A "double solid" fermentation rich in non-volatile acid, ligustrazine and flavone ester old vinegar and its preparation method

Abstract

This invention belongs to the field of vinegar fermentation technology, and provides a "double-solid" fermentation method for producing a fragrant aged vinegar rich in non-volatile acids, tetramethylpyrazine, and flavonoids, as well as its preparation method. Using tannin-rich sorghum as raw material, and based on a heated Daqu (a type of starter culture) process, the alcoholic fermentation stage involves adding *Monascus purpureus* CGMCC 42465 red yeast rice, *Saccharomyces cerevisiae* CGMCC 34907 inoculum, *Saccharomyces cerevisiae* CGMCC 39090 inoculum, and a complex enzyme of xylanase and cellulase. The acetic acid fermentation stage involves adding a high proportion of wheat bran, sorghum husks, sea buckthorn leaf powder, and *Lactobacillus plantarum* CGMCC. The 15731 microbial agent and protease are used in a gradient temperature full-fumigation process. After sun-drying and aging for 1 year and cellaring for 1 year, the resulting aromatic aged vinegar is clear and bright, with a mellow, sweet and sour aroma, and has the aroma of grain, fermentation, smoking and aging. The total acidity is ≥8.50g / 100mL, the non-volatile acidity is ≥2.50g / 100mL, the tetramethylpyrazine is ≥70.00mg / L, the total flavonoids are ≥350.00mg / 100mL, and the total esters are ≥4.00g / 100mL.

Description

Technical Field

[0001] This invention belongs to the field of vinegar fermentation technology, specifically relating to a "double-solid" fermented ester aged vinegar rich in non-volatile acids, tetramethylpyrazine and flavonoids, and its preparation method. Background Technology

[0002] With the upgrading of food consumption and the quality-oriented development of the condiment industry, consumers' demand for vinegar has shifted from basic seasoning function to flavor richness, taste harmony, and ingredient distinctiveness. High-quality vinegar with both signature components and complex flavor layers has become the core of market competition. In the quality system of aged vinegar, non-volatile acids directly determine the mellowness and richness of the sour taste, and are the core material basis for forming the "oily and smooth" taste. Ligustrazine is a characteristic flavor component generated during the smoking stage, giving the product a unique smoky aroma, and is the signature component that distinguishes aged vinegar from other types of vinegar. Amino acid nitrogen, esters, and other components are the basis for constructing the complex flavors of vinegar, such as grain aroma, fermentation aroma, and aged aroma. Flavonoids can enrich the flavor complexity of vinegar. The synergistic optimization of these components has become the core goal of vinegar quality upgrading. Traditional aged vinegar brewing relies heavily on natural fermentation microorganisms, which presents several quality bottlenecks: low efficiency and insufficient proportion of non-volatile acids, leading to a sharp and pungent sour taste and a lack of mellowness and smoothness, affecting the palatability of the product; ligustrazine is generated naturally only during the fumigation stage, resulting in insufficient supply of precursor substances and low precision in reaction control, leading to large fluctuations in content and low enrichment efficiency; flavonoids rely solely on natural introduction from raw materials, lacking targeted enrichment and transformation pathways, resulting in an overall low content.

[0003] Current research on increasing the content of flavonoids, non-volatile acids, and tetramethylpyrazine in vinegar includes the following: Patent application number 202310008889.7, "A brewed vinegar with a rich caramel aroma and its production method," regulates the amount of furfural precursor in vinegar by using compound enzymes, acids, wheat bran, and rice husks, and improves the temperature and time parameters of the vinegar boiling and cooking process to control the amount of furfural generated in vinegar, resulting in a product with a rich caramel aroma; Patent application number 202311555134.5, "A method for increasing the non-volatile acid content of glutinous rice vinegar," uses glutinous rice as raw material to prepare glutinous rice vinegar through alcoholic fermentation and acetic acid fermentation processes, and the non-volatile acid content meets the requirements of GB18187-2000. The patent application number 202111364743.3, entitled "A type of dead Bacillus and its application in vinegar brewing", describes the application of dead Bacillus in the brewing of vinegar in cellars. It can grow and metabolize under acidic conditions and produce α-acetolactate decarboxylase, which converts α-acetolactate into acetoin, thereby providing sufficient precursor substances for tetramethylpyrazine.

[0004] Enhancing the flavonoids, non-volatile acids, and ligustrazine characteristic functional components of Shanxi aged vinegar is a systematic project that should be systematically optimized from the aspects of raw materials, fermentation, and strains.

[0005] Currently, the vinegar industry mostly employs a "liquid-to-solid" fermentation process, involving initial liquid alcohol fermentation followed by solid acetic acid fermentation. This process involves crushing sorghum to 50-60 mesh, mixing it with water, heating the mixture to 90-95℃, adding heat-resistant liquefying enzymes for enzymatic liquefaction, and then adding saccharifying enzymes, Daqu (a type of starter culture), and Angel yeast inoculum. Liquid alcohol fermentation is then carried out in stainless steel fermentation tanks. After alcoholic fermentation, bran, rice husks, and rice husks are added to the mash for aerobic solid acetic acid fermentation. This process is suitable for large-scale, mechanized production, but it suffers from insufficient flavor compound formation and a lack of complexity in flavor profiles. Summary of the Invention

[0006] This invention provides a "dual-solid" fermentation method for producing aged vinegar rich in non-volatile acids, tetramethylpyrazine, and flavonoids, along with its preparation method. Based on traditional dual-solid fermentation technology, this invention systematically optimizes the entire process through precise raw material selection, multi-stage functional microbial enhancement, compounding of special auxiliary ingredients, and gradient smoking parameter control. This achieves targeted enrichment and synergistic flavor optimization of non-volatile acids, tetramethylpyrazine, and flavonoids, resulting in a high-quality aged vinegar with a rich ester aroma, multi-layered complex fragrance, and characteristic components. This aligns with the development trend of the modern condiment industry and possesses significant industrial value and market prospects.

[0007] This invention is achieved by the following technical solution: a method for preparing aged vinegar rich in non-volatile acids, ligustrazine, and flavonoids through "double solid" fermentation. The method uses sorghum rich in tannins as raw material, based on a heated Daqu (a type of starter culture) process. During the alcoholic fermentation stage, *Monascus purpureus* CGMCC 42465 (red yeast), high-yield brewing yeast CGMCC 34907, high-yield ester-producing *Saccharomyces cerevisiae* CGMCC 39090, and a complex enzyme of xylanase and cellulase are added. Using the weight of sorghum as the calculation basis, during the acetic acid fermentation stage, 120% of wheat bran, sorghum husks, sea buckthorn leaf powder, high-yield non-volatile acid *Lactobacillus plantarum* CGMCC 15731, and protease are added. A gradient-heated full-smoking process is used, followed by sun-drying and aging for one year, and then cellar aging for one year to obtain the aged vinegar.

[0008] The specific steps include: Crush sorghum into four to six pieces, add water at a sorghum mass fraction of 350%, and moisten for 10-22 hours. After moistening, mix sorghum, rice husks, rice bran, and water at a mass-to-volume ratio (w / v) of 20:12:3:8. Stir the mixture thoroughly, place it in a steamer, and steam under normal pressure for 90 minutes, then let it sit overnight. After cooling, add water at a sorghum mass fraction of 300%, and add 30% of a warming starter culture, 20% of *Aspergillus oryzae* CGMCC42465 red yeast rice, 0.1% of *Saccharomyces cerevisiae* CGMCC 34907 inoculum, and 0.1% of *Saccharomyces cerevisiae* CGMCC. Mix 39090 microbial agent and 0.1% compound enzymes (xylanase and cellulase) evenly. Transport the mixed mash to the alcohol fermentation tank, flatten it after entering the tank, remove air, and ferment in a solid state for 8 days until the alcohol content reaches 8-9% vol. Then, stop the alcohol fermentation to obtain the mash. Mix the fermented mash with 120% sorghum bran, 10% sorghum husk, and 5% dried and crushed sea buckthorn leaf powder to 40 mesh evenly. Then, put it into the acetic acid fermentation tank and inoculate it with 10% aril mash, 1% protease, and 0.1% *Lactobacillus plantarum* CGMCC. The acetic acid fermentation process is completed after 12 days of fermentation using 15731 microbial agent, resulting in a total acid content of 5-6 g / 100 mL. The fermented vinegar mash is then smoked for 5 days in an electrically heated stainless steel tank, maintaining a moisture content of 68% throughout the process. Temperature is controlled in gradients: 60-65℃ on the first day, 65-75℃ on the second, 75-85℃ on the third, 85-90℃ on the fourth, and 90-95℃ on the fifth. After leaching, the total acid content is reduced to 5 g / 100 mL. The mash is then aged using a combination of summer sun-drying and winter ice-harvesting until the total acid content reaches over 6.5 g / 100 mL. Finally, it is aged in cellars for another year to obtain fragrant aged vinegar.

[0009] The preparation method of the heated Daqu is as follows: the raw materials are crushed and mixed in a mass ratio of barley:wheat:pea = 5.5:1.5:3. After sieving, the coarse flour accounts for 30%-40% and the fine flour accounts for 60%-70%. Water of 37%-50% of the total mass of the Daqu raw materials is added and mixed. The mixture is pressed into brick-shaped Daqu blocks of 28cm×17cm×6cm with a moisture content of 36%. The Daqu is then processed through a mold growth period, a mold drying period, a damp fire period, a high fire period, and a post-fire period to produce room Daqu, which takes a total of 21 days. After room Daqu is produced, it undergoes a 3-month maturation and drying stage to finally form a high-quality finished Daqu with a pure aroma, uniform mycelium distribution on the surface, and rich enzyme system.

