Method for improving flavor and nutritional functional components of solid-state brewed vinegar by using monascus purpureus-eurotium cristatum fructus forsythiae tea koji

Abstract

The invention belongs to the technical field of microbial fermentation, and provides a method for improving flavor and nutritional functional components of solid-state brewed vinegar by using monascus purpureus-eurotium cristatum fructus forsythiae tea koji. The method comprises the following steps: mixing a monascus purpureus strain CGMCC (China General Microbiological Culture Collection Center) 42465 with high yield of gamma-aminobutyric acid and lovastatin with eurotium cristatum CICC 2099, inoculating the mixture into a forsythia suspensa leaf indica rice oat bran solid state fermentation culture medium, and culturing in a wet jute bag to prepare monascus purpureus-eurotium cristatum forsythia suspensa tea koji; the method is applied to solid-state vinegar brewing. The obtained new sprayed vinegar is reddish brown and rich in color, the total acid content is 5.78 g / 100 mL, the total ester content is 2.72 g / 100 mL, the flavone content is 163.48 mg / 100 mL, the gamma-aminobutyric acid content is 42.49 mg / 100 mL, and the lovastatin content is 1.82 mu g / mL, which are respectively increased by 23.50%, 46.24%, 95.67%, 61.31% and 73.33% compared with a control group, and the types and contents of organic acids and volatile aroma components are enriched.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for enhancing the flavor and nutritional components of solid-state brewed vinegar using Aspergillus purpureus-Eurotium cristatum Forsythia tea starter. Background Technology

[0002] Most Chinese grain vinegars are solid-state fermented vinegars, meaning they are made using solid-state fermentation techniques during the acetic acid fermentation stage. Taking Shanxi aged vinegar as an example, the main raw material is sorghum, with Daqu (a type of starter culture) as the saccharification and fermentation agent. It is produced through multiple processes including low-temperature concentrated mash alcoholic fermentation, solid-state acetic acid fermentation, smoking, vinegar extraction, and aging. Currently, consumers have higher demands for the richness of vinegar's flavor and nutritional value, especially for functional vinegars that are both food and medicine. This has become an important direction for driving vinegar's transformation from a traditional condiment to a high-end health product. Currently, most functional vinegars on the market are prepared by soaking finished vinegar or directly adding extracts of functional active ingredients. Not only are the types and contents of these functional active ingredients unclear, but the market acceptance of these exogenous functional additives is also low.

[0003] Forsythia, a traditional Chinese medicine native to Shanxi Province, is recognized as one of the "Ten Great Shanxi Medicines" along with Astragalus membranaceus and Bupleurum chinense. It possesses properties such as clearing heat and detoxifying, reducing swelling and dissipating nodules, and dispelling wind-heat. It is widely distributed in the Zhongtiao Mountains, Taiyue Mountains, Taihang Mountains, Wutai Mountains, and Lüliang Mountains, with Lingchuan County being a major production area nationwide. Forsythia fruit contains active ingredients such as forsythoside A, forsythoside B ester A, and chlorogenic acid, which exhibit significant antibacterial, antiviral, and anti-inflammatory effects and are widely used in the medical field. Forsythia leaves, a processing byproduct of the Forsythia industry, also have significant medicinal value. They contain active ingredients such as forsythoside B ester A, rutin, forsythoside A, and chlorogenic acid, possessing various pharmacological activities including antioxidant and antiviral effects.

[0004] Monascus purpureus is an important fungus used in both food and medicine. It produces various beneficial secondary metabolites, such as monascus pigments, lovastatin, gamma-aminobutyric acid (GABA), and ergosterol, which have significant effects on lowering blood lipids, blood pressure, cholesterol, and blood sugar, as well as inhibiting bacteria. Monascus purpureus secretes various enzymes, such as esterases and saccharifying enzymes, which can break down complex substances in raw materials and promote the generation of metabolites during fermentation. Monascus purpureus is widely used in the preparation of red yeast rice vinegar, with Yongchun aged vinegar being a typical example, its unique flavor and nutritional value being highly favored by consumers. In the preparation of red yeast rice vinegar, Monascus purpureus not only enhances the color and taste of the product but also imparts more health benefits through its metabolites.

[0005] Coronavirus ( E.cristatum*Aspergillus cristatus* is the dominant microbial strain in the "fermentation" process of Fu brick tea. Because it produces golden-yellow cleistothecia, it is known as the "golden flower" fungus and is also a dual-purpose (medicinal and edible) fungus. This strain can secrete various extracellular enzymes such as polyphenol oxidase, protease, pectinase, and cellulase, which can decompose macromolecules such as proteins, pectin, and cellulose into free amino acids and soluble sugars, promoting the conversion of nutrients during fermentation and thus promoting the formation of flavor and functional substances. Studies have shown that *Aspergillus cristatus* has been used to ferment wolfberry leaves, improving the appearance and taste of traditional wolfberry leaf tea. Other studies have used it in the fermentation of medicinal and edible herbs such as Panax notoginseng, kudzu root, yam, and Dendrobium officinale, helping to obtain new processed products with enhanced or increased medicinal effects, further enriching and improving the value of traditional Chinese medicine. Summary of the Invention

