Lactobacillus gasseri NK109 and Its Supplement Alleviate Cognitive Impairment in Mice by Modulating NF-κB Activation, BDNF Expression, and Gut Microbiota Composition.

Aging-related gut microbiota dysbiosis initiates gut inflammation and microbiota dysbiosis, which induce the occurrence of psychiatric disorders including dementia. The alleviation of gut microbiota dysbiosis by probiotics is suggested to be able to alleviate psychiatric disorders including cognitive impairment (CI). Therefore, to understand how probiotics could alleviate CI, we examined the effects of anti-inflammatory Lactobacillus gasseri NK109 and its supplement (NS, mixture of NK109 and soybean embryo ethanol extract) on cognitive function in aged (Ag), 5XFAD transgenic (Tg), or mildly cognition-impaired adult fecal microbiota (MCF)-transplanted mice. Oral administration of NK109 or NS decreased CI-like behaviors in Ag mice. Their treatments suppressed TNF-α and p16 expression and NF-κB-activated cell populations in the hippocampus and colon, while BDNF expression was induced. Moreover, they partially shifted the ß-diversity of gut microbiota in Ag mice to those of young mice: they decreased Bifidobacteriaceae, Lactobacillaceae, and Helicobacteriaceae populations and increased Rikenellaceae and Prevotellaceae populations. Oral administration of NK109 or NS also reduced CI-like behaviors in Tg mice. Their treatments induced BDNF expression in the hippocampus, decreased hippocampal TNF-α and Aß expression and hippocampal and colonic NF-κB-activated cell populations. NK109 and NS partially shifted the ß-diversity of gut microbiota in Tg mice: they decreased Muribaculaceae and Rhodospiraceae populations and increased Helicobacteriaceae population. Oral administration of NK109 or NS decreased MCF transplantation-induced CI-like behaviors in mice. NK109 and NS increased hippocampal BDNF expression, while hippocampal and colonic TNF-α expression and NF-κB-activated cell populations decreased. These findings suggest that dementia can fluctuate the gut microbiota composition and NK109 and its supplement NS can alleviate CI with systemic inflammation by inducing BDNF expression and suppressing NF-κB activation and gut microbiota dysbiosis.

Protective Effects of Lactobacillus gasseri against High-Cholesterol Diet-Induced Fatty Liver and Regulation of Host Gene Expression Profiles.

Fatty liver is one of the most pervasive liver diseases worldwide. Probiotics play an important role in the progression of liver disease, but their effects on host regulation are poorly understood. This study investigated the protective effects of lactobacillus gasseri (L. gasseri) against high-cholesterol diet (HCD)-induced fatty liver injury using a zebrafish larvae model. Liver pathology, lipid accumulation, oxidative stress and hepatic inflammation were evaluated to demonstrate the changes in a spectrum of hepatic injury. Moreover, multiple indexes on host gene expression profiles were comprehensively characterized by RNA screening. The results showed that treatment with L. gasseri ameliorated HCD-induced morphological and histological alterations, lipid regulations, oxidative stress and macrophage aggregation in the liver of zebrafish larvae. Furthermore, the enrichment of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed that the core pathways of L. gasseri regulation were interleukin-17 (IL-17) signaling, phosphoinositide 3-kinase (PI3K)-AKT signaling pathway, the regulation of lipolysis and adipocytes and fatty acid elongation and estrogen signaling. The genes at key junction nodes, hsp90aa1.1, kyat3, hsd17b7, irs2a, myl9b, ptgs2b, cdk21 and papss2a were significantly regulated by L. gasseri administration. To conclude, the current research extends our understanding of the protective effects of L. gasseri against fatty liver and provides potential therapeutic options for fatty liver treatment.

Lactobacillus gasseri RW2014 Ameliorates Hyperlipidemia by Modulating Bile Acid Metabolism and Gut Microbiota Composition in Rats.

