滇南小耳猪源芽孢杆菌分离鉴定及益生特性

Isolation, identification, and probiotic property characterization of Bacillus strains from Diannan small-ear pigs

摘要:

背景 芽孢杆菌(Bacillus sp.)是制备畜禽饲用微生态制剂的重要菌种来源，近年来受到广泛关注。目的 筛选益生特性和安全性良好的滇南小耳猪源芽孢杆菌，为开发畜禽饲用微生态制剂提供候选菌株。方法 采集滇南小耳猪新鲜粪便，通过胰酪大豆胨培养基进行芽孢杆菌候选株分离纯化，测定其模拟胃肠液耐受性、抑菌活性、抗生素敏感性、黏附能力和抗炎性能，综合评估益生特性和安全性。结果 从滇南小耳猪新鲜粪便中共分离得到27株芽孢杆菌，综合评估模拟胃肠液耐受性、抑菌活性及抗生素敏感性，筛选出5株具有益生潜力的分离株(DN2-3, DN3-1, DN3-4, DN3-5, DN3-6)进行肠上皮细胞黏附能力及抗炎能力测定。其中，枯草芽孢杆菌(Bacillus subtilis) DN3-1对HT-29肠上皮细胞黏附性能优良，并且对脂多糖(Lipopolysaccharide, LPS)诱导的HT-29细胞中IL-8基因表达水平的抑制能力最强。对其进行小鼠急性经口毒性实验，灌胃后对小鼠采食量、体重均无负面影响；小鼠空肠、回肠、肝脏、肾脏无病理变化且脏器指数无显著差异；空肠和回肠绒毛高度呈升高趋势，安全性良好。结论 枯草芽孢杆菌DN3-1具有优良的益生特性及安全性，可为畜禽饲用微生态制剂的开发提供优质菌种。

Abstract:

Background Bacillus species represent an important source of microbial strains for developing microecological preparations used in livestock and poultry feeding, which have attracted considerable attention in recent years.Objective This study screened Bacillus strains with favorable probiotic properties and safety profiles from Diannan small-ear pigs, aiming to provide candidate strains for the development of microecological preparations for livestock and poultry.Methods We collected fresh fecal samples from Diannan small-ear pigs and used tryptic soy agar to isolate candidate Bacillus strains. Furthermore, we comprehensively assessed the probiotic properties and safety of the strains based on the strain tolerance to simulated gastrointestinal fluids, antimicrobial activity, antibiotic susceptibility, adhesion capacity, and anti-inflammatory properties.Results A total of 27 Bacillus strains were isolated from the fresh feces. Through comprehensive assessment of tolerance to simulated gastrointestinal fluid, antimicrobial activity, and antibiotic susceptibility, five strains (DN2-3, DN3-1, DN3-4, DN3-5, and DN3-6) with probiotic potential were selected for evaluation of intestinal epithelial cell adhesion and anti-inflammatory capacity. Among them, Bacillus subtilis DN3-1 exhibited strong adhesion to HT-29 intestinal epithelial cells and demonstrated the most potent inhibition of IL-8 expression in HT-29 cells exposed to lipopolysaccharide. Acute oral toxicity tests of this strain in mice showed no adverse effects on feed intake or body weight after gavage administration. No pathological changes were observed in the jejunum, ileum, liver, or kidney, and there were no significant differences in organ indices. Villus height in the jejunum and ileum showed an increasing trend, indicating good safety of the strain.Conclusion B. subtilis DN3-1 possesses excellent probiotic properties and safety, being a high-quality candidate strain for developing microecological preparations for livestock and poultry.