Effects Of Ultrasonic-assisted Aspergillus Niger On Degradation Of Secondary Precipitate Protein And The Taste Of Soy Sauce

Soy sauce is an indispensable condiment for home cooking,catering industry and food industry in China,East Asia and Southeast Asia.However,during the storage and sale of soy sauce,a layer of mud-like secondary sediment is easily formed on the bottom of the bottle.It seriously affects the appearance quality and commercial value of soy sauce,and has become a bottleneck for the development of the soy sauce industry.Preliminary studies have proved that the B₃ subunit in soy sauce derived from glycinin G4 is the main component of soy sauce secondary precipitation protein and the key factor leading to the formation of secondary precipitation in soy sauce.Firstly,this study used the hydrolysis degree of soybean protein B₃ subunit as an indicator to screen microorganisms and proteases from microorganisms generally recognized as safe(GRAS)and food-grade commercial proteases that can effectively degrade this subunit in a simulated soy sauce fermentation environment using polyacrylamide-sodium dodecyl sulfate gel electrophoresis(SDS-PAGE).The results showed that the crude enzyme solution of Aspergillus niger could effectively degrade the B₃ subunit of soybean protein under simulated soy sauce fermentation conditions.The results indicated that 31 kinds of proteases were isolated and identified from the crude enzyme solution from A.niger using Liquid Chromatography Mass Spectrometry/Mass Spectrometry(LC-MS/MS)combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS)and SDS-PAGE analysis.Among them,aspergillin I,aspergillin II,prolyl protease and aspartic protease are the key proteases for degrading the B₃ subunit of soy sauce secondary precipitation protein.Secondly,the effect of ultrasonic frequency,intensity and time on the degradation of soy sauce secondary precipitation protein B₃ subunit was studied under the simulated soy sauce fermentation environment using SDS-PAGE method.The results showed that the ultrasonic frequency had no significant effect on the degradation rate of soybean protein B₃subunit,but the ultrasonic intensity and time had a significant effect on the degradation rate of soybean protein B₃subunit.Considering the factors of energy saving and processing time reduction,the optimal ultrasonic technology for ultrasonic-assisted A.niger enzymatic hydrolysis of soybean protein B3 subunit is:68 kHz,60 W/L,ultrasonic 8times(10 min/time,over 5 s/Stop for 5 s).Validation experiments showed that ultrasound-assisted A.niger could significantly increase the degradation rate of soy protein B₃ subunit in soy sauce and reduce the amount of secondary precipitation of soy sauce.Ultrasound significantly improved the activities of neutral and acidic proteases in the mixed Daqu sauce mash of A.oryzae and A.niger,and significantly destroyed the soybean surface structure,increased its specific surface area,and improved the chance of enzyme to contact with the substrate simultaneously.This should be the preliminary mechanism of ultrasound assisting A.niger to significantly increase the degradation rate of soybean B₃ subunit.Finally,this study analyzed the effect of ultrasound on the taste of A.oryzae and A.niger mixed Daqu fermented soy sauce using high performance liquid chromatography(HPLC)and quantitative description analysis(QDA).The results showed that ultrasound increased the content of organic flavor substances in soy sauce(amino acid nitrogen including aspartic acid and glutamic acid,total nitrogen,total acid,total sugar,reducing sugar and non-salt solids)content,but reduced the NaCl content in soy sauce.QDA analysis confirmed that ultrasound could improve the overall taste of soy sauce.In summary,ultrasound-assisted A.niger can not only significantly increase the degradation rate of soy protein B₃ subunit in soy sauce,reduce the amount of secondary precipitation in soy sauce,but also improve the overall taste of soy sauce.