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Sample records for raffinose

  1. Silencing of Soybean Raffinose Synthase Gene Reduced Raffinose Family Oligosaccharides and Increased True Metabolizable Energy of Poultry Feed

    PubMed Central

    Valentine, Michelle F.; De Tar, Joann R.; Mookkan, Muruganantham; Firman, Jeffre D.; Zhang, Zhanyuan J.

    2017-01-01

    Soybean [Glycine max (L.) Merr.] is the number one oil and protein crop in the United States, but the seed contains several anti-nutritional factors that are toxic to both humans and livestock. RNA interference technology has become an increasingly popular technique in gene silencing because it allows for both temporal and spatial targeting of specific genes. The objective of this research is to use RNA-mediated gene silencing to down-regulate the soybean gene raffinose synthase 2 (RS2), to reduce total raffinose content in mature seed. Raffinose is a trisaccharide that is indigestible to humans and monogastric animals, and as monogastric animals are the largest consumers of soy products, reducing raffinose would improve the nutritional quality of soybean. An RNAi construct targeting RS2 was designed, cloned, and transformed to the soybean genome via Agrobacterium-mediated transformation. Resulting plants were analyzed for the presence and number of copies of the transgene by PCR and Southern blot. The efficiency of mRNA silencing was confirmed by real-time quantitative PCR. Total raffinose content was determined by HPLC analysis. Transgenic plant lines were recovered that exhibited dramatically reduced levels of raffinose in mature seed, and these lines were further analyzed for other phenotypes such as development and yield. Additionally, a precision-fed rooster assay was conducted to measure the true metabolizable energy (TME) in full-fat soybean meal made from the wild-type or transgenic low-raffinose soybean lines. Transgenic low-raffinose soy had a measured TME of 2,703 kcal/kg, an increase as compared with 2,411 kcal/kg for wild-type. As low digestible energy is a major limiting factor in the percent of soybean meal that can be used in poultry diets, these results may substantiate the use of higher concentrations of low-raffinose, full-fat soy in formulated livestock diets. PMID:28559898

  2. Composition and metabolism of fecal microbiota from normal and overweight children are differentially affected by melibiose, raffinose and raffinose-derived fructans.

    PubMed

    Adamberg, Kaarel; Adamberg, Signe; Ernits, Karin; Larionova, Anneli; Voor, Tiia; Jaagura, Madis; Visnapuu, Triinu; Alamäe, Tiina

    2018-06-20

    The aim of the study was to investigate the metabolism of non-digestible oligo- and polysaccharides by fecal microbiota, using isothermal microcalorimetry. The five tested substrates were raffinose, melibiose, a mixture of oligo- and polysaccharides produced from raffinose by levansucrase, levan synthesized from raffinose, and levan from timothy grass. Two inocula were comprised of pooled fecal samples from overweight or normal-weight children, from healthy adult volunteers and a pure culture of Bacteroides thetaiotaomicron as a reference bacterium for colon microbiota. The growth was analyzed based on the heat evolution curves, and the production of organic acids and gases. Taxonomic profiles of the microbiota were assessed by 16S rDNA sequencing. Raffinose and melibiose promoted the growth of bifidobacteria in all fecal pools. Several pool-specific substrate-related responses to raffinose and melibiose were revealed. Lactate-producing bacteria (Streptococcus and Enterococcus) became enriched in the pool of overweight children resulting in lactic acid as the major fermentation product on short saccharides. Acetic and butyric acids were prevalent at fermentation in the normal-weight pool coinciding with the enrichment of Catenibacterium. In the adult pool, the specific promotion of Bacteroides and Lachnospiraceae by levans was disclosed. In the fecal pool of normal-weight children, levans stimulated the growth of Senegalimassilia and Lachnoclostridium and this particular pool also showed the highest maximum heat production rate at levan fermentation. Levans and raffinose-derived oligosaccharides, but not raffinose and melibiose were completely fermented by a pure culture of Bacteroides thetaiotaomicron. The main conclusion from the study is that fecal microbiota of normal and overweight children have different compositions and they respond in specific manners to non-digestible oligo- and polysaccharides: raffinose, melibiose, raffinose-derived oligosaccharides and

  3. Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation.

    PubMed

    Brühlmann, David; Muhr, Anais; Parker, Rebecca; Vuillemin, Thomas; Bucsella, Blanka; Kalman, Franka; Torre, Serena; La Neve, Fabio; Lembo, Antonio; Haas, Tobias; Sauer, Markus; Souquet, Jonathan; Broly, Hervé; Hemberger, Jürgen; Jordan, Martin

    2017-06-20

    Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO 2 -controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Synbiotic promotion of epithelial proliferation by orally ingested encapsulated Bifidobacterium breve and raffinose in the small intestine of rats.

    PubMed

    Ishizuka, Satoshi; Iwama, Ami; Dinoto, Achmad; Suksomcheep, Akarat; Maeta, Kohshi; Kasai, Takanori; Hara, Hiroshi; Yokota, Atsushi

    2009-05-01

    We evaluated the effects of Bifidobacterium breve JCM1192(T )and/or raffinose on epithelial proliferation in the rat small and large intestines. WKAH/Hkm Slc rats (4 wk old) were fed a control diet, a diet supplemented with either encapsulated B. breve (30 g/kg diet, 1.5 x 10(7) colony-forming unit/g capsule) or raffinose (30 g/kg diet), or a diet supplemented with both encapsulated B. breve and raffinose, for 3 wk. Epithelial proliferation in the small intestine, as assessed by bromodeoxyuridine immunohistochemistry, was increased only in the B. breve plus raffinose-fed group. We determined the number of bifidobacteria in cecal contents using fluorescence in situ hybridization and confirmed the presence of ingested B. breve only in the B. breve plus raffinose-fed group. This suggests that the ingested B. breve cells used raffinose and were activated in the small intestine, where they subsequently influenced epithelial proliferation. In conclusion, we found a prominent synbiotic effect of encapsulated B. breve in combination with raffinose on epithelial proliferation in rat small intestine but not in large intestine. To our knowledge, this is the first report of a synbiotic that affects epithelial proliferation.

  5. Glucosylation of Raffinose via Alternansucrase Acceptor Reactions

    USDA-ARS?s Scientific Manuscript database

    The glucansucrase known as alternansucrase [EC 2.4.1.140] can transfer glucosyl units from sucrose to raffinose to give good yields of oligosaccharides, which may serve as prebiotics. The main products were the tetrasaccharides a-D-Glcp-(1-3)-a-D-Galp-(1-6)-a-D-Glcp-(1-2)-ß-D-Fruf and a-D-Glcp-(1-4...

  6. Raffinose, a plant galactoside, inhibits Pseudomonas aeruginosa biofilm formation via binding to LecA and decreasing cellular cyclic diguanylate levels

    NASA Astrophysics Data System (ADS)

    Kim, Han-Shin; Cha, Eunji; Kim, Yunhye; Jeon, Young Ho; Olson, Betty H.; Byun, Youngjoo; Park, Hee-Deung

    2016-05-01

    Biofilm formation on biotic or abiotic surfaces has unwanted consequences in medical, clinical, and industrial settings. Treatments with antibiotics or biocides are often ineffective in eradicating biofilms. Promising alternatives to conventional agents are biofilm-inhibiting compounds regulating biofilm development without toxicity to growth. Here, we screened a biofilm inhibitor, raffinose, derived from ginger. Raffinose, a galactotrisaccharide, showed efficient biofilm inhibition of Pseudomonas aeruginosa without impairing its growth. Raffinose also affected various phenotypes such as colony morphology, matrix formation, and swarming motility. Binding of raffinose to a carbohydrate-binding protein called LecA was the cause of biofilm inhibition and altered phenotypes. Furthermore, raffinose reduced the concentration of the second messenger, cyclic diguanylate (c-di-GMP), by increased activity of a c-di-GMP specific phosphodiesterase. The ability of raffinose to inhibit P. aeruginosa biofilm formation and its molecular mechanism opens new possibilities for pharmacological and industrial applications.

  7. A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

    PubMed Central

    Mollerup, Filip; Parikka, Kirsti; Koutaniemi, Sanna; Boer, Harry; Juvonen, Minna; Master, Emma; Tenkanen, Maija; Kruus, Kristiina

    2017-01-01

    ABSTRACT We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.−) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on d-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family. IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola. As discussed in the present study, the bioinformatics approach using the modular structure of

  8. Hydrophilic interaction liquid chromatography for the separation, purification, and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz.

    PubMed

    Liang, Tu; Fu, Qing; Li, Fangbing; Zhou, Wei; Xin, Huaxia; Wang, Hui; Jin, Yu; Liang, Xinmiao

    2015-08-01

    A systematic strategy based on hydrophilic interaction liquid chromatography was developed for the separation, purification and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz. Methods with enough hydrophilicity and selectivity were utilized to resolve the problems encountered in the separation of oligosaccharides such as low retention, low resolution and poor solubility. The raffinose family oligosaccharides in L. lucidus Turcz. were isolated using solid-phase extraction followed by hydrophilic interaction liquid chromatography at semi-preparative scale to obtain standards of stachyose, verbascose and ajugose. Utilizing the obtained oligosaccharides as standards, a quantitative determination method was developed, validated and applied for the content determination of raffinose family oligosaccharides both in the aerial and root parts of L. lucidus Turcz. There were no oligosaccharides in the aerial parts, while in the root parts, the total content was 686.5 mg/g with the average distribution: raffinose 66.5 mg/g, stachyose 289.0 mg/g, verbascose 212.4 mg/g, and ajugose 118.6 mg/g. The result provided the potential of roots of L. lucidus Turcz. as new raffinose family oligosaccharides sources for functional food. Moreover, since the present systematic strategy is efficient, sensitive and robust, separation, purification and quantification of oligosaccharides by hydrophilic interaction liquid chromatography seems to be possible. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Raffinose Induces Biofilm Formation by Streptococcus mutans in Low Concentrations of Sucrose by Increasing Production of Extracellular DNA and Fructan.

    PubMed

    Nagasawa, Ryo; Sato, Tsutomu; Senpuku, Hidenobu

    2017-08-01

    Streptococcus mutans is the primary etiological agent of dental caries and causes tooth decay by forming a firmly attached biofilm on tooth surfaces. Biofilm formation is induced by the presence of sucrose, which is a substrate for the synthesis of extracellular polysaccharides but not in the presence of oligosaccharides. Nonetheless, in this study, we found that raffinose, which is an oligosaccharide with an intestinal regulatory function and antiallergic effect, induced biofilm formation by S. mutans in a mixed culture with sucrose, which was at concentrations less than those required to induce biofilm formation directly. We analyzed the possible mechanism behind the small requirement for sucrose for biofilm formation in the presence of raffinose. Our results suggested that sucrose contributed to an increase in bacterial cell surface hydrophobicity and biofilm formation. Next, we examined how the effects of raffinose interacted with the effects of sucrose for biofilm formation. We showed that the presence of raffinose induced fructan synthesis by fructosyltransferase and aggregated extracellular DNA (eDNA, which is probably genomic DNA released from dead cells) into the biofilm. eDNA seemed to be important for biofilm formation, because the degradation of DNA by DNase I resulted in a significant reduction in biofilm formation. When assessing the role of fructan in biofilm formation, we found that fructan enhanced eDNA-dependent cell aggregation. Therefore, our results show that raffinose and sucrose have cooperative effects and that this induction of biofilm formation depends on supportive elements that mainly consist of eDNA and fructan. IMPORTANCE The sucrose-dependent mechanism of biofilm formation in Streptococcus mutans has been studied extensively. Nonetheless, the effects of carbohydrates other than sucrose are inadequately understood. Our findings concerning raffinose advance the understanding of the mechanism underlying the joint effects of sucrose and

  10. Environmental Stability of Seed Carbohydrate Profiles in Soybeans Containing Different Alleles of the Raffinose Synthase 2 (RS2) Gene.

    PubMed

    Bilyeu, Kristin D; Wiebold, William J

    2016-02-10

    Soybean [Glycine max (L.) Merr.] is important for the high protein meal used for livestock feed formulations. Carbohydrates contribute positively or negatively to the potential metabolizable energy in soybean meal. The positive carbohydrate present in soybean meal consists primarily of sucrose, whereas the negative carbohydrate components are the raffinose family of oligosaccharides (RFOs), raffinose and stachyose. Increasing sucrose and decreasing raffinose and stachyose are critical targets to improve soybean. In three recently characterized lines, variant alleles of the soybean raffinose synthase 2 (RS2) gene were associated with increased sucrose and decreased RFOs. The objective of this research was to compare the environmental stability of seed carbohydrates in soybean lines containing wild-type or variant alleles of RS2 utilizing a field location study and a date of planting study. The results define the carbohydrate variation in distinct regional and temporal environments using soybean lines with different alleles of the RS2 gene.

  11. Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose - an emerging prebiotic.

    PubMed

    Celebioglu, Hasan Ufuk; Ejby, Morten; Majumder, Avishek; Købler, Carsten; Goh, Yong Jun; Thorsen, Kristian; Schmidt, Bjarne; O'Flaherty, Sarah; Abou Hachem, Maher; Lahtinen, Sampo J; Jacobsen, Susanne; Klaenhammer, Todd R; Brix, Susanne; Mølhave, Kristian; Svensson, Birte

    2016-05-01

    Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Subcellular distribution of raffinose oligosaccharides and other metabolites in summer and winter leaves of Ajuga reptans (Lamiaceae).

    PubMed

    Findling, Sarah; Zanger, Klaus; Krueger, Stephan; Lohaus, Gertrud

    2015-01-01

    In Ajuga reptans, raffinose oligosaccharides accumulated during winter. Stachyose, verbascose, and higher RFO oligomers were exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. The evergreen labiate Ajuga reptans L. can grow at low temperature. The carbohydrate metabolism changes during the cold phase, e.g., raffinose family oligosaccharides (RFOs) accumulate. Additionally, A. reptans translocates RFOs in the phloem. In the present study, subcellular concentrations of metabolites were studied in summer and winter leaves of A. reptans to gain further insight into regulatory instances involved in the cold acclimation process and into the function of RFOs. Subcellular metabolite concentrations were determined by non-aqueous fractionation. Volumes of the subcellular compartments of summer and winter leaves were analyzed by morphometric measurements. The metabolite content varied strongly between summer and winter leaves. Soluble metabolites increased up to tenfold during winter whereas the starch content was decreased. In winter leaves, the subcellular distribution showed a shift of carbohydrates from cytoplasm to vacuole and chloroplast. Despite this, the metabolite concentration was higher in all compartments in winter leaves compared to summer leaves because of the much higher total metabolite content in winter leaves. The different oligosaccharides did show different compartmentations. Stachyose, verbascose, and higher RFO oligomers were almost exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. Apparently, the subcellular distribution of the RFOs differs because they fulfill different functions in plant metabolism during winter. Raffinose might function in protecting chloroplast membranes during freezing, whereas higher RFO oligomers may exert protective effects on vacuolar membranes. In addition, the high content of RFOs in winter leaves may also result from reduced consumption of

  13. Efficiency Analysis and Mechanism Insight of that Whole-Cell Biocatalytic Production of Melibiose from Raffinose with Saccharomyces cerevisiae.

    PubMed

    Zhou, Yingbiao; Zhu, Yueming; Dai, Longhai; Men, Yan; Wu, Jinhai; Zhang, Juankun; Sun, Yuanxia

    2017-01-01

    Melibiose is widely used as a functional carbohydrate. Whole-cell biocatalytic production of melibiose from raffinose could reduce its cost. However, characteristics of strains for whole-cell biocatalysis and mechanism of such process are unclear. We compared three different Saccharomyces cerevisiae strains (liquor, wine, and baker's yeasts) in terms of concentration variations of substrate (raffinose), target product (melibiose), and by-products (fructose and galactose) in whole-cell biocatalysis process. Distinct difference was observed in whole-cell catalytic efficiency among three strains. Furthermore, activities of key enzymes (invertase, α-galactosidase, and fructose transporter) involved in process and expression levels of their coding genes (suc2, mel1, and fsy1) were investigated. Conservation of key genes in S. cerevisiae strains was also evaluated. Results show that whole-cell catalytic efficiency of S. cerevisiae in the raffinose substrate was closely related to activity of key enzymes and expression of their coding genes. Finally, we summarized characteristics of producing strain that offered advantages, as well as contributions of key genes to excellent strains. Furthermore, we presented a dynamic mechanism model to achieve some mechanism insight for this whole-cell biocatalytic process. This pioneering study should contribute to improvement of whole-cell biocatalytic production of melibiose from raffinose.

  14. In vitro fermentation of alternansucrase raffinose acceptor products by human gut bacteria

    USDA-ARS?s Scientific Manuscript database

    In this work, in vitro fermentation of alternansucrase raffinose acceptor products, previously fractionated according to their degree of polymerization (DP; from DP4 to DP10) was carried out using pH-controlled small scale batch cultures at 37ºC under anaerobic conditions with human faeces. Bifidog...

  15. Raffinose from Costus speciosus attenuates lipid synthesis through modulation of PPARs/SREBP1c and improves insulin sensitivity through PI3K/AKT.

    PubMed

    Muthukumaran, Padmanaban; Thiyagarajan, Gopal; Arun Babu, Rajendran; Lakshmi, Baddireddi Subhadra

    2018-03-25

    Among several metabolic disorders, the pathogenesis of insulin resistance is considered to be multifactorial. Raffinose, an oligosaccharide isolated from the rhizome of Costus speciosus showed ≤50% inhibition of lipid accumulation in differentiated HepG2 and 3T3-L1 cells through exhibiting partial agonism to PPARγ, and, an enhanced secretion of adiponectin in 3T3-L1 adipocytes. Raffinose was also observed to attenuate the expression of SREBP1c, ACC and FAS which are involved in the fatty acid synthesis. A corresponding upregulation of PPARα and ACO involved in fatty acid oxidation was observed in steatotic HepG2 hepatocytes and 3T3-L1 adipocytes. In vitro evaluation of its anti-diabetic potential showed a dose dependent enhancement of glucose uptake. Investigation of the insulin sensitizing efficacy of Raffinose revealed an increase in Glut4 translocation via phosphorylation of IRβ/PI3K/Akt in differentiated L6 myocytes and 3T3-L1 preadipocytes. In addition, Raffinose was potentially involved in glycogen synthesis by inhibiting the activation of GSK3β. Hence, Raffinose could be a useful therapeutic agent for metabolic maladies. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Evolutionary diversification of galactinol synthases in Rosaceae: adaptive roles of galactinol and raffinose during apple bud dormancy.

    PubMed

    Falavigna, Vítor da Silveira; Porto, Diogo Denardi; Miotto, Yohanna Evelyn; Santos, Henrique Pessoa Dos; Oliveira, Paulo Ricardo Dias de; Margis-Pinheiro, Márcia; Pasquali, Giancarlo; Revers, Luís Fernando

    2018-01-24

    Galactinol synthase (GolS) is a key enzyme in the biosynthetic pathway of raffinose family oligosaccharides (RFOs), which play roles in carbon storage, signal transduction, and osmoprotection. The present work assessed the evolutionary history of GolS genes across the Rosaceae using several bioinformatic tools. Apple (Malus × domestica) GolS genes were transcriptionally characterized during bud dormancy, in parallel with galactinol and raffinose measurements. Additionally, MdGolS2, a candidate to regulate seasonal galactinol and RFO content during apple bud dormancy, was functionally characterized in Arabidopsis. Evolutionary analyses revealed that whole genome duplications have driven GolS gene evolution and diversification in Rosaceae speciation. The strong purifying selection identified in duplicated GolS genes suggests that differential gene expression might define gene function better than protein structure. Interestingly, MdGolS2 was differentially expressed during bud dormancy, concomitantly with the highest galactinol and raffinose levels. One of the intrinsic adaptive features of bud dormancy is limited availability of free water; therefore, we generated transgenic Arabidopsis plants expressing MdGolS2. They showed higher galactinol and raffinose contents and increased tolerance to water deficit. Our results suggest that MdGolS2 is the major GolS responsible for RFO accumulation during apple dormancy, and these carbohydrates help to protect dormant buds against limited water supply. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  17. Particle engineering of materials for oral inhalation by dry powder inhalers. I-Particles of sugar excipients (trehalose and raffinose) for protein delivery.

    PubMed

    Ogáin, Orla Ní; Li, Jianhe; Tajber, Lidia; Corrigan, Owen I; Healy, Anne Marie

    2011-02-28

    The pulmonary route of delivery offers a potential alternative to parenteral administration of peptides and proteins. Protection of protein structure is essential in both processing and storage of the final formulation. Sugars, such as trehalose and raffinose, have been employed to act as protein stabilisers. Optimisation of the aerodynamic characteristics of microparticles in dry powder inhaler formulations is critical to ensure optimum deposition of the formulation into the respiratory tract. In the present study we examine the adaptation to hydrophilic materials, specifically the disaccharide, trehalose and the trisaccharide, raffinose, of a previously reported spray drying process for producing nanoporous microparticles (NPMPs). We also investigate the feasibility of incorporating a model protein, lysozyme, into these sugar-based NPMPs. While spray drying raffinose or trehalose from aqueous solution or ethanol:water solutions resulted in non-porous microspheres, spray drying from a methanol:n-butyl acetate mixed solvent system resulted in microparticles which appeared to consist of an agglomeration of individual nanoparticles, i.e. nanoporous/nanoparticulate microparticles. NPMPs of trehalose and raffinose were amorphous, with glass transition temperatures (Tgs) that were sufficiently high (124°C and ∼120°C for trehalose and raffinose, respectively) to suggest good physical stability at room temperature and good potential to act as protein carriers and/or stabilisers. NPMPs demonstrated improved aerosolisation properties compared to spray dried non-porous particles. The successful incorporation of lysozyme into these NPMPs at a sugar to protein weight ratio of 1:4 demonstrated the potential of these systems to act as carriers for peptide or protein drugs which could be delivered via the pulmonary route. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. The in vitro biocompatibility of d-(+) raffinose modified chitosan: Two-dimensional and three-dimensional systems for culturing of horse articular chondrocytes.

    PubMed

    De Angelis, Elena; Ravanetti, Francesca; Martelli, Paolo; Cacchioli, Antonio; Ivanovska, Ana; Corradi, Attilio; Nasi, Sonia; Bianchera, Annalisa; Passeri, Benedetta; Canelli, Elena; Bettini, Ruggero; Borghetti, Paolo

    2017-12-01

    The present study investigated the biocompatibility of chitosan films and scaffolds modified with d-(+)raffinose and their capability to support the growth and maintenance of the differentiation of articular chondrocytes in vitro. Primary equine articular chondrocytes were cultured on films and scaffolds of modified d-(+) raffinose chitosan. Their behavior was compared to that of chondrocytes grown in conventional bi- and three-dimensional culture systems, such as micromasses and alginate beads. Chitosan films maintained the phenotype of differentiated chondrocytes (typical round morphology) and sustained the synthesis of cartilaginous extracellular matrix (ECM), even at 4weeks of culture. Indeed, starting from 2weeks of culture, chondrocytes seeded on chitosan scaffolds were able to penetrate the surface pores and to colonize the internal matrix. Moreover they produced ECM expressing the genes of typical chondrocytes differentiation markers such as collagen II and aggrecan. In conclusion, chitosan modified with d-raffinose represents an ideal support for chondrocyte adhesion, proliferation and for the maintenance of cellular phenotypic and genotypic differentiation. This novel biomaterial could potentially be a reliable support for the re-differentiation of dedifferentiated chondrocytes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Raffinose and stachyose metabolism are not required for efficient soybean seed germination.

    PubMed

    Dierking, Emily C; Bilyeu, Kristin D

    2009-08-15

    Raffinose family oligosaccharides (RFOs), which include raffinose and stachyose, are thought to be an important source of energy during seed germination. In contrast to their potential for promoting germination, RFOs represent anti-nutritional units for monogastric animals when consumed as a component of feed. The exact role for RFOs during soybean seed development and germination has not been experimentally determined; but it has been hypothesized that RFOs are required for successful germination. Previously, inhibition of RFO breakdown during imbibition and germination was shown to significantly delay germination in pea seeds. The objective of this study was to compare the germination potential for soybean seeds with either wild-type (WT) or low RFO levels and to examine the role of RFO breakdown in germination of soybean seeds. There was no significant difference in germination between normal and low RFO soybean seeds when imbibed/germinated in water. Similar to the situation in pea, soybean seeds of wild-type carbohydrate composition experienced a delay in germination when treated with a chemical inhibitor of alpha-galactosidase activity (1-deoxygalactonojirimycin or DGJ) during imbibition. However, low RFO soybean seed germination was not significantly delayed or reduced when treated with DGJ. In contrast to the situation in pea, the inhibitor-induced germination delay in wild-type soybean seeds was not partially overcome by the addition of galactose or sucrose. We conclude that RFOs are not an essential source of energy during soybean seed germination.

  20. Multifunctional fructans and raffinose family oligosaccharides

    PubMed Central

    den Ende, Wim Van

    2013-01-01

    Fructans and raffinose family oligosaccharides (RFOs) are the two most important classes of water-soluble carbohydrates in plants. Recent progress is summarized on their metabolism (and regulation) and on their functions in plants and in food (prebiotics, antioxidants). Interest has shifted from the classic inulin-type fructans to more complex fructans. Similarly, alternative RFOs were discovered next to the classic RFOs. Considerable progress has been made in the understanding of structure–function relationships among different kinds of plant fructan metabolizing enzymes. This helps to understand their evolution from (invertase) ancestors, and the evolution and role of so-called “defective invertases.” Both fructans and RFOs can act as reserve carbohydrates, membrane stabilizers and stress tolerance mediators. Fructan metabolism can also play a role in osmoregulation (e.g., flower opening) and source–sink relationships. Here, two novel emerging roles are highlighted. First, fructans and RFOs may contribute to overall cellular reactive oxygen species (ROS) homeostasis by specific ROS scavenging processes in the vicinity of organellar membranes (e.g., vacuole, chloroplasts). Second, it is hypothesized that small fructans and RFOs act as phloem-mobile signaling compounds under stress. It is speculated that such underlying antioxidant and oligosaccharide signaling mechanisms contribute to disease prevention in plants as well as in animals and in humans. PMID:23882273

  1. Intra amniotic administration of raffinose and stachyose affects the intestinal brush border functionality and alters gut microflora populations

    USDA-ARS?s Scientific Manuscript database

    This study investigates the effectiveness of two types of prebiotics, stachyose and raffinose, which are present in staple food crops that are widely consumed in regions where dietary Fe deficiency is a health concern. The hypothesis is that these prebiotics will improve Fe status, intestinal functi...

  2. Effects of dietary β-1,3-glucan, chitosan or raffinose on the growth, innate immunity and resistance of koi (Cyprinus carpio koi).

    PubMed

    Lin, Shimei; Pan, Yu; Luo, Lin; Luo, Li

    2011-12-01

    This study was performed to determine the efficacy of three immunomodulators viz., β-1,3 glucan, chitosan and raffinose on the innate immune response of koi, Cyprinus carpio koi. Kois were divided into 4 groups and each group was fed with diets supplemented with or without immunostimulant for 56 days. Total leukocyte counts (WBC), the non-specific humoral (lysozyme, alternative complement pathway and superoxide dismutase) and cellular (phagocytic capacity and respiratory burst activity) responses were determined and compared with controls (no supplement) after 7, 14, 21 and 56 days of feeding. The results of 8 weeks feeding trial showed that β-1,3 glucan supplementation significantly enhanced koi growth, whereas other immunostimulants did not. Variation in the levels of responses was evident among different supplements. Compared with chitosan or raffinose, β-1,3 glucan could maintain the immunity of kois at a higher level during the experimental period. However, continuously applying β-1,3 glucan, chitosan or raffinose into the diet caused immunity fatigue in koi. No significant change in alternative complement pathway (ACP) activity was observed for any of the three supplements over the four different periods. After feeding for 14 days, the total leukocyte count (WBC), respiratory burst activity and superoxide dismutase (SOD) activity of the kois fed with chitosan or raffinose continuously remained relatively unchanged, subsequently decreased on the 56th day, but SOD did not. Meanwhile, lysozyme activity was no longer significantly higher on the 7th day, and for phagocytic capacity on the 14th day. After 56 days, these three immunostimulants groups also exhibited a decrease in the cumulative symptom rates compared to the controls when challenged with Aeromonas veronii. These results indicated that dietary intake containing immunostimulants could enhance the immune responses of koi and improve its resistance to infection by A.veronii. Especially

  3. Analysis of the Raffinose Family Oligosaccharide Pathway in Pea Seeds with Contrasting Carbohydrate Composition1

    PubMed Central

    Peterbauer, Thomas; Lahuta, Leslaw B.; Blöchl, Andreas; Mucha, Jan; Jones, David A.; Hedley, Cliff L.; Gòrecki, Richard J.; Richter, Andreas

    2001-01-01

    Raffinose family oligosaccharides (RFOs) are synthesized by a set of galactosyltransferases, which sequentially add galactose units from galactinol to sucrose. The accumulation of RFOs was studied in maturing seeds of two pea (Pisum sativum) lines with contrasting RFO composition. Seeds of the line SD1 accumulated stachyose as the predominant RFO, whereas verbascose, the next higher homolog of stachyose, was almost absent. In seeds of the line RRRbRb, a high level of verbascose was accumulated alongside with stachyose. The increase in verbascose in developing RRRbRb seeds was associated with galactinol-dependent verbascose synthase activity. In addition, a galactinol-independent enzyme activity was detected, which catalyzed transfer of a galactose residue from one stachyose molecule to another. The two enzyme activities synthesizing verbascose showed an optimum at pH 7.0. Both activities were almost undetectable in SD1. Maximum activity of stachyose synthase was about 4-fold higher in RRRbRb compared with SD1, whereas the activities of galactinol synthase and raffinose synthase were only about 1.5-fold higher in RRRbRb. The levels of galactinol synthase and stachyose synthase activity were reflected by steady-state levels of corresponding mRNAs. We suggest that the accumulation of verbascose in RRRbRb was controlled by a coordinated up-regulation of the last steps of verbascose biosynthesis. PMID:11743119

  4. Purification an α-galactosidase from Coriolus versicolor with acid-resistant and good degradation ability on raffinose family oligosaccharides.

    PubMed

    Du, Fang; Liu, Qin; Wang, Hexiang; Ng, TziBin

    2014-04-01

    An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg⁻¹. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4-50 °C and at a pH range of 2-5. Among the metal ions tested, Cu²⁺, Cd²⁺ and Hg²⁺ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag⁺ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.

  5. Genotype and growing environment interaction shows a positive correlation between substrates of raffinose family oligosaccharides (RFO) biosynthesis and their accumulation in chickpea ( Cicer arietinum L.) seeds.

    PubMed

    Gangola, Manu P; Khedikar, Yogendra P; Gaur, Pooran M; Båga, Monica; Chibbar, Ravindra N

    2013-05-22

    To develop genetic improvement strategies to modulate raffinose family oligosaccharides (RFO) concentration in chickpea ( Cicer arietinum L.) seeds, RFO and their precursor concentrations were analyzed in 171 chickpea genotypes from diverse geographical origins. The genotypes were grown in replicated trials over two years in the field (Patancheru, India) and in the greenhouse (Saskatoon, Canada). Analysis of variance revealed a significant impact of genotype, environment, and their interaction on RFO concentration in chickpea seeds. Total RFO concentration ranged from 1.58 to 5.31 mmol/100 g and from 2.11 to 5.83 mmol/100 g in desi and kabuli genotypes, respectively. Sucrose (0.60-3.59 g/100 g) and stachyose (0.18-2.38 g/100 g) were distinguished as the major soluble sugar and RFO, respectively. Correlation analysis revealed a significant positive correlation between substrate and product concentration in RFO biosynthesis. In chickpea seeds, raffinose, stachyose, and verbascose showed a moderate broad sense heritability (0.25-0.56), suggesting the use of a multilocation trials based approach in chickpea seed quality improvement programs.

  6. The effect of dehydration conditions on the functionality of anhydrous amorphous raffinose.

    PubMed

    Chamarthy, Sai Prasanth; Khalef, Nawel; Trasi, Niraj; Bakri, Aziz; Carvajal, M Teresa; Pinal, Rodolfo

    2010-06-14

    The purpose of this investigation is to study the effect of dehydration conditions of raffinose pentahydrate (RF.5H2O) on the physical properties and functionality of the resulting material. Crystalline RF.5H2O was dehydrated at two temperatures, 80 degrees C and 110 degrees C, producing the amorphous anhydrous form (RF.am). The dehydration temperature had no effect on a number of physical properties of the obtained RF.am, including X-ray powder diffraction, surface energy and water uptake. However, despite resulting on the same dynamics and extent of water sorption, different dehydration temperatures produced amorphous samples with drastically different recrystallization tendencies. Thermodynamic parameters show that despite the similarities on certain physical attributes, different dehydration temperature results in samples with significantly different free energy, hence stability. The difference in free energy produced by the dehydration temperature is attributed to differences in supramolecular structure that persist even in the liquid domain (above T(g)) of the amorphous samples. Evidence of such effects is observed as fluctuations in heat capacity present in RF.am but absent in the freshly prepared glass and also supported by the presence of molecular mobility modes observed using thermal polarization measurements. Copyright 2010 Elsevier B.V. All rights reserved.

  7. IMP2, a nuclear gene controlling the mitochondrial dependence of galactose, maltose and raffinose utilization in Saccharomyces cerevisiae.

    PubMed

    Donnini, C; Lodi, T; Ferrero, I; Puglisi, P P

    1992-02-01

    The IMP2 gene of Saccharomyces cerevisiae is involved in the nucleo-mitochondrial control of maltose, galactose and raffinose utilization as shown by the inability of imp2 mutants to grow on these carbon sources in respiratory-deficient conditions or in the presence of ethidium bromide and erythromycin. The negative phenotype cannot be scored in the presence of inhibitors of respiration and oxidative phosphorylation, indicating that the role of the mitochondria in the utilization of the above-mentioned carbon sources in imp2 mutants is not at the energetical level. Mutations in the IMP2 gene also confer many phenotypic alterations in respiratory-sufficient conditions, e.g. leaky phenotype on oxidizable carbon sources, sensitivity to heat shock and sporulation deficiency. The IMP2 gene has been cloned, sequenced and disrupted. The phenotype of null imp2 mutants is indistinguishable from that of the originally isolated mutant.

  8. Porous Graphitic Carbon Liquid Chromatography-Mass Spectrometry Analysis of Drought Stress-Responsive Raffinose Family Oligosaccharides in Plant Tissues.

    PubMed

    Jorge, Tiago F; Florêncio, Maria H; António, Carla

    2017-01-01

    Drought is a major limiting factor in agriculture and responsible for dramatic crop yield losses worldwide. The adjustment of the metabolic status via accumulation of drought stress-responsive osmolytes is one of the many strategies that some plants have developed to cope with water deficit conditions. Osmolytes are highly polar compounds, analysis of whcih is difficult with typical reversed-phase chromatography. Porous graphitic carbon (PGC) has shown to be a suitable alternative to reversed-phase stationary phases for the analysis of highly polar compounds typically found in the plant metabolome. In this chapter, we describe the development and validation of a PGC-based liquid chromatography tandem mass spectrometry (LC-MS n ) method suitable for the target analysis of water-soluble carbohydrates, such as raffinose family oligosaccharides (RFOs). We present detailed information regarding PGC column equilibration, LC-MS n system operation, data analysis, and important notes to be considered during the steps of method development and validation.

  9. Phloem loading in Verbascum phoeniceum L. depends on the synthesis of raffinose-family oligosaccharides

    PubMed Central

    McCaskill, Ashlee; Turgeon, Robert

    2007-01-01

    Phloem loading is the initial step in photoassimilate export and the one that creates the driving force for mass flow. It has been proposed that loading occurs symplastically in species that translocate carbohydrate primarily as raffinose family oligosaccharides (RFOs). In these plants, dense fields of plasmodesmata connect bundle sheath cells to specialized companion cells (intermediary cells) in the minor veins. According to the polymer trap model, advanced as a mechanism of symplastic loading, sucrose from the mesophyll diffuses into intermediary cells and is converted there to RFOs. This process keeps the sucrose concentration low and, because of the larger size of the RFOs, prevents back diffusion. To test this model, the RFO pathway was down-regulated in Verbascum phoeniceum L. by suppressing the synthesis of galactinol synthase (GAS), which catalyzes the first committed step in RFO production. Two GAS genes (VpGAS1 and VpGAS2) were cloned and shown to be expressed in intermediary cells. Simultaneous RNAi suppression of both genes resulted in pronounced inhibition of RFO synthesis. Phloem transport was negatively affected, as evidenced by the accumulation of carbohydrate in the lamina and the reduced capacity of leaves to export sugars during a prolonged dark period. In plants with severe down-regulation, additional symptoms of reduced export were obvious, including impaired growth, leaf chlorosis, and necrosis and curling of leaf margins. PMID:18048337

  10. Dietary soy, meat, and fish proteins modulate the effects of prebiotic raffinose on composition and fermentation of gut microbiota in rats.

    PubMed

    Bai, Gaowa; Tsuruta, Takeshi; Nishino, Naoki

    2018-06-01

    Soy, meat (mixture of pork and beef), and fish proteins were fed to rats with and without prebiotic raffinose (RAF), and the composition and fermentation of gut microbiota were examined. Bifidobacterium spp. populations were higher, and propionic acid concentration was lower in soy protein-fed than meat protein-fed rats. Likewise, Enterobacteriaceae populations were higher in fish protein-fed rats than other rats. RAF feeding increased Bifidobacterium spp. and decreased Faecalibacterium prausnitzii populations regardless of the dietary protein source. Interactions between dietary proteins and RAF were shown for Lactobacillus spp. and Clostridium perfringens group; the increase of Lactobacillus spp. populations by RAF was seen only for soy protein-fed rats, whereas the reduction of C. perfringens group by RAF was evident in fish and meat protein-fed rats. It is concluded that dietary proteins may differentially modulate the effects of prebiotic oligosaccharides on gut fermentation and microbiota, with differences observed between plant and animal proteins.

  11. A Fungal α-Galactosidase from Tricholoma matsutake with Broad Substrate Specificity and Good Hydrolytic Activity on Raffinose Family Oligosaccharides.

    PubMed

    Geng, Xueran; Tian, Guoting; Zhao, Yongchang; Zhao, Liyan; Wang, Hexiang; Ng, Tzi Bun

    2015-07-24

    An acidic α-galactosidase designated as TMG was purified from the fruiting bodies The purification protocol entailed ion exchange chromatography on Q-Sepharose and of Tricholoma matsutake with 136-fold purification and a specific activity of 909 units/mg. Mono-Q and fast protein liquid chromatography on Superdex 75. TMG is a monomeric protein exhibiting a molecular mass of 47 kDa in SDS-PAGE and gel filtration. The purified enzyme was identified by LC-MS/MS and three inner amino acid sequences were obtained. The optimum pH and temperature for TMG with pNPGal as substrate were pH 4.5 and 55 °C, respectively. The α-galactosidase activity was strongly inhibited by K+, Ca2+, Cd2+, Hg2+, Ag+ and Zn2+ ions. The enzyme activity was inhibited by the chemical modification agent N-bromosuccinimide (NBS), indicating the importance of tryptophan residue(s) at or near the active site. Besides hydrolyzing pNPGal, TMG also efficaciously catalyzed the degradation of natural substrates such as stachyose, raffinose, and melibiose. Thus TMG can be exploited commercially for improving the nutritional value of soy milk by degradation of indigestible oligosaccharides.

  12. Trehalose, sucrose and raffinose are novel activators of autophagy in human keratinocytes through an mTOR-independent pathway

    PubMed Central

    Chen, Xu; Li, Min; Li, Li; Xu, Song; Huang, Dan; Ju, Mei; Huang, Ju; Chen, Kun; Gu, Heng

    2016-01-01

    Trehalose is a natural disaccharide that is found in a diverse range of organisms but not in mammals. Autophagy is a process which mediates the sequestration, lysosomal delivery and degradation of proteins and organelles. Studies have shown that trehalose exerts beneficial effects through inducing autophagy in mammalian cells. However, whether trehalose or other saccharides can activate autophagy in keratinocytes is unknown. Here, we found that trehalose treatment increased the LC3-I to LC3-II conversion, acridine orange-stained vacuoles and GFP-LC3B (LC3B protein tagged with green fluorescent protein) puncta in the HaCaT human keratinocyte cell line, indicating autophagy induction. Trehalose-induced autophagy was also observed in primary keratinocytes and the A431 epidermal cancer cell line. mTOR signalling was not affected by trehalose treatment, suggesting that trehalose induced autophagy through an mTOR-independent pathway. mTOR-independent autophagy induction was also observed in HaCaT and HeLa cells treated with sucrose or raffinose but not in glucose, maltose or sorbitol treated HaCaT cells, indicating that autophagy induction was not a general property of saccharides. Finally, although trehalose treatment had an inhibitory effect on cell proliferation, it had a cytoprotective effect on cells exposed to UVB radiation. Our study provides new insight into the saccharide-mediated regulation of autophagy in keratinocytes. PMID:27328819

  13. [Changes of transport sugar content in different organs of Rehmannia glutinosa].

    PubMed

    Wang, Dong-Hui; Liao, Na; Sun, Peng; Ji, Xue-Qi; Li, Xian-En; Qin, Min-Jian

    2018-04-01

    Raffinose series oligosaccharides are the transport and storage sugars of many plants, Rehmannia glutinosa is one of the commonly used Chinese herbal medicines, medicinal parts ist he roots. Root and tuber of R. glutinosa contains stachyose, raffinose and other oligosaccharides, but the study about the process of growth and development of other organs in the non-structural changes in sugar content is rare.In this study, leaves, stems and roots of R. glutinosa were used as materials to analyze the diurnal variation and the changes of sugar content of sucrose, raffinose and stachyose in different organs of R. glutinosa. The results showed that the content of sucrose in R. glutinosa leaves gradually increased from seedling stage.However, the content of stachyose did not change much at the early stage of growth, and the stachyose rapidly increased at the later stage of growth. The raffinose content gradually decreased throughout the growing season, young leaves of R. glutinosa have higher ability to sucrose synthesis than mature leaves, while mature leaf has higher raffinose and stachyose synthesis ability than young leaves. Sucrose and stachyose content in stem gradually increased, while there was little change in raffinose content. The content of raffinose and stachyose in root increased rapidly from the beginning of fast growing period, while the content of sucrose did not change much. The content of sucrose in leaves of R. glutinosa did not change much at day and night, while the daily changes of raffinose and stachyose contents were very obvious. The contents of raffinose and stachyose in daytime were higher than those at night. The content of raffinose in root and stem was not changed much, but the change of stachyose in root, stem and leaf was very obvious, especially in stem and leaf. In summary, the leaf is the main synthetic organ of raffinose, leaves, stems and roots are stachyose synthesis organ. Sucrose, raffinose and stachyose are the major transport

  14. Transcriptome Analysis of Leaves, Flowers and Fruits Perisperm of Coffea arabica L. Reveals the Differential Expression of Genes Involved in Raffinose Biosynthesis.

    PubMed

    Ivamoto, Suzana Tiemi; Reis, Osvaldo; Domingues, Douglas Silva; Dos Santos, Tiago Benedito; de Oliveira, Fernanda Freitas; Pot, David; Leroy, Thierry; Vieira, Luiz Gonzaga Esteves; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães; Pereira, Luiz Filipe Protasio

    2017-01-01

    Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages.

  15. Transcriptome Analysis of Leaves, Flowers and Fruits Perisperm of Coffea arabica L. Reveals the Differential Expression of Genes Involved in Raffinose Biosynthesis

    PubMed Central

    dos Santos, Tiago Benedito; de Oliveira, Fernanda Freitas; Pot, David; Leroy, Thierry; Vieira, Luiz Gonzaga Esteves; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães

    2017-01-01

    Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages. PMID:28068432

  16. Low Volume Resuscitation with Cell Impermeants

    DTIC Science & Technology

    2013-10-01

    LVR solution produced the best individual protection. Mixtures of trehalose, raffinose, gluconate, and sorbitol at a specific ratio was optimal. This... Sorbitol  Gluconate  Trehalose  Raffinose These agents have been extensively used in organ preservation solutions so their safety and...10% 5% Sorbitol Na-Gluconate Trehalose Na-Lactobionate Raffinose PEG-8K 122 98

  17. Reduction of α-galactoside content in red gram (Cajanus cajan L.) upon germination followed by heat treatment.

    PubMed

    Devindra, Shekappa; Sreenivasa Rao, Jarapala; Krishnaswamy, Padmanabhan; Bhaskar, Varanasi

    2011-08-15

    Red gram (Cajanus cajan L.) is an important crop for human and animal nutrition. However, raffinose family oligosaccharides present in red gram seed hinder its consumption as it is not digested by normal human carbohydrases and is further fermented by intestinal microflora, which induces flatulence. In order to make the grain legume more amenable for human consumption, we have tried to shed some light on the effect of germination followed by heat treatment methods such as autoclaving, cooking and pressure cooking on the raffinose family of sugars. These techniques, however, are primary prerequisites before consumption of the gram. The percent removal of raffinose, stachyose and verbascose after germinating red gram seeds for 8 h followed by autoclaving was 65.6%, 58.9% and 65.3% respectively; and after cooking was 61.6%, 69.2% and 72.5%. Germinating for 16 h followed by autoclaving led to a mean decrease of 53.3% for raffinose, 60.3% for stachyose and 62.3% for verbascose. Germination of red gram seeds for 16 h followed by cooking led to a mean decrease of 71.7% for raffinose, 76.2% for stachyose and 74.0% for verbascose, respectively. The results for the percent removal of raffinose, stachyose and verbascose after germination of red gram seeds for 16 h followed by pressure cooking was 68.3%, 73.3% and 68.2% respectively. This study demonstrates that local methods of processing reduce raffinose family oligosaccharides in red gram. The technique of germinating the seeds for 16 h followed by autoclaving, cooking and pressure cooking for the reduction of raffinose family oligosaccharides is a promising solution to overcome flatulence and increase the overall acceptance of red gram among general populace. Copyright © 2011 Society of Chemical Industry.

  18. Promising Prebiotic Candidate Established by Evaluation of Lactitol, Lactulose, Raffinose, and Oligofructose for Maintenance of a Lactobacillus-Dominated Vaginal Microbiota

    PubMed Central

    McMillan, Amy; Seney, Shannon; van der Veer, Charlotte; Kort, Remco; Sumarah, Mark W.

    2017-01-01

    ABSTRACT Perturbations to the vaginal microbiota can lead to dysbiosis, including bacterial vaginosis (BV), which affects a large portion of the female population. In a healthy state, the vaginal microbiota is characterized by low diversity and colonization by Lactobacillus spp., whereas in BV, these species are displaced by a highly diverse population of bacteria associated with adverse vaginal health outcomes. Since prebiotic ingestion has been a highly effective approach to invigorate lactobacilli for improved intestinal health, we hypothesized that these compounds could stimulate lactobacilli at the expense of BV organisms to maintain vaginal health. Monocultures of commensal Lactobacillus crispatus, Lactobacillus vaginalis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, and Lactobacillus iners, in addition to BV-associated organisms and Candida albicans, were tested for their ability to utilize a representative group of prebiotics consisting of lactitol, lactulose, raffinose, and oligofructose. The disaccharide lactulose was found to most broadly and specifically stimulate vaginal lactobacilli, including the strongly health-associated species L. crispatus, and importantly, not to stimulate BV organisms or C. albicans. Using freshly collected vaginal samples, we showed that exposure to lactulose promoted commensal Lactobacillus growth and dominance and resulted in healthy acidity partially through lactic acid production. This provides support for further testing of lactulose to prevent dysbiosis and potentially to reduce the need for antimicrobial agents in managing vaginal health. IMPORTANCE Bacterial vaginosis (BV) and other dysbioses of the vaginal microbiota significantly affect the quality of life of millions of women. Antimicrobial therapy is often poorly effective, causes side effects, and does not prevent recurrences. We report one of very few studies that have evaluated how prebiotics—compounds that are selectively fermented

  19. Low Night Temperature Affects the Phloem Ultrastructure of Lateral Branches and Raffinose Family Oligosaccharide (RFO) Accumulation in RFO-Transporting Plant Melon (Cucumismelo L.) during Fruit Expansion

    PubMed Central

    Hao, Jinghong; Gu, Fengying; Zhu, Jie; Lu, Shaowei; Liu, Yifei; Li, Yunfei; Chen, Weizhi; Wang, Liping; Fan, Shuangxi; Xian, Cory J.

    2016-01-01

    Due to the importance and complexity of photo assimilate transport in raffinose family oligosaccharide (RFO)-transporting plants such as melon, it is important to study the features of the transport structure (phloem) particularly of the lateral branches connecting the source leaves and the sink fruits, and its responses to environmental challenges. Currently, it is unclear to what extents the cold environmental temperature stress would alter the phloem ultrastructure and RFO accumulation in RFO-transporting plants. In this study, we firstly utilized electron microscopy to investigate the changes in the phloem ultrastructure of lateral branches and RFO accumulation in melons after being subjected to low night temperatures (12°C and 9°C). The results demonstrated that exposure to 9°C and 12°C altered the ultrastructure of the phloem, with the effect of 9°C being more obvious. The most obvious change was the appearance of plasma membrane invaginations in 99% companion cells and intermediary cells. In addition, phloem parenchyma cells contained chloroplasts with increased amounts of starch grains, sparse cytoplasm and reduced numbers of mitochondria. In the intermediary cells, the volume of cytoplasm was reduced by 50%, and the central vacuole was present. Moreover, the treatment at 9°C during the night led to RFO accumulation in the vascular bundles of the lateral branches and fruit carpopodiums. These ultrastructural changes of the transport structure (phloem) following the treatment at 9°C represented adaptive responses of melons to low temperature stresses. Future studies are required to examine whether these responses may affect phloem transport. PMID:27501301

  20. Low Night Temperature Affects the Phloem Ultrastructure of Lateral Branches and Raffinose Family Oligosaccharide (RFO) Accumulation in RFO-Transporting Plant Melon (Cucumismelo L.) during Fruit Expansion.

    PubMed

    Hao, Jinghong; Gu, Fengying; Zhu, Jie; Lu, Shaowei; Liu, Yifei; Li, Yunfei; Chen, Weizhi; Wang, Liping; Fan, Shuangxi; Xian, Cory J

    2016-01-01

    Due to the importance and complexity of photo assimilate transport in raffinose family oligosaccharide (RFO)-transporting plants such as melon, it is important to study the features of the transport structure (phloem) particularly of the lateral branches connecting the source leaves and the sink fruits, and its responses to environmental challenges. Currently, it is unclear to what extents the cold environmental temperature stress would alter the phloem ultrastructure and RFO accumulation in RFO-transporting plants. In this study, we firstly utilized electron microscopy to investigate the changes in the phloem ultrastructure of lateral branches and RFO accumulation in melons after being subjected to low night temperatures (12°C and 9°C). The results demonstrated that exposure to 9°C and 12°C altered the ultrastructure of the phloem, with the effect of 9°C being more obvious. The most obvious change was the appearance of plasma membrane invaginations in 99% companion cells and intermediary cells. In addition, phloem parenchyma cells contained chloroplasts with increased amounts of starch grains, sparse cytoplasm and reduced numbers of mitochondria. In the intermediary cells, the volume of cytoplasm was reduced by 50%, and the central vacuole was present. Moreover, the treatment at 9°C during the night led to RFO accumulation in the vascular bundles of the lateral branches and fruit carpopodiums. These ultrastructural changes of the transport structure (phloem) following the treatment at 9°C represented adaptive responses of melons to low temperature stresses. Future studies are required to examine whether these responses may affect phloem transport.

  1. Proteomic analysis of common bean seed with storage protein deficiency reveals up-regulation of sulfur-rich proteins and starch and raffinose metabolic enzymes, and down-regulation of the secretory pathway.

    PubMed

    Marsolais, Frédéric; Pajak, Agnieszka; Yin, Fuqiang; Taylor, Meghan; Gabriel, Michelle; Merino, Diana M; Ma, Vanessa; Kameka, Alexander; Vijayan, Perumal; Pham, Hai; Huang, Shangzhi; Rivoal, Jean; Bett, Kirstin; Hernández-Sebastià, Cinta; Liu, Qiang; Bertrand, Annick; Chapman, Ralph

    2010-06-16

    A deficiency in major seed storage proteins is associated with a nearly two-fold increase in sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris). Their mature seed proteome was compared by an approach combining label-free quantification by spectral counting, 2-DE, and analysis of selective extracts. Lack of phaseolin, phytohemagglutinin and arcelin was mainly compensated by increases in legumin, alpha-amylase inhibitors and mannose lectin FRIL. Along with legumin, albumin-2, defensin and albumin-1 were major contributors to the elevated sulfur amino acid content. Coordinate induction of granule-bound starch synthase I, starch synthase II-2 and starch branching enzyme were associated with minor alteration of starch composition, whereas increased levels of UDP-glucose 4-epimerase were correlated with a 30% increase in raffinose content. Induction of cell division cycle protein 48 and ubiquitin suggested enhanced ER-associated degradation. This was not associated with a classical unfolded protein response as the levels of ER HSC70-cognate binding protein were actually reduced in the mutant. Repression of rab1 GTPase was consistent with decreased traffic through the secretory pathway. Collectively, these results have implications for the nutritional quality of common bean, and provide information on the pleiotropic phenotype associated with storage protein deficiency in a dicotyledonous seed. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

  2. Carbohydrate composition of mature and immature faba bean (Vicia faba L.) seeds from diverse origins

    USDA-ARS?s Scientific Manuscript database

    Faba bean (Vicia faba L.) is a valuable pulse crop for human consumption. The low molecular weight carbohydrates (LMWC): glucose, fructose, sucrose (GFS), raffinose, stachyose, and verbascose (RFO- raffinose family oligosaccharides) in faba bean seeds are significant components of human nutrition an...

  3. Environmental stability of carbohydrate profiles in different soybean genotypes

    USDA-ARS?s Scientific Manuscript database

    Soybean is an important annual crop. The raffinose family of oligosaccharides (RFO) raffinose and stachyose are anti-nutritional carbohydrates present in soybean seeds. Consumption of soybean seed products with low RFO reduced flatulence in humans and increased metabolizable energy efficiency in chi...

  4. Quantitative trait loci underlying seed sugars content in MD96-5722 by spencer recombinant inbred line population of soybean

    USDA-ARS?s Scientific Manuscript database

    Sucrose, raffinose, and stachyose are important soluble sugars in soybean [Glycine max (L.) Merr.] seeds, and soybean seeds with higher sucrose and lower raffinose and stachyose are desirable. Therefore, optimizing sugars biosynthesis is a major goal for soy food industry. The objective of this stud...

  5. Enzymatic degradation of oligosaccharides in pinto bean flour.

    PubMed

    Song, Danfeng; Chang, Sam K C

    2006-02-22

    The use of dry edible beans is limited due to the presence of flatulence factors, the raffinose oligosaccharides. Our objective was to investigate the process for the removal of oligosaccharides from pinto bean using enzymatic treatment and to compare it to removal by soaking and cooking methods. Crude enzyme preparation was produced by six fungal species on wheat bran- and okara-based substrates with soy tofu whey. The loss of raffinose oligosaccharides after soaking pinto beans for 16 h at the room temperature was 10%, after cooking for 90 min was 52%, and after autoclaving for 30 min was 58%. On the other hand, the treatment using crude alpha-galactosidase (60 U mL(-1)) produced by Aspergillus awamori NRRL 4869 from wheat bran-based substrate with soy tofu whey on pinto bean flour for 2 h completely hydrolyzed raffinose oligosaccharides. These results supported that the enzymatic treatment was the most effective among various processing methods tested for removing the raffinose oligosaccharides, and hence, crude alpha-galactosidases from fungi have potential use in the food industry.

  6. Compositional variations for alpha-galactosides in different species of leguminosae, brassicaceae, and barley: a chemotaxonomic study based on chemometrics and high-performance capillary electrophoresis.

    PubMed

    Andersen, Keld Ejdrup; Bjergegaard, Charlotte; Møller, Peter; Sørensen, Jens Christian; Sørensen, Hilmer

    2005-07-13

    The contents of raffinose family oligosaccharides (RFO) and sucrose in Brassica, Lupinus, Pisum, and Hordeum species were investigated by chemometric principal component analysis (PCA). Hordeum samples contained sucrose and raffinose, and Brassica samples all contained sucrose, raffinose, and stachyose. In addition to these, the Pisum samples contained verbascose and the Lupinus samples also contained ajugose. High stachyose and low ajugose contents were found in Lupinus albus in contrast to Lupinus angustifolius, having low stachyose and high ajugose contents. Lupinus luteus had average stachyose and ajugose contents, whereas large amounts of verbascose were accumulated in these seeds. Lupinus mutabilis had high stachyose and low ajugose contents, similar to the composition in L. albus but showing higher raffinose content. The Brassica samples also showed compositional RFO variations within the species, and subgroup formations were discovered within the investigated Brassica napus varieties. PCA results indicated compositional variations between the investigated genera and within the various species of value as chemotaxonomic defined parameters and as tools in evaluations of authenticity/falsifications when RFO-containing plants are used as, for example, feed and food additives.

  7. Cloning and Expression Analysis of a UDP-Galactose/Glucose Pyrophosphorylase from Melon Fruit Provides Evidence for the Major Metabolic Pathway of Galactose Metabolism in Raffinose Oligosaccharide Metabolizing Plants1

    PubMed Central

    Dai, Nir; Petreikov, Marina; Portnoy, Vitaly; Katzir, Nurit; Pharr, David M.; Schaffer, Arthur A.

    2006-01-01

    The Cucurbitaceae translocate a significant portion of their photosynthate as raffinose and stachyose, which are galactosyl derivatives of sucrose. These are initially hydrolyzed by α-galactosidase to yield free galactose (Gal) and, accordingly, Gal metabolism is an important pathway in Cucurbitaceae sink tissue. We report here on a novel plant-specific enzyme responsible for the nucleotide activation of phosphorylated Gal and the subsequent entry of Gal into sink metabolism. The enzyme was antibody purified, sequenced, and the gene cloned and functionally expressed in Escherichia coli. The heterologous protein showed the characteristics of a dual substrate UDP-hexose pyrophosphorylase (PPase) with activity toward both Gal-1-P and glucose (Glc)-1-P in the uridinylation direction and their respective UDP-sugars in the reverse direction. The two other enzymes involved in Glc-P and Gal-P uridinylation are UDP-Glc PPase and uridyltransferase, and these were also cloned, heterologously expressed, and characterized. The gene expression and enzyme activities of all three enzymes in melon (Cucumis melo) fruit were measured. The UDP-Glc PPase was expressed in melon fruit to a similar extent as the novel enzyme, but the expressed protein was specific for Glc-1-P in the UDP-Glc synthesis direction and did not catalyze the nucleotide activation of Gal-1-P. The uridyltransferase gene was only weakly expressed in melon fruit, and activity was not observed in crude extracts. The results indicate that this novel enzyme carries out both the synthesis of UDP-Gal from Gal-1-P as well as the subsequent synthesis of Glc-1-P from the epimerase product, UDP-Glc, and thus plays a key role in melon fruit sink metabolism. PMID:16829585

  8. Survey of Some Actinomycetales for α-Galactosidase Activity1

    PubMed Central

    Lyons, A. J.; Pridham, T. G.; Hesseltine, C. W.

    1969-01-01

    The enzyme α-galactosidase offers potential to (i) eliminate possibly the flatus-inducing factor(s) in edible beans, (ii) eliminate raffinose during beet-sugar processing, and (iii) determine raffinose analytically. Accordingly, 20 genera of the order Actinomycetales Buchanan 1917 were tested for evidence of α-galactosidase activity. Test filtrates were prepared with a medium containing D-galactose and soybean meal. Enzyme activity was demonstrated through cellulose thin-layer chromatography. Of 123 strains tested, 28 produced extracellular α-galactosidase. Almost all were streptomycetes. Members of the genera Actinoplanes Couch 1950, Micromonospora ϕOrskov 1923, and Promicromonospora Krasil'nikov et al. 1961 also exhibited α-galactosidase activity. Additional tests led to the selection of five strains whose filtrates degraded melibiose, raffinose, and stachyose but not lactose and sucrose. Tests also were made with several soybean preparations. PMID:5392462

  9. Augmented Dried versus Cryopreserved Amniotic Membrane as an Ocular Surface Dressing

    PubMed Central

    Allen, Claire L.; Clare, Gerry; Stewart, Elizabeth A.; Branch, Matthew J.; McIntosh, Owen D.; Dadhwal, Megha; Dua, Harminder S.; Hopkinson, Andrew

    2013-01-01

    Purpose Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material. Methods AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays. Results Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM. Conclusions Our modified preservation process and our resultant optimised dried AM has enhanced structural properties

  10. A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon

    PubMed Central

    Goh, Yong Jun; Klaenhammer, Todd R

    2013-01-01

    Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP-amy-pgm genes. Expression of the glg operon and glycogen accumulation were carbon source- and growth phase-dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log-phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose-grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen-branching enzyme) mutants are glycogen-deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus. PMID:23879596

  11. Face-by-face growth of sucrose crystals from aqueous solutions in the presence of raffinose—II: Growth morphology and segregation

    NASA Astrophysics Data System (ADS)

    Sgualdino, G.; Aquilano, D.; Pastero, L.; Vaccari, G.

    2007-10-01

    Raffinose segregation into sucrose crystals is experimentally determined along with the modifications of the quantitative sucrose growth morphology, which are in turn related to the different growth conditions. ( Craff, σ) morphodromes nicely represent the conflict between the supersaturation and the raffinose concentration in the solution on the growth morphology, while the overall segregation rate is nearly proportional to the linear overall crystal growth rate. Chernov and Burton-Prim-Slichter models, checked to fit our keff and ln(keff-1-1) coefficients as a function of the supersaturation and of the mean linear overall growth rate, do not allow to know whether the segregation occurs either by a process dominated by surface integration, or by additive transfer dominated by volume diffusion within the boundary layer. The distribution of segregated raffinose strictly depends on the { h k l} growth sectors and doped crystals contain deformed lattice zones, as it comes out from X-ray powder diagrams.

  12. Genetic Analysis of Seed-Soluble Oligosaccharides in Relation to Seed Storability of Arabidopsis1

    PubMed Central

    Bentsink, Leónie; Alonso-Blanco, Carlos; Vreugdenhil, Dick; Tesnier, Karine; Groot, Steven P.C.; Koornneef, Maarten

    2000-01-01

    Seed oligosaccharides (OSs) and especially raffinose series OSs (RSOs) are hypothesized to play an important role in the acquisition of desiccation tolerance and consequently in seed storability. In the present work we analyzed the seed-soluble OS (sucrose, raffinose, and stachyose) content of several Arabidopsis accessions and thus identified the genotype Cape Verde Islands having a very low RSO content. By performing quantitative trait loci (QTL) mapping in a recombinant inbred line population, we found one major QTL responsible for the practically monogenic segregation of seed stachyose content. This locus also affected the content of the two other OSs, sucrose, and raffinose. Two candidate genes encoding respectively for galactinol synthase and raffinose synthase were located within the genomic region around this major QTL. In addition, three smaller-effect QTL were identified, each one specifically affecting the content of an individual OS. Seed storability was analyzed in the same recombinant inbred line population by measuring viability (germination) under two different seed aging assays: after natural aging during 4 years of dry storage at room temperature and after artificial aging induced by a controlled deterioration test. Thus, four QTL responsible for the variation of this trait were mapped. Comparison of the QTL genetic positions showed that the genomic region containing the major OS locus did not significantly affect the seed storability. We concluded that in the studied material neither RSOs nor sucrose content had a specific effect on seed storability. PMID:11115877

  13. Synthesis of Stachyobifiose Using Bifidobacterial α-Galactosidase Purified from Recombinant Escherichia coli.

    PubMed

    Oh, So Young; Youn, So Youn; Park, Myeong Soo; Baek, Nam In; Ji, Geun Eog

    2018-02-07

    The prebiotic effects of GOS (galactooligosaccharides) are known to depend on the glycosidic linkages, degree of polymerization (DP), and the monosaccharide composition. In this study, a novel form of α-GOS with a potentially improved prebiotic effect was synthesized using bifidobacterial α-galactosidase (α-Gal) purified from recombinant Escherichia coli. The carbohydrate produced was identified as α-d-galactopyranosyl-(1→6)-O-α-d-glucopyranosyl-(1→2)-[α-d-galactopyranosyl-(1→6)-O-β-d-fructofuranoside] and was termed stachyobifiose. Among 17 nonprobiotics, 16 nonprobiotics showed lower growth on stachyobifiose than β-GOS. In contrast, among the 16 probiotics, 6 probiotics showed higher growth on stachyobifiose than β-GOS. When compared with raffinose, stachyobifiose was used less by nonprobiotics than raffinose. Moreover, compared with stachyose, stachyobifiose was used less by Escherichia coli, Enterobacter cloacae, and Clostridium butyricum. The average amounts of total short-chain fatty acids (SCFA) produced were in the order of stachyobifiose > stachyose > raffinose > β-GOS. Taken together, stachyobifiose is expected to contribute to beneficial changes of gut microbiota.

  14. Effect of soaking, cooking and germination on the oligosaccharide content of selected Nigerian legume seeds.

    PubMed

    Oboh, H A; Muzquiz, M; Burbano, C; Cuadrado, C; Pedrosa, M M; Ayet, G; Osagie, A U

    2000-01-01

    The identity and quantity of and effect of processing on raffinose oligosaccharides in raw, mature seeds of lima beans (Phaseolus lunatus), pigeon peas (Cajanus cajan), African yam beans (Sphenostylis sternocarpa) and jackbeans (Canavalia ensiformis) were investigated. Sucrose, raffinose, stachyose and verbascose were identified by HPLC in all the legume seeds. The total alpha-galactoside contents of the seeds in decreasing order were African yam beans 3.84 mg/100 mg; white lima beans 3.62 mg/100 mg; cream pigeon peas 3.51 mg/100 mg; red lima beans 3.37 mg/100 mg; jackbeans 2.83 mg/100 mg and brown pigeon peas 2.34 mg/100 mg. The predominant oligosaccharide was verbascose in pigeon peas and stachyose in the other three legumes. Cooking unsoaked seeds brought about a greater reduction in the total alpha-galactoside content than soaking for nine hours. The removal of oligosaccharides was higher in legumes cooked in alkaline solution than in water. Germination quantitatively reduced raffinose, stachyose and verbascose while sucrose was increased in all seeds except red lima beans and jackbeans.

