Oxygen Transfer Effect on the Growth of Limosilactobacillus reuteri ATCC 53608 and on Its Metabolic Capacity.

Limosilactobacillus reuteri is a probiotic microorganism used in the treatment of gastrointestinal disorders. The effect of oxygen transfer on cultures of L. reuteri ATCC 53608 at shake flask and stirred tank bioreactor scales was studied, using MRS and molasses-based media. At shake flask scale, in MRS medium, a maximum bacterial concentration of 2.01 ± 0.02 g L-1 was obtained; the oxygen transfer coefficient was 2.01 ± 0.04 h-1. Similarly, in a 7.5 L bioreactor, in MRS, a maximum bacterial concentration of 2.46 ± 0.16 g L-1 was achieved (kLa = 2.64 ± 0.06 h-1). In contrast, using a molasses-based medium, bacterial concentration reached 3.13 ± 0.17 g L-1 in the 7.5 L bioreactor. A progressive reduction in lactic acid concentration and yield was observed as the oxygen transfer coefficient increased, at shake flask scale. Also, the oxygen transfer coefficient strongly affected the growth of L. reuteri in shake flask and bioreactor and allowed us to successfully scale up L. reuteri culture, producing similar maximum bacterial concentrations in both scales (2.01 g L-1 and 2.46 g L-1 in MRS). This is the first study on oxygen transfer coefficients in L. reuteri, and it is a valuable contribution to the field as it provides important insights about how this organism tolerates oxygen and adapts its metabolism for larger biomass production.

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.

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.

Innovative method to grow the probiotic Lactobacillus reuteri in the omega3-rich microalga Isochrysis galbana.

Microalgae are natural sources of valuable bioactive compounds, such as polyunsaturated fatty acids (PUFAs), that show antioxidant, anti-inflammatory, anticancer and antimicrobial activities. The marine microalga Isochrysis galbana (I. galbana) is extremely rich in ω3 PUFAs, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Probiotics are currently suggested as adjuvant therapy in the management of diseases associated with gut dysbiosis. The Lactobacillus reuteri (L. reuteri), one of the most widely used probiotics, has been shown to produce multiple beneficial effects on host health. The present study aimed to present an innovative method for growing the probiotic L. reuteri in the raw seaweed extracts from I. galbana as an alternative to the conventional medium, under conditions of oxygen deprivation (anaerobiosis). As a result, the microalga I. galbana was shown for the first time to be an excellent culture medium for growing L. reuteri. Furthermore, the gas-chromatography mass-spectrometry analysis showed that the microalga-derived ω3 PUFAs were still available after the fermentation by L. reuteri. Accordingly, the fermented compound (FC), obtained from the growth of L. reuteri in I. galbana in anaerobiosis, was able to significantly reduce the adhesiveness and invasiveness of the harmful adherent-invasive Escherichia coli to intestinal epithelial cells, due to a cooperative effect between L. reuteri and microalgae-released ω3 PUFAs. These findings open new perspectives in the use of unicellular microalgae as growth medium for probiotics and in the production of biofunctional compounds.

Fecal miR-142a-3p from dextran sulfate sodium-challenge recovered mice prevents colitis by promoting the growth of Lactobacillus reuteri.

Feces are enriched with microRNAs (miRNAs) that shape the gut microbiota. These miRNAs are differentially expressed in the feces of healthy and diseased subjects. However, whether fecal miRNAs in subjects with inflammatory bowel diseases are involved in regulating microbiota composition and whether they have any beneficial effects remains unknown. Here, we studied the fecal microbiome composition and miRNA abundance in mice with dextran sulfate sodium (DSS)-induced colitis and mice at the recovery phase to explore different miRNAs expressed, their relations with microbial abundance, and their effects on colitis. We found that miR-142a-3p expression was significantly increased in the feces of mice recovered from colitis and that it could alleviate disease symptoms in mice treated with DSS in a microbiome-dependent manner. Specifically, miR-142a-3p promoted the growth of Lactobacillus reuteri, which had a high abundance in the feces of mice recovered from colitis, by regulating transcripts of polA and locus tag LREU_RS03575. Moreover, L. reuteri, as well as its metabolite reuterin, could alleviate DSS-induced disease symptoms. These results highlight the role of fecal miR-142a-3p in the prevention of colitis. We propose that the feces of subjects who have recovered from diseases might be enriched with miRNAs with preventive effects against those diseases.

