改良PCA培养基检测鸡肉胴体中的污染细菌

Using Modified PCA Medium to Detect Difficult Cultivable Bacteria from Chicken Carcasses

摘要:

通过添加鸡肉浸液改良经典PCA培养基获得模拟鸡肉营养条件的近自然的CEA培养基, 用于检测鸡肉中对营养要求苛刻的污染细菌。根据添加不同浓度的新鲜鸡肉浸液所获得菌落总数的变化获得最佳添加浓度，并利用生化特性及16S rRNA序列对仅生长于CEA培养基的细菌进行了鉴定。在CEA培养基上获得的细菌数量和多样性都显著高于PCA培养基(P < 0.05), 且分离到3株无法在PCA培养基上生长的细菌, 经鉴定其与Enterococcus faecalis、Rothia mucllaginosa, Staphylococcus saprophyticus subsp. saprophyticus的相似性分别为99%、96%、99%。E. faecalis因产生生物胺而被认为是肉品中的腐败菌, 后两者则均是条件致病菌株, 他们的存在对鸡肉产品的安全可能造成隐患。该方法中的近自然培养法能提高对微生物数量和种类的检测灵敏度, 特别适于对营养要求苛刻的污染细菌的检出。

Abstract:

Investigation of microbial spoilage in meats is usually hindered by the lack of suitable growth media and protocols to characterize the causative agents. A near-native medium of chicken extract agar (CEA), developed by the addition of sterile chicken extract into conventional plate count agar (PCA), was utilized to detect difficult cultivable bacteria on chicken carcasses. Suitable additive concentration was selected. Bacteria absent on conventional PCA medium but recovered by CEA were identified based on morphology, biochemical features and 16S rRNA gene sequencing. The colony form unit (CFU) was significantly greater (P < 0.05) and microflora more diversified on CEA than on PCA. CEA containing 3.0% (W/V) chicken extract gave the highest microbial enumeration at 64 CFU/g, almost twice that in PCA. Three species of bacteria absent on conventional PCA medium were recovered by CEA. According to their morphology and biochemical features, three bacteria recovered were identified as Enterococcus sp., Rothia sp., and Staphylococcus sp., respectively. The bacterial identification was confirmed by 16S rRNA sequence analysis with the similarity being 99% for both Enterococcus faecalis and Staphylococcus saprophyticus subsp. saprophyticus, and 96% for Rothia mucllaginosa. Three bacteria recovered by CEA are considered as either spoilage (Enterococcus sp.), opportunistic pathogen (Rothia sp.), or pathogen (Staphylococcus sp.) in foods, thus to cause problems associated with food safety.