Human gut-associated Bifidobacterium species salvage exogenous indole, a uremic toxin precursor, to synthesize indole-3-lactic acid via tryptophan.

Indole in the gut is formed from dietary tryptophan by a bacterial tryptophan-indole lyase. Indole not only triggers biofilm formation and antibiotic resistance in gut microbes but also contributes to the progression of kidney dysfunction after absorption by the intestine and sulfation in the liver. As tryptophan is an essential amino acid for humans, these events seem inevitable. Despite this, we show in a proof-of-concept study that exogenous indole can be converted to an immunomodulatory tryptophan metabolite, indole-3-lactic acid (ILA), by a previously unknown microbial metabolic pathway that involves tryptophan synthase ß subunit and aromatic lactate dehydrogenase. Selected bifidobacterial strains converted exogenous indole to ILA via tryptophan (Trp), which was demonstrated by incubating the bacterial cells in the presence of (2-13C)-labeled indole and l-serine. Disruption of the responsible genes variedly affected the efficiency of indole bioconversion to Trp and ILA, depending on the strains. Database searches against 11,943 bacterial genomes representing 960 human-associated species revealed that the co-occurrence of tryptophan synthase ß subunit and aromatic lactate dehydrogenase is a specific feature of human gut-associated Bifidobacterium species, thus unveiling a new facet of bifidobacteria as probiotics. Indole, which has been assumed to be an end-product of tryptophan metabolism, may thus act as a precursor for the synthesis of a host-interacting metabolite with possible beneficial activities in the complex gut microbial ecosystem.

Sharing of Moonlighting Proteins Mediates the Symbiotic Relationship among Intestinal Commensals.

The human gastrointestinal tract is inhabited by trillions of symbiotic bacteria that form a complex ecological community and influence human physiology. Symbiotic nutrient sharing and nutrient competition are the most studied relationships in gut commensals, whereas the interactions underlying homeostasis and community maintenance are not fully understood. Here, we provide insights into a new symbiotic relationship wherein the sharing of secreted cytoplasmic proteins, called "moonlighting proteins," between two heterologous bacterial strains (Bifidobacterium longum and Bacteroides thetaiotaomicron) was observed to affect the adhesion of bacteria to mucins. B. longum and B. thetaiotaomicron were cocultured using a membrane-filter system, and in this system the cocultured B. thetaiotaomicron cells showed greater adhesion to mucins compared to that shown by monoculture cells. Proteomic analysis showed the presence of 13 B. longum-derived cytoplasmic proteins on the surface of B. thetaiotaomicron. Moreover, incubation of B. thetaiotaomicron with the recombinant proteins GroEL and elongation factor Tu (EF-Tu)-two well-known mucin-adhesive moonlighting proteins of B. longum-led to an increase in the adhesion of B. thetaiotaomicron to mucins, a result attributed to the localization of these proteins on the B. thetaiotaomicron cell surface. Furthermore, the recombinant EF-Tu and GroEL proteins were observed to bind to the cell surface of several other bacterial species; however, the binding was species dependent. The present findings indicate a symbiotic relationship mediated by the sharing of moonlighting proteins among specific strains of B. longum and B. thetaiotaomicron. IMPORTANCE The adhesion of intestinal bacteria to the mucus layer is an important colonization strategy in the gut environment. Generally, the bacterial adhesion process is a characteristic feature of the individual cell surface-associated adhesion factors secreted by a particular bacterium. In this study, coculture experiments between Bifidobacterium and Bacteroides show that the secreted moonlighting proteins adhere to the cell surface of coexisting bacteria and alter the adhesiveness of the bacteria to mucins. This finding indicates that the moonlighting proteins act as adhesion factors for not only homologous strains but also for coexisting heterologous strains. The presence of a coexisting bacterium in the environment can significantly alter the mucin-adhesive properties of another bacterium. The findings from this study contribute to a better understanding of the colonization properties of gut bacteria through the discovery of a new symbiotic relationship between them.

