Screening And Identification Of β-carotene Degrading Strain And Its Effect On Enhancing Aroma Of Cigar Tobacco Leaves During Air Curing

The air curing of cigar tobacco is a key element in the production of raw cigar material,transforming it from an unsmokable to a smokable state.Air curing of tobacco color changes significantly,and from green to yellow and finally brown,and the substances that affect the color are also precursors of aroma components.In the process,air curing humidity has an important effect on the color class aroma precursors(chlorophyll a,chlorophyll b,lutein,β-carotene,chlorogenic acid,rutin,scopoletin,protein,amino acids and reducing sugar),aroma and microorganisms.Based on this,this study mainly adopts a biological approach to investigate the role of microorganisms in the conversion of color class aroma precursors into aroma during tobacco air curing,and at the same time explore and analyze the corresponding mechanism of action.To this end,we first determined the optimal air curing humidity of cigar tobacco leaves.After that,we used gas chromatography-mass spectrometry(GS-MS)and liquid chromatography-mass spectrometry(LC-MS)to determine the aroma components and aroma precursors of tobacco leaves during the air curing process.Meanwhile,in order to understand the role of microorganisms in the process,we used 16 S r RNA bacterial and ITS fungal high-throughput sequencing techniques to comprehensively analyze the tobacco microorganisms during the air curing process.After understanding the changes of bacteria and fungi in cigar tobacco leaves during different periods of drying,I conducted a joint analysis of aroma precursors,aroma substances and microorganisms,and found and screened a strain of β-carotene efficiently degrading Paenibacillus.In addition,we analyzed the effect of its application in the air curing process and found that the bacterium was able to increase the content of carotene degradation products in tobacco leaves.In order to understand the mechanism of action of this bacterium,we determined the genes and metabolites of Paenibacillus by using Illumina Novaseq platform and Waters Acquity I-Class PLUS ultrahigh-performance liquid chromatography in tandem with Waters Xevo G2-XS QTOF highresolution mass spectrometer.The main results obtained from the study are as follows:1.Based on the content of stromal pigments and polyphenols in cigar tobacco,the optimum humidity for air curing cigar tobacco has been determined.Humidity has a great influence on the air curing of cigar tobacco.Appropriate air curing humidity can produce cigar tobacco with better appearance and aroma quality,which in turn improves the usability of the tobacco.The results of the three air curing humidity studies of high humidity,medium humidity and low humidity showed that the degradation of chlorophyll and polyphenols was higher during the air curing process of cigar tobacco treated with high humidity,and the degradation of plastid pigment was higher to avoid the appearance of greenish tobacco after air curing,and more degradation of reducing sugar could improve the non-enzymatic Browning reaction products.However,more degradation of polyphenols will cause the appearance of black phenomenon in the cigar tobacco,which would reduce the quality of the leaf;low humidity treatment of the degradation of plastid pigments and polyphenols was lower,which would easily lead to the appearance of greenish and brownish leaf;low humidity treatment of the degradation of plastid pigments and polyphenols was less,which would easily lead to the appearance of green and brownish leaf.Low humidity treatment of the tobacco less degradation of plastid pigments and polyphenols,prone to contain the phenomenon of green and brown unevenly lighter;medium humidity is conducive to the formation of the color of cigar tobacco,while the degradation of carotenoids and the degradation of the substrate of the Maillard reaction of the cigar tobacco treated with the treatment of the higher,so that the tobacco has a high content of the aroma.At the same time the cigar tobacco leaves treated with medium humidity had rich microorganisms during the air curing process.In conclusion,cigar tobacco leaves are beneficial to air-curing under the conditions of moderate humidity of 80%,75%,70% and 45% at withering stage,yellowing stage,browning stage and dry gluten stage,respectively,which provides a good environmental basis for subsequent microbial screening.2.The microbial changes during the air curing of cigar tobacco were studied and β-carotene biodegradation was determined to increase the aroma substances in tobacco leaves.In the air-drying process,except amino acids,the contents of color aroma precursors(chlorophyll a,chlorophyll b,lutein,β-carotene,chlorogenic acid,rutin,scopolamine,reducing sugar,amino acids and protein)decreased obviously,and the aroma components also changed.In addition,the structure of bacterial and fungal communities varied at each critical stage of the air curing process.Partial least squares discriminant analysis showed that unclassified Burkholderiaceae,Paenibacillus,Bacillus,and Alternaria were the key microorganisms in the formation of tobacco aroma components.Correlation analysis showed that β-carotene showed a significant negative correlation with all three microorganisms,β-carotene as an important aroma precursor substance in tobacco,and its degradation products have an important contribution to the aroma of tobacco,so the screening of β-carotene degrading bacteria was mainly carried out at a later stage.3.A strain of Paenibacillus with high β-carotene degrading efficiency was screened from cigar tobacco leaf and cultured.Taking cigar tobacco as the research object,four β-carotene degrading strains were screened out during the air curing process,and they were numbered as A1,A2,A3,A4.The degradation efficiency and application effect of the four strains were compared and analyzed,and it was determined that the A2 strain had a better effect.Molecular identification showed that A2 belonged to the genus Paenibacillus.The aroma substances produced by the degradation of β-carotene by this strain were determined by GC-MS.It was found that the strain could degrade β-carotene into linalool and tabanone.Then,the culture conditions of this strain were optimized,and the results showed that soluble starch was 4.67 g/L,skim milk powder was 5.55 g/L,Na Cl was 10.85 g/L,culture conditions were p H 7.0,inoculation amount was 6%,temperature was 30 ℃,and time was 24 h.The highest degradation rate was 91.77%.After verifying the culture conditions,the degradation rate of the bacterial liquid was 90.86%,which was little different from the theoretical value.The material is provided for the later application of biological fragrance enhancement.4.The application of Paenibacillus can increase the carotenoid degradation rate and degradation products of cigar tobacco leaves after air curing.The results showed that the content of carotene degradation products in cigar tobacco leaves after air curing by spraying Paenibacillus with OD600 1.0 was significantly higher than that in control treatment without spraying bacillus.The results showed that the application of Paenibacillus can increase the content of carotene aroma substances in tobacco leaves,and provide a reference for the application of biological aroma enhancement in cigar tobacco leaves.5.The potential mechanism of β-carotene degradation by Paenibacillus was clarified.Metabolites in Paenibacillus culture medium were analyzed.Echinone and 9-cis-10’-Apobeta-carotenal related to β-carotene degradation showed different change trends.At the same time,the transcriptome results showed that,metabolic pathways such as oxidative phosphorylation and teichoic acid biosynthesis were mainly enriched in the process of β-carotene degradation by Paenibacillus.Meanwhile,genes related to dioxygenase,oxidoreductase and carbon-carbon lyase were up-regulated in β-carotene degradation by Paenibacillus.Meanwhile,the analysis of the degradation pathway of β-carotene showed that β-carotene was degraded to echinone and 9-cis-10’-Apo-beta-carotenal aldehyde,and 9-cis-10’-Apo-beta-carotenal may be degraded to ketones later by dioxygenase,oxidoreductase and carbon-carbon lyase.This may be a potential mechanism for the degradation of β-carotene by Paenibacillus.