Isolation,Indentification And Molecule Detection Biogenic Amines Producing Bacteria Of Rice Wine Qu

The 45 of strains biogenic amine bacteria that could were screened out from the rice wine.26 strains can produce histamine and putrescine simultaneously,while only three strains can produce tyramine.In order to identify the histamine and putrescine produce species,On this paper,the RAPD-PCR classified identification,16S rRNA combined species for single-genotype strains and Enterobacter sp special primers separated method were used for focus on identificating and classificating 26 strains of histamine and putrescine produce bacteria screened out from the rice wine.Results demonstrated a part of the strains are classified using RAPD-PCR(two pairs of primers M13/D11344)combining 16S rRNA identification,while 17 strains can not identify the species.Due to most of those strains are Enterobacter sp,specific primer DN1-1F/2R for Enterobacter sp in contrast two pairs of primers amplification method directly for a bacterial strains for Cronobacter sp to amplification and sequencing were used.All of the 26 strains of histamine and putrescin bacteria were identified by the three methods,as mentioned above.That demonstrated the 12 strains were Enterobacter cloace,3 strains was Enterobacter asburiae including La7 being Citrobacter amalonaticus,Hc3 being Cronobacter sakazakii,Ha15 being Klebsiella pneumoniae,3 strains tyramine produce bacteria were La5 being Enterococcus sp,La6being Enterococcus sp,La8being Enterococcus durans.This paper established a multiple PCR method that can simultaneously detect histamine,putrescine and tyramine producing bacteria.And this method can be applied to detect the biogenic amine produce bacteria of rice wine Qu and brewing process of rice wine.The multiple PCR method being established was used for identifying biogenic amine produce bacteria that the screened out from the rice wine Qu,The result show 26 strains can produce histamine and putrescine simultaneously and three strains can produce tyramine,which correspond to the result of HPLC.The method was used for detected the Qu of rice wine and the brewing process of rice wine,and the putrescine and tyramine involved gene was amplificated,while histamine involved gene was not amplificated,which was also correspond to the result of HPLC.Further,the Real-Time PCR detection method was established to detecte the brewing process of the rice wine.The result demonstrated that the number of histamine produce bacteria was little 104 copy/?L and nonsignificant of the brewing process of the rice wine.However the number of tyramine and putrescine produce bacteria become much accompanied by brewing process of rice wine,finally reached 1.0×1010 copy/?L,this also correspond to the result of HPLC detected.