Monacolin k high-yield and citrinin non-yield monascus purpureus ncu fhqj-m-38 and application thereof

Abstract

The application provides a high-yield monacolin K and non-citrinin Monascus purpureus NCUFHQJ-M-38 and application thereof, and relates to the technical field of microorganism and food vinegar brewing. The strain is preserved with the number of CCTCC NO: M 20252778, is obtained by using Monascus purpureus NCUFHQ-9 as a starting strain, and is obtained by combining protoplast ARTP mutagenesis and lovastatin resistance plate screening. After liquid culture for 7 days, the monacolin K yield reaches 481.25 mg / L, is increased by 62.3% compared with the starting strain, and no citrinin is detected. The genetic stability of the strain is stable after continuous passage for 6 times. When the strain is applied to the brewing of Monascus vinegar, the monacolin K content in the finished product vinegar reaches 47.27 mg / L, and the volatile aroma components are more abundant.

Description

Technical Field

[0001] This invention relates to the fields of microbial technology and vinegar brewing technology, specifically to a purple red Monascus bacterium NCUFHQJ-M-38 that produces high levels of Monascus K but not citrinin and its applications. Background Technology

[0002] Monascus purpureus is an important functional fungus in red yeast fermented products such as red yeast vinegar, red yeast wine, and red yeast rice. During the saccharification and alcoholic fermentation stages in which Monascus purpureus participates, this strain can not only synthesize Monacolin K, but also produce amylase, saccharifying enzymes, acidic proteases, and metabolites related to ester formation, thereby promoting starch degradation, flavor precursor release, and aroma compound formation.

[0003] In recent years, the prevalence of dyslipidemia among adults in my country has remained at a high level. A 2018 survey showed that the overall prevalence of dyslipidemia among Chinese adults was 35.6%. Monaclin K, as a natural HMG-CoA reductase inhibitor, possesses physiological activities such as inhibiting cholesterol synthesis, lowering blood lipids, and slowing atherosclerosis. It has been widely used in functional foods and health products, therefore, red yeast rice fermented products rich in Monaclin K have high development value.

[0004] However, some Monascus purpureus can produce citrinin during fermentation. Citrinin is a fungal toxin with potential nephrotoxicity, and its presence can affect the food safety and industrial application of red yeast rice-related products. Furthermore, the accumulation level of Monacolin K in existing red yeast rice vinegar products is significantly influenced by factors such as strain performance, fermentation method, and process control conditions. There is still room for further optimization in terms of stable accumulation of functional components, improvement of flavor and quality, and safe control of fungal toxins.

[0005] In existing technologies, Monax monaxatum strains that produce high levels of Monax monaxatum K often carry the risk of simultaneous production of citrinin, while non-citrinin-producing strains obtained through genetic engineering may face problems such as insufficient food application acceptance, genetic stability, or fermentation performance.

[0006] Therefore, developing a purple Monascus strain that simultaneously produces high levels of Monascus K, does not produce citrinin, and is suitable for red yeast rice vinegar brewing is of great significance for improving the functional attributes, safety, and added value of red yeast rice fermented products. Summary of the Invention

[0007] The purpose of this invention is to provide a purple Monascus purpureus NCUFHQJ-M-38 that produces high levels of Monascus K but not citrinin, and its applications.

[0008] In a first aspect, the present invention provides a purple Monascus purpureus NCUFHQJ-M-38 that produces high levels of Monascus K and does not produce citrinin. The purple Monascus purpureus NCUFHQJ-M-38 was deposited on December 4, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252778, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0009] Optionally, the key gene ctnR for citrinin synthesis in the purple red Monascus NCUFHQJ-M-38 undergoes a frameshift mutation, with the mutation site at position 2348554 changing from G to GAAA.

[0010] Optionally, the genome of the purple red Monascus NCUFHQJ-M-38 contains one or more mutations in one of the following groups of genes: MDR5, MAL13, AMT1, abc4, HXT1, and LaeA.