[0010] The preparation method of the *Monascus purpureus* CGMCC42465 red yeast rice is as follows: Select plump indica rice, soak it in water at 23-27℃ for 2 hours, and judge by the criterion that there is no hard core when rubbed between the fingers. After soaking, rinse twice and drain until there is no dripping water on the surface. Pour the drained indica rice into a steamer and steam for 1 hour (steam until the indica rice has no white core, does not crack, is cooked but not sticky, and the rice grains are not sticky when touched with wet hands). Transfer the steamed indica rice to a fermentation tray and spread it out to cool at room temperature. After cooling to 28-32℃, inoculate with a *Monascus purpureus* spore suspension at an inoculation amount of 30% of the dry weight of the raw material, mix thoroughly, and ensure that each grain of rice is inoculated. The rice grains, coated with bacterial solution, are placed in clean, damp burlap sacks and sealed tightly. They are then incubated at 30°C for 48 hours, with water added every 24 hours. When the temperature rises to 45°C and more than 70% of the rice grains have mycelial colonization, the rice is soaked in koji. During soaking, the rice grains with mycelial colonization are wrapped in gauze and soaked in distilled water at 23-27°C for 20 seconds. After draining, the rice grains are poured back into the koji tray and spread evenly. The rice is soaked for 2 days, with soaking every 8 hours. When all the rice grains are uniformly dark red and have a strong red koji aroma, fermentation is stopped. The rice is then dried at 37°C until the moisture content is ≤12%, thus obtaining the purple red koji mold CGMCC42465 red koji. The method for preparing the *Monascus purpureus* spore suspension is as follows: *Monascus purpureus* CGMCC 42465 activated on a slant agar plate was used to scrape mycelia from the surface of the culture medium using a sterile inoculation loop. After scraping three loops consecutively, the mycelia were inoculated into rice flour liquid culture medium to ensure consistent inoculation. The medium was then incubated at 30℃ and 180 rpm for 48 hours on a shaker. During this period, the rice flour liquid culture medium was observed to turn pink. After another 48 hours of incubation, when the medium turned deep red, the suspension was obtained at a concentration of 10... 6 CFU / mL Aspergillus violaceus spore suspension; The formula for the rice flour liquid culture medium is as follows: 2.5 g rice flour, 0.15 g NaNO3, 0.05 g MgSO4·7H2O, 0.075 g KH2PO4, add water to make up to 50 mL, and then sterilize at 0.1 MPa and 121℃ for 20 min. The purple-red Aspergillus ( Monascus purpureus The strain described in CGMCC 42465, application number 202610202716.2, invention patent "A method for enhancing the flavor and nutritional components of solid-state brewed vinegar using Aspergillus purpureus-Aspergillus cristatus and Forsythia suspensa tea starter", was isolated from Shanxi aged vinegar starter. When cultured in rice flour liquid medium at 30℃ and 180 r / min on a shaker for 48 h, it produced 52.92 mg / 100 g of lovastatin and 61.38 mg / 100 g of γ-aminobutyric acid, and also exhibited excellent enzyme activity characteristics, including 22.38 U / g of amylase and 2485.62 U of esterification power. It is deposited at the China General Microbiological Culture Collection Center on December 5, 2025.

[0011] The preparation method of the Saccharomyces cerevisiae CGMCC 34907 inoculum is as follows: Saccharomyces cerevisiae CGMCC 34907 seed culture is inoculated into 75L of yeast extract liquid culture medium at a 3% inoculation rate. The medium is cultured at 30℃ with an aeration rate of 0.6L / min for 24 hours. During this period, 0.2% (v / v) of glucose from the yeast extract liquid culture medium is added every 8 hours until the cell concentration reaches 10... 8 The bacterial sludge was obtained by centrifuging at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-weight ratio (v / w) of 3:1. After thorough mixing, the mixture was spread evenly onto the freeze dryer tray to a thickness of 1 cm. The material was then frozen at -60℃ for 6 hours. The freeze dryer was pre-cooled, and once the cold trap temperature stabilized at -50℃, the frozen material was placed inside. Vacuum freeze drying was performed using a segmented temperature control mode to obtain *Saccharomyces cerevisiae* CGMCC 34907 inoculum with a viable count of 10-1. 9 CFU / g, vacuum packaged and stored at 4℃; The method for preparing the Saccharomyces cerevisiae CGMCC 34907 seed culture is as follows: culture the culture in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Saccharomyces cerevisiae* CGMCC 34907 (CFU / mL) was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium. The fermentation was carried out at 30℃, aeration rate of 0.2L / min, and rotation speed of 200 rpm for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL; The brewing yeast ( Saccharomyces cerevisiae CGMCC 34907, isolated from Shanxi aged vinegar mash, was cultured in sorghum juice medium at 30°C for 72 hours, with an ethanol content of 3.24% vol. It is deposited at the China General Microbiological Culture Collection Center on June 16, 2025.

[0012] The preparation method of *Saccharomyces cerevisiae* CGMCC 39090 inoculum is as follows: *Saccharomyces cerevisiae* CGMCC 39090 seed culture is inoculated at a rate of 3% into 75L of yeast extract liquid culture medium. The medium is cultured at 30℃ with an aeration rate of 0.6L / min for 24 hours. During this period, 0.2% (v / v) of glucose from the yeast extract liquid culture medium is added every 8 hours until the cell concentration reaches 10... 8The bacterial sludge was obtained by centrifuging at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-weight ratio (v / w) of 3:1. After thorough mixing, the mixture was spread evenly onto the freeze dryer tray to a thickness of 1 cm. The material was frozen at -60℃ for 6 hours. The freeze dryer was pre-cooled, and once the cold trap temperature stabilized at -50℃, the frozen material was placed inside. Vacuum freeze drying was performed using a segmented temperature control mode to obtain *Saccharomyces cerevisiae* CGMCC 39090 inoculum with a viable count of 10-1. 9 CFU / g, vacuum packaged and stored at 4℃; The method for preparing the seed culture of *Saccharomyces cerevisiae* CGMCC 39090 is as follows: culture the culture in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Saccharomyces cerevisiae* CGMCC 39090 (CFU / mL) was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium. The culture was incubated at 30℃, aeration rate of 0.2L / min, and rotation speed of 200 rpm for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL; The yeast mentioned (Jieye Mengmaieri) Meyerozyma guilliermondii CGMCC 39090 was obtained from Shanxi aged vinegar mash. In sorghum juice culture medium, it produced 2.93 g / 100 mL of esters. It is deposited at the China General Microbiological Culture Collection Center on December 5, 2025. The preparation method of the *Lactobacillus plantarum* CGMCC 15731 inoculum is as follows: *Lactobacillus plantarum* CGMCC 15731 seed culture is inoculated into 75 L of yeast extract liquid culture medium at a 3% inoculation rate. The medium is cultured at 37℃ with an aeration rate of 0.8 L / min for 48 h. During this period, 0.2% glucose (by volume) of yeast extract liquid culture medium is added every 8 h. The pH value of the fermentation broth is sampled and monitored every 12 h. The pH of the fermentation broth is adjusted to neutral by adding 20% ​​(w / v) sodium carbonate solution until the bacterial concentration reaches 10. 8 The bacterial sludge was obtained by centrifugation at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-weight ratio (v / w) of 3:1. After thorough mixing, the mixture was spread evenly onto the freeze dryer tray to a thickness of 1 cm. The material was then frozen at -60℃ for 6 hours. The freeze dryer was pre-cooled beforehand, and the frozen material was placed in the cold trap after the temperature stabilized at -50℃. Vacuum freeze drying was performed using a segmented temperature control mode to obtain *Lactobacillus plantarum* CGMCC 15731 bacterial agent with a viable count of 10-1. 9 CFU / g, vacuum packaged and stored at 4℃; The method for preparing the *Lactobacillus plantarum* CGMCC 15731 seed culture is as follows: cultured in MRS liquid medium until the bacterial concentration reaches 10... 8 A 3% inoculum of *Lactobacillus plantarum* CGMCC 15731 was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium and cultured at 37℃, aeration rate of 0.2 L / min, and rotation speed of 200 r / min for 48 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL; The *Lactobacillus plantarum* CGMCC 15731 is a high-yield strain of non-volatile acid in the invention patent application No. 201910136447.4 entitled "A method for developing and multi-stage strengthening of Shanxi aged vinegar compound microbial agent based on microbial interaction". The non-volatile acid content is 0.61 g / 100 mL. It is deposited at the China General Microbiological Culture Collection Center on May 7, 2018.

[0013] The method for preparing the yeast extract liquid culture medium is as follows: 1g yeast extract, 2g glucose, 0.3g yeast extract, 100mL water, sterilized at 121℃ for 20 min for later use. The method for preparing the vacuum freeze-drying protectant is as follows: Protectant 1: 5 g skim milk powder, 8 g whey powder, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; Protectant 2: 5 g trehalose, 3 g sucrose, 5 g glucose, 0.5 g monosodium glutamate, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; After sterilization, protectant 1 and protectant 2 are cooled to room temperature and mixed evenly at a volume ratio (v / v) of 1:1 to obtain the bacterial cell vacuum freeze-drying protectant; The segmented temperature-controlled vacuum freeze-drying parameters are set as follows: first segment: -60℃, 15min; second segment: -25℃, 1920min; third segment: 0℃, 420min; fourth segment: 20℃, 120min.

