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.

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.

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

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.

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.

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.

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

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.

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.

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

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

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.

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

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)).

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

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

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

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.

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

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.

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

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.

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.

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

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.

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

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.

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.

Selected Lactobacillus strains isolated from sugary and milk kefir reduce Salmonella infection of epithelial cells in vitro.

PubMed

Zavala, L; Golowczyc, M A; van Hoorde, K; Medrano, M; Huys, G; Vandamme, P; Abraham, A G

2016-09-01

The isolation of potentially probiotic strains and the subsequent study of their properties are very important steps to gain insight in the health benefits ascribed to sugary and milk kefir. The aim of the present study was to characterise fifteen Lactobacillus strains isolated from these beverages by determining some surface properties and their ability to antagonise enterocyte cell damage after Salmonella infection in vitro. Lactobacillus surface properties were determined by hydrophobicity, autoaggregation, and coaggregation assays with Salmonella. In addition, lactobacilli adhesion to Caco-2/TC-7 cells and the effect on Salmonella invasion were evaluated. Finally, the disassembly of F-actin cytoskeleton on intestinal epithelial cells was assayed in vitro when Salmonella infection was performed in the presence of selected Lactobacillus strains. Ten out of the 15 strains showed a high adhesion capacity to Caco-2/TC-7 cells. Most of the strains were hydrophilic and non-autoaggregating. Strains isolated from sugary kefir were non-coaggregating with Salmonella, while strains Lactobacillus paracasei CIDCA 83120, 83121, 83123, 83124, 8339, 83102 isolated from milk kefir were able to coaggregate after 1 h. L. paracasei CIDCA 8339 and Lactobacillus kefiri CIDCA 83102 were able to diminish Salmonella invasion to the enterocytes. An antagonistic effect on cytoskeleton disruption elicited by the pathogen was also demonstrated. Our results suggest that both strains isolated from milk kefir could be considered as appropriate probiotic candidates.

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

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.

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

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

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.

Physicochemical, microbiological and sensory profiles of fermented milk containing probiotic strains isolated from kefir.

PubMed

Kakisu, Emiliano; Irigoyen, Aurora; Torre, Paloma; De Antoni, Graciela L; Abraham, Analía G

2011-11-01

A two-strain starter culture containing Lactobacillus plantarum CIDCA 83114, a potential probiotic strain isolated from kefir grains, and Streptococcus thermophilus CIDCA 321 was tested for the preparation of a fermented milk product. Kluyveromyces marxianus CIDCA 8154, a yeast with immunomodulatory properties was included to formulate a three-strain starter culture. Supernatants of enterohaemorragic Escherichia coli, shiga-toxin-producing strain, along with a two-strain or a three-strain starter culture were included in the medium of Vero-cell surface cultures. The results demonstrated that these combinations of microorganisms antagonize the cytopathic action of shiga toxins. The cell concentration of Lb. plantarum did not decrease during fermentation, indicating that the viability of this strain was not affected by low pH, nor did the number of viable bacteria change during 21 days of storage in either fermented products. The number of viable yeasts increases during fermentation and storage. Trained assessors analyzed the general acceptability of fresh fermented milks and considered both acceptable. The milk fermented with the two-strain starter culture was considered acceptable after two week of storage, while the product fermented with the three-strain starter culture remained acceptable for less than one week. The main changes in sensory attributes detected by the trained panel were in sour taste, milky taste and also in fermented attributes. The correlation between different sensory attributes and acceptability indicated that the panel was positively influenced by milky attributes (taste, odour, and flavour) as well as the intensity of flavour. In conclusion, the two-strain starter culture would be the more promising alternative for inclusion of that potential probiotic lactobacillus in a fermented milk product.

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

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

[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.

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.

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

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

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.

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.

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

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.

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.

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.

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

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

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

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

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

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.

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.

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.

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.

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

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.

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

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

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

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.

Phage-resistance linked to cell heterogeneity in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1.

PubMed

Suárez, Viviana B; Maciel, Natalia; Guglielmotti, Daniela; Zago, Miriam; Giraffa, Giorgio; Reinheimer, Jorge

2008-12-10

The aim of this work was to study the relationship between the cell morphological heterogeneity and the phage-resistance in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1. Two morphological variants (named C and T) were isolated from this strain. Phage-resistant derivatives were isolated from them and the percentage of occurrence of confirmed phage-resistant cells was 0.001% of the total cellular population. Within these phage-resistant cell derivatives there were T (3 out of 4 total isolates) and C (1 out of 4 total isolates) variants. The study of some technological properties (e.g. proteolytic and acidifying activities) demonstrated that most of phage-resistant derivatives were not as good as the parental strain. However, for one derivative (a T variant), the technological properties were better than those of the parental strain. On the other hand, it was possible to determinate that the system of phage-resistance in the T variants was interference in adsorption step, with adsorption rates <15%. For the C variant derivative it was possible to demonstrate the presence of a restriction/modification system and, moreover, to determinate that this system could be Type I R/M.

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.

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)).

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.

Malt sprout, an underused beer by-product with promising potential for the growth and dehydration of lactobacilli strains.

PubMed

Cejas, Luján; Romano, Nelson; Moretti, Ana; Mobili, Pablo; Golowczyc, Marina; Gómez-Zavaglia, Andrea

2017-12-01

Malt sprout (MS), a by-product of the malt industry obtained by removing rootlets and sprouts from the seed of germinated barley ( Hordeum vulgare L.), was used as culture, dehydration and storage medium of three strains of lactobacilli: Lactobacillus salivarius CM-CIDCA 1231B and CM-CIDCA 1232Y and Lactobacillus plantarum CIDCA 83114. The three strains were grown in MS and MS supplemented with 20% w/v fructo-oligosaccharides (MS FOS). Bacterial growth was determined by registering the decrease of pH and by plate counting. Comparable results with those of microorganisms grown in MRS (controls) were observed in terms of lag times, ΔpH and acidification rates. Furthermore, during fermentation, a significant increase of DP6 (FOS with degree of polymerization 6) was observed at expenses of inulin and DP7, probably indicating their hydrolysis. A concomitant decrease of DP3, sucrose and monosaccharides was also observed, as result of their bacterial consumption during growth. The presence of FOS in the fermented media protected microorganisms during freeze-drying and storage, as no decrease of culturability was observed after 60 days at 4 °C (> 10 8 CFU/mL). Using MS appears as an innovative strategy for the production of lactobacilli at large scale, supporting their use for the elaboration of functional foods containing prebiotics and probiotics.

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.

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

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.

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.

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.

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

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

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.

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.

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.

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.

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.

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.

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

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.

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®.

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.

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.

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

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.

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

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.

Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM® and Bifidobacterium animalis subsp. lactis Bl-04 ® via Chip-Based Digital PCR.

PubMed

Hansen, Sarah J Z; Morovic, Wesley; DeMeules, Martha; Stahl, Buffy; Sindelar, Connie W

2018-01-01

The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically documented dose for its associated health benefits. A method is described using chip-based digital PCR (cdPCR) to enumerate Bifidobacterium animalis subsp. lactis Bl-04 and Lactobacillus acidophilus NCFM both as single strains and in combination. Primers and probes were designed to differentiate the target strains against other strains of the same species using known single copy, genetic differences. The assay was optimized to include propidium monoazide pre-treatment to prevent amplification of DNA associated with dead probiotic cells as well as liberation of DNA from cells with intact membranes using bead beating. The resulting assay was able to successfully enumerate each strain whether alone or in multiplex. The cdPCR method had a 4 and 5% relative standard deviation (RSD) for Bl-04 and NCFM, respectively, making it more precise than plate counts with an industry accepted RSD of 15%. cdPCR has the potential to replace traditional plate counts because of its precision, strain specificity and the ability to obtain results in a matter of hours.

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.

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.

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

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.

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.

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.

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.

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.

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.

Selection of a Bifidobacterium strain to complement resistant starch in a synbiotic yoghurt.

PubMed

Crittenden, R G; Morris, L F; Harvey, M L; Tran, L T; Mitchell, H L; Playne, M J

2001-02-01

To employ an in vitro screening regime to select a probiotic Bifidobacterium strain to complement resistant starch (Hi-maizetrade mark) in a synbiotic yoghurt. Of 40 Bifidobacterium isolates examined, only B. lactis Laftitrade mark B94 possessed all of the required characteristics. This isolate hydrolysed Hi-maizetrade mark, survived well in conditions simulating passage through the gastrointestinal tract and possessed technological properties suitable for yoghurt manufacture. It grew well at temperatures up to 45 degrees C, and grew to a high cell yield in an industrial growth medium. In addition to resistant starch, the organism was able to utilize a range of prebiotics including inulin, and fructo-, galacto-, soybean- and xylo-oligosaccharides. Pulse field gel electrophoresis of restriction enzyme cut chromosomal DNA revealed that B. lactis Laftitrade mark B94 was very closely related to the B. lactis Type Strain (DSM 10140), and to the commercial strains B. lactis Bb-12 and B. lactis DS 920. However, B. lactis Laftitrade mark B94 was the only one of these isolates that could hydrolyse Hi-maizetrade mark. This phenotypic difference did not appear to be due to the presence of plasmid encoded amylase. Bifidobacterium lactis Laftitrade mark B94 survived without substantial loss of viability in synbiotic yoghurt containing Hi-maizetrade mark during storage at 4 degrees C for six weeks. Bifidobacterium lactis Laftitrade mark B94 is a promising new yoghurt culture that warrants further investigation to assess its probiotic potential. In vitro screening procedures can be used to integrate complementary probiotic and prebiotic ingredients for new synbiotic functional food products.

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

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

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

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

Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products.

PubMed

Monteagudo-Mera, A; Caro, I; Rodríguez-Aparicio, L B; Rúa, J; Ferrero, M A; García-Armesto, M R

2011-08-01

The present work was aimed at characterizing 12 strains of lactic acid bacteria (LAB) to obtain improved potential starter or probiotic cultures that could be used for making dairy products from ewe's milk and cow's milk. Eight strains with antimicrobial properties, isolated from ewe's milk and from cheese made from ewe's and/or cow's milk, were studied. They were identified as Enterococcus faecalis (five strains), Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides, and Lactobacillus paracasei subsp. paracasei (one strain of each species). Additionally, four strains were obtained from the American Type Culture Collection: Lactobacillus casei 393 (isolated from cheese), L. lactis subsp. lactis 11454 (origin nonspecified and a producer of nisin), and two strains isolated from human feces (L. paracasei subsp. paracasei 27092 and Lactobacillus rhamnosus 53103, antibacterial agent producer). All E. faecalis strains showed at least one virulence factor (either hemolysin or gelatinase), which emphasizes the importance of these studies in this species. Both L. lactis strains and most Lactobacillus spp. were good acidifiers in ewe's milk and cow's milk at 30°C. High β-galactosidase activity, as well as aminopeptidase activities that favor the development of desirable flavors in cheese, were detected in all Lactobacillus spp. strains. Furthermore, L. rhamnosus ATCC 53103 showed α-fucosidase activity (thought to help colonization of the intestine) and lack of α-glucosidase activity (a trait considered positive for diabetic and obese humans). This last enzymatic activity was also lacking in L. lactis ATCC 11454. L. mesenteroides was the only strain D(2)-lactic acid producer. The selection of any particular strain for probiotic or dairy cultures should be performed according to the technological and/or functional abilities needed.

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

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

Yeast Kluyveromyces lactis as host for expression of the bacterial lipase: cloning and adaptation of the new lipase gene from Serratia sp.

PubMed

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

2015-10-01

Many microbial lipases have been successfully expressed in yeasts, but not in industrially attractive Kluyveromyces lactis, which among other benefits can be cultivated on a medium supplemented with whey--cheap and easily available industrial waste. A new bacterial lipase from Serratia sp. was isolated and for the first time expressed into the yeast Kluyveromyces lactis by heterologous protein expression system based on a strong promoter of Kluyveromyces marxianus triosephosphate isomerase gene and signal peptide of Kluyveromyces marxianus endopolygalacturonase gene. In addition, the bacterial lipase gene was synthesized de novo by taking into account a codon usage bias optimal for K. lactis and was expressed into the yeast K. lactis also. Both resulting strains were characterized by high output level of the target protein secreted extracellularly. Secreted lipases were characterized for activity and stability.

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.

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.

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

Addition to thermized milk of Lactococcus lactis subsp. cremoris M104, a wild, novel nisin a-producing strain, replaces the natural antilisterial activity of the autochthonous raw milk microbiota reduced by thermization.

PubMed

Lianou, Alexandra; Samelis, John

2014-08-01

Recent research has shown that mild milk thermization treatments routinely used in traditional Greek cheese production are efficient to inactivate Listeria monocytogenes and other pathogenic or undesirable bacteria, but they also inactivate a great part of the autochthonous antagonistic microbiota of raw milk. Therefore, in this study, the antilisterial activity of raw or thermized (63°C, 30 s) milk in the presence or absence of Lactococcus lactis subsp. cremoris M104, a wild, novel, nisin A-producing (Nis-A+) raw milk isolate, was assessed. Bulk milk samples were taken from a local cheese plant before or after thermization and were inoculated with a five-strain cocktail of L. monocytogenes (approximately 4 log CFU/ml) or with the cocktail, as above, plus the Nis-A+ strain (approximately 6 log CFU/ml) as a bioprotective culture. Heat-sterilized (121°C, 5 min) raw milk inoculated with L. monocytogenes was used as a control treatment. All milk samples were incubated at 37°C for 6 h and then at 18°C for an additional 66 h. L. monocytogenes grew abundantly (>8 log CFU/ml) in heat-sterilized milk, whereas its growth was completely inhibited in all raw milk samples. Conversely, in thermized milk, L. monocytogenes increased by 2 log CFU/ml in the absence of strain M104, whereas its growth was completely inhibited in the presence of strain M104. Furthermore, nisin activity was detected only in milk samples inoculated with strain M104. Thus, postthermal supplementation of thermized bulk milk with bioprotective L. lactis subsp. cremoris cultures replaces the natural antilisterial activity of raw milk reduced by thermization.

Protective immunity induced by an intranasal multivalent vaccine comprising 10 Lactococcus lactis strains expressing highly prevalent M-protein antigens derived from Group A Streptococcus.

PubMed

Wozniak, Aniela; Scioscia, Natalia; García, Patricia C; Dale, James B; Paillavil, Braulio A; Legarraga, Paulette; Salazar-Echegarai, Francisco J; Bueno, Susan M; Kalergis, Alexis M

2018-04-28

Streptococcus pyogenes (group A Streptococcus) causes diseases ranging from mild pharyngitis to severe invasive infections. The N-terminal fragment of Streptococcal M protein elicits protective antibodies and is an attractive vaccine target. However, this N- terminal fragment is hypervariable and there are more than 200 different M types. We are developing an intranasal live bacterial vaccine comprised of 10 strains of Lactococcus lactis, each expressing one N-terminal fagment of M protein. Live bacterial-vectored vaccines have lower associated costs because of its less complex manufacturing processes compared to protein subunit vaccines. Moreover, intranasal administration does not require syringe or specilized personnel. The evaluation of individual vaccine types (M1, M2, M3, M4, M6, M9, M12, M22, M28 and M77) showed that most of them protected mice against challenge with virulent S. pyogenes. All of the 10 strains combined in a 10-valent vaccine (Mx10) induced serum and bronchoalveolar lavages IgG titers that ranged from 3 to 10-fold those of unimmunized mice. Survival of Mx10-immunized mice after intranasal challenge with M28 streptococci is significantly higher than unimmunized mice. In contrast, when mice were challenged with M75 streptococci, survival of Mx10-immunized mice was not significantly different from unimmunized mice. Mx-10 immunized mice were significantly less colonized with S. pyogenes in oropharyngeal washes and developed less severe disease symptoms after challenge compared to unimmunized mice. Our L. lactis-based vaccine may provide an alternative solution to the development of broadly protective group A streptococcal vaccines. © 2018 The Societies and John Wiley & Sons Australia, Ltd.

Simulated gastrointestinal conditions increase adhesion ability of Lactobacillus paracasei strains isolated from kefir to Caco-2 cells and mucin.

PubMed

Bengoa, Ana Agustina; Zavala, Lucía; Carasi, Paula; Trejo, Sebastián Alejandro; Bronsoms, Silvia; Serradell, María de Los Ángeles; Garrote, Graciela Liliana; Abraham, Analía Graciela

2018-01-01

Gastrointestinal conditions along the digestive tract are the main stress to which probiotics administrated orally are exposed because they must survive these adverse conditions and arrive alive to the intestine. Adhesion to epithelium has been considered one of the key criteria for the characterization of probiotics because it extends their residence time in the intestine and as a consequence, can influence the health of the host by modifying the local microbiota or modulating the immune response. Nevertheless, there are very few reports on the adhesion properties to epithelium and mucus of microorganisms after passing through the gastrointestinal tract. In the present work, we evaluate the adhesion ability in vitro of L. paracasei strains isolated from kefir grains after acid and bile stress and we observed that they survive simulated gastrointestinal passage in different levels depending on the strain. L. paracasei CIDCA 8339, 83120 and 83123 were more resistant than L. paracasei CIDCA 83121 and 83124, with a higher susceptibility to simulated gastric conditions. Proteomic analysis of L. paracasei subjected to acid and bile stress revealed that most of the proteins that were positively regulated correspond to the glycolytic pathway enzymes, with an overall effect of stress on the activation of the energy source. Moreover, it is worth to remark that after gastrointestinal passage, L. paracasei strains have increased their ability to adhere to mucin and epithelial cells in vitro being this factor of relevance for maintenance of the strain in the gut environment to exert its probiotic action. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

[Enhanced nisin production by overexpression of nisin immunity gene nisI in the nisin-producing strain].

PubMed

Hu, Hongmei; Jiang, Like; Lin, Yuheng; Huan, Liandong; Zhong, Jin

2010-10-01

Our aim was to enhance nisin production by overexpression of nisin immunity gene nisI in nisin-producing strains. Nisin immunity gene nisI with a strong promoter P59 was cloned into vector pHJ201 and introduced into Lacotococcus lactis NZ9800, resulting in a recombinant strain L. lactis NZ9800/pHMI. Then the differences between the recombinant strain and the control strain L. lactis NZ9800/pHJ201 were analyzed in several aspects, including their growth curves, nisin resistance level and antibacterial activity against indicator strain Microccus flavus NCIB 8166. The overexpression of nisI had no significant difference in growth rate between recombinant strain and contrast strain. However, it promoted recombinant strain tolerance 25% higer nisin resistance level and stronger antibacterial activity against M. flavus NCIB 8166, which was increased by 32% and 25% when fermented for 6 and 8 hours, respectively. These results indicated that overexpression of nisI gene in the nisin producing strain can effectively enhance nisin resistence level and thus improve nisin production.

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.

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.

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.

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.

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

Response of Leuconostoc strains against technological stress factors: Growth performance and volatile profiles.

PubMed

Cicotello, Joaquín; Wolf, Irma V; D'Angelo, Luisa; Guglielmotti, Daniela M; Quiberoni, Andrea; Suárez, Viviana B

2018-08-01

The ability of twelve strains belonging to three Leuconostoc species (Leuconostoc mesenteroides, Leuconostoc lactis and Leuconostoc pseudomesenteroides) to grow under diverse sub-lethal technological stress conditions (cold, acidic, alkaline and osmotic) was evaluated in MRS broth. Two strains, Leuconostoc lactis Ln N6 and Leuconostoc mesenteroides Ln MB7, were selected based on their growth under sub-lethal conditions, and volatile profiles in RSM (reconstituted skim milk) at optimal and under stress conditions were analyzed. Growth rates under sub-lethal conditions were strain- and not species-dependent. Volatilomes obtained from the two strains studied were rather diverse. Particularly, Ln N6 (Ln. lactis) produced more ethanol and acetic acid than Ln MB7 (Ln. mesenteroides) and higher amounts and diversity of the rest of volatile compounds as well, at all times of incubation. For the two strains studied, most of stress conditions applied diminished the amounts of ethanol and acetic acid produced and the diversity and levels of the rest of volatile compounds. These results were consequence of the different capacity of the strains to grow under each stress condition tested. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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

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.

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.

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

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.

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.

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

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.

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

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.

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

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

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.

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.

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

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.

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

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.

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

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

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.

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., nontoxicogenic yeast Kluyveromyces lactis (previously named Saccharomyces lactis). It contains the enzyme β... prepared from yeast that has been grown in a pure culture fermentation and by using materials that are...

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., nontoxicogenic yeast Kluyveromyces lactis (previously named Saccharomyces lactis). It contains the enzyme β... prepared from yeast that has been grown in a pure culture fermentation and by using materials that are...

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.

Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

PubMed Central

Ramos, Ana; Boels, Ingeborg C.; de Vos, Willem M.; Santos, Helena

2001-01-01

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation. PMID:11133425

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.

Grana Padano cheese whey starters: microbial composition and strain distribution.

PubMed

Rossetti, Lia; Fornasari, Maria Emanuela; Gatti, Monica; Lazzi, Camilla; Neviani, Erasmo; Giraffa, Giorgio

2008-09-30

The aim of this work was to evaluate the species composition and the genotypic strain heterogeneity of dominant lactic acid bacteria (LAB) isolated from whey starter cultures used to manufacture Grana Padano cheese. Twenty-four Grana Padano cheese whey starters collected from dairies located over a wide geographic production area in the north of Italy were analyzed. Total thermophilic LAB streptococci and lactobacilli were quantified by agar plate counting. Population structure of the dominant and metabolically active LAB species present in the starters was profiled by reverse transcriptase, length heterogeneity-PCR (RT-LH-PCR), a culture-independent technique successfully applied to study whey starter ecosystems. The dominant bacterial species were Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum. Diversity in the species composition allowed the whey cultures to be grouped into four main typologies, the one containing L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus being the most frequent one (45% of the cultures analyzed), followed by that containing only the two lactobacilli (40%). Only a minor fraction of the cultures contained L. helveticus alone (4%) or all the four LAB species (11%). Five hundred and twelve strains were isolated from the 24 cultures and identified by M13-PCR fingerprinting coupled with 16S rRNA gene sequencing. Most of the strains were L. helveticus (190 strains; 37% of the total), L delbrueckii subsp. lactis (90 strains; 18%) and S. thermophilus (215 strains; 42%). This result was in good agreement with the qualitative whey starter composition observed by RT-LH-PCR. M13-PCR fingerprinting indicated a markedly low infra-species diversity, i.e. the same biotypes were often found in more than one culture. The distribution of the biotypes into the different cultures was mainly dairy plant-specific rather than correlated with the different production areas.

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

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

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.

