A compound koji inoculated with indigenous aspergillus oryzae and aspergillus niger and a method for strengthening shanxi old mature vinegar

Abstract

The application belongs to the technical field of vinegar brewing, and provides a compound koji inoculated with indigenous Aspergillus oryzae and Aspergillus niger and a method for strengthening Shanxi old mature vinegar. Indigenous Aspergillus oryzae CGMCC 42103 with high yield of acid protease and glutaminease and Aspergillus niger CGMCC 15672 with high yield of saccharifying enzyme are mixed and inoculated in bran and soybean powder medium to prepare compound koji, and the prepared compound koji is applied to the fermentation process of Shanxi old mature vinegar to prepare new vinegar, the total acid, amino acid nitrogen, non-volatile acid and glutamic acid content of the new vinegar are 6.18 g / 100 mL, 0.38 g / 100 mL, 2.45 g / 100 mL and 58.83 mg / 100 mL respectively, compared with the comparative example in which only highland millet with a mass fraction of 60% is added in the alcohol fermentation stage to carry out conventional fermentation of food vinegar, the new vinegar is improved by 20.47%, 153.33%, 33.15% and 118.86% respectively, and the content of organic acid and volatile aroma components is enriched.

Description

Technical Field

[0001] This invention belongs to the field of vinegar brewing technology, specifically relating to a compound koji inoculated with a mixture of indigenous Aspergillus oryzae and Aspergillus niger and its method for strengthening Shanxi aged vinegar. Background Technology

[0002] The yeast starter is the backbone of wine, and the yeast starter is the backbone of vinegar. As the premier vinegar among the four famous types of vinegar, Shanxi aged vinegar's unique flavor of "sour, mellow, sweet, fragrant, and fresh" is inseparable from the traditional processes of "steaming, fermentation, smoking, leaching, and aging," but even more so from its core saccharifying agent—daqu (a type of yeast starter). Daqu plays a crucial role in the brewing of Shanxi aged vinegar, performing saccharification, fermentation, and aroma-generating functions. Not only is it used in large quantities, but its quality directly determines the flavor profile and physicochemical properties of the finished vinegar.

[0003] However, traditional Daqu (a type of starter culture) has many limitations: the production process is cumbersome, involves a high proportion of manual labor, is labor-intensive, has a long production cycle, and its output is limited by climate, making it unable to meet the supply needs of large-scale production by enterprises. Daqu relies on natural inoculation to form a microbial community, and the composition of the community is easily affected by fluctuations in the Daqu production environment (temperature and humidity), resulting in poor stability of the activity of key enzymes such as saccharifying enzymes and proteases in different batches of Daqu, leading to fluctuations in the flavor and quality of the finished vinegar and hindering standardized production. At the same time, the activity of key enzymes such as glutaminase and β-glucosidase in traditional Daqu is generally low, which makes it difficult to fully decompose the proteins in the raw materials to generate umami amino acids, and also makes it difficult to efficiently convert alcohols and aldehydes into esters, ultimately resulting in insufficient umami flavor and a single aroma in the finished vinegar, making it difficult to fully showcase the traditional flavor characteristics of Shanxi aged vinegar.

[0004] To improve production efficiency, most vinegar factories use a single strain of yeast to prepare bran koji and apply it to vinegar brewing. Among them, the invention patent CN104312895B, "A Buckwheat Vinegar and Its Preparation Method", uses buckwheat and corn as the main ingredients and produces buckwheat vinegar through biological fermentation. The bran koji used is prepared by a single yeast inoculation, and the enzyme and bacterial systems are limited, which cannot solve the problems of unstable enzyme activity and insufficient flavor of traditional koji. The invention patent CN1467286A, "Solid-state Bran Koji Fermented Vinegar and Its Manufacturing Method", uses bran koji containing broken wheat flakes as a saccharifying agent, combined with rice and other raw materials, and shortens the production cycle through fermentation with compound acetic acid bacteria. However, this technology does not involve the screening and optimization of key enzyme-producing strains. The bran koji used relies on black koji for inoculation, which also suffers from the defect of a limited enzyme system. In addition, it lacks key enzyme systems such as acidic protease and glutaminase to enhance freshness and aroma, ultimately resulting in a product with thin and poor flavor components. Summary of the Invention

[0005] This invention provides a method for enhancing Shanxi aged vinegar using a compound koji inoculated with a mixture of Aspergillus oryzae and Aspergillus niger. A superior native Aspergillus oryzae strain, CGMCC 42103, producing a high amount of acidic protease-glutaminase, was screened from Shanxi aged vinegar koji. This strain was then mixed with another strain from Shanxi aged vinegar koji, Aspergillus niger CGMCC15672, which produces a high amount of saccharifying enzyme, and inoculated onto a wheat bran and soybean flour culture medium to prepare the compound koji. This compound koji was then applied in a two-stage enhancement process during the alcoholic and acetic acid fermentation stages of Shanxi aged vinegar. After fermentation, the physicochemical properties, flavor, and production efficiency of the product were systematically tested to clarify the enhancing effect of the compound koji inoculated with the mixed Aspergillus oryzae and Aspergillus niger in the brewing process of Shanxi aged vinegar.

