Volatile Flavor Compounds And Protease Of High-Salt Liquid-State Soy Sauce Fermentated By Aspergillus Oryzae

Soy sauce is a kind of traditional condiment in China and Aspergillus oryzae is its main strain used for soy sauce fermenting.A variety of enzymes produced by Aspergillus oryzae can make use of carbon source and nitrogen source in raw matirials.The volatile flavor compounds in soy sauce are complex,which can be classified into alcohols,acids,esters,aldehydes,funans and so on.As an important enzyme secreted by Aspergillus oryzae,the function of protease is related to the degradation of raw materials and the formation of amino nitrogen,which directly determines the flavor and quality of soy sauce.At present,researchers concentrate more on the determination of main volatile flavor compounds in soy sauce in stead of soy sauce in fermentation process,and there is a lack of knowing protease secreted by Aspergillus oryzae in soy sauce during fermentation.This study simulated the fermentation process of high-salt liquid-state soy sauce fermentated by Aspergillus oryzae and aimed to know about the changes of specific volatile flavor compounds in soy sauce during fementation,study the expression of Aspergillus oryzae protease and purify it to determine its enzymatic properties.The main results are shown as follows:1. In order to investigate the variety and content of volatile flavor compounds in the fermentation process of high-salt liquid-state soy sauce fermentated by Aspergillus oryzae,the SPME-GC-MS method was used to detect volatile flavor compounds.The result showed that in the earlier stage of fermentation,the main volatile flavor compounds of koji were alcohols and aldehydes.In the middle stage of fermentation,the main volatile flavor compounds of koji were alcohols,acids and aldehydes.In the later stage of fermentation,the main volatile flavor compounds of koji were alcohols,acids,esters and nitrogen compounds.Ethanol,ethanol,acetic acid,3-methyl-butyric acid,ethyl phenyl acetate,palmitic acid ethyl ester,2,5-dimethyl-pyrazine,2,3,5-trimethyl-pyrazine,benzaldehyde and benzene acetaldehyde were important volatile flavor compounds among them,which was similar to sold high-salt liquid-state soy sauce and made the flavor alike.2.Label free was used to study the secretion of Aspergillus oryzae protease in koji when fermentated 0 month,2 month and 6 month.The types and expression of protease decreased during fermentation and neutral protease II may be a key protease during the soy sauce fermentation,which makes important effect on flavor.3.The Aspergillus oryzae protease was isolated and purified by ammonium sulfate salting-out,anion exchange chromatography and gel chromatography.The relative molecular weight of purified protease was 50 k Da and SDS-PAGE proved the sample’s purity.The specific activity of the purified protease was 460.53 U/mg and the yield was 1.70%,which was purified 5.70 fold.4.The enzymatic properties of purified protease were assayed,including the optimum p H,optimum temperature,thermal stability and the effect of metal ions on protease activity.The results showed that the optimum p H of purified protease was 7,which indicated it a kind of neutral protease.The optimum temperature was 50°C and the activity was also high between 45-55°C.The purified protease was stable under the condition of 30°C and 40°C,decreased rapidly at the temperature of 60°C and 70°C.Mn²⁺had a positive effect on purified significant inhibitory effect on protease activity.The optimum conditions of purified protease were consistent with the fermentation environment of soy sauce.It may be the important protease in the early fermentation stage.