Optimizing Levilactobacillus brevis NPS-QW 145 Fermentation for Gamma-Aminobutyric Acid (GABA) Production in Soybean Sprout Yogurt-like Product.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid with various physiological functions. Levilactobacillus brevis NPS-QW 145 strains active in GABA catabolism and anabolism can be used as a microbial platform for GABA production. Soybean sprouts can be treated as a fermentation substrate for making functional products. This study demonstrated the benefits of using soybean sprouts as a medium to produce GABA by Levilactobacillus brevis NPS-QW 145 when monosodium glutamate (MSG) is the substrate. Based on this method, a GABA yield of up to 2.302 g L-1 was obtained with a soybean germination time of one day and fermentation of 48 h with bacteria using 10 g L-1 glucose according to the response surface methodology. Research revealed a powerful technique for producing GABA by fermentation with Levilactobacillus brevis NPS-QW 145 in foods and is expected to be widely used as a nutritional supplement for consumers.

Cysteine protected cells from H2O2-induced damage and promoted long-chain fatty acids synthesis in vivo to improve γ-aminobutyric acid production in Levilactobacillus brevis.

Levilactobacillus brevis NPS-QW-145 isolated from kimchi is deficient in glutamate dehydrogenase-encoding gene (gdhA) to form glutamate, hence it required exogenous supplementation of glutamate/monosodium glutamate (MSG) for decarboxylation reaction to produce γ-aminobutyric acid (GABA). However, GABA conversion rate from MSG was relatively low. The individual effect of 20 amino acids on regulating GABA biosynthesis was investigated. Cysteine was selected to significantly improve GABA production from MSG. It was found that Lb. brevis was capable of producing H2O2, cysteine protected Lb. brevis against H2O2-induced oxidative damage to increase cell viability for the enhancement of GABA production. Moreover, cysteine promoted glucose consumption to produce acetyl-CoA for synthesizing long-chain fatty acids to significantly up-regulate GABA biosynthesis. These findings deciphered antioxidative capability of cysteine in Lb. brevis 145 and provided a theoretical basis for fatty acids synthesis-mediated GABA synthesis in Lb. brevis 145, and possibly in other lactic acid bacteria.

Assessment of Yeasts as Potential Probiotics: A Review of Gastrointestinal Tract Conditions and Investigation Methods.

Probiotics are microorganisms (including bacteria, yeasts and moulds) that confer various health benefits to the host, when consumed in sufficient amounts. Food products containing probiotics, called functional foods, have several health-promoting and therapeutic benefits. The significant role of yeasts in producing functional foods with promoted health benefits is well documented. Hence, there is considerable interest in isolating new yeasts as potential probiotics. Survival in the gastrointestinal tract (GIT), salt tolerance and adherence to epithelial cells are preconditions to classify such microorganisms as probiotics. Clear understanding of how yeasts can overcome GIT and salt stresses and the conditions that support yeasts to grow under such conditions is paramount for identifying, characterising and selecting probiotic yeast strains. This study elaborated the adaptations and mechanisms underlying the survival of probiotic yeasts under GIT and salt stresses. This study also discussed the capability of yeasts to adhere to epithelial cells (hydrophobicity and autoaggregation) and shed light on in vitro methods used to assess the probiotic characteristics of newly isolated yeasts.

Functional oligosaccharide fermentation in the gut: Improving intestinal health and its determinant factors-A review.

The human intestine is characterized by an abundance of nutrients and a complex microbiota that make crucial contributions to overall health. These nutrients facilitate the adaptation of resident commensals to extreme environments and the development of a robust ecological network in host species. Long-term deprivation of microbiota-accessible carbohydrates (MACs) in the gut results in a loss of bacterial diversity, disruption of intestinal barrier function, and inflammatory diseases. Functional oligosaccharides are excellent MACs possessing important prebiotic properties for intestinal health through their fermentation in the gut. Its mechanism of action is predominantly attributed to acting as carbon sources for specific probiotics, promoting short-chain fatty acids production, and regulating the gut microbiota. In this review, we describe the source and structural characteristics of functional oligosaccharides, provide a framework for strategies to improve intestinal health by oligosaccharide fermentation and discuss structural determinants influencing the functional properties of oligosaccharides.

