一种芽孢杆菌广谱细菌素的分离纯化及其性质研究

Purification and characterization of broad-spectrum bacteriocin from Bacillus

目的筛选产细菌素的芽孢杆菌,研究纯化后的细菌素的性质。方法首先采用双层琼脂牛津杯法,从高温酒曲中筛选产细菌素的芽孢杆菌;然后利用加热法、pH沉淀法、盐析法等纯化细菌素;最后使用不同pH、温度、蛋白酶等条件处理细菌素,测定抑菌活性,分析其pH稳定性、热稳定性、抗酶解性及抗菌谱特性。结果分离得到一株产细菌素的芽孢杆菌菌株PL-2,经16S rDNA序列比对鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。纯化后的细菌素,在Tricine-SDS-PAGE电泳中得到的单一条带,分子量约为10kDa。该细菌素在pH 2.0~14.0处理4h,抑菌活力最大降幅为5%;经120℃保温30min,抑菌活力仅降低18%;能抵抗蛋白酶K、胃蛋白酶等多种蛋白酶水解;对革兰氏阴性菌、革兰氏阳性菌、真菌均有较强的抑制能力。结论Bacillus velezensis PL-2产生的细菌素具有优良的特性,可进一步开展其在食品加工、生物防治以及医疗领域等方面的应用研究。

Objective To screen the strain of Bacillus producing bacteriocin and research the characteristics of the purified bacteriocin.Methods First,a strain producing bacteriocin was screened from a high temperature koji by the method of Oxford cup double agar diffusion.Second,the bacteriocin was purified by thermal treatment,pH precipitation and salting-out method.Finally,stabilities of the purified bacteriocin were studied at different pH,temperatures and proteases.Results The screened strain producing bacteriocin was identified by the 16S rDNA sequence alignment and named as Bacillus velezensis PL-2.After purification,a single band with a molecular weight of^10 kDa was obtained in the Tricine-SDS-PAGE electrophoresis.After treatment for 4 h at pH 2.0-14.0,the maximum reduction of bacteriocin activity was 5%.In addition,the reduction of bacteriocin activity was only 18%at 120℃for 30 min.The bacteriocin had an ability of resisting the hydrolysis of protease K,pepsin and other proteases.It was a strong inhibitor on gram-negative bacteria,gram-positive bacteria and fungi.Conclusion The bacteriocin produced by Bacillus velezensis PL-2 has excellent characteristics and a potential application in the fields of food processing,biological control and medicine.