Evidence for formation of heterooligosaccharides by Lactobacillus sanfranciscensis during growth in wheat sourdough

Lactobacillus sanfranciscensis is a key organism of the lactic microflora in traditional and industrial sourdough fermentations. In this paper we provide evidence for the formation of heterooligosaccharides (HeOS) by L. sanfranciscensis during growth in sourdough. To identify the HeOS based on HPAEC-PAD analysis, HeOS standards were synthesized by enzymatic reactions with L. sanfranciscensis levansucrase in a chemically defined system in the presence of raffinose, maltotriose, maltose, xylose, or arabinose as acceptor carbohydrates. The oligosaccharides known to originate from the corresponding acceptor reactions, 1(F)-beta-fructosylraffinose, 1(F)-beta-fructofuranosylmaltotriose, erlose (1(F)-beta-fructofuranosylmaltose), xylsucrose, 1(F)-beta-fructosylxylsucrose, and arabsucrose, were identified by HPAEC-PAD. Evidence for the formation of further tri-, tetra-, and pentasaccharides was provided. Wheat doughs with sucrose were fermented with L. sanfranciscesis TMW 1.392 or the isogenic, levansucrase-negative strain TMW 1.392Deltalev, and the analysis of dough extracts or invertase-treated dough extracts provided evidence for the formation of arabsucrose and erlose in sourdough in addition to 1-kestose and nystose.     

植物乳杆菌抑制黄曲霉活性代谢物的初步研究

植物乳杆菌（Lactobacillus plantarum）能够抑制黄曲霉生长,但起主要抑菌作用的物质尚未明确。该研究采用非靶向代谢组学技术比较分析了8株抑菌活性较好（S组）和8株抑菌活性较差（W组）的L. plantarum发酵上清液。结果显示,两组L. plantarum发酵上清液的代谢组存在显著差异（P<0.05）。通过数据库比对鉴定得到咪唑乙酸、酪氨酸等30个显著差异代谢物（P<0.05）,其中有机酸、脂肪酸等酸性物质较多为22个。通过与已报道的乳酸菌产生的抗真菌物质相比较,找到十八烷酸、吲哚乙酸等结构一致或结构类似物,表明上清液中酸性物质起主要的抑菌作用,且其抑菌活性依赖于低 pH 值的酸性环境。在L. plantarum产生的主要有机酸中,乳酸、乙酸、丙酸的抑菌活性良好,其抑制黄曲霉活性从大到小依次为丙酸、乙酸、乳酸。当乙酸浓度为2.64g/L、丙酸浓度为1.76 g/L时,可完全抑制浓度为106个/mL的黄曲霉孢子生长。综合表明,植物乳杆菌代谢物中有机酸和脂肪酸为主要抑菌物质,且抑菌活性随酸性物质浓度增大而增强。     

Characterization of the p-coumaric acid decarboxylase from Lactobacillus plantarum CECT 748(T)

It was previously reported that cell cultures from Lactobacillus plantarum CECT 748 (T) were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748 (T). Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748 (T) is a heat-labile enzyme, showing optimal activity at 22 degrees C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher K(M) value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry.     

植物乳杆菌对苏尼特羊肠道菌群、血浆代谢物及肉品质的影响

为了研究植物乳杆菌对苏尼特羊肠道菌群、血浆代谢物和肉品质的影响及其作用机理,选取三月龄苏尼特羊为试验对象,随机分为2组:对照组(C组,基础日粮)和植物乳杆菌组(R组,基础日粮、活菌数为3×1010cfu/g植物乳杆菌),进行为期90 d的饲喂试验.屠宰后取其肠道内容物及背最长肌,利用高通量测序技术、代谢组学液相-色谱联...     

