酱油原料蛋白质和酱油中总氮含量的检测

智能文字提取功能测试中

通过添加蛋白酶来提高酱油的蛋白质利用率和发酵品质Improving protein utilization and fermentation quality of soy sauce by adding protease。总氮和蛋白质由德国格哈特公司总氮分析仪测量。The total nitrogen (TN) and protein were measured with a total nitrogen analyzer (Dumatherm® DT Ar/He basic; C. Gerhardt GmbH & Co. KG, Konigswinter, Germany).Journal of Food Composition and Analysis 121 (2023) 105399Contents lists available at ScienceDirectJournal of Food Composition and Analysis Journal of Food Composition and Analysis 121 (2023)105399C. Chen et al. ELSEVIER journal homepage： www.elsevier.com/locate/j fca 通过添加蛋白酶来提高酱油的蛋白质利用率和发酵品质 Improving protein utilization and fermentation quality of soy sauce by adding protease Caifeng Chen a ，b ，Sha Hou ， Changzheng Wu ， Yong Cao ， Xing Tong， Yunjiao Chen a ，** Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods， College of Food Science， South China Agricultural University， Guangzhou 510640，Guangdong， China DFoshan Hai t ian Flavoring and Food Co.， Ltd.， Foshan 528000， China 佛山海 天调味 食品有 限公司 ARTICLEINFO ABSTRACT Keywords：Soy sauce Protein utilization Flavor Moromi fermentation Protease activity Enzymatic hydrolysis Soy sauce is the most popular traditional fermented condiment in Asia . However， some proteins are unutilized during fermentation， causing resource waste. Therefore， this study aimed to improve protein utilization and fermentation quality by adding protease i n the moromi. Firstly， Aspergillus oryzae (A. oryzae) endogenous pro-teases were found to play an important role in nitrogen element utilization， i ncluding increasing total nitrogen (TN)， amino acid nitrogen (AAN)， and relative protein expression. Secondly， among six exogenous proteases，neutral protease (NP) performed better both alone and synergistical l y with A. oryzae crude protease powder (CPP)， because of the enhancement of smal l peptides and enzymatic effect. Importantly， adding NP increased AAN and T N by 7.45 % and 2.21 % respectively ， as wel l as i ncreasing peptides below 500 Da， Maillard peptides and the proportion of lactic to acetic acid， hence i mproving the umami and richness of soy sauce. Overall， adding NP could increase soy sauce protein ut i lization and fermentation quality. Soybean is one of the most widely grown economic crops in the world (Li u e t a l.， 2022； W e i et a l .， 2023). The demand for soybean is increasing year by year as its processed products become growing popular such as soy sauce and soybean paste (Li u e t al .， 2020； Ca i e t a l.，2021). Additionally， soy sauce with its unique and rich flavor has become the most popular traditional fermented soybean product among Asian and Western countries (Liu e t a l.， 2021； Z h ou e t a l.， 2022).However， the low protein uti l ization of soy sauce during fermentation leads to its weak competitiveness i n both domestic and foreign markets (Gao e t a l.， 2018； Zhu e t a l .， 2022). Therefore， how to improve protein utilization is an important problem to be solved in the soy sauce pro-duction industry. Many reports have confirmed that pretreatments and additives could enhance the taste of product and the quality attributes by improving the internal chemical structure or functional activity. Dough is the main ingredient of steamed bread and i t s main component is starch. The dough is hydrolyzed by the amylase present in the dough system during the fermentation process while the ultrasonication treatment could enhance the fermentation quality of the steamed bread by improving the intrinsic starch structure of the dough (Zh an g e t al.， 2022). Another study demonstrated tha t the characteristic viscosity of glutinous rice， the main ingredient in rice dumpl i ngs， could directly influence the quality attributes of dumplings such as taste and texture. However， adding germinated rice flour could lead to the degradation of food polymers such as protein and starch by activating endogenous enzymes， thus enabling rice dumplings to exhibit better storage and textural qualit i es (Wan g e t a l .， 2022). Furthermore， orange peel exhibits antimicrobial properties due to its richness in plant compounds， i ncluding terpenoids，saponins and flavonoids. Adding orange peel extract to func t ional bev-erages could increase the quality and shelf l ife by enhancing the ant i -microbial effect and sensory acceptability of functional beverages (Se lah v a rz i e t al .， 2021).Overall， additives or pretreatment are effective ways to i mprove the fermentation characteristics and quality attributes. Soy sauce is mainly made from a mixture of soybean and wheat，which is divided into two fermentation stages： koji and moromi fermentation (Z hou et al.， 2019). The koji was obtained by mixing soybean， wheat and A. oryzae，and the moromi was obtained by injecting 18 % brine for fermentation for 3 months. The best source of nitrogen for microbial growth i s soybean (Li e t al .， 2021)， which greatly influences the extracel l ular production of proteases dur i ng the koj i fermentation * Corresponding author. ** Correspondence to： College of Food Science ， South China Agricultural University， 483 Wushan Road， Tianhe District， Guangzhou， Guangdong 510642， China. E-mail addresses： 22108970@q q.com (X. Tong)， yunjiaoche n @sca u.edu .cn (Y. Chen). h t tps：//doi.or g/10.1016/j .jfc a.2023.105399 Received 26 March 2023； Received in revised form 5 May 2023； Accepted 10 May 2023Available online 11 May 2023 phase (Zh ao e t a l.， 2019； Z h ou et a l.， 2019). These proteases could degrade soybean proteins into peptides， free amino acids， and other smal l molecules during the moromi fermentation period， which play an important role in the flavor and qual i ty of soy sauce (Ti a n et al .