Role of cysteine in the improvement of γ-aminobutyric acid production by nonproteolytic Levilactobacillus brevis in coculture with Streptococcus thermophilus.

Previous research has showed that nonproteolytic Levilactobacillus brevis 145 (L) in coculture with Streptococcus thermophilus 1275 (S), not Lactobacillus delbrueckii ssp. bulgaricus (Lbu), was able to produce γ-aminobutyric acid (GABA) during milk fermentation in the presence of monosodium glutamate (MSG). It was assumed that differences of casein hydrolysis patterns between Strep. thermophilus 1275 and L. bulgaricus caused the phenomenon. Moreover, the GABA content was low and residual MSG was high in SL-fermented milk. In our research, comparison of peptide profiles determined by liquid chromatography/tandem mass spectrometry showed that αS2-casein, ß-casein, and κ-casein degradation by L. bulgaricus and Strep. thermophilus varied. Importantly, the peptide number in the L and Lbu coculture group increased compared with the Lbu monoculture group, whereas the peptide number in the SL coculture group decreased in comparison with S monoculture group, suggesting that L. bulgaricus was not able to provide peptides for the growth of Lb. brevis 145. Furthermore, we found that after supplementation with cysteine (50 mg/L) during milk fermentation by SL, 10 g/L MSG was converted into 4.8 g/L GABA with a minimum level of residual MSG, viable cell counts of Lb. brevis and lactic acid production were increased, and the casein hydrolysis pattern was not influenced. Moreover, sulfhydryl group-containing chemicals including cystine, reduced glutathione, and oxidized glutathione showed effects similar to that of cysteine in improving GABA production. Finally, when L. bulgaricus YIB2 was combined with SL, supplementation of cysteine was also able to significantly improve GABA production.

L-citrulline enriched fermented milk with Lactobacillus helveticus attenuates dextran sulfate sodium (DSS) induced colitis in mice.

Inflammatory bowel disease (IBD) is a group of chronic inflammatory gastrointestinal diseases that causes worldwide suffering. L. helveticus is a probiotic that can enhance intestinal barrier function via alleviation of excessive inflammatory response. Citrulline, a functional amino acid, has been reported to stimulate muscle synthesis and to function with a prebiotic-like action with certain Lactobacillus strains. The aim of this study was to investigate the potential synergistic effect of combining L. helveticus and citrulline on protection against damage induced by dextran sulfate sodium (DSS) in a mouse model. 6-week-old male C57BL/6J mice were fed with DSS water and randomly divided for administering with different milk treatments: 1) plain milk (control or DSS control), 2) 1% (w/v) citrulline enriched milk (Cit_milk), 3) milk fermented with L. helveticus (LHFM) and 4) DSS+milk fermented with L. helveticus with 1% (w/v) citrulline (Cit_LHFM). The treatment effects on the survival and macroscopic and microscopic signs were examined. All treatments presented different degrees of protective effects on attenuating the damages induced by DSS. All treatments reduced the body weight loss, disease activity index (DAI), histological scores, pro-inflammatory cytokine expression (IL-6, TNF-α and IFN-γ) and production (IL-4) (all P <0.05) and the tight junction (TJ) protein (zonula occluden-1 (ZO-1) expression. LHFM and Cit_LHFM improved survival rate (both at P<0.05). Particularly, Cit_LHFM showed greater effects on protecting the damages induced by DSS, especially in ameliorating colonic permeability, TJ protein (ZO-1, occludin and claudin-1) expression and distribution as well as in reducing IL-4 and IL-17 expression (all P <0.05). Our findings suggested that the combination of and citrulline had significant synergistic effect on protecting against injury from DSS-induced colitis. Therefore, citrulline enriched L. helveticus fermented milk is suggested to be a potential therapy for treating IBD.

Potential Probiotic Pediococcus pentosaceus M41 Modulates Its Proteome Differentially for Tolerances Against Heat, Cold, Acid, and Bile Stresses.

