Bifidobacterium longum subsp. longum BG-L47 boosts growth and activity of Limosilactobacillus reuteri DSM 17938 and its extracellular membrane vesicles.

The aim of this study was to identify a Bifidobacterium strain that improves the performance of Limosilactobacillus reuteri DSM 17938. Initial tests showed that Bifidobacterium longum subsp. longum strains boosted the growth of DSM 17938 during in vivo-like conditions. Further characterization revealed that one of the strains, BG-L47, had better bile and acid tolerance compared to BG-L48, as well as mucus adhesion compared to both BG-L48 and the control strain BB536. BG-L47 also had the capacity to metabolize a broad range of carbohydrates and sugar alcohols. Mapping of glycoside hydrolase (GH) genes of BG-L47 and BB536 revealed many GHs associated with plant-fiber utilization. However, BG-L47 had a broader phenotypic fiber utilization capacity. In addition, B. longum subsp. longum cells boosted the bioactivity of extracellular membrane vesicles (MV) produced by L. reuteri DSM 17938 during co-cultivation. Secreted 5'-nucleotidase (5'NT), an enzyme that converts AMP into the signal molecule adenosine, was increased in MV boosted by BG-L47. The MV exerted an improved antagonistic effect on the pain receptor transient receptor potential vanilloid 1 (TRPV1) and increased the expression of the immune development markers IL-6 and IL-1ß in a peripheral blood mononuclear cell (PBMC) model. Finally, the safety of BG-L47 was evaluated both by genome safety assessment and in a human safety study. Microbiota analysis showed that the treatment did not induce significant changes in the composition. In conclusion, B. longum subsp. longum BG-L47 has favorable physiological properties, can boost the in vitro activity of L. reuteri DSM 17938, and is safe for consumption, making it a candidate for further evaluation in probiotic studies. IMPORTANCE: By using probiotics that contain a combination of strains with synergistic properties, the likelihood of achieving beneficial interactions with the host can increase. In this study, we first performed a broad screening of Bifidobacterium longum subsp. longum strains in terms of synergistic potential and physiological properties. We identified a superior strain, BG-L47, with favorable characteristics and potential to boost the activity of the known probiotic strain Limosilactobacillus reuteri DSM 17938. Furthermore, we demonstrated that BG-L47 is safe for consumption in a human randomized clinical study and by performing a genome safety assessment. This work illustrates that bacteria-bacteria interactions differ at the strain level and further provides a strategy for finding and selecting companion strains of probiotics.

The Lreu_1276 protein from Limosilactobacillus reuteri represents a third family of dihydroneopterin triphosphate pyrophosphohydrolases in bacteria.

Tetrahydrofolate is a cofactor involved in C1 metabolism including biosynthesis pathways for adenine and serine. In the classical tetrahydrofolate biosynthesis pathway, the steps removing three phosphate groups from the precursor 7,8-dihydroneopterin triphosphate (DHNTP) remain unclear in many bacteria. DHNTP pyrophosphohydrolase hydrolyzes pyrophosphate from DHNTP and produces 7,8-dihydroneopterin monophosphate. Although two structurally distinct DHNTP pyrophosphohydrolases have been identified in the intestinal bacteria Lactococcus lactis and Escherichia coli, the distribution of their homologs is limited. Here, we aimed to identify a third DHNTP pyrophosphohydrolase gene in the intestinal lactic acid bacterium Limosilactobacillus reuteri. In a gene operon including genes involved in dihydrofolate biosynthesis, we focused on the lreu_1276 gene, annotated as Ham1 family protein or XTP/dITP diphosphohydrolase, as a candidate encoding DHNTP pyrophosphohydrolase. The Lreu_1276 recombinant protein was prepared using E. coli and purified. Biochemical analyses of the reaction product revealed that the Lreu_1276 protein displays significant pyrophosphohydrolase activity toward DHNTP. The optimal reaction temperature and pH were 35°C and around 7, respectively. Substrate specificity was relatively strict among 17 tested compounds. Although previously characterized DHNTP pyrophosphohydrolases prefer Mg2+, the Lreu_1276 protein exhibited maximum activity in the presence of Mn2+, with a specific activity of 28.2 ± 2.0 µmol min-1 mg-1 in the presence of 1 mM Mn2+. The three DHNTP pyrophosphohydrolases do not share structural similarity to one another, and the distribution of their homologs does not overlap, implying that the Lreu_1276 protein represents a third structurally novel DHNTP pyrophosphohydrolase in bacteria. IMPORTANCE: The identification of a structurally novel DHNTP pyrophosphohydrolase in L. reuteri provides valuable information in understanding tetrahydrofolate biosynthesis in bacteria that possess lreu_1276 homologs. Interestingly, however, even with the identification of a third family of DHNTP pyrophosphohydrolases, there are still a number of bacteria that do not harbor homologs for any of the three genes while possessing other genes involved in the biosynthesis of the pterin ring structure. This suggests the presence of an unrecognized DHNTP pyrophosphohydrolase gene in bacteria. As humans do not harbor DHNTP pyrophosphohydrolase, the high structural diversity of enzymes responsible for a reaction in tetrahydrofolate biosynthesis may provide an advantage in designing inhibitors targeting a specific group of bacteria in the intestinal microbiota.