[0011] Secondly, the present invention provides a method for breeding any of the above-mentioned optional purple Monascus purpureus NCUFHQJ-M-38, comprising the following steps:

[0012] (1) The starting strain of Monascus purpureus NCUFHQ-9 was inoculated into liquid culture medium and cultured, and mycelial balls were collected;

[0013] (2) After washing the mycelial balls with a high-osmotic stabilizing solution, add a compound enzyme for enzymatic hydrolysis to prepare a protoplast suspension;

[0014] (3) Take the protoplast suspension and perform ARTP mutagenesis treatment;

[0015] (4) Spread the mutagenized protoplast suspension on a resistant plate containing lovastatin and an osmotic stabilizer for culture, and select well-grown colonies to transfer to PDA plates for culture;

[0016] (5) The colonies obtained in step (4) were tested for Monacolin K and citrinin, and purple red Monascus NCUFHQJ-M-38 was screened.

[0017] Optionally, the cultivation conditions in step (1) are: temperature 25-35℃, rotation speed 150-250 r / min, and cultivation time 2-5 days.

[0018] Optionally, the high-permeability stabilizing solution in step (2) is a 0.8 mol / L sorbitol solution.

[0019] Optionally, the addition of the complex enzyme in step (2) includes the addition of snail enzyme, wall-lysing enzyme and cellulase; the final concentration of the snail enzyme is 0.4%-0.8%; the final concentration of the wall-lysing enzyme is 0.2%-0.6%; and the final concentration of the cellulase is 0.6%-1.0%.

[0020] Optionally, the mass-to-volume ratio of the mycelial balls to the complex enzyme in step (2) is 1:30.

[0021] Optionally, the power of the ARTP mutagenesis treatment in step (3) is 110-130 W and the treatment time is 100-120 s.

[0022] Optionally, the concentration of lovastatin in step (4) is 350-450 mg / L; the osmotic pressure stabilizer is sucrose; and the concentration of sucrose is 0.4-0.8 mol / L.

[0023] Thirdly, this application provides the application of any of the above-mentioned optional purple Monascus purpureus NCUFHQJ-M-38 in the preparation of red yeast fermentation products.

[0024] Optionally, the red yeast fermented product is one of red yeast vinegar, red yeast wine, or red yeast rice.

[0025] Fourthly, this application provides a red yeast rice vinegar produced by fermentation using any of the aforementioned optional purple red yeast rice strains NCUFHQJ-M-38.

[0026] Optionally, the preparation of the red yeast rice vinegar includes the following steps:

[0027] (1) Soak and steam the glutinous rice, then cool it to room temperature;

[0028] (2) Add yeast and seed liquid of purple red yeast NCUFHQJ-M-38 to the cooled glutinous rice, mix well and carry out solid-state alcohol fermentation;

[0029] (3) After the alcoholic fermentation is completed, vinegar mash is added for solid-state acetic acid fermentation;

[0030] (4) After fermentation, the red yeast rice vinegar is obtained through post-processing.

[0031] Optionally, the soaking time in step (1) is 4-8 h; the steaming time is 15-30 min.

[0032] Optionally, the yeast in step (2) is a yeast starter; the amount of yeast added is 0.2%-0.5% of the mass of the glutinous rice; and the amount of seed liquid added is 5%-15% of the mass of the glutinous rice.

[0033] Optionally, the seed liquid preparation method in step (2) is as follows: inoculate purple red Monascus NCUFHQJ-M-38 into liquid culture medium and culture it at 25-35℃ and 150-250 r / min for 3-7 days.

[0034] Optionally, the conditions for solid-state alcohol fermentation in step (2) are: temperature 25-35℃, fermentation time 3-7 days.

[0035] Optionally, the solid acetic acid fermentation temperature in step (3) is 25-35℃ and the fermentation time is 10-16 days.