Wine volatile and amino acid composition after malolactic fermentation: effect of Oenococcus oeni and Lactobacillus plantarum starter cultures

Red wine amino acids and volatile compounds were analyzed before and after malolactic fermentation carried out by four different starter cultures of the species Oenococcus oeni and Lactobacillus plantarum. The purpose of this study was to determine whether differences can be attributed to the lactic acid bacteria strain used in this important step of the wine-making process. The malolactic cultures selected for this study were indigenous wine lactic acid bacteria strains. The data were evaluated using different multivariate analysis techniques. Results showed different malolactic behaviors for O. oeni and L. plantarum and significant metabolic differences between both species. A degree of diversity was found within each lactic acid bacteria group, since wines presented specific characteristics depending on the lactic acid bacteria strain used. In all cases, malolactic fermentation seemed to modify the amino acid and volatile composition of the wine.     

Comparative survey in Lactobacillus plantarum of the growth and metabolism of arginine and citrulline in different media

Arginine deiminase activity increased in the presence of arginine in Lactobacillus plantarum strains N4 and N8 isolated from orange. The influence of citrulline and ornithine on arginine deiminase and ornithine transcarbamylase activities was strain-dependent. The growth and arginine and citrulline metabolism of L. plantarum were studied in the presence of tomato juice. Its addition enhances the growth in both strains. The specific amino acids utilization was inversely proportional to the initial glucose concentration. Arginine and citrulline addition to basal medium exerted a stimulatory effect on the growth of N4 strain, and this effect was observed only with citrulline in strain N8. The magnitude of this effect was lower in the presence of tomato juice.     

Contribution of the microbial and meat endogenous enzymes to the free amino acid and amine contents of dry fermented sausages.

The role of the starter culture and meat endogenous enzymes on the free amino acid and amine contents of dry fermented sausages was studied. Five batches of sausages were prepared. The control batch was manufactured with aseptic ingredients without microbial inoculation. The other four experimental batches were manufactured with aseptic ingredients inoculated with Lactobacillus plantarum 4045 or Micrococcus-12 or L. plantarum 4045 and Micrococcus-12 or L. plantarum 4045 and Staphylococcus sp. Their effects on pH, a(w), myofibrillar proteins, and free amino acid and amine contents were studied. Sausages inoculated only with L. plantarum 4045 or with this starter combined with a Micrococcaceae had the lowest pH as a result of carbohydrate fermentation. In all batches similar patterns were observed for myofibrillar proteins and free amino acids which could indicate that meat endogenous proteases play an important role in proteolytic phenomena. No changes were observed in the amine fraction, indicating that the strains used as starter cultures did not show amino acid decarboxylase activity.     

Glucan and fructan production by sourdough Weissella cibaria and Lactobacillus plantarum

After a large screening on sourdough lactic acid bacteria, exopolysaccharide (EPS)-forming strains of Weissella cibaria, Lactobacillus plantarum, and Pediococcus pentosaceus were selected. After 6 days of incubation at 30 degrees C, the synthesis of EPS in MRS-based broth ranged from 5.54 to 7.88 mg mL-1. EPS had an apparent molecular mass of ca. 104 Da. As shown by carbohydrate consumption, the synthesis of EPS was found from sucrose only. Two types of homopolysaccharides were synthesized: glucans simultaneously with growth and fructans after 1 day of incubation. Two protein bands of ca. 180-200 kDa were in situ detected on SDS-PAGE gels incubated with sucrose. PCR products of ca. 220 bp were found for L. plantarum PL9 (100% of identity to putative priming glycosyltransferase of L. plantarum WCFS1) and W. cibaria WC4 (80% of identity to putative glycosyltransferase, epsD, of Bacillus cereus G9241) by using hybrid primers for the priming gtf genes. Degenerated primers DexreuR and DexreuV showed a unique PCR product, and the predicted amino acid sequences were identical for W. cibaria WC4 and L. plantarum PL9. The sequence had similarity with polysaccharide biosynthesis glycosyltransferases. W. cibaria WC4 or L. plantarum LP9 synthesized ca. 2.5 g kg-1 EPS during sourdough fermentation with sucrose added. Compared to the sourdough started with an EPS-negative strain, the sourdough started with W. cibaria WC4 or L. plantarum LP9 increased the viscosity, and the resulting bread had higher specific volume and lower firmness. The synthesis of EPS by selected sourdough lactic acid bacteria could be considered as a useful tool to replace the additives for improving the textural properties of baked goods.     