S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 enhances macrophages response to LPS in a Ca+2-dependent manner.

PubMed

Malamud, Mariano; Carasi, Paula; Freire, Teresa; Serradell, María de Los Angeles

2018-01-01

The S-layer is a (glyco)-proteinaceous envelope constituted by self-assembled subunits that form a two-dimensional lattice covering the surface of different species of Bacteria and Archaea. It could be considered as one of the most abundant biopolymers in our planet. Because of their unique self-assembly features, exhibiting repetitive identical physicochemical properties down to the subnanometer scale, as well as their involvement in specific interactions with host cells, the S-layer proteins (SLPs) show a high potential application in different areas of biotechnology, including the development of antigen carriers or new adjuvants. The presence of a glycosylated SLP on potentially probiotic Lactobacillus kefiri strains was previously described by our research group. In this study, we aim to investigate the role of carbohydrates present in the SLP from L. kefiri CIDCA 8348 (SLP-8348) in their internalization by murine macrophages, as well as to analyze their immunomodulatory capacity and their effect on LPS-stimulated macrophages. RAW 264.7 cells internalized the SLP-8348 in a process that was mediated by carbohydrate-receptor interactions since it was inhibited by glucose, mannose or EGTA, a Ca +2 chelating agent. These results correlated with the recognition of SLP-8348 by ConA lectin. We further show that while SLP-8348 was not able to induce the activation of macrophages by itself, it favored the LPS-induced response, since there was a significant increase in the expression of surface cell markers MHC-II, CD86 and CD40, as well as in IL-6 and IL-10 expression at both transcript and protein levels, in comparison with LPS-stimulated cells. The presence of EGTA completely abrogated this synergistic effect. Taken together, these results strongly suggest the involvement of both glycosidic residues and Ca +2 ions in the recognition of SLP-8348 by cellular receptors on murine macrophages. Moreover, these results suggest the potentiality of the SLP-8348 for the

A glycoproteomic approach reveals that the S-layer glycoprotein of Lactobacillus kefiri CIDCA 83111 is O- and N-glycosylated.

PubMed

Cavallero, Gustavo J; Malamud, Mariano; Casabuono, Adriana C; Serradell, M de Los Ángeles; Couto, Alicia S

2017-06-06

In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive β-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequence SSASSASSA already identified as a signature glycosylation motif in L. buchneri, substituted on average with eight glucose residues and decorated with galacturonic acid and another O-glycosylated site on peptide 471-476, with a Glc 5-8 GalA 2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures. To our knowledge, this is the first description of the structure of N-glycans in S-layer glycoproteins from Lactobacillus species. A detailed characterization of protein glycosylation is essential to establish the basis for understanding and investigating its biological role. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. Furthermore, this is the first

Cyclopropanation of membrane unsaturated fatty acids is not essential to the acid stress response of Lactococcus lactis subsp. cremoris.

PubMed

To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

2011-05-01

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-L-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

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

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.

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.

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

Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris ▿

PubMed Central

To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

2011-01-01

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-l-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells. PMID:21421775

Adhesion properties of potentially probiotic Lactobacillus kefiri to gastrointestinal mucus.

PubMed

Carasi, Paula; Ambrosis, Nicolás M; De Antoni, Graciela L; Bressollier, Philippe; Urdaci, María C; Serradell, María de los Angeles

2014-02-01

We investigated the mucus-binding properties of aggregating and non-aggregating potentially probiotic strains of kefir-isolated Lactobacillus kefiri, using different substrates. All the strains were able to adhere to commercial gastric mucin (MUCIN) and extracted mucus from small intestine (SIM) and colon (CM). The extraction of surface proteins from bacteria using LiCl or NaOH significantly reduced the adhesion of three selected strains (CIDCA 8348, CIDCA 83115 and JCM 5818); although a significant proportion (up to 50%) of S-layer proteins were not completely eliminated after treatments. The surface (S-layer) protein extracts from all the strains of Lb. kefiri were capable of binding to MUCIN, SIM or CM, and no differences were observed among them. The addition of their own surface protein extract increased adhesion of CIDCA 8348 and 83115 to MUCIN and SIM, meanwhile no changes in adhesion were observed for JCM 5818. None of the seven sugars tested had the ability to inhibit the adhesion of whole bacteria to the three mucus extracts. Noteworthy, the degree of bacterial adhesion reached in the presence of their own surface protein (S-layer) extract decreased to basal levels in the presence of some sugars, suggesting an interaction between the added sugar and the surface proteins. In conclusion, the ability of these food-isolated bacteria to adhere to gastrointestinal mucus becomes an essential issue regarding the biotechnological potentiality of Lb. kefiri for the food industry.

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.

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.

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

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.

Extraction of the same novel homoglycan mixture from two different strains of Bifidobacterium animalis and three strains of Bifidobacterium breve.

PubMed

Alhudhud, M; Sadiq, S; Ngo, H N; Hidalgo-Cantabrana, C; Ruas-Madiedo, P; van Sinderen, D; Humphreys, P N; Laws, A P

2018-06-15

Three strains of Bifidobacterium breve (JCM 7017, JCM 7019 and JCM 2258) and two strains of Bifidobacterium animalis subsp. lactis (AD011 and A1dOxR) were grown in broth cultures or on plates, and a standard exopolysaccharide extraction method was used in an attempt to recover exocellular polysaccharides. When the extracted materials were analysed by NMR it was clear that mixtures of polysaccharides were being isolated including exopolysaccharides (EPS) cell wall polysaccharides and intracellular polysaccharides. Treatment of the cell biomass from the B. breve strains, or the B. animalis subsp. lactis AD011 strain, with aqueous sodium hydroxide provided a very similar mixture of polysaccharides but without the EPS. The different polysaccharides were partially fractionated by selective precipitation from an aqueous solution upon the addition of increasing percentages of ethanol. The polysaccharides extracted from B. breve JCM 7017 grown in HBM media supplemented with glucose (or isotopically labelled D-glucose-1- 13 C) were characterised using 1D and 2D-NMR spectroscopy. Addition of one volume of ethanol generated a medium molecular weight glycogen (Mw=1×10 5 Da, yield 200 mg/l). The addition of two volumes of ethanol precipitated an intimate mixture of a low molecular weight β-(1→6)-glucan and a low molecular weight β-(1→6)-galactofuranan which could not be separated (combined yield 46 mg/l). When labelled D-glucose-1- 13 C was used as a carbon supplement, the label was incorporated into >95% of the anomeric carbons of each polysaccharide confirming they were being synthesised in situ. Similar 1 H NMR profiles were obtained for polysaccharides recovered from the cells of B. animalis subsp. lactis AD011and A1dOxR (in combination with an EPS), B. breve JCM 7017, B. breve JCM 7019, B. breve JCM 2258 and from an EPS (-ve) mutant of B. breve 7017 (a non-EPS producer).

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.

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

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.

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.

Allergenicity reduction of bovine milk β-lactoglobulin by proteolytic activity of lactococcus lactis BMC12C and BMC19H isolated from Iranian dairy products.

PubMed

Kazemi, Rezvan; Taheri-Kafrani, Asghar; Motahari, Ahmad; Kordesedehi, Reihane

2018-06-01

Nowadays health benefits of bioactive food constituents, known as probiotic microorganisms, are a growing awareness. Cow's milk is a nutritious food containing probiotic bacteria. However, milk allergenicity is one of the most common food allergies. The milk protein, β-lactoglobulin (BLG), is in about 80% of all main cases of milk allergies for children and infants. With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated new proteolytic strains of cocci lactic acid bacteria from traditional Iranian dairy products. The proteases produced by these strains had strong proteolytic activity against BLG. Proteolysis of BLG, observed after sodium dodecyl sulfate-PAGE, was confirmed by the analysis of the peptide profiles by reversed-phase HPLC. The two isolates were submitted to 16S rDNA sequencing and identified as Lactcoccus lactis subsp. cremoris and Lactcoccus lactis subsp. hordniea. The competitive ELISA experiments confirmed that these isolates, with high proteolytic activity, reduce significantly the allergenicity of BLG. Accordingly, these isolates can reduce the immunoreactivity of bovine milk proteins, which can be helpful for the production of low-allergic dairy products. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area

PubMed Central

Corroler, D.; Mangin, I.; Desmasures, N.; Gueguen, M.

1998-01-01

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of “Camembert de Normandie” cheese. PMID:9835555

An ecological study of lactococci isolated from raw milk in the camembert cheese registered designation of origin area.

PubMed

Corroler, D; Mangin, I; Desmasures, N; Gueguen, M

1998-12-01

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of "Camembert de Normandie" cheese.

Characterization of homofermentative lactobacilli isolated from kefir grains: potential use as probiotic.

PubMed

Golowczyc, Marina A; Gugliada, Maria J; Hollmann, Axel; Delfederico, Lucrecia; Garrote, Graciela L; Abraham, Analía G; Semorile, Liliana; De Antoni, Graciela

2008-05-01

Considering that several health promoting properties are associated with kefir consumption and a reliable probiotic product requires a complete identification of the bacterial species, the present work evaluates several proved markers of probiotic potential of eleven isolates of homofermentative lactobacilli isolated from kefir grains and molecular identification and genotypic diversity. Using restriction analysis of amplified ribosomal DNA (ARDRA) and analysis of the 16S-23S rRNA internal spacer region we confirmed that all homofermentative lactobacilli belong to the species Lactobacillus plantarum. RAPD-PCR analysis allowed the discrimination of lactobacilli in five clusters. All isolates exhibited high resistance to bile salt. High survival after one hour of exposure to pH 2.5 was observed in Lb. plantarum CIDCA 8313, 83210, 8327 and 8338. All isolates were hydrophilic and non autoaggregative. Isolate CIDCA 8337 showed the highest percentage of adhesion among strains. All tested lactobacilli had strong inhibitory power against Salmonella typhimurium and Escherichia coli. Seven out of eleven isolates showed inhibition against Sal. enterica and five isolates were effective against Sal. gallinarum. Only CIDCA 8323 and CIDCA 8327 were able to inhibit Sal. sonnei. We did not find any correlation between the five clusters based on RAPD-PCR and the probiotic properties, suggesting that these isolates have unique characteristics.

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.

Three new insertion sequence elements ISLdl2, ISLdl3, and ISLdl4 in Lactobacillus delbrueckii: isolation, molecular characterization, and potential use for strain identification.

PubMed

Ravin, Victor; Alatossava, Tapani

2003-05-01

A group of new insertion sequence (IS) elements, ISLdl2, ISLdl3, and ISLdl4, from Lactobacillus delbrueckii subsp. lactis ATCC 15808 was isolated, characterized, and used for strain identification together with ISLdl1, recently characterized as an L. delbrueckii IS element belonging to the ISL3 family. ISLdl2 was 1367 bp in size and had a 24 bp IR and an 8 bp DR. The single ORF of ISLdl2 encoded a protein of 392 aa similar to transposases of the IS256 family. ISLdl3 had a single ORF encoding a protein of 343 aa similar to transposases of the IS30 family. Finally, ISLdl4 had a single ORF encoding a protein of 406 aa and displayed homology to the transposases of the IS110 family. ISLdl4 was only slight different from ISL4 (Accession No. AY040213). ISLdl1, ISLdl2, and ISLdl4 were present in all of the 10 L. delbrueckii subsp. lactis and subsp. delbrueckii strains tested, as well as in three of the 11 L. delbrueckii subsp. bulgaricus strains tested. ISLdl3 was present only in four closely related strains of L. delbrueckii subsp. lactis. These IS elements were not observed in Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, or Lactobacillus plantarum. A cluster of IS elements, ISLdl1, ISLdl2, ISLdl3, ISLdl4, and ISL6, was observed in L. delbrueckii subsp. lactis strain ATCC 15808. Within this cluster, ISLdl4 was inserted into ISLdl1 between the left IR and the start codon of ORF455, encoding a putative transposase. Most of the integration sites of the IS elements were strain-specific. We have observed that IS elements can migrate from one strain to another as integral parts of bacterial DNA by using phage LL-H as a vehicle. We demonstrate for the first time that inverse PCR and vectorette PCR methods with primers based on sequences of the IS elements could be used for identification of L. delbrueckii strains.

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

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.

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

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.

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.

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.

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

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.

The suitability of different probiotic strains for the production of fruit-whey beverages.

PubMed

Sady, Marek; Najgebauer-Lejko, Dorota; Domagała, Jacek

2017-01-01

When designing new probiotic products, one of the most important aspects is the selection of bacterial strains with high survival rates in the matrix of the product concerned. The aim of the present research was to evaluate the potential of selected strains of probiotic bacteria for the production of fruit-whey beverages. Orange, apple and blackcurrant whey beverages were produced, and each was inoculated with one of the following probiotic strains: Bifidobacterium lactis HN019TM; Lactobacillus aci- dophilus NCFM®; Lactobacillus paracasei Lpc-37TM; Lactobacillus rhamnosus HN001TM. The count of probiotic bacteria as well as pH and total acidity were evaluated at the 1st, 7th, 14th, 21st and 28th day of storage. Beverages containing L. paracasei Lpc-37TM or L. rhamnosus HN001TM were characterized by a sig- nificantly higher average number of viable cells (7.02 or 7.05 log cfu/g, respectively) than products with lactis HN019TM or L. acidophilus NCFM® (6.43 or 6.37 log cfu/g, respectively). The use of L. paracasei Lpc-37 and L. rhamnosus HN001 strains in orange and apple drinks allows the recommended count for probiotic products, 106 cfu/g for 28 days of storage, to be exceeded. Survival of the B. lactis HN019 strain fulfills the above requirements only in the orange drink. The L. acidophilus NCFM® strain was found to be the least suitable for the production of beverages, as it did not reach 6 log cfu/g in any products after 28 days of stor- age. The highest average number of bacteria was found in the orange beverages (7.14 log cfu/g). In terms of bacteria viability, blackcurrant juice was the least suitable for the production of whey probiotic drinks, due to its high acidity. The results of the present study indicate that careful selection of the fruit juice component, especially in terms of its acidity, is key to designing successful probiotic fruit-whey beverages. Other factors which should be taken into account to ensure a sufficient number of live

Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese.

PubMed

Cremonesi, Paola; Vanoni, Laura; Morandi, Stefano; Silvetti, Tiziana; Castiglioni, Bianca; Brasca, Milena

2011-03-30

A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10(3)CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species. Copyright © 2011 Elsevier B.V. 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.

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.

Antibacterial activity of selected standard strains of lactic acid bacteria producing bacteriocins--pilot study.

PubMed

Bodaszewska-Lubas, Malgorzata; Brzychczy-Wloch, Monika; Gosiewski, Tomasz; Heczko, Piotr B

2012-10-25

In this paper, an attempt was made to evaluate the antibacterial potential of standard strains of lactic acid bacteria (LAB) producing bacteriocins of various classes, thus demonstrating various mechanisms of cell membrane damages against the Streptococcus agalactiae strains (Group B Streptococcus, GBS), depending on surface polysaccharides and surface alpha-like protein genes. Antimicrobial property of the strains of L. plantarum C 11, L. sakei DSMZ 6333, and L. lactis ATCC 11454 producing bacteriocins: JK and EF plantaricins, sakacin and nisin, respectively, against the GBS strains was evaluated. The chosen to the study GBS strains were represented by serotypes Ia, Ib, II, III, V and they had bca, epsilon, rib, alp2 or alp3 alpha-like protein genes. The experiment was conducted by means of suspension culture and the bacteria count was determined using the serial dilution method. A great ability of L. plantarum C 11 strain was proven to inhibit the GBS growth. The strain of L. sakei DSMZ 6333 did not demonstrate any ability to inhibit the growth of GBS, whereas L. lactis ATCC 11454 inhibited the growth of S. agalactiae indicator strains to a minor extent. Statistically significant differences were demonstrated between the GBS strains representing various serotypes against the antimicrobial activity of model LAB strains. The least sensitive to the activity of bacteriocins were the strains representing serotypes Ib and III, whereas the strains representing serotype II were the most sensitive. The sensitivity of the GBS strains to the antimicrobial activity of LAB was not dependent on alpha-like protein genes. Among the LAB standard strains producing bacteriocins, the strongest antimicrobial property was observed in the strain of L. plantarum C 11. Because of the generally known and verified strong antagonistic property of the strains of L. plantarum species against indicator bacteria, it is necessary to further pursue the research presented in this paper.

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 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

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

Safety Characterization and Antimicrobial Properties of Kefir-Isolated Lactobacillus kefiri

PubMed Central

Carasi, Paula; Racedo, Silvia M.; De Antoni, Graciela; Urdaci, María C.

2014-01-01

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused α- or β-hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use. PMID:24955346

Safety characterization and antimicrobial properties of kefir-isolated Lactobacillus kefiri.

PubMed

Carasi, Paula; Díaz, Mariángeles; Racedo, Silvia M; De Antoni, Graciela; Urdaci, María C; Serradell, María de los Angeles

2014-01-01

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused α- or β-hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(-) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 10(8) CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.

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

Antibiotic susceptibility of bifidobacterial strains distributed in the Japanese market.

PubMed

Xiao, Jin-Zhong; Takahashi, Sachiko; Odamaki, Toshitaka; Yaeshima, Tomoko; Iwatsuki, Keiji

2010-01-01

The aim of the present study was to analyze the antibiotic susceptibility of bifidobacterial strains distributed in the Japanese market. A total of 23 strains, including probiotic isolates from foods, supplements, pharmaceuticals and reference strains of each species (or subspecies), were tested for susceptibility to 15 antibiotics by the broth microdilution method and examined for the presence of possible resistant determinants. The strains were susceptible overall to chloramphenicol, ampicillin, vancomycin and linezolid, and were intrinsically resistant to aminoglycoside group agents. Susceptibility to erythromycin, clindamycin, rifampicin, tetracycline and trimethoprim varied among the strains. All strains of Bifidobacterium animalis subsp. lactis were resistant to tetracycline and appeared to harbor tet(W) genes. No risk factor for safety was found for bifidobacterial strains distributed in the Japanese market in respect of their antimicrobial resistance, although the presence of the tet(W) gene in some strains stresses the need for future evaluation.

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.

Characterization of lactococci isolated from milk produced in the Camembert region of Normandy.

PubMed

Desmasures, N; Mangin, I; Corroler, D; Guéguen, M

1998-12-01

Thirty-eight Lactococcus strains, isolated from raw milk produced in two dairy areas in Normandy, were identified at the phenotypic level. Only Lactococcus lactis strains with the lactis phenotype were found in the milk samples. Most strains fermented lactose (97%) and showed proteinase activity (76%). Isolates were characterized by RAPD technique and rRNA gene restriction analysis. More L. lactis strains with the lactis genotype were found in the first area, while L. lactis strains with the cremoris genotype predominated in the second area. RAPD was more efficient than rRNA gene restriction analysis in differentiating between strains with the subsp. lactis genotype. For L. lactis with the subsp. cremoris genotype, the second method gave a better result but there was poor discrimination between strains. Plasmid profiles were determined. Patterns ranged in size from 1.3 to 16.5 kbp, and 29 different profiles were found. Six groups of strains were determined, five of which were specific for the area of origin. It is suggested that the region of manufacture could influence organoleptic properties of cheeses because of different Lactococcus strains in the raw milk used for cheese making.

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.

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.

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.

Lactobacillus plantarum isolated from kefir protects vero cells from cytotoxicity by type-II shiga toxin from Escherichia coli O157:H7.

PubMed

Kakisu, Emiliano; Abraham, Analía G; Farinati, Carla Tironi; Ibarra, Cristina; De Antoni, Graciela L

2013-02-01

Kefir is a fermented-milk beverage originating and widely consumed in the Caucasus as well as in Eastern Europe and is a source of bacteria with potential probiotic properties. Enterohaemorrhagic Escherichia coli producing Shiga toxin is commonly associated with food-transmitted diseases; the most prevalent serotype causing epidemics is Esch. coli O157:H7. The aim of this study was to evaluate the antagonism of Lactobacillus plantarum isolated from kefir against the action on Vero cells of supernatants of the Esch. coli O157:H7 strain 69160 expressing the type-II Shiga toxin (Stx2) and to study the role of the Lactobacillus cell wall in that inhibition. Spent culture supernatants of Esch. coli O157:H7 strain 69160 led to cytotoxic effects on cultured eukaryotic cells as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide-cleavage assay or by lactate-dehyrogenase release. Lb. plantarum CIDCA 83114 reduced the cytotoxic activity of Stx present in strain-69160 supernatants, and this protection was markedly higher than those of Lactobacillus kefir CIDCA 83113 and 8348 and Lb. delbrueckii subsp. bulgaricus CIDCA 333. This antagonism of cytotoxicity was mimicked by Lb. plantarum cell walls but was reduced after heating or protease treatments, thus indicating a protein or peptide as being involved in the protection mechanism. The cell surface of the lactobacilli bound the subunit B of Stx thereby decreasing the cytotoxicity. These interactions could constitute the first step in preventing the damage induced by Esch. coli O157:H7 supernatants, thus representing a valuable means of potentially mitigating the noxious effects of this food pathogen.

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) This enzyme preparation is derived from the nonpathogenic, nontoxicogenic yeast Kluyveromyces lactis... 683), which converts lactose to glucose and galactose. It is prepared from yeast that has been grown...

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

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.

Genomic features of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid.

PubMed

Shimizu-Kadota, Mariko; Kato, Hiroaki; Shiwa, Yuh; Oshima, Kenshiro; Machii, Miki; Araya-Kojima, Tomoko; Zendo, Takeshi; Hattori, Masahira; Sonomoto, Kenji; Yoshikawa, Hirofumi

2013-01-01

Lactococcus lactis IO-1 (JCM7638) produces L-lactic acid predominantly when grown at high xylose concentrations, and its utilization is highly desired in the green plastics industry. Therefore it is worthwhile studying its genomic traits. In this study, we focused on (i) genes of possible horizontal transfer derivation (prophages, the nisin-sucrose transposon, and several restriction-modification systems), and (ii) genes for the synthetic pathways of amino acids and vitamins in the IO-1 genome. In view of the results of this analysis, we consider their meanings in strain IO-1.

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.

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

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.

Growth, nisA Gene Expression, and In Situ Activity of Novel Lactococcus lactis subsp. cremoris Costarter Culture in Commercial Hard Cheese Production.