，2022).Consequent l y， the koji stage is considered the predominant phase of protease secretion by A. oryzae (Zh a o et al.， 2019). Meanwhile， the moromi period is recognized as a cr i tical period for the protease to function as a protein degrader， as well as determining the quality of soy sauce (D iez-Simon e t a l.， 2020； Zho u et al.，2022). Therefore， protease activity is closely related to amino acid nitrogen content and soy sauce production costs (Di ng et a l .， 2019； T i an e t al .， 2022). Current re-searchers have mainly applied optimization of strains， screening strains with high protease yields and mixing koji to i mprove protein ut il ization of soy sauce by enhancing the unit enzyme activ i ty of koji (C h en e t a l.，2015； Ga o et al .， 2019； X ie et al.，2019)， but their protease activity would be greatly reduced under the high-salt and low-pH fermentation envi-ronment of moromi ， which caused some proteins to be underut i lized.Nevertheless， the moromi phase serves as an important period for pro-tein hydrolysis to generate amino acid nitrogen， less research has been conducted on the exogenous addition of proteases during moromi fermentation. Furthermore， the relationship between protease and amino acid nitrogen and total nitrogen throughout the fermentation stage of soy sauce has not been fully reported， nor has the influences of adding exogenous protease on protein uti l ization and qual i ty during the moromi phase within soy sauce been thoroughly explored. Hence， this study focuses on the relationship between protease and nitrogen elements such as amino acid nitrogen and total nitrogen during soy sauce fermentation from koji to moromi， as well as the influence of exogenous protease addition on the change in soy sauce quality during the moromi period. The purpose of this study was to promote the protein utilization and fermentation quality of soy sauce by adding exogenous protease and to understand the under l ying mechanism. The research provided theoretical guidance for the optimization of soy sauce fermentation and the improvement of soy sauce quality. 2. Materials and methods 2.1. Materials and chemicals Soy protein isolate (SPI) (protein content： 90.0%) was purchased from Maya Reagent (Zhejiang， China). Soybean， wheat and edible salt were supplied from Foshan Haitian (Gaoming) Flavoring Food Co.， Ltd.Aspergillus oryzae (A. oryzae) was purchased from Yishui Jinrun Bio-logical Technology Co.， Ltd (Shandong， China). Neutral protease (NP)，acid protease (AcP)， alkal i ne protease (AlP)， papain and bromelin with act i vities of 1 ×10u/g，1×10U/g，2×10U/g，2×10U/g and 1×10 U/g， respectively， were purchased from Nanning Pangbo Bioengi-neering Co.， Ltd (Guangxi， China). Aminopeptidase (AP) with activity of 3 × 10 u/g was purchased from Nanning Doing Higher Bio-tech Co.，Ltd. Most of the chemicals and reagents used were of analytical grade. 2.2. Soy sauce fermentation First， the koji was obtained by mixing soybean：wheat at a 5.5：1 (w/w) ratio and inoculating with 0.1 %(w/w) A. oryzae for 40 h at 28-33C.The koji sample was collected at f ive t i me periods： 12， 18，24， 36， and 40h. Second， the moromi was prepared by mix i ng koji：18 % (w/w) brine at a 1：2 ratio (Fi g. S1). At 30 °C fermentation， neutral protease (NP) at concentrations of 275， 550 and 825 U/ (per gram of koji) were added as experimental groups while using non-added protease as a control (CK).Samples were collected periodically on D5， D15， D30， D45， D60 and D90. The supernatant was obtained by centrifugal filtration and then stored at -20 °C unti l analysis. 2.3. Preparation of crude proteases extract The koj i sample was mixed wi t h phosphate buffer (1：10， w/w) in a water bath at 40 °C for 60 min， f iltered through gauze， and the super-natant collected by centrifugation was the crude protease extract. 2.4. Protein hydrolysate preparation 2.4.1. Enzymatic hydrolysis experiments with proteases acting alone The 1 % SPI solution was used by boiling at 95 °C for 10 min，adding protease (4000 U/g) after cooling and incubating at 30 C for 4 h. The protease was i nactivated at 95°C for 10 min and centrifuged at 10，000rpm for 10 min， and the supernatant was collected for analysis. 2.4.2. Enzymatic hydrolysis experiments with exogenous proteases cooperating with CPP in the simulated late fermentation moromi environment The 2 % SPI solution was used by boi l ing at 95 °C for 10 min. Adding protease (8000 U/g) after cooling and incubate at 30 °C for 2 h. The brine was subsequently added to make the NaCl f inal concentration was 18 % and 2000 U/g of protease was added . The solution was adjusted to pH 5.5 and incubated at 30 °C for 6 h. The protease was inactivated at 95 °C for 10 min and centri f uged at 10，000 rpm for 10 min， and the supernatant was collected for analysis. 2.5. Enzyme activity analysis The activities of neutral protease， acid protease and alkaline protease were determined as follows： 1 % Casein was prepared in 0.1 M buffer at appropriate pH (Na2HPO4·12H2O-NaH2PO4·2H2O for pH 7.5， C3H603-C3HsNaO3 for pH 3.0 and Na2B407·10H2O-NaOH for pH 10.5). The enzyme solution was mixed with casein in equal quantity，incubated for 10 min at 40 °C and 2.0 mL of 0.4 M trichloroacetic acid was added， then centrifuged at 10，000 rpm for 2 min (Eppendorf，Centri f uge 5424， Germany). 