Aspergillus oryzae strain a08 and application thereof

Abstract

The application discloses an Aspergillus oryzae strain A08 and application thereof, the Aspergillus oryzae A08 is preserved in the China General Microbiological Culture Collection Center on January 20, 2026, the suggested classification name is Aspergillus oryzae, and the preservation number is CGMCC No.42436.The koji-making process of the strain can simultaneously promote saccharifying enzyme and acid protease, the saccharifying enzyme activity is 1479 U / g, is increased by 31% than the original strain 3.042, the acid protease activity is also increased by 18.92% than the original strain 3.042, and raw material starch and protein are simultaneously degraded; the strain is used for high-salt dilute-state soy sauce fermentation, the D-anhydrous glucose yield is 2.51 times of the original strain, and the contents of free amino acid, sweet amino acid, glutamic acid and amino acid nitrogen are increased by 4.52%, 15.56%, 12.46% and 3.75% respectively.The application solves the problems of insufficient starch saccharification and insufficient protein degradation of the Aspergillus oryzae strain from the source, and has industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an Aspergillus oryzae strain AO8 and its applications. Background Technology

[0002] Soy sauce is made from soybeans, soybean meal, wheat, and other raw materials. Through microbial enzymatic hydrolysis, macromolecules are converted into flavor compounds such as amino acids and sugars. With the large-scale development of the food industry, market demands for increased soy sauce production and quality stability are rising. The increasing cost of raw materials is also driving the industry to seek technological solutions to improve raw material utilization.

[0003] Aspergillus oryzae is the core microorganism in the solid-state fermentation of soy sauce, and its metabolic activity directly determines the quality of the koji and the fermentation efficiency. This strain can secrete hydrolytic enzymes such as proteases and saccharifying enzymes, which degrade proteins and starches into small molecule products, serving as flavor precursors for soy sauce and carbon and nitrogen sources for microbial metabolism. It is key to influencing the quality of soy sauce and the conversion of raw materials.

[0004] However, the current industrial fermentation of soy sauce faces technical bottlenecks that restrict the improvement of raw material utilization. Traditional Aspergillus oryzae strains have problems such as insufficient decomposition of raw materials and low saccharifying enzyme activity, resulting in the easy residue of starchy substances. Moreover, fluctuations in fermentation parameters can easily lead to unstable spore germination rates and decreased enzyme production efficiency, exacerbating raw material waste and potentially causing fluctuations in soy sauce quality.

[0005] Saccharifying enzymes are a key indicator in the early stages of soy sauce fermentation. Their activity directly determines the degradation and conversion efficiency of starchy raw materials. They catalyze the hydrolysis of polysaccharides into glucose, providing a carbon source for subsequent fermentation and flavor synthesis. Insufficient saccharifying enzyme activity leads to raw material waste and inhibited microbial metabolism, thus affecting soy sauce yield and quality. Enhancing their activity is crucial for optimizing the fermentation process. Acidic proteases are the core functional enzymes that degrade soybean protein and generate amino acid nitrogen during soy sauce fermentation. They are essential for increasing the amino acid nitrogen content in soy sauce, optimizing the richness and umami flavor of the product, improving the overall utilization rate of raw materials, and reducing production costs. They demonstrate significant potential for quality and efficiency improvement in the industrial production of soy sauce and other condiments.

[0006] Currently, the problems of weak umami flavor and poor raw material degradation efficiency in soy sauce fermentation urgently need to be addressed. Based on this, a strain of *Aspergillus oryzae* with synergistically enhanced saccharifying enzyme and acidic protease activity, and improved spore quantity and morphology, was obtained. This provides valuable insights for strengthening the starch and protein degradation capacity during the koji-making stage and improving the stability of koji preparation. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide Aspergillus oryzae AO8, which was deposited at the China General Microbiological Culture Collection Center on January 20, 2026, with the proposed classification name Aspergillus oryzae and the accession number CGMCC No. 42436.

[0010] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of Aspergillus oryzae in soy sauce fermentation.

[0011] As a preferred embodiment of the application described in this invention, the process includes inoculating the Aspergillus oryzae during the preparation of the starter culture to obtain the starter culture;

[0012] Then, koji (a type of starter culture) is prepared and soy sauce is produced through brine fermentation.

[0013] Among them, Aspergillus oryzae has the effect of simultaneously enhancing the activity of saccharifying enzymes and proteases during the koji-making process.

[0014] As a preferred embodiment of the application described in this invention, the Aspergillus oryzae in the soy sauce fermentation system increases the total amount of free amino acids and the proportion of sweet amino acids and glutamic acid, thereby enhancing the umami intensity and richness of the product.