[0014] The compound enzyme, model SFQ200, contains xylanase and cellulase, wherein the xylanase activity is ≥2000 U / g and the cellulase activity is ≥100 U / g; the protease activity is ≥8000 U / g, and the optimal pH is 4.0-5.0. All of them were purchased from Hangzhou Baoankang Biotechnology Co., Ltd.

[0015] The prepared aged vinegar is clear and transparent, with a mellow, sweet and sour taste, combining the aromas of grain, yeast, smokiness and aging. It has no off-flavors or odors, and contains ≥8.50 g / 100mL of total acid, ≥2.50 g / 100mL of non-volatile acid, ≥70.00mg / L of tetramethylpyrazine, ≥350.00mg / 100mL of total flavonoids, and ≥4.00g / 100mL of total esters.

[0016] This invention employs a "pre-solid and post-solid" fermentation process, namely, solid-state alcoholic fermentation in the early stage and solid-state acetic acid fermentation in the later stage, achieving solid-state production throughout the entire vinegar production process. This process involves crushing sorghum into four or six pieces to avoid over-crushing, which could damage the starch granule structure and cause material sticking during fermentation. The crushed sorghum is moistened with water for 10-22 hours, allowing the water to fully penetrate into the endosperm, promoting the starch granules to absorb water and expand, which is beneficial for subsequent steaming and gelatinization. The moistened sorghum is then mixed with rice husks and rice bran as auxiliary materials and steamed for 90 minutes. After steaming, it is left to sit overnight. The rice husks and rice bran enhance the looseness and permeability of the material, resulting in more even heating and ensuring thorough steaming. During steaming, the sorghum starch undergoes full gelatinization, releasing its aroma fully. After steaming, the mixture is cooled, and appropriate amounts of water are added, along with Daqu (a type of starter culture) and bran starter. The mixture is thoroughly stirred and mixed, then transferred to an alcoholic fermentation tank for solid-state alcoholic fermentation. This earthen tank process is characterized by its warm winter and cool summer conditions, easy temperature control, stable fermentation, and low energy consumption. Adding wheat bran as an adjunct to solid acetic acid fermentation improves the looseness of the mash. Aerobic fermentation provides suitable conditions for the oxidative metabolism of acetic acid bacteria, promoting the conversion of ethanol to acetic acid and completing the solid-state acetic acid fermentation process. The pre-solid and post-solid processes maximize the preservation of the raw material's flavor precursors, resulting in a richer and more mellow product flavor.

[0017] This invention selects sorghum, rich in tannins, as the brewing raw material. The abundant tannins in sorghum grains are gradually released during fermentation and participate in microbial metabolism and flavor transformation, promoting the generation and coordination of aroma compounds such as alcohols, acids, and esters, thus enhancing the flavor profile of the vinegar. Sorghum husks and sea buckthorn leaves are rich in natural flavonoids, which can be stably dissolved during fermentation, directly increasing the flavonoid content in the vinegar. A high proportion of wheat bran provides ample attachment sites and nutritional support for microbial growth, promoting the transformation and generation of flavor compounds.

[0018] Compared with existing technologies, it has the following significant advantages: (1) This invention uses high-tannin sorghum as raw material, combined with sorghum husk and sea buckthorn leaves as special auxiliary materials. Sorghum tannin can promote the conversion of alcohol and ester flavor substances and enhance the mellowness of vinegar; sorghum husk and sea buckthorn leaves are rich in natural flavonoids and polyphenols, which are continuously dissolved during fermentation, increasing the total flavonoid content, while providing sufficient precursors for the Maillard reaction of the mash and promoting the formation of tetramethylpyrazine; the three work together to simultaneously enrich the flavor base, increase the content of functional components, and optimize the overall flavor and nutritional quality of aged vinegar.

[0019] (2) The present invention adopts a dual solid-state fermentation process of solid-state fermentation followed by solid-state fermentation, namely solid-state alcoholic fermentation and solid-state acetic acid fermentation. Compared with the liquid-liquid-solid process, the solid-state-solid process can retain the flavor precursors in the raw materials to the greatest extent, promote the metabolic interaction of the microbial community, make the raw materials more thoroughly transformed and the flavor substances more fully accumulated, and improve the flavor richness and aroma persistence of aged vinegar from the bottom of the process, providing a process basis for high-quality aromatic ester aged vinegar.

[0020] (3) In the solid alcohol fermentation stage, this invention strengthens the addition of warming Daqu, red yeast rice CGMCC 42465, high alcohol-producing brewing yeast CGMCC 34907, high ester-producing yeast CGMCC 39090 and compound enzyme preparation. Compared with traditional vinegar-making koji, the temperature of the koji room and koji blocks in the heated koji are increased to 39℃ and 53℃ respectively during the high-fire period, significantly improving the activity of multiple enzymes such as saccharification, esterification, and protein hydrolysis, providing sufficient precursors for ester synthesis; the red koji mold CGMCC42465 enhances saccharification efficiency and promotes the accumulation of esters and functional substances such as γ-aminobutyric acid and lovastatin; the high-ethanol-producing brewer's yeast CGMCC 34907 efficiently converts fermentable sugars into ethanol; the high-ester-producing ester-producing yeast Mayer's yeast CGMCC39090 catalyzes alcohol and acid esterification, significantly increasing the total ester content; the xylanase and cellulase complex enzyme preparation decomposes raw materials, releasing polysaccharide and protein small molecule substrates, synergistically increasing the organic acid and total ester content of the system.

[0021] (4) In the acetic acid fermentation stage, the present invention adds 120% of the sorghum bran by weight, which is higher than the 80%~100% addition ratio of the traditional process. The high proportion of bran can optimize the pore structure of the mash and adapt it to the aerobic fermentation environment. It is rich in vitamins, free amino acids and other lactic acid bacteria growth factors, which can accelerate the reproduction of bacteria and increase the amount of lactic acid produced. The compound acidic protease can decompose the large molecular protein of the raw materials and release a large amount of flavor precursor free amino acids. Combined with the high-yield non-volatile acid Bacillus plantarum CGMCC 15731 bacterial agent for synergistic metabolism, it further improves the non-volatile acid content of the system, softens the sour taste of vinegar, enriches the mellow taste of vinegar base, and promotes the accumulation of flavor substances at the same time.

[0022] (5) This invention is based on the natural "summer sun-drying and winter ice-catching" aging process specified by the geographical indication of Shanxi aged vinegar for 1 year, and then carries out a cellar aging process for 1 year. Sun-drying and ice-catching can concentrate the vinegar body and initially adjust the acidity; the subsequent cellar aging environment has stable temperature and humidity, and the organic acids, esters and amino acids in the system continue to undergo reversible reactions such as esterification, condensation and oxidation. Acetic acid is gradually converted into mild esters, free amino acids are continuously enriched, and impurities in the vinegar slowly settle. In the end, the acidity is mellow, the taste is sweet and clear, and the multi-layered grain aroma, smoky aroma and aged aroma are fully integrated, which significantly improves the mellowness of the finished product and the long-term storage stability.

[0023] (6) The aged vinegar prepared by this invention is clear and transparent, with a mellow, sweet and sour taste that combines the aroma of grain, yeast, smoky and aged flavors. The total acid content is ≥8.50 g / 100mL, the non-volatile acid content is ≥2.50 g / 100mL, the ligustrazine content is ≥70.00mg / L, the total flavonoid content is ≥350.00mg / 100mL, and the total ester content is ≥4.00g / 100mL.

[0024] The yeast mentioned (Jieye Mengmaieri) Meyerozyma guilliermondii JY1422 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC 39090 and deposit date of December 5, 2025.

[0025] The brewing yeast ( Saccharomyces cerevisiae JY311 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC 34907 and deposit date of June 16, 2025. Attached Figure Description

[0026] Figure 1 In the middle: A represents the temperature change in the koji room during the preparation of Shanxi aged vinegar koji; B represents the temperature change of the koji blocks. Figure 2 The images show the colony and cell morphology of yeast JY311 on PDA medium. In the figure: the left image shows the colony morphology; the right image shows the cell morphology. Figure 3 This is the ITS sequencing phylogenetic tree of yeast JY311. Figure 4 The images show the colony and cell morphology of yeast JY1422 on PDA medium. In the figure: the left image shows the colony morphology; the right image shows the cell morphology. Figure 5 This is a phylogenetic tree diagram of the ITS sequencing of yeast JY1422. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] Equivalent techniques of the specific embodiments described herein, which can be understood through routine experiments and are recognized by those skilled in the art, are all included in this application. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the instruments and equipment used in the following embodiments are conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores; the thermostable α-amylase and saccharifying enzyme used in the following embodiments were purchased from Shandong Longket Enzyme Preparation Co., Ltd.; the fungal DNA extraction kit used in the following embodiments was purchased from Beijing Solarbio Technology Co., Ltd.; the compound enzyme used in the following embodiments, model SFQ200 (with xylanase activity ≥2000U / g, cellulase activity ≥100U / g), and the protease (with enzyme activity ≥8000U / g, optimal pH 4.0–5.0), were all purchased from Hangzhou Baoankang Biotechnology Co., Ltd.