[0006] This invention provides a method for enhancing the flavor and nutritional components of solid-state brewed vinegar using *Aspergillus purpureus*-*Aureomyces cristatus* Forsythia suspensa tea starter. A superior *Aspergillus purpureus* strain CGMCC 42465, which is highly productive in γ-aminobutyric acid (GABA) and lovastatin, isolated from Shanxi aged vinegar starter, is mixed with *Aureomyces cristatus* CICC 2099 and inoculated into a solid-state fermentation medium of forsythia leaves, indica rice, and oat bran. After cultivation in wet burlap sacks, *Aspergillus purpureus*-*Aureomyces cristatus* Forsythia suspensa tea starter is produced. This tea starter is then applied to the vinegar brewing process. After fermentation, the physicochemical indicators, functional substances, organic acids, and volatile aroma components of the newly extracted vinegar are systematically analyzed to clarify the effect of *Aspergillus purpureus*-*Aureomyces cristatus* Forsythia suspensa tea starter on enhancing the flavor and functional components of solid-state brewed vinegar.

[0007] This invention is achieved by the following technical solution: a method for enhancing the flavor and nutritional components of solid-state brewed vinegar using *Aspergillus purpureus*-*Aspergillus cristatus* *Forsythia suspensa* tea starter, wherein the *Aspergillus purpureus* strain (… Purple monkfish CGMCC42465, mixed with *Aspergillus cristatus* CICC 2099, was inoculated into a solid-state fermentation medium of *Forsythia suspensa* leaves, indica rice, oats, and bran, and cultured in wet burlap sacks to produce *Aspergillus purpureus*-*Aspergillus cristatus* *Forsythia suspensa* tea starter; this *Aspergillus purpureus*-*Aspergillus cristatus* *Forsythia suspensa* tea starter was then applied to solid-state vinegar brewing.

[0008] The preparation method of the *Aspergillus purpureus*-*Eurotium cristatum* Forsythia tea starter is as follows: Forsythia leaves with a moisture content of ≤10% are crushed to 40 mesh, whole indica rice is soaked in water at 15-20℃ for 2 hours, drained and steamed for 1 hour, and then spread on a starter tray to cool to 34-44℃. Mix forsythia leaf powder, steamed and cooled indica rice, and oat bran in a mass ratio of 60:25:15. Add 1-1.5 times the mass of water to the mixture and stir well to obtain the forsythia leaf-indica rice-oat bran solid-state fermentation medium. Mix the seed liquid of *Monascus purpureus* CGMCC42465 and the seed liquid of *Aspergillus cristatus* CICC 2099 in a volume ratio of 1:1 to prepare a mixed seed liquid. Inoculate the mixed seed liquid into the above forsythia leaf-indica rice-oat bran solid-state fermentation medium at an inoculation amount of 30% (v / w), mix thoroughly, and place in a damp burlap bag for incubation at 30°C for 48 hours. When more than 50% of the solid-state fermentation medium turns light red and the surface is dry, start soaking the koji (fermentation starter). Soak the koji every 12 hours for a total of 4 times. After the incubation is completed, dry the koji at 37°C until the moisture content is ≤12%, pulverize and pass through a 20-mesh sieve to obtain *Monascus purpureus*-*Aspergillus cristatus* forsythia tea koji.

[0009] The method for preparing the *Aspergillus purpureus* CGMCC 42465 seed culture is as follows: *Aspergillus purpureus* CGMCC 42465 activated on a slant was used to scrape the 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 culture medium to ensure consistent inoculation. The medium was then cultured at 30℃ and 180 r / min on a shaker for 48 h. During this period, the rice flour culture medium was observed to turn pink. Cultured for another 48 h until the medium turned purplish-red was obtained, thus obtaining the *Aspergillus purpureus* CGMCC 42465 seed culture. The rice flour culture medium formula was: 2.5 g rice flour, 0.15 g NaNO3, 0.05 g MgSO4·7H2O, and 0.075 g KH2PO4. Water was added to bring the volume to 50 mL, and the medium was sterilized at 121℃ for 20 min. The method for preparing the *Aurogonium cristatum* CICC 2099 seed culture is as follows: *Aurogonium cristatum* CICC 2099 activated on an slant culture is cultured with a spore count ≥ 1 × 10⁻⁶. 6 CFU / g was inoculated into PDA liquid culture medium and cultured at 30℃ and 180 r / min for 72 h. The PDA liquid culture medium formula was: 20 g potato, 2 g glucose, 0.2 g peptone, 0.2 g KH2PO4, 0.1 g MgSO4, 100 mL distilled water, and sterilized at 121℃ for 20 min.