Hyperlipidemia is a leading risk of cardiovascular and cerebrovascular disease. Dietary supplementation with probiotics has been suggested as an alternative intervention to lower cholesterol. In the current study, we isolated a strain of Lactobacillus gasseri RW2014 (LGA) from the feces of a healthy infant fed with breast milk, and it displayed bile salt hydrolase (BSH) activity. Using this strain we determined its cholesterol-lowering and fatty liver-improving functions. SD rats were randomly divided into four groups. The control rats were fed a commercial chow diet and the other three groups were fed a high-fat diet (HFD) for a 7-week experiment period. After two weeks of feeding, the rats in PBS, simvastin, and LGA group were daily administered through oral gavage with 2 mL PBS, simvastin (1 mg/mL), and 2 × 109 CFU/mouse live LGA in PBS, respectively. After five weeks of such treatment, the rats were euthanized and tissue samples were collected. Blood lipid and inflammatory factors were measured by ELISA, gut microbiota was determined by 16S rRNA sequencing, and bile acids profiles were detected by metabolomics. We found that LGA group had lower levels of blood cholesterol and liver steatosis compared to the simvastin group. LGA also significantly reducedthe levels of inflammatory factors in the serum, including TNFα, IL-1ß, MCP-1, IL-6, and exotoxin (ET), and increased the levels of short-chain fatty acids in feces, including isobutyric acid, butyric acid, isovaleric acid, valeric acid, and hexanoic acid. In addition, LGA altered the compositions of gut microbiota as manifested by the increased ratio of Firmicutes/Bacteroides and the relative abundance of Blautia genus. Targeted metabolomics results showed that bile acids, especially free bile acids and secondary bile acids in feces, were increased in LGA rats compared with the control rats. Accordingly, the rats administrated with LGA also had a higher abundance of serum bile acids, including 23-norcholic acid, 7-ketolithocholic acid, ß-muricholic acid, cholic acid, and deoxycholic acid. Together, this study suggests that LGA may exert a cholesterol-lowering effect by modulating the metabolism of bile acids and the composition of gut microbiota.

The Lactobacillus gasseri G098 Strain Mitigates Symptoms of DSS-Induced Inflammatory Bowel Disease in Mice.

Inflammatory bowel disease (IBD) is a recurring inflammatory disease of the gastrointestinal tract with unclear etiology, but it is thought to be related to factors like immune abnormalities and gut microbial dysbiosis. Probiotics can regulate host immunity and gut microbiota; thus, we investigated the alleviation effect and mechanism of the strain Lactobacillus gasseri G098 (G098) on dextran sodium sulfate (DSS)-induced colitis in mice. Three groups of mice (n = 8 per group) were included: normal control (NC), DSS-induced colitis mice (DSS), and colitis mice given strain (G098). Our results showed that administering G098 effectively reversed DSS-induced colitis-associated symptoms (mitigating weight loss, reducing disease activity index and pathology scores; p < 0.05 in all cases) and prevented DSS-induced mortality (62.5% in DSS group; 100% in G098 group). The mortality rate and symptom improvement by G098 administration was accompanied by a healthier serum cytokine balance (significant decreases in serum pro-inflammatory factors, interleukin (IL)-6 [p < 0.05], IL-1ß [p < 0.01], and tumor necrosis factor (TNF)-α [p < 0.001], and significant increase in the serum anti-inflammatory factor IL-13 [p < 0.01], compared with DSS group) and gut microbiome modulation (characterized by a higher gut microbiota diversity [p < 0.05], significantly more Firmicutes and Lachnoclostridium [p < 0.05], significantly fewer Bacteroidetes [p < 0.05], and significant higher gene abundances of sugar degradation-related pathways [p < 0.05], compared with DSS-treated group). Taken altogether, our results suggested that G098 intake could mitigate DSS-induced colitis through modulating host immunity and gut microbiome, and strain treatment is a promising strategy for managing IBD.

Lactobacillus gasseri PA-3 reduces serum uric acid levels in patients with marginal hyperuricemia.

Several studies have reported that Lactobacillus gasseri PA-3 reduces the level of serum uric acid (SUA) in patients with hyperuricemia. However, it remains unknown how PA-3 affects uric acid metabolism. In the present study, we examined effects of PA-3-containing yoghurt on uric acid metabolism in patients with marginal hyperuricemia. Sixteen patients with SUA > 357 µmol/L (marginal hyperuricemia) were enrolled. PA-3-containing yoghurt was administered for 8 weeks. Uric acid metabolism was evaluated just before and 8 weeks after the administration and at 4 weeks after the administration ended (post-administration). SUA levels after the administration were significantly lower than that before the administration and remained low at post-administration. Urinary uric acid concentration (Uur) after the administration were significantly lower than that before the administration. However, post-administration Uur levels were comparable to those before the administration. Therefore, PA-3-containing yoghurt significantly reduced the levels of SUA and Uur in patients with marginal hyperuricemia.