Effect of soybean proteins and peptides on the growth and adhesive ability of Limosilactobacillus Reuteri DSM17938.

Limosilactobacillus reuteri DSM17938 is one of the most pivotal probiotics, whose general beneficial effects on the intestinal microbiota are well recognized. Enhancing their growth and metabolic activity can effectively regulate the equilibrium of intestinal microbiota, leading to improved physical health. A common method to promote the growth of Lactobacillus is the addition of prebiotics. Current research suggests that proteins and their hydrolysates from different sources with potential prebiotic activity can also promote the growth of probiotics. In this study, soybean proteins and peptides were effective in promoting the growth, organic acid secretion, and adhesive properties of Limosilactobacillus reuteri DSM17938 to Caco-2 cells. These results illustrate the feasibility of soybean proteins and peptides as prebiotics, providing theoretical and practical advantages for their application.

The prevention effect of Limosilactobacillus reuteri on acute kidney injury by regulating gut microbiota.

Acute kidney injury (AKI) has considerably high morbidity and mortality but we do not have proper treatment for it. There is an urgent need to develop new prevention or treatment methods. Gut microbiota has a close connection with renal diseases and has become the new therapy target for AKI. In this study, we found the oral administration of the probiotic Limosilactobacillus reuteri had a prevention effect on the AKI induced by lipopolysaccharide (LPS). It reduced serum concentration of creatinine and urea nitrogen and protected the renal cells from necrosis and apoptosis. Meanwhile, L. reuteri improved the gut barrier function, which is destroyed in AKI, and modulated the gut microbiota and relevant metabolites. Compared with the LPS group, L. reuteri increased the proportion of Proteobacteria and reduced the proportion of Firmicutes, changing the overall structure of the gut microbiota. It also influenced the fecal metabolites and changed the metabolite pathways, such as tyrosine metabolism, pentose and glucuronate interconversions, galactose metabolism, purine metabolism, and insulin resistance. These results showed that L. reuteri is a potential therapy for AKI as it helps in sustaining the gut barrier integrity and modulating gut microbiota and related metabolites.

Taking SCFAs produced by Lactobacillus reuteri orally reshapes gut microbiota and elicits antitumor responses.

BACKGROUND: Colorectal cancer (CRC) incidence is increasing in recent years due to intestinal flora imbalance, making oral probiotics a hotspot for research. However, numerous studies related to intestinal flora regulation ignore its internal mechanisms without in-depth research. RESULTS: Here, we developed a probiotic microgel delivery system (L.r@(SA-CS)2) through the layer-by-layer encapsulation technology of alginate (SA) and chitosan (CS) to improve gut microbiota dysbiosis and enhance anti-tumor therapeutic effect. Short chain fatty acids (SCFAs) produced by L.r have direct anti-tumor effects. Additionally, it reduces harmful bacteria such as Proteobacteria and Fusobacteriota, and through bacteria mutualophy increases beneficial bacteria such as Bacteroidota and Firmicutes which produce butyric acid. By binding to the G protein-coupled receptor 109A (GPR109A) on the surface of colonic epithelial cells, butyric acid can induce apoptosis in abnormal cells. Due to the low expression of GPR109A in colon cancer cells, MK-6892 (MK) can be used to stimulate GPR109A. With increased production of butyrate, activated GPR109A is able to bind more butyrate, which further promotes apoptosis of cancer cells and triggers an antitumor response. CONCLUSION: It appears that the oral administration of L.r@(SA-CS)2 microgels may provide a treatment option for CRC by modifying the gut microbiota.