[0010] The specific steps for solid-state vinegar brewing are as follows: 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 mass) 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℃. Another 0.1% (by mass) of saccharifying enzyme from the sorghum is added, and the mixture is kept at this temperature for 20-30 minutes. Once the temperature drops to around 30℃, the mixture is poured into an alcohol fermentation tank. 30-40% (by mass) of Daqu (a type of starter culture) and 10-20% (by mass) of *Aspergillus violaceus*-*Eurotium cristatum*-*Forsythia tea starter culture are added. 0.1%-0.5% (by mass) of Angel yeast inoculant from the sorghum is added. The mixture is fermented openly for 2 days, then sealed for 6-14 days until the alcohol content reaches 8-10% vol. The alcohol fermentation is then complete, yielding the mash. The fermented mash is thoroughly mixed with sorghum bran (120%–140% by weight), rice bran (70%–80% by weight), and rice husk (60%–70% by weight). Then, 10% sorghum mash is added to the mixture for solid-state acetic acid fermentation. The fermentation lasts for 12–15 days, and the total acid content is 5–6 g / 100 mL. After the acetic acid fermentation is completed, the mash is smoked and then leached to obtain new leached vinegar. The physicochemical properties, organic acid content, and volatile aroma substance content of the new leached vinegar are measured.

[0011] The purple-red Aspergillus ( Purple monkfish CGMCC 42465, deposited at the China General Microbiological Culture Collection Center on December 5, 2025, was isolated from Shanxi aged vinegar koji. When cultured in rice flour medium at 30℃ and 180 r / min for 48 h, it produced lovastatin at a content of 52.92 mg / 100 g and γ-aminobutyric acid at a content of 61.38 mg / 100 g, and also exhibited excellent enzyme activity characteristics, with amylase activity of 22.38 U / g and esterification power of 2485.62 U.

[0012] This invention uses Forsythia leaves as a substrate carrier to co-culture superior indigenous Monascus purpureus and Eurotium cristatum to prepare a novel medicinal and edible functional starter—Monascus purpureus-Eurotium cristatum Forsythia tea starter. Through the synergistic metabolic action of Monascus purpureus and Eurotium cristatum, the efficient transformation of medicinal and edible components and the generation of flavor substances are achieved. Introducing this functional starter into the solid-state fermentation process of vinegar replaces the traditional single-strain fermentation mode, promoting the decomposition of macromolecules and the enrichment of functional factors in the raw materials, ultimately forming a novel vinegar product rich in flavor substances and with clearly defined nutritional functions.

[0013] The new acetic acid prepared by this invention is reddish-brown with a rich color, total acid ≥ 5.50 g / 100 mL, total ester ≥ 2.50 g / 100 mL, flavonoids ≥ 150 mg / 100 mL, γ-aminobutyric acid ≥ 40.00 mg / 100 mL, lovastatin ≥ 1.50 μg / mL, and also enriches the types and contents of organic acids and volatile aroma components.

[0014] Compared with the prior art, the present invention has the following advantages: (1) This invention uses *Monascus purpureus* CGMCC 42465 and *Aspergillus cristatus* CICC 2099 to prepare *Monascus purpureus*-*Aspergillus cristatus* Forsythia tea starter. *Monascus purpureus* CGMCC 42465 is an excellent indigenous strain isolated from Shanxi aged vinegar starter, exhibiting good adaptability to the fermentation environment and possessing excellent characteristics such as high production of γ-aminobutyric acid, lovastatin, and pigments, while also possessing high amylase activity and esterification power. *Aspergillus cristatus* CICC 2099 is rich in metabolites, including various extracellular polysaccharides and tea polyphenols, and possesses a rich and efficient enzyme system, including cellulase, pectinase, and amylase, which promotes the conversion of active ingredients in the raw materials. The synergistic effect of the two bacteria not only enhances the flavor of the Forsythia tea starter but also significantly increases the proportion of its functional components.

[0015] (2) This invention uses “Forsythia leaves-Indica rice-Oat bran” as raw materials to construct a nutritionally balanced fermentation system. Forsythia leaves contain polyphenols and flavonoids, which provide basic functional components for the fermentation system. Indica rice can provide sufficient carbon and nitrogen sources, providing core nutritional support for the growth and metabolism of the strain. Oat bran can not only make the fermentation system loose and breathable, ensuring the respiration and metabolic efficiency of the strain, but also contains rich vitamins, minerals, growth factors and β-glucan, further enriching the nutritional dimensions of the fermentation system. At the same time, this raw material combination can effectively improve the comprehensive utilization rate of agricultural product processing by-products such as forsythia leaves and oat bran.

[0016] (3) This invention innovatively applies Aspergillus purpureus-Eurotium cristatum Forsythia tea starter to the alcoholic fermentation stage of Shanxi aged vinegar, thereby improving the nutritional components and functional active substances content of the product from the perspective of fermentation, enriching the flavor layers, and empowering the innovation of aged vinegar fermentation technology and the development of products towards diversification, functionalization and high quality.

[0017] The purple-red Aspergillus (Aspergillus violaceus) described in this invention Purple monkfish QM308, with accession number CGMCC 42465, is deposited at the China General Microbiological Culture Collection Center on December 5, 2025. Attached Figure Description

[0018] Figure 1 This image shows the colony morphology of Monascus purpureus strain QM308 on PDA agar plates. Figure 2 This is an ITS sequencing phylogenetic tree diagram of Monascus purpureus strain QM308. Detailed Implementation

[0019] 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.

