Edible and medicinal monascus purpureus with high yield of monascus pigment and application of monascus purpureus

Abstract

The invention relates to monascus purpureus M23067Z4Fd12 with high yield of monascus pigment, a leavening agent and a composition containing the monascus purpureus M23067Z4Fd12 and application of the monascus purpureus M23067Z4Fd12 in preparation of fermented food or medicine. The monascus purpureus has the following remarkable advantages that the monascus purpureus can produce monascus pigment at high yield and does not produce mycotoxins such as citrinin, the content of functional components such as total flavonoids can be remarkably increased in the fermentation process of traditional Chinese medicinal materials including pericarpium citri reticulatae, the antioxidant activity of the traditional Chinese medicinal materials is enhanced, the monascus purpureus has excellent fermentation performance, and the monascus purpureus can be used for preparing the monascus purpureus. Comprising relatively strong temperature adaptability, pH adaptability and alcohol tolerance; the monascus purpureus has the advantages that the nutritional value of a traditional product can be improved, the product can be endowed with unique health attributes, important strain resources and technical support are provided for innovative development of monascus purpureus related industries, and the monascus purpureus has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine resource microbial strains and fermentation engineering, and in particular to a purple monascus strain with high yield of monascus pigment and application thereof. Background Art

[0002] Monascus is an important resource microorganism for both medicinal and edible purposes. Monascus pigment is a natural pigment produced by Monascus, mainly used for food coloring, winemaking, etc. Long-term use practice has shown that Monascus pigment is a natural and safe pigment. In addition to food coloring, Monascus pigment also has the following functions: (1) Antioxidant effect: Monascus pigment can scavenge free radicals in the body, reduce oxidative stress, reduce oxidative damage, and help prevent aging and oxidation-related diseases such as cardiovascular disease and cancer; (2) Regulating blood lipids: Certain components in Monascus pigment may have a certain effect on regulating blood lipids and is beneficial to cardiovascular health; (3) Antibacterial effect: Monascus pigment has a certain inhibitory effect on certain bacteria, molds and yeasts. It can inhibit the growth and reproduction of microorganisms by affecting the cell membrane permeability and metabolic process of microorganisms, and has certain application potential in food preservation and antiseptic treatment; (4) Improving intestinal flora: Monascus pigment has a certain regulatory effect on intestinal flora, helps maintain the balance of intestinal microecology, promotes the growth of beneficial bacteria, and inhibits the reproduction of harmful bacteria, thereby having a positive impact on intestinal health. In addition, Monascus can also produce functional substances such as lovastatin, which helps lower blood lipids.

[0003] Functional substances related to red yeast rice have the ability to lower blood lipids. As an important source of effective substances, red yeast rice and its related industries will also usher in new development opportunities in this process.

[0004] The "Opinions on Improving the Quality of Traditional Chinese Medicine and Promoting the High-Quality Development of the Traditional Chinese Medicine Industry" emphasizes the need to maintain the security of industrial development and vigorously strengthen the protection of traditional Chinese medicine resources and core technologies and processes through various means, such as the protection of traditional knowledge. Red yeast rice is an important edible and medicinal microbial product, and the red yeast rice strain is the key to its efficacy.

[0005] Given the significant value of red yeast rice products in Traditional Chinese Medicine and food and drug applications, and given the diverse metabolite types and yields of different red yeast rice strains, highly active, superior strains are prioritized for protection. These differences in properties determine the quality and efficacy of red yeast rice products, profoundly impacting the development of related industries. Protecting superior strains will help ensure the stable quality of red yeast rice products, promote industry innovation and upgrading, and enable red yeast rice to continue to play a greater role in maintaining health and promoting economic development. Summary of the Invention

[0006] Purpose of the Invention

[0007] In view of the defects or needs in the above-mentioned prior art, the object of the present invention is to provide a purple Monascus with high yield of Monascus pigment, a starter and composition containing the same, and the use of the same in the preparation of fermented foods or medicines.

[0008] Solution

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] In the first aspect, the present invention provides a purple Monascus M23067Z4Fd12, which is taxonomically named Monascus purpureus and was deposited in the China Center for Type Culture Collection (CCTCC) on September 25, 2024, with a deposit number of CCTCC NO: M20242078.

[0011] The suitable culture medium of the purple Monascus M23067Z4Fd12 comprises: glucose, peptone, NaNO3, MgSO4, and KH2PO4; in a feasible embodiment, each 1000 mL of culture medium comprises: 40-60 g, preferably 50 g glucose, 10-20 g, preferably 15 g peptone, 1-3 g, preferably 2 g NaNO3, 0.8-1.2 g, preferably 1 g MgSO4·7H2O, 1-2 g, preferably 1.5 g KH2PO4, and the rest is water.

[0012] The inventors have discovered that adding specific types of amino acids and / or organic acids to the culture medium can significantly improve the ability of the purple Monascus sp. M23067Z4Fd12 to produce Monascus pigment.