  15. Reduction of soybean oligosaccharides and properties of alpha-D-galactosidase from Lactobacillus curvatus R08 and Leuconostoc mesenteroides [corrected] JK55.

    PubMed

    Yoon, Mi Young; Hwang, Han-Joon

    2008-09-01

    This study was undertaken to investigate the potential for reducing non-digestive oligosaccharides (NDO) in soy foods, as well as the influence of exogenous conditions on intracellular alpha-galactosidase (alpha-Gal) producing lactic acid bacteria. Two strains, Lactobacillus curvatus R08 and Leuconostoc mesenteroides [corrected]JK55, showed the highest levels of raffinose degrading activity at over 40 U mL(-1), and presented maximum activities during the stationary phase in a medium where raffinose was the only carbon source. Raffinose was the most effective inducer, followed by melibiose, and galactose; the enzymes were partially inhibited by fructose and sucrose. On the other hand, limited activity was observed in glucose. The strains displayed optimum activity levels at neutral pH and a 35-37 degrees C temperature range. The alpha-Gal activities of L. curvatus R08 and Leu. mesenteroides [corrected] JK55 were maintained at pH 6.5-10.0. The activity of the alpha-Gal enzyme was stable in a relatively broad range of temperatures from 0 to 40 degrees C for 3h. In soymilk, Leu. mesenteroides [corrected] JK55 and L. curvatus R08 completely hydrolyzed the NDO after 18-24h of fermentation. The abilities of L. curvatus R08 and Leu. mesenteroides [corrected] JK55 to degrade raffinose sugars and, particularly, to produce organic acids from sugar, could contribute to reductions in the anti-nutritional properties of soy, and to the accumulation of compounds with beneficial properties during food processing. Furthermore, this study provides the optimum conditions to induce alpha-Gal from these strains.

  16. Down-Regulating α-Galactosidase Enhances Freezing Tolerance in Transgenic Petunia1

    PubMed Central

    Pennycooke, Joyce C.; Jones, Michelle L.; Stushnoff, Cecil

    2003-01-01

    α-Galactosidase (α-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the α-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of α-Gal was modified in transgenic petunia (Petunia × hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that α-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of α-Gal mRNAs. α-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from –4°C for cold-acclimated wild-type plants to –8°C for the most tolerant antisense line. Down-regulating α-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the α-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with α-Gal provides an additional method for improving the freezing tolerance of plants. PMID:14500789

  17. An ATP Binding Cassette Transporter Mediates the Uptake of α-(1,6)-Linked Dietary Oligosaccharides in Bifidobacterium and Correlates with Competitive Growth on These Substrates*

    PubMed Central

    Fredslund, Folmer; Vujičić Žagar, Andreja; Andersen, Thomas Lars; Svensson, Birte; Slotboom, Dirk Jan

    2016-01-01

    The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria. PMID:27502277

  18. The influence of soy oligosaccharides on apparent and true ileal amino acid digestibilities and fecal consistency in growing pigs.

    PubMed

    Smiricky, M R; Grieshop, C M; Albin, D M; Wubben, J E; Gabert, V M; Fahey, G C

    2002-09-01

    Fourteen ileally cannulated pigs (BW = 35 +/- 2 kg) were randomly allotted to a replicated 7 x 7 Latin square design experiment to evaluate the influence of the soybean oligosaccharides (OS), raffinose and stachyose, on ileal nutrient digestibility and fecal consistency. Semipurified diets containing soy protein concentrate (SPC) or soybean meal (SBM) as the sole protein sources were fed. Soy solubles (SS), a by-product of SBM processing containing 3.5% raffinose and 11.5% stachyose, were used to increase dietary raffinose and stachyose concentrations. The seven dietary treatments were SPC, SPC + 9% SS, SBM, SBM + 9% SS, SBM + 18% SS, SBM + 24,000 U alpha-galactosidase enzyme preparation/kg diet, and a low-protein casein (LPC) diet used to calculate true digestibility. Diets, with the exception of the LPC diet, were formulated to contain 17% CP. All diets contained 0.5% chromic oxide as a marker for ileal digestibility determination. The experimental periods were divided into a 5-d diet adaptation followed by 2-d of ileal digesta collection. Diets and digesta were analyzed for DM, N, Cr, amino acids (AA), raffinose, and stachyose. Fecal consistency was determined on d 6 and 7 of each experimental period. The apparent and true ileal AA digestibilities were not different (P < 0.05) for the SPC and SBM control diets. When SS was added to the SPC diet, apparent and true N and AA digestibilities were depressed (P < 0.05) with the exception of Trp and Pro. The apparent and true ileal N and AA digestibilities were not different (P > 0.05) between the SBM control and SBM + 9% SS diets with the exception of Glu. There was a linear decrease (P < 0.05) in apparent and true DM, Val, Gly, and Tyr digestibilities when increasing levels of SS were added to the SBM diet. The addition of alpha-galactosidase did not improve apparent or true ileal N or AA digestibilities except for apparent and true Val and Tyr. Ileal raffinose digestibility was improved (P < 0.05) by addition of a

  19. Solubility of carbohydrates in heavy water.

    PubMed

    Cardoso, Marcus V C; Carvalho, Larissa V C; Sabadini, Edvaldo

    2012-05-15

    The solubility of several mono-(glucose and xylose), di-(sucrose and maltose), tri-(raffinose) and cyclic (α-cyclodextrin) saccharides in H(2)O and in D(2)O were measured over a range of temperatures. The solution enthalpies for the different carbohydrates in the two solvents were determined using the vant' Hoff equation and the values in D(2)O are presented here for the first time. Our findings indicate that the replacement of H(2)O by D(2)O remarkably decreases the solubilities of the less soluble carbohydrates, such as maltose, raffinose and α-cyclodextrin. On the other hand, the more soluble saccharides, glucose, xylose, and sucrose, are practically insensitive to the H/D replacement in water. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Predicting the behavior of novel sugar carriers for dry powder inhaler formulations via the use of a cohesive-adhesive force balance approach.

    PubMed

    Hooton, Jennifer C; Jones, Matthew D; Price, Robert

    2006-06-01

    The aim of this work was to utilize the recently developed cohesive-adhesive balance (CAB) technique for analyzing quantitative AFM measurements to compare the relative forces of interaction of micronized salbutamol sulfate particles and a selection of specifically grown sugar substrates (beta cyclodextrin, lactose, raffinose, trehalose and xylitol). The interfacial behavior was subsequently related to the in-vitro delivery performance of these sugars as carrier particles in dry powder inhalation (DPI) formulations. The CAB analysis indicated that the rank order of adhesion between salbutamol sulfate and the sugars was beta cyclodextrin < lactose < trehalose < raffinose < xylitol. The beta cyclodextrin was the only substrate with which salbutamol sulfate demonstrated a greater cohesive behavior. All other sugars exhibited an adhesive dominance. In-vitro deposition performance of the salbutamol sulfate based carrier DPI formulations showed that the rank order of the fine particle fraction (FPF) was beta cyclodextrin > lactose > raffinose > trehalose > xylitol. A linear correlation (R(2) = 0.9572) was observed between the FPF and cohesive-adhesive ratios of the AFM force measurements. The observed link between CAB analysis of the interactive forces and in-vitro performance of carrier based formulations suggested a fundamental understanding of the relative balance of the various forces of interaction within a dry powder formulation may provide a critical insight into the behavior of these formulations. (c) 2006 Wiley-Liss, Inc. and the American Pharmacists Association

  1. Quantification of anti-nutritional factors and their correlations with protein and oil in soybeans.

    PubMed

    Bueno, Rafael D; Borges, Leandro L; God, Pedro I V Good; Piovesan, Newton D; Teixeira, Arlindo I; Cruz, Cosme Damião; Barros, Everaldo G DE

    2018-01-01

    Soybeans contain about 30% carbohydrate, mainly consisting of non-starch polysaccharides (NSP) and oligosaccharides. NSP are not hydrolyzed in the gastrointestinal tract of monogastric animals. These NSP negatively affect the development of these animals, especially the soluble fraction. This work aimed to establish a method to quantify NSP in soybeans, using high performance liquid chromatography (HPLC), and to estimate correlations between NSP, oligosaccharides, protein and oil. Sucrose, raffinose + stachyose, soluble and insoluble NSP contents were determined by HPLC. Oil and protein contents were determined by near-infrared spectroscopy (NIRS). The soluble PNAs content showed no significant correlation with protein, oil, sucrose and raffinose + stachyose contents, but oligosaccharides showed a negative correlation with protein content. These findings open up the possibility of developing cultivars with low soluble NSP content, aiming to develop feed for monogastric animals.

  2. Enzymatic breakdown of raffinose oligosaccharides in pea seeds.

    PubMed

    Blöchl, Andreas; Peterbauer, Thomas; Hofmann, Julia; Richter, Andreas

    2008-06-01

    Both alkaline and acidic alpha-galactosidases (alpha-D: -galactoside galactohydrolases, E.C.3.2.1.22) isolated from various plant species have been described, although little is known about their co-occurrence and functions in germinating seeds. Here, we report on the isolation of two cDNAs, encoding for alpha-galactosidases from maturing and germinating seeds of Pisum sativum. One was identified as a member of the acidic alpha-galactosidase of the family 27 glycosyl hydrolase cluster and the other as a member of the family of alkaline alpha-galactosidases, which are highly homologous to seed imbibition proteins (SIPs). PsGAL1 transcripts, encoding for the ACIDIC alpha-GALACTOSIDASE, were predominately expressed during seed maturation and acidic enzyme activities were already present in dry seeds, showing little changes during seed germination. Compartmentation studies revealed that acidic alpha-galactosidases were located in protein storage vacuoles (PSVs). PsAGA1, encoding for the ALKALINE alpha-GALACTOSIDASE, was only expressed after radicle protrusion, when about 50% of RFOs have already been broken down. RFO breakdown was markedly decreased when the translation of the alkaline enzyme was inhibited, providing evidence that PsAGA1 indeed functioned in RFO degradation. Based on these data, we present an integrated model of RFO breakdown by two sequentially active alpha-galactosidases in pea seeds.

  3. Differential Metabolic Rearrangements after Cold Storage Are Correlated with Chilling Injury Resistance of Peach Fruits

    PubMed Central

    Bustamante, Claudia A.; Monti, Laura L.; Gabilondo, Julieta; Scossa, Federico; Valentini, Gabriel; Budde, Claudio O.; Lara, María V.; Fernie, Alisdair R.; Drincovich, María F.

    2016-01-01

    Reconfiguration of the metabolome is a key component involved in the acclimation to cold in plants; however, few studies have been devoted to the analysis of the overall metabolite changes after cold storage of fruits prior to consumption. Here, metabolite profiling of six peach varieties with differential susceptibility to develop mealiness, a chilling-injury (CI) symptom, was performed. According to metabolic content at harvest; after cold treatment; and after ripening, either following cold treatment or not; peach fruits clustered in distinct groups, depending on harvest-time, cold treatment, and ripening state. Both common and distinct metabolic responses among the six varieties were found; common changes including dramatic galactinol and raffinose rise; GABA, Asp, and Phe increase; and 2-oxo-glutarate and succinate decrease. Raffinose content after long cold treatment quantitatively correlated to the degree of mealiness resistance of the different peach varieties; and thus, raffinose emerges as a candidate biomarker of this CI disorder. Xylose increase after cold treatment was found only in the susceptible genotypes, indicating a particular cell wall reconfiguration of these varieties while being cold-stored. Overall, results indicate that peach fruit differential metabolic rearrangements due to cold treatment, rather than differential metabolic priming before cold, are better related with CI resistance. The plasticity of peach fruit metabolism renders it possible to induce a diverse metabolite array after cold, which is successful, in some genotypes, to avoid CI. PMID:27746802

  4. Processing, cooking, and cooling affect prebiotic concentrations in lentil (Lens culinaris Medikus)

    USDA-ARS?s Scientific Manuscript database

    Lentil is an important staple food crop in many regions world-wide and is a good source of protein (20-30%) and various micronutrients. Lentil contains raffinose-family oligosaccharides (RFO), resistant starch (RS), and other prebiotic compounds essential for maintenance of healthy gastrointestinal ...

  5. Immobilized alpha-Galactosidase in the Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Mulimani, V. H.; Dhananjay, K.

    2007-01-01

    This laboratory experiment was designed to demonstrate the application of immobilized galactosidase in food industry to hydrolyze raffinose family oligosaccharides in soymilk. This laboratory experiment was conducted for postgraduate students of biochemistry and developed for graduate and undergraduate students of biochemistry, biotechnology,…

  6. Metabolism of Four α-Glycosidic Linkage-Containing Oligosaccharides by Bifidobacterium breve UCC2003

    PubMed Central

    O'Connell, Kerry Joan; O'Connell Motherway, Mary; O'Callaghan, John; Fitzgerald, Gerald F.; Ross, R. Paul; Ventura, Marco; Stanton, Catherine

    2013-01-01

    Members of the genus Bifidobacterium are common inhabitants of the gastrointestinal tracts of humans and other mammals, where they ferment many diet-derived carbohydrates that cannot be digested by their hosts. To extend our understanding of bifidobacterial carbohydrate utilization, we investigated the molecular mechanisms by which 11 strains of Bifidobacterium breve metabolize four distinct α-glucose- and/or α-galactose-containing oligosaccharides, namely, raffinose, stachyose, melibiose, and melezitose. Here we demonstrate that all B. breve strains examined possess the ability to utilize raffinose, stachyose, and melibiose. However, the ability to metabolize melezitose was not common to all B. breve strains tested. Transcriptomic and functional genomic approaches identified a gene cluster dedicated to the metabolism of α-galactose-containing carbohydrates, while an adjacent gene cluster, dedicated to the metabolism of α-glucose-containing melezitose, was identified in strains that are able to use this carbohydrate. PMID:23913435

  7. Metabolism of four α-glycosidic linkage-containing oligosaccharides by Bifidobacterium breve UCC2003.

    PubMed

    O'Connell, Kerry Joan; O'Connell Motherway, Mary; O'Callaghan, John; Fitzgerald, Gerald F; Ross, R Paul; Ventura, Marco; Stanton, Catherine; van Sinderen, Douwe

    2013-10-01

    Members of the genus Bifidobacterium are common inhabitants of the gastrointestinal tracts of humans and other mammals, where they ferment many diet-derived carbohydrates that cannot be digested by their hosts. To extend our understanding of bifidobacterial carbohydrate utilization, we investigated the molecular mechanisms by which 11 strains of Bifidobacterium breve metabolize four distinct α-glucose- and/or α-galactose-containing oligosaccharides, namely, raffinose, stachyose, melibiose, and melezitose. Here we demonstrate that all B. breve strains examined possess the ability to utilize raffinose, stachyose, and melibiose. However, the ability to metabolize melezitose was not common to all B. breve strains tested. Transcriptomic and functional genomic approaches identified a gene cluster dedicated to the metabolism of α-galactose-containing carbohydrates, while an adjacent gene cluster, dedicated to the metabolism of α-glucose-containing melezitose, was identified in strains that are able to use this carbohydrate.

  8. Formulation, stability and pharmacokinetics of sugar-based salmon calcitonin-loaded nanoporous/nanoparticulate microparticles (NPMPs) for inhalation.

    PubMed

    Amaro, Maria Inês; Tewes, Frederic; Gobbo, Oliviero; Tajber, Lidia; Corrigan, Owen I; Ehrhardt, Carsten; Healy, Anne Marie

    2015-04-10

    A challenge exists to produce dry powder inhaler (DPI) formulations with appropriate formulation stability, biological activity and suitable physicochemical and aerosolisation characteristics that provide a viable alternative to parenteral formulations. The present study aimed to produce sugar-based nanoporous/nanoparticulate microparticles (NPMPs) loaded with a therapeutic peptide - salmon calcitonin (sCT). The physicochemical properties of the powders and their suitability for pulmonary delivery of sCT were determined. Production of powders composed of sCT loaded into raffinose or trehalose with or without hydroxypropyl-β-cyclodextrin was carried out using a laboratory scale spray dryer. Spray dried microparticles were spherical, porous and of small geometric size (≤2 μm). Aerodynamic assessment showed that the fine particle fraction (FPF) less than 5 μm ranged from 45 to 86%, depending on the formulation. The mass median aerodynamic diameter (MMAD) varied between 1.9 and 4.7 μm. Compared to unprocessed sCT, sCT:raffinose composite systems presented a bioactivity of approximately 100% and sCT:trehalose composite systems between 70-90% after spray drying. Storage stability studies demonstrated composite systems with raffinose to be more stable than those containing trehalose. These sugar-based salmon calcitonin-loaded NPMPs retain reasonable sCT bioactivity and have micromeritic and physicochemical properties which indicate their suitability for pulmonary delivery. Formulations presented a similar pharmacokinetic profile to sCT solution. Hence the advantage of a dry powder formulation is its non-invasive delivery route and ease of administration of the sCT. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Changes in carbohydrate content in zucchini fruit (Cucurbita pepo L.) under low temperature stress.

    PubMed

    Palma, Francisco; Carvajal, Fátima; Lluch, Carmen; Jamilena, Manuel; Garrido, Dolores

    2014-03-01

    The postharvest handling of zucchini fruit includes low-temperature storage, making cold stress unavoidable. We have investigated the changes of soluble carbohydrates under this stress and its relation with weight loss and chilling injury in zucchini fruit during postharvest storage at 4 °C and 20 °C for up to 14 days. Two varieties with different degrees of chilling tolerance were compared: Natura, the more tolerant variety, and Sinatra, the variety that suffered more severe chilling-injury symptoms and weight loss. In both varieties, total soluble carbohydrates, reducing soluble carbohydrates and polyols content was generally higher during storage at 4 °C than at 20 °C, thus these parameters are related to the physiological response of zucchini fruit to cold stress. However, the raffinose content increased in Natura and Sinatra fruits during storage at 4 °C and 20 °C, although at 20 °C the increase in raffinose was more remarkable than at 4 °C in both varieties, so that the role of raffinose could be more likely related to dehydration than to chilling susceptibility of zucchini fruit. Glucose, fructose, pinitol, and acid invertase activity registered opposite trends in both varieties against chilling, increasing in Natura and decreasing in Sinatra. The increase in acid invertase activity in Natura fruit during cold storage could contribute in part to the increase of these reducing sugars, whose metabolism could be involved in the adaptation to postharvest cold storage. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. Molecularly imprinted polymers for separation of various sugars from human urine.

    PubMed

    Okutucu, Burcu; Onal, Seçil

    2011-12-15

    Molecularly imprinted polymers were the new, simple and unexpensive materials that can be used in several clinical applications. Phenylboronic acid has been frequently used as functional monomer for the covalent imprinting of diols. In this study, the phenylboronic acid esters of fructose, galactose, glucose and raffinose were synthesized and then used as template analytes. The adsorption capacities of fructose, galactose and glucose-phenylboronic acid imprinted polymers were 75, 10 and 30%, respectively. The batch rebinding studies and Scatchard analysis were done for all sugar imprinted polymer. Glucose is one of the mostly found sugar in the urine. The glucose:phenylboronic acid imprinted polymer was used for the analysis of glucose, fructose, galactose, sucrose, maltose, lactose and raffinose in spiked urine. The selectivity of glucose:phenylboronic acid imprinted polymer to urine monosaccharides was found as nearly 45-55% and to di- and polysaccharides was found as 30-35%, respectively. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Effect of gamma irradiation on microbial load, physicochemical and sensory characteristics of soybeans (Glycine max L. Merrill)

    NASA Astrophysics Data System (ADS)

    Yun, Juan; Li, Xihong; Fan, Xuetong; Tang, Yao; Xiao, Yao; Wan, Sen

    2012-08-01

    Gamma irradiation is highly effective in inactivating microorganisms in various foods and offers a safe alternative method of food decontamination. In the present study, soybeans (Glycine max L. Merrill) were treated with 0, 1.0, 3.0, 5.0 and 10.0 KGy of gamma irradiation. Microbial populations on soybeans, isoflavone, tocopherol contents, raffinose family oligosaccharides, color and sensory properties were evaluated as a function of irradiation dose. The results indicated that gamma irradiation reduced aerobic bacterial and fungal load. Irradiation at the doses applied did not cause any significant change (p>0.05) in the contents of isoflavone of soybeans, but decreased tocopherol contents. The content of key flatulence-producing raffinose family oligosaccharides in irradiated soybeans (10.0 kGy) decreased by 82.1% compared to the control. Sensory analysis showed that the odor of the soybeans was organoleptically acceptable at doses up to 5.0 kGy and no significant differences were observed between irradiated and nonirradiated samples in flavor, texture and color after irradiation.

  12. Shared and novel molecular responses of mandarin to drought.

    PubMed

    Gimeno, Jacinta; Gadea, José; Forment, Javier; Pérez-Valle, Jorge; Santiago, Julia; Martínez-Godoy, María A; Yenush, Lynne; Bellés, José M; Brumós, Javier; Colmenero-Flores, José M; Talón, Manuel; Serrano, Ramón

    2009-07-01

    Drought is the most important stress experienced by citrus crops. A citrus cDNA microarray of about 6.000 genes has been utilized to identify transcriptomic responses of mandarin to water stress. As observed in other plant species challenged with drought stress, key genes for lysine catabolism, proline and raffinose synthesis, hydrogen peroxide reduction, vacuolar malate transport, RCI2 proteolipids and defence proteins such as osmotin, dehydrins and heat-shock proteins are induced in mandarin. Also, some aquaporin genes are repressed. The osmolyte raffinose could be detected in stressed roots while the dehydrin COR15 protein only accumulated in stressed leaves but not in roots. Novel drought responses in mandarin include the induction of genes encoding a new miraculin isoform, chloroplast beta-carotene hydroxylase, oleoyl desaturase, ribosomal protein RPS13A and protein kinase CTR1. These results suggest that drought tolerance in citrus may benefit from inhibition of proteolysis, activation of zeaxanthin and linolenoyl synthesis, reinforcement of ribosomal structure and down-regulation of the ethylene response.

  13. Dehydration accelerates root respiration and impacts sugarbeet raffinose metabolism

    USDA-ARS?s Scientific Manuscript database

    Sugarbeet roots lose water during storage and often become severely dehydrated after prolonged storage and at the outer portions of piles which have greater wind and sun exposure. Sucrose loss is known to be elevated in dehydrated roots, although the metabolic processes responsible for this loss ar...

  14. Efficient free fatty acid production in engineered Escherichia coli strains using soybean oligosaccharides as feedstock.

    PubMed

    Wang, Dan; Wu, Hui; Thakker, Chandresh; Beyersdorf, Jared; Bennett, George N; San, Ka-Yiu

    2015-01-01

    To be competitive with current petrochemicals, microbial synthesis of free fatty acids can be made to rely on a variety of renewable resources rather than on food carbon sources, which increase its attraction for governments and companies. Industrial waste soybean meal is an inexpensive feedstock, which contains soluble sugars such as stachyose, raffinose, sucrose, glucose, galactose, and fructose. Free fatty acids were produced in this report by introducing an acyl-ACP carrier protein thioesterase and (3R)-hydroxyacyl-ACP dehydratase into E. coli. Plasmid pRU600 bearing genes involved in raffinose and sucrose metabolism was also transformed into engineered E. coli strains, which allowed more efficient utilization of these two kinds of specific oligosaccharide present in the soybean meal extract. Strain ML103 (pRU600, pXZ18Z) produced ~1.60 and 2.66 g/L of free fatty acids on sucrose and raffinose, respectively. A higher level of 2.92 g/L fatty acids was obtained on sugar mixture. The fatty acid production using hydrolysate obtained from acid or enzyme based hydrolysis was evaluated. Engineered strains just produced ~0.21 g/L of free fatty acids with soybean meal acid hydrolysate. However, a fatty acid production of 2.61 g/L with a high yield of 0.19 g/g total sugar was observed on an enzymatic hydrolysate. The results suggest that complex mixtures of oligosaccharides derived from soybean meal can serve as viable feedstock to produce free fatty acids. Enzymatic hydrolysis acts as a much more efficient treatment than acid hydrolysis to facilitate the transformation of industrial waste from soybean processing to high value added chemicals. © 2015 American Institute of Chemical Engineers.

  15. Antinutritional factors and in vitro protein digestibility of improved haricot bean (Phaseolus vulgaris L.) varieties grown in Ethiopia.

    PubMed

    Admassu Shimelis, Emire; Kumar Rakshit, Sudip

    2005-09-01

    The antinutrient (raffinose oligosaccharides, tannins, phytic acid and trypsin inhibitors) composition and in vitro protein digestibility of eight improved varieties of Phaseolus vulgaris grown in Ethiopia were determined. Stachyose was the predominant alpha-galactosides in all haricot bean samples. Raffinose was also present in significant quantities but verbascose, glucose and fructose were not detected at all in the samples. The concentrations observed for the protein digestibility and antinutritional factors, varied significantly (P<0.05) between varieties investigated in this study. Mean values for protein digestibility ranged from 80.66% (in Roba variety) to 65.64% (in Beshbesh variety). Mean values for raffinose, stachyose, sucrose, trypsin inhibitors, tannins and phytic acid were 3.14 mg/g, 14.86 mg/g, 24.22 mg/g, 20.68 TUIx10(3)/g, 17.44 mg, catechin equivalents/g and 20.54 mg/g respectively. Statistical analyses of data revealed that antinutritional factors and protein digestibility were influenced by variety (genotype). Relationships between antinutritional factors and protein digestibility were also observed. The possibility of selecting varieties to be used for large-scale cultivation in Ethiopia on the basis of these data is discussed. Among the improved varieties studied, Roba, Redwolaita, Mexican and Awash were found to be the best food and export type of haricot beans in the Ethiopian context, because of their higher protein digestibility, lower antinutrtional factors and other beneficial nutritional parameters. Roba variety can be used by local food processors for the production of value-added bean-based products especially to combat the problem of protein energy malnutrition and related diseases which are very common in developing countries.

  16. Hypolipidemic Effect of Red Gram (Cajanus cajan L.) Prebiotic Oligosaccharides in Wistar NIN Rats.

    PubMed

    Shakappa, Devindra; Talari, Aruna; Rajkumar, Hemalatha; Shujauddin, Mohammed

    2017-08-24

    The hypolipidemic effect of red gram prebiotics of raffinose family oligosaccharides was studied in Wistar National Institute of Nutrition male rat strain. The study consisted of 36 rats randomly divided into three groups of 12 rats each. For 16 weeks, Group I was fed with the control diet; Group II was fed with a diet containing 3% standard raffinose as the reference group; Group III received the diet containing 3% red gram prebiotics. The results showed that the gain in body weight was low in the red gram prebiotics-supplemented group followed by the control group; highest increase of body weight was seen in the raffinose standard-fed group. Serum glucose levels of the red gram prebiotic-fed group decreased 14.92% compared to the control group and increased 2.07% compared to the reference group. The decrease in serum triglycerides (TG) levels of the red gram prebiotic-fed groups was 32.76% compared to the control group and 33.64% compared to the reference group. Decrease in the serum TC of the red gram-fed animals was 18.51% and 4.63% compared to the control group and the reference group, respectively. Increase in the level of serum high-density lipoprotein cholesterol (HDL-C) in the red gram-fed animals was 18.51% compared to the control group and 4.63% compared to the reference group. The present study can be a proof for the use of prebiotics as a preventive measure for overweight and obesity in humans, and legume prebiotics can be explored as a novel prebiotic product in the consumer market.

  17. Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification, characterization, and insights towards thermal denaturation.

    PubMed

    M, Ramadevi; Panwar, Deepesh; Joseph, Juby Elsa; Kaira, Gaurav Singh; Kapoor, Mukesh

    2018-06-20

    A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal) from germinating black gram (Vigna mungo) seeds was purified to homogeneity (432 U/mg specific activity, 1542-fold purification) using ion-exchange (DEAE-cellulose, CM-sepharose) and affinity (Con-A Sepharose 4B) chromatography. VM-αGal appeared as a single band ~ 45 kDa on SDS-PAGE and showed optimal activity at pH 5 and 55 °C. Hg 2+ and SDS completely inhibited VM-αGal activity. The K m, V max and catalytic efficiency (k cat /K m ) of VM-αGal for pNPG and raffinose was found to be 0.99, 17.23 mM, 0.413 s -1  mM -1 and 1.66, 0.146 mM ml -1  min -1 , 0.0026 s -1  mM -1 , respectively. VM-αGal was competitively inhibited by galactose (K i 7.70 mM). Thermodynamic parameters [activation enthalpy (ΔH), activation entropy (ΔS) and free energy (ΔG)] of VM-αGal at 45-51 °C showed that the enzyme was in a less energetic state and had susceptibility towards denaturation. Temperature-induced structural unfolding studies of VM-αGal probed by fluorescence, and far-UV CD spectroscopy revealed significant loss in tertiary structure and a steep decline in β-sheet content at 45-65 °C, and above 55 °C, respectively. VM-αGal improved the nutritional quality of soymilk by hydrolyzing the flatulence-causing raffinose family oligosaccharides (26.5% and 18.45% decrease in stachyose and raffinose, respectively). Copyright © 2018. Published by Elsevier B.V.

  18. The effect of the osmolality of sugar-containing media, the type of sugar, and the mass and molar concentration of sugar on the survival of frozen-thawed mouse sperm.

    PubMed

    Koshimoto, Chihiro; Mazur, Peter

    2002-08-01

    Several factors have contributed to problems in mouse sperm cryopreservation, and we and others have found ways to ameliorate them. These include high sensitivity to several types of mechanical stresses and to oxygen-derived free radicals, low tolerance to osmotic cell volume changes, and rather rigorous requirements for cooling and warming rates. Another important factor is the cryoprotective agent. Mouse sperm are unusual in that our best results have been obtained in media containing the nonpermeating sugar raffinose (18% w/v) and lacking glycerol. This paper deals with questions about the basis of the protective action of sugars, and whether raffinose is unusual or unique in its ability to confer protection. More specifically, we investigated whether protection was more related to the total osmolality of the freezing solution, to the mass concentration of sugar, or to the molarity of the sugar, and we looked to see whether there are effects attributable to specific sugars. To investigate these questions, mouse sperm were frozen at the optimal rate of 25 degrees C/min in solutions prepared with different proportions of three sugars-raffinose, sucrose, and glucose-dissolved in 1/4x PBS. In the first experimental series, the total osmolality and the total sugar molarity were varied from 400 to 700 mOsm and from 300 to 530 mM, respectively, while holding the mass concentration of sugar constant at 18% (w/v). In the second experimental series, the mass concentration of sugars was varied from 10 to 18% while the sugar molarity and solution osmolality remained constant at 300 mM and 420 mOsm, respectively. The results suggest that protection against freezing and thawing depends more on the mass concentration of the sugar than on its molar concentration, a conclusion that has mechanistic implications.

  19. Physiological, biochemical and molecular responses in four Prunus rootstocks submitted to drought stress.

    PubMed

    Jiménez, Sergio; Dridi, Jihène; Gutiérrez, Diego; Moret, David; Irigoyen, Juan J; Moreno, María A; Gogorcena, Yolanda

    2013-10-01

    An understanding of the mechanisms that determine plant response to reduced water availability is essential to improve water-use efficiency (WUE) of stone fruit crops. The physiological, biochemical and molecular drought responses of four Prunus rootstocks (GF 677, Cadaman, ROOTPAC 20 and ROOTPAC(®) R) budded with 'Catherina' peach cultivar were studied. Trees were grown in 15-l containers and subjected to a progressive water stress for 26 days, monitoring soil moisture content by time domain reflectometry. Photosynthetic and gas exchange parameters were determined. Root and leaf soluble sugars and proline content were also measured. At the end of the experiment, stressed plants showed lower net photosynthesis rate, stomatal conductance and transpiration rate, and higher intrinsic leaf WUE (AN/gs). Soluble sugars and proline concentration changes were observed, in both root and leaf tissues, especially in an advanced state of stress. The accumulation of proline in roots and leaves with drought stress was related to the decrease in osmotic potential and increase in WUE, whereas the accumulation of sorbitol in leaves, raffinose in roots and proline in both tissues was related only to the increase in the WUE. Owing to the putative role of raffinose and proline as antioxidants and their low concentration, they could be ameliorating deleterious effects of drought-induced oxidative stress by protecting membranes and enzymes rather than acting as active osmolytes. Higher expression of P5SC gene in roots was also consistent with proline accumulation in the tolerant genotype GF 677. These results indicate that accumulation of sorbitol, raffinose and proline in different tissues and/or the increase in P5SC expression could be used as markers of drought tolerance in peach cultivars grafted on Prunus rootstocks.

  20. Genotype evaluation of cowpea seeds (Vigna unguiculata) using 1H qNMR combined with exploratory tools and solid-state NMR.

    PubMed

    Alves Filho, Elenilson G; Silva, Lorena M A; Teofilo, Elizita M; Larsen, Flemming H; de Brito, Edy S

    2017-01-01

    The ultimate aim of this study was to apply a non-targeted chemometric analysis (principal component analysis and hierarchical clustering analysis using the heat map approach) of NMR data to investigate the variability of organic compounds in nine genotype cowpea seeds, without any complex pre-treatment. In general, both exploratory tools show that Tvu 233, CE-584, and Setentão genotypes presented higher amount mainly of raffinose and Tvu 382 presented the highest content of choline and least content of raffinose. The evaluation of the aromatic region showed the Setentão genotype with highest content of niacin/vitamin B3 whereas Tvu 382 with lowest amount. To investigate rigid and mobile components in the seeds cotyledon, 13 C CP and SP/MAS solid-state NMR experiments were performed. The cotyledon of the cowpea comprised a rigid part consisting of starch as well as a soft portion made of starch, fatty acids, and protein. The variable contact time experiment suggests the presence of lipid-amylose complexes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Metabolic profiling of a range of peach fruit varieties reveals high metabolic diversity and commonalities and differences during ripening.

    PubMed

    Monti, Laura L; Bustamante, Claudia A; Osorio, Sonia; Gabilondo, Julieta; Borsani, Julia; Lauxmann, Martin A; Maulión, Evangelina; Valentini, Gabriel; Budde, Claudio O; Fernie, Alisdair R; Lara, María V; Drincovich, María F

    2016-01-01

    Peach (Prunus persica) fruits from different varieties display differential organoleptic and nutritional properties, characteristics related to their chemical composition. Here, chemical biodiversity of peach fruits from fifteen varieties, at harvest and after post-harvest ripening, was explored by gas chromatography-mass spectrometry. Metabolic profiling revealed that metabolites involved in organoleptic properties (sugars, organic and amino acids), stress tolerance (raffinose, galactinol, maltitol), and with nutritional properties (amino, caffeoylquinic and dehydroascorbic acids) displayed variety-dependent levels. Peach varieties clustered into four groups: two groups of early-harvest varieties with higher amino acid levels; two groups of mid- and late-harvest varieties with higher maltose levels. Further separation was mostly dependent on organic acids/raffinose levels. Variety-dependent and independent metabolic changes associated with ripening were detected; which contribute to chemical diversity or can be used as ripening markers, respectively. The great variety-dependent diversity in the content of metabolites that define fruit quality reinforces metabolomics usage as a tool to assist fruit quality improvement in peach. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Effect of pH on subunit association and heat protection of soybean alpha-galactosidase

    NASA Technical Reports Server (NTRS)

    Porter, J. E.; Sarikaya, A.; Herrmann, K. M.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

    1992-01-01

    Soybeans contain the enzyme alpha-galactosidase, which hydrolyzes alpha-1, 6 linkages in stachyose and raffinose to give sucrose and galactose. We have found that galactose, a competitive product inhibitor of alpha-galactosidase, strongly promotes the heat stability of the tetrameric form of the enzyme at pH 4.0 and at temperatures of up to 70 degrees C for 60 min. Stachyose and raffinose also protect alpha-galactosidase from denaturation at pH 4.0 although to a lesser extent. Glucose and mannose have little effect. At pH 7.0 the enzyme is a monomer, and galactose has no effect on the heat stability of the enzyme. In the absence of heat protection of the enzyme by added sugars, a series deactivation mechanism was found to describe the deactivation data. In comparison, a unimolecular, non-first order deactivation model applies at pH 4.0, where heat protection effects were observed. At a temperature above 60 degrees C, simple deactivation is a suitable model. The results suggest that alpha-galactosidase conformation and heat stability are directly related.

  3. A novel α-galactosidase from the thermophilic probiotic Bacillus coagulans with remarkable protease-resistance and high hydrolytic activity

    PubMed Central

    Zhao, Ruili; Zhao, Rui; Tu, Yishuai; Zhang, Xiaoming; Deng, Liping

    2018-01-01

    A novel α-galactosidase of glycoside hydrolase family 36 was cloned from Bacillus coagulans, overexpressed in Escherichia coli, and characterized. The purified enzyme Aga-BC7050 was 85 kDa according to SDS-PAGE and 168 kDa according to gel filtration, indicating that its native structure is a dimer. With p-nitrophenyl-α-d- galactopyranoside (pNPGal) as the substrate, optimal temperature and pH were 55 °C and 6.0, respectively. At 60 °C for 30 min, it retained > 50% of its activity. It was stable at pH 5.0–10.0, and showed remarkable resistance to proteinase K, subtilisin A, α-chymotrypsin, and trypsin. Its activity was not inhibited by glucose, sucrose, xylose, or fructose, but was slightly inhibited at galactose concentrations up to 100 mM. Aga-BC7050 was highly active toward pNPGal, melibiose, raffinose, and stachyose. It completely hydrolyzed melibiose, raffinose, and stachyose in < 30 min. These characteristics suggest that Aga-BC7050 could be used in feed and food industries and sugar processing. PMID:29738566

  4. Identification of a Novel α-Galactosidase from the Hyperthermophilic Archaeon Sulfolobus solfataricus†

    PubMed Central

    Brouns, Stan J. J.; Smits, Nicole; Wu, Hao; Snijders, Ambrosius P. L.; Wright, Phillip C.; de Vos, Willem M.; van der Oost, John

    2006-01-01

    Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable α-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for α-linked galactosides, which are optimally hydrolyzed at pH 5 and 90°C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable α-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea. PMID:16547025

  5. 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.

  6. 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

  7. Cellulosimicrobium funkei: first report of infection in a nonimmunocompromised patient and useful phenotypic tests for differentiation from Cellulosimicrobium cellulans and Cellulosimicrobium terreum.

    PubMed

    Petkar, Hawabibee; Li, Anthony; Bunce, Nicholas; Duffy, Kim; Malnick, Henry; Shah, Jayesh J

    2011-03-01

    Cellulosimicrobium funkei is a rare, opportunistic pathogen. We describe a case of bacteremia and possibly prosthetic valve endocarditis by this organism in a nonimmunocompromised patient. Useful phenotypic tests for differentiating C. funkei from Cellulosimicrobium cellulans and Cellulosimicrobium terreum include motility, raffinose fermentation, glycogen, D-xylose, and methyl-α-D-glucopyranoside assimilation, and growth at 35°C.

  8. Application of ultrasound for enhanced extraction of prebiotic oligosaccharides from selected fruits and vegetables.

    PubMed

    Jovanovic-Malinovska, Ruzica; Kuzmanova, Slobodanka; Winkelhausen, Eleonora

    2015-01-01

    Ultrasound assisted extraction (UAE) was used to extract oligosaccharides from selected fruits (blueberry, nectarine, raspberry, watermelon) and vegetables (garlic, Jerusalem artichoke, leek, scallion, spring garlic and white onion). The individual fractions of the oligosaccharides were analyzed: 1-kestose (GF2), nystose (GF3) and 1F-β-fructofuranosylnystose (GF4) from the fructo-oligosaccharides (FOS), and raffinose and stachyose from the raffinose family oligosaccharides (RFO). Extraction parameters including solvent concentration (35-85% v/v), extraction temperature (25-50°C) and sonication time (5-15min) were examined using response surface methodology (RSM). Ethanol concentration of 63% v/v, temperature of 40°C and extraction time of 10min gave maximal concentration of the extracted oligosaccharides. The experimental values under optimal conditions were consistent with the predicted values. UAE increased the concentration of extracted oligosaccharides in all fruits and vegetables from 2 to 4-fold compared to conventional extraction. The highest increase of total oligosaccharides extracted by UAE was detected in Jerusalem artichoke, 7.17±0.348g/100gFW, compared to 1.62±0.094g/100gFW with conventional method. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Wood decay by brown-rot fungi : changes in pore structure and cell wall volume

    Treesearch

    Douglas S. Flournoy; T. Kent Kirk; T.L. Highley

    1991-01-01

    Sweetgum (Liquidambar styraciflua L.) wood blocks were decayed by Postia (= Poria) placenta in soilblock cultures. Decay was terminated at various weight losses, and the pore volumes available to four low molecular weight molecules, (water, 4 Å,; glucose, 8 Å,; maltose, 10 Å; and raffinose, 128,) and three dextrans (Mr 6,000, 38 Å; 11,200, 51 Å; nd 17,500, 61 Å) were...

  10. Optimisation of spray drying process conditions for sugar nanoporous microparticles (NPMPs) intended for inhalation.

    PubMed

    Amaro, Maria Inês; Tajber, Lidia; Corrigan, Owen I; Healy, Anne Marie

    2011-12-12

    The present study investigated the effect of operating parameters of a laboratory spray dryer on powder characteristics, in order to optimise the production of trehalose and raffinose powders, intended to be used as carriers of biomolecules for inhalation. The sugars were spray dried from 80:20 methanol:n-butyl acetate (v/v) solutions using a Büchi Mini Spray dryer B-290. A 2(4) factorial design of experiment (DOE) was undertaken. Process parameters studied were inlet temperature, gas flow rate, feed solution flow rate (pump setting) and feed concentration. Resulting powders where characterised in terms of yield, particle size (PS), residual solvent content (RSC) and outlet temperature. An additional outcome evaluated was the specific surface area (SSA) (by BET gas adsorption), and a relation between SSA and the in vitro deposition of the sugar NPMPs powders was also investigated. The DOE resulted in well fitted models. The most significant factors affecting the characteristics of the NPMPs prepared, at a 95% confidence interval, were gas flow: yield, PS and SSA; pump setting: yield; inlet temperature: RSC. Raffinose NPMPs presented better characteristics than trehalose NPMPs in terms of their use for inhalation, since particles with larger surface area resulting in higher fine particle fraction can be produced. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Acceptability and characterization of extruded pinto, navy and black beans.

    PubMed

    Simons, Courtney W; Hall, Clifford; Tulbek, Mehmet; Mendis, Mihiri; Heck, Taylor; Ogunyemi, Samuel

    2015-08-30

    Consumption of dry beans has been relatively flat over the last decade. Creating new bean products may increase the consumption of beans and allow more consumers to obtain the health benefits of beans. In this study, pinto, navy and black beans were milled and the resulting flours extruded into puffs. Unflavored extruded puffs were evaluated by untrained panelists using a hedonic scale for appearance, flavor, texture and overall acceptability. The compositions of raw flours and extrudates were characterized. Sensory results indicated that all beans met or exceeded the minimum requirement for acceptability. Overall acceptability of navy and pinto beans was not significantly different, while acceptability of black bean puffs was significantly lower. Total protein (198-217 g kg(-1)) in extrudates was significantly different among the three beans. Total starch ranged from 398 to 406 g kg(-1) and was not significantly different. Resistant starch, total extractable lipid and raffinose contents were significantly reduced by extrusion. Extrusion did not affect crude fiber and phytic acid contents. The minimal effects on protein and fiber contents, the significant reduction in raffinose content and the acceptability of the unflavored extruded puffs support using various bean flours as ingredients in extruded puffed products. © 2014 Society of Chemical Industry.

  12. Transcription of two adjacent carbohydrate utilization gene clusters in Bifidobacterium breve UCC2003 is controlled by LacI- and repressor open reading frame kinase (ROK)-type regulators.

    PubMed

    O'Connell, Kerry Joan; Motherway, Mary O'Connell; Liedtke, Andrea; Fitzgerald, Gerald F; Paul Ross, R; Stanton, Catherine; Zomer, Aldert; van Sinderen, Douwe

    2014-06-01

    Members of the genus Bifidobacterium are commonly found in the gastrointestinal tracts of mammals, including humans, where their growth is presumed to be dependent on various diet- and/or host-derived carbohydrates. To understand transcriptional control of bifidobacterial carbohydrate metabolism, we investigated two genetic carbohydrate utilization clusters dedicated to the metabolism of raffinose-type sugars and melezitose. Transcriptomic and gene inactivation approaches revealed that the raffinose utilization system is positively regulated by an activator protein, designated RafR. The gene cluster associated with melezitose metabolism was shown to be subject to direct negative control by a LacI-type transcriptional regulator, designated MelR1, in addition to apparent indirect negative control by means of a second LacI-type regulator, MelR2. In silico analysis, DNA-protein interaction, and primer extension studies revealed the MelR1 and MelR2 operator sequences, each of which is positioned just upstream of or overlapping the correspondingly regulated promoter sequences. Similar analyses identified the RafR binding operator sequence located upstream of the rafB promoter. This study indicates that transcriptional control of gene clusters involved in carbohydrate metabolism in bifidobacteria is subject to conserved regulatory systems, representing either positive or negative control.

  13. Transcription of Two Adjacent Carbohydrate Utilization Gene Clusters in Bifidobacterium breve UCC2003 Is Controlled by LacI- and Repressor Open Reading Frame Kinase (ROK)-Type Regulators

    PubMed Central

    O'Connell, Kerry Joan; O'Connell Motherway, Mary; Liedtke, Andrea; Fitzgerald, Gerald F.; Ross, R. Paul; Stanton, Catherine; Zomer, Aldert

    2014-01-01

    Members of the genus Bifidobacterium are commonly found in the gastrointestinal tracts of mammals, including humans, where their growth is presumed to be dependent on various diet- and/or host-derived carbohydrates. To understand transcriptional control of bifidobacterial carbohydrate metabolism, we investigated two genetic carbohydrate utilization clusters dedicated to the metabolism of raffinose-type sugars and melezitose. Transcriptomic and gene inactivation approaches revealed that the raffinose utilization system is positively regulated by an activator protein, designated RafR. The gene cluster associated with melezitose metabolism was shown to be subject to direct negative control by a LacI-type transcriptional regulator, designated MelR1, in addition to apparent indirect negative control by means of a second LacI-type regulator, MelR2. In silico analysis, DNA-protein interaction, and primer extension studies revealed the MelR1 and MelR2 operator sequences, each of which is positioned just upstream of or overlapping the correspondingly regulated promoter sequences. Similar analyses identified the RafR binding operator sequence located upstream of the rafB promoter. This study indicates that transcriptional control of gene clusters involved in carbohydrate metabolism in bifidobacteria is subject to conserved regulatory systems, representing either positive or negative control. PMID:24705323

  14. Amborella trichopoda, plasmodesmata, and the evolution of phloem loading.

    PubMed

    Turgeon, Robert; Medville, Richard

    2011-01-01

    Phloem loading is the process by which photoassimilates synthesized in the mesophyll cells of leaves enter the sieve elements and companion cells of minor veins in preparation for long distance transport to sink organs. Three loading strategies have been described: active loading from the apoplast, passive loading via the symplast, and passive symplastic transfer followed by polymer trapping of raffinose and stachyose. We studied phloem loading in Amborella trichopoda, a premontane shrub that may be sister to all other flowering plants. The minor veins of A. trichopoda contain intermediary cells, indicative of the polymer trap mechanism, forming an arc on the abaxial side and subtending a cluster of ordinary companion cells in the interior of the veins. Intermediary cells are linked to bundle sheath cells by highly abundant plasmodesmata whereas ordinary companion cells have few plasmodesmata, characteristic of phloem that loads from the apoplast. Intermediary cells, ordinary companion cells, and sieve elements form symplastically connected complexes. Leaves provided with (14)CO(2) translocate radiolabeled sucrose, raffinose, and stachyose. Therefore, structural and physiological evidence suggests that both apoplastic and polymer trapping mechanisms of phloem loading operate in A. trichopoda. The evolution of phloem loading strategies is complex and may be difficult to resolve.

  15. Sugars of the unfermented sap and the wine from the oil palm, Elaeis guinensis, tree.

    PubMed

    Eze, M O; Ogan, U

    1988-01-01

    The sugar composition of the unfermented sap from oil palm (Elaeis guinensis) trees growing in the plantations of the Nigerian Institute for Oil Palm Research, Benin City, has been determined. While sucrose concentration ranges from 9.59 to 10.59% (w/v) in the pure unfermented sap, that of either glucose or fructose is much less than 1% (w/v) (0.13-0.73% w/v). Raffinose occurs in traces only (0.13-0.35 w/v). These results were derived from our improved methods which eliminate completely, or reduce to a bare minimum, fermentation of the sap during collection. The variation with time of storage of the individual sugars in the sap during fermentation to form palm wine reveals that, as sucrose steadily decreases, fructose reaches a peak at 1.51% (w/v) at the 9th hour, and thereafter declines, while glucose and raffinose remain continuously low; all sugars disappear beyond the 33rd hour. Concomittantly, pH decreases from pH 6.60 at zero time and stabilizes at pH 3.30 after 48 h, while titrable acidity increases continuously up until the 96th hour. These changes account for the variations in the quality of palm wine during storages.

  16. The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm

    PubMed Central

    Varisli, Omer; Scott, Hollie; Agca, Cansu; Agca, Yuksel

    2013-01-01

    Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10–12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4°C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150 °C by using various cooling rates (10, 40, 70, and 100 °C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37 °C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P<0.05). Sperm motility increased as cooling rate was increased for both strains (P<0.05). Highest cryosurvival was achieved when 100 °C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1 M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70

  17. Spray drying from organic solvents to prepare nanoporous/nanoparticulate microparticles of protein: excipient composites designed for oral inhalation.

    PubMed

    Ní Ógáin, Orla; Tajber, Lidia; Corrigan, Owen I; Healy, Anne Marie

    2012-09-01

    The aim of this study was to determine if spray-drying could successfully produce microparticles containing the model protein trypsin in a form suitable for inhalation. Trypsin was spray-dried with raffinose from a methanol : n-butyl acetate solvent system (MeOH : BA). The solvent system was then adjusted to include water, and trypsin was co-spray-dried with raffinose, trehalose or hydroxpropyl-β-cyclodextrin. The spray-dried products were characterised by SEM, XRD, DSC, TGA and FTIR. Protein biological activity and in-vitro deposition of trypsin : excipient nanoporous/nanoparticulate microparticles (NPMPs) was also assessed. The inclusion of water in a MeOH : BA solvent system allowed for the successful production of NPMPs of trypsin : excipient by spray-drying. Trypsin formulated as trypsin : excipient NPMPs retained biological activity on processing and showed no deterioration in activity or morphological characteristics when stored with desiccant at either 4 or 25°C. Hydroxpropyl-β-cyclodextrin showed advantages over the sugars in terms of producing powders with appropriate density and with greater physical stability under high-humidity conditions. Fine particle fractions of between 41 and 45% were determined for trypsin : excipient NPMPs. NPMPs of trypsin : excipient systems can be produced by spray-drying by adjustment of the solvent system to allow for adequate solubility of trypsin. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

  18. Hexoses as phloem transport sugars: the end of a dogma?

    PubMed

    van Bel, Aart J E; Hess, Paul H

    2008-01-01

    According to most textbooks, only non-reducing carbohydrate species such as sucrose, sugar alcohols, and raffinose-family sugars function as phloem translocates. Occasional abundance of reducing sugar species (such as hexoses) in sieve-tube sap has been discarded as an experimental artefact. This study, however, discloses a widespread occurrence of hexoses in the sieve-tube sap. Phloem exudation facilitated by EDTA provided evidence that many of the members of two plant families (Ranunculaceae and Papaveraceae) investigated translocate >80% of carbohydrates in the form of hexoses. Representatives of other families also appear to translocate appreciable amounts of hexoses in the sieve tubes. Promoting effects of EDTA, activities of sucrose-degrading enzymes, and sugar uptake by micro-organisms on hexose contents of phloem exudates were checked. The rate of sucrose degradation is far too low to explain the large proportions of hexoses measured in phloem exudates; nor did other factors tested seem to stimulate the occurrence of hexoses. The validity of the approach is further supported by the virtual absence of hexoses in exudates from species that were known as exclusive sucrose transporters. This study urges a rethink of the existing views on carbohydrate transport species in the phloem stream. Hexose translocation is to be regarded as a normal mode of carbohydrate transfer by the phloem equivalent to that of sucrose, raffinose-family sugars, or sugar alcohols.

  19. Initial water deficit effects on Lupinus albus photosynthetic performance, carbon metabolism, and hormonal balance: metabolic reorganization prior to early stress responses.

    PubMed

    Pinheiro, Carla; António, Carla; Ortuño, Maria Fernanda; Dobrev, Petre I; Hartung, Wolfram; Thomas-Oates, Jane; Ricardo, Cândido Pinto; Vanková, Radomira; Chaves, M Manuela; Wilson, Julie C

    2011-10-01

    The early (2-4 d) effects of slowly imposed soil water deficit on Lupinus albus photosynthetic performance, carbon metabolism, and hormonal balance in different organs (leaf blade, stem stele, stem cortex, and root) were evaluated on 23-d-old plants (growth chamber assay). Our work shows that several metabolic adjustments occurred prior to alteration of the plant water status, implying that water deficit is perceived before the change in plant water status. The slow, progressive decline in soil water content started to be visible 3 d after withholding water (3 DAW). The earliest plant changes were associated with organ-specific metabolic responses (particularly in the leaves) and with leaf conductance and only later with plant water status and photosynthetic rate (4 DAW) or photosynthetic capacity (according to the Farquhar model; 6 DAW). Principal component analysis (PCA) of the physiological parameters, the carbohydrate and the hormone levels and their relative values, as well as leaf water-soluble metabolites full scan data (LC-MS/MS), showed separation of the different sampling dates. At 6 DAW classically described stress responses are observed, with plant water status, ABA level, and root hormonal balance contributing to the separation of these samples. Discrimination of earlier stress stages (3 and 4 DAW) is only achieved when the relative levels of indole-3-acetic acid (IAA), cytokinins (Cks), and carbon metabolism (glucose, sucrose, raffinose, and starch levels) are taken into account. Our working hypothesis is that, in addition to single responses (e.g. ABA increase), the combined alterations in hormone and carbohydrate levels play an important role in the stress response mechanism. Response to more advanced stress appears to be associated with a combination of cumulative changes, occurring in several plant organs. The carbohydrate and hormonal balance in the leaf (IAA to bioactive-Cks; soluble sugars to IAA and starch to IAA; relative abundances of the

  20. Quantification of fructans, galacto-oligosacharides and other short-chain carbohydrates in processed grains and cereals.

    PubMed

    Biesiekierski, J R; Rosella, O; Rose, R; Liels, K; Barrett, J S; Shepherd, S J; Gibson, P R; Muir, J G

    2011-04-01

    Wholegrain grains and cereals contain a wide range of potentially protective factors that are relevant to gastrointestinal health. The prebiotics best studied are fructans [fructooligosaccharides (FOS), inulin] and galactooligosaccharides (GOS). These and other short-chain carbohydrates can also be poorly absorbed in the small intestine (named fermentable oligo-, di- and monosaccharides and polyols; FODMAPs) and may have important implications for the health of the gut. In the present study, FODMAPs, including fructose in excess of glucose, FOS (nystose, kestose), GOS (raffinose, stachyose) and sugar polyols (sorbitol, mannitol), were quantified using high-performance liquid chromatography with an evaporative light scattering detector. Total fructan was quantified using an enzymic hydrolysis method. Fifty-five commonly consumed grains, breakfast cereals, breads, pulses and biscuits were analysed. Total fructan were the most common short-chain carbohydrate present in cereal grain products and ranged (g per portion as eaten) from 1.12 g in couscous to 0 g in rice; 0.6 g in dark rye bread to 0.07 g in spelt bread; 0.96 g in wheat-free muesli to 0.11 g in oats; and 0.81 g in muesli fruit bar to 0.05 g in potato chips. Raffinose and stachyose were most common in pulses.   Composition tables including FODMAPs and prebiotics (FOS and GOS) that are naturally present in food will greatly assist research aimed at understanding their physiological role in the gut. © 2011 The Authors. Journal compilation © 2011 The British Dietetic Association Ltd.

  1. Mutations in the Arabidopsis Homolog of LST8/GβL, a Partner of the Target of Rapamycin Kinase, Impair Plant Growth, Flowering, and Metabolic Adaptation to Long Days[C][W

    PubMed Central

    Moreau, Manon; Azzopardi, Marianne; Clément, Gilles; Dobrenel, Thomas; Marchive, Chloé; Renne, Charlotte; Martin-Magniette, Marie-Laure; Taconnat, Ludivine; Renou, Jean-Pierre; Robaglia, Christophe; Meyer, Christian

    2012-01-01

    The conserved Target of Rapamycin (TOR) kinase forms high molecular mass complexes and is a major regulator of cellular adaptations to environmental cues. The Lethal with Sec Thirteen 8/G protein β subunit-like (LST8/GβL) protein is a member of the TOR complexes, and two putative LST8 genes are present in Arabidopsis thaliana, of which only one (LST8-1) is significantly expressed. The Arabidopsis LST8-1 protein is able to complement yeast lst8 mutations and interacts with the TOR kinase. Mutations in the LST8-1 gene resulted in reduced vegetative growth and apical dominance with abnormal development of flowers. Mutant plants were also highly sensitive to long days and accumulated, like TOR RNA interference lines, higher amounts of starch and amino acids, including proline and glutamine, while showing reduced concentrations of inositol and raffinose. Accordingly, transcriptomic and enzymatic analyses revealed a higher expression of genes involved in nitrate assimilation when lst8-1 mutants were shifted to long days. The transcriptome of lst8-1 mutants in long days was found to share similarities with that of a myo-inositol 1 phosphate synthase mutant that is also sensitive to the extension of the light period. It thus appears that the LST8-1 protein has an important role in regulating amino acid accumulation and the synthesis of myo-inositol and raffinose during plant adaptation to long days. PMID:22307851

  2. Inulin and levan synthesis by probiotic Lactobacillus gasseri strains: characterization of three novel fructansucrase enzymes and their fructan products.

    PubMed

    Anwar, Munir A; Kralj, Slavko; Piqué, Anna Villar; Leemhuis, Hans; van der Maarel, Marc J E C; Dijkhuizen, Lubbert

    2010-04-01

    Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. Here, we report an evaluation of fructan synthesis in three Lactobacillus gasseri strains, identification of the fructansucrase-encoding genes and characterization of the recombinant proteins and fructan (oligosaccharide) products. High-performance anion-exchange chromatography and nuclear magnetic resonance analysis of the fructo-oligosaccharides (FOS) and polymers produced by the L. gasseri strains and the recombinant enzymes revealed that, in situ, L. gasseri strains DSM 20604 and 20077 synthesize inulin (and oligosaccharides) and levan products, respectively. L. gasseri DSM 20604 is only the second Lactobacillus strain shown to produce inulin polymer and FOS in situ, and is unique in its distribution of FOS synthesized, ranging from DP2 to DP13. The probiotic bacterium L. gasseri DSM 20243 did not produce any fructan, although we identified a fructansucrase-encoding gene in its genome sequence. Further studies showed that this L. gasseri DSM 20243 gene was prematurely terminated by a stop codon. Exchanging the stop codon for a glutamine codon resulted in a recombinant enzyme producing inulin and FOS. The three recombinant fructansucrase enzymes characterized from three different L. gasseri strains have very similar primary protein structures, yet synthesize different fructan products. An interesting feature of the L. gasseri strains is that they were unable to ferment raffinose, whereas their respective recombinant enzymes converted raffinose into fructan and FOS.

  3. Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice

    PubMed Central

    Horta, Fabrizzio; Alzobi, Hamida; Jitanantawittaya, Sutthipat; Catt, Sally; Chen, Penny; Pangestu, Mulyoto; Temple-Smith, Peter

    2017-01-01

    This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling (18% w/v raffinose, RS-C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS-C (n = 86), and FC (n = 79). The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS-C]; P < 0.01). Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection. PMID:27427551

  4. Ion chromatography separation of cotton surface melezitose and raffinose: entomological vs. plant sugars

    USDA-ARS?s Scientific Manuscript database

    According to previous studies, certain levels of the carbohydrates melezitose and trehalulose deposited on the surface of cotton are indicative of either whitefly or aphid contamination, which may cause problems during cotton processing. Obtaining reliable IC values for those surface sugars is para...

  5. Desiccation and freezing tolerance of embryonic axes from Citrus sinensis [L.] osb. pretreated with sucrose.

    PubMed

    Santos, Izulmé R I; Stushnoff, Cecil

    2003-01-01

    Embryonic axes of Citrus sinensis L. were successfully cryopreserved. While fully hydrated unfrozen axes germinated 100%, survival decreased as axes water content dropped, and total loss of viability was observed when the water content dropped to 0.04 and 0.10 mg H2O/mg dry mass, for axes without and with sucrose preculture, respectively. Fully hydrated axes did not survive exposure to liquid nitrogen. Highest seedling recovery (93-100%) for untreated axes was observed at 0.26 to 0.15 mg H2O/mg dry mass. Differential scanning calorimetry revealed the presence of broad melting peaks in fully hydrated embryonic axes. The size of the melting peak diminished as water was removed by desiccation. Minimum melting of water was observed at the point axes survived cryopreservation. Occurrence of a glass transition upon warming was not a condition for axes to survive liquid nitrogen exposure. In untreated axes, glucose, increased with desiccation to 0.2 mg H2O/mg dry mass, and decreased as the axes were desiccated to lower water contents. Fructose and sucrose levels did not increase when untreated samples were desiccated for the same periods of time. Raffinose and stachyose levels decreased as untreated and precultured embryonic axes were desiccated. In sucrose precultured axes, sucrose and fructose levels increased when they were dehydrated, reaching maximum levels at 0.2 mg H2O/mg dry mass. Tissue glucose did not change significantly with desiccation. Raffinose and stachyose levels dropped as precultured embryonic axes were dried.

  6. Mrt, a Gene Unique to Fungi, Encodes an Oligosaccharide Transporter and Facilitates Rhizosphere Competency in Metarhizium robertsii1[C][W

    PubMed Central

    Fang, Weiguo; St. Leger, Raymond J.

    2010-01-01

    The symbiotic associations between rhizospheric fungi and plants have enormous environmental impact. Fungi are crucial to plant health as antagonists of pathogens and herbivores and facilitate the uptake of soil nutrients. However, little is known about the plant products obtained by fungi in exchange or how they are transported through the symbiotic interface. Here, we demonstrate that sucrose and raffinose family oligosaccharides in root exudates are important for rhizosphere competence in the insect pathogen Metarhizium robertsii (formerly known as Metarhizium anisopliae). We identified mutants in the Metarhizium raffinose transporter (Mrt) gene of M. robertsii that grew poorly in root exudate and were greatly reduced in rhizosphere competence on grass roots. Studies on sugar uptake, including competition assays, revealed that MRT was a sucrose and galactoside transporter. Disrupting MRT resulted in greatly reduced or no growth on sucrose and galactosides but did not affect growth on monosaccharides or oligosaccharides composed entirely of glucose subunits. Consistent with this, expression of Mrt is exclusively up-regulated by galactosides and sucrose. Expressing a green fluorescent protein gene under the control of the Mrt promoter confirmed that MRT was expressed by germlings in the vicinity of grass roots but not in surrounding bulk soil. Disrupting Mrt did not reduce virulence to insects, demonstrating that Mrt is exclusively involved in M. robertsii’s interactions with plants. To our knowledge, MRT is the first oligosaccharide transporter identified and characterized in a fungus and is unique to filamentous fungi, but homologous genes in Magnaporthe, Ustilago, Aspergillus, Fusarium, Epichloe, and Penicillium species indicate that oligosaccharide transport is of widespread significance. PMID:20837701

  7. Contributions of fluid convection and electrical migration to transport in cartilage: relevance to loading.

    PubMed

    Garcia, A M; Frank, E H; Grimshaw, P E; Grodzinsky, A J

    1996-09-15

    We have studied the contributions of diffusion, fluid flow and electrical migration to molecular transport through adult articular cartilage explants using neutral and charged solutes that were either radiolabeled (3H2O, [35S]sulfate, [3H]thymidine, [3H]raffinose, and a synthetic matrix metalloproteinase inhibitor) or fluorescently tagged (NSPA and Lissamine-dextran). In order to induce fluid flow within the cartilage matrix without mechanical deformation, electric current densities were applied across cartilage disks. These currents produced electroosmotic fluid velocities of 1-2 microns/s, magnitudes that have been reported to exist during joint loading in vivo. This fluid convection enhanced neutral solute flux relative to passive diffusion alone by a factor that increased with the size of the solute. While the enhancement factor for 3H2O was 2.3-fold, that for [3H]raffinose (594 Da) and similar sized neutral solutes was 10-fold, suggesting that the effect of fluid flow is important even for small solutes. The largest enhancement (25-fold) was seen for the neutral 10-kDa Lissamine-dextran, confirming that fluid convection is most important for large solutes. We also studied the electrophoretic contribution to solute flux, which is relevant to the presence of intratissue streaming potentials induced during loading in vivo. Using the negatively charged [35S]sulfate ion with a range of current densities, as much as a 10-fold enhancement in flux was observed. Values for the intrinsic transport properties of the solutes (e.g., diffusivity, electrical mobility, hydrodynamic hindrance factor) can be obtained from the data.