[0020] 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.

[0021] 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 all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores; the Shanxi aged vinegar starter used in the following embodiments was from Shanxi Fuyuanchang Aged Vinegar Co., Ltd.; the *Aspergillus cristatus* CICC2099 used in the following embodiments was purchased from the China Industrial Microbiological Culture Collection Center; the *Monascus purpureus* 3.4629 used in the following embodiments was purchased from the China General Microbiological Culture Collection Center; the thermostable α-amylase and saccharifying enzyme used in the following embodiments were purchased from Shandong Longket Enzyme Preparation Co., Ltd.; the Angel Yeast inoculum used in the following embodiments was purchased from Angel Yeast Co., Ltd.; and the fungal DNA extraction kit used in the following embodiments was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0022] Example 1: Screening of superior Monascus strains Screening of superior Monascus purpureus strains: Ten Monascus purpureus strains isolated from Shanxi aged vinegar starter in the laboratory were compared with the fermentation characteristics of the commonly available Monascus purpureus 3.4629 to screen for superior strains. The Monascus purpureus strains were inoculated onto PDA solid slant agar and cultured at 30℃ for 5 days. The activated slant agar was then used to scrape the mycelia from the surface of the culture medium with a sterile inoculation loop and inoculated into rice flour culture medium. The medium was then cultured at 30℃ and 180 r / min in a shaker. During this period, the rice flour culture medium was observed to turn pink. The fermentation broth was obtained when the medium turned purplish-red after 48 hours. The rice flour culture medium formula was: 2.5 g rice flour, 0.15 g NaNO3, 0.05 g MgSO4·7H2O, and 0.075 g KH2PO4. Water was added to bring the volume to 50 mL, and the medium was sterilized at 121℃ for 20 min. The Monascus purpureus fermentation broth was filtered to obtain the test solution. The levels of γ-aminobutyric acid (GABA), lovastatin, pigments, polysaccharides, amylase activity, saccharification power, and esterification power in the test solution were determined using the following methods: 1. Determination method of γ-aminobutyric acid Sample pretreatment: Take 1.0 mL of the test solution and add 10 mL of 75% ethanol. Extract by sonication for 30 min, vortex for 2 min, and let stand for 5 min. Centrifuge at 8000 r / min for 5 min and collect the supernatant. Add 0.2 μL of 4% sodium bicarbonate solution and 0.4 μL of 0.2% 4-dimethylaminoazobenzene-4-sulfonyl chloride derivatizing reagent to 1 mL of the sample solution. Mix well and derivatize in a 70℃ water bath in the dark for 20 min. Filter through a 0.45 μm filter and perform HPLC analysis.

[0023] Preparation of γ-aminobutyric acid (GABA) standard curve: Dilute the prepared 1000.0 g / L GABA standard solution to make working standard solutions with mass concentrations of 10.0 mg / L, 20.0 mg / L, 40.0 mg / L, 80.0 mg / L, and 160.0 mg / L. Add 0.2 μL of 4% sodium bicarbonate solution and 0.4 μL of 0.2% 4-dimethylaminoazobenzene-4-sulfonyl chloride derivatizing reagent, mix well, and incubate in a water bath at 70℃ in the dark for 20 min. Filter through a 0.45 μm filter membrane for later use. High performance night phase chromatography detection conditions: Use C 18 The chromatographic column was 4.6 mm × 250 mm; the detection wavelength was 436 nm; the column temperature was 30℃; the injection volume was 10 μL; the flow rate was 1.0 mL / min; and the mobile phase was acetonitrile + 0.68% sodium acetate trihydrate solution (35%:65%, v / v). A standard curve for γ-aminobutyric acid (GABA) was plotted with the mass concentration (x) of the GABA standard solution as the abscissa and the peak area (y) as the ordinate. The GABA content in the sample was calculated according to the regression equation of the standard curve.

[0024] 2. Method for determining lovastatin content Sample pretreatment: Take 1.0 mL of the test solution, add 75% ethanol (v / v), mix well, and bring the volume to 50 mL. Sonicate at room temperature for 1 h. Centrifuge at 8000 r / min for 5 min, and filter the supernatant through a 0.45 μm filter membrane for HPLC analysis.

[0025] Preparation of the standard curve for lactone-type (closed-ring) lovastatin: Accurately weigh a certain amount of lovastatin standard and prepare lovastatin standard solutions with mass concentrations of 0.005 mg / mL, 0.030 mg / mL, 0.045 mg / mL, 0.090 mg / mL, and 0.270 mg / mL using 75% ethanol as the solvent. High-performance liquid chromatography (HPLC) detection conditions: The chromatographic column was C10. 18 A chromatographic column (4.6 mm × 250 mm) was used, with a mobile phase of methanol:water:phosphoric acid = 385:115:0.14 (v / v / v), a detection wavelength of 238 nm, a flow rate of 1 mL / min, a column temperature of 25 ℃, and an injection volume of 20 μL. A lovastatin standard curve was plotted with the mass concentration (x) of the lovastatin standard solution as the abscissa and the peak area (y) as the ordinate.