[0030] Example 1: Preparation of heated Daqu: The barley:wheat:pea mass ratio (w / w) of 5.5:1.5:3 was crushed and mixed evenly. The mixture was then sieved through a grading sieve, with 30%-40% coarse flour and 60%-70% fine flour. Water of 37%-50% of the total mass of the raw materials was added and mixed evenly. The mixed material was then placed into a Daqu making machine and pressed into brick-shaped Daqu blocks of 28 cm × 17 cm × 6 cm with a moisture content of about 36%. The koji blocks are arranged in three layers in a koji room lined with reeds and sprinkled with rice husks. The room is covered with reed mats and enters the koji-laying stage. The koji is then processed through five stages: the mold-forming stage (SM) (0-2 days), the mold-drying stage (LM) (2-3 days), the high-temperature firing stage (CH) (3-9 days), the high-temperature firing stage (DH) (9-15 days), and the low-temperature firing stage (HH) (15-21 days). The entire process takes 21 days. During this period, the temperature and humidity of the koji room are regulated by turning the koji, adjusting the arrangement / spacing of the koji blocks, and opening and closing the windows. After the koji is removed from the koji room, it undergoes a 3-month maturation and drying stage, ultimately forming a high-quality finished koji with a pure aroma, uniform mycelium distribution on the surface, and rich enzyme system.

[0031] Traditional Daqu preparation method: The raw materials are prepared in a barley:pea mass ratio of 7:3 (w / w). The remaining preparation steps, parameters and post-ripening drying process are the same as those for the heated Daqu preparation method.

[0032] The specific measurement method is as follows: (1) Determination of liquefaction power, saccharification power, fermentation power and esterification power, moisture, total acid and amino acid nitrogen: Refer to QB / T4257—2011 "General Analytical Methods for Brewing Daqu"; (2) Determination of protease activity: Refer to SB / T 10317.1999 "Determination of Protease Activity"; (3) Cellulase activity determination: The activity was determined by the DNS colorimetric method according to GB / T 23881—2009 "Determination of cellulase activity in feed by filter paper method"; (4) Determination of reducing sugar content: The content was determined by direct titration according to GB 5009.7-2016.

[0033] The temperature changes in the koji room and koji blocks during the preparation of Shanxi aged vinegar koji are shown in the figure. Figure 1 Compared to traditional Daqu (a type of starter culture), the temperatures of the fermentation room and the starter blocks in the heated Daqu rise to 39℃ and 53℃ respectively during the high-heat stage. As fermentation progresses, the temperatures of the fermentation room and the starter blocks decrease to 28℃ and 36℃ respectively. Adding wheat can increase the temperature of the aged vinegar Daqu during the damp and high-heat stages, and the temperature drop in the fermentation room is gradual during the later stages, which is beneficial to the formation and stability of the Daqu quality. Table 1 shows that there are significant differences in physicochemical and enzyme activity indicators between the heated Daqu and traditional Daqu. Among them, the saccharification power of the heated Daqu was increased by 20.49% and the esterification power was increased by 27.16% compared with the traditional Daqu, which reflects the enrichment effect of the heated process on the activity of heat-resistant enzymes such as saccharification and esterification. However, the liquefaction power was reduced by 18.52% compared with the traditional Daqu. This is because the high temperature caused partial inactivation of heat-sensitive α-amylase. At the same time, the total acid, amino acid nitrogen and reducing sugar of the heated Daqu were increased by 45.00%, 44.44% and 40.00% respectively compared with the traditional Daqu, indicating that its functional microbial community is more active, and the efficiency of protein hydrolysis and starch conversion is higher, making it more suitable for the fermentation requirements of aged vinegar brewing.

[0034] Table 1. Results of physicochemical and enzyme activity assays of heated Daqu and traditional Daqu Example 2: Screening and identification of high-yield alcohol-producing yeasts 1. Screening of high-yield alcohol-producing yeasts: Ten yeast strains isolated from the mash of various Shanxi aged vinegar factories were selected and inoculated into PDA liquid medium using an inoculation loop. The cultures were incubated at 30°C for 24 hours to achieve a concentration of 10⁻⁶. 8 The yeast culture solution was inoculated into 150 mL of sorghum juice fermentation medium at a 3% inoculation rate and placed in a 30 ℃ constant temperature incubator for 72 h (the incubator was sealed with six layers of gauze for the first 24 h and with plastic sheeting for the next 48 h). After the fermentation broth had fermented for 72 h, the alcohol content was measured.

[0035] The PDA liquid culture medium is prepared as follows: 200g potato, 20g glucose, 2g peptone, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 1000mL water, natural pH, sterilized at 121℃ for 20min.

[0036] The method for preparing the sorghum juice culture medium is as follows: 200g of sorghum is crushed into four to six pieces, 800mL of water and 200μL of high-temperature amylase are added, and the mixture is cooked at 100℃ for 60 min. When the mixture is cooled to 55℃, 1mL of saccharifying enzyme is added. After keeping the mixture at 55℃ for 4 h, the mixture is filtered to obtain the filtrate, and then sterilized at 121℃ under high pressure for 20 min for later use.

[0037] The determination method is as follows: Alcohol content was determined by distillation.

[0038] Table 2 shows that there were significant differences in alcohol content among the 10 tested yeast strains. Among them, strain JY311 had an alcohol content of 3.24% vol, which was significantly better than the other strains.

[0039] Table 2. Screening results of high-alcohol-producing yeasts 2. Identification of high-yield alcohol-producing yeast JY311: Morphological identification: A small amount of yeast JY311 was picked up with an inoculation loop and streaked onto a PDA agar plate to isolate single colonies. The colonies were photographed and recorded, and then their cell morphology was observed under a microscope using simple staining. See below for colony and cell morphology of yeast JY311 on PDA agar. Figure 2 The colonies are smooth and moist, opaque and milky white, medium-sized, uniform in texture, raised, and with very rounded edges. They are oval in shape and arranged individually in close clusters.

[0040] ITS Sequencing: Yeast strain JY311 cells were freeze-ground, and DNA was extracted using a kit for ITS sequencing and strain identification. Primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used. BLAST was employed for sequence homology analysis, and an ITS phylogenetic tree was constructed. The ITS phylogenetic tree for yeast strain JY311 is shown below. Figure 3 The yeast strain JY311 was identified as *Saccharomyces cerevisiae*. Saccharomyces cerevisiae It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 34907 and deposit date of June 16, 2025.

[0041] Example 3: Preparation of high-yield Saccharomyces cerevisiae CGMCC 34907 inoculum: Saccharomyces cerevisiae CGMCC 34907 seed culture was inoculated at a rate of 3% into 75L of yeast extract liquid culture medium. The medium was cultured at 30℃ with an aeration rate of 0.6L / min for 24 hours. During this period, 0.2% (v / v) of glucose from the yeast extract liquid culture medium was added every 8 hours until the cell concentration reached 10...8 The bacterial sludge was obtained by centrifuging at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-weight ratio (v / w) of 3:1. After thorough mixing, the mixture was spread evenly onto the freeze dryer tray to a thickness of 1 cm. The material was then frozen at -60℃ for 6 hours. The freeze dryer was pre-cooled, and once the cold trap temperature stabilized at -50℃, the frozen material was placed inside. Vacuum freeze drying was performed using a segmented temperature control mode to obtain *Saccharomyces cerevisiae* CGMCC 34907 inoculum with a viable count of 10-1. 9 CFU / g, vacuum packaged and stored at 4℃.

[0042] The method for preparing the Saccharomyces cerevisiae CGMCC 34907 seed culture is as follows: culture the culture in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Saccharomyces cerevisiae* CGMCC 34907 (CFU / mL) was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium. The culture was incubated at 30℃, aeration rate of 0.2L / min, and rotation speed of 200 r / min for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL.

[0043] The method for preparing the yeast extract liquid culture medium is as follows: 1g yeast extract, 2g glucose, 0.3g yeast extract, 100mL water, sterilized at 121℃ for 20 min for later use. The method for preparing the vacuum freeze-drying protectant is as follows: Protectant 1: 5 g skim milk powder, 8 g whey powder, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; Protectant 2: 5 g trehalose, 3 g sucrose, 5 g glucose, 0.5 g monosodium glutamate, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; After sterilization, protectant 1 and protectant 2 are cooled to room temperature and mixed evenly at a volume ratio (v / v) of 1:1 to obtain the bacterial cell vacuum freeze-drying protectant; The segmented temperature-controlled vacuum freeze-drying parameters are set as follows: first segment: -60℃, 15min; second segment: -25℃, 1920min; third segment: 0℃, 420min; fourth segment: 20℃, 120min.

[0044] Example 4: Screening and identification of high-ester-producing yeasts (1) Screening of high ester-producing yeast strains: Ten yeast strains isolated from the mash of various vinegar factories in Shanxi were selected in the laboratory. Their ester production capacity was measured to screen for high ester-producing yeast strains. The above 10 yeast strains were activated by inoculating them into PDA liquid medium. Then, they were inoculated into 150 mL of sorghum juice medium at an inoculation rate of 3% and placed in a constant temperature incubator at 30 ℃ for 72 h (the incubator was sealed with six layers of gauze for the first 24 h and with plastic sheeting for the last 48 h). After the fermentation broth had fermented for 72 h, the total ester content of the fermentation broth was measured.

[0045] The method for preparing the sorghum juice culture medium is as follows: 200 g of sorghum is crushed into four to six pieces, 800 mL of water and 200 μL of high-temperature amylase are added, and the mixture is cooked at 100 ℃ for 60 min. When the mixture is cooled to 55 ℃, 1 mL of saccharifying enzyme is added. After keeping the mixture at 55 ℃ for 4 h, the mixture is filtered to obtain the filtrate, which is then sterilized at 0.1 MPa and 121 ℃ for 20 min for later use.

[0046] The determination method is as follows: Determination of total esters: The determination was carried out in accordance with the method of the national standard GB / T 19777—2013 "Geographical Indication Product Shanxi Aged Vinegar".

[0047] As shown in Table 3, there were significant differences in the ester production capacity of different yeast strains in the sorghum juice fermentation system. Among them, strain JY1422 had the best fermentation performance and was the strain with the best high ester production characteristics among the 10 yeast strains: its total ester content was 2.93 g / 100 mL.