  8. Metabolite profiling during cold acclimation of Lolium perenne genotypes distinct in the level of frost tolerance.

    PubMed

    Bocian, Aleksandra; Zwierzykowski, Zbigniew; Rapacz, Marcin; Koczyk, Grzegorz; Ciesiołka, Danuta; Kosmala, Arkadiusz

    2015-11-01

    Abiotic stresses, including low temperature, can significantly reduce plant yielding. The knowledge on the molecular basis of stress tolerance could help to improve its level in species of relatively high importance to agriculture. Unfortunately, the complex research performed so far mainly on model species and also, to some extent, on cereals does not fully cover the demands of other agricultural plants of temperate climate, including forage grasses. Two Lolium perenne (perennial ryegrass) genotypes with contrasting levels of frost tolerance, the high frost tolerant (HFT) and the low frost tolerant (LFT) genotypes, were selected for comparative metabolomic research. The work focused on the analysis of leaf metabolite accumulation before and after seven separate time points of cold acclimation. Gas chromatography-mass spectrometry (GC/MS) was used to identify amino acids (alanine, proline, glycine, glutamic and aspartic acid, serine, lysine and asparagine), carbohydrates (fructose, glucose, sucrose, raffinose and trehalose) and their derivatives (mannitol, sorbitol and inositol) accumulated in leaves in low temperature. The observed differences in the level of frost tolerance between the analysed genotypes could be partially due to the time point of cold acclimation at which the accumulation level of crucial metabolite started to increase. In the HFT genotype, earlier accumulation was observed for proline and asparagine. The increased amounts of alanine, glutamic and aspartic acids, and asparagine during cold acclimation could be involved in the regulation of photosynthesis intensity in L. perenne. Among the analysed carbohydrates, only raffinose revealed a significant association with the acclimation process in this species.

  9. Longitudinal development of the gut microbiome and metabolome in preterm neonates with late onset sepsis and healthy controls.

    PubMed

    Stewart, Christopher J; Embleton, Nicholas D; Marrs, Emma C L; Smith, Daniel P; Fofanova, Tatiana; Nelson, Andrew; Skeath, Tom; Perry, John D; Petrosino, Joseph F; Berrington, Janet E; Cummings, Stephen P

    2017-07-12

    Late onset sepsis (LOS) in preterm infants is associated with considerable morbidity and mortality. While studies have implicated gut bacteria in the aetiology of the disease, functional analysis and mechanistic insights are generally lacking. We performed temporal bacterial (n = 613) and metabolomic (n = 63) profiling on extensively sampled stool from 7 infants with LOS and 28 matched healthy (no LOS or NEC) controls. The bacteria isolated in diagnostic blood culture usually corresponded to the dominant bacterial genera in the gut microbiome. Longitudinal changes were monitored based on preterm gut community types (PGCTs), where control infants had an increased number of PGCTs compared to LOS infants (P = 0.011). PGCT 6, characterised by Bifidobacteria dominance, was only present in control infants. Metabolite profiles differed between LOS and control infants at diagnosis and 7 days later, but not 7 days prior to diagnosis. Bifidobacteria was positively correlated with control metabolites, including raffinose, sucrose, and acetic acid. Using multi-omic analysis, we show that the gut microbiome is involved in the pathogenesis of LOS. While the causative agent of LOS varies, it is usually abundant in the gut. Bifidobacteria dominance was associated with control infants, and the presence of this organism may directly protect, or act as a marker for protection, against gut epithelial translocation. While the metabolomic data is preliminary, the findings support that gut development and protection in preterm infants is associated with increased in prebiotic oligosaccharides (e.g. raffinose) and the growth of beneficial bacteria (e.g. Bifidobacterium).

  10. Integrative “omic” analysis reveals distinctive cold responses in leaves and roots of strawberry, Fragaria × ananassa ‘Korona’

    PubMed Central

    Koehler, Gage; Rohloff, Jens; Wilson, Robert C.; Kopka, Joachim; Erban, Alexander; Winge, Per; Bones, Atle M.; Davik, Jahn; Alsheikh, Muath K.; Randall, Stephen K.

    2015-01-01

    To assess underlying metabolic processes and regulatory mechanisms during cold exposure of strawberry, integrative “omic” approaches were applied to Fragaria × ananassa Duch. ‘Korona.’ Both root and leaf tissues were examined for responses to the cold acclimation processes. Levels of metabolites, proteins, and transcripts in tissues from plants grown at 18°C were compared to those following 1–10 days of cold (2°C) exposure. When leaves and roots were subjected to GC/TOF-MS-based metabolite profiling, about 160 compounds comprising mostly structurally annotated primary and secondary metabolites, were found. Overall, ‘Korona’ showed a modest increase of protective metabolites such as amino acids (aspartic acid, leucine, isoleucine, and valine), pentoses, phosphorylated and non-phosphorylated hexoses, and distinct compounds of the raffinose pathway (galactinol and raffinose). Distinctive responses were observed in roots and leaves. By 2DE proteomics a total of 845 spots were observed in leaves; 4.6% changed significantly in response to cold. Twenty-one proteins were identified, many of which were associated with general metabolism or photosynthesis. Transcript levels in leaves were determined by microarray, where dozens of cold associated transcripts were quantitatively characterized, and levels of several potential key contributors (e.g., the dehydrin COR47 and GADb) to cold tolerance were confirmed by qRT-PCR. Cold responses are placed within the existing knowledge base of low temperature-induced changes in plants, allowing an evaluation of the uniqueness or generality of Fragaria responses in photosynthetic tissues. Overall, the cold response characteristics of ‘Korona’ are consistent with a moderately cold tolerant plant. PMID:26528299

  11. System level analysis of cacao seed ripening reveals a sequential interplay of primary and secondary metabolism leading to polyphenol accumulation and preparation of stress resistance.

    PubMed

    Wang, Lei; Nägele, Thomas; Doerfler, Hannes; Fragner, Lena; Chaturvedi, Palak; Nukarinen, Ella; Bellaire, Anke; Huber, Werner; Weiszmann, Jakob; Engelmeier, Doris; Ramsak, Ziva; Gruden, Kristina; Weckwerth, Wolfram

    2016-08-01

    Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti-depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity-dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N-phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N-phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  12. Acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans.

    PubMed

    de Matos, Bruno Mello; Brighenti, Fernanda Lourenção; Do, Thuy; Beighton, David; Koga-Ito, Cristiane Yumi

    2017-06-01

    The aim of this study was to evaluate the acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans. The following strains were tested: Bifidobacterium dentium DSM20436, Parascardovia denticolens DSM10105, and Scardovia inopinata DSM10107. Streptococcus mutans UA159 and Lactobacillus acidophilus ATCC4356 were used as control. Bifidobacteria were studied planktonically as they were not able to form monospecies biofilm, they were grown in biofilms associated with S. mutans. Endogenous polysaccharide reserves of cultures at log phase were depleted. Standardized suspensions of the microorganisms were incubated in growth media supplemented with 10 mM glucose, lactose, raffinose, glucose, or xylitol. S. mutans biofilms were grown on glass cover slips for 24 h to which bifidobacteria were added. After 24 h, the dual-species biofilms were exposed to the same carbon sources, and after 3 h, the pH of spent culture media and concentrations of organic acids were measured. Statistical analyses were carried out using ANOVA and Tukey's test (α = 0.05). A higher pH drop was observed when S. mutans was associated with P. denticolens or S. inopinata, in either planktonic or biofilm cultures, than with S. mutans alone. Bifidobacteria showed a higher pH drop in the presence of raffinose than S. mutans or L. acidophilus. Dual-species biofilms of bifidobacteria and S. mutans produced more acid and greater pH drops than biofilms of S. mutans alone. New insights on the complex process of caries pathogenicity contribute to the establishment of preventive and therapeutic measures, in particular in specific cases, such as in early childhood caries.

  13. Comparison of melibiose utilizing baker's yeast strains produced by genetic engineering and classical breeding.

    PubMed

    Vincent, S F; Bell, P J; Bissinger, P; Nevalainen, K M

    1999-02-01

    Yeast strains currently used in the baking industry cannot fully utilize the trisaccharide raffinose found in beet molasses due to the absence of melibiase (alpha-galactosidase) activity. To overcome this deficiency, the MEL1 gene encoding melibiase enzyme was introduced into baker's yeast by both classical breeding and recombinant DNA technology. Both types of yeast strains were capable of vigorous fermentation in the presence of high levels of sucrose, making them suitable for the rapidly developing Asian markets where high levels of sugar are used in bread manufacture. Melibiase expression appeared to be dosage-dependent, with relatively low expression sufficient for complete melibiose utilization in a model fermentation system.

  14. Antinutritional factors in anasazi and other pinto beans (Phaseolus vulgaris L.).

    PubMed

    Weder, J K; Telek, L; Vozári-Hampe, M; Saini, H S

    1997-01-01

    Antinutritional factors of anasazi bean were compared to traditional pinto bean (Phaseolus vulgaris L.). Anasazi beans contained less (p<0.001) soluble and bound condensed tannins compared to pinto beans. No differences (p>0.05) in stachyose and raffinose content were found between the two bean types; verbascose was not detected at all. Significant (p<0.05) differences in lectin content were observed between anasazi and pinto bean. The lectins of anasazi beans were classified as non toxic and those of the pinto beans as toxic types. No differences (p>0.05) in inhibitor activity against human and bovine trypsin and chymotrypsin were found between the two bean types.

  15. The pH Dependence of Saccharides' Influence on Thermal Denaturation of Two Model Proteins Supports an Excluded Volume Model for Stabilization Generalized to Allow for Intramolecular Electrostatic Interactions*

    PubMed Central

    Beg, Ilyas; Islam, Asimul; Hassan, Md. Imtaiyaz; Ahmad, Faizan

    2017-01-01

    The reversible thermal denaturation of apo α-lactalbumin (α-LA) and lysozyme was measured in the absence and presence of multiple concentrations of each of seven saccharides (glucose, galactose, fructose, sucrose, trehalose, raffinose, and stachyose) at multiple pH values. It was observed that with increasing pH, the absolute stability of α-LA decreased, whereas the stabilizing effect per mole of all saccharides increased, and that the absolute stability of lysozyme increased, whereas the stabilizing effect per mole of all saccharides decreased. All of the data may be accounted for quantitatively by straightforward electrostatic generalization of a previously introduced coarse-grained model for stabilization of proteins by sugars. PMID:27909048

  16. Comparative study of invertases of Streptococcus mutans.

    PubMed

    Tanzer, J M; Brown, A T; McInerney, M F; Woodiel, F N

    1977-04-01

    Sucrase activity was studied in 13 strains of Streptococcus mutans representing the five Bratthall serotypes. Sucrose-adapted cells have sucrase activity in the 37,000 x g-soluble fraction of all strains. The enzyme was identified as invertase (beta-d-fructofuranoside fructohydrolase; EC 3.2.1.26) because it hydrolyzed the beta-fructofuranoside trisaccharide raffinose, giving fructose and melibiose as its products, and because it hydrolyzed the beta-fructofuranoside dissacharide sucrose, giving equimolar glucose and fructose as its products. Invertases of c and e strains exhibit two activity peaks by molecular exclusion chromatography with molecular weights of 45,000 to 50,000 and about 180,000; those of serotypes a, b, and d strains exhibit only a single component of 45,000 to 50,000 molecular weight. The electrophoretic mobility of invertases is different between the serotypes and the same within them. Inorganic orthophosphate (P(i)) has a weak positive effect on the V(max) of invertases of serotypes c and e cells but a strong positive effect on the invertases of serotype b cells; P(i) has a strong positive effect on the apparent K(m) of the invertases of serotype d cells, but has no effect on the V(max); P(i) has a strong positive effect on both the apparent K(m) and V(max) of the invertases of serotype a cells. Thus, the invertases were different between all of the serotypes but similar within the serotypes. These findings support the taxonomic schemes of Coykendall and of Bratthall. It was additionally noted that 37,000 x g-soluble fractions of only serotypes b and c but not serotypes a, d, and e cells have melibiase activity, and it could be deduced that serotype d cells lack an intact raffinose permease system.

  17. Poplar trees reconfigure the transcriptome and metabolome in response to drought in a genotype- and time-of-day-dependent manner.

    PubMed

    Hamanishi, Erin T; Barchet, Genoa L H; Dauwe, Rebecca; Mansfield, Shawn D; Campbell, Malcolm M

    2015-04-21

    Drought has a major impact on tree growth and survival. Understanding tree responses to this stress can have important application in both conservation of forest health, and in production forestry. Trees of the genus Populus provide an excellent opportunity to explore the mechanistic underpinnings of forest tree drought responses, given the growing molecular resources that are available for this taxon. Here, foliar tissue of six water-deficit stressed P. balsamifera genotypes was analysed for variation in the metabolome in response to drought and time of day by using an untargeted metabolite profiling technique, gas chromatography/mass-spectrometry (GC/MS). Significant variation in the metabolome was observed in response the imposition of water-deficit stress. Notably, organic acid intermediates such as succinic and malic acid had lower concentrations in leaves exposed to drought, whereas galactinol and raffinose were found in increased concentrations. A number of metabolites with significant difference in accumulation under water-deficit conditions exhibited intraspecific variation in metabolite accumulation. Large magnitude fold-change accumulation was observed in three of the six genotypes. In order to understand the interaction between the transcriptome and metabolome, an integrated analysis of the drought-responsive transcriptome and the metabolome was performed. One P. balsamifera genotype, AP-1006, demonstrated a lack of congruence between the magnitude of the drought transcriptome response and the magnitude of the metabolome response. More specifically, metabolite profiles in AP-1006 demonstrated the smallest changes in response to water-deficit conditions. Pathway analysis of the transcriptome and metabolome revealed specific genotypic responses with respect to primary sugar accumulation, citric acid metabolism, and raffinose family oligosaccharide biosynthesis. The intraspecific variation in the molecular strategies that underpin the responses to drought

  18. Global analysis of gene expression profiles in physic nut (Jatropha curcas L.) seedlings exposed to drought stress.

    PubMed

    Zhang, Chao; Zhang, Lin; Zhang, Sheng; Zhu, Shuang; Wu, Pingzhi; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2015-01-21

    Physic nut (Jatropha curcas L.) is a small perennial tree or large shrub, which is well-adapted to semi-arid regions and is considered to have potential as a crop for biofuel production. It is now regarded as an excellent model for studying biofuel plants. However, our knowledge about the molecular responses of this species to drought stress is currently limited. In this study, genome-wide transcriptional profiles of roots and leaves of 8-week old physic nut seedlings were analyzed 1, 4 and 7 days after withholding irrigation. We observed a total of 1533 and 2900 differentially expressed genes (DEGs) in roots and leaves, respectively. Gene Ontology analysis showed that the biological processes enriched in droughted plants relative to unstressed plants were related to biosynthesis, transport, nucleobase-containing compounds, and cellular protein modification. The genes found to be up-regulated in roots were related to abscisic acid (ABA) synthesis and ABA signal transduction, and to the synthesis of raffinose. Genes related to ABA signal transduction, and to trehalose and raffinose synthesis, were up-regulated in leaves. Endoplasmic reticulum (ER) stress response genes were significantly up-regulated in leaves under drought stress, while a number of genes related to wax biosynthesis were also up-regulated in leaves. Genes related to unsaturated fatty acid biosynthesis were down-regulated and polyunsaturated fatty acids were significantly reduced in leaves 7 days after withholding irrigation. As drought stress increased, genes related to ethylene synthesis, ethylene signal transduction and chlorophyll degradation were up-regulated, and the chlorophyll content of leaves was significantly reduced by 7 days after withholding irrigation. This study provides us with new insights to increase our understanding of the response mechanisms deployed by physic nut seedlings under drought stress. The genes and pathways identified in this study also provide much information of

  19. Oligosaccharides are a key factor in prediction of amino acid digestibility in soybean meal of different origins when fed to growing pigs

    PubMed Central

    Li, Zhongchao; Wang, Qiuyun; Xie, Fei; Liu, Dewen; Li, Yakui; Lyu, Zhiqian; Lai, Changhua

    2017-01-01

    Objective The objective of this experiment was to determine apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of crude protein (CP) and amino acid (AA) in 15 sources of soybean meal (SBM) produced from soybeans from different countries and subsequently to establish equations for predicting the AID and SID in SBM based on their chemical composition. Methods Eighteen barrows (57.9±6.1 kg) fitted with a simple T-cannula were allotted into three 6×6 Latin square designs. Each period comprised a 6-d adaption period followed by a 2-d collection of ileal digesta. The 15 test diets included SBM as a sole source of AA in the diet. Another nitrogen-free diet was used to measure basal endogenous losses of CP and AA. Chromic oxide (0.3%) was used as an inert marker in each diet. Results The AID of lysine in SBM from China and USA tended to be greater than in SBM from Brazil (p<0.10). The SID of valine and proline in SBM from China was greater than in SBM from Brazil (p<0.05). The SID of lysine, threonine, cysteine and glycine in SBM from China tended to be greater than in SBM from Brazil (p<0.10). From a stepwise regression analysis, a series of AID and SID prediction equations were generated. The best fit equations for lysine in SBM were: AID lysine = 1.16 sucrose−1.81 raffinose+82.10 (R2 = 0.69, p<0.01) and SID lysine = 1.14 sucrose−1.93 raffinose−0.99 ether extract (EE)+85.26 (R2 = 0.77, p<0.01). Conclusion It was concluded that under the conditions of this experiment, the oligosaccharides (such as sucrose and raffinose) can be used to predict the AID and SID of AA in SBM with reasonable accuracy. PMID:28427255

  20. Influence of fungi associated with bananas on nutritional content during storage.

    PubMed

    Odebode, A C; Sanusi, J

    1996-06-01

    Botryodiplodia theobromae, Rhizopus oryzae, Aspergillus niger, A. flavus and Fusarium equiseti were found to be associated with the ripening of bananas and also caused rot during storage. Bananas stored in baskets with ash fire wood ripened 2-3 days earlier than bananas stored in fibre sacks and under constant light. The infected bananas showed a decrease in the quantity of total soluble sugars, protein, lipid, crude fibre, ash, ascorbic acid and mineral elements when compared with the control fruit. Paper chromatographic studies showed the presence of glucose, sucrose, fructose, maltose and raffinose in healthy control fruit, while only sucrose appeared during storage in bananas infected with B. theobromae. The total soluble sugar and crude protein contents increased during ripening.

  1. Isolation of Clostridium absonum and its cultural and biochemical properties.

    PubMed

    Hayase, M; Mitsui, N; Tamai, K; Nakamura, S; Nishida, S

    1974-01-01

    A new procedure for isolation of Clostridium absonum was devised. Sixtyseven strains of C. absonum were isolated from 135 soil samples, but no strain of C. absonum could be found from human fecal samples. The lecithinase, hemolysin, and lethal toxin in the culture filtrates of this species exhibited low avidity for C. perfringens type A antitoxin. The three activities were inseparable by the present method of purification. A reinvestigation of biochemical properties revealed that incomplete suppression of lecithinase reaction by C. perfringens type A antitoxin and no fermentation of raffinose, melibiose, and starch are useful criteria to differentiate C. absonum from C. perfringens, and that positive, although weak, gelatin liquefaction and fermentation of trehalose are useful to differentiate it from C. paraperfringens.

  2. Influence of in ovo prebiotic and synbiotic administration on meat quality of broiler chickens.

    PubMed

    Maiorano, G; Sobolewska, A; Cianciullo, D; Walasik, K; Elminowska-Wenda, G; Slawinska, A; Tavaniello, S; Zylinska, J; Bardowski, J; Bednarczyk, M

    2012-11-01

    A trial was conducted to evaluate the effect of in ovo injection of prebiotic and synbiotics on growth performance, meat quality traits (cholesterol content, intramuscular collagen properties, fiber measurements), and the presence of histopathological changes in the pectoral muscle (PS) of broiler chickens. On d 12 of incubation, 480 eggs were randomly divided into 5 experimental groups treated with different bioactives, in ovo injected: C, control with physiological saline; T1 with 1.9 mg of raffinose family oligosaccharides; T2 and T3 with 1.9 mg of raffinose family oligosaccharides enriched with different probiotic bacteria, specifically 1,000 cfu of Lactococcus lactis ssp. lactis SL1 and Lactococcus lactis ssp. cremoris IBB SC1, respectively; T4 with commercially available synbiotic Duolac, containing 500 cfu of both Lactobacillus acidophilus and Streptococcus faecium with the addition of lactose (0.001 mg/embryo). Among the hatched chickens, 60 males were randomly chosen (12 birds for each group) and were grown to 42 d in collective cages (n = 3 birds in each 4 cages: replications for experimental groups). Broilers were fed ad libitum commercial diets according to age. In ovo prebiotic and synbiotic administration had a low effect on investigated traits, but depend on the kind of bioactives administered. Commercial synbiotic treatment (T4) reduced carcass yield percentage, and the feed conversion ratio was higher in T3 and T4 groups compared with other groups. The abdominal fat, the ultimate pH, and cholesterol of the PS were not affected by treatment. Broiler chickens of the treated groups with both slightly greater PS and fiber diameter had a significantly lower amount of collagen. The greater thickness of muscle fibers (not significant) and the lower fiber density (statistically significant), observed in treated birds in comparison with those of the C group, are not associated with histopathological changes in the PS of broilers. The incidence of

  3. Protective Response Mechanisms to Heat Stress in Interaction with High [CO2] Conditions in Coffea spp.

    PubMed Central

    Martins, Madlles Q.; Rodrigues, Weverton P.; Fortunato, Ana S.; Leitão, António E.; Rodrigues, Ana P.; Pais, Isabel P.; Martins, Lima D.; Silva, Maria J.; Reboredo, Fernando H.; Partelli, Fábio L.; Campostrini, Eliemar; Tomaz, Marcelo A.; Scotti-Campos, Paula; Ribeiro-Barros, Ana I.; Lidon, Fernando J. C.; DaMatta, Fábio M.; Ramalho, José C.

    2016-01-01

    Modeling studies have predicted that coffee crop will be endangered by future global warming, but recent reports highlighted that high [CO2] can mitigate heat impacts on coffee. This work aimed at identifying heat protective mechanisms promoted by CO2 in Coffea arabica (cv. Icatu and IPR108) and Coffea canephora cv. Conilon CL153. Plants were grown at 25/20°C (day/night), under 380 or 700 μL CO2 L−1, and then gradually submitted to 31/25, 37/30, and 42/34°C. Relevant heat tolerance up to 37/30°C for both [CO2] and all coffee genotypes was observed, likely supported by the maintenance or increase of the pools of several protective molecules (neoxanthin, lutein, carotenes, α-tocopherol, HSP70, raffinose), activities of antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT), and the upregulated expression of some genes (ELIP, Chaperonin 20). However, at 42/34°C a tolerance threshold was reached, mostly in the 380-plants and Icatu. Adjustments in raffinose, lutein, β-carotene, α-tocopherol and HSP70 pools, and the upregulated expression of genes related to protective (ELIPS, HSP70, Chape 20, and 60) and antioxidant (CAT, CuSOD2, APX Cyt, APX Chl) proteins were largely driven by temperature. However, enhanced [CO2] maintained higher activities of GR (Icatu) and CAT (Icatu and IPR108), kept (or even increased) the Cu,Zn-SOD, APX, and CAT activities, and promoted a greater upregulation of those enzyme genes, as well as those related to HSP70, ELIPs, Chaperonins in CL153, and Icatu. These changes likely favored the maintenance of reactive oxygen species (ROS) at controlled levels and contributed to mitigate of photosystem II photoinhibition at the highest temperature. Overall, our results highlighted the important role of enhanced [CO2] on the coffee crop acclimation and sustainability under predicted future global warming scenarios. PMID:27446174

  4. Cryopreservation of mouse spermatozoa. I. Effect of seeding on fertilizing ability of cryopreserved spermatozoa.

    PubMed

    Songsasen, N; Leibo, S P

    1997-11-01

    To examine the effect of seeding to induce ice formation during cryopreservation on their survival, spermatozoa from B6D2F1 mice were cooled to and held at -4 degrees C for 30 min in phosphate-buffered saline (PBS) alone, in egg yolk-supplemented PBS, or in PBS with raffinose + glycerol as cryoprotective additives (CPAs). Seeding and holding spermatozoa at -4 degrees C did not affect their viability as judged by vital staining. Egg yolk protected spermatozoa against chilling injury, as cooling them to -4 degrees C in the presence of egg yolk yielded higher survivals than those cooled without egg yolk (34.4 +/- 3.4 v 9.0 +/- 1.3% in three replicates of >400 spermatozoa/replicate). To study effects of seeding on their fertilizing ability, spermatozoa in the raffinose-glycerol-egg yolk solution were frozen to -196 degrees C either without seeding or after seeding at -4 degrees C. Development of 222 oocytes into two-cell embryos after in vitro fertilization (IVF) with spermatozoa frozen without seeding was 43%; development rates of 186, 186, and 207 oocytes after IVF with spermatozoa frozen after seeding and being held at -4 degrees C for 5, 10, or 30 min were 46, 44, and 9%, respectively. In a direct comparison, after IVF with seeded or unseeded spermatozoa the respective cleavage rates into two-cell embryos were 83% of 275 oocytes and 69% of 304 oocytes, a difference that was small but significant by chi2 analysis. An additional 925 oocytes were fertilized with spermatozoa after being seeded and frozen to -196 degrees C in four separate batches of CPA solutions. Overall, after IVF with frozen sperm, 68% of those oocytes cleaved into two-cell embryos and 59% developed into 544 blastocysts. Based on these results, we concluded that embryo production by IVF seemed to be improved using spermatozoa frozen after being seeded. Mouse spermatozoa cryopreserved by the method described here are capable of fertilizing oocytes at a rather high rate. Copyright 1997 Academic Press.

  5. Impact of warming and drought on carbon balance related to wood formation in black spruce.

    PubMed

    Deslauriers, Annie; Beaulieu, Marilène; Balducci, Lorena; Giovannelli, Alessio; Gagnon, Michel J; Rossi, Sergio

    2014-08-01

    Wood formation in trees represents a carbon sink that can be modified in the case of stress. The way carbon metabolism constrains growth during stress periods (high temperature and water deficit) is now under debate. In this study, the amounts of non-structural carbohydrates (NSCs) for xylogenesis in black spruce, Picea mariana, saplings were assessed under high temperature and drought in order to determine the role of sugar mobilization for osmotic purposes and its consequences for secondary growth. Four-year-old saplings of black spruce in a greenhouse were subjected to different thermal conditions with respect to the outside air temperature (T0) in 2010 (2 and 5 °C higher than T0) and 2011 (6 °C warmer than T0 during the day or night) with a dry period of about 1 month in June of each year. Wood formation together with starch, NSCs and leaf parameters (water potential and photosynthesis) were monitored from May to September. With the exception of raffinose, the amounts of soluble sugars were not modified in the cambium even if gas exchange and photosynthesis were greatly reduced during drought. Raffinose increased more than pinitol under a pre-dawn water potential of less than -1 Mpa, presumably because this compound is better suited than polyol for replacing water and capturing free radicals, and its degradation into simple sugar is easier. Warming decreased the starch storage in the xylem as well the available hexose pool in the cambium and the xylem, probably because of an increase in respiration. Radial stem growth was reduced during drought due to the mobilization of NSCs for osmotic purposes and due to the lack of cell turgor. Thus plant water status during wood formation can influence the NSCs available for growth in the cambium and xylem. © The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Cytoplasmic genome substitution in wheat affects the nuclear-cytoplasmic cross-talk leading to transcript and metabolite alterations

    PubMed Central

    2013-01-01

    Background Alloplasmic lines provide a unique tool to study nuclear-cytoplasmic interactions. Three alloplasmic lines, with nuclear genomes from Triticum aestivum and harboring cytoplasm from Aegilops uniaristata, Aegilops tauschii and Hordeum chilense, were investigated by transcript and metabolite profiling to identify the effects of cytoplasmic substitution on nuclear-cytoplasmic signaling mechanisms. Results In combining the wheat nuclear genome with a cytoplasm of H. chilense, 540 genes were significantly altered, whereas 11 and 28 genes were significantly changed in the alloplasmic lines carrying the cytoplasm of Ae. uniaristata or Ae. tauschii, respectively. We identified the RNA maturation-related process as one of the most sensitive to a perturbation of the nuclear-cytoplasmic interaction. Several key components of the ROS chloroplast retrograde signaling, together with the up-regulation of the ROS scavenging system, showed that changes in the chloroplast genome have a direct impact on nuclear-cytoplasmic cross-talk. Remarkably, the H. chilense alloplasmic line down-regulated some genes involved in the determination of cytoplasmic male sterility without expressing the male sterility phenotype. Metabolic profiling showed a comparable response of the central metabolism of the alloplasmic and euplasmic lines to light, while exposing larger metabolite alterations in the H. chilense alloplasmic line as compared with the Aegilops lines, in agreement with the transcriptomic data. Several stress-related metabolites, remarkably raffinose, were altered in content in the H. chilense alloplasmic line when exposed to high light, while amino acids, as well as organic acids were significantly decreased. Alterations in the levels of transcript, related to raffinose, and the photorespiration-related metabolisms were associated with changes in the level of related metabolites. Conclusion The replacement of a wheat cytoplasm with the cytoplasm of a related species affects

  7. Brain Barrier Disruption and Region-Specific Neuronal Degeneration during Necrotizing Enterocolitis in Preterm Pigs.

    PubMed

    Brunse, Anders; Abbaspour, Afrouz; Sangild, Per Torp

    2018-06-06

    Necrotizing enterocolitis (NEC) increases the risk of brain injury and impaired neurodevelopment. Rapid brain maturation prior to birth may explain why preterm brains are particularly vulnerable to serious infections. Using pigs as models, we hypothesized that preterm birth was associated with altered blood-cerebrospinal fluid (CSF) barrier (BCSFB) function and cerebral structural deficits, and that NEC was associated with systemic inflammation, BCSFB disruption, and neuroinflammation. First, cesarean-delivered preterm and term pigs (n = 43-44) were euthanized at birth to investigate BCSFB function and markers of brain structural maturation, or on day 5 to measure markers of blood-brain barrier maturation in the hippocampus and striatum (experiment 1). Next, preterm pigs (n = 162) were fed increasing volumes of infant formula to assess NEC lesions, systemic inflammation, BCSFB permeability, cerebral histopathology, hippocampal micro-glial density, and cytokine levels on day 5 (experiments 2 and 3). In experiment 1, preterm newborns had increased CSF-plasma ratios of albumin and raffinose, reduced CSF glucose levels, as well as increased cerebral hydration and reduced white matter myelination compared with term animals. We observed lower hippocampal (but not striatal) perivascular astrocyte coverage for the first 5 days after preterm birth, accompanied by altered cell junction protein levels. In experiments 2 and- 3, piglets with severe NEC lesions showed reduced blood thrombocytes and increased plasma C-reactive protein and interleukin-6 levels. NEC was associated with increased CSF-plasma albumin and raffinose ratios, reduced CSF leukocyte numbers, and increased cerebral hydration. In the hippocampus, NEC was associated with pyramidal neuron loss and increased interleukin-6 levels. In the short term, NEC did not affect cerebral myelination or microglia density. In conclusion, altered BCSFB properties and brain structural deficits were observed in pigs after

  8. Protective Response Mechanisms to Heat Stress in Interaction with High [CO2] Conditions in Coffea spp.

    PubMed

    Martins, Madlles Q; Rodrigues, Weverton P; Fortunato, Ana S; Leitão, António E; Rodrigues, Ana P; Pais, Isabel P; Martins, Lima D; Silva, Maria J; Reboredo, Fernando H; Partelli, Fábio L; Campostrini, Eliemar; Tomaz, Marcelo A; Scotti-Campos, Paula; Ribeiro-Barros, Ana I; Lidon, Fernando J C; DaMatta, Fábio M; Ramalho, José C

    2016-01-01

    Modeling studies have predicted that coffee crop will be endangered by future global warming, but recent reports highlighted that high [CO2] can mitigate heat impacts on coffee. This work aimed at identifying heat protective mechanisms promoted by CO2 in Coffea arabica (cv. Icatu and IPR108) and Coffea canephora cv. Conilon CL153. Plants were grown at 25/20°C (day/night), under 380 or 700 μL CO2 L(-1), and then gradually submitted to 31/25, 37/30, and 42/34°C. Relevant heat tolerance up to 37/30°C for both [CO2] and all coffee genotypes was observed, likely supported by the maintenance or increase of the pools of several protective molecules (neoxanthin, lutein, carotenes, α-tocopherol, HSP70, raffinose), activities of antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT), and the upregulated expression of some genes (ELIP, Chaperonin 20). However, at 42/34°C a tolerance threshold was reached, mostly in the 380-plants and Icatu. Adjustments in raffinose, lutein, β-carotene, α-tocopherol and HSP70 pools, and the upregulated expression of genes related to protective (ELIPS, HSP70, Chape 20, and 60) and antioxidant (CAT, CuSOD2, APX Cyt, APX Chl) proteins were largely driven by temperature. However, enhanced [CO2] maintained higher activities of GR (Icatu) and CAT (Icatu and IPR108), kept (or even increased) the Cu,Zn-SOD, APX, and CAT activities, and promoted a greater upregulation of those enzyme genes, as well as those related to HSP70, ELIPs, Chaperonins in CL153, and Icatu. These changes likely favored the maintenance of reactive oxygen species (ROS) at controlled levels and contributed to mitigate of photosystem II photoinhibition at the highest temperature. Overall, our results highlighted the important role of enhanced [CO2] on the coffee crop acclimation and sustainability under predicted future global warming scenarios.

  9. Cryopreservation of epididymal sperm collected postmortem in the Tasmanian devil (Sarcophilus harrisii).

    PubMed

    Keeley, T; McGreevy, P D; O'Brien, J K

    2012-07-15

    Effective sperm cryopreservation protocols are limited to a small number of marsupial species. In this study, postmortem gamete rescue (PMGR) epididymal sperm samples from Tasmanian devils (N=34) euthanized due to the fatal Devil Facial Tumor Disease were used to develop long-term sperm storage techniques for the species. Cryoprotectant toxicity associated with equilibration of sperm samples in a TEST yolk diluent (TEST; 189 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 85 mM Trizma base [Tris], 11 mM glucose, 20% vol/vol egg yolk; pH 7.1, and 315.0±5.0 mOsm/kg) with a final concentration of 0.06 M trehalose, or 4%, 10%, and 18% vol/vol of either glycerol or dimethyl sulfoxide (DMSO), was examined over 12 h at 15 °C. Trehalose supplementation resulted in an immediate decline (P<0.05) of total motility. After 12 h, total motility was reduced (P<0.05) in treatments containing 18% glycerol, and 10% and 18% dimethyl sulfoxide. The effects of final glycerol concentration (4% and 10%), glycerol equilibration duration (10 min 1 h, or 3 h) prefreeze, freezing rate and the addition of 0.10 M lactose or a combination of 0.10 M lactose and 0.11 M raffinose were assessed during three experiments on the cryopreservation of postmortem gamete rescue samples in TEST. In all experiments, motility and viability were reduced (P<0.01 postthaw). Samples cryopreserved in TEST supplemented with lactose or lactose with raffinose using a fast freezing rate (-8 °C/min from 4 to -40 °C, then -65 °C/min until -165 °C) produced the highest (P<0.05) postthaw motility (18.6±5.5% and 16.9±8.5%, respectively), which represented 35% to 48% retention of prefreeze motility. These results apparently were the best postthaw results of dasyurid sperm reported to date and will help lay the foundations for developing assisted reproductive technologies for marsupial species. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Assessment of Maillard reaction evolution, prebiotic carbohydrates, antioxidant activity and α-amylase inhibition in pulse flours.

    PubMed

    Moussou, Nadia; Corzo-Martínez, Marta; Sanz, María Luz; Zaidi, Farid; Montilla, Antonia; Villamiel, Mar

    2017-03-01

    In this paper, the quality of bean, chickpea, fava beans, lentil and pea flours from Algeria has been evaluated. Maillard reaction (MR) indicators, modifications in the carbohydrate and protein fractions, antioxidant activity and α-amylase inhibitor of raw, toasted and stored samples were evaluated. Fava beans, beans and peas showed higher content of raffinose family oligosaccharides while chickpeas and lentils showed higher polyol content. Toasting and storage caused slightly change in pulse quality; MR showed slight losses of lysine but increased antioxidant activity. Moreover, inhibition of α-amylase was slightly augmented during processing; this could increase the undigested carbohydrates that reach the colon, modulating the glycemic response. These results point out the suitability of these flours for preparing high-quality foodstuffs intended for a wide spectrum of the population, including hyperglycemic and gluten intolerant individuals.

  11. Actinomyces hominis sp. nov., isolated from a wound swab.

    PubMed

    Funke, Guido; Englert, Ralf; Frodl, Reinhard; Bernard, Kathryn A; Stenger, Steffen

    2010-07-01

    A coryneform bacterium (strain 1094(T)) was isolated from a wound swab taken from an 89-year-old female patient. Chemotaxonomic investigations suggested that this bacterium was related to the genera Actinomyces, Arcanobacterium and Actinobaculum. Phylogenetic analysis of 16S rRNA gene sequences showed that strain 1094(T) was most closely related to Actinomyces europaeus CCUG 32789 A(T) (94.3 % similarity). Phenotypically, the isolate could be separated from its closest phylogenetic neighbours on the basis of being positive for catalase, CAMP reaction, acid phosphatase, N-acetyl-beta-glucosaminidase and raffinose fermentation. Based on the data presented, it is proposed that strain 1094(T) should be classified in a novel species, Actinomyces hominis sp. nov. The type strain is 1094(T) (=CCUG 57540(T) =DSM 22168(T)).

  12. Determination of soluble sugar profile in rice.

    PubMed

    Hu, Xianqiao; Fang, Changyun; Lu, Lin; Hu, Zhanqiang; Shao, Yafang; Zhu, Zhiwei

    2017-07-15

    Soluble sugars in rice are the main components affecting sweetness taste of rice. In this paper, an accurate, precise and rapid method for simultaneous determination of multi soluble sugars in rice by using ion chromatography equipped with pulsed amperometric detector was presented. Pretreatment and parameters of ion chromatography and pulsed amperometric detector were optimized. Regression coefficients (R) of 0.9998, 1.0000, 0.9979, 0.9998 and 0.9998 were obtained for glucose, fructose, sucrose, raffinose and maltose, respectively. The recovery ranges of five sugars were 92.9-112.0% for milled rice matrix. Repeatability and reproducibility of the method were 0.8-9.7% and 1.9-7.6%, respectively. Method LODs of 3.1-34.6μgg -1 were obtained for soluble sugars in milled rice matrix. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Effect of a rice bran fiber diet on serum glucose levels of diabetic patients in Brazil.

    PubMed

    Rodrigues Silva, Cecilia; Dutra de Oliveira, José Eduardo; de Souza, Rui Augusto Hudari Gonçalves; Silva, Hugo Candido

    2005-03-01

    Eleven diabetic patients: 5, type 1 and 6, type 2 received a low-fiber diet (I) during 1 week and during the next 7 days the same diet, enriched with 40 g of fiber (30.6% insoluble and 11.7% soluble components) from rice bran (II) per day. Results showed that mean fasting and postprandial serum glucose levels were reduced, but values of high fiber diet were significantly lower (p < 0.001) than that of the lower fiber diet. For all patients, the high-fiber diet increased fecal weight. This increase was due to the fiber excreted, rather than water retained. There was no relationship between the increase in fiber intake and its fecal excretion. Sucrose and raffinose were found in the bran, but not in the feces. Lactose was present in the stools of the patients receiving enriched diet.

  14. Sugarbeet as a renewable resource

    SciTech Connect

    Edye, L.A.; Clarke, M.A.

    1995-12-01

    Sugarbeet (Beta vulgaris) is produced annually on the order of 400 million tonnes, in temperate climates. The primary product is sugar (sucrose); other products include feeds (molasses and beet pulp), and raffinose, pectin and arabinan. Recently, production of paper from sugarbeet pulp has begun. A wide range of non-food products is available through microbial and chemical reactions on sugarbeet juices, molasses and sugars. Products of microbial processes (chemical transformations are discussed in the companion presentation on sugarcane) include polymers to use as biodegradable plastics (pullulans, polyhydroxyalkanoates, polylactide) and others for food and non food use (levan, dextran). Basic chemicals, includingmore » citric acid and lactic acid, and amino acids, notably lysine, are produced from sugarbeet sources. The production of ethanol, as fuel or as beverage, is well known. Products and processes are outlined, and recent developments are emphasized.« less

  15. STREPTOCOCCI OCCURRING IN SOUR MILK

    PubMed Central

    Jones, F. S.

    1921-01-01

    A well defined group of rod-like and coccoid organisms arranged in pairs and chains has been encountered in sour milk. The group comprises at least three species; the largest number ferment dextrose, lactose, maltose, mannitol, and salicin, and fail to ferment saccharose, raffinose, and inulin. A smaller number ferment saccharose in addition to dextrose, lactose, maltose, mannitol, and salicin. A few fail to attack mannitol. All three types grow luxuriantly at room temperature, coagulate milk, reduce litmus, and produce large amounts of acid in fermented bouillon containing dextrose. Specific morphological and cultural differences exist between the lactic acid streptococci and those associated with mastitis and those occurring in the udder. The lactic acid organisms outgrow the udder streptococci in the milk-souring process. When both types are implanted in sterile milk the udder type soon disappears. PMID:19868477

  16. Experimental evidence for excess entropy discontinuities in glass-forming solutions.

    PubMed

    Lienhard, Daniel M; Zobrist, Bernhard; Zuend, Andreas; Krieger, Ulrich K; Peter, Thomas

    2012-02-21

    Glass transition temperatures T(g) are investigated in aqueous binary and multi-component solutions consisting of citric acid, calcium nitrate (Ca(NO(3))(2)), malonic acid, raffinose, and ammonium bisulfate (NH(4)HSO(4)) using a differential scanning calorimeter. Based on measured glass transition temperatures of binary aqueous mixtures and fitted binary coefficients, the T(g) of multi-component systems can be predicted using mixing rules. However, the experimentally observed T(g) in multi-component solutions show considerable deviations from two theoretical approaches considered. The deviations from these predictions are explained in terms of the molar excess mixing entropy difference between the supercooled liquid and glassy state at T(g). The multi-component mixtures involve contributions to these excess mixing entropies that the mixing rules do not take into account. © 2012 American Institute of Physics

  17. Study of the principal constituents of tropical angico (Anadenanthera sp.) honey from the atlantic forest.

    PubMed

    Santos, A; Moreira, R F A; De Maria, C A B

    2015-03-15

    Free proline was significantly (p<0.05) lower compared to that of other honeys from the atlantic forest, caatinga and cerrado biomes. Honeys from the atlantic forest and cerrado had a significantly (p<0.05) lower HMF than angico. Fructose and glucose in angico honeys were significantly (p<0.05) higher than those from caatinga. Mean values for turanose, nigerose, sucrose, isomaltose, maltotriose, panose and raffinose in angico were significantly (p<0.05) different from honeys from the atlantic forest and caatinga. Use of cluster analysis permitted the three kinds of honey to be grouped independently. Angico was closest to caatinga honey, but both were significantly (p<0.05) different from other atlantic forest honey. GC/SNIFFING showed that linalool oxide, 2-ethyl hexanol, phenylethanol, and phenylacetic acid may be important contributors to the flavour of angico honey. Published by Elsevier Ltd.

  18. Partial Purification and Properties of an Alkaline α-Galactosidase from Mature Leaves of Cucurbita pepo1

    PubMed Central

    Gaudreault, Pierre-Richard; Webb, John A.

    1983-01-01

    A fourth molecular from of α-galactosidase, designated LIV, an alkaline α-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-α-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. Km determinations in McIlvaine buffer (200 millimolar Na2-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-α-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. LIV was partially inhibited by Ca2+, Mg2+, and Mn2+, more so by Ni2+, Zn2+, and Co2+, and highly so by Cu2+, Ag2+, Hg2+ and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-α-d-galactoside or by myo-inositol, but α-d-galactose was a strong inhibitor. As observed for most other forms of α-galactosidase, LIV only catalyzed the hydrolysis of glycosides possessing the α-d-galactose configuration at C1, C2, and C4, and did not hydrolyze p-nitrophenyl-α-d-fucoside (α-d-galactose substituted at C6). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-α-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of LIV in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of LIVin vivo. It was possible that the

  19. Molecular characterization and analysis of the acrB gene of Aspergillus nidulans: a gene identified by genetic interaction as a component of the regulatory network that includes the CreB deubiquitination enzyme.

    PubMed Central

    Boase, Natasha A; Lockington, Robin A; Adams, Julian R J; Rodbourn, Louise; Kelly, Joan M

    2003-01-01

    Mutations in the acrB gene, which were originally selected through their resistance to acriflavine, also result in reduced growth on a range of sole carbon sources, including fructose, cellobiose, raffinose, and starch, and reduced utilization of omega-amino acids, including GABA and beta-alanine, as sole carbon and nitrogen sources. The acrB2 mutation suppresses the phenotypic effects of mutations in the creB gene that encodes a regulatory deubiquitinating enzyme, and in the creC gene that encodes a WD40-repeat-containing protein. Thus AcrB interacts with a regulatory network controlling carbon source utilization that involves ubiquitination and deubiquitination. The acrB gene was cloned and physically analyzed, and it encodes a novel protein that contains three putative transmembrane domains and a coiled-coil region. AcrB may play a role in the ubiquitination aspect of this regulatory network. PMID:12750323

  20. Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection

    PubMed Central

    Theriot, Casey M.; Koenigsknecht, Mark J.; Carlson, Paul E.; Hatton, Gabrielle E.; Nelson, Adam M.; Li, Bo; Huffnagle, Gary B.; Li, Jun; Young, Vincent B.

    2014-01-01

    Antibiotics can have significant and long lasting effects on the gastrointestinal tract microbiota, reducing colonization resistance against pathogens including Clostridium difficile. Here we show that antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome of mice susceptible to C. difficile infection. Levels of secondary bile acids, glucose, free fatty acids, and dipeptides decrease, whereas those of primary bile acids and sugar alcohols increase, reflecting the modified metabolic activity of the altered gut microbiome. In vitro and ex vivo analyses demonstrate that C. difficile can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including primary bile acid taurocholate for germination, and carbon sources mannitol, fructose, sorbitol, raffinose and stachyose for growth. Our results indicate that antibiotic-mediated alteration of the gut microbiome converts the global metabolic profile to one that favors C. difficile germination and growth. PMID:24445449

  1. Effects of extrusion cooking on the chemical composition and functional properties of dry common bean powders.

    PubMed

    Ai, Yongfeng; Cichy, Karen A; Harte, Janice B; Kelly, James D; Ng, Perry K W

    2016-11-15

    The impact of extrusion cooking on the chemical composition and functional properties of bean powders from four common bean varieties was investigated. The raw bean powders were extruded under eight different conditions, and the extrudates were then dried and ground (particle size⩽0.5mm). Compared with corresponding non-extruded (raw) bean powders (particle size⩽0.5mm), the extrusion treatments did not substantially change the protein and starch contents of the bean powders and showed inconsistent effects on the sucrose, raffinose and stachyose contents. The extrusion cooking did cause complete starch gelatinization and protein denaturation of the bean powders and thus changed their pasting properties and solvent-retention capacities. The starch digestibilities of the cooked non-extruded and cooked extruded bean powders were comparable. The extruded bean powders displayed functional properties similar to those of two commercial bean powders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Cryptococcus socialis sp. nov. and Cryptococcus consortionis sp. nov., Antarctic basidioblastomycetes

    NASA Technical Reports Server (NTRS)

    Vishniac, H. S.

    1985-01-01

    New yeasts from the Ross Desert (dry valley area) of Antarctica include Cryptococcus socialis sp. nov. and Cryptococcus consortionis sp. nov. Cryptococcus socialis MYSW A801-3aY1 (= ATCC 56685) requires no vitamins, assimilates L-arabinose, cellobiose, D-glucuronate, maltose, melezitose, raffinose, soluble starch, sucrose, and trehalose, and may be distinguished from all other basidioblastomycetes by the combination of amylose production, cellobiose assimilation, and failure to utilize nitrate, D-galactose, myo-inositol, and mannitol. Its guanine-plus-cytosine content is 56 mol%. Cryptococcus consortionis MYSW A801-3aY92 (= ATCC 56686) requires thiamine, assimilates L-arabinose, D-glucuronate, 2-ketogluconate, salicin, succinate, sucrose, trehalose, and D-xylose, and may be distinguished from all other basidioblastomycetes by the combination of amylose production and failure to utilize nitrate, cellobiose, D-galactose, myo-inositol, and mannitol. Its guanine-plus-cytosine content is 56 mol%.

  3. Comparison of the thermal stabilization of proteins by oligosaccharides and monosaccharide mixtures: Measurement and analysis in the context of excluded volume theory.

    PubMed

    Beg, Ilyas; Minton, Allen P; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2018-06-01

    The thermal stability of apo α-lactalbumin (α-LA) and lysozyme was measured in the presence of mixtures of glucose, fructose, and galactose. Mixtures of these monosaccharides in the appropriate stoichiometric ratio were found to have a greater stabilizing effect on each of the two proteins than equal weight/volume concentrations of di- tri- and tetrasaccharides with identical subunit composition (sucrose, trehalose, raffinose, and stachyose). The excluded volume model for the effect of a single saccharide on the stability of a protein previously proposed by Beg et al. [Biochemistry 54 (2015) 3594] was extended to treat the case of saccharide mixtures. The extended model predicts quantitatively the stabilizing effect of all monosaccharide mixtures on α-LA and lysozyme reported here, as well as previously published results obtained for ribonuclease A [Biophys. Chem. 138 (2008) 120] to within experimental uncertainty. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Environmental stability of seed carbohydrate profiles in soybeans containing different alleles of the raffinose synthase 2 (RS2) gene

    USDA-ARS?s Scientific Manuscript database

    Soybean [Glycine max (L.) Merr] is an important crop because of the vegetable oil used for human consumption and the high protein meal used mainly for livestock feed formulations. For the highest quality soybean meal, the content of protein as well as the level of carbohydrates contributing positiv...

  5. O-Raffinose Crosslinking Substantially Ameliorates the Vasoconstrictive and Nitric-Oxide-Inactivating Effects of Unmodified Human Hemoglobin in the Rat

    DTIC Science & Technology

    1997-07-11

    severity of the increase in MAP and SVR is accounted for by the profound depressant effect of Ao SFH on cardie output (Table III). We have not examined...1985. N- acetylcysteine potentiates inhibition of platelet aggregation by nitroglycerin. / Clin Invest 76:703-708. 34. Mendelsohn, M., S. O’Neill...D. George, and J. Loscalzo. 1990. Inhibition of fibrinogen binding to human platelets by S-nitroso-N- acetylcysteine . / Biol Chem 265:19028-19034. 27

  6. Development by extrusion of soyabari snack sticks: a nutritionally improved soya-maize product based on the Nigerian snack (kokoro).

    PubMed

    Omueti, O; Morton, I D

    1996-01-01

    A nutritionally improved local snack compared to existing kokoro has been developed by extrusion cooking of different formulations of maize, soybean and condiments such as pepper, onion, salt, palm oil, plantain and banana. The improved snack was named as the 'soyabari snack stick'. The chemical composition of representative extruded products indicates a high level of crude protein, fat, energy, available lysine and improved in vitro digestibility compared to the usual maize-based products. The level of stachyose and raffinose were greatly reduced in the extruded products compared to raw soya. Formulations using various additives yielded products suitable for different consumers' preferences such as hot, sweet, bland, gritty or crispy and acceptable to taste assessors. Soyabari snack sticks were equally acceptable as Bombay mix, a product on the market in London. Sensory analysis showed no significant differences in the two products but the crude fibre content of Bombay mix was higher while the protein was slightly lower than for soyabari sticks. Local ingredients can produce acceptable extrudates.

  7. Novel fiber-rich lentil flours as snack-type functional foods: an extrusion cooking effect on bioactive compounds.

    PubMed

    Morales, P; Berrios, J De J; Varela, A; Burbano, C; Cuadrado, C; Muzquiz, M; Pedrosa, M M

    2015-09-01

    Novel snack-type functional foods based on extruded lentil flours could convey the related health benefit of their bioactive compounds, provide a gluten-free alternative to consumers, and potentially increase the consumption of pulses. Extrusion treatment promoted an increase in galactopinitol, ciceritol, raffinose, stachyose and total α-galactoside content, in most lentil flours. As α-galactosides may act as prebiotics, they could convey beneficial effects to human and monogastric animals. Conversely, extrusion significantly (p < 0.05) reduced the inositol hexaphosphate content to less phosphorylated phytates (inositol pentaphosphate and inositol tetraphosphate), which provide health effects. The gluten-free formulation (control formulation #3) presented the highest significant (p < 0.05) drop in the inositol hexaphosphate of 14.7-fold decrease, but had a large increase in inositol pentaphosphate, due to extrusion processing. These two results are desirable in the finished product. Extrusion also caused a significant (p < 0.05) reduction in the trypsin content and completely inactivated lectin, in all processed samples.

  8. MsmK, an ATPase, Contributes to Utilization of Multiple Carbohydrates and Host Colonization of Streptococcus suis.

    PubMed

    Tan, Mei-Fang; Gao, Ting; Liu, Wan-Quan; Zhang, Chun-Yan; Yang, Xi; Zhu, Jia-Wen; Teng, Mu-Ye; Li, Lu; Zhou, Rui

    2015-01-01

    Acquisition and metabolism of carbohydrates are essential for host colonization and pathogenesis of bacterial pathogens. Different bacteria can uptake different lines of carbohydrates via ABC transporters, in which ATPase subunits energize the transport though ATP hydrolysis. Some ABC transporters possess their own ATPases, while some share a common ATPase. Here we identified MsmK, an ATPase from Streptococcus suis, an emerging zoonotic bacterium causing dead infections in pigs and humans. Genetic and biochemistry studies revealed that the MsmK was responsible for the utilization of raffinose, melibiose, maltotetraose, glycogen and maltotriose. In infected mice, the msmK-deletion mutant showed significant defects of survival and colonization when compared with its parental and complementary strains. Taken together, MsmK is an ATPase that contributes to multiple carbohydrates utilization and host colonization of S. suis. This study gives new insight into our understanding of the carbohydrates utilization and its relationship to the pathogenesis of this zoonotic pathogen.

  9. Deep freezing of Angora goat semen: effects of diluent composition and method and rate of dilution on survival of spermatozoa.

    PubMed

    Salamon, S; Ritar, A J

    1982-01-01

    Five factorial experiments were conducted to examine the effects of concentration of tris(hydroxymethyl)aminomethane (Tris), type and concentration of sugar in the diluent, rate and method of dilution on the survival of goat spermatozoa after freezing by the pellet method. Spermatozoa tolerated a relatively wide range in concentration of Tris, but the cell survival depended on the type of sugar included in the Tris diluent. Glucose and fructose were more suitable components than lactose or raffinose. Survival of spermatozoa after thawing was better for three- to fivefold prefreezing dilution. There was interaction between method of semen dilution (one-step, two-step), holding time at 5 degrees C, and glycerol concentration. The best result was obtained after one-step dilution at 30 degrees C (Tris 375 mM-glucose 41.625 mM-citric acid 124 mM), 1.5 h holding at 5 degrees C, and with 4% (v/v) glycerol concentration in the diluted semen.

  10. Reduction of soybean meal non-starch polysaccharides and α-galactosides by solid-state fermentation using cellulolytic bacteria obtained from different environments.

    PubMed

    Opazo, Rafael; Ortúzar, Felipe; Navarrete, Paola; Espejo, Romilio; Romero, Jaime

    2012-01-01

    Soybean meal (SBM) is an important protein source in animal feed. However, the levels of SBM inclusion are restricted in some animal species by the presence of antinutritional factors (ANFs), including non-starch polysaccharides (NSPs) and α-galactosides (GOSs). The aim of this study was to reduce the soybean meal NSPs and GOSs by solid-state fermentation (SSF) using a combination of cellulolytic bacteria isolated from different environments (termites, earthworms, corn silage and bovine ruminal content). To analyse the key enzymatic activities, the isolates were grown in minimal media containing NSPs extracted from SBM. The selected bacterial strains belonged to the genera Streptomyces, Cohnella and Cellulosimicrobium. SSF resulted in a reduction of nearly 24% in the total NSPs, 83% of stachyose and 69% of raffinose and an increase in the protein content. These results suggest that cellulolytic bacteria-based SSF processing facilitates SBM nutritional improvement. In addition, the use of fermented SBM in animal diets can be recommended.

  11. Establishing the relative importance of damaged starch and fructan as sources of fermentable sugars in wheat flour and whole meal bread dough fermentations.

    PubMed

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

    2017-03-01

    It is generally believed that maltose drives yeast-mediated bread dough fermentation. The relative importance of fructose and glucose, released from wheat fructan and sucrose by invertase, compared to maltose is, however, not documented. This is surprising given the preference of yeast for glucose and fructose over maltose. This study revealed that, after 2h fermentation of wheat flour dough, about 44% of the sugars consumed were generated by invertase-mediated degradation of fructan, raffinose and sucrose. The other 56% were generated by amylases. In whole meal dough, 70% of the sugars consumed were released by invertase activity. Invertase-mediated sugar release seems to be crucial during the first hour of fermentation, while amylase-mediated sugar release was predominant in the later stages of fermentation, which explains why higher amylolytic activity prolonged the productive fermentation time only. These results illustrate the importance of wheat fructan and sucrose content and their degradation for dough fermentations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Influence of cold stress on contents of soluble sugars, vitamin C and free amino acids including gamma-aminobutyric acid (GABA) in spinach (Spinacia oleracea).

    PubMed

    Yoon, Young-Eun; Kuppusamy, Saranya; Cho, Kye Man; Kim, Pil Joo; Kwack, Yong-Bum; Lee, Yong Bok

    2017-01-15

    The contents of soluble sugars (sucrose, fructose, glucose, maltose and raffinose), vitamin C and free amino acids (34 compounds, essential and non-essential) were quantified in open-field and greenhouse-grown spinaches in response to cold stress using liquid chromatography. In general, greenhouse cultivation produced nutritionally high value spinach in a shorter growing period, where the soluble sugars, vitamin C and total amino acids concentrations, including essential were in larger amounts compared to those grown in open-field scenarios. Further, low temperature exposure of spinach during a shorter growth period resulted in the production of spinach with high sucrose, ascorbate, proline, gamma-aminobutyric acid, valine and leucine content, and these constitute the most important energy/nutrient sources. In conclusion, cultivation of spinach in greenhouse at a low temperature (4-7°C) and exposure for a shorter period (7-21days) before harvest is recommended. This strategy will produce a high quality product that people can eat. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Effects of Organic and Conventional Crop Nutrition on Profiles of Polar Metabolites in Grain of Wheat.

    PubMed

    Shewry, Peter; Rakszegi, Marianna; Lovegrove, Alison; Amos, Dominic; Corol, Delia-Irina; Tawfike, Ahmed; Mikó, Péter; Ward, Jane L

    2018-05-30

    The profiles of polar metabolites were determined in wholemeal flours of grain from the Broadbalk wheat experiment and from plants grown under organic and low-input systems to study the effects of nutrition on composition. The Broadbalk samples showed increased amino acids, acetate, and choline and decreased fructose and succinate with increasing nitrogen fertilization. Samples receiving farm yard manure had similar grain nitrogen to those receiving 96 kg of N/ha but had higher contents of amino acids, sugars, and organic acids. A comparison of the profiles of grain from organic and low-input systems showed only partial separation, with clear effects of climate and agronomy. However, supervised multivariate analysis showed that the low-input samples had higher contents of many amino acids, raffinose, glucose, organic acids, and choline and lower sucrose, fructose, and glycine. Consequently, although differences between organic and conventional grain occur, these cannot be used to confirm sample identity.

  14. Dehydration Preparation of Mouse Sperm for Vitrification and Rapid Laser Warming.

    PubMed

    Paredes, E; Mazur, P

    Mice are fundamental models of study due to their ease of breeding, manipulation, and the well-studied genome. There has been extensive research focused on the cryopreservation of mouse germaplasm, as a way to help maintain the different transgenic mouse breeds. The first protocols for mouse sperm were developed in the 90's using slow cooling and a mixture of raffinose and glycerol. Since then, the rate of success reported remains highly variable. The Aim of this work is to study factors that are key for developing vitrification protocols for ultra-rapid laser warming of mouse sperm. Our results show that due to the exquisite sensitivity of sperm cells to osmotic excursions, our target levels of dehydration (~85% water content) cannot be achieved without causing a significant decrease in sperm motility and membrane fusion. It seems likely that mouse sperm vitrification is going to be difficult to develop due to the exquisite sensitivity of mouse sperm cells to handling and dehydration.

  15. Condensation Kinetics of Water on Amorphous Aerosol Particles.

    PubMed

    Rothfuss, Nicholas E; Marsh, Aleksandra; Rovelli, Grazia; Petters, Markus D; Reid, Jonathan P

    2018-06-25

    Responding to changes in the surrounding environment, aerosol particles can grow by water condensation changing rapidly in composition and changing dramatically in viscosity. The timescale for growth is important to establish for particles undergoing hydration processes in the atmosphere or during inhalation. Using an electrodynamic balance, we report direct measurements at -7.5, 0, and 20 °C of timescales for hygroscopic condensational growth on a range of model hygroscopic aerosol systems. These extend from viscous aerosol particles containing a single saccharide solute (sucrose, glucose, raffinose, or trehalose) and a starting viscosity equivalent to a glass of ∼10 12 Pa·s, to nonviscous (∼10 -2 Pa·s) tetraethylene glycol particles. The condensation timescales observed in this work indicate that water condensation occurs rapidly at all temperatures examined (<10 s) and for particles of all initial viscosities spanning 10 -2 to 10 12 Pa·s. Only a marginal delay (<1 order of magnitude) is observed for particles starting as a glass.

  16. Acid-catalyzed conversion of mono- and poly-sugars into platform chemicals: effects of molecular structure of sugar substrate.

    PubMed

    Hu, Xun; Wu, Liping; Wang, Yi; Song, Yao; Mourant, Daniel; Gunawan, Richard; Gholizadeh, Mortaza; Li, Chun-Zhu

    2013-04-01

    Hydrolysis/pyrolysis of lignocellulosic biomass always produces a mixture of sugars with distinct structures as intermediates or products. This study tried to elucidate the effects of molecular structure of sugars on their acid-catalyzed conversions in ethanol/water. Location of carbonyl group in sugars (fructose versus glucose) and steric configuration of hydroxyl groups (glucose versus galactose) significantly affected yields of levulinic acid/ester (fructose>glucose>galactose). The dehydration of fructose to 5-(hydroxymethyl)furfural produces much less soluble polymer than that from glucose and galactose, which results in high yields of levulinic acid/ester from fructose. Anhydrate sugar such as levoglucosan tends to undergo the undesirable decomposition to form less levulinic acid/ester. Catalytic behaviors of the poly-sugars (sucrose, maltose, raffinose, β-cyclodextrins) were determined much by their basic units. However, their big molecular sizes create the steric hindrance that significantly affects their followed conversion over solid acid catalyst. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. MsmK, an ATPase, Contributes to Utilization of Multiple Carbohydrates and Host Colonization of Streptococcus suis

    PubMed Central

    Tan, Mei-Fang; Gao, Ting; Liu, Wan-Quan; Zhang, Chun-Yan; Yang, Xi; Zhu, Jia-Wen; Teng, Mu-Ye; Li, Lu; Zhou, Rui

    2015-01-01

    Acquisition and metabolism of carbohydrates are essential for host colonization and pathogenesis of bacterial pathogens. Different bacteria can uptake different lines of carbohydrates via ABC transporters, in which ATPase subunits energize the transport though ATP hydrolysis. Some ABC transporters possess their own ATPases, while some share a common ATPase. Here we identified MsmK, an ATPase from Streptococcus suis, an emerging zoonotic bacterium causing dead infections in pigs and humans. Genetic and biochemistry studies revealed that the MsmK was responsible for the utilization of raffinose, melibiose, maltotetraose, glycogen and maltotriose. In infected mice, the msmK-deletion mutant showed significant defects of survival and colonization when compared with its parental and complementary strains. Taken together, MsmK is an ATPase that contributes to multiple carbohydrates utilization and host colonization of S. suis. This study gives new insight into our understanding of the carbohydrates utilization and its relationship to the pathogenesis of this zoonotic pathogen. PMID:26222651

  18. Isolation and properties of an endo-β-mannanase-producing Bacillus sp. LX114 capable of degrading guar gum.

    PubMed

    Jiang, Baohang; Sun, Zhen; Hou, Yingmin; Yang, Lan; Yang, Fan; Chen, Xiaoyi; Li, Xianzhen

    2016-07-03

    Endo-β-mannanase, catalyzing the random hydrolysis of β-1,4-mannosidic linkage in the backbone of (hetero) mannan, can increase feed conversion efficiency of animal feed or form functional mannanooligosaccharides. In this study, a gram-positive, straight-rod, facultative anaeorobic bacterium producing endo-β-mannanase was isolated from soil sample. The isolate only fermented glucose, galactose, sorbose, and raffinose to acid. The test in hydrogen sulfide production was positive. Combining the data acquired from phenotypic analysis and phylogenetic analysis based on 16S rRNA gene sequences, this strain presumably represented a novel species of the genus Bacillus and was designated as LX114. The strain LX114 could break down guar gum molecules, leading to a rapid decrease of the viscosity of guar gum solutions. Endo-β-mannanase activity was also detected in the culture supernatant. The isolate LX114 would be useful for potential application in degrading plant cell walls for increasing feed conversion efficiency and formation of functional oligosaccharides.

  19. Glycosidases induced in Aspergillus tamarii. Mycelial alpha-D-galactosidases.

    PubMed Central

    Civas, A; Eberhard, R; Le Dizet, P; Petek, F

    1984-01-01

    Two alpha-D-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) produced by Aspergillus tamarii were purified from the mycelial extract by a procedure including chromatography on hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose. Each of these enzymes showed a single protein band corresponding to the alpha-D-galactosidase activity when examined by polyacrylamide-gel electrophoresis. They catalysed the hydrolysis of o-nitrophenyl alpha-D-galactoside, melibiose, raffinose and stachyose, but did not attack the galactomannans. Their Mr values were respectively 265000 +/- 5000 and 254000 +/- 5000 by the method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate in each case showed a single protein band, with Mr 88000 and 77500 respectively. The purified enzymes contained carbohydrate, consisting of N-acetylglucosamine, mannose, glucose and galactose in the estimated molar proportions of 1:9:5:8 in alpha-galactosidase I. Images Fig. 1. PMID:6331398

  20. Candida xylanilytica sp. nov., a xylan-degrading yeast species isolated from Thailand.

    PubMed

    Boonmak, Chanita; Limtong, Savitree; Jindamorakot, Sasitorn; Am-In, Somjit; Yongmanitchai, Wichien; Suzuki, Ken-ichiro; Nakase, Takashi; Kawasaki, Hiroko

    2011-05-01

    Xylan is a major component of hemicellulose, which constitutes about 40 % of plant biomass. Hydrolysis of xylan into simple sugars is one of the important steps in the conversion of lignocellulosic material to value-added products. During an investigation of cellulose- and xylan-degrading yeasts, two yeast strains that were able to use cellulose and xylan as sole carbon source were found to represent a phylogenetically distinct species in the Spathaspora clade. The closest species in terms of pairwise sequence similarity in the D1/D2 domain of the LSU rRNA gene was Candida subhashii. The novel species can be distinguished from the other species in the Spathaspora clade based on the ability to assimilate methanol and raffinose, growth in medium containing 60 % glucose, and growth at 42 °C. It ferments glucose but not other carbohydrates. The name Candida xylanilytica sp. nov. is proposed for this species. The type strain is KU-Xn11(T) ( = NBRC 106499(T)  = BCC 34694(T)  = CBS 11761(T)).