[0006] This invention is achieved by the following technical solution: a method for preparing a compound koji inoculated with a mixture of indigenous Aspergillus oryzae and Aspergillus niger. The method involves mixing Aspergillus oryzae CGMCC42103 koji, a native koji with high production of acidic protease and glutaminase isolated from Shanxi aged vinegar koji, with Aspergillus niger CGMCC15672 koji, also isolated from Shanxi aged vinegar koji, at a mass ratio of 1:1 to prepare the koji starter. Wheat bran and soybean flour ground to 20 mesh are mixed evenly at a mass ratio of 9:1. Water, at 0.5 times the dry weight of the culture medium, is added to prepare a wheat bran and soybean flour culture medium. The koji starter is inoculated at 0.4% w / w into the wheat bran and soybean flour culture medium. The medium is then spread evenly on a koji tray at 30℃, with a koji thickness of 5–6 cm. Humidity is controlled at ≥95% throughout the process. The medium is cultured for 12–16 h. When mycelial growth, clumping, and the temperature of the koji reaches 38℃, the koji is turned and cooled to 28–32℃ for further culture for 6–8 hours. h, when the temperature of the koji material rises to 38℃ again, the koji is turned over for the second time; then the koji is cultured at 28℃ for 42-48 h to produce the koji, and dried at a low temperature of 40-45℃ until the moisture content is ≤12%, which is the compound koji; The aforementioned Aspergillus oryzae ( Aspergillus oryzae CGMCC 42103 is a strain isolated and screened from Shanxi aged vinegar koji (a type of starter culture) that exhibits high acid protease-glutaminase activity. After culturing in barley and pea medium for 3 days, its acid protease activity and glutaminase activity were 630.25 U / g and 7.03 U / g, respectively. It also possesses amylase, β-glucosidase, aminopeptidase, cellulase, and xylanase. It is deposited at the China General Microbiological Culture Collection Center on June 16, 2025. The Aspergillus niger CGMCC 15672 is the strain described in the invention application number 201910136401.2, entitled "Method for producing Shanxi aged vinegar using fast koji made from superior Aspergillus niger, Rhizopus, and yeast and its combination with daqu". This strain has high saccharifying enzyme activity, and after being cultured in barley and pea medium for 8 days, its saccharifying enzyme activity is 2549.66 U / g.

[0007] The preparation method of the Aspergillus oryzae seed koji is as follows: the concentration is 10 7 ~10 8 Add 4 mL of CFU / mL Aspergillus oryzae CGMCC 42103 spore suspension to 30 g wheat bran and soybean powder culture medium, incubate at 30℃, knock the bottle every 12 h, and after 72 h of incubation, dry at 40℃ for 3 h to obtain Aspergillus oryzae inoculum. The preparation method of the Aspergillus niger inoculum: concentration is 10 7 ~10 8 Add 4 mL of CFU / mL Aspergillus niger CGMCC 15672 spore suspension to 30 g wheat bran medium and incubate at 30℃. Knock the bottle every 12 h. After 72 h of incubation, dry at 40℃ for 3 h to obtain Aspergillus niger inoculum. The preparation method of the bran and soybean powder culture medium is as follows: soybeans are crushed into 20 mesh, bran and soybean powder are mixed evenly at a mass ratio of 9:1, water is added at 0.5 times the dry weight of the culture medium, and the mixture is sterilized at 0.1 MPa and 121℃ for 30 min. After sterilization, the temperature is lowered to 40℃. The method for preparing the bran culture medium is as follows: mix bran and water at a mass-to-volume ratio of 1:1.5, sterilize at 0.1 MPa and 121°C for 30 min, and then allow the raw materials to cool down to 40°C after sterilization.

[0008] The present invention also provides a composite culture prepared by mixing Aspergillus oryzae and Aspergillus niger according to the above method.

[0009] This invention also provides a method for strengthening Shanxi aged vinegar using a compound koji inoculated with a mixture of the indigenous Aspergillus oryzae and Aspergillus niger, comprising the following steps: Sorghum is crushed to 50-60 mesh. Sorghum and water are mixed at a mass-to-volume ratio of 1:4.5-5. The mixture is heated to 90-95℃, and 0.1% (by weight) of heat-resistant α-amylase from the sorghum is added. The mixture is kept at this temperature for 80-90 minutes, then cooled to 60℃. 0.1% (by weight) of saccharifying enzyme from the sorghum is added, and the mixture is kept at this temperature for 20-30 minutes. When the temperature drops to 28-32℃, the mixture is placed in an alcohol fermentation tank. 30-40% (by weight) of Daqu (a type of starter culture) and 10-20% (by weight) of a compound starter culture inoculated with a mixture of Aspergillus oryzae and Aspergillus niger are added. 0.1-0.3% (by weight) of yeast inoculum from the sorghum is added. The mixture is fermented openly for 2 days, then sealed for 6-14 days until the alcohol content reaches 8-9%. %vol, after the alcoholic fermentation is completed, the mash is obtained; add 120%–140% wheat bran, 70%–80% rice bran, 60%–70% rice husk and 10%–20% of a compound koji inoculated with native Aspergillus oryzae and Aspergillus niger, mix thoroughly, and transfer to solid-state acetic acid fermentation for 12–15 days. At the same time, add 10% of fire mash for acetic acid fermentation. When the total acid is 5–6 g / 100 mL, the acetic acid fermentation is completed. After smoking the mash and leaching the vinegar, new vinegar is obtained.