[0026] Preparation of acid (open-ring) lovastatin: Weigh 4 mg of lovastatin (lactone) standard, dilute to 100 mL with 0.2 mol / L sodium hydroxide solution, sonicate at 50℃ for 1 h, and allow to stand at room temperature for another 1 h. Add the peak areas of acid (open-ring) lovastatin and lactone (closed-ring) lovastatin in the sample, and substitute the sum of the peak areas into the standard curve regression equation to calculate the lovastatin content in the sample.

[0027] 3. Color value determination method Transfer 1 mL of the test solution to 9 mL of 70% ethanol solution, place in a constant temperature shaker at 60℃, extract at 200 r / min for 1 h, centrifuge at 4000 r / min for 15 min, take the supernatant and dilute to 20 times, and measure the absorbance of the yellow pigment at 410 nm. The result is calculated as the OD value multiplied by the dilution factor.

[0028] 4. Polysaccharide determination method Add 1 mL of the test solution to 1 mL of 1 mol / L NaOH solution, shake thoroughly, incubate at 60℃ for 1 h, centrifuge at 10,000 r / min for 10 min, and the supernatant is the polysaccharide sample. Take 0.5 mL of the polysaccharide sample diluted 400 times, add 0.75 mL of 5% phenol solution and 3.75 mL of concentrated sulfuric acid, mix well, let stand at room temperature for 30 min, and measure the absorbance at 490 nm. Using glucose as a standard, plot a standard curve using the same method. Substitute the sample absorbance value into the standard curve to calculate the polysaccharide content.

[0029] 5. Enzyme activity assay method Sample pretreatment: Weigh 5 mL of the test solution, add 6 mL of acetate-sodium acetate buffer, add distilled water to 60 mL, stir well and place in a 35℃ water bath; keep at constant temperature for 1 h (stir for the first 30 min, then let stand for the next 30 min), centrifuge and take the supernatant as enzyme solution.

[0030] Method for determining amylase activity: Add 0.02 mL of enzyme solution to 0.18 mL of 1% soluble starch, react at 60℃ for 10 min, add 0.175 mL of DNS reagent, boil for color development for 5 min, then terminate the reaction. After cooling, add distilled water to make a final volume of 2.5 mL. Measure the OD value of each sample at a wavelength of 510 nm.

[0031] Method for determining saccharification power: Refer to the saccharification power determination method in the light industry standard QB / T4257—2011 "General Analytical Methods for Brewing Daqu". Method for determining esterification power: Refer to the esterification power determination method in QB / T5188—2017 "Brewing Red Yeast Rice".

[0032] The results are shown in Table 1. As can be seen from Table 1, among the 10 strains, the γ-aminobutyric acid content, lovastatin content, color value, and esterification power of Monascus purpureus strain QM308 reached 61.38 mg / 100 g, 52.92 mg / 100 g, 50.67 U, and 2485.62 U, respectively, which were significantly higher than those of other strains. Compared with the commonly available Monascus purpureus 3.4629, the γ-aminobutyric acid content, lovastatin content, color value, and esterification power were increased by 73.93%, 15.34%, 19.22%, and 42.87%, respectively.

[0033] Table 1. Results of functional substance and enzyme activity assays of different Monascus purpureus strains 2. Identification of superior Monascus purpureus strain QM308 Morphological identification: Mycelia of Monascus purpureus strain QM308 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 colony morphology of Monascus purpureus strain QM308 on PDA agar plates is shown in the figure. Figure 1The colonies are regular and round, dense and thick, without spreading, and with clear and neat edges; the colony color is bright orange-red to typical purplish-red, with a uniform and full color; the surface is covered with abundant aerial hyphae, which are soft and fluffy, and have a loose texture.

[0034] Sequencing: Hyphae of strain QM308, cultured on slant agar, 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 comparisons were performed using BLAST software, and an ITS sequencing phylogenetic tree was constructed. The ITS sequencing phylogenetic tree of Monascus purpureus strain QM308 is shown below. Figure 2 Strain QM308 was identified as *Aspergillus violaceus* (…). Monk purple It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 42465 and deposit date of December 5, 2025.