  1. Presumptive speciation of Streptococcus bovis and other group D streptococci from human sources by using arginine and pyruvate tests.

    PubMed Central

    Gross, K C; Houghton, M P; Senterfit, L B

    1975-01-01

    A simplified method for speciation of group D streptococci is described. A total of 4,156 streptococcal isolates from human clinical material was tested for ability to hydrolyze esculin in the presence of 40% bile, ferment pyruvate, hydrolyze arginine, and grow in media containing 40% bile or 6.5% NaCl. Streptococci which hydrolyzed esculin in 40% bile, but which did not hydrolyze arginine, were also tested for their ability to ferment raffinose or sorbose. Sixty percent (2,503) of the isolates hydrolyzed esculin in the presence of 40% bile and were thus presumptively identified as group D. By application of the other criteria, 84% of these were speciated as Streptococcus faecalis, 7% were speciated as S. faecium, 6% were speciated as S. bovis, 2% were speciated as S. avium, and 1% were not identified. This scheme was shown to be both reliable and practical for use in the diagnostic laboratory. S. avium and S. bovis isolates were characterized, and 18 S. bovis isolates from patients with bacterial endocarditis were compared physiologically with 151 isolates of this species from other sources. PMID:1176592

  2. Transport of Stachyose and Sucrose by Vacuoles of Japanese Artichoke (Stachys sieboldii) Tubers 1

    PubMed Central

    Keller, Felix

    1992-01-01

    Vacuoles are the stores for large amounts of stachyose [αgal (1,6) αgal (1,6) αglc (1,2) βfru] in tubers of Japanese artichoke (Stachys sieboldii). The uptake of stachyose by these vacuoles was examined and compared with that of sucrose. The uptake mechanisms of both sugars were quite similar. The kinetics showed a single saturable response to increasing external concentrations of 14C-sugars with similar apparent Km values of about 50 and 30 millimolar for stachyose and sucrose, respectively. The uptake rates, however, were always higher for stachyose than for sucrose. Stachyose and sucrose uptake was inhibited by fructose and raffinose, and, reciprocally, by sucrose and stachyose, but not by glucose or galactose. The main structural feature common to all sugars recognized by the uptake systems seems to be a terminal fructosyl residue. The uptake of both sugars was stimulated by Mg-ATP and inorganic pyrophosphate, suggesting a proton-sugar antiport system. The possibility that stachyose and sucrose might be transported by the same carrier is discussed. PMID:16668659

  3. Physiological Changes of Surface Membrane in Lactobacillus with Prebiotics.

    PubMed

    Pan, Mingfang; Kumaree, Kishore K; Shah, Nagendra P

    2017-03-01

    Synbiotics are always considered to be beneficial in healthy manipulation of gut environment; however, the purpose of this research was to investigate the dominance of synbiotic over the individual potential of probiotics and prebiotics. Four different types of prebiotics, fructo-oligosaccharides, raffinose, inulin, and cellobiose, were evaluated based on their varying degree of polymerization, combined each with 2 different Lactobacilli strains, including Lactobacillus paracasei 276 and Lactobacillus plantarum WCFS1. The effects of synbiotics combination on the surface structure were evaluated by analyzing auto-aggregation, membrane hydrophobicity, and adhesion to Caco-2 cells. Our results showed that both Lactobacilli exhibited significantly greater degree of attachment to Caco-2 cells (23.31% and 16.85%, respectively) when using cellobiose as a substrate than with other prebiotics (P < 0.05). Intestinal adhesion ability was in correlation with the percent of auto-aggregation, both Lactobacillus exhibited higher percent of auto-aggregation in cellobiose compared to other prebiotics. These behavioral changes in terms of attachment and auto-aggregation were further supported with the changes noticed from infrared spectra (FT-IR). © 2017 Institute of Food Technologists®.

  4. Galactosyltransferase and Concanavalin A Agglutination of Cells

    PubMed Central

    Podolsky, Daniel K.; Weiser, Milton M.; Mont, J. Thomas La; Isselbacher, Kurt J.

    1974-01-01

    A correlation has been observed between concanavalin A agglutination of various cell types and the presence of surface membrane galactosyltransferase (1-O-α-D-Galactosyl-myo-inositol:raffinose galactosyltransferase, EC 2.4.1.67) activity. Moreover, a reduction to less than 50% of cell surface galactosyltransferase activity occurred after treatment with concanavalin A; other cell surface glycosyltransferase enzyme activities examined were unaffected by concanavalin A treatment. To confirm the participation of cell surface galactosyltransferase in concanavalin A-induced cell agglutination, the enzyme from rabbit erythrocytes was solubilized by sonication and purified by preparative polyacrylamide gel electrophoresis. It was possible to achieve a purified preparation of rabbit erythrocyte galactosyltransferase by separation on concanavalin A-Sepharose. The purified enzyme showed visible immunoprecipitation (Ouchterlony) with concanavalin A. Furthermore, human erythrocytes, which are not normally agglutinated by concanavalin A, became agglutinable by the lectin when the erythrocytes were preincubated with purified galactosyltransferase. These experiments suggest a direct and possible specific role of cell surface galactosyltransferase enzyme in the mechanism of concanavalin A agglutination of cells. Images PMID:4522801

  5. Effect of gamma irradiation and cooking on cowpea bean grains ( Vigna unguiculata L. Walp)

    NASA Astrophysics Data System (ADS)

    Lima, Keila dos Santos Cople; Souza, Luciana Boher e.; Godoy, Ronoel Luiz de Oliveira; França, Tanos Celmar Costa; Lima, Antônio Luís dos Santos

    2011-09-01

    Leguminous plants are important sources of proteins, vitamins, carbohydrates, fibers and minerals. However, some of their non-nutritive elements can present undesirable side effects like flatulence provoked by the anaerobic fermentation of oligosaccharides, such as raffinose and stachyose, in the gut. A way to avoid this inconvenience, without any change in the nutritional value and post-harvesting losses, is an irradiation process. Here, we evaluated the effects of gamma irradiation on the amino acids, thiamine and oligosaccharide contents and on the fungi and their toxin percentages in cowpea bean ( Vigna unguiculata L. Walp) samples. For irradiation doses of 0.0, 0.5, 1.0, 2.5, 5.0 and 10.0 kGy the results showed no significant differences in content for the uncooked samples. However, the combination of irradiation and cooking processes reduced the non-nutritive factors responsible for flatulence. Irradiation also significantly reduced the presence of Aspergillus, Penicilium, Rhizopus and Fusarium fungi and was shown to be efficient in grain conservation for a storage time of 6 months.

  6. A Metabolomic Approach to the Study of Wine Micro-Oxygenation

    PubMed Central

    Arapitsas, Panagiotis; Scholz, Matthias; Vrhovsek, Urska; Di Blasi, Stefano; Biondi Bartolini, Alessandra; Masuero, Domenico; Perenzoni, Daniele; Rigo, Adelio; Mattivi, Fulvio

    2012-01-01

    Wine micro-oxygenation is a globally used treatment and its effects were studied here by analysing by untargeted LC-MS the wine metabolomic fingerprint. Eight different procedural variations, marked by the addition of oxygen (four levels) and iron (two levels) were applied to Sangiovese wine, before and after malolactic fermentation. Data analysis using supervised and unsupervised multivariate methods highlighted some known candidate biomarkers, together with a number of metabolites which had never previously been considered as possible biomarkers for wine micro-oxygenation. Various pigments and tannins were identified among the known candidate biomarkers. Additional new information was obtained suggesting a correlation between oxygen doses and metal contents and changes in the concentration of primary metabolites such as arginine, proline, tryptophan and raffinose, and secondary metabolites such as succinic acid and xanthine. Based on these findings, new hypotheses regarding the formation and reactivity of wine pigment during micro-oxygenation have been proposed. This experiment highlights the feasibility of using unbiased, untargeted metabolomic fingerprinting to improve our understanding of wine chemistry. PMID:22662221

  7. Properties of hemagglutination by Prevotella melaninogenica.

    PubMed

    Haraldsson, Gunnsteinn; Meurman, Jukka H; Könönen, Eija; Holbrook, W Peter

    2005-10-01

    Although Prevotella melaninogenica belongs to the commensal oral microbiota, some strains possess putative virulence factors. For example, we have previously described fimbriated, hemagglutinating strains of P. melaninogenica, isolated from patients with periodontal disease. The aim of this investigation was to compare some chemical and physical properties of hemagglutination (HA) of P. melaninogenica with those of other pigmented gram-negative anaerobes. HA of 13 P. melaninogenica strains proved to be considerably weaker than that of the major periodontal pathogen, Porphyromonas gingivalis. Vigorous shaking reduced HA of shaken cells but the shaken supernatant had the same hemagglutinating activity as non-shaken cells. The hemagglutinating agent on P. melaninogenica seemed to be a protein, which can be separated from the cell and binds to lactose-, galactose-, and raffinose-containing carbohydrates on the erythrocytes. Adherence to epithelial cells did not differ significantly between the hemagglutinating and non-hemagglutinating strains of P. melaninogenica. Although P. melaninogenica is able to agglutinate erythrocytes, this potential virulence factor is of a considerably lower magnitude than that of major periodontal pathogens.

  8. Biologically active peptides of the vesicular stomatitis virus glycoprotein.

    PubMed Central

    Schlegel, R; Wade, M

    1985-01-01

    A peptide corresponding to the amino-terminal 25 amino acids of the mature vesicular stomatitis virus glycoprotein has recently been shown to be a pH-dependent hemolysin. In the present study, we analyzed smaller constituent peptides and found that the hemolytic domain resides within the six amino-terminal amino acids. Synthesis of variant peptides indicates that the amino-terminal lysine can be replaced by another positively charged amino acid (arginine) but that substitution with glutamic acid results in the total loss of the hemolytic function. Peptide-induced hemolysis was dependent upon buffer conditions and was inhibited when isotonicity was maintained with mannitol, sucrose, or raffinose. In sucrose, all hemolytic peptides were also observed to mediate hemagglutination. The large 25-amino acid peptide is also a pH-dependent cytotoxin for mammalian cells and appears to effect gross changes in cell permeability. Conservation of the amino terminus of vesicular stomatitis virus and rabies virus suggests that the membrane-destabilizing properties of this domain may be important for glycoprotein function. Images PMID:2981356

  9. Measurements of thermodynamic and optical properties of selected aqueous organic and organic-inorganic mixtures of atmospheric relevance.

    PubMed

    Lienhard, Daniel M; Bones, David L; Zuend, Andreas; Krieger, Ulrich K; Reid, Jonathan P; Peter, Thomas

    2012-10-11

    Atmospheric aerosol particles can exhibit liquid solution concentrations supersaturated with respect to the dissolved organic and inorganic species and supercooled with respect to ice. In this study, thermodynamic and optical properties of sub- and supersaturated aqueous solutions of atmospheric interest are presented. The density, refractive index, water activity, ice melting temperatures, and homogeneous ice freezing temperatures of binary aqueous solutions containing L(+)-tartaric acid, tannic acid, and levoglucosan and ternary aqueous solutions containing levoglucosan and one of the salts NH(4)HSO(4), (NH(4))(2)SO(4), and NH(4)NO(3) have been measured in the supersaturated concentration range for the first time. In addition, the density and refractive index of binary aqueous citric acid and raffinose solutions and the glass transition temperatures of binary aqueous L(+)-tartaric acid and levoglucosan solutions have been measured. The data presented here are derived from experiments on single levitated microdroplets and bulk solutions and should find application in thermodynamic and atmospheric aerosol models as well as in food science applications.

  10. Cryptococcus nanyangensis sp. nov., a new basidiomycetous yeast isolated from the gut of wood-boring larvae.

    PubMed

    Hui, Feng-Li; Niu, Qiu-Hong; Ke, Tao; Li, Ying-Xia

    2012-11-01

    Two strains of a novel basidiomycetous yeast species were isolated from the gut of wood-boring larvae collected in the Baotianman Nature Reserve, the central China. Sequence analysis of the D1/D2 domains of the large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) regions showed that these yeasts belong to the Bulleromyces clade and formed a cluster together with eleven undescribed Cryptococcus species. The novel species differed from its closest known species, Cryptococcus rajasthanensis, by 3.3 % divergence (15 substitutions and 6 gaps over 630 bases) in the D1/D2 domains, and by 13.4 % divergence (41 substitutions and 27 gaps over 508 bases) in the ITS regions. Physiologically, the fermentation of glucose, galactose, sucrose, trehalose, and raffinose in Durham tubes was observed for the strains of this new yeast. Based on the phenotypical and molecular characteristics presented, the two strains are proposed as a new species, Cryptococcus nanyangensis sp. nov., with the type strain KCY-1(T) (=CICC 1976(T) = CBS 12474(T)).

  11. Influence of ozone on cold acclimation in sugar maple seedlings.

    PubMed

    Bertrand, Annick; Robitaille, Gilles; Nadeau, Paul; Castonguay, Yves

    1999-07-01

    During summer 1994, sugar maple (Acer saccharum Marsh.) seedlings were grown in open-top chambers supplied with air containing near ambient ozone concentration (control, low O(3)) or three times the ambient ozone concentration (high O(3)). The rate of CO(2) assimilation was significantly reduced by chronic exposure to a high concentration of ozone during the summer. During fall, seedlings were removed from the open-top chambers and acclimated to cold under natural conditions. In both species during cold acclimation, the starch concentration decreased, whereas the sucrose concentration increased. There was no treatment effect on the freezing tolerance of roots, even though roots in the high-O(3) treatment accumulated higher concentrations of the cryoprotective oligosaccharides raffinose and stachyose than control roots. Cold acclimation occurred earlier and stachyose concentration of stems was higher in high-O(3)-treated seedlings than in low-O(3)-treated seedlings. Cold acclimation was associated with an earlier accumulation of ABA in the xylem sap of high-O(3)-treated seedlings compared with low-O(3)-treated seedlings.

  12. Polymalic acid fermentation by Aureobasidium pullulans for malic acid production from soybean hull and soy molasses: Fermentation kinetics and economic analysis.

    PubMed

    Cheng, Chi; Zhou, Yipin; Lin, Meng; Wei, Peilian; Yang, Shang-Tian

    2017-01-01

    Polymalic acid (PMA) production by Aureobasidium pullulans ZX-10 from soybean hull hydrolysate supplemented with corn steep liquor (CSL) gave a malic acid yield of ∼0.4g/g at a productivity of ∼0.5g/L·h. ZX-10 can also ferment soy molasses, converting all carbohydrates including the raffinose family oligosaccharides to PMA, giving a high titer (71.9g/L) and yield (0.69g/g) at a productivity of 0.29g/L·h in fed-batch fermentation under nitrogen limitation. A higher productivity of 0.64g/L·h was obtained in repeated batch fermentation with cell recycle and CSL supplementation. Cost analysis for a 5000 MT plant shows that malic acid can be produced at $1.10/kg from soy molasses, $1.37/kg from corn, and $1.74/kg from soybean hull. At the market price of $1.75/kg, malic acid production from soy molasses via PMA fermentation offers an economically competitive process for industrial production of bio-based malic acid. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Extracellular inulinases from Penicillium janczewskii, a fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae).

    PubMed

    Pessoni, R A; Figueiredo-Ribeiro, R C; Braga, M R

    1999-07-01

    Extracellular inulinases from Penicillium janczewskii were obtained from the filtrate of 12 day-old cultures supplemented with inulin from Vernonia herbacea. Crude filtrates and partially-purified enzyme preparations (peaks I and II) were active on inulin, sucrose and raffinose. The apparent M(r) of the enzymes from peaks I and II were 48 and 66 kDa, respectively. The apparent K(m) (mmol l-1) values of peak I were 0.43 for inulin and 18.7 for sucrose; for peak II they were 0.87 and 18.5 for inulin and sucrose, respectively. Their temperature and pH optima were 55 degrees C and 5.0, respectively. Both peaks catalysed the hydrolysis of beta-(2,1) fructans more rapidly than beta-(2,6) fructans. Free fructose was the predominant product released from inulin, indicating that these enzymes display exo-inulinase activity. In view of these characteristics, the yield and the high specific activity towards beta-(2,1) fructans, inulinases from P. janczewskii can be utilized for the preparation of fructose syrup from inulin.

  14. Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192

    PubMed Central

    Keung, Hoi Yee; Li, Tsz Kai; Sham, Lok To; Cheung, Man Kit; Cheung, Peter Chi Keung

    2017-01-01

    ABSTRACT Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast (Saccharomyces cerevisiae) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1→3,1→6)-β-glucans and (1→4,1→6)-α-glucans. Although the YCWG were composed of 78.3% β-glucans and 21.7% α-glucans, only α-glucans were consumed by the B. breve strain. The ABC transporter (malEFG1) and pullulanase (aapA) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics. IMPORTANCE In general, Bifidobacterium strains are genetically

  15. Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192.

    PubMed

    Keung, Hoi Yee; Li, Tsz Kai; Sham, Lok To; Cheung, Man Kit; Cheung, Peter Chi Keung; Kwan, Hoi Shan

    2017-04-01

    Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast ( Saccharomyces cerevisiae ) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1 H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1→3,1→6)-β-glucans and (1→4,1→6)-α-glucans. Although the YCWG were composed of 78.3% β-glucans and 21.7% α-glucans, only α-glucans were consumed by the B. breve strain. The ABC transporter ( malEFG1 ) and pullulanase ( aapA ) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics. IMPORTANCE In general, Bifidobacterium strains are genetically intractable

  16. Use of adenovirus as a model system to illustrate a simple method using standard equipment and inexpensive excipients to remove live virus dependence on the cold-chain.

    PubMed

    Stewart, M; Ward, S J; Drew, J

    2014-05-19

    Thermolability of complex biological molecules is a major consideration for the long-term maintenance of titer during periods of storage. The development of a simple, cost effective method for long term storage of virus samples, which maintains viral titer would prove useful for a wide variety of applications including the preservation of viral vaccines, and is paramount for alleviating the reliance upon the cold chain. We have investigated the potential use of a method adapted for this purpose originating from a natural mechanism used by plants which helps to maintain the integrity of seeds, enabling them to overcome extensive periods of temperature elevation and desiccation. As maturation of a seed progresses, many complex biological macromolecules are laid down which maintain the germination potential. Sucrose and raffinose (in addition to other oligosaccharides) are commonly found to accumulate. In addition highly charged protein molecules accumulate, Late Embryogenesis Abundant (LEA) proteins, reaching their maximal level when the seed is most desiccation and thermally tolerant, and indeed are among the first molecules to be lost when germination is initiated. We have examined the potential use of sucrose and raffinose in concert with chemical replacements for the LEA, which when dried with the active product forms an amorphous solid able to maintain the titer of infectious Adenovirus at elevated temperatures for extended periods, in the case of lyophilized presentations several months at 37 °C, or as liquid, stability for several weeks at 37 °C was achieved. We demonstrate that after embedding the active product in the matrix, the viral titer is preserved even at temperatures for relatively extended periods at temperatures significantly greater than ambient. In addition we believe that these results could open the way for a new type of vaccine which we refer to as a hybrid stability vaccine, whereby for the first time the same excipient components are used

  17. Gene regulation of atrial natriuretic peptide A, B, and C receptors in rat glomeruli.

    PubMed

    Itoh, K; Nonoguchi, H; Shiraishi, N; Tomita, K

    1999-01-01

    Atrial natriuretic peptide (ANP) has three types of receptor. We investigated the gene regulation of three types of ANP receptors (ANPR-A, B, and C) in rat glomeruli using reverse transcription coupled with competitive polymerase chain reaction (PCR). Competitive PCR revealed that ANPR-C mRNA expression was most abundant (ANPR-C > A > B) in glomeruli from control rats among mRNA expressions of three receptors, which were 20- to 15,000-fold higher than those in inner medullary collecting ducts. Two days' dehydration caused reversible decreases of ANPR-A, B, and C mRNAs by 50-80%. To determine the mechanisms of down-regulation of mRNA expression, isolated glomeruli were incubated in isotonic or hypertonic solution. Hyperosmolality induced by NaCl, mannitol or raffinose caused significant increases of ANPR-A, B, and C mRNA expression. Hypertonicity by urea showed smaller effects. ANP stimulated the expression of ANPR-A, B, and C mRNA in vitro. These results indicate that dehydration caused reversible decreases of ANPR-A, B, and C mRNA expression in glomeruli, and these decreases were not caused by increased plasma osmolality but probably by lower circulating levels of ANP.

  18. MIG1-dependent and MIG1-independent regulation of GAL gene expression in Saccharomyces cerevisiae: role of Imp2p.

    PubMed

    Alberti, Adriana; Lodi, Tiziana; Ferrero, Iliana; Donnini, Claudia

    2003-10-15

    Imp2p (Yil154c) is a transcriptional activator involved in glucose derepression of the maltose, galactose and raffinose utilization pathways and in resistance to thermal, oxidative or osmotic stress. We analysed the role of Imp2 in the regulation of GAL genes. Imp2 was shown to have a positive effect on glucose derepression of Leloir pathway genes and their activator gene GAL4. The effect of Imp2 on galactose metabolism was shown to be partially dependent on Mig1p. The Mig1-independent role depends on Nrg1p. However, disruption of both MIG1 and NRG1 only partially relieves the glucose repression of GAL genes in the Deltaimp2 mutant, indicating that Imp2 must also have other function(s). Moreover, the interaction between IMP2 and GAL6/BLH1, a recently isolated gene involved in the regulation of GAL genes that shares with Imp2 the ability to protect cells from the glycopeptide bleomycin, was also analysed. The results suggest a major role of Imp2 in a GAL6-independent pathway. Copyright 2003 John Wiley & Sons, Ltd.

  19. Valorization of Cheese and Tofu Whey through Enzymatic Synthesis of Lactosucrose.

    PubMed

    Corzo-Martinez, Marta; Luscher, Alice; de Las Rivas, Blanca; Muñoz, Rosario; Moreno, F Javier

    2015-01-01

    This work deals with the development of a new bioprocess for the efficient synthesis of lactosucrose, a potential prebiotic oligosaccharide with a high value-added, from two important and inexpensive agro-industrial by-products such as tofu whey and cheese whey permeate. The bioconversion is driven by the ability of the enzyme levansucrase SacB from Bacillus subtilis CECT 39 to transfructosylate lactose contained in the cheese whey permeate by using not only sucrose but also raffinose and stachyose, which are present in considerable amounts in the tofu whey, as suitable donors of fructosyl moieties. The maximum lactosucrose concentration obtained from both by-products was 80.1 g L-1 after a short reaction time 120 min at 37°C, leading to productivity and specific productivity values of 40.1 g lactosucrose L-1 h-1 and 80.1 mg lactosucrose U enzyme-1 h-1, respectively. Findings contained in this work could provide a new strategy to valorize agro-industrial by-products as cheese whey permeate and, specially, tofu whey by means of their use as renewable resources in the enzymatic synthesis of bioactive oligosaccharides.

  20. Valorization of Cheese and Tofu Whey through Enzymatic Synthesis of Lactosucrose

    PubMed Central

    Corzo-Martinez, Marta; Luscher, Alice; de las Rivas, Blanca; Muñoz, Rosario; Moreno, F. Javier

    2015-01-01

    This work deals with the development of a new bioprocess for the efficient synthesis of lactosucrose, a potential prebiotic oligosaccharide with a high value-added, from two important and inexpensive agro-industrial by-products such as tofu whey and cheese whey permeate. The bioconversion is driven by the ability of the enzyme levansucrase SacB from Bacillus subtilis CECT 39 to transfructosylate lactose contained in the cheese whey permeate by using not only sucrose but also raffinose and stachyose, which are present in considerable amounts in the tofu whey, as suitable donors of fructosyl moieties. The maximum lactosucrose concentration obtained from both by-products was 80.1 g L-1 after a short reaction time 120 min at 37°C, leading to productivity and specific productivity values of 40.1 g lactosucrose L-1 h-1 and 80.1 mg lactosucrose U enzyme−1 h−1, respectively. Findings contained in this work could provide a new strategy to valorize agro-industrial by-products as cheese whey permeate and, specially, tofu whey by means of their use as renewable resources in the enzymatic synthesis of bioactive oligosaccharides. PMID:26406885

  1. Expanded metabolomics approach to profiling endogenous carbohydrates in the serum of ovarian cancer patients.

    PubMed

    Cheng, Yu; Li, Li; Zhu, Bangjie; Liu, Feng; Wang, Yan; Gu, Xue; Yan, Chao

    2016-01-01

    We applied hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry to the quantitative analysis of serum from 58 women, including ovarian cancer patients, ovarian benign tumor patients, and healthy controls. All of these ovarian cancer and ovarian benign tumor patients have elevated cancer antigen 125, which makes them clinically difficult to differentiate the malignant from the benign. All of the 16 endogenous carbohydrates were quantitatively detected in the human sera, of which, eight endogenous carbohydrates were significantly different (P-value < 0.05) between the ovarian cancer and healthy control. According to the receiver operating characteristic curve analysis, arabitol was the most potentially specific biomarker for discriminating ovarian cancer from healthy control, having an area under the curve of 0.911. A panel of metabolite markers composed of maltose, maltotriose, raffinose, and mannitol was selected, which was able to discriminate the ovarian cancer from the benign ovarian tumor counterparts, with an area under concentration-time curve value of 0.832. Endogenous carbohydrates in the expanded metabolomics approach after the global metabolic profiling are characterized and are potential biomarkers for the early diagnosis of ovarian cancer. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Enhancement of Phloem Exudation from Fraxinus uhdei Wenz. (Evergreen Ash) using Ethylenediaminetetraacetic Acid 1

    PubMed Central

    Costello, L. R.; Bassham, James A.; Calvin, Melvin

    1982-01-01

    Ethylenediaminetetraacetic acid (EDTA) enhanced the exudation of 14C-labeled assimilates from excised leaflets and whole plant specimens of Fraxinus uhdei Wenz. A 2 millimolar EDTA concentration was found to be most effective in promoting exudation from excised leaflets, while 10 millimolar EDTA was most effective in whole plants experiments. Exudation rate reached a maximum after 24 hours in both experiments. The continuous presence of EDTA throughout the treatment period was required for maximum exudation from excised leaflets. Stachyose, raffinose, verbascose, and sucrose were the principal compounds found to occur in exudate samples. These compounds are typically transported in sieve elements of various Fraxinus species suggesting the exudate was of phloem origin. Electron microscope studies of petiolule sieve plate pores from excised leaflets showed substantially less callose appearing after treatment with EDTA than after H2O treatment. It is suggested that EDTA enhances phloem exudation by inhibiting or reducing callose formation in sieve plate pores. The exudation enhancement technique described for whole plant specimens is suggested as a useful means of collecting phloem sap and studying translocation in woody plants. Images PMID:16662189

  3. Sporothrix brasiliensis, S. globosa, and S. mexicana, Three New Sporothrix Species of Clinical Interest▿

    PubMed Central

    Marimon, Rita; Cano, Josep; Gené, Josepa; Sutton, Deanna A.; Kawasaki, Masako; Guarro, Josep

    2007-01-01

    Sporothrix schenckii is the species responsible for sporotrichosis, a fungal infection caused by the traumatic implantation of this dimorphic fungus. Recent molecular studies have demonstrated that this species constitutes a complex of numerous phylogenetic species. Since the delineation of such species could be of extreme importance from a clinical point of view, we have studied a total of 127 isolates, most of which were received as S. schenckii, including the available type strains of species currently considered synonyms, and also some close morphological species. We have phenotypically characterized all these isolates using different culture media, growth rates at different temperatures, and numerous nutritional tests and compared their calmodulin gene sequences. The molecular analysis revealed that Sporothrix albicans, S. inflata, and S. schenckii var. luriei are species that are clearly different from S. schenckii. The combination of these phenetic and genetic approaches allowed us to propose the new species Sporothrix brasiliensis, S. globosa, and S. mexicana. The key phenotypic features for recognizing these species are the morphology of the sessile pigmented conidia, growth at 30, 35, and 37°C, and the assimilation of sucrose, raffinose, and ribitol. PMID:17687013

  4. Transfer of the potato plant isolates of Pectobacterium wasabiae to Pectobacterium parmentieri sp. nov.

    PubMed

    Khayi, Slimane; Cigna, Jérémy; Chong, Teik Min; Quêtu-Laurent, Angélique; Chan, Kok-Gan; Hélias, Valérie; Faure, Denis

    2016-12-01

    Pectobacterium wasabiae was originally isolated from Japanese horseradish (Eutrema wasabi), but recently some Pectobacterium isolates collected from potato plants and tubers displaying blackleg and soft rot symptoms were also assigned to P. wasabiae. Here, combining genomic and phenotypical data, we re-evaluated their taxonomic position. PacBio and Illumina technologies were used to complete the genome sequences of P. wasabiae CFBP 3304T and RNS 08-42-1A. Multi-locus sequence analysis showed that the P. wasabiae strains RNS 08-42-1A, SCC3193, CFIA1002 and WPP163, which were collected from potato plant environment, constituted a separate clade from the original Japanese horseradish P. wasabiae. The taxonomic position of these strains was also supported by calculation of the in-silico DNA-DNA hybridization, genome average nucleotide indentity, alignment fraction and average nucleotide indentity values. In addition, they were phenotypically distinguished from P. wasabiae strains by producing acids from (+)-raffinose, α-d(+)-α-lactose, d(+)-galactose and (+)-melibiose but not from methyl α-d-glycopyranoside, (+)-maltose or malonic acid. The name Pectobacterium parmentieri sp. nov. is proposed for this taxon; the type strain is RNS 08-42-1AT (=CFBP 8475T=LMG 29774T).

  5. Species Differentiation of Group D Streptococci

    PubMed Central

    Papavassiliou, J.

    1962-01-01

    Three hundred and fourteen strains of group D streptococci were studied by means of a number of tests. The majority of the strains were identified as Streptococcus faecalis (83 strains), Streptococcus faecium (131 strains), or Streptococcus bovis (32 strains). Several strains (47 or nearly 15%) either shared characteristics of two species or were completely atypical. S. faecalis and S. bovis were more easily identified than S. faecium, which is not sharply defined from the other species and could be subdivided into several fermentative types on the basis of fermentation of arabinose, mannitol, sorbitol, glycerol, and sucrose. The value of some characteristics in species identification is discussed. Growth in the presence of potassium tellurite 1:2,500 and in the presence of 6.5% NaCl and fermentation of arabinose, glycerol, and raffinose are very important tests for the identification of the three species. The reduction of tetrazolium salts, the reduction of litmus milk, and the fermentation of sorbitol may serve as complementary tests for the same purpose. For the differentiation of these three species the “pattern of reactions” is more important than single tests. PMID:14483707

  6. Multi-Spectroscopic Analysis of Seed Quality and 13C-Stable-Iotopologue Monitoring in Initial Growth Metabolism of Jatropha curcas L.

    PubMed Central

    Komatsu, Takanori; Ohishi, Risa; Shino, Amiu; Akashi, Kinya; Kikuchi, Jun

    2014-01-01

    In the present study, we applied nuclear magnetic resonance (NMR), as well as near-infrared (NIR) spectroscopy, to Jatropha curcas to fulfill two objectives: (1) to qualitatively examine the seeds stored at different conditions, and (2) to monitor the metabolism of J. curcas during its initial growth stage under stable-isotope-labeling condition (until 15 days after seeding). NIR spectra could non-invasively distinguish differences in storage conditions. NMR metabolic analysis of water-soluble metabolites identified sucrose and raffinose family oligosaccharides as positive markers and gluconic acid as a negative marker of seed germination. Isotopic labeling patteren of metabolites in germinated seedlings cultured in agar-plate containg 13C-glucose and 15N-nitrate was analyzed by zero-quantum-filtered-total correlation spectroscopy (ZQF-TOCSY) and 13C-detected 1H-13C heteronuclear correlation spectroscopy (HETCOR). 13C-detected HETOCR with 13C-optimized cryogenic probe provided high-resolution 13C-NMR spectra of each metabolite in molecular crowd. The 13C-13C/12C bondmer estimated from 1H-13C HETCOR spectra indicated that glutamine and arginine were the major organic compounds for nitrogen and carbon transfer from roots to leaves. PMID:25401292

  7. Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation.

    PubMed

    Matthews, Jennifer L; Murphy, Joy M; Carmichael, Carrie; Yang, Huiping; Tiersch, Terrence; Westerfield, Monte; Varga, Zoltan M

    2018-01-25

    Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.

  8. Chemometric Data Analysis for Deconvolution of Overlapped Ion Mobility Profiles

    NASA Astrophysics Data System (ADS)

    Zekavat, Behrooz; Solouki, Touradj

    2012-11-01

    We present the details of a data analysis approach for deconvolution of the ion mobility (IM) overlapped or unresolved species. This approach takes advantage of the ion fragmentation variations as a function of the IM arrival time. The data analysis involves the use of an in-house developed data preprocessing platform for the conversion of the original post-IM/collision-induced dissociation mass spectrometry (post-IM/CID MS) data to a Matlab compatible format for chemometric analysis. We show that principle component analysis (PCA) can be used to examine the post-IM/CID MS profiles for the presence of mobility-overlapped species. Subsequently, using an interactive self-modeling mixture analysis technique, we show how to calculate the total IM spectrum (TIMS) and CID mass spectrum for each component of the IM overlapped mixtures. Moreover, we show that PCA and IM deconvolution techniques provide complementary results to evaluate the validity of the calculated TIMS profiles. We use two binary mixtures with overlapping IM profiles, including (1) a mixture of two non-isobaric peptides (neurotensin (RRPYIL) and a hexapeptide (WHWLQL)), and (2) an isobaric sugar isomer mixture of raffinose and maltotriose, to demonstrate the applicability of the IM deconvolution.

  9. Organ-specific defence strategies of pepper (Capsicum annuum L.) during early phase of water deficit.

    PubMed

    Sziderics, Astrid Heide; Oufir, Mouhssin; Trognitz, Friederike; Kopecky, Dieter; Matusíková, Ildikó; Hausman, Jean-Francois; Wilhelm, Eva

    2010-03-01

    Drought is one of the major factors that limits crop production and reduces yield. To understand the early response of plants under nearly natural conditions, pepper plants (Capsicum annuum L.) were grown in a greenhouse and stressed by withholding water for 1 week. Plants adapted to the decreasing water content of the soil by adjustment of their osmotic potential in root tissue. As a consequence of drought, strong accumulation of raffinose, glucose, galactinol and proline was detected in the roots. In contrast, in leaves the levels of fructose, sucrose and also galactinol increased. Due to the water deficit cadaverine, putrescine, spermidine and spermine accumulated in leaves, whereas the concentration of polyamines was reduced in roots. To study the molecular basis of these responses, a combined approach of suppression subtractive hybridisation and microarray technique was performed on the same material. A total of 109 unique ESTs were detected as responsive to drought, while additional 286 ESTs were selected from the bulk of rare transcripts on the array. The metabolic profiles of stressed pepper plants are discussed with respect to the transcriptomic changes detected, while attention is given to the differences between defence strategies of roots and leaves.

  10. [Streptoverticillium griseoviridum n. sp., a producer of the candidin-amphotericin B group, antifungal heptaene nonaromatic antibiotic 0185].

    PubMed

    Konev, Iu E; Efimova, V M; Etingov, E D; Zaval'naia, N M

    1978-02-01

    An actinomyceteous strain LIA-0185 producing a heptaenic non-aromatic antibiotic of the candidin type was isolated from a soil sample taken in the Georgian SSR under the programme of screening antifungal antibiotics. The taxonomic study of the strain showed that it belonged to the series of viridoflavum and had the following main taxonomic features: the sporophores in the whorls, straight, remote: the aerial mycelium from yellow to dark-olive-grey; the substrate mycelium olive; the soluble pigment absent; the melanine pigment was produced on the peptone medium; the culture formed H2S; assimilated glucose, mannose, inozide and to a lesser extent fructose; did not assimilate arabinose, xylose, sucrose, lactose, ramnose and raffinose. The strain inhibited the growth of yeast and fungi, grampositive bacteria and actinomycetes and produced a complex of non-aromatic heptaenic antibiotics. The actinomycete differed from the other whorl cultures. It was classified as a new species Sv. griseoviridum sp. nov. The antibiotic complex was a mixture of 2 components, i. e. I and II present approximately in equal amounts. Component II was analogous to candidin. Component I was a new original substance.

  11. Reclassification of Lactobacillus kimchii and Lactobacillus bobalius as later subjective synonyms of Lactobacillus paralimentarius.

    PubMed

    Pang, Huili; Kitahara, Maki; Tan, Zhongfang; Wang, Yanping; Qin, Guangyong; Ohkuma, Moriya; Cai, Yimin

    2012-10-01

    Characterization and identification of strain CW 1 ( = JCM 17161) isolated from corn silage were performed. Strain CW 1 was a Gram-positive, catalase-negative and homofermentative rod that produced the DL-form of lactic acid. This strain exhibited more than 99.6% 16S rRNA gene sequence similarity and greater than 82% DNA-DNA reassociation with type strains of Lactobacillus kimchii, L. bobalius and L. paralimentarius. To clarify the taxonomic positions of these type strains, phenotypic characterization, 16S rRNA gene sequencing, ribotyping and DNA-DNA relatedness were examined. The three type strains displayed different L-arabinose, lactose, melibiose, melezitose, raffinose and N-acetyl-β-glucosaminidase fermentation patterns. Phylogenetic analysis showed that L. paralimentarius is a closer neighbour of L. kimchii and L. bobalius, sharing 99.5-99.9% 16S rRNA gene sequence similarity, which was confirmed by the high DNA-DNA relatedness (≥82%) between L. paralimentarius JCM 10415(T), L. bobalius JCM 16180(T) and L. kimchii JCM 10707(T). Therefore, it is proposed that L. kimchii and L. bobalius should be reclassified as later synonyms of L. paralimentarius.

  12. Characterization of a Glycoside Hydrolase Family 27 α-Galactosidase from Pontibacter Reveals Its Novel Salt-Protease Tolerance and Transglycosylation Activity.

    PubMed

    Zhou, Junpei; Liu, Yu; Lu, Qian; Zhang, Rui; Wu, Qian; Li, Chunyan; Li, Junjun; Tang, Xianghua; Xu, Bo; Ding, Junmei; Han, Nanyu; Huang, Zunxi

    2016-03-23

    α-Galactosidases are of great interest in various applications. A glycoside hydrolase family 27 α-galactosidase was cloned from Pontibacter sp. harbored in a saline soil and expressed in Escherichia coli. The purified recombinant enzyme (rAgaAHJ8) was little or not affected by 3.5-30.0% (w/v) NaCl, 10.0-100.0 mM Pb(CH3COO)2, 10.0-60.0 mM ZnSO4, or 8.3-100.0 mg mL(-1) trypsin and by most metal ions and chemical reagents at 1.0 and 10.0 mM concentrations. The degree of synergy on enzymatic degradation of locust bean gum and guar gum by an endomannanase and rAgaAHJ8 was 1.22-1.54. In the presence of trypsin, the amount of reducing sugars released from soybean milk treated by rAgaAHJ8 was approximately 3.8-fold compared with that treated by a commercial α-galactosidase. rAgaAHJ8 showed transglycosylation activity when using sucrose, raffinose, and 3-methyl-1-butanol as the acceptors. Furthermore, potential factors for salt adaptation of the enzyme were presumed.

  13. Purification and characterization of thermostable alpha-galactosidase from Aspergillus terreus (GR).

    PubMed

    Shankar, S K; Dhananjay, S K; Mulimani, V H

    2009-02-01

    An extracellular thermostable alpha-galactosidase producing Aspergillus terreus (GR) strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by acetone precipitation, gel filtration followed by DEAE-Sephacel chromatographic step. The purified enzyme showed a single band after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme after SDS-PAGE was 108 kDa. The enzyme showed optimum pH and temperature of 5.0 and 65 degrees C, respectively, for artificial substrate pNPalphaGal. alpha-Galactosidase from A. terreus (GR) is found to be thermostable, as it was not inactivated after heating at 65 degrees C for 40 min. The K (m) for pNPalphaGal, oNPalphaGal, raffinose, and stachyose are 0.1, 0.28, 0.42, and 0.33 mM, respectively. Inhibitors such as 1,10-phenanthroline, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, mercaptoethanol, and urea have no effect, whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg(2+), Ni(2+), Ca(2+), Co(2+), and Mn(2+) had no effect on enzyme activity, but Ag(+), Hg(2+), and Cu(2+) have inhibited complete activity.

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

    PubMed

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

    2007-05-01

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

  15. Effect of gamma irradiation on lipoxygenases, trypsin inhibitor, raffinose family oligosaccharides and nutritional factors of different seed coat colored soybean (Glycine max L.)

    NASA Astrophysics Data System (ADS)

    Kumar Dixit, Amit; Kumar, Vineet; Rani, Anita; Manjaya, J. G.; Bhatnagar, Deepak

    2011-04-01

    Three soybean genotypes Kalitur, Hara soya and NRC37 with black, green and yellow seed coat color, respectively, were gamma irradiated at 0.5, 2.0 and 5.0 kGy and tested for antinutritional and nutritional factors. Gamma irradiation at all doses reduced the level of lipoxygenase isomers, trypsin inhibitor (TI) and ascorbic acid in all the 3 soybean genotypes as compared to the unirradiated control. However, irradiation dose of 5.0 kGy increased the sucrose content of the soybean genotypes. No significant change was observed in oil, protein fatty acids and total tocopherol content of the 3 genotypes at any irradiation dose. It is suggested that inhibition of lipoxygenase, reduction in TI and ascorbic acid may be due to the breakage or oxidation of protein structure by the gamma irradiation. Similarly, gamma irradiation at higher doses may break glycosidic linkages in oligosaccharides to produce more sucrose and decrease the content of flatulence causing oligosaccharides.

  16. Candida aechmeae sp. nov. and Candida vrieseae sp. nov., novel yeast species isolated from the phylloplane of bromeliads in Southern Brazil.

    PubMed

    Landell, Melissa Fontes; Billodre, Raisa; Ramos, Jesus P; Leoncini, Orílio; Vainstein, Marilene H; Valente, Patrícia

    2010-01-01

    Two novel yeast species, Candida aechmeae sp. nov. and Candida vrieseae sp. nov., were isolated from bromeliads in Itapuã Park, Rio Grande do Sul, Brazil. These species are genetically isolated from all other currently recognized ascomycetous yeasts based on their sequence divergence in the D1/D2 domain of the LSU rRNA gene. C. aechmeae sp. nov. is phylogenetically close to Candida ubatubensis, a species also isolated from bromeliads in Brazil, but the novel species can be differentiated on the basis of differences in the D1/D2 domain and positive results for the assimilation of l-arabinose, raffinose, inulin and citrate. Candida vrieseae sp. nov. is phylogenetically placed in a clade near Candida membranifaciens that is composed of several species associated with insects, but the novel species can be differentiated from them by the D1/D2 and ITS gene sequences, positive results for the assimilation of nitrite and a negative result for the assimilation of ethylamine. The type strain for Candida aechmeae sp. nov. is BI153(T) (=CBS 10831(T)=NRRL Y-48456(T)) and the type strain for C. vrieseae sp. nov. is BI146(T) (=CBS 10829(T)=NRRL Y-48461(T)).

  17. Alkaline β-fructofuranosidases of tuberous roots: Possible physiological function.

    PubMed

    Ricardo, C P

    1974-12-01

    Alkaline invertase of roots of carrot (Daucus carota L.) did not hydrolyze raffinose while the acid invertase from the same tissue showed with this sugar ca. 60% of the activity found with sucrose. The activity of the two invertases was inhibited by fructose to a different extent, the K i value being ca. 4×10(-2) M and 3×10(-1)M, respectively, for the alkaline and the acid invertases from the roots of both carrot and turnip (Brassica rapa L.). It is proposed that fructose inhibition of acid invertase is of no physiological significance but that, in contrast, hexoses might regulate the activity of alkaline invertase.Comparing several species and cultivars, it was found that the content of reducing sugars and the activity of alkaline invertase of mature tuberous roots showed a positive correlation. This indicates that alkaline invertase may participate in the regulation of the hexose level of the cell, as was previously suggested for sugar-cane. A scheme is presented which proposes a way of participation of alkaline invertase in such a regulation, assuming that this enzyme is located in the cytoplasm and acid invertase is membrane-bound and mainly located at the cell surface.

  18. Evaluation of hydrophilic permeant transport parameters in the localized and non-localized transport regions of skin treated simultaneously with low-frequency ultrasound and sodium lauryl sulfate.

    PubMed

    Kushner, Joseph; Blankschtein, Daniel; Langer, Robert

    2008-02-01

    The porosity (epsilon), the tortuosity (tau), and the hindrance factor (H) of the aqueous pore channels located in the localized transport regions (LTRs) and the non-LTRs formed in skin treated simultaneously with low-frequency ultrasound (US) and the surfactant sodium lauryl sulfate (SLS), were evaluated for the delivery of four hydrophilic permeants (urea, mannitol, raffinose, and inulin) by analyzing dual-radiolabeled diffusion masking experiments for three different idealized cases of the aqueous pore pathway hypothesis. When epsilon and tau were assumed to be independent of the permeant radius, H was found to be statistically larger in the LTRs than in the non-LTRs. When a distribution of pore radii was assumed to exist in the skin, no statistical differences in epsilon, tau, and H were observed due to the large variation in the pore radii distribution shape parameter (3 A to infinity). When infinitely large aqueous pores were assumed to exist in the skin, epsilon was found to be 3-8-fold greater in the LTRs than in the non-LTRs, while little difference was observed in the LTRs and in the non-LTRs for tau. This last result suggests that the efficacy of US/SLS treatment may be enhanced by increasing the porosity of the non-LTRs.

  19. A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter.

    PubMed

    Wang, Zhi; Zhu, Yan; Wang, Lili; Liu, Xia; Liu, Yongxiu; Phillips, Jonathan; Deng, Xin

    2009-11-01

    Accumulation of compatible osmolytes, such as soluble sugars, in plants is an important osmoprotective mechanism. Sugars play a role in osmotic adjustment and are associated with stabilization of proteins and cell structures, reactive oxygen species scavenging, signaling functions or induction of adaptive pathways. Galactinol is the galactosyl donor for the synthesis of raffinose family oligosaccharides (RFOs) and its synthesis by galactinol synthase (GolS) is the first committed step of the RFOs biosynthetic pathway. GolS genes are induced by a variety of stresses in both stress-sensitive and tolerant-plant species; however, the mechanism of transcriptional regulation is not fully established. In this paper, we characterized a GolS gene (BhGolS1) that was dehydration and ABA-inducible in the resurrection plant Boea hygrometrica and conferred dehydration tolerance in a transgenic tobacco system. Four W-box cis-elements were identified in the BhGolS1 promoter and shown to be bound by an early dehydration and ABA-inducible WRKY gene (BhWRKY1). These data suggest a mechanism where BhWRKY1 is likely to function in an ABA-dependent signal pathway to regulate BhGolS1 expression, which leads to the accumulation of RFOs in desiccation-tolerant B. hygrometrica leaves.

  20. Production of dissolvable microneedles using an atomised spray process: effect of microneedle composition on skin penetration.

    PubMed

    McGrath, Marie G; Vucen, Sonja; Vrdoljak, Anto; Kelly, Adam; O'Mahony, Conor; Crean, Abina M; Moore, Anne

    2014-02-01

    Dissolvable microneedles offer an attractive delivery system for transdermal drug and vaccine delivery. They are most commonly formed by filling a microneedle mold with liquid formulation using vacuum or centrifugation to overcome the constraints of surface tension and solution viscosity. Here, we demonstrate a novel microneedle fabrication method employing an atomised spray technique that minimises the effects of the liquid surface tension and viscosity when filling molds. This spray method was successfully used to fabricate dissolvable microneedles (DMN) from a wide range of sugars (trehalose, fructose and raffinose) and polymeric materials (polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose, hydroxypropylmethylcellulose and sodium alginate). Fabrication by spraying produced microneedles with amorphous content using single sugar compositions. These microneedles displayed sharp tips and had complete fidelity to the master silicon template. Using a method to quantify the consistency of DMN penetration into different skin layers, we demonstrate that the material of construction significantly influenced the extent of skin penetration. We demonstrate that this spraying method can be adapted to produce novel laminate-layered as well as horizontally-layered DMN arrays. To our knowledge, this is the first report documenting the use of an atomising spray, at ambient, mild processing conditions, to create dissolvable microneedle arrays that can possess novel, laminate layering. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. A comparative study of the extracellular glucosyl- and fructosyltransferases from cariogenic and non-cariogenic Streptococcus mutans strains of two different serotypes.

    PubMed

    Asem, K; Cornish-Bowden, A J; Cole, J A

    1986-01-01

    Extracellular proteins from continuous cultures of serotype c and g Streptococcus mutans strains were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Gels stained with raffinose after electrophoresis revealed that although serotype c strains secrete two fructosyltransferases of molecular mass 68 kDa and 79 kDa, no fructosyltransferase was secreted by the serotype g strain K1. A sucrose activity stain was used to detect two glucosyltransferases (GTF) of molecular mass 162 kDa (bifunctional 1,6-alpha-D-glucan 3-alpha- and 6-alpha GTF or 'dextransucrase') and 153 kDa (a 1,3-alpha-D-glucan 3-alpha-GTF) in samples from cariogenic serotype c strains. Neither the 153 kDa protein nor the corresponding GTF activity was secreted by the non-cariogenic mutant C 67-25. The molecular masses of the corresponding 1,3-alpha and 1,6-alpha-GTF proteins from the serotype g strain K1 were 164 kDa and 158 kDa, respectively. All of the GTF proteins were degraded to discrete bands of lower molecular mass on storage at 4 degrees C even after extensive purification. The results provide an explanation for several outstanding controversies in the GTF literature.

  2. Antioxidants, low molecular weight carbohydrates, and total antioxidant capacity in strawberries (Fragaria x ananassa): effects of cultivar, ripening, and storage.

    PubMed

    Olsson, Marie E; Ekvall, Jimmy; Gustavsson, Karl-Erik; Nilsson, Jessica; Pillai, Deepa; Sjöholm, Ingegerd; Svensson, Ulla; Akesson, Björn; Nyman, Margareta G L

    2004-05-05

    Four cultivars of strawberries (Senga Sengana, BFr77111, Elsanta, and Honeoye) were studied for their content of antioxidants, total antioxidant capacity, and low molecular weight carbohydrates in relation to harvest year, ripening stage, and cold storage. For ascorbic acid, chlorogenic acid, ellagic acid, and total antioxidative capacity, measured in both water-soluble and water-insoluble extracts, there was a 2-5-fold variation among cultivars. Unripe berries contained lower concentrations of chlorogenic acid and p-coumaric acid and also quercetin and kaempferol compared with riper berries. During cold storage for up to 3 days, relatively few changes in the concentration of the different antioxidants occurred. The concentrations of several investigated parameters were interrelated, for example, for ascorbic acid and water-soluble antioxidant capacity and for ellagic acid and water-insoluble antioxidant capacity. The dominating sugars in strawberries were fructose and glucose, but considerable amounts of sucrose were also present, and their contents varied among cultivars, giving a predicted glycemic index of approximately 81. Verbascose, raffinose, and stachyose were found in only minor amounts. The study shows that the concentration of a number of bioactive compounds in strawberries varied according to cultivar, ripening stage, and storage. This information should make it possible to select strawberries with an optimal content of bioactive compounds.

  3. Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae

    PubMed Central

    Paulo, Joao A.; O’Connell, Jeremy D.; Gaun, Aleksandr; Gygi, Steven P.

    2015-01-01

    The global proteomic alterations in the budding yeast Saccharomyces cerevisiae due to differences in carbon sources can be comprehensively examined using mass spectrometry–based multiplexing strategies. In this study, we investigate changes in the S. cerevisiae proteome resulting from cultures grown in minimal media using galactose, glucose, or raffinose as the carbon source. We used a tandem mass tag 9-plex strategy to determine alterations in relative protein abundance due to a particular carbon source, in triplicate, thereby permitting subsequent statistical analyses. We quantified more than 4700 proteins across all nine samples; 1003 proteins demonstrated statistically significant differences in abundance in at least one condition. The majority of altered proteins were classified as functioning in metabolic processes and as having cellular origins of plasma membrane and mitochondria. In contrast, proteins remaining relatively unchanged in abundance included those having nucleic acid–related processes, such as transcription and RNA processing. In addition, the comprehensiveness of the data set enabled the analysis of subsets of functionally related proteins, such as phosphatases, kinases, and transcription factors. As a resource, these data can be mined further in efforts to understand better the roles of carbon source fermentation in yeast metabolic pathways and the alterations observed therein, potentially for industrial applications, such as biofuel feedstock production. PMID:26399295

  4. Suppression of cucumber stachyose synthase gene (CsSTS) inhibits phloem loading and reduces low temperature stress tolerance.

    PubMed

    Lü, Jianguo; Sui, Xiaolei; Ma, Si; Li, Xin; Liu, Huan; Zhang, Zhenxian

    2017-09-01

    Stachyose is the main transporting sugar in phloem of Raffinose family oligosaccharides-transporting species. Stachyose synthase (STS) is a key enzyme for stachyose biosynthesis, but the gene encoding STS is poorly characterized in cucumber (Cucumis sativus L.), which is a model plant for studying stachyose metabolism and phloem function. In this research, stachyose synthase gene (CsSTS) from cucumber was isolated and its physiological functions were analyzed. CsSTS expressed mainly in the phloem of the minor veins in mature leaves and localized to companion cells. Reverse genetics with CsSTS RNAi lines revealed obviously reductions in STS activity and stachyose content along with a small amount of starch accumulation in leaves, suggesting that CsSTS is involved in phloem loading of cucumber leaves. After 6 °C low temperature stress, malondialdehyde content and electrical conductivity increased, especially in CsSTS-RNAi plants. But CsSTS expression was up-regulated, STS activity and stachyose level increased, the activities of reactive-oxygen-scavenging enzyme in cucumber seedlings improved significantly and starch accumulation reduced, especially in CsSTS-OE lines. These results demonstrate clearly that CsSTS is involved in phloem loading, carbohydrate distribution and tolerance of cucumber seedlings to low temperature stress.

  5. Structural confirmation of oligosaccharides newly isolated from sugar beet molasses.

    PubMed

    Abe, Tatsuya; Horiuchi, Kenichi; Kikuchi, Hiroto; Aritsuka, Tsutomu; Takata, Yusuke; Fukushi, Eri; Fukushi, Yukiharu; Kawabata, Jun; Ueno, Keiji; Onodera, Shuichi; Shiomi, Norio

    2012-08-27

    Sugar beet molasses is a viscous by-product of the processing of sugar beets into sugar. The molasses is known to contain sucrose and raffinose, a typical trisaccharide, with a well-established structure. Although sugar beet molasses contains various other oligosaccharides as well, the structures of those oligosaccharides have not been examined in detail. The purpose of this study was isolation and structural confirmation of these other oligosaccharides found in sugar beet molasses. Four oligosaccharides were newly isolated from sugar beet molasses using high-performance liquid chromatography (HPLC) and carbon-Celite column chromatography. Structural confirmation of the saccharides was provided by methylation analysis, matrix-assisted laser desorption/ionaization time of flight mass spectrometry (MALDI-TOF-MS), and nuclear magnetic resonance (NMR) measurements. The following oligosaccharides were identified in sugar beet molasses: β-D-galactopyranosyl-(1- > 6)-β-D-fructofuranosyl-(2 <-> 1)-α-D-glucopyranoside (named β-planteose), α-D-galactopyranosyl-(1- > 1)-β-D-fructofuranosyl-(2 <-> 1)-α-D-glucopyranoside (named1-planteose), α-D-glucopyranosyl-(1- > 6)-α-D-glucopyranosyl-(1 <-> 2)-β-D-fructofuranoside (theanderose), and β-D-glucopyranosyl-(1- > 3)-α-D-glucopyranosyl-(1 <-> 2)-β-D-fructofuranoside (laminaribiofructose). 1-planteose and laminaribiofructose were isolated from natural sources for the first time.

  6. Thermostability of Probiotics and Their α -Galactosidases and the Potential for Bean Products.

    PubMed

    Liu, Xiaoli; Champagne, Claude P; Lee, Byong H; Boye, Joyce I; Casgrain, Michel

    2014-01-01

    Soybeans and other pulses contain oligosaccharides which may cause intestinal disturbances such as flatulence. This study was undertaken to investigate α -galactosidase-producing probiotics added to frozen foods which can survive warming treatments used in thawing and consumption of the pulses. The maximum α -galactosidase activity (1.26 U/mg protein) was found in Bifidobacterium breve S46. Lactobacillus casei had the highest α -galactosidase thermostability among the various strains, with D values of 35, 29, and 9.3 minutes at 50°C, 55°C, and 60°C, respectively. The enzyme activity was less affected than viable cells by heating. However, the D values of two bacterial enzymes were lower than those of three commercial α -galactosidase-containing products. Freshly grown cells and their enzymes were more stable than the rehydrated cultures and their enzymes. Practical Application. Enzymes and cultures can be added to foods in order to enhance the digestibility of carbohydrates in the gastrointestinal tract. However since many foods are warmed, it is important that the thermostability of the enzymes be assessed. This paper provides data on the stability of α -galactosidase, which could potentially be added to food matrices containing stachyose or raffinose, such as beans.

  7. Assessment of probiotic, antifungal and cholesterol lowering properties of Pediococcus pentosaceus KCC-23 isolated from Italian ryegrass.

    PubMed

    Ilavenil, Soundharrajan; Vijayakumar, Mayakrishnan; Kim, Da Hye; Valan Arasu, Mariadhas; Park, Hyung Su; Ravikumar, Sivanesan; Choi, Ki Choon

    2016-01-30

    Lactic acid bacteria (LAB) are important for the processing of various food products. Although genetically modified organisms have contributed to improvements in various food products, there are some limitations. Thus, the discovery of wild strains from natural sources must be considered as the most suitable approach for identifying new LAB. Therefore, we planned to isolate and characterise the LAB from Italian ryegrass forage and evaluate their biological potential. A total of 28 strains were isolated and screened for their anti-fungal and probiotic properties. A single strain was selected due to its antifungal and probiotic efficiency. The strain was identified as Pediococcus pentosaceus KCC-23. The strain KCC-23 showed effective inhibition against Aspergillus fumigatus, Pencillium chrysogenum, Pencillium roqueforti, Botrytis elliptica and Fusarium oxysporum. Further, it survived low pH, and the presence of bile salts and gastric juice. It exhibited significant aggregation and hydrophobicity properties. The KCC-23 effectively assimilated cholesterol and had the ability to utilise pre-biotics such as raffinose and inulin. Finally, KCC-23 exhibited significant free radical scavenging activity. P. pentosaceus KCC-23 showed effective anti-fungal, probiotic and anti-oxidant properties and would be a promising isolate for exploitation in the formulation of food for ruminants and humans. © 2015 Society of Chemical Industry.

  8. Video imaging analysis of the plasma membrane permeabilizing effects of Bacillus thuringiensis insecticidal toxins in Sf9 cells.

    PubMed

    Villalon, M; Vachon, V; Brousseau, R; Schwartz, J L; Laprade, R

    1998-01-05

    The size and ionic selectivity of the pores formed by the insecticidal crystal protein Cry1C from Bacillus thuringiensis in the plasma membrane of Sf9 cells, an established cell line derived from the fall armyworm Spodoptera frugiperda, were analyzed with a video imaging technique. Changes in the permeability of the membrane were estimated from the rate of osmotic swelling of the cells. In the presence of Cry1C, which is toxic to Sf9 cells, the permeability of the cell membrane to KCl and glucose increased in a dose-dependent manner. In contrast, Cry1Aa, Cry1Ab and Cry1Ac, toxins to which Sf9 cells are not susceptible, had no detectable effect. Pores formed by Cry1C allowed the diffusion of sucrose, but were impermeable to the trisaccharide raffinose. On the basis of the hydrodynamic radii of these substances, the diameter of the pores was estimated to be 1.0-1.2 nm. In the presence of salts, the rate of swelling of cells exposed to Cry1C was about equally influenced by the size of the anion as by that of the cation, indicating that the ionic selectivity of the pores is low.

  9. In situ synthesis of exopolysaccharides by Leuconostoc spp. and Weissella spp. and their rheological impacts in fava bean flour.

    PubMed

    Xu, Yan; Wang, Yaqin; Coda, Rossana; Säde, Elina; Tuomainen, Päivi; Tenkanen, Maija; Katina, Kati

    2017-05-02

    Fava bean flour is regarded as a potential plant-based protein source, but the addition of it at high concentration is restricted by its poor texture-improving ability and by anti-nutritional factors (ANF). Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) are regarded as good texture modifiers. In this study, fava bean flour was fermented with Leuconostoc spp. and Weissella spp. with or without sucrose addition, in order to evaluate their potential in EPS production. The contents of free sugars, organic acids, mannitol and EPS in all fermented fava bean doughs were measured. Rheological properties of sucrose-enriched doughs, including viscosity flow curves, hysteresis loop and dynamic oscillatory sweep curves, were measured after fermentation. As one of the ANF, the degradation of raffinose family oligosaccharides (RFO) was also studied by analyzing RFO profiles of different doughs. Quantification of EPS revealed the potential of Leuconostoc pseudomesenteroides DSM 20193 in EPS production, and the rheological analysis showed that the polymers produced by this strain has the highest thickening and gelling capability. Furthermore, the viscous fava bean doughs containing plant proteins and synthesized in situ EPS may have a potential application in the food industry and fulfill consumers' increasing demands for "clean labels" and plant-originated food materials. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Galactinol synthase transcriptional profile in two genotypes of Coffea canephora with contrasting tolerance to drought.

    PubMed

    Santos, Tiago Benedito Dos; de Lima, Rogério Barbosa; Nagashima, Getúlio Takashi; Petkowicz, Carmen Lucia de Oliveira; Carpentieri-Pípolo, Valéria; Pereira, Luiz Filipe Protasio; Domingues, Douglas Silva; Vieira, Luiz Gonzaga Esteves

    2015-05-01

    Increased synthesis of galactinol and raffinose family oligosaccharides (RFOs) has been reported in vegetative tissues in response to a range of abiotic stresses. In this work, we evaluated the transcriptional profile of a Coffea canephora galactinol synthase gene (CcGolS1) in two clones that differed in tolerance to water deficit in order to assess the contribution of this gene to drought tolerance. The expression of CcGolS1 in leaves was differentially regulated by water deficit, depending on the intensity of stress and the genotype. In clone 109A (drought-susceptible), the abundance of CcGolS1 transcripts decreased upon exposure to drought, reaching minimum values during recovery from severe water deficit and stress. In contrast, CcGolS1 gene expression in clone 14 (drought-tolerant) was stimulated by water deficit. Changes in galactinol and RFO content did not correlate with variation in the steady-state transcript level. However, the magnitude of increase in RFO accumulation was higher in the tolerant cultivar, mainly under severe water deficit. The finding that the drought-tolerant coffee clone showed enhanced accumulation of CcGolS1 transcripts and RFOs under water deficit suggests the possibility of using this gene to improve drought tolerance in this important crop.

  11. Intraspecies differences in cold hardiness, carbohydrate content and β-amylase gene expression of Vaccinium corymbosum during cold acclimation and deacclimation.

    PubMed

    Lee, Jun Hyung; Yu, Duk Jun; Kim, Su Jin; Choi, Doil; Lee, Hee Jae

    2012-12-01

    Changes in cold hardiness, carbohydrate content and β-amylase gene expression were monitored in the shoots of the highbush blueberry (Vaccinium corymbosum L.) cultivars 'Sharpblue' and 'Jersey' during cold acclimation (CA) and deacclimation (DA). The seasonal patterns were similar in both cultivars, but the levels of cold hardiness determined by electrolyte leakage analysis were significantly different; 'Jersey' was hardier than 'Sharpblue'. Cold hardiness was closely related to total soluble sugar content (r = -0.98** and -0.99** for 'Sharpblue' and 'Jersey', respectively). In 'Jersey', more soluble sugars accumulated during CA. Of the detected soluble sugars, glucose, fructose and raffinose contents were significantly associated with cold hardiness in both cultivars. Sucrose was abundant in both cultivars, and stachyose content changed significantly during CA and DA. However, they were not associated with cold hardiness. A sharp decrease in starch contents in the middle of CA coincided with β-amylase gene (VcBMY) expression, indicating the conversion of starch into soluble sugars. During CA, VcBMY was expressed up to twofold higher in 'Jersey' than in 'Sharpblue'. These results suggest that intraspecies differences in the cold hardiness of highbush blueberries are associated with total soluble sugar content, which is driven partly by differential expression of VcBMY.

  12. Transcriptional Analysis of Prebiotic Uptake and Catabolism by Lactobacillus acidophilus NCFM

    PubMed Central

    Andersen, Joakim Mark; Barrangou, Rodolphe; Hachem, Maher Abou; Lahtinen, Sampo J.; Goh, Yong-Jun; Svensson, Birte; Klaenhammer, Todd R.

    2012-01-01

    The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β- linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota. PMID:23028535

  13. Phloem unloading follows an extensive apoplasmic pathway in cucumber (Cucumis sativus L.) fruit from anthesis to marketable maturing stage.