Newly Isolated Limosilactobacillus reuteri B1/1 Modulates the Expression of Cytokines and Antimicrobial Proteins in a Porcine ex Vivo Model.

BACKGROUND: The epithelia of the intestine perform various functions, playing a crucial role in providing a physical barrier and an innate immune defense against infections. By generating a "three-dimensional" (3D) model of cell co-cultures using the IPEC-J2 cell line and porcine blood monocyte-derived macrophages (MDMs), we are getting closer to mimicking the porcine intestine ex vivo.Methods: The effect of Limosilactobacillus reuteri B1/1 and Limosilactobacillus fermentum CCM 7158 (indicator strain) on the relative gene expression of interleukins (IL-1ß, IL-6, IL-8, IL-18 and IL-10), genes encoding receptors for TLR4 and TLR2, tight junction proteins such as claudin-1 (CLDN1), occludin (OCLN) and important antimicrobial proteins such as lumican (LUM) and olfactomedin-4 (OLMF-4) was monitored in this model. RESULTS: The results obtained from this pilot study point to the immunomodulatory potential of newly isolated L. reuteri B1/1, as it was able to suppress the enhanced pro-inflammatory response to lipopolysaccharide (LPS) challenge in both cell types. L. reuteri B1/1 was even able to up-regulate the mRNA levels of genes encoding antimicrobial proteins LUM and OLFM-4 and to increase tight junction (TJ)-related genes CLDN1 and OCLN, which were significantly down-regulated in LPS-induced IPEC-J2 cells. Conversely, L. fermentum CCM 7158, chosen as an indicator lactic acid bacteria (LAB) strain, increased the mRNA levels of the investigated pro-inflammatory cytokines (IL-18, IL-6, and IL-1ß) in MDMs when LPS was simultaneously applied to basally deposited macrophages. Although L. fermentum CCM 7158 induced the production of pro-inflammatory cytokines, synchronous up-regulation of the anti-inflammatory cytokine IL-10 was detected in both LAB strains used in both cell cultures. CONCLUSIONS: The obtained results suggest that the recently isolated LAB strain L. reuteri B1/1 has the potential to alleviate epithelial disruption caused by LPS and to influence the production of antimicrobial molecules by enterocytes.

Effects of Limosilactobacillus reuteri strains PTA-126787 and PTA-126788 on intestinal barrier integrity and immune homeostasis in an alcohol-induced leaky gut model.

Intestinal barrier is a first line of defense that prevents entry of various harmful substances from the lumen into the systemic environment. Impaired barrier function with consequent translocation of harmful substances into systemic circulation ("leaky gut") is a central theme in many gastrointestinal, autoimmune, mental, and metabolic diseases. Probiotics have emerged as a promising strategy to maintain intestinal integrity and address "leaky gut". Using in silico, in vitro and avian in vivo analyses, we previously showed that two novel L. reuteri strains, PTA-126787 (L. reuteri 3630) and PTA-126788 (L. reuteri 3632), isolated from broiler chickens possess favorable safety profiles. Consistent with a recent study, here we show that L. reuteri 3630 and 3632 are phylogenetically similar to human L. reuteri strains. Daily administration of high doses of L. reuteri 3630 and 3632 to Sprague Dawley rats for 28 days was found to be safe with no adverse effects. More importantly, administration of L. reuteri 3630 and 3632 significantly reduced markers associated with alcohol-induced leaky gut, by downregulating inflammatory cytokines and upregulating anti-inflammatory cytokines in an alcohol model of leaky gut in mice. While L. reuteri 3630 cells and supernatant showed no activation, L. reuteri 3632 cells but not supernatant showed activation of AhR, a key transcription factor that regulates gut and immune homeostasis. L. reuteri 3630 is creamish white in morphology typical of Lactobacillus species and L. reuteri 3632 displays a unique orange pigmentation, which was stable even after passaging for 480 generations. We identified a rare polyketide biosynthetic gene cluster in L. reuteri 3632 that likely encodes for the orange-pigmented secondary metabolite. Similar to L. reuteri 3632 cells, the purified orange metabolite activated AhR. All together, these data provide evidence on the phylogenetic relatedness, safety, efficacy, and one of the likely mechanisms of action of L. reuteri 3630 and 3632 for potential probiotic applications to address "leaky gut" and associated pathologies in humans.