[0035] Example 2: Preparation method of Forsythia tea starter from Aspergillus purpureus-Eurotium cristatum (1) Preparation of Forsythia tea starter from Aspergillus purpureus-Eurotium cristatum Forsythia leaves with a moisture content ≤10% are pulverized to 40 mesh. Whole indica rice is soaked in water at 15-20℃ for 2 hours, drained, and steamed for 1 hour. It is then spread evenly on a fermentation tray and cooled to 34-44℃. Forsythia leaf powder, steamed and cooled indica rice, and oat bran are mixed at a mass ratio of 60:25:15. Simultaneously, water is added in an equal volume ratio to the above materials and mixed thoroughly. This is the solid-state fermentation medium for forsythia leaves, indica rice, and oat bran. A mixed seed solution is prepared by mixing Aspergillus purpureus CGMCC 42465 seed solution and Aspergillus cristatus CICC 2099 seed solution at a volume ratio of 1:1. This mixed seed solution is inoculated into the above solid-state fermentation medium for forsythia leaves, indica rice, and oat bran at an inoculation rate of 30% (v / w). The mixture is thoroughly mixed and placed in a damp burlap sack for incubation at 30℃ for 48 hours. Fermentation begins when more than 50% of the solid-state fermentation medium turns pale red and the surface is dry. Fermentation is carried out every 12 hours. Soak the starter once, for a total of 4 times; after the culture is completed, dry it at a low temperature of 37℃ until the moisture content is ≤12%, pulverize it and pass it through a 20-mesh sieve to obtain the purple-red Aspergillus-Eurotium cristatum Forsythia tea starter.

[0036] The method for preparing the *Aspergillus purpureus* CGMCC 42465 seed culture is as follows: *Aspergillus purpureus* CGMCC 42465 activated on a slant was used to scrape the 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 culture medium to ensure consistent inoculation. The medium was then cultured at 30℃ and 180 r / min on a shaker for 48 h. During this period, the rice flour culture medium was observed to turn pink. Cultured for another 48 h until the medium turned purplish-red was obtained, thus obtaining the *Aspergillus purpureus* CGMCC 42465 seed culture. The rice flour culture medium formula was: 2.5 g rice flour, 0.15 g NaNO3, 0.05 g MgSO4·7H2O, and 0.075 g KH2PO4. Water was added to bring the volume to 50 mL, and the medium was sterilized at 121℃ for 20 min.

[0037] The method for preparing the *Aurogonium cristatum* CICC 2099 seed culture is as follows: *Aurogonium cristatum* CICC2099 activated on an slant culture is prepared with a spore count ≥1×10⁻⁶. 6 CFU / g was inoculated into PDA liquid medium and cultured at 30℃ and 180 r / min for 72 h. The formula of the above PDA liquid medium was: 20 g potato, 2 g glucose, 0.2 g peptone, 0.2 g KH2PO4, 0.1 g MgSO4, and sterilized at 121℃ for 20 min.

[0038] (2) Preparation of Forsythia suspensa tea starter from Coronavirus cristatum Inoculate the *Aspergillus cristatus* CICC 2099 seed culture into the solid-state fermentation medium of *Forsythia suspensa* leaves, rice, and oat bran at a rate of 30% (v / w) and mix thoroughly. The remaining procedures are the same as described above.

[0039] Table 2 shows that the content of functional components and enzyme activity in *Monascus purpureus*-*Aspergillus cristatus* Forsythia suspensa tea koji were significantly superior to those in *Aspergillus cristatus* Forsythia suspensa tea koji. Regarding functional components, the contents of γ-aminobutyric acid (GABA), flavonoids, and polyphenols in *Monascus purpureus*-*Aspergillus cristatus* Forsythia suspensa tea koji were 96.78 mg / 100 g, 58.67 mg / g, and 15.68 mg / g, respectively, representing increases of 98.89%, 64.53%, and 59.03% compared to *Aspergillus cristatus* Forsythia suspensa tea koji. Furthermore, its lovastatin content reached 78.48 mg / 100 g, while it was not detected in *Aspergillus cristatus* Forsythia suspensa tea koji. In terms of enzyme activity, the amylase activity, saccharification power, and esterification power of *Monascus purpureus*-*Aspergillus cristatus* Forsythia suspensa tea koji were increased by 57.04%, 85.51%, and 58.54% compared to *Aspergillus cristatus* Forsythia suspensa tea koji. This is due to the synergistic effect between *Monascus purpureus* and *Eurotium cristatum* during the fermentation of *Forsythia suspensa* tea. Enzymes secreted by *Monascus purpureus* break down complex substances in the raw materials, providing nutritional precursors for *Eurotium cristatum*; conversely, the metabolites of *Eurotium cristatum* enhance the enzyme activity of *Monascus purpureus*. This interaction significantly increases the content of functional substances in the system and optimizes the fermentation environment, thereby enhancing key indicators such as saccharification power, amylase activity, and esterification power.

[0040] Table 2. Results of determination of functional components and enzyme activity of different Forsythia tea starter. Note: "-" indicates that it was not detected.

[0041] Example 3: Enhanced application of Aspergillus purpureus-Eurotium cristatum Forsythia tea starter in the brewing process of Shanxi aged vinegar. 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 mass) 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℃. Another 0.1% (by mass) 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 around 30℃, the mixture is pumped into an alcohol fermentation tank. 40% (by mass) of Daqu (a type of starter culture) and 10% (by mass) of *Aspergillus violaceus*-*Aspergillus cristatus*-*Forsythia tea starter culture from the sorghum are added. 0.1% (by mass) of Angel yeast inoculum is added. The mixture is fermented openly for 2 days, then sealed for 6-14 days until the alcohol content reaches 8-10% vol. The alcohol fermentation is then complete, yielding the mash. The fermented mash is thoroughly mixed with sorghum bran (120% by weight), rice bran (80% by weight), and rice husk (60% by weight), and then mixed with vinegar mash (10% by weight) for solid-state acetic acid fermentation. The fermentation lasts for 12 to 15 days, and the total acid content is 5 to 6 g / 100 mL. After the acetic acid fermentation is completed, the new vinegar is obtained through fumigation and vinegar leaching.