1.0 mL supernatant was added to 5.0 mL 0.4 M sodium bicarbonate and 1.0 mL Forinol. Absorbance was read at 680 nm (Eppendorf BioSpectrometer， Germany) after standing at 40℃for 20 min. 2.6. Enzymatic properties analysis The effect of pH on the activity of CPP was measured at pH ranging from 3.0 to 10.0 in different buffers， including C3H603/C3HsNaO3 (pH 3.0)，C6HsNa3072H2O (pH 4.0-5.0)， Na2HPO4·12H2O/NaH2PO4·2H2O (pH 6.0-8.0) and Na2B407·10H2O/NaOH (pH 9.0-10.0). To study pH stability ， the enzyme was preincubated in these buffers at 40 °C for 4 h. The effect of temperature on the activity of CPP was assayed at pH 7.5 in the range of 20-60 °C. The thermal stabil i ty of CPP was deter-mined by treatment at the above temperatures for 60 min. To examine the effect of different NaCl on CPP activity， reaction systems were prepared at final concentrations of 0-24 %. The NaCl stabil i ty for CPP was assayed at 40 °C after incubat i ng in 18 % NaCl for 7days. 2.7. Physicochemical properties analysis 2.7.1. Determination of pH， TN， protein， AAN The pH was measured with a pH meter (Bante， pHscan40， China).The total nitrogen (TN) and protein were measured with a tota l nitrogen analyzer (DumathermQ DT Ar/He basic； C. Gerhardt GmbH & Co. KG，Konigswinter， Germany). Amino acid nitrogen (AAN) was determined using an automatic potentiometric titrator (905； Metrohm， Herisau，Switzerland). 2.7.2. Determination of molecular weight The molecular weight was determined using a GPC-HPLC system (Agilent 1260； Agilent， Santa Clara， CA) equipped with a HPLC column (Bio SEC-3， 4.6×150 mm； Agilent). The determination conditions were as follows： mobile phase： ultrapure water， i njection volume： 20 pL， f low rate： 0.4 mL/min， column temperature and RID detector temperature：35°C. 2.7.3. Determination of free amino acid The free amino acid was analyzed by an amino acid analyzer (HITACHI， LA8080， Japan). The total free amino acid content was calculated as the sum of the free amino acid contents of Asp， Glu， Ser，Gly， Thr， Ala， Pro， Lys， His， Val， Met， Arg， Ile， Leu， Tyr， Phe and Cys. 2.8. Analysis of indices in enzymolysis experiment The supernatant recovery referred to the percentage of supernatant enzymolysis solution in the reaction mixture. The degree of hydrolysis (DH) was expressed as the ratio of amino acid nitrogen to total ni t rogen.The protein recovery (PR) was calculated with the ratio of the protein content of the supernatant hydrolysates to the enzymatic mixture. The peptide nitrogen (PN) was determined according to (Tk a cze w s ka e t al.，2020； Zhu et al.， 2021) with some modifications . An equal amount of supernatant hydrolysate was placed in 10 % TCA for 10 min， cent r ifuged and the supernatant was collected to determine TN and AAN. Addi-tionally， the extraction rate of the polypeptide (EP) was determined by color reaction of one part of the supernatant with four parts of the biuret reagent for 30 min and then the absorbance was measured at 540 nm. 2.9. SDS-PAGE analysis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed for protein separation using a 10 % fixed gel . The samples were mixed with loading buffer and heated for 10 min at 95°℃in a dry heating bath. The electrophoresis procedure was first run at 100V for 30 min and then at 150 V for 30 min. Staining with Coomassie Bril l iant Blue for 60 min took place after completing electrophoresis，followed by elution with a decolorizing solution until the bands were clear and photographed using the imaging gel system (EBOX CX5；VILBER，Marne-la -Vallee， France). 2.10. Organic acids analysis The soy sauce was diluted and filtered through a 0.22 um micropo-rous cellulose membrane， and was ready for UHPLC analysis (Agilent 1290 Infinity I I ； Agilent， Santa Clara， CA). The conditions of the l iquid chromatography were as follows： the samples were separated by an HPLC column (3 um， 4.6 mm x 250 mm， osaka soda， 550-0011，Japan)，using H2PO4 (0.2 %) as the mobile phase at a flow rate of 0.2 mL/min.The detection wavelength was set at 215 nm. 2.11. Volatile compounds analysis Soy sauce (5.0 mL) was t ransferred into a 20.0 mL headspace vial，and 25 pL 2-octanol (3.35 ppm in methanol)， as an internal standard，were added and mixed. Samples were equilibrated at 40 °C for 10 min in an incubator. The volatile compounds were extracted for 30 min at 40 °C with an SPME fibre (CAR/PDMS； Supelco， Bellefonte， PA). After extraction， the fiber was inserted in the GC-MS injector and desorbed for 1 min at 250°C.Separation was performed with a HP-INNOWAX column (60 m ×250um x0.25 um). Helium was used as carrier gas with a flow rate of 1.2mL/min. The analysis conditions were as follows： the oven temperature was started at 40 °C for 5 min， raised by 5°C/min to 240 °C and held for 15 min. The mass spectrometer was operated in electron impact mode.The ion source temperature was set to 250 °C. The mass scanning range ranged from m/z 35-500 and electron energy was 70 eV. MassHunter software was used for data analysis. The identification of volatile compounds was achieved by matching both the mass spectra of the reference standards and retention indices (RI) (F ig . S 2A &Tab l e S 2). The RI values were calculated using a C6-C33 n-alkane mixture as the external reference under the same chromatographic conditions. The preliminary identification was based on comparison with the mass spectra and retention indices of known compounds in the NIST 17.0 standard reference database when no reference component was available. For the semi-quantitative analysis 2-octanol was selected as the internal standard (F ig. S2B ). The semi-quantities of the volatile compounds were calculated based on their peak areas relative to that of the internal standard. 2.12. Sensory evaluation Quantitative descript i ve analysis (QDA) was used for the sensory evaluation based on the analytical method (Zh u e t a l .， 2021) and modified. All samples were coded with random three-digi t numbers， and served to panel members at random placement. The pane l cons i sted of 20 members (10 women and 10 men， aged 20-45 years) who were trained in descriptive sensory analysis and had some experience with the sensory properties of soy sauce. The experimental group with added protease was evaluated by using no added enzyme as a reference solu-tion. The score was measured on a scale of 0-10 (from weak to strong)and the reference solution was given a score of 5. 