[0015] Beneficial effects of this invention:

[0016] This invention provides an Aspergillus oryzae AO8, which was deposited at the China General Microbiological Culture Collection Center on January 20, 2026, with the suggested classification name Aspergillus oryzae and the accession number CGMCC No. 42436.

[0017] The Aspergillus oryzae AO8 strain of this invention exhibits a saccharifying enzyme activity of 1479 U / g during koji making, representing a 31% increase compared to the original strain Hu Niang 3.042, and an 18.92% increase in acidic protease activity. When applied to soy sauce fermentation, it produces 2.51 times more glucose than the original strain, with an amino acid nitrogen content 3.75% higher than the original strain 3.042, and a free amino acid content of 50.69 mg / mL, a 4.92% increase compared to the original strain. Among these, the content of sweet amino acids is 15.56% higher, and the content of core fresh glutamic acid significantly increases by 12.46%. In soy sauce fermentation, it can efficiently degrade carbohydrates in raw materials to produce glucose, enhancing the flavor richness of soy sauce and other fermented condiments, and has broad application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0019] Figure 1 This is a colony morphology diagram of Aspergillus oryzae on PDA agar medium in an embodiment of the present invention;

[0020] Figure 2 This is a microscope image of Aspergillus oryzae in an embodiment of the present invention;

[0021] Figure 3 This is a graph showing the results of the initial and secondary screening of the saccharifying enzyme production capacity of Aspergillus oryzae in this invention embodiment;

[0022] Figure 4 This is a graph showing the results of the initial and secondary screening of the protease production capacity of Aspergillus oryzae in this invention.

[0023] Figure 5 This is a graph showing the determination of Aspergillus oryzae spore count in an embodiment of the present invention;

[0024] Figure 6 This is a graph showing the change in sugar content in soy sauce after Aspergillus oryzae fermentation in an embodiment of the present invention;

[0025] Figure 7 This is a graph showing the change in amino acid nitrogen content after Aspergillus oryzae fermentation in an embodiment of the present invention;

[0026] Figure 8 This is a graph showing the change in free amino acid content after Aspergillus oryzae fermentation in an embodiment of the present invention. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0028] The present invention relates to Aspergillus oryzae AO8, which was deposited at the China Center for Type Culture Collection (CGMCC) on January 20, 2026, and is classified as Aspergillus oryzae AO8, with accession number CGMCC No. 42436, and deposited at the Institute of Microbiology, Chinese Academy of Sciences.

[0029] Example 1

[0030] Mutagenesis and selection of Aspergillus oryzae:

[0031] The Aspergillus oryzae strain AO8 was obtained by screening the Aspergillus oryzae strain Hu Niang 3.042, which was used for making soy sauce koji, after ARTP mutagenesis in our laboratory. It was deposited at the China General Microbiological Culture Collection Center on January 20, 2026, with the suggested classification name Aspergillus oryzae and the accession number CGMCC No.42436.

[0032] Example 2

[0033] Assay for the saccharifying enzyme production capacity of Aspergillus oryzae:

[0034] Initial screening was performed using the transparent ring method:

[0035] PDA slant culture medium: Boil 200g of potatoes in water, filter the liquid, add distilled water to 1L, add 20g of glucose and 15g of agar.

[0036] Initial screening medium: 12g soluble starch, 8g yeast extract, 5g NaCl, 20g agar, 1L water. After preparation, sterilize at 121℃ and 0.1MPa for 20min. Pour the hot medium into sterile petri dishes and allow it to solidify completely.

[0037] Prepare spore suspension and activate the strain. Add 10 mL of sterile physiological saline containing Tween-80 to the activated Aspergillus oryzae slant culture medium. Gently scrape spores from the slant culture medium with an inoculation loop. Transfer the scraped spore suspension to a sterile Erlenmeyer flask and place it in a constant temperature shaker at 180 rpm for 10 min to disperse the spores evenly. Filter the mycelium using 6 layers of sterile gauze, count the mycelium using a hemocytometer, and dilute the spore suspension to 1×10⁻⁶ with sterile physiological saline. 7 ~1×10 8 Quantity / mL, keep for later use.

[0038] Prepare for the spotting operation. Use a pipette to draw 10 μL of spore suspension and spot it evenly on the surface of the starch selection medium plate (with a blank control set up at the same time). After the spore suspension is fully absorbed into the medium, incubate it upside down in a 30℃ constant temperature incubator for 48~72 days to observe its status.

[0039] For strain screening, the diameter (d) of each colony and the diameter (D) of the surrounding clear zone were measured using calipers, and the ratio of the clear zone diameter to the colony diameter (D / d) was calculated. Strains with a larger D / d ratio and a clear zone edge were selected. Single colonies were picked up with an inoculation loop in a clean bench and transferred to fresh PDA slant medium for activation culture to obtain purified primary screening dominant strains.

