Study On Quantitative Detection And Mutant Breeding Of Dominant Bacteria On Aging Flue-Cuerd Tobacco Leaves

This article quantitative detected the change of DNA copies of Bacillus megaterium which was dominante strain on surface of tobacco during the aging period by Real-Time fluorescent quantitative PCR technique. Enzyme activity in different aging period were determined by microbiology, enzymatic and molecular biological methods and technologies too. We studied effects of tobacco quality by spraying dominant microbe to the surface of tobacco leaves. Spores of Bacillus megatherium were mutagenized by the physical and chemical mutagenic agents,which were screened from tobacco surface.Results as follow:1. Quantitative detection the change of DNA copies of Bacillus megaterium and dynamic changes of several enzymatic activity of redried flue-cured tobacco leaves aged for different periods.Results showed the DNA copies of Bacillus megaterium increased at first and decreased subsequently with the aging time.The DNA copies per gram tobacco leaves were most at aging 6 monthes. Enzyme activity increased at first and decreased subsequently with the aging time,too. The content of chemical composition decreased by the aging time.Microbe was active during tobacco aging because of multiplication of Bacillus megaterium on surface of tobacco. Enzyme activity and content of chemical composition could be affected by DNA copies of Bacillus megaterium, which indicated that Bacillus megaterium was the internal motivation of flue-cured tobacco natural aging.2. Effects of microbial preparation on tobacco quality.We quantitative detected the change of DNA copies of Bacillus megaterium which was dominante strain on surface of treatment group and the control group tobacco during the aging period by Real-Time fluorescent quantitative PCR technique,also we determined enzyme activity,the content of chemical composition,and the content of volatile aroma composition of tobacco leaves. Results showed the DNA copies of Bacillus megaterium of treatment group was more than the control group,the enzyme activity of treatment group was higher than the control group, the content of chemical composition of treatment group was better than the control group, more importantly, the content of volatile aroma composition of treatment group was more than the control group.To enhanced the quality and shorten the fermentation cycle of redried flue-cured by microbial preparation was feasible and efficient.3. Study on mutation breeding of dominante strain on surface of tobacco .Spores of BCK were mutagenized by the physical and chemical mutagenic agents,which were screened from tobacco surface.From the two selected mediums containing soluble starch and casein, we selected 17 strains with lager Hc value(ratio of diameter of clearing zones and colony)than that of BCK.These selected strains were repeatedly screened by measuringα-amylase and protease activity of fermentating liquid and we obtained 1 strain,whoseα-amylase and protease activity are 1.905 and 2.227 times of that of BCK.The stability study ahowed that the strain have a good genetic stability and after subculturing for 5 generations, theα-amylase activity was 3.201u/mL (It was 1.908 times of BCK),and the protease activity was 23.79u/mL (It was 1.926 times of BCK).