Lactobacillus gasseri PA-3 directly incorporates purine mononucleotides and utilizes them for growth.

Lactococcus lactis has been reported unable to directly incorporate mononucleotides but instead requires their external dephosphorylation by nucleotidases to the corresponding nucleosides prior to their incorporation. Although Lactobacillus gasseri PA-3 (PA-3), a strain of lactic acid bacteria, has been found to incorporate purine mononucleotides such as adenosine 5'-monophosphate (AMP), it remains unclear whether these bacteria directly incorporate these mononucleotides or incorporate them after dephosphorylation to the corresponding nucleosides. This study evaluated whether PA-3 incorporated radioactively-labeled mononucleotides in the presence or absence of the 5'-nucleotidase inhibitor α,ß-methylene ADP (APCP). PA-3 took up 14C-AMP in the presence of APCP, as well as incorporating 32P-AMP. Furthermore, radioactivity was detected in the RNA/DNA of bacterial cells cultured in the presence of 32P-AMP. Taken together, these findings indicated that PA-3 incorporated purine mononucleotides directly rather than after their dephosphorylation to purine nucleosides and that PA-3 utilizes these purine mononucleotides in the synthesis of RNA and DNA. Although additional studies are required to identify purine mononucleotide transporters in PA-3, this study is the first to show that some lactic acid bacteria directly incorporate purine mononucleotides and use them for growth.

Homeostasis effects of fermented Maillard reaction products by Lactobacillus gasseri 4M13 in dextran sulfate sodium-induced colitis mice.

BACKGROUND: The incidence of inflammatory bowel disease (IBD) continues to increase worldwide. Multiple factors, including diet, loss of the intestinal barrier function, and imbalance of the immune system can cause IBD. A balanced diet is important for maintaining a healthy bowel and preventing IBD from occurring. The effects of probiotic Lactobacillus gasseri-fermented Maillard reaction products (MRPs) prepared by reacting whey protein with galactose on anti-inflammation and intestinal homeostasis were investigated in this study, which compared MPRs and probiotics separately. RESULTS: In an animal colitis model induced by 2% dextran sulfate sodium (DSS), FWG administration alleviated colon length loss and maintained intestinal immune system homeostasis as reflected by down-regulated interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α output, and metallopeptidase-9, and epithelial barrier balance as reflected by up-regulated occludin, E-cadherin, and zonula occludens-1 production in the colon. Furthermore, the expression of splenic cytokines such as IL-6, TNF-α, and IL-10 was up-regulated in the FWG-treated mice in a comparable amount to the control group to ensure the balance of immune responses. CONCLUSION: This study showed that the use of FWG protects the intestines from colitis caused by DSS and maintains immune balance. FWG increased antioxidant enzyme activity, increased intestinal permeability, and regulated the balance of pro- and anti-inflammatory cytokines in the intestines and spleen. Continued intake of FWG can alleviate IBD symptoms through the preservation of mucosal immune responses, epithelial junction and homeostasis through the regulated splenic cytokines. © 2021 Society of Chemical Industry.

Lactobacillus gasseri JM1 Isolated from Infant Feces Alleviates Colitis in Mice via Protecting the Intestinal Barrier.

Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease, and the intestinal barrier is an important line of defense against intestinal disease. Herein, we investigated the effect of Lactobacillus gasseri JM1 at different doses (1 × 106, 1 × 107, 1 × 108 CFU/day) on colitis mice and explored the possible mechanism. The results showed that L. gasseri JM1 alleviated DSS-induced colitis in mice, with reductions in disease activity index (DAI), histological scores and myeloperoxidase activity as well as alleviation of colonic shortening. Furthermore, L. gasseri JM1 regulated the levels of inflammatory cytokines TNF-α, IL-6, IL-1ß, and IL-10; restored the expression of Claudin-3, Occludin, ZO-1, and MUC2; and increased the number of goblet cells and acidic mucin. The 16S rDNA sequencing results indicated that intervention with L. gasseri JM1 balanced the gut microbiota structure by elevating the abundance of beneficial bacteria (Oscillospira, Clostridium and Ruminococcus) and decreasing that of harmful bacteria (Shigella and Turicibacter). Meanwhile, the contents of short-chain fatty acids (SCFAs) increased. In conclusion, L. gasseri JM1 could alleviate intestinal barrier damage in colitis mice by modulating the tight junction structures, intestinal mucus layer, inflammatory cytokines, gut microbiota, and SCFAs. It can be considered a potential preventive strategy to alleviate colitis injury.