[0040] Figure 1This is a colony morphology diagram of Aspergillus oryzae AO8, showing vigorous mycelial growth and bright color.

[0041] Figure 2 This is a microscopic image of Aspergillus oryzae (AO8), showing plump spores, a thick sporophyte layer, and uniform spore attachment.

[0042] Figure 3 In the middle, on the left of the image, the original strain Hu Niang 3.042 is on the left with a D / d ratio of 1.006, while the strain AO8 of Aspergillus oryzae preserved in this invention is on the right with a D / d ratio of 1.159. The difference in the D / d ratio directly reflects the enzyme production capacity of the strain. The higher D / d ratio of AO8 indicates that the clear zone formed during the initial screening process is significantly larger than that of the original strain. This indicates that the target enzyme synthesis and secretion capacity of this mutagenic strain is significantly better than that of the original strain, which meets the initial screening characteristics of high enzyme activity strains and can be further screened.

[0043] Secondary screening was performed using DNS colorimetry.

[0044] Secondary screening culture medium: Soybeans and flour are mixed in a 7:3 mass ratio.

[0045] The strains obtained from the initial screening were cultured in PDA, and then inoculated with three or more rings into the seed culture medium (wheat bran:water = 1:1.1) for 72 h. The number of spores in the seed culture was determined using a hemocytometer; the original strain 3.042 had 8.504 billion / g (dry basis), and AO8 had 9.876 billion / g (dry basis). The spore germination rate of the original strain 3.042 was 86.5%, and that of AO8 was 90.05%. Simultaneously, seed culture was inoculated at 0.3% (w / w) of the total dry weight of soybeans and flour, and fermentation was carried out at 32℃ for 72 h to obtain the finished koji.

[0046] The crude enzyme solution was obtained by extracting the koji with pure water, and the activity of the saccharifying enzyme was measured.

[0047] The method for detecting glucoamylase activity is as follows:

[0048] (1) Reagent preparation

[0049] Preparation of pH 4.6 acetate buffer solution (pH 4.6 acetic acid-sodium acetate buffer).

[0050] 0.1 mol / L acetic acid solution: Pipette 5.7 mL of glacial acetic acid, dilute to 1000 mL with distilled water, and mix well;

[0051] 0.1 mol / L sodium acetate solution: Weigh 8.2 g of anhydrous sodium acetate, dissolve it, and bring the volume to 1000 mL. Mix well.

[0052] Take 780 mL of 0.1 mol / L acetic acid stock solution and 220 mL of 0.1 mol / L sodium acetate stock solution, pour them into a beaker and mix thoroughly. After calibrating the pH meter at room temperature, it is ready for use.

[0053] 1% soluble starch solution: 1 g starch, diluted to 100 ml with pH 4.6 acetate buffer solution.

[0054] (2) Drawing the standard curve

[0055] Table 1 Standard curve of saccharifying enzyme

[0056]

[0057] Using tube 0 as a blank control, the absorbance (OD) of each tube was measured at a wavelength of 550 nm using a UV-Vis spectrophotometer. 550 A standard curve for glucose was plotted with glucose concentration (mg / mL) on the x-axis and absorbance value on the y-axis, yielding the regression equation.

[0058] (3) Determination of enzyme activity in samples

[0059] Sample enzyme activity assay:

[0060] Prepare a test tube and add 0.5 mL of 1.0% (w / v) soluble starch solution and 0.45 mL of 0.1 mol / L acetate buffer (pH 4.6) sequentially. Then add 0.05 mL of crude enzyme solution diluted to an appropriate concentration and mix thoroughly using a vortex mixer. Place the test tube in a 40℃ water bath and incubate for 20 min. Add 1.5 mL of DNS colorimetric reagent to a colorimetric tube. After the reaction is complete, take 0.5 mL of the reaction solution and add it to the colorimetric tube containing DNS, mixing thoroughly. Place the test tube in a boiling water bath and heat for 15 min. Immediately remove the test tube and cool it to room temperature with running water. Add 10.5 mL of distilled water to the cooled test tube, mix thoroughly by shaking, and then measure the absorbance at 550 nm using a UV-Vis spectrophotometer.

[0061] The crude enzyme solution inactivated by high temperature was used as a blank control: After the crude enzyme solution was inactivated, the same operating procedure as the above sample determination was performed, and the resulting absorbance value was used as a blank reference.

