Rapid Detection Of Spoilage Bacteria In Beer Production By The Combined Approach Of FISH And Microcolony

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.