[0036] Optionally, the post-processing described in step (4) includes vinegar rinsing, sterilization, and aging.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The purple red yeast strain NCUFHQJ-M-38, which produces high levels of Monacolin K and does not produce citrinin, provided by the present invention was obtained by a synergistic screening strategy of ARTP mutagenesis combined with lovastatin resistance plates. Under liquid culture conditions, the Monacolin K yield reached 481.25 mg / L, which is 62.3% higher than the starting strain. At the same time, citrinin was not detected within the detection limit adopted, which helps to reduce the safety risks related to citrinin in red yeast fermentation products.

[0039] (2) The purple red yeast rice NCUFHQJ-M-38 provided by the present invention has a frameshift mutation in the key transcriptional activation gene ctnR for citrinin synthesis (mutation site: the 2348554th site is mutated from G to GAAA). This mutation can affect the biosynthetic pathway of citrinin, so that citrinin is not detected under the detection conditions used, thereby improving the food safety of red yeast rice fermented products and reducing the dependence on the post-processing to remove citrinin.

[0040] (3) The purple red Monascus provided by the present invention, after being passaged 6 times, showed no statistically significant difference in Monacolin K production and citrinin non-production trait, and has good genetic stability.

[0041] (4) The breeding method provided by the present invention utilizes the cross-resistance mechanism between lovastatin resistance and Monaclin K synthesis ability to establish an efficient positive screening system, which overcomes the lack of specific screening markers in traditional mutation breeding and greatly improves the screening efficiency of target mutant strains; purple red Monascus NCUFHQJ-M-38 is obtained through non-transgenic physical mutation breeding methods without the introduction of exogenous genes;

[0042] (5) The purple Monascus purpureus NCUFHQJ-M-38 provided by this invention was applied to the brewing of red yeast rice vinegar. During the alcoholic fermentation stage, seed liquid of purple Monascus purpureus NCUFHQJ-M-38 was added at 10% of the weight of glutinous rice. The content of Monacolin K in the finished vinegar reached 47.27 mg / L, and citrinin was not detected within the detection limit adopted. Red yeast rice vinegar with high Monacolin K content and good safety was obtained. At the same time, the volatile aroma components were richer, achieving a synergistic effect of functional improvement and flavor enhancement. Attached Figure Description

[0043] Figure 1 The results of protoplast extraction of Monascus purpleis NCUFHQ-9 in Example 1 of this invention;

[0044] Figure 2 The colony morphology of the mutagenized strain in Example 1 of this invention;

[0045] Figure 3 This demonstrates the ability of the mutagenic strain in Example 2 of this invention to produce Monacolin K and Citrulline;

[0046] Figure 4 The curve showing the change in Monacolin K content during the brewing process of red yeast rice vinegar in Example 4 of this invention;

[0047] Figure 5 This is the curve showing the change in citrinin content during the brewing process of red yeast rice vinegar in Example 4 of the present invention;

[0048] Figure 6 This is a heat map of volatile aroma components during the acetic acid fermentation stage in Example 4 of the present invention.

[0049] Figure 7 This is a colony morphology diagram of the purple Monascus purpureus NCUFHQJ-M-38 strain of the present invention. Detailed Implementation

[0050] 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 only some embodiments of the present invention, 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. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0051] This invention provides a purple red Monascus strain NCUFHQJ-M-38 that produces high levels of Monacolin K but not citrinin. It is classified as *Monascus purpureus* NCUFHQJ-M-38 and was deposited on December 4, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252778. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0052] In fact, Monascus purpureus NCUFHQJ-M-38 was obtained by protoplast ARTP mutagenesis combined with lovastatin resistance plate screening, using Monascus purpureus NCUFHQ-9 as the starting strain. After 7 days of liquid culture, the Monacolin K yield reached 481.25 mg / L, which was 62.3% higher than the starting strain, and citrinin was not detected. The strain remained genetically stable after 6 consecutive passages. When this strain was applied to the brewing of red yeast rice vinegar, the finished vinegar contained 47.27 mg / L of Monacolin K, and the volatile aroma components were more abundant.

[0053] In some embodiments, the key gene ctnR for citrinin synthesis in Monascus purpureus NCUFHQJ-M-38 undergoes a frameshift mutation, with the mutation site at position 2348554 changing from G to GAAA.