2.13. Stat i stical analysis All experimental data were reported as mean ± standard deviation (SD)， and were subjected to one-way ANOVA with Duncan’s test (p<0.05)， using IBM SPSS (version 22.0 for Windows； SPSS， USA). Graphs were mainly plotted using Origin (OriginPro 2021， USA) and Prism (GraphPad Prism 8.0.1， USA). Analysis of the heat map between samples was conducted using Hiplot online platform (https ：//h i p lot .co m.c n /)and Chiplot online platform (h t tps ：//www.chiplot .o n l ine/). Redun-dancy analysis (RDA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed in Canoco 5 and Simca 14.1，respectively. 3. Results and discussion 3.1. The endogenous neutral protease secreted by A. oryzae exerted an important role i n the utilization of the nitrogen element Soybean is the best nitrogen source for A. oryzae to grow， with various nitrogen sources affecting the extracel l ular production of pro-tease (Z ha o et al .， 2018)， whi l e nitrogen elements such as ANN and TN could ref l ect the consumption of ni t rogen sources in the koji as well as an important parameter to evaluate the quality of soy sauce (Ruan e t al.，2022). Analysi s of AAN， TN， relative protein expression and free amino acids was performed to further investigate the relationship between A. oryzae protease and nitrogen sources. First ， the AAN and TN contents increased rapidly at 12-24 h， followed by insignificant change (F ig . 1B).Furthermore， a clearly visible protein band was seen on the SDS-PAGE profi l e from 12 h onwards (F ig. 1C)， demonstrating that A. oryzae Spo ein the peak growth period and secreted A 1500- B 7→- Neutral protease --Acid protease 品 Fig. 1. The endogenous neutral protease secreted by A. oryzae exerted an important role in the utilization of the nitrogen element. (A) The tendency of protease secreted by Aspergillus oryzae (A. oryzae) and i ts distribution. (B) Variation of amino acid ni t rogen (AAN) and total nitrogen (TN) in different koji fermentation time.(C) The SDS-PAGE analysis of koji in different fermentation time， lanes 1-7 were respectively：(1) Marker (2) Marker (3) 12 h (4) 18 h (5) 24h (6) 36 h (7) 40 h. (D)The SDS-PAGE prof i les were quant i tatively analyzed using ImageJ software.(E) Visualization of heat map of the changes in free amino acids of the koji in di f ferent fermentation time. Different letters were significantly different among these groups (p <0.05). continuously. Besides， the relative protein expression exhibited a remarkable elevation at 12-24 h (F ig . 1D). Also， the content of both total free amino acids (TFAA) and hydrophobic amino acids (HAA)showed a rapid increase at 12-24h (Fi g. 1E)， which was because A. oryzae could rapidly secrete protease dur i ng this period， simulta-neously causing the protein to be rapidly degraded into more free amino acids. Nevertheless， the content started to decrease slowly after 24 h，which was linked to these reasons. Firstly， free amino acids were consumed supplying the nitrogen source to A. oryzae for growing and better secreting proteases. Secondly， when A. oryzae grew faster than i ts ability to secrete protease， the protease degradation rate would decrease， causing a lower free amino acid content. In addition， the in-crease in hydrophobic amino acids could cause bitterness (Z h a o e t a l .，2021). It was found that the bitter amino acids content showed a trend of rapid increase followed by a slowdown (F ig .S 3). Taken together， there was a rapid increase in AAN， TN， relative protein expression and free amino acid during 12-24 h， which was generally consistent with the trend of the neutra l protease secreted by A. oryzae. Therefore， A. oryzae endogenous proteases played an important role in the util i zation of ni-trogen elements， which would further affect the f lavor of soy sauce. The level of protease activity plays an i nfluentia l role in the protein hydrolysis efficiency (Ti a n e t a l .， 2022)， as wel l as directly or indirectly influencing the flavor and quality of soy sauce. High-salt and low-pH environments were the main characteristics of soy sauce fermentation；however ， unsuitable environments such as pH， temperature， and NaCl could reduce the activity of proteases (G a o et a l.， 2018). Thus， it is necessary to investigate the enzymatic properties of A. oryzae protease to understand the effect of the actual environment on the enzyme ac-tivity during the later phase of moromi fermentation. To reduce the amount of protease addition in the system and to obtain protease with high unit enzyme activity for l ater enzymatic experiments， crude enzyme powder (CPP) was obtained after the crude proteases extract of A. oryzae was treated with ultrafi l tration and freeze-drying and its specific activity was i ncreased from 21.96 U/mg to 62.69 U/mg (Ta b l e S 1). Then the enzymatic properties were investigated. Firstly，CPP displayed both optima l activity and stability at pH 6.0， where its stability remained high in the pH range of 5.0-9.0 (F ig. S4A )， demon-strating that CPP was appropriate for utilization across a wide pH range.It is worth noting that the moromi was generally kept i n the pH range of 4.0-6.0 during fermentation. But the stability of CPP decreased rapidly when the pH was below 5.0. Secondly， CPP exhibited the best activity and stability at 50°C and 30°C， respectively (F ig. S4B ). Finally， the relative activity of CPP was diminished with increasing the NaCl mass fraction (Fi g . S4C -D). Since the salt concentration was generally controlled at about 18 % during the fermentation of high-salt dilute soy sauce， the storage stabi l ity of t he protease treated with 18 % NaCl was analyzed， and i t was found to have li t tle effect on the stability， exhibit i ng a dynamic equilibrium state within 7 days of storage. I t followed that the activity and stabi l ity of CPP were susceptible to different environments，such as pH， temperature and NaCl. To summarize， the endogenous neutral protease secreted by A. oryzae exerted an important role in the uti l ization of nitrogen element， including the enhancement of AAN， TN and relative protein expression. Furthermore， CPP， concentrated proteases from A. oryzae，exhibited an optimum pH ac t ivity and stability at 6.0， and when CPP was at 18 % NaCl ， over 50 % enzyme activity was lost. It i mplied that the enzymatic activity of the moromi could be inhibited in the later stages of fermentation， thus reducing t he abi l ity to degrade proteins. Therefore， it was necessary to screen for an exogenous protease that could be adapted to the soy moromi environment and could synergize with CPP for better degradation. 3.2. NP performed best in enzymatic hydrolysis experiments in the simulated moromi environment The enzymatic effects of exogenous proteases both alone and synergistically with endogenous proteases was investigated to compre-hensively analyze the protein utilization by adding commercial proteases. 3.2.1. The enzymatic effect of exogenous protease acting alone in the environment without adjustment of the enzymatic conditions To screen exogenous proteases quickly， a simulated environment for the enzymatic assay was constructed using SPI. CPP was used as the control and as the experimental group for six commercial proteases，including neutral protease (NP)， acid protease (AcP)， alkaline protease (AlP)， papain， bromelin and aminopeptidase (AP). The supernatant re-covery was measured to understand the overall enzymatic efficiency. As displayed in Fig . 2A， the supernatant recovery when t reated with CPP，NP， AlP and AP was considerably reduced compared to CK. SDS-PAGE was used to better visualize the effect of protease degradation. The re-sults showed differences in the degradation effect by various proteases (F ig. 2B). Of these， bands were st i ll present i n the high molecular weight region after treatment with AcP， papain and bromelin， suggesting that there were sti l l undegraded macromolecular proteins. Nevertheless，treatment with CPP， NP， AlP and AP promoted their enzymatic hydro-lysis of 7 S globulin i n SPI， with the majority of the bands accumulating 11 S globulin. The above outcomes displayed that the degradation effect of CPP， NP， AlP and AP was dramatically increased， but their enzymatic ef f iciency became lower. Likely explanations were as follows： firstly，CPP， NP， AlP， and AP preferentially hydrolyzed 7 S globulins to form more 11 S globulins， thus increasing the degradation effect， but the binding ef f iciency to proteins was worse， resulting in a lower superna-tant recovery. Secondly， more disulfide bonds exist in the internal structure of 11 S globulin， making its molecular structure more compact (K an g et a l .， 2022)， which made it harder to effectively combine with the enzyme， resulting in poor supernatant recovery. The protein recovery (PR) and the degree of hydrolysis (DH) were analyzed to observe protein utilization from di f ferent perspectives.Firstly， the result of PR displayed that the hydrolysates treated with NP，AlP and bromelain were all substantially higher compared to CPP (F ig . 2 C)， meaning that these proteases were more conducive to improving the protein recovery. Besides， the content of DH was considerably enhanced in hydrolysates treated with CPP， NP， AlP and AP， especially CPP and AP (F i g . 2 C). Probably the reasons were as follows： CPP had the highest DH content because it contained an abundant protease system and exerted di f ferent cleavage s i tes. Next， the AP was an exonuclease (Matka w a l a et a l.， 2021)， which could cut the peptide chain of the amino-terminal to form free amino acids and therefore exhibit an increased DH content. Overall， CPP， NP， AlP， and AP were more beneficia l in increasing the degree of hydrolysis. The free amino acids were determined to further characterize the degradation of small molecules by CPP， NP， AlP and AP. First l y， the CPP and AP treatment displayed higher total free amino acid and hydro-phobic amino acid contents in agreement wi t h the trend of DH (F i g. 2D)，but their bitter amino acid content was remarkably higher than that treated with NP and AlP (F ig . S5). Furthermore， the NP-treated hydro-lysate had noticeably higher sweet amino acids than AlP-treated. This was because di f ferent proteases had different cleavage sites as well as releasing different amino acid sequences. NP could cleave proteins and may increase the number of target sites for flavor enzymes (Huang e t al .，2018； T a c ia s -P a s c a c io e t a l .， 2021). AlP is an endo-protease (Bu e t al.