[0062] (4) Calculation of enzyme activity

[0063]

[0064] In the formula:

[0065] X — Saccharifying enzyme activity per gram of koji material (U / g);

[0066] A—The glucose concentration of the sample reaction solution (mg / mL) obtained from the standard curve;

[0067] V1—Total final volume of sample (ml);

[0068] n—dilution factor;

[0069] m—mass of the sampled material (g);

[0070] V2—The volume of sample liquid added to the reaction system (ml);

[0071] t — reaction time (min);

[0072] Control group: Shanghai-brewed 3.042 koji-making sample;

[0073] Experimental group: Aspergillus oryzae AO8 preserved strain koji preparation sample, Aspergillus oryzae A22.

[0074] The measurement results are as follows Figure 3 As shown in the right figure, the saccharifying enzyme activities of the original Aspergillus oryzae 3.042 and the mutagenized screening strain AO8 both showed a dynamic characteristic of first increasing and then decreasing, and the enzyme activity peaks both occurred at 48h of koji preparation.

[0075] Among them, the peak activity of saccharifying enzyme in the original strain 3.042 was 1130 U / g at 48 h; the peak activity of enzyme in the mutant strain AO8 at the same time was 1479 U / g, which was about 31% higher than that of the original strain 3.042, indicating that the mutagenesis treatment of 3.042 effectively enhanced the saccharifying enzyme synthesis ability of the strain.

[0076] Another Aspergillus oryzae strain, A22, obtained in the laboratory through ARTP mutagenesis, had a saccharifying enzyme activity of 989 U / g, lower than that of the original strain. The Aspergillus oryzae strain AO8 preserved in this invention showed significantly higher saccharifying enzyme activity in its fermentation koji material after 48 h of koji preparation compared to the control strain Hu Niang 3.042, indicating that this strain possesses superior saccharification performance and has significant technical advantages in fermentation koji preparation applications.

[0077] Example 3

[0078] Assay for Aspergillus oryzae protease activity:

[0079] (1) Initial screening

[0080] Referring to the above-mentioned method for initial screening of saccharifying enzymes, the casein clear zone method was used for initial screening of high-yielding protease strains. The specific screening culture medium is as follows:

[0081] Upper layer: MgSO4·7H2O 0.05%, KH2PO4 0.036%, Na2HPO4·7H2O 0.107%, NaCl 0.016%, ZnCl2 0.0014%, CaCl2 0.0002%, hydrolyzed casein 0.005%, casein 0.4%, agar 1.5%, pH adjusted to 5.5. Lower layer: agar aqueous solution, 2% by mass. Sterilize at 121℃ and 0.1 MPa for 20 min. After sterilization, pour into sterile petri dishes and allow to solidify completely. The preparation of spore suspension and plate-forming procedures are the same as the saccharifying enzyme screening steps in Example 2 above.

[0082] Figure 4 This image shows a comparison of initial screening plates for Aspergillus oryzae strains. The left side shows the original strain, Hu Niang 3.042, and the right side shows the Aspergillus oryzae AO8 strain preserved in this invention. The ratio of the clear zone diameter to the colony diameter (D / d) of the original strain 3.042 is 1.100, while the D / d ratio of the AO8 strain reaches 1.271, which is about 15.5% higher than that of the original strain. This result indicates that the AO8 strain of this invention has a significant advantage in protease synthesis and secretion capabilities, and can be further verified through secondary screening.

[0083] (2) Secondary screening

[0084] Reagent preparation:

[0085] 0.4 mol / L sodium carbonate solution: Weigh 42.4 g of anhydrous Na₂CO₃, dissolve in distilled water, and bring the volume to 1 L. 0.4 mol / L TCA solution: Weigh 65.4 g of trichloroacetic acid (CCl₃COOH), dissolve in distilled water, and bring the volume to 1 L.

[0086] 0.1 mol / L pH 7.2 phosphate buffer: Solution A: 31.2 g NaH2PO4・2H2O, dissolved in distilled water and brought to a final volume of 1 L (0.2 mol / L); Solution B: 71.63 g Na2HPO4・12H2O, dissolved in distilled water and brought to a final volume of 1 L (0.2 mol / L); Take 28 mL of solution A and 72 mL of solution B, and dilute with distilled water by half to obtain 0.1 mol / L pH 7.2 phosphate buffer.

[0087] 2% acidic casein solution: When preparing a 2% acidic casein solution, first add a few drops of concentrated lactic acid to moisten the casein to accelerate dissolution, and then bring the volume to 100 mL with pH 3.0 phosphate buffer.

[0088] 100 μg / ml L-tyrosine standard solution: Accurately weigh 0.1000 g of L-tyrosine dried to constant weight in an oven at 105℃, gradually add 6 mL of 1 mol / L hydrochloric acid solution and stir until completely dissolved, then dilute to 100 mL with 0.2 mol / L hydrochloric acid solution to obtain a concentration of 1000 μg / mL; pipette 10 mL of this solution, dilute to 100 mL with 0.2 mol / L hydrochloric acid solution, shake well, and store at 4℃. Use within 48 h after preparation.

