounds as a result of the fermentation. These products were subsequently identified in the experimental fermentations carried out. Thus, the genome-scale metabolic model couples the carbon and nitrogen metabolism in L. lactis MG1363 with complete known catabolic pathways leading to flavor formation. The model provided valuable insights into the metabolic networks underlying flavor formation and has the potential to contribute to new developments in dairy industries and cheese-flavor research.

Evolution of the carboxylate Jen transporters in fungi.

PubMed

Lodi, Tiziana; Diffels, Julie; Goffeau, André; Baret, Philippe V

2007-08-01

Synteny analysis is combined with sequence similarity and motif identification to trace the evolution of the putative monocarboxylate (lactate/pyruvate) transporters Jen1p and the dicarboxylate (succinate/fumarate/malate) transporters Jen2p in Hemiascomycetes yeasts and Euascomycetes fungi. It is concluded that a precursor form of Jen1p, named here preJen1p, arose by the duplication of an ancestral Jen2p, during the speciation of Yarrowia lipolytica, which was transferred into a new syntenic context. The Jen1p transporters differentiated from preJen1p in Kluyveromyces lactis, before the Whole Genome Duplication (WGD), and are conserved as a single copy in the Saccharomyces species. In contrast, the ancestral Jen2p was definitively lost just prior to the WGD and is absent in Saccharomyces.

Licheniocin 50.2 and Bacteriocins from Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 Inhibit Biofilms of Coagulase Negative Staphylococci and Listeria monocytogenes Clinical Isolates.

PubMed

Cirkovic, Ivana; Bozic, Dragana D; Draganic, Veselin; Lozo, Jelena; Beric, Tanja; Kojic, Milan; Arsic, Biljana; Garalejic, Eliana; Djukic, Slobodanka; Stankovic, Slavisa

2016-01-01

Coagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates. The effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates. Licheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200-400 AU/ml for licheniocin 50.2 and 400-3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001). This study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes.

Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor

PubMed Central

2010-01-01

Background Staphylococcal (or micrococcal) nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in Staphylococcus aureus spread in the infected host. Purified Nuc protein can be used as an exogenous reagent to clear cellular extracts and improve protein purification. Here, a recombinant form of Nuc was produced and secreted in a Gram-positive host, Lactococcus lactis, and purified from the culture medium. Results The gene segment corresponding to the S. aureus nuclease without its signal peptide was cloned in an expression-secretion vector. It was then fused to a lactococcal sequence encoding a signal peptide, and expressed under the control of a lactococcal promoter that is inducible by zinc starvation. An L. lactis subsp cremoris model strain (MG1363) transformed with the resulting plasmid was grown in either of two media (GM17v and CDM) that are free of animal compounds, allowing GMP (Good Manufacturing Practice) production. Induction conditions (concentration of the metal chelator EDTA and timing of addition) in small-scale pH-regulated fermentors were optimized using LacMF (Lactis Multi-Fermentor), a home-made parallel fermentation control system able to monitor 12 reactors simultaneously. Large amounts of recombinant Nuc (rNuc) were produced and secreted in both media, and rNuc was purified from GM17v medium in a single-step procedure. Conclusions In L. lactis, rNuc production and secretion were optimal after induction by 0.5 mM EDTA in small scale (200 mL) GM17v exponential phase cultures (at an OD600 of 2), leading to a maximal protein yield of 210 mg per L of culture medium. Purified rNuc was highly active, displaying a specific activity of 2000 U/mg. PMID:20492646

Strain improvement of Lactobacillus lactis for D-lactic acid production.

PubMed

Joshi, D S; Singhvi, M S; Khire, J M; Gokhale, D V

2010-04-01

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased D: -lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest D: -lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into D-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain D-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.

Controlled production of camembert-type cheeses: part III role of the ripening microflora on free fatty acid concentrations.

PubMed

Leclercq-Perlat, Marie-Noëlle; Corrieu, Georges; Spinnler, Henry-Eric

2007-05-01

Phenomena generating FFAs, important flavour precursors, are significant in cheese ripening. In Camembert-like cheeses, it was intended to establish the relationships between the dynamics of FFA concentrations changes and the succession of ripening microflora during ripening. Experimental Camembert-type cheeses were prepared in duplicate from pasteurised milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti, and Brevibacterium aurantiacum under aseptic conditions. For each cheese and each cheesy medium, concentrations of FFAs with odd-numbered carbons, except for 9:0 and 13:0, did not change over time. For long-chain FFAs, concentrations varied with the given cheese part (rind or core). K. lactis produced only short or medium-chain FFAs during its growth and had a minor influence on caproic, caprylic, capric, and lauric acids in comparison with G. candidum, the most lipolytic of the strains used here. It generated all short or medium-chain FFAs (4:0-12:0) during its exponential and slowdown growth periods and only long-chain ones (14:0-18:0) during its stationary phase. Pen. camemberti produced more long-chain FFAs (14:0-18:0) during its sporulation. Brev. aurantiacum did not generate any FFAs. The evidence of links between specific FFAs and the growth of a given microorganism is shown.

Effect of yogurt containing polydextrose, Lactobacillus acidophilus NCFM and Bifidobacterium lactis HN019: a randomized, double-blind, controlled study in chronic constipation.

PubMed

Magro, Daniéla Oliveira; de Oliveira, Lais Mariana R; Bernasconi, Isabela; Ruela, Marilia de Souza; Credidio, Laura; Barcelos, Irene K; Leal, Raquel F; Ayrizono, Maria de Lourdes Stesuko; Fagundes, João José; Teixeira, Leandro de B; Ouwehand, Arthur C; Coy, Claudio S R

2014-07-24

Constipation is a frequent complaint and the combination of a prebiotic and probiotics could have a potentially synergic effect on the intestinal transit. The present study therefore aims to investigate the combination of polydextrose (Litesse), L. acidophilus NCFM® and B. lactis HN019 in a yogurt on intestinal transit in subjects who suffer from constipation. Patients with constipation were randomly divided into two groups, Control Group (CG) and Treatment Group (TG), and had to eat 180 ml of unflavored yogurt every morning for 14 days. Those in the CG received only yogurt, while the TG received yogurt containing polydextrose, L. acidophilus NCFM (ATCC 700396) and B. lactis HN019 (AGAL NM97/09513). Favourable clinical response was assessed since Agachan score had a significant reduction at the end of the study in both groups and tended to be better in the TG. The subjects in the treatment group also had a shorter transit time at the end of the intervention compared to the control group (p = 0.01). The product containing yogurt with polydextrose, B. lactis HN019 and L. acidophilus NCFM® significantly shortened colonic transit time after two weeks in the TG compared to CG and may be an option for treatment of constipation.

Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis*

PubMed Central

Solopova, Ana; Formosa-Dague, Cécile; Courtin, Pascal; Furlan, Sylviane; Veiga, Patrick; Péchoux, Christine; Armalyte, Julija; Sadauskas, Mikas; Kok, Jan; Hols, Pascal; Dufrêne, Yves F.; Kuipers, Oscar P.; Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius

2016-01-01

To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of l-aspartate (l-Asp) to N-carbamoyl-l-aspartate by PyrB may reduce the amount of l-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of l-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that l-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes. PMID:27022026

A surprisingly large RNase P RNA in Candida glabrata

PubMed Central

KACHOURI, RYM; STRIBINSKIS, VILIUS; ZHU, YANGLONG; RAMOS, KENNETH S.; WESTHOF, ERIC; LI, YONG

2005-01-01

We have found an extremely large ribonuclease P (RNase P) RNA (RPR1) in the human pathogen Candida glabrata and verified that this molecule is expressed and present in the active enzyme complex of this hemiascomycete yeast. A structural alignment of the C. glabrata sequence with 36 other hemiascomycete RNase P RNAs (abbreviated as P RNAs) allows us to characterize the types of insertions. In addition, 15 P RNA sequences were newly characterized by searching in the recently sequenced genomes Candida albicans, C. glabrata, Debaryomyces hansenii, Eremothecium gossypii, Kluyveromyces lactis, Kluyveromyces waltii, Naumovia castellii, Saccharomyces kudriavzevii, Saccharomyces mikatae, and Yarrowia lipolytica; and by PCR amplification for other Candida species (Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida stellatoidea, and Candida tropicalis). The phylogenetic comparative analysis identifies a hemiascomycete secondary structure consensus that presents a conserved core in all species with variable insertions or deletions. The most significant variability is found in C. glabrata P RNA in which three insertions exceeding in total 700 nt are present in the Specificity domain. This P RNA is more than twice the length of any other homologous P RNAs known in the three domains of life and is eight times the size of the smallest. RNase P RNA, therefore, represents one of the most diversified noncoding RNAs in terms of size variation and structural diversity. PMID:15987816

The RNA polymerase III-dependent family of genes in hemiascomycetes: comparative RNomics, decoding strategies, transcription and evolutionary implications

PubMed Central

Marck, Christian; Kachouri-Lafond, Rym; Lafontaine, Ingrid; Westhof, Eric; Dujon, Bernard; Grosjean, Henri

2006-01-01

We present the first comprehensive analysis of RNA polymerase III (Pol III) transcribed genes in ten yeast genomes. This set includes all tRNA genes (tDNA) and genes coding for SNR6 (U6), SNR52, SCR1 and RPR1 RNA in the nine hemiascomycetes Saccharomyces cerevisiae, Saccharomyces castellii, Candida glabrata, Kluyveromyces waltii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and the archiascomycete Schizosaccharomyces pombe. We systematically analysed sequence specificities of tRNA genes, polymorphism, variability of introns, gene redundancy and gene clustering. Analysis of decoding strategies showed that yeasts close to S.cerevisiae use bacterial decoding rules to read the Leu CUN and Arg CGN codons, in contrast to all other known Eukaryotes. In D.hansenii and C.albicans, we identified a novel tDNA-Leu (AAG), reading the Leu CUU/CUC/CUA codons with an unusual G at position 32. A systematic ‘p-distance tree’ using the 60 variable positions of the tRNA molecule revealed that most tDNAs cluster into amino acid-specific sub-trees, suggesting that, within hemiascomycetes, orthologous tDNAs are more closely related than paralogs. We finally determined the bipartite A- and B-box sequences recognized by TFIIIC. These minimal sequences are nearly conserved throughout hemiascomycetes and were satisfactorily retrieved at appropriate locations in other Pol III genes. PMID:16600899

Genome Sequence of Lactobacillus delbrueckii subsp. lactis CNRZ327, a Dairy Bacterium with Anti-Inflammatory Properties.

PubMed

El Kafsi, Hela; Binesse, Johan; Loux, Valentin; Buratti, Julien; Boudebbouze, Samira; Dervyn, Rozenn; Hammani, Amal; Maguin, Emmanuelle; van de Guchte, Maarten

2014-07-17

Lactobacillus delbrueckii subsp. lactis CNRZ327 is a dairy bacterium with anti-inflammatory properties both in vitro and in vivo. Here, we report the genome sequence of this bacterium, which appears to contain no less than 215 insertion sequence (IS) elements, an exceptionally high number regarding the small genome size of the strain. Copyright © 2014 El Kafsi et al.

Potential aquaculture probiont Lactococcus lactis TW34 produces nisin Z and inhibits the fish pathogen Lactococcus garvieae.

PubMed

Sequeiros, Cynthia; Garcés, Marisa E; Vallejo, Marisol; Marguet, Emilio R; Olivera, Nelda L

2015-04-01

Bacteriocin-producing Lactococcus lactis TW34 was isolated from marine fish. TW34 bacteriocin inhibited the growth of the fish pathogen Lactococcus garvieae at 5 AU/ml (minimum inhibitory concentration), whereas the minimum bactericidal concentration was 10 AU/ml. Addition of TW34 bacteriocin to L. garvieae cultures resulted in a decrease of six orders of magnitude of viable cells counts demonstrating a bactericidal mode of action. The direct detection of the bacteriocin activity by Tricine-SDS-PAGE showed an active peptide with a molecular mass ca. 4.5 kDa. The analysis by MALDI-TOF-MS detected a strong signal at m/z 2,351.2 that corresponded to the nisin leader peptide mass without the initiating methionine, whose sequence STKDFNLDLVSVSKKDSGASPR was confirmed by MS/MS. Sequence analysis of nisin structural gene confirmed that L. lactis TW34 was a nisin Z producer. This nisin Z-producing strain with probiotic properties might be considered as an alternative in the prevention of lactococcosis, a global disease in aquaculture systems.

Licheniocin 50.2 and Bacteriocins from Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 Inhibit Biofilms of Coagulase Negative Staphylococci and Listeria monocytogenes Clinical Isolates

PubMed Central

Draganic, Veselin; Lozo, Jelena; Beric, Tanja; Kojic, Milan; Arsic, Biljana; Garalejic, Eliana; Djukic, Slobodanka; Stankovic, Slavisa

2016-01-01

Background Coagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates. Methods The effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates. Results Licheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200–400 AU/ml for licheniocin 50.2 and 400–3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001). Conclusions This study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes. PMID:27930711

Taggiasca extra virgin olive oil colonization by yeasts during the extraction process.

PubMed

Ciafardini, G; Cioccia, G; Zullo, B A

2017-04-01

The opalescent appearance of the newly produced olive oil is due to the presence of solid particles and microdrops of vegetation water in which the microorganisms from the olives' carposphere are trapped. Present research has demonstrated that the microbiota of the fresh extracted olive oil, produced in the mills, is mainly composed of yeasts and to a lesser extent of molds. The close link between the composition of the microbiota of the olives' carposphere undergoing to processing, and that of the microbiota of the newly produced olive oil, concerns only the yeasts and molds, given that the bacterial component is by and large destroyed mainly in the kneaded paste during the malaxation process. Six physiologically homogenous yeast groups were highlighted in the wash water, kneaded paste and newly produced olive oil from the Taggiasca variety which had been collected in mills located in the Liguria region. The more predominant yeasts of each group belonged to a single species called respectively: Kluyveromyces marxianus, Candida oleophila, Candida diddensiae, Candida norvegica, Wickerhamomyces anomalus and Debaryomyces hansenii. Apart from K. marxianus, which was found only in the wash water, all the other species were found in the wash water and in the kneaded paste as well as in the newly produced olive oil, while in the six-month stored olive oil, was found only one physiologically homogeneous group of yeast represented by the W. anomalus specie. These findings in according to our previous studies carried out on other types of mono varietal olive oils, confirms that the habitat of the Taggiascas' extra virgin olive oil, had a strong selective pressure on the yeast biota, allowing only to a few member of yeast species, contaminating the fresh product, to survive and reproduce in it during storage. Copyright © 2016 Elsevier Ltd. All rights reserved.

Safety, potential biotechnological and probiotic properties of bacteriocinogenic Enterococcus lactis strains isolated from raw shrimps.

PubMed

Ben Braïek, Olfa; Morandi, Stefano; Cremonesi, Paola; Smaoui, Slim; Hani, Khaled; Ghrairi, Taoufik

2018-04-01

The aims of this study are to isolate new bacteriocinogenic lactic acid bacterial strains from white (Penaeus vannamei) and pink (Palaemon serratus) raw shrimps and evaluate their technological and probiotic potentialities. Seven strains were selected, among fifty active isolates, as producing interesting antimicrobial activity. Identified as Enterococcus lactis, these isolates were able to produce enterocins A, B and/or P. The safety aspect, assessed by microbiological and molecular tests, demonstrated that the strains were susceptible to relevant antibiotics such as vancomycin, negative for haemolysin and gelatinase activities, and did not harbour virulence and antibiotic resistance genes. The assessment of potential probiotic and technological properties showed a low or no lipolytic activity, moderate milk-acidifying ability, high reducing power, proteolytic activity and tolerance to bile (P < 0.05) and good autoaggregation and coaggregation capacities. Two strains designated as CQ and C43 exhibiting high enzymatic activities and bile salt hydrolase activity were found to display high survival under simulated in vitro oral cavity and gastrointestinal tract conditions caused by presence of lysozyme, pepsin, pancreatin, bile salts and acidic pH. This study highlights safe Enterococcus lactis strains with great technological and probiotic potentials for future application as new starter, adjunct, protective or probiotic cultures in food industry. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of the mature cell surface proteinase of Lactobacillus delbrueckii subsp. lactis CRL 581.

