基于PCR-DGGE方法分析榨菜中乳酸菌群落结构

摘要

为了深入了解榨菜中的乳酸菌多样性以及影响因素，以市场销售含有辣椒和不含辣椒两种袋装榨菜为研究对象，测定榨菜中的食盐浓度以及亚硝酸盐含量；通过变性梯度凝胶电泳（denaturing gradient gel electrophoresis， DGGE）法分离榨菜总微生物混合16S rDNA基因V7～V8片段，采用Quantity One软件分析乳酸菌物种丰富度、均匀度及物种多样性指数。结果表明：含辣椒的榨菜食盐浓度和亚硝酸盐含量均略低于不含有辣椒的榨菜；含有辣椒和不含辣椒的榨菜两者间物种多样性指数、丰富度和均匀度无显著差异（P＞0.05）。通过回收DGGE电泳带，经克隆后测定碱基序列、与GenBank库序列对比鉴定，不含有辣椒的榨菜5条回收聚合酶链式反应（polymerase chain reaction，PCR）-DGGE泳带a、b、c、d、e经鉴定分别与Pediococcus argentinicus CRL 776、Uncultured Lactobacillus sp. isolate DGGE gel band lx12、Uncultured bacterium clone 11.02-12、Uncultured bacterium clone 11.02-9、Uncultured Lactobacillus sp. clone PxSC03相似度为98%、96%、97%、97%、97%。含有辣椒的榨菜4条回收PCR-DGGE泳带1、2、3、4经鉴定分别与Uncultured Lactobacillus sp.、Lactobacillus sakei A156、Lactobacillus sakei YY1、Lactobacillus sakei WJ1相似度为96%、97%、98%、97%。研究结果表明辣椒对榨菜中微生物群落结构无显著影响。%In order to understand the diversity of lactic acid bacteria in pickled mustard tuber and its influencing factors, two commercial bagged pickled mustard tubers (with and without hot pepper) were determined for the contents of salt and nitrite, and the V7-V8 region of total bacterial 16S rDNA sequences was separated by denaturing gradient gel electrophoresis (DGGE). Furthermore, the species richness, evenness and diversity of lactic acid bacteria were analyzed by the Quantity One software. Results indicated that the contents of salt and nitrite in the first sample were slightly lower than in the second sample. However, no significant difference in species richness, evenness and diversity was observed between both samples (P>0.05). The target DGGE bands were recovered, cloned and sequenced by comparison with the sequences published in the GenBank after PCR amplification. A total of 5 bands from the DGGE gel of the second sample were recovered and identified as Pediococcus argentinicus CRL 776, uncultured Lactobacillus sp. isolate DGGE gel band lx12, uncultured bacterium clone 11.02-12, uncultured bacterium clone 11.02-9, and uncultured Lactobacillus sp. clone PxSC03 with a similarity of 98%, 96%, 97%, 97%, and 97%, respectively. Four bands from the first sample were recovered and identified as uncultured Lactobacillus sp., Lactobacillus sakei A156, Lactobacillus sakei YY1, and Lactobacillus sakei WJ1 with a similarity of 96%, 97%, 98%and 97%, respectively. These results show that hot pepper has little effect on the microbial community structure of pickled mustard tuber.