[0089] Plot the standard curve:

[0090] Referring to the Folin method of SB / T 10317-1999, a series of tyrosine standard solutions of different concentrations were prepared using blank tubes as controls. After colorimetric reaction, the absorbance was measured at a wavelength of 660 nm. A standard curve was plotted with tyrosine concentration as the abscissa and absorbance as the ordinate to obtain the linear regression equation.

[0091] Table 2. Preparation of L-tyrosine standard solutions of different concentrations

[0092]

[0093] The specific operating procedures are shown in Table 3.

[0094] Table 3. Procedure for protease activity assay

[0095]

[0096] The definition of a protease activity unit is: the enzyme activity level corresponding to the hydrolysis of casein and the generation of 1 μg of tyrosine per minute at 40°C is counted as 1 protease activity unit.

[0097]

[0098] In the formula:

[0099] A—The OD value measured from the sample is used to find the equivalent number of micrograms of tyrosine (or OD value × K) from the standard curve.

[0100] Take 1 mL of 4-4 mL of the reaction solution for testing (i.e., 4 times the original volume).

[0101] 10 — Reaction time: 10 min;

[0102] W—Percentage of moisture content in the sample.

[0103] The steps of preparing the sample for rescreening, the proportion of Aspergillus oryzae inoculation, and the extraction of crude enzyme solution are the same as the saccharifying enzyme operation process in Example 2 above.

[0104] Control group: Hu Niang 3.042 koji-making sample.

[0105] Experimental group: koji samples prepared from the preserved strain Aspergillus oryzae AO8 and Aspergillus oryzae A22.

[0106] like Figure 4 As shown, the acidic protease activity of the original strain of Aspergillus oryzae Hu Niang 3.042 and the Aspergillus oryzae AO8 strain preserved in this invention was measured during the koji-making process. The results showed that the acidic protease activity of both strains first increased and then decreased with the koji-making time, and the enzyme activity reached the peak at 48 h. Among them, the peak acidic protease activity of strain AO8 reached 1213.15 U / g, which was about 18.86% higher than that of the original strain 3.042. In addition, another strain of Aspergillus oryzae A22 obtained by ARTP mutagenesis had an acidic protease activity of 1207 U / g, which was 9.71% higher than that of the original strain. This result highly corresponds to the initial screening results of the strain protease.

[0107] Therefore, the Aspergillus oryzae AO8 strain preserved in this invention achieves a synergistic enhancement of the activities of saccharifying enzymes and acidic proteases during the koji-making process, and has both efficient starch degradation and protein hydrolysis capabilities, thus ensuring the efficient generation of reducing sugars and amino acids during the subsequent soy sauce fermentation process.

[0108] Example 4

[0109] Determination of Aspergillus oryzae spore count:

[0110] (1) Sample preparation

[0111] Accurately weigh 1 g of seed culture, add 5 mL of 95% ethanol, 20 mL of sterile water, 10 mL of 10% dilute sulfuric acid, and a number of glass beads in sequence, and shake thoroughly for 5-10 min to completely disperse the conidia into a single-cell suspension. Filter the solution using several layers of sterile gauze, and repeatedly rinse the filter residue with sterile water to ensure complete spore elution. Finally, bring the filtrate to a final volume of 500 mL to obtain the spore dilution.

[0112] (2) Production

[0113] Take 1 drop of the diluent and place it on the counting grid of the hemocytometer. Then, gently press the coverslip down from one side to the other to ensure a complete seal between the coverslip and the counting chamber. Use filter paper to absorb any excess spore suspension and let it stand for several minutes until the spores settle.

[0114] Take one drop of the above diluent and add it to the center of the counting area of ​​a 25×16 hemocytometer. Slowly cover the counting area with a coverslip from one side, ensuring that the coverslip is completely in contact with the surface of the counting chamber and that there are no air bubbles. Use filter paper to absorb any suspension that overflows from the edge of the counting area, and let it stand at room temperature for 5 minutes to allow the spores to settle naturally to the bottom of the counting chamber grid.

[0115] (3) Observation and counting

[0116] The spores were observed and counted under a microscope with a magnification of 10×40. The counting area consisted of four large squares at the corners and one large square in the center of a 25×16 counting plate (a total of 80 small squares). If spores were located on the grid lines, only spores on the adjacent sides and in the included corners were counted to reduce systematic error. Each sample was counted at least twice in parallel, and the average value was taken as the spore count result for that sample.

