Monitoring bacterial communities in raw milk and cheese by culture-dependent and -independent 16S rRNA gene-based analyses
Monitoring bacterial communities in raw milk and cheese by culture-dependent and -independent 16S rRNA gene-based analyses
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DOI：
10.1128/aem.01716-06
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发表时间：
2007-03-01
影响因子：
4.4
通讯作者：
Montel, Marie-Christine
中科院分区：
文献类型：
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作者：
Delbes, Celine;Ali-Mandjee, Leila;Montel, Marie-Christine
采用单链构象多态性（SSCP）指纹图谱和16 S rRNA基因测序相结合的双重培养依赖性和直接分子方法，研究了圣内克泰尔（Saint-Nectaire），一种生牛奶，半硬奶酪中细菌种群的多样性和动态。在从牛奶中分离的125个16 S rRNA基因中，占优势的克隆属于厚壁菌门（占总克隆的58%），主要隶属于梭菌目和乳杆菌目，其次是变形菌门（21.6%），放线菌门（16.8%）和拟杆菌门（4%）。从四种培养基中收集的126个牛奶分离株的16 S rRNA基因测序显示，存在36个不同的物种，表现出更广泛的多样性，在γ-变形菌门和葡萄球菌属之间的克隆。从170株干酪中分离到21种细菌，优势种为乳杆菌目，亚优势种为放线菌门、拟杆菌门（拟金杆菌属）、或γ-变形菌（Stenotrophomonasp.）。通过SSCP分析从牛奶中分离的DNA指纹图谱产生了复杂的模式，而分析从奶酪中分离的DNA导致由对应于乳酸菌的单峰组成的模式。从平板计数琼脂收获的所有菌落的混合物，补充结晶紫和万古霉素的SSCP指纹显示出良好的潜力，用于监测牛奶和奶酪中的亚优势变形菌门和拟杆菌门（黄杆菌门）生物体。同样，分析可培养的亚群落从奶酪成熟的细菌培养基允许评估的多样性，耐盐放线菌和葡萄球菌生物。直接和培养依赖的方法产生了互补的信息，从而产生了更准确的牛奶和奶酪微生物生态的看法。
The diversity and dynamics of bacterial populations in Saint-Nectaire, a raw-milk, semihard cheese, were investigated using a dual culture-dependent and direct molecular approach combining single-strand conformation polymorphism (SSCP) fingerprinting and sequencing of 16S rRNA genes. The dominant clones, among 125 16S rRNA genes isolated from milk, belonged to members of the Firmicutes (58% of the total clones) affiliated mainly with the orders Clostridiales and the Lactobacillales, followed by the phyla Proteobacteria (21.6%), Actinobacteria (16.8%), and Bacteroidetes (4%). Sequencing the 16S rRNA genes of 126 milk isolates collected from four culture media revealed the presence of 36 different species showing a wider diversity in the Gammaproteobacteria phylum and Staphylococcus genus than that found among clones. In cheese, a total of 21 species were obtained from 170 isolates, with dominant species belonging to the Lactobacillales and subdominant species affiliated with the Actinobacteria, Bacteroidetes (Chryseobacterium sp.), or Gammaproteobacteria (Stenotrophomonas sp.). Fingerprinting DNA isolated from milk by SSCP analysis yielded complex patterns, whereas analyzing DNA isolated from cheese resulted in patterns composed of a single peak which corresponded to that of lactic acid bacteria. SSCP fingerprinting of mixtures of all colonies harvested from plate count agar supplemented with crystal violet and vancomycin showed good potential for monitoring the subdominant Proteobacteria and Bacteroidetes (Flavobacteria) organisms in milk and cheese. Likewise, analyzing culturable subcommunities from cheese-ripening bacterial medium permitted assessment of the diversity of halotolerant Actinobacteria and Staphylococcus organisms. Direct and culture-dependent approaches produced complementary information, thus generating a more accurate view of milk and cheese microbial ecology.