[0010] The total acid, amino acid nitrogen, non-volatile acid, and glutamic acid content of the new leached vinegar prepared by this invention are 6.18 g / 100 mL, 0.38 g / 100 mL, 2.45 g / 100 mL, and 58.83 mg / 100 mL, respectively. Compared with the control group, which only added 60% sorghum Daqu (a type of starter culture) during the alcoholic fermentation stage for conventional vinegar fermentation, the content of these components was increased by 20.47%, 153.33%, 33.15%, and 118.86%, respectively. The content of organic acids and volatile aroma components was also enriched.

[0011] This invention screened a superior indigenous Aspergillus oryzae strain, CGMCC 42103, from Shanxi aged vinegar koji (a type of starter culture), which is a high-yield producer of acidic protease-glutaminase. Compared to the commonly used Aspergillus oryzae strain AS 3.042, this strain has a richer protease system, containing abundant acidic protease in addition to neutral protease. It can adapt to the acidic environment of the acetic acid fermentation stage of Shanxi aged vinegar and enhance the umami flavor of the product through the combined action of glutaminase. This Aspergillus oryzae strain CGMCC 42103 was mixed with another high-yield saccharifying enzyme Aspergillus niger strain CGMCC 15672, also from Shanxi aged vinegar koji, and inoculated on a wheat bran and soybean flour culture medium to prepare a compound koji. This compound koji was then applied in a two-stage enhanced process during the alcoholic fermentation and acetic acid fermentation stages of Shanxi aged vinegar. This not only improved the fermentation efficiency and total acid content but also significantly increased the amino acid nitrogen content in the vinegar, especially the glutamic acid content, enriching the product flavor and enhancing its market competitiveness.

[0012] Compared with the prior art, the present invention has the following significant advantages: (1) The Aspergillus oryzae CGMCC 42103 and Aspergillus niger CGMCC 15672 used in this invention are both excellent native strains isolated from Shanxi aged vinegar Daqu, which have better adaptability to raw materials. Among them, Aspergillus oryzae CGMCC 42103 is rich in protease system, and contains abundant acidic protease in addition to neutral protease. It can adapt to the acidic environment of the acetic acid fermentation stage of Shanxi aged vinegar and can enhance the umami flavor of the product through the complex action of glutaminase. Aspergillus niger CGMCC 15672 has the characteristic of high production of saccharifying enzyme, which helps to improve fermentation efficiency.

[0013] (2) This invention uses a dual-strain synergistic approach to prepare a composite koji inoculated with a mixture of indigenous Aspergillus oryzae and Aspergillus niger. Soybeans are added to the matrix of this composite koji to further enrich the protein substrate source and provide a guarantee for the subsequent amino acid generation. At the same time, the synergistic effect of the two strains achieves the complementarity and enhancement of the enzyme system. The prepared composite koji not only has rich neutral protease activity, acid protease activity, amylase activity, saccharifying enzyme activity, and glutaminase activity, but also has enzyme systems such as β-glucosidase, aminopeptidase, and xylanase. The rich enzyme system plays a crucial role in improving the nutritional, functional and flavor quality of vinegar.

[0014] (3) The present invention applies the compound koji dual-stage enhancement to the alcohol fermentation stage and the acetic acid fermentation stage of Shanxi aged vinegar. Compared with the comparative example, which only adds 60% of sorghum koji to the alcohol fermentation stage for conventional vinegar fermentation, it significantly improves production efficiency. The total acid, amino acid nitrogen, non-volatile acid and glutamic acid content in the new vinegar are increased by 20.47%, 153.33%, 33.15% and 118.86% respectively, while enriching the types and contents of volatile aroma substances.

[0015] The Aspergillus oryzae described in this invention ( Aspergillus oryzae QM81 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 42103 and deposit date of June 16, 2025. Attached Figure Description

[0016] Figure 1 Results of protease and glutaminase activity assays for 11 Aspergillus oryzae strains; Figure 2 This image shows the colony morphology of Aspergillus oryzae strain QM81 on a PDA plate. Figure 3 This is an ITS sequencing phylogenetic tree diagram of Aspergillus oryzae strain QM81. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0019] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are standard laboratory instruments and equipment. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. The AS 3.042 Aspergillus oryzae strain used in the following examples was purchased from Wuhan Huana Biotechnology Co., Ltd. The Shanxi aged vinegar starter used in the following examples was from Shanxi Fuyuanchang Aged Vinegar Co., Ltd. The glutaminase activity assay kit used in the following examples was purchased from Beijing Solarbio Technology Co., Ltd. The thermostable α-amylase and saccharifying enzyme used in the following examples were purchased from Shandong Longket Enzyme Preparation Co., Ltd. The Angel Yeast inoculum used in the following examples was purchased from Angel Yeast Co., Ltd. The fungal DNA extraction kit used in the following examples was purchased from Beijing Solarbio Technology Co., Ltd.