[0117] like Figure 5 As shown, the spore count of strain 3.042 was 8.504 billion / g, while the spore count of the mutant strain AO8 was 9.876 billion / g, an increase of approximately 16.13% compared to the original strain. This result indicates that AO8, while maintaining good growth characteristics, also possesses the previously verified excellent traits such as high saccharifying enzyme activity, further supporting the application potential of the Aspergillus oryzae strain AO8 preserved in this invention.

[0118] Example 5

[0119] Changes in sugar content after Aspergillus oryzae fermentation of soy sauce:

[0120] Gas chromatography-mass spectrometry (GC-MS) was used for detection and analysis.

[0121] Sample pretreatment: Take 50 μL of soy sauce sample into a centrifuge tube, pre-freeze at -80℃ for 12 h, and then freeze-dry under vacuum for 12 h to remove moisture. Add 100 μL of chromatographic grade N,N-dimethylformamide (DMF) to the freeze-dried sample, sonicate for 35 min to dissolve and vortex to mix, centrifuge at 12000 rpm for 5 min, and take 50 μL of the clear supernatant to a new centrifuge tube. Add 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (containing 1% trimethylchlorosilane, BSTFA + 1% TMCS) derivatization reagent and 5 μL of 100 μg / mL methyl heptadecanate internal standard solution to the tube, vortex to mix for 1 min, and then derivatize in an 80℃ constant temperature water bath for 2 h. After derivatization, cool to room temperature, and inject 1 μL of the derivatized product into a gas chromatography-mass spectrometry (GC-MS) instrument for detection and analysis.

[0122] GC-MS detection conditions:

[0123] Table 4 GC-MS Detection Condition Parameters

[0124]

[0125] Qualitative and quantitative analysis were performed. Qualitative analysis employed a spectral library search and matching method, comparing the mass spectra corresponding to each unknown peak in the acquired total ion current chromatogram with the NIST11 standard spectral library. A matching degree of ≥80% was used as the qualitative criterion for identifying the target compound. Quantitative analysis combined semi-quantitative and relative quantitative methods: semi-quantitative analysis calculated the relative content by the ratio of the peak area of ​​the target compound to the total peak area of ​​all detected substances.

[0126] like Figure 6 As shown, in the fermentation system of the original strain 3.042, the D-anhydrous glucose content was 1.60 mg / mL, accounting for approximately 14.6% of the total sugar. In the fermentation system of the mutant strain AO8, the D-anhydrous glucose content reached 4.02 mg / mL, accounting for approximately 16.1% of the total sugar. The glucose production was significantly higher than that of the original strain, and the proportion was also slightly increased. The comparison shows that AO8's glucose production capacity is significantly better than that of the original strain 3.042, with its D-anhydrous glucose content being 2.51 times that of the original strain, and its proportion being relatively higher. Combined with the characteristic of AO8 being a high-saccharifying enzyme-producing mutant strain, this indicates that high-saccharifying enzyme production can enhance the hydrolysis efficiency of carbohydrates during fermentation, promote the conversion of polysaccharides to monosaccharides such as glucose, thereby increasing the accumulation of glucose in the fermentation system. In conclusion, the Aspergillus oryzae strain AO8 preserved in this invention can effectively enhance the glucose production capacity in soy sauce fermentation, providing a sufficient carbon source basis for subsequent microbial metabolism and flavor compound synthesis.

[0127] Example 6

[0128] Changes in amino acid nitrogen content after Aspergillus oryzae fermentation of soy sauce:

[0129] Soy sauce koji-making and fermentation process:

[0130] (1) Preparation of starter culture: After moistening the wheat bran at a material-to-water ratio of 1.2:1, it is packaged and sterilized, cooled to room temperature, and inoculated with three or more rings of Aspergillus oryzae spores. It is then cultured at 32-34℃. When the starter culture turns white in the early stage, it is broken up. When the mycelium is fully grown and the spores are dispersed in a green mist, the starter culture is obtained.

[0131] (2) Preparation of Daqu: After soaking soybeans for 4 hours, wrap and sterilize them, mix them with flour in a ratio of 7:3, coat them with powder, cool them, and then add 0.3% of the dry material mass of seed koji. Set the temperature of the incubator to 30~35℃. The first turning of koji is about 14 hours after koji preparation, and the second turning is about 22 hours after koji preparation. Add salt water when the enzyme activity peak is about 48 hours after koji preparation.

[0132] (3) Salt water fermentation: Prepare a 20% salt water solution; mix the koji and salt water at a ratio of 1:2.5 and ferment at a constant temperature of 32℃, stirring regularly during the fermentation process.

