抑制食源性致病菌脂肽的分离鉴定及生物学特性分析

Isolation and identification of lipopeptides from Bacillus pumilus inhibiting foodborne pathogens and analysis of its biological characteristics

从传统香肠中分离拮抗芽孢杆菌菌株,并对菌株所产脂肽进行分离纯化和抗菌特性分析。采取平板对峙法筛选产生抗菌脂肽的芽孢杆菌菌株。抗菌脂肽采用有机溶剂萃取、Sephadex LH-20柱层析、半制备反向高效液相色谱(reverse-phase high performance liquid chromatography,RP-HPLC)分离纯化,傅里叶红外光谱(Fourier transform infrared spectroscopy,FT-IR)扫描、薄层层析(thin-layer chromatography,TLC)-生物自显影鉴定,扫描电镜分析脂肽抗菌机理。分离筛选到1株产广谱抗菌脂肽的菌株,鉴定为短小芽孢杆菌(命名为XZK-18)。菌株XZK-18发酵8 h开始合成脂肽,24 h产量达到最大。TLC初步鉴定抑菌活性成分为脂肽,且主要由脂肽家族中的表面活性素(surfactin)及少量伊枯草菌素(iturin)组成,TLC-生物自显影检测到表面活性素对金黄色葡萄球菌和大肠杆菌有显著抑菌活性。FT-IR进一步证实抑菌活性成分为脂肽。扫描电镜结果表明,纯化的脂肽通过破坏大肠杆菌细胞膜完整性、使细胞壁穿孔、增大细胞膜通透性杀灭大肠杆菌。抗菌脂肽有广谱抑菌活性,对革兰氏阳性菌如蜡样芽孢杆菌、枯草芽孢杆菌、金黄色葡萄球菌、藤黄微球菌和单核增生李斯特菌有显著抑菌活性,对革兰氏阴性菌如铜绿假单胞菌、大肠杆菌、伤寒沙门氏菌、副溶血性弧菌和福氏志贺氏菌也有一定抑菌作用,对所试真菌无抑菌活性。脂肽在110℃以下保温30 min稳定性好,在pH 3~11下处理10 d抑菌活性保持稳定。以上特性表明菌株XZK-18合成的脂肽在食品生产与加工中有作为天然食品防腐剂的潜力。

To isolate antagonistic Bacillus strains from traditional sausage,and to purify antibacterial substances produced by the strains and analyze their antibacterial characteristics,Bacillus strains producing antibacterial substances were screened by plate confrontation method.The antibacterial substances were separated by organic solvent extraction,Sephadex LH-20 column chromatography,semi preparation reverse high performance liquid chromatography(RP-HPLC),and identified by Fourier transform infrared spectroscopy(FT-IR)and thin layer chromatography(TLC)-bioautography.A strain producing broad-spectrum antibacterial substance was isolated and identified as Bacillus pumilus(named XZK-18).Strain XZK-18 began to synthesize antibacterial substances after 8 h of fermentation,and the yield reached the maximum at 24 h.The antimicrobial active component was identified as lipopeptide by TLC,and it was mainly composed of surfactant and a small amount of iturin.TLC-bioautography showed that the surfactant had significant antibacterial activity against Staphylococcus aureus and Escherichia coli.FT-IR further confirmed that the antibacterial active component was lipopeptide.The results of scanning electron microscope demonstrated that the lipopeptide killed E.coli by destroying the integrity of cell envelope,perforating the cell wall and increasing the permeability of cell membrane.The lipopeptide had broad-spectrum antibacterial activity,and had significant antibacterial activity against Gram-positive bacteria such as Bacillus cereus,Bacillus subtilis,S.aureus,Micrococcus gaminerae and Listeria monocytogenes;it also had certain antibacterial effect on Gram-negative bacteria such as Pseudomonas aeruginosa,E.coli,Salmonella typhi,Vibrio parahaemolyticus and Shigella flexneri.It had no antimicrobial activity to the tested fungi.The lipopeptide was stable below 110℃for 30 min,and its antibacterial activity remained stable at pH 3-11 for 10 d.These characteristics indicate that the lipopeptide synthesized by strain XZK-18 has potential as a natural food preservative in food production and processing.