PubMed

Villegas, Josefina M; Brown, Lucía; Savoy de Giori, Graciela; Hebert, Elvira M

2015-05-01

The cell envelope-associated proteinase (CEP) of Lactobacillus delbrueckii subsp. lactis CRL 581 (PrtL) has an essential role in bacterial growth, contributes to the flavor and texture development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. The genome of L. delbrueckii subsp. lactis CRL 581 possesses only one gene that encodes PrtL, which consists of 1924 amino acids and is a multidomain protein anchored to the cell via its W domain. PrtL was extracted from the cell under high ionic strength conditions using NaCl, suggesting an electrostatic interaction between the proteinase and the cell envelope. The released PrtL was purified and biochemically characterized; its activity was maximal at temperatures between 37 and 40 °C and at pH between 7 and 8. Under optimal conditions, PrtL exhibited higher affinity for succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide than for succinyl-alanyl-glutamyl-prolyl-phenylalanine-p-nitroanilide, while methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide was not degraded. A similar α- and β-casein degradation pattern was observed with the purified and the cell envelope-bound proteinase. Finally, on the basis of its specificity towards caseins and the unique combination of amino acids at residues thought to be involved in substrate specificity, PrtL can be classified as a representative of a new group of CEP.

Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium.

PubMed

Hugo, Ayelén A; Rolny, Ivanna S; Romanin, David; Pérez, Pablo F

2017-03-01

Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.

D-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum L-arabinose isomerase.

PubMed

Salonen, Noora; Salonen, Kalle; Leisola, Matti; Nyyssölä, Antti

2013-04-01

Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum L-AI were used for production of D-tagatose from D-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of D-galactose to D-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L⁻¹ substrate and at 37.5 °C after 5 days. The D-tagatose production rate of 185 g L⁻¹ day ⁻¹ was obtained at 300 g L⁻¹ galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial D-tagatose production rate was 290 g L⁻¹ day⁻¹ under these conditions.

Immunogenicity and immunoprotection of porcine circovirus type 2 (PCV2) Cap protein displayed by Lactococcus lactis.

PubMed

Li, Peng-Cheng; Qiao, Xu-Wen; Zheng, Qi-Sheng; Hou, Ji-Bo

2016-01-27

The capsid (Cap) protein, an important immunoprotective protein of porcine circovirus type 2 (PCV2), was expressed on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. Cap protein was fused to the peptidoglycan binding domain (known as the protein anchor domain, PA) of the lactococcal AcmA cell-wall hydrolase. The Cap protein fusion was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells (designated Gram-positive enhancer matrix (GEM) particles). Expression of the recombinant GEM-displaying capsid protein (GEM-PA-Cap) was verified by Western blotting and immunofluorescence and transmission electron microscopy assays. To evaluate the immunogenicity of the recombinant Cap protein (rCap), 20 PCV2-seronegative piglets were immunized with the GEM-PA-Cap subunit vaccine, GEM alone, or phosphate-buffered saline (PBS, challenge control and empty control). Each group consisted of five piglets. The results showed that the level of PCV2-specific antibodies in piglets immunized with the GEM-PA-Cap subunit vaccine was significantly higher than that of the piglets immunized with GEM alone or the control group at all the time points post-vaccination (P<0.01). After challenge with the PCV2 wild-type strain, piglets that received the GEM-PA-Cap subunit vaccine showed significantly higher average daily weight gain (DWG) and shorter fever duration than the other two groups (P<0.001). Furthermore, a significant reduction in the gross lung lesion scores and lymph node lesion scores was noted in the GEM-PA-Cap-immunized group compared with the scores of the GEM or PBS-treated group (P<0.01). The results suggest that recombinant rCap displayed by L. lactis GEM particles provided the piglets with significant immunoprotection from PCV2-associated disease. Thus, the novel GEM-PA-Cap subunit vaccine has potential to be considered an effective and safe candidate vaccine against PCV2 infection in piglets

Isolation of a bacteriocin-producing Lactococcus lactis subsp. lactis and application to control Listeria monocytogenes in Moroccan jben.

PubMed

Benkerroum, N; Oubel, H; Zahar, M; Dlia, S; Filali-Maltouf, A

2000-12-01

Use of a bacteriocin-producing lactococcal strain to control Listeria monocytogenes in jben. A Lactococcus lactis strain isolated from lben was shown, by the spot technique, to produce a bacteriocin different from nisin. Inhibitory activity of the bacteriocin-producing strain against Listeria monocytogenes was investigated in jben, made from cow's milk fermented with the producer organism and contaminated with 104 or 107 cfu ml-1. Listeria counts were monitored during manufacture, and during conservation at room and at refrigeration temperatures. Results showed that the pathogen was reduced by 2.7 logarithmic units after 30 h of jben processing when the initial inoculum of 107 cfu ml(-1) was used. For the initial inoculum of 104 cfu ml(-1), the bacterium was completely eliminated at 24 h. Furthermore, the use of the bacteriocin-producing starter culture extended the shelf-life of jben by 5 days. In situ production of the lactococcal bacteriocin is an efficient biological means of controlling L. monocytogenes in jben and of allowing shelf-life extension. The proposed technology will essentially benefit minimally processed dairy products and those made with raw milk.

Extraction of genomic DNA from yeasts for PCR-based applications.

PubMed

Lõoke, Marko; Kristjuhan, Kersti; Kristjuhan, Arnold

2011-05-01

We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp.

Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose.

PubMed

Ishchuk, Olena P; Voronovsky, Andriy Y; Stasyk, Oleh V; Gayda, Galina Z; Gonchar, Mykhailo V; Abbas, Charles A; Sibirny, Andriy A

2008-11-01

Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.

Cloning and Expression of Plantaricin W Produced by Lactobacillus plantarum U10 Isolate from "Tempoyak" Indonesian Fermented Food as Immunity Protein in Lactococcus lactis.

PubMed

Lages, Aksar Chair; Mustopa, Apon Zaenal; Sukmarini, Linda; Suharsono

2015-10-01

Plantaricins, one of bacteriocin produced by Lactobacillus plantarum, are already known to have activities against several pathogenic bacterium. L. plantarum U10 isolated from "tempoyak," an Indonesian fermented food, produced one kind of plantaricin designated as plantaricin W (plnW). The plnW is suggested as a putative membrane location of protein and has similar conserved motif which is important as immunity to bacteriocin itself. Thus, due to study about this plantaricin, several constructs have been cloned and protein was analyzed in Lactococcus lactis. In this study, plnW gene was successfully cloned into vector NICE system pNZ8148 and created the transformant named L. lactis NZ3900 pNZ8148-WU10. PlnW protein was 25.3 kDa in size. The concentration of expressed protein was significantly increased by 10 ng/mL nisin induction. Furthermore, PlnW exhibited protease activity with value of 2.22 ± 0.05 U/mL and specific activity about 1.65 ± 0.03 U/mg protein with 50 ng/mL nisin induction. Immunity study showed that the PlnW had immunity activity especially against plantaricin and rendered L. lactis recombinant an immunity broadly to other bacteriocins such as pediocin, fermentcin, and acidocin.

Mucosal and systemic immune responses elicited by recombinant Lactococcus lactis expressing a fusion protein composed of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis.

PubMed

Torkashvand, Ali; Bahrami, Fariborz; Adib, Minoo; Ajdary, Soheila

2018-05-05

We constructed a food-grade expression system harboring a F1S1 fusion protein of Bordetella pertussis to be produced in Lactococcus lactis NZ3900 as a new oral vaccine model against whooping cough, caused by B. pertussis. F1S1 was composed of N-terminally truncated S1 subunit of pertussis toxin and type I immunodominant domain of filamentous hemagglutinin which are both known as protective immunogens against pertussis. The recombinant L. lactis was administered via oral or intranasal routes to BALB/c mice and the related specific systemic and mucosal immune responses were then evaluated. The results indicated significantly higher levels of specific IgA in the lung extracts and IgG in sera of mucosally-immunized mice, compared to their controls. It was revealed that higher levels of IgG2a, compared to IgG1, were produced in all mucosally-immunized mice. Moreover, immunized mice developed Th1 responses with high levels of IFN-γ production by the spleen cells. These findings provide evidence for L. lactis to be used as a suitable vehicle for expression and delivery of F1S1 fusion protein to mucosa and induction of appropriate systemic and mucosal immune responses against pertussis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Kluyveromyces wickerhamii killer toxin: purification and activity towards Brettanomyces/Dekkera yeasts in grape must.

PubMed

Comitini, Francesca; Ciani, Maurizio

2011-03-01

Brettanomyces/Dekkera yeasts have been identified as part of the grape yeast flora. They are well known for colonizing the cellar environmental and spoiling wines, causing haze, turbidity and strong off-flavours in wines and enhancing the volatile acidity. As the general practices applied to combat Brettanomyces/Dekkera yeasts are not particularly appropriate during wine ageing and storage, a biological alternative to curtailing their growth would be welcomed in winemaking. In this study, we investigated the Kluyveromyces wickerhamii killer toxin (Kwkt) that is active against Brettanomyces/Dekkera spoilage yeasts. Purification procedures allowed the identification of Kwkt as a protein with an apparent molecular mass of 72 kDa and without any glycosyl residue. Interestingly, purified Kwkt has fungicidal effects at low concentrations under the physicochemical conditions of winemaking. The addition of 40 and 80 mg L(-1) purified Kwkt showed efficient antispoilage effects, controlling both growth and metabolic activity of sensitive spoilage yeasts. At these two killer toxin concentrations, compounds known to contribute to the 'Brett' character of wines, such as ethyl phenols, were not produced. Thus, purified Kwkt appears to be a suitable biological strategy to control Brettanomyces/Dekkera yeasts during fermentation, wine ageing and storage. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Safety evaluation of HOWARU® Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

PubMed

Morovic, Wesley; Roper, Jason M; Smith, Amy B; Mukerji, Pushkor; Stahl, Buffy; Rae, Jessica Caverly; Ouwehand, Arthur C

2017-12-01

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM ® , Lactobacillus paracasei Lpc-37 ® , Bifidobacterium animalis subsp. lactis strains Bl-04 ® , and Bi-07 ® , and their combination (HOWARU ® Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 10 12  CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 10 12  CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Complete Genome Sequence of Lactococcus lactis Strain AI06, an Endophyte of the Amazonian Açaí Palm

PubMed Central

de Oliveira, Viviane Matoso; de Almeida Pina, André Vicioli; Pérez-Chaparro, Paula Juliana; de Almeida, Lara Mendes; de Vasconcelos, Janaina Mota; de Oliveira, Layanna Freitas; da Silva, Daisy Elaine Andrade; Rogez, Hervé Louis Ghislain; Cretenet, Marina; Mamizuka, Elsa Masae; Nunes, Marcio Roberto Teixeira

2014-01-01

We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the açaí fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the açaí palm and also a component of the microbiota of the edible food product. PMID:25414513

Multiplex PCR to detect bacteriophages from natural whey cultures of buffalo milk and characterisation of two phages active against Lactococcus lactis, ΦApr-1 and ΦApr-2.

PubMed

Aprea, Giuseppe; Mullan, William Michael; Murru, Nicoletta; Fitzgerald, Gerald; Buonanno, Marialuisa; Cortesi, Maria Luisa; Prencipe, Vincenza Annunziata; Migliorati, Giacomo

2017-09-30

This work investigated bacteriophage induced starter failures in artisanal buffalo Mozzarella production plants in Southern Italy. Two hundred and ten samples of whey starter cultures were screened for bacteriophage infection. Multiplex polymerase chain reaction (PCR) revealed phage infection in 28.56% of samples, all showing acidification problems during cheese making. Based on DNA sequences, bacteriophages for Lactococcus lactis (L. lactis), Lactobacillus delbruekii (L. delbruekii) and Streptococcus thermophilus (S. thermophilus) were detected. Two phages active against L. lactis, ΦApr-1 and ΦApr-2, were isolated and characterised. The genomes, approximately 31.4 kb and 31 kb for ΦApr-1 and ΦApr-2 respectively, consisted of double-stranded linear DNA with pac-type system. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑PAGE) showed one major structural protein of approximately 32.5 kDa and several minor proteins. This is the first report of phage isolation in buffalo milk and of the use of multiplex PCR to screen and study the diversity of phages against Lactic Acid Bacteria (LAB) strains in artisanal Water Buffalo Mozzarella starters.

Dynamics in copy numbers of five plasmids of a dairy Lactococcus lactis in dairy-related conditions including near-zero growth rates.

PubMed

van Mastrigt, Oscar; Lommers, Marcel M A N; de Vries, Yorick C; Abee, Tjakko; Smid, Eddy J

2018-03-23

Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-encoded genes and the activity of the corresponding proteins is severely affected by changes in the number of plasmid copies. We studied the impact of growth rate on dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected which varied in size (3 to 39 kb), in replication mechanism (theta or rolling-circle) and in putative (dairy-associated) functions. Copy numbers ranged from 1.5 to 40.5 and the copy number of theta-type replicating plasmids were negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h -1 to 0.6 h -1 ), copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates showing that the plasmid replication rate was strictly controlled. One low-copy number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations reflected in complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation or presence of citrate (maximum 2.2-fold) signifying the stability in copy number of the plasmids. Importance Lactococcus lactis is extensively used in starter cultures for dairy fermentations. Important traits for growth and survival of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation and oligopeptide uptake and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-encoded genes, it is important to know the factors that influence the plasmid copy numbers. We monitored plasmid copy numbers of L

One-pot synthesis of GDP-l-fucose by a four-enzyme cascade expressed in Lactococcus lactis.

PubMed

Li, Ling; Kim, Seul-Ah; Heo, Ji Eun; Kim, Tae-Jip; Seo, Jin-Ho; Han, Nam Soo

2017-12-20

GDP-l-fucose is an l-fucose donor to synthesize fucosylated compounds such as human milk oligosaccharides or Lewis antigen. In this study, we used Lactococcus lactis subsp. cremoris NZ9000 to express 4 enzymes, ManB, ManC, Gmd, and WcaG and produced GDP-l-fucose by using one-pot synthesis method with mannose-6-phosphate as substrate and the enzymes as biocatalyst. For preparation of enzyme mixture, 4 genes (manB, manC, gmd, and wcaG) cloned from Escherichia coli were transformed into L. lactis strains using pNZ8008 and the recombinant cell lysates were obtained after cultivation. When mannose-6-phosphate was used as the substrate, the consecutive reactions with ManB, ManC, Gmd, and WcaG resulted in the successful production of GDP-l-fucose (0.13mM). When GDP-d-mannose was used as the substrate, it was entirely converted to GDP-l-fucose (0.2mM; 0.12g/L) via 2 enzymatic reactions mediated by Gmd and WcaG. This is the first report of GDP-l-fucose production by using multiple enzymes expressed in lactic acid bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri

PubMed Central

van Pijkeren, Jan-Peter; Neoh, Kar Mun; Sirias, Denise; Findley, Anthony S.; Britton, Robert A.