    PubMed

    Hu, Liping; Sun, Huihui; Li, Ruifu; Zhang, Lingyun; Wang, Shaohui; Sui, Xiaolei; Zhang, Zhenxian

    2011-11-01

    The phloem unloading pathway remains unclear in fruits of Cucurbitaceae, a classical stachyose-transporting species with bicollateral phloem. Using a combination of electron microscopy, transport of phloem-mobile symplasmic tracer carboxyfluorescein, assays of acid invertase and sucrose transporter, and [(14)C]sugar uptake, the phloem unloading pathway was studied in cucumber (Cucumis sativus) fruit from anthesis to the marketable maturing stage. Structural investigations showed that the sieve element-companion cell (SE-CC) complex of the vascular bundles feeding fruit flesh is apparently symplasmically restricted. Imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the vascular bundles in the whole fruit throughout the stages examined. A 37 kDa acid invertase was located predominantly in the cell walls of SE-CC complexes and parenchyma cells. Studies of [(14)C]sugar uptake suggested that energy-driven transporters may be functional in sugar trans-membrane transport within symplasmically restricted SE-CC complex, which was further confirmed by the existence of a functional plasma membrane sucrose transporter (CsSUT4) in cucumber fruit. These data provide a clear evidence for an apoplasmic phloem unloading pathway in cucumber fruit. A presumption that putative raffinose or stachyose transporters may be involved in soluble sugars unloading was discussed. © 2011 Blackwell Publishing Ltd.

  14. Effect of cooking time on some nutrient and antinutrient components of bambaragroundnut seeds.

    PubMed

    Omoikhoje, Stanley Omoh; Aruna, Mohammed Bashiru; Bamgbose, Adeyemi Mustapha

    2009-02-01

    The proximate composition, gross energy, mineral composition, percentage sugar, oligosaccharides and antinutrient substances of bambaragroundnut seeds subjected to different cooking times were determined. The seeds were cooked for 30, 60, 90 and 120 min. Results of the proximate analysis showed that only the ether extract and ash were significantly (P < 0.05) reduced as the cooking time increased. In contrast, gross energy values significantly (P < 0.05) increased with increased cooking time. Amongst, the mineral elements assayed, calcium, magnesium and iron were significantly (P < 0.05) increased, while phosphorous, potassium, sodium and copper were reduced significantly (P > 0.05) with inreased cooking time. Percentage sucrose and glucose of bambaragroundnut seeds were significantly (P < 0.05) lowest in the raw form, but increased progressively with increased of cooking time. Raffinose and stachyose levels were reduced significantly by increased cookinf time (P < 0.05) with the least value in seeds cooked for 120 min. Trypsin inhibitor, hemagglutinin and tannin were completely eliminated in seeds cooked for 60 min or longer, but the phytin level was reduced significantly (P < 0.05) by cooking. For a significant detoxification of antinutrient substances and for optimal bioavailability of the component nutrients of bambaragroundnut seeds, an optimum cooking time of 60 min at 100 degrees C is therefore recommended.

  15. Metabolism of Oligosaccharides and Starch in Lactobacilli: A Review

    PubMed Central

    Gänzle, Michael G.; Follador, Rainer

    2012-01-01

    Oligosaccharides, compounds that are composed of 2–10 monosaccharide residues, are major carbohydrate sources in habitats populated by lactobacilli. Moreover, oligosaccharide metabolism is essential for ecological fitness of lactobacilli. Disaccharide metabolism by lactobacilli is well understood; however, few data on the metabolism of higher oligosaccharides are available. Research on the ecology of intestinal microbiota as well as the commercial application of prebiotics has shifted the interest from (digestible) disaccharides to (indigestible) higher oligosaccharides. This review provides an overview on oligosaccharide metabolism in lactobacilli. Emphasis is placed on maltodextrins, isomalto-oligosaccharides, fructo-oligosaccharides, galacto-oligosaccharides, and raffinose-family oligosaccharides. Starch is also considered. Metabolism is discussed on the basis of metabolic studies related to oligosaccharide metabolism, information on the cellular location and substrate specificity of carbohydrate transport systems, glycosyl hydrolases and phosphorylases, and the presence of metabolic genes in genomes of 38 strains of lactobacilli. Metabolic pathways for disaccharide metabolism often also enable the metabolism of tri- and tetrasaccharides. However, with the exception of amylase and levansucrase, metabolic enzymes for oligosaccharide conversion are intracellular and oligosaccharide metabolism is limited by transport. This general restriction to intracellular glycosyl hydrolases differentiates lactobacilli from other bacteria that adapted to intestinal habitats, particularly Bifidobacterium spp. PMID:23055996

  16. Galactinol synthase transcriptional profile in two genotypes of Coffea canephora with contrasting tolerance to drought

    PubMed Central

    Santos, Tiago Benedito Dos; de Lima, Rogério Barbosa; Nagashima, Getúlio Takashi; Petkowicz, Carmen Lucia de Oliveira; Carpentieri-Pípolo, Valéria; Pereira, Luiz Filipe Protasio; Domingues, Douglas Silva; Vieira, Luiz Gonzaga Esteves

    2015-01-01

    Increased synthesis of galactinol and raffinose family oligosaccharides (RFOs) has been reported in vegetative tissues in response to a range of abiotic stresses. In this work, we evaluated the transcriptional profile of a Coffea canephora galactinol synthase gene (CcGolS1) in two clones that differed in tolerance to water deficit in order to assess the contribution of this gene to drought tolerance. The expression of CcGolS1 in leaves was differentially regulated by water deficit, depending on the intensity of stress and the genotype. In clone 109A (drought-susceptible), the abundance of CcGolS1 transcripts decreased upon exposure to drought, reaching minimum values during recovery from severe water deficit and stress. In contrast, CcGolS1 gene expression in clone 14 (drought-tolerant) was stimulated by water deficit. Changes in galactinol and RFO content did not correlate with variation in the steady-state transcript level. However, the magnitude of increase in RFO accumulation was higher in the tolerant cultivar, mainly under severe water deficit. The finding that the drought-tolerant coffee clone showed enhanced accumulation of CcGolS1 transcripts and RFOs under water deficit suggests the possibility of using this gene to improve drought tolerance in this important crop. PMID:26273221

  17. Comparative analysis of the response and gene regulation in cold resistant and susceptible tea plants

    PubMed Central

    Pan, Cheng; Wang, Yiwei; Kong, Lei; Jiang, Huiguang; Xu, Yiqun; Wang, Wenzhi; Pan, Yuting; Li, Yeyun; Jiang, Changjun

    2017-01-01

    Cold environment is the main constraint for tea plants (Camellia sinensis) distribution and tea farming. We identified two tea cultivars, called var. sinensis cv. Shuchazao (SCZ) with a high cold-tolerance and var. assamica cv. Yinghong9 (YH9) with low cold-tolerance. To better understand the response mechanism of tea plants under cold stress for improving breeding, we compared physiological and biochemical responses, and associated genes expression in response to 7-day and 14-day cold acclimation, followed by 7-day de-acclimation in these two tea cultivars. We found that the low EL50, low Fv/Fm, and high sucrose and raffinose accumulation are responsible for higher cold tolerance in SCZ comparing with YH9. We then measured the expression of 14 key homologous genes, known as involved in these responses in other plants, for each stages of treatment in both cultivars using RT-qPCR. Our results suggested that the increased expression of CsCBF1 and CsDHNs coupling with the accumulation of sucrose play key roles in conferring higher cold resistance in SCZ. Our findings have revealed key genes regulation responsible for cold resistance, which help to understand the cold-resistant mechanisms and guide breeding in tea plants. PMID:29211766

  18. Comparative analysis of the response and gene regulation in cold resistant and susceptible tea plants.

    PubMed

    Ban, Qiuyan; Wang, Xuewen; Pan, Cheng; Wang, Yiwei; Kong, Lei; Jiang, Huiguang; Xu, Yiqun; Wang, Wenzhi; Pan, Yuting; Li, Yeyun; Jiang, Changjun

    2017-01-01

    Cold environment is the main constraint for tea plants (Camellia sinensis) distribution and tea farming. We identified two tea cultivars, called var. sinensis cv. Shuchazao (SCZ) with a high cold-tolerance and var. assamica cv. Yinghong9 (YH9) with low cold-tolerance. To better understand the response mechanism of tea plants under cold stress for improving breeding, we compared physiological and biochemical responses, and associated genes expression in response to 7-day and 14-day cold acclimation, followed by 7-day de-acclimation in these two tea cultivars. We found that the low EL50, low Fv/Fm, and high sucrose and raffinose accumulation are responsible for higher cold tolerance in SCZ comparing with YH9. We then measured the expression of 14 key homologous genes, known as involved in these responses in other plants, for each stages of treatment in both cultivars using RT-qPCR. Our results suggested that the increased expression of CsCBF1 and CsDHNs coupling with the accumulation of sucrose play key roles in conferring higher cold resistance in SCZ. Our findings have revealed key genes regulation responsible for cold resistance, which help to understand the cold-resistant mechanisms and guide breeding in tea plants.

  19. Transport behavior of hairless mouse skin during constant current DC iontophoresis, part 2: iontophoresis of nonionic molecules with cotransport of polystyrene sulfonate oligomers.

    PubMed

    Liddell, Mark R; Li, S Kevin; Higuchi, William I

    2011-07-01

    The purpose of this study was to characterize changes that occur in the iontophoretic transport of nonionic probe permeants in hairless mouse skin epidermal membrane from the anode to cathode when polystyrene sulfonate (PSS) oligomers are cotransported from the cathode to anode. The experiments were conducted with trace levels of the nonionic probe permeants: urea, mannitol, and raffinose. In order to systematically assess changes that occur as a result of having PSS in the cathodal chamber, the steady-state transport parameters of the membrane and the experimental permeability coefficients of the probe permeants were determined and compared with results obtained from earlier baseline experiments where both the cathodal and anodal chamber media were phosphate buffered saline. In addition, the physicochemical properties of the PSS solutions were determined including the solution viscosity and conductance as well as the mobilities of individual PSS oligomers. The effective pore radii of the transport pathways were calculated using a theoretical expression based on simultaneous diffusion and electroosmosis. Compared with the baseline results, the calculated radii were found to have increased up to around twofold and the iontophoretic fluxes of the probe permeants increased by as much sixfold. Copyright © 2011 Wiley-Liss, Inc. and the American Pharmacists Association

  20. Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit

    PubMed Central

    Liu, Ling; Sansing, Steven R.; Morse, Iva S.; Pritchett-Corning, Kathleen R.

    2011-01-01

    Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18% raffinose and 3% skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains. Here we demonstrate a newly developed I•Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (> 60%) and rapid progressive motility (> 45%) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I•Cryo kit and later recovered by IVF. PMID:22214993

  1. [Expression and characterization of a neutral Enterobacter cloacae GX-3 invertase].

    PubMed

    Zhao, Yingli; Wu, Qianqian; Zhang, Zhikai; Wang, Zilong; Wei, Yutuo; Huang, Ribo; Du, Liqin

    2015-04-04

    To characterize a neutral invertase from Enterobacter cloacae GX-3. By searching GenBank database, we found the genes encoding invertase from the same genus Enterobacter. These sequences were aligned and analyzed. Then, a gene encoding neutral invertase was amplified by PCR. The recombinant plasmid pQE-Einv was constructed. We purified the expressed protein Einv with nickel-nitrilotriacetic acid chromatography. At last, the characterics of the recombinant protein Einv were studied in detail. A gene encoding neutral invertase was discovered and cloned from E. cloacae GX-3. The recombinant enzyme Einv was characterized. Einv had an optimum pH of 6.5 and an optimum temperature of 40 degrees C. The results of sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and gel permeation chromatography ( GPC) showed that Einv was a homo-dimer protein. Einv retained 80% activity at sucrose concentrations up to 1170 mmol/L. But, Einv had no transglycosylation activity at high sucrose concentration. It could hydrolyze raffinose, 1-kestose, nystose, fructofuranosylnystose and stachyose. It is first reported that an invertase from Enterobacter cloacae is a beta-fructofuranosidase at neutral pH range. It only has hydrolysis activity without tranglycosylation activity. These characteristics indicate that the neutral invertase Einv has important applications in food industry.

  2. Molecular and functional characterization of an invertase secreted by Ashbya gossypii.

    PubMed

    Aguiar, Tatiana Q; Dinis, Cláudia; Magalhães, Frederico; Oliveira, Carla; Wiebe, Marilyn G; Penttilä, Merja; Domingues, Lucília

    2014-06-01

    The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-μm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii.

  3. Thermostability of Probiotics and Their α-Galactosidases and the Potential for Bean Products

    PubMed Central

    Liu, Xiaoli; Champagne, Claude P.; Lee, Byong H.; Boye, Joyce I.; Casgrain, Michel

    2014-01-01

    Soybeans and other pulses contain oligosaccharides which may cause intestinal disturbances such as flatulence. This study was undertaken to investigate α-galactosidase-producing probiotics added to frozen foods which can survive warming treatments used in thawing and consumption of the pulses. The maximum α-galactosidase activity (1.26 U/mg protein) was found in Bifidobacterium breve S46. Lactobacillus casei had the highest α-galactosidase thermostability among the various strains, with D values of 35, 29, and 9.3 minutes at 50°C, 55°C, and 60°C, respectively. The enzyme activity was less affected than viable cells by heating. However, the D values of two bacterial enzymes were lower than those of three commercial α-galactosidase-containing products. Freshly grown cells and their enzymes were more stable than the rehydrated cultures and their enzymes. Practical Application. Enzymes and cultures can be added to foods in order to enhance the digestibility of carbohydrates in the gastrointestinal tract. However since many foods are warmed, it is important that the thermostability of the enzymes be assessed. This paper provides data on the stability of α-galactosidase, which could potentially be added to food matrices containing stachyose or raffinose, such as beans. PMID:24744923

  4. Stachyose synthesis in seeds of adzuki bean (Vigna angularis): molecular cloning and functional expression of stachyose synthase.

    PubMed

    Peterbauer, T; Mucha, J; Mayer, U; Popp, M; Glössl, J; Richter, A

    1999-12-01

    Stachyose is the major soluble carbohydrate in seeds of a number of important crop species. It is synthesized from raffinose and galactinol by the action of stachyose synthase (EC 2.4.1.67). We report here on the identification of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). Based on internal amino acid sequences of the enzyme purified from adzuki bean, oligonucleotides were designed and used to amplify corresponding sequences from adzuki bean cDNA by RT-PCR, followed by rapid amplification of cDNA ends (RACE-PCR). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was amplified by PCR, engineered into the baculovirus expression vector pVL1393 and introduced into Spodoptera frugiperda (Sf21) insect cells for heterologous expression. The recombinant protein was immunologically reactive with polyclonal antibodies raised against stachyose synthase purified from adzuki bean and was shown to be a functional stachyose synthase with the same catalytic properties as its native counterpart. High levels of stachyose synthase mRNA were transiently accumulated midway through seed development, and the enzyme was also present in mature seeds and during germination.

  5. Highly defined 3D printed chitosan scaffolds featuring improved cell growth.

    PubMed

    Elviri, Lisa; Foresti, Ruben; Bergonzi, Carlo; Zimetti, Francesca; Marchi, Cinzia; Bianchera, Annalisa; Bernini, Franco; Silvestri, Marco; Bettini, Ruggero

    2017-07-12

    The augmented demand for medical devices devoted to tissue regeneration and possessing a controlled micro-architecture means there is a need for industrial scale-up in the production of hydrogels. A new 3D printing technique was applied to the automation of a freeze-gelation method for the preparation of chitosan scaffolds with controlled porosity. For this aim, a dedicated 3D printer was built in-house: a preliminary effort has been necessary to explore the printing parameter space to optimize the printing results in terms of geometry, tolerances and mechanical properties of the product. Analysed parameters included viscosity of the starting chitosan solution, which was measured with a Brookfield viscometer, and temperature of deposition, which was determined by filming the process with a cryocooled sensor thermal camera. Optimized parameters were applied to the production of scaffolds from solutions of chitosan alone or with the addition of raffinose as a viscosity modifier. Resulting hydrogels were characterized in terms of morphology and porosity. In vitro cell culture studies comparing 3D printed scaffolds with their homologous produced by solution casting evidenced an improvement in biocompatibility deriving from the production technique as well as from the solid state modification of chitosan stemming from the addition of the viscosity modifier.

  6. Effect of controlled lactic acid fermentation on selected bioactive and nutritional parameters of tempeh obtained from unhulled common bean (Phaseolus vulgaris) seeds.

    PubMed

    Starzyńska-Janiszewska, Anna; Stodolak, Bożena; Mickowska, Barbara

    2014-01-30

    Tempeh is a traditional Indonesian food of high nutritional quality obtained by fungal fermentation of dehulled, soaked and cooked legumes. The aim of this research was to study the effect of Lactobacillus plantarum DSM 20174 activity on selected parameters of tempeh made from unhulled seeds of common bean (Phaseolus vulgaris). Lactobacillus plantarum cells were applied during soaking of seeds (submerged fermentation) or during solid state fermentation with Rhizopus microsporus var. chinensis (co-cultivation). Tempeh obtained from common beans contained 200 g kg⁻¹ protein of 34% in vitro bioavailability. Fungal fermentation caused decomposition of raffinose, stachyose and verbascose levels in seeds, on average by 93, 84 and 73% respectively. Enhanced antiradical (DPPH•, ABTS•+) capacity was accompanied by increased soluble phenol content. Application of Lactobacillus in the fermentation procedure increased tempeh protein and in vitro protein bioavailability by 18 and 17% respectively. Mixed culture tempeh contained lower levels of stachyose (25%), verbascose (64%) and condensed tannins (20%). Co-cultivation enhanced both DPPH•-scavenging activity and antioxidant capacity. The application of Lactobacillus in most cases improved the nutritional parameters of tempeh from unhulled common beans. It may also be recommended to obtain products with diverse antioxidant properties as compared with fungal fermentation alone. © 2013 Society of Chemical Industry.

  7. Seed desiccation mechanisms co-opted for vegetative desiccation in the resurrection grass Oropetium thomaeum.

    PubMed

    VanBuren, Robert; Wai, Ching Man; Zhang, Qingwei; Song, Xiaomin; Edger, Patrick P; Bryant, Doug; Michael, Todd P; Mockler, Todd C; Bartels, Dorothea

    2017-10-01

    Resurrection plants desiccate during periods of prolonged drought stress, then resume normal cellular metabolism upon water availability. Desiccation tolerance has multiple origins in flowering plants, and it likely evolved through rewiring seed desiccation pathways. Oropetium thomaeum is an emerging model for extreme drought tolerance, and its genome, which is the smallest among surveyed grasses, was recently sequenced. Combining RNA-seq, targeted metabolite analysis and comparative genomics, we show evidence for co-option of seed-specific pathways during vegetative desiccation. Desiccation-related gene co-expression clusters are enriched in functions related to seed development including several seed-specific transcription factors. Across the metabolic network, pathways involved in programmed cell death inhibition, ABA signalling and others are activated during dehydration. Oleosins and oil bodies that typically function in seed storage are highly abundant in desiccated leaves and may function for membrane stability and storage. Orthologs to seed-specific LEA proteins from rice and maize have neofunctionalized in Oropetium with high expression during desiccation. Accumulation of sucrose, raffinose and stachyose in drying leaves mirrors sugar accumulation patterns in maturing seeds. Together, these results connect vegetative desiccation with existing seed desiccation and drought responsive pathways and provide some key candidate genes for engineering improved drought tolerance in crop plants. © 2017 John Wiley & Sons Ltd.

  8. Novel process of fermenting black soybean [Glycine max (L.) Merrill] yogurt with dramatically reduced flatulence-causing oligosaccharides but enriched soy phytoalexins.

    PubMed

    Feng, Shengbao; Saw, Chin Lee; Lee, Yuan Kun; Huang, Dejian

    2008-11-12

    Black soybeans [Glycine max (L.) Merrill] were germinated under fungal stress with food grade R. oligosporus for 3 days and were homogenized and fermented with lactic acid bacteria (LAB) to produce soy yogurt. Fungal stress led to the generation of oxylipins [oxooctadecadienoic acids (KODES) isomers and their respective glyceryl esters] and glyceollins--a type of phytoalexins unique to soybeans. In soy yogurt, the concentrations of total KODES and total glyceollins were 0.678 mg/g (dry matter) and 0.953 mg/g, respectively. The concentrations of other isoflavones (mainly genistein and daidzein and their derivatives) in soy yogurt remained largely unchanged after the processes compared with the control soy yogurt. Germination of black soybean under fungal stress for 3 days was sufficient to reduce stachyose and raffinose (which cause flatulence) by 92 and 80%, respectively. With a pH value of 4.42, a lactic acid content of 0.262%, and a maximum viable cell count of 2.1 x 10 (8) CFU/mL in the final soy yogurt, soy milk from germinated soybeans under fungal stress was concluded to be a suitable medium for yogurt-making. The resulting soy yogurt had significantly altered micronutrient profiles with significantly reduced oligosaccharides and enriched glyceollins.

  9. The effect of poorly absorbed solute on intestinal absorption.

    PubMed

    Menzies, I S; Jenkins, A P; Heduan, E; Catt, S D; Segal, M B; Creamer, B

    1990-12-01

    To determine the effects of poorly absorbed solute on intestinal absorption, the urinary recovery of ingested lactulose, L-rhamnose, D-xylose, and 3-O-methyl-D-glucose was measured after simultaneous ingestion of various 'loads' of mannitol given in iso-osmolar solution. Mannitol reduced intestinal uptake of the poorly absorbed test sugars, lactulose and L-rhamnose; uptake of D-xylose and 3-O-methyl-D-glucose, which are absorbed by carrier-mediated transport largely from the jejunum, was less affected. The dose-response effect of mannitol on the absorption of L-rhamnose was approximately exponential; doses of 5, 10, and 20 g mannitol reduced the average urinary excretion of L-rhamnose by 34.7%, 51.7%, and 61.2%, respectively. In this respect, an osmotically equivalent load of lactulose, ingested as 'solute', was approximately twice as effective as mannitol in reducing L-rhamnose absorption, probably because lactulose is more poorly absorbed than mannitol (less than 1.0% versus 32-41%). Ingestion of other poorly absorbed solutes such as raffinose, sorbitol, xylitol, magnesium sulphate, and sodium sulphate also significantly depressed the absorption of L-rhamnose; in contrast, more efficiently absorbed solutes, such as sodium chloride, glucose, glycerol, and urea had little effect.

  10. First results from a novel methodological approach for δ18O analyses of sugars using GC-Py-IRMS

    NASA Astrophysics Data System (ADS)

    Zech, Michael; Saurer, Matthias; Tuthorn, Mario; Rinne, Katja; Werner, Roland; Juchelka, Dieter; Siegwolf, Rolf; Glaser, Bruno

    2013-04-01

    Although the instrumental coupling of gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-Py-IRMS) for compound-specific δ18O analyses is commercially available for more than 10 years, this method is hardly applied by isotope researchers so far. Using GC-Py-IRMS, Zech and Glaser (2009) and Zech et al. (2013; 2012) developed and applied a method, which allows determining δ18O of hemicellulose-derived sugar biomarkers extracted from soils and sediments. However, the used methylboronic acid (MBA) derivatization is suitable only for pentoses and deoxyhexoses, not for hexoses. Here we present first GC-Py-IRMS results for TMS-(trimethylsilyl)-derivatives of plant sap-relevant sugars (glucose, fucose, sucrose, raffinose) and a polyalkohol (pinitol) produced using BSTFA (N,O-Bis(trimethylsilyl)trifluoroacetamide) as the derivatization reagent. Particularly, we focus on sucrose, which is the most important transport sugar in plants and hence of utmost relevance in plant physiology and in tree-ring studies. Replicate analyses of sucrose standards with known δ18O values suggest that the δ18O measurements are not stable over several days. A calibration (including a drift correction) against an external sucrose standard is hence essential when measuring sample batches. Furthermore, we observed a large dependence of the δ18O values on the analyte amount (area), which needs to be considered by a respective correction procedure. Tests with 18O-enriched water do not provide any evidence for oxygen exchange reactions between water and sucrose, glucose and raffinose. Finally we present the first application of compound-specific δ18O analyses from natural samples, namely from seven needle extracts (soluble carbohydrates) from a Siberian study area. Both the δ18O amplitude and values of sucrose are considerably higher (32.1‰ to 40.1‰) compared to the δ18O amplitude and values of bulk needle extract (24.6‰ to 27.2‰). We found positive correlation (although

  11. Overexpression of the Arabidopsis CBF3 Transcriptional Activator Mimics Multiple Biochemical Changes Associated with Cold Acclimation1

    PubMed Central

    Gilmour, Sarah J.; Sebolt, Audrey M.; Salazar, Maite P.; Everard, John D.; Thomashow, Michael F.

    2000-01-01

    We further investigated the role of the Arabidopsis CBF regulatory genes in cold acclimation, the process whereby certain plants increase in freezing tolerance upon exposure to low temperature. The CBF genes, which are rapidly induced in response to low temperature, encode transcriptional activators that control the expression of genes containing the C-repeat/dehydration responsive element DNA regulatory element in their promoters. Constitutive expression of either CBF1 or CBF3 (also known as DREB1b and DREB1a, respectively) in transgenic Arabidopsis plants has been shown to induce the expression of target COR (cold-regulated) genes and to enhance freezing tolerance in nonacclimated plants. Here we demonstrate that overexpression of CBF3 in Arabidopsis also increases the freezing tolerance of cold-acclimated plants. Moreover, we show that it results in multiple biochemical changes associated with cold acclimation: CBF3-expressing plants had elevated levels of proline (Pro) and total soluble sugars, including sucrose, raffinose, glucose, and fructose. Plants overexpressing CBF3 also had elevated P5CS transcript levels suggesting that the increase in Pro levels resulted, at least in part, from increased expression of the key Pro biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthase. These results lead us to propose that CBF3 integrates the activation of multiple components of the cold acclimation response. PMID:11115899

  12. Comparative metabolomic analysis of seed metabolites associated with seed storability in rice (Oryza sativa L.) during natural aging.

    PubMed

    Yan, Shijuan; Huang, Wenjie; Gao, Jiadong; Fu, Hua; Liu, Jun

    2018-06-01

    Seed storability is an important trait for crop breeding, however, the mechanism underlying seed storability remains largely unknown. Here, a mass spectrometry-based comparative metabolomic study was performed for rice seeds before and after 24-month natural storage between two hybrid rice cultivars, IIYou 998 (IIY) with low storability and BoYou 998 (BY) with relative high storability. A total of 48 metabolites among 90 metabolite peaks detected were conclusively identified, and most of them are involved in the primary metabolism. During the 24-month storage, 19 metabolites with significant changes in abundance were found in the storage-sensitive IIY seeds, but only 8 in the BY seeds, most of which are free amino acids and soluble sugars. The observed changes of the metabolites in IIY seeds that are consistent with our protoemics results are likely to be involved in its sensitivity to storage. Levels of all identified 18 amino acid-related metabolites and most sugar-related metabolites were significantly higher in IIY seeds both before and after storage. However the level of raffinose was lower in IIY seeds before and after storage, and did not change significantly throughout the storage period in both two cultivars, suggesting its potential role in seed storability. Taken together, these results may help to improve our understanding of seed storability. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  13. Effect of photoperiod prior to cold acclimation on freezing tolerance and carbohydrate metabolism in alfalfa (Medicago sativa L.).

    PubMed

    Bertrand, Annick; Bipfubusa, Marie; Claessens, Annie; Rocher, Solen; Castonguay, Yves

    2017-11-01

    Cold acclimation proceeds sequentially in response to decreases in photoperiod and temperature. This study aimed at assessing the impact of photoperiod prior to cold acclimation on freezing tolerance and related biochemical and molecular responses in two alfalfa cultivars. The fall dormant cultivar Evolution and semi-dormant cultivar 6010 were grown in growth chambers under different photoperiods (8, 10, 12, 14 or 16h) prior to cold acclimation. Freezing tolerance was evaluated as well as carbohydrate concentrations, levels of transcripts encoding enzymes of carbohydrate metabolism as well as a K-3dehydrin, before and after cold acclimation. The fall dormant cultivar Evolution had a better freezing tolerance than the semi-dormant cultivar 6010. The effect of photoperiod prior to cold acclimation on the level of freezing tolerance differed between the two cultivars: an 8h-photoperiod induced the highest level of freezing tolerance in Evolution and the lowest in 6010. In Evolution, the 8h-induced superior freezing tolerance was associated with higher concentration of raffinose-family oligosaccharides (RFO). The transcript levels of sucrose synthase (SuSy) decreased whereas those of sucrose phosphatase synthase (SPS) and galactinol synthase (GaS) increased in response to cold acclimation in both cultivars. Our results indicate that RFO metabolism could be involved in short photoperiod-induced freezing tolerance in dormant alfalfa cultivars. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  14. Feeding response of Ips paraconfusus to phloem and phloem metabolites of Heterobasidion annosum-inoculated ponderosa pine, Pinus ponderosa.

    PubMed

    McNee, William R; Bonello, Pierluigi; Storer, Andrew J; Wood, David L; Gordon, Thomas R

    2003-05-01

    In studies of feeding by the bark beetle, Ips paraconfusus, two pine stilbenes (pinosylvin and pinosylvin methyl ether), ferulic acid glucoside, and enantiomers of the four most common sugars present in ponderosa pine phloem (sucrose, glucose, fructose, and raffinose) did not stimulate or reduce male feeding when assayed on wet alpha-cellulose with or without stimulatory phloem extractives present. When allowed to feed on wet alpha-cellulose containing sequential extracts (hexane, methanol, and water) of ponderosa pine phloem, methanol and water extractives stimulated feeding, but hexane extractives did not. Males confined in wet alpha-cellulose containing aqueous or organic extracts of culture broths derived from phloem tissue and containing the root pathogen. Heterobasidion annosum, ingested less substrate than beetles confined to control preparations. In an assay using logs from uninoculated ponderosa pines, the mean lengths of phloem in the digestive tracts increased as time spent feeding increased. Males confined to the phloem of basal logs cut from ponderosa pines artificially inoculated with H. annosum ingested significantly less phloem than beetles in logs cut from trees that were (combined) mock-inoculated or uninoculated and did not contain the pathogen. However, individual pathogen-containing treatments were not significantly different from uninoculated controls. It was concluded that altered feeding rates are not a major factor which may explain why diseased ponderosa pines are colonized by I. paraconfusus.

  15. Change in oligosaccharides during processing of soybean sheet.

    PubMed

    Wang, Qiushuang; Ke, Leqin; Yang, Dongmei; Bao, Bili; Jiang, Jianmei; Ying, Tiejin

    2007-01-01

    Oligosaccharides have been credited with many health-promoting functions, which had been identified in many clinical studies, such as promoting the growth of Bifidobacterium in human intestine and balance of intestinal bacteria, modulating the immune response, inhibition of cancer and tumor, stimulation of mineral absorption. In this study the effect of processing unit operations on the levels of soybean oligosaccharides during production of soybean sheet were investigated. The concentrations of oligosaccharide in initial raw soybean were: sucrose 43.05 g/kg, raffinose 7.52 g/kg and stachyose 41.32 g/kg (in dry matter). Oligosaccharide losses in the soaking water, in the first filtrating stage, in the second filtrating stage and finally in the sheet formation stage were 0.68, 10.3, 8.15 and 47.22 g/kg (initial dry soybean) respectively, representing 0.74, 11.21, 8.87 and 51.39% of the total oligosaccharides present in the initial soybeans. The recovery of oligosaccharides in the final soybean sheet from the initial soybean was 27.92%. The loss of soybean oligosaccharides in different processing stages, especially in the by-product, the sweet slurry, was considerable. The loss of oligosaccharides was mainly associated with water/matter removal in production process. The analysis of loss profile implied possible ways to improve the technology for production of oligosaccharides-enriched soy-sheets.

  16. Effects of two low phytic acid mutations on seed quality and nutritional traits in soybean (Glycine max L. Merr).

    PubMed

    Yuan, Feng-Jie; Zhu, Dan-Hua; Deng, Bo; Fu, Xu-Jun; Dong, De-Kun; Zhu, Shen-Long; Li, Bai-Quan; Shu, Qing-Yao

    2009-05-13

    Reduction of phytic acid in soybean seeds has the potential to improve the nutritional value of soybean meal and lessen phosphorus pollution in large scale animal farming. The objective of this study was to assess the effect of two new low phytic acid (LPA) mutations on seed quality and nutritional traits. Multilocation/season comparative analyses showed that the two mutations did not affect the concentration of crude protein, any of the individual amino acids, crude oil, and individual saturated fatty acids. Among other traits, Gm-lpa-TW75-1 had consistently higher sucrose contents (+47.4-86.1%) and lower raffinose contents (-74.2 to -84.3%) than those of wild type (WT) parent Taiwan 75; Gm-lpa-ZC-2 had higher total isoflavone contents (3038.8-4305.4 microg/g) than its parent Zhechun # 3 (1583.6-2644.9 microg/g) in all environments. Further tests of homozygous F(3) progenies of the cross Gm-lpa-ZC-2 x Wuxing # 4 (WT variety) showed that LPA lines had a mean content of total isoflavone significantly higher than WT lines. This study demonstrated that two LPA mutant genes have no negative effects on seed quality and nutritional traits; they instead have the potential to improve a few other properties. Therefore, these two mutant genes are valuable genetic resources for breeding high quality soybean varieties.

  17. Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean).

    PubMed

    Wong, Jack H; Wan, Chung T; Ng, Tzi B

    2010-01-15

    A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity. Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells. Copyright (c) 2009 Society of Chemical Industry.

  18. Sporothrix brunneoviolacea and Sporothrix dimorphospora, two new members of the Ophiostoma stenoceras-Sporothrix schenckii complex.

    PubMed

    Madrid, H; Gené, J; Cano, J; Silvera, C; Guarro, J

    2010-01-01

    Sporothrix inflata is a saprobic member of the Ophiostoma stenoceras-Sporothrix schenckii species complex, reported mainly from soil. Ophiostoma bragantinum, an ascomycete described from Brazil, has been proposed as its possible teleomorph. Previous studies revealed that Sporothrix inflata is phenotypically and genetically variable, suggesting the existence of cryptic species. During a continued survey on the biodiversity of microfungi from different countries, seven isolates morphologically similar to S. inflata were obtained from soil samples collected in Spain and USA. In this study their phenotypic features and phylogenetic relationships were assessed. DNA sequence data of two nuclear loci revealed that these isolates correspond to two unnamed clades in S. inflata s.l., one of which also included the type strain of Humicola dimorphospora, a species that traditionally has been considered a synonym of S. inflata. These two groups are proposed herein as Sporothrix brunneoviolacea sp. nov. and Sporothrix dimorphospora comb. nov. S. brunneoviolacea is characterized phenotypically by the production of a diffusible violet-brown pigment in culture and mostly globose, pigmented, lateral blastoconidia. On the other hand S. dimorphospora lacks diffusible pigments and shows mostly subglobose to obovoid pigmented lateral blastoconidia. In contrast to the type strain of S. inflata S. brunneoviolacea and S. dimorphospora assimilate raffinose. The phylogenetic analysis suggested that the proposed anamorph-teleomorph connection between S. inflata and O. bragantinum might not be correct.

  19. Validation of an LC-MS/MS Method for Urinary Lactulose and Mannitol Quantification: Results in Patients with Irritable Bowel Syndrome.

    PubMed

    Gervasoni, Jacopo; Schiattarella, Arcangelo; Giorgio, Valentina; Primiano, Aniello; Russo, Consuelo; Tesori, Valentina; Scaldaferri, Franco; Urbani, Andrea; Zuppi, Cecilia; Persichilli, Silvia

    2016-01-01

    Aim . Lactulose/mannitol ratio is used to assess intestinal barrier function. Aim of this work was to develop a robust and rapid method for the analysis of lactulose and mannitol in urine by liquid chromatography coupled to tandem mass spectrometry. Lactulose/mannitol ratio has been measured in pediatric patients suffering from irritable bowel syndrome. Methods . Calibration curves and raffinose, used as internal standard, were prepared in water : acetonitrile 20 : 80. Fifty μ L of urine sample was added to 450  μ L of internal standard solution. The chromatographic separation was performed using a Luna NH 2 column operating at a flow rate of 200  μ L/min and eluted with a linear gradient from 20% to 80% water in acetonitrile. Total run time is 9 minutes. The mass spectrometry operates in electrospray negative mode. Method was fully validated according to European Medicine Agency guidelines. Results and Conclusions . Linearity ranged from 10 to 1000 mg/L for mannitol and 2.5 to 1000 mg/L for lactulose. Imprecision in intra- and interassay was lower than 15% for both analytes. Accuracy was higher than 85%. Lactulose/mannitol ratio in pediatric patients is significantly higher than that measured in controls. The presented method, rapid and sensitive, is suitable in a clinical laboratory.

  20. Protection by naringin and some other flavonoids of hepatocytic autophagy and endocytosis against inhibition by okadaic acid.

    PubMed

    Gordon, P B; Holen, I; Seglen, P O

    1995-03-17

    In isolated rat hepatocytes, the protein phosphatase inhibitor okadaic acid exerts a strong inhibitory effect on autophagy, which can be partially overcome by certain protein kinase inhibitors like the isoflavone genistein. To see if other, more specific okadaic acid antagonists could be found among the flavonoids, 55 different flavonoids were tested for their effect on okadaic acid-inhibited autophagy, measured as the sequestration of electroinjected [3H]raffinose. Naringin (naringenin 7-hesperidoside) and several other flavanone and flavone glycosides (prunin, neoeriocitrin, neohesperidin, apiin, rhoifolin, kaempferol 3-rutinoside) offered virtually complete protection against the autophagy-inhibitory effect of okadaic acid. Unlike genistein, these compounds had little or no autophagy-inhibitory effect of their own. Their innocuousness appeared to be related to glycosylation, because the corresponding aglycones (naringenin, eriodictyol, hesperetin, apigenin, kaempferol) were all inhibitory, in particular apigenin (80% inhibition at 100 microM). Naringin, the most potent okadaic acid-antagonistic flavonoid, gave half-maximal protection at 5 microM and maximal effect at 100 microM. Naringin also prevented the okadaic acid-induced inhibition of endogenous, autophagic lysosomal protein degradation and of receptor-mediated asialoglycoprotein uptake and degradation. Naringin and other okadaic acid-antagonistic flavonoids may be useful tools in the study of intracellular protein phosphorylation and could have potential therapeutic value as protectants against pathological hyperphosphorylations, environmental toxins, or side effects of chemotherapeutic drugs.

  1. Studies on the incorporation of velvet bean (Mucuna pruriens var. utilis) as an alternative protein source in poultry feed and its effect on growth performance of broiler chickens.

    PubMed

    Vadivel, Vellingiri; Pugalenthi, Muthiah

    2010-10-01

    The effect of replacement of soybean meal by the velvet bean meal as an alternative protein ingredient on the growth performance of broiler chickens was investigated. The raw seeds of velvet bean [Mucuna pruriens (L.) DC. var. utilis (Wall. ex Wight) Baker ex Burck], an under-utilized food legume collected from South India, was found to contain appreciable levels of crude protein (273.2 g/kg DM), lipid (60.61 g/kg DM), neutral detergent fiber (84.3 g/kg DM), and ash content (56.04 g/kg DM). Soaking in 0.2% sodium bicarbonate solution + autoclaving treatment caused a substantial reduction on the levels of various antinutritional compounds such as tannins (84%), L: -Dopa (79%), phytic acid (87%), raffinose (93%), stachyose (83%), verbascose (73%), haemagglutinating activity (84%), trypsin inhibitor activity (77%), and alpha-amylase inhibitor activity (78%) without affecting the nutritional quality of velvet bean seeds. The processed velvet bean meal was incorporated as an alternative protein source by replacing soybean meal protein at 0, 20%, 40%, 60%, 80%, and 100% levels in the broiler diets. Replacement of soybean meal protein up to 40% level, which corresponds to the inclusion of velvet bean meal up to 15.7% and 11% in the starter and finisher phase poultry feeds, respectively, exhibited better growth performance of broiler birds without any adverse effects.

  2. Contrasting strategies used by lichen microalgae to cope with desiccation-rehydration stress revealed by metabolite profiling and cell wall analysis.

    PubMed

    Centeno, Danilo C; Hell, Aline F; Braga, Marcia R; Del Campo, Eva M; Casano, Leonardo M

    2016-05-01

    Most lichens in general, and their phycobionts in particular, are desiccation tolerant, but their mechanisms of desiccation tolerance (DT) remain obscure. The physiological responses and cell wall features of two putatively contrasting lichen-forming microalgae, Trebouxia sp. TR9 (TR9), isolated from Ramalina farinacea (adapted to frequent desiccation-rehydration cycles), and Coccomyxa solorina-saccatae (Csol), obtained from Solorina saccata (growing in usually humid limestone crevices, subjected to seasonal dry periods) was characterized. Microalgal cultures were desiccated under 25%-30% RH and then rehydrated. Under these conditions, RWC and ψw decreased faster and simultaneously during dehydration in Csol, whereas TR9 maintained its ψw until 70% RWC. The metabolic profile indicated that polyols played a key role in DT of both microalgae. However, TR9 constitutively accumulated higher amounts of polyols, whereas Csol induced the polyol synthesis under desiccation-rehydration. Csol also accumulated ascorbic acid, while TR9 synthesized protective raffinose-family oligosaccharides (RFOs) and increased its content of phenolics. Additionally, TR9 exhibited thicker and qualitatively different cell wall and extracellular polymeric layer compared with Csol, indicating higher water retention capability. The findings were consistent with the notion that lichen microalgae would have evolved distinct strategies to cope with desiccation-rehydration stress in correspondence with the water regime of their respective habitats. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  3. Use of yeast spores for microencapsulation of enzymes.

    PubMed

    Shi, Libing; Li, Zijie; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

    2014-08-01

    Here, we report a novel method to produce microencapsulated enzymes using Saccharomyces cerevisiae spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the α-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher α-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose.This suggests that substrate selectivity could be altered by the encapsulation.

  4. Changes in nutrient and antinutrient composition of Vigna racemosa flour in open and controlled fermentation.

    PubMed

    Difo, V H; Onyike, E; Ameh, D A; Njoku, G C; Ndidi, U S

    2015-09-01

    This study was conducted to investigate the effect of open and controlled fermentation on the proximate composition, mineral elements, antinutritional factors and flatulence-causing oligosaccharides in Vigna racemosa. The open fermentation was carried out using the microorganisms present in the atmosphere while the controlled fermentation was carried out using Aspergillus niger as a starter. The proximate composition of the Vigna racemosa, some anti-nutrients and the mineral elements were analyzed using standard procedures. The protein content was increased by 12.41 ± 1.73 % during open fermentation while it decreased by 29.42 ± 0.1 % during controlled fermentation. The lipids, carbohydrates, crude fibre and ash content were all reduced in both types of fermentation except the moisture content which increased in controlled fermentation. Apart from calcium, the other elements (Fe, Na, Mg, Zn, and K) suffered reduction in both types of fermentation. The phytate, tannin, alkaloids, hydrogen cyanide, lectins, trypsin inhibitors and oxalate content all had drastic reductions in both types of fermentation. Open and controlled fermentation reduced the levels of both raffinose and stachyose. The percentages of reduction due to controlled fermentation were higher than those of open fermentation in the antinutrients studied. Fermentation is an efficient method for detoxifying the antinutrients in the Vigna racemosa studied in this work.

  5. NMR Quantification of Carbohydrates in Complex Mixtures. A Challenge on Honey.

    PubMed

    Schievano, Elisabetta; Tonoli, Marco; Rastrelli, Federico

    2017-12-19

    The knowledge of carbohydrate composition is greatly important to determine the properties of natural matrices such as foodstuff and food ingredients. However, because of the structural similarity and the multiple isomeric forms of carbohydrates in solution, their analysis is often a complex task. Here we propose an NMR analytical procedure based on highly selective chemical shift filters followed by TOCSY, which allows us to acquire specific background-free signals for each sugar. The method was tested on raw honey samples dissolved in water with no other pretreatment. In total, 22 sugars typically found in honey were quantified: 4 monosaccharides (glucose, fructose, mannose, rhamnose), 11 disaccharides (sucrose, trehalose, turanose, maltose, maltulose, palatinose, melibiose and melezitose, isomaltose, gentiobiose nigerose, and kojibiose), and 7 trisaccharides (raffinose, isomaltotriose, erlose, melezitose, maltotriose, panose, and 1-kestose). Satisfactory results in terms of limit of quantification (0.03-0.4 g/100g honey), precision (% RSD: 0.99-4.03), trueness (bias % 0.4-4.2), and recovery (97-104%) were obtained. An accurate control of the instrumental temperature and of the sample pH endows an optimal chemical shift reproducibility, making the procedure amenable to automation and suitable to routine analysis. While validated on honey, which is one of the most complex natural matrices in terms of saccharides composition, this innovative approach can be easily transferred to other natural matrices.

  6. Effect of stachyose supplementation on growth performance, nutrient digestibility and caecal fermentation characteristics in broilers.

    PubMed

    Jiang, H Q; Gong, L M; Ma, Y X; He, Y H; Li, D F; Zhai, H X

    2006-08-01

    1. The objective of this study was to evaluate whether the oligosaccharide stachyose enhances gastrointestinal tract health by fermentation and proliferation of desirable bacteria species and thus affects growth performance and nutrient digestibility in broilers. 2. A total of 432 1-d-old male Arbor Acres (AA) broilers were randomly allocated to one of 6 treatments, with 12 replicate pens per treatment and 6 birds per pen. Chicks were fed a maize-hamlet protein 300 (HP300) basal diet with 0, 4.0, 8.0, 12.0 or 16.0 g/kg stachyose. A sixth diet contained no HP300 but soybean meal (SBM) and provided 8.7 g/kg stachyose and 3.1 g/kg raffinose. The duration of the study was 42 d. 3. Stachyose contents above 12.0 g/kg depressed group body weights, average daily gain and feed/gain but not feed intake during the whole experimental period. Broiler growth decreased linearly and quadratically with increasing stachyose content. No differences were detected between diets supplemented with 12.0 g/kg stachyose and SBM. 4. Nutrient digestibility tended to decrease but not significantly with increasing stachyose. 5. Stachyose content had no significant positive effects on caecal pH, microflora population and the resulting short-chain fatty acid (SCFA) metabolites during the 42 d experiment, with only butyrate differing significantly in the initial period.

  7. Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing

    PubMed Central

    Zhou, Xiaojin; Zhang, Wei; Xu, Xiaolu; Chen, Rumei; Meng, Qingchang; Yuan, Jianhua; Yang, Peilong; Yao, Bin

    2015-01-01

    Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50°C) and lower stability over alkaline pH range, but better thermal stability at 60°C to 70°C and resistance to feed pelleting inactivation (80°C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing. PMID:26053048

  8. Preservation of sperm within the mouse cauda epididymidis in salt or sugars at room temperature.

    PubMed

    Ono, Tetsuo; Mizutani, Eiji; Li, Chong; Wakayama, Teruhiko

    2010-08-01

    The development of preservation techniques for male gametes at room temperature might allow us to store them in a simple and cost-effective manner. In this study, we studied the use of pure salt or sugar to preserve the whole cauda epididymidis, because it is known that food can be preserved in this way at room temperature for long periods. Mouse epididymides were placed directly in powdered salt (NaCl) or sugars (glucose or raffinose) for 1 day to 1 year at room temperature. Spermatozoa were recovered from the preserved organs after being rehydrated with medium and then isolated sperm heads were microinjected into fresh oocytes. Importantly, the oocyte activation capacity of spermatozoa was maintained after epididymal storage in NaCl for 1 year, whereas most untreated spermatozoa failed to activate oocytes within 1 month of storage. Pronuclear morphology, the rate of extrusion of a second polar body and the methylation status of histone H3 lysine 9 (H3K9me3) in those zygotes were similar to those of zygotes fertilized with fresh spermatozoa. However, the developmental ability of the zygotes decreased within 1 day of sperm storage. This effect led to nuclear fragmentation at the 2-cell embryo stage, irrespective of the storage method used. Thus, although the preserved sperm failed to allow embryo development, their oocyte activation factors were maintained by salt storage of the epididymis for up to 1 year at room temperature.

  9. Altering carbon allocation in hybrid poplar ( Populus alba × grandidentata ) impacts cell wall growth and development

    SciTech Connect

    Unda, Faride; Kim, Hoon; Hefer, Charles

    Galactinol synthase is a pivotal enzyme involved in the synthesis of the raffinose family of oligosaccharides (RFOs) that function as transport carbohydrates in the phloem, as storage compounds in sink tissues and as soluble metabolites that combat both abiotic and biotic stress in several plant species. For hybrid poplar (Populus alba 9 grandidentata) overexpressing the Arabidopsis thaliana GolS3 (AtGolS3) gene showed clear effects on development; the extreme overexpressing lines were stunted and had cell wall traits characteristic of tension wood, whereas lines with only moderate up-regulation grew normally and had moderately altered secondary cell wall composition and ultrastructure. Stem cross-sectionsmore » of the developing xylem revealed a significant increase in the number of vessels, as well as the clear presence of a G-layer in the fibres. Furthermore, AtGolS3-OE lines possessed higher cellulose and lower lignin contents, an increase in cellulose crystallinity, and significantly altered hemicellulose-derived carbohydrates, notably manifested by their mannose and xylose contents. Additionally, the transgenic plants displayed elevated xylem starch content. Transcriptome interrogation of the transgenic plants showed a significant up-regulation of genes involved in the synthesis of myo-inositol, along with genes involved in sucrose degradation. Our results suggest that the over expression of GolS and its product galactinol may serve as a molecular signal that initiates metabolic changes, culminating in a change in cell wall development and potentially the formation of tension wood.« less

  10. Altering carbon allocation in hybrid poplar ( Populus alba × grandidentata ) impacts cell wall growth and development

    DOE PAGES

    Unda, Faride; Kim, Hoon; Hefer, Charles; ...

    2017-03-04

    Galactinol synthase is a pivotal enzyme involved in the synthesis of the raffinose family of oligosaccharides (RFOs) that function as transport carbohydrates in the phloem, as storage compounds in sink tissues and as soluble metabolites that combat both abiotic and biotic stress in several plant species. For hybrid poplar (Populus alba 9 grandidentata) overexpressing the Arabidopsis thaliana GolS3 (AtGolS3) gene showed clear effects on development; the extreme overexpressing lines were stunted and had cell wall traits characteristic of tension wood, whereas lines with only moderate up-regulation grew normally and had moderately altered secondary cell wall composition and ultrastructure. Stem cross-sectionsmore » of the developing xylem revealed a significant increase in the number of vessels, as well as the clear presence of a G-layer in the fibres. Furthermore, AtGolS3-OE lines possessed higher cellulose and lower lignin contents, an increase in cellulose crystallinity, and significantly altered hemicellulose-derived carbohydrates, notably manifested by their mannose and xylose contents. Additionally, the transgenic plants displayed elevated xylem starch content. Transcriptome interrogation of the transgenic plants showed a significant up-regulation of genes involved in the synthesis of myo-inositol, along with genes involved in sucrose degradation. Our results suggest that the over expression of GolS and its product galactinol may serve as a molecular signal that initiates metabolic changes, culminating in a change in cell wall development and potentially the formation of tension wood.« less

  11. Flower abscission in Vitis vinifera L. triggered by gibberellic acid and shade discloses differences in the underlying metabolic pathways

    PubMed Central

    Domingos, Sara; Scafidi, Pietro; Cardoso, Vania; Leitao, Antonio E.; Di Lorenzo, Rosario; Oliveira, Cristina M.; Goulao, Luis F.

    2015-01-01

    Understanding abscission is both a biological and an agronomic challenge. Flower abscission induced independently by shade and gibberellic acid (GAc) sprays was monitored in grapevine (Vitis vinifera L.) growing under a soilless greenhouse system during two seasonal growing conditions, in an early and late production cycle. Physiological and metabolic changes triggered by each of the two distinct stimuli were determined. Environmental conditions exerted a significant effect on fruit set as showed by the higher natural drop rate recorded in the late production cycle with respect to the early cycle. Shade and GAc treatments increased the percentage of flower drop compared to the control, and at a similar degree, during the late production cycle. The reduction of leaf gas exchanges under shade conditions was not observed in GAc treated vines. The metabolic profile assessed in samples collected during the late cycle differently affected primary and secondary metabolisms and showed that most of the treatment-resulting variations occurred in opposite trends in inflorescences unbalanced in either hormonal or energy deficit abscission-inducing signals. Particularly concerning carbohydrates metabolism, sucrose, glucose, tricarboxylic acid metabolites and intermediates of the raffinose family oligosaccharides pathway were lower in shaded and higher in GAc samples. Altered oxidative stress remediation mechanisms and indolacetic acid (IAA) concentration were identified as abscission signatures common to both stimuli. According to the global analysis performed, we report that grape flower abscission mechanisms triggered by GAc application and C-starvation are not based on the same metabolic pathways. PMID:26157448

  12. Moorella stamsii sp. nov., a new anaerobic thermophilic hydrogenogenic carboxydotroph isolated from digester sludge.

    PubMed

    Alves, J I; van Gelder, A H; Alves, M M; Sousa, D Z; Plugge, C M

    2013-11-01

    A novel anaerobic, thermophilic, carbon monoxide-utilizing bacterium, strain E3-O(T), was isolated from anaerobic sludge from a municipal solid waste digester. Cells were straight rods, 0.6-1 µm in diameter and 2-3 µm in length and grew as single cells or in pairs. Cells formed round terminal endospores. The temperature range for growth was 50-70 °C, with an optimum at 65 °C. The pH range for growth was 5.7-8.0, with an optimum at 7.5. Strain E3-O(T) had the ability to ferment various sugars, such as fructose, galactose, glucose, mannose, raffinose, ribose, sucrose and xylose, producing mainly H2 and acetate. In addition, the isolate was able to grow with CO as the sole carbon and energy source. CO oxidation was coupled to H2 and CO2 formation. The G+C content of the genomic DNA was 54.6 mol%. Based on 16S rRNA gene sequence analysis, this bacterium is most closely related to Moorella glycerini (97 % sequence identity). Based on the physiological features and phylogenetic analysis, it is proposed that strain E3-O(T) should be classified in the genus Moorella as a representative of a novel species, Moorella stamsii. The type strain of Moorella stamsii is E3-O(T) ( = DSM 26271(T) = CGMCC 1.5181(T)).

  13. Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing.

    PubMed

    Yang, Wenxia; Zhang, Yuhong; Zhou, Xiaojin; Zhang, Wei; Xu, Xiaolu; Chen, Rumei; Meng, Qingchang; Yuan, Jianhua; Yang, Peilong; Yao, Bin

    2015-01-01

    Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50 °C) and lower stability over alkaline pH range, but better thermal stability at 60 °C to 70 °C and resistance to feed pelleting inactivation (80 °C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing.

  14. Cowpea fresh pods - a new legume for the market: assessment of their quality and dietary characteristics of 37 cowpea accessions grown in southern Europe.

    PubMed

    Karapanos, Ioannis; Papandreou, Anastasia; Skouloudi, Marianna; Makrogianni, Despoina; Fernández, Juan A; Rosa, Eduardo; Ntatsi, Georgia; Bebeli, Penelope J; Savvas, Dimitrios

    2017-10-01

    Cowpea is traditionally cultivated in some regions of southern Europe for its dried seeds; however, there is a scarcity of information on the quality and dietary characteristics of fresh pods, which are occasionally used in folk diets. This paper aims at covering this gap in knowledge, thereby contributing to the dissemination of fresh cowpea pods as a novel product for the market. The quality and dietary characteristics of pods from 37 accessions (Vigna unguiculata ssp. unguiculata and ssp. sesquipedalis) grown in southern Europe were assessed in an attempt to provide information on pod quality and nutritional properties and to identify relationships between quality traits and accession origin. Pods from the sesquipedalis accessions were heavier and larger, and reached commercial maturity 2 days later, than those from the unguiculata accessions. There were also large differences in the quality and dietary characteristics of the accessions. The pods of most accessions were rich in proteins, chlorophylls, carotenoids and phenolics, and showed high antioxidant activity and low concentrations of nitrates and raffinose-family oligosaccharides. Cluster analysis based on quality, dietary or antinutritional traits did not reveal any apparent grouping among the accessions. All the quality characteristics were independent of accession origin and subspecies. Most of the accessions produced fresh pods of good quality and high dietary value, suitable for introduction in the market and/or for use as valuable genetic material for the development of new improved varieties. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  15. Metabolomics Characterization of Two Apocynaceae Plants, Catharanthus roseus and Vinca minor, Using GC-MS and LC-MS Methods in Combination.

    PubMed

    Chen, Qi; Lu, Xueyan; Guo, Xiaorui; Guo, Qingxi; Li, Dewen

    2017-06-17

    Catharanthus roseus ( C. roseus ) and Vinca minor ( V. minor ) are two common important medical plants belonging to the family Apocynaceae. In this study, we used non-targeted GC-MS and targeted LC-MS metabolomics to dissect the metabolic profile of two plants with comparable phenotypic and metabolic differences. A total of 58 significantly different metabolites were present in different quantities according to PCA and PLS-DA score plots of the GC-MS analysis. The 58 identified compounds comprised 16 sugars, eight amino acids, nine alcohols and 18 organic acids. We subjected these metabolites into KEGG pathway enrichment analysis and highlighted 27 metabolic pathways, concentrated on the TCA cycle, glycometabolism, oligosaccharides, and polyol and lipid transporter (RFOS). Among the primary metabolites, trehalose, raffinose, digalacturonic acid and gallic acid were revealed to be the most significant marker compounds between the two plants, presumably contributing to species-specific phenotypic and metabolic discrepancy. The profiling of nine typical alkaloids in both plants using LC-MS method highlighted higher levels of crucial terpenoid indole alkaloid (TIA) intermediates of loganin, serpentine, and tabersonine in V. minor than in C. roseus . The possible underlying process of the metabolic flux from primary metabolism pathways to TIA synthesis was discussed and proposed. Generally speaking, this work provides a full-scale comparison of primary and secondary metabolites between two medical plants and a metabolic explanation of their TIA accumulation and phenotype differences.

  16. Cell culture media supplementation of infrequently used sugars for the targeted shifting of protein glycosylation profiles.

    PubMed

    Hossler, Patrick; Racicot, Christopher; Chumsae, Christopher; McDermott, Sean; Cochran, Keith

    2017-03-01

    Mammalian cells in culture rely on sources of carbohydrates to supply the energy requirements for proliferation. In addition, carbohydrates provide a large source of the carbon supply for supporting various other metabolic activities, including the intermediates involved in the protein glycosylation pathway. Glucose and galactose, in particular, are commonly used sugars in culture media for these purposes. However, there exists a very large repertoire of other sugars in nature, and many that have been chemically synthesized. These sugars are particularly interesting because they can be utilized by cells in culture in distinct ways. In the present work it has been found that many infrequently used sugars, and the corresponding cellular response towards them as substrates, led to differences in the protein N-glycosylation profile of a recombinant glycoprotein. The selective media supplementation of raffinose, trehalose, turanose, palatinose, melezitose, psicose, lactose, lactulose, and mannose were found to be capable of redirecting N-glycan oligosaccharide profiles. Despite this shifting of protein glycosylation, there were no other adverse changes in culture performance, including both cell growth and cellular productivity over a wide range of supplemented sugar concentrations. The approach presented highlights a potential means towards both the targeted shifting of protein glycosylation profiles and ensuring recombinant protein comparability, which up to this point in time has remained under-appreciated for these under-utilized compounds. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:511-522, 2017. © 2017 American Institute of Chemical Engineers.

  17. Passive driving forces of proximal tubular fluid and bicarbonate transport: gradient dependence of H+ secretion.

    PubMed

    Chan, Y L; Malnic, G; Giebisch, G

    1983-11-01

    The effect of oncotic pressure changes on fluid (Jv) and net bicarbonate transport (JHCO-3) and the transepithelial bicarbonate permeability (PHCO-3) were measured by an improved luminal and capillary microperfusion method that allows paired experiments on the same tubule. Rat proximal tubules were pump-perfused and Jv and [HCO-3] measured with [14C]inulin and a pH glass electrode. Raising peritubular protein (0-8-15 g/100 ml bovine serum albumin) stimulated Jv and HCO-3 reabsorption. The response to oncotic pressure changes was asymmetrical since changes of the luminal protein concentration had no significant effects. Whereas transepithelial solvent drag effects on HCO-3 must be minimal, peritubular protein most likely stimulates translocation of fluid and bicarbonate from intercellular spaces into peritubular capillaries. PHCO-3 was measured from HCO-3 net flux along a lumen-to-capillary-directed electrochemical potential gradient. In these experiments active H+ transport and Jv were minimized by 10(-4) M acetazolamide and luminal raffinose. PHCO-3 was 1.77 X 10(-5) cm X s-1 and was unaffected by increasing luminal flow rate from 10 to 45 nl X min-1. Since bicarbonate backflux is only a small fraction of physiological rates of JHCO-3, net transport alterations at varying [HCO-3] in the lumen must be due to changes in active HCO-3 (H+) transport. Thus, active H+ ion secretion across the luminal membrane of the proximal tubule is gradient dependent.

  18. Sugars profiles of different chestnut (Castanea sativa Mill.) and almond (Prunus dulcis) cultivars by HPLC-RI.

    PubMed

    Barreira, João C M; Pereira, José Alberto; Oliveira, M Beatriz P P; Ferreira, Isabel C F R

    2010-03-01

    Sugar profiles of different almond and chestnut cultivars were obtained by high-performance liquid chromatography (HPLC), by means of a refractive index (RI) detector. A solid-liquid extraction procedure was used in defatted and dried samples. The chromatographic separation was achieved using a Eurospher 100-5 NH(2) column using an isocratic elution with acetonitrile/water (70:30, v/v) at a flow rate of 1.0 ml/min. All the compounds were separated in 16 min. The method was optimized and proved to be reproducible and accurate. Generally, more than 95% of sugars were identified for both matrixes. Sugars profiles were quite homogeneous for almond cultivars; sucrose was the main sugar (11.46 +/- 0.14 in Marcona to 22.23 +/- 0.59 in Ferragnes g/100 g of dried weight), followed by raffinose (0.71 +/- 0.05 in Ferraduel to 2.11 +/- 0.29 in Duro Italiano), glucose (0.42 +/- 0.12 in Pegarinhos two seeded to 1.47 +/- 0.19 in Ferragnes) and fructose (0.11 +/- 0.02 in Pegarinhos two seeded to 0.59 +/- 0.05 in Gloriette). Commercial cultivars proved to have higher sucrose contents, except in the case of Marcona. Nevertheless, chestnut cultivars revealed a high heterogeneity. Sucrose was the main sugar in Aveleira (22.05 +/- 1.48), Judia (23.30 +/- 0.83) and Longal (9.56 +/- 0.91), while glucose was slightly prevalent in Boa Ventura (6.63 +/- 0.49). The observed variance could serve for inter-cultivar discrimination.

  19. ABI5 Is a Regulator of Seed Maturation and Longevity in Legumes

    PubMed Central

    Zinsmeister, Julia; Lalanne, David; Terrasson, Emmanuel; Chatelain, Emilie; Vandecasteele, Céline; Vu, Benoit Ly; Gutbrod, Katharina; Dörmann, Peter; Bendahmane, Abdelhafid

    2016-01-01

    The preservation of our genetic resources and production of high-quality seeds depends on their ability to remain viable and vigorous during storage. In a quantitative trait locus analysis on seed longevity in Medicago truncatula, we identified the bZIP transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5). Characterization of Mt-abi5 insertion mutant seeds revealed that both the acquisition of longevity and dormancy were severely impaired. Using transcriptomes of developing Mt-abi5 seeds, we created a gene coexpression network and revealed ABI5 as a regulator of gene modules with functions related to raffinose family oligosaccharide (RFO) metabolism, late embryogenesis abundant (LEA) proteins, and photosynthesis-associated nuclear genes (PhANGs). Lower RFO contents in Mt-abi5 seeds were linked to the regulation of SEED IMBIBITION PROTEIN1. Proteomic analysis confirmed that a set of LEA polypeptides was reduced in mature Mt-abi5 seeds, whereas the absence of repression of PhANG in mature Mt-abi5 seeds was accompanied by chlorophyll and carotenoid retention. This resulted in a stress response in Mt-abi5 seeds, evident from an increase in α-tocopherol and upregulation of genes related to programmed cell death and protein folding. Characterization of abi5 mutants in a second legume species, pea (Pisum sativum), confirmed a role for ABI5 in the regulation of longevity, seed degreening, and RFO accumulation, identifying ABI5 as a prominent regulator of late seed maturation in legumes. PMID:27956585

  20. Expression of a Streptococcus mutans glucosyltransferase gene in Escherichia coli.

    PubMed

    Robeson, J P; Barletta, R G; Curtiss, R

    1983-01-01

    Chromosomal DNA from Streptococcus mutans strain UAB90 (serotype c) was cloned into Escherichia coli K-12. The clone bank was screened for any sucrose-hydrolyzing activity by selection for growth on raffinose in the presence of isopropyl-beta-D-thiogalactoside. A clone expressing an S. mutans glucosyltransferase was identified. The S. mutans DNA encoding this enzyme is a 1.73-kilobase fragment cloned into the HindIII site of plasmid pBR322. We designated the gene gtfA. The plasmid-encoded gtfA enzyme, a 55,000-molecular-weight protein, is synthesized at 40% the level of pBR322-encoded beta-lactamase in E. coli minicells. Using sucrose as substrate, the gtfA enzyme catalyzes the formation of fructose and a glucan with an apparent molecular weight of 1,500. We detected the gtfA protein in S. mutans cells with antibody raised against the cloned gtfA enzyme. Immunologically identical gtfA protein appears to be present in S. mutans cells of serotypes c, e, and f, and a cross-reacting protein was made by serotype b cells. Proteins from serotype a, g, and d S. mutans cells did not react with antibody to gtfA enzyme. The gtfA activity was present in the periplasmic space of E. coli clones, since 15% of the total gtfA activity was released by cold osmotic shock and the clones were able to grow on sucrose as sole carbon source.

