产抗菌肽乳酸菌的分离、鉴定及培养条件优化  

Isolation and identification of antimicrobial peptides-producing lactic acid bacteria and optimization of the culture conditions

作　　者：杨云喜[1] 李佩[1] 徐岳松 郭洪秀[1] 郭明月[1] 曹毅[1] 乔代蓉[1] 徐辉[1] 

机构地区：[1]四川大学生命科学学院微生物与代谢工程四川省重点实验室,成都610065

出　　处：《应用与环境生物学报》2014年第5期817-824,共8页Chinese Journal of Applied and Environmental Biology

基　　金："十二五"国家科技支撑计划项目(2013BAD10B01;2014BAD02B02);国家科技基础条件平台项目(NIMR-2014-8);四川省科技厅项目(2013HH0007;2013GZX0161-3;2013GZ0058;2013JCPT003)资助~~

摘　　要：乳酸菌(Lactic acid bacteria,LAB)抗菌肽对食品生物防腐、疾病防治等有着重要意义,本研究直接从食物中分离抗菌肽高产乳酸菌,并研究其活性和发酵条件.采用溶钙圈法分离得到14株产抗菌肽乳酸菌,用琼脂孔穴扩散法测定抑菌活性,菌株9-4抑菌效果最显著,抑菌圈直径为17.09 mm,并用盐析和凝胶层析法初步鉴定抑菌物质,显示其为抗菌肽;经生理生化和16S rDNA分子鉴定,该菌株为Lactobacillus paraplantarum,所产抗菌肽对细菌有广谱抑菌性,而对真菌无抑菌活性.利用大肠杆菌Escherich coli JM109为指示菌,对菌株9-4发酵条件进行单因素优化,发现葡萄糖、酵母提取物+胰化蛋白胨和锰离子浓度对其产抗菌肽影响最为显著;对3个因素进行正交优化,得到该菌产抗菌肽的最佳发酵条件:葡萄糖浓度3%,酵母提取物浓度3%,胰化蛋白胨浓度2%,M n2+浓度0.003 mol/L,pH 6.0,在此条件下进行发酵,抗菌肽产量比优化前提高47.36%,抑菌圈直径大小为25.18 mm.本研究表明菌株9-4在产抗菌肽方面具有很大开发潜力,在食品、农业、医药等行业具有广阔的应用前景.Antimicrobial peptides（AMPs） produced by lactic acid bacteria（LAB） are important in food biopreservation, disease prevention and treatment. The purpose of this study was to directly isolate AMPs from food high-yielding LAB and to study its activity and fermentation conditions. We used dissolving calcium circle to screen the AMPs-producing strains, 16 S rDNA sequence analysis to identify the object strain, salting-out and Sphadex-50 chromatography to purify the AMPs, single factor experiment and orthogonal test to optimize the fermentation conditions. As a result, fourteen AMPs-producing LAB were isolated from diverse samples. Comparison of inhibition zone size showed that the strain 9-4 had the most significant inhibitory effect, with an inhibition zone diameter of 17.09 mm. The identification results showed that it was Lactobacillus paraplantarum. The peptide produced by the strain 9-4 was broad-spectrum antimicrobial against bacteria, without antibacterial activity against fungi. With E. coli as indicator bacteria, the single factor optimization of fermentation conditions showed that the concentration of nitrogen, carbon concentration, ion concentration significantly affected the inhibitory effect of fermentation supernatant. By orthogonal optimization of the above three factors, we obtained the optimum fermentation conditions for the bacteria to produce antimicrobial peptides： 3% of glucose content, 3% of yeast extract content, 2% of tryptone content, 0.003 mol/L of Mn2＋ concentration, pH 6.0. Under these conditions, the inhibition zone diameter size was 25.18 mm, 47.36% greater than before. In the aspect of AMPs production, the strain 9-4 has a great potential as biopreservatives in food, agriculture and medicine.

关 键 词：乳酸菌 抗菌肽 抑菌活性 发酵条件优化 

分 类 号：Q93[生物学—微生物学]