2012-01-01

Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution. PMID:22750793

Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

PubMed Central

Bernasconi, Eric; Germond, Jacques-Edouard; Delley, Michèle; Fritsché, Rodolphe; Corthésy, Blaise

2002-01-01

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB∂, PrtB∂-BLG, and PrtB∂-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB∂-BLG yielded up to 170 μg per 109 CFU in the culture supernatant and 9 μg per 109 CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue. PMID:12039750

High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling ▿ †

PubMed Central

Gaspar, Paula; Neves, Ana Rute; Gasson, Michael J.; Shearman, Claire A.; Santos, Helena

2011-01-01

Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds. PMID:21841021

Maximization of beta-galactosidase production: a simultaneous investigation of agitation and aeration effects.

PubMed

Alves, Fernanda Germano; Filho, Francisco Maugeri; de Medeiros Burkert, Janaína Fernandes; Kalil, Susana Juliano

2010-03-01

In this work, the agitation and aeration effects in the maximization of the beta-galactosidase production from Kluyveromyces marxianus CCT 7082 were investigated simultaneously, in relation to the volumetric enzyme activity and the productivity, as well as the analysis of the lactose consumption and production of glucose, and galactose of this process. Agitation and aeration effects were studied in a 2 L batch stirred reactor. A central composite design (2(2) trials plus three central points) was carried out. Agitation speed varied from 200 to 500 rpm and aeration rate from 0.5 to 1.5 vvm. It has been shown in this study that the volumetric enzyme production was strongly influenced by mixing conditions, while aeration was shown to be less significant. Linear models for activity and productivity due to agitation and aeration were obtained. The favorable condition was 500 rpm and 1.5 vvm, which lead to the best production of 17 U mL(-1) for enzymatic activity, 1.2 U mL(-1) h(-1) for productivity in 14 h of process, a cellular concentration of 11 mg mL(-1), and a 167.2 h(-1) volumetric oxygen transfer coefficient.

Microbial communities and chemical changes during fermentation of sugary Brazilian kefir.

PubMed

Magalhães, Karina Teixeira; de M Pereira, G V; Dias, Disney Ribeiro; Schwan, Rosane Freitas

2010-07-01

The microorganisms associated with sugary Brazilian kefir beverage were investigated using a combination of culture-dependent and -independent methods. A total of 289 bacteria and 129 yeasts were identified via phenotypic and genotypic methods. Lb. paracasei (23.8%) was the major bacterial isolate identified, followed by Acetobacter lovaniensis (16.31%), Lactobacillus parabuchneri (11.71%), Lactobacillus kefir (10.03%) and Lactococcus lactis (10.03%). Saccharomyces cerevisiae (54.26%) and Kluyveromyces lactis (20.15%) were the most common yeast species isolated. Scanning electron microscopy showed that the microbiota was dominated by lemon-shaped yeast cells growing in close association with Lactobacillus (long and curved). Some lactic acid bacteria detected by sequence analysis of DGGE (denaturing gradient gel electrophoresis) bands were not recovered at any time through fermentation by plating. Conversely, DGGE fingerprints did not reveal bands corresponding to some of the species isolated by culturing methods. The bacteria Acetobacter lovaniensis and the yeast Kazachstania aerobia are described for the first time in sugary kefir. During the 24 h of fermentation, the concentration of lactic acid ranged from 0.2 to 1.80 mg/ml, and that of acetic acid increased from 0.08 to 1.12 mg/ml. The production of ethanol was limited, reaching a final mean value of 1.24 mg/ml.

Evaluation of a select strain of Lactobacillus delbrueckii subsp. lactis as a biological control agent for pathogens on fresh-cut vegetables stored at 7 degrees C.

PubMed

Harp, E; Gilliland, S E

2003-06-01

Raw vegetables inoculated with selected pathogenic bacteria were treated with a strain of Lactobacillus delbrueckii subsp. lactis, which was selected for its ability to produce hydrogen peroxide at refrigerated temperatures. The vegetables inoculated included broccoli, cabbage, carrots, and lettuce. Each vegetable was rinsed, chopped, and stored under conditions similar to those used for ready-to-eat vegetables sold at retail. Portions of each vegetable were separately inoculated with one of two pathogenic bacteria, Escherichia coli O157:H7 or Listeria monocytogenes. Prior to packaging, one portion of each inoculated vegetable was treated with a cell suspension of the selected strain of L. delbrueckii subsp. lactis. The vegetables were stored at 7 degrees C for 6 days. The populations of pathogens and lactobacilli on each sample were enumerated on storage days 0, 3, and 6. Although populations of L. delbrueckii subsp. lactis remained at high levels during storage, there was no noticeable antagonistic action against the pathogens under conditions similar to those used for these products at the retail level. Each pathogen survived on all vegetables throughout storage. Further testing revealed that there was apparently sufficient catalase activity in the cut vegetables to destroy enough of the hydrogen peroxide to prevent antagonistic action against the pathogens.

Biological characteristics and probiotic effect of Leuconostoc lactis strain isolated from the intestine of black porgy fish

PubMed Central

Zhang, Wei; Liu, Mingqi; Dai, Xianjun

2013-01-01

A strain of lactic acid bacteria, Leuconostoc lactis, was isolated from the intestinal tract of black porgy, Sparus macrocephalus, and identified by conventional biochemical characteristics and 16S rDNA gene sequence analysis. The isolated strain had the ability of bile tolerance and resistance to low pH, and survived well in the trypsinase and pepsin solution. But the highly concentrated dose of trypsinase and pepsin affect the viability of the isolated strain. The isolate was resistant to several antibiotics, including Cephalothin, Ceftriaxone, Imipenem and Tobramycin. The isolate could auto-aggregate itself and coaggregate with other bacteria in vitro. The autoaggregation percentage increased to 23.29% after 20 h of incubation. The percentage of coaggregation were respectively 31.21%, 29.44%, 10.74%, 16.49%, 24.36%, 24.41% and 20.99% for Vibrio parahaemolyticus, Listeria monocytogenes, Escherichia coli O157, Salmonella typhimurium, Shigella, Staphylococcus aureus and Proteusbacillus vulgaris after 20 h incubation of a mixed suspension. The supernatant of the strain inhibited the growth of several pathogens, such as V.parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Staphylococcus aureus, Escherichia coli O157, Salmonella typhimurium, Bacillus subtilis, Proteusbacillus vulgaris and Shigella. These results indicated that the isolate, Leuconostoc lactis, might be an attractive candidate for perspectival strain for probiotics in marine aquaculture. PMID:24516418

Adaptation of Lactococcus lactis to high growth temperature leads to a dramatic increase in acidification rate

PubMed Central

Chen, Jun; Shen, Jing; Ingvar Hellgren, Lars; Ruhdal Jensen, Peter; Solem, Christian

2015-01-01

Lactococcus lactis is essential for most cheese making, and this mesophilic bacterium has its growth optimum around 30 °C. We have, through adaptive evolution, isolated a mutant TM29 that grows well up to 39 °C, and continuous growth at 40 °C is possible if pre-incubated at a slightly lower temperature. At the maximal permissive temperature for the wild-type, 38 °C, TM29 grows 33% faster and has a 12% higher specific lactate production rate than its parent MG1363, which results in fast lactate accumulation. Genome sequencing was used to reveal the mutations accumulated, most of which were shown to affect thermal tolerance. Of the mutations with more pronounced effects, two affected expression of single proteins (chaperone; riboflavin transporter), two had pleiotropic effects (RNA polymerase) which changed the gene expression profile, and one resulted in a change in the coding sequence of CDP-diglyceride synthase. A large deletion containing 10 genes was also found to affect thermal tolerance significantly. With this study we demonstrate a simple approach to obtain non-GMO derivatives of the important L. lactis that possess properties desirable by the industry, e.g. thermal robustness and increased rate of acidification. The mutations we have identified provide a genetic basis for further investigation of thermal tolerance. PMID:26388459

Simultaneous hydrolysis and co-fermentation of whey lactose with wheat for ethanol production.

PubMed

Jin, Yiqiong; Parashar, Archana; Mason, Beth; Bressler, David C

2016-12-01

Whey permeate was used as a co-substrate to replace part of the wheat for ethanol production by Saccharomyces cerevisiae. The simultaneous saccharification and fermentation was achieved with β-galactosidase added at the onset of the fermentation to promote whey lactose hydrolysis. Aspergillus oryzae and Kluyveromyces lactis β-galactosidases were two enzymes selected and used in the co-fermentation of wheat and whey permeate for the comparison of their effectiveness on lactose hydrolysis. The possibility of co-fermentations in both STARGEN and jet cooking systems was investigated in 5L bioreactors. Ethanol yields from the co-fermentations of wheat and whey permeate were evaluated. It was found that A. oryzae β-galactosidase was more efficient for lactose hydrolysis during the co-fermentation and that whey permeate supplementation can contribute to ethanol yield in co-fermentations with wheat. Copyright © 2016 Elsevier Ltd. All rights reserved.

New insights into the complex regulation of the glycolytic pathway in Lactococcus lactis. I. Construction and diagnosis of a comprehensive dynamic model.

PubMed

Dolatshahi, Sepideh; Fonseca, Luis L; Voit, Eberhard O

2016-01-01

This article and the companion paper use computational systems modeling to decipher the complex coordination of regulatory signals controlling the glycolytic pathway in the dairy bacterium Lactococcus lactis. In this first article, the development of a comprehensive kinetic dynamic model is described. The model is based on in vivo NMR data that consist of concentration trends in key glycolytic metabolites and cofactors. The model structure and parameter values are identified with a customized optimization strategy that uses as its core the method of dynamic flux estimation. For the first time, a dynamic model with a single parameter set fits all available glycolytic time course data under anaerobic operation. The model captures observations that had not been addressed so far and suggests the existence of regulatory effects that had been observed in other species, but not in L. lactis. The companion paper uses this model to analyze details of the dynamic control of glycolysis under aerobic and anaerobic conditions.

Bifidobacterium lactis B94 plus inulin for Treatment of Helicobacter pylori infection in children: does it increase eradication rate and patient compliance?

PubMed

Islek, A; Sayar, E; Yilmaz, A; Artan, R

2015-01-01

The aim of this study is to investigate the effects of Bifidobacterium lactis B94 and inulin (synbiotic) treatment on eradication rate and patient compliance in subjects treated for symptomatic H. pylori infection. Patients with symptomatic H. pylori infection were divided into two groups. One group was treated with standard triple therapy (lansoprazole, amoxicillin, and clarithromycin) and B. lactis B94 (5 × 109 CFU/dose) plus inulin (900 mg) twice daily for seven days. The control group was treated with standard triple therapy and placebo. The side effects and eradication rates were evaluated at the end of the study. Ninety-three patients with H. pylori infection were treated with either synbiotic plus triple therapy (n = 47) or placebo plus triple therapy (n = 46). The infection eradication rates were not significantly different between the synbiotic and placebo groups [intent-to-treat (ITT), 80.8% and 67.3%, p = 0.13, respectively; per-protocol (PP), 86.3% and 81.5%, p = 0.55, respectively]. The drug side effects were significantly higher in the placebo group than in the synbiotic group (63% and 17%, respectively, p < 0.01). Although no intolerable adverse side effects were observed in the synbiotic group, intolerable adverse side effects were observed in 13% of the placebo group (p = 0.01). Our results suggest that twice daily 5 × 109 CFU/dose B. lactis B94 plus 900 mg inulin treatment did not have a direct positive effect on the H. pylori eradication rate. However, this treatment had significantly reduced side effects and indirectly increased eradication rates by increasing patient compliance. © Acta Gastro-Enterologica Belgica.

Lactococcus lactis, causative agent of an endocarditis valvularis and parietalis thromboticans in the allis shad, Alosa alosa (L.).

PubMed

Wünnemann, H; Eskens, U; Prenger-Berninghoff, E; Ewers, C; Lierz, M

2018-05-28

Since the 1940s, the anadromous allis shad, Alosa alosa (L.), has suffered population declines throughout its distribution range in Europe. In context of EU-LIFE projects for the reintroduction of the allis shad in the Rhine system, a comprehensive study was started in 2012 to investigate infectious diseases occurring in allis shad. In course of the study, 217 mature and young-of-the-year allis shad originating from the wild population from the Gironde-Garonne-Dordogne system (GGD-system) and the Rhine system as well as 38 allis shad from the breeding population were examined by use of bacteriological and histological methods. In 2012 and 2014, an endocarditis valvularis thromboticans caused by a coccoid bacterium was detected in 16% and 25% of mature allis shad originating from the GGD-system. Results of microbiologic examinations, including biochemical characteristics, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence analysis, revealed Lactococcus lactis as causative agent of this infection. This is the first report of an endocarditis valvularis and parietalis thromboticans caused by Lactococcus lactis in fish. Possible sources of infection as well as the impact for the reintroduction programme are discussed. © 2018 John Wiley & Sons Ltd.

Specific Identification and Targeted Characterization of Bifidobacterium lactis from Different Environmental Isolates by a Combined Multiplex-PCR Approach

PubMed Central

Ventura, Marco; Reniero, Roberto; Zink, Ralf

2001-01-01

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach. PMID:11375192

Promoting acid resistance and nisin yield of Lactococcus lactis F44 by genetically increasing D-Asp amidation level inside cell wall.

PubMed

Hao, Panlong; Liang, Dongmei; Cao, Lijie; Qiao, Bin; Wu, Hao; Caiyin, Qinggele; Zhu, Hongji; Qiao, Jianjun

2017-08-01

Nisin fermentation by Lactococcus lactis requires a low pH to maintain a relatively higher nisin activity. However, the acidic environment will result in cell arrest, and eventually decrease the relative nisin production. Hence, constructing an acid-resistant L. lactis is crucial for nisin harvest in acidic nisin fermentation. In this paper, the first discovery of the relationship between D-Asp amidation-associated gene (asnH) and acid resistance was reported. Overexpression of asnH in L. lactis F44 (F44A) resulted in a sevenfold increase in survival capacity during acid shift (pH 3) and enhanced nisin desorption capacity compared to F44 (wild type), which subsequently contributed to higher nisin production, reaching 5346 IU/mL, 57.0% more than that of F44 in the fed-batch fermentation. Furthermore, the engineered F44A showed a moderate increase in D-Asp amidation level (from 82 to 92%) compared to F44. The concomitant decrease of the negative charge inside the cell wall was detected by a newly developed method based on the nisin adsorption amount onto cell surface. Meanwhile, peptidoglycan cross-linkage increased from 36.8% (F44) to 41.9% (F44A), and intracellular pH can be better maintained by blocking extracellular H + due to the maintenance of peptidoglycan integrity, which probably resulted from the action of inhibiting hydrolases activity. The inference was further supported by the acmC-overexpression strain F44C, which was characterized by uncontrolled peptidoglycan hydrolase activity. Our results provided a novel strategy for enhancing nisin yield through cell wall remodeling, which contributed to both continuous nisin synthesis and less nisin adsorption in acidic fermentation (dual enhancement).

Strain improvement and metabolic flux analysis in the wild-type and a mutant Lactobacillus lactis strain for L(+)-lactic acid production.