Probiotics containing functional food confer health benefits in addition to their nutritional properties. In this study, we have evaluated the differential proteomic responses of a potential novel probiotic Pediococcus pentosaceus M41 under heat, cold, acid, and bile stress conditions. We identified stress response proteins that could provide tolerances against these stresses and could be used as probiotic markers for evaluating stress tolerance. Pediococcus pentosaceus M41 was exposed for 2 h to each condition: 50°C (heat stress), 4°C (cold stress), pH 3.0 (acid stress) and 0.05% bile (bile stress). Proteomic analysis was carried out using 2D-IEF SDS PAGE and LC-MS/MS. Out of 60 identified proteins, 14 upregulated and 6 downregulated proteins were common among all the stress conditions. These proteins were involved in different biological functions such as translation-related proteins, carbohydrate metabolism (phosphoenolpyruvate phosphotransferase), histidine biosynthesis (imidazole glycerol phosphate synthase) and cell wall synthesis (tyrosine-protein kinase CapB). Proteins such as polysaccharide deacetylase, lactate oxidase, transcription repressor NrdR, dihydroxyacetone kinase were upregulated under three out of the four stress conditions. The differential expression of these proteins might be responsible for tolerance and protection of P. pentosaceus M41 against different stress conditions.

Exopolysaccharides as Antimicrobial Agents: Mechanism and Spectrum of Activity.

Exopolysaccharides (EPSs) are metabolites synthesized and excreted by a variety of microorganisms, including lactic acid bacteria (LAB). EPS serve several biological functions such as interactions between bacteria and their environments, protection against hostile conditions including dehydration, the alleviation of the action of toxic compounds (bile salts, hydrolyzing enzymes, lysozyme, gastric, and pancreatic enzymes, metal ions, antibiotics), and stresses (changing pH, osmolarity), and evasion of the immune response and phage attack. Bacterial EPSs are considered valuable by the food, pharmaceutical, and nutraceutical industries, owing to their health-promoting benefits and rheological impacts. Numerous studies have reported the unusual antimicrobial activities of various EPS against a wide variety of pathogenic microbes (bacteria, virus, and fungi). This review aims to provide a comprehensive examination of the in vitro and in vivo antimicrobial activities of different EPSs, mainly against foodborne bacterial, fungal, and viral pathogens. The mechanism of EPS action against these pathogens as well as the methods used to measure antimicrobial activities are critically reviewed.

Invited review: Characterization of new probiotics from dairy and nondairy products-Insights into acid tolerance, bile metabolism and tolerance, and adhesion capability.

The selection of potential probiotic strains that possess the physiological capacity of performing successfully in the gastrointestinal tract (GIT) is a critical challenge. Probiotic microorganisms must tolerate the deleterious effects of various stresses to survive passage and function in the human GIT. Adhesion to the intestinal mucosa is also an important aspect. Recently, numerous studies have been performed concerning the selection and evaluation of novel probiotic microorganisms, mainly probiotic bacteria isolated from dairy and nondairy products. Therefore, it would be crucial to critically review the assessment methods employed to select the potential probiotics. This article aims to review and discuss the recent approaches, methods used for the selection, and outcomes of the evaluation of novel probiotic strains with the main purpose of supporting future probiotic microbial assessment studies. The findings and approaches used for assessing acid tolerance, bile metabolism and tolerance, and adhesion capability are the focus of this review. In addition, probiotic bile deconjugation and bile salt hydrolysis are explored. The selection of a new probiotic strain has mainly been based on the in vitro tolerance of physiologically related stresses including low pH and bile, to ensure that the potential probiotic microorganism can survive the harsh conditions of the GIT. However, the varied experimental conditions used in these studies (different types of media, bile, pH, and incubation time) hamper the comparison of the results of these investigations. Therefore, standardization of experimental conditions for characterizing and selecting probiotics is warranted.

Starch properties of high and low amylose proso millet (Panicum miliaceum L.) genotypes are differentially affected by varying salt and pH.

Variation in salt content and in pH are common in starch-based foods and can affect starch properties and final product texture. Fifteen accessions of proso millet starch with diverse amylose content were selected to investigate single factor and interaction effects of pH and NaCl on thermal, pasting, and textural properties. Pasting properties and gelatinization temperatures were markedly altered by salt addition. Changes in pH only had substantial effects on ΔH, but other properties were generally stable under different pH conditions. From two-way ANOVA, interactive effects of salt and pH were found to affect ΔH. The response of starch of different genotypes in terms of thermal and pasting properties differed under the same pH and salinity conditions. The reason is likely that ions in the starch-water system performed the roles of both reducing water activity and building of hydrogen bonds, which will have opposite effects on starch gelatinization.