Flow cytometric analysis reveals culture condition dependent variations in phenotypic heterogeneity of Limosilactobacillus reuteri.

Optimisation of cultivation conditions in the industrial production of probiotics is crucial to reach a high-quality product with retained probiotic functionality. Flow cytometry-based descriptors of bacterial morphology may be used as markers to estimate physiological fitness during cultivation, and can be applied for online monitoring to avoid suboptimal growth. In the current study, the effects of temperature, initial pH and oxygen levels on cell growth and cell size distributions of Limosilactobacillus reuteri DSM 17938 were measured using multivariate flow cytometry. A pleomorphic behaviour was evident from the measurements of light scatter and pulse width distributions. A pattern of high growth yielding smaller cells and less heterogeneous populations could be observed. Analysis of pulse width distributions revealed significant morphological heterogeneities within the bacterial cell population under non-optimal growth conditions, and pointed towards low temperature, high initial pH, and high oxygen levels all being triggers for changes in morphology towards cell chain formation. However, cell size did not correlate to survivability after freeze-thaw or freeze-drying stress, indicating that it is not a key determinant for physical stress tolerance. The fact that L. reuteri morphology varies depending on cultivation conditions suggests that it can be used as marker for estimating physiological fitness and responses to its environment.

Exoproteome Analysis of Antagonistic Interactions between the Probiotic Bacteria Limosilactobacillus reuteri LR1 and Lacticaseibacillus rhamnosus F and Multidrug Resistant Strain of Klebsiella pneumonia.

The expansion of multiple drug resistant (MDR) strains of Klebsiella pneumoniae presents an immense threat for public health. Annually, this microorganism causes thousands of lethal nosocomial infections worldwide. Currently, it has been shown that certain strains of lactic acid bacteria (LAB) can efficiently inhibit growth of K. pneumoniae and the formation of its biofilms; however, the active principle of such action remains unknown. In the current article, the growth inhibition of MDR K. pneumoniae by two LAB-Limosilactobacillus reuteri LR1 and Lacticaseibacillus rhamnosus F-is demonstrated, and the nature of this inhibition studied at the level of exoproteome. This article shows that the exoproteomes of studied LAB contains both classically and non-classically secreted proteins. While for L. reuteri LR1 the substantial portion of classically secreted proteins was presented by cell-wall-degrading enzymes, for L. rhamnosus F only one out of four classically secreted proteins was presented by cell-wall hydrolase. Non-classically secreted proteins of both LAB were primarily metabolic enzymes, for some of which a possible moonlighting functioning was proposed. These results contribute to knowledge regarding antagonistic interaction between LAB and pathogenic and opportunistic microorganisms and set new perspectives for the use of LAB to control the spread of these microorganisms.

Genome-scale insights into the metabolic versatility of Limosilactobacillus reuteri.

