Mehanism Of Aroma Characteristics Improvement In Soy Sauce Fermented By Mixed Koji

Aroma characteristic is one of the most important indicators for the quality of high-salt liquid fermentation soy sauce.Mixed-strain koji of Aspergillus oryzae(A.oryzae)and Monascus purpureus(M.purpureus)was used to improve the flavor of soy sauce,however,the reasons behind the improvement of soy sauce aroma were still unclear.Therefore,nine samples were prepared to explore the reason,and the results showed that metabolites of matured koji was main reason responsible for the formation of volatile compounds.Gas chromatographymass spectrometry(GC-MS)combined with derivatization analysis was used and optimized to detect non-volatiles of koji,the changes of metabolites of different kojis during koji-making process were revealed.The main contents and results of this research are as follows:(1)The quantitative description analysis of soy sauce prepared with A.oryzae koji(ASS)and mixed-strain koji(AMSS)was carried out,the intensities of caramal-like,roasty,cook potato-like,flowery,fruity and ethanolic were all significantly(p<0.05)stronger in AMSS than ASS,while the intensity of sour was lower.Then,the enzyme profiles and metabolites of two kojis were detected.Compared with A.oryzae koji,the activities of acid protease,aminopeptidase,glucoamylase,xylanase and cellulose were significantly(p<0.05)higher in mixed-strain koji.Besides,the relative contents of sugars,sugar alcohols,organic acids,fatty acids and other compounds were significantly(p<0.05)higher in mixed-strain koji than A.oryzae koji.(2)Both metabolites accumulated during koji-making and enzyme profiles during moromi fermentation could affect the aroma formation of soy sauce,so nine samples were prepared to explore the main reason responsible for aroma improvement of AMSS,the result indicated that compared with enzyme profiles during moromi fermentation,the metabolites accumulated in matured koji had a greater impact on the aroma of soy sauce.There were obvious differences in volatiles families between the samples fermented by different matrix of kojis.The percentage of alcohol furan(one)s,pyran(one)s,esters,sulfur-containing compounds,pyrroles and pyrazines in the sample fermented based on matrix of mixed-stain koji were higher than these compounds in the sample fermented based on matrix of A.oryzae koji,while the percentage of acids was lower.(3)NMR technology and GC-MS combined with derivatization analysis were used to detect the non-volatiles of koji,24 and 63 compounds were detected,respectively.Then,GCMS combined with derivatization analysis was chose to optimize by three factors including sample extraction methods,addition amount of samples and amount of derivatization reagent.The best conditions were as followed: freeze-dried kojis were sonicated with 80% methanol for 10 min,then shaken for 20 h,after that,100 ?L of supernatant was derivatized with 120 ?L of derivatization reagent.Standard curves of 51 non-volatile compounds were established,the correlation coefficient of standard curves and relative standard deviation were between 0.990-0.999 and 2%-15% detected by GC-MS combined with derivatization analysis,which suggested that the latter method was reliable with good linearity and repeatability.(4)Qualitative and quantitative analysis of 51 non-volatile compounds of kojis during kojimaking were determined by GC-MS combined with derivatization analysis.The contents of amino acids,sugars,sugar alcohols,fatty acids and biogenic amines showed the rising trend first and then falling,while contents of organic acids and other compounds showed an overall upward trend during koji-making.Koji was divided into four stages according to the changes of non-volatiles: during 0-12 h,most compounds showed low contents;during 12-24 h,amino acids and reducing sugars were accumulated;during 24-36 h,sugar alcohols showed the highest content;during 36-44 h,content of organic acids showed the highest content.(5)There was remarkable difference in the contents of non-volatiles among three kojis,and 17 compounds were identified as key different compounds(VIP>1,p<0.05).Metabolites mainly underwent sugars metabolism,amino acids metabolism,fatty acids metabolism and ricarboxylic acid cycle during koji-making.The trends of sugars metabolism-related metabolites and tricarboxylic acid cycle-related metabolites were quite similar,while the highest contents of amino acids metabolism-related metabolites in M.purpureus koji appeared later than A.oryzae koji and mixed-strain koji.Noteworthy,mixed-strain koji was more conducive to glucose metabolite than A.oryzae koji.The contents of sugars metabolism-related metabolites and some key amino aicd(phenylalanine,leucine and isoleucine),the important aroma precursors,were higher in mixed-strain koji than these compounds in A.oryzae koji.