[0048] Table 3 Screening of high-ester-producing yeast strains (2) Identification of superior yeast strain JY1422 Morphological identification: A small amount of yeast JY1422 was picked up with an inoculation loop and streaked onto PDA agar plates to isolate single colonies. The colonies were photographed and recorded, and then their cell morphology was observed under a microscope using simple staining. See below for colony and cell morphology of yeast JY1422 on PDA agar. Figure 4 The colonies are round, 2.0-3.0 mm in diameter, milky white, slightly raised, with neat edges, smooth, moist and glossy surface, and soft, creamy texture; cell morphology: elliptical, oval or oblong, arranged singly or in pairs.

[0049] ITS Sequencing: Yeast strain JY1422 cells were freeze-ground, and DNA was extracted using a kit for ITS sequencing and strain identification. Primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used. BLAST was employed for sequence homology analysis, and an ITS phylogenetic tree was constructed. The ITS phylogenetic tree for yeast strain JY1422 is shown below. Figure 5 The yeast strain JY1422 was identified as *Saccharomyces giardi* (Jymnema einsteinii). Meyerozyma guilliermondii It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 39090 and deposit date of December 5, 2025.

[0050] Example 5: Preparation of *Saccharomyces cerevisiae* CGMCC 39090 inoculum: The preparation method of *Saccharomyces cerevisiae* CGMCC 39090 inoculum was as follows: *Saccharomyces cerevisiae* CGMCC 39090 seed culture was inoculated into 75L of yeast extract liquid culture medium at an inoculation rate of 3%. The culture was incubated at 30℃ and an aeration rate of 0.6L / min for 24h. During this period, 0.2% (v / v) of glucose from the yeast extract liquid culture medium was added every 8h until the cell concentration reached 10... 8 The bacterial sludge was obtained by centrifuging at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-weight ratio (v / w) of 3:1. After thorough mixing, the mixture was spread evenly onto the freeze dryer tray to a thickness of 1 cm. The material was frozen at -60℃ for 6 hours. The freeze dryer was pre-cooled, and once the cold trap temperature stabilized at -50℃, the frozen material was placed inside. Vacuum freeze drying was performed using a segmented temperature control mode to obtain *Saccharomyces cerevisiae* CGMCC 39090 inoculum with a viable count of 10-1. 9 CFU / g, vacuum packaged and stored at 4℃.

[0051] The method for preparing the seed culture of *Saccharomyces cerevisiae* CGMCC 39090 is as follows: culture the culture in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Saccharomyces cerevisiae* CGMCC 39090 (CFU / mL) was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium. The culture was incubated at 30℃, aeration rate of 0.2L / min, and rotation speed of 200 rpm for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL.

[0052] The method for preparing the yeast extract liquid culture medium is as follows: 1g yeast extract, 2g glucose, 0.3g yeast extract, 100mL water, sterilized at 121℃ for 20 min for later use. The method for preparing the vacuum freeze-drying protectant is as follows: Protectant 1: 5 g skim milk powder, 8 g whey powder, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; Protectant 2: 5 g trehalose, 3 g sucrose, 5 g glucose, 0.5 g monosodium glutamate, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; After sterilization, protectant 1 and protectant 2 are cooled to room temperature and mixed evenly at a volume ratio (v / v) of 1:1 to obtain the bacterial cell vacuum freeze-drying protectant; The segmented temperature-controlled vacuum freeze-drying parameters are set as follows: first segment: -60℃, 15min; second segment: -25℃, 1920min; third segment: 0℃, 420min; fourth segment: 20℃, 120min.

[0053] Example 6: Preparation of *Lactobacillus plantarum* CGMCC 15731 inoculum: *Lactobacillus plantarum* CGMCC 15731 seed culture was inoculated into 75 L of yeast extract liquid culture medium at a 3% inoculation rate. The medium was cultured at 37℃ and an aeration rate of 0.8 L / min for 48 h. During this period, 0.2% glucose (v / v) of yeast extract liquid culture medium was added every 8 h. The pH of the fermentation broth was monitored every 12 h, and the pH was adjusted to neutral by adding 20% ​​(w / v) sodium carbonate solution until the bacterial concentration reached 10. 8 The bacterial sludge was obtained by centrifugation at 8000 rpm for 10 minutes at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-weight ratio (v / w) of 3:1. After thorough mixing, the mixture was spread evenly onto the freeze dryer tray to a thickness of 1 cm. The material was then frozen at -60°C for 6 hours. The freeze dryer was pre-cooled beforehand, and the frozen material was placed in the cold trap after the temperature stabilized at -50°C. Vacuum freeze drying was then performed using a segmented temperature control mode to obtain *Lactobacillus plantarum* CGMCC 15731 bacterial agent with a viable count of 10-1. 9 CFU / g, vacuum packaged and stored at 4℃.

[0054] The method for preparing the *Lactobacillus plantarum* CGMCC 15731 seed culture is as follows: cultured in MRS liquid medium until the bacterial concentration reaches 10... 8 A 3% inoculum of *Lactobacillus plantarum* CGMCC 15731 was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium and cultured at 37℃, aeration rate of 0.2 L / min, and rotation speed of 200 r / min for 48 hours, achieving a cell concentration of 10⁻⁶. 8CFU / mL.

[0055] The method for preparing the yeast extract liquid culture medium is as follows: 1g yeast extract, 2g glucose, 0.3g yeast extract, 100mL water, sterilized at 121℃ for 20 min for later use. The method for preparing the vacuum freeze-drying protectant is as follows: Protectant 1: 5 g skim milk powder, 8 g whey powder, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; Protectant 2: 5 g trehalose, 3 g sucrose, 5 g glucose, 0.5 g monosodium glutamate, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; After sterilization, protectant 1 and protectant 2 are cooled to room temperature and mixed evenly at a volume ratio (v / v) of 1:1 to obtain the bacterial cell vacuum freeze-drying protectant; The segmented temperature-controlled vacuum freeze-drying parameters are set as follows: first segment: -60℃, 15min; second segment: -25℃, 1920min; third segment: 0℃, 420min; fourth segment: 20℃, 120min.

[0056] The *Lactobacillus plantarum* CGMCC 15731 is a high-yield strain of non-volatile acid in the invention patent application No. 201910136447.4 entitled "A method for developing and multi-stage strengthening of Shanxi aged vinegar compound microbial agent based on microbial interaction". The non-volatile acid content is 0.61 g / 100 mL. It is deposited at the China General Microbiological Culture Collection Center on May 7, 2018.

[0057] Example 7: Preparation of Monascus purpureus CGMCC42465 red yeast rice: Select plump indica rice and soak it in water at about 25℃ for 2 hours. The rice is judged by the absence of a hard core when rubbed between the fingers. After soaking, rinse twice and drain until no water drips from the surface. Pour the drained rice into a steamer and steam for 1 hour (until the rice is cooked without a white core, does not crack, and is cooked but not sticky; the rice grains should not stick to the touch with a wet hand). Transfer the steamed rice to a yeast tray and let it cool to room temperature. When it cools to about 30℃, inoculate it with a Monascus purpureus spore suspension at an inoculation rate of 30% of the raw material's dry weight. Mix thoroughly to ensure each grain of rice is coated with the bacterial solution. Pack the rice into clean, damp burlap sacks and tie the openings tightly. Incubate at 30℃ for 48 hours, adding water every 24 hours. Continue incubation until the temperature reaches 45℃. When the rice is at a certain temperature and more than 70% of the rice has mycelial colonization, the soaking process begins. During soaking, the rice with mycelial colonization is wrapped in gauze and soaked in distilled water at about 25℃ for 20 seconds. After draining, it is poured back into the koji tray and spread evenly. Soaking is carried out for 2 days, once every 8 hours. When all the rice turns a uniform dark red color and has a strong red koji aroma, fermentation is stopped. The rice is then dried at a low temperature of 37℃ until the moisture content is ≤12%, thus obtaining the purple red koji mold CGMCC42465 red koji.

[0058] The method for preparing the *Monascus purpureus* spore suspension is as follows: *Monascus purpureus* CGMCC 42465 activated on a slant culture medium is used to scrape mycelia from the surface of the medium using a sterile inoculation loop. Three loops of mycelia are scraped and inoculated into rice flour liquid medium to ensure consistent inoculation. The medium is then incubated at 30℃ and 180 r / min on a shaker for 48 h. During this period, the rice flour liquid medium is observed to turn pink. After another 48 h of incubation, if the medium turns deep red, the suspension is considered to have a concentration of 10... 6 CFU / mL Monascus purpureus spore suspension.

[0059] The formula for the rice flour liquid culture medium is as follows: 2.5 g rice flour, 0.15 g NaNO3, 0.05 g MgSO4·7H2O, 0.075 g KH2PO4, add water to make up to 50 mL, and then sterilize at 0.1 MPa and 121℃ for 20 min.

[0060] The purple-red Aspergillus ( Monascus purpureus The strain described in CGMCC 42465, application number 202610202716.2, invention patent "A method for enhancing the flavor and nutritional components of solid-state brewed vinegar using Aspergillus purpureus-Aspergillus cristatus and Forsythia suspensa tea starter", was isolated from Shanxi aged vinegar starter. When cultured in rice flour liquid medium at 30℃ and 180 r / min on a shaker for 48 h, it produced 52.92 mg / 100 g of lovastatin and 61.38 mg / 100 g of γ-aminobutyric acid, and also exhibited excellent enzyme activity characteristics, including 22.38 U / g of amylase and 2485.62 U of esterification power. It is deposited at the China General Microbiological Culture Collection Center on December 5, 2025.