[0054] In some embodiments, the genome of Monascus purpureus NCUFHQJ-M-38 contains one or more mutations in one of the following groups of genes: MDR5, MAL13, AMT1, abc4, HXT1, and LaeA.

[0055] The present invention also provides a method for breeding the purple Monascus NCUFHQJ-M-38 in any of the above embodiments, comprising the following steps:

[0056] (1) The starting strain of Monascus purpureus NCUFHQ-9 was inoculated into liquid culture medium and cultured, and mycelial balls were collected;

[0057] (2) After washing the mycelial balls with a high-osmotic stabilizing solution, add a compound enzyme for enzymatic hydrolysis to prepare a protoplast suspension;

[0058] (3) Take the protoplast suspension and perform ARTP mutagenesis treatment;

[0059] (4) Spread the mutagenized protoplast suspension on a resistant plate containing lovastatin and an osmotic stabilizer for culture, and select well-grown colonies to transfer to PDA plates for culture;

[0060] (5) The colonies obtained in step (4) were tested for Monacolin K and citrinin, and purple red Monascus NCUFHQJ-M-38 was screened.

[0061] In some embodiments, the culture conditions used in step (1) are: temperature 25-35℃, rotation speed 150-250 r / min, and culture time 2-5 days.

[0062] In some embodiments, the high-permeability stabilizing solution used in step (2) is a 0.8 mol / L sorbitol solution.

[0063] In some embodiments, the addition of the complex enzyme in step (2) includes the addition of snail enzyme, lysozyme and cellulase; the final concentration of the snail enzyme used is 0.4%-0.8%; the final concentration of the lysozyme used is 0.2%-0.6%; and the final concentration of the cellulase used is 0.6%-1.0%.

[0064] In some embodiments, the mass-to-volume ratio of mycelial balls to complex enzyme in step (2) is 1:30.

[0065] In some embodiments, the power of the ARTP mutagenesis treatment used in step (3) is 110-130 W and the treatment time is 100-120 s.

[0066] In some embodiments, the concentration of lovastatin used in step (4) is 350-450 mg / L; the osmotic pressure stabilizer used is sucrose; and the concentration of sucrose used is 0.4-0.8 mol / L.

[0067] This application also provides the application of purple Monascus purpureus NCUFHQJ-M-38 in any of the above embodiments in the preparation of red yeast fermentation products.

[0068] In some embodiments, the prepared red yeast fermented product is one of red yeast vinegar, red yeast wine, or red yeast rice.

[0069] This application also provides a red yeast rice vinegar prepared by fermentation using purple red yeast rice NCUFHQJ-M-38 in any of the above embodiments.

[0070] In some embodiments, the preparation of red yeast rice vinegar includes the following steps:

[0071] (1) Soak and steam the glutinous rice, then cool it to room temperature;

[0072] (2) Add yeast and seed liquid of purple red yeast NCUFHQJ-M-38 to the cooled glutinous rice, mix well and carry out solid-state alcohol fermentation;

[0073] (3) After the alcoholic fermentation is completed, vinegar mash is added for solid-state acetic acid fermentation;

[0074] (4) After fermentation, the red yeast rice vinegar is obtained through post-processing.

[0075] In some embodiments, the soaking time used in step (1) is 4-8 h; the steaming time used is 15-30 min.

[0076] In some embodiments, the yeast used in step (2) is a yeast starter; the amount of yeast added is 0.2%-0.5% of the weight of glutinous rice; and the amount of seed liquid added is 5%-15% of the weight of glutinous rice.

[0077] In some embodiments, the method for preparing the seed culture of Monascus purpureus NCUFHQJ-M-38 in step (2) is as follows: Monascus purpureus NCUFHQJ-M-38 is inoculated into a liquid culture medium and cultured at 25-35℃ and 150-250 r / min for 3-7 days.

[0078] In some embodiments, the conditions for solid-state alcohol fermentation in step (2) are: temperature 25-35℃, fermentation time 3-7 days.