PubMed

Noutsopoulos, Dimitrios; Kakouri, Athanasia; Kartezini, Eleftheria; Pappas, Dimitrios; Hatziloukas, Efstathios; Samelis, John

2017-12-01

This study evaluated in situ expression of the nisA gene by an indigenous, nisin A-producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A-mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

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

Influence of Cofermentation by Amylolytic Lactobacillus plantarum and Lactococcus lactis Strains on the Fermentation Process and Rheology of Sorghum Porridge

PubMed Central

Byaruhanga, Yusuf B.; Muyanja, Charles M. B. K.; Aijuka, Matthew; Schüller, Reidar B.; Sahlstrøm, Stefan; Langsrud, Thor; Narvhus, Judith A.

2012-01-01

Amylolytic lactic acid bacteria (ALAB) can potentially replace malt in reducing the viscosity of starchy porridges. However, the drawback of using ALAB is their low and delayed amylolytic activity. This necessitates searching for efficient ALAB and strategies to improve their amylolytic activity. Two ALAB, Lactobacillus plantarum MNC 21 and Lactococcus lactis MNC 24, isolated from Obushera, were used to ferment starches in MRS broth: sorghum, millet, sweet potato, and commercial soluble starch. The amylolytic activity of MNC 21 was comparable to that of the ALAB collection strain Lb. plantarum A6, while that of MNC 24 was extremely low. MNC 21, MNC 24, and their coculture were compared to A6 and sorghum malt for ability to ferment and reduce the viscosity of sorghum porridge (11.6% dry matter). ALAB and the coculture lowered the pH from 6.2 to <4.5 within 12 h, while malt as a carrier of wild starter took about 20 h. Coculturing increased lactic acid yield by 46% and 76.8% compared to the yields of MNC 21 and MNC 24 monocultures, respectively. The coculture accumulated significantly larger (P < 0.05) amounts of maltose and diacetyl than the monocultures. Sorghum malt control and the coculture hydrolyzed more starch in sorghum porridge than the monocultures. The coculture initiated changes in the rheological parameters storage modulus (G′), loss modulus (G″), phase angle (δ), and complex viscosity (η*) earlier than its constituent monocultures. The shear viscosity of sorghum porridge was reduced significantly (P < 0.05) from 1950 cP to 110 cP (malt), 281 cP (coculture), 382 cP (MNC 21), 713 cP (MNC 24), and 722 cP (A6). Coculturing strong ALAB with weak ALAB or non-ALAB can be exploited for preparation of nutrient-dense weaning foods and increasing lactic acid yield from starchy materials. PMID:22610432

Influence of cofermentation by amylolytic Lactobacillus plantarum and Lactococcus lactis strains on the fermentation process and rheology of sorghum porridge.

PubMed

Mukisa, Ivan M; Byaruhanga, Yusuf B; Muyanja, Charles M B K; Aijuka, Matthew; Schüller, Reidar B; Sahlstrøm, Stefan; Langsrud, Thor; Narvhus, Judith A

2012-08-01

Amylolytic lactic acid bacteria (ALAB) can potentially replace malt in reducing the viscosity of starchy porridges. However, the drawback of using ALAB is their low and delayed amylolytic activity. This necessitates searching for efficient ALAB and strategies to improve their amylolytic activity. Two ALAB, Lactobacillus plantarum MNC 21 and Lactococcus lactis MNC 24, isolated from Obushera, were used to ferment starches in MRS broth: sorghum, millet, sweet potato, and commercial soluble starch. The amylolytic activity of MNC 21 was comparable to that of the ALAB collection strain Lb. plantarum A6, while that of MNC 24 was extremely low. MNC 21, MNC 24, and their coculture were compared to A6 and sorghum malt for ability to ferment and reduce the viscosity of sorghum porridge (11.6% dry matter). ALAB and the coculture lowered the pH from 6.2 to <4.5 within 12 h, while malt as a carrier of wild starter took about 20 h. Coculturing increased lactic acid yield by 46% and 76.8% compared to the yields of MNC 21 and MNC 24 monocultures, respectively. The coculture accumulated significantly larger (P < 0.05) amounts of maltose and diacetyl than the monocultures. Sorghum malt control and the coculture hydrolyzed more starch in sorghum porridge than the monocultures. The coculture initiated changes in the rheological parameters storage modulus (G'), loss modulus (G″), phase angle (δ), and complex viscosity (η*) earlier than its constituent monocultures. The shear viscosity of sorghum porridge was reduced significantly (P < 0.05) from 1950 cP to 110 cP (malt), 281 cP (coculture), 382 cP (MNC 21), 713 cP (MNC 24), and 722 cP (A6). Coculturing strong ALAB with weak ALAB or non-ALAB can be exploited for preparation of nutrient-dense weaning foods and increasing lactic acid yield from starchy materials.

Dual-Color Bioluminescence Imaging for Simultaneous Monitoring of the Intestinal Persistence of Lactobacillus plantarum and Lactococcus lactis in Living Mice

PubMed Central

Poiret, Sabine; Dennin, Véronique; Boutillier, Denise; Lacorre, Delphine Armelle; Foligné, Benoit; Pot, Bruno

2015-01-01

Lactic acid bacteria are found in the gastrointestinal tract of mammals and have received tremendous attention due to their health-promoting properties. We report the development of two dual-color luciferase-producing Lactobacillus (Lb.) plantarum and Lactococcus (Lc.) lactis strains for noninvasive simultaneous tracking in the mouse gastrointestinal tract. We previously described the functional expression of the red luciferase mutant (CBRluc) from Pyrophorus plagiophthalamus in Lb. plantarum NCIMB8826 and Lc. lactis MG1363 (C. Daniel, S. Poiret, V. Dennin, D. Boutillier, and B. Pot, Appl Environ Microbiol 79:1086–1094, 2013, http://dx.doi.org/10.1128/AEM.03221-12). In this study, we determined that CBRluc is a better-performing luciferase for in vivo localization of both lactic acid bacteria after oral administration than the green click beetle luciferase mutant construct developed in this study. We further established the possibility to simultaneously detect red- and green-emitting lactic acid bacteria by dual-wavelength bioluminescence imaging in combination with spectral unmixing. The difference in spectra of light emission by the red and green click beetle luciferase mutants and dual bioluminescence detection allowed in vitro and in vivo quantification of the red and green emitted signals; thus, it allowed us to monitor the dynamics and fate of the two bacterial populations simultaneously. Persistence and viability of both strains simultaneously administered to mice in different ratios was studied in vivo in anesthetized mice and ex vivo in mouse feces. The application of dual-luciferase-labeled bacteria has considerable potential to simultaneously study the interactions and potential competitions of different targeted bacteria and their hosts. PMID:26025906

The ltp gene of temperate Streptococcus thermophilus phage TP-J34 confers superinfection exclusion to Streptococcus thermophilus and Lactococcus lactis

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

Sun Xingmin; Goehler, Andre; Heller, Knut J.

2006-06-20

The ltp gene, located within the lysogeny module of temperate Streptococcus thermophilus phage TP-J34, has been shown to be expressed in lysogenic strain S. thermophilus J34. It codes for a lipoprotein, as demonstrated by inhibition of cleavage of the signal sequence by globomycin. Exposure of Ltp on the surface of Lactococcus lactis protoplasts bearing a plasmid-encoded copy of ltp has been demonstrated by immunogold labeling and electron microscopy. Expression of ltp in prophage- and plasmid-cured S. thermophilus J34-6f interfered with TP-J34 infection. While plating efficiency was reduced by a factor of about 40 and lysis of strain J34-6f in liquidmore » medium was delayed considerably, phage adsorption was not affected at all. Intracellular accumulation of phage DNA was shown to be inhibited by Ltp. This indicates interference of Ltp with infection at the stage of triggering DNA release and injection into the cell, indicating a role of Ltp in superinfection exclusion. Expression of ltp in L. lactis Bu2-60 showed that the same superinfection exclusion mechanism was strongly effective against phage P008, a member of the lactococcal 936 phage species: no plaque-formation was detectable with even 10{sup 9} phage per ml applied, and lysis in liquid medium did not occur. In Lactococcus also, Ltp apparently inhibited phage DNA release and/or injection. Ltp appears to be a member of a family of small, secreted proteins with a 42 amino acids repeat structure encoded by genes of Gram-positive bacteria. Some of these homologous genes are part of the genomes of prophages.« less

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.

Transport of beta-casein-derived peptides by the oligopeptide transport system is a crucial step in the proteolytic pathway of Lactococcus lactis.

PubMed

Kunji, E R; Hagting, A; De Vries, C J; Juillard, V; Haandrikman, A J; Poolman, B; Konings, W N

1995-01-27

In the proteolytic pathway of Lactococcus lactis, milk proteins (caseins) are hydrolyzed extracellularly to oligopeptides by the proteinase (PrtP). The fate of these peptides, i.e. extracellular hydrolysis followed by amino acid uptake or transport followed by intracellular hydrolysis, has been addressed. Mutants have been constructed that lack a functional di-tripeptide transport system (DtpT) and/or oligopeptide transport system (Opp) but do express the P1-type proteinase (specific for hydrolysis of beta- and to a lesser extent kappa-casein). The wild type strain and the DtpT- mutant accumulate all beta-casein-derived amino acids in the presence of beta-casein as protein substrate and glucose as a source of metabolic energy. The amino acids are not accumulated significantly inside the cells by the Opp- and DtpT- Opp- mutants. When cells are incubated with a mixture of amino acids mimicking the composition of beta-casein, the amino acids are taken up to the same extent in all four strains. Analysis of the extracellular peptide fraction, formed by the action of PrtP on beta-casein, indicates that distinct peptides disappear only when the cells express an active Opp system. These and other experiments indicate that (i) oligopeptide transport is essential for the accumulation of all beta-casein-derived amino acids, (ii) the activity of the Opp system is sufficiently high to support high growth rates on beta-casein provided leucine and histidine are present as free amino acids, and (iii) extracellular peptidase activity is not present in L. lactis.

Evaluation of technological properties of Enterococcus faecium CECT 8849, a strain isolated from human milk, for the dairy industry.

PubMed

Cárdenas, Nivia; Arroyo, Rebeca; Calzada, Javier; Peirotén, Ángela; Medina, Margarita; Rodríguez, Juan Miguel; Fernández, Leonides

2016-09-01

In this work, a variety of biochemical properties of Enterococcus faecium CECT 8849, which had been isolated from breast milk, were analyzed. Its acidifying capacity and proteolytic activity were low but, in contrast, remarkable peptidase and esterase activities were observed. Ethanol and 3-hydroxy-2-butanone were the most abundant volatile compounds found in experimental model cheese manufactured with E. faecium CECT 8849. This strain inhibited the growth of several Listeria monocytogenes and Listeria innocua strains in vitro. Enterocin A and B structural genes were detected in E. faecium CECT 8849. Model fermented milk and cheeses were manufactured from milk inoculated or not with L. innocua CECT 8848 (2.5-3 log10 colony forming units mL(-1)) using E. faecium CECT 8849 or Lactococcus lactis ESI 153 as starter cultures. Although E. faecium CECT 8849 controlled Listeria growth in both dairy models, it led to lower reduction in Listeria counts when compared with L. lactis ESI 153.

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

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.

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).

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.

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.

Potential of functional strains, isolated from traditional Maasai milk, as starters for the production of fermented milks.

PubMed

Patrignani, Francesca; Lanciotti, Rosalba; Mathara, Julius Maina; Guerzoni, Maria Elisabetta; Holzapfel, Wilhelm H

2006-03-01

The purpose of this research was the evaluation of technological features and of the ability of functional LAB strains with desirable sensory characteristics, to produce fermented milk. Eight strains of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei and Lactococcus lactis, isolated from Maasai traditional fermented milk in Kenya and previously tested for their probiotic properties, were selected for this investigation. Technological features such as growth kinetics in fresh heat-treated whole milk medium and survival in the final product during storage at 4 degrees C, were studied. The strains Lb. acidophilus BFE 6,059, Lb. paracasei BFE 5,264 and Lc. lactis BFE 6,049 showed the best potential and were thus selected for use as starter cultures in further trials with the objective to improve their technological performance and to optimise the sensory features of fermented milk obtained. The effects of fat (F), non-fat milk solids (S) and fermentation temperature (T), modulated according to a Central Composite Design, on fermentation rates and viability losses during refrigerated storage of the chosen starters, and on product texture parameters, were studied. From the data analysis, it was possible to select optimum conditions for enhancing positive sensory traits of final products and for improving the survival of these potentially probiotic cultures.

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.

Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Spray-Drying

PubMed Central

Sosa, Natalia; Gerbino, Esteban; Golowczyc, Marina A.; Schebor, Carolina; Gómez-Zavaglia, Andrea; Tymczyszyn, E. Elizabeth

2016-01-01

In this work maltodextrins were added to commercial galacto-oligosaccharides (GOS) in a 1:1 ratio and their thermophysical characteristics were analyzed. GOS:MD solutions were then used as matrices during spray-drying of Lactobacillus plantarum CIDCA 83114. The obtained powders were equilibrated at different relative humidities (RH) and stored at 5 and 20°C for 12 weeks, or at 30°C for 6 weeks. The Tgs of GOS:MD matrices were about 20–30°C higher than those of GOS at RH within 11 and 52%. A linear relation between the spin-spin relaxation time (T2) and T-Tg parameter was observed for GOS:MD matrices equilibrated at 11, 22, 33, and 44% RH at 5, 20, and 30°C. Spray-drying of L. plantarum CIDCA 83114 in GOS:MD matrices allowed the recovery of 93% microorganisms. In contrast, only 64% microorganisms were recovered when no GOS were included in the dehydration medium. Survival of L. plantarum CIDCA 83114 during storage showed the best performance for bacteria stored at 5°C. In a further step, the slopes of the linear regressions provided information about the rate of microbial inactivation for each storage condition (k values). This information can be useful to calculate the shelf-life of spray-dried starters stored at different temperatures and RH. Using GOS:MD matrices as a dehydration medium enhanced the recovery of L. plantarum CIDCA 83114 after spray-drying. This strategy allowed for the first time the spray-drying stabilization of a potentially probiotic strain in the presence of GOS. PMID:27199918

DNA–DNA hybridization study of strains of Chryseobacterium, Elizabethkingia and Empedobacter and of other usually indole-producing non-fermenters of CDC groups IIc, IIe, IIh and IIi, mostly from human clinical sources, and proposals of Chryseobacterium bernardetii sp. nov., Chryseobacterium carnis sp. nov., Chryseobacterium lactis sp. nov., Chryseobacterium nakagawai sp. nov. and Chryseobacterium taklimakanense comb. nov

PubMed Central

Holmes, B.; Steigerwalt, A. G.; Nicholson, A. C.

2015-01-01

The taxonomic classification of 182 phenotypically similar isolates was evaluated using DNA–DNA hybridization and 16S rRNA gene sequence analysis. These bacterial isolates were mainly derived from clinical sources; all were Gram-negative non-fermenters and most were indoleproducing. Phenotypically, they resembled species from the genera Chryseobacterium, Elizabethkingia or Empedobacter or belonged to CDC groups IIc, IIe, IIh and IIi. Based on these analyses, four novel species are described: Chryseobacterium bernardetii sp. nov. (type strain NCTC 13530T=CCUG 60564T=CDC G229T), Chryseobacterium carnis sp. nov. (type strain NCTC 13525T=CCUG 60559T=CDC G81T), Chryseobacterium lactis sp. nov. (type strain NCTC 11390T=CCUG 60566T=CDC KC1864T) and Chryseobacterium nakagawai sp. nov. (type strain NCTC 13529T=CCUG 60563T=CDC G41T). The new combination Chryseobacterium taklimakanense comb. nov. (type strain NCTC 13490T=X-65T=CCTCC AB 208154T=NRRL B-51322T) is also proposed to accommodate the reclassified Planobacterium taklimakanense. PMID:23934253

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.

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.

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.

Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic.

PubMed

Mirkovic, Nemanja; Polovic, Natalija; Vukotic, Goran; Jovcic, Branko; Miljkovic, Marija; Radulovic, Zorica; Diep, Dzung B; Kojic, Milan

2016-04-01

Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins.Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes,lmgA ,lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes,lmgF,lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization-time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic

PubMed Central

Mirkovic, Nemanja; Polovic, Natalija; Vukotic, Goran; Jovcic, Branko; Miljkovic, Marija; Radulovic, Zorica; Diep, Dzung B.

2016-01-01

Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins. Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes, lmgA, lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes, lmgF, lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization–time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid. PMID:26896142

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.

The impact of a consortium of fermented milk strains on the gut microbiome of gnotobiotic mice and monozygotic twins

PubMed Central

McNulty, Nathan P.; Yatsunenko, Tanya; Hsiao, Ansel; Faith, Jeremiah J.; Muegge, Brian D.; Goodman, Andrew L.; Henrissat, Bernard; Oozeer, Raish; Cools-Portier, Stéphanie; Gobert, Guillaume; Chervaux, Christian; Knights, Dan; Lozupone, Catherine A.; Knight, Rob; Duncan, Alexis E.; Bain, James R.; Muehlbauer, Michael J.; Newgard, Christopher B.; Heath, Andrew C.; Gordon, Jeffrey I.

2012-01-01

Understanding how the human gut microbiota and host are impacted by probiotic bacterial strains requires carefully controlled studies in humans and in mouse models of the gut ecosystem where potentially confounding variables that are difficult to control in humans can be constrained. Therefore, we characterized the fecal microbiomes and metatranscriptomes of adult female monozygotic twin pairs through repeated sampling 4 weeks prior to, 7 weeks during, and 4 weeks following consumption of a commercially available fermented milk product (FMP) containing a consortium of Bifidobacterium animalis subsp. lactis, two strains of Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis subsp. cremoris, and Streptococcus thermophilus. In addition, gnotobiotic mice harboring a 15-species model human gut microbiota whose genomes contain 58,399 known or predicted protein-coding genes were studied prior to and after gavage with all five sequenced FMP strains. No significant changes in bacterial species composition or in the proportional representation of genes encoding known enzymes were observed in the feces of humans consuming the FMP. Only minimal changes in microbiota configuration were noted in mice following single or repeated gavage with the FMP consortium. However, RNA-Seq analysis of fecal samples and follow-up mass spectrometry of urinary metabolites disclosed that introducing the FMP strains into mice results in significant changes in expression of microbiome-encoded enzymes involved in numerous metabolic pathways, most prominently those related to carbohydrate metabolism. B. animalis subsp. lactis, the dominant persistent member of the FMP consortium in gnotobiotic mice, upregulates a locus in vivo that is involved in the catabolism of xylooligosaccharides, a class of glycans widely distributed in fruits, vegetables and other foods, underscoring the importance of these sugars to this bacterial species. The human fecal metatranscriptome exhibited significant

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

Characterisation of the Poly-(Vinylpyrrolidone)-Poly-(Vinylacetate-Co-Crotonic Acid) (PVP:PVAc-CA) Interpolymer Complex Matrix Microparticles Encapsulating a Bifidobacterium lactis Bb12 Probiotic Strain.

PubMed

Mamvura, C I; Moolman, F S; Kalombo, L; Hall, A N; Thantsha, M S

2011-06-01

The method of producing poly-(vinylpyrrolidone)-poly-(vinylacetate-co-crotonic acid) (PVP:PVAc-CA) interpolymer complex matrix microparticles in supercritical carbon dioxide (scCO2), encapsulating bacteria, has recently been developed. This study was aimed at probing the external and internal structure of these microparticles, which can be used in food. The encapsulation efficiency and distribution of encapsulated Bifidobacterium lactis Bb12 within these microparticles were also investigated. Scanning electron microscopy (SEM) revealed irregular, mostly small, smooth microparticles with no visible bacterial cells on the surface. However, some of the microparticles appeared to have porous surfaces. The results of a Microtrac S3500 particle size analyzer showed that the PVP:PVAc-CA interpolymer complex matrix microparticles encapsulating B. lactis Bb12 had an average particle size of 166.1 μm (<350 μm designated standard size for microparticles). The D 10, D 50 and D 90 values for these microparticles were 48.16, 166.06 and 382.55 μm, respectively. Both SEM and confocal laser scanning microscopy showed a high density of bacterial cells within the microparticles. An average encapsulation efficiency of 96% was achieved. Consequently, the microparticles have the potential to be evenly distributed in foods, deliver adequate amounts of probiotics and produce minimal adverse effects on the texture and mouth feel of the foods into which they are incorporated.

Proteolytic enzyme activities in Cheddar cheese juice made using lactococcal starters of differing autolytic properties.

PubMed

Sheehan, A; Cuinn, G O'; Fitzgerald, R J; Wilkinson, M G

2006-04-01

To determine proteolytic enzyme activities released in Cheddar cheese juice manufactured using lactococcal starter strains of differing autolytic properties. The activities of residual chymosin, cell envelope proteinase and a range of intracellular proteolytic enzymes were determined during the first 70 days of ripening when starter lactococci predominate the microbial flora. In general, in cell free extracts (CFE) of the strains, the majority of proteolytic activities was highest for Lactococcus lactis HP, intermediate for L. lactis AM2 and lowest for L. lactis 303. However, in cheese juice, as ripening progressed, released proteolytic activities were highest for the highly autolytic strain L. lactis AM2, intermediate for L. lactis 303 and lowest for L. lactis HP. These results indicate that strain related differences in autolysis influence proteolytic enzyme activities released into Cheddar cheese during ripening. No correlation was found between proteolytic potential of the starter strains measured in CFE prior to cheese manufacture and levels of activities released in cheese juice. The findings further support the importance of autolysis of lactococcal starters in determining the levels of proteolytic activities present in cheese during initial stages of ripening.

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.

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

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

DNA-DNA hybridization study of strains of Chryseobacterium, Elizabethkingia and Empedobacter and of other usually indole-producing non-fermenters of CDC groups IIc, IIe, IIh and IIi, mostly from human clinical sources, and proposals of Chryseobacterium bernardetii sp. nov., Chryseobacterium carnis sp. nov., Chryseobacterium lactis sp. nov., Chryseobacterium nakagawai sp. nov. and Chryseobacterium taklimakanense comb. nov.

PubMed

Holmes, B; Steigerwalt, A G; Nicholson, A C

2013-12-01

The taxonomic classification of 182 phenotypically similar isolates was evaluated using DNA-DNA hybridization and 16S rRNA gene sequence analysis. These bacterial isolates were mainly derived from clinical sources; all were Gram-negative non-fermenters and most were indole-producing. Phenotypically, they resembled species from the genera Chryseobacterium, Elizabethkingia or Empedobacter or belonged to CDC groups IIc, IIe, IIh and IIi. Based on these analyses, four novel species are described: Chryseobacterium bernardetii sp. nov. (type strain NCTC 13530(T) = CCUG 60564(T) = CDC G229(T)), Chryseobacterium carnis sp. nov. (type strain NCTC 13525(T) = CCUG 60559(T) = CDC G81(T)), Chryseobacterium lactis sp. nov. (type strain NCTC 11390(T) = CCUG 60566(T) = CDC KC1864(T)) and Chryseobacterium nakagawai sp. nov. (type strain NCTC 13529(T) = CCUG 60563(T) = CDC G41(T)). The new combination Chryseobacterium taklimakanense comb. nov. (type strain NCTC 13490(T) = X-65(T) = CCTCC AB 208154(T) = NRRL B-51322(T)) is also proposed to accommodate the reclassified Planobacterium taklimakanense.

