Screening Of Microorganisms Suitable For Fermentation And Flavor Enhancement Of Tobacco-containing Raw Materials And Preparation Of Starter

WHO’s restrictions and prohibitions on the prohibition of the spread of tobacco smoke in public places have led to the rapid development and popularization of smokeless tobacco represented by oral smoke,which is a type of smoke that does not produce smoke and does not cause second-hand smoke hazards.The product.Because the smoke contained in the mouth is directly put in the mouth for smoking,without the high-temperature release process of tobacco combustion,this places high requirements on the quality of the raw material containing smoke.The use of appropriate technological methods to improve the quality of tobacco-containing tobacco raw materials can effectively improve the quality of tobacco-containing tobacco.In this experiment,the high-throughput sequencing technology was used to analyze the microbial flora of Dalihongda CL314 and Yuxi K326 tobacco leaves,and the dominant bacteria were selected.The high-density cultivation process and vacuum freezing of the Bacillus amyloliquefaciens GUHP86 and GZU03 screened by the research team were conducted.The drying protection agent is optimized and then applied to tobacco leaves for fermentation,which provides an experimental basis for further industrialization.The research results are as follows:（1）Using high-throughput sequencing technology to detect microbial diversity in Dalihongda CL314 and Yuxi K326.It was found that the tobacco leaves of Dalihongda CL314 and Yuxi K326 were mainly Microbacterium,Acinetobacter,and Pseudomonas,but Yuxi K326 tobacco had a higher proportion of Pseudomonas,while Pseudomonas most of them are harmful bacteria,and Dalihongda CL314 tobacco leaves were selected as raw materials for the next step.（2）Select Bacillus amyloliquefaciens GUHP86 and GZU03 as the test strains,and explore the high-density culture process of Bacillus amyloliquefaciens GUHP86and GZU03 through single factor,PB and Box-Benhnken response surface experiments to obtain the best culture conditions.The optimal culture conditions for the strain GUHP86 are:the medium is soy peptone 1%,starch 1.5%,yeast extract0.5%,MgSO₄ 0.02%,culture conditions:inoculation amount 0.2%,initial pH 6.2,temperature 37℃,culture time 27 h The liquid volume is 80/250 mL,and the optimized viable cell count reaches 9.57 log CFU/m L,the actual number of viable bacteria was 7 times higher than that of the initial fermentation conditions,;the optimal culture conditions for strain GZU03 are:the medium is soybean peptone 1%,glucose 1%,yeast extract 0.5%,MgSO₄ 0.01%,culture conditions:inoculation volume 0.6%,initial p H 8.0,temperature 30℃,culture time 24 h,filling volume80/250 mL,and the number of viable bacteria after optimization reaches 9.58 log CFU/mL,the actual number of viable bacteria was 13 times higher than that of the initial fermentation conditions,and the protease activity is also significantly improved.（3）Ice crystals formed at low temperature during vacuum freeze-drying have a great impact on the survival rate of the strain.Adding a suitable protective agent can effectively reduce the influence of the strain on the low temperature and improve the survival rate of the strain during the drying process.Through the single factor experiment and Box-Benhnken response surface experiment,the protective agent added in the vacuum freeze-drying process was optimized to obtain the most suitable compound protective agent.The best protective agent combination of strain GUHP86is:gelatin 11.5%,skimmed milk powder 11%,sodium glutamate 6.3%,the survival rate after optimization is 90%,the survival rate is about 37 times higher than that without adding protective agent;strain GZU03 is the most The best combination of protective agents is:gelatin 11.5%,gum arabic 12%,mannitol 6.3%,and the survival rate after optimization is 92%,which is about 20 times higher than that without the protection agent.（4）Taking Dalihongda CL314 tobacco leaves as raw materials,using Bacillus amyloliquefaciens strains GUHP86 and GZU03 to carry out mixed fermentation of tobacco leaves,and using single factor,PB and CCD response surface methods to explore the fermentation conditions to obtain the best fermentation process:Inoculation amount 24%,glucose addition amount 1.2%,moisture content 40%,compound ratio（GZU03:GUHP86）1:1,soybean protein addition amount 1%.After optimization,the sensory performance of the fermented tobacco leaves was increased by 40.66%compared with the initial natural fermentation.The initial natural fermentation leaves did not produce protease activity,and the optimized process had29.53 U/g protease activity.The GC-MS detection found that the relative content of nicotine in the tobacco leaves after fermentation decreased,the relative contents of various tobacco leaf aroma substances such as isobutyl isobutyrate,linalool,β-damascone andβ-ionone are increased,and the overall is more harmonious,the fragrance is complex and strong,the energy is strong,and the irritation is small,Tobacco leaves with rich flavor and better flavor.