| Taking Henan Flue-Cured tobacco origined in Henan as material, we adopt chemical techniques together with biochemical means to analyse the variation laws of enzymatic activity and chemical constitutions of some enzymes appeared in various periods of the process of ageing. Under the PCR-DGGE, dominant microorganisms on fermentation tobacco leaves were identified, and their diversities were studied. With the help of FQ-PCR, quantitative detection are done to get the copy numbers in the DNA of dominant microorganisms on fermentation tobacco leaves in various periods of ageing. At the same time, complex inducing agents are used in the mutagenic breeding of dominant microorganisms on fermentation tobacco leaves. By researching the factors such as culture medium compositions, growing temperature, environmental pH, we come to the best fermentation conditions of efficient fragrance-enhancing strains. The microorganisms fermentation fragrance-enhancing preparation by using bacteria enzyme mixtures modeling technology researched the fragrance-enhancing effect and mechanism of tobacco fermentation, which revealed the main fragrance-enhancing mechanism of microorganisms fermentation. Here are the results:1. Dynamic changes of enzymatic activity and chemical constitutions in various periods of ageing. It is shown that regardless of species there are generally slow declines in the contents of total sugar, reducing sugar, starch, total nitrogen, soluble protein, nicotine, pigments and total phenols; while the trend varies in species. Meanwhile, changes of enzymatic activity of polyphenol oxidase, protease, amylase, peroxidase obey the same variation law. They all increase in the early period of ageing to get the summit at the 6th or 9th month, and then decrease gradually. The fact that different specises obeying the same variation law indicates that there is little relationship between the changes of enzymatic activity and the genes of tobacco.2. Studied on the vbariation of microogranisms fauna ,screened and identified the dominant microorganisms on aging flue-cured tobacco leaves. The results showed that , whether natural or artificial fermentation, there are bacteria, and mould without yeast on flue-cured tobacco leaves. Of all the strains, bacteria, the dominanting microorganisms on aging flue-cured tobacco leaves, has the maximum number,then mould accounts for fewer percentage and actinobacteria accounts less. Bacillus of bacteria, aspergillus and penicillium of mould are dominant microflora while actinobacteria is mainly streptomyces. Moreover, the species and quantity of dominanting microorganisms on aging flue-cured tobacco leaves both reduced and its biological activity also decreased.3. Screening and Identification of Dominant Microorganisms of Tobacco Leaves during ageing. Under the PCR-DGGE, dominant microorganisms on fermentation tobacco leaves were screened and identified. The results imply that the types and numbers of microorganisms on the same species of tobacco leaves vary in different periods of ageing. When the time goes in ageing, some microorganisms increase in numbers while other declines. Seen from the general quantity, numbers of microorganisms gradually decrease with the time. Much more dominant bands are shared in the whole process of ageing of the same species while there are great differences among various species. However, there are still some shared groups of dominant microorganisms in different species of tobacco leaves. According to sequence analysis, the shared groups include Bacteriovorax sp. EPC3,Bacillus megaterium,Uncultured Sphingomonas sp,Uncultured bacterium and Uncultured yard-trimming-compost bacterium clone S-12. Besides, we use flat separation to screen out and identify the culturable microorganisms, which is shown to be bacillus including bacillus megaterium, bacillus subtilis, bacillus mycoides, condensate bacillus, bacillus firmus and bacillus cereus.4.The mutation breeding and heredity synergy of dominant strains on cured tobacco leaves.Taking Bacillus megatherium BCK selected from the tobacco leaf surface as starting strain, by means of physically and chemically mutagenizing, using transparent circle method to do preliminary selection. That is to say, selecting the strain with larger Hc value from the starch culture medium and protein culture medium plates, before shaking the bottles which contain the strains to re-select and to detect the enzyme activity ofα-Amylase and protease in zymotic fluid, with the hope to obtain the strains B8 with higher activity. Theα-Amylase activity of B8 is 0.905 times higher than BCK, while the protease activity is 1.227 times higher. This strain has good genetic stability. After five generations cultivation, theα-Amylase activity of the strain can be 0.908 times higher than the starting one; while the protease activity can reach to 0.926 times.5. Quantitative detection of the copy numbers in the DNA and the variation laws of enzymatic activity of dominant microorganisms on fermentation tobacco leaves in various periods of ageing. It is shown that the copy numbers of bacillus megaterium increase to the summit at the 6th month and then decline with the time. The activity the four enzymes also ascends to the peak at the 6th month and then drops. The chemical constitutions of the enzymes have a downward trend. The influence of the change in copy numbers of bacillus megaterium on enzymatic activity and the chemical constitution indicates that bacillus megaterium is the inner power for natural ageing. 6. Research on best culture situations of dominant strains on cured tobacco leaves. The result is as follows: The quality-improved strains have the greatest growth at 40℃; and the propagation reaches its peak at pH 8.0; conditions of weak acidic or alkalescent are not conducive to the growth or breeding of the strains. Corn flour is the best carbon source for the quality-improved strains, while the best nitrogen source is yeast extract, and the best metallic ion is Mg2+.7. Studied the fragrance-enhancing effect and mechanism of fermented fragrance-enhancing strains and the preparation. The result shows that: the tobacco fermentation fragrance-enhancing preparation which was made up with dominant and fragrance-enhancing strains and high biological activity substance can speedup the tobacco fermentation process so as to shrink the fermentation cycle. And that, the fragrance an flavor of tobacco which was worked by fermentation fragrance-enhancing preparation was improved obviously. But, different compositions lead to different effects. This result shows, the tobacco fermentation fragrance-enhancing preparation with multiplicate fragrance-enhancing strains affected greater than the one with single. The research result also shows that, most of the fragrance lead-ups content in cigarettes worked by fermentation fragrance-enhancing preparation increased, while few fragrance lead-ups content reduced. Among them, the good performance-type substance had the biggest amplification, as benzaldehyde, phenylacetaldehyde, phenethyl alcohol, solanone,β-damascenone, 3 isomers of megastigmatrienone , terpene ketone, (1S,2E,4R,6E,8R,11S,12E)-8,11-oxide-2,6,12-duvatriene-4-alcohol and so on. It indicated that the fact of that microorganisms fermentation fragrance-enhancing preparation can increase the fragrance lead-ups in cigarettes is definitely obvious.
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