[0061] Comparative Example 1: Solid-state fermentation process (50% of the weight of ordinary sorghum koji and 0.1% brewing yeast CGMCC 34907 inoculant were added during koji mixing) Crush sorghum into four or six pieces, add water at a sorghum mass fraction of 350%, and moisten for 10-22 hours. After moistening, mix sorghum, rice husks, rice bran, and water at a mass-to-volume ratio (w / v) of 20:12:3:8. Stir the mixture thoroughly, place it in a steamer, and steam under normal pressure for 90 minutes, then let it sit overnight. After cooling, add water at a sorghum mass fraction of 300%, add ordinary koji (fermented rice starter) at a sorghum mass fraction of 50%, and brewing yeast CGMCC 34907 inoculum, and stir thoroughly. Transport the mixed mash to an alcoholic fermentation tank, flatten it after placement, remove air, and ferment in a solid state for 8 days until the alcohol content reaches 8-9%. The alcoholic fermentation process is completed at %vol, yielding the mash. The fermented mash is then mixed evenly with 100% sorghum bran and placed in an acetic acid fermentation tank. A 10% sorghum-based fermenting agent is added, and fermentation continues for 12 days until the total acid content reaches 5–6 g / 100 mL, at which point the acetic acid fermentation is complete. The fermented mash is then smoked for 5 days in an electrically heated stainless steel tank, maintaining a moisture content of 68% throughout. Temperature is controlled in gradients: 60–65℃ on the first day, 65–75℃ on the second, 75–85℃ on the third, 85–90℃ on the fourth, and 90–95℃ on the fifth. After leaching, the total acid content is reduced to 5 g / 100 mL. The mash is then aged using a process of "summer sun-drying and winter ice-harvesting" until the total acid content reaches over 6.5 g / 100 mL. Finally, it is aged in cellars for another year to obtain aged vinegar.

[0062] Example 7: Enhanced application during alcoholic fermentation Example 7-(1): During the alcohol fermentation stage, 50% of the sorghum mass of the warming koji and 0.1% of the brewing yeast CGMCC 34907 were added, and the rest of the process was the same as in Comparative Example 1.

[0063] Example 7-(2): During the alcohol fermentation stage, 50% of sorghum mass of warming Daqu, 0.1% of brewing yeast CGMCC 34907 and 0.1% of yeast CGMCC 39090 were added, and the rest of the process was the same as in Comparative Example 1.

[0064] Example 7-(3): During the alcohol fermentation stage, 50% of sorghum mass of warming Daqu, 0.1% of brewing yeast CGMCC 34907, 0.1% of yeast CGMCC 39090 and 0.1% of compound enzyme were added. The rest of the process was the same as Comparative Example 1.

[0065] Example 7-(4): During the alcohol fermentation stage, 30% of sorghum mass fraction of warming Daqu, 20% of purple red Monascus CGMCC 42465 red yeast, 0.1% of brewing yeast CGMCC 34907 inoculum, 0.1% of guillemocma CGMCC 39090 inoculum and 0.1% of compound enzyme were added. The remaining process operations were the same as those in Comparative Example 1.

[0066] The composite enzymes have the following activity: xylanase activity ≥2000 U / g and cellulase activity ≥100 U / g.

[0067] The specific measurement method is as follows: (1) Determination methods of physicochemical indicators and functional components in aged vinegar: Determination methods of total acid, amino acid nitrogen, non-volatile acid, total ester, total soluble solids, total flavonoids, ligustrazine, reducing sugar and alcohol content: refer to the national standard GB / T19777—2013 "Geographical Indication Product Shanxi Aged Vinegar" for determination; Determination method of starch: refer to the national standard GB5009.9-2023 "National Food Safety Standard Starch in Food" for determination.

[0068] (2) Methods for determining flavor compounds in aged vinegar Determination of organic acids: Take 1000 μL of the test solution, dilute to 10 mL with ultrapure water, filter through a 0.22 μm microporous membrane, and inject the sample to determine the content of eight organic acids (oxalic acid, tartaric acid, pyruvic acid, malic acid, lactic acid, acetic acid, citric acid, and succinic acid). High performance liquid chromatography conditions: Agilent ZORBAX-C 18 A chromatographic column (4.6 mm × 150 mm × 5 μm) was used; 20 mmol / L NaH₂PO₄ was used as the mobile phase; the injection volume was 20 μL; the flow rate was 0.8 mL / min; the wavelength was 210 nm; and the column temperature was 30℃. The external standard method was used for quantitative analysis of organic acids in aged vinegar. Qualitative analysis was based on the retention time of each organic acid, while quantification was performed using the external standard method with peak area, with standard solutions of different concentrations of organic acids injected separately.

[0069] Amino acids: Take 2 mL of the test solution, 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 the supernatant and pass it through a membrane for testing using a fully automated amino acid analyzer.

[0070] Determination of volatile aroma components: The extraction head was aged at 270 ℃ in the injection port until no impurity peaks were observed. 100 μL of a mixed internal standard (tert-amyl alcohol, n-amyl acetate, and 2-ethylbutyric acid, with mass concentrations of 297.88 mg / L, 296.10 mg / L, and 295.87 mg / L, respectively) was added to aged vinegar. The mixture was equilibrated in a 45 ℃ incubator for 30 min, followed by headspace adsorption for 30 min. The extraction head was then inserted into the injection port, and desorption was performed at 270 ℃ for 5 min to extract the volatile aroma components from the sample. Chromatographic conditions: VF-5MS column (30 m × 0.25 mm × 0.25 μm), carrier gas: helium, purity 99.999%, flow rate 1 mL / min, splitless. Temperature program: initial temperature 40 ℃, hold for 3 min, then increase to 160 ℃ at a rate of 4 ℃, hold for 1 min. The temperature was then increased to 270 °C at a rate of 10 °C / min and held for 5 min. Mass spectrometry conditions: interface temperature 280 °C, ion source temperature 280 °C, electron energy 70 eV, scan mass range 41-500 amu, and internal standard method was used for quantitative analysis.

[0071] As shown in Table 4, compared with Comparative Example 1, all indicators of the mash in Example 7-(4) were significantly improved. Among the improvements, the alcohol content increased from 7.12% vol to 9.08% vol, an increase of 27.53%, mainly attributed to the enhanced starch saccharification ability of the heated Daqu (a type of starter culture), while the high-ethanol-producing brewer's yeast CGMCC 34907 improved the conversion efficiency of fermentable sugars to ethanol; the total ester content increased from 1.07 g / 100g to 2.15 g / 100g, an increase of 100.93%, mainly due to the high ester-producing characteristics of *Saccharomyces cerevisiae* CGMCC39090, while the saccharifying and esterifying enzymes provided by *Monascus purpureus* CGMCC 42465 promoted the release of alcohol precursors, jointly promoting the accumulation of esters; the amino acid nitrogen increased from 0.07 g / 100g to 0.16 g / 100g, an increase of 128.57%, indicating that the complex enzymes promoted the degradation of raw materials and increased the release of macromolecules such as proteins and polysaccharides; and γ-aminobutyric acid (GABA) increased from 3.21 mg / 100g to 7.86 mg / 100g. The concentration of lovastatin increased from undetectable to 3.76 μg / g, indicating that Monascus purpureus CGMCC 42465 promoted the accumulation of functional components.

[0072] Table 4. Results of determination of physicochemical indicators and functional components of mash at the end of alcoholic fermentation. Note: "ND" indicates not detected.

[0073] Comparative Example 2: Based on Example 7-(4), alcoholic fermentation was used to obtain mash, and the remaining acetic acid fermentation process was the same as that of Comparative Example 1.

[0074] Example 8: Enhanced application of acetic acid fermentation stage based on Example 7-(4) Example 8-(1): During the acetic acid fermentation stage, 120% sorghum bran was added, and the rest of the process was the same as in Comparative Example 2.

[0075] Example 8-(2): During the acetic acid fermentation stage, 120% sorghum bran and 1% vinegar mash protease were added. The remaining process operations were the same as those in Comparative Example 2.

[0076] Example 8-(3): During the acetic acid fermentation stage, 120% sorghum bran, 1% vinegar mash protease, and 0.1% Bacillus plantarum CGMCC 15731 inoculant were added. The remaining process operations were the same as those in Comparative Example 2.

[0077] Example 8-(4): During the acetic acid fermentation stage, 120% wheat bran, 10% sorghum husk, and 5% sea buckthorn leaf powder (dried and pulverized to 40 mesh) were added to the acetic acid mash. 1% protease and 0.1% Bacillus plantarum CGMCC 15731 inoculant were added to the acetic acid mash. The remaining process operations were the same as those in Comparative Example 2.

[0078] The protease has an enzyme activity of ≥8000 U / g and an optimal pH of 4.0–5.0.