The Effect of Continuous Intake of Lactobacillus gasseri OLL2716 on Mild to Moderate Delayed Gastric Emptying: A Randomized Controlled Study.

Probiotics have been suggested to be effective for functional dyspepsia, but their effect on gastric motility is not clear. We evaluated the effect of Lactobacillus gasseri OLL2716 (LG21 strain) on mild to moderate delayed gastric emptying by a double-blind, parallel-group, placebo-controlled, randomized trial. Participants (n = 28) were randomly assigned to ingest LG21 strain-containing yogurt (LG21 strain group) or LG21 strain-free yogurt (placebo group) for 12 weeks. The 13C gastric emptying breath test was performed to measure the gastric emptying rate over time following ingestion of a liquid meal, and the time to reach the peak (Tmax) was used as an indicator of gastric emptying. We also measured the salivary amylase concentration, an indicator of autonomic dysfunction under stress. The per-protocol population (n = 27, male n = 4, female n = 23) was evaluated for efficacy. When a ≥30% reduction in the difference between participant's Tmax and the Japanese mean Tmax was defined as an improvement, the odds ratio of improvement in delayed gastric emptying compared to placebo after 12 weeks was 4.1 (95% confidence interval, 0.8 to 20.2). Moreover, salivary amylase concentrations were significantly lower than in the placebo group, indicating an improvement in autonomic function. The present data were not enough to support the beneficial effects of the LG21 strain on delayed gastric emptying. However, if we define the odds ratio in further study investigated with a larger number of participants, LG21 strain might be expected to have some impact on delayed gastric emptying.

Oral probiotic activities and biosafety of Lactobacillus gasseri HHuMIN D.

BACKGROUND: Lactobacillus spp. have been researched worldwide and are used in probiotics, but due to difficulties with laboratory cultivation of and experimentation on oral microorganisms, there are few reports of Lactobacillus spp. being isolated from the oral cavity and tested against oral pathogens. This research sought to isolate and determine the safety and inhibitory capabilities of a Lactobacillus culture taken from the human body. RESULTS: One organism was isolated, named "L. gasseri HHuMIN D", and evaluated for safety. A 5% dilution of L. gasseri HHuMIN D culture supernatant exhibited 88.8% inhibition against halitosis-producing anaerobic microorganisms and the organism itself exhibited powerful inhibitory effects on the growth of 11 oral bacteria. Hydrogen peroxide production reached 802 µmol/L after 12 h and gradually diminished until 24 h, it efficiently aggregated with P. catoniae and S. sanguinis, and it completely suppressed S. mutans-manufactured artificial dental plaque. L. gasseri HHuMIN D's KB cell adhesion capacity was 4.41 cells per cell, and the cell adhesion of F. nucleatum and S. mutans diminished strongly in protection and displacement assays. CONCLUSION: These results suggest that L. gasseri HHuMIN D is a safe, bioactive, lactobacterial food ingredient, starter culture, and/or probiotic microorganism for human oral health.

Effects of multi-species probiotic supplementation on alcohol metabolism in rats.

Probiotics are known to protect against liver damage induced by the alcohol and acetaldehyde accumulation associated with alcohol intake. However, there have been few studies of the direct effect of probiotics on alcohol metabolism, and the types of probiotics that were previously analyzed were few in number. Here, we investigated the effects of 19 probiotic species on alcohol and acetaldehyde metabolism. Four probiotic species that had a relatively high tolerance to alcohol and metabolized alcohol and acetaldehyde effectively were identified: Lactobacillus gasseri CBT LGA1, Lactobacillus casei CBT LC5, Bifidobacterium lactis CBT BL3, and Bifidobacterium breve CBT BR3. These species also demonstrated high mRNA expression of alcohol and acetaldehyde dehydrogenases. ProAP4, a mixture of these four probiotics species and excipient, was then administered to rats for 2 weeks in advance of acute alcohol administration. The serum alcohol and acetaldehyde concentrations were significantly lower in the ProAP4-administered group than in the control and excipient groups. Thus, the administration of ProAP4, containing four probiotic species, quickly lowers blood alcohol and acetaldehyde concentrations in an alcohol and acetaldehyde dehydrogenasedependent manner. Furthermore, the serum alanine aminotransferase activity, which is indicative of liver damage, was significantly lower in the ProAP4 group than in the control group. The present findings suggest that ProAP4 may be an effective means of limiting alcohol-induced liver damage.