Updates on understanding of probiotic lactic acid bacteria responses to environmental stresses and highlights on proteomic analyses.

Probiotics are defined as live microorganisms that improve the health of the host when administered in adequate quantities. Nonetheless, probiotics encounter extreme environmental conditions during food processing or along the gastrointestinal tract. This review discusses different environmental stresses that affect probiotics during food preparation, storage, and along the alimentary canal, including high temperature, low temperature, low and alkaline pH, oxidative stress, high hydrostatic pressure, osmotic pressure, and starvation. The understanding of how probiotics deal with environmental stress and thrive provides useful information to guide the selection of the strains with enhanced performance in specific situations, in food processing or during gastrointestinal transit. In most cases, multiple biological functions are affected upon exposure of the cell to environmental stress. Sensing of sublethal environmental stress can allow for adaptation processes to occur, which can include alterations in the expression of specific proteins.

Comparative Peptidomic and Metatranscriptomic Analyses Reveal Improved Gamma-Amino Butyric Acid Production Machinery in Levilactobacillus brevis Strain NPS-QW 145 Cocultured with Streptococcus thermophilus Strain ASCC1275 during Milk Fermentation.

The high-gamma-amino butyric acid (GABA)-producing bacterium Levilactobacillus brevis strain NPS-QW 145, along with Streptococcus thermophilus (one of the two starter bacteria used to make yogurt for its proteolytic activity), enhances GABA production in milk. However, a mechanistic understanding of how Levilactobacillus brevis cooperates with S. thermophilus to stimulate GABA production has been lacking. Comparative peptidomic and metatranscriptomic analyses were carried out to unravel the casein and lactose utilization patterns during milk fermentation with the coculture. We found that particular peptides hydrolyzed by S. thermophilus ASCC1275 were transported and biodegraded with peptidase in Lb. brevis 145 to meet the growth needs of the latter. In addition, amino acid synthesis and metabolism in Lb. brevis 145 were activated to further support its growth. Glucose, as a result of lactose hydrolysis by S. thermophilus 1275, but not available lactose in milk, was metabolized as the main carbon source by Lb. brevis 145 for ATP production. In the stationary phase, under acidic conditions due to the accumulation of lactic acid produced by S. thermophilus 1275, the expression of genes involved in pyridoxal phosphate (coenzyme of glutamic acid decarboxylase) metabolism and glutamic acid decarboxylase (Gad) in Lb. brevis 145 was induced for GABA production.SIGNIFICANCE A huge market for GABA-rich milk as a dietary therapy for the management of hypertension is anticipated. The novelty of this work lies in applying peptide profiles supported by metatranscriptomics to elucidate (i) the pattern of casein hydrolysis by S. thermophilus 1275, (ii) the supply of peptides and glucose by S. thermophilus 1275 to Lb. brevis 145, (iii) the transportation of peptides in Lb. brevis and the degradation of peptides by this organism, which was reported to be nonproteolytic, and (iv) GABA production by Lb. brevis 145 under acidic conditions. Based on the widely reported contribution of lactic acid bacteria (LAB) and GABA to human health, the elucidation of interactions between the two groups of bacterial communities in the production of GABA-rich milk is important for promoting the development of functional dairy food and may provide new insight into the development of industrial GABA production.

The potential application of probiotics and prebiotics for the prevention and treatment of COVID-19.

COVID-19 is a pandemic disease caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This new viral infection was first identified in China in December 2019, and it has subsequently spread globally. The lack of a vaccine or curative treatment for COVID-19 necessitates a focus on other strategies to prevent and treat the infection. Probiotics consist of single or mixed cultures of live microorganisms that can beneficially affect the host by maintaining the intestinal or lung microbiota that play a major role in human health. At present, good scientific evidence exists to support the ability of probiotics to boost human immunity, thereby preventing colonization by pathogens and reducing the incidence and severity of infections. Herein, we present clinical studies of the use of probiotic supplementation to prevent or treat respiratory tract infections. These data lead to promising benefits of probiotics in reducing the risk of COVID-19. Further studies should be conducted to assess the ability of probiotics to combat COVID-19.

