Research On Detection Of Beer-Spoilage Microorganisms Using RAPD-PCR

Contamination of beer by unwanted microorganisms can seriously affects the quality of beers due to peculiar tastes, cloudy texture and precipitation caused by metabolites of the microorganisms. Currently, traditional techniques.for detecting beer-contaminating microorganisms all have many problems, such as time-consuming, low accuracy and resistance to automation. PCR technique for gene detection provides a fast, sensitive and accurate method for detecting beer-contaminating microorganisms, because it could completely analyses in relatively short time (about 6-10hr).Mixed microbes in the beer ferment liquid, cultivated in culture media, were isolated as a beer yeast (S.cerevisiae) and three beer-contaminating bacteria (W, Y, T) that belong to gram-positive bacteria. The whole genome of mixed microbes in beer ferment was extracted by various methods, produced different cell-disruption efficacy For example, glass bead-beating had the lysis efficiency beyond 90%; through lysozyme or freeze-thaw more than 99% for lysis of bacteria, while less than 30% of yeast cells were disrupted by these methods. The use of phCH2Cl had lysis efficiency for bacterial and yeast cells all reaching 100%. The total genomic DNA that was extracted by glass beads, lysozyme or phCH2Cl all had beyond 23kb in size and good integrity when checked in gel electrophoresis. The fingerprinting patterns of the genomic DNA that was extracted through phCH2Cl were the most clear and sharp. This method was thus used for all following experiments.The genomic DNA of S.cerevisiae was respectively amplified in a thermal cycle for a random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with 76 primers, and the PCR amplification products were detected by gel electrophoresis. 16 primers had no amplification products and 60 produced different fingerprints. First, we selected 16 primers based on clear fingerprints, modest number of bands and distinct predominant band. Further, we selected 4 primers for distinguishing yeast and bacteria based on fingerprint patterns that produced, using each primer for PCR amplification of the genomes of 12 stains.W bacterium and S.cerevisiae both had unique and reproducible RAPD-PCR fingerprinting patterns. With primer S106 for PCR amplification of the genomes of W bacteria and S.cerevisiae, a very significant band was present inbetween the two predominant bands in RAPD-PCR fingerprint pattern of S.cerevisiae in that of Wbacteria. The fingerprint pattern of PCR amplification products of mixed genomes by W bacteria and S.cerevisiae extracted using lysozyme and phCH2Cl showed that when W bacteria reached 0.1 %of the total microbial population, it was detectable by gel electrophoresis. The same was also valid for the mixture of As2.399 and S.cerevisiae, an indication that the lower detection limit of RAPD-PCR for beer-contaminating bacteria was 0.1%.RAPD-PCR distinguished yeast for beer production from those of contaminating yeasts and bacteria. It is possible to monitor the beer fermentation process for contamination control. It is also possible to identify unknown contaminants obtained during regular testing by the quality control laboratories.