一株与中草药五倍子协同抑菌的芽孢杆菌筛选及体外益生效果评价

Isolation and identification of Bacillus subtilis with synergic antibacterial effect with Galla chinensis and assessment of its probiotic effect in vitro

目的利用分子生物学方法对从猪肠道食糜分离的芽孢杆菌进行鉴定,并通过体外法评价其益生效果。方法实验从健康猪肠道食糜中筛选到1株能抑制大肠埃希菌、副伤寒沙门菌、金黄色葡萄球菌和巴氏杆菌生长,并与中草药五倍子具有协同抑菌作用的芽孢杆菌AP139。结果经16SrDNA同源性序列分析,结合形态和生理生化特点,鉴定为枯草芽孢杆菌（B.subtilis）。芽孢杆菌AP139联合中草药五倍子发酵结果表明：5%组对4种致病菌的抑菌圈直径分别扩大了1.20、14.45、23.54和6.09 mm,其抑菌效果优于未发酵,对巴氏杆菌PM2010和猪大肠埃希菌抑菌圈极显著提高（P〈0.01）,对金黄色葡萄球菌抑菌圈有显著提高（P〈0.05）。经体外益生效果评价得知,AP139菌株在pH=5.0-8.0的条件下活菌数均较高,NaCl浓度为1%时生长较好,0.3%胆盐和人工肠液分别处理3h后存活率分别达84.26%和89.35%。结论芽孢杆菌AP139具有良好的协同抑菌作用,并具有益生菌的耐酸、耐胆盐、耐胃肠液等特性,可适应动物胃肠道内环境。

Objective To identify a Bacillus strain AP139 isolated from the chime in healthy pig intestine, and evaluate its probiotic effect in vitro. Methods Molecular biological method was used to screen a Bacillus strain AP139 from the chyme in pig intestine, which was able to inhibit the growth of pathogenic bacteria Escherichia coli, Paratyphoid salmonella, Staphylococcus aureus and Pasteurella, and had synergic inhibitory effect with Chinese herbal medicine Galla chinensis. Results Based on homological analysis of 16S rDNA sequence together with morphological, physical and biochemical characteristics, AP139 was identified as Bacillus subtilis （B. subtilis）. After fermented with Bacillus AP139, the inhibition zone diameters of 5% Galla chinensis against the four pathogens were increased by 1.2 mm, 14.45 mm, 23.54 mm and 6.09 mm respectively. The inhibitory effects of 5% Galla Chinensis fermented with Bacillus AP139 against Pasteurella PM2010 and swine Escherichia coli were significantly increased （P〈0.01）, and that against Staphylococcus aureus was improved significantly （P〈0.05）. High countings of live Bacillus AP139 were seen under the conditions of pH 5.0 - 8.0 and 1% NaCl. After treated with 0.3% bile salts or artificial intestinal juice for 3 hours, the survival rate of Bacillus AP139 was 84.26% and 89.35% respectively. Conclusion Bacillus AP139 has good synergic antibacterial effect with Galla chinensis, and is resistant to acid, bile salt and gastrointestinal fluid, which can adapt to the environment of animals＇ gastrointestinal tract.