Study Of High Moisture Solid-state Fermentation To Improve The Quality Of Upper Tobacco Leaves

The quality of upper tobacco leaves is relatively low, which is reflected in the high starchand protein content, poor aroma quality, heavy miscellaneous gas, more irritating, thus resultsin a low value in use. So far, artificial fermentation of processing tobacco in factory isgenerally low moisture (less than20%) solid-state fermentation, in which the tobaccofermentation is very slow and the fermentation cycle is too long. Moreover, it takes up a lot ofwarehouse area. Although increasing the moisture of tobacco can contribute to acceleratingfermentation, it will easily lead to tobacco mildew under high moisture condition. Therefore,during solid-state fermentation of upper tobacco leaves at the high moisture condition, theresearch focus on how to meet rapid fermentation, reducing tobacco starch and proteincontent to improve the quality of their smoking and avoiding tobacco mildew.In this study, the changes of the amount of microorganisms and chemical compositionsof tobacco leaves during high moisture solid-state fermentation were firstly studied, and thefeasibility of fermenting tobacco was determined. Secondly, how to effectively reducetobacco protein and starch content, so as to enhance the quality of tobacco without mildew byraising the temperature of fermentation, anaerobic fermentation, artificial inoculatedfermentation and enzyme fermentation were systematically investigated under the highmoisture condition. Furthermore, the impact of fried-rice leachate on microorganisms intobacco during fermentation was also studied. The details of the above experiments are asfollows:The changes of the amount of microorganisms and chemical compositions of tobaccoleaves during high moisture solid-state fermentation were studied. The results showed that innormal temperature aerobic solid-state fermentation, the tobacco leaves became moldy after2~3days, and the aerobic bacteria gradually reduced while the yeasts and moulds grewrapidly; In normal temperature anaerobic solid-state fermentation, the amount of anaerobicbacteria had a slight reduction while the amount of yeasts increased substantially at the earlydays and stabilized after4days; In high temperature aerobic and anaerobic solid-statefermentations, yeasts and moulds did not grow while both the aerobic and anaerobic bacteriareduced significantly; In the four different fermentation conditions, the content of starch andprotein in tobacco decreased with time.High moisture anaerobic solid-state fermentation was used to decrease the content ofstarch and protein of tobacco. Monofactorial and orthogonal experiments were adopted for theoptimization of the condition of solid-state fermentation. The results showed that the bestcondition for solid-state fermentation was that fermentation temperature was45oC, moisturecontent of the tobacco was50%and fermentation time was9d. Under these conditions ofsolid-state fermentation, the starch and protein decomposition rates of tobacco were20.41%and12.35%. Therefore, the content of starch and protein of tobacco can be quickly decreasedin a short time by this solid-state fermentation under optimum conditions.Two thermotolerant beneficial strains were isolated from tobacco leaves, one is anamylase producing bacterial strain and another is a protease producing bacterial strain. Both of them were identified by morphological observation of colonies,16S rRNA sequencehomology analysis. We initially identified them as Bacillus subtilis and Bacillusamyloliquefaciens. They were called Bacillus amyloliquefaciens JDF and Bacillus subtilisJDB Then two strains were used for tobacco solid-state fermentation, and the condition wasoptimized by monofactorial experiments. The experimental results showed that the bestconditions for solid-state fermentation were that: JDF and JDB’s inoculum(v:w) of bacterialsuspension(OD600=1) were7.5%and10%, tobacco filling degree was50g·(500mL)-1,fermentation temperature was48oC, moisture content of the tobacco was45%andfermentation time was5d. Under these conditions of solid-state fermentation, the starch andprotein decomposition rates of tobacco were22.33%and16.74%. The content of starch andprotein of tobacco were close to the standard of the good quality tobacco. In this method, wecan greatly shorten the fermentation time than traditional storage fermentation.In this study, enzyme dosage used in tobacco fermentation was optimized. The complexenzymes recipe are as follows: cellulose200U·g-1, amylase200U·g-1, glucoamylase300U·g-1and protease500U·g-1. After high-moisture anaerobic solid-state fermentation with thecomplex enzymes, the aroma quality of the tobacco was increased while the miscellaneousgas and irritation were reduced.In conclusion, under the conditions of high temperature and high moisture, the content ofstarch and protein of upper tobacco leaves was significantly degraded, and the quality ofsmoking was improved by anaerobic fermentation, double bacteria fermentation and complexenzyme fermentation.