[0079] In some embodiments, the solid acetic acid fermentation used in step (3) is carried out at a temperature of 25-35°C for 10-16 days.

[0080] In some embodiments, the post-treatment used in step (4) includes vinegar rinsing, sterilization, and aging.

[0081] I. Experimental Materials and Methods

[0082] 1.1 Main Culture Medium Formulation

[0083] The culture medium formula used in this invention is as follows:

[0084] (1) Protoplast regeneration plate: Add 205.374 g of sucrose per liter of PDA medium and sterilize at 121℃ for 20 min.

[0085] (2) Liquid culture medium: 3% glucose, 7% glycerol, 3.8% peptone, 0.2% sodium nitrate, 0.1% magnesium sulfate heptahydrate and 0.25% potassium dihydrogen phosphate, sterilized at 121℃ for 20 min.

[0086] (3) Lovastatin resistance plate: Lovastatin solution was added to PDA medium to make the concentration of lovastatin in the medium 400 mg / L.

[0087] (4) PDA solid culture medium: Take 200 g of potato, add water and boil for 30 min, filter with four layers of gauze, add 2% glucose and 2% agar to each liter of filtrate, and sterilize at 121℃ for 20 min.

[0088] 1.2 Main Detection Methods

[0089] (1) Monaclin K determination method

[0090] Take 1 mL of fermentation broth, add 9 mL of 75% ethanol, mix well, and incubate in a water bath at 50℃ for 4 h. After centrifugation at 5000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm filter membrane. The filtrate was detected by high performance liquid chromatography (Water E2695) at a wavelength of 238 nm.

[0091] The chromatographic column was a Symmetry C18 (4.6 mm × 250 mm), the injection volume was 10 µL, the column temperature was 30℃, the flow rate was 0.8 mL / min, the mobile phase A was water, the mobile phase B was acetonitrile, and the gradient elution conditions are shown in Table 1.

[0092] Table 1: Elution conditions for Monacolin K assay

[0093]

[0094] (2) Method for determining citrinin

[0095] Take 2 mL of fermentation broth and add an equal volume of organic extractant (toluene-ethyl acetate-formic acid, volume ratio 7:3:1). Mix thoroughly and centrifuge at 10000 r / min for 10 min to separate the organic phase. Evaporate the organic phase to dryness in a 65℃ water bath, then redissolve it with 80% methanol, mix well, filter through a 0.22 μm filter membrane, and detect it using an ultra-high performance liquid chromatograph (Water ACQUITY UPLCH-Class PLUS) at an excitation wavelength of 331 nm and an emission wavelength of 500 nm.

[0096] The chromatographic column was a Poroshell 120 SB-C18 (4.6 mm × 150 mm), the column temperature was 40℃, the injection volume was 10 µL, the flow rate was 0.4 mL / min, the mobile phase A was 0.1% phosphoric acid water, the mobile phase B was acetonitrile, and the gradient elution conditions are shown in Table 2.

[0097] Table 2: Elution conditions for citrinin assay

[0098]

[0099] II. Implementation Examples

[0100] Example 1: Breeding of Monascus purpureus NCUFHQJ-M-38

[0101] 1.1 Starting strain

[0102] The starting strain used in this embodiment was Monascus purpureus NCUFHQ-9, screened from red yeast rice, and stored at 4°C for later use. ITS rDNA sequence analysis of Monascus purpureus NCUFHQ-9 was performed, and the sequencing results are shown in SEQ ID NO: 9.

[0103] 1.2 Protoplast Preparation

[0104] The starting strain NCUFHQ-9 was inoculated into liquid culture medium and cultured in a shaker at 30℃ and 200 r / min for 3 days. Mycelial balls were collected by filtration through sterile gauze, washed twice with sterile high-osmotic stabilizing solution, resuspended, and centrifuged at 4000 r / min and 4℃ for 10 min.