[0042] Comparative Example 1: In the brewing process of Shanxi aged vinegar, only 60% Daqu was added during the alcoholic fermentation stage, and the remaining process operations were the same as in Example 3.

[0043] The application of *Aspergillus purpureus*-*Eurotium cristatum* Forsythia tea koji fortification in the production of Shanxi aged vinegar significantly improved the overall quality of the product. Table 3 shows that *Aspergillus purpureus*-*Eurotium cristatum* Forsythia tea koji significantly improved the basic physicochemical properties of the newly extracted vinegar. Specifically, the contents of total acid, non-volatile acid, reducing sugar, total ester, and amino acid nitrogen reached 5.78 g / 100 mL, 2.21 g / 100 mL, 2.25 g / 100 mL, 2.72 g / 100 mL, and 0.18 g / 100 mL, respectively, representing increases of 23.50%, 49.32%, 63.04%, 46.24%, and 80.00% compared to Comparative Example 1.

[0044] Table 3. Effects of *Monascus purpureus*-*Eurotium cristatum* fortified application of *Forsythia suspensa* and *Tea yeast* on the physicochemical properties of freshly processed vinegar. As shown in Table 4, the total flavonoids, total phenols, γ-aminobutyric acid, and lovastatin contents of the new ester prepared by the enhanced application of Aspergillus violaceus-Aspergillus cristatus and Forsythia suspensa tea koji in Example 3 were 163.48 mg / 100 mL, 186.44 mg / 100 mL, 42.49 mg / 100 mL, and 1.82 μg / mL, respectively, which were 95.67%, 53.26%, 61.31%, and 73.33% higher than those in Comparative Example 1.

[0045] Table 4. Effects of *Monascus purpureus*-*Eurotium cristatum* fortified application of *Forsythia suspensa* tea starter on functional substances in fresh vinegar. As shown in Table 5, the total organic acid content in the fresh leached vinegar of Example 3 increased to 4.4680 g / 100 mL, which is 36.88% higher than that of Comparative Example 1. Among them, the contents of tartaric acid, succinic acid, malic acid, lactic acid, and acetic acid increased by 105.56%, 91.85%, 76.38%, 51.80%, and 22.34%, respectively. This is because *Monascus purpureus* decomposes starch to provide precursors, *Aspergillus cristatus* activates the enzyme system to promote the accumulation of succinic acid and malic acid in the tricarboxylic acid cycle, and the interaction between the two bacteria optimizes the fermentation environment. Together with acetic acid bacteria and lactic acid bacteria, the contents of acetic acid, tartaric acid, and lactic acid are increased, resulting in a significant increase in the total organic acid content.

[0046] Table 5. Effects of *Monascus purpureus*-*Eurotium cristatum* fortified application of *Forsythia suspensa* and *Tea yeast* on organic acids in fresh lye. As shown in Table 6, a total of 58 volatile aroma components were detected in the fresh acetic acid in Example 3 (26 esters, 7 alcohols, 7 acids, 7 aldehydes, 8 ketones, 2 pyrazines, and 1 other). The total amounts of esters, alcohols, and acids reached 2439.56 μg / 100 mL, 677.53 μg / 100 mL, and 2228.07 μg / 100 mL, respectively, which were 21.62%, 40.45%, and 17.07% higher than those in Comparative Example 1. Typical volatile flavor substances such as ethyl acetate, ethyl lactate, phenylethanol, and furfural were significantly increased.

[0047] Table 6. Effects of *Monascus purpureus*-*Eurotium cristatum* Forsythia suspensa* tea yeast fortification on volatile aroma compounds in fresh vinegar. Note: "-" indicates that it was not detected.

[0048] 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 enhancing the flavor and nutritional components of solid-state brewed vinegar using *Aspergillus purpureus*-*Eurotium cristatum* *Forsythia suspensa* tea starter, characterized in that: The superior Aspergillus violaceus strain isolated from Shanxi aged vinegar koji, which produces high levels of γ-aminobutyric acid and lovastatin, was used. Monascus purpureus CGMCC 42465, mixed with *Aspergillus cristatus* CICC 2099, was inoculated into a solid-state fermentation medium of forsythia leaves, indica rice, oats, and bran. After being cultured in wet burlap sacks, *Aspergillus cristatus*-*Aspergillus cristatus* forsythia tea starter was produced. This *Aspergillus cristatus*-*Aspergillus cristatus* forsythia tea starter was then applied to solid-state vinegar brewing.