BACKGROUND: Limosilactobacillus reuteri (earlier known as Lactobacillus reuteri) is a well-studied lactic acid bacterium, with some specific strains used as probiotics, that exists in different hosts such as human, pig, goat, mouse and rat, with multiple body sites such as the gastrointestinal tract, breast milk and mouth. Numerous studies have confirmed the beneficial effects of orally administered specific L. reuteri strains, such as preventing bone loss and promoting regulatory immune system development. L. reuteri ATCC PTA 6475 is a widely used strain that has been applied in the market as a probiotic due to its positive effects on the human host. Its health benefits may be due, in part, to the production of beneficial metabolites. Considering the strain-specific effects and genetic diversity of L. reuteri strains, we were interested to study the metabolic versatility of these strains. RESULTS: In this study, we aimed to systematically investigate the metabolic features and diversities of L. reuteri strains by using genome-scale metabolic models (GEMs). The GEM of L. reuteri ATCC PTA 6475 was reconstructed with a template-based method and curated manually. The final GEM iHL622 of L. reuteri ATCC PTA 6475 contains 894 reactions and 726 metabolites linked to 622 metabolic genes, which can be used to simulate growth and amino acids utilization. Furthermore, we built GEMs for the other 35 L. reuteri strains from three types of hosts. The comparison of the L. reuteri GEMs identified potential metabolic products linked to the adaptation to the host. CONCLUSIONS: The GEM of L. reuteri ATCC PTA 6475 can be used to simulate metabolic capabilities and growth. The core and pan model of 35 L. reuteri strains shows metabolic capacity differences both between and within the host groups. The GEMs provide a reliable basis to investigate the metabolism of L. reuteri in detail and their potential benefits on the host.

A concerted probiotic activity to inhibit periodontitis-associated bacteria.

Periodontitis can result in tooth loss and the associated chronic inflammation can provoke several severe systemic health risks. Adjunctive to mechanical treatment of periodontitis and as alternatives to antibiotics, the use of probiotic bacteria was suggested. In this study, the inhibitory effect of the probiotic Streptococcus salivarius subsp. salivarius strains M18 and K12, Streptococcus oralis subsp. dentisani 7746, and Lactobacillus reuteri ATCC PTA 5289 on anaerobic periodontal bacteria and Aggregatibacter actinomycetemcomitans was tested. Rarely included in other studies, we also quantified the inverse effect of pathogens on probiotic growth. Probiotics and periodontal pathogens were co-incubated anaerobically in a mixture of autoclaved saliva and brain heart infusion broth. The resulting genome numbers of the pathogens and of the probiotics were measured by quantitative real-time PCR. Mixtures of the streptococcal probiotics were also used to determine their synergistic, additive, or antagonistic effects. The overall best inhibitor of the periodontal pathogens was L. reuteri ATCC PTA 5289, but the effect is coenzyme B12-, anaerobiosis-, as well as glycerol-dependent, and further modulated by L. reuteri strain DSM 17938. Notably, in absence of glycerol, the pathogen-inhibitory effect could even turn into a growth spurt. Among the streptococci tested, S. salivarius M18 had the most constant inhibitory potential against all pathogens, followed by K12 and S. dentisani 7746, with the latter still having significant inhibitory effects on P. intermedia and A. actinomycetemcomitans. Overall, mixtures of the streptococcal probiotics did inhibit the growth of the pathogens equally or-in the case of A. actinomycetemcomitans- better than the individual strains. P. gingivalis and F. nucleatum were best inhibited by pure cultures of S. salivarius K12 or S. salivarius M18, respectively. Testing inverse effects, the growth of S. salivarius M18 was enhanced when incubated with the periodontal pathogens minus/plus other probiotics. In contrast, S. oralis subsp. dentisani 7746 was not much influenced by the pathogens. Instead, it was significantly inhibited by the presence of other streptococcal probiotics. In conclusion, despite some natural limits such as persistence, the full potential for probiotic treatment is by far not utilized yet. Especially, further exploring concerted activity by combining synergistic strains, together with the application of oral prebiotics and essential supplements and conditions, is mandatory.

Resveratrol-mediated attenuation of superantigen-driven acute respiratory distress syndrome is mediated by microbiota in the lungs and gut.