Metabolites of Latilactobacillus curvatus BYB3 and Indole Activate Aryl Hydrocarbon Receptor to Attenuate Lipopolysaccharide-Induced Intestinal Barrier Dysfunction.

This study aimed to investigate the effects of the metabolites of Latilactobacillus curvatus BYB3 and indole-activated aryl hydrocarbon receptor (AhR) to increase the tight junction (TJ) proteins in an in vitro model of intestinal inflammation. In a Western blot assay, the metabolites of L. curvatus BYB3 reduced the TJ demage in lipoploysaccharide (LPS) stimulated-Caco-2 cells. This reduction was a result of upregulating the expression of TJ-associated proteins and suppressing the nuclear factor-κB signaling. Immunofluorescence images consistently revealed that LPS disrupted and reduced the expression of TJ proteins, while the metabolites of L. curvatus BYB3 and indole reversed these alterations. The protective effects of L. curvatus BYB3 were observed on the intestinal barrier function when measuring transepithelial electrical resistance. Using high-performance liquid chromatography analysis the metabolites, the indole-3-latic acid and indole-3-acetamide concentrations were found to be 1.73±0.27 mg/L and 0.51±0.39 mg/L, respectively. These findings indicate that the metabolites of L. curvatus BYB3 have increasing mRNA expressions of cytochrome P450 1A1 (CYP1A1) and AhR, and may thus be applicable for therapy of various inflammatory gut diseases as postbiotics.

Genome analysis of Limosilactobacillus fermentum JN2019 applied to tumeric fermentation for animal feed.

Limosilactobacillus fermentum JN2019, formerly named Lactobacillus fermentum JN2019, was isolated from kimchi. Its genome was completely sequenced using the PacBio RSII sequencing system to explore beneficial phenotypes. In a previous study, L. fermentum JN2019 was used to ferment the by-product of tumeric for use in livestock feed. The 2.3 Mb genome had a high guanine (G) + cytosine (C) content of 50.6% and a 30 kb plasmid. The data will inform the comprehensive understanding of JN2019 and provide insights for potential applications.

Optimizing the composition of the medium for the viable cells of Bifidobacterium animalis subsp. lactis JNU306 using response surface methodology.

This research improved the growth potential of Bifidobacterium animalis subsp lactis strain JNU306, a commercial medium that is appropriate for large-scale production, in yeast extract, soy peptone, glucose, L-cysteine, and ferrous sulfate. Response surface methodology (RSM) was used to optimize the components of this medium, using a central composite design and subsequent analyses. A second-order polynomial regression model, which was fitted to the data at first, significantly lacked fitness. Thus, through further analyses, the model with linear and quadratic terms plus two-way, three-way, and four-way interactions was selected as the final model. Through this model, the optimized medium composition was found as 2.8791% yeast extract, 2.8030% peptone soy, 0.6196% glucose, 0.2823% L-cysteine, and 0.0055% ferrous sulfate, w/v. This optimized medium ensured that the maximum biomass was no lower than the biomass from the commonly used blood-liver (BL) medium. The application of RSM improved the biomass production of this strain in a more cost-effective way by creating an optimum medium. This result shows that B. animalis subsp lactis JNU306 may be used as a commercial starter culture in manufacturing probiotics, including dairy products.

A Non-yeast Kefir-like Fermented Milk Development with Lactobacillus acidophilus KCNU and Lactobacillus brevis Bmb6.

