Dynamic Analysis Of Flue-Cured Tobacco In Aging Process And Study Of Functional Microorganisms

The spontaneous aging process is the degradation and transformation of large molecules and forming of aroma-causing compounds in flue-cured tobacco(FCT)through the synergistic action of microorganisms,enzymes and chemical oxidation.The aging process is an essential part of improving the FCT quality.However,so far,it still needs to be clarified about the critical factors,their interrelationships affecting the FCT quality,and the process mechanism affecting the quality of FCT in the aging process.Meanwhile,the traditional spontaneous aging process is time-con-suming and low efficiency,so the analysis of the mechanism of spontaneous aging and the profil-ing of critical factors affecting the quality can provide the theoretical basis for reforming the tra-ditional aging process.Thus,our study took the superior,medium,and inferior quality of strong-and light-flavor FCT as the research object,dynamically studied the changes of microorganisms,enzymes,flavors and sensory quality in the aging process,analyzed the difference factors,ex-plored the common difference factors in the different sample,profiled the correlation between the factors,analyzed the mechanism of the aging process,and lied the theoretical foundation for the targeted screening of functional microorganisms to biological intervention in the aging process.The main contents and results are as follows.(1)Study of the dynamic changes of microbial diversity during agingUsing high-throughput sequencing technology,bacterial and fungal diversity was investigated.The common dominant genera with relative abundance greater than 1%for different flavor types of FCT were bacteria such as Sphingomonas,Pseudomonas,Methylobacterium,Acinetobac-ter and fungi such as Alternaria、Aspergillus、Sampaiozyma and so on.The dominant bacterial genera in the two types FCT were significantly correlated with environmental temperature(P<0.05)by the redundancy analysis.Based on theβ-diversity analysis,there are 21 species of bacteria and9 species of fungi in different samples during the aging process.The function of the bacterial and fungal flora was predicted.Flavor formation-related pathways have a high abundance,such as fatty acid synthesis and degradation,aromatic amino acid degradation,fatty acid synthesis,sec-ondary metabolite degradation,carbohydrate degradation and fermentation in bacteria and fatty acid degradation pathways in fungi.(2)Study of the dynamic changes in the composition and quality characteristics of FCT during agingThe contents of starch and total phenol in FCT were significantly decreased.The starch con-tent in strong-flavor(SFT)was higher than that in the light-flavor tobacco(LFT),but the contents of total phenol,reducing sugar,and total sugar were lower than that in LFT.The contents of re-ducing sugar,total sugar and starch were positively related to the quality grade of FCT.The total phenol and total nitrogen contents were inversely related to the grades in both flavor types FCT.Our study focused onα-amylase and glycolytic enzymes related to starch degradation and poly-phenol oxidase,lipoxygenase and peroxidase related to aroma formation.The change of enzyme activities in the two types FCT showed different fluctuations.The volatile components in samples were analyzed using non-targeted techniques based on HP-SPME-GC-TOF-MS.82 differential compounds in SFT and 69 differential compounds in LFT were identified.There are 34 different common compounds.During aging,FCT color became darker and browner according to the change in the colourimetric index.The color of LFT was brighter and more yellow than SFT at the same aging time.The quality of FCT continued to improve with aging.After aging,the total evaluation scores of the superior,middle and inferior grades of SFT increased by 11,11 and 13.5points,respectively;the total scores of the three grades of LFT increased by 6.5,10.5 and 8 points,respectively.(3)Analysis of the correlation between the key factorsThe relationship model between chemical composition and quality evaluation was constructed using a hybrid algorithm combining a genetic algorithm(GA)and a feedback neural network(BPANN).The R~2 value of the model for SFT was 0.97075 and 0.96832 for LFT,indicating that the model has a good prediction.The GA-BPANN output was coupled with MIV algorithm to quantify the degree of importance.There were eight common substances:phenylethyl alcohol,benzyl alcohol,safranal,(E)-solanone,(E)-β-damascenone,3-ethyl-4-methyl-1H-pyrrole-2,5-di-one,3-oxo-α-violanol andβ-springene,who were shared in the top 30 MIV values in the two mod-els.Further,the correlation analysis of the critical factors of aging process,with the help of the cumulative explanatory rate of each RDA ranking chart and the contribution of factors with sig-nificant influence,showed a significant correlation(P<0.05)between the factors in SFT and LFT.The significant factors related to the changes in the abundance of dominant bacteria and fungi in SFT were lipoxygenase(23.6%of the total explanation)and glycolytic enzymes(39.4%),which increase the content of the aroma-causing components through the degradation of starch and the formation of reducing sugar,in turn significantly affected the color and sensory quality(smoke strength,total score,and reduction of impurities)of SFT.The significant factors related to the changes in the abundance of dominant bacteria and fungi genera in LFT were amylase(57.0%)and lipoxygenase(53.7%),which promoted the aroma of FCT through the degradation of starch and polyphenolic substances,in turn significantly affected the color and sensory quality of LFT(aroma quality,aftertaste,and irritation).(4)Targeted screening and application of functional strainsBased on the aging mechanism,three functional strains,Bacillus amyloliquefaciens LB,Ba-cillus kochii SC and Filobasidium magnum F7,were obtained by targeted screening.The func-tional strains were used for solid-state tobacco fermentation by bioaugmentation,and all samples obtained the best quality improvement by fermenting for 2-2.5 d.Strain LB and SC were incubated for 2 d at an initial addition ratio of 3:1,37°C,and 80%relative humidity,and the total sensory score improved by 8 points.Strain F7 and SC were co-cultured for 2 d at 30°C and 80%relative humidity at an initial addition ratio of 3:1,and the total sensory score improved by 10 points,obtaining better results than monoculture.The study investigated the differences in microbial di-versity,enzymatic activity and volatile components between monoculture and co-culture.The crit-ical role of starch degradation contributing to the formation of the Maillard reaction products and lipoxygenase promoting the degradation of terpenoids and higher fatty acids in aging was con-firmed.