[0105] The compound enzyme solution (containing 0.6% snailase, 0.4% lysozyme, and 0.8% cellulase) was added at a mass-to-volume ratio of mycelium to compound enzyme solution of 1:30. Enzymatic hydrolysis was carried out at 30℃ for 2 hours. The enzymatic hydrolysis process is as follows: Figure 1 As shown. After enzymatic hydrolysis, the precipitate was collected by centrifugation and washed twice with a high-osmotic stabilizing solution to obtain a protoplast suspension.

[0106] 1.3 ARTP mutagenesis treatment

[0107] Dilute protoplasts to 1×10⁻⁶ 6 CFU / mL, 10 μL of the suspension was spread onto the surface of the slide and mutagenesis was performed using an ARTP mutagenesis breeding instrument. The mutagenesis conditions were 120 W power and 110 s treatment time.

[0108] 1.4 Resistance Screening

[0109] The mutagenic protoplast suspension was spread onto antibiotic-resistant plates containing 0.6 mol / L sucrose and 400 mg / L lovastatin, and incubated at 30°C. Well-grown colonies from the antibiotic-resistant plates were picked and transferred to PDA plates for further incubation for 7 days. Some mutagenic strains exhibited colony morphology as shown below. Figure 2 .

[0110] 1.5 Obtaining the target strain

[0111] The colonies obtained from the above culture were subjected to the determination of Monacolin K and citrinin content, and a target strain that produced high levels of Monacolin K and did not produce citrinin was screened out and named Monascus purpureus NCUFHQJ-M-38.

[0112] 1.6 Strain Identification

[0113] ITS rDNA sequence analysis was performed on strain NCUFHQJ-M-38, and the sequencing results are shown in SEQ ID NO: 1.

[0114] BLAST comparison results showed that the strain had 99.84% homology with the standard strain Monascus purpureus NRRL1596, and was confirmed as Monascus purpureus (GenBank accession number OQ694412.1).

[0115] Example 2: Performance characterization of strain NCUFHQJ-M-38

[0116] 2.1 Monacolin K yield and citrinin detection

[0117] Under the same culture conditions, the target strain NCUFHQJ-M-38 was cultured in liquid. After 7 days of fermentation, samples were taken to detect the contents of Monacolin K and Citrinin, and the results are as follows: Figure 3 The Monacolin K yield was 481.25 mg / L, and citrinin was not detected within the limits of detection used.

[0118] 2.2 Genetic stability test

[0119] The target strain NCUFHQJ-M-38 was passaged six times, and the Monacolin K yield and citrinin content of each generation were measured. The results are shown in Table 3. There were no significant differences between the generations, indicating that the strain has good genetic stability.

[0120] Table 3: Genetic stability results of Monascus purpureus NCUFHQJ-M-38

[0121]

[0122] Note: nd indicates that it was not detected under the detection conditions used.

[0123] 2.3 Analysis of Mutant Genes

[0124] Whole-genome sequencing analysis of the target strain NCUFHQJ-M-38 and comparison with the originating strain NCUFHQ-9 identified seven key mutant genes. The mutation types, mutation sites, and functional descriptions of each gene are shown in Table 4. Among them, the frameshift mutation of ctnR, a key transcriptional activator gene for citrinin synthesis, occurred: guanine nucleotides (G) were inserted at position 2348554 of the originating strain's genome sequence, while the mutant strain inserted three adenine nucleotides (AAA) at this position, i.e., a mutation from G to GAAA. This frameshift mutation resulted in the loss of function of the key transcriptional activator for citrinin synthesis, consistent with the mutant strain's phenotype of not producing citrinin, which is the molecular mechanism underlying this strain's lack of citrinin production. Information on the seven key mutant genes is shown in Table 4. The gene sequences of the key mutant gene ctnR are shown in SEQ ID NO: 2; the gene sequence of MDR5 is shown in SEQ ID NO: 3; the gene sequence of MAL13 is shown in SEQ ID NO: 4; the gene sequence of AMT1 is shown in SEQ ID NO: 5; the gene sequence of LaeA is shown in SEQ ID NO: 6; the gene sequence of abc4 is shown in SEQ ID NO: 7; and the gene sequence of HXT1 is shown in SEQ ID NO: 8.