[0079] As shown in Table 5, compared with Comparative Example 2, Example 8-(4) showed the best overall effect. The total acid content increased from 5.12 g / 100g to 6.58 g / 100g, an increase of 28.52%; the non-volatile acid content increased from 1.02 g / 100g to 2.19 g / 100g, an increase of 114.71%; and the amino acid nitrogen content increased from 0.15 g / 100g to 0.43 g / 100g, an increase of 186.67%. At the same time, the contents of total flavonoids, tetramethylpyrazine, and γ-aminobutyric acid were 252.36 mg / 100g, 58.62 mg / kg, and 17.42 mg / 100g, respectively, which increased by 90.35%, 106.33%, and 121.91%. The above results indicate that adding a high proportion of wheat bran during the acetic acid fermentation stage provided sufficient nitrogen source and nutrient matrix for microbial metabolism; proteases promoted the hydrolysis of raw material proteins, increasing the release of free amino acids and flavor precursors; the *Lactobacillus plantarum* CGMCC 15731 inoculant further promoted amino acid conversion and the accumulation of functional components such as organic acids and γ-aminobutyric acid; the abundant flavonoids in sorghum husks and sea buckthorn leaf powder were fully dissolved during solid-state acetic acid fermentation, increasing the total flavonoid content in the vinegar mash, and synergistically promoting the formation of nitrogen-containing aroma substances such as tetramethylpyrazine with the fermentation system, achieving simultaneous improvement in physicochemical quality and functional components during the acetic acid fermentation stage. The stable lovastatin content indicates that it mainly originated from the addition of *Monascus purpureus* during the alcoholic fermentation stage, rather than from the acetic acid fortification process.

[0080] Table 5. Results of determination of physicochemical indicators and functional components of vinegar mash at the end of acetic acid fermentation. Example 8-(4): In the alcoholic fermentation stage, 30% of the sorghum mass fraction of warming Daqu, 20% of purple red yeast rice, 0.1% of brewing yeast CGMCC 34907 inoculum, 0.1% of yeast kiwifruit CGMCC 39090 inoculum, and 0.1% of compound enzyme were added; in the acetic acid fermentation stage, 120% of the sorghum mass fraction of wheat bran, 10% of sorghum husk, and 5% of sea buckthorn leaf powder (dried and pulverized to 40 mesh) were added, and the vinegar mash mass fraction of 1% of protease and 0.1% of Bacillus plantarum CGMCC 15731 inoculum were added. The remaining process operations were the same as those in Comparative Example 1.

[0081] As shown in Table 6, in Example 8-(4), the total acid content increased from 5.06 g / 100mL to 6.32 g / 100mL, and the contents of non-volatile acid, amino acid nitrogen, total ester, total flavonoids and ligustrazine increased by 33.88%, 176.92%, 107.41%, 41.34% and 122.46%, respectively. The contents of γ-aminobutyric acid and lovastatin reached 10.86 mg / 100mL and 2.86 μg / mL, respectively. The above results indicate that the use of warming Daqu (a type of starter culture), *Monascus purpureus* CGMCC 42465 (red yeast rice), *Saccharomyces cerevisiae* CGMCC 39090 (yeast extract), and compound enzymes to enhance alcoholic fermentation improved the accumulation of flavor precursors. High proportions of wheat bran, protease, *Lactobacillus plantarum* CGMCC 15731 (inoculum extract), sorghum husk, and sea buckthorn leaf powder synergistically promoted the formation of acids, amino acids, flavonoids, and flavor compounds, achieving a simultaneous improvement in the flavor quality and functional components of the new vinegar.

[0082] Table 6. Determination results of physicochemical properties and functional components of balsamic ester acetic acid As shown in Table 7, after one year of natural sun-drying and one year of cellar aging, the total acid of the aged vinegar in Example 8-(4) reached 8.64 g / 100mL, while the non-volatile acid, amino acid nitrogen, and total esters reached 2.72 g / 100mL, 0.46 g / 100mL, and 4.29 g / 100mL, respectively. During the aging process, acid-alcohol esterification, protein hydrolysis, and continuous release of functional components resulted in the levels of tetramethylpyrazine, γ-aminobutyric acid, and lovastatin reaching 76.53 mg / L, 18.65 mg / 100mL, and 4.25 μg / mL, respectively. This indicates that the dual-solid-state enhanced fermentation combined with the aging process can effectively promote the accumulation of flavor substances and functional components, thereby improving the quality of aged vinegar.

[0083] Table 7. Results of determination of physicochemical properties and functional components of aged vinegar with aromatic esters obtained by natural sun-drying for 1 year and cellar aging for 1 year. As shown in Table 8, after a two-stage intensified fermentation process involving alcohol and acetic acid, followed by long-term aging, the aged vinegar developed a rich composition of organic acids and amino acids. The total amount of organic acids reached 6.7124 g / 100 mL, with acetic acid and lactic acid accounting for the majority, at 4.8265 g / 100 mL and 0.9825 g / 100 mL respectively, giving the product a harmonious sour taste and a mellow flavor.

[0084] Table 8. Results of determination of organic acid content in aged vinegar with aromatic esters after 1 year of natural sun-drying and 1 year of cellar aging. Table 9 shows that the total amino acid content reached 646.51 mg / 100 mL. Alanine, glutamic acid, and aspartic acid were present in relatively high amounts, reaching 105.28 mg / 100 mL, 96.37 mg / 100 mL, and 82.46 mg / 100 mL, respectively, accounting for a large proportion of the total amino acid content. Alanine imparts a mellow sweet aroma to the vinegar, while glutamic acid and aspartic acid enhance its umami characteristics. The results indicate that the proteins derived from sorghum, daqu (a type of starter culture), and wheat bran are fully degraded through microbial metabolism and enzymatic hydrolysis, promoting the accumulation of free amino acids and improving the flavor harmony of aged vinegar.

[0085] Table 9. Results of determination of amino acid content in aged vinegar with aromatic esters obtained by natural sun-drying for 1 year and cellar aging for 1 year. As shown in Table 10, the aged vinegar of Example 8-(4) contained 31 esters and 8 alcohols, which are 8 major categories of volatile flavor substances. The total amount of esters was 3.84 mg / 100mL, which is the core of the aroma. Ethyl acetate and ethyl lactate contribute to the rich fruit aroma. The high proportion of bran and gradient smoke mash enriches pyrazine substances and gives it a typical smoke aroma. Fermentation enhances alcohols, aldehydes and phenols to form a complex aroma of grain, yeast and aged aroma. Compared with the traditional process, the aroma components are richer and the flavor is more full.

[0086] Table 10 Results of determination of volatile aroma compounds in aged vinegar obtained by natural sun-drying for 1 year and cellar aging for 1 year. 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 aged vinegar rich in non-volatile acids, tetramethylpyrazine, and flavonoids through "double-solid" fermentation, characterized in that: Using tannin-rich sorghum as raw material and based on the high-temperature Daqu process, the alcoholic fermentation stage adds Monascus purpureus CGMCC 42465 red yeast, high-yield alcohol brewing yeast CGMCC 34907 inoculum, high-yield ester-producing Saccharomyces meyerii CGMCC 39090 inoculum, and a complex enzyme of xylanase and cellulase. Based on the weight of sorghum, the acetic acid fermentation stage adds 120% wheat bran, sorghum husk, sea buckthorn leaf powder, high-yield non-volatile acid Bacillus plantarum CGMCC 15731 inoculum, and protease. The process is carried out using a gradient temperature full-smoking process, followed by sun-drying and aging for 1 year and cellar aging to produce fragrant ester aged vinegar.

2. The method for preparing aged vinegar rich in non-volatile acids, tetramethylpyrazine, and flavonoids through "double-solid" fermentation according to claim 1, characterized in that: Includes the following steps: Crush sorghum into four or six pieces, add water at a sorghum mass fraction of 350%, and moisten for 10-22 hours. After moistening, mix sorghum, rice husks, rice bran, and water at a mass-volume ratio of 20:12:3:

8. Stir the mixture thoroughly, place it in a steamer, and steam under normal pressure for 90 minutes, then let it sit overnight. After cooling, add water at a sorghum mass fraction of 300%, and add 30% of a warming starter culture, 20% of *Aspergillus oryzae* CGMCC42465 red yeast rice, 0.1% of *Saccharomyces cerevisiae* CGMCC 34907 inoculum, and 0.1% of *Saccharomyces cerevisiae* CGMCC. Mix 39090 microbial agent and a compound enzyme of xylanase and cellulase (0.1%) evenly. Transport the mixed mash to the alcohol fermentation tank, flatten it after entering the tank, remove air, and ferment in a solid state for 8 days until the alcohol content reaches 8-9% vol. After alcohol fermentation, obtain the mash. Mix the fermented mash with 120% sorghum bran, 10% sorghum husk, and 5% dried and crushed sea buckthorn leaf powder (40 mesh). Then, put it into an acetic acid fermentation tank and inoculate it with 10% aril mash, 1% protease, and 0.1% *Lactobacillus plantarum* CGMCC. The acetic acid fermentation process is completed after 12 days of fermentation using 15731 microbial agent, resulting in a total acid content of 5-6 g / 100 mL. The fermented vinegar mash is then smoked for 5 days in an electrically heated stainless steel tank, with the moisture content controlled at 68% throughout. Temperature is controlled in gradients: 60-65℃ on the first day, 65-75℃ on the second, 75-85℃ on the third, 85-90℃ on the fourth, and 90-95℃ on the fifth. After leaching, the total acid content is reduced to 5 g / 100 mL. The mixture is then aged using a process of "summer sun-drying and winter ice-harvesting" until the total acid content reaches ≥6.5 g / 100 mL, followed by another year of cellar aging to obtain fragrant aged vinegar.

3. The method for preparing aged vinegar rich in non-volatile acids, tetramethylpyrazine, and flavonoids through "double-solid" fermentation according to claim 2, characterized in that: The preparation method of the heated Daqu is as follows: the raw materials are crushed and mixed in a mass ratio of barley:wheat:pea = 5.5:1.5:

3. After sieving, the coarse flour accounts for 30%-40% and the fine flour accounts for 60%-70%. Water of 37%-50% of the total mass of the Daqu raw materials is added and mixed. The mixture is pressed into brick-shaped Daqu blocks of 28cm×17cm×6cm with a moisture content of 36%. The Daqu is then processed through a mold growth period, a mold drying period, a damp fire period, a high fire period, and a post-fire period to produce room Daqu, which takes a total of 21 days. After room Daqu is produced, it undergoes a 3-month maturation and drying stage to finally form a high-quality finished Daqu with a pure aroma, uniform mycelium distribution on the surface, and rich enzyme system.