Crystal structure of the long Rib domain of the LPXTG-anchored surface protein from Limosilactobacillus reuteri.

The Rib domain, which is often found as tandem-repeat structural modules in surface proteins of Gram-positive bacteria, plays important roles in mediating interactions of bacteria with their environments and hosts. A comprehensive structural analysis of various Rib domains is essential to fully understand their impact on the structure and functionality of these bacterial adhesins. To date, structural information has been limited for this expansive group of domains. In this study, the high-resolution crystal structure of the second member of the long Rib domain, a unique subclass within the Rib-domain family, derived from Limosilactobacillus reuteri is presented. The data not only demonstrate a highly conserved structure within the long Rib domain, but also highlight an evolutionary convergence in structural architecture with other modular domains found in cell-adhesion molecules.

Prophylactic Supplementation with Lactobacillus Reuteri or Its Metabolite GABA Protects Against Acute Ischemic Cardiac Injury.

The gut microbiome has emerged as a potential target for the treatment of cardiovascular disease. Ischemia/reperfusion (I/R) after myocardial infarction is a serious complication and whether certain gut bacteria can serve as a treatment option remains unclear. Lactobacillus reuteri (L. reuteri) is a well-studied probiotic that can colonize mammals including humans with known cholesterol-lowering properties and anti-inflammatory effects. Here, the prophylactic cardioprotective effects of L. reuteri or its metabolite γ-aminobutyric acid (GABA) against acute ischemic cardiac injury caused by I/R surgery are demonstrated. The prophylactic gavage of L. reuteri or GABA confers cardioprotection mainly by suppressing cardiac inflammation upon I/R. Mechanistically, GABA gavage results in a decreased number of proinflammatory macrophages in I/R hearts and GABA gavage no longer confers any cardioprotection in I/R hearts upon the clearance of macrophages. In vitro studies with LPS-stimulated bone marrow-derived macrophages (BMDM) further reveal that GABA inhibits the polarization of macrophages toward the proinflammatory M1 phenotype by inhibiting lysosomal leakage and NLRP3 inflammasome activation. Together, this study demonstrates that the prophylactic oral administration of L. reuteri or its metabolite GABA attenuates macrophage-mediated cardiac inflammation and therefore alleviates cardiac dysfunction after I/R, thus providing a new prophylactic strategy to mitigate acute ischemic cardiac injury.

Gut microbiota metabolite indole-3-acetic acid maintains intestinal epithelial homeostasis through mucin sulfation.

The global incidence and prevalence of inflammatory bowel disease (IBD) are gradually increasing. A high-fat diet (HFD) is known to disrupt intestinal homeostasis and aggravate IBD, yet the underlying mechanisms remain largely undefined. Here, a positive correlation between dietary fat intake and disease severity in both IBD patients and murine colitis models is observed. A HFD induces a significant decrease in indole-3-acetic acid (IAA) and leads to intestinal barrier damage. Furthermore, IAA supplementation enhances intestinal mucin sulfation and effectively alleviates colitis. Mechanistically, IAA upregulates key molecules involved in mucin sulfation, including 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (Papss2) and solute carrier family 35 member B3 (Slc35b3), the synthesis enzyme and the transferase of 3'-phosphoadenosine-5'-phosphosulfate (PAPS), via the aryl hydrocarbon receptor (AHR). More importantly, AHR can directly bind to the transcription start site of Papss2. Oral administration of Lactobacillus reuteri, which can produce IAA, contributes to protecting against colitis and promoting mucin sulfation, while the modified L. reuteri strain lacking the iaaM gene (LactobacillusΔiaaM) and the ability to produce IAA fail to exhibit such effects. Overall, IAA enhances intestinal mucin sulfation through the AHR-Papss2-Slc35b3 pathway, contributing to the protection of intestinal homfeostasis.

