| The quality of tobacco raw materials determines the quality of cigarette products.As an agricultural product,the quality of tobacco leaves is affected by ecology,cultivation technology,preparation technology,management and other aspects,and it is inevitable that there are defects.At present,the problem of tobacco leaf maturity is widespread in various producing areas,which have become the main contradictory factors restricting the improvement of tobacco leaf quality in this producing area,mainly in protein,starch,and pectin.The high content of other materials,uncoordinated chemical composition,and incomplete material conversion restrict the industrial availability of tobacco leaves.At present,this type of problem is mainly solved by improving harvesting maturity.However,improving harvesting maturity is affected by technology,management,policy and other aspects.Although initial results have been achieved,it is difficult to solve the problem fundamentally.Microorganisms have unparalleled advantages in tobacco material conversion and macromolecular degradation.They can improve tobacco leaf fumes and irritation in a targeted manner,improve the level of smoke fineness,tobacco leaf quality and industrial availability.This study conducted a targeted screening of Henan tobacco leaf surface starch degradation bacteria,studied the enzyme production conditions of the strains and tobacco leaf fermentation technology,and aimed to improve the quality of tobacco leaves by increasing the enzyme production activity of the strains and the degradation rate of tobacco leaf starch,and improve the quality and industrial availability of tobacco leaves,which is the industrialization of the strains.Application provides theoretical basis.The results of the study are as follows:1.Using the method of preliminary screening of soluble starch medium and re-screening of amylase activity,53 strains of starch degrading bacteria were screened from the surface of tobacco leaves in Jia Xian,Luo Ning,Xiang Xian,and Ling Bao,Henan Province,and 10 strains produced transparent circles.The ratio is greater than 1.5,respectively named HX-6,HX19,HX15,HX37,FN29,YC26,YC31,FN20,YC-3,YC19.Ten strains were further screened for amylase activity to obtain the strain with the highest amylase activity HX-6 has an enzyme activity of 38.96 U/mL.Combining the observation of colony morphology,physiological and biochemical determination and molecular identification,the strain was determined to be Bacillus subtilis,and its similarity to Bacillus subtilis reached 99%.2.Using single factor test and response surface test to optimize the amylase production conditions of HX-6 strain,it was determined that the best enzyme production medium was:soluble starch and sweet potato starch mixed powder(m soluble starch:m sweet potato starch=1:1)The mass concentration is 17.0 g/L,the mixed powder of peptone and yeast powder(m peptone:m yeast powder=1:1)the mass concentration is 21.89 g/L,and the KNO3 mass concentration is 2.58 g/L.Through model verification,the predicted enzyme activity is 66.23 U/mL,and the actual enzyme activity is 66.82 U/mL,which is in good agreement with the predicted enzyme activity,and its enzyme activity is 71.51%higher than before optimization.The single factor method was used to optimize the enzyme production conditions of the HX-6 strain,and the optimal fermentation conditions were determined as follows:35? pH 7.5,inoculum size 6%.rotation speed 180 r/min,and fermentation time 36 h.Under these conditions,the maximum amylase activity is as high as 87.56 U/mL,which is 1.25 times higher than the initial enzyme activity.3.In order to clarify the optimal fermentation process of tobacco leaves,with starch content as an indicator,three factors of fermentation time,the amount of crude enzyme solution added and the fermentation temperature were set.Through single factor test and orthogonal test,different starches in four regions of Henan were tested.The content of tobacco raw materials is researched on fermentation technology.Combining the starch content and the optimal fermentation process parameters,when the starch content is less than 5%,the optimal fermentation process parameters for tobacco leaves are fermentation time 30 h,strain crude enzyme liquid addition amount 8%,fermentation temperature 30?;starch content is 5%?6%,the optimal fermentation process parameters for tobacco leaves are fermentation time 54 h,the amount of crude enzyme solution is 4%,and the fermentation temperature is 33?;when the starch content is 6%?8%,the optimal fermentation process parameters for tobacco leaves are fermentation time 54 h.The addition amount of the strain crude enzyme solution is 8%,and the fermentation temperature is 30?;when the starch content is greater than 8%,the optimal fermentation process parameters for tobacco leaves are the tobacco fermentation time of 54 h,the addition amount of the strain crude enzyme solution is 6%,and the fermentation temperature is 35?.After fermentation by Bacillus subtilis HX-6,the starch content of the four groups of tobacco leaves were significantly reduced,and the starch degradation rates were 41.57%,43.05%,35.93%,and 39.50%,respectively;the total sugar and reducing sugar content increased significantly,and the total sugar content changed.The larger ones were Xiang Xianflue-cured tobacco C3F and Ling Bao flue-cured tobacco C2F,with an increase of 25.18%and 19.63%,respectively;the larger reducing sugar content increased in Xiang Yang cured tobacco C3F and Luo Ning flue-cured tobacco C3F,respectively 28.13%and 23.80%.The nicotine and total nitrogen content of the four groups of tobacco were reduced,among which the decrease of nicotine was small;the ratio of sugar to nitrogen and sugar to nicotine increased,the disaccharide difference was reduced,and the C3F disaccharide difference of Luo Ning flue-cured tobacco decreased the most;the fourth group of tobacco leaves After fermentation of the crude enzyme liquid of strain HX-6,the tobacco leaf starch,total sugar,reducing sugar,nicotine,total nitrogen and the difference between the two sugars are all close to the appropriate value,the internal chemical composition of the tobacco leaf is more coordinated,and the internal quality of the tobacco leaf is improved.Comparing the sensory quality of tobacco leaves before and after fermentation,the sensory quality of tobacco leaves is significantly improved after being treated with the crude enzyme solution of strain HX-6,the aroma quality and amount of aroma are significantly improved,the impurity and irritation are reduced,the quality of tobacco leaves is improved,and the industrial usability is enhanced. |
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