Umami Taste Soy Sauce Prepared By Aspergillus Oryzae Autolysis Under Low Temperature Stress And Isolation And Identification Of Flavor Peptides

Soy sauce is a traditional condiment fermented with beans,wheat and bran.It originated in China and is an excellent source of nutrients and bioactivity substances.Aspergillus oryzae is a common microorganism during soy sauce fermentation process,which produces abundant enzymes.Glutaminase is a key enzyme to control the flavor of fermented food and it can catalyze the decomposition of glutamine into glutamic acid(Glu)and ammonia to produce umami taste.Currently on the market,the activity of glutaminase during soy sauce fermentation seems very low and less Glu is produced,which lead to poor flavor of final soy sauce products.Meanwhile,the cost of glutaminase artificial adding is too high,which is not acceptable to industrial production.Low temperature stress is one kind of environmental stress,which can induce microbial autolysis and cause significant changes in biochemical characteristics and physiological functions of organisms,such as cell membranes,enzyme activities,etc.Still,the effect of low temperature stress on glutaminase production by Aspergillus oryzae in high-salt and diluted-state soy sauce fermentation has not been reported.The effects of low temperature stress on the quality of high-salt dilute fermented soy sauce were systematically studied,including changes of several enzyme activities and nucleic acid content,formation and release of soy sauce flavor substances during the initial moromi fermentation under low temperature stress(salt-free or salt-free).Also,the effects of different pH moromi fermentation on soy sauce color,aroma and flavor were explored.High performance liquid chromatography-mass spectrometry(LC-MS)was used to isolate,purify and characterize the umami and kokumi peptides in soy sauce.All of these researches were aimed to providing a theoretical basis and methodological guidance for fermentation of soy sauce under low temperature stress.Results are as follows:The changes of several enzyme activities,nucleic acid content,pH during initial moromi fermentation process and the basic physical-chemical indexes,free amino acid composition and sensory evaluation of final soy sauce products under different conditions were analyzed.Greater extent of autolysis in samples was found under low temperature stress conditions(4 °C),was much lower than the normal temperature for natural microbial growth(25 °C).Compared to Control,LTSF had a 3-fold increase of glutaminase activity in dregs and 65.17% increase in supernatant,and after 60 days of moromi fermentation,a 5.73% and 3.47% increase,respectively,in the contents of glutamic acid(Glu)and aspartic acid(Asp).LTSF had the highest total free amino acid content.The intensity ranking of umaminess and kokumi sensation as LTSF > LTSH > Control.However,samples under low temperature stress seems a slight decrease in red and yellow index that lead to a lighter color of soy sauce.The changes of flavor substances during fermentation of soy sauce with different moromi pH were monitored and their effects on the flavor were analyzed.The results showed that the red and yellow index,dark intensity and color ratio of soy sauce fermented by adjusting the pH of moromi near to 6.5 were significantly increased(P < 0.05)compared with the natural moromi pH sample,the increase were 32.23%,18.29%,14.74% and 21.05%,respectively.The activities of glutaminase and protease retained were higher,but there was no significant difference in total nitrogen,amino acid nitrogen and total sugar.After 90 days of fermentation,the proportion of >5 kDa peptide decreased in regulated pH sample,while the proportion of 1-5 kDa peptide increased significantly.Furthermore,the Quantitative Description Analysis showed that the umami,kokumi and salty taste were the strongest in regulated moromi pH sample,while the bitterness was the weakness.A total of 106 volatile aroma compounds were identified by HS-SPME and GC-MS for analysis.Alcohols,esters,aldehydes,ketones and acids were abundant in kinds,while the contents of pyrazines,alcohols,acids,aldehydes and esters were higher,which were the main volatile components in soy sauce.Except for furan and phenolic substances,other aroma substances in soy sauce fermented by regulated the pH of moromi showed a significant increase(P < 0.05).Substances like pyrazines,alcohols and aldehydes increased by 258%,211% and 166%,respectively.As a result,the overall aroma was enhanced,with more prominent caramel,alcohol and potato aroma,but weaker smoked aroma.UPLC-MS/MS was used to purify and identify the acidic components separated from Oasis MAX mixed anionic solid-phase extraction column.18 flavor peptides,less than 700 Da molecular weight,were identified.Except for three pentapeptides,the others were dipeptides,tripeptides or tetrapeptides.The sensory evaluation showed that peptides ED?EE?EF?ESAY?AELY?FELT have umami and sour taste with low threshold value,peptide ESAY also presents kokumi taste.Peptides EM?EDD?QLLN have slight umami taste with higher threshold value.Peptide QNM shows sour and astringent taste with highest threshold value.Peptides SV?LLVVQ?ALVLL have a strong sense of kokumi.Peptides FELT?FLTW?LL?QVELF?NVP?LDP exhibit sour and bitterness.Selected synthetic peptides with stronger ionic response and added them back to market soy sauce.It seems that peptides contain one or more Glu,Asp,Gln or Asn had umami taste,peptides contain val had kokumi taste.