PubMed

Bai, Dong-Mei; Zhao, Xue-Ming; Li, Xin-Gang; Xu, Shi-Min

2004-12-20

The effects of initial glucose concentration and calcium lactate concentration on the lactic acid production by the parent strain, Lactobacillus lactis BME5-18, were studied. The results of the experiments indicated that glucose and lactate repressed the cell growth and the lactic acid production by Lactobacillus lactis BME5-18. A L(+)-lactic acid overproducing strain, Lactobacillus lactis BME5-18M, was screened by mutagenizing the parent strain with ultraviolet (UV) light irradiation and selecting the high glucose and lactate calcium concentration repression resistant mutant. Starting with a concentration of 100g L(-1) glucose, the mutant produced 98.6 g L(-1) lactic acid after 60 h in flasks, 73.9% higher than that of the parent strain. The L(+)-lactic acid purity was 98.1% by weight based on the amount of total lactic acid. The culture of the parent strain could not be analyzed well by conventional metabolic flux analysis techniques, since some pyruvate were accumulated intracellularly. Therefore, a revised flux analysis method was proposed by introducing intracellular pyruvate pool. Further studies demonstrate that there is a high level of NADH oxidase activity (12.11 mmol mg(-1) min(-1)) in the parent strain. The molecular mechanisms of the strain improvement were proposed, i.e., the high level of NADH oxidase activity was eliminated and the uptake rate of glucose was increased from 82.1 C-mmol (g DW h)(-1) to 98.9 C-mmol (g DW h)(-1) by mutagenizing the parent strain with UV, and therefore the mutant strain converts mostly pyruvate to lactic acid with a higher productivity (1.76 g L(-1) h(-1)) than the parent strain (0.95 g L(-1) h(-1)).

Technological characterization and survival of the exopolysaccharide-producing strain Lactobacillus delbrueckii subsp. lactis 193 and its bile-resistant derivative 193+ in simulated gastric and intestinal juices.

PubMed

Burns, Patricia; Vinderola, Gabriel; Reinheimer, Jorge; Cuesta, Isabel; de Los Reyes-Gavilán, Clara G; Ruas-Madiedo, Patricia

2011-08-01

The capacity of lactic acid bacteria to produce exopolysaccharides (EPS) conferring microorganisms a ropy phenotype could be an interesting feature from a technological point of view. Progressive adaptation to bile salts might render some lactobacilli able to overcome physiological gut barriers but could also modify functional properties of the strain, including the production of EPS. In this work some technological properties and the survival ability in simulated gastrointestinal conditions of Lactobacillus delbrueckii subsp. lactis 193, and Lb. delbrueckii subsp. lactis 193+, a strain with stable bile-resistant phenotype derived thereof, were characterized in milk in order to know whether the acquisition of resistance to bile could modify some characteristics of the microorganism. Both strains were able to grow and acidify milk similarly; however the production of ethanol increased at the expense of the aroma compound acetaldehyde in milk fermented by the strain 193+, with respect to milk fermented by the strain 193. Both microorganisms produced a heteropolysaccharide composed of glucose and galactose, and were able to increase the viscosity of fermented milks. In spite of the higher production yield of EPS by the bile-resistant strain 193+, it displayed a lower ability to increase viscosity than Lb. delbrueckii subsp. lactis 193. Milk increased survival in simulated gastric juice; the presence of bile improved adhesion to the intestinal cell line HT29-MTX in both strains. However, the acquisition of a stable resistance phenotype did not improve survival in simulated gastric and intestinal conditions or the adhesion to the intestinal cell line HT29-MTX. Thus, Lb. delbrueckii subsp. lactis 193 presents suitable technological properties for the manufacture of fermented dairy products; the acquisition of a stable bile-resistant phenotype modified some properties of the microorganism. This suggests that the possible use of bile-resistant derivative strains should be

Design of aqueous two-phase systems for purification of hyaluronic acid produced by metabolically engineered Lactococcus lactis.

PubMed

Rajendran, Vivek; Puvendran, Kirubhakaran; Guru, Bharath Raja; Jayaraman, Guhan

2016-02-01

Hyaluronic acid has a wide range of biomedical applications and its commercial value is highly dependent on its purity and molecular weight. This study highlights the utility of aqueous two-phase separation as a primary recovery step for hyaluronic acid and for removal of major protein impurities from fermentation broths. Metabolically engineered cultures of a lactate dehydrogenase mutant strain of Lactococcus lactis (L. lactis NZ9020) were used to produce high-molecular-weight hyaluronic acid. The cell-free fermentation broth was partially purified using a polyethylene glycol/potassium phosphate system, resulting in nearly 100% recovery of hyaluronic acid in the salt-rich bottom phase in all the aqueous two-phase separation experiments. These experiments were optimized for maximum removal of protein impurities in the polyethylene glycol rich top phase. The removal of protein impurities resulted in substantial reduction of membrane fouling in the subsequent diafiltration process, carried out with a 300 kDa polyether sulfone membrane. This step resulted in considerable purification of hyaluronic acid, without any loss in recovery and molecular weight. Diafiltration was followed by an adsorption step to remove minor impurities and achieve nearly 100% purity. The final hyaluronic acid product was characterized by Fourier-transform IR and NMR spectroscopy, confirming its purity. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Continuous D-lactic acid production by a novel thermotolerant Lactobacillus delbrueckii subsp. lactis QU 41.

PubMed

Tashiro, Yukihiro; Kaneko, Wataru; Sun, Yanqi; Shibata, Keisuke; Inokuma, Kentaro; Zendo, Takeshi; Sonomoto, Kenji

2011-03-01

We isolated and characterized a D-lactic acid-producing lactic acid bacterium (D-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 (T) and L. delbrueckii subsp. lactis JCM 1248 (T), which are also known as D-LAB, the QU 41 strain exhibited a high thermotolerance and produced D-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on D-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l D-lactic acid was acquired with high optical purity (>99.9% of D-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve D-lactic acid productivity. At a dilution rate of 0.87 h(-1), the high cell density continuous culture exhibited the highest D-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.

An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.

PubMed Central

Ryan, M P; Rea, M C; Hill, C; Ross, R P

1996-01-01

Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147. The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials. Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products. PMID:8593062

Bile resistance in Lactococcus lactis strains varies with cellular fatty acid composition: analysis by using different growth media.

PubMed

Kimoto-Nira, Hiromi; Kobayashi, Miho; Nomura, Masaru; Sasaki, Keisuke; Suzuki, Chise

2009-05-31

Bile resistance is one of the basic characteristics of probiotic bacteria. The aim of this study was to investigate the characteristics of bile resistance in lactococci by studying the relationship between bile resistance and cellular fatty acid composition in lactococcci grown on different media. We determined the bile resistance of 14 strains in lactose-free M17 medium supplemented with either glucose only (GM17) or lactose only (LM17). Gas chromatographic analyses of free lipids extracted from the tested strains were used for determining their fatty acid composition. A correlation analysis of all strains grown in both media revealed significant positive correlations between bile resistance and relative contents of hexadecanoic acid and octadecenoic acid, and negative correlations between bile resistance and relative contents of hexadecenoic acid and C-19 cyclopropane fatty acid. It is also a fact that the fatty acids associated with bile resistance depended on species, strain, and/or growth medium. In L. lactis subsp. cremoris strains grown in GM17 medium, the bile-resistant strains had significantly more octadecenoic acid than the bile-sensitive strains. In LM17 medium, bile-resistant strains had significantly more octadecenoic acid and significantly less C-19 cyclopropane fatty acid than the bile-sensitive strains. In L. lactis subsp. lactis strains, bile resistances of some of the tested strains were altered by growth medium. Some strains were resistant to bile in GM17 medium but sensitive to bile in LM17 medium. Some strains were resistant in both media tested. The strains grown in GM17 medium had significantly more hexadecanoic acid and octadecenoic acid, and significantly less tetradecanoic acid, octadecadienoic acid and C-19 cyclopropane fatty acid than the strains grown in LM17 medium. In conclusion, the fatty acid compositions of the bile-resistant lactococci differed from those of the bile-sensitive ones. More importantly, our data suggest that

Development of a rapid SNP-typing assay to differentiate Bifidobacterium animalis ssp. lactis strains used in probiotic-supplemented dairy products.

PubMed

Lomonaco, Sara; Furumoto, Emily J; Loquasto, Joseph R; Morra, Patrizia; Grassi, Ausilia; Roberts, Robert F

2015-02-01

Identification at the genus, species, and strain levels is desirable when a probiotic microorganism is added to foods. Strains of Bifidobacterium animalis ssp. lactis (BAL) are commonly used worldwide in dairy products supplemented with probiotic strains. However, strain discrimination is difficult because of the high degree of genome identity (99.975%) between different genomes of this subspecies. Typing of monomorphic species can be carried out efficiently by targeting informative single nucleotide polymorphisms (SNP). Findings from a previous study analyzing both reference and commercial strains of BAL identified SNP that could be used to discriminate common strains into 8 groups. This paper describes development of a minisequencing assay based on the primer extension reaction (PER) targeting multiple SNP that can allow strain differentiation of BAL. Based on previous data, 6 informative SNP were selected for further testing, and a multiplex preliminary PCR was optimized to amplify the DNA regions containing the selected SNP. Extension primers (EP) annealing immediately adjacent to the selected SNP were developed and tested in simplex and multiplex PER to evaluate their performance. Twenty-five strains belonging to 9 distinct genomic clusters of B. animalis ssp. lactis were selected and analyzed using the developed minisequencing assay, simultaneously targeting the 6 selected SNP. Fragment analysis was subsequently carried out in duplicate and demonstrated that the assay yielded 8 specific profiles separating the most commonly used commercial strains. This novel multiplex PER approach provides a simple, rapid, flexible SNP-based subtyping method for proper characterization and identification of commercial probiotic strains of BAL from fermented dairy products. To assess the usefulness of this method, DNA was extracted from yogurt manufactured with and without the addition of B. animalis ssp. lactis BB-12. Extracted DNA was then subjected to the minisequencing

Distinct contributions of the nisin biosynthesis enzymes NisB and NisC and transporter NisT to prenisin production by Lactococcus lactis.

PubMed

van den Berg van Saparoea, H Bart; Bakkes, Patrick J; Moll, Gert N; Driessen, Arnold J M

2008-09-01

Several Lactococcus lactis strains produce the lantibiotic nisin. The dedicated enzymes NisB and NisC and the transporter NisT modify and secrete the ribosomally synthesized nisin precursor peptide. NisB can function in the absence of the cyclase NisC, yielding the dehydrated prenisin that lacks the thioether rings. A kinetic analysis of nisin production by L. lactis NZ9700 demonstrated that the prenisin was released from the cell into the medium before the processing of the leader sequence occurred. Upon the deletion of nisC, the production of prenisin was reduced by 70%, while in the absence of nisB, the production of prenisin was nearly completely abolished. In cells lacking nisT, no secretion was observed, while the expression of nisABC in these cells resulted in considerable growth rate inhibition caused by the intracellular accumulation of active nisin. Overall, these data indicate that the efficiency of prenisin transport by NisT is markedly enhanced by NisB, suggesting a channeling mechanism of prenisin transfer between the nisin modification enzymes and the transporter.

Synergistic cooperation promotes multicellular performance and unicellular free-rider persistence

PubMed Central

Driscoll, William W; Travisano, Michael

2017-01-01

The evolution of multicellular life requires cooperation among cells, which can be undermined by intra-group selection for selfishness. Theory predicts that selection to avoid non-cooperators limits social interactions among non-relatives, yet previous evolution experiments suggest that intra-group conflict is an outcome, rather than a driver, of incipient multicellular life cycles. Here we report the evolution of multicellularity via two distinct mechanisms of group formation in the unicellular budding yeast Kluyveromyces lactis. Cells remain permanently attached following mitosis, giving rise to clonal clusters (staying together); clusters then reversibly assemble into social groups (coming together). Coming together amplifies the benefits of multicellularity and allows social clusters to collectively outperform solitary clusters. However, cooperation among non-relatives also permits fast-growing unicellular lineages to ‘free-ride' during selection for increased size. Cooperation and competition for the benefits of multicellularity promote the stable coexistence of unicellular and multicellular genotypes, underscoring the importance of social and ecological context during the transition to multicellularity. PMID:28580966

Impact of ultrasound on galactooligosaccharides and gluconic acid production throughout a multienzymatic system.

PubMed

Rico-Rodríguez, Fabián; Serrato, Juan Carlos; Montilla, Antonia; Villamiel, Mar

2018-06-01

Galactooligosaccharides (GOS), recognised prebiotic, can be industrially produced from lactose and commercial β-galactosidase (β-gal) from Kluyveromyces lactis. Residual lactose and glucose limit GOS applications. To handle this problem, a multienzymatic system, with β-gal and glucose oxidase (Gox), was proposed to reduce glucose content in reaction media through its oxidation to gluconic acid (GA). Besides, ultrasound (US) probe effect over the multienzymatic system to produce GOS and GA has been evaluated. A production around 40% of GOS was found in all treatments after the first hour of reaction. However, glucose consumption and GA production was significantly higher (P < 0.05) for sequential reaction assisted by US, obtaining the best production of GOS (49%) and GA (28%) after 2 h of reaction. The conformational and residual activity changes of enzymes under US conditions were also evaluated, Gox being positively affected whereas in β-gal hardly any change was found. Copyright © 2018 Elsevier B.V. All rights reserved.

Genetic instability of an oligomycin resistance mutation in yeast is associated with an amplification of a mitochondrial DNA segment.

PubMed Central

Ragnini, A; Fukuhara, H

1989-01-01

In the yeast Kluyveromyces lactis, mutations affecting mitochondrial functions are often highly unstable. In order to understand the basis of this genetic instability, we examined the case of an oligomycin resistant mutant. When the mutant was grown in the absence of the drug, the resistance was rapidly lost. This character showed a typical cytoplasmic inheritance. The unstable resistance was found to be associated with the presence of a repetitive DNA in which the repeating unit was a specific segment of the mitochondrial DNA. The amplified molecules were co-replicating with the wild type genome in the mutant cells. The spontaneous loss of the drug resistance was accompanied by the disappearance of the amplified DNA. The repetitive sequence came from a 405 base-pair segment immediately downstream of a cluster of two transfer RNA genes (threonyl 2 and glutamyl). Modified processing of these tRNAs was detected in the mutant. A possible mechanism by which these events could lead to drug resistance is discussed. Images PMID:2780315

Synthetic biology and molecular genetics in non-conventional yeasts: Current tools and future advances.

PubMed

Wagner, James M; Alper, Hal S

2016-04-01

Coupling the tools of synthetic biology with traditional molecular genetic techniques can enable the rapid prototyping and optimization of yeast strains. While the era of yeast synthetic biology began in the well-characterized model organism Saccharomyces cerevisiae, it is swiftly expanding to include non-conventional yeast production systems such as Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. These yeasts already have roles in the manufacture of vaccines, therapeutic proteins, food additives, and biorenewable chemicals, but recent synthetic biology advances have the potential to greatly expand and diversify their impact on biotechnology. In this review, we summarize the development of synthetic biological tools (including promoters and terminators) and enabling molecular genetics approaches that have been applied in these four promising alternative biomanufacturing platforms. An emphasis is placed on synthetic parts and genome editing tools. Finally, we discuss examples of synthetic tools developed in other organisms that can be adapted or optimized for these hosts in the near future. Copyright © 2015 Elsevier Inc. All rights reserved.