Capability of Lactobacillus plantarum IFPL935 to catabolize flavan-3-ol compounds and complex phenolic extracts

Lactobacillus plantarum IFPL935 was incubated with individual monomeric flavan-3-ols and dimeric A- and B-type procyanidins to identify new metabolites and to determine the effect of compound structural features on bacterial growth and catabolism. Complex extracts rich in A-type proanthocyanidins and phenolic acids from cranberry were also tested. The results showed that L. plantarum IFPL935 exhibited higher resistance to nongalloylated monomeric flavan-3-ols, A-type dimeric procyanidins, and cranberry extract than to (-)-epicatechin-3-O-gallate and B-type dimeric procyanidins. Despite these findings, the strain was capable of rapidly degrading (-)-epicatechin-3-O-gallate, but not A- or B-type dimeric procyanidins. However, it was able to produce large changes in the phenolic profile of the cranberry extract mainly due to the catabolism of hydroxycinnamic and hydroxybenzoic acids. Of most relevance was the fact that L. plantarum IFPL935 cleaved the heterocyclic ring of monomeric flavan-3-ols, giving rise to 1-(3',4'-dihydroxyphenyl)-3-(2″,4″,6″-trihydroxyphenyl)propan-2-ol, activity exhibited by only a few human intestinal bacteria.     

Sucrose metabolism and exopolysaccharide production in wheat and rye sourdoughs by Lactobacillus sanfranciscensis.

The exopolysaccharide (EPS) produced from sucrose by Lactobacillus sanfranciscensis LTH2590 is predominantly composed of fructose. EPS production during sourdough fermentation has the potential to affect rheological properties of the dough as well as the volume, texture, and keepability of bread. Its in situ production by L. sanfranciscensis LTH2590 was demonstrated during sourdough fermentation after the hydrolysis of water soluble polysaccharides. In wheat and rye doughs with sucrose addition the concentration of fructose in the hydrolysate of polysaccharides was significantly higher than that in the hydrolysate of control doughs or doughs without sucrose addition. EPS production by L. sanfranciscensis in wheat doughs was confirmed by the determination of delta (13)C values of water soluble polysaccharides after the addition of naturally labeled sucrose, originating from C(3)- and C(4)-plants. In rye doughs, evidence for EPS production with the isotope technique could be demonstrated only by the determination of delta (13)C values of fructose from water soluble polysaccharides. In addition to EPS formation from sucrose, sucrose hydrolysis by L. sanfranciscensis in wheat and rye sourdoughs resulted in an increase of mannitol and acetate concentrations and in accumulation of glucose. It was furthermore observed that flour arabinoxylans were solublized during the fermentation.     

Proteolysis and bioconversion of cereal proteins to glutamate and γ-Aminobutyrate (GABA) in Rye malt sourdoughs

This study aimed to achieve the conversion of cereal proteins to the alternative end products glutamate or γ-aminobutyrate (GABA). Rye malt, fungal proteases, and lactobacilli were employed to convert wheat gluten or barley proteins. Glutamate and GABA formations were strain-dependent. Lactobacillus reuteri TMW1.106 and Lactobacillus rossiae 34J accumulated glutamate; L. reuteri LTH5448 and LTH5795 accumulated GABA. Glutamate and GABA accumulation by L. reuteri TMW1.106 and LTH5448 increased throughout fermentation time over 96 h, respectively. Peptides rather than amino acids were the main products of proteolysis in all doughs, and barley proteins were more resistant to degradation by rye malt proteases than wheat gluten. However, addition of fungal protease resulted in comparable degradation of both substrates. Glutamate and GABA accumulated to concentrations up to 63 and 90 mmol kg(-1) DM, respectively. Glutamate levels obtained through bioconversion of cereal proteins enable the use of hydrolyzed cereal protein as condiment.     

High-level expression of recombinant beta-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a Sakacin P-based expression system

This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( S?rvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.     