The use of yeast assist kefir fermentation, but also can cause food spoilage if uncontrolled. Hence, in this study, the microbial composition of an existing commercial kefir starter was modified to produce a functional starter, where Lactobacillus acidophilus KCNU and Lactobacillus brevis Bmb6 were used to replace yeast in the original starter to produce non-yeast kefir-like fermented milk. The functional starter containing L. acidophilus KCNU and L. brevis Bmb6 demonstrated excellent stability with 1010 CFU/g of total viable cells throughout the 12 weeks low-temperature storage. The newly developed functional starter also displayed a similar fermentation efficacy as the yeast-containing control starter, by completing the milk fermentation within 12 h, with a comparable total number of viable cells (108 CFU/mL) in the final products, as in control. Sensory evaluation revealed that the functional starter-fermented milk highly resembled the flavor of the control kefir, with enhanced sourness. Furthermore, oral administration of functional starter-fermented milk significantly improved the disease activity index score by preventing drastic weight-loss and further deterioration of disease symptoms in DSS-induced mice. Altogether, L. acidophilus KCNU and L. brevis Bmb6 have successfully replaced yeast in a commercial starter pack to produce a kefir-like fermented milk beverage with additional health benefits. The outcome of this study provides an insight that the specific role of yeast in the fermentation process could be replaced with suitable probiotic candidates.

Regulatory Effect of Lactobacillus brevis Bmb6 on Gut Barrier Functions in Experimental Colitis.

The integrity of gut barrier functions is closely associated with the pathogenesis of colitis. It is speculated that Lactobacillus brevis Bmb6 alleviates colitis by improving the tight junction (TJ) of the inflamed intestinal epithelial layer. In the present study, the regulatory effects of L. brevis Bmb6 on the TJ barrier to ameliorate colitis-symptoms were investigated. Preliminary screening showed that L. brevis Bmb6 exhibited strong acid and bile acid tolerance, along with antioxidants and ß-galactosidase activities. In a 14-day dextran sulfate sodium (DSS)-induced colitis mouse model, treatment with L. brevis Bmb6 significantly decreased in the disease activity index score. In addition, histological analyses showed that treatment with L. brevis Bmb6 protected the structural integrity of the intestinal epithelial layer and mucin-secreting goblet cells from DSS-induced damage, with only slight infiltration of immune cells. Interestingly, western blotting analyses showed that the expression of the TJ protein, zona occluden-1, was restored in Bmb6-treated mice, but not in DSS-induced mice. Consistently, the gene expression of inflammatory cytokines (tumor necrosis factor-α and interferon-γ) was also suppressed in the Bmb6-treated mice. Hence, our findings suggest that suppression of inflammatory conditions enhanced expression of TJ protein, ZO-1, or vice versa, contributing to a colitis-ameliorating effect in L. brevis Bmb6.

Lactobacillus plantarum USM8613 Aids in Wound Healing and Suppresses Staphylococcus aureus Infection at Wound Sites.

This study aimed to elucidate the targets and mechanisms of anti-staphylococcal effects from bioactive metabolites produced by lactic acid bacteria. We aimed to better understand the safety and efficacy of these bioactive metabolites in in vivo systems, typically at topical sites. The cell-free supernatant and protein-rich fraction from Lactobacillus plantarum USM8613 inhibited staphyloxanthin biosynthesis, reduced (p < 0.05) the cell number of Staphylococcus aureus by 106 CFU/mL and reduced biofilm thickness by 55% in S. aureus-infected porcine skins. Genome-wide analysis and gene expression analysis illustrated the production of several plantaricins, especially the plantaricins EF and JK that enhanced the anti-staphylococcal effects of L. plantarum USM8613. In vivo data using rats showed that the protein-rich fraction from L. plantarum USM8613 exerted wound healing properties via direct inhibition of S. aureus and promoted innate immunity, in which the expression of ß-defensin was significantly (p < 0.05) upregulated by 3.8-fold. The protein fraction from L. plantarum USM8613 also significantly enhanced (p < 0.05) the production of cytokines and chemokines through various stages of wound recovery. Using ∆atl S. aureus, the protein-rich fraction from L. plantarum USM8613 exerted inhibitory activity via targeting the atl gene in S. aureus. Taken altogether, our present study illustrates the potential of L. plantarum USM8613 in aiding wound healing, suppressing of S. aureus infection at wound sites and promoting host innate immunity.

