Study On The Optimization Of Detection Culture Medium Of Draught Beer Spoilage Bacteria And Ripid Detection Method

Draught is produced by the process of aseptic brewing, aseptic filtration and aseptic packaging, as well as non-pasteurization technology. If the draught beer is contaminated by beer spoilage bacteria in the production, the quality of draught beer will have serious repercussions, so we should strictly control and prevent the contamination of beer spoilage bacteria. So far, the brewery still use technology of medium to detect beer spoilage bacteria, such as NBB,MRS etc. However, those media are used by brewery now are made in overseas ane imported.Fourthermore, the characteristic of domestic and foreign beer is quite different, there may be a difference between the varieties and quantities of beer spoilage bacteria as far as domestic beer and foreign beer is concerned. There are some rapid detection methods, such as bioluminescence assay, PCR, immunology assay etc. However, those are not widely used in the brewery for the lack of the detection sensitivity and specificity or the expensive equipment and reagents, as a result, it is necessary to develop the new detection medium of beer spoilage bacteria in order to be suitable for the characteristic of demestic beer and exploit a new rapid detection method.MRS and NBB, which were widespreadly used to detect and analyze beer spoilage bacteria in world brewing industry, were optimized by malt rootlet extract, arginine and folic acid according to the nutritional demands of beer spoilage bacteria in beer. The ingredients of the two media were optimized by single factor experimentation and quadratic design respectively. The optimum addition dosage of the three nutritional factors in MRS was as follow: 20ppm malt rootlet extract, 0.75g/L arginine and 220ppm folic acid. The optimum addition in NBB was as follow: 40ppm malt rootlet extract, 0.75g/L arginine and 140ppm folic acid. The diameter of colonies incubated with the optimized MRS media was bigger, and the number of colonies of beer spoilage bacteria was more than that of original medium. The allochroism time of optimized NBB was approximately 12h shorter than that of original NBB medium, the number of beer spoilage bacteria were also obtained by optimized NBB is similar to the phenomena of the optimized.This study established a rapid detection method of beer anaerobic bacteria, and the following procedures were taken to analyze samples. Firstly, beer samples were filtrated by polycarbonate membrane. Secondly, the microcolonies which grew on the polycarbonate membrane were stained with CFDA for 10min under the dark environment after anaerobic culturing for 36h, based on using fluorescence microscopy for counting. All of the repetitive tests and the results of the two analysis were not significantly different (P> 0.05) after corresponding test results were in comparison to the routine plate culture counting method. The minimum detection limit of the fluorescent microcolony method was 4 cfu/mL.It takes 30 hours to detect beerspoilage bacteria based on fluorescent microcolony method in combination with the optimized medium. The superiority of MRS optimized is showed that cell growth rate and number of formation colonies are obviously better than the original MRS on the production-scale test of brewery. It proved that the fluorescent microcolony method can rapidly detect beer spoilage bacteria in the brewery and is more fast, economic, accurate and easy to performance to be applied.