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.

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.

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.

Ultraviolet light-induced mutants of Streptococcus lactis subspecies diacetylactis with enhanced acid- or flavor-producing abilities

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

Kuila, R.K.; Ranganathan, B.

1978-04-01

A strain of Streptococcus lactis subspecies diacetylactis S/sub 1/ isolated from fresh milk was exposed to 7200 ergs/mm/sup 2/ of ultraviolet radiation. Over 8100 colonies surviving from 7.4 x 10/sup 6/ cells exposed to radiation were screened on citrate agar for detection and isolation of mutants with increased flavor and/or acid production. Of the survivors, 960 were type-I mutants that exhibited clear zone on citrate agar after 18 h (presumed to be high diacetyl producers), and 288 were type-II mutants which did not exhibit clear zones on citrate agar for up to 72 h (high acid producers). Type-II mutants producedmore » an average .93 percent titratable acidity which was 34 percent more than the .69 percent of the parent. Reduction in titratable acidity (56 percent less) was considerable in type-I mutants, compared with the parent culture. Diacetyl + acetoin production by type-I mutants was 137.9 ppM which has 4.5 times more than that of the parental strain. Acetaldehyde production in the mutants varied from 1.5 to 34.5 ppM (parent culture 3.0 ppM). The mutants with increased acid and high acetoin plus diacetyl production were stable after 50 subcultures in milk.« less

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...

Altering textural properties of fermented milk by using surface-engineered Lactococcus lactis.

PubMed

Tarazanova, Mariya; Huppertz, Thom; Kok, Jan; Bachmann, Herwig

2018-05-09

Lactic acid bacteria are widely used for the fermentation of dairy products. While bacterial acidification rates, proteolytic activity and the production of exopolysaccharides are known to influence textural properties of fermented milk products, little is known about the role of the microbial surface on microbe-matrix interactions in dairy products. To investigate how alterations of the bacterial cell surface affect fermented milk properties, 25 isogenic Lactococcus lactis strains that differed with respect to surface charge, hydrophobicity, cell chaining, cell-clumping, attachment to milk proteins, pili expression and EPS production were used to produce fermented milk. We show that overexpression of pili increases surface hydrophobicity of various strains from 3-19% to 94-99%. A profound effect of different cell surface properties was an altered spatial distribution of the cells in the fermented product. Aggregated cells tightly fill the cavities of the protein matrix, while chaining cells seem to be localized randomly. A positive correlation was found between pili overexpression and viscosity and gel hardness of fermented milk. Gel hardness also positively correlated with clumping of cells in the fermented milk. Viscosity of fermented milk was also higher when it was produced with cells with a chaining phenotype or with cells that overexpress exopolysaccharides. Our results show that alteration of cell surface morphology affects textural parameters of fermented milk and cell localization in the product. This is indicative of a cell surface-dependent potential of bacterial cells as structure elements in fermented foods. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

PubMed Central

Overkamp, Wout; Beilharz, Katrin; Detert Oude Weme, Ruud; Solopova, Ana; Karsens, Harma; Kovács, Ákos T.; Kok, Jan

2013-01-01

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria. PMID:23956387

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

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®

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.

16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae, Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis.

PubMed

Blaiotta, Giuseppe; Pepe, Olimpia; Mauriello, Gianluigi; Villani, Francesco; Andolfi, Rosamaria; Moschetti, Giancarlo

2002-12-01

The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.

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.

Leuconostoc strains isolated from dairy products: Response against food stress conditions.

PubMed

D'Angelo, Luisa; Cicotello, Joaquín; Zago, Miriam; Guglielmotti, Daniela; Quiberoni, Andrea; Suárez, Viviana

2017-09-01

A systematic study about the intrinsic resistance of 29 strains (26 autochthonous and 3 commercial ones), belonging to Leuconostoc genus, against diverse stress factors (thermal, acidic, alkaline, osmotic and oxidative) commonly present at industrial or conservation processes were evaluated. Exhaustive result processing was made by applying one-way ANOVA, Student's test (t), multivariate analysis by Principal Component Analysis (PCA) and Matrix Hierarchical Cluster Analysis. In addition, heat adaptation on 4 strains carefully selected based on previous data analysis was assayed. The strains revealed wide diversity of resistance to stress factors and, in general, a clear relationship between resistance and Leuconostoc species was established. In this sense, the highest resistance was shown by Leuconostoc lactis followed by Leuconostoc mesenteroides strains, while Leuconostoc pseudomesenteroides and Leuconostoc citreum strains revealed the lowest resistance to the stress factors applied. Heat adaptation improved thermal cell survival and resulted in a cross-resistance against the acidic factor. However, all adapted cells showed diminished their oxidative resistance. According to our knowledge, this is the first study regarding response of Leuconostoc strains against technological stress factors and could establish the basis for the selection of "more robust" strains and propose the possibility of improving their performance during industrial processes. Copyright © 2017 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.

Genome analysis of food-processing stressful-resistant probiotic Bifidobacterium animalis subsp. lactis BF052, and its potential application in fermented soymilk.

PubMed

Charnchai, Pattra; Jantama, Sirima Suvarnakuta; Jantama, Kaemwich

2017-09-15

In this study, Bifidobacterium animalis subsp. lactis BF052 was demonstrated the growth capability in soymilk and could be thus supplemented as a probiotic starter that employed soymilk as one of its food vehicles. The complete genome sequence of BF052 was therefore determined to understand the genetic basis of BF052 as a technological and functional probiotic starter. The whole genome sequence of BF052 consists of a circular genome of 1938 624 bp with a G+C content of 60.50%. This research highlights relevant genes involving in its adaptive responses to industrial and/or environmental stresses and utilization of α-galacto-oligosaccharides in BF052 strain compared with other representative bifidobacterial genomes. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effect of the consumption of a fermented dairy product containing Bifidobacterium lactis DN-173 010 on constipation in childhood: a multicentre randomised controlled trial (NTRTC: 1571).

PubMed

Tabbers, Merit M; Chmielewska, Ania; Roseboom, Maaike G; Boudet, Claire; Perrin, Catherine; Szajewska, Hania; Benninga, Marc A

2009-03-18

Constipation is a frustrating symptom affecting 3% of children worldwide. Randomised controlled trials show that both polyethylene glycol and lactulose are effective in increasing defecation frequency in children with constipation. However, in 30-50%, these children reported abdominal pain, bloating, flatulence, diarrhoea, nausea and bad taste of the medication. Two recent studies have shown that the fermented dairy product containing Bifidobacterium lactis strain DN-173 010 is effective in increasing stool frequency in constipation-predominant irritable bowel syndrome patients with a defecation frequency < 3/week and in constipated women with a defecation frequency < 3/week. Goal of this study is to determine whether this fermented dairy product is effective in the treatment of constipated children with a defecation frequency < 3/week. It is a two nation (The Netherlands and Poland) double-blind, placebo-controlled randomised multicentre trial in which 160 constipated children (age 3-16 years) with a defecation frequency <3/week will be randomly allocated to consume a fermented dairy product containing Bifidobacterium lactis DN-173 010 or a control product, twice a day, for 3 weeks. During the study all children are instructed to try to defecate on the toilet for 5-10 minutes after each meal (3 times a day) and daily complete a standardized bowel diary. Primary endpoint is stool frequency. Secondary endpoints are stool consistency, faecal incontinence frequency, pain during defecation, digestive symptoms (abdominal pain, flatulence), adverse effects (nausea, diarrhoea, bad taste) and intake of rescue medication (Bisacodyl). Rate of success and rate of responders are also evaluated, with success defined as > or = 3 bowel movements per week and < or =1 faecal incontinence episode over the last 2 weeks of product consumption and responder defined as a subject reporting a stool frequency > or = 3 on the last week of product consumption. To demonstrate that the success

Effect of the consumption of a fermented dairy product containing Bifidobacterium lactis DN-173 010 on constipation in childhood: a multicentre randomised controlled trial (NTRTC: 1571)

PubMed Central

Tabbers, Merit M; Chmielewska, Ania; Roseboom, Maaike G; Boudet, Claire; Perrin, Catherine; Szajewska, Hania; Benninga, Marc A

2009-01-01

Background Constipation is a frustrating symptom affecting 3% of children worldwide. Randomised controlled trials show that both polyethylene glycol and lactulose are effective in increasing defecation frequency in children with constipation. However, in 30–50%, these children reported abdominal pain, bloating, flatulence, diarrhoea, nausea and bad taste of the medication. Two recent studies have shown that the fermented dairy product containing Bifidobacterium lactis strain DN-173 010 is effective in increasing stool frequency in constipation-predominant irritable bowel syndrome patients with a defecation frequency < 3/week and in constipated women with a defecation frequency < 3/week. Goal of this study is to determine whether this fermented dairy product is effective in the treatment of constipated children with a defecation frequency < 3/week. Methods/design It is a two nation (The Netherlands and Poland) double-blind, placebo-controlled randomised multicentre trial in which 160 constipated children (age 3–16 years) with a defecation frequency <3/week will be randomly allocated to consume a fermented dairy product containing Bifidobacterium lactis DN-173 010 or a control product, twice a day, for 3 weeks. During the study all children are instructed to try to defecate on the toilet for 5–10 minutes after each meal (3 times a day) and daily complete a standardized bowel diary. Primary endpoint is stool frequency. Secondary endpoints are stool consistency, faecal incontinence frequency, pain during defecation, digestive symptoms (abdominal pain, flatulence), adverse effects (nausea, diarrhoea, bad taste) and intake of rescue medication (Bisacodyl). Rate of success and rate of responders are also evaluated, with success defined as ≥ 3 bowel movements per week and ≤1 faecal incontinence episode over the last 2 weeks of product consumption and responder defined as a subject reporting a stool frequency ≥ 3 on the last week of product consumption. To

Assessment of probiotic properties of lactic acid bacteria isolated from Indonesian naturally fermented milk

NASA Astrophysics Data System (ADS)

Jatmiko, Yoga Dwi; Howarth, Gordon S.; Barton, Mary D.

2017-11-01

This study aimed to characterize the probiotic properties of lactic acid bacteria from the naturally fermented milk of Indonesia, namely dangke and dadih. Fifty-one representative lactic acid bacteria belonging to the species Lactobacillus Plantarum, Lactococcus lactis subsp. lactis and Enterococcus faecium were evaluated in vitro for potential probiotic properties based on their bile salt resistance, low pH tolerance, antimicrobial activity, antibiotic susceptibility and adherence to Caco-2 colon cancer cells. In addition, bacteriocin related gene (plantaricin A), bile salt hydrolase (bsh) and mannose-specific adhesin (msa) genes in the genome of lactobacilli were also examined. None of the dangke isolates, which belonged to the species L. lactis subsp. lactis tolerated low pH. However, eight of the isolates were able to grow in the presence of bile salts. It was observed that L. plantarum strain S1.30 and SL2.7 from dadih tolerated low pH, survived bile salt concentrations and were resistant to vancomycin. Furthermore, these strains also contained bacteriocin regulating gene (plantaricin A) and msa and bsh genes in their genome. However, only the strain S1.30 exhibited optimal antimicrobial activity against the selected pathogens and was able to adhere to Caco-2 cells by up to 82.24±0.14%. Antagonistic activity of L. lactis subsp. lactis from dadih and dangke was not detected. However, 73.94±1.26% adherence to Caco-2 cells was demonstrated by L. lactis subsp. lactis strain SL3.34 sourced from dangke. These results suggest that Lactobacillus plantarum strain S1.30 associated with dadih fulfilled the in vitro probiotic criteria and could be exploited for further in vivo evaluation. In addition, dadih was an effective probiotic carrier compared to dangke.

Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice.

PubMed

Shigemori, Suguru; Watanabe, Takafumi; Kudoh, Kai; Ihara, Masaki; Nigar, Shireen; Yamamoto, Yoshinari; Suda, Yoshihito; Sato, Takashi; Kitazawa, Haruki; Shimosato, Takeshi

2015-11-25

Mucosal delivery of therapeutic proteins using genetically modified strains of lactic acid bacteria (gmLAB) is being investigated as a new therapeutic strategy. We developed a strain of gmLAB, Lactococcus lactis NZ9000 (NZ-HO), which secretes the anti-inflammatory molecule recombinant mouse heme oxygenase-1 (rmHO-1). The effects of short-term continuous oral dosing with NZ-HO were evaluated in mice with dextran sulfate sodium (DSS)-induced acute colitis as a model of inflammatory bowel diseases (IBD). We identified the secretion of rmHO-1 by NZ-HO. rmHO-1 was biologically active as determined with spectroscopy. Viable NZ-HO was directly delivered to the colon via oral administration, and rmHO-1 was secreted onto the colonic mucosa in mice. Acute colitis in mice was induced by free drinking of 3 % DSS in water and was accompanied by an increase in the disease activity index score and histopathological changes. Daily oral administration of NZ-HO significantly improved these colitis-associated symptoms. In addition, NZ-HO significantly increased production of the anti-inflammatory cytokine interleukin (IL)-10 and decreased the expression of pro-inflammatory cytokines such as IL-1α and IL-6 in the colon compared to a vector control strain. Oral administration of NZ-HO alleviates DSS-induced acute colitis in mice. Our results suggest that NZ-HO may be a useful mucosal therapeutic agent for treating IBD.

In vitro evaluation of the probiotic and functional potential of Lactobacillus strains isolated from fermented food and human intestine.

PubMed

Ren, Dayong; Li, Chang; Qin, Yanqing; Yin, Ronglan; Du, Shouwen; Ye, Fei; Liu, Cunxia; Liu, Hongfeng; Wang, Maopeng; Li, Yi; Sun, Yang; Li, Xiao; Tian, Mingyao; Jin, Ningyi

2014-12-01

This study aims to evaluate the functional and probiotic characteristics of eight indigenous Lactobacillus strains in vitro. The selected lactobacilli include strains of Lactobacillus casei subsp. casei, Lactobacillus salivarius subsp. salicinius, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus rhamnosus. All strains tolerated both pH 2 for 3 h and 1% bile salt for 24 h. The strains CICC 23174 and CGMCC 1.557 were the most adhesive strains producing the highest quantity of EPS. Although a wide variation in the ability of the eight strains to deplete cholesterol and nitrite, antagonize pathogens, scavenge free radical, and stimulate innate immune response were observed, the strains CICC 23174 and CGMCC 1.557 showed the widest range of these useful traits. Taken together, the strains CICC 23174 and CGMCC 1.557 exhibited the best probiotic properties with the potential for use in the production of probiotic fermented foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

Impact of bile salt adaptation of Lactobacillus delbrueckii subsp. lactis 200 on its interaction capacity with the gut.

PubMed

Burns, Patricia; Reinheimer, Jorge; Vinderola, Gabriel

2011-10-01

In a previous work, bile-salt-resistant derivatives were obtained from non-intestinal lactobacilli. The aim of this work was to investigate the impact of bile adaptation of Lactobacillus delbrueckii subsp. lactis 200 on morphology, surface properties, in vivo interaction capacity with the gut and ability to activate the gut immune response. Electron microscopy studies, growth kinetics in the presence of bovine and porcine bile, the capacity to deconjugate bile acids, hydrophobicity, autoaggregation and co-aggregation capacities were studied for the parental strain and its bile-resistant derivative in vitro. Additionally, survival in intestinal fluid, the interaction with the gut and the immunomodulating capacities were studied in mice. Bile salt adaptation conferred upon the adapted strain a higher capacity to withstand physiological concentrations of bile salts and greater survival capacity in intestinal fluid. However, bile salt exposure reduced cell hydrophobicity, autoaggregation and adhesion capacities, resulting in reduced persistence in the intestinal lumen and delayed capacity to activate the gut immune response. Insight into the effects of bile salts upon the interaction and immunomodulating capacity of lactobacilli with the gut is provided, relating in vitro and in vivo results. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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

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

In Vitro Characterization of Lactic Acid Bacteria Isolated from Bovine Milk as Potential Probiotic Strains to Prevent Bovine Mastitis.

PubMed

Pellegrino, Matías S; Frola, Ignacio D; Natanael, Berardo; Gobelli, Dino; Nader-Macias, María E F; Bogni, Cristina I

2018-01-02

Bovine mastitis causes economic losses on dairy farms worldwide. Lactic acid bacteria (LAB) in animal health are an alternative tool to avoid antibiotic therapy on the prevention of bovine mastitis. In previous studies, 12 LAB isolated from bovine milk were selected taking into account some of the following characteristics: hydrophobicity, auto aggregative capability, inhibition of indicator pathogens, hydrogen peroxide, and capsular polysaccharide production. These LAB were considered because of their beneficial properties. In this work, we also analyzed the antimicrobial activity and the co-aggregation against mastitis causing bacteria, auto-inhibition, adhesion to bovine teat canal epithelial cells (BTCEC), and growth kinetic curves for the 12 LAB. Two of them, Lactococcus lactis subsp. lactis CRL 1655 and Lactobacillus perolens CRL 1724, were selected because they had an interesting pattern of adhesion to BTEC, the inhibition of pathogens and the co-aggregation with the 100% of the assayed pathogens. They showed a predictable difference in the PFGE genomic pattern bands. The kinetic growth of these two strains was similar between them and with the rest of the assayed LAB. The strains selected in the present study showed indispensable characteristics for their inclusion in a probiotic formulation to be used at dry-off period for the prevention of bovine mastitis.

Novel Phage Group Infecting Lactobacillus delbrueckii subsp. lactis, as Revealed by Genomic and Proteomic Analysis of Bacteriophage Ldl1

PubMed Central

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio

2014-01-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 ± 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species. PMID:25501478

Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

PubMed

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2015-02-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

Lacticin LC14, a new bacteriocin produced by Lactococcus lactis BMG6.14: isolation, purification and partial characterization.

PubMed

Lasta, Samar; Ouzari, Hadda; Andreotti, Nicolas; Fajloun, Ziad; Mansuelle, Pascal; Boudabous, Abdellatif; Sampieri, Francois; Sabatier, Jean Marc

2012-08-01

A new bacteriocin, lacticin LC14, produced by Lactococcus lactis BMG6.14, was isolated and characterized. It was purified to homogeneity from overnight broth culture by ammonium sulfate precipitation, Sep-Pak chromatography, and two steps of reversed-phase HPLC. Lacticin LC14 showed bactericidal-type antimicrobial activity against several lactic acid bacteria and pathogenic strains including Listeria monocytogenes. It was inactivated by proteinase K and pronase E, but was resistant to papain, lysozyme, lipase and catalase. Lacticin LC14 was heat resistant, stable over a wide range of pH (2-10) and after treatment by solvents and detergents. Its N-terminal end was found unreactive towards Edman sequencing. Based on MALDI-TOF mass spectrometry, its molecular mass was 3333.7 Da. LC14 amino acid composition revealed a high proportion of hydrophobic residues, but no modified ones. LC14 may be able to challenge other well known other bacteriocins in probiotic and therapeutic applications.

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.

Technical note: development of a quantitative PCR method for monitoring strain dynamics during yogurt manufacture.

PubMed

Miller, D M; Dudley, E G; Roberts, R F

2012-09-01

Yogurt starter cultures may consist of multiple strains of Lactobacillus delbrueckii ssp. bulgaricus (LB) and Streptococcus thermophilus (ST). Conventional plating methods for monitoring LB and ST levels during yogurt manufacture do not allow for quantification of individual strains. The objective of the present work was to develop a quantitative PCR method for quantification of individual strains in a commercial yogurt starter culture. Strain-specific primers were designed for 2 ST strains (ST DGCC7796 and ST DGCC7710), 1 LB strain (DGCC4078), and 1 Lactobacillus delbrueckii ssp. lactis strain (LL; DGCC4550). Primers for the individual ST and LB strains were designed to target unique DNA sequences in clustered regularly interspersed short palindromic repeats. Primers for LL were designed to target a putative mannitol-specific IIbC component of the phosphotransferase system. Following evaluation of primer specificity, standard curves relating cell number to cycle threshold were prepared for each strain individually and in combination in yogurt mix, and no significant differences in the slopes were observed. Strain balance data was collected for yogurt prepared at 41 and 43°C to demonstrate the potential application of this method. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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.

A Multibacteriocin Cheese Starter System, Comprising Nisin and Lacticin 3147 in Lactococcus lactis, in Combination with Plantaricin from Lactobacillus plantarum

PubMed Central

Mills, S.; Griffin, C.; O'Connor, P. M.; Serrano, L. M.; Meijer, W. C.; Hill, C.

2017-01-01

ABSTRACT Functional starter cultures demonstrating superior technological and food safety properties are advantageous to the food fermentation industry. We evaluated the efficacies of single- and double-bacteriocin-producing starters of Lactococcus lactis capable of producing the class I bacteriocins nisin A and/or lacticin 3147 in terms of starter performance. Single producers were generated by mobilizing the conjugative bacteriophage resistance plasmid pMRC01, carrying lacticin genetic determinants, or the conjugative transposon Tn5276, carrying nisin genetic determinants, to the commercial starter L. lactis CSK2775. The effect of bacteriocin coproduction was examined by superimposing pMRC01 into the newly constructed nisin transconjugant. Transconjugants were improved with regard to antimicrobial activity and bacteriophage insensitivity compared to the recipient strain, and the double producer was immune to both bacteriocins. Bacteriocin production in the starter was stable, although the recipient strain proved to be a more efficient acidifier than transconjugant derivatives. Overall, combinations of class I bacteriocins (the double producer or a combination of single producers) proved to be as effective as individual bacteriocins for controlling Listeria innocua growth in laboratory-scale cheeses. However, using the double producer in combination with the class II bacteriocin producer Lactobacillus plantarum or using the lacticin producer with the class II producer proved to be most effective for reducing bacterial load. As emergence of bacteriocin tolerance was reduced 10-fold in the presence of nisin and lacticin, we suggest that the double producer in conjunction with the class II producer could serve as a protective culture providing a food-grade, multihurdle approach to control pathogenic growth in a variety of industrial applications. IMPORTANCE We generated a suite of single- and double-bacteriocin-producing starter cultures capable of generating the

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.

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

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...

