5株解淀粉芽孢杆菌的益生特性及抑菌活性研究

Study on the Probiotic Properties and Antimicrobial Activity of 5 Strains of Bacillus amyloliquefaciens

【目的】探究5株自筛解淀粉芽孢杆菌(YN-BA1、YN-BA2、YN-BA3、YN-BA4、YN-BA5)的生长曲线、pH和胆盐耐受性、抑菌活性和抗生素敏感性等特性,为将其开发成微生态制剂应用于养殖中提供理论依据。【方法】采用比浊法测定解淀粉芽孢杆菌生长曲线,将5株解淀粉芽孢杆菌分别接种于经盐酸配制成pH为2.0、3.0、4.0和7.0的LB肉汤中培养2 h和0.3%胆盐环境中培养5 h,以评价其在人工胃肠液中的耐受性;采用琼脂打孔法测定体外抑菌活性,采用KB纸片法测定抗生素敏感性。【结果】5株解淀粉芽孢杆菌在经过0~2 h的缓慢生长和6~8 h生长速度降低后,其生长量快速上升,进入对数生长期;36 h后生长量下降,进入衰退期。5株解淀粉芽孢杆菌均具有不同的耐酸、耐胆盐能力,其中YN-BA2菌株在pH 2.0、3.0、4.0存活率分别为42.10%、51.50%和74.67%,均高于另外4株菌;在0.3%胆盐处理5 h后,YN-BA2菌株存活率为51.00%,高于另外4株菌,表明YN-BA2菌株的耐受力最高。5株解淀粉芽孢杆菌对肠炎沙门氏菌(YN-S1、YN-S2)、鸡白痢沙门菌(YN-SP9、YN-SP10)、大肠杆菌DO157均有抑菌作用,YN-BA2菌株对鸡白痢沙门氏菌YN-SP10具有高敏感性,其余4株解淀粉芽孢杆菌对实验室保存的肠道致病菌均为中敏,表明YN-BA2菌株抑菌效果最好。5株解淀粉芽孢杆菌对四环素(除YN-BA5外)、诺氟沙星有耐药性,对链霉素、青霉素G、头孢氨苄有不同程度的耐药性,对红霉素、卡那霉素(除YN-BA5外)、左氧氟沙星、环丙沙星、庆大霉素、阿奇霉素(除YN-BA1外)、氨苄西林、林可霉素敏感。【结论】解淀粉芽孢杆菌能够快速活化,对胆盐及pH的耐受性良好,对大肠杆菌、鸡白痢沙门氏菌和肠炎沙门氏菌等具有一定的抑制作用,对四环素、诺氟沙星耐药。因此,解淀粉芽孢杆菌具有开发为微生态制剂的潜能,以用于畜禽养殖生产。

【Objective】This study was aimed to explore the growth curve,pH,bile salt tolerance,antimicrobial activity,antibiotic sensitivity and other characteristics of 5 strains of self screening Bacillus amyloliquefaciens(YN-BA1,YN-BA2,YN-BA3,YN-BA4 and YN-BA5),and provide theoretical basis for the development of microecological agents in aquaculture.【Method】In this test,the growth curve of Bacillus amyloliquefaciens was determined by turbidimetry.The 5 strains of Bacillus amyloliquefaciens were inoculated into LB broth prepared with hydrochloric acid,pH 2.0,3.0,4.0 and 7.0 for 2 h,and cultured 5 h in an environment with 0.3%bile salt concentration to evaluate their tolerance in artificial gastric juice intestinal juice.The antibacterial activity in vitro was determined by agar drilling method,and the antibiotic sensitivity was determined by KB paper method.【Result】The growth rates of 5 strains of Bacillus amyloliquefaciens increased rapidly and entered the logarithmic growth phase after the slow growth 0-2 and 6-8 h.After 36 h,the growth decreased and entered the recession period.The 5 strains had different abilities of acid resistance and bile salt tolerance.Among them,the survival rates of YN-BA2 strain at pH 2.0,3.0 and 4.0 were 42.10%,51.50%and 74.67%,respectively,which were higher than the other 4 strains.After being treated for 5 h at 0.3%bile salt concentration,the survival rate of YN-BA2 was 51.00%,which was higher than the other 4 strains,indicating that YN-BA2 strain had the highest tolerance.The 5 strains of Bacillus amyloliquefaciens had antibacterial effect on Salmonella Enteritidis YN-S1 and YN-S2,Salmonella Pullorum YN-SP9 and YN-SP10,and Escherichia coli DO157.YN-BA2 had high sensitivity to Salmonella Pullorum YN-SP10.The other 4 strains of Bacillus amyloliquefaciens were moderately sensitive to intestinal pathogens preserved in the laboratory,indicating that YN-BA2 had the best antibacterial effect.The 5 strains of Bacillus amyloliquefaciens were resistant to tetracycline(except for YN-BA5)and norfloxacin,ofloxacin,peillin G and cephalexin to varying degrees,and were sensitive to erythromycin,kanamycin(except for YN-BA5),levofloxacin,ciprofloxacin,gentamicin,azithromycin(except for YN-BA1),ampicillin and lincomycin.【Conclusion】Bacillus amyloliquefaciens could be activated rapidly,had good tolerance to bile salt and pH,had certain inhibitory effects on Escherichia coli,Salmonella Pullorum and Salmonella Enteritidis,and was resistant to tetracycline and norfloxacin.Therefore,Bacillus amyloliquefaciens had the potential to be used in the development of microecological agents for livestock and poultry production.