Contribution of Sourdough Lactobacilli,Yeast, and Cereal Enzymes to the Generation of Amino Acids in Dough Relevant for Bread Flavor

The amino acid release was determined in wheat doughs supplied with salt, acid, dithiothreitol, or starter cultures to evaluate the relevance of the amino acid concentration on bread flavor. Wheat flour proteinases almost linearly released amino acids and the highest activity of wheat flour proteinases was found in acidified and reduced doughs. The effects of starter cultures on amino acid concentrations depended on their composition. Yeasts exhibited a high demand for amino acids, however, the total amino acid concentrations were not markedly affected by lactic acid bacteria. The individual amino acid contents were determined by the pH during fermentation and microbial metabolism. The formation of proline was favored by values higher than pH 5.5, whereas release of phenylalanine, leucine and cysteine mainly occurred at lower pH. Ornithine was found only in doughs fermented with Lactobacillus pontis. To determine effects of the amino acid concentration on bread aroma, fermented doughs were evaluated in baking experiments. An increased intensity of bread flavor was obtained by preferments prepared with lactic acid bacteria. The roasty note of wheat bread crust could be markedly enhanced by L. pontis. This results support the assumption that flavor of wheat bread is enhanced by increasing the concentration of free amino acids and especially ornithine in dough.     

Influence and Interactions of Processing Conditions and Starter Culture on Formation of Acids,Volatile Compounds,and Amino Acids in Wheat Sourdoughs

The aim of this work was to study the influence of process parameters and the starter culture on the characteristics of wheat sourdough by using response surface methodology. Influence of fermentation temperature (16–32°C), ash content of flour (0.6–1.8%), and fermentation time (6–20 hr) were considered as independent factors and their effects were studied in sourdough fermented with Lactobacillus plantarum, L. brevis, Saccharomyces cerevisiae, or with a combination of yeast and lactic acid bacteria. Formation of acidity, free amino acids, and volatile compounds were considered the main responses. A possibility to enhance formation of potential flavor compounds and precursors without excessive acidity formation in wheat sourdoughs was established. The total amount of amino acids increased by 25–50%, depending on the strain and fermentation conditions. The total amount of volatile compounds increased seven‐ to 100‐fold, depending on the strain and fermentation conditions. Sourdough started with S. cerevisiae was an effective way to optimize the amount of volatile compounds without excessive acidity formation in appropriate processing conditions. Ash content of flour and fermentation time were the most significant factors to modify metabolic activity of wheat sourdoughs. Frequent interactions between the studied factors were observed on the formation of acidity, amino acids, and volatile compounds with most of the strains studied. Possibility to improve current industrial fermentation processes and control flavor attributes of breads by using optimized sourdough was established.     

Cucumber performance is improved by inoculation with plant growth-promoting microorganisms

We investigated the effects of inoculation of Rhodobacter sphaeroides, Lactobacillus plantarum, and Saccharomyces cerevisiae on cucumber plant growth promotion and on the contents of plant hormones, amino acids, and mineral nutrients. We showed that treatment with all three bio-inoculants significantly increased the shoot length, root length, shoot fresh weight, shoot dry weight, and chlorophyll content, via secretion of indole acetic acid and/or organic acids. Inoculation with R. sphaeroides had more favorable effect on plant growth than did inoculation with L. plantarum or S. cerevisiae, by significantly enhancing the gibberellin and reducing the abscisic acid contents. The results of amino acid analysis revealed that inoculation with R. sphaeroides, L. plantarum, and S. cerevisiae generally increased the contents of 17 amino acids, namely, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, and proline. With the exception of cysteine, all these amino acids were present in higher concentrations in plants inoculated with R. sphaeroides than in control plants or in plants inoculated with L. plantarum and S. cerevisiae. Furthermore, inoculation with R. sphaeroides significantly increased the calcium, potassium, magnesium, and phosphate contents. Our results suggest that the use of R. sphaeroides, L. plantarum, and S. cerevisiae in agricultural fields can improve plant growth. Moreover, inoculation of cucumber plants with R. sphaeroides regulates plant functional metabolites, thereby promoting plant growth.     

