广谱抗菌乳酸菌的分离鉴定及细菌素的提取和纯化

Isolation and Identification of Lactic Acid Bacterial Strain with Broad-Spectrum Antibacterial Activity and Extraction and Purification of Bacteriocin Produced by It

本研究从黑龙江传统自制酸菜中分离到1株产抑菌活性物质的乳酸菌HLJ-174,其发酵产物排除有机酸、H2O2等干扰因素后对大肠杆菌、荧光假单胞菌、蜡样芽孢杆菌和藤黄微球菌等食品中常见的致病菌有广谱抑菌活性。通过形态学、生理生化和16S r RNA序列分析鉴定该菌株为植物乳杆菌(Lactobacillus plantarum),命名为Lactobacillus plantarum HLJ-174。研究了利用有机溶剂萃取的方法提取细菌素,发现正丁醇、乙酸乙酯萃取效果较好,正己烷、二氯甲烷、三氯甲烷、四氯化碳、丙酮、乙醇等没有效果。使用不同体积的正丁醇和乙酸乙酯(0.5、1.0、1.5、2.0、2.5、3.0倍发酵液体积)萃取,发现1.5倍发酵液体积的正丁醇效果最好。利用Sephadex LH-20对萃取产物进行了进一步分离,得到的活性物质对大肠杆菌和蜡样芽孢杆菌等具有较强的抑菌活性。

A lactic acid bacterial strain, Lactobacillus HLJ-174, was isolated from naturally fermented Chinese sauerkraut from Heilongjiang province. The bacteriocin produced by it showed a strong antibacterial activity against Escherichia coli, Pseudomonas fluorescens, Bacillus cereus and Micrococcus aureus after elimination of some interference factors such as organic acid and hydrogen peroxide. Based on morphological, physiological and biochemical characteristics and 16 S r DNA sequence analysis, the strain HLJ-174 was identified as Lactobacillus plantarum. The bacteriocin from HLJ-174 was extracted with organic solvent, and higher extraction efficiency was obtained using butanol and ethyl acetate, but it was not extractable with other organic solvents such as hexane, methylene chloride, chloroform, carbon tetrachloride, acetone and ethanol. Furthermore, the addition of different volumes(0.5-, 1.0-, 1.5-, 2.0-, 2.5- and 3.0-fold) of butanol or ethyl acetate to the fermentation broth was applied to extract the bacteriocin, and the results showed that the best extraction efficiency was achieved by using 1.5-fold volume of butanol as the extraction solvent. The extract was further purified by Sephadex LH-20 chromatography, and the purified product exhibited a strong antibacterial activity against Escherichia coli and Bacillus cereus.