兔源益生菌Tu-569菌株鉴定与其胞外产抑菌物性质分析

Identification of probiotics strain Tu-569 from rabbit cecum and property analysis of its extracellular antibacterial product

目的对兔源益生菌Tu-569菌株进行菌落形态和菌体形态观察、生理生化特性测定及16S rDNA序列系统发育分析以确定其种属。方法以大肠埃希菌为指示菌,通过琼脂打孔扩散法分析Tu-569菌株胞外产抑菌物性质,初步评价该抗菌物活性。结果 Tu-569菌株为Bacillus velezensis;Tu-569菌株胞外产抑菌物为分子量3.5～8.0kD的多肽,可被饱和度60%的硫酸铵盐析;该抗菌肽热稳定性较好,但可被蛋白酶降解;采用正丁醇萃取该抗菌肽不会降低其活性;其活性可被Mn2+离子增强,但在强酸(pH 3)强碱(pH11)环境中会大幅度降低;当存在表面活性剂和β-巯基乙醇时,抗菌肽抑菌活性完全丧失。每1mg粗蛋白中含有的抗菌肽抑制大肠埃希菌的能力相当于2μg硫酸庆大霉素、10μg硫酸链霉素或50μg土霉素。结论该抗菌肽性质稳定,具有较高的抗菌活性,在畜牧业、养殖业、食品等领域中有一定的应用价值。

Objective Works were done to identify probiotic strain Tu-569 and to analyze properties of its extracellular antibacterial product. Methods Identification was carried out by observations for colony morphology and cell shape, determinations of physiological, biochemical characteristics and sequence analysis. Properties analysis for extracellular antibacterial product were done with various treatments, such as salting out with ammonium sulfate, extraction with different organic solvent, heating, different pH environment, proteases, ions, protein protective and denaturant agents. The activity of antibacterial product was evaluated by agar diffusion assay with Escherichia coli as indicator bacteria. Results Strain Tu-569 was identified as Bacillus velezensis. The extracellular antibacterial product was polypeptide with molecular weight between 3.5kD to 8.0kD. The majority antibacterial polypeptide was salted out by 60% ammonium sulfate. The antibacterial polypeptid~ showed a good stability with heat, but could be degraded by proteases. The antibacterial polypeptide could be concentrated from butanol extraction without loss of antibacterial activity. The antibacterial activity of antibacterial polypeptide could be enhanced by Mn2＋, but would be reduced in lower pH （pH3） or higher pH （pill 1） buffer solutions. When surfactants or β-mercaptoethanol was present in buffer solutions, antimicrobial peptides would show complete loss of the antibacterial activity. The antibacterial activity against E. coli of antimicrobial peptides in lmg of crude protein was equivalent to 2μg of gentamicin sulfate, 10~tg of streptomycin sulfate or 50μg of oxytetracycline. Conclusion Antimicrobial peptides of Tu-569 strain had a high and stable antibacterial activity against Escherichia coli, which was useful in applications of animal husbandry, aquaculture, food and other fields.