，2020)， which typically breaks peptide bonds of hydrophobic amino acids， increasing the release of bitter substances. AP could cleave hy-drophobic amino acids and enhance the flavor of the hydrolysate. I t demonstrated that the hydrolysates treated with CPP， NP， AlP， and AP increased the free amino acids content and formed differences in flavor-presenting amino acids by distinct proteases. Overal l ， NP， AlP and AP were the better exogenous proteases acting alone for the enzymatic degradation， but NP had the greater effect on flavor presentation according to the outcome of free amino acids. These were the results of the exogenous protease acting alone， and then we will A C (CK CPP 二 NP AcP AIP 口 I Papain 一 Bromel i n AP 2 Fig. 2. The enzymatic effect of exogenous protease acting alone in the environment without adjustment of the enzymatic conditions. (A) Effect of different proteases on the supernatant recovery. (B) The SDS-PAGE was used to visualize the degradation of proteases， lanes 1-10 were respectively： (1) Marker (2) Marker (3) CK (4)A. oryzae crude protease powder (CPP) (5) neutral protease (NP) (6) acid protease (AcP) (7) alkal i ne protease (AlP) (8) papain (9) bromel i n (10) aminopeptidase (AP). (C) Effect of different proteases on the protein recovery (PR) and degree of hydrolysis (DH). (D) Visualization of heat map of the effect of different proteases on free amino acids. Different letters were signi f icantly di f ferent among these groups (p <0.05). continue to explore the degradation effect of exogenous protease added onto endogenous protease from the perspective of synergistic effect. 3.2.2. The enzymatic effect of exogenous protease synergistic with CPP in the simulated high-salt moromi environment Proteases could exert different degradation effects according to their cleavage sites (Hana f i et a l .， 2018； Zh ao et a l .， 2021)， which function differently alone or synergistically with other proteases. Additionally，proteases were greatly affected by di f ferent environments， so it was necessary to further analyze the protein ut i lization by exogenous pro-teases acting synergistically with CPP under the moromi simulation environment. Firstly， CPP+NP exhibited markedly lower supernatant Was；recovery than CPP+CPP (Fi g. S6A ). There was a clear degradation i n the bands with different protease t reatments， but there was a non-significant difference among the groups (F i g . S6B ). Likewise， the results of PR and DH revealed that CPP or NP combined with CPP acted significantly better than the other groups (Fi g . 3A).Besides， PN and EP were used to assess the effect of hydrolysis.Compared to CPP+CPP， the CPP+NP and CPP+AlP groups displayed obviously higher PN content， while CPP +AP showed a clear advantage in EP. Based on the above data analysis， NP was the exogenous protease that could simultaneously improve protein recovery， hydrolysis and peptide nitrogen content after synergistic action with CPP， and its enhancement effect was superior to CPP +CK alone. Proteases could hydrolyze protein to form small molecules such as peptides and amino acids (L i u e t a l.， 2022； Ren &Li， 2022). Thus， the molecular weight and f ree amino acids were assayed to fur t her analyze the protease degradation towards small molecules. Firstly， the content of macromolecular peptides above 10 kDa was dist i nctly reduced after protease treatment (F ig .3B)， i ndicating that protease exerted its effect of degrading proteins. Moreover， the CPP+NP group had significantly higher peptide content below 500 Da， indicating that adding NP had the best effect on the hydrolysis of SPI into small molecule peptides.Furthermore， t he hydrolysates after protease synergy performed well i n the total free amino acid content， but the synergy exerted by NP， AlP and AP was not significant (F i g. 3C & S6C). Taken together ， NP was the most effective exogenous protease that could cooperate with CPP for enzymatic degradation under the simu-lated sauce moromi fermentation environment， including the enhance-ment of DH， PR， PN， and small molecule peptide content. A 80 CPP+CK 60一 a CPP+CPP a ab 十 CPP+NP d CPP+A1P C d CPP+AP CK+CK b b a d 0 PR DH PN EP B >10kDa 3-10kDa 1-3kDa 500-1000Da <500Da b b b b b a 0 20 40 C Hab c be abc Hab b C a ab abc b C 3 b bc b b b b a a ab hc C C Fig. 3. The enzymatic effect of exogenous protease synergistic with CPP in the simulated high-salt moromi environment. (A) Effect of different proteases on the protein recovery (PR)， degree of hydrolysis (DH)， peptide nitrogen (PN) and the extraction rate of peptide (EP). (B) Effect of different proteases on the molecular weight. (C) Visualization of heat map of the effect of different proteases on free amino acids. Different letters were significantly different among these groups (p <0.05). 3.3. Adding NP was beneficial to improve the protein utilization during the moromi stage NP was added to the moromi to verify the degradation effect of exogenous protease in the real soy sauce fermentation environment.Meanwhile， for the consideration of cost and the appropriate level of protease addition， different NP addi t ion was set up as experimental groups， inc l uding low enzyme activity (LEA)， medium enzyme activity (MEA) and high enzyme act i vity (HEA)， while no NP addition was taken as the control. The TN and AAN are important indicators for classifying the quality grade of soy sauce in China (Hu et a l .， 2022). As demonstrated in Fi g . 4A-B， the change in TN and AAN contents was mainly divided into two stages. The f i rst stage was the rapid increase phase in the pre-fermentation period (D5--D15)， where the MEA group treated with medium enzyme activity increased the TN and AAN content by 1.03 %and 5.80 % compared to CK on D15， respectively. The other stage was a slowly increasing stage from middle to end of fermentation (D30-D90).The MEA group treated with moderate enzyme activity enhanced the TN and AAN contents by 2.21 % and 7.45 % compared to CK on D90，respectively. Among them ， the AAN content was 0.833 g/100 mL， which reached t the national standard for special grade soy ysauce (>0.80 g/100 mL) (L i u e t a l.， 2021). Notably， the enhancement of TN and AAN content on D90 was better than on D15 after protease treat-ment ， illustrat i ng that adding NP st il l played an influential role in improving protein uti l ization towards the end of moromi fermentation. The heat map displayed the abundance of AAN and TN advanced with the extension of fermentation ti me (Fig . 