Combined effect of sourdough lactic acid bacteria and additives on bread firmness and staling

The effect of various sourdoughs and additives on bread firmness and staling was studied. Compared to the bread produced with Saccharomyces cerevisiae 141, the chemical acidification of dough fermented by S. cerevisiae 141 or the use of sourdoughs increased the volume of the breads. Only sourdough fermentation was effective in delaying starch retrogradation. The effect depended on the level of acidification and on the lactic acid bacteria strain. The effect of sourdough made of S. cerevisiae 141-Lactobacillus sanfranciscensis 57-Lactobacillus plantarum 13 was improved when fungal alpha-amylase or amylolytic strains such as L. amylovorus CNBL1008 or engineered L. sanfranciscensis CB1 Amy were added. When pentosans or pentosans, endoxylanase enzyme, and L. hilgardii S32 were added to the same sourdough, a greater delay of the bread firmness and staling was found. When pentosans were in part hydrolyzed by the endoxylanase enzyme, the bread also had the highest titratable acidity, due to the fermentation of pentoses by L. hilgardii S32. The addition of the bacterial protease to the sourdough increased the bread firmness and staling.     

Immunoreactivity and amino acid content of fermented soybean products

Food allergy has become a public health problem that continues to challenge both the public and the food industry. The objective of this research was the detection and quantification of the major human allergenic soy proteins and to study the reduction in immunoreactivity and improvement of amino acid content after fermentation of soybean flour. Fermentation was carried out in the solid state of cracked seeds inoculated with Aspergillus oryzae, Rhizopus oryzae, and Bacillus subtilis and in the liquid state of milled soybean flours fermented naturally by microorganisms present only in the seeds or by inoculation with Lactobacillus plantarum. ELISA and Western blot were used to quantify IgE antibody response, and HPLC was used to identify and quantify total amino acids. L. plantarum fermented soy flour showed the highest reduction in IgE immunoreactivity (96-99%) depending upon the sensitivity of the plasma used. Among the solid fermented products, the lowest reduction in immunoreactivity was obtained when mold strains, R. oryzae and A. oryzae, were used (66 and 68%, respectively, for human plasma 97.5 kUA/L). Among the solid fermented products, those inoculated with B. subtilis yielded a 81 and 86% reduction in immunoreactivity against both human plasma 97.5 IgE kUA/L and human pooled plasma samples, respectively. When soybean was subjected to liquid fermentation, most of the total amino acids increased significantly ( p < or = 0.05). In solid fermentation with R. oryzae, only Ala and Thr content improved. Fermentation can decrease soy immunoreactivity, and there is potential of developing nutritious hypoallergenic soy products.     

A food-grade system for inducible gene expression in Lactobacillus plantarum using an alanine racemase-encoding selection marker

Food-grade gene expression systems for lactic acid bacteria are useful for applications in the food industry. We describe a new food-grade host/vector system for Lactobacillus plantarum based on pSIP expression vectors and the use of the homologous alanine racemase gene (alr) as selection marker. A new series of expression vectors were constructed by exchanging the erythromycin resistance gene (erm) in pSIP vectors by the L. plantarum WCFS1 alr gene. The vectors were applied for the overexpression of β-galactosidase genes from L. reuteri L103 and L. plantarum WCFS1 in an alr deletion mutant of L. plantarum WCFS1. The expression levels obtained in this way, i.e. without the use of antibiotics, were comparable to the levels obtained with the conventional system based on selection for erythromycin resistance. The new system is suitable for the production of ingredients and additives for the food industry.     

Kinetics of interaction of vanillin with amino acids and peptides in model systems

Model systems were used to study the reaction kinetics of vanillin and pentalysine, lysine, glutathione, cysteine, aspartame, or phenylalanine (molar ratio 1:1) in phosphate buffer. The buffer pH was adjusted to the pK(a)(2) of the available alpha-amino group of each amino acid or peptide. Reductions of vanillin followed first-order kinetics at 55, 65, and 75 degrees C in the presence of each of the amino acids or peptides used. The reaction rates were accelerated as the temperature increased. The rate constants were highest for pentalysine followed by lysine, phenylalanine, glutathione/cysteine, and aspartame. The reduction of phenylalanine followed first-order kinetics, whereas the formation of its reaction product followed zero-order kinetics. The activation energy (E(a)) for the reaction ranged from 5.6 to 14.5 kcal/mol.