  1. Source-to-sink transport of sugar and regulation by environmental factors

    PubMed Central

    Lemoine, Remi; Camera, Sylvain La; Atanassova, Rossitza; Dédaldéchamp, Fabienne; Allario, Thierry; Pourtau, Nathalie; Bonnemain, Jean-Louis; Laloi, Maryse; Coutos-Thévenot, Pierre; Maurousset, Laurence; Faucher, Mireille; Girousse, Christine; Lemonnier, Pauline; Parrilla, Jonathan; Durand, Mickael

    2013-01-01

    Source-to-sink transport of sugar is one of the major determinants of plant growth and relies on the efficient and controlled distribution of sucrose (and some other sugars such as raffinose and polyols) across plant organs through the phloem. However, sugar transport through the phloem can be affected by many environmental factors that alter source/sink relationships. In this paper, we summarize current knowledge about the phloem transport mechanisms and review the effects of several abiotic (water and salt stress, mineral deficiency, CO2, light, temperature, air, and soil pollutants) and biotic (mutualistic and pathogenic microbes, viruses, aphids, and parasitic plants) factors. Concerning abiotic constraints, alteration of the distribution of sugar among sinks is often reported, with some sinks as roots favored in case of mineral deficiency. Many of these constraints impair the transport function of the phloem but the exact mechanisms are far from being completely known. Phloem integrity can be disrupted (e.g., by callose deposition) and under certain conditions, phloem transport is affected, earlier than photosynthesis. Photosynthesis inhibition could result from the increase in sugar concentration due to phloem transport decrease. Biotic interactions (aphids, fungi, viruses…) also affect crop plant productivity. Recent breakthroughs have identified some of the sugar transporters involved in these interactions on the host and pathogen sides. The different data are discussed in relation to the phloem transport pathways. When possible, the link with current knowledge on the pathways at the molecular level will be highlighted. PMID:23898339

  2. Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings.

    PubMed

    Kim, Donggiun; Lee, Gunsup; Chang, Man; Park, Jongbum; Chung, Youngjae; Lee, Sukchan; Lee, Taek-Kyun

    2011-10-26

    Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.

  3. The specificity of induction of alpha-galactosidase from Saccharomyces carlsbergensis.

    PubMed

    Flórez, I G; Lazo, P S; Ochoa, A G; Gascón, S

    1981-04-17

    A number of sugars and derivatives have been tested for their ability to induce the synthesis of alpha-galactosidase from Saccharomyces carlsbergensis. Besides galactose and the substrates of the enzyme melibiose, raffinose and stachyose, D-galacturonic acid, L-arabinose, D-tagatose, methyl-alpha-D-galactoside, lactose and isopropyl-beta-D-thiogalactoside were able to act as inducers. Of these, methyl-alpha-D-galactoside, lactose, isopropyl-beta-D-thiogalactoside and L-arabinose have been shown to be gratuitous inducers with which kinetic studies of induction have been carried out. Lactose was the most efficient inducer, giving a maximal differential rate of synthesis of the enzyme of 110 mU/10(7) cells at a concentration of 180 mM, followed by L-arabinose (60 mU/10(7) cells at 40 mM), isopropyl-beta-D-thiogalactoside (43 mU/10(7) cells at 60 mM) and methyl-alpha-D-galactoside (25 mU/10(7) cells at 150 mM). The concentration of inducer required to obtain half-maximal induction was similar for lactose, L-arabinose and isopropyl-beta-D-thiogalactoside and about 5-fold higher for methyl-alpha-D-galactoside. The property of the compounds to act as inducers was compared to their ability to interact with the enzyme and the results discussed in terms of the molecular structures which are recognized by the enzyme and by the induction machinery.

  4. Phloem Loading through Plasmodesmata: A Biophysical Analysis1[OPEN

    PubMed Central

    2017-01-01

    In many species, Suc en route out of the leaf migrates from photosynthetically active mesophyll cells into the phloem down its concentration gradient via plasmodesmata, i.e. symplastically. In some of these plants, the process is entirely passive, but in others phloem Suc is actively converted into larger sugars, raffinose and stachyose, and segregated (trapped), thus raising total phloem sugar concentration to a level higher than in the mesophyll. Questions remain regarding the mechanisms and selective advantages conferred by both of these symplastic-loading processes. Here, we present an integrated model—including local and global transport and kinetics of polymerization—for passive and active symplastic loading. We also propose a physical model of transport through the plasmodesmata. With these models, we predict that (1) relative to passive loading, polymerization of Suc in the phloem, even in the absence of segregation, lowers the sugar content in the leaf required to achieve a given export rate and accelerates export for a given concentration of Suc in the mesophyll and (2) segregation of oligomers and the inverted gradient of total sugar content can be achieved for physiologically reasonable parameter values, but even higher export rates can be accessed in scenarios in which polymers are allowed to diffuse back into the mesophyll. We discuss these predictions in relation to further studies aimed at the clarification of loading mechanisms, fitness of active and passive symplastic loading, and potential targets for engineering improved rates of export. PMID:28794259

  5. Fermented nondigestible fraction from common bean (Phaseolus vulgaris L.) cultivar Negro 8025 modulates HT-29 cell behavior.

    PubMed

    Cruz-Bravo, R K; Guevara-Gonzalez, R; Ramos-Gomez, M; Garcia-Gasca, T; Campos-Vega, R; Oomah, B D; Loarca-Piña, G

    2011-03-01

    The aim of the study was to evaluate the effect of a fermented nondigestible fraction (FNDF) of cooked bean (Phaseolus vulgaris L.) cultivar Negro 8025 on human colon adenocarcinoma HT-29 cell survival. Negro 8025 was chosen for in vitro fermentation based on comparison of chemical composition with 2 other cultivars: Azufrado Higuera and Pinto Durango. Negro 8025 had 58% total dietary fiber, 27% resistant starch, and 20 mg of (+)-catechin equivalents per gram of sample. Short-chain fatty acids (SCFAs) production and pH of the medium were measured after fermentation as indicators of colon protection through induced arrest on cell culture and apoptosis. Butyrate and pH of FNDF of Negro 8025 were higher than the control fermented raffinose extract. The FNDF inhibited HT-29 cell survival in a time- and concentration-dependent manner. The lethal concentration 50 (LC(50)) was 13.63% FNDF (equivalent to 7.36, 0.33, and 3.31 mmol of acetic, propionic, and butyric acids, respectively). DNA fragmentation, an apoptosis indicator, was detected by the TdT-mediated dUTP nick end labeling method in cells treated with the LC(50)-FNDF and a synthetic mixture of SCFAs mimicking LC(50)-FNDF. Our results suggest that common bean is a reliable source of fermentable substrates in colon, producing compounds with potential chemoprotective effect on HT-29 colon adenocarcinoma cells, so it may present an effective alternative to mitigate colon cancer development.

  6. Adaptation of the AOAC 2011.25 integrated total dietary fiber assay to determine the dietary fiber and oligosaccharide content of dry edible beans.

    PubMed

    Kleintop, Adrienne E; Echeverria, Dimas; Brick, Leslie A; Thompson, Henry J; Brick, Mark A

    2013-10-09

    Dietary fiber (DF) has important health benefits in the human diet. Developing dry edible bean (Phaseolus vulgaris L.) cultivars with improved DF and reduced nondigestible oligosaccharide content is an important goal for dry bean breeders to increase consumer acceptance. To determine if genetic variation exists among dry bean cultivars for DF, two populations of diverse dry bean cultivars/lines that represent two centers of dry bean domestication were evaluated for dietary fiber using the Integrated Total Dietary Fiber Assay (AOAC 2011.25). This assay was adapted to measure water insoluble dietary fiber, water soluble dietary fiber, oligosaccharides raffinose and stachyose, and the calculated total dietary fiber (TDF) content of cooked dry bean seed. The AOAC 2011.25 protocol was modified by using a quick, simple, and sensitive high-performance liquid chromatography method paired with an electrochemical detection method to separate and quantify specific oligosaccharides, and using duplicate samples as replicates to generate statistical information. The TDF of dry bean entries ranged from 20.0 to 27.0% in population I and from 20.6 to 25.7% in population II. Total oligosaccharides ranged from 2.56 to 4.65% in population I and from 2.36 to 3.84% in population II. The results suggest that significant genetic variation exists among dry bean cultivars/lines to allow for genetic selection for improved DF content in dry beans and that the modifications to the AOAC 2011.25 method were suitable for estimating DF in cooked dry edible beans.

  7. Source-to-sink transport of sugar and regulation by environmental factors.

    PubMed

    Lemoine, Remi; La Camera, Sylvain; Atanassova, Rossitza; Dédaldéchamp, Fabienne; Allario, Thierry; Pourtau, Nathalie; Bonnemain, Jean-Louis; Laloi, Maryse; Coutos-Thévenot, Pierre; Maurousset, Laurence; Faucher, Mireille; Girousse, Christine; Lemonnier, Pauline; Parrilla, Jonathan; Durand, Mickael

    2013-01-01

    Source-to-sink transport of sugar is one of the major determinants of plant growth and relies on the efficient and controlled distribution of sucrose (and some other sugars such as raffinose and polyols) across plant organs through the phloem. However, sugar transport through the phloem can be affected by many environmental factors that alter source/sink relationships. In this paper, we summarize current knowledge about the phloem transport mechanisms and review the effects of several abiotic (water and salt stress, mineral deficiency, CO2, light, temperature, air, and soil pollutants) and biotic (mutualistic and pathogenic microbes, viruses, aphids, and parasitic plants) factors. Concerning abiotic constraints, alteration of the distribution of sugar among sinks is often reported, with some sinks as roots favored in case of mineral deficiency. Many of these constraints impair the transport function of the phloem but the exact mechanisms are far from being completely known. Phloem integrity can be disrupted (e.g., by callose deposition) and under certain conditions, phloem transport is affected, earlier than photosynthesis. Photosynthesis inhibition could result from the increase in sugar concentration due to phloem transport decrease. Biotic interactions (aphids, fungi, viruses…) also affect crop plant productivity. Recent breakthroughs have identified some of the sugar transporters involved in these interactions on the host and pathogen sides. The different data are discussed in relation to the phloem transport pathways. When possible, the link with current knowledge on the pathways at the molecular level will be highlighted.

  8. [Proposed neotype Streptomyces ruber (Krainsky, 1914) Waksman et Henrici, 1948].

    PubMed

    Kuznetsov, V D; Filippova, S N; Poltorak, V A

    1987-01-01

    Culture 78 was proposed as a neotype of Streptomyces ruber. It was isolated from the soils of the Baikal region and was closest, in its taxonomic properties, to the original description of the species [13] whose representative had been lost. Cultures from different microbial collections designated as S. ruber were shown to be unlike the original description. The neotype had the following taxononic properties: the cell wall of type I; spiral sporophores with extended spirals having 2-3 coils; oval spores with a smooth envelope; greyish pink aerial and dark-red substrate mycelia; a red pigment not passing into the medium; slow gelatin liquefaction and milk peptonization; weak starch hydrolysis; assimilation of glucose, xylose, rammose, fructose, and inositol; weak growth on arabinose, raffinose and mannitol, but not on sucrose; no formation of melanoid pigments; synthesis of riboflavin and prodigiosin pigments; inhibition of Gram-positive bacterial and acid-resistant mycobacterial growth; no inhibition of yeast and fungal growth. The culture was sensitive to streptomycin, neomycin, gentamycin, monomycin, tetracycline,erythromycin, oleandomycin, lincomycin, ristomycin, levomycetin, polymyxin and fusidin, but resistant in penicillin. The population was composed of six variants [3]: main, faded, asporogenic red, asporogenic yellow, asporogenic white and nocardia-like. The latter two were not capable of riboflavin and prodigiosin formation. The asporogenic yellow variant was a monosynthetic organism: it formed riboflavin, but could not synthesize prodigiosin. The neotype of S. ruber 78 is deposited withthe national Collection of Microorganisms (the reference number is VKM A-611).

  9. Tolerance to high soil temperature in foxtail millet (Setaria italica L.) is related to shoot and root growth and metabolism.

    PubMed

    Aidoo, Moses Kwame; Bdolach, Eyal; Fait, Aaron; Lazarovitch, Naftali; Rachmilevitch, Shimon

    2016-09-01

    Roots play important roles in regulating whole-plant carbon and water relations in response to extreme soil temperature. Three foxtail millet (Setaria italica L.) lines (448-Ames 21521, 463-P1391643 and 523-P1219619) were subjected to two different soil temperatures (28 and 38 °C). The gas exchange, chlorophyll fluorescence, root morphology and central metabolism of leaves and roots were studied at the grain-filling stage. High soil temperature (38 °C) significantly influenced the shoot transpiration, stomatal conductance, photosynthesis, root growth and metabolism of all lines. The root length and area were significantly reduced in lines 448 and 463 in response to the stress, while only a small non-specific reduction was observed in line 523 in response to the treatment. The shift of root metabolites in response to high soil temperature was also genotype specific. In response to high soil temperature, glutamate, proline and pyroglutamate were reduced in line 448, and alanine, aspartate, glycine, pyroglutamate, serine, threonine and valine were accumulated in line 463. In the roots of line 523, serine, threonine, valine, isomaltose, maltose, raffinose, malate and itaconate were accumulated. Root tolerance to high soil temperature was evident in line 523, in its roots growth potential, lower photosynthesis and stomatal conductance rates, and effective utilization and assimilation of membrane carbon and nitrogen, coupled with the accumulation of protective metabolites. Copyright © 2016. Published by Elsevier Masson SAS.

  10. The influence of crystal habit on the prediction of dry powder inhalation formulation performance using the cohesive-adhesive force balance approach.

    PubMed

    Hooton, Jennifer C; Jones, Matthew D; Harris, Haggis; Shur, Jagdeep; Price, Robert

    2008-09-01

    The aim of this investigation was to study the influence of crystalline habit of active pharmaceutical ingredients on the cohesive-adhesive force balance within model dry powder inhaler (DPI) formulations and the corresponding affect on DPI formulation performance. The cohesive-adhesive balance (CAB) approach to colloid probe atomic force microscopy (AFM) was employed to determine the cohesive and adhesive interactions of micronized budesonide particles against the {102} and {002} faces of budesonide single crystals and crystalline substrates of different sugars (cyclodextrin, lactose, trehalose, raffinose, and xylitol), respectively. These data were used to measure the relative level of cohesion and adhesion via CAB and the possible influence on in vitro performance of a carrier-based DPI formulation. Varying the crystal habit of the drug had a significant effect on the cohesive measurement of micronized budesonide probes, with the cohesive values on the {102} faces being approximately twice that on the {002} crystal faces. However, although different CAB values were measured with the sugars with respect to the crystal faces chosen for the cohesive-based measurement, the overall influence on the rank order of the CAB values was not directly influenced. For these data sets, the CAB gradient indicated that a decrease in the dominance of the adhesive forces led to a concomitant increase in fine particle delivery, reaching a plateau as the cohesive forces became dominant. The study suggested that crystal habit of the primary drug crystals influences the cohesive interactions and the resulting force balance measurements of colloid probe CAB analysis.

  11. Exploring the Microbiota of Faba Bean: Functional Characterization of Lactic Acid Bacteria.

    PubMed

    Verni, Michela; Wang, Changyin; Montemurro, Marco; De Angelis, Maria; Katina, Kati; Rizzello, Carlo G; Coda, Rossana

    2017-01-01

    This study investigated the metabolic traits of 27 lactic acid bacteria (LAB) strains belonging to different species, previously isolated from faba bean. The activities assayed, related to technological and nutritional improvement of fermented faba bean, included peptidases, β-glucosidase, phytase, as well as exopolysaccharides synthesis and antimicrobial properties. In addition, the bacteria performance as starter cultures during faba bean fermentation on proteolysis, antioxidant potential, and degradation of condensed tannins were assessed. Fermentative profiling showed that only 7 out of 27 strains were able to metabolize D-raffinose, particularly Leuc. mesenteroides I01 and I57. All strains of Pediococcus pentosaceus exerted high PepN activity and exhibited β-glucosidase activity higher than the median value of 0.015 U, while phytase activity was largely distributed among the different strains. All the weissellas, and in lower amount leuconostocs, showed ability to produce EPS from sucrose. None of the strains did not survive the simulated gastrointestinal tract with the exception of P. pentosaceus I56, I76, 147, I214, having a viability of 8-9 log CFU/ml at the end of the treatment. None of the strains showed antimicrobial activity toward Staphylococcus aureus , while eight strains of P. pentosaceus exhibited a strong inhibitory activity toward Escherichia coli and Listeria monocytogenes . Generally, the doughs fermented with pediococci exhibited high amount of total free amino acids, antioxidant activity, and condensed tannins degradation. These results allowed the identification of LAB biotypes as potential starter cultures for faba bean bioprocessing, aiming at the enhancement of faba bean use in novel food applications.

  12. Comparative transcriptome analysis reveals key genes potentially related to soluble sugar and organic acid accumulation in watermelon

    PubMed Central

    Gao, Lei; Zhao, Shengjie; Lu, Xuqiang; He, Nan; Zhu, Hongju; Dou, Junling

    2018-01-01

    Soluble sugars and organic acids are important components of fruit flavor and have a strong impact on the overall organoleptic quality of watermelon (Citrullus lanatus) fruit. Several studies have analyzed the expression levels of the genes related to soluble sugar accumulation and the dynamic changes in their content during watermelon fruit development and ripening. Nevertheless, to date, there have been no reports on the organic acid content in watermelon or the genes regulating their synthesis. In this study, the soluble sugars and organic acids in watermelon were measured and a comparative transcriptome analysis was performed to identify the key genes involved in the accumulation of these substances during fruit development and ripening. The watermelon cultivar ‘203Z’ and its near-isogenic line (NIL) ‘SW’ (in the ‘203Z’ background) were used as experimental materials. The results suggested that soluble sugar consist of fructose, glucose and sucrose while malic-, citric-, and oxalic acids are the primary organic acids in watermelon fruit. Several differentially expressed genes (DEGs) related to soluble sugar- and organic acid accumulation and metabolism were identified. These include the DEGs encoding raffinose synthase, sucrose synthase (SuSy), sucrose-phosphate synthase (SPSs), insoluble acid invertases (IAI), NAD-dependent malate dehydrogenase (NAD-cyt MDH), aluminum-activated malate transporter (ALMT), and citrate synthase (CS). This is the first report addressing comparative transcriptome analysis via NILs materials in watermelon fruit. These findings provide an important basis for understanding the molecular mechanism that leads to soluble sugar and organic acid accumulation and metabolism during watermelon fruit development and ripening. PMID:29324867

  13. Characterization of Atypical Isolates of Yersinia intermedia and Definition of Two New Biotypes▿ †

    PubMed Central

    Martin, Liliane; Leclercq, Alexandre; Savin, Cyril; Carniel, Elisabeth

    2009-01-01

    The species Yersinia intermedia is a member of the genus Yersinia which belongs to the Enterobacteriaceae family. This species is divided into eight biotypes, according to Brenner's biotyping scheme. This scheme relies on five tests (utilization of Simmons citrate and acid production from d-melibiose, d-raffinose, α-methyl-d-glucoside [αMG], and l-rhamnose). The collection of the French Yersinia Reference Laboratory (Institut Pasteur, Paris, France) contained 44 strains that were originally identified as Y. intermedia but whose characteristics did not fit into the biotyping scheme. These 44 strains were separated into two biochemical groups: variant 1 (positive for acid production from l-rhamnose and αMG and positive for Simmons citrate utlization) and variant 2 (positive for acid production from l-rhamnose and αMG). These atypical strains could correspond to new biotypes of Y. intermedia, to Y. frederiksenii strains having the atypical property of fermenting αMG, or to new Yersinia species. These strains did not exhibit growth or phenotypic properties different from those of Y. intermedia and Y. frederiksenii and did not harbor any of the virulence traits usually found in pathogenic species. DNA-DNA hybridizations performed between one strain each of variants 1 and 2 and the Y. intermedia and Y. frederiksenii type strains demonstrated that these variants do belong to the Y. intermedia species. We thus propose that Brenner's biotyping scheme be updated by adding two new biotypes: 9 (for variant 1) and 10 (for variant 2) to the species Y. intermedia. PMID:19494062

  14. Combinational effects of prebiotic oligosaccharides on bifidobacterial growth and host gene expression in a simplified mixed culture model and neonatal mice.

    PubMed

    Ehara, Tatsuya; Izumi, Hirohisa; Tsuda, Muneya; Nakazato, Yuki; Iwamoto, Hiroshi; Namba, Kazuyoshi; Takeda, Yasuhiro

    2016-07-01

    It is important to provide formula-fed infants with a bifidobacteria-enriched gut microbiota similar to those of breastfed infants to ensure intestinal health. Prebiotics, such as certain oligosaccharides, are a useful solution to this problem, but the combinational benefits of these oligosaccharides have not been evaluated. This study investigated the benefits of oligosaccharide combinations and screened for an optimal combination of oligosaccharides to promote healthy gut microbiota of formula-fed infants. In vitro and in vivo experiments were performed to assess the bifidogenic effects of lactulose (LAC) alone and LAC combined with raffinose (RAF) and/or galacto-oligosaccharide (GOS), using a mixed culture model and neonatal mice orally administered with these oligosaccharides and Bifidobacterium breve. In the in vitro culture model, the combination of the three oligosaccharides (LAC-RAF-GOS) significantly increased cell numbers of B. breve and Bifidobacterium longum (P<0·05) compared with either LAC alone or the combination of two oligosaccharides, and resulted in the production of SCFA under anaerobic conditions. In the in vivo experiment, the LAC-RAF-GOS combination significantly increased cell numbers of B. breve and Bacteroidetes in the large intestinal content (P<0·05) and increased acetate concentrations in the caecal content and serum of neonatal mice. Genes related to metabolism and immune responses were differentially expressed in the liver and large intestine of mice administered with LAC-RAF-GOS. These results indicate a synergistic effect of the LAC-RAF-GOS combination on the growth of bifidobacteria and reveal possible benefits of this combination to the gut microbiota and health of infants.

  15. Effect of oligosaccharides on the adhesion of gut bacteria to human HT-29 cells.

    PubMed

    Altamimi, M; Abdelhay, O; Rastall, R A

    2016-06-01

    The influence of five oligosaccharides (cellobiose, stachyose, raffinose, lactulose and chito-oligosaccharides) on the adhesion of eight gut bacteria (Bifidobacterium bifidum ATCC 29521, Bacteroides thetaiotaomicron ATCC 29148D-5, Clostridium leptum ATCC 29065, Blautia coccoides ATCC 29236, Faecalibacterium prausnitzii ATCC 27766, Bacteroides fragilis ATCC 23745, Clostridium difficile ATCC 43255 and Lactobacillus casei ATCC 393) to mucous secreting and non-mucous secreting HT-29 human epithelial cells, was investigated. In pure culture, the bacteria showed variations in their ability to adhere to epithelial cells. The effect of oligosaccharides diminished adhesion and the presence of mucus played a major factor in adhesion, likely due to high adhesiveness to mucins present in the native human mucus layer covering the whole cell surface. However, clostridia displayed almost the same level of adhesion either with or without mucus being present. Bl. coccoides adhesion was decreased by stachyose and cellobiose in non-mucus-secreting cells in pure culture, while in mixed faecal culture cellobiose displayed the highest antiadhesive activity with an overall average of 65% inhibition amongst tested oligomers and lactulose displayed the lowest with an average of 47.4%. Bifidobacteria, Bacteroides, lactobacilli and clostridia were inhibited within the following ranges 47-78%, 32-65%, 11.7-58% and 64-85% respectively. This means that clostridia were the most strongly influenced members of the microflora amongst the bacterial groups tested in mixed culture. In conclusion, introducing oligosaccharides which are candidate prebiotics into pure or mixed cultures has affected bacterial adhesion. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Metabolomics Reveals How Cucumber (Cucumis sativus) Reprograms Metabolites to Cope with Silver Ions and Silver Nanoparticle-Induced Oxidative Stress.

    PubMed

    Zhang, Huiling; Du, Wenchao; Peralta-Videa, Jose R; Gardea-Torresdey, Jorge L; White, Jason C; Keller, Arturo A; Guo, Hongyan; Ji, Rong; Zhao, Lijuan

    2018-06-14

    Due to their well-known antifungal activity, the intentional use of Ag nanoparticle (NPs) as sustainable nano-fungicides is expected to increase in agriculture. However, the impacts of AgNPs on plants must be critically evaluated to guarantee their safe use in food production. In this study, 4-week-old cucumber (Cucumis sativus) plants received a foliar application of AgNPs (4 or 40 mg per plant) or Ag+ (0.04 or 0.4 mg per plant) for seven days. Gas chromatography-mass spectrometry (GC-MS) based non-target metabolomics enabled the identification and quantification of 268 metabolites in cucumber leaves. Multivariate analysis revealed that all the treatments significantly altered the metabolite profile. Exposure to AgNPs resulted in metabolic reprogramming, including activation of antioxidant defense systems (up-regulation of phenolic compounds) and down-regulation of photosynthesis (up-regulation of phytol). Additionally, AgNPs enhanced respiration (up-regulation of TCA cycle intermediates), inhibited photorespiration (down-regulation of glycine/serine ratio), altered membrane properties (up-regulation of pentadecanoic and arachidonic acid, down-regulation of linoleic and linolenic acid), and reduced of inorganic nitrogen fixation (down-regulation of glutamine and asparagine). Although Ag ions induced some of the same metabolic changes, alterations in the levels of carbazole, indoleactate, raffinose, adenosine, lactamide, erythrose, and p-benzoquinone were AgNPs-specific. The results of this study offer new insight into the molecular mechanisms by which cucumber responds to AgNPs exposure and provide important information to support the sustainable use of AgNPs in agriculture.

  17. Lactobacillus brantae sp. nov., isolated from faeces of Canada geese (Branta canadensis).

    PubMed

    Volokhov, Dmitriy V; Amselle, Megan; Beck, Brian J; Popham, David L; Whittaker, Paul; Wang, Hua; Kerrigan, Elizabeth; Chizhikov, Vladimir E

    2012-09-01

    Three strains of lactic acid bacteria (LAB) were isolated from the faeces of apparently healthy wild Canada geese (Branta canadensis) in 2010 by cultivating faecal LAB on Rogosa SL agar under aerobic conditions. These three isolates were found to share 99.9 % gene sequence similarity of their 16S rRNA, their 16S-23S intergenic transcribed spacer region (ITS), partial 23S rRNA, rpoB, rpoC, rpoA and pheS gene sequences. However, the three strains exhibited lower levels of sequence similarity of these genetic targets to all known LAB, and the phylogenetically closest species to the geese strains were Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus saniviri. In comparison to L. casei ATCC 393(T), L. paracasei ATCC 25302(T), L. rhamnosus ATCC 7469(T) and L. saniviri DSM 24301(T), the novel isolates reacted uniquely in tests for cellobiose, galactose, mannitol, citric acid, aesculin and dextrin, and gave negative results in tests for l-proline arylamidase and l-pyrrolydonyl-arylamidase, and in the Voges-Proskauer test. Biochemical tests for cellobiose, aesculin, galactose, gentiobiose, mannitol, melezitose, ribose, salicin, sucrose, trehalose, raffinose, turanose, amygdalin and arbutin could be used for differentiation between L. saniviri and the novel strains. On the basis of phenotypic and genotypic characteristics, and phylogenetic data, the three isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus brantae sp. nov. is proposed. The type strain is SL1108(T) (= ATCC BAA-2142(T) = LMG 26001(T) = DSM 23927(T)) and two additional strains are SL1170 and SL60106.

  18. Comparative transcriptome analysis reveals key genes potentially related to soluble sugar and organic acid accumulation in watermelon.

    PubMed

    Gao, Lei; Zhao, Shengjie; Lu, Xuqiang; He, Nan; Zhu, Hongju; Dou, Junling; Liu, Wenge

    2018-01-01

    Soluble sugars and organic acids are important components of fruit flavor and have a strong impact on the overall organoleptic quality of watermelon (Citrullus lanatus) fruit. Several studies have analyzed the expression levels of the genes related to soluble sugar accumulation and the dynamic changes in their content during watermelon fruit development and ripening. Nevertheless, to date, there have been no reports on the organic acid content in watermelon or the genes regulating their synthesis. In this study, the soluble sugars and organic acids in watermelon were measured and a comparative transcriptome analysis was performed to identify the key genes involved in the accumulation of these substances during fruit development and ripening. The watermelon cultivar '203Z' and its near-isogenic line (NIL) 'SW' (in the '203Z' background) were used as experimental materials. The results suggested that soluble sugar consist of fructose, glucose and sucrose while malic-, citric-, and oxalic acids are the primary organic acids in watermelon fruit. Several differentially expressed genes (DEGs) related to soluble sugar- and organic acid accumulation and metabolism were identified. These include the DEGs encoding raffinose synthase, sucrose synthase (SuSy), sucrose-phosphate synthase (SPSs), insoluble acid invertases (IAI), NAD-dependent malate dehydrogenase (NAD-cyt MDH), aluminum-activated malate transporter (ALMT), and citrate synthase (CS). This is the first report addressing comparative transcriptome analysis via NILs materials in watermelon fruit. These findings provide an important basis for understanding the molecular mechanism that leads to soluble sugar and organic acid accumulation and metabolism during watermelon fruit development and ripening.

  19. Exploring the Microbiota of Faba Bean: Functional Characterization of Lactic Acid Bacteria

    PubMed Central

    Verni, Michela; Wang, Changyin; Montemurro, Marco; De Angelis, Maria; Katina, Kati; Rizzello, Carlo G.; Coda, Rossana

    2017-01-01

    This study investigated the metabolic traits of 27 lactic acid bacteria (LAB) strains belonging to different species, previously isolated from faba bean. The activities assayed, related to technological and nutritional improvement of fermented faba bean, included peptidases, β-glucosidase, phytase, as well as exopolysaccharides synthesis and antimicrobial properties. In addition, the bacteria performance as starter cultures during faba bean fermentation on proteolysis, antioxidant potential, and degradation of condensed tannins were assessed. Fermentative profiling showed that only 7 out of 27 strains were able to metabolize D-raffinose, particularly Leuc. mesenteroides I01 and I57. All strains of Pediococcus pentosaceus exerted high PepN activity and exhibited β-glucosidase activity higher than the median value of 0.015 U, while phytase activity was largely distributed among the different strains. All the weissellas, and in lower amount leuconostocs, showed ability to produce EPS from sucrose. None of the strains did not survive the simulated gastrointestinal tract with the exception of P. pentosaceus I56, I76, 147, I214, having a viability of 8–9 log CFU/ml at the end of the treatment. None of the strains showed antimicrobial activity toward Staphylococcus aureus, while eight strains of P. pentosaceus exhibited a strong inhibitory activity toward Escherichia coli and Listeria monocytogenes. Generally, the doughs fermented with pediococci exhibited high amount of total free amino acids, antioxidant activity, and condensed tannins degradation. These results allowed the identification of LAB biotypes as potential starter cultures for faba bean bioprocessing, aiming at the enhancement of faba bean use in novel food applications. PMID:29312174

  20. Serratia glossinae sp. nov., isolated from the midgut of the tsetse fly Glossina palpalis gambiensis.

    PubMed

    Geiger, A; Fardeau, M-L; Falsen, E; Ollivier, B; Cuny, G

    2010-06-01

    We report the isolation of a novel bacterium, strain C1(T), from the midgut of the tsetse fly Glossina palpalis gambiensis, one of the vector insects responsible for transmission of the trypanosomes that cause sleeping sickness in sub-Saharan African countries. Strain C1(T) is a motile, facultatively anaerobic, rod-like bacterium (0.8-1.0 microm in diameter; 2-6 microm long) that grows as single cells or in chains. Optimum growth occurred at 25-35 degrees C, at pH 6.7-8.4 and in medium containing 5-20 g NaCl l(-1). The bacterium hydrolysed urea and used L-lysine, L-ornithine, citrate, pyruvate, D-glucose, D-mannitol, inositol, D-sorbitol, melibiose, amygdalin, L-arabinose, arbutin, aesculin, D-fructose, D-galactose, glycerol, maltose, D-mannose, raffinose, trehalose and d-xylose; it produced acetoin, reduced nitrate to nitrite and was positive for beta-galactosidase and catalase. The DNA G+C content was 53.6 mol%. It was related phylogenetically to members of the genus Serratia, family Enterobacteriaceae, the type strain of Serratia fonticola being its closest relative (99 % similarity between 16S rRNA gene sequences). However, DNA-DNA relatedness between strain C1(T) and S. fonticola DSM 4576(T) was only 37.15 %. Therefore, on the basis of morphological, nutritional, physiological and fatty acid analysis and genetic criteria, strain C1(T) is proposed to be assigned to a novel Serratia species, Serratia glossinae sp. nov. (type strain C1(T) =DSM 22080(T) =CCUG 57457(T)).

  1. Expression and characterization of a β-fructofuranosidase from the parasitic protist Trichomonas vaginalis

    PubMed Central

    2014-01-01

    Background Trichomonas vaginalis, a flagellated protozoan, is the agent responsible for trichomoniasis, the most common nonviral sexually transmitted infection worldwide. A reported 200 million cases are documented each year with far more cases going unreported. However, T. vaginalis is disproportionality under studied, especially considering its basic metabolism. It has been reported that T. vaginalis does not grow on sucrose. Nevertheless, the T. vaginalis genome contains some 11 putative sucrose transporters and a putative β-fructofuranosidase (invertase). Thus, the machinery for both uptake and cleavage of sucrose appears to be present. Results We amplified the β-fructofuranosidase from T. vaginalis cDNA and cloned it into an Escherichia coli expression system. The expressed, purified protein was found to behave similarly to other known β-fructofuranosidases. The enzyme exhibited maximum activity at pH close to 5.0, with activity falling off rapidly at increased or decreased pH. It had a similar Km and Vmax to previously characterized enzymes using sucrose as a substrate, was also active towards raffinose, but had no detectable activity towards inulin. Conclusions T. vaginalis has the coding capacity to produce an active β-fructofuranosidase capable of hydrolyzing di- and trisaccharides containing a terminal, non-reducing fructose residue. Since we cloned this enzyme from cDNA, we know that the gene in question is transcribed. Furthermore, we could detect β-fructofuranosidase activity in T. vaginalis cell lysates. Therefore, the inability of the organism to utilize sucrose as a carbon source cannot be explained by an inability to degrade sucrose. PMID:24972630

  2. Expression and characterization of a β-fructofuranosidase from the parasitic protist Trichomonas vaginalis.

    PubMed

    Dirkx, Michael; Boyer, Michael P; Pradhan, Prajakta; Brittingham, Andrew; Wilson, Wayne A

    2014-06-28

    Trichomonas vaginalis, a flagellated protozoan, is the agent responsible for trichomoniasis, the most common nonviral sexually transmitted infection worldwide. A reported 200 million cases are documented each year with far more cases going unreported. However, T. vaginalis is disproportionality under studied, especially considering its basic metabolism. It has been reported that T. vaginalis does not grow on sucrose. Nevertheless, the T. vaginalis genome contains some 11 putative sucrose transporters and a putative β-fructofuranosidase (invertase). Thus, the machinery for both uptake and cleavage of sucrose appears to be present. We amplified the β-fructofuranosidase from T. vaginalis cDNA and cloned it into an Escherichia coli expression system. The expressed, purified protein was found to behave similarly to other known β-fructofuranosidases. The enzyme exhibited maximum activity at pH close to 5.0, with activity falling off rapidly at increased or decreased pH. It had a similar K(m) and V(max) to previously characterized enzymes using sucrose as a substrate, was also active towards raffinose, but had no detectable activity towards inulin. T. vaginalis has the coding capacity to produce an active β-fructofuranosidase capable of hydrolyzing di- and trisaccharides containing a terminal, non-reducing fructose residue. Since we cloned this enzyme from cDNA, we know that the gene in question is transcribed. Furthermore, we could detect β-fructofuranosidase activity in T. vaginalis cell lysates. Therefore, the inability of the organism to utilize sucrose as a carbon source cannot be explained by an inability to degrade sucrose.

  3. Apoplastic and symplastic phloem loading in Quercus robur and Fraxinus excelsior

    PubMed Central

    Lohaus, Gertrud

    2014-01-01

    Whereas most of the research on phloem loading is performed on herbaceous plants, less is known about phloem loading strategies in trees. In this study, the phloem loading mechanisms of Quercus robur and Fraxinus excelsior were analysed. The following features were examined: the minor vein structure, the sugar concentrations in phloem sap by the laser–aphid–stylet technique, the distribution of photoassimilates in the mesophyll cells by non-aqueous fractionation, gradients of sugar concentrations and osmotic pressure, and the expression of sucrose transporters. The minor vein configurations of Q. robur and F. excelsior belong to the open type. Quercus robur contained companion cells in the minor veins whereas F. excelsior showed intermediary cells in addition to ordinary companion cells. The main carbon transport form in Q. robur was sucrose (~1M). In F. excelsior high amounts of raffinose and stachyose were also transported. However, in both tree species, the osmolality of phloem sap was higher than the osmolality of the mesophyll cells. The concentration gradients between phloem sap and the cytoplasm of mesophyll cells for sucrose were 16-fold and 14-fold for Q. robur and F. excelsior, respectively. Independent of the type of translocated sugars, sucrose transporter cDNAs were cloned from both species. The results indicate that phloem loading of sucrose and other metabolites must involve active loading steps in both tree species. Quercus robur seems to be an apoplastic phloem loader while F. excelsior shows indications of being a symplastic or mixed symplastic–apoplastic phloem loader. PMID:24591056

  4. NMR metabolomics of thrips (Frankliniella occidentalis) resistance in Senecio hybrids.

    PubMed

    Leiss, Kirsten A; Choi, Young H; Abdel-Farid, Ibrahim B; Verpoorte, Robert; Klinkhamer, Peter G L

    2009-02-01

    Western flower thrips (Frankliniella occidentalis) has become a key insect pest of agricultural and horticultural crops worldwide. Little is known about host plant resistance to thrips. In this study, we investigated thrips resistance in F (2) hybrids of Senecio jacobaea and Senecio aquaticus. We identified thrips-resistant hybrids applying three different bioassays. Subsequently, we compared the metabolomic profiles of these hybrids applying nuclear magnetic resonance spectroscopy (NMR). The new developments of NMR facilitate a wide range coverage of the metabolome. This makes NMR especially suitable if there is no a priori knowledge of the compounds related to herbivore resistance and allows a holistic approach analyzing different chemical compounds simultaneously. We show that the metabolomes of thrips-resistant and -susceptible hybrids differed considerably. Thrips-resistant hybrids contained higher amounts of the pyrrolizidine alkaloids (PA), jacobine, and jaconine, especially in younger leaves. Also, a flavanoid, kaempferol glucoside, accumulated in the resistant plants. Both PAs and kaempferol are known for their inhibitory effect on herbivores. In resistant and susceptible F (2) hybrids, young leaves showed less thrips damage than old leaves. Consistent with the optimal plant defense theory, young leaves contained increased levels of primary metabolites such as sucrose, raffinose, and stachyose, but also accumulated jacaranone as a secondary plant defense compound. Our results prove NMR as a promising tool to identify different metabolites involved in herbivore resistance. It constitutes a significant advance in the study of plant-insect relationships, providing key information on the implementation of herbivore resistance breeding strategies in plants.

  5. Comprehensive Dissection of Spatiotemporal Metabolic Shifts in Primary, Secondary, and Lipid Metabolism during Developmental Senescence in Arabidopsis1[W

    PubMed Central

    Watanabe, Mutsumi; Balazadeh, Salma; Tohge, Takayuki; Erban, Alexander; Giavalisco, Patrick; Kopka, Joachim; Mueller-Roeber, Bernd; Fernie, Alisdair R.; Hoefgen, Rainer

    2013-01-01

    Developmental senescence is a coordinated physiological process in plants and is critical for nutrient redistribution from senescing leaves to newly formed sink organs, including young leaves and developing seeds. Progress has been made concerning the genes involved and the regulatory networks controlling senescence. The resulting complex metabolome changes during senescence have not been investigated in detail yet. Therefore, we conducted a comprehensive profiling of metabolites, including pigments, lipids, sugars, amino acids, organic acids, nutrient ions, and secondary metabolites, and determined approximately 260 metabolites at distinct stages in leaves and siliques during senescence in Arabidopsis (Arabidopsis thaliana). This provided an extensive catalog of metabolites and their spatiotemporal cobehavior with progressing senescence. Comparison with silique data provides clues to source-sink relations. Furthermore, we analyzed the metabolite distribution within single leaves along the basipetal sink-source transition trajectory during senescence. Ceramides, lysolipids, aromatic amino acids, branched chain amino acids, and stress-induced amino acids accumulated, and an imbalance of asparagine/aspartate, glutamate/glutamine, and nutrient ions in the tip region of leaves was detected. Furthermore, the spatiotemporal distribution of tricarboxylic acid cycle intermediates was already changed in the presenescent leaves, and glucosinolates, raffinose, and galactinol accumulated in the base region of leaves with preceding senescence. These results are discussed in the context of current models of the metabolic shifts occurring during developmental and environmentally induced senescence. As senescence processes are correlated to crop yield, the metabolome data and the approach provided here can serve as a blueprint for the analysis of traits and conditions linking crop yield and senescence. PMID:23696093

  6. Forestry alters foraging efficiency and crop contents of aphid-tending red wood ants, Formica aquilonia.

    PubMed

    Johansson, Therese; Gibb, Heloise

    2012-01-01

    Forest management alters species behaviours, distributions and interactions. To evaluate forestry effects on ant foraging performance, we compared the quality and quantity of honeydew harvested by ants among clear-cuts, middle-aged and mature spruce-dominated stands in boreal forests in Sweden. Honeydew quality was examined using honeydew collected by squeezing the gasters of laden Formica aquilonia workers. We used fifteen laden individuals at each study site (four replicates of each stand age) and analysed honeydew chemical composition with gas chromatography-mass spectroscopy. To compare the quantity of honeydew collected by individual ants, we collected and weighed five ants moving up and five ants moving down each of ten trees at the twelve sites (totally 1200 ants). The concentration of trehalose in honeydew was lower in clear-cuts compared with middle aged and mature stands, and similar trends were shown for sucrose, raffinose and melezitose, indicating poorer honeydew quality on clear cuts. Concentrations of the amino acid serine were higher on clear-cuts. The same trend occurred for glutamine, suggesting that increased N-uptake by the trees after clear cutting is reflected in the honeydew of aphids. Ants in mature stands had larger heads and carried proportionally more honeydew and may therefore be more efficient foragers. Human alternation of habitats through clear-cutting thus affects food quality and worker condition in F. aquilonia. This is the first study to show that honeydew quality is affected by anthropogenic disturbances, likely contributing to the reduction in size and abundance of F. aquilonia workers and mounds after clear cutting.

  7. Characteristics and composition of watermelon, pumpkin, and paprika seed oils and flours.

    PubMed

    El-Adawy, T A; Taha, K M

    2001-03-01

    The nutritional quality and functional properties of paprika seed flour and seed kernel flours of pumpkin and watermelon were studied, as were the characteristics and structure of their seed oils. Paprika seed and seed kernels of pumpkin and watermelon were rich in oil and protein. All flour samples contained considerable amounts of P, K, Mg, Mn, and Ca. Paprika seed flour was superior to watermelon and pumpkin seed kernel flours in content of lysine and total essential amino acids. Oil samples had high amounts of unsaturated fatty acids with linoleic and oleic acids as the major acids. All oil samples fractionated into seven classes including triglycerides as a major lipid class. Data obtained for the oils' characteristics compare well with those of other edible oils. Antinutritional compounds such as stachyose, raffinose, verbascose, trypsin inhibitor, phytic acid, and tannins were detected in all flours. Pumpkin seed kernel flour had higher values of chemical score, essential amino acid index, and in vitro protein digestibility than the other flours examined. The first limiting amino acid was lysine for both watermelon and pumpkin seed kernel flours, but it was leucine in paprika seed flour. Protein solubility index, water and fat absorption capacities, emulsification properties, and foam stability were excellent in watermelon and pumpkin seed kernel flours and fairly good in paprika seed flour. Flour samples could be potentially added to food systems such as bakery products and ground meat formulations not only as a nutrient supplement but also as a functional agent in these formulations.

  8. Metabolic Profiling during Peach Fruit Development and Ripening Reveals the Metabolic Networks That Underpin Each Developmental Stage1[C][W

    PubMed Central

    Lombardo, Verónica A.; Osorio, Sonia; Borsani, Julia; Lauxmann, Martin A.; Bustamante, Claudia A.; Budde, Claudio O.; Andreo, Carlos S.; Lara, María V.; Fernie, Alisdair R.; Drincovich, María F.

    2011-01-01

    Fruit from rosaceous species collectively display a great variety of flavors and textures as well as a generally high content of nutritionally beneficial metabolites. However, relatively little analysis of metabolic networks in rosaceous fruit has been reported. Among rosaceous species, peach (Prunus persica) has stone fruits composed of a juicy mesocarp and lignified endocarp. Here, peach mesocarp metabolic networks were studied across development using metabolomics and analysis of key regulatory enzymes. Principal component analysis of peach metabolic composition revealed clear metabolic shifts from early through late development stages and subsequently during postharvest ripening. Early developmental stages were characterized by a substantial decrease in protein abundance and high levels of bioactive polyphenols and amino acids, which are substrates for the phenylpropanoid and lignin pathways during stone hardening. Sucrose levels showed a large increase during development, reflecting translocation from the leaf, while the importance of galactinol and raffinose is also inferred. Our study further suggests that posttranscriptional mechanisms are key for metabolic regulation at early stages. In contrast to early developmental stages, a decrease in amino acid levels is coupled to an induction of transcripts encoding amino acid and organic acid catabolic enzymes during ripening. These data are consistent with the mobilization of amino acids to support respiration. In addition, sucrose cycling, suggested by the parallel increase of transcripts encoding sucrose degradative and synthetic enzymes, appears to operate during postharvest ripening. When taken together, these data highlight singular metabolic programs for peach development and may allow the identification of key factors related to agronomic traits of this important crop species. PMID:22021422

  9. A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6.

    PubMed Central

    Liang, H; Gaber, R F

    1996-01-01

    We show that cells deleted for SNF3, HXT1, HXT2, HXT3, HXT4, HXT6, and HXT7 do not take up glucose and cannot grow on media containing glucose as a sole carbon source. The expression of Hxt1, Hxt2, Hxt3, Hxt6, or Gal2 in these cells resulted in glucose transport and allowed growth on glucose media. In contrast, the expression of Snf3 failed to confer glucose uptake or growth on glucose. HXT6 is highly expressed on raffinose, low glucose, or nonfermentable carbon sources but is repressed in the presence of high concentrations of glucose. The maintenance of HXT6 glucose repression is strictly dependent on Snf3 and not on intracellular glucose. In snf3 delta cells expression of HXT6 is constitutive even when the entire repertoire of HXT genes is present and glucose uptake is abundant. In addition, glucose repression of HXT6 does not require glucose uptake by HXT1, HXT2, HXT3 or HXT4. We show that a signal transduction pathway defined by the Snf3-dependent hexose regulation of HXT6 is distinct from but also overlaps with general glucose regulation pathways in Saccharomyces cerevisiae. Finally, glucose repression of ADH2 and SUC2 is intact in snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta hxt6 delta hxt7 delta gal2 cells, suggesting that the sensing and signaling mechanism for general glucose repression is independent from glucose uptake. Images PMID:8970157

  10. Knocking down mitochondrial iron transporter (MIT) reprograms primary and secondary metabolism in rice plants

    PubMed Central

    Vigani, Gianpiero; Bashir, Khurram; Ishimaru, Yasuhiro; Lehmann, Martin; Casiraghi, Fabio Marco; Nakanishi, Hiromi; Seki, Motoaki; Geigenberger, Peter; Zocchi, Graziano; Nishizawa, Naoko K.

    2016-01-01

    Iron (Fe) is an essential micronutrient for plant growth and development, and its reduced bioavailability strongly impairs mitochondrial functionality. In this work, the metabolic adjustment in the rice (Oryza sativa) mitochondrial Fe transporter knockdown mutant (mit-2) was analysed. Biochemical characterization of purified mitochondria from rice roots showed alteration in the respiratory chain of mit-2 compared with wild-type (WT) plants. In particular, proteins belonging to the type II alternative NAD(P)H dehydrogenases accumulated strongly in mit-2 plants, indicating that alternative pathways were activated to keep the respiratory chain working. Additionally, large-scale changes in the transcriptome and metabolome were observed in mit-2 rice plants. In particular, a strong alteration (up-/down-regulation) in the expression of genes encoding enzymes of both primary and secondary metabolism was found in mutant plants. This was reflected by changes in the metabolic profiles in both roots and shoots of mit-2 plants. Significant alterations in the levels of amino acids belonging to the aspartic acid-related pathways (aspartic acid, lysine, and threonine in roots, and aspartic acid and ornithine in shoots) were found that are strictly connected to the Krebs cycle. Furthermore, some metabolites (e.g. pyruvic acid, fumaric acid, ornithine, and oligosaccharides of the raffinose family) accumulated only in the shoot of mit-2 plants, indicating possible hypoxic responses. These findings suggest that the induction of local Fe deficiency in the mitochondrial compartment of mit-2 plants differentially affects the transcript as well as the metabolic profiles in root and shoot tissues. PMID:26685186

  11. Use of an alpha-galactosidase gene as a food-grade selection marker for Streptococcus thermophilus.

    PubMed

    Labrie, S; Bart, C; Vadeboncoeur, C; Moineau, S

    2005-07-01

    The alpha-galactosidase gene (aga) of Lactococcus raffinolactis ATCC 43920 was previously shown to be an efficient food-grade selection marker in Lactococcus lactis and Pediococcus acidilactici but not in Streptococcus thermophilus. In this study, we demonstrated that the alpha-galactosidase of L. raffinolactis is thermolabile and inoperative at 42 degrees C, the optimal growth temperature of S. thermophilus. An in vitro assay indicated that the activity of this alpha-galactosidase at 42 degrees C was only 3% of that at 30 degrees C, whereas the enzyme retained 23% of its activity at 37 degrees C. Transformation of Strep. thermophilus RD733 with the shuttle-vector pNZ123 bearing the aga gene of L. raffinolactis (pRAF301) generated transformants that were stable and able to grow on melibiose and raffinose at 37 degrees C or below. The transformed cells possessed 6-fold more alpha-galactosidase activity after growth on melibiose than cells grown on lactose. Slot-blot analyses of aga mRNA indicated that repression by lactose occurred at the transcriptional level. The presence of pRAF301 did not interfere with the lactic acid production when the transformed cells of Strep. thermophilus were grown at the optimal temperature in milk. Using the recombinant plasmid pRAF301, which carries a chloramphenicol resistance gene in addition to aga, we showed that both markers were equally efficient at differentiating transformed from nontransformed cells. The aga gene of L. raffinolactis can be used as a highly efficient selection marker in Strep. thermophilus.

  12. Model-driven analysis of experimentally determined growth phenotypes for 465 yeast gene deletion mutants under 16 different conditions

    PubMed Central

    Snitkin, Evan S; Dudley, Aimée M; Janse, Daniel M; Wong, Kaisheen; Church, George M; Segrè, Daniel

    2008-01-01

    Background Understanding the response of complex biochemical networks to genetic perturbations and environmental variability is a fundamental challenge in biology. Integration of high-throughput experimental assays and genome-scale computational methods is likely to produce insight otherwise unreachable, but specific examples of such integration have only begun to be explored. Results In this study, we measured growth phenotypes of 465 Saccharomyces cerevisiae gene deletion mutants under 16 metabolically relevant conditions and integrated them with the corresponding flux balance model predictions. We first used discordance between experimental results and model predictions to guide a stage of experimental refinement, which resulted in a significant improvement in the quality of the experimental data. Next, we used discordance still present in the refined experimental data to assess the reliability of yeast metabolism models under different conditions. In addition to estimating predictive capacity based on growth phenotypes, we sought to explain these discordances by examining predicted flux distributions visualized through a new, freely available platform. This analysis led to insight into the glycerol utilization pathway and the potential effects of metabolic shortcuts on model results. Finally, we used model predictions and experimental data to discriminate between alternative raffinose catabolism routes. Conclusions Our study demonstrates how a new level of integration between high throughput measurements and flux balance model predictions can improve understanding of both experimental and computational results. The added value of a joint analysis is a more reliable platform for specific testing of biological hypotheses, such as the catabolic routes of different carbon sources. PMID:18808699

  13. Metabolomic response of Calotropis procera growing in the desert to changes in water availability.

    PubMed

    Ramadan, Ahmed; Sabir, Jamal S M; Alakilli, Saleha Y M; Shokry, Ahmed M; Gadalla, Nour O; Edris, Sherif; Al-Kordy, Magdy A; Al-Zahrani, Hassan S; El-Domyati, Fotouh M; Bahieldin, Ahmed; Baker, Neil R; Willmitzer, Lothar; Irgang, Susann

    2014-01-01

    Water availability is a major limitation for agricultural productivity. Plants growing in severe arid climates such as deserts provide tools for studying plant growth and performance under extreme drought conditions. The perennial species Calotropis procera used in this study is a shrub growing in many arid areas which has an exceptional ability to adapt and be productive in severe arid conditions. We describe the results of studying the metabolomic response of wild C procera plants growing in the desert to a one time water supply. Leaves of C. procera plants were taken at three time points before and 1 hour, 6 hours and 12 hours after watering and subjected to a metabolomics and lipidomics analysis. Analysis of the data reveals that within one hour after watering C. procera has already responded on the metabolic level to the sudden water availability as evidenced by major changes such as increased levels of most amino acids, a decrease in sucrose, raffinose and maltitol, a decrease in storage lipids (triacylglycerols) and an increase in membrane lipids including photosynthetic membranes. These changes still prevail at the 6 hour time point after watering however 12 hours after watering the metabolomics data are essentially indistinguishable from the prewatering state thus demonstrating not only a rapid response to water availability but also a rapid response to loss of water. Taken together these data suggest that the ability of C. procera to survive under the very harsh drought conditions prevailing in the desert might be associated with its rapid adjustments to water availability and losses.

  14. Metabolomic Response of Calotropis procera Growing in the Desert to Changes in Water Availability

    PubMed Central

    Ramadan, Ahmed; Sabir, Jamal S. M.; Alakilli, Saleha Y. M.; Shokry, Ahmed M.; Gadalla, Nour O.; Edris, Sherif; Al-Kordy, Magdy A.; Al-Zahrani, Hassan S.; El-Domyati, Fotouh M.; Bahieldin, Ahmed; Baker, Neil R.; Willmitzer, Lothar; Irgang, Susann

    2014-01-01

    Water availability is a major limitation for agricultural productivity. Plants growing in severe arid climates such as deserts provide tools for studying plant growth and performance under extreme drought conditions. The perennial species Calotropis procera used in this study is a shrub growing in many arid areas which has an exceptional ability to adapt and be productive in severe arid conditions. We describe the results of studying the metabolomic response of wild C procera plants growing in the desert to a one time water supply. Leaves of C. procera plants were taken at three time points before and 1 hour, 6 hours and 12 hours after watering and subjected to a metabolomics and lipidomics analysis. Analysis of the data reveals that within one hour after watering C. procera has already responded on the metabolic level to the sudden water availability as evidenced by major changes such as increased levels of most amino acids, a decrease in sucrose, raffinose and maltitol, a decrease in storage lipids (triacylglycerols) and an increase in membrane lipids including photosynthetic membranes. These changes still prevail at the 6 hour time point after watering however 12 hours after watering the metabolomics data are essentially indistinguishable from the prewatering state thus demonstrating not only a rapid response to water availability but also a rapid response to loss of water. Taken together these data suggest that the ability of C. procera to survive under the very harsh drought conditions prevailing in the desert might be associated with its rapid adjustments to water availability and losses. PMID:24520340

  15. Identification of PEG-induced water stress responsive transcripts using co-expression network in Eucalyptus grandis.

    PubMed

    Ghosh Dasgupta, Modhumita; Dharanishanthi, Veeramuthu

    2017-09-05

    Ecophysiological studies in Eucalyptus have shown that water is the principal factor limiting stem growth. Effect of water deficit conditions on physiological and biochemical parameters has been extensively reported in Eucalyptus. The present study was conducted to identify major polyethylene glycol induced water stress responsive transcripts in Eucalyptus grandis using gene co-expression network. A customized array representing 3359 water stress responsive genes was designed to document their expression in leaves of E. grandis cuttings subjected to -0.225MPa of PEG treatment. The differentially expressed transcripts were documented and significantly co-expressed transcripts were used for construction of network. The co-expression network was constructed with 915 nodes and 3454 edges with degree ranging from 2 to 45. Ninety four GO categories and 117 functional pathways were identified in the network. MCODE analysis generated 27 modules and module 6 with 479 nodes and 1005 edges was identified as the biologically relevant network. The major water responsive transcripts represented in the module included dehydrin, osmotin, LEA protein, expansin, arabinogalactans, heat shock proteins, major facilitator proteins, ARM repeat proteins, raffinose synthase, tonoplast intrinsic protein and transcription factors like DREB2A, ARF9, AGL24, UNE12, WLIM1 and MYB66, MYB70, MYB 55, MYB 16 and MYB 103. The coordinated analysis of gene expression patterns and coexpression networks developed in this study identified an array of transcripts that may regulate PEG induced water stress responses in E. grandis. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Erwinia gerundensis sp. nov., a cosmopolitan epiphyte originally isolated from pome fruit trees.

    PubMed

    Rezzonico, Fabio; Smits, Theo H M; Born, Yannick; Blom, Jochen; Frey, Jürg E; Goesmann, Alexander; Cleenwerck, Ilse; de Vos, Paul; Bonaterra, Anna; Duffy, Brion; Montesinos, Emilio

    2016-03-01

    A survey to obtain potential antagonists of pome fruit tree diseases yielded two yellow epiphytic bacterial isolates morphologically similar to Pantoea agglomerans , but showing no biocontrol activity. Whole-cell MALDI-TOF mass spectrometry and analysis of 16S rRNA gene and gyrB sequences suggested the possibility of a novel species with a phylogenetic position in either the genus Pantoea or the genus Erwinia . Multi-locus sequence analysis (MLSA) placed the two strains in the genus Erwinia and supported their classification as a novel species. The strains showed general phenotypic characteristics typical of this genus and results of DNA-DNA hybridizations confirmed that they represent a single novel species. Both strains showed a DNA G+C content, as determined by HPLC, of 54.5 mol% and could be discriminated from phylogenetically related species of the genus Erwinia by their ability to utilize potassium gluconate, potassium 2-ketogluconate, maltose, melibiose and raffinose. Whole-genome sequencing of strain EM595 T revealed the presence of a chromosomal carotenoid biosynthesis gene cluster similar to those found in species of the genera Cronobacter and Pantoea that explains the pigmentation of the strain, which is atypical for the genus Erwinia . Additional strains belonging to the same species were recovered from different plant hosts in three different continents, revealing the cosmopolitan nature of this epiphyte. The name Erwinia gerundensis sp. nov. is proposed, with EM595 T ( = LMG 28990 T  = CCOS 903 T ) as the designated type strain.

  17. Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer.

    PubMed

    Drake, Andrew C; Lee, Youngjoo; Burgess, Emma M; Karlsson, Jens O M; Eroglu, Ali; Higgins, Adam Z

    2018-01-01

    Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants.

  18. Identification of metabolic signatures linked to anti-inflammatory effects of Faecalibacterium prausnitzii

    SciTech Connect

    Miquel, Sylvie; Leclerc, Marion; Martin, Rebeca

    Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified on the basis of human clinical data. The mechanisms underlying its beneficial effects are still unknown. Gnotobiotic mice harboring F. prausnitzii (A2-165) and Escherichia coli (K-12 JM105) were subjected to 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute colitis. The inflammatory colitis scores and a gas chromatography-time of flight (GC/TOF) mass spectrometry-based metabolomic profile were monitored in blood, ileum, cecum, colon, and feces in gnotobiotic mice. The potential anti-inflammatory metabolites were tested in vitro. We obtained stable E. coli and F. prausnitzii-diassociated mice in which E. coli primed the gastrointestinal tract (GIT), allowing a durable andmore » stable establishment of F. prausnitzii. The disease activity index, histological scores, myeloperoxidase (MPO) activity, and serum cytokine levels were significantly lower in the presence of F. prausnitzii after TNBS challenge. The protective effect of F. prausnitzii against colitis was correlated to its implantation level and was linked to overrepresented metabolites along the GIT and in serum. Among 983 metabolites in GIT samples and serum, 279 were assigned to known chemical reactions. Some of them, belonging to the ammonia (α-ketoglutarate), osmoprotective (raffinose), and phenolic (including anti-inflammatory shikimic and salicylic acids) pathways, were associated with a protective effect of F. prausnitzii, and the functional link was established in vitro for salicylic acid. We show for the first time that F. prausnitzii is a highly active commensal bacterium involved in reduction of colitis through in vivo modulation of metabolites along the GIT and in the peripheral blood.« less

  19. Knocking down mitochondrial iron transporter (MIT) reprograms primary and secondary metabolism in rice plants.

    PubMed

    Vigani, Gianpiero; Bashir, Khurram; Ishimaru, Yasuhiro; Lehmann, Martin; Casiraghi, Fabio Marco; Nakanishi, Hiromi; Seki, Motoaki; Geigenberger, Peter; Zocchi, Graziano; Nishizawa, Naoko K

    2016-03-01

    Iron (Fe) is an essential micronutrient for plant growth and development, and its reduced bioavailability strongly impairs mitochondrial functionality. In this work, the metabolic adjustment in the rice (Oryza sativa) mitochondrial Fe transporter knockdown mutant (mit-2) was analysed. Biochemical characterization of purified mitochondria from rice roots showed alteration in the respiratory chain of mit-2 compared with wild-type (WT) plants. In particular, proteins belonging to the type II alternative NAD(P)H dehydrogenases accumulated strongly in mit-2 plants, indicating that alternative pathways were activated to keep the respiratory chain working. Additionally, large-scale changes in the transcriptome and metabolome were observed in mit-2 rice plants. In particular, a strong alteration (up-/down-regulation) in the expression of genes encoding enzymes of both primary and secondary metabolism was found in mutant plants. This was reflected by changes in the metabolic profiles in both roots and shoots of mit-2 plants. Significant alterations in the levels of amino acids belonging to the aspartic acid-related pathways (aspartic acid, lysine, and threonine in roots, and aspartic acid and ornithine in shoots) were found that are strictly connected to the Krebs cycle. Furthermore, some metabolites (e.g. pyruvic acid, fumaric acid, ornithine, and oligosaccharides of the raffinose family) accumulated only in the shoot of mit-2 plants, indicating possible hypoxic responses. These findings suggest that the induction of local Fe deficiency in the mitochondrial compartment of mit-2 plants differentially affects the transcript as well as the metabolic profiles in root and shoot tissues. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  20. Metabolic survey of Botryococcus braunii: Impact of the physiological state on product formation.

    PubMed

    Blifernez-Klassen, Olga; Chaudhari, Swapnil; Klassen, Viktor; Wördenweber, Robin; Steffens, Tim; Cholewa, Dominik; Niehaus, Karsten; Kalinowski, Jörn; Kruse, Olaf

    2018-01-01

    The microalga Botryococcus braunii is widely regarded as a potential renewable and sustainable source for industrial applications because of its capability to produce large amounts of metabolically expensive (exo-) polysaccharides and lipids, notably hydrocarbons. A comprehensive and systematic metabolic characterization of the Botryococcus braunii race A strain CCAP 807/2 was conducted within the present study, including the detailed analysis of growth-associated and physiological parameters. In addition, the intracellular metabolome was profiled for the first time and showed growth- and product-specific fluctuations in response to the different availability of medium resources during the cultivation course. Among the identified metabolites, a constant expression of raffinose was observed for the first time under standard conditions, which has until now only been described for higher plants. Overall, the multilayered analysis during the cultivation of strain CCAP 807/2 allowed the differentiation of four distinct physiological growth phases and revealed differences in the production profiles and content of liquid hydrocarbons and carbohydrates with up to 84% of organic dry weight (oDW). In the process, an enhanced production of carbohydrates with up to 63% of oDW (1.36±0.03 g L-1) could be observed during the late linear growth phase, whereas the highest accumulation of extracellular hydrocarbons with up to 24% of oDW (0.66±0.12 g L-1) occurred mainly during the stationary growth phase. Altogether, the knowledge obtained is potentially useful for the general understanding of the overall physiology of Botryococcus braunii and provide important insights into the growth behavior and product formation of this microalga, and is thus relevant for large scale biofuel production and industrial applications.

  1. History of cryobiology, with special emphasis in evolution of mouse sperm cryopreservation.

    PubMed

    Sztein, Jorge M; Takeo, Toru; Nakagata, Naomi

    2018-06-01

    Confucius said study the past if you would define the future and a popular statement says that history depends on who writes it. To talk about history it is necessary to find and define a milestone where to start the narration. The intention of this quick review is to take the reader through moments and selected publications; part and pieces of memories showing how the concept of cryopreservation, specifically for mouse sperm, was conceived and sustained as we know it today. Beginning with the development of the microscope (1677) and continuing through the 17th century with the first documented observation by L. Spallanzani describing that sperm could maintain the motility under cold conditions. As J. Sherman suggested, we divide the cryopreservation evolution into two sequences, previous to and after 1949 when Polge, Smith and Parkes discovered the property of glycerol as cryoprotectant. Later, in 1972, D. Whittingham, S. Leibo, and P. Mazur applying a slow freezing process achieved the first embryo freezing (mouse). During that time many theories were scientifically confirmed. Among those, Peter Mazur demonstrated the relation between the speed of freezing and intracellular ice formation, and Stanley Leibo that each cell type has their unique freezing curve. In 1950, after the discovery of the protective aspect of glycerol, sperm from many mammals were frozen, except from the mouse. It was in the early 90's when the mouse sperm freezing becomes important and it was a real challenge for many groups, nevertheless, the technique using skim milk and raffinose modified by Dr Nakagata was the beginning of a different story …. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer

    PubMed Central

    Burgess, Emma M.; Karlsson, Jens O. M.; Eroglu, Ali

    2018-01-01

    Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants. PMID:29304068

  3. Sugar fermentation in probiotic bacteria--an in vitro study.

    PubMed

    Hedberg, M; Hasslöf, P; Sjöström, I; Twetman, S; Stecksén-Blicks, C

    2008-12-01

    Food supplemented with probiotic bacteria is a rapidly growing sector of the market. The aim of the present study was to evaluate and compare the acid production of selected probiotic strains available in commercial products. Six Lactobacillus strains (Lactobacillus plantarum 299v and 931; Lactobacillus rhamnosus GG and LB21; Lactobacillus paracasei subsp. paracasei F19, and Lactobacillus reuteri PTA 5289) were cultivated at 37 degrees C in an anaerobic atmosphere on Man, Rogosa, Shape (MRS) agar for 48 h or MRS broth for 16 h. After centrifugation, the cells were washed and resuspended in sterile phosphate-buffered saline and immediately subjected to a fermentation assay with 12 different carbohydrates (nine sugars and three sugar alcohols) in microtiter plates with a pH indicator. The plates were examined for color changes after 24, 48, and 72 h of incubation under aerobic and anaerobic conditions. Three scores were used: negative (pH > 6.8); weak (pH 5.2-6.8), and positive (pH < 5.2). The strains were characterized with the API 50 CH system to confirm their identity. L. plantarum fermented all the sugars except for melibiose, raffinose, and xylitol. Both L. rhamnosus strains were generally less active although L. rhamnosus GG was slightly more active than strain LB21 in the 5% CO(2) setting. The latter strain exhibited negative reactions for sucrose, maltose, arabinose, and sorbitol under anaerobic conditions. The assays with L. paracasei and L. reuteri had negative or weak reactions for all tested sugars under both aerobic and anaerobic conditions. The metabolic capacity to form acid from dietary sugars differed significantly between the various probiotic strains.