[0125] Table 4: Information on key mutant genes

[0126]

[0127] Example 3: Brewing process of red yeast rice vinegar

[0128] 3.1 Seed liquid preparation

[0129] The purple red Monascus purpureus NCUFHQJ-M-38 was inoculated into liquid culture medium and cultured at 30℃ and 200 r / min for 7 days to obtain seed culture.

[0130] 3.2 Alcoholic fermentation

[0131] Select plump, mold-free, and insect-free glutinous rice grains, soak them for 6 hours, and then steam them for 20 minutes to fully gelatinize them. After steaming, rinse them quickly with sterile cold water to reduce stickiness and create gaps. After cooling to room temperature, add 0.3% yeast and 10% seed liquid according to the weight of the glutinous rice, mix well, and then carry out solid-state alcoholic fermentation at 30℃ for 5 days.

[0132] 3.3 Acetic acid fermentation

[0133] After alcoholic fermentation, vinegar mash (10% of the mash weight) is added, and the process transitions to acetic acid fermentation. The fermentation temperature is 30℃, and the mash is turned once daily for 13 days of solid-state acetic acid fermentation.

[0134] 3.4 Post-processing

[0135] After fermentation, the product is red yeast rice vinegar through rinsing, sterilization, and aging.

[0136] Example 4: Application effect of strain NCUFHQJ-M-38 in red yeast rice vinegar brewing

[0137] Red yeast rice vinegar was brewed according to the process described in Example 3, while a brewing system with the addition of the starting strain NCUFHQ-9 was used as a control.

[0138] 4.1 Changes in Monacolin K content, safety evaluation of citrinin, and analysis of volatile flavor components

[0139] The contents of Monacolin K and Citrinin were measured at different time points during alcoholic fermentation (0 d, 1 d, 2 d, 3 d, 4 d, 5 d) and acetic acid fermentation (1 d, 3 d, 5 d, 7 d, 9 d, 11 d, 13 d), and volatile components were measured after fermentation. The results are as follows: Figures 4-6 As shown: During the alcoholic fermentation stage, the Monacolin K content in each group rose rapidly in the early stage and then stabilized. After 5 days of fermentation, the Monacolin K content in the system with added NCUFHQJ-M-38 strain reached 60.00 mg / L. After entering the acetic acid fermentation stage, the Monacolin K content decreased slightly and gradually stabilized. After the fermentation was completed, the Monacolin K content in the finished red yeast rice vinegar reached 47.27 mg / L.

[0140] Meanwhile, no citrinin was detected in any of the groups fermented with the NCUFHQJ-M-38 strain. Compared with red yeast rice vinegar fermented with the original strain NCUFHQ-9, the red yeast rice vinegar fermented with the NCUFHQJ-M-38 strain showed a significant increase in the variety of volatile aroma components, indicating that this strain has the potential to improve the flavor and quality of red yeast rice vinegar.

[0141] In summary, the selection of superior strains, performance evaluation of strains, and verification of red yeast rice vinegar brewing in this invention have demonstrated that the purple red yeast rice strain NCUFHQJ-M-38 possesses the excellent characteristics of having high-monacolin K, not producing citrinin, and being suitable for red yeast rice vinegar brewing, and has good prospects for promotion and application.

[0142] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A purple Monascus purpureus NCUFHQJ-M-38 that produces high levels of Monacolin K but not citrinin, characterized in that, The purple red Monascus strain NCUFHQJ-M-38 was deposited at the China Center for Type Culture Collection (CCTCC) on December 4, 2025, with accession number CCTCC NO: M 20252778, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The purple Monascus purpureus NCUFHQJ-M-38 according to claim 1, characterized in that, The key gene for citrinin synthesis in the purple red Monascus purpureus NCUFHQJ-M-38 ctnR A frameshift mutation occurred at position 2348554, where G was mutated to GAAA.

