Screening And Application Of High-yielding Protease Microorganisms In Traditional Fermented Douchi In Chuxiong And Its Quality Formation Mechanism

Douchi is an important traditional fermented soybean seasoning food in China,which is more widely distributed in China and favored by many countries around the world because it is both nutritious and functional.The quality and flavor of Douchi varies greatly from region to region and from processing to processing.Yunnan Chuxiong traditional Douchi is popular among consumers because of its rich nutritional value,unique taste and flavor,and its role in improving human immunity,but the processing of Yunnan Chuxiong traditional Douchi is mostly natural fermentation,which has problems such as instability of key flavor substances,difficulty in regulating production,and food safety risks in an open fermentation environment.In this thesis,three strains of protease-producing bacteria were selected and mixed to prepare direct-fermentation Douchi fermenters,which were applied to Douchi fermentation to optimize the production process,The physical and chemical parameters,microorganisms,color,and volatile flavor substances of direct-fermented Douchi were measured under optimal process conditions,and correlation analysis of quality and flavor was performed to provide some theoretical guidance and data support for the industrial production of traditional Douchi in Chuxiong,Yunnan.The main findings are as follows:(1)High-producing protease microorganisms from traditional fermented Douchi in Chuxiong,Yunnan were isolated and purified,primed,re-screened,morphologically observed,physiologically and biochemically identified,and molecularly identified,and six high-producing protease strains were obtained,Bacillus subtilis(X9),Kurthia gibsonii(X14),Bacillus velezensis(X19),Meyerozyma smithsonii(J8),Meyerozyma caribbica(J13),Aspergillus tamarii(M24),3 bacterial,2 yeast and 1 mycobacterial strains among the 6 high protease producing strains.The growth characteristics of the six high protease producing strains were studied,and it was found that M24,J8 and J13 were the most salt tolerant with the highest salt tolerance concentration of 12%;M24 had the strongest protease production capacity,reaching 423 U/g at 72 h,which was the highest among all the strains screened.x9 was the most stable and had higher protease production capacity among the three bacterial strains,and J13 was the most stable among the two yeast strains.J13 had the highest protease production capacity among the two yeast strains,therefore,the three strains X9,J13 and M24 were more suitable for further mixed strain fermentation studies.(2)The optimum growth temperature of M24 at 32 ℃,X9 at 36 ℃ and J8 at28 ℃ was used for the fermentation of steamed soybean for 48 h.The best ratio of M24(1×10~6 CFU/m L):X9(1×10~7 CFU/m L):J8(1×10~7 CFU/m L)was used as the indicator of protease activity.The optimized suspension was fermented at 32 ℃ for 48 h,and the protease activity was up to 434 U/g.Using the optimized suspension to make fermentative agent,the optimal process was: the amount of mixed bacterial suspension access 4 %,the moisture content of the semi-solid medium was 80%,the shaking bed incubation temperature was 32 ℃,and the time was 3 d.The logarithmic value of the live bacterial count of M24 was 8.80,and that of X9 was 9.0.Under this condition,the logarithmic values of M24,X9 and J13 reached 8.80,9.07 and 8.37,respectively,and then lyophilized and made into bacterial powder for Douchi fermentation.The optimum process conditions were: 0.60% inoculum,33 ℃,3 d,86.00% relative humidity,and 373 U/g of protease activity.The fermentation temperature was 33 ℃ and the fermentation time was 8 d.The highest amino acid nitrogen content of 0.82 g/100 g and sensory score of 86.33 were obtained in the Douchi.(3)The dynamic changes of microorganisms,basic nutrient components and flavor substances during the fermentation of Douchi with mixed strain fermenters were measured.The moisture content of steamed soybeans(S0)was 58.47%,which gradually decreased during the period of making music,and was 45.28% at the completion of making music(S2),and rose sharply to 63.35% at S3 because of the absorption of water by Douchi after washing,until the completion of fermentation(S5),when the moisture content was 23.76%;the total acid content The total acid content was 0.22 g/100 g at the end of fermentation;the total sugar and reducing sugar content also decreased significantly to 5.77 mg/g and 2.17 mg/g,respectively;throughout the fermentation process,the color of Douchi gradually deepened as fermentation proceeded,and the L*,a*,and b* values of Douchi were significantly different between the S0 stage and the other stages,with fermentation significantly changing the soybean color and luster.The amino acid nitrogen content was significantly higher than in steamed soybeans(S0),increasing by 0.43 g/100 g by the end of fermentation;the total free amino acid content reached a maximum of 7.98g/100 g at stage S2 and was 0.32 g/100 g by the end of fermentation;the enzyme activity first increased and then decreased as fermentation progressed;the analysis of flavor substances revealed that aldehydes,esters,ketones The analysis of flavor substances revealed that aldehydes,esters,ketones,alcohols and phenols were the main flavor compounds in the fermentation process of Douchi,and the flavor substances gradually increased with the extension of fermentation time and stabilized at the end of fermentation.(4)Multiple correlation analysis was established for the factors related to the quality formation during the fermentation of Douchi,and it was found that the correlations among most of the Douchi quality indicators reached significant or highly significant levels,and the color,physical and chemical composition of Douchi,free amino acids and flavor substances had significant effects on each other,all showing significant positive or negative correlation effects.