Biocidal Inactivation of Lactococcus lactis Bacteriophages: Efficacy and Targets of Commonly Used Sanitizers

PubMed Central

Hayes, Stephen; Murphy, James; Mahony, Jennifer; Lugli, Gabriele A.; Ventura, Marco; Noben, Jean-Paul; Franz, Charles M. A. P.; Neve, Horst; Nauta, Arjen; Van Sinderen, Douwe

2017-01-01

Lactococcus lactis strains, being intensely used in the dairy industry, are particularly vulnerable to members of the so-called 936 group of phages. Sanitization and disinfection using purpose-made biocidal solutions is a critical step in controlling phage contamination in such dairy processing plants. The susceptibility of 36 936 group phages to biocidal treatments was examined using 14 biocides and commercially available sanitizers. The targets of a number of these biocides were investigated by means of electron microscopic and proteomic analyses. The results from this study highlight significant variations in phage resistance to biocides among 936 phages. Furthermore, rather than possessing resistance to specific biocides or biocide types, biocide-resistant phages tend to possess a broad tolerance to multiple classes of antimicrobial compounds. PMID:28210242

Controlled production of Camembert-type cheeses. Part I: Microbiological and physicochemical evolutions.

PubMed

Leclercq-Perlat, Marie-Noëlle; Buono, Frédéric; Lambert, Denis; Latrille, Eric; Spinnler, Henry-Eric; Corrieu, Georges

2004-08-01

A holistic approach of a mould cheese ripening is presented. The objective was to establish relationships between the different microbiological and biochemical changes during cheese ripening. Model cheeses were prepared from pasteurized milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti and Brevibacterium linens under aseptic conditions. Two cheese-making trials with efficient control of environmental parameters were carried out and showed similar ripening characteristics. K. lactis grew rapidly between days 1 and 6 (generation time around 48 h). G. candidum grew exponentially between days 4 and 10 (generation time around 4.6 d). Brevi. linens also grew exponentially but after day 6 when Pen. camemberti mycelium began developing and the pH of the rind was close to 7. Its exponential growth presented 3 phases in relation to carbon and nitrogen substrate availability. Concentrations of Pen. camemberti mycelium were not followed by viable cell count but they were evaluated visually. The viable microorganism concentrations were well correlated with the carbon substrate concentrations in the core and in the rind. The lactose concentrations were negligible after 10 d ripening, and changes in lactate quantities were correlated with fungi flora. The pH of the inner part depended on NH3. Surface pH was significantly related to NH3 concentration and to fungi growth. The acid-soluble nitrogen (ASN) and non-protein nitrogen (NPN) indexes and NH3 concentrations of the rind were low until day 6, and then increased rapidly to follow the fungi concentrations until day 45. The ASN and NPN indexes and NH3 concentrations in the core were lower than in the rind and they showed the same evolution. G. candidum and Pen. camemberti populations have a major effect on proteolysis; nevertheless, K. lactis and Brevi. linens cell lysis also had an impact on proteolysis. Viable cell counts of K. lactis, G. candidum, Pen. camemberti and Brevi. linens were

Predominant genera of fecal microbiota in children with atopic dermatitis are not altered by intake of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bi-07.

PubMed

Larsen, Nadja; Vogensen, Finn K; Gøbel, Rikke; Michaelsen, Kim F; Abu Al-Soud, Waleed; Sørensen, Søren J; Hansen, Lars H; Jakobsen, Mogens

2011-03-01

The effect of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 on the composition of the Lactobacillus group, Bifidobacterium and the total bacterial population in feces from young children with atopic dermatitis was investigated. The study included 50 children randomized to intake of one of the probiotic strain or placebo. Microbial composition was characterized by denaturing gradient gel electrophoresis, quantitative PCR and, in a subset of subjects, by pyrosequencing of the 16S rRNA gene. The core population of the Lactobacillus group was identified as Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus oris, Leuconostoc mesenteroides, while the bifidobacterial community included Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium catenulatum. The fecal numbers of L. acidophilus and B. lactis increased significantly after intervention, indicating survival of the ingested bacteria. The levels of Bifidobacterium correlated positively (P=0.03), while the levels of the Lactobacillus group negatively (P=0.01) with improvement of atopic eczema evaluated by the Severity Scoring of Atopic Dermatitis index. This correlation was observed across the whole study cohort and not attributed to the probiotic intake. The main conclusion of the study is that administration of L. acidophilus NCFM and B. lactis Bi-07 does not affect the composition and diversity of the main bacterial populations in feces. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism.

PubMed Central

Crow, V L; Davey, G P; Pearce, L E; Thomas, T D

1983-01-01

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway. Images PMID:6294064

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies

Engineering of EPA/DHA omega-3 fatty acid production by Lactococcus lactis subsp. cremoris MG1363.

PubMed

Amiri-Jami, Mitra; Lapointe, Gisele; Griffiths, Mansel W

2014-04-01

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be of major importance in human health. Therefore, these essential polyunsaturated fatty acids have received considerable attention in both human and farm animal nutrition. Currently, fish and fish oils are the main dietary sources of EPA/DHA. To generate sustainable novel sources for EPA and DHA, the 35-kb EPA/DHA synthesis gene cluster was isolated from a marine bacterium, Shewanella baltica MAC1. To streamline the introduction of the genes into food-grade microorganisms such as lactic acid bacteria, unnecessary genes located upstream and downstream of the EPA/DHA gene cluster were deleted. Recombinant Escherichia coli harboring the 20-kb gene cluster produced 3.5- to 6.1-fold more EPA than those carrying the 35-kb DNA fragment coding for EPA/DHA synthesis. The 20-kb EPA/DHA gene cluster was cloned into a modified broad-host-range low copy number vector, pIL252m (4.7 kb, Ery) and expressed in Lactococcus lactis subsp. cremoris MG1363. Recombinant L. lactis produced DHA (1.35 ± 0.5 mg g(-1) cell dry weight) and EPA (0.12 ± 0.04 mg g(-1) cell dry weight). This is believed to be the first successful cloning and expression of EPA/DHA synthesis gene cluster in lactic acid bacteria. Our findings advance the future use of EPA/DHA-producing lactic acid bacteria in such applications as dairy starters, silage adjuncts, and animal feed supplements.

Elucidation of new condition-dependent roles for fructose-1,6-bisphosphatase linked to cofactor balances

PubMed Central

Kilian, Stephanus G.; du Preez, James C.

2017-01-01

The cofactor balances in metabolism is of paramount importance in the design of a metabolic engineering strategy and understanding the regulation of metabolism in general. ATP, NAD+ and NADP+ balances are central players linking the various fluxes in central metabolism as well as biomass formation. NADP+ is especially important in the metabolic engineering of yeasts for xylose fermentation, since NADPH is required by most yeasts in the initial step of xylose utilisation, including the fast-growing Kluyveromyces marxianus. In this simulation study of yeast metabolism, the complex interplay between these cofactors was investigated; in particular, how they may affect the possible roles of fructose-1,6-bisphosphatase, the pentose phosphate pathway, glycerol production and the pyruvate dehydrogenase bypass. Using flux balance analysis, it was found that the potential role of fructose-1,6-bisphosphatase was highly dependent on the cofactor specificity of the oxidative pentose phosphate pathway and on the carbon source. Additionally, the excessive production of ATP under certain conditions might be involved in some of the phenomena observed, which may have been overlooked to date. Based on these findings, a strategy is proposed for the metabolic engineering of a future xylose-fermenting yeast for biofuel production. PMID:28542187

Selection of enhanced antimicrobial activity posing lactic acid bacteria characterised by (GTG)5-PCR fingerprinting.

PubMed

Šalomskienė, Joana; Abraitienė, Asta; Jonkuvienė, Dovilė; Mačionienė, Irena; Repečkienė, Jūratė

2015-07-01

The aim of the study was a detail evaluation of genetic diversity among the lactic acid bacteria (LAB) strains having an advantage of a starter culture in order to select genotypically diverse strains with enhanced antimicrobial effect on some harmfull and pathogenic microorganisms. Antimicrobial activity of LAB was performed by the agar well diffusion method and was examined against the reference strains and foodborne isolates of Bacillus cereus, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium. Antifungal activity was tested against the foodborne isolates of Candida parapsilosis, Debaromyces hansenii, Kluyveromyces marxianus, Pichia guilliermondii, Yarowia lipolytica, Aspergillus brasiliensis, Aspergillus versicolor, Cladosporium herbarum, Penicillium chrysogenum and Scopulariopsis brevicaulis. A total 40 LAB strains representing Lactobacillus (23 strains), Lactococcus (13 strains) and Streptococcus spp. (4 strains) were characterised by repetitive sequence based polymerase chain reaction fingerprinting which generated highly discriminatory profiles, confirmed the identity and revealed high genotypic heterogeneity among the strains. Many of tested LAB demonstrated strong antimicrobial activity specialised against one or few indicator strains. Twelve LAB strains were superior in suppressing growth of the whole complex of pathogenic bacteria and fungi. These results demonstrated that separate taxonomic units offered different possibilities of selection for novel LAB strains could be used as starter cultures enhancing food preservation.

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

PubMed Central

Zadravec, Petra; Štrukelj, Borut

2015-01-01

Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities. PMID:25576617

Analysis of volatile compounds produced by 2 strains of Lactococcus lactis isolated from leben (Tunisian fermented milk) using solid-phase microextraction-gas chromatography.

PubMed

Ziadi, M; Wathelet, J P; Marlier, M; Hamdi, M; Thonart, P

2008-08-01

The volatile compounds that characterize Leben during fermentation with 2 Lactococcus lactis strains (SLT6 and SLT10) in flasks, in a 100-L fermentor, and during storage at 4 degrees C, were investigated and compared to those from commercial Leben. Volatile compounds from Leben were concentrated by a Carboxen-PDMS fiber and analyzed by GC-MS. These compounds include acids, alcohols, aldehydes, ketones, sulfur compounds, and hydrocarbons. Commercial Leben presented a poor volatile profile compared to the laboratory-made Leben. The mixed culture of 2 Lactococcus lactis strains resulted in higher volatile compound formation than the single strain culture. The GC volatile profiles of Leben produced in flask and in the 100-L fermentor were similar. Changes in volatile compounds were observed during storage at 4 degrees C. The effect of culture conditions on production of volatiles by SLT6 strain was studied. Aeration (0.1 mL/min) and agitation enhanced the production of diacetyl, acetoin, 3-methylbutanal, and 3-methylbutanol. Fermentation at pH 5 had no effect on volatile production.

Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment.

PubMed

Qiao, Jian-Jun; Zhang, Yan-Fei; Sun, Li-Fan; Liu, Wei-Wei; Zhu, Hong-Ji; Zhang, Zhijun

2011-09-01

Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (3(4)) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120°C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40°C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.9×10(11)CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS. Copyright © 2011 Elsevier Ltd. All rights reserved.

Recombinant Invasive Lactococcus lactis Carrying a DNA Vaccine Coding the Ag85A Antigen Increases INF-γ, IL-6, and TNF-α Cytokines after Intranasal Immunization.

PubMed

Mancha-Agresti, Pamela; de Castro, Camila Prosperi; Dos Santos, Janete S C; Araujo, Maíra A; Pereira, Vanessa B; LeBlanc, Jean G; Leclercq, Sophie Y; Azevedo, Vasco

2017-01-01

Tuberculosis (TB) remains a major threat throughout the world and in 2015 it caused the death of 1.4 million people. The Bacillus Calmette-Guérin is the only existing vaccine against this ancient disease; however, it does not provide complete protection in adults. New vaccines against TB are eminently a global priority. The use of bacteria as vehicles for delivery of vaccine plasmids is a promising vaccination strategy. In this study, we evaluated the use of, an engineered invasive Lactococcus lactis (expressing Fibronectin-Binding Protein A from Staphylococcus aureus ) for the delivery of DNA plasmid to host cells, especially to the mucosal site as a new DNA vaccine against tuberculosis. One of the major antigens documented that offers protective responses against Mycobacterium tuberculosis is the Ag85A. L. lactis FnBPA + (pValac: Ag85A) which was obtained and used for intranasal immunization of C57BL/6 mice and the immune response profile was evaluated. In this study we observed that this strain was able to produce significant increases in the amount of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-6) in the stimulated spleen cell supernatants, showing a systemic T helper 1 (Th1) cell response. Antibody production (IgG and sIgA anti-Ag85A) was also significantly increased in bronchoalveolar lavage, as well as in the serum of mice. In summary, these findings open new perspectives in the area of mucosal DNA vaccine, against specific pathogens using a Lactic Acid Bacteria such as L. lactis.

Effect of yogurt containing Bifidobacterium animalis subsp . lactis DN-173010 probiotic on dental plaque and saliva in orthodontic patients.

PubMed

Pinto, G S; Cenci, M S; Azevedo, M S; Epifanio, M; Jones, M H

2014-01-01

To assess how consumption of yogurt containing Bifidobacterium animalis subsp. lactis DN-173010 probiotic for a period of 2 weeks affects salivary and dental plaque levels of mutans streptococci and lactobacilli in patients undergoing orthodontic treatment. A crossover, double-blind, randomized and placebo-controlled clinical trial was performed with 26 volunteers. The study was divided into four periods. During periods 2 and 4, the volunteers ingested yogurt containing probiotic or control yogurt daily for 2 weeks. Periods 1 and 3 were a 1-week run-in period and 4-week washout period, respectively. Saliva and dental plaque samples were collected from each participant at the end of each period. Mutans streptococci, lactobacilli, and total cultivable microorganisms were counted. Values were compared between groups and across periods with the Wilcoxon's test. There was no difference between the yogurt containing probiotic and the control yogurt for any of the studied variables (all p > 0.05). A reduction in counts of total cultivable microorganisms was observed in dental plaque samples after ingestion of either yogurts (both p < 0.05 vs. baseline), but not in saliva (p < 0.05). Daily ingestion of yogurt with or without B. animalis subsp. lactis for a period of 2 weeks was beneficial in reducing total microbial counts in dental plaque. Therefore, no additional benefits were achieved by the use of the tested probiotic strain.

Spatial Distribution of Lactococcus lactis Colonies Modulates the Production of Major Metabolites during the Ripening of a Model Cheese.

PubMed

Le Boucher, Clémentine; Gagnaire, Valérie; Briard-Bion, Valérie; Jardin, Julien; Maillard, Marie-Bernadette; Dervilly-Pinel, Gaud; Le Bizec, Bruno; Lortal, Sylvie; Jeanson, Sophie; Thierry, Anne

2016-01-01

In cheese, lactic acid bacteria are immobilized at the coagulation step and grow as colonies. The spatial distribution of bacterial colonies is characterized by the size and number of colonies for a given bacterial population within cheese. Our objective was to demonstrate that different spatial distributions, which lead to differences in the exchange surface between the colonies and the cheese matrix, can influence the ripening process. The strategy was to generate cheeses with the same growth and acidification of a Lactococcus lactis strain with two different spatial distributions, big and small colonies, to monitor the production of the major ripening metabolites, including sugars, organic acids, peptides, free amino acids, and volatile metabolites, over 1 month of ripening. The monitored metabolites were qualitatively the same for both cheeses, but many of them were more abundant in the small-colony cheeses than in the big-colony cheeses over 1 month of ripening. Therefore, the results obtained showed that two different spatial distributions of L. lactis modulated the ripening time course by generating moderate but significant differences in the rates of production or consumption for many of the metabolites commonly monitored throughout ripening. The present work further explores the immobilization of bacteria as colonies within cheese and highlights the consequences of this immobilization on cheese ripening. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Fine Structure of Tibetan Kefir Grains and Their Yeast Distribution, Diversity, and Shift

PubMed Central

Lu, Man; Wang, Xingxing; Sun, Guowei; Qin, Bing; Xiao, Jinzhou; Yan, Shuling; Pan, Yingjie; Wang, Yongjie

2014-01-01

Tibetan kefir grains (TKGs), a kind of natural starter for fermented milk in Tibet, China, host various microorganisms of lactic acid bacteria, yeasts, and occasionally acetic acid bacteria in a polysaccharide/protein matrix. In the present study, the fine structure of TKGs was studied to shed light on this unusual symbiosis with stereomicroscopy and thin sections. The results reveal that TKGs consist of numerous small grain units, which are characterized by a hollow globular structure with a diameter between 2.0 and 9.0 mm and a wall thickness of approximately 200 µm. A polyhedron-like net structure, formed mainly by the bacteria, was observed in the wall of the grain units, which has not been reported previously to our knowledge. Towards the inside of the grain unit, the polyhedron-like net structures became gradually larger in diameter and fewer in number. Such fine structures may play a crucial role in the stability of the grains. Subsequently, the distribution, diversity, and shift of yeasts in TKGs were investigated based on thin section, scanning electron microscopy, cloning and sequencing of D1/D2 of the 26S rRNA gene, real-time quantitative PCR, and in situ hybridization with specific fluorescence-labeled oligonucleotide probes. These show that (i) yeasts appear to localize on the outer surface of the grains and grow normally together to form colonies embedded in the bacterial community; (ii) the diversity of yeasts is relatively low on genus level with three dominant species – Saccharomyces cerevisiae, Kluyveromyces marxianus, and Yarrowia lipolytica; (iii) S. cerevisiae is the stable predominant yeast species, while the composition of Kluyveromyces and Yarrowia are subject to change over time. Our results indicate that TKGs are relatively stable in structure, and culture conditions to some extent shape the microbial community and interaction in kefir grains. These findings pave the way for further study of the specific symbiotic associations between S

Some Factors Influencing Acid Production by an Oxytetracycline-Resistant Strain of Streptococcus lactis1

PubMed Central

Mikolajcik, E. M.; Harper, W. J.; Gould, I. A.