Survival, Physiology, and Lysis of Lactococcus lactis in the Digestive Tract

PubMed Central

Drouault, Sophie; Corthier, Gérard; Ehrlich, S. Dusko; Renault, Pierre

1999-01-01

The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration. For this purpose, we used strains marked with reporter genes, the luxA-luxB gene of Vibrio harveyi and the gfp gene of Aequora victoria, that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria. Luciferase was chosen to measure the metabolic activity of Lactococcus lactis in the digestive tract because it requires NADH, which is available only in metabolically active cells. The green fluorescent protein was used to assess the bacterial lysis independently of death. We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact. Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival). In contrast, only 10 to 30% of bacteria survive in the duodenum. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm. This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies. PMID:10543799

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.

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.

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.

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.

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.

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.

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.

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

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.

Heterologous Coproduction of Enterocin A and Pediocin PA-1 by Lactococcus lactis: Detection by Specific Peptide-Directed Antibodies

PubMed Central

Martínez, José M.; Kok, Jan; Sanders, Jan W.; Hernández, Pablo E.

2000-01-01

Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martínez, M. I. Martínez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodríguez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536–4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available. PMID:10919819

A Nisin Bioassay Based on Bioluminescence

PubMed Central

Wahlström, G.; Saris, P. E. J.

1999-01-01

A Lactococcus lactis subsp. lactis strain that can sense the bacteriocin nisin and transduce the signal into bioluminescence was constructed. By using this strain, a bioassay based on bioluminescence was developed for quantification of nisin, for detection of nisin in milk, and for identification of nisin-producing strains. As little as 0.0125 ng of nisin per ml was detected within 3 h by this bioluminescence assay. This detection limit was lower than in previously described methods. PMID:10427078

Anticandidal activity of cell extracts from 13 probiotic Lactobacillus strains and characterisation of lactic acid and a novel fatty acid derivative from one strain.

PubMed

Nyanzi, Richard; Awouafack, Maurice D; Steenkamp, Paul; Jooste, Piet J; Eloff, Jacobus N

2014-12-01

This study investigated the anti-Candida activity of methanol extracts from freeze-dried probiotic cells and the isolation of some constituents in the extracts. The MIC values of the probiotic methanol cell extracts against Candida albicans ranged between 1.25 and 5mg/ml after 48 h of incubation. However, Lactococcus latics subsp. lactis strain X and Lactobacillus casei strain B extracts had an MIC of 10mg/ml after 48 h of incubation. The extracts had fungistatic rather than fungicidal activity. These extracts had a much higher antifungal activity than antifungal compounds isolated from the growth medium by many other authors. This indicates that probiotics may also release antifungal compounds in their cells that could contribute to a therapeutic effect. Lactic acid (1) and 6-O-(α-D-glucopyranosyl)-1,6-di-O-pentadecanoyl-α-D-glucopyranose a novel fatty acid derivative (2) were isolated from methanol probiotic extracts and the structure of these compounds were elucidated using NMR (1 and 2D) and mass spectrometry (MS). Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of a fermented milk containing Bifidobacterium lactis DN-173010 on Chinese constipated women.

PubMed

Yang, Yue-Xin; He, Mei; Hu, Gang; Wei, Jie; Pages, Philippe; Yang, Xian-Hua; Bourdu-Naturel, Sophie

2008-10-28

To investigate the effect of a fermented milk containing Bifidobacterium lactis DN-173010 and yogurt strains (BIO(R)) on adult women with constipation in Beijing. A total of 135 adult females with constipation were randomly allocated to consume for 2 wk either 100 g of the test fermented milk or 100 g of an acidified milk containing non-living bacteria (control). Stool frequency, defecation condition scores, stool consistency and food intake were recorded at baseline and after 1 and 2 wk in an intention-to-treat population of 126 subjects. In parallel, safety evaluation parameters were performed. At baseline, no differences were found between groups. Following consumption of test product, stool frequency was significantly increased after 1 wk (3.5 +/- 1.5 vs 2.4 +/- 0.6, P < 0.01) and 2 wk (4.1 +/- 1.7 vs 2.4 +/- 0.6, P < 0.01), vs baseline. Similarly, after 1 and 2 wk, of test product consumption, defecation condition (1.1 +/- 0.9 vs 1.9 +/- 1.2, P < 0.01 and 0.8 +/- 1.0 vs 1.9 +/- 1.2, P < 0.01, respectively) and stool consistency (1.0 +/- 0.8 vs 1.5 +/- 1.1, P < 0.01 and 0.6 +/- 0.8 vs 1.5 +/- 1.1, P < 0.01, respectively) were significantly improved. Compared with the control group, stool frequency was also significantly increased (3.5 +/- 1.5 vs 2.5 +/- 0.9, P < 0.01 and 4.1 +/- 1.7 vs 2.6 +/- 1.0, P < 0.01, respectively), and defecation condition (1.1 +/- 0.9 vs 1.6 +/- 1.1, P < 0.01 and 0.8 +/- 1.0 vs 1.6 +/- 1.1, P < 0.01, respectively) and stool consistency (1.0 +/- 0.8 vs 1.4 +/- 1.0, P < 0.05 and 0.6 +/- 0.8 vs 1.3 +/- 1.0, P < 0.01, respectively) significantly decreased after 1 and 2 wk of product consumption. During the same period, food intake did not change between the two groups, and safety parameters of the subjects were within normal ranges. This study suggests a beneficial effect of a fermented milk containing B. lactis DN-173010 on stool frequency, defecation condition and stool consistency in adult women with constipation constipated women after

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.

Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations.

PubMed

Hansen, Gunda; Johansen, Claus Lindvald; Marten, Gunvor; Wilmes, Jacqueline; Jespersen, Lene; Arneborg, Nils

2016-07-01

In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points.

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.

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.

Phenotypic and genotypic characterization of lactic acid bacteria isolated from raw goat milk and effect of farming practices on the dominant species of lactic acid bacteria.

PubMed

Tormo, Hélène; Ali Haimoud Lekhal, Djamila; Roques, C

2015-10-01

Lactic acid bacteria, in particular Lactococcus lactis, play a decisive role in the cheese making process and more particularly in lactic cheeses which are primarily produced on goat dairy farms. The objective of this study was therefore to identify the main lactic acid bacteria found in raw goats' milk from three different regions in France and evaluate if certain farming practices have an effect on the distribution of species of lactic acid bacteria in the various milk samples. Identification at genus or species level was carried out using phenotypic tests and genotypic methods including repetitive element REP-PCR, species-specific PCR and 16S rRNA gene sequencing. The distribution of the main bacterial species in the milk samples varied depending on farms and their characteristics. Out of the 146 strains identified, L. lactis was the dominant species (60% of strains), followed by Enterococcus (38%) of which Enterococcus faecalis and Enterococcus faecium. Within the species L. lactis, L. lactis subsp lactis was detected more frequently than L. lactis subsp cremoris (74% vs. 26%). The predominance of L. lactis subsp cremoris was linked to geographical area studied. It appears that the animals' environment plays a role in the balance between the dominance of L. lactis and enterococci in raw goats' milk. The separation between the milking parlor and the goat shed (vs no separation) and only straw in the bedding (vs straw and hay) seems to promote L. lactis in the milk (vs enterococci). Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of dissolved oxygen on redox potential and milk acidification by lactic acid bacteria isolated from a DL-starter culture.

PubMed

Larsen, Nadja; Werner, Birgit Brøsted; Vogensen, Finn Kvist; Jespersen, Lene

2015-03-01

Milk acidification by DL-starter cultures [cultures containing Lactococcus lactis diacetylactis (D) and Leuconostoc (L) species] depends on the oxidation-reduction (redox) potential in milk; however, the mechanisms behind this effect are not completely clear. The objective of this study was to investigate the effect of dissolved oxygen on acidification kinetics and redox potential during milk fermentation by lactic acid bacteria (LAB). Fermentations were conducted by single strains isolated from mixed DL-starter culture, including Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, and Leuconostoc mesenteroides ssp. cremoris, by the DL-starter culture, and by the type strains. High and low levels of oxygen were produced by flushing milk with oxygen or nitrogen, respectively. The kinetics of milk acidification was characterized by the maximum rate and time of acidification (Vamax and Tamax), the maximum rate and time of reduction (Vrmax and Trmax), the minimum redox potential (Eh7 final), and time of reaching Eh7 final (Trfinal). Variations in kinetic parameters were observed at both the species and strain levels. Two of the Lc. lactis ssp. lactis strains were not able to lower redox potential to negative values. Kinetic parameters of the DL-starter culture were comparable with the best acidifying and reducing strains, indicating their additive effects. Acidification curves were mostly diauxic at all oxygen levels, displaying 2 maxima of acidification rate: before (aerobic maximum) and after (anaerobic maximum) oxygen depletion. The redox potential decreased concurrently with oxygen consumption and continued to decrease at slower rate until reaching the final values, indicating involvement of both oxygen and microbiological activity in the redox state of milk. Oxygen flushing had a negative effect on reduction and acidification capacity of tested LAB. Reduction was significantly delayed at high initial oxygen, exhibiting longer Trmax, Trfinal, or both

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

Effects of synbiotic fermented milk containing Lactobacillus acidophilus La-5 and Bifidobacterium animalis ssp. lactis BB-12 on the fecal microbiota of adults with irritable bowel syndrome: A randomized double-blind, placebo-controlled trial.

PubMed

Bogovič Matijašić, Bojana; Obermajer, Tanja; Lipoglavšek, Luka; Sernel, Tjaša; Locatelli, Igor; Kos, Mitja; Šmid, Alenka; Rogelj, Irena

2016-07-01

global profile of the fecal microbiota of patients was not altered by consumption of the synbiotic or placebo. In conclusion, daily consumption of a synbiotic fermented milk had a short-term effect on the amount and proportion of La-5-like strains and B. animalis ssp. lactis in the fecal microbiome of IBS patients. Furthermore, both synbiotic and placebo products caused a temporary increase in fecal Strep. thermophilus. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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 secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

PubMed

Rhimi, Moez; Bermudez-Humaran, Luis G; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, Héla; Langella, Philippe; Maguin, Emmanuelle

2015-12-21

The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food

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

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.

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

PubMed Central

Forsman, Päivi; Alatossava, Tapani

1991-01-01

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages. Images PMID:16348513

Prevention of gastrointestinal lead poisoning using recombinant Lactococcus lactis expressing human metallothionein-I fusion protein.

PubMed

Xiao, Xue; Zhang, Changbin; Liu, Dajun; Bai, Weibin; Zhang, Qihao; Xiang, Qi; Huang, Yadong; Su, Zhijian

2016-04-05

Low-level lead poisoning is an insidious disease that affects millions of children worldwide, leading to biochemical and neurological dysfunctions. Blocking lead uptake via the gastrointestinal tract is an important prevention strategy. With this in mind, we constructed the recombinant Lactococcus lactis strain pGSMT/MG1363, which constitutively expressed the fusion protein glutathione S-transferase (GST)-small molecule ubiquitin-like modifier protein (SUMO)-metallothionein-I (GST-SUMO-MT). The thermodynamic data indicated that the average number of lead bound to a GST-SUMO-MT molecule was 3.655 and this binding reaction was a spontaneous, exothermic and entropy-increasing process. The total lead-binding capacity of pGSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass. Oral administration of pGSMT/MG1363 (1 × 10(10) Colony-Forming Units) to pubertal male rats that were also treated with 5 mg/kg of lead acetate daily significantly inhibited the increase of blood lead levels, the impairment of hepatic function and the decrease of testosterone concentration in the serum, which were all impaired in rats treated by lead acetate alone. Moreover, the administration of pGSMT/MG1363 for 6 weeks did not affect the serum concentration of calcium, magnesium, potassium or sodium ions. This study provides a convenient and economical biomaterial for preventing lead poisoning via the digestive tract.

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.

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.

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

Removal of cadmium by Lactobacillus kefir as a protective tool against toxicity.

PubMed

Gerbino, Esteban; Carasi, Paula; Tymczyszyn, E Elizabeth; Gómez-Zavaglia, Andrea

2014-08-01

The aim of this work was to evaluate the capacity of Lactobacillus kefir strains to remove cadmium cations and protect eukaryotic cells from cadmium toxicity. Lb. kefir CIDCA 8348 and JCM 5818 were grown in a 1/2 dilution of MRS broth supplemented with Cd(NO3)2 ranging 0 to 1 mM. Growth kinetics were followed during 76 h at 30 °C by registering optical density at 600 nm every 4-10 h. The accumulated concentration of cadmium was determined on cultures in the stationary phase by atomic absorption. The viability of a human hepatoma cell line (HepG2) upon exposure to (a) free cadmium and (b) cadmium previously incubated with Lb. kefir strains was evaluated by determining the mitochondrial dehydrogenase activity. Lb. kefir strains were able to grow and tolerate concentrations of cadmium cations up to 1 mM. The addition of cadmium to the culture medium increased the lag time in all the concentrations used. However, a decrease of the total biomass (maximum Absorbance) was observed only at concentrations above 0.0012 and 0.0011 mM for strains CIDCA 8348 and JCM 5818, respectively. Shorter and rounder lactobacilli were observed in both strains upon microscopic observations. Moreover, dark precipitates compatible with intracellular precipitation of cadmium were observed in the cytoplasm of both strains. The ability of Lb. kefir to protect eukaryotic cells cultures from cadmium toxicity was analysed using HepG2 cells lines. Concentrations of cadmium greater than 3×10(-3) mM strongly decreased the viability of HepG2 cells. However, when the eukaryotic cells were exposed to cadmium pre-incubated 1 h with Lb. kefir the toxicity of cadmium was considerably lower, Lb. kefir JCM 5818 being more efficient. The high tolerance and binding capacity of Lb. kefir strains to cadmium concentrations largely exceeding the tolerated weekly intake (TWI) of cadmium for food (2.5 μg per kg of body weight) and water (3 μg/l) addressed to human consumption, is an important added value when

Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91.

PubMed

Wladyka, Benedykt; Wielebska, Katarzyna; Wloka, Marcin; Bochenska, Oliwia; Dubin, Grzegorz; Dubin, Adam; Mak, Pawel

2013-08-01

Staphylococcus aureus strain CH-91, isolated from a broiler chicken with atopic dermatitis, has a highly proteolytic phenotype that is correlated with the disease. We describe the isolation and biochemical and molecular characterization of the AI-type lantibiotic BacCH91 from S. aureus CH-91 culture medium. The bacteriocin was purified using a three-stage procedure comprising precipitation with ammonium sulfate, extraction with organic solvents, and reversed-phase HPLC. The BacCH91 peptide is thermostable and highly resistant to cleavage by both prokaryotic and eukaryotic peptidases. The MIC for the Gram-positive bacteria ranged from 2.5 nM for Microococcus luteus through 1.3-6.0 μM for staphylococcal strains up to more than 100 μM for Lactococcus lactis. BacCH91 was ineffective against the Gram-negative strains tested at the maximal concentration (100 μM). The amino acid sequence of BacCH91 is similar to that of epidermin and gallidermin. The encoding gene (bacCH91) occurred in two allelic variants distinguishable in the restriction fragment length polymorphism assay. Variant I, identified in S. aureus CH-91, dominated in S. aureus strains of poultry origin, although strains with variant II were also identified in this group. S. aureus strains of human origin were characterized exclusively by variant II.

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

FTIR spectroscopy structural analysis of the interaction between Lactobacillus kefir S-layers and metal ions

NASA Astrophysics Data System (ADS)

Gerbino, E.; Mobili, P.; Tymczyszyn, E.; Fausto, R.; Gómez-Zavaglia, A.

2011-02-01

FTIR spectroscopy was used to structurally characterize the interaction of S-layer proteins extracted from two strains of Lactobacillus kefir (the aggregating CIDCA 8348 and the non-aggregating JCM 5818) with metal ions (Cd +2, Zn +2, Pb +2 and Ni +2). The infrared spectra indicate that the metal/protein interaction occurs mainly through the carboxylate groups of the side chains of Asp and Glut residues, with some contribution of the NH groups belonging to the peptide backbone. The frequency separation between the νCOO - anti-symmetric and symmetric stretching vibrations in the spectra of the S-layers in presence of the metal ions was found to be ca. 190 cm -1 for S-layer CIDCA 8348 and ca. 170 cm -1 for JCM 5818, denoting an unidentate coordination in both cases. Changes in the secondary structures of the S-layers induced by the interaction with the metal ions were also noticed: a general trend to increase the amount of β-sheet structures and to reduce the amount of α-helices was observed. These changes allow the proteins to adjust their structure to the presence of the metal ions at minimum energy expense, and accordingly, these adjustments were found to be more important for the bigger ions.

[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.

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.

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.

Use of the alr gene as a food-grade selection marker in lactic acid bacteria.

PubMed

Bron, Peter A; Benchimol, Marcos G; Lambert, Jolanda; Palumbo, Emmanuelle; Deghorain, Marie; Delcour, Jean; De Vos, Willem M; Kleerebezem, Michiel; Hols, Pascal

2002-11-01

Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.

Identification of Lactococcus-Specific Bacteriocins Produced by Lactococcal Isolates, and the Discovery of a Novel Bacteriocin, Lactococcin Z.

PubMed

Ishibashi, Naoki; Seto, Hiromi; Koga, Shoko; Zendo, Takeshi; Sonomoto, Kenji

2015-09-01

Lactic acid bacteria that produce Lactococcus-specific bacteriocins were isolated and identified as Lactococcus lactis from fresh corn or lettuce. Among them, four isolates were identified as lactococcin Q producers. Seven isolates showed antimicrobial activity against a lactococcin Q producer, L. lactis QU 4, as well as against nisin Z and lacticin Q producers belonging to L. lactis. Strain QU 7 was selected as a standard strain and showed no cross-immunity to lactococcin Q or other lactococcal bacteriocins. The bacteriocin produced by strain QU 7 was purified in three chromatographic steps, and its molecular mass was determined to be 5041.35 Da. The amino acid sequence analysis revealed that it is a novel class IId bacteriocin, referred to as lactococcin Z. It consisted of 45 amino acid residues. The lczA gene encoding the prepeptide of lactococcin Z showed homology to lactococcins A, B, and M. Thus, this report demonstrates a new example of Lactococcus-specific bacteriocins.

Prevention of gastrointestinal lead poisoning using recombinant Lactococcus lactis expressing human metallothionein-I fusion protein

PubMed Central

Xiao, Xue; Zhang, Changbin; Liu, Dajun; Bai, Weibin; Zhang, Qihao; Xiang, Qi; Huang, Yadong; Su, Zhijian

2016-01-01

Low-level lead poisoning is an insidious disease that affects millions of children worldwide, leading to biochemical and neurological dysfunctions. Blocking lead uptake via the gastrointestinal tract is an important prevention strategy. With this in mind, we constructed the recombinant Lactococcus lactis strain pGSMT/MG1363, which constitutively expressed the fusion protein glutathione S-transferase (GST)–small molecule ubiquitin-like modifier protein (SUMO)–metallothionein-I (GST-SUMO-MT). The thermodynamic data indicated that the average number of lead bound to a GST-SUMO-MT molecule was 3.655 and this binding reaction was a spontaneous, exothermic and entropy-increasing process. The total lead-binding capacity of pGSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass. Oral administration of pGSMT/MG1363 (1 × 1010 Colony-Forming Units) to pubertal male rats that were also treated with 5 mg/kg of lead acetate daily significantly inhibited the increase of blood lead levels, the impairment of hepatic function and the decrease of testosterone concentration in the serum, which were all impaired in rats treated by lead acetate alone. Moreover, the administration of pGSMT/MG1363 for 6 weeks did not affect the serum concentration of calcium, magnesium, potassium or sodium ions. This study provides a convenient and economical biomaterial for preventing lead poisoning via the digestive tract. PMID:27045906

The investigation of probiotic potential of lactic acid bacteria isolated from traditional Mongolian dairy products.

PubMed

Takeda, Shiro; Yamasaki, Keiko; Takeshita, Masahiko; Kikuchi, Yukiharu; Tsend-Ayush, Chuluunbat; Dashnyam, Bumbein; Ahhmed, Abdulatef M; Kawahara, Satoshi; Muguruma, Michio

2011-08-01

The aims of this study were to investigate the diversity of lactic acid bacteria (LAB) isolated from traditional Mongolian dairy products, and to estimate the probiotic potential of the isolated strains. We collected 66 samples of the traditional Mongolian dairy products tarag (n = 45), airag (n = 7), aaruul (n = 8), byasulag (n = 1) and eezgii (n = 5), from which 543 LAB strains were isolated and identified based on 16S ribosomal DNA sequence. The predominant species of those products were Lactobacillus (L.) delbrueckii ssp. bulgaricus, L. helveticus, L. fermentum, L. delbrueckii ssp. lactis and Lactococcus lactis ssp. lactis. However, we could not detect any LAB strains from eezgii. All LAB isolates were screened for tolerance to low pH and to bile acid, gas production from glucose, and adherence to Caco-2 cells. In vitro, we found 10 strains possess probiotic properties, and almost identified them as L. plantarum or L. paracasei subspecies, based on 16S ribosomal DNA and carbohydrate fermentation pattern. These strains were differentiated from each other individually by randomly amplified polymorphic DNA analysis. Additionally, it was notable that 6/10 strains were isolated from camel milk tarag from the Dornogovi province. 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

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.

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.

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

Inhibition of Listeria monocytogenes in Hot Dogs by Surface Application of Freeze-Dried Bacteriocin-Containing Powders from Lactic Acid Bacteria.

PubMed

Ünlü, Gülhan; Nielsen, Barbara; Ionita, Claudia

2016-06-01

Six lactic acid bacteria (LAB) strains, Lactococcus lactis BFE 920, L. lactis subsp. lactis ATCC 11454, L. lactis subsp. cremoris ATCC 14365, Lactobacillus curvatus L442, Lact. curvatus LTH 1174, and Lact. bavaricus MN, were grown in cheddar cheese whey supplemented with complex nutrient sources. Cell-free culture supernatants were freeze-dried, and the resulting bacteriocin-containing powders were applied on the surface of hot dogs that were inoculated (~4 log cfu/hot dog) with a five-strain Listeria monocytogenes cocktail. Hot dogs were vacuum-sealed and stored at 4 °C for 4 weeks. L. monocytogenes was enumerated, using both tryptic soy agar (TSA) and oxford listeria agar (OXA), on day 0 and at 1, 2, 3, and 4 weeks of the refrigerated storage. In hot dogs containing only the L. monocytogenes inoculum, L. monocytogenes counts increased from 4 up to 7 log cfu/hot dog. All samples containing freeze-dried bacteriocin-containing powders exhibited significantly lowered (P < 0.05) L. monocytogenes populations on the surface of hot dogs throughout the 4-week study except for bavaricin MN powder. Bacterial counts on hot dogs packed without any powder were statistically equal on day 0 when enumerated on OXA. Freeze-dried bacteriocin-containing powders from Lact. curvatus L442 and L. lactis subsp. cremoris ATCC 14365 decreased L. monocytogenes populations on the surface of hot dogs by greater than 2 log cfu/hot dog throughout the 4-week study. For the powdered bacteriocin preparations from L. lactis BFE 920, L. lactis subsp. lactis ATCC 11454, and Lact. curvatus LTH 1174, L. monocytogenes populations were determined to be approximately 3-log cfu/hot dog after 4 weeks of storage.