Acute Respiratory Distress Syndrome (ARDS) is triggered by a variety of agents, including Staphylococcal Enterotoxin B (SEB). Interestingly, a significant proportion of patients with COVID-19, also develop ARDS. In the absence of effective treatments, ARDS results in almost 40% mortality. Previous studies from our laboratory demonstrated that resveratrol (RES), a stilbenoid, with potent anti-inflammatory properties can attenuate SEB-induced ARDS. In the current study, we investigated the role of RES-induced alterations in the gut and lung microbiota in the regulation of ARDS. Our studies revealed that SEB administration induced inflammatory cytokines, ARDS, and 100% mortality in C3H/HeJ mice. Additionally, SEB caused a significant increase in pathogenic Proteobacteria phylum and Propionibacterium acnes species in the lungs. In contrast, RES treatment attenuated SEB-mediated ARDS and mortality in mice, and significantly increased probiotic Actinobacteria phylum, Tenericutes phylum, and Lactobacillus reuteri species in both the colon and lungs. Colonic Microbiota Transplantation (CMT) from SEB-injected mice that were treated with RES as well as the transfer of L. reuteri into recipient mice inhibited the production of SEB-mediated induction of pro-inflammatory cytokines such as IFN-γ and IL-17 but increased that of anti-inflammatory IL-10. Additionally, such CMT and L. reuteri recipient mice exposed to SEB, showed a decrease in lung-infiltrating mononuclear cells, cytotoxic CD8+ T cells, NKT cells, Th1 cells, and Th17 cells, but an increase in the population of regulatory T cells (Tregs) and Th3 cells, and increase in the survival of mice from SEB-mediated ARDS. Together, the current study demonstrates that ARDS induced by SEB triggers dysbiosis in the lungs and gut and that attenuation of ARDS by RES may be mediated, at least in part, by alterations in microbiota in the lungs and the gut, especially through the induction of beneficial bacteria such as L. reuteri.

Culture conditions affect Lactobacillus reuteri DSM 17938 ability to perform glycerol bioconversion into 3-hydroxypropionic acid.

The platform molecule 3-hydroxypropionic acid (3-HP) can be produced using Lactobacillus reuteri through a two-step bioprocess that involves a growth phase followed by a bioconversion phase. The bioproduction is performed by resting cells that convert glycerol into 3-HP and 1,3-propanediol in fed-batch mode. This work aimed at studying the effect of the growth conditions of L. reuteri DSM 17938 during the first step, on the glycerol bioconversion into 3-HP during the second step. A Plackett and Burman design was carried out to test, in controlled bioreactors, the effect of 11 growth conditions simultaneously, at fixed bioconversion conditions. The supplementation of the growth medium with vitamin B12 and cysteine displayed a negative effect on the 3-HP bioproduction. The addition of glucose, phytone peptone, Tween 80, 1,2-propanediol and betaine in the growth medium, together with a low temperature and an optimal pH of 6.0 during the growth phase increased the bioconversion duration from 56 h to 89 h at a glycerol feeding rate of 0.5 g·h-1. A validating experiment displayed that the 3-HP titer, 3-HP production yield and 3-HP specific production rate were significantly improved by 25 %, 150 % and 61 %, respectively.

Characterization of Lactobacillus reuteri WQ-Y1 with the ciprofloxacin degradation ability.

OBJECT: As a broad-spectrum fluoroquinolone antibiotic drug, ciprofloxacin (CIP) is frequently used in the treatment of a wide variety of infections. However, the residues of this antibiotic pose a big threat to the aquatic environment and human health. In this research, Lactobacillus reuteri WQ-Y1 with CIP degradation ability was screened and identified. RESULTS: L. reuteri WQ-Y1 with a degradation rate of 65.1% for 4 µg mL-1 CIP was screened from 17 lactic acid bacteria (LAB), and cytochrome P450 enzyme was confirmed to promote the degradation of CIP by L. reuteri WQ-Y1. Meanwhile, the CIP degradation rate were also higher in 48 h' culture time when co-cultured with 1 mg/mL of glucose in the culture media. Furthermore, result also proved that fluoroquinolone antibiotics with the similar piperazine ring structures could be degraded by L. reuteri WQ-Y1. CONCLUSIONS: L. reuteri WQ-Y1 could degrade fluoroquinolone antibiotics with the similar piperazine ring structure. However, future work still needs to be done on the confirmation of the key enzymes in the cytochrome P450 enzymes family in the biodegradation. The isolated ciprofloxacin-degrading strain L. reuteri WQ-Y1 had a CIP degradation rate of 65.1% at 24 hours, and one biodegradation metabolite was identified and proved to be an important metabolite of CIP from cytochrome P450 enzymes family hydrolysis with UPLC-MS/MS spectrograms approach.