3. The purple Monascus purpureus NCUFHQJ-M-38 according to claim 1, characterized in that, The genome of the purple red Monascus NCUFHQJ-M-38 contains one or more mutations in one of the following groups of genes: MDR5, MAL13, AMT1 abc4, HXT1, LaeA .

4. A method for breeding the purple Monascus purpureus NCUFHQJ-M-38 as described in claim 1, characterized in that, Includes the following steps: (1) The starting strain of Monascus purpureus NCUFHQ-9 was inoculated into liquid culture medium and cultured, and mycelial balls were collected; (2) After washing the mycelial balls with a high-osmotic stabilizing solution, add a compound enzyme for enzymatic hydrolysis to prepare a protoplast suspension; (3) Take the protoplast suspension and perform ARTP mutagenesis treatment; (4) Spread the mutagenized protoplast suspension on a resistant plate containing lovastatin and an osmotic stabilizer for culture, and select well-grown colonies to transfer to PDA plates for culture; (5) The colonies obtained in step (4) were tested for Monacolin K and citrinin, and purple red Monascus NCUFHQJ-M-38 was screened.

5. The method according to claim 4, characterized in that, The cultivation conditions described in step (1) are: temperature 25-35℃, rotation speed 150-250 r / min, and cultivation time 2-5 days; and / or, the high-osmotic stabilizing solution described in step (2) is a 0.8 mol / L sorbitol solution; and / or, the addition of the complex enzyme described in step (2) includes the addition of snail enzyme, lysozyme, and cellulase; the final concentration of the snail enzyme is 0.4%-0.8%; the final concentration of the lysozyme is 0.2%-0.6%; the final concentration of the cellulase is 0.6%-1.0%; and / or, the mass-to-volume ratio of mycelial balls to the complex enzyme described in step (2) is 1:30; and / or, the power of the ARTP mutagenesis treatment described in step (3) is 110-130 W, and the treatment time is 100-120 s; and / or, the concentration of lovastatin described in step (4) is 350-450 W. mg / L; the osmotic pressure stabilizer is sucrose; the concentration of the sucrose is 0.4-0.8 mol / L.

6. The use of the purple red yeast rice strain NCUFHQJ-M-38 as described in any one of claims 1-3 in the preparation of red yeast rice fermented products.

7. The application according to claim 6, characterized in that, The red yeast fermented product is one of red yeast vinegar, red yeast wine, or red yeast rice.

8. A red yeast rice vinegar prepared by fermentation using the purple red yeast rice strain NCUFHQJ-M-38 as described in any one of claims 1-3.

9. The red yeast rice vinegar according to claim 8, characterized in that, The preparation of the red yeast rice vinegar includes the following steps: (1) Soak and steam the glutinous rice, then cool it to room temperature; (2) Add yeast and seed liquid of purple red yeast NCUFHQJ-M-38 to the cooled glutinous rice, mix well and carry out solid-state alcohol fermentation; (3) After the alcoholic fermentation is completed, vinegar mash is added for solid-state acetic acid fermentation; (4) After fermentation, the red yeast rice vinegar is obtained through post-processing.

10. The red yeast rice vinegar according to claim 9, characterized in that, The soaking time in step (1) is 4-8 h; the steaming time is 15-30 min; and / or, the yeast in step (2) is a yeast starter; the amount of yeast added is 0.2%-0.5% of the mass of the glutinous rice; the amount of seed liquid added is 5%-15% of the mass of the glutinous rice; and / or, the seed liquid in step (2) is prepared by inoculating purple red spore NCUFHQJ-M-38 into a liquid culture medium and culturing it at 25-35℃ and 150-250 r / min for 3-7 days; and / or, the conditions for solid-state alcohol fermentation in step (2) are: temperature 25-35℃ and fermentation time 3-7 days; and / or, the temperature for solid-state acetic acid fermentation in step (3) is 25-35℃ and the fermentation time is 10-16 days; and / or, the post-treatment in step (4) includes vinegar leaching, sterilization and aging.

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