Use of Potential Probiotic Lactic Acid Bacteria (LAB) Biofilms for the Control of Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli O157:H7 Biofilms Formation

PubMed Central

Gómez, Natacha C.; Ramiro, Juan M. P.; Quecan, Beatriz X. V.; de Melo Franco, Bernadette D. G.

2016-01-01

Use of probiotic biofilms can be an alternative approach for reducing the formation of pathogenic biofilms in food industries. The aims of this study were (i) to evaluate the probiotic properties of bacteriocinogenic (Lactococcus lactis VB69, L. lactis VB94, Lactobacillus sakei MBSa1, and Lactobacillus curvatus MBSa3) and non-bacteriocinogenic (L. lactis 368, Lactobacillus helveticus 354, Lactobacillus casei 40, and Weissela viridescens 113) lactic acid bacteria (LAB) isolated from Brazilian’s foods and (ii) to develop protective biofilms with these strains and test them for exclusion of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella Typhimurium. LAB were tested for survival in acid and bile salt conditions, surface properties, biosurfactant production, β-galactosidase and gelatinase activity, antibiotic resistance and presence of virulence genes. Most strains survived exposure to pH 2 and 4% bile salts. The highest percentages of auto-aggregation were obtained after 24 h of incubation. Sixty-seven percentage auto-aggregation value was observed in W. viridescens 113 and Lactobacillus curvatus MBSa3 exhibited the highest co-aggregation (69% with Listeria monocytogenes and 74.6% with E. coli O157:H7), while the lowest co-aggregation was exhibited by W. viridescens 113 (53.4% with Listeria monocytogenes and 38% with E. coli O157:H7). Tests for hemolytic activity, bacterial cell adherence with xylene, and drop collapse confirmed the biosurfactant-producing ability of most strains. Only one strain (L. lactis 368) produced β-galactosidase. All strains were negative for virulence genes cob, ccf, cylLL, cylLs, cyllM, cylB, cylA and efaAfs and gelatinase production. The antibiotic susceptibility tests indicated that the MIC for ciprofloxacin, clindamycin, gentamicin, kanamycin, and streptomycin did not exceed the epidemiological cut-off suggested by the European Food Safety Authority. Some strains were resistant to one or more antibiotics and

Requirement of Autolytic Activity for Bacteriocin-Induced Lysis

PubMed Central

Martínez-Cuesta, M. Carmen; Kok, Jan; Herranz, Elisabet; Peláez, Carmen; Requena, Teresa; Buist, Girbe

2000-01-01

The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co2+ and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmAΔ1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmAΔ1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA. PMID:10919766

Biochemical patterns in ovine cheese: influence of probiotic strains.

PubMed

Albenzio, M; Santillo, A; Caroprese, M; Marino, R; Trani, A; Faccia, M

2010-08-01

This study was undertaken to evaluate the effect of lamb rennet paste containing probiotic strains on proteolysis, lipolysis, and glycolysis of ovine cheese manufactured with starter cultures. Cheeses included control cheese made with rennet paste, cheese made with rennet paste containing Lactobacillus acidophilus culture (LA-5), and cheese made with rennet paste containing a mix of Bifidobacterium lactis (BB-12) and Bifidobacterium longum (BB-46). Cheeses were sampled at 1, 7, 15, and 30 d of ripening. Starter cultures coupled with probiotics strains contained in rennet paste affected the acidification and coagulation phases leading to the lowest pH in curd and cheese containing probiotics during ripening. As consequence, maturing cheese profiles were different among cheese treatments. Cheeses produced using rennet paste containing probiotics displayed higher percentages of alpha(S1)-I-casein fraction than traditional cheese up to 15 d of ripening. This result could be an outcome of the greater hydrolysis of alpha-casein fraction, attributed to higher activity of the residual chymosin. Further evidence for this trend is available in chromatograms of water-soluble nitrogen fractions, which indicated a more complex profile in cheeses made using lamb paste containing probiotics versus traditional cheese. Differences can be observed for the peaks eluted in the highly hydrophobic zone being higher in cheeses containing probiotics. The proteolytic activity of probiotic bacteria led to increased accumulation of free amino acids. Their concentrations in cheese made with rennet paste containing Lb. acidophilus culture and cheese made with rennet paste containing a mix of B. lactis and B. longum were approximately 2.5 and 3.0 times higher, respectively, than in traditional cheese. Principal component analysis showed a more intense lipolysis in terms of both free fatty acids and conjugated linoleic acid content in probiotic cheeses; in particular, the lipolytic pattern of

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.

Characterization of lactic acid bacteria isolated from a Thai low-salt fermented fish product and the role of garlic as substrate for fermentation.

PubMed

Paludan-Müller, C; Huss, H H; Gram, L

1999-02-18

Lactic acid bacteria (LAB) isolated from raw materials (fish, rice, garlic and banana leaves) and processed som-fak (a Thai low-salt fermented fish product) were characterized by API 50-CH and other phenotypic criteria. Lactococcus lactis subsp. lactis and Leuconostoc citreum were specifically associated with fish fillet and minced fish, Lactobacillus paracasei subsp. paracasei with boiled rice and Weisella confusa with garlic mix and banana leaves. In addition, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus pentosaceus were isolated from raw materials. A succession of aciduric, homofermentative lactobacillus species, dominated by Lb. plantarum/pentosus, was found during fermentation. In total, 9% of the strains fermented starch and 19% fermented garlic, the two main carbohydrate components in som-fak. The ability to ferment garlic was paralleled by a capacity to ferment inulin. An increased percentage of garlic fermenting strains was found during fermentation of som-fak, from 8% at day 1 to 40% at day 5. No starch fermenting strains were isolated during fermentation. Three mixed LAB cultures, composed of either starch fermenting Lc. lactis subsp. lactis and Lb. paracasei subsp. paracasei, or garlic fermenting Lb. plantarum and Pd. pentosaceus, or a combination of these strains were inoculated into laboratory prepared som-fak with or without garlic. In som-fak without garlic, pH was above 4.8 after three days, irrespective of addition of mixed LAB cultures. The starch fermenting LAB were unable to ferment som-fak and sensory spoilage occurred after three days. Fermentation with the combined mix of starch and garlic fermenting strains led to production of 2.5% acid and a decrease in pH to 4.5 in two days. The fermentation was slightly slower with the garlic fermenting strains alone. This is the first report describing the role of garlic as carbohydrate source for LAB in fermented fish products.

Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression.

PubMed

van Rooijen, R J; Dechering, K J; Niek, C; Wilmink, J; de Vos, W M

1993-02-01

Site-directed mutagenesis of the Lactococcus lactis lacR gene was performed to identify residues in the LacR repressor that are involved in the induction of lacABCDFEGX operon expression by tagatose-6-phosphate. A putative inducer binding domain located near the C-terminus was previously postulated based on homology studies with the Escherichia coli DeoR family of repressors, which all have a phosphorylated sugar as inducer. Residues within this domain and lysine residues that are charge conserved in the DeoR family were changed into alanine or arginine. The production of the LacR mutants K72A, K80A, K80R, D210A, K213A and K213R in the LacR-deficient L.lactis strain NZ3015 resulted in repressed phospho-beta-galactosidase (LacG) activities and decreased growth rates on lactose. Gel mobility shift assays showed that the complex between a DNA fragment carrying the lac operators and LacR mutants K72A, K80A, K213A and D210A did not dissociate in the presence of tagatose-6-phosphate, in contrast to wild type LacR. Other mutations (K62A/K63A, K72R, K73A, K73R, T212A, F214R, R216R and R216K) exhibited no gross effects on inducer response. The results strongly suggest that the lysines at positions 72, 80 and 213 and aspartic acid at position 210 are involved in the induction of lac operon expression by tagatose-6-phosphate.

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

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.

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

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.

Screening lactic acid bacteria strains with ability to bind di-n-butyl phthalate via Turbiscan technique.

PubMed

Lili, Zhao; Hongfei, Zhao; Shoukat, Sana; Xiaochen, Zhang; Bolin, Zhang

2017-06-01

Di-n-butyl phthalate (DBP) is a ubiquitous environmental contaminant that poses a risk to humans. Previous work indicates that the ability of lactic acid bacteria (LAB) to bind phthalic acid esters is strain-specific. As cell suspensions of LAB strains in aqueous solution are likely to be colloidal dispersions, this study provided a technique to efficiently screen LAB strains that bind DBP via Turbiscan, which has been widely used to measure the stability of emulsions or colloidal dispersions. Eleven LAB strains belonging to Lactobacillus plantarum, Lb. pentosus, Lb. paralimentarius, Lb. helveticus, Leuconostoc mesenteroides, Lb. acidophilus, Bifidobacterium lactis, and Bifidobacterium bifidum species were used in this study, and seven of them were selected to test in an earlier stage of exploring the process for finding a screening method; others were used for a validation test. It was observed that the various values of the 10 h Turbiscan Stability Index (TSI) of the cell suspension from each strain, at the equilibrium time of dispersed particles according to the peak thickness of cell-suspensions as measured by Turbiscan, had significant negative correlations with the DBP-binding percentage of LAB strains. Higher TSI values are correlated with lower binding of bacteria strains to DBP with a correlation coefficient of 0.8292. Cell surface hydrocarbons of LAB strains and their adherence were observed to correlate with DBP-binding percentages and may lead to the different states of aggregation or equilibrium of bacterial cell-suspensions, and the aggregation of bacterial cells resulted in fewer binding sites in the cell wall for DBP. Finally, four LAB strains were randomly selected to verify the feasibility of the method. In all, the findings demonstrate that TSI might be used as a tool to quickly screen strains that bind DBP. The present work could be extended to the removal of other toxic compounds, when screening of high-efficiency strains is required.

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.

New probiotic strains for inflammatory bowel disease management identified by combining in vitro and in vivo approaches.

PubMed

Alard, J; Peucelle, V; Boutillier, D; Breton, J; Kuylle, S; Pot, B; Holowacz, S; Grangette, C

2018-02-27

Alterations in the gut microbiota composition play a key role in the development of chronic diseases such as inflammatory bowel disease (IBD). The potential use of probiotics therefore gained attention, although outcomes were sometimes conflicting and results largely strain-dependent. The present study aimed to identify new probiotic strains that have a high potential for the management of this type of pathologies. Strains were selected from a large collection by combining different in vitro and in vivo approaches, addressing both anti-inflammatory potential and ability to improve the gut barrier function. We identified six strains with an interesting anti-inflammatory profile on peripheral blood mononuclear cells and with the ability to restore the gut barrier using a gut permeability model based on Caco-2 cells sensitized with hydrogen peroxide. The in vivo evaluation in two 2,4,6-trinitrobenzene sulfonic acid-induced murine models of colitis highlighted that some of the strains exhibited beneficial activities against acute colitis while others improved chronic colitis. Bifidobacterium bifidum PI22, the strain that exhibited the most protective capacities against acute colitis was only slightly efficacious against chronic colitis, while Bifidobacterium lactis LA804 which was less efficacious in the acute model was the most protective against chronic colitis. Lactobacillus helveticus PI5 was not anti-inflammatory in vitro but the best in strengthening the epithelial barrier and as such able to significantly dampen murine acute colitis. Interestingly, Lactobacillus salivarius LA307 protected mice significantly against both types of colitis. This work provides crucial clues for selecting the best strains for more efficacious therapeutic approaches in the management of chronic inflammatory diseases. The strategy employed allowed us to identify four strains with different characteristics and a high potential for the management of inflammatory diseases, such as IBD.

Changes in the microbial composition of raw milk induced by thermization treatments applied prior to traditional Greek hard cheese processing.

PubMed

Samelis, John; Lianou, Alexandra; Kakouri, Athanasia; Delbès, Céline; Rogelj, Irena; Bogovic-Matijasić, Bojana; Montel, Marie-Christine

2009-04-01

The microbiological quality, safety, and composition of mixtures of ewe's and goat's milk (90:10) used for cheesemaking were evaluated before and after thermization at 60 and 67 degrees C for 30 s. Such mild thermal treatments are commonly applied to reduce natural contaminants of raw milk before processing for traditional hard Greek cheeses. Raw milk samples had an average total bacterial count of 7.3 log CFU/ml; most of these bacteria were lactic acid bacteria (LAB) and pseudomonads. The LAB flora of raw milk was dominated by enterococci (40.8%), followed by lactococci (20.4%), leuconostocs (18.4%), and mesophilic lactobacilli (10.2%). Enterococcus faecalis (30.1%) and Enterococcus faecium (13.7%) were the most common LAB isolates, followed by Enterococcus durans, Lactococcus lactis subsp. lactis, Lactobacillus plantarum, and Leuconostoc lactis. Thermization at 60 degrees C for 30 s was effective for reducing raw milk contamination by enterobacteria (5.1 log CFU/ml), coagulase-positive staphylococci (3.3 log CFU/ml), and Listeria (present in 25-ml samples) to safe levels, but it also reduced mesophilic lactococci, leuconostocs, lactobacilli, and selected enterococci (72.0%) in thermized milk. Thermization at 67 degrees C for 30 s had a major inactivation effect on all bacterial groups. Two nisin-producing L. lactis subsp. lactis strains (M78 and M104) were isolated from raw milk, but neither nisin-producing nor other bacteriocin-producing LAB strains were isolated from thermized milk. Thus, thermization treatments control harmful bacteria but also may have a negative impact on milk quality by reducing desirable LAB and the biodiversity of raw milk bacteria overall, inactivating potentially protective LAB strains and enhancing the ability of potentially pathogenic enterococci to grow in fresh cheese curds.

Lactococcus lactis and Lactobacillus salivarius differently modulate early immunological response of Wistar rats co-administered with Listeria monocytogenes.

PubMed

Lukic, J; Jancic, I; Mirkovic, N; Bufan, B; Djokic, J; Milenkovic, M; Begovic, J; Strahinic, I; Lozo, J

2017-10-13

In the light of the increasing resistance of bacterial pathogens to antibiotics, one of the main global strategies in applied science is development of alternative treatments, which would be safe both for the host and from the environmental perspective. Accordingly, the aim of this study was to test whether two lactic acid bacteria (LAB) strains, Lactococcus lactis BGBU1-4 and Lactobacillus salivarius BGHO1, could be applied as safe supplements for Listeria infection. Two major research objectives were set: to compare the effects of BGBU1-4 and BGHO1 on early immune response in gut tissue of Wistar rats co-administered with Listeria monocytogenes ATCC19111 and next, to test how this applies to their usage as therapeutics in acute ATCC19111 infection. Intestinal villi (IV), Peyer's patches (PP) and mesenteric lymph nodes (MLN) were used for the analysis. The results showed that BGHO1 increased the mRNA expression of innate immune markers CD14, interleukin (IL)-1β and tumour necrosis factor (TNF)-α in PP and IV, and, in parallel, caused a decrease of listeriolysin O (LLO) mRNA expression in same tissues. In MLN of BGHO1 treated rats, LLO expression was increased, along with an increase of the expression of OX-62 mRNA and CD69, pointing to the activation of adaptive immunity. On the other hand, in BGBU1-4 treated rats, there was no reduction of LLO mRNA expression and no induction of innate immunity markers in intestinal tissue. Additionally, CD14 and IL-1β, as well as LLO, but not OX-62 mRNA and CD69 expression, were elevated in MLN of BGBU1-4 treated rats. However, when applied therapeutically, both, BGBU1-4 and BGHO1, lowered Listeria count in spleens of infected rats. Our results not only reveal the potential of LAB to ameliorate Listeria infections, but suggest different immunological effects of two different LAB strains, both of which could be effective in Listeria elimination.

Identification of Lactic Acid Bacteria in Fruit Pulp Processing Byproducts and Potential Probiotic Properties of Selected Lactobacillus Strains

PubMed Central

Garcia, Estefânia F.; Luciano, Winnie A.; Xavier, Danilo E.; da Costa, Whyara C. A.; de Sousa Oliveira, Kleber; Franco, Octávio L.; de Morais Júnior, Marcos A.; Lucena, Brígida T. L.; Picão, Renata C.; Magnani, Marciane; Saarela, Maria; de Souza, Evandro L.

2016-01-01

This study aimed to identify lactic acid bacteria (LAB) in byproducts of fruit (Malpighia glabra L., Mangifera indica L., Annona muricata L., and Fragaria vesca L.) pulp processing. Fifty strains of LAB were identified using matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence (16S rRNA) analysis. Species belonging to Lactobacillus genus were the predominant LAB in all fruit pulp processing byproducts. The average congruency between the MALDI-TOF MS and 16S rRNA in LAB species identification reached 86%. Isolates of L. plantarum, L. brevis, L. pentosus, L. lactis and L. mesenteroides were identified with 100% congruency. MALDI-TOF MS and 16S rRNA analysis presented 86 and 100% efficiency of LAB species identification, respectively. Further, five selected Lactobacillus strains (L. brevis 59, L. pentosus 129, L. paracasei 108, L. plantarum 49, and L. fermentum 111) were evaluated for desirable probiotic-related properties and growth behavior on two different cultivation media. The exposure to pH 2.0 sharply decreased the counts of the different Lactobacillus strains after a 1 or 2 h incubation, while varied decreases were noted after 3 h of exposure to pH 3.0. Overall, the exposure to pH 5.0 and to bile salts (0.15, 0.30, and 1.00%) did not decrease the counts of the Lactobacillus strains. All tested Lactobacillus strains presented inhibitory activity against Staphylococcus aureus, Salmonella Typhimurium, Salmonella Enteritidis, Listeria monocytogenes and Escherichia coli, and presented variable susceptibility to different antibiotics. The selected Lactobacillus strains presented satisfactory and reproducible growth behavior. In conclusion, MALDI-TOF MS and 16S rRNA analysis revealed high efficiency and congruency for LAB species identification, and the selected Lactobacillus strains may be candidates for further investigation of novel probiotic strains. PMID:27625647

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.

Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia.

PubMed

Yu, J; Wang, W H; Menghe, B L G; Jiri, M T; Wang, H M; Liu, W J; Bao, Q H; Lu, Q; Zhang, J C; Wang, F; Xu, H Y; Sun, T S; Zhang, H P

2011-07-01

Spontaneous milk fermentation has a long history in Mongolia, and beneficial microorganisms have been handed down from one generation to the next for use in fermented dairy products. The objective of this study was to investigate the diversity of lactic acid bacteria (LAB) communities in fermented yak, mare, goat, and cow milk products by analyzing 189 samples collected from 13 different regions in Mongolia. The LAB counts in these samples varied from 3.41 to 9.03 log cfu/mL. Fermented yak and mare milks had almost identical mean numbers of LAB, which were significantly higher than those in fermented goat milk but slightly lower than those in fermented cow milk. In total, 668 isolates were obtained from these samples using de Man, Rogosa, and Sharpe agar and M17 agar. Each isolate was considered to be presumptive LAB based on gram-positive and catalase-negative properties, and was identified at the species level by 16S rRNA gene sequencing, multiplex PCR assay, and restriction fragment length polymorphism analysis. All isolates from Mongolian dairy products were accurately identified as Enterococcus faecalis (1 strain), Enterococcus durans (3 strains), Lactobacillus brevis (3 strains), Lactobacillus buchneri (2 strains), Lactobacillus casei (16 strains), Lactobacillus delbrueckii ssp. bulgaricus (142 strains), Lactobacillus diolivorans (17 strains), Lactobacillus fermentum (42 strains), Lactobacillus helveticus (183 strains), Lactobacillus kefiri (6 strains), Lactobacillus plantarum ssp. plantarum (7 strains), Lactococcus lactis ssp. lactis (7 strains), Leuconostoc lactis (22 strains), Leuconostoc mesenteroides (21 strains), Streptococcus thermophilus (195 strains), and Weissella cibaria (1 strain). The predominant LAB were Strep. thermophilus and Lb. helveticus, which were isolated from all sampling sites. The results demonstrate that traditional fermented dairy products from different regions of Mongolia have complex compositions of LAB species. Such diversity of

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.

Dominant lactic acid bacteria and their technological properties isolated from the Himalayan ethnic fermented milk products.

PubMed

Dewan, Sailendra; Tamang, Jyoti Prakash

2007-10-01

Ethnic people of the Himalayan regions of India, Nepal, Bhutan and China consume a variety of indigenous fermented milk products made from cows milk as well as yaks milk. These lesser-known ethnic fermented foods are dahi, mohi, chhurpi, somar, philu and shyow. The population of lactic acid bacteria (LAB) ranged from 10(7) to 10(8) cfu/g in these Himalayan milk products. A total of 128 isolates of LAB were isolated from 58 samples of ethnic fermented milk products collected from different places of India, Nepal and Bhutan. Based on phenotypic characterization including API sugar test, the dominant lactic acid bacteria were identified as Lactobacillus bifermentans, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus kefir, Lactobacillus hilgardii, Lactobacillus alimentarius, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Enterococcus faecium. LAB produced a wide spectrum of enzymes and showed high galactosidase, leucine-arylamidase and phosphatase activities. They showed antagonistic properties against selected Gram-negative bacteria. None of the strains produced bacteriocin and biogenic amines under the test conditions used. Most strains of LAB coagulated skim milk with a moderate drop in pH. Some strains of LAB showed a high degree of hydrophobicity, suggesting these strains may have useful adhesive potential. This paper is the first report on functional lactic acid bacterial composition in some lesser-known ethnic fermented milk products of the Himalayas.

Evaluation of the probiotic properties of new Lactobacillus and Bifidobacterium strains and their in vitro effect.