2. The method according to claim 1, characterized in that: The preparation method of the *Aspergillus purpureus*-*Eurotium cristatum* forsythia tea fermentation medium is as follows: Forsythia leaves with a moisture content ≤10% are pulverized to 40 mesh. Whole indica rice is soaked in water at 15-20℃ for 2 hours, drained, and steamed for 1 hour. The rice is then spread evenly on a fermentation tray and cooled to 34-44℃. Forsythia leaf powder, steamed and cooled indica rice, and oat bran are mixed at a mass ratio of 60:25:

15. Simultaneously, 1-1.5 times the mass of water is added and mixed thoroughly to form the forsythia leaf, indica rice, and oat bran solid-state fermentation medium. *Aspergillus purpureus* CGMCC 42465 seed liquid and *Eurotium cristatum* CICC 2099 seed liquid are mixed at a volume ratio of 1:1 to prepare a mixed seed liquid. This mixed seed liquid is inoculated into the above forsythia leaf, indica rice, and oat bran solid-state fermentation medium at an inoculation amount of 30%. Mix v / w thoroughly and place in a wet burlap bag for 30°C incubation for 48 hours. When more than 50% of the solid fermentation medium turns light red and the surface is dry, start soaking the koji. Soak the koji once every 12 hours for a total of 4 times. After the incubation is completed, dry at 37°C until the moisture content is ≤12%. Grind and pass through a 20-mesh sieve to obtain the purple-red Aspergillus-Eurotium cristatum Forsythia tea koji.

3. The method according to claim 2, characterized in that: The method for preparing the seed culture of *Aspergillus purpureus* CGMCC 42465 is as follows: *Aspergillus purpureus* CGMCC 42465 activated on a slant is used to scrape the mycelia from the surface of the culture medium using a sterile inoculation loop. After scraping three loops consecutively, the mycelia are inoculated into rice flour culture medium to ensure consistency in inoculation. The medium is then cultured at 30℃ and 180 r / min on a shaker for 48 h. During this period, the rice flour culture medium is observed to turn pink. Culture is continued for another 48 h until the medium turns purplish-red, at which point the seed culture of *Aspergillus purpureus* CGMCC 42465 is obtained. The rice flour culture medium formula is: 2.5 g rice flour, 0.15 g NaNO3, 0.05 g MgSO4·7H2O, and 0.075 g KH2PO4. Water is added to bring the volume to 50 mL, and the medium is then sterilized at 121℃ for 20 min. The method for preparing the seed culture of *Aspergillus cristatus* CICC 2099 is as follows: *Aspergillus cristatus* CICC 2099 activated on an slant agar plate is cultured with a spore count ≥1×10⁻⁶. 6 CFU / g was inoculated into PDA liquid medium and cultured at 30℃ and 180 r / min for 72 h. The formula of the above PDA liquid medium was: 20 g potato, 2 g glucose, 0.2 g peptone, 0.2 g KH2PO4, 0.1 g MgSO4, 100 mL distilled water, and sterilized at 121℃ for 20 min.

4. The method according to claim 1, characterized in that: The specific steps for solid-state vinegar brewing are as follows: 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 mass) 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℃. Next, 0.1% (by mass) of saccharifying enzyme from the sorghum is added, and the mixture is kept at this temperature for 20-30 minutes. Once the temperature drops to 30℃, the mixture is poured into an alcohol fermentation tank, and 30%-40% (by mass) of sorghum koji (a type of starter culture) and 10%-20% (by mass) of... Aspergillus purpureus-Eurotium cristatum Forsythia tea starter is added to sorghum with 0.1%~0.3% Angel yeast inoculum. The mixture is fermented in an open container for 2 days, then sealed for 6~14 days until the alcohol content reaches 8~10% vol. The fermentation process ends to obtain mash. The fermented mash is then thoroughly mixed with sorghum bran (120%~140%), rice bran (70%~80%), and rice husk (60%~70%). This mixture is then inoculated with 10% arsenic mash for solid-state acetic acid fermentation for 12~15 days until the total acid content reaches 5~6 g / 100 mL. The fermentation process ends, and the mash is then smoked and leached to obtain fresh leached vinegar.

5. The method according to claim 1, characterized in that: The purple-red Aspergillus ( Monascus purpureus CGMCC 42465, deposited at the China General Microbiological Culture Collection Center on December 5, 2025, was isolated from Shanxi aged vinegar koji. When cultured in rice flour medium at 30℃ and 180 r / min for 48 h, it produced lovastatin at a content of 52.92 mg / 100 g and γ-aminobutyric acid at a content of 61.38 mg / 100 g, and also exhibited excellent enzyme activity characteristics, with amylase activity of 22.38 U / g and esterification power of 2485.62 U.

6. The method according to claim 4, characterized in that: The prepared new lye is reddish-brown with a rich color, and contains ≥5.50 g / 100 mL of total acid, ≥2.50 g / 100 mL of total esters, ≥150 mg / 100 mL of flavonoids, ≥40.00 mg / 100 mL of γ-aminobutyric acid, and ≥1.50 μg / mL of lovastatin.

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