Functional Screening Of Starch-degrading Bacteria And The Application In Flue-cured Tobacco Leaves

In order to reduce the content of starch in flue-cured tobacco and improve the quality and safety of tobacco leaves,the starch-degrading bacteria were screened in this study.Tobacco leaves and its rhizosphere soils were used as materials to isolate microorganisms with starch degradation functionfrom the experimental base of Tobacco Research Institute of Guangdong Nanxiong.Through the functional screening of the isolates,we obtained predominant strains with both high temperature and high-yielding amylase function.Morphological observation,physiological and biochemical identification and 16S rRN A gene sequence analysis were identified to determine the type of strains.The optimum fermentation conditions were determined by liquid fermentation under different pH values,inoculation amount,temperature and ionic composition.The obtained predominant strains were prepared as microbial inoculum YTK1,B5221and YTK1+B5221,and then sprayed at 15 d,10 d,5 d and 0 d before flue-cured.The amylase activity,nitrate reductase and sucrose invertases of tobacco leaves were measured before sprayed at different time periods.Alpha-amylase,Neutral protease and its mixed solution were sprayed on the untreated middle tobacco leaves before flue-cured.Before baking,the above-mentioned bacterial treatment and exogenous enzyme treatment were subjected to orthogonal test.The content of starch,protein,total sugar and reducing sugar of the treated tobacco leaves were measured after baking.At last,the difference was statistically analyzed by the determination results and the optimum spraying scheme was selected for balancing the intrinsic chemical composition of tobacco leaves effectively.The results showed as follow:1.Isolation and screening of predominant strains:64 thermostable strains were isolated from tobacco leaves and its rhizosphere soils,38 strains were isolated from tobacco leaves and 26 strains were isolated from its rhizosphere soil.Two predominant strains YTK1 and B5221 were obtained by high temperature and amylase selection.Through the identification of strains,strain YTK1 was identified as Bacillus amyloliquefaciens and strain YTK1 was identified as Bacillus subtilis.Two strains can grow at 60?with strong ability of starch hydrolysis.Two Bacillus strains mainly produce alpha-amylase.The amylase activities were 9.67 U·mL^-1 and 9.94 U·mL^-1,respectively.2.The study of enzyme production:The changes of cell concentration and extracellular amylase activity of two Bacillusstrains were consistent under different culture conditions.The best fermentation program for strain YTK1 is that p H is 6,inoculation amount is 2.5%,culture temperature is 35?and ion Additive is about Ca²⁺,K⁺and Mg²⁺.After optimization of the conditions,the cell concentration and extracellular amylase activity increased by86.70%and 338.52%.The best fermentation program for strain B5221 is that pH is 6,inoculation amount is 1.5%,culture temperature is 40?and ionAdditive is about Ca²⁺,K⁺and Mg²⁺.After optimization of the conditions,the cell concentration and extracellular amylase activity increased by 53.62%and 316.28%.3.The changes of enzyme in fresh tobacco leaves:The microbial inoculum YTK1,B5221 and YTK1+B5221(OD₆₀₀=1)were sprayed on tobacco surface for 15d,10 d,5 d and0 d before flue-cured.From 15 days before baking to the pre-bake stage,the activity of amylase in tobacco leaves decreased first and then increased,both nitrate reductase and sucrose convertase showed a decreasing trend.Compared with the control,The activity of amylase and sucrose convertase were increased and the activity of nitrate reductase were decreased after sprayed.4.The spraying treatment of microbial inoculum and exogenous enzyme:The results showed that the content of starch and protein were significantly lower than those of CK after sprayed by YTK1,B5221 and YTK1+B5221 treatment,and the content of total sugar and reducing sugar were significantly higher than CK.In this compare,treatment YTK1 is the best combination.The degradation rate of starch and protein was 39.89%and 16.33%,respectively.The increase rate of total sugar and reducing sugar was 25.54%and 19.97%,respectively.According tospraying treatment with?,D and?+D,the content of tobacco starch after flue-curedby treatment?and?+D were significantly different,and the difference of treatment D was not significant.The content of protein after flue-curedby treatment D and?+D Were lower than CK,the treatment?difference was not significant.The results showed that the trend of the content of total sugar and reducing sugar in the flue-cured tobacco leaves was the same,and the content of total sugar and reducing sugar in the treatment?and?+D were higher than CK,the difference in treatment D was not significant.The difference between the treatments was significant after orthogonal treatment with the microbial inoculum and exogenous enzyme.The degradation rates of starch and protein were the highest in T6 and T5,namely,treatment YTK1+?+D and YTK1+D,the degradation rates were 43.89%and 19.27%,respectively.The increase of total sugar and reducing sugar content in tobacco leaves was T4,namely,treatment YTK1+?,and the rate of increase was 28.39%and 22.01%,respectively.Based on the comprehensive data analysis,T6 is more coordinated in the chemical composition of tobacco leaves,namely,treatment YTK1+?+D.The degradation rate of starch and protein was 43.89%and 15.42%,respectively.The increase rate of total sugar and reducing sugar was 27.83%and 21.88%,respectively.