[0021] Example 1: Screening, Identification and Preservation of Superior Aspergillus oryzae Strains (1) Screening of superior Aspergillus oryzae strains: Ten Aspergillus oryzae strains isolated from Shanxi aged vinegar koji were selected in the laboratory. The commercially available Hu Niang AS 3.042 strain was used as the control group. The enzyme activities of acidic protease, neutral protease, glutaminase, amylase, saccharifying enzyme, aminopeptidase, cellulase, xylanase and β-glucosidase were measured. The specific measurement methods are as follows: Screening of Aspergillus oryzae strains: Aspergillus oryzae strains were activated by slant agar inoculation, and spores were scraped off using an inoculation spatula and placed in sterile physiological saline to prepare 10... 8CFU / mL Aspergillus oryzae spore suspension was transferred to a 4 mL Erlenmeyer flask containing barley-pea medium. After shaking, the flask was incubated at 30°C for 72 h, shaking once every 12 h. The above barley-pea medium was prepared as follows: barley and peas were crushed into granules and mixed evenly at a mass ratio of 7:3. Water was added to the mixture at a mass-volume ratio of 1:1 and mixed well. 30 g of the mixture was placed into a 250 mL Erlenmeyer flask and sterilized at 121°C for 20 min.

[0022] Crude enzyme extraction: Accurately weigh 2.50 g of the cultured Aspergillus oryzae strain solid culture in an Erlenmeyer flask and immerse it in 50 mL of the corresponding buffer solution. Incubate at 150 pm and 40℃ with constant temperature and shaking for 60 min. Pass the crude enzyme solution through a 200-mesh double-layer filter cloth and dilute to 250 mL in a volumetric flask with the corresponding buffer solution for later use.

[0023] Methods for determining enzyme activity: Protease activity assay: Protease activity was determined by ultraviolet spectrophotometry according to GB / T 23527.1—2023 "Quality requirements for enzyme preparations Part 1: Protease preparations". Neutral protease activity was determined using sodium phosphate buffer at pH 7.2, and acidic protease activity was determined using lactate-sodium lactate buffer at pH 3.0.

[0024] Glutaminase activity assay: A glutaminase activity assay kit was used. Glutaminase is a key functional enzyme for enhancing the flavor of vinegar. Its activity determines the conversion efficiency of glutamine into glutamic acid, a flavor substance, while also increasing the amino acid nitrogen content of vinegar, ensuring that the finished vinegar meets the standards for flavor and physicochemical indicators.

[0025] Amylase activity assay: Extraction, filtration, and volume adjustment were performed using phosphate-citrate buffer at pH 6.0. Under constant temperature water bath conditions of 60℃, amylase extract, soluble starch solution, and buffer were reacted accurately for 5 min. The reaction was then terminated by adding 0.5 mL of 0.1 mol / L HCl, followed by adding 2.5 mL of dilute iodine solution and vortexing. The absorbance was then measured at 660 nm.

[0026] Saccharifying enzyme activity assay: Extraction was performed using an acetate-sodium acetate buffer solution at pH 4.6, followed by filtration and volume adjustment. Under constant temperature water bath conditions of 40℃, the saccharifying enzyme extract was accurately reacted with a soluble starch solution for 30 min. NaOH was then added to terminate the reaction. The terminated solution was vortexed with DNS, and the reaction was terminated by boiling in a water bath for 5 min. The absorbance was measured at 540 nm.

[0027] Aminopeptidase activity assay: Extraction was performed using a Trimethylolamine buffer solution at pH 8.0, followed by filtration and volume adjustment. At a constant temperature of 40℃, 90 μL of the aminopeptidase extract was reacted with 10 μL of leucine-p-nitroaniline solution in a 96-well plate for 10 min. The reaction was then terminated with 100 μL of anhydrous ethanol, and the result was measured at 405 nm using a microplate reader. Aminopeptidase hydrolyzes peptides to generate free amino acids, enriching the variety of amino acids in vinegar and increasing the amino acid nitrogen content. This provides precursors for the formation of umami and flavor compounds, ensuring the umami flavor profile and physicochemical quality of the finished vinegar.

[0028] Cellulase and xylanase activity determination: Cellulase activity was determined according to GB / T 23881—2009 "Determination of Cellulase Activity in Feed - Filter Paper Method"; xylanase activity was determined according to GB / T 23874—2009 "Determination of Xylanase Activity in Feed Additives - Spectrophotometric Method". Cellulase and xylanase synergistically hydrolyze cellulose, xylan and other polysaccharides in vinegar brewing raw materials into fermentable sugars, thereby improving raw material utilization and fermentation efficiency.

[0029] β-glucosidase activity assay: Extraction was performed using an acetate-sodium acetate buffer solution at pH 4.6, followed by filtration and volume adjustment. Under constant temperature water bath conditions at 50℃, 30 μL of the glucosidase extract was accurately reacted with 270 μL of 0.15% p-Nitrophenol-Glucoside (pNPG) solution for 10 min. Then, 600 μL of Na2CO3 was added to terminate the reaction. The absorbance was measured at 410 nm using an enzyme-linked immunosorbent assay (ELISA) reader. β-glucosidase can hydrolyze bound glycosides and flavor precursors in vinegar brewing raw materials, releasing free aromatic substances, enriching the aroma layers of the finished vinegar. Simultaneously, it assists in the degradation of carbohydrates to generate fermentable sugars, improving the utilization rate of raw materials and fermentation efficiency.

[0030] The enzyme activity assay results for different Aspergillus oryzae strains were as follows: Figure 1 As shown in Table 1, Aspergillus oryzae strain QM81 exhibits the strongest enzyme production capacity, producing high levels of acidic protease and glutaminase. Its acidic protease activity and glutaminase activity reach 630.25 U / g and 7.03 U / g, respectively, which are 19.96% and 48.00% higher than those of commercially available Hu Niang AS 3.042. It also possesses a rich enzyme system including amylase, saccharifying enzyme, aminopeptidase, cellulase, β-glucosidase, pectinase, and xylanase.