PubMed

Presti, I; D'Orazio, G; Labra, M; La Ferla, B; Mezzasalma, V; Bizzaro, G; Giardina, S; Michelotti, A; Tursi, F; Vassallo, M; Di Gennaro, P

2015-07-01

Probiotic ingestion is recommended as a preventive approach to maintain the balance of the intestinal microbiota and to enhance the human well-being. During the whole life of each individual, the gut microbiota composition could be altered by lifestyle, diet, antibiotic therapies and other stress conditions, which may lead to acute and chronic disorders. Hence, probiotics can be administered for the prevention or treatment of some disorders, including lactose malabsorption, acute diarrhoea, irritable bowel syndrome, necrotizing enterocolitis and mild forms of inflammatory bowel disease. The probiotic-mediated effect is an important issue that needs to be addressed in relation to strain-specific probiotic properties. In this work, the probiotic properties of new Lactobacillus and Bifidobacterium strains were screened, and their effects in vitro were evaluated. They were screened for probiotic properties by determining their tolerance to low pH and to bile salts, antibiotic sensitivity, antimicrobial activity and vitamin B8, B9 and B12 production, and by considering their ability to increase the antioxidant potential and to modulate the inflammatory status of systemic-miming cell lines in vitro. Three out of the examined strains presenting the most performant probiotic properties, as Lactobacillus plantarum PBS067, Lactobacillus rhamnosus PBS070 and Bifidobacterium animalis subsp. lactis PBSO75, were evaluated for their effects also on human intestinal HT-29 cell line. The obtained results support the possibility to move to another level of study, that is, the oral administration of these probiotical strains to patients with acute and chronic gut disorders, by in vivo experiments.

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.

Nisin Z Production by Lactococcus lactis subsp. cremoris WA2-67 of Aquatic Origin as a Defense Mechanism to Protect Rainbow Trout (Oncorhynchus mykiss, Walbaum) Against Lactococcus garvieae.

PubMed

Araújo, Carlos; Muñoz-Atienza, Estefanía; Pérez-Sánchez, Tania; Poeta, Patrícia; Igrejas, Gilberto; Hernández, Pablo E; Herranz, Carmen; Ruiz-Zarzuela, Imanol; Cintas, Luis M

2015-12-01

Probiotics represent an alternative to chemotherapy and vaccination to control fish diseases, including lactococcosis caused by Lactococcus garvieae. The aims of this study were (i) to determine the in vitro probiotic properties of three bacteriocinogenic Lactococcus lactis subsp. cremoris of aquatic origin, (ii) to evaluate in vivo the ability of L. cremoris WA2-67 to protect rainbow trout (Oncorhynchus mykiss, Walbaum) against infection by L. garvieae, and (iii) to demonstrate the role of nisin Z (NisZ) production as an anti-infective mechanism. The three L. cremoris strains survived in freshwater at 18 °C for 7 days, withstood exposure to pH 3.0 and 10 % (v/v) rainbow trout bile, and showed different cell surface hydrophobicity (37.93-58.52 %). The wild-type NisZ-producer L. cremoris WA2-67 and its non-bacteriocinogenic mutant L. cremoris WA2-67 ∆nisZ were administered orally (10(6) CFU/g) to rainbow trout for 21 days and, subsequently, fish were challenged with L. garvieae CLG4 by the cohabitation method. The fish fed with the bacteriocinogenic strain L. cremoris WA2-67 reduced significantly (p < 0.01) the mortality (20 %) compared to the fish treated with its non-bacteriocinogenic knockout isogenic mutant (50 %) and the control (72.5 %). We demonstrated the effectiveness of L. cremoris WA2-67 to protect rainbow trout against infection with the invasive pathogen L. garvieae and the relevance of NisZ production as an anti-infective mechanism. This is the first report demonstrating the effective in vivo role of LAB bacteriocin (NisZ) production as a mechanism to protect fish against bacterial infection. Our results suggest that the wild-type NisZ-producer strain L. cremoris WA2-67 could be used in fish farming to prevent lactococcosis in rainbow trout.

Molecular identification and cluster analysis of homofermentative thermophilic lactobacilli isolated from dairy products.

PubMed

Andrighetto, C; De Dea, P; Lombardi, A; Neviani, E; Rossetti, L; Giraffa, G

1998-10-01

Twenty-five strains of thermophilic lactobacilli isolated from yoghurt and from semi-hard and hard cheeses (in parallel with nine type or reference strains) were identified and grouped according to their genetic relatedness. Strains were identified by sugar fermentation patterns using the "API 50 CHL" galleries, by species-specific DNA probes in dot-blot hybridization experiments, by amplification and restriction analysis of the 16S rRNA gene (ARDRA) and by polymerase chain reaction (PCR) using species-specific oligonucleotide primers. Strains were classified as Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus, L. helveticus, and L. acidophilus. Strains which were atypical by sugar fermentation patterns were also identified. Most of the strains could not be grouped using carbohydrate fermentation profiles. PCR fingerprinting was used to identify DNA profiles for the 25 lactobacilli. Experimentally obtained PCR profiles enabled discrimination of all strains, which were grouped according to the similarities in their combined patterns. In general, the clustering of the strains corresponded well with species delineation obtained by molecular identification. The dendrogram of genetic relatedness enabled the unambiguous identification of most of the strains which were shown to be atypical by the sugar fermentation profile, except for a discrepancy in one L. delbrueckii subsp. lactis strain and one atypical Lactobacillus sp. strain.

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...

Batch culture characterization and metabolic flux analysis of succinate-producing Escherichia coli strains.

PubMed

Sánchez, Ailen M; Bennett, George N; San, Ka-Yiu

2006-05-01

This study presents an in-depth analysis of the anaerobic metabolic fluxes of various mutant strains of Escherichia coli overexpressing the Lactococcus lactis pyruvate carboxylase (PYC) for the production of succinate. Previously, a metabolic network design that includes an active glyoxylate pathway implemented in vivo increased succinate yield from glucose in an E. coli mutant to 1.6 mol/mol under fully anaerobic conditions. The design consists of a dual succinate synthesis route, which diverts required quantities of NADH through the traditional fermentative pathway and maximizes the carbon converted to succinate by balancing the carbon flux through the fermentative pathway and the glyoxylate pathway (which has a lower NADH requirement). Mutant strains previously constructed during the development of high-yield succinate-producing strains were selected for further characterization to understand their metabolic response as a result of several genetic manipulations and to determine the significance of the fermentative and the glyoxylate pathways in the production of succinate. Measured fluxes obtained under batch cultivation conditions were used to estimate intracellular fluxes and identify critical branch point flux split ratios. The comparison of changes in branch point flux split ratios to the glyoxylate pathway and the fermentative pathway at the oxaloacetate (OAA) node as a result of different mutations revealed the sensitivity of succinate yield to these manipulations. The most favorable split ratio to obtain the highest succinate yield was the fractional partition of OAA to glyoxylate of 0.32 and 0.68 to the fermentative pathway obtained in strains SBS550MG (pHL413) and SBS990MG (pHL413). The succinate yields achieved in these two strains were 1.6 and 1.7 mol/mol, respectively. In addition, an active glyoxylate pathway in an ldhA, adhE, ack-pta mutant strain is shown to be responsible for the high succinate yields achieved anaerobically. Furthermore, in vitro

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).

Quantitative analyses of the bacterial microbiota of rearing environment, tilapia and common carp cultured in earthen ponds and inhibitory activity of its lactic acid bacteria on fish spoilage and pathogenic bacteria.

PubMed

Kaktcham, Pierre Marie; Temgoua, Jules-Bocamdé; Ngoufack Zambou, François; Diaz-Ruiz, Gloria; Wacher, Carmen; Pérez-Chabela, María de Lourdes

2017-02-01

The present study aimed to evaluate the bacterial load of water, Nile Tilapia and common Carp intestines from earthen ponds, isolate lactic acid bacteria (LAB) and assess their antimicrobial activity against fish spoilage and pathogenic bacteria. Following enumeration and isolation of microorganisms the antimicrobial activity of the LAB isolates was evaluated. Taxonomic identification of selected antagonistic LAB strains was assessed, followed by partial characterisation of their antimicrobial metabolites. Results showed that high counts (>4 log c.f.u ml -1 or 8 log c.f.u g -1 ) of total aerobic bacteria were recorded in pond waters and fish intestines. The microbiota were also found to be dominated by Salmonella spp., Vibrio spp., Staphylococcus spp. and Escherichia coli. LAB isolates (5.60%) exhibited potent direct and extracellular antimicrobial activity against the host-derived and non host-derived spoilage and pathogenic bacteria. These antagonistic isolates were identified and Lactococcus lactis subsp. lactis was found as the predominant (42.85%) specie. The strains displayed the ability to produce lactic, acetic, butyric, propionic and valeric acids. Bacteriocin-like inhibitory substances with activity against Gram-positive and Gram-negative (Vibrio spp. and Pseudomonas aeruginosa) bacteria were produced by three L. lactis subsp. lactis strains. In this study, the LAB from the microbiota of fish and pond water showed potent antimicrobial activity against fish spoilage or pathogenic bacteria from the same host or ecological niche. The studied Cameroonian aquatic niche is an ideal source of antagonistic LAB that could be appropriate as new fish biopreservatives or disease control agents in aquaculture under tropical conditions in particular or worldwide in general.

Proteolysis in goat "coalho" cheese supplemented with probiotic lactic acid bacteria.

PubMed

Bezerra, Taliana Kênia Alves; de Araujo, Ana Rita Ribeiro; do Nascimento, Edilza Santos; de Matos Paz, José Eduardo; Gadelha, Carlos Alberto; Gadelha, Tatiane Santi; Pacheco, Maria Teresa Bertoldo; do Egypto Queiroga, Rita de Cássia Ramos; de Oliveira, Maria Elieidy Gomes; Madruga, Marta Suely

2016-04-01

This study aimed to analyse the proteolytic effects of adding isolated and combined probiotic strains to goat "coalho" cheese. The cheeses were: QS - with culture Start, composed by Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris (R704); QLA - with Lactobacillus acidophilus (LA-5); QLP - with Lactobacillus paracasei subsp. paracasei (L. casei 01); QB - with Bifidobacterium animalis subsp. lactis (BB 12); and QC, co-culture with the three probiotic microorganisms. The cheeses were analysed during 28 days of storage at 10°C. The probiotic cell count was higher than 6.5 and 7 log colony-forming units (CFU) g(-1) of cheese at the 1st and 28th days of storage, respectively. The addition of co-culture influenced (p<0.01) proteolysis in the cheese and resulted in a higher content of soluble protein and release of amino acids at the 1st day after processing. However, over all 28 days, the cheese supplemented with Bifidobacterium lactis in its isolated form showed the highest proteolytic activity, particularly in the hydrolysis of the alpha-s2 and kappa-casein fractions. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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

Thompson, J.; Chassy, B.M.; Egan, W.

A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of (/sup 14/C)lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same.more » During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution /sup 31/P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM.« less

Selection, application and monitoring of Lactobacillus paracasei strains as adjunct cultures in the production of Gouda-type cheeses.

PubMed

Van Hoorde, Koenraad; Van Leuven, Isabelle; Dirinck, Patrick; Heyndrickx, Marc; Coudijzer, Kathleen; Vandamme, Peter; Huys, Geert

2010-12-15

Raw milk cheeses have more intense flavours than cheeses made from pasteurized milk and harbour strains with potential adjunct properties. Two Lactobacillus paracasei strains, R-40926 and R-40937, were selected as potential adjunct cultures from a total of 734 isolates from good quality artisan raw milk Gouda-type cheeses on the basis of their prevalence in different cheese types and/or over several production batches, safety and technological parameters. Conventional culturing, isolation and identification and a combined PCR-DGGE approach using total cheese DNA extracts and DNA extracts obtained from culturable fractions were employed to monitor viability of the introduced adjuncts and their effect on the cheese microbiota. The control cheese made without adjuncts was dominated by members of the starter, i.e. Lactococcus lactis and Leuconostoc pseudomesenteroides. In the cheeses containing either R-40926 or R-40937, the respective adjuncts increased in number as ripening progressed indicating that both strains are well adapted to the cheese environment and can survive in a competitive environment in the presence of a commercial starter culture. Principal component analysis of cheese volatiles determined by steam distillation-extraction and gas chromatography-mass spectrometry could differentiate cheeses made with different concentrations of adjunct R-40926 from the control cheese, and these differences could be correlated to the proteolytic and lipolytic properties of this strain. Collectively, results from microbiological and metabolic analyses indicate that the screening procedure followed throughout this study was successful in delivering potential adjunct candidates to enrich or extend the flavour palette of artisan Gouda-type cheeses under more controlled conditions. Copyright © 2010 Elsevier B.V. All rights reserved.

Some Technological Properties of Lactic Acid Bacteria Isolated from Dahi and Datshi, Naturally Fermented Milk Products of Bhutan

PubMed Central

Shangpliang, H. N. J.; Sharma, Sharmila; Rai, Ranjita; Tamang, Jyoti P.

2017-01-01

Dahi and datshi are common naturally fermented milk (NFM) products of Bhutan. Population of lactic acid bacteria (LAB) in dahi (pH 3.7) and datshi (pH 5.2) was 1.4 × 107 and 3.9 × 108 cfu/ml, respectively. Based on 16S rRNA gene sequencing isolates of LAB from dahi and datshi were identified as Enterococcus faecalis, E. faecium, Lactococcus lactis subsp. lactis. LAB strains were tested for some technological properties. All LAB strains except E. faecalis CH2:17 caused coagulation of milk at both 30°C for 48 h. Only E. faecium DH4:05 strain was resistant to pH 3. No significant difference (P > 0.05) of viable counts was observed in MRS broth with and without lysozyme. All LAB strains grew well in 0.3% bile showing their ability to tolerate bile salt. None of the LAB strains showed >70% hydrophobicity. This study, being the first of its microbiological analysis of the NFM of Bhutan, has opened up to an extent of research work that gives a new insight to the products. PMID:28203227

Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products.

PubMed

Valerio, Francesca; Favilla, Mara; De Bellis, Palmira; Sisto, Angelo; de Candia, Silvia; Lavermicocca, Paola

2009-09-01

Thirty samples of Italian durum wheat semolina and whole durum wheat semolina, generally used for the production of Southern Italy's traditional breads, were subjected to microbiological analysis in order to explore their lactic acid bacteria (LAB) diversity and to find strains with antifungal activity. A total of 125 presumptive LAB isolates (Gram-positive and catalase-negative) were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and sequence analysis of the 16S rRNA gene, leading to the identification of the following species: Weissella confusa, Weissella cibaria, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus rossiae and Lactobacillus plantarum. The REP-PCR results delineated 17 different patterns whose cluster analysis clearly differentiated W. cibaria from W. confusa isolates. Seventeen strains, each characterized by a different REP-PCR pattern, were screened for their antifungal properties. They were grown in a flour-based medium, comparable to a real food system, and the resulting fermentation products (FPs) were tested against fungal species generally contaminating bakery products, Aspergillus niger, Penicillium roqueforti and Endomyces fibuliger. The results of the study indicated a strong inhibitory activity - comparable to that obtained with the common preservative calcium propionate (0.3% w/v) - of ten LAB strains against the most widespread contaminant of bakery products, P. roqueforti. The screening also highlighted the unexplored antifungal activity of L. citreum, L. rossiae and W. cibaria (1 strain), which inhibited all fungal strains to the same or a higher extent compared with calcium propionate. The fermentation products of these three strains were characterized by low pH values, and a high content of lactic and acetic acids.

Protective effects of Lactococcus chungangensis CAU 28 on alcohol-metabolizing enzyme activity in rats.

PubMed

Konkit, Maytiya; Kim, Kiyoung; Kim, Jong-Hwa; Kim, Wonyong

2018-04-19

In this study, we investigated the beneficial effects of Lactococcus chungangensis CAU 28, a bacterial strain of nondairy origin, on alcohol metabolism in rats treated with ethanol, focusing on alcohol elimination and prevention of damage and comparing the effects with those observed for Lactococcus lactis ssp. lactis ATCC 19435. Male Sprague-Dawley rats were orally administered 20% ethanol and 3 substrates (freeze-dried cells, cream cheese, and yogurt) containing Lc. chungangensis CAU 28 or Lc. lactis ssp. lactis ATCC 19435, which were provided 1 h before or 1 h after ethanol ingestion. Blood samples were collected from the tail veins of the rats at 1, 3, 6, 12, and 24 h after ingestion of ethanol, Lc. chungangensis CAU 28 substrate, or Lc. lactis ssp. lactis ATCC 19435 substrate. Alcohol and acetaldehyde concentrations in the Lc. chungangensis CAU 28 substrate-treated rats were significantly reduced in a time-dependent manner compared with those in the Lc. lactis ssp. lactis ATCC 19435 substrate-treated rats. Among the experimental groups, treatment with cream cheese before ingestion of 20% ethanol was found to be the most effective method for reducing both alcohol and acetaldehyde levels in the blood. Alanine aminotransferase and aspartate aminotransferase activities in the Lc. chungangensis CAU 28 substrate-treated rats were significantly lower than those in the positive controls. Moreover, in the Lc. chungangensis CAU 28 cream cheese-treated group, rats showed a reduction of liver enzymes by up to 60%, with good effectiveness observed for both pre- and post-ethanol ingestion. These results suggested that intake of lactic acid bacteria, particularly in Lc. chungangensis CAU 28-supplemented dairy products, may reduce blood alcohol and acetaldehyde concentrations, thereby mitigating acute alcohol-induced hepatotoxicity by altering alcohol-metabolizing enzyme activities. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights

On the microbiological profile of traditional Portuguese sourdough.

PubMed

Rocha, J M; Malcata, F X

1999-12-01

Traditional manufacture of bread from maize has been noted to play important roles from both economic and social standpoints; however, enforcement of increasingly strict hygiene standards requires thorough knowledge of the adventitious microbiota of the departing dough. To this goal, sourdough as well as maize and rye flours from several geographic locations and in two different periods within the agricultural year were assayed for their microbiota in sequential steps of quantification and identification. More than 400 strains were isolated and taxonomic differentiation between them was via Biomerieux API galleries (375 of which were successfully identified) following preliminary biochemical and morphological screening. The dominant groups were yeasts and lactic acid bacteria (LAB). The most frequently isolated yeasts were Saccharomyces cerevisiae and Candida pelliculosa. The most frequently isolated LAB were (heterofermentative) Leuconostoc spp. and (homofermentative) Lactobacillus spp.; L. brevis, L. curvatus, and L. lactis ssp. lactis were the dominant species for the Lactobacillus genera; Lactococcus lactis ssp. lactis for lactococci; Enterococcus casseliflavus, E. durans, and E. faecium for enterococci; and Streptococcus constellantus and S. equinus for streptococci.

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

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

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.

Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines

PubMed Central

2011-01-01

Food-grade Lactic Acid Bacteria (LAB) have been safely consumed for centuries by humans in fermented foods. Thus, they are good candidates to develop novel oral vectors, constituting attractive alternatives to attenuated pathogens, for mucosal delivery strategies. Herein, this review summarizes our research, up until now, on the use of LAB as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Most of our work has been based on the model LAB Lactococcus lactis, for which we have developed efficient genetic tools, including expression signals and host strains, for the heterologous expression of therapeutic proteins such as antigens, cytokines and enzymes. Resulting recombinant lactococci strains have been tested successfully for their prophylactic and therapeutic effects in different animal models: i) against human papillomavirus type 16 (HPV-16)-induced tumors in mice, ii) to partially prevent a bovine β-lactoglobulin (BLG)-allergic reaction in mice and iii) to regulate body weight and food consumption in obese mice. Strikingly, all of these tools have been successfully transposed to the Lactobacillus genus, in recent years, within our laboratory. Notably, anti-oxidative Lactobacillus casei strains were constructed and tested in two chemically-induced colitis models. In parallel, we also developed a strategy based on the use of L. lactis to deliver DNA at the mucosal level, and were able to show that L. lactis is able to modulate the host response through DNA delivery. Today, we consider that all of our consistent data, together with those obtained by other groups, demonstrate and reinforce the interest of using LAB, particularly lactococci and lactobacilli strains, to develop novel therapeutic protein mucosal delivery vectors which should be tested now in human clinical trials. PMID:21995317

Short communication: Salt tolerance of Lactococcus lactis R-604 as influenced by mild stresses from ethanol, heat, hydrogen peroxide, and UV light.

PubMed

Gonzalez, Ernesto E; Olson, Douglas; Aryana, Kayanush

2017-06-01

Lactococcus lactis is a culture widely used in salt-containing dairy products. Salt hinders bacterial growth, but exposure to environmental stress may protect cells against subsequent stress, including salt. The objective of this study was to evaluate the salt tolerance of L. lactis R-604 after exposure to various stresses. The culture was subjected to 10% (vol/vol) ethanol for 30 min, mild heat at 52°C for 30 min, 15 mM hydrogen peroxide for 30 min, or UV light (254 nm) for 5 min and compared with a control. Starting with 5 log cfu/mL for all treatments, growth was determined in M17 broth with 5 NaCl concentrations (0, 1, 3, 5, and 7% wt/vol). Plating was conducted daily for 5 d. Salt tolerance was enhanced with mild heat exposure before growth in M17 broth with 5% (wt/vol) NaCl on d 3, 4, and 5, and with exposure to hydrogen peroxide and ethanol stresses before growth in M17 broth with 5% (wt/vol) NaCl on d 4 and 5. Exposure of this culture to mild heat, hydrogen peroxide, or ethanol before growth in M17 broth containing 5% (wt/vol) salt can enhance its survival, which could be beneficial when using it in salt-containing dairy products. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Effect of wild strains used as starter cultures and adjunct cultures on the volatile compounds of the Pecorino Siciliano cheese.

PubMed

Randazzo, C L; Pitino, I; De Luca, S; Scifò, G O; Caggia, C

2008-03-20

The effect of six wild strains on the volatile profile of the PS cheese was investigated and compared to that generated from industrial starters generally used to produce PS cheese. All cheeses were subjected to microbiological, physicochemical, and volatile compounds analyses. The DGGE of the 16S rDNA analysis was also applied. The volatile compounds generated during ripening were studied through the SPME and the GC-MS methods. No difference was detected between the experimental and control cheeses throughout chemical and microbiological analyses, while the DGGE results showed the presence of Streptococcus thermophilus in all cheeses, and the dominance of Enterococcus durans, Lactobacillus rhamnosus, and Lactobacillus casei in most of the experimental cheeses. Moreover, the presence of Lactococcus lactis species as in the control and in the experimental P2 and P4 cheeses was also revealed. The SPME results showed more pronounced volatile compounds in the experimental cheese samples than in the control ones.

Technological properties of bacteriocin-producing lactic acid bacteria isolated from Pico cheese an artisanal cow's milk cheese.