1963-01-01

Induction of oxytetracycline resistance in a strain of Streptococcus lactis caused this organism to display reduced acid production, salt tolerance, pyruvate synthesis, growth at alkaline pH, and a loss in ability to produce ammonia from arginine. α-Ketoglutaric and oxaloacetic acids were found to accumulate in the growth medium of resistant cells, in contrast to none in the medium of susceptible cells. No free arginine could be detected in the intracellular fraction of resistant cells, but arginine was present in the intracellular fraction of susceptible cells and decreased in concentration upon the addition of oxytetracycline to the growth medium. Depressed acid production in milk by the oxytetracycline resistant strain is evidently a consequence of the inability of this organism to metabolize arginine effectively. PMID:14063784

Novel conjugative plasmids from the natural isolate Lactococcus lactis subspecies cremoris DPC3758: a repository of genes for the potential improvement of dairy starters.

PubMed

Fallico, V; Ross, R P; Fitzgerald, G F; McAuliffe, O

2012-07-01

A collection of 17 natural lactococcal isolates from raw milk cheeses were studied in terms of their plasmid distribution, content, and diversity. All strains in the collection harbored an abundance of plasmids, including Lactococcus lactis ssp. cremoris DPC3758, whose 8-plasmid complement was selected for sequencing. The complete sequences of pAF22 (22,388 kb), pAF14 (14,419 kb), pAF12 (12,067 kb), pAF07 (7,435 kb), and pAF04 (3,801 kb) were obtained, whereas gene functions of technological interest were mapped to pAF65 (65 kb) and pAF45 (45 kb) by PCR. The plasmids of L. lactis DPC3758 were found to encode many genes with the potential to improve the technological properties of dairy starters. These included 3 anti-phage restriction/modification (R/M) systems (1 of type I and 2 of type II) and genes for immunity/resistance to nisin, lacticin 481, cadmium, and copper. Regions encoding conjugative/mobilization functions were present in 6 of the 8 plasmids, including those containing the R/M systems, thus enabling the food-grade transfer of these mechanisms to industrial strains. Using cadmium selection, the sequential stacking of the R/M plasmids into a plasmid-free host provided the recipient with increased protection against 936- and c2-type phages. The association of food-grade selectable markers and mobilization functions on L. lactis DPC3758 plasmids will facilitate their exploitation to obtain industrial strains with enhanced phage protection and robustness. These natural plasmids also provide another example of the major role of plasmids in contributing to host fitness and preservation within its ecological niche. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Improved Acid Stress Survival of Lactococcus lactis Expressing the Histidine Decarboxylation Pathway of Streptococcus thermophilus CHCC1524*

PubMed Central

Trip, Hein; Mulder, Niels L.; Lolkema, Juke S.

2012-01-01

Degradative amino acid decarboxylation pathways in bacteria generate secondary metabolic energy and provide resistance against acid stress. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 was functionally expressed in the heterologous host Lactococcus lactis NZ9000, and the benefits of the newly acquired pathway for the host were analyzed. During growth in M17 medium in the pH range of 5–6.5, a small positive effect was observed on the biomass yield in batch culture, whereas no growth rate enhancement was evident. In contrast, a strong benefit for the engineered L. lactis strain was observed in acid stress survival. In the presence of histidine, the pathway enabled cells to survive at pH values as low as 3 for at least 2 h, conditions under which the host cells were rapidly dying. The flux through the histidine decarboxylation pathway in cells grown at physiological pH was under strict control of the electrochemical proton gradient (pmf) across the membrane. Ionophores that dissipated the membrane potential (ΔΨ) and/or the pH gradient (ΔpH) strongly increased the flux, whereas the presence of glucose almost completely inhibited the flux. Control of the pmf over the flux was exerted by both ΔΨ and ΔpH and was distributed over the transporter HdcP and the decarboxylase HdcA. The control allowed for a synergistic effect between the histidine decarboxylation and glycolytic pathways in acid stress survival. In a narrow pH range around 2.5 the synergism resulted in a 10-fold higher survival rate. PMID:22351775

Bifidobacterium animalis subspecies lactis modulates the local immune response and glucose uptake in the small intestine of juvenile pigs infected with the parasitic nematode Ascaris suum

USDA-ARS?s Scientific Manuscript database

The probiotic bacteria Bifidobacterium animalis subspecies lactis (Bb12) or a placebo containing vehicle without Bb12 was administered orally to pregnant sows during the last trimester of pregnancy, and to their offspring from birth through the termination of the study three months later. Weaned-pig...

Use of Taiwanese ropy fermented milk (TRFM) and Lactococcus lactis subsp. cremoris isolated from TRFM in manufacturing of functional low-fat cheeses.

PubMed

Chiang, Ming-Lun; Chen, Hsi-Chia; Wang, Sheng-Yao; Hsieh, Yueh-Ling; Chen, Ming-Ju

2011-09-01

The purpose of this study was to manufacture new functional low-fat cheeses using Taiwanese ropy fermented milk (TRFM) and Lactococcus lactis subsp. cremoris strains isolated from TRFM. After 28 d of ripening and storage, the viable populations of lactic acid bacteria (LAB) in the low-fat cheeses made with L. lactis subsp. cremoris TL1 (TL1), L. lactis subsp. cremoris TL4 (TL4), and TRFM still maintained above 10(8) CFU/g. The low-fat cheeses made with TL1 and TRFM showed higher moisture contents than the cheeses made with TL4, full-fat, and low-fat cheese controls. The low-fat cheeses made with TL1 and TL4 had higher customer preferential scores similar to full-fat cheese control in the sensory evaluation. Additionally, the low-fat cheeses fermented with TL1, TL4, and TRFM for 4 h had higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging and ferrous ion-chelating abilities than the cheeses fermented with the starters for 8 h, full-fat, and low-fat cheese controls. A better angiotensin-converting enzyme (ACE) inhibition activity was also observed in the low-fat cheeses made with TL1, TL4, and TRFM than that in the full-fat and low-fat cheese controls during ripening and storage period. As health-conscious consumers continue to seek low-fat alternatives in their diets, there remain strong interests for the dairy industry to develop low-fat cheeses to meet the demands. This study clearly demonstrated that the low-fat cheeses fermented with TL1 for 4 h showed a better overall acceptability and possessed antioxidative abilities and ACE inhibitory activities than other cheeses tested in this study. By improving its flavor and investigating the possible mechanisms of its functionalities in the future, this low-fat cheese might possibly be commercialized and give a positive impact on cheese consumption in the future. © 2011 Institute of Food Technologists®

Lactococcus lactis carrying the pValac eukaryotic expression vector coding for IL-4 reduces chemically-induced intestinal inflammation by increasing the levels of IL-10-producing regulatory cells.

PubMed

Souza, Bianca Mendes; Preisser, Tatiane Melo; Pereira, Vanessa Bastos; Zurita-Turk, Meritxell; de Castro, Camila Prósperi; da Cunha, Vanessa Pecini; de Oliveira, Rafael Pires; Gomes-Santos, Ana Cristina; de Faria, Ana Maria Caetano; Machado, Denise Carmona Cara; Chatel, Jean-Marc; Azevedo, Vasco Ariston de Carvalho; Langella, Philippe; Miyoshi, Anderson

2016-08-30

Inflammatory bowel diseases are characterized by chronic intestinal inflammation that leads to severe destruction of the intestinal mucosa. Therefore, the understanding of their aetiology as well as the development of new medicines is an important step for the treatment of such diseases. Consequently, the development of Lactococcus lactis strains capable of delivering a eukaryotic expression vector encoding the interleukin 4 (IL-4) of Mus musculus would represent a new strategy for the elaboration of a more effective alternative therapy against Crohn's disease. The murine IL-4 ORF was cloned into the eukaryotic expression vector pValac::dts. The resulting plasmid-pValac::dts::IL-4-was transfected into CHO cells so that its functionality could be evaluated in vitro. With fluorescent confocal microscopy, flow cytometry and ELISA, it was observed that pValac::dts::IL-4-transfected cells produced IL-4, while non-transfected cells and cells transfected with the empty vector did not. Then, pValac::dts::IL-4 was inserted into L. lactis MG1363 FnBPA(+) in order to evaluate the therapeutic potential of the recombinant strain against TNBS-induced colitis. Intragastric administration of L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) was able to decrease the severity of colitis, with animals showing decreased levels of IL-12, IL-6 and MPO activity; and increased levels of IL-4 and IL-10. Finally, LP-isolated cells from mice administered TNBS were immunophenotyped so that the main IL-4 and IL-10 producers were identified. Mice administered the recombinant strain presented significantly higher percentages of F4/80(+)MHCII(+)Ly6C(-)IL-4(+), F4/80(+)MHCII(+)Ly6C(-)IL-10(+), F4/80(+)MHCII(+)Ly6C(-)CD206(+)CD124(+)IL-10(+) and CD4(+)Foxp3(+)IL10(+) cells compared to the other groups. This study shows that L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) is a good candidate to maintain the anti-inflammatory and proinflammatory balance in the gastrointestinal tract, increasing the levels

Dynamic modeling of lactic acid fermentation metabolism with Lactococcus lactis.

PubMed

Oh, Euhlim; Lu, Mingshou; Park, Changhun; Park, Changhun; Oh, Han Bin; Lee, Sang Yup; Lee, Jinwon

2011-02-01

A dynamic model of lactic acid fermentation using Lactococcus lactis was constructed, and a metabolic flux analysis (MFA) and metabolic control analysis (MCA) were performed to reveal an intensive metabolic understanding of lactic acid bacteria (LAB). The parameter estimation was conducted with COPASI software to construct a more accurate metabolic model. The experimental data used in the parameter estimation were obtained from an LC-MS/ MS analysis and time-course simulation study. The MFA results were a reasonable explanation of the experimental data. Through the parameter estimation, the metabolic system of lactic acid bacteria can be thoroughly understood through comparisons with the original parameters. The coefficients derived from the MCA indicated that the reaction rate of L-lactate dehydrogenase was activated by fructose 1,6-bisphosphate and pyruvate, and pyruvate appeared to be a stronger activator of L-lactate dehydrogenase than fructose 1,6-bisphosphate. Additionally, pyruvate acted as an inhibitor to pyruvate kinase and the phosphotransferase system. Glucose 6-phosphate and phosphoenolpyruvate showed activation effects on pyruvate kinase. Hexose transporter was the strongest effector on the flux through L-lactate dehydrogenase. The concentration control coefficient (CCC) showed similar results to the flux control coefficient (FCC).

Safety evaluation of Lactobacillus delbrueckii subsp. lactis UO 004, a probiotic bacterium.

PubMed

Fernández, M Fernanda; Boris, Soledad; Barbés, Covadonga

2005-03-01

Lactobacillus delbrueckii subsp. lactis UO 004 was evaluated for its use as a potential probiotic from a safety point of view. The strain did not exhibit mucinolytic or other enzymatic activities that might be detrimental, such as those involving glycosidases (beta-D-glucosaminidase or alpha-D-galactosidase) or arylamidases (factor Xa and quimotrypsin-like activities), frequently present in Lactobacillus strains isolated from patients with endocarditis, although it was able to express protein Ca and kallikrein-like activities. On the other hand, the presence of the strain did not interfere with the growth of certain species of normal intestinal microbiota, such as Enterococcus fecalis, Escherichia coli, Bifidobacterium bifidum or Bacteroides fragilis. Moreover, the potential probiotic strain UO 004 is sensitive to antibiotics with transmissible resistance mechanisms in Lactobacillus such as chloramphenicol, erythromycin, tetracycline and vancomycin. In addition, strain L. delbrueckii UO 004 was not able to translocate towards the intestinal barrier of mice or produce changes in their activity or general health status.

Survival of cheese-ripening microorganisms in a dynamic simulator of the gastrointestinal tract.

PubMed

Adouard, Nadège; Magne, Laurent; Cattenoz, Thomas; Guillemin, Hervé; Foligné, Benoît; Picque, Daniel; Bonnarme, Pascal

2016-02-01

A mixture of nine microorganisms (six bacteria and three yeasts) from the microflora of surface-ripened cheeses were subjected to in vitro digestive stress in a three-compartment "dynamic gastrointestinal digester" (DIDGI). We studied the microorganisms (i) grown separately in culture medium only (ii) grown separately in culture medium and then mixed, (iii) grown separately in culture medium and then included in a rennet gel and (iv) grown together in smear-ripened cheese. The yeasts Geotrichum candidum, Kluyveromyces lactis and Debaryomyces hansenii, were strongly resistant to the whole DIDGI process (with a drop in viable cell counts of less than <1 log CFU mL(-1)) and there were no significant differences between lab cultures and cheese-grown cultures. Ripening bacteria such as Hafnia alvei survived gastric stress less well when grown in cheese (with no viable cells after 90 min of exposure of the cheese matrix, compared with 6 CFU mL(-1) in lab cultures). The ability of Corynebacterium casei and Staphylococcus equorum to withstand digestive stress was similar for cheese and pure culture conditions. When grow in a cheese matrix, Brevibacterium aurantiacum and Arthrobacter arilaitensis were clearly more sensitive to the overall digestive process than when grown in pure cultures. Lactococcus lactis displayed poorer survival in gastric and duodenal compartments when it had been grown in cheese. In vivo experiments in BALB/c mice agreed with the DIDGI experiments and confirmed the latter's reliability. Copyright © 2015. Published by Elsevier Ltd.

Biodiversity of antifungal lactic acid bacteria isolated from raw milk samples from cow, ewe and goat over one-year period.

