Screening And Identification Of Flavor-enhancing Microorganisms In Rice Wine Qu And Its Application In Tobacco Fermentation

Nowadays,people are paying more attention to the health problem caused by smoking.Tobacco companies has to adopt various technologies to reduce the tar content in cigarettes,which leads to a decrease in the transmission of aroma in the smoke.Insufficient aroma has consequently become the primary issue to be solved in the low tar tobacco products,which puts higher requirements on the aroma quality of cigarette raw materials.Based on the promotion effect of yellow wine koji fermentation on the aroma quality of tobacco leaves,this paper analyzed microbial diversity and function in the co-fermentation system,and isolated the aroma enhancing strains to ferment tobacco leaves for the aromatic properties enhancement.Furthermore,the functional components and degradation mechanism of efficient carotenoid degrading strain L-4 were explored.The main results are as follows:(1)The fermentation of tobacco leaves with yellow wine koji significantly improved the aroma quality,and the co-fermentation system contained potential aroma enhancing strains that can degrade macromolecular substances.Compared with natural fermentation,yellow wine koji fermentation of tobacco leaves is more superior at shortening the fermentation cycle,improving sensory scores,and increasing the types and content of aroma components.Microbial diversity and function enrichment analysis found that a large number of microorganisms with macromolecular substances degrading capability were enriched in the tobacco substrates,such as those belong to genera Bacillus,Sphingomonas,Novosphingobium,Pseudomonas,and Staphylococcus,etc.which are potential aroma enhancing microorganisms.(2)The strains Bacillus cereus L-4,Staphylococcus pasteuri L-8,and Novosphingobium sp.D-4 with high degrading activity on starch,protein or β-carotene were isolated from yellow wine koji and identified.The β-carotene degrading mechanism of Bacillus cereus L-4was illustrated.The β-carotene degrading active component FS1 of strain L-4 was isolated and characterized structurally.By properties analysis,hydrolysis amino acid spectrum determination,peptides identification,and molecular docking simulation,it was confirmed that the hydrophobic amino acids in the active component are the key factors involved in its interaction with β-carotene molecules.It is speculated that the active component is an aqueous phase rich in hydrophobic amino acids.When added to the β-carotene lotion,the composition of the aqueous phase changes,which destroys the thermodynamic stability of the lotion,leading to the double bond cleavage under the action of hydrophobic amino acids,free radicals,metal ions,etc.Strain L-4 and its active components have great potential to be applied in spices production,such as β-ionone.(3)After optimal fermentation model,the 3 isolated aroma enhancing strains were prepared as fermentation agent to be applied in 30 kg-scale tobacco leaves fermentation in the cigarette factory.In the laboratory small-scale fermentation with single strain,B.cereus L-4showed greater effects on improving the aroma quality than the other two strains,with sensory score increasment of 2.5.The optimal mixed strains fermentation method was to inoculate L-4 for the first 72 h of fermentation,and then inoculate L-8:D-4 in a volume ratio of 2:1 for the rest fermentation process.Both varieties and contents of volatile components in the fermented tobacco leaves were significantly improved.The contents of phenylethyl alcohol,solanone,geranyl acetone,and macrostigmatrienone increased by 1.82-,1.37-,1.27-and 4-fold,respectively.Based on this,fermentation in pilot(30 kg)in cigarette factory was performed,using the microbial agent consisted of the 3 aroma enhancing strains.The sensory scores of tobacco leaves fermented by agents consisting of pure B.cereus L-4 strain and the optimal mixed strains were increased by 2.5 and 1,respectively,with sensory properties of aroma quality and quantity significantly improved.The contents of ciberane and β-carotene degradation products,such as solanone,dalmartensone,and geranylacetone,were significantly increased.