Lactobacillus reuteri DSM 17938 and ATCC PTA 5289 ameliorates chemotherapy-induced oral mucositis.

Oral mucositis (OM) is a common complication of cancer therapy, however OM management remains unsatisfactory. There is a growing interest in the therapeutic potential of probiotics in OM due to positive findings of its use in intestinal mucositis. This study aimed to determine the efficacy and safety of the probiotic combination Lactobacillus reuteri DSM 17938 and ATCC PTA 5289 strains in chemotherapy-induced OM. Mice were divided into 4 groups. PBS/water and PBS/LR groups comprised of mice injected with PBS intraperitoneally (i.p.), and were given water or the mixture of L. reuteri (LR) DSM 17938 and ATCC PTA 5289 in water respectively. The 5-FU/water and 5-FU/LR groups comprised of mice injected with 5-FU i.p., and were given water or L. reuteri DSM 17938 and ATCC PTA 5289 in water respectively. Histopathological analysis revealed that the oral epithelia of the 5-FU/water and 5-FU/LR groups were thinner compared to PBS/water and PBS/LR groups. However, epithelial damage was significantly reduced in the 5-FU/LR compared to 5-FU/water group. Additionally, the 5-FU/LR group showed reduced oxidative stress and inflammation in the oral mucosa. We further showed that L. reuteri reduced oxidative stress through the nuclear factor E2-related factor-2 (Nrf-2) signalling. There was no evidence of translocation of L. reuteri systemically. This study demonstrated for the first time that L. reuteri protected oral mucosa against damage induced by chemotherapy.

A modified reinforced clostridial medium for the isolation and enumeration of Lactobacillus delbrueckii ssp. bulgaricus in a mixed culture.

In this study, we modified reinforced clostridial medium (RCM) to selectively enumerate and isolate Lactobacillus delbrueckii ssp. bulgaricus, a probiotic and important starter culture in the dairy industry. The disparity in the reported carbohydrate fermentation pattern of L. delbrueckii ssp. bulgaricus was used to develop a growth medium not only selective for L. delbrueckii ssp. bulgaricus but significantly inhibitory to the growth of other lactic acid bacteria. A recently modified RCM (mRCM) was optimized for this study by the addition of 0.5% fructose, 0.5% dextrose, 1% maltose, and 0.25% sodium pyruvate while replacing lactose as a carbohydrate source. The cell recovery and bacterial counts of L. delbrueckii ssp. bulgaricus in tested products (pure L. delbrueckii ssp. bulgaricus strains, starter culture, probiotic supplements, and yogurt) using our mRCM with sodium pyruvate (mRCM-PYR) were significantly higher than in the recently modified RCM and the common de Man, Rogosa, and Sharpe (MRS) culture medium. The growth of other lactic acid bacteria (Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus reuteri) and Bifidobacteria was retarded in this modified medium compared with their growth in MRS and mRCM. This result is a significant improvement in the enumeration and differentiation of L. delbrueckii ssp. bulgaricus in mRCM-PYR compared with the results in MRS and mRCM where the high background growth of similar species interferes with the accuracy of bacterial population counts. Our results thus suggest that mRCM-PYR could be recommended as a reliable alternative growth medium for the selective enumeration and isolation of L. delbrueckii ssp. bulgaricus in a mixed culture.

Growth of Lactobacillus reuteri DSM17938 Under Two Simulated Microgravity Systems: Changes in Reuterin Production, Gastrointestinal Passage Resistance, and Stress Genes Expression Response.