4C). With the exception of the HEA group treated with high enzyme activity， the blue color basi-cally faded and turned red from D5 to D15， demonstrating that i t was a period of remarkable change. Besides， the TN and AAN contents i n MEA were consistently greater than in CK， suggesting that the addition of NP at a medium enzyme activity of 550 U/ (per gram of koji) was beneficial for increasing the TN and AAN contents during the moromi period，which also implied an i mprovement in the protein ut i l i zation of the soy sauce. 3.4. Adding NP was beneficial to improve the soy sauce f lavor during the moromi phase Organic acids played an important role in the flavor of soy sauce (Zhu et a l .， 2021). The contents of tartaric acid， malic acid， lactic acid，acetic acid， citric acid and succinic acid in soy sauce were determined in this study. Lactic acid and acetic acid were the dominant organic acids (Fi g . 5A). Studies had shown that the higher the ratio of lactate to acetic acid ， the softer and more mellow the flavor of soy sauce (Rua n et a l.，2022). Adding NP was beneficial in i ncreasing the ratio of lactic acid to acetic acid (F ig . S7). Besides， citric acid， succinic acid and tartaric acid had a unique sour taste (Wa n g et al .， 2019； L i u et al .， 2021； X u et al.，2022). Yet the results displayed that the content of citric and succinic acids was considerably lower in the HEA group treated with high enzyme activity than in CK， while there was no significant difference in tartaric acid. This demonstrated that the soy sauce flavor was the result of the combined effect of multiple organic acids， while adding NP strengthened the richness of the soy sauce by enhancing the ratio of lactic to acetic acid. Altogether 381 volatile flavor compounds were detected in the soy sauce crude oil using SPME-GC-MS. There were 242， 236，251 and 214compounds detected for the control and low， medium and high enzyme activity treatment groups， respectively (F ig .5B)， among which the HEA group wi t h high enzyme activity treatment had much less variety. Also，the OPLS-DA score plot displayed that the different treatment groups were divided into different quadrants (F i g . 5C)， demonstrating differ-ences in aroma types， particularly in the HEA group. Studies have re-ported that acetic acid (L u l f e t a l .， 2021) was the key acid compound.Ethanol (Lulf et al.， 2021)， 1-octen-3-ol (Ga o et al.， 2021) and The results of the sensory evaluation are presented in Fi g . 5D. The soy sauces treated with low and medium enzyme activity enhanced their comprehensive preference and umami， respectively， as well as their excellent performance in richness， presumably due to their considerably high l actic/acetic acid ratio. It is worth mentioning that adding NP did not i ncrease bitterness or astringency. Besides， there was no significant change in aroma， which h was inconsistent with the finding of SPME-GC-MS， presumably because human olfactory perception was not as sensitive as that of the instrument. In summary， adding NP at a level of 550 U/ (per gram of koji) to the MEA group remarkably improved the umami and richness ， whilst at a level of 275 U/ (per gram of koji)， the LEA group dramatically enhanced the umami and comprehensive preference. It meant that the addi t ion of NP below 550 U/(per gram of koji) could contribute to the overall flavor of the soy sauce. 3.5. Adding NP played an i mportant part i n improving protein utilization and fermentation quality of soy sauce The degradation products of proteins had a considerable impact on the taste of soy sauce and serve indirectly as the precursor of soy sauce aroma (C hen e t al.， 2015； Zho u et a l.， 2019； Z ho u et al .， 2022). The molecular weight and free amino acids were assayed to further observe the effect of adding NP on small molecule degradation and f lavor of soy sauce. First l y， the MEA treated with medium enzyme activity showed a dramatically increased peptide content below 500 Da and 1-5kDa compared to CK (Fig .6A). Previous studies have demonstrated that the below 500 Da and Mai l lard peptides (1-5 kDa) possess stronger umami in soy sauce， which could endow food with a mellow and persistent flavor(Og asa w a r a e t a l.， 2006； Zha o et al .， 2021). Therefore， adding NP improved the content of both smal l molecule peptides and Maillard peptides， thus increasing the protein ut il ization and umami substances.Secondly， the HEA group treated with high enzyme activity had mark-edly lower total f ree amino acids compared to CK (F i g. 6C & S8A)， which may account for the decrease in total nitrogen. It demonstrated that adding NP could not dramatically enhance the total free amino acid content. Overall ， the interaction of NP with A. oryzae protease resulted in the degradation of the protein into more peptides less than 500 Da，which enhanced the flavor of the soy sauce. It implied that adding NP was an effective means to improve the protein utilization and f lavor of soy sauce. The protease activity of moromi during the late fermentation phase was conducted to further investigate whether adding NP improved protein ut i lization and flavor by enhancing enzyme ac t ivity. The dis-tribution of the mixed proteases remained dominated by neutral pro-teases (F i g . 