Genomic Analysis for Antioxidant Property of Lactobacillus plantarum FLPL05 from Chinese Longevity People.

Antioxidant activity is one of the important probiotic characteristics for lactic acid bacteria including Lactobacillus plantarum, which is used for food fermentation or as a probiotic supplement. L. plantarum FLPL05 is a novel strain originally isolated from a healthy elderly individual of longevity. The organism has been demonstrated to exhibit high antioxidant property. However, there are limited genomic insights into the antioxidant properties of this organism. In this study, we performed whole-genome analysis regarding its antioxidant property. L. plantarum FLPL05 exhibited higher antioxidant activity compared with that of L. plantarum strains ATCC14917, ATCC8014, and WCFS1. The antioxidant capacity of L. plantarum FLPL05 was genetically linked to its antioxidant system, i.e., glutathione and thioredoxin involved in global regulation of defense against hydrogen peroxide challenge. L. plantarum FLPL05 was further examined for its antioxidant potential in D-Gal-induced aging mice and exhibited a significant increase in the activity of serum glutathione peroxidase (GSH-PX) and a decrease in the level of malondialdehyde (MDA). Moreover, our analyses exhibited a complete gene cluster including plnA, plnB, plnC, plnD, plnE, plnF, plnG, plnH, plnI, plnJ, plnK, plnM, plnN, plnO, plnP, plnQ, plnST, plnU, plnV, plnW, plnX, and plnY for production of bacteriocin. Our results suggest that L. plantarum FLPL05 could be a probiotic candidate.

Structural characterization of exopolysaccharide from Streptococcus thermophilus ASCC 1275.

In this study, we purified and characterized exopolysaccharide (EPS) produced by a high-EPS-producing dairy starter bacterium, Streptococcus thermophilus ASCC 1275. Crude EPS was extracted from S. thermophilus ASCC 1275 and partially purified using dialysis. Further purification and fractionation of exopolysaccharide was conducted using HPLC on a Superose 6 column (Cytiva/Global Life Sciences Solutions, Marlborough, MA). Glycosyl composition analysis, linkage analysis along with 1-dimensional and 2-dimensional nuclear magnetic resonance spectroscopy were performed to deduce the structure of EPS. Three fractions (F) obtained from gel permeation chromatography were termed F1 (2.6%), F2 (45.8%), and F3 (51.6%) with average molecular weights of approximately 511, 40, and 5 kDa, respectively. Monosaccharide composition analysis revealed the dominance of glucose, galactose, and mannose in all 3 fractions. Major linkages observed in F3 were terminal galactopyranosyl (t-Gal), 3-linked glucopyranosyl (3-Glc), 3-linked galactofuranosyl (3-Galf), and 3,6-linked glucopyranosyl (3,6-Glc) and major linkages present in F2 were 4-Glc (48 mol%), followed by terminal mannopyranosyl (t-Man), 2- + 3-linked mannopyranosyl (2-Man+3-Man), and 2,6-linked mannopyranosyl (2,6-Man; total ∼28 mol%). The 1-dimensional and 2-dimensional nuclear magnetic resonance spectroscopy revealed that F2 comprised mannans linked by (1→2) linkages and F3 consisted of linear chains of α-d-glucopyranosyl (α-d-Glcp), ß-d-glucopyranosyl (ß-d-Glcp), and ß-d-galactofuranosyl (ß-d-Galf) connected by (1→3) linkages; branching was through (1→6) linkage in F3. A possible structure of EPS in F2 and F3 was proposed.

Enterococcus hirae WEHI01 isolated from a healthy Chinese infant ameliorates the symptoms of type 2 diabetes by elevating the abundance of Lactobacillales in rats.

