摘要
在食品发酵行业,需要一种简捷有效的方法检测发酵产品中的有益微生物.聚合酶链式反应-变性梯度凝胶电泳(Ploymerase chain reaction and denaturing gredient gel electrophoresis,PCR-DGGE)技术广泛应用于微生物生态学的研究.采用PCR-DGGE对5种含有乳酸菌的乳制品进行了菌群组成分析,并通过传统培养方法和核酸序列分析进行了验证.结果表明,所有测试样品中的活菌数量都在106～109 cfu/mL之间,通过平板培养法分离出了形态明显的杆状菌和链球状菌,对其进行16S rDNA核酸序列测定及同源性分析,将其鉴定为德氏乳杆菌(Lactobacillus delbrueckii)和嗜热链球菌(Streptococcus thermophilus).建立了由已知乳酸菌组成的DGGE参考阶梯(Reference ladder),可用其对相应菌株进行DGGE鉴定.用两对不同引物进行PCR-DGGE分析,除样品1外,试样中均检测到德氏乳杆菌和嗜热链球菌.对DGGE参考阶梯未能直接鉴定的样品1的电泳条带进行回收、序列分析,分别鉴定为卷曲乳杆菌(L.crispatus)和鼠李糖乳杆菌(L.rhamnosus).同时发现,引物R518-F357对嗜热链球菌模板的识别效率高于引物Lac1GC-HDA2.以上结果表明,RCR-DGGE技术可以对样品中的乳酸菌进行快速的分析和鉴定.
For food fermentation industry, it is often desirable to monitor the probiotic microorganisms present in commercial products for quality control. Simple, reliable and rapid detection methods with low cost are required to achieve this goal. Denaturing gradient gel electrophoresis analysis of 16S rDNA has been widely used in identification of microbial communities without any culturing step. We have modified and tailored this technique, independently confirmed by sequencing and culture analyses, for detecting the lactic acid bacteria in commercial yoghurt products. All sampled yoghurt products contained bacteria ranging from 106 to 109-cfu/mL. Two strains, further identified as Streptococcus thermophilus and Lactobacillus delbrueckii, with typical morphological characters of Bacillus sp. and Streptococcus sp. were isolated. A reference ladder derived from amplification products of known lactic acid bacteria was constructed and characterized. L. delbrueckii and S. thermophilus were detected in four out of five yoghurt products by comparison of the reference ladder, while L. crispatus and L. rhamnosus were identified in another product by sequencing analysis. Furthermore, the preferential amplification was readily found between primers R518-F357 and LaclGC-HDA2. Those results suggested that DGGE could be considered a reliable approach for rapid detection of bacteria in commercial yoghurt products. Fig 4, Tab 1, Ref 30
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2009年第4期534-539,共6页
Chinese Journal of Applied and Environmental Biology
基金
Supported by the National High-tech R&D Program of China(No.2006AA10Z344)
the National Natural Science Foundation of China(No.30570043)
