【作者】 陈利娜；

【导师】 田瑞华；

【作者基本信息】 内蒙古农业大学 ， 发酵工程， 2008， 硕士

【摘要】 乳酸菌是造成啤酒腐败的最常见微生物,对啤酒的危害很大。而纯生啤酒的厌氧菌污染危害更大,会使其质量在短时间内发生改变。目前常用的啤酒有害菌检测方法是厌氧平板培养法,即待长出菌落后通过菌落形态、细胞形态、革兰氏染色、过氧化氢酶阳性以及一系列生理生化实验来鉴定这些腐败菌。这种方法最大的缺陷是费时, 5～7d才可看到结果,不能够及时反馈微生物的污染状况。现代啤酒工业非常需要一种快速微生物检测技术,以解决传统微生物检测结果大大滞后于工艺生产的尴尬状况。PCR技术应用于啤酒厌氧菌的鉴定,不仅快速、准确,而且可以预测污染菌对啤酒的腐败能力,及时为生产提供指导性信息,具有一定的推广价值。PCR技术在快速鉴定啤酒污染菌中的应用,需要对样品中的菌体细胞进行增殖培养以提高检测的灵敏度,为此,本课题比较了目前广泛用于啤酒有害菌检测的选择性培养基对啤酒有害菌检出的效果和菌体在培养基上的生长速度,为啤酒污染乳酸菌选择合适的富集培养基。本文比较了短乳杆菌、林氏乳杆菌、布氏乳杆菌、植物乳杆菌和四联球菌在NBB、MRS和自制UBA培养基上的菌落生量。同时比较了几种DNA提取的方法的提取效果。根据乳酸菌16s rDNA序列的特征,设计合成了针对啤酒有害乳酸菌的通用引物,同时根据乳酸菌16s rDNA序列的高变区设计了5种乳酸菌的特征引物,PCR技术验证结果表明该5种引物能够准确鉴定乳酸菌。在以上研究的基础上,本文建立了PCR快速检测啤酒有害菌的新方法,用基于16s rDNA部分序列的特征引物对啤酒污染乳酸菌进行PCR检测,灵敏度可达到15个细胞(CFU)/250mL,样品的预培养需12～18h。啤酒有害菌检测所需时间从传统的5d减少到24h。更多还原

【Abstract】 Microbial contaminants of the brewing process cause product spoilage. The recent increase in the consumption of draft beer as opposed to pasteurized beer has made the biological control of beer spoilage microorganisms even more important. The plate count method for enumerating microbiological contamination has remained unchanged for over a century, but it requires several days before the microorganisms are detected.The PCR method compares favorably in sensitivity with the present plate count methods (which require several days) for the detection of lactic acid bacteria and with many rapid detection methods.Colony growth of Lactobacillus brevis,Lactobacillus lindneri,Lactobacillus buchneri, Lactobacillus plantarum,Pediococcus damnosus on five culture media (NBB ,MRS and self-made UBA) was compared. Furthermore, The effects of different factors ,including DNA templates and primers , on the quality and reproducibility of PCR were investigated. According to the characteristic of 16S ribosomal RNA gene (16s rDNA),a pair of universal primers were designed for beer spoilage lactic acid bacteria (LAB). Furthermore, 5 other special primers were designed based on identifying results. It was showed that all the primers acted well in accurate identification of LAB by PCR.A new method for rapid detecting beer spoilage microorganisms by PCR was developed,This method is based on the sequence of 16s rDNA, on which a pair of specific primers were designed .Using the primers , beer spoilage LAB species were detected by PCR,The sensitivity was reached to 15CFU/250mL, The pre-enrichment of samples was needed only 12-18h,The duration for beer spoilage determination by PCR (24h) was markly shorter than by conventional method (5-7d) .更多还原