A HFD can lead to the development of colitis by disrupting tryptophan metabolism in the gut microbiome and lowering levels of IAA. Supplementation with IAA has been shown to alleviate colitis in mice and improve intestinal barrier function. It is believed that IAA may activate the AHR to upregulate the expression of Papss2 and Slc35b3, promoting sulfation modification of mucins and protecting the intestinal barrier. HFD, high-fat diet; AHR, aryl hydrocarbon receptor; IAA, indole-3-acetic acid; Papss2, 3'-phosphoadenosine 5'-phosphosulfate synthase 2; Slc35b3, solute carrier family 35 member B3.

Polygonatum kingianum Polysaccharides Enhance the Preventive Efficacy of Heat-Inactivated Limosilactobacillus reuteri WX-94 against High-Fat-High-Sucrose-Induced Liver Injury and Gut Dysbacteriosis.

Limosilactobacillus reuteri (L. reuteri) is an efficacious probiotic that could reduce inflammation and prevent metabolic disorders. Here, we innovatively found that Polygonatum kingianum polysaccharides (PKP) promoted proliferation and increased stability of L. reuteri WX-94 (a probiotic strain showing anti-inflammation potentials) in simulated digestive fluids in vitro. PKP was composed of galactose, glucose, mannose, and arabinose. The cell-free supernatant extracted from L. reuteri cultured with PKP increased ABTS•+, DPPH•, and FRAP scavenging capacities compared with the supernatant of the medium without PKP and increased metabolites with health-promoting activities, e.g., 3-phenyllactic acid, indole-3-lactic acid, indole-3-carbinol, and propionic acid. Moreover, PKP enhanced alleviating effects of heat-inactivated L. reuteri on high-fat-high-sucrose-induced liver injury in rats via reducing inflammation and regulating expressions of protein and genes involved in fatty acid metabolism (such as HIF1-α, FAßO, CPT1, and AMPK) and fatty acid profiles in liver. Such benefits correlated with its prominent effects on enriching Lactobacillus and short-chain fatty acids while reducing Dubosiella, Fusicatenilacter, Helicobacter, and Oscillospira. Our work provides novel insights into the probiotic property of PKP and emphasizes the great potential of the inactivated L. reuteri cultured with PKP in contracting unhealthy diet-induced liver dysfunctions and gut dysbacteriosis.

Milk with Juçara (Euterpe edulis Martius) Pulp: Fermentation by L. reuteri LR92 and Reuterin Production in Situ

Abstract This study aimed to develop and evaluate fermented milk by Lactobacillus reuteri LR92 with addition of juçara pulp (FMJ) and reuterin production in situ. The fermentation process was analyzed for 24 hours and the storage of FMJ for 30 days at 4 °C. During the fermentation, there was consumption of 25% (w / v) of lactose and increase of 0.01 to 0.85% (w / v) of lactic acid. The FMJ presented 0.43 ± 0.01 mM of reuterin, inhibiting Staphylococcus aureus strains under in vitro test. For the carbohydrates, the percentages (g.100g-1) found were 7.31 ± 1.07; 9.19 ± 0.82; 1.60 ± 0.50 and 0.08 ± 0.00 for sucrose, lactose, galactose and fructose respectively. The survival of L. reuteri, present in FMJ, was 2.47 log CFU / mL after 6 hours of gastrointestinal simulation. In sensory analysis FMJ received a grade 7 for global acceptance indicating good acceptance of the product.

Molecular characterisation and biomass and metabolite production of Lactobacillus reuteri LPB P01-001: a potential probiotic

Lactobacillus reuteri LPB P01-001 was isolated from the gastrointestinal tract of wild swine and was characterised by biochemical testing and sequencing of gene 16S rRNA. A simple and low-cost culture medium based on cane sugar (2.5% p/v) and yeast extract (1% p/v) was used in the production of this probiotic. The fermentative conditions were a) pH control at 6.5 and b) no pH control; both were set at 37°C in a 12 L slightly stirred tank bioreactor. Fermentation parameters such as the specific growth rate, productivity and yield of biomass, lactic and acetic acid levels were determined. L. reuteri LPB P01-001 behaves as an aciduric bacteria because it grows better in a low pH medium without pH control. However, the lactic acid production yield was practically half (9.22 g.L-1) of that obtained under a constant pH of 6.5, which reached 30.5 g.L-1 after 28 hours of fermentation. The acetic acid production was also higher under pH-controlled fermentation, reaching 10.09 g.L-1 after 28 hours of fermentation. These parameters may raise the interest of those committed to the efficient production of a probiotic agent for swine.