[0133] The fermentation period for high-salt dilute soy sauce was 6 months, with samples taken periodically at 30-day intervals to determine its physicochemical properties. Following GB5009.235-2016, 5.0 g of soy sauce sample was accurately weighed, washed several times with water into a 100 mL volumetric flask, and diluted to the mark, then thoroughly mixed. 20.0 mL of the diluted soy sauce solution was titrated, first to pH 8.2 (for total acid calculation), then 10.0 mL of neutral formaldehyde solution was added, and titration continued to pH 9.2. A blank test was performed simultaneously, and the amino acid nitrogen content was calculated based on the sodium hydroxide consumption and the formula.

[0134] The measurement results are as follows Figure 7 As shown, the amino acid nitrogen content of both the original strain Hu Niang 3.042 and the Aspergillus oryzae strain AO8 preserved in this invention showed a continuous upward trend with the extension of high-salt dilute fermentation time in soy sauce samples; among them. Within 0-90 days, the amino acid nitrogen accumulation rate of Hu Niang 3.042 was faster, and at 90 days, its content was 0.751 g / 100 mL, slightly higher than that of AO8. However, in the middle and late stages of fermentation, from 90 to 180 days, the accumulation rate of AO8 significantly increased and gradually surpassed that of AO8. At the end of fermentation at 180 days, the amino acid nitrogen content of AO8 reached 0.831 g / 100 mL, which was about 3.75% higher than that of the original strain 3.042. This indicates that the AO8 strain has a better ability to degrade proteins and generate amino acids in the middle and late stages of fermentation, and its amino acid nitrogen accumulation effect is significantly better than that of the original strain. At the same time, it shows that the increased activity of acidic protease can more efficiently degrade proteins in raw materials, promote the generation of peptides and free amino acids, and thus directly increase the accumulation level of amino acid nitrogen, which is beneficial to improving the umami flavor, enhancing the mellowness and quality stability of fermented soy sauce.

[0135] Example 7

[0136] Changes in free amino acid content after Aspergillus oryzae fermentation of soy sauce:

[0137] (1) Sample pretreatment

[0138] Accurately pipette 1 mL of soy sauce sample into a centrifuge tube, dilute appropriately, add 9 mL of 5% trichloroacetic acid solution, mix thoroughly, seal, and incubate at room temperature for acid extraction for 12 h, gently shaking once every 2 h to ensure complete extraction of free amino acids. After acid extraction, slowly adjust the pH of the sample solution to 7.2 with saturated sodium hydroxide solution, being careful to mix thoroughly to avoid localized excessive alkalinity affecting amino acid stability. Pipe 200 μL of the sample solution into a new centrifuge tube, add 20 μL of ortholeucine internal standard solution, 100 μL of triethylamine-acetonitrile solution, and 100 μL of PITC-acetonitrile solution sequentially, vortex to mix, seal, and incubate at room temperature for 1 h to complete the derivatization reaction. After derivatization, add 800 μL of n-hexane to the centrifuge tube, vortex for 20-30 s, let stand for 10 min, wait for the layers to separate, aspirate the lower derivatized solution, filter through a 0.22 μm organic phase filter membrane, take the filtrate, dilute it appropriately, and place it in a sample vial for later use, to be detected by high performance liquid chromatography.

[0139] (2) Chromatographic conditions and detection

[0140] High-performance liquid chromatography (HPLC) was used for qualitative and quantitative analysis of amino acid components in the samples. Specific detection conditions were as follows: the selected column was an Ultimate Amino Acid column with a particle size of 5 μm and column dimensions of 4.6 mm × 250 mm; the mobile phase consisted of two systems: the main mobile phase was a buffer solution composed of 0.1 mol / L sodium acetate solution (pH adjusted to 6.5) and acetonitrile at a volume ratio of 93:7, with 80% acetonitrile solution used as the auxiliary elution solvent; the injection volume was 10 μL; the elution flow rate was controlled at 1.0 mL / min, which balanced analysis time and peak broadening effect while ensuring separation efficiency; the column temperature was maintained at a constant 40℃ to reduce fluctuations in chromatographic retention time and improve the stability and reliability of the analytical results.

[0141] The composition and content of free amino acids during the koji-making and fermentation process of the Aspergillus oryzae AO8 strain and the starting strain Hu Niang 3.042 described in this invention were determined, and the results are as follows: Figure 8As shown in the figure, the total free amino acid content of Aspergillus oryzae AO8 described in this invention is 4.92% higher than that of the original strain Hu Niang 3.042, indicating a higher overall degree of protein hydrolysis. Specifically, the total umami amino acid content increased by 1.92%, with a significant increase in the core umami glutamic acid content (12.46%) and a slight decrease in aspartic acid (15.76%), resulting in a stable increase in overall umami amino acid levels. The contents of serine, glycine, threonine, and alanine, all sweet amino acids, were significantly increased, with the total sweet amino acid content increasing by 15.56%, effectively softening the flavor of the soy sauce and enhancing its sweetness and palatability. The internal components of bitter amino acids were optimized and adjusted, with an overall decrease of 6.83%, a gradual increase without obvious bitterness or off-flavors, while maintaining the full-bodied texture of the soy sauce. The cysteine ​​content reached 2.27 mg / ml, significantly increasing the content of aroma precursors. These results indicate that strain AO8 can efficiently degrade raw material proteins, promote the targeted generation and accumulation of flavor amino acids, optimize amino acid composition, and significantly improve the flavor harmony and palatability of soy sauce.