Characterisation of EFV12 a bio-active small peptide produced by the human intestinal isolate Lactobacillus gasseri SF1109.

EFV12 is a small bioactive peptide produced by Lactobacillus gasseri SF1109, a human intestinal isolate with probiotic features. In this study, EFV12 antimicrobial and anti-inflammatory properties are characterised. In particular, we propose a possible mechanism of action for EFV12 involving bacterial membranes targeting. Moreover, we show that this small peptide is able to bind lipopolysaccharides (LPS) and to counteract its inflammatory insult preventing LPS action on Toll-like receptor 4, thus interfering with extracellular signal-regulated kinase, p38 and Jun N-terminal kinase, mitogen-activated protein kinases signalling pathways. Altogether these observations suggest that the bioactive peptide EFV12 is a good candidate to promote L. gasseri induced gut homeostasis and counteracting intestinal pathogens.

Metabolically engineered Lactobacillus gasseri JCM 1131 as a novel producer of optically pure L- and D-lactate.

High-quality environmentally-friendly bioplastics can be produced by mixing poly-L-lactate with poly-D-lactate. On an industrial scale, this process simultaneously consumes large amounts of both optically pure lactate stereoisomers. However, because optimal growth conditions of L-lactate producers often differ from those of D-lactate producers, each stereoisomer is produced in a specialised facility, which raises cost and lowers sustainability. To address this challenge, we metabolically engineered Lactobacillus gasseri JCM 1131T, a bioprocess-friendly and genetically malleable strain of homofermentative lactic acid bacterium, to efficiently produce either pure L- or pure D-lactate under the same bioprocess conditions. Transformation of L. gasseri with plasmids carrying additional genes for L- or D-lactate dehydrogenases failed to affect the ratio of produced stereoisomers, but inactivation of the endogenous genes created strains which yielded 0.96 g of either L- or D-lactate per gram of glucose. In this study, the plasmid pHBintE, routinely used for gene disruption in Bacillus megaterium, was used for the first time to inactivate genes in lactobacilli. Strains with inactivated genes for endogenous lactate dehydrogenases efficiently fermented sugars released by enzymatic hydrolysis of alkali pre-treated wheat straw, an abundant lignocellulose-containing raw material, producing 0.37-0.42 g of lactate per gram of solid part of alkali-treated wheat straw. Thus, the constructed strains are primed to serve as producers of both optically pure L-lactate and D-lactate in the next-generation biorefineries.

Production of multiple bacteriocins, including the novel bacteriocin gassericin M, by Lactobacillus gasseri LM19, a strain isolated from human milk.

Bacteriocins are antimicrobial peptides produced by bacteria, and their production is regarded as a desirable probiotic trait. We found that Lactobacillus gasseri LM19, a strain isolated from human milk, produces several bacteriocins, including a novel bacteriocin, gassericin M. These bacteriocins were purified from culture and synthesised to investigate their activity and potential synergy. L. gasseri LM19 was tested in a complex environment mimicking human colon conditions; it not only survived, but expressed the seven bacteriocin genes and produced short-chain fatty acids. Metagenomic analysis of these in vitro colon cultures showed that co-inoculation of L. gasseri LM19 with Clostridium perfringens gave 16S ribosomal RNA metagenomic profiles with more similarity to controls than to vessels inoculated with C. perfringens alone. These results indicate that L. gasseri LM19 could be an interesting candidate for maintaining homeostasis in the gut environment.

A synbiotic combination of Lactobacillus gasseri 505 and Cudrania tricuspidata leaf extract prevents hepatic toxicity induced by colorectal cancer in mice.