Enterococcus hirae WEHI01 is a potential probiotic strain isolated from a healthy Chinese infant. This strain has previously been characterized as having cholesterol-lowering potential and good dairy fermentation performance. In this study, we used rat models with obesity and type 2 diabetes mellitus (T2DM) induced by a high fat and sucrose diet and low-dose streptozotocin, respectively, and we evaluated the effect of E. hirae WEHI01 on glycolipid metabolism, glycolipid-related gene expression, organ histopathology, and intestinal flora changes in the 2 models. Our results showed that administration of 5.0 × 109 cfu of E. hirae WEHI01 for 4 wk decreased serum lipid levels and regulated glycolipid metabolism in the liver of obese rats. Following continuous administration of the same concentration of E. hirae WEHI01 to a T2DM rat model for another 5 wk, E. hirae WEHI01 improved glucose tolerance, recovered body weight loss, and led to significant decreases in tumor necrosis factor-α, IL-6, IL-10, and total bile acid in serum. We also found that E. hirae WEHI01 restored the morphology of the pancreas, kidney, and liver, and changed the composition of the gut microbiota (i.e., decreased the Shannon index, increased the Simpson index, and substantially increased the abundance of Lactobacillales). Combining the results for the obese model and the T2DM model, we speculated that beneficial effects of E. hirae WEHI01 on T2DM could be due to (1) a significant increase in PPARA expression and a tendency for increased CYP7A1 expression in the liver of obese rats, promoting the conversion of cholesterol into bile acid and reducing serum total bile acid levels in T2DM model rats; or (2) a change in gut microbial diversity, especially elevated Lactobacillales abundance, which reduced the total bile acid in T2DM model rats. These results demonstrated that E. hirae WEHI01 has the potential to ameliorate type 2 diabetes in rats and provide a promising rationale for further research into the prevention and treatment of T2DM.

Proteomic analysis reveals potential factors associated with enhanced EPS production in Streptococcus thermophilus ASCC 1275.

Streptococcus thermophilus ASCC 1275 has two chain length determining genes - epsC and epsD- in its eps gene cluster, and produces two times more EPS in sucrose medium than that in glucose and lactose. Hence, we investigated the influence of sugars (glucose, sucrose and lactose), at log phase (5 h) and stationary phase (10 h), on the global proteomics of S. thermophilus 1275 to understand the differentially expressed proteins (DEPs) during EPS production using isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis. Among 98 DEPs in sucrose medium, most of them were mapped into EPS biosynthesis pathway and other related metabolisms. There was an upregulation of several proteins involved in sugar transport (phosphoenolpyruvate (PEP) phosphotransferase system), EPS assembly (epsG1D) and amino acid metabolism (methionine, cysteine/arginine metabolism) in sucrose medium. This study showed that increased EPS production in S. thermophilus 1275 requires a well-co-ordinated regulation of pathway involved in both EPS assembly and amino acid metabolism along with the availability of sugars. Thus, it provided valuable insights into the biosynthesis and regulation of EPS in S. thermophilus 1275, and potential gene targets for understanding high-EPS strains.

The protective effects of enriched citrulline fermented milk with Lactobacillus helveticus on the intestinal epithelium integrity against Escherichia coli infection.

This study examined the protective effects of citrulline enriched-fermented milk with live Lactobacillus helveticus ASCC 511 (LH511) on intestinal epithelial barrier function and inflammatory response in IPEC-J2 cells caused by pathogenic Escherichia coli. Five percent (v/v) of fermented milk with live LH511 and 4 mM citrulline (5%LHFM_Cit-4mM) significantly stimulated the population of IPEC-J2 cells by 36% as determined by MTT assay. Adhesion level of LH511 was significantly increased by 9.2% when incubated with 5%LHFM_Cit-4mM and 5%LHFM_Cit-4mM reduced the adhesion of enterohemorrhagic (EHEC) and entero-invasive (EIEC) E. coli in IPEC-J2 cells by 35.79% and 42.74%, respectively. Treatment with 5%LHFM_Cit-4mM ameliorated lipopolysaccharide (LPS) from E. coli O55:B5 induced activated inflammatory cytokines expression (TNF-α, IL-6 and IL-8) and concentration (IL-6 and IL-8) and early apoptosis. It restored the transepithelial electrical resistance (TEER) and regulated the expression and distribution of tight junction (TJ) proteins (zonula occluden-1 (ZO-1), occludin and claudin-1), toll-like receptors (TLRs) (TLR2 and TLR4) and negative regulators of TLRs signalling pathway (A20 and IRAK-M). In conclusion, our findings suggested that 5%LHFM_Cit-4mM might have the positive effects on improving and maintaining the intestinal epithelial cell integrity and inflammatory response under both normal and pathogenic LPS-stimulated conditions.

