Screening And Identification Of Tannin-degrading Bacteria And Their Application In The Fermentation Of Cigar Tobacco Leaves

In order to reduce the content of tannic acid in cigar tobacco and solve the problem of heavy bitterness in tobacco leaves,this study used Indonesian cigar tobacco leaves as raw material,screened the microorganisms with degradation effect on tannic acid on their surface,and optimized the tannase production process by medium composition and culture conditions to improve the tannase production capacity of the strain and prepare crude enzyme solution for cigar tobacco fermentation.The optimized crude enzyme solution was used to ferment cigar tobacco leaves,and the effects of fermentation time,crude enzyme solution addition and fermentation temperature on the fermentation effect of cigar tobacco leaves were studied to clarify the best fermentation process for crude enzyme solution to improve the quality of tobacco leaves,so as to lay the foundation for biotechnology to improve the quality of cigar tobacco leaves.The main research results are as follows:1.The tannin identification medium was used to screen 16 bacterial strains with degradation effect on tannic acid from Indonesian cigar tobacco leaves,among which 5 strains with D/d greater than 1.5 were named DT-1,B-3,DT-3,YCYM-04 and YCYM-09,and the strain was further re-screened with tannase activity as the index,and the strain with the highest initial tannase activity was B-3 with tannase activity of 25.23 U/m L.The strain B-3 was identified by morphological observation,its surface was yellow,opaque,with irregular edges,size was1.62 μm×0.48 μm,and its body was rod-shaped with uneven surface by electron microscopy,combined with physiological and biochemical tests and molecular identification,strain B-3was identified as(Pantoea agglomerans)2.The optimal conditions for tannase production were: sucrose mass concentration 15.45 g/L,yeast paste mass concentration 17.00 g/L,Na Cl mass concentration 4.94 g/L,and the tannase activity reached 55.83 U/m L.The optimal tannase production process was optimized on the basis of the optimal medium formulation,and the optimal tannase production process was 18 h fermentation time,180 rpm shaker speed,4% inoculum,30 ℃ fermentation temperature,p H 6,and the tannase activity was 76.81 U/m L,which was 2.04 times higher than the initial tannase activity.In addition,the strain also had 13 enzyme activities such as cellulase,pectinase,amylase,xylanase,acidic protease,neutral protease,alkaline protease,peroxidase,manganese peroxidase,laccase,nicotinase and β-carotene enzyme,and the enzyme activities of cellulase,pectinase,amylase,protease and laccase of the strain were higher under the optimal tannase production conditions.3.Under the optimal tannase production conditions,the fermentation process conditions were determined as follows: fermentation time of 9 d,crude enzyme solution addition of 10%,and fermentation temperature of 45 °C,using the tannic acid content in tobacco as the index and fermentation time,crude enzyme solution addition and fermentation temperature as the independent variables,respectively.The optimal fermentation conditions were determined as follows: fermentation time of 7 d,crude enzyme concentration of 10% and fermentation temperature of 45 °C.The tannic acid content of the fermented tobacco was determined to be9.36 mg/g,with a degradation rate of 39.69% compared with that before fermentation(15.52mg/g).The conventional chemical composition of Indonesian cigar tobacco leaves before and after the optimal fermentation process was determined,and it was found that the total and reducing sugar contents of cigar tobacco leaves increased and the contents of cellulose,pectin,starch,lignin and protein decreased after fermentation by tannic acid degrading bacteria B-3,indicating that the improvement of tobacco leaf quality by strain B-3 was the result of the combined action of various enzymes,which made the endochemical composition of tobacco leaves more harmonious and enhanced the intrinsic quality of tobacco leaves.The neutral aroma-causing substances of Indonesian cigar tobacco were measured and the dihydrokiwi lactone content of the tobacco leaves was significantly higher.The total aroma content of the tobacco leaves increased by 32.39 ug/g compared to the control,and the aromatic amino acid degradation products,melatonin,carotenoid degradation products,siderophore-like degradation products and neophytodiene increased in the fermented tobacco leaves of strain B-3.The sensory quality of the tobacco leaves before and after fermentation was also evaluated,and the results showed that the sensory quality of the tobacco leaves treated with the crude enzyme solution of strain B-3 was improved,mainly in the form of reduced bitterness and impurities,better permeability,mellowness and aroma quality,significantly improved quality of Indonesian cigar tobacco,and enhanced industrial availability.