[0031] Table 1. Results of enzyme activity assays for different Aspergillus oryzae strains (2) Identification of superior Aspergillus oryzae strains Morphological identification: Aspergillus oryzae QM81 spores were picked up with an inoculation loop and spot-inoculated onto PDA agar plates. The colonies growing on the plates were photographed and recorded. The colonies of Aspergillus oryzae QM81 on PDA agar plates are shown below. Figure 2 The colonies of Aspergillus oryzae QM81 on PDA medium are greenish-yellow in the middle, white at the edge, loose on the surface, and flocculent in texture.

[0032] ITS sequencing: After the strain was cultured on an slant, mycelia were scraped, frozen, and ground. DNA was extracted using a kit for ITS sequencing and strain identification. Primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used. Homology comparison was performed using BLAST software, and an ITS sequencing phylogenetic tree was constructed. The ITS sequencing phylogenetic tree of Aspergillus oryzae strain QM81 is shown below. Figure 3 QM81 strain and Aspergillus oryzae ( Aspergillus oryzae CBD-OH-S1 is highly homologous to Aspergillus flavus (CBD-OH-S1). Aspergillus flavus Glinf027 also showed high homology, indicating that ITS sequence alignment alone cannot definitively distinguish strain QM81 as Aspergillus oryzae. This strain was isolated from brewing koji and its morphological characteristics matched those of Aspergillus oryzae; combining morphological observation and phylogenetic analysis, strain QM81 was identified as Aspergillus oryzae (…). Aspergillus oryzae It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 42103 and deposit date of June 16, 2025.

[0033] Example 2: Preparation of a composite starter culture inoculated with a mixture of indigenous Aspergillus oryzae and Aspergillus niger. Preparation method of compound koji inoculated with native Aspergillus oryzae and Aspergillus niger: Aspergillus oryzae CGMCC 42103 koji, a native koji with high production of acidic protease and glutaminase isolated from Shanxi aged vinegar koji, and Aspergillus niger CGMCC 15672 koji, a koji with high production of saccharifying enzyme isolated from Shanxi aged vinegar koji, are mixed evenly at a mass ratio of 1:1 to form the koji starter. Wheat bran and soybean flour ground to 20 mesh are mixed evenly at a mass ratio of 9:1. Water is added at 0.5 times the dry weight of the culture medium to prepare a wheat bran and soybean flour culture medium. The koji starter is inoculated into the wheat bran and soybean flour culture medium at 0.4% of the total dry weight of the culture medium. The medium is then spread evenly on koji trays at 30℃, with a koji thickness of 5-6 cm. The humidity is controlled at ≥95% throughout the process. The culture time is 12-16 hours. When mycelial growth, clumping, and the temperature of the koji reaches 38℃, the koji is turned over and cooled to 28-32℃ for another 6-8 hours. h, when the temperature of the koji material rises to 38℃ again, the koji is turned over for the second time; then the koji is cultured at 28℃ for 42-48 h to produce the koji, and dried at a low temperature of 40-45℃ until the moisture content is ≤12%, which is the compound koji; The Aspergillus oryzae CGMCC 42103 described above was isolated and screened from Shanxi aged vinegar koji. It has high acid protease-glutaminase activity. After being cultured in barley and pea medium for 3 days, its acid protease activity and glutaminase activity were 630.25 U / g and 7.03 U / g, respectively. It also has amylase, β-glucosidase, aminopeptidase, cellulase and xylanase. It is deposited at the China General Microbiological Culture Collection Center on June 16, 2025. The Aspergillus niger CGMCC 15672 is the strain described in the invention application number 201910136401.2, entitled "Method for producing Shanxi aged vinegar using fast koji made from superior Aspergillus niger, Rhizopus, and yeast and its combination with daqu". This strain has high saccharifying enzyme activity, and after being cultured in barley and pea medium for 8 days, its saccharifying enzyme activity is 2549.66 U / g.

[0034] The preparation method of the Aspergillus oryzae seed koji is as follows: the concentration is 10 8 Add 4 mL of CFU / mL Aspergillus oryzae CGMCC 42103 spore suspension to 30 g wheat bran and soybean powder culture medium, incubate at 30℃, knock the bottle every 12 h, and after 72 h of incubation, dry at 40℃ for 3 h to obtain Aspergillus oryzae inoculum. The preparation method of the Aspergillus niger inoculum: concentration is 10 8 Add 4 mL of CFU / mL Aspergillus niger CGMCC 15672 spore suspension to 30 g wheat bran medium and incubate at 30℃. Knock the bottle every 12 h. After 72 h of incubation, dry at 40℃ for 3 h to obtain Aspergillus niger inoculum. The preparation method of the bran and soybean powder culture medium is as follows: soybeans are crushed into 20 mesh, bran and soybean powder are mixed evenly at a mass ratio of 9:1, water is added at 0.5 times the dry weight of the culture medium, and the mixture is sterilized at 0.1 MPa and 121℃ for 30 min. After sterilization, the temperature is lowered to 40℃. The method for preparing the bran culture medium is as follows: mix bran and water at a mass-to-volume ratio of 1:1.5, sterilize at 0.1 MPa and 121°C for 30 min, and then allow the raw materials to cool down to 40°C after sterilization.