Extreme factors such as space microgravity, radiation, and magnetic field differ from those that occur on Earth. Microgravity may induce and select some microorganisms for physiological, metabolic, and/or genetic variations. This study was conducted to determine the effects of simulated microgravity conditions on the metabolism and gene expression of the probiotic bacterium Lactobacillus reuteri DSM17938. To investigate microbial response to simulated microgravity, two devices-the rotating wall vessel (RWV) and the random positioning machine (RPM)-were used. Microbial growth, reuterin production, and resistance to gastrointestinal passage were assessed, and morphological characteristics were analyzed by scanning electron microscopy. The expression of some selected genes that are responsive to stress conditions and to bile salts stress was evaluated through real-time quantitative polymerase chain reaction assay. Monitoring of bacterial growth, cell size, and shape under simulated microgravity did not reveal differences compared with 1 × g controls. On the contrary, an enhanced production of reuterin and a greater tolerance to the gastrointestinal passage were observed. Moreover, some stress genes were upregulated under RWV conditions, especially after 24 h of treatment, whereas RPM conditions seemed to determine a downregulation over time of the same stress genes. These results show that simulated microgravity could alter some physiological characteristics of L. reuteri DSM17938 with regard to tolerance toward stress conditions encountered on space missions and could be useful to elucidate the adaptation mechanisms of microbes to the space environment.

Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source.

We report a method for suppression of lactate production by lactic acid bacteria (LAB) in culture. LAB produce lactate to regenerate NAD+ that is consumed during glycolysis. Glucose suppresses NAD+ regeneration pathways other than lactate dehydrogenase and non-glycolytic ATP production pathways. Therefore, the carbon source was changed to sucrose, and fed-batch culture was performed to limit the glycolytic flux and thus suppress lactate production. As a result, lactate productivity (i.e., the amount of lactate produced per amount of grown cell) in the sucrose/fed-batch culture was decreased compared to that in glucose/batch culture, in all five LAB strains examined. The productivity level decreased to 24% and 46% in Lactobacillus reuteri JCM 1112 and Lactococcus lactis JCM 7638, respectively. Metabolic flux analysis of Lactobacillus reuteri JCM 1112 revealed increased contributions of the mannitol production pathway to NAD+ regeneration and the arginine deiminase pathway to ATP production in the sucrose/fed-batch culture.

Levan from Bacillus amyloliquefaciens JN4 acts as a prebiotic for enhancing the intestinal adhesion capacity of Lactobacillus reuteri JN101.

Improving intrinsic adhesion performance of the known probiotics facilitates their residence and colonization, and therefore exerts more beneficial effects on the human or animal host. In this study, through adaptive culture with levan, Lactobacillus reuteri JN101 achieved the same biomass and exhibited 2.6 times higher adhesion capacity to HT-29 cells than those grown with glucose. The mechanism study related to this adhesion enhancement showed that the elevated proportion of unsaturated fatty acids facilitated the bacterial cells to overcome repulsive forces to approach the intestinal epithelial cell. At the same time, and the greater amounts of cell membrane proteins, such as S-layer protein (3.2 folds), elongation factor Tu (2.6 folds) and phosphoglycerate kinase (2.4 folds) probably enhanced the complementary interactions to the receptor on the epithelial cell. These results presented here indicated levan could be used as a potential prebiotic to regulate the adhesion capacity of probiotics, and provide ground for developing the specific-probiotics oriented functional food.

A metabolic reconstruction of Lactobacillus reuteri JCM 1112 and analysis of its potential as a cell factory.

