Improvement Of The Quality Of Soy Sauce By Reducing Enzyme Activity In Aspergillus Oryzae

Aspergillus oryzae was widely used in the soy sauce fermentation industry because of its ability to produce enzymes efficiently.At present,previous studies had improved the quality of soy sauce by improving the capacity of enzyme production in A.oryzae,such as increasing the expression level of proteases.However,not the more enzymes secreted by A.oryzae,the better the quality of soy sauce.In this study,the quality of soy sauce was improved by reducing the expression levels of cellulase and pectinase produced by A.oryzae.The multinuclear nature of A.oryzae was one of the difficulties to construct the stable genetic strains.Therefore,A.oryzae 3.042-3,which can stably produce mononuclear spores,was constructed using A.oryzae 3.042 as the starting strain in this study.The fragment cis-CreA was obtained by double-digesting the constructed plasmid pUC18-cis-CreA and was transformed into A.oryzae 3.042-3 to obtain A.oryzae 3.042-3-c.A.oryzae 3.042-3-c was observed on the growth in the medium with only 1%glucose as the carbon source and the medium with only 0.9%CMC as the carbon source.It had been proved that A oryzae 3.042 contained cis-CreA at the level of gene and morphology.A.oryzae 3.042-3-c contained a fragment of cis-CreA which was confirmed that was ligated to the VII chromosome of the constructed strain A.oryzae 3.042-3-c.After that,A.oryzae 3.042,A.oryzae 3.042-3 and A.oryzae 3.042-3-c were inoculated with koji,and three samples of koji were sampled at 24 h,30 h,36 h,42 h,48 h.The difference in expression levels of cellulase and pectinase genes was followed by RT-qPCR,and the activities of cellulase and pectinase of the three stains was tested.The results showed that the cellulase activity of A.oryzae 3.042-3-c was reduced by 58.6%and the pectinase activity was decreased by 16.1%compared with A.oryzae 3.042,which was also consistent with the result of RT-qPCR.Then,A.oryzae 3.042,A.oryzae 3.042-3 and A.oryzae 3.042-3-c were applied to the low-salt solid soy sauce fermentation to track the changes of amino acid nitrogen and total nitrogen content.At the end of the soy sauce fermentation,the salt-free solid content and of the soy sauce brewed by the three strains was tested.The results showed that the salt-free solid content of A.oryzae 3.042-3-c was 58.9%higher than that of A.oryzae 3.042 and A.oryzae 3.042.The kinds and contents of the flavor components of the soy sauce from the fermentation by A.oryzae 3.042-3-c were higher than those of the A.oryzae 3.042 and A.oryzae 3.042-3,especially in alcohols and esters.HEMF was only found in the soy sauce from A.oryzae 3.042-3-c.The results indicated that the new strain A.oryzae 3.042-3-c could improve the flavor of soy sauce from the low-salt solid fermentation by decreasing enzyme activity of cellulase and pectinase.