PubMed

Ribeiro, S C; Coelho, M C; Todorov, S D; Franco, B D G M; Dapkevicius, M L E; Silva, C C G

2014-03-01

Evaluate technologically relevant properties from bacteriocin-producing strains to use as starter/adjunct cultures in cheese making. Eight isolates obtained from Pico cheese produced in Azores (Portugal) were found to produce bacteriocins against Listeria monocytogenes and three isolates against Clostridium perfringens. They were identified as Lactococcus lactis and Enterococcus faecalis and submitted to technological tests: growth at different conditions of temperature and salt, acid production, proteolysis, lipolysis, coexistence, enzymatic profile and autolytic capacity. Safety evaluation was performed by evaluating haemolytic, gelatinase and DNase activity, resistance to antibiotics and the presence of virulence genes. Some isolates presented good technological features such as high autolytic activity, acid and diacetyl production. Lactococcus lactis was negative for all virulence genes tested and inhibit the growth of all Lactic acid bacteria (LAB) isolates. Enterococci were positive for the presence of some virulence genes, but none of the isolates were classified as resistant to important antibiotics. The bacteriocin-producing Lc. lactis present good potential for application in food as adjunct culture in cheese production. The study also reveals good technological features for some Enterococcus isolates. Bacteriocin-producing strains presented important technological properties to be exploited as new adjunct culture for the dairy industry, influencing flavour development and improve safety. © 2013 The Society for Applied Microbiology.

In vitro evidence for efficacy in food intolerance for the multispecies probiotic formulation Ecologic® Tolerance (Syngut™).

PubMed

Besseling-van der Vaart, I; Heath, M D; Guagnini, F; Kramer, M F

2016-02-01

The beneficial effects of probiotics are currently the subject of extensive studies in health and medical research. The aim of this research was to specifically design a new probiotic formulation for supplementation in people suffering from food intolerance. The selection of strains was focussed on the capacity to influence mechanisms of action that are important in development of food intolerance with the following parameters measure: in vitro capacity to produce β-galactosidase, in vitro strengthening of the epithelial barrier, in vitro stimulation of cytokines produced by regulatory T cells, in addition to assessing fundamental quality criteria (stability, gastrointestinal (GI)-survival, multispecies concept, allergen-free). Ecologic®Tolerance/Syngut™ was subsequently developed consisting of a multispecies concept using 4 different probiotic strains (Bifidobacterium lactis W51, Lactobacillus acidophilus W22, Lactobacillus plantarum W21 and Lactococcus lactis W19). Each of these strains demonstrated ability to survive the GI-tract and strain specific effects in producing β-galactosidase, strengthening the gut barrier function after immunological-induced stress and inhibiting Th2 cytokines (IL-4, IL-5 and IL-13 (≥50%), in addition to stimulating interleukin-10 levels; thus, providing in vitro evidence for the efficacy of the selected strains to provide beneficial effects in patients suffering from food intolerance.

Innovative approaches to nisin production.

PubMed

Özel, Burcu; Şimşek, Ömer; Akçelik, Mustafa; Saris, Per E J

2018-05-30

Nisin is a bacteriocin produced by Lactococcus lactis that has been approved by the Food Drug Administration for utilization as a GRAS status food additive. Nisin can inhibit spore germination and demonstrates antimicrobial activity against Listeria, Clostridium, Staphylococcus, and Bacillus species. Under some circumstances, it plays an immune modulator role and has a selective cytotoxic effect against cancer cells, although it is notable that the high production cost of nisin-a result of the low nisin production yield of producer strains-is an important factor restricting intensive use. In recent years, production of nisin has been significantly improved through genetic modifications to nisin producer strains and through innovative applications in the fermentation process. Recently, 15,400 IU ml -1 nisin production has been achieved in L. lactis cells following genetic modifications by eliminating the factors that negatively affect nisin biosynthesis or by increasing the cell density of the producing strains in the fermentation medium. In this review, innovative approaches related to cell and fermentation systems aimed at increasing nisin production are discussed and interpreted, with a view to increasing industrial nisin production.

Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii.

PubMed

Tanigawa, Kana; Watanabe, Koichi

2011-03-01

Currently, the species Lactobacillus delbrueckii is divided into four subspecies, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. indicus and L. delbrueckii subsp. lactis. These classifications were based mainly on phenotypic identification methods and few studies have used genotypic identification methods. As a result, these subspecies have not yet been reliably delineated. In this study, the four subspecies of L. delbrueckii were discriminated by phenotype and by genotypic identification [amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST)] methods. The MLST method developed here was based on the analysis of seven housekeeping genes (fusA, gyrB, hsp60, ileS, pyrG, recA and recG). The MLST method had good discriminatory ability: the 41 strains of L. delbrueckii examined were divided into 34 sequence types, with 29 sequence types represented by only a single strain. The sequence types were divided into eight groups. These groups could be discriminated as representing different subspecies. The results of the AFLP and MLST analyses were consistent. The type strain of L. delbrueckii subsp. delbrueckii, YIT 0080(T), was clearly discriminated from the other strains currently classified as members of this subspecies, which were located close to strains of L. delbrueckii subsp. lactis. The MLST scheme developed in this study should be a useful tool for the identification of strains of L. delbrueckii to the subspecies level.

Recombinant Lactococcus lactis can make the difference in antigen-specific immune tolerance induction, the Type 1 Diabetes case

PubMed Central

2014-01-01

Especially in western civilizations, immune diseases that are driven by innocuous (auto- or allo-) antigens are gradually evolving to become pandemic threats. A particularly poignant example is type 1 diabetes, where young children are confronted with the perspective and consequences of total pancreatic β-cell destruction. Along these disquieting observations we find ourselves equipped with impressively accumulating molecular immunological knowledge on the ins and outs of these pathologies. Often, however, it is difficult to translate this wealth into efficacious medicines. The molecular understanding, the concept of oral tolerance induction, the benefit of using recombinant Lactococcus lactis therein and recent openings towards their clinical use may well enable turning all colors to their appropriate fields on this Rubik's cube. PMID:25185797

Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1

PubMed Central

2011-01-01

Background Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast Hansenula polymorpha is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of H. polymorpha DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (GSH2) and its precursor cysteine (MET4) was studied. Results Glutathione producing capacity of H. polymorpha DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSGin, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSGex, per liter of the culture medium. Conclusions H. polymorpha is an competitive glutathione producer as compared to other known yeast and bacteria strains (Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione. PMID:21255454

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.

A food-grade fimbrial adhesin FaeG expression system in Lactococcus lactis and Lactobacillus casei.

PubMed

Lu, W W; Wang, T; Wang, Y; Xin, M; Kong, J

2016-03-01

Enterotoxigenic Escherichia coli (ETEC) infection is the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factor in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Lactic acid bacteria (LAB) are attractive tools to deliver antigens at the mucosal level. With the safety of genetically modified LAB in mind, a food-grade secretion vector (pALRc or pALRb) was constructed with DNA entirely from LAB, including the replicon, promoter, signal peptide, and selection marker alanine racemase gene (alr). To evaluate the feasibility of the system, the nuclease gene (nuc) from Staphylococcus aureus was used as a reporter to be expressed in both Lactococcus lactis and Lactobacillus casei. Subsequently, the extracellular secretion of the fimbrial adhesin FaeG of ETEC was confirmed by Western blot analysis. These results showed that this food-grade expression system has potential as the delivery vehicle for the safe use of genetically modified LAB for the development of vaccines against ETEC infection.

Fermentation performance of lactic acid bacteria in different lupin substrates-influence and degradation ability of antinutritives and secondary plant metabolites.

PubMed

Fritsch, C; Vogel, R F; Toelstede, S

2015-10-01

The main objectives were to determine the influence of secondary plant metabolites and antinutritives in lupin seeds on the fermentation performance of lactic acid bacteria and to study their ability to degrade these substances. The suitability of lupin raw materials as fermentation substrates was examined. To evaluate the fermentation performance, microbial growth, metabolite formation and substrate uptake in three different lupin substrates was monitored. On the one hand, a lupin protein isolate, which contained only trace amounts of phytochemicals was used in the study. On the other hand, the flour of Lupinus angustifolius cv. Boregine and the flour of the alkaloid rich lupin Lupinus angustifolius cv. Azuro were inoculated with Bifidobacterium animalis subsp. lactis, Pediococcus pentosaceus, Lactobacillus plantarum and Lactococcus lactis subsp. lactis. The micro-organisms showed no significant differences in the fermentation performance on the different lupin flours. Similarly, the growth of most strains on lupin protein isolate was comparable to that on the lupin flours. The fermentation with Bifidobacterium animalis subsp. lactis led to a significant decrease in flatulence causing oligosaccharides. During fermentation with Lactobacillus plantarum the phytic acid content was partially degraded. Neither the secondary plant metabolites nor the antinutritives of lupin flour inhibited the growth or metabolic activity of the tested micro-organisms. Therefore, lupin flour is suitable for lactic fermentation. Some strains showed the ability to degrade oligosaccharides or phytic acid. This work contributes to the fundamental knowledge of the metabolism of lactic acid bacteria during fermentation of lupin substrates. Fermentation of lupin raw materials could be used to improve the nutritional value of the substrates due to the reduction of antinutritives. © 2015 The Society for Applied Microbiology.

Production of enterocins L50A, L50B, and IT, a new enterocin, by Enterococcus faecium IT62, a strain isolated from Italian ryegrass in Japan.

PubMed

Izquierdo, Esther; Bednarczyk, Audrey; Schaeffer, Christine; Cai, Yimin; Marchioni, Eric; Van Dorsselaer, Alain; Ennahar, Saïd

2008-06-01

Enterococcus faecium IT62, isolated from ryegrass in Japan, was shown to produce three different bacteriocins, two of which had molecular masses and amino acid sequences that corresponded to those of enterocin L50A and enterocin L50B. These peptides existed, however, as chemically modified forms that were either N formylated or N formylated and oxidized at Met(24). The third bacteriocin, named enterocin IT, had a molecular mass of 6,390 Da, was made up of 54 amino acids, and did not correspond to any known bacteriocin. However, enterocin IT was identical to the C-terminal part of the 16-amino-acid-longer bacteriocin 32 (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). For the first time, the antimicrobial activity spectra for enterocins L50A and L50B were determined separately and included a wide range of gram-positive bacteria but also a few gram-negative strains that were weakly sensitive. Slight differences in the activities of enterocins L50A and L50B were observed, as gram-positive bacteria showed an overall higher level of sensitivity to L50A than to L50B, as opposed to gram-negative ones. Conversely, enterocin IT showed a very narrow antimicrobial spectrum that was limited to E. faecium strains, one strain of Bacillus subtilis, and one strain of Lactococcus lactis. This study showed that E. faecium IT62, a grass-borne strain, produces bacteriocins with very different activity features and structures that may be found in strains associated with food or those of clinical origin, which demonstrates that a particular enterocin structure may be widespread and not related to the producer's origin.

Production of Enterocins L50A, L50B, and IT, a New Enterocin, by Enterococcus faecium IT62, a Strain Isolated from Italian Ryegrass in Japan▿

PubMed Central

Izquierdo, Esther; Bednarczyk, Audrey; Schaeffer, Christine; Cai, Yimin; Marchioni, Eric; Van Dorsselaer, Alain; Ennahar, Saïd

2008-01-01

Enterococcus faecium IT62, isolated from ryegrass in Japan, was shown to produce three different bacteriocins, two of which had molecular masses and amino acid sequences that corresponded to those of enterocin L50A and enterocin L50B. These peptides existed, however, as chemically modified forms that were either N formylated or N formylated and oxidized at Met24. The third bacteriocin, named enterocin IT, had a molecular mass of 6,390 Da, was made up of 54 amino acids, and did not correspond to any known bacteriocin. However, enterocin IT was identical to the C-terminal part of the 16-amino-acid-longer bacteriocin 32 (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). For the first time, the antimicrobial activity spectra for enterocins L50A and L50B were determined separately and included a wide range of gram-positive bacteria but also a few gram-negative strains that were weakly sensitive. Slight differences in the activities of enterocins L50A and L50B were observed, as gram-positive bacteria showed an overall higher level of sensitivity to L50A than to L50B, as opposed to gram-negative ones. Conversely, enterocin IT showed a very narrow antimicrobial spectrum that was limited to E. faecium strains, one strain of Bacillus subtilis, and one strain of Lactococcus lactis. This study showed that E. faecium IT62, a grass-borne strain, produces bacteriocins with very different activity features and structures that may be found in strains associated with food or those of clinical origin, which demonstrates that a particular enterocin structure may be widespread and not related to the producer's origin. PMID:18391036

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2.

PubMed

Neviani, E; Boquien, C Y; Monnet, V; Thanh, L P; Gripon, J C

1989-09-01

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.

Probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 versus placebo for the symptoms of bloating in patients with functional bowel disorders: a double-blind study.

PubMed

Ringel-Kulka, Tamar; Palsson, Olafur S; Maier, Danielle; Carroll, Ian; Galanko, Joseph A; Leyer, Gregory; Ringel, Yehuda

2011-07-01

Recent data suggest a role for the intestinal microbiota in the pathogenesis of functional bowel disorders (FBDs). Probiotic studies in FBDs generated inconsistent results suggesting a strain-specific and product-specific effect. To investigate the clinical efficacy of Lactobacillus acidophilus NCFM (L-NCFM) and Bifidobacterium lactis Bi-07 (B-LBi07) in nonconstipation FBDs. A double-blind, placebo-control clinical trial of the probiotic bacterias L-NCFM and B-LBi07 twice a day (2×10(11) CFU/d) versus placebo over 8 weeks. Primary endpoints were global relief of gastrointestinal symptoms and satisfaction with treatment. Secondary endpoints were change in symptoms severity, well-being, and quality of life. Microbiological effect was assessed by quantitative real time polymerase chain reaction on fecal samples. Sixty patients (probiotic, n=31; placebo, n=29), 72% females, 84% whites, mean age 37 years. Abdominal bloating improved in the probiotics compared with the placebo group at 4 weeks (4.10 vs 6.17, P=0.009; change in bloating severity P=0.02) and 8 weeks (4.26 vs 5.84, P=0.06; change in bloating severity P<0.01). Analyses on the irritable bowel syndrome subgroup (n=33) showed similar results. L-NCFM and B-LBi07 twice a day improve symptoms of bloating in patients with FBDs. These data supports the role of intestinal bacteria in the pathophysiology of FBD and the role for probiotic bacteria in the management of these disorders.

Inactivation of a gene that is highly conserved in Gram-positive bacteria stimulates degradation of non-native proteins and concomitantly increases stress tolerance in Lactococcus lactis.

PubMed

Frees, D; Varmanen, P; Ingmer, H

2001-07-01

Exposure of cells to elevated temperatures triggers the synthesis of chaperones and proteases including components of the conserved Clp protease complex. We demonstrated previously that the proteolytic subunit, ClpP, plays a major role in stress tolerance and in the degradation of non-native proteins in the Gram-positive bacterium Lactococcus lactis. Here, we used transposon mutagenesis to generate mutants in which the temperature- and puromycin-sensitive phenotype of a lactococcal clpP null mutant was partly alleviated. In all mutants obtained, the transposon was inserted in the L. lactis trmA gene. When analysing a clpP, trmA double mutant, we found that the expression normally induced from the clpP and dnaK promoters in the clpP mutant was reduced to wild-type level upon introduction of the trmA disruption. Additionally, the degradation of puromycyl-containing polypeptides was increased, suggesting that inactivation of trmA compensates for the absence of ClpP by stimulating an as yet unidentified protease that degrades misfolded proteins. When trmA was disrupted in wild-type cells, both stress tolerance and proteolysis of puromycyl peptides was enhanced above wild-type level. Based on our results, we propose that TrmA, which is well conserved in several Gram-positive bacteria, affects the degradation of non-native proteins and thereby controls stress tolerance.

Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine.

PubMed

Sreekumar, R; Al-Attabi, Z; Deeth, H C; Turner, M S

2009-06-01

To investigate the abilities of various probiotic bacteria to produce volatile sulfur compounds (VSCs) relevant to food flavour and aroma. Probiotic strains (Lactobacillus acidophilus NCFM, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC55730 and L. reuteri BR11), Lactobacillus delbrueckii ATCC4797, L. plantarum ATCC14917 and Lactococcus lactis MG1363 were incubated with either cysteine or methionine. Volatile compounds were captured, identified and quantified using a sensitive solid-phase microextraction (SPME) technique combined with gas chromatography coupled to a pulsed flame photometric detector (SPME/GC/PFPD). Several VSCs were identified including H(2)S, methanethiol, dimethyldisulfide and dimethyltrisulfide. The VSC profiles varied substantially for different strains of L. plantarum and L. reuteri and it was found that L. reuteri ATCC55730 and L. lactis MG1363 produced the lowest levels of VSCs (P < 0.05). Levels of VSCs generated by bacteria were found to be equivalent to, or higher than, that found in commercial cheeses. Several probiotic strains are able to generate considerable levels of VSCs and substantial variations in VSC generating potential exists between different strains from the same species. This study demonstrates that probiotic bacteria are able to efficiently generate important flavour and aroma compounds and therefore has implications for the development of probiotic containing foods.

A qualified presumption of safety approach for the safety assessment of Grana Padano whey starters.

PubMed

Rossetti, Lia; Carminati, Domenico; Zago, Miriam; Giraffa, Giorgio

2009-03-15

A Qualified Presumption of Safety (QPS) approach was applied to dominant lactic acid bacteria (LAB) associated with Grana Padano cheese whey starters. Thirty-two strains belonging to Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum, and representing the overall genotypic LAB diversity associated with 24 previously collected whey starters [Rossetti, L., Fornasari, M.E., Gatti, M., Lazzi, C., Neviani, E., Giraffa, G., 2008. Grana Padano cheese whey starters: microbial composition and strain distribution. International Journal of Food Microbiology 127, 168-171], were analyzed. All L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus isolates were susceptible to four (i.e. vancomycin, gentamicin, tetracycline, and erythromycin) of the clinically most relevant antibiotics. One L. fermentum strain displayed phenotypic resistance to tetracycline (Tet(R)), with MIC of 32 microg/ml, and gentamycin (Gm(R)), with MIC of 32 microg/ml. PCR was applied to this strain to test the presence of genes tet(L), tet(M), tet(S), and aac(6')-aph(2')-Ia, which are involved in horizontal transfer of Tet(R) and Gm(R), respectively but no detectable amplification products were observed. According to QPS criteria, we conclude that Grana cheese whey starters do not present particular safety concerns.

An in vitro study on bacterial growth interactions and intestinal epithelial cell adhesion characteristics of probiotic combinations.

PubMed

Moussavi, Mahta; Adams, Michelle Catherine

2010-05-01

The aims of this study were to examine long-term growth interactions of five probiotic strains (Lactobacillus casei 01, Lactobacillus plantarum HA8, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC 55730 and Bifidobacterium lactis Bb12) either alone or in combination with Propionibacterium jensenii 702 in a co-culture system and to determine their adhesion ability to human colon adenocarcinoma cell line Caco-2. Growth patterns of probiotic Lactobacillus strains were not considerably affected by the presence of P. jensenii 702, whereas lactobacilli exerted a strong antagonistic action against P. jensenii 702. In the co-culture of Bif. lactis Bb12 and P. jensenii 702, a significant synergistic influence on growth of both bacteria was observed (P < 0.05). The results of adhesion assay showed that when probiotic strains were tested in combination, there was evidence of an associated effect on percentage adherence. However, in most cases these differences were not statistically significant (P < 0.05). Adhesion percentage of Lb. casei 01 and Lb. rhamnosus GG both decreased significantly in the presence of P. jensenii 702 compared to their adhesion levels when alone (P < 0.05). These results show that the survival and percentage adhesion of some probiotic strains may be influenced by the presence of other strains and this should be considered when formulating in the probiotic products.

Molecular discrimination of lactobacilli used as starter and probiotic cultures by amplified ribosomal DNA restriction analysis.

PubMed

Roy, D; Sirois, S; Vincent, D

2001-04-01

Lactic acid bacteria such as Lactobacillus helveticus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. acidophilus, and L. casei related taxa which are widely used as starter or probiotic cultures can be identified by amplified ribosomal DNA restriction analysis (ARDRA). The genetic discrimination of the related species belonging to these groups was first obtained by PCR amplifications by using group-specific or species-specific 16S rDNA primers. The numerical analysis of the ARDRA patterns obtained by using CfoI, HinfI, Tru9I, and ScrFI was an efficient typing tool for identification of species of the L. acidophilus and L. casei complex. ARDRA by using CfoI was a reliable method for differentiation of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Finally, strains ATCC 393 and ATCC 15820 exhibited unique ARDRA patterns with CfoI and Tru9I restriction enzymes as compared with the other strains of L. casei, L. paracasei, and L. rhamnosus.

Influence of starters on chemical, biochemical, and sensory changes in Turkish White-brined cheese during ripening.

PubMed

Hayaloglu, A A; Guven, M; Fox, P F; McSweeney, P L H

2005-10-01

Turkish White-brined cheese was manufactured using Lactococcus strains (Lactococcus lactis ssp. lactis NCDO763 plus L. lactis ssp. cremoris SK11 and L. lactis ssp. lactis UC317 plus L. lactis ssp. cremoris HP) or without a starter culture, and ripened for 90 d. It was found that the use of starters significantly influenced the physical, chemical, biochemical, and sensory properties of the cheeses. Chemical composition, pH, and sensory properties of cheeses made with starter were not affected by the different starter bacteria. The levels of soluble nitrogen fractions and urea-PAGE of the pH 4.6-insoluble fractions were found to be significantly different at various stages of ripening. Urea-PAGE patterns of the pH 4.6-insoluble fractions of the cheeses showed that considerable degradation of alpha(s1)-casein occurred and that beta-casein was more resistant to hydrolysis. The use of a starter culture significantly influenced the levels of 12% trichloroacetic acid-soluble nitrogen, 5% phosphotungstic acid-soluble nitrogen, free amino acids, total free fatty acids, and the peptide profiles (reverse phase-HPLC) of 70% (vol/vol) ethanol-soluble and insoluble fractions of the pH 4.6-soluble fraction of the cheeses. The levels of peptides in the cheeses increased during the ripening period. Principal component and hierarchical cluster analyses of electrophoretic and chromatographic results indicated that the cheeses were significantly different in terms of their peptide profiles and they were grouped based on the use and type of starter and stage of ripening. Levels of free amino acid in the cheeses differed; Leu, Glu, Phe, Lys, and Val were the most abundant amino acids. Nitrogen fractions, total free amino acids, total free fatty acids, and the levels of peptides resolved by reverse phase-HPLC increased during ripening. No significant differences were found between the sensory properties of cheeses made using a starter, but the cheese made without starter received lower

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

Variations in the Degree of d-Alanylation of Teichoic Acids in Lactococcus lactis Alter Resistance to Cationic Antimicrobials but Have No Effect on Bacterial Surface Hydrophobicity and Charge▿

PubMed Central