[0035] Example 3: Preparation of pure culture koji from indigenous Aspergillus oryzae Preparation method of pure Aspergillus oryzae koji: Inoculate 0.4% of the total mass of the mixed dry materials with wheat bran and soybean flour culture medium, mix thoroughly, and spread evenly on koji trays at 30℃, with a koji thickness of 5-6 cm. Incubate for 12-16 hours. When mycelial growth and clumping are normal and the koji temperature reaches 38℃, perform the first turning of the koji to ensure the temperature drops to around 30℃. Maintain humidity above 95% during the incubation process. After the first turning, continue incubation for approximately 6-8 hours. When the koji temperature further rises to 38℃, perform the second turning. Then, maintain the temperature at 28℃ for 48 hours to produce the koji. Dry at 40℃ until the moisture content is ≤12%.

[0036] Example 4: Preparation of pure culture koji from indigenous Aspergillus niger Preparation method of pure Aspergillus niger koji: Mix 0.4% (by weight of wheat bran) of Aspergillus niger koji into the wheat bran culture medium, mix thoroughly, and spread evenly on a koji tray at 30℃, with a koji thickness of 5-6 cm. Incubate for 12-16 hours. When mycelial growth and clumping are normal, and the koji temperature reaches 38℃, perform the first turning of the koji to ensure the temperature drops to around 30℃. Maintain humidity above 95% during the incubation process. After the first turning, continue incubation for approximately 6-8 hours. When the koji temperature further rises to 38℃, perform the second turning. Then, maintain the temperature at 28℃ for further incubation for 48 hours to produce the koji. Dry at 40℃ until the moisture content is ≤12%.

[0037] Example 5 Preparation of indigenous Aspergillus oryzae-Aspergillus niger mixed koji: The indigenous Aspergillus oryzae pure koji and indigenous Aspergillus niger pure koji prepared in Examples 3 and 4 respectively were mixed in a mass ratio of 1:1 to obtain indigenous Aspergillus oryzae-Aspergillus niger mixed koji.

[0038] Compared to Example 3, Example 5 showed increases in acidic protease activity, glutaminase activity, amylase activity, and cellulase activity of 94.81%, 15.51%, 26.83%, and 454.18%, respectively. Compared to Example 4, neutral protease and amylase activities increased by 61.16% and 16.85%, respectively, indicating that the Aspergillus oryzae-Aspergillus niger mixed koji prepared by mixing two pure koji strains can achieve enzyme complementarity to a certain extent, thereby improving the overall enzyme activity level. Compared to the mixed koji of Example 5, the composite koji of Example 2 showed increases in acidic protease, glutaminase, amylase, cellulase, β-glucosidase activity, and xylanase activity of 42.65%, 23.33%, 24.30%, 84.55%, 72.80%, and 25.35%, respectively. This significant improvement is due to the metabolic interaction and synergistic effect formed between Aspergillus oryzae and Aspergillus niger during growth, rather than a simple enzyme superposition, which stimulated the enzyme production potential of the strains.

[0039] Table 2. Results of enzyme activity assays for purebred, mixed, and compound koji. Example 6: Enhanced application of compound koji inoculated with native Aspergillus oryzae and Aspergillus niger in the brewing process of Shanxi aged vinegar (30% by weight of sorghum koji and 10% by weight of compound koji inoculated with native Aspergillus oryzae and Aspergillus niger were added during the alcoholic fermentation stage; 10% by weight of compound koji inoculated with native Aspergillus oryzae and Aspergillus niger was added during the acetic acid fermentation stage). Crush sorghum to 50-60 mesh. Mix sorghum and water at a mass-to-volume ratio of 1:4.5. Heat the mixture to 90-95°C, add 0.1% (by mass) of heat-resistant α-amylase from the sorghum, and maintain the temperature for 80-90 minutes. Cool the mixture to 60°C, then add 0.1% (by mass) of saccharifying enzyme from the sorghum, and maintain the temperature for 20-30 minutes. After the temperature drops to 28-32℃, pump the mixture into an alcohol fermentation tank, add 30% sorghum-based Daqu (a type of starter culture) and 10% compound Daqu (a mixture of Aspergillus oryzae and Aspergillus niger), and 0.1% sorghum-based yeast. Ferment openly for 2 days, then seal and ferment for 6-14 days until the alcohol content reaches 8-9% vol. End the alcohol fermentation to obtain the mash. Add 120% sorghum bran, 80% rice bran, 60% rice husk, and 10% compound Daqu (a mixture of Aspergillus oryzae and Aspergillus niger) and mix thoroughly. Transfer to solid-state acetic acid fermentation for 12-15 days, simultaneously adding 10% sorghum-based Huomai (a type of fermented mash) for acetic acid fermentation. When the total acid content reaches 5-6 g / 100 mL, end the acetic acid fermentation. After fumigation and leaching, obtain fresh leached vinegar. Comparative Example 1: Daqu (60% by weight of sorghum) was added only during the alcoholic fermentation stage, and the rest of the process was the same as in Example 6.

[0040] Comparative Example 2: During the alcoholic fermentation stage, 30% of the sorghum mass fraction of Daqu and 20% of commercial bran Daqu (prepared using pure Aspergillus niger) were added, and the remaining process operations were the same as in Example 6.