4. The method for preparing aged vinegar rich in non-volatile acids, tetramethylpyrazine, and flavonoids through "double-solid" fermentation according to claim 2, characterized in that: The preparation method of the *Monascus purpureus* CGMCC42465 red yeast rice is as follows: Select plump indica rice, soak it in water at 23-27℃ for 2 hours, and judge by the criterion that there is no hard core when rubbed between the fingers. After soaking, rinse twice and drain until there is no dripping water on the surface. Pour the drained indica rice into a steamer and steam for 1 hour, until the rice is cooked without white core, does not crack, and is not sticky. Touch the rice with wet hands and it will not stick. Transfer the steamed indica rice to a fermentation tray and let it cool at room temperature to 28-32℃. Inoculate with a *Monascus purpureus* spore suspension at an inoculation rate of 30% of the dry weight of the raw material, mix thoroughly, and ensure that each grain of rice is inoculated. The rice grains, coated with bacterial solution, are placed in clean, damp burlap sacks and sealed tightly. They are then incubated at 30°C for 48 hours, with water added every 24 hours. When the temperature rises to 45°C and more than 70% of the rice grains have mycelial colonization, the rice is soaked in koji. During soaking, the rice grains with mycelial colonization are wrapped in gauze and soaked in distilled water at 23-27°C for 20 seconds. After draining, the rice grains are poured back into the koji tray and spread evenly. The rice is soaked for 2 days, with soaking every 8 hours. When all the rice grains are uniformly dark red and have a strong red koji aroma, fermentation is stopped. The rice is then dried at 37°C until the moisture content is ≤12%, thus obtaining the purple red koji mold CGMCC42465 red koji. The method for preparing the *Monascus purpureus* spore suspension is as follows: *Monascus purpureus* CGMCC 42465 activated on a slant culture medium is used to scrape mycelia from the surface of the medium using a sterile inoculation loop. After scraping three loops consecutively, the mycelia are inoculated into rice flour liquid medium to ensure consistent inoculation. The medium is then incubated at 30℃ and 180 r / min on a shaker for 48 hours. During this period, the rice flour liquid medium is observed to turn pink. After another 48 hours of incubation, if the medium turns deep red, the suspension is obtained at a concentration of 10%. 6 CFU / mL Aspergillus violaceus spore suspension; The formula for the rice flour liquid culture medium is as follows: 2.5 g rice flour, 0.15 g NaNO3, 0.05 g MgSO4·7H2O, 0.075 g KH2PO4, add water to make up to 50 mL, and then sterilize at 0.1 MPa and 121℃ for 20 min.

5. The method for preparing aged vinegar rich in non-volatile acids, tetramethylpyrazine, and flavonoids through "double-solid" fermentation according to claim 2, characterized in that: The preparation method of the Saccharomyces cerevisiae CGMCC 34907 inoculum is as follows: Saccharomyces cerevisiae CGMCC34907 seed culture is inoculated into 75L of yeast extract liquid culture medium at a 3% inoculation rate. The medium is cultured at 30℃ with an aeration rate of 0.6L / min for 24 hours. During this period, 0.2% (v / v) of glucose from the yeast extract liquid culture medium is added every 8 hours until the cell concentration reaches 10... 8 The bacterial sludge was obtained by centrifuging at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-mass ratio of 3:1, and the mixture was thoroughly mixed. The mixture was then spread evenly on the freeze dryer tray to a thickness of 1 cm. The material was frozen at -60℃ for 6 hours. The freeze dryer was pre-cooled, and once the cold trap temperature stabilized at -50℃, the frozen material was placed inside. Vacuum freeze drying was performed using a segmented temperature control mode to obtain the *Saccharomyces cerevisiae* CGMCC 34907 inoculum with a viable count of 10-1. 9 CFU / g, vacuum packaged and stored at 4℃; The method for preparing the Saccharomyces cerevisiae CGMCC 34907 seed culture is as follows: culture the culture in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Saccharomyces cerevisiae* CGMCC 34907 (CFU / mL) was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium. The fermentation was carried out at 30℃, aeration rate of 0.2L / min, and rotation speed of 200 rpm for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL.

6. The method for preparing aged vinegar rich in non-volatile acids, tetramethylpyrazine, and flavonoids through "double-solid" fermentation according to claim 2, characterized in that: The preparation method of *Saccharomyces cerevisiae* CGMCC 39090 inoculum is as follows: *Saccharomyces cerevisiae* CGMCC 39090 seed culture is inoculated at a rate of 3% into 75L of yeast extract liquid culture medium. The medium is cultured at 30℃ with an aeration rate of 0.6L / min for 24 hours. During this period, 0.2% (v / v) of glucose from the yeast extract liquid culture medium is added every 8 hours until the cell concentration reaches 10... 8 The bacterial sludge was obtained by centrifuging at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-mass ratio of 3:1, and the mixture was thoroughly mixed. The mixture was then spread evenly on the freeze dryer tray to a thickness of 1 cm. The material was frozen at -60℃ for 6 hours. The freeze dryer was pre-cooled, and once the cold trap temperature stabilized at -50℃, the frozen material was placed inside. Vacuum freeze drying was performed using a segmented temperature control mode to obtain *Saccharomyces cerevisiae* CGMCC39090 bacterial inoculum with a viable count of 10-1. 9 CFU / g, vacuum packaged and stored at 4℃; The method for preparing the seed culture of *Saccharomyces cerevisiae* CGMCC 39090 is as follows: culture the culture in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Saccharomyces cerevisiae* CGMCC 39090 (CFU / mL) was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium. The culture was incubated at 30℃, aeration rate of 0.2L / min, and rotation speed of 200 rpm for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL.

7. The method for preparing aged vinegar rich in non-volatile acids, tetramethylpyrazine, and flavonoids through "double-solid" fermentation according to claim 2, characterized in that: The preparation method of the *Lactobacillus plantarum* CGMCC 15731 inoculum is as follows: *Lactobacillus plantarum* CGMCC 15731 seed culture is inoculated into 75 L of yeast extract liquid culture medium at a 3% inoculation rate. The medium is cultured at 37℃ and an aeration rate of 0.8 L / min for 48 h. During this period, 0.2% glucose (by volume) of yeast extract liquid culture medium is added every 8 h. The pH value of the fermentation broth is sampled and monitored every 12 h. The pH of the fermentation broth is adjusted to neutral by adding 20% ​​w / v sodium carbonate solution until the bacterial concentration reaches 10. 8 The bacterial sludge was obtained by centrifuging at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-mass ratio of 3:1 (bacterial cell freeze-drying protectant to bacterial sludge). After thorough mixing, the mixture was spread evenly onto the freeze dryer tray to a thickness of 1 cm. The material was then frozen at -60℃ for 6 hours. The freeze dryer was pre-cooled beforehand, and the frozen material was placed in the cold trap after the temperature stabilized at -50℃. Vacuum freeze-drying was performed using a segmented temperature control mode to obtain *Lactobacillus plantarum* CGMCC 15731 bacterial agent with a viable count of 10-1. 9 CFU / g, vacuum packaged and stored at 4℃; The method for preparing the *Lactobacillus plantarum* CGMCC 15731 seed culture is as follows: cultured in MRS liquid medium until the bacterial concentration reaches 10... 8 A 3% inoculum of *Lactobacillus plantarum* CGMCC 15731 was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium and cultured at 37℃, aeration rate of 0.2 L / min, and rotation speed of 200 r / min for 48 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL.

8. The method for preparing the microbial agent according to claims 5, 6, and 7, characterized in that: The method for preparing the yeast extract liquid culture medium is as follows: 1g yeast extract, 2g glucose, 0.3g yeast extract, 100mL water, sterilized at 121℃ for 20min for later use. The method for preparing the vacuum freeze-drying protectant is as follows: Protectant 1: 5 g skim milk powder, 8 g whey powder, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; Protectant 2: 5 g trehalose, 3 g sucrose, 5 g glucose, 0.5 g monosodium glutamate, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; After sterilization, protectant 1 and protectant 2 are cooled to room temperature and mixed evenly at a volume ratio of 1:1 to obtain the bacterial cell vacuum freeze-drying protectant; The parameters for the segmented temperature-controlled vacuum freeze-drying process are set as follows: First segment: -60℃, 15min; Second segment: -25℃, 1920min; Third segment: 0℃, 420min; Fourth segment: 20℃, 120min.

9. The preparation method according to claim 1, characterized in that: The complex enzyme comprises xylanase and cellulase, wherein the xylanase activity is ≥2000 U / g and the cellulase activity is ≥100 U / g; the protease activity is ≥8000 U / g and the optimal pH is 4.0-5.

0.

10. The method for preparing aged vinegar rich in non-volatile acids, tetramethylpyrazine, and flavonoids through "double-solid" fermentation according to claim 1, characterized in that: The prepared aged vinegar is clear and transparent, with a mellow, sweet and sour taste, combining the aromas of grain, yeast, smokiness and aging. It has no off-flavors or odors, and contains ≥8.50 g / 100mL of total acid, ≥2.50 g / 100mL of non-volatile acid, ≥70.00mg / L of tetramethylpyrazine, ≥350.00mg / 100mL of total flavonoids, and ≥4.00g / 100mL of total esters.

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