为探究发酵乳杆菌LBM97细菌素的适宜提取方法,采用(NH4)2SO4沉淀法、乙酸乙酯抽提法和pH吸附解吸法分别提取发酵乳杆菌LBM97所产的细菌素,以样品的抑菌圈直径和蛋白质含量作为评价各方法的指标。试验确定了发酵乳杆菌LBM97的适宜培养时间为60 h。优化试验表明,在(NH4)2SO4溶解度80%、V(乙酸乙酯)∶V(发酵上清液)=7∶5、吸附pH 6.1、解吸pH 1.8时,得到的细菌素粗样具有较好的抑菌性与较高的蛋白质含量,对金黄色葡萄球菌的抑菌圈直径分别为13.67、17.00和16.33 mm;对大肠杆菌的抑菌圈直径分别为15.20、17.27和16.87 mm;蛋白质质量浓度分别为4 804.70、396.35和543.96 μg/mL。对比研究发现,3种方法得到的细菌素样品具有不同的抑菌效果,用pH吸附解吸法得到的细菌素样品表现出最稳定的抑菌性,这与该方法可以提取到细菌保留于菌体周围的细菌素有关。综上所述,pH吸附解吸法为提取发酵乳杆菌LBM97细菌素的较适方法。该研究为细菌素提取方法的选择提供了数据支持与参考,为细菌素的全面获取和产率提升提供了理论基础。
To develop a suitable extraction method of bacteriocins from bacterial culture of Lactobacillus fermentum LBM97, different methods were compared and optimized, including ammonium sulfate precipitation, ethyl acetate extraction, and pH adsorption/desorption. The results showed that the appropriate culturing time of L. fermentum LBM97 was 60 h. By adjusting conditions of each method, the optimal conditions for antibacterial activity and protein content were: 80% saturation of ammonium sulfate; V(ethyl acetate)∶V(fermentation supernatant)=7∶5; adsorption pH 6.1 and desorption pH 1.8, respectively. The protein concentrations for the optimized methods were 4 804.70, 396.35 and 543.96 μg/mL, respectively. The diameters of inhibition zone were 13.67, 17.00 and 16.33 mm against Staphylococcus aureus, and 15.20, 17.27 and 16.87 mm against Escherichia coli, respectively. The bacteriocins prepared by the pH adsorption/desorption method possess the most stable antibacterial activity, which could be explained by its high efficiency to extract bacteriocins that attached to the bacterial cells. Thus, the pH adsorption/desorption method was suggested as the preferred method for the extraction of bacteriocins from L. fermentum LBM97. The data provided in this study can be used as reference for selecting suitable method of bacteriocin extraction, which is of great significance for the large-scale extraction and yield improvement of bacteriocins.
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