[0041] The specific measurement method is as follows: 1. Physicochemical indicators: Total acidity was determined according to GB / T 5009.41—2003; non-volatile acidity was determined according to the single-boiling distillation method in GB 18187-2000 "Brewing Vinegar"; total esters were determined by the sodium hydroxide saponification method (referring to GB 19777-2005); reducing sugars were determined according to GB / T 5009.7—2003; amino acid nitrogen, total soluble solids, total flavonoids, and ligustrazine content were all determined according to GB / T 19777-2013; clarity was determined using a portable turbidimeter.

[0042] 2. Organic Acids: The organic acid content of the fresh leached vinegar sample was determined by HPLC. The chromatographic conditions were as follows: Welch Ultimate AQ-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: V (0.1% phosphoric acid aqueous solution, pH 2.6):V (methanol) = 97:3, isocratic elution; flow rate: 0.5 mL / min; column temperature: 40°C; detection wavelength: 210 nm; injection volume: 20 μL.

[0043] 3. Amino acid detection method: Take 2 mL of fresh acetic acid sample, add 30 mL of anhydrous ethanol, vortex for 2 min, then make up to 50 mL with ultrapure water, freeze at -20°C for 30 min, centrifuge at 10,000 r / min for 5 min, then take 1 mL of supernatant and pass it through a membrane for analysis using a fully automated amino acid analyzer.

[0044] 4. Volatile aroma compounds: Separation was performed using a DB-FFAP polar capillary column (30 m × 0.25 mm × 0.25 μm) with high-purity helium (≥99.999%) as the carrier gas at a flow rate of 1 mL / min. The injection system was manual, with an injection volume of 1 μL and a non-split flow program. The column temperature gradient was set as follows: initial temperature of 40 °C for 5 min, followed by a programmed increase to 240 °C at a rate of 10 °C / min and a hold for 2 min. The mass spectrometry detection system parameters were set as follows: conduction line temperature 280 °C, ion source temperature 230 °C, electron impact ionization (EI) mode, electron energy 70 eV, and mass scan range 20–350 amu.

[0045] As shown in Table 3, Example 6 had the highest levels of total acid, non-volatile acid, amino acid nitrogen, total ester, reducing sugar, and soluble solids among the three groups, increasing by 20.47%, 33.15%, 153.33%, 35.09%, 61.49%, and 22.31% respectively compared to Comparative Example 1, and by 8.80%, 8.89%, 46.15%, 25.59%, 9.24%, and 5.10% respectively compared to Comparative Example 2. It also had the lowest turbidity, decreasing by 53.83% and 35.43% compared to Comparative Examples 1 and 2, respectively. The synergistic metabolism of the compound koji inoculated with a mixture of native Aspergillus oryzae and Aspergillus niger can efficiently degrade proteins and polysaccharides, enriching flavor precursors. The dual-stage enhancement supplements a complete enzyme system, overcoming the limitations of traditional single-enzyme systems and improving raw material utilization and fermentation efficiency. The interaction of strains inhibits contaminating bacteria, effectively reducing turbidity. In contrast, Comparative Example 2 only contained pure Aspergillus niger commercial bran koji, lacking the Aspergillus oryzae flavor-enhancing enzyme system, resulting in a significantly weaker effect.

[0046] Table 3. Effects of Compound Intensification Application on Physicochemical Properties of Newly Added Vinegar As shown in Table 4, the total amount of organic acids in Example 6 was 6.0382 g / 100 mL, which was 21.14% and 6.41% higher than that in Comparative Example 1 and Comparative Example 2, respectively, with lactic acid and citric acid being particularly prominent.

[0047] Table 4. Effects of compound koji strengthening application on the organic acid content of fresh lye. As shown in Table 5, the total amino acid content of Example 6 was 439.25 mg / 100 mL, which was 33.56% and 19.05% higher than that of Comparative Example 1 and Comparative Example 2, respectively. Among them, the contents of glutamic acid, aspartic acid and valine were significantly increased, with contents of 58.83 mg / 100 mL, 16.18 mg / 100 mL and 63.02 mg / 100 mL, respectively, which helped to enhance the umami flavor of the vinegar.

[0048] Table 5. Effects of compound koji strengthening application on amino acid content of neolymum lye. In Example 6, the total ester content was 2449.90 μg / 100 mL, significantly higher than Comparative Examples 1 and 2. The content of characteristic aroma esters such as ethyl acetate and ethyl lactate was significantly increased, imparting a rich fruity and ester aroma to the new vinegar, making the flavor more harmonious. The total alcohol content was 660.00 μg / 100 mL, far higher than Comparative Examples 1 and 2. The content of characteristic alcohols such as phenylethanol was significantly enriched, adding a mellow rose aroma and a mellow taste to the vinegar, further enriching its flavor. Meanwhile, the furfural content remained at a high level, giving the vinegar a unique caramel and nutty aroma. The Aspergillus oryzae and Aspergillus niger strains, through complementary secretion of rich enzyme systems, efficiently degraded macromolecules such as proteins, glutamine, starch, and cellulose in the raw materials, generating flavor precursors. Simultaneously, the metabolic interactions between the strains inhibited interference from other microorganisms, promoting the synthesis and accumulation of aroma substances, ultimately leading to a significant increase in flavor components such as esters, alcohols, and aldehydes.

