Isolation Purification The Beer-Spoilage Bacteria And Fast Detection And Identification Of Them

Beer-spoilage bacteria are inevitable in beer. Detecting and identifyingthem specially and rapidly can not only guarantee the quality of beer, butalso indicate the level of technology in the company. As the development ofmodem biology technology, the ways of detecting beer-spoilage bacteriumbecome more variously. The methods of selective culture mediums for eachgenus and other five ways of bacteria detection and identification haveapplied in the practice, which are base on ATP, antigen-specific antibodies,PCR, situ hybridization and electrophoretic characterization of D-Lactatedehydrogenase.In this study, the selective culture mediums are used to isolate andpurification the beer-spoilage bacteria and 10 strains are obtained, whichare named after their characteristic as Ls, Lm, Ll, Lsuper, Lw, P, 4-1, 5221,5222, 928. By the means of API50CHL kits the strains above areidentificated as L. plantarum, L. lactis spp lactis, L. brevis3, L. brevis3, L.brevisl, L. plantarum2, L. buchneri, L. fructivorans, L. delbruechii spp delbruechii, uncertain strain respectively.In order to confirm the results above, the primer 27F-907R quotedform the literature are used to sequence the part of 16S rRNA, and theproducts of PCR show clearly, uniquely and brightly in the electrophoresis.The results of sequencing compared to the standard Lactobacillus strainsusing BLAST software in internet. Then it's known that Ls belongs toLactobacillusplantarum, 4-1 and 522 belongs to Lactobacillusparabuchneri, Lm, Lw, P and 5222 belongs to Lactobacillusperolens, 928belongs to C.beijerinckii. However the comparison result of Ll is"Uncultured bacterium clone rRNA228 16S ribosomal RNA gene". Butaccording the result of API50CHL kits and the PCR results ofLactobacillus brevis species primers both designed by myself and quotedfrom literature, strain Ll belongs to Lactobacillus brevis.In this study three sets of species special primers are designed, whichare Lactobacillus brevis, Lactobacillu parabuchneri and Lactobacillusperolens species special primers. Then their most proper annealtemperature and time are determined, which are 5℃1min forLactobacillus brevis, 55℃2min for Lactobacillu parabuchneri and 56℃2min for Lactobacillus perolens. This three sets of species special primersand other two sets quoted from literature build up series of speciesidentification.Then, the ability of hop resistance and beer spoilage is identified, which are arranged below: Ll＞928＞Lw＞5221＞Lm, 4-1＞Ls, P＞5222 andLl, Lw＞5221＞P＞Ls, Lm＞5222＞4-1 respectively. The order of maximalhop-adaptation is Ll, Lw＞P＞Lm＞5221, 928＞4-1, Ls＞5222. it's obviousthat the maximal hop-adaptation of Lw and P have improved much. Thecopies' numbers of amplification of horA, horB and horC are compared,the result are showed that Ll, Lw, P, 5221＞4-1, Ls＞5222, Lm, whichaccords to the ability of beer spoilage very well. As above said, it's certainthat the increasing of hop-adaptation accords the increasing of horA'scopies number. Finally, the lowest concentration of strains could bedetected by the modified primer of horA is 21 colony per millitier...