【作者】 孟思；

【导师】 刘晓宇；

【作者基本信息】 华中农业大学 ， 食品科学， 2011， 硕士

【摘要】 本研究是一种应用荧光原位杂交和荧光微菌落结合的技术为啤酒厂快速定性及定量检测有害菌的方法。这个方法不但将检测时间从常规的7d缩短到22h,而且其检测结果与常规检测具有一致性。该研究包括了样品和培养基的制备,以及荧光微菌落和荧光原位杂交方法的结合。根据荧光微菌落法进行检测和计数,用荧光探针与CFDA染料所被激发的不同颜色的荧光来定性及定量分析啤酒厂生产过程中所带来的有害菌。本研究首先应用纯培养的菌进行实验方法的建立,而后再通过对实际生产样品进行检测,从而很好地把两种方法相结合,达到啤酒厂生产所需要的快速检测,并且定性及定量的要求。1.初步建立应用荧光原位杂交技术检测啤酒厂中污染有害菌的实验方法,并对实验方法进行优化。根据实验结果显示,FISH技术检测啤酒中乳酸菌、四联球菌及短乳杆菌时,较好的杂交条件为：全细菌探针条件为：杂交温度46℃,杂交时间2.5h,甲酰胺浓度为20%,洗脱液中c(NaCl)为260 mmol/L；乳酸杆菌探针条件为：杂交温度46℃,杂交时间2.5 h,甲酰胺浓度为25%,洗脱液中100mmol/L；四联球菌探针条件为：杂交温度46℃,杂交时间2.5 h,甲酰胺浓度为25%,洗脱液中150mmol/L;短乳杆菌探针条件为：杂交温度46℃,杂交时间2.5 h,甲酰胺浓度为25%,洗脱液中c(NaCl)为159mmo1/L2.微菌落技术是一项可以通过计算定量的技术,FISH技术则是根据其探针设计不同达到定性目的的技术。当两种方法在一定条件下结合后可以达到啤酒厂检测微生物所需的快速定性定量的要求。3.为了达到快速检测的目的,本实验将FISH技术与微菌落技术结合,结合后的检测使得很多难于在培养基上培养的菌同样可以通过次方法检测得到。而且微菌落技术缺少对有害菌鉴定的技术,只能依赖于所选择的培养基,但是有很多有害菌在普通培养基上难于生长,因此就造成了检测不完全的结果。当把FISH技术与之连用后,可以根据荧光探针来判断有害菌,大大减少了检测不完全的可能。是对微菌落技术的有益补充。4从浇注实验、过滤培养实验、FISH及微菌落结合实验比较中,可以看出三种方法所得结果基本相吻合,进一步证明了应用FISH技术以及FISH联用微菌落技术的准确与快捷。更多还原

【Abstract】 This study is an approach that combines FISH and fluorescence microcolony technology for rapid qualitative and quantitative detection of spoilage bacteria for breweries. This method can shorten testing time from 7d to 22 h and the detection rate is the same as conventional or traditional results. This method includes the preparation of sample and culture medium, as well as laboratory practice of fluorescence microcolony detection and fluorescence in situ hybridization (FISH). The sample is examined with fluorescence microscopic detection and counting of bacteria numbers, performing qualitative and quantitative analysis according to the fluorescence colors after reaction with fluorescent probe and CFDA marker. This study establishes the experimental method by applying to cultured bacteria at first, and then examines the samples from production, to combine the two procedures together to meet the demand of rapid test in beer production, as well as the qualitative and quantitative requirements.1. Initially established by fluorescence in situ hybridization to detect beer spoilage Lactic acid bacteria, and optimized the experimental method. According to the experimental results, the best optimized conditions for FISH detection of Lactic acid bacteria、Pediococcus and L. brevis in beer are as follows, general Bacteria:temperature is 46℃,hybridization time is 2.5 h, formamide concentration is 20% and sodium chloride concentration in the eluate is 260 mmol/L. Lactic acid bacteria:temperature is 48℃,hybridization time is 2.5 h, formamide concentration is 25% and sodium chloride concentration in the eluate is 100 mmol/L. Pediococcus:temperature is 46℃,hybridization time is 2.5 h, formamide concentration is 25% and sodium chloride concentration in the eluate is 150 mmol/L. L. brevis:temperature is 46℃,hybridization time is 2.5 h, formamide concentration is 25% and sodium chloride concentration in the eluate is 159 mmol/L. 2. Microcolony is a calculable quantitative technology, while FISH is a qualitative technology, the aim of which is subject to the probe design. When the two methods are combined in certain condition, we can meet the demand of qualitative and quantitative rapid microbe detection of the breweries.3. For the aim of rapid detection, this experiment combines the FISH and microcolony technology. The combined method applies to many kinds of bacteria that are difficult to be cultivated on culture medium. Besides, microcolony technology lacks the capacity to identify spoilage bacteria, and is subject to the selected culture medium. But many kinds of spoilage bacteria is hard to grow on ordinary medium, thus the results usually turn out to be incomplete. When it is combination with FISH technology, the spoilage bacteria can be identified by fluorescence probe. This greatly reduces the chance of incomplete test, which is a beneficial complement of the microcolony technology.4. Similar results are obtained from the injection experiment, filtration and culture experiment, and combination of FISH and microcolony experiment, proving the accuracy and quickness of FISH and the combination of FISH and microcolony.更多还原