[0049] Table 6. Effects of compound brocade enhancement application on the content of volatile aroma compounds. Note: "-" indicates that it was not detected.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a composite starter culture inoculated with a mixture of indigenous Aspergillus oryzae and Aspergillus niger, characterized in that: A 1:1 mixture of *Aspergillus oryzae* CGMCC 42103, a native strain of *Aspergillus oryzae* with high production of acidic protease and glutaminase, isolated from Shanxi aged vinegar koji, and *Aspergillus niger* CGMCC 15672, a strain of *Aspergillus niger* with high production of saccharifying enzyme, also isolated from Shanxi aged vinegar koji, was used as the starter culture. Wheat bran and soybean flour (crushed to 20 mesh) were mixed at a 9:1 ratio. Water (0.5 times the dry weight of the culture medium) was added to prepare a wheat bran and soybean flour culture medium. The starter culture was inoculated at 0.4% w / w into the medium and cultured at 30℃, spread evenly on koji trays with a thickness of 5–6 cm. Humidity was controlled at ≥95% throughout the culture process for 12–16 hours. When mycelial growth, clumping, and the temperature of the koji reached 38℃, the koji was turned and cooled to 28–32℃ for another 6–8 hours. h, when the temperature of the koji material rises to 38℃ again, the koji is turned over for the second time; then the koji is cultured at 28℃ for 42-48 h to produce the koji, and dried at a low temperature of 40-45℃ until the moisture content is ≤12%, which is the compound koji; The aforementioned Aspergillus oryzae ( Aspergillus oryzae CGMCC 42103 is a strain isolated and screened from Shanxi aged vinegar koji (a type of starter culture). It has high acid protease-glutaminase activity. After being cultured in barley and pea medium for 3 days, its acid protease activity and glutaminase activity were 630.25 U / g and 7.03 U / g, respectively. It also has amylase, β-glucosidase, aminopeptidase, cellulase and xylanase. It is deposited at the China General Microbiological Culture Collection Center on June 16, 2025.

2. The preparation method according to claim 1, characterized in that: The preparation method of the Aspergillus oryzae seed koji is as follows: the concentration is 10 7 ~10 8 Add 4 mL of CFU / mL Aspergillus oryzae CGMCC 42103 spore suspension to 30 g wheat bran and soybean powder culture medium, incubate at 30℃, knock the bottle every 12 h, and after 72 h of incubation, dry at 40℃ for 3 h to obtain Aspergillus oryzae inoculum. The preparation method of the Aspergillus niger inoculum: concentration is 10 7 ~10 8 Add 4 mL of CFU / mL Aspergillus niger CGMCC 15672 spore suspension to 30 g wheat bran medium and incubate at 30℃. Knock the bottle every 12 h. After 72 h of incubation, dry at 40℃ for 3 h to obtain Aspergillus niger inoculum. The preparation method of the bran and soybean powder culture medium is as follows: soybeans are crushed into 20 mesh, bran and soybean powder are mixed evenly at a mass ratio of 9:1, water is added at 0.5 times the dry weight of the culture medium, and the mixture is sterilized at 0.1 MPa and 121℃ for 30 min. After sterilization, the temperature is lowered to 40℃. The method for preparing the bran culture medium is as follows: mix bran and water at a mass-to-volume ratio of 1:1.5, sterilize at 0.1 MPa and 121°C for 30 min, and then allow the raw materials to cool down to 40°C after sterilization.

3. The composite starter culture prepared by the method according to claim 1 or 2, which is inoculated with a mixture of Aspergillus oryzae and Aspergillus niger.

4. The method for strengthening Shanxi aged vinegar using a compound koji inoculated with a mixture of indigenous Aspergillus oryzae and Aspergillus niger as described in claim 3, characterized in that: The process includes the following steps: Sorghum is crushed to 50-60 mesh; sorghum and water are mixed at a mass-to-volume ratio of 1:4.5-5; the mixture is heated to 90-95℃; 0.1% (by mass) of heat-resistant α-amylase from the sorghum is added; the mixture is kept at this temperature for 80-90 minutes; the temperature is lowered to 60℃; 0.1% (by mass) of saccharifying enzyme from the sorghum is added; the mixture is kept at this temperature for 20-30 minutes; once the temperature drops to 28-32℃, the mixture is placed in an alcohol fermentation tank; 30%-40% (by mass) of Daqu (a type of starter culture) and 10%-20% (by mass) of a compound starter culture inoculated with a mixture of Aspergillus oryzae and Aspergillus niger are added; 0.1%-0.3% (by mass) of yeast inoculum is added; the mixture is fermented openly for 2 days, then sealed for 6-14 days, until the alcohol content reaches 8-9%. %vol, after alcoholic fermentation, the mash is obtained; 120%–140% wheat bran, 70%–80% rice bran, 60%–70% rice husk, and 10%–20% of a compound koji inoculated with native Aspergillus oryzae and Aspergillus niger are added and thoroughly mixed, then transferred to solid-state acetic acid fermentation for 12–15 days. At the same time, 10% of fire mash is added to the vinegar mash for acetic acid fermentation. When the total acid is 5–6 g / 100 mL, the acetic acid fermentation is stopped, and new vinegar is obtained through smoking and vinegar leaching.

5. The method according to claim 4, characterized in that: The resulting new leached acetic acid is reddish-brown, with a rich color and good clarity, and has a total acid content ≥6.00 g / 100 mL, an amino acid nitrogen content ≥0.30 g / 100 mL, a non-volatile acid content ≥2.30 g / 100 mL, and a glutamic acid content ≥55.00 mg / 100 mL.

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