6B)， which is consistent with the phenomenon in the koji phase. Furthermore， protease activity slowly declined on D5-D15，maintaining high activity， while it decreased significantly at D30-D90and remained lower. This corresponded to the rapidly growing and then slowly decl i ning results of TN and AAN. Moreover， the MEA group with medium enzyme activity had increased enzyme activity， reflecting a Fermentation time (days) B Fermentation time (days) C Fig. 4. Adding NP was beneficial to improve the protein uti l ization during the moromi stage. Effect of adding protease on (A) TN and (B) AAN. (C) Visualization of heat map between different exper i menta l groups and AAN， TN i n moromi fermentation. Different letters were significantly different among these groups (p <0.05). A B D C Fig. 5. Adding NP was beneficial to improve the soy sauce flavor during the moromi phase. Effect of adding protease on (A) organic acid and (B) volat i le compounds.(C) The score plot was obtained by OPLS-DA analysis. (D) Effect of adding protease on sensory evaluation. Different letters were signi f icantly different among these groups (p <0.05). higher degradation rate， which was also responsible for the increase in TN and AAN. However， the total enzyme activity was rather reduced with higher additions than 550 U/ (per gram of koji)， which may be due to autolysis caused by excess protease. Notably， the HEA with high enzymatic activity exhibited significantl y lower acid protease activity on D90， which is consistent with the result of free amino acids. It was because acid protease was a peptidase that further catalyzes the decomposition of peptides into amino acids. Above results demonstrated that the level of protease activity played an important part in improving the protein hydrolysis ef f iciency. Next， the pH was assayed to track changes i n the moromi environment during fermentation. The pH of moromi trended downwards with increasing fermentation time and started to drop below 5.0 on D15 (F ig. S8B ). Previously we have demonstrated that the stability of protease activity decl i ned rapidly at pH below 5.0. It i mplied that the decline in enzyme activity of the moromi was due to low pH， reducing the efficiency of degrading protein and leading to slowly increasing the AAN and T N content . Furthermore，the pH dropped significantly with adding above 550 U/ (per gram of koji)， which may also be responsible for the lower enzyme activity. Redundancy analysis was used to analyze the correlation between protease and physicochemical index (Fi g . 6D). The mixed neutral pro-tease was predominantly associated with AAN and TN， especially in the MEA group treated with medium enzyme activity， while mixed acid protease was mostly associated with FAA. Of these， the mixed neutral protease mainly degraded the protein into peptides， such as below 500 Da and the Mai l lard peptides， thereby increasing the content of AAN and TN， which further interacted with organic acids and volatile flavor compounds to affec t the flavor of the soy sauce， such as t he enhancement of umami and richness. Hence， adding NP was beneficial to improve the protein ut il ization and flavor of soy sauce， which provided theoretical guidance for industrial production. 4. Conclusion Overall， the role of exogenous proteases in i mproving protein utili-zation and fermentation quality of soy sauce was systematically explored in this study . First， the endogenous protease secreted by A. oryzae was found to act an important role in the utilization of nitrogen elements. Moreover， NP was the exogenous protease that synergized with A. oryzae endogenous protease to perform the best enzymatic >10kDa 5-10kDa 3-5kDa 1-3kDa 500-1000Da ≤500Da 1-5kDa E lab b b 2500 B Day 5 Day 15 Day 30 D ay 45 Day 60 Day 90 C D T P HEA MEA LEA CK Leu U m ami -1一 Swee tn ess 0- I l e Bitterness 1- U nf lavor FAA Asp ANB Glu 心 多 Fig. 6. Adding NP played an important part in improving protein utilization and fermentation quality of soy sauce. Variation of the (A) molecular weight and (B)enzymatic activity during moromi fermentation after adding protease. (C) Visual i zation of heat map between different experimental groups with f ree amino acids in moromi fermentation. (D) Correlations of protease activity with ni t rogen element were constructed by redundancy analysis (RDA). Di f feren t letters were signi f icantly different among these groups (p <0.05). degradation in the simulation experiments. Importantly， adding NP to the moromi enhanced the degradation rate by increasing enzyme ac-tivity， releasing more peptides. Further improving protein ut i lization and shortening the fermentation cycle by boosting the TN and AAN content. Also enhancing flavor expression and fermentation quality by increasing the Mi l lard peptides and the ratio of lactic to acetic acid. This research provided a novel idea and theoretical guidance for improving protein utilization and optimizing the quality of soy sauce. CRediT authorship contribution statement Caifeng Chen： Conceptualization， Data curation， Formal analysis，Methodology， Software， Wri t ing - original draft. Sha Hou： Data cura-tion， Formal analysis， Methodology， Project administration. Changz-heng Wu： Visualization， Investigation， Supervision. Yong Cao： Writ i ng - review & editing， Investigation， Validation， Supervision. Xing Tong：Review & editing， Validation， Supervision， Funding acquisition， Project administration， Resources. Yunjiao Chen： Writing- review & editing，Validation， Supervision， Funding acquisition， Project administration，Resources. Declaration of Competing Interest The authors declare no competing financial interest. Data availability Data will be made available on request. Acknowledgements This work was f inancial l y supported by the Core Technology Project of Foshan City， China (Grant number 1920001000824) and General project of the Natural Science Foundation of Guangdong Province，China (2022A1515010907). Appendix A. 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