  4. Raman and infrared spectroscopy of carbohydrates: A review

    NASA Astrophysics Data System (ADS)

    Wiercigroch, Ewelina; Szafraniec, Ewelina; Czamara, Krzysztof; Pacia, Marta Z.; Majzner, Katarzyna; Kochan, Kamila; Kaczor, Agnieszka; Baranska, Malgorzata; Malek, Kamilla

    2017-10-01

    Carbohydrates are widespread and naturally occurring compounds, and essential constituents for living organisms. They are quite often reported when biological systems are studied and their role is discussed. However surprisingly, up till now there is no database collecting vibrational spectra of carbohydrates and their assignment, as has been done already for other biomolecules. So, this paper serves as a comprehensive review, where for selected 14 carbohydrates in the solid state both FT-Raman and ATR FT-IR spectra were collected and assigned. Carbohydrates can be divided into four chemical groups and in the same way is organized this review. First, the smallest molecules are discussed, i.e. monosaccharides (D-(-)-ribose, 2-deoxy-D-ribose, L-(-)-arabinose, D-(+)-xylose, D-(+)-glucose, D-(+)-galactose and D-(-)-fructose) and disaccharides (D-(+)-sucrose, D-(+)-maltose and D-(+)-lactose), and then more complex ones, i.e. trisaccharides (D-(+)-raffinose) and polysaccharides (amylopectin, amylose, glycogen). Both Raman and IR spectra were collected in the whole spectral range and discussed looking at the specific regions, i.e. region V (3600-3050 cm- 1), IV (3050-2800 cm- 1) and II (1200-800 cm- 1) assigned to the stretching vibrations of the OH, CH/CH2 and C-O/C-C groups, respectively, and region III (1500-1200 cm- 1) and I (800-100 cm- 1) dominated by deformational modes of the CH/CH2 and CCO groups, respectively. In spite of the fact that vibrational spectra of saccharides are significantly less specific than spectra of other biomolecules (e.g. lipids or proteins), marker bands of the studied molecules can be identified and correlated with their structure.

  5. Conserving, Distributing and Managing Genetically Modified Mouse Lines by Sperm Cryopreservation

    PubMed Central

    Farley, Jane S.; Taft, Robert A.

    2008-01-01

    Background Sperm from C57BL/6 mice are difficult to cryopreserve and recover. Yet, the majority of genetically modified (GM) lines are maintained on this genetic background. Methodology/Principal Findings Reported here is the development of an easily implemented method that consistently yields fertilization rates of 70±5% with this strain. This six-fold increase is achieved by collecting sperm from the vas deferens and epididymis into a cryoprotective medium of 18% raffinose (w/v), 3% skim milk (w/v) and 477 µM monothioglycerol. The sperm suspension is loaded into 0.25 mL French straws and cooled at 37±1°C/min before being plunged and then stored in LN2. Subsequent to storage, the sperm are warmed at 2,232±162°C/min and incubated in in vitro fertilization media for an hour prior to the addition of oocyte cumulus masses from superovulated females. Sperm from 735 GM mouse lines on 12 common genetic backgrounds including C57BL/6J, BALB/cJ, 129S1/SvImJ, FVB/NJ and NOD/ShiLtJ were cryopreserved and recovered. C57BL/6J and BALB/cByJ fertilization rates, using frozen sperm, were slightly reduced compared to rates involving fresh sperm; fertilization rates using fresh or frozen sperm were equivalent in all other lines. Developmental capacity of embryos produced using cryopreserved sperm was equivalent, or superior to, cryopreserved IVF-derived embryos. Conclusions/Significance Combined, these results demonstrate the broad applicability of our approach as an economical and efficient option for archiving and distributing mice. PMID:18665210

  6. Identification of metabolic signatures linked to anti-inflammatory effects of Faecalibacterium prausnitzii

    DOE PAGES

    Miquel, Sylvie; Leclerc, Marion; Martin, Rebeca; ...

    2015-04-21

    Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified on the basis of human clinical data. The mechanisms underlying its beneficial effects are still unknown. Gnotobiotic mice harboring F. prausnitzii (A2-165) and Escherichia coli (K-12 JM105) were subjected to 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute colitis. The inflammatory colitis scores and a gas chromatography-time of flight (GC/TOF) mass spectrometry-based metabolomic profile were monitored in blood, ileum, cecum, colon, and feces in gnotobiotic mice. The potential anti-inflammatory metabolites were tested in vitro. We obtained stable E. coli and F. prausnitzii-diassociated mice in which E. coli primed the gastrointestinal tract (GIT), allowing a durable andmore » stable establishment of F. prausnitzii. The disease activity index, histological scores, myeloperoxidase (MPO) activity, and serum cytokine levels were significantly lower in the presence of F. prausnitzii after TNBS challenge. The protective effect of F. prausnitzii against colitis was correlated to its implantation level and was linked to overrepresented metabolites along the GIT and in serum. Among 983 metabolites in GIT samples and serum, 279 were assigned to known chemical reactions. Some of them, belonging to the ammonia (α-ketoglutarate), osmoprotective (raffinose), and phenolic (including anti-inflammatory shikimic and salicylic acids) pathways, were associated with a protective effect of F. prausnitzii, and the functional link was established in vitro for salicylic acid. We show for the first time that F. prausnitzii is a highly active commensal bacterium involved in reduction of colitis through in vivo modulation of metabolites along the GIT and in the peripheral blood.« less

  7. The tonoplast-localized sucrose transporter in Populus (PtaSUT4) regulates whole-plant water relations, responses to water stress, and photosynthesis.

    PubMed

    Frost, Christopher J; Nyamdari, Batbayar; Tsai, Chung-Jui; Harding, Scott A

    2012-01-01

    The Populus sucrose (Suc) transporter 4 (PtaSUT4), like its orthologs in other plant taxa, is tonoplast localized and thought to mediate Suc export from the vacuole into the cytosol. In source leaves of Populus, SUT4 is the predominantly expressed gene family member, with transcript levels several times higher than those of plasma membrane SUTs. A hypothesis is advanced that SUT4-mediated tonoplast sucrose fluxes contribute to the regulation of osmotic gradients between cellular compartments, with the potential to mediate both sink provisioning and drought tolerance in Populus. Here, we describe the effects of PtaSUT4-RNA interference (RNAi) on sucrose levels and raffinose family oligosaccharides (RFO) induction, photosynthesis, and water uptake, retention and loss during acute and chronic drought stresses. Under normal water-replete growing conditions, SUT4-RNAi plants had generally higher shoot water contents than wild-type plants. In response to soil drying during a short-term, acute drought, RNAi plants exhibited reduced rates of water uptake and delayed wilting relative to wild-type plants. SUT4-RNAi plants had larger leaf areas and lower photosynthesis rates than wild-type plants under well-watered, but not under chronic water-limiting conditions. Moreover, the magnitude of shoot water content, height growth, and photosynthesis responses to contrasting soil moisture regimes was greater in RNAi than wild-type plants. The concentrations of stress-responsive RFOs increased in wild-type plants but were unaffected in SUT4-RNAi plants under chronically dry conditions. We discuss a model in which the subcellular compartmentalization of sucrose mediated by PtaSUT4 is regulated in response to both sink demand and plant water status in Populus.

  8. Altering carbon allocation in hybrid poplar (Populus alba × grandidentata) impacts cell wall growth and development.

    PubMed

    Unda, Faride; Kim, Hoon; Hefer, Charles; Ralph, John; Mansfield, Shawn D

    2017-07-01

    Galactinol synthase is a pivotal enzyme involved in the synthesis of the raffinose family of oligosaccharides (RFOs) that function as transport carbohydrates in the phloem, as storage compounds in sink tissues and as soluble metabolites that combat both abiotic and biotic stress in several plant species. Hybrid poplar (Populus alba × grandidentata) overexpressing the Arabidopsis thaliana GolS3 (AtGolS3) gene showed clear effects on development; the extreme overexpressing lines were stunted and had cell wall traits characteristic of tension wood, whereas lines with only moderate up-regulation grew normally and had moderately altered secondary cell wall composition and ultrastructure. Stem cross-sections of the developing xylem revealed a significant increase in the number of vessels, as well as the clear presence of a G-layer in the fibres. Furthermore, AtGolS3-OE lines possessed higher cellulose and lower lignin contents, an increase in cellulose crystallinity, and significantly altered hemicellulose-derived carbohydrates, notably manifested by their mannose and xylose contents. In addition, the transgenic plants displayed elevated xylem starch content. Transcriptome interrogation of the transgenic plants showed a significant up-regulation of genes involved in the synthesis of myo-inositol, along with genes involved in sucrose degradation. The results suggest that the overexpression of GolS and its product galactinol may serve as a molecular signal that initiates metabolic changes, culminating in a change in cell wall development and potentially the formation of tension wood. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  9. Purification and some properties of rose (Fructus cynosbati) hips invertase.

    PubMed

    Sacan, Ozlem; Yanardag, Refiye

    2012-04-01

    Invertase was purified from rose (Fructus cynosbati) hips by ammonium sulfate fractionation and hydroxyapatite column chromatography. The enzyme was obtained with a yield of 4.25% and about 10.48-fold purification and had a specific activity of 8.59 U/mg protein. The molecular mass of invertase was estimated to be 66.51 kDa by PAGE and 34 kDa by SDS-PAGE, indicating that the native enzyme was a homodimer. The enzyme was a glycoprotein and contained 5.86% carbohydrate. The K(m) for sucrose was 14.55 mM and the optimum pH and temperature of the enzyme were 4.5 and 40 degrees C, respectively. Sucrose was the most preferred substrate of the enzyme. The enzyme also hydrolyzed D(+) raffinose, D(+) trehalose and inulin (activity 39.88, 8.12 and 4.94%, respectively of that of sucrose), while D(+) lactose, cellobiose and D(+) maltose showed no effect on the enzyme. The substrate specificity was consistent with that for a beta-fructofuranoside, which is the most popular type in the higher plants. The enzyme was completely inhibited by HgCl2, MnCl2, MnSO4, FeCl3, Pb(NO3)2, ammonium heptamolybdate, iodoacetamide and pyridoxine hydrochloride. It was also inhibited by Ba(NO3)2 (86.32%), NH4Cl (84.91%), MgCl2 (74.45%), urea (71.63%), I2 (69.64%), LiCl (64.99%), BaCl2 (50.30%), Mg(NO3)2 (49.90%), CrCl3 (31.90%) and CuSO4 (21.45%) and but was activated by Tris (73.99%) and methionine (12.47%).

  10. Characterization, localization, and seasonal changes of the sucrose transporter FeSUT1 in the phloem of Fraxinus excelsior

    PubMed Central

    Öner-Sieben, Soner; Rappl, Christine; Sauer, Norbert; Stadler, Ruth; Lohaus, Gertrud

    2015-01-01

    Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker’s yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior. PMID:26022258

  11. Whole-plant versus leaf-level regulation of photosynthetic responses after partial defoliation in Eucalyptus globulus saplings.

    PubMed

    Eyles, Alieta; Pinkard, Elizabeth A; Davies, Noel W; Corkrey, Ross; Churchill, Keith; O'Grady, Anthony P; Sands, Peter; Mohammed, Caroline

    2013-04-01

    Increases in photosynthetic capacity (A1500) after defoliation have been attributed to changes in leaf-level biochemistry, water, and/or nutrient status. The hypothesis that transient photosynthetic responses to partial defoliation are regulated by whole-plant (e.g. source-sink relationships or changes in hydraulic conductance) rather than leaf-level mechanisms is tested here. Temporal variation in leaf-level gas exchange, chemistry, whole-plant soil-to-leaf hydraulic conductance (KP), and aboveground biomass partitioning were determined to evaluate mechanisms responsible for increases in A1500 of Eucalyptus globulus L. potted saplings. A1500 increased in response to debudding (B), partial defoliation (D), and combined B&D treatments by up to 36% at 5 weeks after treatment. Changes in leaf-level factors partly explained increases in A1500 of B and B&D treatments but not for D treatment. By week 5, saplings in B, B&D, and D treatments had similar leaf-specific KP to control trees by maintaining lower midday water potentials and higher transpiration rate per leaf area. Whole-plant source:sink ratios correlated strongly with A1500. Further, unlike KP, temporal changes in source:sink ratios tracked well with those observed for A1500. The results indicate that increases in A1500 after partial defoliation treatments were largely driven by an increased demand for assimilate by developing sinks rather than improvements in whole-plant water relations and changes in leaf-level factors. Three carbohydrates, galactional, stachyose, and, to a lesser extent, raffinose, correlated strongly with photosynthetic capacity, indicating that these sugars may function as signalling molecules in the regulation of longer term defoliation-induced gas exchange responses.

  12. Whole-plant versus leaf-level regulation of photosynthetic responses after partial defoliation in Eucalyptus globulus saplings

    PubMed Central

    Eyles, Alieta

    2013-01-01

    Increases in photosynthetic capacity (A1500) after defoliation have been attributed to changes in leaf-level biochemistry, water, and/or nutrient status. The hypothesis that transient photosynthetic responses to partial defoliation are regulated by whole-plant (e.g. source–sink relationships or changes in hydraulic conductance) rather than leaf-level mechanisms is tested here. Temporal variation in leaf-level gas exchange, chemistry, whole-plant soil-to-leaf hydraulic conductance (KP), and aboveground biomass partitioning were determined to evaluate mechanisms responsible for increases in A1500 of Eucalyptus globulus L. potted saplings. A1500 increased in response to debudding (B), partial defoliation (D), and combined B&D treatments by up to 36% at 5 weeks after treatment. Changes in leaf-level factors partly explained increases in A1500 of B and B&D treatments but not for D treatment. By week 5, saplings in B, B&D, and D treatments had similar leaf-specific KP to control trees by maintaining lower midday water potentials and higher transpiration rate per leaf area. Whole-plant source:sink ratios correlated strongly with A1500. Further, unlike KP, temporal changes in source:sink ratios tracked well with those observed for A1500. The results indicate that increases in A1500 after partial defoliation treatments were largely driven by an increased demand for assimilate by developing sinks rather than improvements in whole-plant water relations and changes in leaf-level factors. Three carbohydrates, galactional, stachyose, and, to a lesser extent, raffinose, correlated strongly with photosynthetic capacity, indicating that these sugars may function as signalling molecules in the regulation of longer term defoliation-induced gas exchange responses. PMID:23382548

  13. Viscous organic aerosol particles in the upper troposphere: diffusivity-controlled water uptake and ice nucleation?

    DOE PAGES

    Lienhard, D. M.; Huisman, A. J.; Krieger, U. K.; ...

    2015-01-01

    New measurements of water diffusion in secondary organic aerosol (SOA) material produced by oxidation of α-pinene and in a number of organic/inorganic model mixtures (3-methylbutane-1,2,3-tricarboxylic acid (3-MBTCA), levoglucosan, levoglucosan/NH 4HSO 4, raffinose) are presented. These indicate that water diffusion coefficients are determined by several properties of the aerosol substance and cannot be inferred from the glass transition temperature or bouncing properties. Our results suggest that water diffusion in SOA particles is faster than often assumed and imposes no significant kinetic limitation on water uptake and release at temperatures above 220 K. The fast diffusion of water suggests that heterogeneous icemore » nucleation on a glassy core is very unlikely in these systems. At temperatures below 220 K, model simulations of SOA particles suggest that heterogeneous ice nucleation may occur in the immersion mode on glassy cores which remain embedded in a liquid shell when experiencing fast updraft velocities. The particles absorb significant quantities of water during these updrafts which plasticize their outer layers such that these layers equilibrate readily with the gas phase humidity before the homogeneous ice nucleation threshold is reached. Glass formation is thus unlikely to restrict homogeneous ice nucleation. Only under most extreme conditions near the very high tropical tropopause may the homogeneous ice nucleation rate coefficient be reduced as a consequence of slow condensed-phase water diffusion. Since the differences between the behavior limited or non limited by diffusion are small even at the very high tropical tropopause, condensed-phase water diffusivity is unlikely to have significant consequences on the direct climatic effects of SOA particles under tropospheric conditions.« less

  14. SciTech Connect

    Lienhard, D. M.; Huisman, A. J.; Krieger, U. K.

    New measurements of water diffusion in secondary organic aerosol (SOA) material produced by oxidation of α-pinene and in a number of organic/inorganic model mixtures (3-methylbutane-1,2,3-tricarboxylic acid (3-MBTCA), levoglucosan, levoglucosan/NH 4HSO 4, raffinose) are presented. These indicate that water diffusion coefficients are determined by several properties of the aerosol substance and cannot be inferred from the glass transition temperature or bouncing properties. Our results suggest that water diffusion in SOA particles is faster than often assumed and imposes no significant kinetic limitation on water uptake and release at temperatures above 220 K. The fast diffusion of water suggests that heterogeneous icemore » nucleation on a glassy core is very unlikely in these systems. At temperatures below 220 K, model simulations of SOA particles suggest that heterogeneous ice nucleation may occur in the immersion mode on glassy cores which remain embedded in a liquid shell when experiencing fast updraft velocities. The particles absorb significant quantities of water during these updrafts which plasticize their outer layers such that these layers equilibrate readily with the gas phase humidity before the homogeneous ice nucleation threshold is reached. Glass formation is thus unlikely to restrict homogeneous ice nucleation. Only under most extreme conditions near the very high tropical tropopause may the homogeneous ice nucleation rate coefficient be reduced as a consequence of slow condensed-phase water diffusion. Since the differences between the behavior limited or non limited by diffusion are small even at the very high tropical tropopause, condensed-phase water diffusivity is unlikely to have significant consequences on the direct climatic effects of SOA particles under tropospheric conditions.« less

  15. Inbred mouse strains C57BL/6J and DBA/2J vary in sensitivity to a subset of bitter stimuli

    PubMed Central

    Boughter, John D; Raghow, Sandeep; Nelson, Theodore M; Munger, Steven D

    2005-01-01

    Background Common inbred mouse strains are genotypically diverse, but it is still poorly understood how this diversity relates to specific differences in behavior. To identify quantitative trait genes that influence taste behavior differences, it is critical to utilize assays that exclusively measure the contribution of orosensory cues. With a few exceptions, previous characterizations of behavioral taste sensitivity in inbred mouse strains have generally measured consumption, which can be confounded by post-ingestive effects. Here, we used a taste-salient brief-access procedure to measure taste sensitivity to eight stimuli characterized as bitter or aversive in C57BL/6J (B6) and DBA/2J (D2) mice. Results B6 mice were more sensitive than D2 mice to a subset of bitter stimuli, including quinine hydrochloride (QHCl), 6-n-propylthiouracil (PROP), and MgCl2. D2 mice were more sensitive than B6 mice to the bitter stimulus raffinose undecaacetate (RUA). These strains did not differ in sensitivity to cycloheximide (CYX), denatonium benzoate (DB), KCl or HCl. Conclusion B6-D2 taste sensitivity differences indicate that differences in consumption of QHCl, PROP, MgCl2 and RUA are based on immediate orosensory cues, not post-ingestive effects. The absence of a strain difference for CYX suggests that polymorphisms in a T2R-type taste receptor shown to be differentially sensitive to CYX in vitro are unlikely to differentially contribute to the CYX behavioral response in vivo. The results of these studies point to the utility of these common mouse strains and their associated resources for investigation into the genetic mechanisms of taste. PMID:15967025

  16. Raman and infrared spectroscopy of carbohydrates: A review.

    PubMed

    Wiercigroch, Ewelina; Szafraniec, Ewelina; Czamara, Krzysztof; Pacia, Marta Z; Majzner, Katarzyna; Kochan, Kamila; Kaczor, Agnieszka; Baranska, Malgorzata; Malek, Kamilla

    2017-10-05

    Carbohydrates are widespread and naturally occurring compounds, and essential constituents for living organisms. They are quite often reported when biological systems are studied and their role is discussed. However surprisingly, up till now there is no database collecting vibrational spectra of carbohydrates and their assignment, as has been done already for other biomolecules. So, this paper serves as a comprehensive review, where for selected 14 carbohydrates in the solid state both FT-Raman and ATR FT-IR spectra were collected and assigned. Carbohydrates can be divided into four chemical groups and in the same way is organized this review. First, the smallest molecules are discussed, i.e. monosaccharides (d-(-)-ribose, 2-deoxy-d-ribose, l-(-)-arabinose, d-(+)-xylose, d-(+)-glucose, d-(+)-galactose and d-(-)-fructose) and disaccharides (d-(+)-sucrose, d-(+)-maltose and d-(+)-lactose), and then more complex ones, i.e. trisaccharides (d-(+)-raffinose) and polysaccharides (amylopectin, amylose, glycogen). Both Raman and IR spectra were collected in the whole spectral range and discussed looking at the specific regions, i.e. region V (3600-3050cm -1 ), IV (3050-2800cm -1 ) and II (1200-800cm -1 ) assigned to the stretching vibrations of the OH, CH/CH 2 and C-O/C-C groups, respectively, and region III (1500-1200cm -1 ) and I (800-100cm -1 ) dominated by deformational modes of the CH/CH 2 and CCO groups, respectively. In spite of the fact that vibrational spectra of saccharides are significantly less specific than spectra of other biomolecules (e.g. lipids or proteins), marker bands of the studied molecules can be identified and correlated with their structure. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Determination of carbohydrates in medicinal plants--comparison between TLC, mf-MELDI-MS and GC-MS.

    PubMed

    Qureshi, Muhammad Nasimullah; Stecher, Guenther; Sultana, Tahira; Abel, Gudrun; Popp, Michael; Bonn, Guenther K

    2011-01-01

    Quality control in the pharmaceutical and phytopharmaceutical industries requires fast and reliable methods for the analysis of raw materials and final products. This study evaluates different analytical approaches in order to recognise the most suitable technique for the analysis of carbohydrates in herbal drug preparations. The specific focus of the study is on thin-layer chromatography (TLC), gas chromatography (GC), and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption/ionisation time of flight mass spectrometry (mf-MELDI-MS). Samples employed in the study were standards and microwave-assisted water extracts from Quercus. TLC analysis proved the presence of mono-, di- and trisaccharides within the biological sample and hinted at the existence of an unknown carbohydrate of higher oligomerisation degree. After evaluation of different derivatisation techniques, GC-MS confirmed data obtained via TLC for mono- to trisaccharides, delivering additionally quantified values under a considerable amount of time. A carbohydrate of higher oligomerisation degree could not be found. The application of mf-MELDI-MS further confirmed the presence of carbohydrates up to trisaccharides, also hinting at the presence of a form of tetrasaccharide. Besides this information, mf-MELDI-MS delivered further data about other substances present in the extract. Quantitative determination resulted in 1.750, 1.736 and 0.336 mg/mL for glucose, sucrose and raffinose respectively. Evaluation of all three techniques employed, clearly proved the heightened performance of mf-MELDI-MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. In addition, GC-MS is suitable for quantitative analysis. Copyright © 2011 John Wiley & Sons, Ltd.

  18. [The low molecular weight carbohydrates and polyols in the cambial sap of beech (Fagus silvatica L.) and a number of other deciduous trees].

    PubMed

    Oesch, F

    1969-12-01

    The low molecular-weight carbohydrates sucrose, D-glucose, D-fructose, myoinositol, O-α-D-galactopyranosyl-(1→1)-myoinositol, raffinose, D,L-inositol, stachyose, α,α-trehalose and D-galactose (in decreasing order of percentage by weight) were isolated from the cambial sap of the beech (Fagus silvatica L.). The acidic compounds glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-diphosphate, malic acid and oxalic acid were also tentatively identified.Practically the only difference between the cambial saps of beech trees felled at different times during the period of most active secondary growth was in their content of myoinositol and an as yet unidentified extremely labile basic substance (BF 2). The content of both these substances increased greatly towards the end of the growing season. The cambial sap of any tree differed little from that of the xylem which had already begun to differentiate. On the other hand the cambial sap next to the bark and the phloem sap had a completely different composition. In the former sucrose constituted ca. 90% and in the latter practically 100% of the neutral, water-soluble fraction.A comparison of the cambial saps of beech and a number of other deciduous trees brought to light a number of characteristic differences that appeared to be chemotaxonomically significant. Trehalose and D,L-inositol were detected only in the genus Fagus and never in the closely related genus Nothofagus. The presence of large amounts of stachyose and mannitol was characteristic for Fraxinus, and a high content of 2-O-methyl-L-inositol was typical of Acer. Galactose was not detected in all trees studied.

  19. Vibrio japonicus sp. nov., a novel member of the Nereis clade in the genus Vibrio isolated from the coast of Japan

    PubMed Central

    2017-01-01

    A novel Vibrio strain, JCM 31412T, was isolated from seawater collected from the Inland Sea (Setonaikai), Japan, and characterized as a Gram-negative, oxidase-positive, catalase-negative, facultatively anaerobic, motile, ovoid-shaped bacterium with one polar flagellum. Based on 16S rDNA gene identity, strain JCM 31412T showed a close relationship with type strains of Vibrio brasiliensis (LMG 20546T, 98.2% identity), V. harveyi (NBRC 15634T, 98.2%), V. caribbeanicus (ATCC BAA-2122T, 97.8%) and V. proteolyticus (NBRC 13287T, 97.8%). The G+C content of strain JCM 31412T DNA was 46.8%. Multi-locus sequence analysis (MLSA) of eight loci (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA; 5535bp) further clustered strain JCM 31412T in the Nereis clade, genus Vibrio. Phenotypically, strain JCM 31412T differed from the closest related Vibrio species in its utilization of melibiose and raffinose, and its lack of casein and gelatin hydrolysis. It was further differentiated based on its fatty acid composition, specifically properties of C12:03OH and summed features, which were significantly different from those of V. brasiliensis, V. nigripulchritudo and V. caribbeanicus type strains. Overall, the results of DNA-DNA hybridization, and physiological and biochemical analysis differentiated strain JCM 31412T from other described species of the genus Vibrio. Based on these polyphasic taxonomic findings, it was therefore concluded that JCM 31412T was a novel Vibrio species, for which the name Vibrio japonicus sp. nov. was proposed, with JCM 31412T (= LMG 29636T = ATCC TSD-62T) as the type strain. PMID:28231272

  20. Comparison of Nitrogen Fixation Activity in Tall and Short Spartina alterniflora Salt Marsh Soils 1

    PubMed Central

    Hanson, Roger B.

    1977-01-01

    A comparison of the N2 fixers in the tall Spartina alterniflora and short S. alterniflora marsh soils was investigated. Zero-order kinetics and first-order kinetics of acetylene reduction were used to describe the activity of the N2 fixers in marsh soil slurries. It was found that the Vmax values were approximately 10 times greater for the N2 fixers in the tall Spartina than in the short Spartina marsh when raffinose was used as the energy source. In addition, the (Ks + Sn) values were approximately 4 to 15 times lower for the N2 fixers in the tall Spartina than in short Spartina marsh. First-order kinetics of nitrogen fixation for several substrates indicate that the N2 fixers in the tall Spartina marsh were two to seven times more active than those in the short Spartina marsh. Ammonium chloride (25 μg/ml) did not inhibit nitrogen fixation in the tall Spartina marsh, but there was a 50% inhibition in nitrogen fixation in the short Spartina marsh. On the other hand, sodium nitrate inhibited nitrogen fixation almost 100% at 25 μg/ml in both soil environments. Amino nitrogen (25 to 100 μg/ml) had little or no effect on nitrogen fixation. The results indicate that the N2 fixers in the tall Spartina marsh were physiologically more responsive to nutrient addition than those in the short Spartina marsh. This difference in the two populations may be related to the difference in daily tidal influence in the respective areas and thus provide another explanation for the enhanced S. alterniflora production in the creek bank soil system. PMID:16345213

  1. Growth platform-dependent and -independent phenotypic and metabolic responses of Arabidopsis and its halophytic relative, Eutrema salsugineum, to salt stress.

    PubMed

    Kazachkova, Yana; Batushansky, Albert; Cisneros, Aroldo; Tel-Zur, Noemi; Fait, Aaron; Barak, Simon

    2013-07-01

    Comparative studies of the stress-tolerant Arabidopsis (Arabidopsis thaliana) halophytic relative, Eutrema salsugineum, have proven a fruitful approach to understanding natural stress tolerance. Here, we performed comparative phenotyping of Arabidopsis and E. salsugineum vegetative development under control and salt-stress conditions, and then compared the metabolic responses of the two species on different growth platforms in a defined leaf developmental stage. Our results reveal both growth platform-dependent and -independent phenotypes and metabolic responses. Leaf emergence was affected in a similar way in both species grown in vitro but the effects observed in Arabidopsis occurred at higher salt concentrations in E. salsugineum. No differences in leaf emergence were observed on soil. A new effect of a salt-mediated reduction in E. salsugineum leaf area was unmasked. On soil, leaf area reduction in E. salsugineum was mainly due to a fall in cell number, whereas both cell number and cell size contributed to the decrease in Arabidopsis leaf area. Common growth platform-independent leaf metabolic signatures such as high raffinose and malate, and low fumarate contents that could reflect core stress tolerance mechanisms, as well as growth platform-dependent metabolic responses were identified. In particular, the in vitro growth platform led to repression of accumulation of many metabolites including sugars, sugar phosphates, and amino acids in E. salsugineum compared with the soil system where these same metabolites accumulated to higher levels in E. salsugineum than in Arabidopsis. The observation that E. salsugineum maintains salt tolerance despite growth platform-specific phenotypes and metabolic responses suggests a considerable degree of phenotypic and metabolic adaptive plasticity in this extremophile.

  2. [Isolation and characterization of Thermopirellula anaerolimosa gen. nov., sp. nov., an obligate anaerobic hydrogen-producing bacterium of the phylum Planctomycetes].

    PubMed

    Liu, Dongying; Liu, Yi; Men, Xuehui; Guo, Qunqun; Guo, Rongbo; Qiu, Yanling

    2012-08-04

    To cultivate various yet-to-be cultured heterotrophs from anaerobic granule sludge, we used a selective culture medium with low concentrations of substrates supplemented a variety of antibiotics. An obligate anaerobic, thermophilic, hydrogen-producing bacterium, strain VM20-7(T), was isolated from an upflow anaerobic sludge blanket (UASB) reactor treating high-strength organic wastewater from isomerized sugar production processes. Cells of strain VM20-7(T) are non-motile, spherical, pear or teardrop shaped, occurring singly(o)r as aggregates (0.7 - 2.0 microm x 0.7 - 2.0 microm). Spore formation was not observed. Growth temperature ranges from 35 - 50 degrees C (optimum 45 degrees C), pH ranges from 6.0 - 8.3 (optimum 7.0 - 7.5) , NaCl tolerant concentration ranges from 0% - 0.5% (w/v, optimum 0% ). Nitrate, sulfate, thiosulfate, sulfite, elemental sulfur and Fe (III)-NTA were not used as terminal electron acceptors. Strain VM20-7(T) utilizes a wide range of carbohydrates, including glucose, maltose, ribose, xylose, sucrose, galactose, mannose, raffinose, pectin, yeast extract and xylan. Acetate and H2 are the main end products of glucose fermentation. The G + C content of the genomic DNA was 60.9 mol%. 16S rRNA gene sequence analysis revealed that it is related to the Pirellula-Rhodopirellula-Blastopirellula (PRB) clade within the order Planctomycetales (82.7 - 84.3% similarity with 16S rRNA genes of other known related species). The first obligate anaerobic bacterium within the phylum Planctomycetes was isolated with low concentration of carbohydrates and antibiotics. On the basis of the physiological and phylogenetic data, the name Thermopirellula anaerolimosa gen. nov. , sp. nov. is proposed for strain VM20-7(T) (= CGMCC 1.5169(T) = JCM 17478(T) = DSM 24165(T)).

  3. Global Analysis of Gene Expression Profiles in Physic Nut (Jatropha curcas L.) Seedlings Exposed to Salt Stress

    PubMed Central

    Wu, Pingzhi; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2014-01-01

    Background Salt stress interferes with plant growth and production. Plants have evolved a series of molecular and morphological adaptations to cope with this abiotic stress, and overexpression of salt response genes reportedly enhances the productivity of various crops. However, little is known about the salt responsive genes in the energy plant physic nut (Jatropha curcas L.). Thus, excavate salt responsive genes in this plant are informative in uncovering the molecular mechanisms for the salt response in physic nut. Methodology/Principal Findings We applied next-generation Illumina sequencing technology to analyze global gene expression profiles of physic nut plants (roots and leaves) 2 hours, 2 days and 7 days after the onset of salt stress. A total of 1,504 and 1,115 genes were significantly up and down-regulated in roots and leaves, respectively, under salt stress condition. Gene ontology (GO) analysis of physiological process revealed that, in the physic nut, many “biological processes” were affected by salt stress, particular those categories belong to “metabolic process”, such as “primary metabolism process”, “cellular metabolism process” and “macromolecule metabolism process”. The gene expression profiles indicated that the associated genes were responsible for ABA and ethylene signaling, osmotic regulation, the reactive oxygen species scavenging system and the cell structure in physic nut. Conclusions/Significance The major regulated genes detected in this transcriptomic data were related to trehalose synthesis and cell wall structure modification in roots, while related to raffinose synthesis and reactive oxygen scavenger in leaves. The current study shows a comprehensive gene expression profile of physic nut under salt stress. The differential expression genes detected in this study allows the underling the salt responsive mechanism in physic nut with the aim of improving its salt resistance in the future. PMID:24837971

  4. Phenotypic and genotypic diversity of Lactobacillus buchneri strains isolated from spoiled, fermented cucumber.

    PubMed

    Daughtry, Katheryne V; Johanningsmeier, Suzanne D; Sanozky-Dawes, Rosemary; Klaenhammer, Todd R; Barrangou, Rodolphe

    2018-09-02

    Lactobacillus buchneri is a Gram-positive, obligate heterofermentative, facultative anaerobe commonly affiliated with spoilage of food products. Notably, L. buchneri is able to metabolize lactic acid into acetic acid and 1,2-propanediol. Although beneficial to the silage industry, this metabolic capability is detrimental to preservation of cucumbers by fermentation. The objective of this study was to characterize isolates of L. buchneri purified from both industrial and experimental fermented cucumber after the onset of secondary fermentation. Genotypic and phenotypic characterization included 16S rRNA sequencing, DiversiLab® rep-PCR, colony morphology, API 50 CH carbohydrate analysis, and ability to degrade lactic acid in modified MRS and fermented cucumber media. Distinct groups of isolates were identified with differing colony morphologies that varied in color (translucent white to opaque yellow), diameter (1 mm-11 mm), and shape (umbonate, flat, circular or irregular). Growth rates in MRS revealed strain differences, and a wide spectrum of carbon source utilization was observed. Some strains were able to ferment as many as 21 of 49 tested carbon sources, including inulin, fucose, gentiobiose, lactose, mannitol, potassium ketogluconate, saccharose, raffinose, galactose, and xylose, while others metabolized as few as eight carbohydrates as the sole source of carbon. All isolates degraded lactic acid in both fermented cucumber medium and modified MRS, but exhibited differences in the rate and extent of lactate degradation. Isolates clustered into eight distinct groups based on rep-PCR fingerprints with 20 of 36 of the isolates exhibiting >97% similarity. Although isolated from similar environmental niches, significant phenotypic and genotypic diversity was found among the L. buchneri cultures. A collection of unique L. buchneri strains was identified and characterized, providing the basis for further analysis of metabolic and genomic capabilities of this

  5. Diffusion and bulk flow in phloem loading: A theoretical analysis of the polymer trap mechanism for sugar transport in plants

    NASA Astrophysics Data System (ADS)

    Dölger, Julia; Rademaker, Hanna; Liesche, Johannes; Schulz, Alexander; Bohr, Tomas

    2014-10-01

    Plants create sugar in the mesophyll cells of their leaves by photosynthesis. This sugar, mostly sucrose, has to be loaded via the bundle sheath into the phloem vascular system (the sieve elements), where it is distributed to growing parts of the plant. We analyze the feasibility of a particular loading mechanism, active symplasmic loading, also called the polymer trap mechanism, where sucrose is transformed into heavier sugars, such as raffinose and stachyose, in the intermediary-type companion cells bordering the sieve elements in the minor veins of the phloem. Keeping the heavier sugars from diffusing back requires that the plasmodesmata connecting the bundle sheath with the intermediary cell act as extremely precise filters, which are able to distinguish between molecules that differ by less than 20% in size. In our modeling, we take into account the coupled water and sugar movement across the relevant interfaces, without explicitly considering the chemical reactions transforming the sucrose into the heavier sugars. Based on the available data for plasmodesmata geometry, sugar concentrations, and flux rates, we conclude that this mechanism can in principle function, but that it requires pores of molecular sizes. Comparing with the somewhat uncertain experimental values for sugar export rates, we expect the pores to be only 5%-10% larger than the hydraulic radius of the sucrose molecules. We find that the water flow through the plasmodesmata, which has not been quantified before, contributes only 10%-20% to the sucrose flux into the intermediary cells, while the main part is transported by diffusion. On the other hand, the subsequent sugar translocation into the sieve elements would very likely be carried predominantly by bulk water flow through the plasmodesmata. Thus, in contrast to apoplasmic loaders, all the necessary water for phloem translocation would be supplied in this way with no need for additional water uptake across the plasma membranes of the

  6. Molecular characterization of the acquisition of longevity during seed maturation in soybean

    PubMed Central

    Lalanne, David; Rossi, Rubiana Falopa; Pelletier, Sandra; da Silva, Edvaldo Aparecido Amaral

    2017-01-01

    Seed longevity, defined as the ability to remain alive during storage, is an important agronomic factor. Poor longevity negatively impacts seedling establishment and consequently crop yield. This is particularly problematic for soybean as seeds have a short lifespan. While the economic importance of soybean has fueled a large number of transcriptome studies during embryogenesis and seed filling, the mechanisms regulating seed longevity during late maturation remain poorly understood. Here, a detailed physiological and molecular characterization of late seed maturation was performed in soybean to obtain a comprehensive overview of the regulatory genes that are potentially involved in longevity. Longevity appeared at physiological maturity at the end of seed filling before maturation drying and progressively doubled until the seeds reached the dry state. The increase in longevity was associated with the expression of genes encoding protective chaperones such as heat shock proteins and the repression of nuclear and chloroplast genes involved in a range of chloroplast activities, including photosynthesis. An increase in the raffinose family oligosaccharides (RFO)/sucrose ratio together with changes in RFO metabolism genes was also associated with longevity. A gene co-expression network analysis revealed 27 transcription factors whose expression profiles were highly correlated with longevity. Eight of them were previously identified in the longevity network of Medicago truncatula, including homologues of ERF110, HSF6AB, NFXL1 and members of the DREB2 family. The network also contained several transcription factors associated with auxin and developmental cell fate during flowering, organ growth and differentiation. A transcriptional transition occurred concomitant with seed chlorophyll loss and detachment from the mother plant, suggesting the activation of a post-abscission program. This transition was enriched with AP2/EREBP and WRKY transcription factors and genes

  7. Nutritional Properties Assessment of Endogenous and Improved Varieties of Maize (Zea mays L.) Grown in Southern Benin.

    PubMed

    Josiane, Semassa Adjobignon; Bienvenu, Anihouvi Victor; Wilfried, Padonou Segla; Adolphe, Adjanohoun; Djima, Aly; Joachin, Gbenou; Lamine, Baba-Moussa

    2017-01-01

    A wide range of maize varieties is used in Benin but information on the nutritional characteristics of these varieties are not well known. This study aims to assess the nutritional composition of maize varieties in use in the southern region of Benin with the purpose of providing consumers accurate information for better choice. Moisture, ash, protein, fiber and fat contents were determined according to Association of Official Analytical Chemists and American Association of Cereal Chemists methods. Sugar and organic acids were assessed using High Performance Liquid Chromatography methods and amino acids profile was established according to Rosen method using glutamic acid. The maize varieties were classified into 5 clusters according to their macro nutrients composition and 4 clusters based on their sugar and organic acids contents. Varieties of group 5 were very rich in protein (14.34 g/100 g), while the highest fat content (7.22 g/100 g) was observed for group 2 varieties. The highest carbohydrate contents obtained were 80.64 g/100 g, 80.11 g/100 g and 79.15 g/100 g for groups 1, 4 and 5 varieties respectively. Moreover the dendrogram gave four homogeneous clusters according to sugars and organic acids composition. Varieties of groups 2, 3 and 4 had almost the same fructose contents ranging between 0.04 and 0.06%; varieties of group 1 contained the highest contents of raffinose, sucrose and glucose; those of group 2 were very rich in propionate and fructose. It is concluded that some of maize varieties investigated contained high level of protein. Furthermore glutamic acid was the predominant amino acid while the least amino acid was methionine. Those varieties, owing to their protein and amino acids contents could have many benefits by providing vital constituents to the body.

  8. The new species Enterobacter oryziphilus sp. nov. and Enterobacter oryzendophyticus sp. nov. are key inhabitants of the endosphere of rice

    PubMed Central

    2013-01-01

    Background Six independent Gram-negative, facultatively anaerobic, non-spore-forming, nitrogen-fixing rod-shaped isolates were obtained from the root endosphere of rice grown at the International Rice Research Institute (IRRI) and investigated in a polyphasic taxonomic study. Results The strains produced fatty acid patterns typical for members of the family Enterobacteriaceae. Comparative sequence analyses of the 16S rRNA as well as rpoB genes allocated the strains to two well-defined groups within the genus Enterobacter, family Enterobacteriaceae. The analyses indicated Enterobacter radicincitans, Enterobacter arachidis and Enterobacter oryzae to be the closest related species. An RpoB (translated) protein comparison supported the placement in the genus Enterobacter and the relatedness of our isolates to the aforementioned species. Genomic DNA:DNA hybridization analyses and biochemical analyses provided further evidence that the novel strains belong to two new species within the genus Enterobacter. The two species can be differentiated from each other and from existing enteric species by acid production from L-rhamnose and D-melibiose, decarboxylation of ornithine and utilization of D-alanine, D-raffinose L-proline and L-aspartic acid, among other characteristics. Members of both species revealed capacities to colonise rice roots, including plant-growth-promoting capabilities such as an active supply of fixed nitrogen to the plant and solubilisation of inorganic phosphorus, next to traits allowing adaptation to the plant. Conclusions Two novel proposed enterobacterial species, denominated Enterobacter oryziphilus sp. nov. (type strain REICA_142T=LMG 26429T=NCCB 100393T) and Enterobacter oryzendophyticus sp. nov. (type strain REICA_082T=LMG 26432T =NCCB 100390T) were isolated from rice roots. Both species are capable of promoting rice growth by supplying nitrogen and phosphorus. PMID:23865888

  9. Norway spruce embryogenesis: changes in carbohydrate profile, structural development and response to polyethylene glycol

    PubMed Central

    Hudec, Lukáš; Konrádová, Hana; Hašková, Anna; Lipavská, Helena

    2016-01-01

    Two unrelated, geographically distinct, highly embryogenic lines of Norway spruce (Picea abies (L.) Karst.) were analysed to identify metabolic traits characteristic for lines with good yields of high-quality embryos. The results were compared with corresponding characteristics of a poorly productive line (low embryo yield, scarce high-quality embryos). The following carbohydrate profiles and spectra during maturation, desiccation and germination were identified as promising characteristics for line evaluation: a gradual decrease in total soluble carbohydrates with an increasing sucrose : hexose ratio during maturation; accumulation of raffinose family oligosaccharides resulting from desiccation and their rapid degradation at the start of germination; and a decrease in sucrose, increase in hexoses and the appearance of pinitol with proceeding germination. We propose that any deviation from this profile in an embryonic line is a symptom of inferior somatic embryo development. We further propose that a fatty acid spectrum dominated by linoleic acid (18 : 2) was a common feature of healthy spruce somatic embryos, although it was quite different from zygotic embryos mainly containing oleic acid (18 : 1). The responses of the lines to osmotic stress were evaluated based on comparison of control (without osmoticum) and polyethylene glycol (PEG)-exposed (PEG 4000) variants. Although genetically distinct, both highly embryogenic lines responded in a very similar manner, with the only difference being sensitivity to high concentrations of PEG. At an optimum PEG concentration (3.75 and 5%), which was line specific, negative effects of PEG on embryo germination were compensated for by a higher maturation efficiency so that the application of PEG at an appropriate concentration improved the yield of healthy germinants per gram of initial embryonal mass and accelerated the process. Polyethylene glycol application, however, resulted in no improvement of the poorly

  10. Isolation of marine fungi Aspergillus sp. and its in vitro antifouling activity against marine bacteria.

    PubMed

    Thiyagarajan, Santhananmari; Bavya, Manoharan; Jamal, Alruwaili

    2016-09-01

    Biofouling is considered as a main issue of concern in aquatic environment causing severe economic loss and pollution. The aim of the present study was to isolate marine fungus antagonistic to biofouling bacteria and to define antifouling compounds present in it. Using standard plate method five predominant biofouling bacteria viz., Methylococcus sp., Flavobacterium sp., Marinococcus sp., Serratia sp. and Pseudomonas sp. were isolated from marine solid substances on Zobell's agar. Tolerance range of these bacteria to NaCl was 2-10%. Isolation of fungi from mangrove and estuarine sediments and their screening identified Aspergillus sp. EF4 as a potential isolate. This isolate caused inhibition of all the five test bacterial cultures measuring zone diameters respectively of 11, 16, 12, 13 and 11mm.? Subsequent to submerged fermentation using shaking flask method this fungus produced bioactive compounds within 5 days. The culture parameters optimized were raffinose as carbon source, yeast extract as lone nitrogen source, pH up to 9.0 and temperature up to 40?C. Antifouling compounds of culture filtrate were separated and detected by a three-step procedure involving thin layer chromatography, bioautography and preparative TLC. The in vitro assay involving glass slide-wooden stick-biofilm method revealed that these compounds could cause inhibition and destruction of bacteria to an extent of 2.16 x 104 CFU ml-1 and 2.46 x 104 CFU ml-1 respectively while growth of bacteria in control beaker was enumerated to be 4.41 x 104 CFU ml-1. High performance liquid chromatography of culture filtrate indicated probable principal antifouling compound as Fumonisin B2. Isolation of antagonistic marine fungus from Indian coast and detection of its antifouling compound would help in planning effective strategies for controlling biofouling in marine environment.

  11. Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments.

    PubMed

    Fletcher, Eugene; Feizi, Amir; Bisschops, Markus M M; Hallström, Björn M; Khoomrung, Sakda; Siewers, Verena; Nielsen, Jens

    2017-01-01

    Tolerance of yeast to acid stress is important for many industrial processes including organic acid production. Therefore, elucidating the molecular basis of long term adaptation to acidic environments will be beneficial for engineering production strains to thrive under such harsh conditions. Previous studies using gene expression analysis have suggested that both organic and inorganic acids display similar responses during short term exposure to acidic conditions. However, biological mechanisms that will lead to long term adaptation of yeast to acidic conditions remains unknown and whether these mechanisms will be similar for tolerance to both organic and inorganic acids is yet to be explored. We therefore evolved Saccharomyces cerevisiae to acquire tolerance to HCl (inorganic acid) and to 0.3M L-lactic acid (organic acid) at pH 2.8 and then isolated several low pH tolerant strains. Whole genome sequencing and RNA-seq analysis of the evolved strains revealed different sets of genome alterations suggesting a divergence in adaptation to these two acids. An altered sterol composition and impaired iron uptake contributed to HCl tolerance whereas the formation of a multicellular morphology and rapid lactate degradation was crucial for tolerance to high concentrations of lactic acid. Our findings highlight the contribution of both the selection pressure and nature of the acid as a driver for directing the evolutionary path towards tolerance to low pH. The choice of carbon source was also an important factor in the evolutionary process since cells evolved on two different carbon sources (raffinose and glucose) generated a different set of mutations in response to the presence of lactic acid. Therefore, different strategies are required for a rational design of low pH tolerant strains depending on the acid of interest. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  12. Expression of a GALACTINOL SYNTHASE Gene in Tomato Seeds Is Up-Regulated before Maturation Desiccation and Again after Imbibition whenever Radicle Protrusion Is Prevented1

    PubMed Central

    Downie, Bruce; Gurusinghe, Sunitha; Dahal, Petambar; Thacker, Richard R.; Snyder, John C.; Nonogaki, Hiroyuki; Yim, Kyuock; Fukanaga, Keith; Alvarado, Veria; Bradford, Kent J.

    2003-01-01

    Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance. PMID:12644684

  13. Exploring traditional aus-type rice for metabolites conferring drought tolerance.

    PubMed

    Casartelli, Alberto; Riewe, David; Hubberten, Hans Michael; Altmann, Thomas; Hoefgen, Rainer; Heuer, Sigrid

    2018-01-25

    Traditional varieties and landraces belonging to the aus-type group of rice (Oryza sativa L.) are known to be highly tolerant to environmental stresses, such as drought and heat, and are therefore recognized as a valuable genetic resource for crop improvement. Using two aus-type (Dular, N22) and two drought intolerant irrigated varieties (IR64, IR74) an untargeted metabolomics analysis was conducted to identify drought-responsive metabolites associated with tolerance. The superior drought tolerance of Dular and N22 compared with the irrigated varieties was confirmed by phenotyping plants grown to maturity after imposing severe drought stress in a dry-down treatment. Dular and N22 did not show a significant reduction in grain yield compared to well-watered control plants, whereas the intolerant varieties showed a significant reduction in both, total spikelet number and grain yield. The metabolomics analysis was conducted with shoot and root samples of plants at the tillering stage at the end of the dry-down treatment. The data revealed an overall higher accumulation of N-rich metabolites (amino acids and nucleotide-related metabolites allantoin and uridine) in shoots of the tolerant varieties. In roots, the aus-type varieties were characterised by a higher reduction of metabolites representative of glycolysis and the TCA cycle, such as malate, glyceric acid and glyceric acid-3-phosphate. On the other hand, the oligosaccharide raffinose showed a higher fold increase in both, shoots and roots of the sensitive genotypes. The data further showed that, for certain drought-responsive metabolites, differences between the contrasting rice varieties were already evident under well-watered control conditions. The drought tolerance-related metabolites identified in the aus-type varieties provide a valuable set of protective compounds and an entry point for assessing genetic diversity in the underlying pathways for developing drought tolerant rice and other crops.

  14. Diffusion and bulk flow in phloem loading: a theoretical analysis of the polymer trap mechanism for sugar transport in plants.

    PubMed

    Dölger, Julia; Rademaker, Hanna; Liesche, Johannes; Schulz, Alexander; Bohr, Tomas

    2014-10-01

    Plants create sugar in the mesophyll cells of their leaves by photosynthesis. This sugar, mostly sucrose, has to be loaded via the bundle sheath into the phloem vascular system (the sieve elements), where it is distributed to growing parts of the plant. We analyze the feasibility of a particular loading mechanism, active symplasmic loading, also called the polymer trap mechanism, where sucrose is transformed into heavier sugars, such as raffinose and stachyose, in the intermediary-type companion cells bordering the sieve elements in the minor veins of the phloem. Keeping the heavier sugars from diffusing back requires that the plasmodesmata connecting the bundle sheath with the intermediary cell act as extremely precise filters, which are able to distinguish between molecules that differ by less than 20% in size. In our modeling, we take into account the coupled water and sugar movement across the relevant interfaces, without explicitly considering the chemical reactions transforming the sucrose into the heavier sugars. Based on the available data for plasmodesmata geometry, sugar concentrations, and flux rates, we conclude that this mechanism can in principle function, but that it requires pores of molecular sizes. Comparing with the somewhat uncertain experimental values for sugar export rates, we expect the pores to be only 5%-10% larger than the hydraulic radius of the sucrose molecules. We find that the water flow through the plasmodesmata, which has not been quantified before, contributes only 10%-20% to the sucrose flux into the intermediary cells, while the main part is transported by diffusion. On the other hand, the subsequent sugar translocation into the sieve elements would very likely be carried predominantly by bulk water flow through the plasmodesmata. Thus, in contrast to apoplasmic loaders, all the necessary water for phloem translocation would be supplied in this way with no need for additional water uptake across the plasma membranes of the

  15. Use of Bacillus subtilis isolates from Tua-nao towards nutritional improvement of soya bean hull for monogastric feed application.

    PubMed

    Wongputtisin, P; Khanongnuch, C; Kongbuntad, W; Niamsup, P; Lumyong, S; Sarkar, P K

    2014-09-01

    Soya bean hull (SBH) is a cheap and high-fibre content feed ingredient that obtained after soya bean oil extraction. Microbial fermentation was expected to improve SBH qualities before applying to animals, especially monogastric animals. Two bacterial strains, Bacillus subtilis MR10 and TK8 that were isolated from Tua-nao, a traditional fermented soya bean in northern Thailand, were used for fermented soya bean hull (FSBH) production. Both could easily grow at 37°C in SBH as the sole substrate. MR10 produced the highest β-mannanase activity (400 U g(-1) SBH) on day 2, while TK8 produced the highest cellulase activity (14·5 U g(-1) SBH) on day 3. After fermentation, the nutritional quality of SBH was obviously improved by an increase in soluble sugars, soluble proteins, crude protein and crude lipid, and a decrease in the content of raffinose family oligosaccharides. Scavenging activity (%) of SBH against ABTS radical cation was also increased from 14 to 27 and 20% by MR10 and TK8 fermentation, respectively. According to the GRAS property of these both strains and various improvements of nutritional values, the fermented SBH proved to be a potential feed ingredient, especially for the monogastric animals. Normally, soya bean hull has been recognized as only a worthless by-product from soya bean oil production process because of its low utilizable nutrients. Our study introduced an alternative way to utilize this worthless residue using biotechnological knowledge. The nutritional quality of soya bean hull was improved by microbial fermentation. Fermented soya bean hull can be used as a cheap, safe and high-nutrient feed ingredient for livestock production, especially monogastric animals, to promote their growth performances, instead of using antibiotics in some regions of the world. © 2014 The Society for Applied Microbiology.

  16. The effects of pressure on the water permeability of the descending limb of Henle's loops of rabbits.

    PubMed

    Stoner, L C; Roch-Ramel, F

    1979-10-01

    Descending limbs of Henle's loops from rabbits were perfused in vitro. Using techniques where the collecting pipets permitted cannulation of the tubule, we were able to maintain reasonable flow rates at lower perfusion reservoir heights than are required with a conventional "Sylgard seal" pipet. The bath was either isosmotic to the perfusate, or was made 300 mOsm hyperosmotic using urea. Net water reabsorption did not occur in tubules perfused at low pressure (average reservoir height = 26 cm H2O) even when the bath was hyperosmotic: delta Jv = -0.06 +/- 0.18 nl/min (n = 7). Observed increases in sodium concentration and osmolality of collected fluid, when the bath was made hyperosmotic, were 16 +/- 8 mM (n - 7) and 254 +/- 38 mOsm (n = 7), respectively. Presumably the large increase in osmolality of the collected fluid was due to entrance of urea. When the "Sylgard seal" collecting end was utilized higher perfusion reservoir heights had to be used to maintain flow (mean height 66 cm H2O). These tubules were highly permeable to water as reported by others for this tubule segment. In the presence of a hyperosmotic bath water extrusion resulted in a dramatic increase in the osmolality of the collected fluid (312 +/- 5 mOsm; 7 tubules) which was almost completely accounted for by an increase in sodium concentration (153 +/- 8 mmole/l; 6 tubules). The 14C urea permeability (measured lumen to bath) of descending limbs in a 300 mOsm bath was 0.64 x 10(-7) cm2 . s-1 +/- 0.23 x 10(-7) (11 tubules). When the bath was made hyperosmotic using urea or raffinose the 14C urea permeability increased significantly.

  17. Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated Caco-2, LLC-PK1 and rat renal SKPT cells.

    PubMed

    Rasmussen, Rune Nørgaard; Lagunas, Candela; Plum, Jakob; Holm, René; Nielsen, Carsten Uhd

    2016-01-20

    The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0μM. β-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was β-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of β-alanine appear to have higher affinities for TauT than GABA-analogues. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Global analysis of gene expression profiles in physic nut (Jatropha curcas L.) seedlings exposed to salt stress.

    PubMed

    Zhang, Lin; Zhang, Chao; Wu, Pingzhi; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2014-01-01

    Salt stress interferes with plant growth and production. Plants have evolved a series of molecular and morphological adaptations to cope with this abiotic stress, and overexpression of salt response genes reportedly enhances the productivity of various crops. However, little is known about the salt responsive genes in the energy plant physic nut (Jatropha curcas L.). Thus, excavate salt responsive genes in this plant are informative in uncovering the molecular mechanisms for the salt response in physic nut. We applied next-generation Illumina sequencing technology to analyze global gene expression profiles of physic nut plants (roots and leaves) 2 hours, 2 days and 7 days after the onset of salt stress. A total of 1,504 and 1,115 genes were significantly up and down-regulated in roots and leaves, respectively, under salt stress condition. Gene ontology (GO) analysis of physiological process revealed that, in the physic nut, many "biological processes" were affected by salt stress, particular those categories belong to "metabolic process", such as "primary metabolism process", "cellular metabolism process" and "macromolecule metabolism process". The gene expression profiles indicated that the associated genes were responsible for ABA and ethylene signaling, osmotic regulation, the reactive oxygen species scavenging system and the cell structure in physic nut. The major regulated genes detected in this transcriptomic data were related to trehalose synthesis and cell wall structure modification in roots, while related to raffinose synthesis and reactive oxygen scavenger in leaves. The current study shows a comprehensive gene expression profile of physic nut under salt stress. The differential expression genes detected in this study allows the underling the salt responsive mechanism in physic nut with the aim of improving its salt resistance in the future.

  19. Defluviitalea raffinosedens sp. nov., a thermophilic, anaerobic, saccharolytic bacterium isolated from an anaerobic batch digester treating animal manure and rice straw.

    PubMed

    Ma, Shichun; Huang, Yan; Wang, Cong; Fan, Hui; Dai, Lirong; Zhou, Zheng; Liu, Xing; Deng, Yu

    2017-05-01

    A thermophilic, anaerobic, fermentative bacterium, strain A6T, was obtained from an anaerobic batch digester treating animal manure and rice straw. Cells were Gram-stain-positive, slightly curved rods with a size of 0.6-1×2.5-8.2 µm, non-motile and produced terminal spores. The temperature, pH and NaCl concentration ranges for growth were 40-60 °C, 6.5-8.0 and 0-15.0 g l-1, with optimum growth noted at 50-55 °C, pH 7.5 and in the absence of NaCl, respectively. Yeast extract was required for growth. d-Glucose, maltose, d-xylose, d-galactose, d-fructose, d-ribose, lactose, raffinose, sucrose, d-arabinose, cellobiose, d-mannose and yeast extract were used as carbon and energy sources. The fermentation products from glucose were ethanol, lactate, acetate, propionate, butyrate, valerate, iso-butyrate, iso-valerate, H2 and CO2. The G+C content of the genomic DNA was 36.6 mol%. The predominant fatty acids were C16 : 0, iso-C17 : 1, C14 : 0, C16 : 1ω7c, C16 : 0 N-alcohol and C13 : 0 3-OH. Respiratory quinones were not detected. The polar lipid profile comprised phosphoglycolipids, phospholipids, glycolipids, a diphosphatidylglycerol, a phosphatidylglycerol and an unidentified lipid. Phylogenetic analyses of the 16S rRNA gene sequence indicated that the strain was closely related to Defluviitalea saccharophila DSM 22681T with a similarity of 96.0 %. Based on the morphological, physiological and taxonomic characterization, strain A6T is considered to represent a novel species of the genus Defluviitalea, for which the name Defluviitalea raffinosedens sp. nov. is proposed. The type strain is A6T (=DSM 28090T=ACCC 19951T).

  20. Validation of a thin-layer chromatography/densitometry method for the characterization of invertase activity.

    PubMed

    Ferey, Justine; Da Silva, David; Bravo-Veyrat, Sophie; Lafite, Pierre; Daniellou, Richard; Maunit, Benoît

    2016-12-16

    This paper presents a kinetic study of invertase, a specific fructofuranosidase cloned from the Leishmania major genome. The kinetic parameters of the β-d-fructofuranosidase from Leishmania major (BfrA) were determined using Thin-Layer Chromatography (TLC) and UV-densitometry (TLC@UV) specifically developed for the separation and detection of three carbohydrates namely sucrose, glucose and fructose. Separation was performed on TLC silica gel 60 F254 plates impregnated with sodium bisulphate and citrate and heated prior to development. This fast and easy separation was performed with two successive developments using ACN/H 2 O 80/20 (v/v) as mobile phase. Sensitive and repeatable derivatization of sugars was achieved by dipping the plates in a solution of 4-aminobenzoic acid. Quantification was performed by UV-detection. The method was validated according to ICH guidelines Q2(R1) in terms of specificity, limits of detection and quantification, precision and robustness (with n=3 replicates and CV ≤10%). The characterization of BfrA reaction kinetic was performed by monitoring the accumulation of either glucose or fructose detected by TLC@UV. Hydrolysis of sucrose was described by the Michaelis-Menten kinetic parameters (K M ; V max ) respectively equal to 63.09±7.590mM; 0.037±0.00094mM/min using glucose production and 83.01±14.39mM; 0.031±0.0021mM/min monitoring fructose. Hydrolyses of three alternative substrates, raffinose, stachyose and inulin, were also compared and the regiospecificity of the reaction was characterized. This TLC@UV method is shown to be suitable for the refined kinetic analysis of different reactions related to the hydrolysis of sugars. Copyright © 2016. Published by Elsevier B.V.

  1. Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus.

    PubMed

    Giraldo, Marielle Aleixo; Gonçalves, Heloísa Bressan; Furriel, Rosa Dos Prazeres Melo; Jorge, João Atílio; Guimarães, Luis Henrique Souza

    2014-05-01

    The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0-6.0 and 55-65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0-10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²⁺ (161 %), Co²⁺ (68 %) and Mg²⁺ (61 %) and was inhibited by Al³⁺, Ag⁺, Fe²⁺ and Fe³⁺. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K(M) = 22 mM). However, in the presence of Mn²⁺, the apparent affinity and V(max) for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V(max) was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.

  2. Manninotriose is a major carbohydrate in red deadnettle (Lamium purpureum, Lamiaceae)

    PubMed Central

    dos Santos, Raquel; Vergauwen, Rudy; Pacolet, Pieter; Lescrinier, Eveline; Van den Ende, Wim

    2013-01-01

    Background and Aims There is a great need to search for natural compounds with superior prebiotic, antioxidant and immunostimulatory properties for use in (food) applications. Raffinose family oligosaccharides (RFOs) show such properties. Moreover, they contribute to stress tolerance in plants, acting as putative membrane stabilizers, antioxidants and signalling agents. Methods A large-scale soluble carbohydrate screening was performed within the plant kingdom. An unknown compound accumulated to a high extent in early-spring red deadnettle (Lamium purpureum) but not in other RFO plants. The compound was purified and its structure was unravelled with NMR. Organs and organ parts of red deadnettle were carefully dissected and analysed for soluble sugars. Phloem sap content was analysed by a common EDTA-based method. Key Results Early-spring red deadnettle stems and roots accumulate high concentrations of the reducing trisaccharide manninotriose (Galα1,6Galα1,6Glc), a derivative of the non-reducing RFO stachyose (Galα1,6Galα1,6Glcα1,2βFru). Detailed soluble carbohydrate analyses on dissected stem and leaf sections, together with phloem sap analyses, strongly suggest that stachyose is the main transport compound, but extensive hydrolysis of stachyose to manninotriose seems to occur along the transport path. Based on the specificities of the observed carbohydrate dynamics, the putative physiological roles of manninotriose in red deadnettle are discussed. Conclusions It is demonstrated for the first time that manninotriose is a novel and important player in the RFO metabolism of red dead deadnettle. It is proposed that manninotriose represents a temporary storage carbohydrate in early-spring deadnettle, at the same time perhaps functioning as a membrane protector and/or as an antioxidant in the vicinity of membranes, as recently suggested for other RFOs and fructans. This novel finding urges further research on this peculiar carbohydrate on a broader array of RFO

  3. Characterisation and biochemical properties of predominant lactic acid bacteria from fermenting cassava for selection as starter cultures.

