Table_1_Production, Storage Stability, and Susceptibility Testing of Reuterin and Its Impact on the Murine Fecal Microbiome and Volatile Organic Compound Profile.docx

Background: Probiotics are generally considered as safe, but infections may rarely occur in vulnerable patients. Alternatives to live microorganisms to manage dysbiosis may be of interest in these patients. Reuterin is a complex component system exhibiting broad spectrum antimicrobial activity and a possible candidate substance in these cases.
Methods: Reuterin supernatant was cultured from Lentilactobacillus diolivorans in a bioreactor in a two-step process. Storage stability at −20°C and effect of repeated freeze-thaw cycles were assessed by high performance liquid chromatography (HPLC). Antimicrobial activity was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus (S.) aureus, Staphylococcus epidermidis, Streptococcus (S.) agalactiae, Propionibacterium acnes, and Pseudomonas aeruginosae. Male BALBc mice were gavage fed with reuterin supernatant (n = 10) or culture medium (n = 10). Fecal volatile organic compounds (VOC) were assessed by gas chromatography mass spectroscopy; the microbiome was examined by 16S rRNA gene sequencing.
Results: The supernatant contained 13.4 g/L reuterin (3-hydroxypropionaldehyde; 3-HPA). 3-HPA content remained stable at −20°C for 35 days followed by a slow decrease of its concentration. Repeated freezing/thawing caused a slow 3-HPA decrease. Antimicrobial activity was encountered against S. aureus, S. epidermidis, and S. agalactiae. Microbiome analysis showed no differences in alpha and beta diversity markers. Linear discriminant effect size (LEfSe) analysis identified Lachnospiraceae_bacterium_COE1 and Ruminoclostridium_5_uncultured_Clostridiales_ bacterium (in the reuterin medium group) and Desulfovibrio_uncultured_ bacterium, Candidatus Arthromitus, Ruminococcae_NK4A214_group, and Eubacterium_xylanophilum_group (in the reuterin group) as markers for group differentiation. VOC analysis showed a significant decrease of heptane and increase of 3-methylbutanal in the reuterin group.
Conclusion: The supernatant produced in this study contained acceptable amounts of 3-HPA remaining stable for 35 days at −20°C and exhibiting an antimicrobial effect against S. aureus, S. agalactiae, and S. epidermidis. Under in vivo conditions, the reuterin supernatant caused alterations of the fecal microbiome. In the fecal, VOC analysis decreased heptane and increased 3-methylbutanal were encountered. These findings suggest the high potential of the reuterin system to influence the intestinal microbiome in health and disease, which needs to be examined in detail in future projects.

背景：益生菌（Probiotics）通常被认为具有良好安全性，但在免疫功能低下的易感患者中，仍可能罕见发生感染相关并发症。针对这类人群，寻找可替代活微生物、用于调控菌群失调的制剂具有重要研究价值。罗伊菌素（Reuterin）是一种具有广谱抗菌活性的复合组分体系，可作为此类场景下的潜在候选制剂。
方法：本研究通过两步法在生物反应器中由解二醇扁豆乳杆菌（Lentilactobacillus diolivorans）培养得到罗伊菌素上清液。采用高效液相色谱（High Performance Liquid Chromatography, HPLC）检测-20℃条件下的储存稳定性，以及反复冻融循环对其含量的影响。针对艰难梭菌（Clostridium difficile）、单核细胞增生李斯特菌（Listeria monocytogenes）、大肠埃希菌（Escherichia coli）、粪肠球菌（Enterococcus faecium）、金黄色葡萄球菌（Staphylococcus aureus，S. aureus）、表皮葡萄球菌（Staphylococcus epidermidis）、无乳链球菌（Streptococcus agalactiae，S. agalactiae）、痤疮丙酸杆菌（Propionibacterium acnes）以及铜绿假单胞菌（Pseudomonas aeruginosa）开展抗菌活性检测。将10只雄性BALB/c小鼠经口灌胃给予罗伊菌素上清液（实验组，n=10），另10只给予基础培养基（对照组，n=10）。采用气相色谱-质谱联用法（Gas Chromatography Mass Spectroscopy）检测粪便挥发性有机物（Volatile Organic Compounds, VOC），并通过16S rRNA基因测序分析粪便微生物组。
结果：所获上清液中罗伊菌素的活性成分为3-羟基丙醛（3-hydroxypropionaldehyde，3-HPA），含量为13.4 g/L。3-HPA在-20℃储存35天内含量保持稳定，此后呈现缓慢下降趋势；反复冻融循环同样会导致3-HPA浓度缓慢降低。抗菌活性检测显示，该上清液对金黄色葡萄球菌、表皮葡萄球菌及无乳链球菌具有抑制活性。微生物组分析表明，两组的α多样性与β多样性指标均无显著差异。线性判别分析效应量（Linear Discriminant Effect Size, LEfSe）分析筛选出两组的差异标志物：罗伊菌素培养基组的差异标志物为未培养的毛螺菌科细菌COE1（Lachnospiraceae_bacterium_COE1）及未培养的梭菌目5属瘤胃梭菌（Ruminoclostridium_5_uncultured_Clostridiales_bacterium）；罗伊菌素组的差异标志物为未培养的脱硫弧菌属细菌（Desulfovibrio_uncultured_bacterium）、候选节杆菌属（Candidatus Arthromitus）、瘤胃球菌科NK4A214群（Ruminococcae_NK4A214_group）及木糖降解真杆菌群（Eubacterium_xylanophilum_group）。粪便VOC分析显示，罗伊菌素组的庚烷含量显著降低，而3-甲基丁醛含量显著升高。
结论：本研究制备的罗伊菌素上清液中3-HPA含量符合应用要求，在-20℃条件下可稳定储存35天，且对金黄色葡萄球菌、无乳链球菌及表皮葡萄球菌具有明确抗菌活性。体内实验表明，罗伊菌素上清液可改变小鼠粪便微生物组组成；粪便VOC分析显示该组庚烷含量降低、3-甲基丁醛含量升高。上述结果提示，罗伊菌素体系在调控健康与疾病状态下肠道微生物组方面具有较高应用潜力，相关机制有待后续研究进一步阐明。