[0142] The Aspergillus oryzae AO8 strain of this invention was obtained by mutagenesis screening of strain 3.042 of Hu Niang using laboratory ambient pressure room temperature plasma (ARTP) mutagenesis technology. Its spores have regular morphology and a higher spore quantity. During the koji-making process, the activities of saccharifying enzymes and proteases are synergistically enhanced, which can efficiently hydrolyze starch and protein, significantly improving raw material utilization and soy sauce fermentation quality.

[0143] Compared with the original strain and existing technologies, the Aspergillus oryzae AO8 described in this invention mainly exhibits the following three changes: First, in terms of saccharifying enzyme activity, the saccharifying enzyme activity of AO8 in soy sauce koji making is 1479 U / g, which is 31% higher than that of the original strain Hu Niang 3.042. Compared with the 1352 U / g reported in the existing patent CN 107828666 A, which is also a UV-mutated Aspergillus oryzae ZA160 of Hu Niang 3.042, it is about 9.40% higher. This enzyme activity level helps to improve the saccharification efficiency of starchy raw materials in soy sauce koji making, providing relatively sufficient substrate for subsequent fermentation. Secondly, regarding the synergistic changes in enzyme activity, the acidic protease activity of AO8 was 1213 U / g, an increase of 18.92% compared to the original strain. This indicates a simultaneous increase in both saccharifying enzyme activity and protease activity. As a reference, another Aspergillus oryzae strain, A22, obtained during the same period through ARTP mutagenesis, had an acidic protease activity of 1207 U / g, an increase of 9.71% compared to the original strain, but its saccharifying enzyme activity was 989 U / g, lower than the original strain, failing to show a simultaneous increase in both saccharifying enzyme and protease activities. The increased activity of both enzymes in AO8 is beneficial for the simultaneous degradation of both protein and starchy raw materials in soy sauce brewing. Thirdly, regarding spore morphology and quantity, under an optical microscope, AO8 spores were observed to be plump with a relatively concentrated particle size distribution, and the spore count reached 9.876 billion / g, an increase of approximately 16.13% compared to the original strain. This higher spore count and better morphology are beneficial for improving germination uniformity and the evenness of mycelial coverage in the soy sauce koji preparation and inoculation stages.

[0144] In the fermentation system of the AO8 mutagenized strain of this invention, the D-anhydrous glucose content reached 4.02 mg / mL, accounting for approximately 16.1% of the total sugar. Its glucose production was significantly higher than that of the original strain 3.042, with a slightly increased proportion. Furthermore, the D-anhydrous glucose content was 2.51 times that of the original strain 3.042, indicating a significantly superior glucose production capacity. In the AO8 mutagenized strain fermentation system, the amino acid nitrogen content increased by 3.75% compared to the original strain 3.042. The free amino acid content in the AO8 mutagenized strain fermentation system was 50.69 mg / mL, an increase of 4.52% compared to 3.042, with the sweet amino acid content increasing by 15.56% and the core fresh glutamic acid content increasing by 12.46%. The increase in the total free amino acid content and the proportion of sweet amino acids effectively improved the flavor profile of the fermentation system, enhanced the sweet and savory flavor of the fermented product, and demonstrated excellent fermentation potential and industrial value.

[0145] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. Aspergillus oryzae AO8 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 20, 2026. The proposed classification name is Aspergillus oryzae, and the accession number is CGMCC No. 42436.

2. The application of Aspergillus oryzae as described in claim 1 in soy sauce fermentation.

3. The application as described in claim 2, characterized in that: include, The *Aspergillus oryzae* strain is inoculated during the koji preparation process to obtain the koji. Then, koji (a type of starter culture) is prepared and soy sauce is produced through brine fermentation. Among them, Aspergillus oryzae has the effect of simultaneously enhancing the activity of saccharifying enzymes and proteases during the koji-making process.

4. The application as described in claim 3, characterized in that: The Aspergillus oryzae in the soy sauce fermentation system increases the total amount of free amino acids and the proportion of umami amino acids, thereby enhancing the umami intensity and richness of the product.

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