    PubMed

    Kostinek, M; Specht, I; Edward, V A; Pinto, C; Egounlety, M; Sossa, C; Mbugua, S; Dortu, C; Thonart, P; Taljaard, L; Mengu, M; Franz, C M A P; Holzapfel, W H

    2007-03-20

    A total of 375 lactic acid bacteria were isolated from fermenting cassava in South Africa, Benin, Kenya and Germany, and were characterised by phenotypic and genotypic tests. These could be divided into five main groups comprising strains of facultatively heterofermentative rods, obligately heterofermentative rods, heterofermentative cocci, homofermentative cocci and obligately homofermentative rods, in decreasing order of predominance. Most of the facultatively heterofermentative rods were identified by phenotypic tests as presumptive Lactobacillus plantarum-group strains, which also comprised the most predominant bacteria (54.4% of strains) isolated in the study. The next predominant group of lactic acid bacteria (14.1% of total isolates) consisted of obligately heterofermentative rods belonging either to the genus Lactobacillus or Weissella, followed by the heterofermentative cocci (13.9% of isolates) belonging to the genera Weissella or Leuconostoc. Homofermentative cocci were also isolated (13.3% of isolates). Biochemical properties such as production of alpha-amylase, beta-glucosidase, tannase, antimicrobials (presumptive bacteriocin and H(2)O(2)-production), acidification and fermentation of the indigestible sugars raffinose and stachyose, were evaluated in vitro for selection of potential starter strains. A total of 32 strains with one or more desirable biochemical properties were pre-selected and identified using rep-PCR fingerprinting in combination with 16S rRNA sequencing of representative rep-PCR cluster isolates. Of these strains, 18 were identified as L. plantarum, four as Lactobacillus pentosus, two each as Leuconostoc fallax, Weissella paramesenteroides and Lactobacillus fermentum, one each as Leuconostoc mesenteroides subsp. mesenteroides and Weissella cibaria, while two remained unidentified but could be assigned to the L. plantarum-group. These strains were further investigated for clonal relationships, using RAPD-PCR with three primers, and of

  4. Differential Regulation of Sorbitol and Sucrose Loading into the Phloem of Plantago major in Response to Salt Stress1[OA

    PubMed Central

    Pommerrenig, Benjamin; Papini-Terzi, Flavia Stal; Sauer, Norbert

    2007-01-01

    Several plant families generate polyols, the reduced form of monosaccharides, as one of their primary photosynthetic products. Together with sucrose (Suc) or raffinose, these polyols are used for long-distance allocation of photosynthetically fixed carbon in the phloem. Many species from these families accumulate these polyols under salt or drought stress, and the underlying regulation of polyol biosynthetic or oxidizing enzymes has been studied in detail. Here, we present results on the differential regulation of genes that encode transport proteins involved in phloem loading with sorbitol and Suc under salt stress. In the Suc- and sorbitol-translocating species Plantago major, the mRNA levels of the vascular sorbitol transporters PmPLT1 and PmPLT2 are rapidly up-regulated in response to salt treatment. In contrast, mRNA levels for the phloem Suc transporter PmSUC2 stay constant during the initial phase of salt treatment and are down-regulated after 24 h of salt stress. This adaptation in phloem loading is paralleled by a down-regulation of mRNA levels for a predicted sorbitol dehydrogenase (PmSDH1) in the entire leaf and of mRNA levels for a predicted Suc phosphate synthase (PmSPS1) in the vasculature. Analyses of Suc and sorbitol concentrations in leaves, in enriched vascular tissue, and in phloem exudates of detached leaves revealed an accumulation of sorbitol and, to a lesser extent, of Suc within the leaves of salt-stressed plants, a reduced rate of phloem sap exudation after NaCl treatment, and an increased sorbitol-to-Suc ratio within the phloem sap. Thus, the up-regulation of PmPLT1 and PmPLT2 expression upon salt stress results in a preferred loading of sorbitol into the phloem of P. major. PMID:17434995

  5. Metagenomic and Metatranscriptomic Analyses of Diverse Watermelon Cultivars Reveal the Role of Fruit Associated Microbiome in Carbohydrate Metabolism and Ripening of Mature Fruits.

    PubMed

    Saminathan, Thangasamy; García, Marleny; Ghimire, Bandana; Lopez, Carlos; Bodunrin, Abiodun; Nimmakayala, Padma; Abburi, Venkata L; Levi, Amnon; Balagurusamy, Nagamani; Reddy, Umesh K

    2018-01-01

    The plant microbiome is a key determinant of plant health and productivity, and changes in the plant microbiome can alter the tolerance to biotic and abiotic stresses and the quality of end produce. Little is known about the microbial diversity and its effect on carbohydrate metabolism in ripe fruits. In this study, we aimed to understand the diversity and function of microorganisms in relation to carbohydrate metabolism of ripe watermelon fruits. We used 16S metagenomics and RNAseq metatranscriptomics for analysis of red (PI459074, Congo, and SDRose) and yellow fruit-flesh cultivars (PI227202, PI435990, and JBush) of geographically and metabolically diverse watermelon cultivars. Metagenomics data showed that Proteobacteria were abundant in SDRose and PI227202, whereas Cyanobacteria were most abundant in Congo and PI4559074. In the case of metatranscriptome data, Proteobacteria was the most abundant in all cultivars. High expression of genes linked to infectious diseases and the expression of peptidoglycan hydrolases associated to pathogenicity of eukaryotic hosts was observed in SDRose, which could have resulted in low microbial diversity in this cultivar. The presence of GH28, associated with polygalacturonase activity in JBush and SDRose could be related to cell wall modifications including de-esterification and depolymerization, and consequent loss of galacturonic acid and neutral sugars. Moreover, based on the KEGG annotation of the expressed genes, nine α-galactosidase genes involved in key processes of galactosyl oligosaccharide metabolism, such as raffinose family were identified and galactose metabolism pathway was reconstructed. Results of this study underline the links between the host and fruit-associated microbiome in carbohydrate metabolism of the ripe fruits. The cultivar difference in watermelon reflects the quantum and diversity of the microbiome, which would benefit watermelon and other plant breeders aiming at the holobiont concept to

  6. Screening and Identification of Cryopreservative Agents for Human Cellular Biotechnology Experiments in Microgravity

    NASA Technical Reports Server (NTRS)

    Love,J.; Elliott, T.; Das, G. C.; Hammond, D. K.; Schwarzkopf, R. J.; Jones, L. B.; Baker, T. L.

    2006-01-01

    Dimethyl sulfoxide (DMSO) has been used as a standard cryopreservative agent for mammalian cell culture; however, prolonged exposure of thawed cells to DMSO can alter cell growth. While DMSO is easily eliminated in ground-based experiments, removal of DMSO in flight-based experiments is more difficult due to various on-orbit constraints. Failure of cryopreservation is due to a number of factors, including intracellular ice formation, solute effect, and apoptotic cell death following thawing. One objective of this study is to identify and characterize an alternative cryopreservative that could be used on the International Space Station (ISS). We systematically screened for potential permeating and non-permeating agents using a human colorectal carcinoma cell line, MIP-101. Cells were suspended in cryopreservation solution and frozen either following a two-step procedure involving initial cooling at -1 C/min overnight followed by storage in liquid nitrogen (LN2) vapor, or by freezing cells directly in the LN2 vapor phase at -10 C/min. Ability to preserve cellular function after one cycle of freeze-thawing was assessed by the recovery of viable cells in short and long-term cell culture experiments. Results showed that permeating preservatives glycerol (G) and ethylene glycol (EG) had an efficacy (80-110%) comparable to, if not better than, 7.5% DMSO; but, propylene glycol (PG) had a somewhat lesser efficacy. Among the non-permeating preservatives, trehalose, raffinose, and dextran exhibited significant protective effect (50-80%) relative to that offered by 7.5% DMSO, but at -10 C and not at -1 C/min cooling rate. Preliminary data thus suggest that a combination of permeating and non-permeating agents may have improved efficacy as a cryoprotectant and serve as an alternate to DMSO for experimentation on ISS.

  7. Metagenomic and Metatranscriptomic Analyses of Diverse Watermelon Cultivars Reveal the Role of Fruit Associated Microbiome in Carbohydrate Metabolism and Ripening of Mature Fruits

    PubMed Central

    Saminathan, Thangasamy; García, Marleny; Ghimire, Bandana; Lopez, Carlos; Bodunrin, Abiodun; Nimmakayala, Padma; Abburi, Venkata L.; Levi, Amnon; Balagurusamy, Nagamani; Reddy, Umesh K.

    2018-01-01

    The plant microbiome is a key determinant of plant health and productivity, and changes in the plant microbiome can alter the tolerance to biotic and abiotic stresses and the quality of end produce. Little is known about the microbial diversity and its effect on carbohydrate metabolism in ripe fruits. In this study, we aimed to understand the diversity and function of microorganisms in relation to carbohydrate metabolism of ripe watermelon fruits. We used 16S metagenomics and RNAseq metatranscriptomics for analysis of red (PI459074, Congo, and SDRose) and yellow fruit-flesh cultivars (PI227202, PI435990, and JBush) of geographically and metabolically diverse watermelon cultivars. Metagenomics data showed that Proteobacteria were abundant in SDRose and PI227202, whereas Cyanobacteria were most abundant in Congo and PI4559074. In the case of metatranscriptome data, Proteobacteria was the most abundant in all cultivars. High expression of genes linked to infectious diseases and the expression of peptidoglycan hydrolases associated to pathogenicity of eukaryotic hosts was observed in SDRose, which could have resulted in low microbial diversity in this cultivar. The presence of GH28, associated with polygalacturonase activity in JBush and SDRose could be related to cell wall modifications including de-esterification and depolymerization, and consequent loss of galacturonic acid and neutral sugars. Moreover, based on the KEGG annotation of the expressed genes, nine α-galactosidase genes involved in key processes of galactosyl oligosaccharide metabolism, such as raffinose family were identified and galactose metabolism pathway was reconstructed. Results of this study underline the links between the host and fruit-associated microbiome in carbohydrate metabolism of the ripe fruits. The cultivar difference in watermelon reflects the quantum and diversity of the microbiome, which would benefit watermelon and other plant breeders aiming at the holobiont concept to

  8. The influence of narrow-leafed lupin seed fermentation on their chemical composition and ileal digestibility and microbiota in growing pigs.

    PubMed

    Zaworska, Anita; Frankiewicz, Andrzej; Kasprowicz-Potocka, Małgorzata

    2017-08-01

    The aims of this study were to provide a controlled fermentation process of blue lupin seeds (Lupinus angustifolius, cv. Neptun), monitor the changes in seed composition and determine the influence of the fermentation on the apparent ileal digestibility (AID) of crude protein (CP) and amino acids in growing pigs, compared with raw lupin seeds. The fermentation with bacteria and yeast was conducted for 24 h at 25ºC under aerobic conditions. Seed fermentation increased the contents of CP, fibre, fat and ash and most of the analysed amino acids but reduced the levels of the nitrogen-free extractives. Furthermore, fermentation decreased the contents of raffinose family oligosaccharides and phytic acids but increased the alkaloid content. The AID was estimated on three barrows (mean initial body weight 25 kg), surgically fitted with a T-cannula in the distal ileum. The pigs received three diets, each for 6 d, within three experimental periods (3 × 3 Latin Square design). The diets contained soybean meal (Group SBM), raw lupin seeds (Group RL) or fermented lupin seeds (Group FL) as solely protein sources. Fermentation had a positive impact on the AID of CP and methionine, cysteine, isoleucine, leucine, phenylalanine and valine (p < 0.05). Feeding raw or fermented lupin seeds did not affect the microbial status of the ileal digesta. Moreover, ammonia content in the caecal digesta did not differ between Groups RL and FL, although it was significantly higher than in Group SBM. It can be concluded that the fermentation process modified the chemical composition of nutrients in seeds, which can influence the digestibility and utilisation of the fermentation product in animal diets compared to raw seeds.

  9. Effect of Germination and Fermentation on Carbohydrate Composition of Australian Sweet Lupin and Soybean Seeds and Flours.

    PubMed

    Kaczmarska, Kornelia T; Chandra-Hioe, Maria V; Zabaras, Dimitrios; Frank, Damian; Arcot, Jayashree

    2017-11-22

    This study investigated the effect of germination and fermentation on the composition of carbohydrates in Australian sweet lupin. Specifically, the amount of sugars (sucrose, fructose, and glucose), starch, oligosaccharides (verbascose, stachyose, and raffinose), and dietary fiber were measured in germinated lupin seeds and fermented lupin flour, and compared with those in soy. High performance liquid chromatography coupled with refractive index was employed for quantitation of sugars, starch, and oligosaccharides, and gas chromatography coupled with a flame ionization detector was used for quantitation of simple sugars in total, and soluble, and insoluble dietary fiber. The enzyme activities of α-amylase and α-glucosidase were compared before and after germination or fermentation. The α-amylase activity in germinated lupin increased to  ∼17 nmol/mL/min/0.1 g and in germinated soy∼32; in fermented lupin, the activity increased to ∼52, while in fermented soy it decreased to ∼20. In general, germination or fermentation decreased the oligosaccharide content, and increased the total sugar in samples (p < 0.05). Total oligosaccharides in lupin after uncontrolled germination were reduced by 98% to 6 mg/g, and after controlled germination reduced by 44% to 86 mg/g. Fermentation with yogurt culture lowered the content of total oligosaccharides due to 94% decrease in stachyose. Total oligosaccharides in soy flour prior to fermentation were 180 mg/g and significantly decreased to ∼124 mg/g in fermented soy. Germination did not affect the starch content. There was no significant change in the amounts of total, soluble, and insoluble dietary fiber after germination or fermentation of lupin except for galactose, which was significantly reduced in germinated lupin seeds. Soluble dietary fiber in germinated soy significantly increased. Germination and fermentation are simple and effective techniques to reduce the oligosaccharides while maintaining the composition of

  10. Molecular Uptake of Chitooligosaccharides through Chitoporin from the Marine Bacterium Vibrio harveyi

    PubMed Central

    Suginta, Wipa; Chumjan, Watcharin; Mahendran, Kozhinjampara R.; Janning, Petra; Schulte, Albert; Winterhalter, Mathias

    2013-01-01

    Background Chitin is the most abundant biopolymer in marine ecosystems. However, there is no accumulation of chitin in the ocean-floor sediments, since marine bacteria Vibrios are mainly responsible for a rapid turnover of chitin biomaterials. The catabolic pathway of chitin by Vibrios is a multi-step process that involves chitin attachment and degradation, followed by chitooligosaccharide uptake across the bacterial membranes, and catabolism of the transport products to fructose-6-phosphate, acetate and NH3. Principal Findings This study reports the isolation of the gene corresponding to an outer membrane chitoporin from the genome of Vibrio harveyi. This porin, expressed in E. coli, (so called VhChiP) was found to be a SDS-resistant, heat-sensitive trimer. Immunoblotting using anti-ChiP polyclonal antibody confirmed the expression of the recombinant ChiP, as well as endogenous expression of the native protein in the V. harveyi cells. The specific function of VhChiP was investigated using planar lipid membrane reconstitution technique. VhChiP nicely inserted into artificial membranes and formed stable, trimeric channels with average single conductance of 1.8±0.13 nS. Single channel recordings at microsecond-time resolution resolved translocation of chitooligosaccharides, with the greatest rate being observed for chitohexaose. Liposome swelling assays showed no permeation of other oligosaccharides, including maltose, sucrose, maltopentaose, maltohexaose and raffinose, indicating that VhChiP is a highly-specific channel for chitooligosaccharides. Conclusion/Significance We provide the first evidence that chitoporin from V. harveyi is a chitooligosaccharide specific channel. The results obtained from this study help to establish the fundamental role of VhChiP in the chitin catabolic cascade as the molecular gateway that Vibrios employ for chitooligosaccharide uptake for energy production. PMID:23383078

  11. Streptococcus caviae sp. nov., isolated from guinea pig faecal samples.

    PubMed

    Palakawong Na Ayudthaya, Susakul; Hilderink, Loes J; Oost, John van der; Vos, Willem M de; Plugge, Caroline M

    2017-05-01

    A novel cellobiose-degrading and lactate-producing bacterium, strain Cavy grass 6T, was isolated from faecal samples of guinea pigs (Cavia porcellus). Cells of the strain were ovalshaped, non-motile, non-spore-forming, Gram-stain-positive and facultatively anaerobic. The strain gr at 25-40 °C (optimum 37 °C) and pH 4.5-9.5 (optimum 8.0). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Cavy grass 6T belongs to the genus Streptococcus with its closest relative being Streptococcus devriesei CCUG 47155T with only 96.5 % similarity. Comparing strain Cavy grass 6T and Streptococcus devriesei CCUG 47155T, average nucleotide identity and level of digital DNA-DNA hybridization dDDH were only 86.9 and 33.3 %, respectively. Housekeeping genes groEL and gyrA were different between strain Cavy grass 6T and other streptococci. The G+C content of strain Cavy grass 6T was 42.6±0.3 mol%. The major (>10 %) cellular fatty acids of strain Cavy grass 6T were C16:0, C20 : 1ω9c and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). Strain Cavy grass 6T ferment a range of plant mono- and disaccharides as well as polymeric carbohydrates, including cellobiose, dulcitol, d-glucose, maltose, raffinose, sucrose, l-sorbose, trehalose, inulin and dried grass extract, to lactate, formate, acetate and ethanol. Based on phylogenetic and physiological characteristics, Cavy grass 6T can be distinguished from other members of the genus Streptococcus. Therefore, a novel species of the genus Streptococcus, family Streptococcaceae, order Lactobacillales is proposed, Streptococcuscaviae sp. nov. (type strain Cavy grass 6T=TISTR 2371T=DSM 102819T).

  12. The metabolic fate of doubly stable isotope labelled heat-killed Lactobacillus johnsonii in humans.

    PubMed

    Wutzke, K D; Berg, D; Haffner, D

    2008-02-01

    In this study, heat-killed Lactobacillus johnsonii (La1), doubly labelled with (13)C and (15)N (hk-dlLa1), was used to follow the metabolic fate after oral administration in humans. Experimental study. University of Rostock, Children's Hospital, Research Laboratory. Ten healthy adults aged 23-26 years. The subjects received 74.6 mg/kg body weight hk-dlLa1 and 10 g alpha-D-raffinose together with breakfast. A sample of venous blood was taken after 2 h. Expired air samples were taken over 14 h, whereas urine and faeces were collected over a period of 48 h. (13)C- and (15)N-enrichments were measured by isotope ratio mass spectrometry. Hydrogen concentrations were measured by electrochemical detection. The orocaecal transit time (OCTT) was reached after 3.4 h. After 2 h, (13)C- and (15)N-enrichment of fibrinogen amounted to 2 and 25 p.p.m. excess, respectively. The (13)CO(2)-exhalation amounted to 9.2% of the ingested dose. The urinary excretion of (13)C and (15)N was 2.1 and 10.4% of the ingested dose, respectively, whereas the faecal excretion was 47.9 and 43.7% of the ingested dose, respectively. In comparison to OCTT of 3.4 h, both stable isotopes appear after 30 min in breath and urine, indicating that hk-dlLa1 is rapidly digested in the small bowel before reaching the caecum. This is confirmed by (13)C-and (15)N-enrichments of blood plasma fractions. The ingestion of hk-dlLa1 led to a (13)C- and (15)N-excretion of 59.2 and 54.1% of the ingested dose, respectively, of both stable isotopes.

  13. Periplasmic binding protein-based detection of maltose using liposomes: a new class of biorecognition elements in competitive assays.

    PubMed

    Edwards, Katie A; Baeumner, Antje J

    2013-03-05

    A periplasmic binding protein (PBP) was investigated as a novel binding species in a similar manner to an antibody in a competitive enzyme linked immunosorbent assay (ELISA), resulting in a highly sensitive and specific assay utilizing liposome-based signal amplification. PBPs are located at high concentrations (10(-4) M) between the inner and outer membranes of gram negative bacteria and are involved in the uptake of solutes and chemotaxis of bacteria toward nutrient sources. Previous sensors relying on PBPs took advantage of the change in local environment or proximity of site-specific fluorophore labels resulting from the significant conformational shift of these proteins' two globular domains upon target binding. Here, rather than monitoring conformational shifts, we have instead utilized the maltose binding protein (MBP) in lieu of an antibody in an ELISA. To our knowledge, this is the first PBP-based sensor without the requirement for engineering site-specific modifications within the protein. MBP conjugated fluorescent dye-encapsulating liposomes served to provide recognition and signal amplification in a competitive assay for maltose using amylose magnetic beads in a microtiter plate-based format. The development of appropriate binding buffers and competitive surfaces are described, with general observations expected to extend to PBPs for other analytes. The resulting assay was specific for d-(+)-maltose versus other sugar analogs including d-(+)-raffinose, sucrose, d-trehalose, d-(+)-xylose, d-fructose, 1-thio-β-d-glucose sodium salt, d-(+)-galactose, sorbitol, glycerol, and dextrose. Cross-reactivity with d-lactose and d-(+)-glucose occurred only at concentrations >10(4)-fold greater than d-(+)-maltose. The limit of detection was 78 nM with a dynamic range covering over 3 orders of magnitude. Accurate detection of maltose as an active ingredient in a pharmaceutical preparation was demonstrated. This method offers a significant improvement over existing

  14. Synbiotics for Broiler Chickens—In Vitro Design and Evaluation of the Influence on Host and Selected Microbiota Populations following In Ovo Delivery

    PubMed Central

    Dunislawska, Aleksandra; Slawinska, Anna; Stadnicka, Katarzyna; Bednarczyk, Marek; Gulewicz, Piotr; Jozefiak, Damian; Siwek, Maria

    2017-01-01

    Synbiotics are synergistic combinations of prebiotics and probiotics. In chickens, synbiotics can be delivered in ovo to expedite colonization of the gut by beneficial bacteria. We therefore aimed to design synbiotics in vitro and validate them in broiler chickens upon in ovo delivery. The probiotic components of the synbiotics were Lactobacillus salivarius and Lactobacillus plantarum. Their growth was assessed in MRS medium supplemented with different prebiotics. Based on in vitro results (hatchability and growth curve), two synbiotics were designed: S1 –Lactobacillus salivarius with galactooligosaccarides (GOS) and S2 –Lactobacillus plantarum with raffinose family oligosaccharides (RFO). These synbiotics were delivered to Cobb broiler chicken embryos on day 12 of incubation at optimized doses (105 cfu egg-1 of probiotic, 2 mg egg-1 of prebiotic). Post hatching, 2,400 roosters were reared (600 individuals group-1 divided into eight replicate pens). Microbial communities were analyzed in ileal and cecal digesta on day 21 using FISH. Gene expression analysis (IL1β, IL4, IL6, IL8, IL12, IL18, IFNβ, and IFNγ) was performed on days 7, 14, 21, and 42 for the spleen and cecal tonsils with RT-qPCR. Body weight and feed intake of the roosters did not differ by the treatments. Microbial populations of Lactobacillus spp. and Enterococcus spp. in the ileum were higher in S1 and S2 than in the control. In the cecum, the control had the highest bacterial counts. S1 caused significant up-regulation of IL6, IL18, IL1β, IFNγ, and IFNβ in the spleen on day 21 and IL1β on day 7 (P < 0.05). In cecal tonsils, S1 caused significant down-regulation of IL12, IL8, and IL1β on day 42 and IFNβ on day 14 (P < 0.05). S2 did not elicit such patterns in any tissues investigated. Thus, we demonstrate that divergent effects of synbiotics in broiler chickens were reflected in in vitro tests. PMID:28045927

  15. Influence of different prebiotics and mode of their administration on broiler chicken performance.

    PubMed

    Bednarczyk, M; Stadnicka, K; Kozłowska, I; Abiuso, C; Tavaniello, S; Dankowiakowska, A; Sławińska, A; Maiorano, G

    2016-08-01

    In the post-antibiotics era, prebiotics are proposed as alternatives to antibiotic growth promoters in poultry production. The goal of this study was to compare in ovo method of prebiotic delivery with in-water supplementation and with both methods combined (in ovo+in-water) in broiler chickens. Two trials were conducted. Trial 1 was carried out to optimize the doses of two prebiotics, DN (DiNovo®, extract of beta-glucans) and BI (Bi2tos, trans-galactooligosaccharides), for in ovo delivery. The estimated parameters were hatchability and bacteriological status of the newly hatched chicks. Prebiotics were dissolved in 0.2 ml of physiological saline, at the doses: 0.18, 0.88, 3.5 and 7.0 mg/embryo; control group (C) was injected in ovo with 0.2 ml of physiological saline. Trial 2 was conducted to evaluate effects of different prebiotics (DN, BI and raffinose family oligosaccharides (RFO)) delivered in ovo, in-water and in a combined way (in ovo+in-water) on broiler chickens performance. The results of the Trial 1 indicated that the optimal dose of DN and BI prebiotics delivered in ovo, that did not reduce chicks' hatchability, was 0.88 mg/embryo (DN) and 3.5 mg/embryo (BI). Both prebiotics numerically increased number of lactobacilli and bifidobacteria in chicken feces (P>0.05). In Trial 2, all prebiotics (DN, BI and RFO) significantly increased BW gain compared with the C group (P<0.05), especially during the first 21 days of life. However, feed intake and feed conversion ratio were increased upon prebiotics delivery irrespective of method used. Injection of prebiotics in ovo combined with in-water supplementation did not express synergistic effects on broilers performance compared with in ovo injection only. Taken together, those results confirm that single in ovo prebiotics injection into the chicken embryo can successfully replace prolonged in-water supplementation post hatching.

  16. Acidic α-galactosidase is the most abundant nectarin in floral nectar of common tobacco (Nicotiana tabacum)

    PubMed Central

    Zha, Hong-Guang; Flowers, V. Lynn; Yang, Min; Chen, Ling-Yang; Sun, Hang

    2012-01-01

    Background and Aims To date, most floral nectarins (nectar proteins) are reported to function in nectar defence, particularly for insect-pollinated outcrossing species. We compared nectarin composition and abundance in selfing common tobacco (Nicotiana tobaccum) with outcrossing ornamental tobacco plants to elucidate the functional difference of nectarins in different reproductive systems. Methods Common tobacco (CT) nectarins were separated by SDS-PAGE and the N terminus of the most abundant nectarin was sequenced via Edman degradation. The full-length nectarin gene was amplified and cloned from genomic DNA and mRNA with hiTail-PCR and RACE (rapid amplification of cDNA ends), and expression patterns were then investigated in different tissues using semi-quantitative reverse transcriptase PCR. Additionally, high-performance liquid chromatography and enzymatic analyses of nectar sugar composition, and other biochemical traits and functions of the novel nectarin were studied. Key Results The most abundant nectarin in CT nectar is an acidic α-galactosidase, here designated NTα-Gal. This compound has a molecular mass of 40 013 Da and a theoretical pI of 5·33. NTα-Gal has a conserved α-Gal characteristic signature, encodes a mature protein of 364 amino acids and is expressed in different organs. Compared with 27 other melliferous plant species from different families, CT floral nectar demonstrated the highest α-Gal activity, which is inhibited by d-galactose. Raffinose family oligosaccharides were not detected in CT nectar, indicating that NTα-Gal does not function in post-secretory hydrolysis. Moreover, tobacco plant fruits did not develop intact skin with galactose inhibition of NTα-Gal activity in nectar, suggesting that NTα-Gal induces cell-wall surface restructuring during the initial stages of fruit development. Conclusions α-Gal was the most abundant nectarin in selfing CT plants, but was not detected in the nectar of strictly outcrossing sister tobacco

  17. Prioritization of Candidate Genes in QTL Regions for Physiological and Biochemical Traits Underlying Drought Response in Barley (Hordeum vulgare L.)

    PubMed Central

    Gudys, Kornelia; Guzy-Wrobelska, Justyna; Janiak, Agnieszka; Dziurka, Michał A.; Ostrowska, Agnieszka; Hura, Katarzyna; Jurczyk, Barbara; Żmuda, Katarzyna; Grzybkowska, Daria; Śróbka, Joanna; Urban, Wojciech; Biesaga-Koscielniak, Jolanta; Filek, Maria; Koscielniak, Janusz; Mikołajczak, Krzysztof; Ogrodowicz, Piotr; Krystkowiak, Karolina; Kuczyńska, Anetta; Krajewski, Paweł; Szarejko, Iwona

    2018-01-01

    Drought is one of the most adverse abiotic factors limiting growth and productivity of crops. Among them is barley, ranked fourth cereal worldwide in terms of harvested acreage and production. Plants have evolved various mechanisms to cope with water deficit at different biological levels, but there is an enormous challenge to decipher genes responsible for particular complex phenotypic traits, in order to develop drought tolerant crops. This work presents a comprehensive approach for elucidation of molecular mechanisms of drought tolerance in barley at the seedling stage of development. The study includes mapping of QTLs for physiological and biochemical traits associated with drought tolerance on a high-density function map, projection of QTL confidence intervals on barley physical map, and the retrievement of positional candidate genes (CGs), followed by their prioritization based on Gene Ontology (GO) enrichment analysis. A total of 64 QTLs for 25 physiological and biochemical traits that describe plant water status, photosynthetic efficiency, osmoprotectant and hormone content, as well as antioxidant activity, were positioned on a consensus map, constructed using RIL populations developed from the crosses between European and Syrian genotypes. The map contained a total of 875 SNP, SSR and CGs, spanning 941.86 cM with resolution of 1.1 cM. For the first time, QTLs for ethylene, glucose, sucrose, maltose, raffinose, α-tocopherol, γ-tocotrienol content, and catalase activity, have been mapped in barley. Based on overlapping confidence intervals of QTLs, 11 hotspots were identified that enclosed more than 60% of mapped QTLs. Genetic and physical map integration allowed the identification of 1,101 positional CGs within the confidence intervals of drought response-specific QTLs. Prioritization resulted in the designation of 143 CGs, among them were genes encoding antioxidants, carboxylic acid biosynthesis enzymes, heat shock proteins, small auxin up-regulated RNAs

  18. A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass

    PubMed Central

    Vija, Heiki; Aasamets, Anneli; Viigand, Katrin

    2017-01-01

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed. PMID:28103254

  19. The role of carbohydrates in seed germination and seedling establishment of Himatanthus sucuuba, an Amazonian tree with populations adapted to flooded and non-flooded conditions

    PubMed Central

    da Silva Ferreira, Cristiane; Piedade, Maria Teresa Fernandez; Tiné, Marco Aurélio Silva; Rossatto, Davi Rodrigo; Parolin, Pia; Buckeridge, Marcos Silveira

    2009-01-01

    Background and Aims In the Amazonian floodplains plants withstand annual periods of flooding which can last 7 months. Under these conditions seedlings remain submerged in the dark for long periods since light penetration in the water is limited. Himatanthus sucuuba is a tree species found in the ‘várzea’ (VZ) floodplains and adjacent non-flooded ‘terra-firme’ (TF) forests. Biochemical traits which enhance flood tolerance and colonization success of H. sucuuba in periodically flooded environments were investigated. Methods Storage carbohydrates of seeds of VZ and TF populations were extracted and analysed by HPAEC/PAD. Starch was analysed by enzyme (glucoamylase) degradation followed by quantification of glucose oxidase. Carbohydrate composition of roots of VZ and TF seedlings was studied after experimental exposure to a 15-d period of submersion in light versus darkness. Key Results The endosperm contains a large proportion of the seed reserves, raffinose being the main non-structural carbohydrate. Around 93 % of the cell wall storage polysaccharides (percentage dry weight basis) in the endosperm of VZ seeds was composed of mannose, while soluble sugars accounted for 2·5%. In contrast, 74 % of the endosperm in TF seeds was composed of galactomannans, while 22 % of the endosperm was soluble sugars. This suggested a larger carbohydrate allocation to germination in TF populations whereas VZ populations allocate comparatively more to carbohydrates mobilized during seedling development. The concentration of root non-structural carbohydrates in non-flooded seedlings strongly decreased after a 15-d period of darkness, whereas flooded seedlings were less affected. These effects were more pronounced in TF seedlings, which showed significantly lower root non-structural carbohydrate concentrations. Conclusions There seem to be metabolic adjustments in VZ but not TF seedlings that lead to adaptation to the combined stresses of darkness and flooding. This seems to be

  20. Metabolic Responses to Low Temperature of Three Peach Fruit Cultivars Differently Sensitive to Cold Storage

    PubMed Central

    Brizzolara, Stefano; Hertog, Maarten; Tosetti, Roberta; Nicolai, Bart; Tonutti, Pietro

    2018-01-01

    Refrigerated storage is widely applied in order to maintain peach quality but it can also induce chilling injuries (CIs) such as flesh browning and bleeding, and mealiness. Peach fruit from three cultivars (‘Red Haven’, RH, ‘Regina di Londa’, RL, and ‘Flaminia’, FL) were stored for 4 weeks under low temperatures (0.5 and 5.5°C). GC-MS was employed to study changes in both metabolome and volatilome induced by cold storage in the mesocarp. CIs were assessed both at the end of each week of storage and after subsequent shelf-life (SL) at 20°C. Flesh browning and mealiness appeared to be more related to 5.5°C storage, while flesh bleeding revealed high incidence following 0.5°C storage. Compared to RL and FL, RH showed a marked lower incidence of CIs. Multivariate statistical analyses indicate that RH peaches indeed differ from RL and FL in particular when considering data from samples collected at the end of the cold storage. Common and divergent responses have been identified in terms of metabolic responses to the applied low temperatures. In all three cultivars raffinose, glucose-6P, fucose, xylose, sorbitol, GABA, epicatechin, catechin, and putrescine markedly increased during cold storage, while citramalic, glucuronic, mucic and shikimic acids decreased. Among volatile organic compounds (VOCs), aldehydes and alcohols generally accumulated more under low temperature conditions while esters and lactones evolved during subsequent SL. The main cultivar differences developed after cold storage during SL although some common responses (e.g., an increased production of ethyl acetate) were observed. The lower levels of flesh browning and bleeding displayed by RH peaches were related to compounds with antioxidant activity, or acting as osmotic protectants and membrane stabilizer. Indeed, RH showed higher levels of amino acids and urea, together with a marked increase in putrescine, sorbitol, maltitol, myoinositol and sucrose detected during storage and SL

  1. De Novo Transcriptional Analysis of Alfalfa in Response to Saline-Alkaline Stress.

    PubMed

    An, Yi-Min; Song, Li-Li; Liu, Ying-Rui; Shu, Yong-Jun; Guo, Chang-Hong

    2016-01-01

    Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress that affects crop productivity. Alfalfa is a widely cultivated perennial forage legume with some tolerance to biotic and abiotic stresses, especially to saline-alkaline stress. To elucidate the mechanism underlying plant saline-alkaline tolerance, we conducted transcriptome analysis of whole alfalfa seedlings treated with saline-alkaline solutions for 0 day (control), 1 day (short-term treatment), and 7 days (long-term treatment) using ion torrent sequencing technology. A transcriptome database dataset of 53,853 unigenes was generated, and 2,286 and 2,233 genes were differentially expressed in the short-term and long-term treatment, respectively. Gene ontology analysis revealed 14 highly enriched pathways and demonstrated the differential response of metabolic pathways between the short-term and long-term treatment. The expression levels of 109 and 96 transcription factors were significantly altered significantly after 1 day and 7 days of treatment, respectively. Specific responses of peroxidase, flavonoids, and the light pathway component indicated that the antioxidant capacity was one of the central mechanisms of saline-alkaline stress tolerance response in alfalfa. Among the 18 differentially expressed genes examined by real time PCR, the expression levels of eight genes, including inositol transporter, DNA binding protein, raffinose synthase, ferritin, aldo/keto reductase, glutathione S-transferase, xyloglucan endotrans glucosylase, and a NAC transcription factor, exhibited different patterns in response to saline and alkaline stress. The expression levels of the NAC transcription factor and glutathione S-transferase were altered significantly under saline stress and saline-alkaline stress; they were upregulated under saline-alkaline stress and downregulated under salt stress. Physiology assays showed an increased concentration of reactive oxygen

  2. De Novo Transcriptional Analysis of Alfalfa in Response to Saline-Alkaline Stress

    PubMed Central

    An, Yi-Min; Song, Li-Li; Liu, Ying-Rui; Shu, Yong-Jun; Guo, Chang-Hong

    2016-01-01

    Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress that affects crop productivity. Alfalfa is a widely cultivated perennial forage legume with some tolerance to biotic and abiotic stresses, especially to saline-alkaline stress. To elucidate the mechanism underlying plant saline-alkaline tolerance, we conducted transcriptome analysis of whole alfalfa seedlings treated with saline-alkaline solutions for 0 day (control), 1 day (short-term treatment), and 7 days (long-term treatment) using ion torrent sequencing technology. A transcriptome database dataset of 53,853 unigenes was generated, and 2,286 and 2,233 genes were differentially expressed in the short-term and long-term treatment, respectively. Gene ontology analysis revealed 14 highly enriched pathways and demonstrated the differential response of metabolic pathways between the short-term and long-term treatment. The expression levels of 109 and 96 transcription factors were significantly altered significantly after 1 day and 7 days of treatment, respectively. Specific responses of peroxidase, flavonoids, and the light pathway component indicated that the antioxidant capacity was one of the central mechanisms of saline-alkaline stress tolerance response in alfalfa. Among the 18 differentially expressed genes examined by real time PCR, the expression levels of eight genes, including inositol transporter, DNA binding protein, raffinose synthase, ferritin, aldo/keto reductase, glutathione S-transferase, xyloglucan endotrans glucosylase, and a NAC transcription factor, exhibited different patterns in response to saline and alkaline stress. The expression levels of the NAC transcription factor and glutathione S-transferase were altered significantly under saline stress and saline-alkaline stress; they were upregulated under saline-alkaline stress and downregulated under salt stress. Physiology assays showed an increased concentration of reactive oxygen

  3. Metabolic Responses to Low Temperature of Three Peach Fruit Cultivars Differently Sensitive to Cold Storage.

    PubMed

    Brizzolara, Stefano; Hertog, Maarten; Tosetti, Roberta; Nicolai, Bart; Tonutti, Pietro

    2018-01-01

    Refrigerated storage is widely applied in order to maintain peach quality but it can also induce chilling injuries (CIs) such as flesh browning and bleeding, and mealiness. Peach fruit from three cultivars ('Red Haven', RH, 'Regina di Londa', RL, and 'Flaminia', FL) were stored for 4 weeks under low temperatures (0.5 and 5.5°C). GC-MS was employed to study changes in both metabolome and volatilome induced by cold storage in the mesocarp. CIs were assessed both at the end of each week of storage and after subsequent shelf-life (SL) at 20°C. Flesh browning and mealiness appeared to be more related to 5.5°C storage, while flesh bleeding revealed high incidence following 0.5°C storage. Compared to RL and FL, RH showed a marked lower incidence of CIs. Multivariate statistical analyses indicate that RH peaches indeed differ from RL and FL in particular when considering data from samples collected at the end of the cold storage. Common and divergent responses have been identified in terms of metabolic responses to the applied low temperatures. In all three cultivars raffinose, glucose-6P, fucose, xylose, sorbitol, GABA, epicatechin, catechin, and putrescine markedly increased during cold storage, while citramalic, glucuronic, mucic and shikimic acids decreased. Among volatile organic compounds (VOCs), aldehydes and alcohols generally accumulated more under low temperature conditions while esters and lactones evolved during subsequent SL. The main cultivar differences developed after cold storage during SL although some common responses (e.g., an increased production of ethyl acetate) were observed. The lower levels of flesh browning and bleeding displayed by RH peaches were related to compounds with antioxidant activity, or acting as osmotic protectants and membrane stabilizer. Indeed, RH showed higher levels of amino acids and urea, together with a marked increase in putrescine, sorbitol, maltitol, myoinositol and sucrose detected during storage and SL.

  4. Elevated Atmospheric CO2 and Strain of Rhizobium Alter Freezing Tolerance and Cold-induced Molecular Changes in Alfalfa (Medicago sativa)

    PubMed Central

    Bertrand, Annick; Prévost, Danielle; Bigras, Francine J.; Castonguay, Yves

    2007-01-01

    Background and Aims The objective of the study was to assess the impact of elevated CO2 in interaction with rhizobial strains on freezing tolerance and cold-induced molecular changes in alfalfa. Methods Alfalfa inoculated with two different strains of rhizobium (A2 and NRG34) was grown and cold acclimated (2 weeks at 2 °C) under either 400 (ambient) or 800 µmol mol−1 (elevated) CO2. Key Results Plants acclimated under 400 µmol mol−1 CO2 were more freezing tolerant than those maintained under 800 µmol mol−1. Cryoprotective sugars typically linked with the acquisition of freezing tolerance such as sucrose, stachyose and raffinose increased in roots in response to low temperature but did not differ between CO2 treatments. Similarly high CO2 did not alter the expression of many cold-regulated (COR) genes although it significantly increased the level of transcripts encoding a COR gene homologous to glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). A significant effect of rhizobial strain was observed on both freezing tolerance and gene expression. Plants of alfalfa inoculated with strain A2 were more freezing tolerant than those inoculated with strain NRG34. Transcripts of COR genes homologous to a pathogenesis-related protein (PR-10) and to a nuclear-targeted protein were markedly enhanced in roots of alfalfa inoculated with strain A2 as compared with strain NRG34. Transcripts encoding the vegetative storage proteins (VSPs) β-amylase and chitinase were more abundant in roots of non-acclimated plants inoculated with strain NRG34 than with strain A2. Conclusions Taken together, the results suggest that elevated CO2 stimulates plant growth and reduces freezing tolerance. The acquisition of cold tolerance is also influenced by the rhizobial strain, as indicated by lower levels of expression of COR genes and sustained accumulation of VSP-encoding transcripts in alfalfa inoculated with strain NRG34 as compared with strain A2. PMID:17218341

  5. Influence of Three Contrasting Detrital Carbon Sources on Planktonic Bacterial Metabolism in a Mesotrophic Lake.

    PubMed

    Wehr; Petersen; Findlay

    1999-01-01

    greater use of raffinose, pyroglutamic acid, and sebacic acid. Results suggest that pelagic bacterial communities respond to changes in organic carbon source rapidly and by different routes, including shifts in per-cell production rates and variations in degradation of a variety of compounds comprising the DOC pool.

  6. Unraveling Key Metabolomic Alterations in Wheat Embryos Derived from Freshly Harvested and Water-Imbibed Seeds of Two Wheat Cultivars with Contrasting Dormancy Status

    PubMed Central

    Das, Aayudh; Kim, Dea-Wook; Khadka, Pramod; Rakwal, Randeep; Rohila, Jai S.

    2017-01-01

    Untimely rains in wheat fields during harvest season can cause pre-harvest sprouting (PHS), which deteriorates the yield and quality of wheat crop. Metabolic homeostasis of the embryo plays a role in seed dormancy, determining the status of the maturing grains either as dormant (PHS-tolerant) or non-dormant (PHS-susceptible). Very little is known for direct measurements of global metabolites in embryonic tissues of dormant and non-dormant wheat seeds. In this study, physiologically matured and freshly harvested wheat seeds of PHS-tolerant (cv. Sukang, dormant) and PHS-susceptible (cv. Baegjoong, non-dormant) cultivars were water-imbibed, and the isolated embryos were subjected to high-throughput, global non-targeted metabolomic profiling. A careful comparison of identified metabolites between Sukang and Baegjoong embryos at 0 and 48 h after imbibition revealed that several key metabolic pathways [such as: lipids, fatty acids, oxalate, hormones, the raffinose family of oligosaccharides (RFOs), and amino acids] and phytochemicals were differentially regulated between dormant and non-dormant varieties. Most of the membrane lipids were highly reduced in Baegjoong compared to Sukang, which indicates that the cell membrane instability in response to imbibition could also be a key factor in non-dormant wheat varieties for their untimely germination. This study revealed that several key marker metabolites (e.g., RFOs: glucose, fructose, maltose, and verbascose), were highly expressed in Baegjoong after imbibition. Furthermore, the data showed that the key secondary metabolites and phytochemicals (vitexin, chrysoeriol, ferulate, salidroside and gentisic acid), with known antioxidant properties, were comparatively low at basal levels in PHS-susceptible, non-dormant cultivar, Baegjoong. In conclusion, the results of this investigation revealed that after imbibition the metabolic homeostasis of dormant wheat is significantly less affected compared to non-dormant wheat. The

  7. Transcriptomics of Desiccation Tolerance in the Streptophyte Green Alga Klebsormidium Reveal a Land Plant-Like Defense Reaction

    PubMed Central

    Holzinger, Andreas; Kaplan, Franziska; Blaas, Kathrin; Zechmann, Bernd; Komsic-Buchmann, Karin; Becker, Burkhard

    2014-01-01

    Background Water loss has significant effects on physiological performance and survival rates of algae. However, despite the prominent presence of aeroterrestrial algae in terrestrial habitats, hardly anything is known about the molecular events that allow aeroterrestrial algae to survive harsh environmental conditions. We analyzed the transcriptome and physiology of a strain of the alpine aeroterrestrial alga Klebsormidium crenulatum under control and strong desiccation-stress conditions. Principal Findings For comparison we first established a reference transcriptome. The high-coverage reference transcriptome includes about 24,183 sequences (1.5 million reads, 636 million bases). The reference transcriptome encodes for all major pathways (energy, carbohydrates, lipids, amino acids, sugars), nearly all deduced pathways are complete or missing only a few transcripts. Upon strong desiccation, more than 7000 transcripts showed changes in their expression levels. Most of the highest up-regulated transcripts do not show similarity to known viridiplant proteins, suggesting the existence of some genus- or species-specific responses to desiccation. In addition, we observed the up-regulation of many transcripts involved in desiccation tolerance in plants (e.g. proteins similar to those that are abundant in late embryogenesis (LEA), or proteins involved in early response to desiccation ERD), and enzymes involved in the biosynthesis of the raffinose family of oligosaccharides (RFO) known to act as osmolytes). Major physiological shifts are the up-regulation of transcripts for photosynthesis, energy production, and reactive oxygen species (ROS) metabolism, which is supported by elevated cellular glutathione content as revealed by immunoelectron microscopy as well as an increase in total antiradical power. However, the effective quantum yield of Photosystem II and CO2 fixation decreased sharply under the applied desiccation stress. In contrast, transcripts for cell integrative

  8. A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass.

    PubMed

    Mardo, Karin; Visnapuu, Triinu; Vija, Heiki; Aasamets, Anneli; Viigand, Katrin; Alamäe, Tiina

    2017-01-01

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5-6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.

  9. Changes in the proteomic and metabolic profiles of Beta vulgaris root tips in response to iron deficiency and resupply

    PubMed Central

    2010-01-01

    Background Plants grown under iron deficiency show different morphological, biochemical and physiological changes. These changes include, among others, the elicitation of different strategies to improve the acquisition of Fe from the rhizosphere, the adjustment of Fe homeostasis processes and a reorganization of carbohydrate metabolism. The application of modern techniques that allow the simultaneous and untargeted analysis of multiple proteins and metabolites can provide insight into multiple processes taking place in plants under Fe deficiency. The objective of this study was to characterize the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply. Results Root tip extract proteome maps were obtained by 2-D isoelectric focusing polyacrylamide gel electrophoresis, and approximately 140 spots were detected. Iron deficiency resulted in changes in the relative amounts of 61 polypeptides, and 22 of them were identified by mass spectrometry (MS). Metabolites in root tip extracts were analyzed by gas chromatography-MS, and more than 300 metabolites were resolved. Out of 77 identified metabolites, 26 changed significantly with Fe deficiency. Iron deficiency induced increases in the relative amounts of proteins and metabolites associated to glycolysis, tri-carboxylic acid cycle and anaerobic respiration, confirming previous studies. Furthermore, a protein not present in Fe-sufficient roots, dimethyl-8-ribityllumazine (DMRL) synthase, was present in high amounts in root tips from Fe-deficient sugar beet plants and gene transcript levels were higher in Fe-deficient root tips. Also, a marked increase in the relative amounts of the raffinose family of oligosaccharides (RFOs) was observed in Fe-deficient plants, and a further increase in these compounds occurred upon short term Fe resupply. Conclusions The increases in DMRL synthase and in RFO sugars were the major changes induced by Fe deficiency and resupply

  10. A novel α-galactosidase from Fusarium oxysporum and its application in determining the structure of the gum arabic side chain.

    PubMed

    Maruta, Akiho; Yamane, Mirei; Matsubara, Midori; Suzuki, Shiho; Nakazawa, Masami; Ueda, Mitsuhiro; Sakamoto, Tatsuji

    2017-08-01

    We previously reported that Fusarium oxysporum 12S produces two bifunctional proteins, FoAP1 and FoAP2, with α-d-galactopyranosidase (GPase) and β-l-arabinopyranosidase (APase) activities. The aim of this paper was to purify a third GPase, FoGP1, from culture supernatant of F. oxysporum 12S, to characterize it, and to determine its mode of action towards gum arabic. A cDNA encoding FoGP1 was cloned and the protein was overexpressed in Escherichia coli. Module sequence analysis revealed the presence of a GH27 domain in FoGP1. The recombinant enzyme (rFoGP1) showed a GPase/APase activity ratio of 330, which was quite different from that of FoAP1 (1.7) and FoAP2 (0.2). Among the natural substrates tested, rFoGP1 showed the highest activity towards gum arabic. In contrast to other well-characterized GPases, rFoGP1 released a small amount of galactose from α-galactosyl oligosaccharides such as raffinose and exhibited no activity toward galactomannans, which are highly substituted with α-galactosyl side chains. This indicated that FoGP1 is an unusual type of GPase. rFoGP1 released 30% of the total galactose from gum arabic, suggesting the existence of a large number of α-galactosyl residues at the non-reducing ends of gum arabic side chains. Together, rFoGP1 and α-l-arabinofuranosidase released four times more arabinose than α-l-arabinofuranosidase acting alone. This suggested that a large number of α-l-arabinofuranosyl residues is capped by α-galactosyl residues. 1 H NMR experiments revealed that rFoGP1 hydrolyzed the α-1,3-galactosidic linkage within the side chain structure of [α-d-Galp-(1→3)-α-l-Araf-(1→] in gum arabic. In conclusion, rFoGP1 is highly active toward α-1,3-galactosyl linkages but negligibly or not active toward α-1,6-galactosyl linkages. The novel FoGP1 might be used to modify the physical properties of gum arabic, which is an industrially important polysaccharide used as an emulsion stabilizer and coating agent. Copyright © 2017

  11. Thermodynamic nonequilibrium phase change behavior and thermal properties of biological solutions for cryobiology applications.

    PubMed

    Han, Bumsoo; Bischof, John C

    2004-04-01

    Understanding the phase change behavior of biomaterials during freezing/thawing including their thermal properties at low temperatures is essential to design and improve cryobiology applications such as cryopreservation and cryosurgery. However, knowledge of phase change behavior and thermal properties of various biomaterials is still incomplete, especially at cryogenic temperatures (< or = -40 degrees C). Moreover, in these applications, chemicals are often added to improve their outcome, which can result in significant variation in the phase change behavior and thermal properties from those of the original biomaterials. These chemical additives include cryoprotective agents (CPAs), antifreeze protein (AFP), or cryosurgical adjuvants like sodium chloride (NaCl). In the present study, phase change behavior and thermal properties of saline solutions--either water-NaCl or phosphate buffered saline (PBS)--with various chemical additives were investigated. The chemical additives studied are glycerol and raffinose as CPAs, an AFP (Type III, molecular weight = 6500), and NaCl as a cryosurgical adjuvant. The phase change behavior was investigated using a differential scanning calorimeter (DSC) and a cryomicroscope. The specific and latent heat of these solutions were also measured with the DSC. The saline solutions have two distinct phase changes--water/ice and eutectic phase changes. During freezing, eutectic solidification of both water-NaCl and PBS are significantly supercooled below their thermodynamic equilibrium eutectic temperatures. However, their melting temperatures are close to thermodynamic equilibrium during thawing. These eutectic phase changes disappear when even a small amount (0.1 M glycerol) of CPA was added, but they are still observed after the addition of an AFP. The specific heats of these solutions are close to that of ice at very low temperatures (< or = -100 degrees C) regardless of the additives, but they increase between -100 degrees C and -30

  12. Above and below ground carbohydrate allocation differs between ash (Fraxinus excelsior L.) and beech (Fagus sylvatica L.)

    PubMed Central

    Thoms, Ronny; Köhler, Michael; Gessler, Arthur

    2017-01-01

    We investigated soluble carbohydrate transport in trees that differed in their phloem loading strategies in order to better understand the transport of photosynthetic products into the roots and the rhizosphere as this knowledge is needed to better understand the respiratory processes in the rhizosphere. We compared beech, which is suggested to use mainly passive loading of transport sugars along a concentration gradient into the phloem, with ash that uses active loading and polymer trapping of raffinose family oligosaccharides (RFOs). We pulse-labeled 20 four-year old European beech and 20 four-year old ash trees with 13CO2 and tracked the fate of the label within different plant compartments. We extracted soluble carbohydrates from leaves, bark of stems and branches, and fine roots, measured their amount and isotopic content and calculated their turnover times. In beech one part of the sucrose was rapidly transported into sink tissues without major exchange with storage pools whereas another part of sucrose was strongly exchanged with unlabeled possibly stored sucrose. In contrast the storage and allocation patterns in ash depended on the identity of the transported sugars. RFO were the most important transport sugars that had highest turnover in all shoot compartments. However, the turnover of RFOs in the roots was uncoupled from the shoot. The only significant relation between sugars in the stem base and in the roots of ash was found for the amount (r2 = 0.50; p = 0.001) and isotopic content (r2 = 0.47; p = 0.01) of sucrose. The negative relation of the amounts suggested an active transport of sucrose into the roots of ash. Sucrose concentration in the root also best explained the concentration of RFOs in the roots suggesting that RFO in the roots of ash may be resynthesized from sucrose. Our results interestingly suggest that in both tree species only sucrose directly entered the fine root system and that in ash RFOs are transported indirectly into the fine

  13. Transport of water in proximal kidney tubules from whole tubules to single channels: length and section of the selectivity filter of aquaporin-1.

    PubMed

    Whittembury, G; González, E; Hernández, C S; Gutiérrez, A M; Echevarría, M

    1997-06-27

    Proximal straight tubule (PST) were dissected from rabbit kidneys, held with crimping pipettes in a chamber bathed in a buffered mannitol isosmotic solution (MBS, 295 mOsm/kg). Tubule cell volume changes with time (dV/Adt) after steps in MBS osmolality (delta Cs) were monitored on line with an inverted microscope, a TV camera and an image processor. Reflection coefficients sigma and osmotic permeability coefficients, Pos, for several solutes were measured using two methods. Method 1: sigma was calculated from the delta Csiso of impermeant and permeant solutes at which (dV/Adt)t-->0 = 0 (i.e., by a null point method). It is denoted as sigma 1. sigma 1 = 1.00 for mannitol (M), raffinose (R), sucrose (S), glycerol (G), acetamide (A) and urea (U). With formamide (F), sigma 1, Formamide = 0.62 +/- 0.05. These findings confirm our previous value of dp = 4.5 A for the diameter of the selectivity filter of the basolateral PST cell membrane water channel AQP1. Method 2: PST were exposed for 20 s to MBS made hyperosmotic by addition of a delta Cs of 35 mOsm/kg of R, S, M, G, A and U. Cells shrunk within 500 ms of t = 0 to their osmometric volume and remained shrunk for the 20 s of the osmotic challenge. Pos was measured from the shrinking curves. P(os) = 3000 +/- 25 microns/s with R, S, M, G, A and U. Method 2 also allowed to calculate sigma, denoted as sigma 2. sigma 2 = 1.00 for R, S, M, G, A and U. By contrast, the shrinking curve produced by a delta Cs of 35 mOsm/kg F was 1/5th to 1/6th slower and smaller (i.e., subosmometric) than that produced by a delta Cs of 35 mOsm/kg R, S, M, G, A and U. Furthermore, with F cells did not remain shrunk but recovered their original volume within 3 s. P(os) (measured with F) is denoted as P(os)*, P(os)* = 480 +/- 30 microns/s. sigma 2, Formamide = 0.16 +/- 0.01. Use of sigma 1, sigma 2 and P(os)* values in Hill's equations for the bimodal theory of osmosis leads to n = 2-9. Where n is the number of water molecules single filling

  14. The effect of hydroxyl functional groups and molar mass on the viscosity of non-crystalline organic and organic-water particles

    NASA Astrophysics Data System (ADS)

    Grayson, James W.; Evoy, Erin; Song, Mijung; Chu, Yangxi; Maclean, Adrian; Nguyen, Allena; Upshur, Mary Alice; Ebrahimi, Marzieh; Chan, Chak K.; Geiger, Franz M.; Thomson, Regan J.; Bertram, Allan K.

    2017-07-01

    The viscosities of three polyols and three saccharides, all in the non-crystalline state, have been studied. Two of the polyols (2-methyl-1,4-butanediol and 1,2,3-butanetriol) were studied under dry conditions, the third (1,2,3,4-butanetetrol) was studied as a function of relative humidity (RH), including under dry conditions, and the saccharides (glucose, raffinose, and maltohexaose) were studied as a function of RH. The mean viscosities of the polyols under dry conditions range from 1.5 × 10-1 to 3.7 × 101 Pa s, with the highest viscosity being that of the tetrol. Using a combination of data determined experimentally here and literature data for alkanes, alcohols, and polyols with a C3 to C6 carbon backbone, we show (1) there is a near-linear relationship between log10 (viscosity) and the number of hydroxyl groups in the molecule, (2) that on average the addition of one OH group increases the viscosity by a factor of approximately 22 to 45, (3) the sensitivity of viscosity to the addition of one OH group is not a strong function of the number of OH functional groups already present in the molecule up to three OH groups, and (4) higher sensitivities are observed when the molecule has more than three OH groups. Viscosities reported here for 1,2,3,4-butanetetrol particles are lower than previously reported measurements using aerosol optical tweezers, and additional studies are required to resolve these discrepancies. For saccharide particles at 30 % RH, viscosity increases by approximately 2-5 orders of magnitude as molar mass increases from 180 to 342 g mol-1, and at 80 % RH, viscosity increases by approximately 4-5 orders of magnitude as molar mass increases from 180 to 991 g mol-1. These results suggest oligomerization of highly oxidized compounds in atmospheric secondary organic aerosol (SOA) could lead to large increases in viscosity, and may be at least partially responsible for the high viscosities observed in some SOA. Finally, two quantitative structure

  15. Fervidicola ferrireducens gen. nov., sp. nov., a thermophilic anaerobic bacterium from geothermal waters of the Great Artesian Basin, Australia.

    PubMed

    Ogg, Christopher D; Patel, Bharat K C

    2009-05-01

    A strictly anaerobic, thermophilic bacterium, designated strain Y170(T), was isolated from a microbial mat colonizing thermal waters of a run-off channel created by the free-flowing waters of a Great Artesian Basin (GAB) bore well (New Lorne bore; registered number 17263). Cells of strain Y170(T) were slightly curved rods (1.2-12x0.8-1.1 mum) and stained Gram-negative. The strain grew optimally in tryptone-yeast extract-glucose medium at 70 degrees C (temperature range for growth was 55-80 degrees C) and pH 7 (pH range for growth was 5-9). Strain Y170(T) grew poorly on yeast extract as a sole carbon source, but not on tryptone (0.2 %). Yeast extract could not be replaced by tryptone and was obligately required for growth on tryptone, peptone, glucose, fructose, galactose, cellobiose, mannose, sucrose, xylose, mannitol, formate, pyruvate, Casamino acids and threonine. No growth was observed on arabinose, lactose, maltose, raffinose, chitin, xylan, pectin, starch, acetate, benzoate, lactate, propionate, succinate, myo-inositol, ethanol, glycerol, amyl media, aspartate, leucine, glutamate, alanine, arginine, serine and glycine. End products detected from glucose fermentation were acetate, ethanol and presumably CO(2) and H(2). Iron(III), manganese(IV), thiosulfate and elemental sulfur, but not sulfate, sulfite, nitrate or nitrite, were used as electron acceptors in the presence of 0.2 % yeast extract. Iron(III) in the form of amorphous Fe(III) oxhydroxide and Fe(III) citrate was also reduced in the presence of tryptone, peptone and Casamino acids, but not with chitin, xylan, pectin, formate, starch, pyruvate, acetate, benzoate, threonine, lactate, propionate, succinate, inositol, ethanol, glycerol, mannitol, aspartate, leucine, glutamate, alanine, arginine, serine or glycine. Strain Y170(T) was not able to utilize molecular hydrogen and/or carbon dioxide in the presence or absence of iron(III). Chloramphenicol, streptomycin, tetracycline, penicillin and ampicillin and

  16. A novel β-fructofuranosidase in Coleoptera: Characterization of a β-fructofuranosidase from the sugarcane weevil, Sphenophorus levis.

    PubMed

    Pedezzi, Rafael; Fonseca, Fernando P P; Santos Júnior, Célio Dias; Kishi, Luciano T; Terra, Walter R; Henrique-Silva, Flávio

    2014-12-01

    β-fructofuranosidases or invertases (EC 3.2.1.26) catalyze the hydrolysis of sucrose into fructose and glucose. β-fructofuranosidases have been widely described in microorganisms, but were not known in the animal kingdom until very recently. There are studies reporting lepidopteran β-fructofuranosidases, but no β-fructofuranosidase gene sequence or encoding transcript has previously been identified in beetles. Considering the scarcity of functional studies on insect β-fructofuranosidases and their apparent non-occurrence among coleopterans, the aim of the present study was to investigate the occurrence and characterize a β-fructofuranosidase transcript identified in a cDNA library from the sugarcane weevil, Sphenophorus levis (Curculionidae). To validate that the β-fructofuranosidase sequence (herein denominated Sl-β-fruct) is indeed encoded by the S. levis genome, PCRs were performed using genomic DNA extracted from the larval fat body as well as DNA from the midgut with microbial content. Amplification of Sl-β-fruct gene using larval fat body DNA indicated its presence in the insect's genomic DNA. The Sl-β-fruct gene was cloned in Pichia pastoris to produce the recombinant enzyme (rSl-β-fruct). Molecular weight of the recombinant protein was about 64 kDa, indicating possible glycosylation, since the theoretical weight was 54.8 kDa. The substrate specificity test revealed that rSl-β-fruct hydrolyzes sucrose and raffinose, but not melibiose or maltose, thereby confirming invertase activity. The pH curve revealed greatest activity at pH 5.0, demonstrating rSl-β-fruct to be an acidic β-fructofuranosidase. Quantitative PCR (qRT-PCR) analyses indicated that the production of mRNA only occurs in the midgut and reaches the greatest expression level in 30-day-old larvae, which is the expected pattern for digestive enzymes. Chromatography of glycosidases from S. levis midguts showed two enzymes acting as β-fructofuranosidase, indicating the presence of a

  17. Sodium Chloride and Water Transport in the Medullary Thick Ascending Limb of Henle. EVIDENCE FOR ACTIVE CHLORIDE TRANSPORT

    PubMed Central

    Rocha, Antonino S.; Kokko, Juha P.

    1973-01-01

    Transport of NaCl and water was examined in the rabbit medullary thick ascending limb of Henle (ALH) by perfusing isolated segments of these nephrons in vitro. Osmotic water permeability was evaluated by perfusing tubules against imposed osmotic gradients. In these experiments the net transport of fluid remained at zero when segments of thick ALH were perfused with isotonic ultrafiltrate in a bath of rabbit serum in which the serum osmolality was increased by the addition of either 239±8 mosmol/liter of raffinose or 232±17 mosmol of NaCl indicating that the thick ascending limb of Henle is impermeant to osmotic flow of water. When these tubules were perfused at slow rates with isosmolal ultrafiltrate of same rabbit serum as used for the bath, the effluent osmolality was consistently lowered to concentrations less than the perfusate and the bath. That this decrease in collected fluid osmolality represented salt transport was demonstrated in a separate set of experiments in which it was shown that the sodium and chloride concentrations decreased to 0.79±0.02 and 0.77±0.02 respectively when compared with the perfusion fluid concentrations. In each instance the simultaneously determined transtubular potential difference (PD) revealed the lumen to be positive with the magnitude dependent on the perfusion rate. At flow rates above 2 nl·min-1, the mean transtubular PD was stable and equal to 6.70±0.34 mv. At stop-flow conditions this PD became more positive. Ouabain and cooling reversibly decreased the magnitude of this PD. The transtubular PD remained positive, 3.3±0.2 mV, when complete substitution of Na by choline was carried out in both the perfusion fluid and the bathing media. These results are interpreted to indicate that the active transport process is primarily an electrogenic chloride mechanism. The isotopic permeability coefficient for Na was 6.27±0.38 × 10-5 cm·s-1 indicating that the thick ALH is approximately as permeable to Na as the proximal

  18. Trabulsiella guamensis, a new genus and species of the family Enterobacteriaceae that resembles Salmonella subgroups 4 and 5.

    PubMed

    McWhorter, A C; Haddock, R L; Nocon, F A; Steigerwalt, A G; Brenner, D J; Aleksić, S; Bockemühl, J; Farmer, J J

    1991-07-01

    , alpha-methyl-D-glucoside, raffinose, and sucrose. There were delayed positive reactions for gelatin liquefaction (22 degrees C), which was positive at 12 to 23 days, esculin hydrolysis (13% positive at day 1, 50% positive at 7 days), lactose fermentation (13% positive at 3 to 7 days, 100% positive at 8 to 10 days), glycerol fermentation (88% positive at 7 days), and salicin fermentation (13% positive at day 1, 88% positive at 7 days). All strains were susceptible by the disk diffusion method to colistin, nalidixic acid, gentamicin, streptomycin, kanamycin, chloramphenicol, and trimethoprim-sulfamethoxazole, and most strains were susceptible to sulfadiazine (75% susceptible), tetracycline (88%), and carbenicillin (75%). The strains were resistant to penicillin, cephalothin, and ampicillin. The strains were isolated from vacuum cleaner dust (five strains), soil (one strain), and human feces (two strains). Although T. guamensis can occur in human diarrheal stools, there is no evidence that it actually causes diarrhea. Its main interest to clinical microbiologists may be its possible misidentification as a strain Salmonella.

  19. Key mechanisms of metabolic changes in mountain pine and larch under drought in the Swiss National Park

    NASA Astrophysics Data System (ADS)

    Churakova, Olga; Bigler, Christof; Bryukhanova, Marina; Siegwolf, Rolf

    2014-05-01

    intrinsic water use efficiency. Carbon isotopes ratios of specific sugar compounds such as sucrose, raffinose, fructose, ribose and pinitol in needle and wood samples will help to evaluate the impact of drought on the carbohydrate balance and carbon allocation. Analyzing the oxygen isotopic compositions of soil water, sap water (water in branches) and precipitation we will determine the role of the different water sources for tree growth under drought conditions. The proposed work will provide a unique opportunity to assess the survival potential of mountain pine and larch trees exposed to drought that will help to estimate risks of modern climatic changes and forest damage. This project was supported by Marie Heim-Vögtlin Programme PMPDP2_145507/1 granted to Olga Churakova (Sidorova). References: Allen CD et al. (2010) A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests Forest Ecology and Management 259:660-684 Bigler C, Rigling A (2013) Precision and accuracy of tree-ring-based death dates of mountain pines in the Swiss National Park. Trees - Structure and Function. 27: 1703-1712 CH 2011 Swiss Climate Change Scenarios CH2011. In. C2SM, MeteoSwiss, ETH, NCCR Climate, and OcCC, p 88