PubMed

Delavenne, E; Mounier, J; Déniel, F; Barbier, G; Le Blay, G

2012-04-16

Antifungal lactic acid bacteria (ALAB) biodiversity was evaluated in raw milk from ewe, cow and goat over one year period. Lactic acid bacteria were enumerated using 8 semi-selective media, and systematically screened for their antifungal activity against 4 spoilage fungi commonly encountered in dairy products. Depending on the selective medium, between 0.05% (Elliker agar) and 5.5% (LAMVAB agar) screened colonies showed an antifungal activity. The great majority of these active colonies originated from cow (49%) and goat (43%) milks, whereas only 8% were isolated from ewe milk. Penicillium expansum was the most frequently inhibited fungus with 48.5% of colonies active against P. expansum among the 1235 isolated, followed by Mucor plumbeus with 30.6% of active colonies, Kluyveromyces lactis with only 12.1% of active colonies and Pichia anomala with 8.7% of active colonies. In the tested conditions, 94% of the sequenced active colonies belonged to Lactobacillus. Among them, targeted fungal species differed according to the Lactobacillus group, whose presence largely depended on year period and milk origin. The Lb. casei and Lb. reuteri groups, predominantly recovered in summer/fall, were overrepresented in the population targeting M. plumbeus, whereas isolates from the Lb. plantarum group, predominantly recovered in spring, were overrepresented in the population targeting K. lactis, the ones belonging to the Lb. buchneri group, predominantly recovered in spring, were overrepresented in the population targeting P. anomala. Raw milk, especially cow and goat milks from the summer/fall period appeared to be a productive reservoir for antifungal lactobacilli. Copyright © 2012 Elsevier B.V. All rights reserved.

Engineering a cardosin B-derived rennet for sheep and goat cheese manufacture.

PubMed

Almeida, Carla Malaquias; Gomes, David; Faro, Carlos; Simões, Isaura

2015-01-01

Different sheep and goat cheeses with world-renowned excellence are produced using aqueous extracts of Cynara cardunculus flowers as coagulants. However, the use of this vegetable rennet is mostly limited to artisanal scale production, and no effective solutions to large-scale industrial applications have been reported so far. In this sense, the development of a synthetic rennet based on the most abundant cardoon milk-clotting enzymes (cardosins) would emerge as a solution for scalability of production and for application of these proteases as alternative rennets in dairy industry. In this work, we report the development of a new cardosin B-derived rennet produced in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. Using a stepwise optimization strategy-consisting of culture media screening, complemented with a protein engineering approach with removal of the plant-specific domain, and a codon optimization step-we successfully improved cardosin B production yield (35×) in K. lactis. We demonstrated that the secreted enzyme displays similar proteolytic properties, such as casein digestion profiles as well as optimum pH (pH 4.5) and temperature (40 °C), with those of native cardosin B. From this optimization process resulted the rennet preparation Vegetable Rennet (VRen), requiring no downstream protein purification steps. The effectiveness of VRen in cheese production was demonstrated by manufacturing sheep, goat, and cow cheeses. Interestingly, the use of VRen resulted in a higher cheese yield for all three types of cheese when compared with synthetic chymosin. Altogether, these results clearly position VRen as an alternative/innovative coagulant for the cheese-making industry.

Transcriptional responses in Lactococcus lactis subsp. cremoris to the changes in oxygen and redox potential during milk acidification.

PubMed

Larsen, N; Brøsted Werner, B; Jespersen, L

2016-08-01

Milk acidification and metabolic activity of the starter cultures are affected by oxygen; however, molecular factors related to the redox changes are poorly defined. The objective of the study was to investigate transcriptional responses in Lactococcus lactis subsp. cremoris CHCCO2 grown in milk to the shifts of oxygen and redox potential (Eh7 ). Transcriptomic studies were performed with the use of Illumina HiSeq 2000 mRNA sequencing and validated by the real-time quantitative PCR. In total 105 differentially expressed genes were assigned functional gene names. Most of the differentially expressed genes were detected during aerobic reduction phase. Upregulated genes were implicated in lactose utilization, glycogen biosynthesis, amino sugar metabolism, oxidation-reduction, pyrimidine biosynthesis and DNA integration processes. Genes of purine nucleotide biosynthesis and genes encoding amino acid, multidrug resistance and ion ABC transporters were mostly downregulated, while oligopeptide transporter genes were reduced during oxygen depletion and induced at minimum Eh7 . Understanding of gene responses in starter cultures to the changes of oxidation-reduction state is important for the better control and reproducibility of dairy fermentations. We applied mRNA sequencing by Illumina HiSeq 2000 to investigate gene expression profile in a dairy strain of Lactococcus lactis subsp. cremoris during milk acidification. Novelty of this study lies in linking transcriptional responses to oxygen depletion and the changes of redox potential with the fermentation kinetics and clarification of molecular factors specifically expressed in milk which might be essential for bacterial performance and the final quality of cheeses. © 2016 The Society for Applied Microbiology.

In Vitro Assessment of the Probiotic Potential of Lactococcus lactis LMG 7930 against Ruminant Mastitis-Causing Pathogens.

PubMed

Armas, Federica; Camperio, Cristina; Marianelli, Cinzia

2017-01-01

Mastitis in dairy ruminants is considered to be the most expensive disease to farmers worldwide. Recently, the intramammary infusion of lactic acid bacteria has emerged as a potential new alternative to antibiotics for preventing and treating bovine mastitis. In this study we have investigated in vitro the probiotic potential of Lactococcus lactis LMG 7930, a food-grade and nisin-producing strain, against mastitis-causing pathogens. We have characterized its carbohydrate fermentation and antibiotic susceptibility profiles, cell surface properties and antimicrobial activity, as well as its capabilities to adhere to and inhibit the invasion of pathogens into the bovine mammary epithelial cell line BME-UV1d. We found that L. lactis LMG 7930 was sensitive to tested drugs, according to the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), and showed an improved carbohydrate fermentation capacity compared to starter strains. Moreover, the strain exhibited antagonistic properties towards many of the pathogens tested. It presented medium surface hydrophobicity, a low basic property and no electron acceptor capability. It showed low auto-aggregation and no co-aggregation abilities towards any of the tested pathogens. The strain was one of the most adhesive to bovine mammary epithelial cells among tested bacteria, but its internalisation was low. The strain did not affect significantly pathogen invasion; however, a trend to decrease internalization of some pathogens tested was observed. In conclusion, our results suggest that this strain might be a promising candidate for the development of new strategies of mastitis control in ruminants. Future investigations are needed to evaluate its safety and efficacy under field conditions.

In Vitro Assessment of the Probiotic Potential of Lactococcus lactis LMG 7930 against Ruminant Mastitis-Causing Pathogens

PubMed Central

Armas, Federica; Camperio, Cristina

2017-01-01

Mastitis in dairy ruminants is considered to be the most expensive disease to farmers worldwide. Recently, the intramammary infusion of lactic acid bacteria has emerged as a potential new alternative to antibiotics for preventing and treating bovine mastitis. In this study we have investigated in vitro the probiotic potential of Lactococcus lactis LMG 7930, a food-grade and nisin-producing strain, against mastitis-causing pathogens. We have characterized its carbohydrate fermentation and antibiotic susceptibility profiles, cell surface properties and antimicrobial activity, as well as its capabilities to adhere to and inhibit the invasion of pathogens into the bovine mammary epithelial cell line BME-UV1d. We found that L. lactis LMG 7930 was sensitive to tested drugs, according to the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), and showed an improved carbohydrate fermentation capacity compared to starter strains. Moreover, the strain exhibited antagonistic properties towards many of the pathogens tested. It presented medium surface hydrophobicity, a low basic property and no electron acceptor capability. It showed low auto-aggregation and no co-aggregation abilities towards any of the tested pathogens. The strain was one of the most adhesive to bovine mammary epithelial cells among tested bacteria, but its internalisation was low. The strain did not affect significantly pathogen invasion; however, a trend to decrease internalization of some pathogens tested was observed. In conclusion, our results suggest that this strain might be a promising candidate for the development of new strategies of mastitis control in ruminants. Future investigations are needed to evaluate its safety and efficacy under field conditions. PMID:28068371

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells

PubMed Central

Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

2015-01-01

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest. PMID:26370773

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

PubMed

Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

2015-09-15

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest.

AguR, a Transmembrane Transcription Activator of the Putrescine Biosynthesis Operon in Lactococcus lactis, Acts in Response to the Agmatine Concentration.

PubMed

Linares, Daniel M; Del Rio, Beatriz; Redruello, Begoña; Ladero, Victor; Martin, M Cruz; de Jong, Anne; Kuipers, Oscar P; Fernandez, Maria; Alvarez, Miguel A

2015-09-01

Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

AguR, a Transmembrane Transcription Activator of the Putrescine Biosynthesis Operon in Lactococcus lactis, Acts in Response to the Agmatine Concentration

PubMed Central

Linares, Daniel M.; del Rio, Beatriz; Redruello, Begoña; Martin, M. Cruz; de Jong, Anne; Kuipers, Oscar P.; Fernandez, Maria

2015-01-01

Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins. PMID:26116671

Structure-function insights into direct lipid transfer between membranes by Mmm1-Mdm12 of ERMES.

PubMed

Kawano, Shin; Tamura, Yasushi; Kojima, Rieko; Bala, Siqin; Asai, Eri; Michel, Agnès H; Kornmann, Benoît; Riezman, Isabelle; Riezman, Howard; Sakae, Yoshitake; Okamoto, Yuko; Endo, Toshiya

2018-03-05

The endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES) physically links the membranes of the ER and mitochondria in yeast. Although the ER and mitochondria cooperate to synthesize glycerophospholipids, whether ERMES directly facilitates the lipid exchange between the two organelles remains controversial. Here, we compared the x-ray structures of an ERMES subunit Mdm12 from Kluyveromyces lactis with that of Mdm12 from Saccharomyces cerevisiae and found that both Mdm12 proteins possess a hydrophobic pocket for phospholipid binding. However in vitro lipid transfer assays showed that Mdm12 alone or an Mmm1 (another ERMES subunit) fusion protein exhibited only a weak lipid transfer activity between liposomes. In contrast, Mdm12 in a complex with Mmm1 mediated efficient lipid transfer between liposomes. Mutations in Mmm1 or Mdm12 impaired the lipid transfer activities of the Mdm12-Mmm1 complex and furthermore caused defective phosphatidylserine transport from the ER to mitochondrial membranes via ERMES in vitro. Therefore, the Mmm1-Mdm12 complex functions as a minimal unit that mediates lipid transfer between membranes. © 2018 Kawano et al.

DOE Office of Scientific and Technical Information (OSTI.GOV)

J Chang; S Xiang; K Xiang

The 5' {yields} 3' exoribonucleases (XRNs) have important functions in transcription, RNA metabolism and RNA interference. The structure of Rat1 (also known as Xrn2) showed that the two highly conserved regions of XRNs form a single, large domain that defines the active site of the enzyme. Xrn1 has a 510-residue segment after the conserved regions that is required for activity but is absent from Rat1/Xrn2. Here we report the crystal structures of Kluyveromyces lactis Xrn1 (residues 1-1,245, E178Q mutant), alone and in complex with a Mn{sup 2+} ion in the active site. The 510-residue segment contains four domains (D1-D4), locatedmore » far from the active site. Our mutagenesis and biochemical studies show that their functional importance results from their ability to stabilize the conformation of the N-terminal segment of Xrn1. These domains might also constitute a platform that interacts with protein partners of Xrn1.« less

Mitochondrial Genome Integrity Mutations Uncouple the Yeast Saccharomyces cerevisiae ATP Synthase*║

PubMed Central

Wang, Yamin; Singh, Usha; Mueller, David M.

2013-01-01

The mitochondrial ATP synthase is a molecular motor, which couples the flow of rotons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the α-, β-, and γ-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the γ-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk. PMID:17244612

Utilization of whey powder as substrate for low-cost preparation of β-galactosidase as main product, and ethanol as by-product, by a litre-scale integrated process.

PubMed

You, Shengping; Chang, Hongxing; Yin, Qingdian; Qi, Wei; Wang, Mengfan; Su, Rongxin; He, Zhimin

2017-12-01

Whey powder, a by-product of dairy industry, is an attractive raw material for value-added products. In this study, utilization of whey powder as substrate for low-cost preparation of β-galactosidase as main product and ethanol as by-product were investigated by a litre-scale integrated strategy, encompassing fermentation, isolation, permeabilization and spray drying. Firstly, through development of low-cost industrial culture and fed-batch strategies by Kluyveromyces lactis, 119.30U/mL β-galactosidase activity and 16.96mg/mL by-product ethanol were achieved. Afterward, an up-dated mathematic model for the recycling permeabilization was established successfully and 30.4g cells sediment isolated from 5L fermentation broth were permeabilized completely by distilled ethanol from broth supernatant. Then β-galactosidase product with 5.15U/mg from protection of gum acacia by spray drying was obtained. Furthermore, by-product ethanol with 31.08% (v/v) was achieved after permeabilization. Therefore, the integrated strategy using whey powder as substrate is a feasible candidate for industrial-scale implementation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Yeast Biomass Production in Brewery's Spent Grains Hemicellulosic Hydrolyzate

NASA Astrophysics Data System (ADS)

Duarte, Luís C.; Carvalheiro, Florbela; Lopes, Sónia; Neves, Ines; Gírio, Francisco M.

Yeast single-cell protein and yeast extract, in particular, are two products which have many feed, food, pharmaceutical, and biotechnological applications. However, many of these applications are limited by their market price. Specifically, the yeast extract requirements for culture media are one of the major technical hurdles to be overcome for the development of low-cost fermentation routes for several top value chemicals in a biorefinery framework. A potential biotechnical solution is the production of yeast biomass from the hemicellulosic fraction stream. The growth of three pentose-assimilating yeast cell factories, Debaryomyces hansenii, Kluyveromyces marxianus, and Pichia stipitis was compared using non-detoxified brewery's spent grains hemicellulosic hydrolyzate supplemented with mineral nutrients. The yeasts exhibited different specific growth rates, biomass productivities, and yields being D. hansenii as the yeast species that presented the best performance, assimilating all sugars and noteworthy consuming most of the hydrolyzate inhibitors. Under optimized conditions, D. hansenii displayed a maximum specific growth rate, biomass yield, and productivity of 0.34 h-1, 0.61 g g-1, and 0.56 g 1-1 h-1, respectively. The nutritional profile of D. hansenii was thoroughly evaluated, and it compares favorably to others reported in literature. It contains considerable amounts of some essential amino acids and a high ratio of unsaturated over saturated fatty acids.

Identification and Characterization of Oleaginous Yeast Isolated from Kefir and Its Ability to Accumulate Intracellular Fats in Deproteinated Potato Wastewater with Different Carbon Sources

PubMed Central

Kieliszek, Marek; Jermacz, Karolina; Błażejak, Stanisław

2017-01-01

The search for efficient oleaginous microorganisms, which can be an alternative to fossil fuels and biofuels obtained from oilseed crops, has been going on for many years. The suitability of microorganisms in this regard is determined by their ability to biosynthesize lipids with preferred fatty acid profile along with the concurrent utilization of energy-rich industrial waste. In this study, we isolated, characterized, and identified kefir yeast strains using molecular biology techniques. The yeast isolates identified were Candida inconspicua, Debaryomyces hansenii, Kluyveromyces marxianus, Kazachstania unispora, and Zygotorulaspora florentina. We showed that deproteinated potato wastewater, a starch processing industry waste, supplemented with various carbon sources, including lactose and glycerol, is a suitable medium for the growth of yeast, which allows an accumulation of over 20% of lipid substances in its cells. Fatty acid composition primarily depended on the yeast strain and the carbon source used, and, based on our results, most of the strains met the criteria required for the production of biodiesel. In particular, this concerns a significant share of saturated fatty acids, such as C16:0 and C18:0, and unsaturated fatty acids, such as C18:1 and C18:2. The highest efficiency in lipid biosynthesis exceeded 6.3 g L−1. Kazachstania unispora was able to accumulate the high amount of palmitoleic acid. PMID:29098157

Identification and Characterization of Oleaginous Yeast Isolated from Kefir and Its Ability to Accumulate Intracellular Fats in Deproteinated Potato Wastewater with Different Carbon Sources.