Bioconversion of Gamma-Aminobutyric Acid from Monosodium Glutamate by Lactobacillus brevis Bmb5.

Gamma-aminobutyric acid (GABA) plays important roles in host physiology. However, the effects of GABA are greatly restricted due to its low bioavailability in the human body. Here, a high acid-tolerance GABA-producing strain, Lactobacillus brevis Bmb5, was isolated from kimchi. Bmb5 converted glutamate to GABA (7.23 ± 0.68 µg/µl) at a rate of 72.3%. The expression of gadB gene, encoding the enzyme involved in the decarboxylation of glutamate to GABA, was decreased upon incubation. Our findings indicate GABA production in Bmb5 is not directly correlated with gadB gene expression, providing new insight into the mechanisms underlying GABA production in Lactobacillus.

Effect of Lactobacillus Fermentation on the Anti-Inflammatory Potential of Turmeric.

Curcumin, the major bioactive constituent of turmeric, has been reported to have a wide range of pharmacological benefits; however, the low solubility in water has restricted its systemic bioavailability and therapeutic potential. Therefore, in the current study, we aimed to investigate the effect of turmeric fermentation on its curcumin content and anti-inflammatory activity by using several lactic acid bacteria. Fermentation with Lactobacillus fermentum significantly increased the curcumin content by 9.76% while showing no cytotoxicity in RAW 246.7 cells, as compared to the unfermented turmeric, regardless of the concentration of L. fermentum-fermented turmeric. The L. fermentum-fermented turmeric also promoted cells survival; a significantly higher number of viable cells in lipopolysaccharide (LPS)-induced RAW 264.7 cells were observed as compared to those treated with unfermented turmeric. It also displayed promising DPPH scavenging activity (7.88 ± 3.36%) and anti-inflammatory activity by significantly reducing the nitrite level and suppressing the expression of the pro-apoptotic tumor necrosis factor-alpha (TNF-α) and Toll-like receptor-4 (TLR4) in LPS-induced RAW 264.7 cells. Western blot analysis further revealed that the anti-inflammatory activity of the fermented turmeric was exerted through suppression of the c-Jun N-terminal kinase (JNK) signal pathway, but not in unfermented turmeric. Taken together, the results suggested that fermentation with lactic acid bacteria increases the curcumin content of turmeric without increasing its cytotoxicity, while strengthening the specific pharmacological activity, thus, highlighting its potential application as a functional food ingredient.

Transcriptional Response and Enhanced Intestinal Adhesion Ability of Lactobacillus rhamnosus GG after Acid Stress.

Lactobacillus rhamnosus GG (LGG) is a probiotic commonly used in fermented dairy products. In this study, RNA-sequencing was performed to unravel the effects of acid stress on LGG. The transcriptomic data revealed that the exposure of LGG to acid at pH 4.5 (resembling the final pH of fermented dairy products) for 1 h or 24 h provoked a stringent-type transcriptomic response wherein stress response- and glycolysis-related genes were upregulated, whereas genes involved in gluconeogenesis, amino acid metabolism, and nucleotide metabolism were suppressed. Notably, the pilus-specific adhesion genes, spaC, and spaF were significantly upregulated upon exposure to acid-stress. The transcriptomic results were further confirmed via quantitative polymerase chain reaction analysis. Moreover, acid-stressed LGG demonstrated an enhanced mucin-binding ability in vitro, with 1 log more LGG cells (p < 0.05) bound to a mucin layer in a 96-well culture plate as compared to the control. The enhanced intestinal binding ability of acid-stressed LGG was confirmed in an animal study, wherein significantly more viable LGG cells (≥ 2 log CFU/g) were observed in the ileum, caecum, and colon of acid-stressed LGG-treated mice as compared with a non-acid-stressed LGG-treated control group. To our knowledge, this is the first report showing that acid stress enhanced the intestine-binding ability of LGG through the induction of pili-related genes.