Colorectal cancer (CRC) is known to be a life-threatening disease and commonly leads to metastasis in the liver. Fermented milk acts as an effective carrier for probiotic strains, whose consumption improves host health. Our previous study indicated that fermented milk that included a synbiotic combination of Lactobacillus gasseri 505 (505) and Cudrania tricuspidata leaf extract (CT) resulted in significantly greater anti-oxidative effects than fermented milk without CT. Therefore, we hypothesized that fermented milk containing CT and 505 (FCT) could result in hepatoprotective effects against CRC-induced liver metastasis. Liver inflammation and CRC were induced in male C57BL/6J mice, using azoxymethane/dextran sodium sulfate, and 505, CT, and FCT were administered to the 3 sample-treated 505, CT, and FCT groups, respectively, for 10 wk. The results showed that FCT treatment significantly reduced serum aspartate aminotransferase and alanine aminotransferase concentrations and elevated albumin concentrations. Moreover, the results of histological analysis showed that hepatic steatosis was notably reduced in the FCT group. Among the 3 sample-treated groups, the expression of mRNA associated with enzymes showing anti-oxidative activities, such as superoxide dismutase, catalase, and glutathione reductase, was the highest in the FCT-treated mice. In addition, FCT administration resulted in the greatest anti-inflammatory activity, as inflammatory marker levels (i.e., tumor necrosis factor-α, cyclooxygenase-2, myeloperoxidase, and nuclear factor kappa-light-chain enhancer of activated B cells) were significantly downregulated at the mRNA level and the expression of proteins associated with the nuclear factor kappa-light-chain enhancer of activated B cells and mitogen-activated protein kinase signaling pathways was suppressed by FCT. Therefore, this study demonstrated that fermented milk containing novel synbiotics has the potential to prevent hepatic toxicity induced because of CRC owing to its enhanced anti-oxidative and anti-inflammatory activities.

Structural analysis of the lipoteichoic acid anchor glycolipid: Comparison of methods for degradation of the glycerophosphate backbone polymer.

Acetic acid treatment [98% (v/v), 100 °C, 3 h] was proposed as a new method for degrading the glycerophosphate polymer moiety of Gram-positive bacterial lipoteichoic acid. We demonstrated that this method resulted in partial O-acetylation on the carbohydrate residues of the anchor glycolipid. Hence, the acetic acid treatment is not suitable for the chemical structural analysis of lipoteichoic acid.

Metabolomic profiling of oxalate-degrading probiotic Lactobacillus acidophilus and Lactobacillus gasseri.

Oxalate, a ubiquitous compound in many plant-based foods, is absorbed through the intestine and precipitates with calcium in the kidneys to form stones. Over 80% of diagnosed kidney stones are found to be calcium oxalate. People who form these stones often experience a high rate of recurrence and treatment options remain limited despite decades of dedicated research. Recently, the intestinal microbiome has become a new focus for novel therapies. Studies have shown that select species of Lactobacillus, the most commonly included genus in modern probiotic supplements, can degrade oxalate in vitro and even decrease urinary oxalate in animal models of Primary Hyperoxaluria. Although the purported health benefits of Lactobacillus probiotics vary significantly between species, there is supporting evidence for their potential use as probiotics for oxalate diseases. Defining the unique metabolic properties of Lactobacillus is essential to define how these bacteria interact with the host intestine and influence overall health. We addressed this need by characterizing and comparing the metabolome and lipidome of the oxalate-degrading Lactobacillus acidophilus and Lactobacillus gasseri using ultra-high-performance liquid chromatography-high resolution mass spectrometry. We report many species-specific differences in the metabolic profiles of these Lactobacillus species and discuss potential probiotic relevance and function resulting from their differential expression. Also described is our validation of the oxalate-degrading ability of Lactobacillus acidophilus and Lactobacillus gasseri, even in the presence of other preferred carbon sources, measuring in vitro 14C-oxalate consumption via liquid scintillation counting.

Anti-inflammatory activities of Maillard reaction products from whey protein isolate fermented by Lactobacillus gasseri 4M13 in lipopolysaccharide-stimulated RAW264.7 cells.