PubMed

Gientka, Iwona; Kieliszek, Marek; Jermacz, Karolina; Błażejak, Stanisław

2017-01-01

The search for efficient oleaginous microorganisms, which can be an alternative to fossil fuels and biofuels obtained from oilseed crops, has been going on for many years. The suitability of microorganisms in this regard is determined by their ability to biosynthesize lipids with preferred fatty acid profile along with the concurrent utilization of energy-rich industrial waste. In this study, we isolated, characterized, and identified kefir yeast strains using molecular biology techniques. The yeast isolates identified were Candida inconspicua , Debaryomyces hansenii , Kluyveromyces marxianus , Kazachstania unispora , and Zygotorulaspora florentina . We showed that deproteinated potato wastewater, a starch processing industry waste, supplemented with various carbon sources, including lactose and glycerol, is a suitable medium for the growth of yeast, which allows an accumulation of over 20% of lipid substances in its cells. Fatty acid composition primarily depended on the yeast strain and the carbon source used, and, based on our results, most of the strains met the criteria required for the production of biodiesel. In particular, this concerns a significant share of saturated fatty acids, such as C16:0 and C18:0, and unsaturated fatty acids, such as C18:1 and C18:2. The highest efficiency in lipid biosynthesis exceeded 6.3 g L -1 . Kazachstania unispora was able to accumulate the high amount of palmitoleic acid.

Occurrence of mycotoxins and yeasts and moulds identification in corn silages in tropical climate.

PubMed

Carvalho, B F; Ávila, C L S; Krempser, P M; Batista, L R; Pereira, M N; Schwan, R F

2016-05-01

This study was aimed to identify yeasts and moulds as well as to detect mycotoxin in corn silages in southern Minas Gerais, Brazil. Corn silages from 36 farms were sampled to analyse dry matter, crude protein, ether extract, ash, neutral detergent fibre, nonfibre carbohydrates and mycotoxins contents, yeasts and moulds population, pH and temperature values. The mycotoxins found in high frequency were aflatoxin in 77·7% of analysed samples, ochratoxin (33·3%) and zearalenone (22·2%). There was no significant correlation between the mycotoxin concentration and the presence of moulds. The pH was negatively correlated with ochratoxin concentration. Aspergillus fumigatus was identified in all silages that presented growth of moulds. Ten different yeast species were identified using the culture-dependent method: Candida diversa, Candida ethanolica, Candida rugosa, Issatchenkia orientalis, Kluyveromyces marxianus, Pichia manshurica, Pichia membranifaciens, Saccharomyces cerevisiae, Trichosporon asahii and Trichosporon japonicum. Another six different yeast species were identified using the culture-independent method. A high mycotoxin contamination rate (91·6% of the analysed silages) was observed. The results indicated that conventional culturing and PCR-DGGE should be combined to optimally describe the microbiota associated with corn silage. This study provides information about the corn silage fermentation dynamics and our findings are relevant to optimization of this silage fermentation. © 2016 The Society for Applied Microbiology.

Modeling of an integrated fermentation/membrane extraction process for the production of 2-phenylethanol and 2-phenylethylacetate.

PubMed

Adler, Philipp; Hugen, Thorsten; Wiewiora, Marzena; Kunz, Benno

2011-03-07

An unstructured model for an integrated fermentation/membrane extraction process for the production of the aroma compounds 2-phenylethanol and 2-phenylethylacetate by Kluyveromyces marxianus CBS 600 was developed. The extent to which this model, based only on data from the conventional fermentation and separation processes, provided an estimation of the integrated process was evaluated. The effect of product inhibition on specific growth rate and on biomass yield by both aroma compounds was approximated by multivariate regression. Simulations of the respective submodels for fermentation and the separation process matched well with experimental results. With respect to the in situ product removal (ISPR) process, the effect of reduced product inhibition due to product removal on specific growth rate and biomass yield was predicted adequately by the model simulations. Overall product yields were increased considerably in this process (4.0 g/L 2-PE+2-PEA vs. 1.4 g/L in conventional fermentation) and were even higher than predicted by the model. To describe the effect of product concentration on product formation itself, the model was extended using results from the conventional and the ISPR process, thus agreement between model and experimental data improved notably. Therefore, this model can be a useful tool for the development and optimization of an efficient integrated bioprocess. Copyright © 2010 Elsevier Inc. All rights reserved.

Yeast derived from lignocellulosic biomass as a sustainable feed resource for use in aquaculture.

PubMed

Øverland, Margareth; Skrede, Anders

2017-02-01

The global expansion in aquaculture production implies an emerging need of suitable and sustainable protein sources. Currently, the fish feed industry is dependent on high-quality protein sources of marine and plant origin. Yeast derived from processing of low-value and non-food lignocellulosic biomass is a potential sustainable source of protein in fish diets. Following enzymatic hydrolysis, the hexose and pentose sugars of lignocellulosic substrates and supplementary nutrients can be converted into protein-rich yeast biomass by fermentation. Studies have shown that yeasts such as Saccharomyces cerevisiae, Candida utilis and Kluyveromyces marxianus have favourable amino acid composition and excellent properties as protein sources in diets for fish, including carnivorous species such as Atlantic salmon and rainbow trout. Suitable downstream processing of the biomass to disrupt cell walls is required to secure high nutrient digestibility. A number of studies have shown various immunological and health benefits from feeding fish low levels of yeast and yeast-derived cell wall fractions. This review summarises current literature on the potential of yeast from lignocellulosic biomass as an alternative protein source for the aquaculture industry. It is concluded that further research and development within yeast production can be important to secure the future sustainability and economic viability of intensive aquaculture. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

The evaluation of kefir pure culture starter: Liquid-core capsule entrapping microorganisms isolated from kefir grains.

PubMed

Wang, Liang; Zhong, Hao; Liu, Keying; Guo, Aizhen; Qi, Xianghui; Cai, Meihong

2016-10-01

The main purpose of this study was to develop a pure culture starter for producing kefir. In order to accomplish starter recycling, yeasts (Kluyveromyces marxianus strain, Pichia kudriavzevii clone), lactic acid bacteria (Lactobacillus kefiri strain F4Aa, Lactobacillus kefiri strain NM131-7, Lactobacillus kefiri strain NM132-3, Lactobacillus kefiri strain NM180-3, respectively), and acetic acid bacteria (Acetobacter lovaniensis strain) were entrapped in liquid core capsules based on the distribution ratio in kefir grains. The microbiological, antimicrobial, and chemical properties of kefir made with capsules (M) and kefir grains (K) were measured and compared. According to the results of plate counts in different selective medium, the number of yeasts and bacteria in the liquid core capsules gradually increased and stabilized after eight fermentation cycles. The results of gas chromatography-mass spectrometry showed that almost all the aroma components existed in the two type of kefir, except the ethyl lactate. There was no significant difference in alcohol content, protein content, and fat content, except the acidity and sugar content. Water holding capacity of kefir K was higher than kefir M. There were 14 same free amino acids in kefir M and kefir K, and the content of most free amino acids was similar. In antimicrobial test, there was no significant difference in both kefirs. © The Author(s) 2016.

NMR resonance assignments of the lantibiotic immunity protein NisI from Lactococcus lactis.

PubMed

Hacker, Carolin; Christ, Nina Alexandra; Duchardt-Ferner, Elke; Korn, Sophie; Berninger, Lucija; Kötter, Peter; Entian, Karl-Dieter; Wöhnert, Jens

2015-10-01

The lantibiotic nisin is a small antimicrobial peptide which acts against a wide range of Gram-positive bacteria. Nisin-producing Lactococcus lactis strains express four genes for self-protection against their own antimicrobial compound. This immunity system consists of the lipoprotein NisI and the ABC transporter NisFEG. NisI is attached to the outside of the cytoplasmic membrane via a covalently linked diacylglycerol anchor. Both the lipoprotein and the ABC transporter are needed for full immunity but the exact immunity mechanism is still unclear. To gain insights into the highly specific immunity mechanism of nisin producing strains on a structural level we present here the backbone resonance assignment of NisI (25.8 kDa) as well as the virtually complete (1)H,(15)N,(13)C chemical shift assignments for the isolated 12.7 kDa N-terminal and 14.6 kDa C-terminal domains of NisI.

Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice.

PubMed

Joan, Stella Siaw Xiu; Pui-Fong, Jee; Song, Adelene Ai-Lian; Chang, Li-Yen; Yusoff, Khatijah; AbuBakar, Sazaly; Rahim, Raha Abdul

2016-05-01

An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

Indigenous Georgian Wine-Associated Yeasts and Grape Cultivars to Edit the Wine Quality in a Precision Oenology Perspective.

PubMed

Vigentini, Ileana; Maghradze, David; Petrozziello, Maurizio; Bonello, Federica; Mezzapelle, Vito; Valdetara, Federica; Failla, Osvaldo; Foschino, Roberto

2016-01-01

In Georgia, one of the most ancient vine-growing environment, the homemade production of wine is still very popular in every rural family and spontaneous fermentation of must, without addition of chemical preservatives, is the norm. The present work investigated the yeast biodiversity in five Georgian areas (Guria, Imereti, Kakheti, Kartli, Ratcha-Lechkhumi) sampling grapes and wines from 22 different native cultivars, in 26 vineyards and 19 family cellars. One hundred and eighty-two isolates were ascribed to 15 different species by PCR-ITS and RFLP, and partial sequencing of D1/D2 domain 26S rDNA gene. Metschnikowia pulcherrima (F' = 0.56, I' = 0.32), Hanseniaspora guilliermondii (F' = 0.49, I' = 0.27), and Cryptococcus flavescens (F' = 0.31, I' = 0.11) were the dominant yeasts found on grapes, whereas Saccharomyces cerevisiae showed the highest prevalence into wine samples. Seventy four isolates with fermentative potential were screened for oenological traits such as ethanol production, resistance to SO2, and acetic acid, glycerol and H2S production. Three yeast strains (Kluyveromyces marxianus UMY207, S. cerevisiae UMY255, Torulaspora delbrueckii UMY196) were selected and separately inoculated in vinifications experiments at a Georgian cellar. Musts were prepared from healthy grapes of local varieties, Goruli Mtsvane (white berry cultivar) and Saperavi (black berry cultivar). Physical (°Brix) and microbial analyses (plate counts) were performed to monitor the fermentative process. The isolation of indigenous S. cerevisiae yeasts beyond the inoculated strains indicated that a co-presence occurred during the vinification tests. Results from quantitative GC-FID analysis of volatile compounds revealed that the highest amount of fermentation flavors, such as 4-ethoxy-4-oxobutanoic acid (monoethyl succinate), 2-methylpropan-1-ol, ethyl 2-hydroxypropanoate, and 2-phenylethanol, were significantly more produced in fermentation conducted in Saperavi variety inoculated

Neonatal mucosal immunization with a non-living, non-genetically modified Lactococcus lactis vaccine carrier induces systemic and local Th1-type immunity and protects against lethal bacterial infection

PubMed Central

Ramirez, Karina; Ditamo, Yanina; Rodriguez, Liliana; Picking, Wendy L.; van Roosmalen, Maarten L.; Leenhouts, Kees; Pasetti, Marcela F.

2010-01-01

Safe and effective immunization of newborns and infants can significantly reduce childhood mortality, yet conventional vaccines have been largely unsuccessful in stimulating the neonatal immune system. We explored the capacity of a novel mucosal antigen delivery system consisting of non-living, non-genetically modified Lactococcus lactis particles, designated Gram-positive Enhancer Matrix (GEM), to induce immune responses in the neonatal setting. Yersinia pestis LcrV, used as model protective antigen, was displayed on the GEM particles. Newborn mice immunized intranasally with GEM-LcrV developed LcrV-specific antibodies, Th1-type cell-mediated immunity, and were protected against lethal Y. pestis (plague) infection. The GEM particles activated and enhanced the maturation of neonatal dendritic cells both in vivo and in vitro. These dendritic cells showed increased capacities for secretion of pro-inflammatory and Th1-cell polarizing cytokines, antigen presentation and stimulation of CD4+ and CD8+ T cells. These data show that mucosal immunization with L. lactis GEM particles carrying vaccine antigens represents a promising approach to prevent infectious diseases early in life. PMID:19924118

Metabolic characterization and transformation of the non-dairy Lactococcus lactis strain KF147, for production of ethanol from xylose.

PubMed

Petersen, Kia Vest; Liu, Jianming; Chen, Jun; Martinussen, Jan; Jensen, Peter Ruhdal; Solem, Christian

2017-08-01

The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, the authors characterized growth and product formation for KF147 when grown on xylose. In a defined medium KF147 was found to co-metabolize xylose and arginine, resulting in bi-phasic growth. Especially at low xylose concentrations, arginine significantly improved growth rate. To facilitate further studies of the xylose metabolism, the authors eliminated arginine catabolism by deleting the arcA gene encoding the arginine deiminase. The fermentation product profile suggested two routes for xylose degradation, the phosphoketolase pathway and the pentose phosphate pathway. Inactivation of the phosphoketolase pathway redirected the entire flux through the pentose phosphate pathway whereas over-expression of phosphoketolase increased the flux through the phosphoketolase pathway. In general, significant amounts of the mixed-acid products, including lactate, formate, acetate and ethanol, were formed irrespective of xylose concentrations. To demonstrate the potential of KF147 for converting xylose into useful chemicals the authors chose to redirect metabolism towards ethanol production. A synthetic promoter library was used to drive the expression of codon-optimized versions of the Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase, and the outcome was a strain producing ethanol as the sole fermentation product with a high yield corresponding to 83% of the theoretical maximum. The results clearly indicate the great potential of using the more metabolically diverse non-dairy L. lactis strains for bio-production based on xylose containing feedstocks. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Controlled production of Camembert-type cheeses. Part II. Changes in the concentration of the more volatile compounds.

PubMed

Leclercq-Perlat, Marie-Noëlle; Latrille, Eric; Corrieu, Georges; Spinnler, Henry-Eric

2004-08-01

Flavour generation in cheese is a major aspect of ripening. In order to enhance aromatic qualities it is necessary to better understand the chemical and microbiological changes. Experimental Camembert-type cheeses were prepared in duplicate from pasteurized milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti and Brevibacterium linens under aseptic conditions. Two replicates performed under controlled conditions of temperature (12 degrees C), relative humidity (95 +/- 2%), and atmosphere showed similar ripening characteristics. The evolutions of metabolite concentrations were studied during ripening. The volatile components were extracted by dynamic headspace extraction, separated and quantified by gas chromatography and identified by mass spectrometry. For each cheese the volatile concentrations varied with the part considered (rind or core). Except for ethyl acetate and 2-pentanone, the volatile quantities observed were higher than their perception thresholds. The flavour component production was best correlated with the starter strains. During the first 10 days the ester formations (ethyl, butyl and isoamyl acetates) were associated with the concentrations of K. lactis and G. candidum. The rind quantity of esters was lower than that observed in core probably due to (1) a diffusion from the core to the surface and (2) evaporation from the surface to the chamber atmosphere. G. candidum and Brev. linens association produced 3 methyl butanol and methyl 3-butanal from leucine, respectively. DMDS came from the methionine catabolism due to Brev. linens. Styrene production was attributed to Pen. camemberti. 2-Pentanone evolution was associated with Pen. camemberti spores and G. candidum. 2-Heptanone changes were not directly related to flora activities while 2-octanone production was essentially due to G. candidum. This study also demonstrates the determining role of volatile component diffusion.