Functional Genomic Analyses of Exopolysaccharide-Producing Streptococcus thermophilus ASCC 1275 in Response to Milk Fermentation Conditions.

Exopolysaccharide (EPS) produced from dairy bacteria improves texture and functionalities of fermented dairy foods. Our previous study showed improved EPS production from Streptococcus thermophilus ASCC1275 (ST1275) by simple alteration of fermentation conditions such as pH decrease (pH 6.5 → pH 5.5), temperature increase (37°C → 40°C) and/or whey protein isolate (WPI) supplementation. The iTRAQ-based proteomics in combination with transcriptomics were applied to understand cellular protein expression in ST1275 in response to above shifts during milk fermentation. The pH decrease induced the most differentially expressed proteins (DEPs) that are involved in cellular metabolic responses including glutamate catabolism, arginine biosynthesis, cysteine catabolism, purine metabolism, lactose uptake, and fatty acid biosynthesis. Temperature increase and WPI supplementation did not induce much changes in global protein express profiles of ST1275 between comparisons of pH 5.5 conditions. Comparative proteomic analyses from pairwise comparisons demonstrated enhanced glutamate catabolism and purine metabolism under pH 5.5 conditions (Cd2, Cd3, and Cd4) compared to that of pH 6.5 condition (Cd1). Concordance analysis for differential expressed genes (DEGs) and DEPs highlighted down-regulated glutamate catabolism and up-regulated arginine biosynthesis in pH 5.5 conditions. Down regulation of glutamate catabolism was also confirmed by pathway enrichment analysis. Down-regulation of EpsB involved in EPS assembly was observed at both mRNA and protein level in pH 5.5 conditions compared to that in pH 6.5 condition. Medium pH decreased to mild acidic level induced cellular changes associated with glutamate catabolism, arginine biosynthesis and regulation of EPS assembly in ST1275.

Effects of Lactic Acid Bacteria-Fermented Soymilk on Isoflavone Metabolites and Short-Chain Fatty Acids Excretion and Their Modulating Effects on Gut Microbiota.

Lactobacillus rhamnosus strain ASCC 1520 with high soy isoflavone transformation ability was used to ferment soymilk and added to the diet of mice. The impact of L. rhamnosus fermentation on soy isoflavone metabolites and intestinal bacterial community, in conjunction with fecal enzyme activity and short-chain fatty acids (SCFA) excretion was evaluated. Antibiotics intervention resulted in a decrease in fecal enzyme activities and SCFA. Although long-term intake of soymilk or L. rhamnosus-fermented soymilk did not affect the fecal ß-glucuronidase and ß-galactosidase activities, it improved the ß-glucosidase activity when antibiotics were concomitantly administered. Soymilk or fermented soymilk administration increased the isoflavone metabolites (O-DMA and equol) excreted in urine. Antibiotics decreased the daidzein excretion and its metabolites but showed little effect on glycitein and genistein excretion. Principal coordinates analysis (PCoA) of the 16s rRNA gene sequencing data found a remarkable shift in gut microbiota after soymilk administration and antibiotics treatment. Matastats test of the relative abundance of bacterial taxa revealed Odoribacter (Bacteroidales family), Lactobacillus (Lactobacillales order), and Alistipes (Rikenellaceae family) were enriched in soymilk while bacterial taxa from Bacteroides and Lactobacillus were enriched in L. rhamnosus-fermented soymilk. Furthermore, there was less decrease in bacterial taxa with fermented soymilk group even when antibiotics were concomitantly administered. Overall, this study revealed that the gut microbiota of a healthy host is enough for the whole isoflavone metabolism under normal conditions. Feeding mice with L. rhamnosus-fermented soymilk improved fecal enzyme activity and kept the balance of the gut mirobiota when antibiotics were used. PRACTICAL APPLICATION: Feeding mice with L. rhamnosus-fermented soymilk improved fecal enzyme activity and kept the balance of the gut mirobiota when antibiotics were used.