Maillard reaction products formed from whey protein isolate (WPI) and sugar have been shown to have an anti-inflammatory effect in vitro. Here, we incubated WPI and galactose (GWA) in an aqueous solution at 65°C for 24 h to produce a glycated conjugate, which was then fermented using Lactobacillus gasseri 4M13 to obtain the fermented product (F-GWA). We demonstrated that F-GWA had an anti-inflammatory effect on lipopolysaccharide (LPS)-stimulated RAW264.7 cells. It reduced both LPS-stimulated nitric oxide production and LPS-stimulated increases in the gene expression levels of tumor necrosis factor-α and cyclooxygenase-2 in a dose-dependent manner. Furthermore, F-GWA inhibited the LPS-induced phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase, members of the mitogen-activated protein kinase family. The glycation process was evaluated by measuring fluorescence intensity and the furosine concentration during the Maillard reaction to form GWA. The protein modifications of WPI were analyzed using MALDI-TOF tandem mass spectrometry. We found that the combination of the Maillard reaction and L. gasseri 4M13 fermentation increased the prebiotic properties of GWA as well as organic acid production, compared with the nonreacted WPI and galactose.

Homologous Expression and Characterization of Gassericin T and Gassericin S, a Novel Class IIb Bacteriocin Produced by Lactobacillus gasseri LA327.

Lactobacillus gasseri LA327, isolated from the large intestine tissue in humans, is a bacteriocinogenic strain with two kinds of class IIb bacteriocin structural genes, i.e., those for gassericin T (GT) and acidocin LF221A (Acd LF221A). In this study, DNA sequencing of the genes for GT and Acd LF221A from L. gasseri LA327 revealed that the amino acid sequences for GT corresponded with those for GT genes, except for GatK (histidine kinase). However, Acd LF221A genes had analogues which differed in at least one amino acid residue, to encode a class IIb bacteriocin designated gassericin S (GS). The LA327 strain retained antimicrobial activity after the deletion of the GT structural genes (gatAX); however, both GS and GT activities were lost by deletion of the putative ABC transporter gene (gatT). This indicates that the LA327 strain produces GS and GT and that GS secretion is performed via GT genes with the inclusion of gatT Homologous expression using deletion mutants of GS and GT, each containing a single peptide, elucidated that GS (GasAX) and GT (GatAX) showed synergistic activity as class IIb bacteriocins and that no synergistic activity was observed between GS and GT peptides. The molecular mass of GS was estimated to be theoretical ca. 5,400 Da by in situ activity assay after SDS-PAGE, clarifying that GS was actually expressed as an active class IIb bacteriocin. Furthermore, the stability of expressed GS to pH, heat, and protease was determined.IMPORTANCE Bacteriocins are regarded as potential alternatives for antibiotics in the absence of highly resistant bacteria. In particular, two-peptide (class IIb) bacteriocins exhibit the maximum activity through the synergy of two components, and their antimicrobial spectra are known to be relatively wide. However, there are few reports of synergistic activity of class IIb bacteriocins determined by isolation and purification of individual peptides. Our results clarified the interaction of each class IIb component peptide for GT and GS via the construction of homologous mutants, which were not dependent on the purification. These data may contribute to understanding the mechanisms of action by which class IIb bacteriocins exhibit wide antibacterial spectra.

The membrane phospholipid cardiolipin plays a pivotal role in bile acid adaptation by Lactobacillus gasseri JCM1131T.

Bile acids exhibit strong antimicrobial activity as natural detergents, and are involved in lipid digestion and absorption. We investigated the mechanism of bile acid adaptation in Lactobacillus gasseri JCM1131T. Exposure to sublethal concentrations of cholic acid (CA), a major bile acid in humans, resulted in development of resistance to otherwise-lethal concentrations of CA by this intestinal lactic acid bacterium. As this adaptation was accompanied by decreased cell-membrane damage, we analyzed the membrane lipid composition of L. gasseri. Although there was no difference in the proportions of glycolipids (~70%) and phospholipids (~20%), adaptation resulted in an increased abundance of long-sugar-chain glycolipids and a 100% increase in cardiolipin (CL) content (to ~50% of phospholipids) at the expense of phosphatidylglycerol (PG). In model vesicles, the resistance of PG vesicles to solubilization by CA increased with increasing CL/PG ratio. Deletion of the two putative CL synthase genes, the products of which are responsible for CL synthesis from PG, decreased the CL content of the mutants, but did not affect their ability to adapt to CA. Exposure to CA restored the CL content of the two single-deletion mutants, likely due to the activities of the remaining CL synthase. In contrast, the CL content of the double-deletion mutant was not restored, and the lipid composition was modified such that PG predominated (~45% of total lipids) at the expense of glycolipids. Therefore, CL plays important roles in bile acid resistance and maintenance of the membrane lipid composition in L. gasseri.