BACKGROUND: Lactobacillus reuteri is a heterofermentative Lactic Acid Bacterium (LAB) that is commonly used for food fermentations and probiotic purposes. Due to its robust properties, it is also increasingly considered for use as a cell factory. It produces several industrially important compounds such as 1,3-propanediol and reuterin natively, but for cell factory purposes, developing improved strategies for engineering and fermentation optimization is crucial. Genome-scale metabolic models can be highly beneficial in guiding rational metabolic engineering. Reconstructing a reliable and a quantitatively accurate metabolic model requires extensive manual curation and incorporation of experimental data. RESULTS: A genome-scale metabolic model of L. reuteri JCM 1112T was reconstructed and the resulting model, Lreuteri_530, was validated and tested with experimental data. Several knowledge gaps in the metabolism were identified and resolved during this process, including presence/absence of glycolytic genes. Flux distribution between the two glycolytic pathways, the phosphoketolase and Embden-Meyerhof-Parnas pathways, varies considerably between LAB species and strains. As these pathways result in different energy yields, it is important to include strain-specific utilization of these pathways in the model. We determined experimentally that the Embden-Meyerhof-Parnas pathway carried at most 7% of the total glycolytic flux. Predicted growth rates from Lreuteri_530 were in good agreement with experimentally determined values. To further validate the prediction accuracy of Lreuteri_530, the predicted effects of glycerol addition and adhE gene knock-out, which results in impaired ethanol production, were compared to in vivo data. Examination of both growth rates and uptake- and secretion rates of the main metabolites in central metabolism demonstrated that the model was able to accurately predict the experimentally observed effects. Lastly, the potential of L. reuteri as a cell factory was investigated, resulting in a number of general metabolic engineering strategies. CONCLUSION: We have constructed a manually curated genome-scale metabolic model of L. reuteri JCM 1112T that has been experimentally parameterized and validated and can accurately predict metabolic behavior of this important platform cell factory.

Genes Involved in Galactooligosaccharide Metabolism in Lactobacillus reuteri and Their Ecological Role in the Gastrointestinal Tract.

Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS, while a second unidentified protein may function as a galactoside transporter. Two ß-galactosidases, encoded by lacA and lacLM, sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ΔlacS ΔlacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ΔlacS ΔlacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that α-galactosides may constitute alternate substrates of the GOS pathway.IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as α-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including α-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies.

Multidisciplinary and Comparative Investigations of Potential Psychobiotic Effects of Lactobacillus Strains Isolated From Newborns and Their Impact on Gut Microbiota and Ileal Transcriptome in a Healthy Murine Model.

Psychobiotics are probiotic microorganisms that may exert positive influence on the psychological status of the host. Studies have revealed immunological and microbiological correlations of gut microbiota and the gut-brain axis, and have investigated psychobiotics based on the findings of the gut-brain axis. Considering their mode of actions, the present study sets anti-inflammatory effect, neurotransmitter modulation, and gut microbiota modulation as three essential criteria to evaluate Lactobacillus casei ATG-F1 (F1), L. reuteri ATG-F3 (F3), and L. reuteri ATG-F4 (F4) isolated from newborns as psychobiotics candidates in a healthy mouse model and compares the results with a non-treated control group and an ampicillin-induced gut dysbiosis (Amp) group as a negative control. The F3 and F4 strains showed anti-inflammatory effects in vitro in RAW264.7 murine macrophages, and the level of anti-inflammatory cytokine interleukin (IL)-10 increased in ileums of mice orally administered with the F4 strain. Serum dopamine level significantly increased only in the F4-treated group as compared with the control group. Serum serotonin level was unaffected in Lactobacillus-treated groups, while a significant decrease in serum serotonin level was observed in the Amp group. Bacteroidetes population increased in fecal samples of the F4-treated group as compared with the control, and Bacteroidales S24-7 and Prevotellaceae population significantly increased at family level in fecal samples from the F4-treated group as compared with the control. In contrast, the Amp group showed an increase in the level of Proteobacteria and a decrease in the level of Bacteroidetes as compared with the control group. Transcriptome analysis revealed a distinctive clustering in ileums from the F4-treated group as compared to other experimental groups. In addition, the circadian rhythm pathway showed maximum enrichment in ileums of Lactobacillus-treated mice, and the F4-treated group showed the highest fold changes in circadian rhythm-related genes (Dbp, Per1, Per2, and Per3). Conclusively, L. reuteri ATG-F4 is suggested as a potential psychobiotics through demonstrations of anti-inflammatory effects, serum dopamine modulation, and gut microbiota modulation in a healthy murine model in the present study. Moreover, we carefully suggest gut circadian rhythm modulation as another important criterion of psychobiotics, which may have an important role in the gut-brain axis.