Insights into the Mechanisms of Reuterin against Staphylococcus aureus Based on Membrane Damage and Untargeted Metabolomics.

Reuterin is a dynamic small-molecule complex produced through glycerol fermentation by Limosilactobacillus reuteri and has potential as a food biopreservative. Despite its broad-spectrum antimicrobial activity, the underlying mechanism of action of reuterin is still elusive. The present paper aimed to explore the antibacterial mechanism of reuterin and its effects on membrane damage and the intracellular metabolome of S. aureus. Our results showed that reuterin has a minimum inhibitory concentration of 18.25 mM against S. aureus, based on the 3-hydroxypropionaldehyde level. Key indicators such as extracellular electrical conductivity, membrane potential and permeability were significantly increased, while intracellular pH, ATP and DNA were markedly decreased, implying that reuterin causes a disruption to the structure of the cell membrane. The morphological damage to the cells was confirmed by scanning electron microscopy. Subsequent metabolomic analysis identified significant alterations in metabolites primarily involved in lipid, amino acid, carbohydrate metabolism and phosphotransferase system, which is crucial for cell membrane regulation and energy supply. Consequently, these findings indicated that the antibacterial mechanism of reuterin initially targets lipid and amino acid metabolism, leading to cell membrane damage, which subsequently results in energy metabolism disorder and, ultimately, cell death. This paper offers innovative perspectives on the antibacterial mechanism of reuterin, contributing to its potential application as a food preservative.

Application of the Reuterin System as Food Preservative or Health-Promoting Agent: A Critical Review.

The reuterin system is a complex multi-component antimicrobial system produced by Limosilactobacillus reuteri by metabolizing glycerol. The system mainly includes 3-hydroxypropionaldehyde (3-HPA, reuterin), 3-HPA dimer, 3-HPA hydrate, acrolein and 3-hydroxypropionic acid, and has great potential to be applied in the food and medical industries due to its functional versatility. It has been reported that the reuterin system possesses regulation of intestinal flora and anti-infection, anti-inflammatory and anti-cancer activities. Typically, the reuterin system exerts strong broad-spectrum antimicrobial properties. However, the antimicrobial mechanism of the reuterin system remains unclear, and its toxicity is still controversial. This paper presents an updated review on the biosynthesis, composition, biological production, antimicrobial mechanisms, stability, toxicity and potential applications of the reuterin system. Challenges and opportunities of the use of the reuterin system as a food preservative or health-promoting agent are also discussed. The present work will allow researchers to accelerate their studies toward solving critical challenges obstructing industrial applications of the reuterin system.

Pretreatment with Lactobacillus reuteri F-9-35 attenuates ethanol-induced gastric injury in rats.

BACKGROUND: Previous studies suggested that probiotics intervention may be one of the methods for preventing and/or treating gastric ulcer. OBJECTIVE: The aim of the study was to compare the preventive effects of a spaceflight mutant Lactobacillus reuteri F-9-35 and its wild type on ethanol-induced gastric injury in rats. DESIGN: Forty rats were randomly allocated into five groups: a normal group (NOR), ethanol group (EtOH), skim milk group (MILK), L. reuteri F-9-35 group (F935) and wild-type group (WT). The NOR and EtOH groups received 1 ml of distilled water by daily gavage for 14 days. The MILK group received 1 ml of skim milk alone, while the F935 and WT groups were administered 1 ml of skim milk containing the mutant and wild type (1 × 1010 colony-forming unit/ml) by daily gavage for 14 days, respectively. Acute gastric injury was induced by absolute alcohol 1 h after the final administration of different treatments, except for the NOR group. RESULTS: Pretreatment with L. reuteri F-9-35, but not milk alone or milk with the L. reuteri wild type, showed significant reduction of ethanol-induced gastric injury, as evidenced by lowering of ulcer index, ulcer area (%), and histological lesion. F-9-35 decreased the levels of lipid peroxidation and myeloperoxidase and increased mucus, glutathione, and nitric oxide levels in gastric tissue. Moreover, F-9-35 inhibited the expression of proinflammatory genes including gastric tumor necrosis factor-α, interleukin-1ß, and cyclooxygenase-2 and decreased the activity of nuclear factor kappa B (NF-κB). CONCLUSION: These findings indicated that L. reuteri F-9-35 pretreatment can attenuate ethanol-induced gastric injury in rats by inhibiting oxidative stress and inflammatory response. Together, L. reuteri F-9-35 has potential preventive efficacy on gastric ulcer.

Lactobacillus reuteri F-9-35 Prevents DSS-Induced Colitis by Inhibiting Proinflammatory Gene Expression and Restoring the Gut Microbiota in Mice.

Probiotics are considered to be a potential treatment for ulcerative colitis (UC). The aim of this study was to compare the preventive effect of a space flight-induced mutant L. reuteri F-9-35 and its wild type on UC in vivo. Female mice were randomly assigned to five groups: one normal and four colitic. Mice from colitis groups were daily gavaged with 0.2 mL 12% (w/v) skim milk containing the mutant or wild type (1 × 1011 CFU/mL), skim milk alone or distilled water for the whole experiment period, starting 7 days before colitis induction. UC was induced by administrating mice with 3.5% (w/v) dextran sulfate sodium (DSS) in drinking water for 7 days, after which DSS was removed and maintained for 3 days as a recovery phase. The results showed that the mice fed with L. reuteri F-9-35 had less inflammatory phenotype according to macroscopic and histological analysis, reduced myeloperoxidase activity, and lower expression of proinflammatory genes (Tumor necrosis factor-α, cyclooxygenase-2 and interleukin-6) in colonic tissue compared with control. Furthermore, L. reuteri F-9-35 protected the mice from gut microbiota dysbiosis from DDS induced colitis. Neither wild type nor the milk alone had such beneficial effects. From above we conclude that L. reuteri F-9-35 has great potential in the prevention of UC as a dietary supplement. PRACTICAL APPLICATION: Ulcerative colitis (UC) is the most common inflammatory bowel diseases and there is still a lack of safe and effective treatments. Consumption of L. reuteri F-9-35 may effective in preventing human UC.

Optimization of the quenching method for metabolomics analysis of Lactobacillus bulgaricus.

This study proposed a quenching protocol for metabolite analysis of Lactobacillus delbrueckii subsp. bulgaricus. Microbial cells were quenched with 60% methanol/water, 80% methanol/glycerol, or 80% methanol/water. The effect of the quenching process was assessed by the optical density (OD)-based method, flow cytometry, and gas chromatography-mass spectrometry (GC-MS). The principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were employed for metabolite identification. The results indicated that quenching with 80% methanol/water solution led to less damage to the L. bulgaricus cells, characterized by the lower relative fraction of prodium iodide (PI)-labeled cells and the higher OD recovery ratio. Through GC-MS analysis, higher levels of intracellular metabolites (including focal glutamic acid, aspartic acid, alanine, and AMP) and a lower leakage rate were detected in the sample quenched with 80% methanol/water compared with the others. In conclusion, we suggested a higher concentration of cold methanol quenching for L. bulgaricus metabolomics due to its decreasing metabolite leakage.