牛源益生性屎肠球菌的筛选与安全性评价

Screening and safety evaluation of probiotic Enterococcus faecium from cattle

摘要:

背景 随着畜牧业进入无抗养殖新时代，寻找安全有效的抗生素替代品已成为保障该行业健康可持续发展的关键。益生菌具有调节肠道微生态平衡、抑制病原菌、增强免疫力及提高饲料转化率等多重功能，被视为最具潜力的替代抗生素策略之一。目的 从健康黄牛粪便中分离出具有优良益生特性且安全性高的益生菌菌株。方法 将健康黄牛新鲜粪便样本在肠球菌选择培养基上分离纯化，结合形态学观察和16S rRNA基因测序进行菌株鉴定。通过在模拟胃肠道环境中的存活率、耐NaCl能力、生长曲线、疏水性、自聚集能力及抑菌活性评估分离菌的益生特性。进一步通过溶血性、明胶酶活性、药敏试验及小鼠亚毒性试验，综合评价其体外与体内安全性。结果 共分离获得5株屎肠球菌(Enterococcus faecium) F1-F5。在模拟肠液中，所有菌株存活率均超过80%；而在pH 3.0的模拟胃液中，仅菌株F5可存活，存活率为(83.38±1.83)%；菌株F5在5% NaCl溶液中存活率仍高达(72.45±1.99)%。菌株F5培养2 h后进入对数生长期，6 h后进入稳定期。该菌株表现出良好的表面疏水性和自聚集能力，并对铜绿假单胞菌(Pseudomonas aeruginosa)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)具有抑制作用。安全性评价显示，菌株F5无溶血活性和明胶酶活性，7种毒力基因中仅检测到efaA；对万古霉素、青霉素、氨苄西林、四环素敏感，对红霉素耐药。小鼠亚毒性试验表明，菌株F5灌胃14 d后未引起异常临床表现，剖检脏器无眼观病理变化，并可显著降低血清中促炎因子IL-1β水平，同时显著提高十二指肠绒毛长度和绒毛长度/隐窝深度比值，并且显著降低隐窝深度值。结论 从健康黄牛新鲜粪便中分离获得5株屎肠球菌，其中菌株F5具备优良的益生潜力，有望作为牛源益生菌制剂用于饲料添加剂的开发。

Abstract:

Background As China’s animal husbandry enters a new era of antibiotic-free farming, finding safe and effective alternatives to antibiotics has become the key to ensuring the healthy and sustainable development of this industry. Probiotics are considered one of the most promising alternatives because of their multiple functions, such as regulating intestinal microecological balance, inhibiting pathogens, enhancing immunity, and improving the feed conversion rate.Objective To isolate a novel probiotic strain from healthy cattle feces that exhibited excellent probiotic characteristics and was highly safe.Methods Fresh fecal samples from healthy cattle were isolated on Pfizer selective Enterococcus agar, and the strains were identified based on morphological characteristics and 16S rRNA gene sequences. The probiotic potential of the isolated strains was assessed by measuring their tolerance to simulated gastrointestinal conditions, NaCl tolerance, hydrophobicity, auto-aggregation capacity, growth characteristics, and antibacterial activity. The safety in vitro and in vivo was evaluated by hemolysis, gelatinase activity, antimicrobial susceptibility, and mouse toxicity tests.Results A total of 5 strains (F1-F5) of Enterococcus faecium were isolated. In simulated intestinal fluid, the survival rate of all strains was more than 80%. In the simulated gastric juice of pH 3.0, only F5 could survive, with the survival rate of (83.38±1.83)%. The survival rate of F5 in 5% NaCl solution was as high as (72.45±1.99)%. F5 entered the logarithmic growth phase after 2 h of culture and entered the plateau phase after 6 h. The strain showed good surface hydrophobicity and auto-aggregation ability and exerted inhibitory effects on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Safety evaluation showed that F5 had no hemolytic activity or gelatinase activity, and only efaA was detected among 7 virulence genes. F5 was sensitive to vancomycin, penicillin, ampicillin, and tetracycline and resistant to erythromycin. The toxicity test in mice showed that F5 did not cause abnormal clinical manifestations after 14 days of gavage, and necropsy showed no visual pathological change in organs. F5 significantly lowered the level of the pro-inflammatory factor IL-1β, significantly increased the length of duodenal villus and the villus length/crypt depth ratio, and significantly reduced the crypt depth.Conclusion Five E. faecium strains were isolated from the fresh feces of healthy cattle. Among them, strain F5 exhibits outstanding probiotic potential and is thus a promising candidate for the development and application of bovine probiotic preparations as feed additives.