[0013] Therefore, preferably, the suitable culture medium for Monascus purpurogenum M23067Z4Fd12 further comprises amino acid and / or organic acid additives; in a preferred embodiment, the culture medium further comprises any one or more additives selected from the following: cysteine, histidine, glycine, tyrosine, valine, tartaric acid, ascorbic acid, citric acid and malic acid, preferably selected from: cysteine, histidine, glycine and tyrosine.

[0014] In addition, the inventors also found that the purple Monascus M23067Z4Fd12 has strong fermentation characteristics, including temperature adaptability, pH adaptability, and alcohol tolerance.

[0015] In a second aspect, the present invention provides a starter culture comprising the Monascus purpurogenum M23067Z4Fd12 or a metabolite thereof as described in the first aspect above.

[0016] Preferably, the fermentation agent is a fermentation culture of Monascus purpurogenum M23067Z4Fd12;

[0017] Preferably, the starter culture is a fermentation culture of Monascus purpurogenum M23067Z4Fd12 in a culture medium containing glucose, peptone, NaNO3, MgSO4, and KH2PO4; further preferably, the culture medium further contains amino acid and / or organic acid additives; even more preferably, the culture medium further contains any one or more additives selected from the following: cysteine, histidine, glycine, tyrosine, valine, tartaric acid, ascorbic acid, citric acid, and malic acid, preferably selected from the following: cysteine, histidine, glycine, and tyrosine;

[0018] Optionally, the fermentation agent is a liquid or solid preparation; optionally, the dosage form of the fermentation agent is selected from the group consisting of: solution, suspension, emulsion, powder, granule, pill, tablet, gel.

[0019] In a third aspect, the present invention provides a method for preparing the starter agent as described in the second aspect above, the preparation method comprising: fermenting the Monascus purpurogenum M23067Z4Fd12 as described in the first aspect above in a culture medium;

[0020] Preferably, the culture medium comprises glucose, peptone, NaNO3, MgSO4, and KH2PO4;

[0021] Further preferably, the culture medium further comprises amino acid and / or organic acid additives; even more preferably, the culture medium further comprises any one or more additives selected from the group consisting of cysteine, histidine, glycine, tyrosine, valine, tartaric acid, ascorbic acid, citric acid and malic acid, preferably selected from the group consisting of cysteine, histidine, glycine and tyrosine;

[0022] Preferably, the fermentation culture temperature is 25-35°C, preferably 30°C;

[0023] Preferably, the fermentation culture time is 7-15 days, preferably 8-12 days.

[0024] In a fourth aspect, the present invention provides a composition comprising the Monascus purpurogenum M23067Z4Fd12 as described in the first aspect above, or comprising the fermentation agent as described in the second aspect above.

[0025] In a feasible embodiment, the composition is a food or a pharmaceutical composition, which further comprises a food or a pharmaceutically acceptable carrier.

[0026] In a fifth aspect, the present invention provides use of the Monascus purpurogenum M23067Z4Fd12 described in the first aspect, the starter described in the second aspect, and / or the composition described in the third aspect in preparing fermented foods.

[0027] In a feasible embodiment, the fermented food is made from cereals, beans or Chinese medicinal materials;

[0028] Preferably, the raw material of the fermented food is rice, sorghum or tangerine peel;

[0029] Further preferably, the fermented food comprises: red yeast rice, red yeast sorghum, red yeast wine, red yeast tangerine peel, and red yeast tangerine peel beverage.

[0030] In a sixth aspect, the present invention provides a method for preparing fermented food, the method comprising: using the purple Monascus M23067Z4Fd12 described in the first aspect above, the starter described in the second aspect above and / or the composition described in the fourth aspect above, and fermenting cereals, beans or Chinese medicinal materials as raw materials.

[0031] Preferably, the raw material is selected from: rice, grains (such as sorghum) or tangerine peel;

[0032] Preferably, the fermented food comprises: red yeast rice, red yeast sorghum, red yeast wine, red yeast tangerine peel, and red yeast tangerine peel beverage;

[0033] Preferably, the fermentation temperature is 25-35°C, preferably 30°C;

[0034] Preferably, the fermentation time is 5-10 days, preferably 6-8 days.

[0035] By fermenting rice using the above method, red yeast rice with high red yeast rice pigment content and no toxicity or harm can be obtained;

[0036] By fermenting sorghum using the above method, red yeast rice sorghum with fermented aroma and high red yeast rice pigment content can be produced;

[0037] The red yeast rice wine obtained by the above method is orange-red in color and uniform and natural, has rich aroma, smooth taste, and is non-toxic and harmless.

[0038] By fermenting tangerine peel using the above method, the total flavonoids content and antioxidant activity of tangerine peel can be greatly increased.

[0039] In a seventh aspect, the present invention provides a method for producing red yeast rice pigment, the method comprising: fermenting the purple Monascus sp. M23067Z4Fd12 according to claim 1 in a culture medium containing glucose, peptone, NaNO3, MgSO4, and KH2PO4 to produce red yeast rice pigment;

[0040] In a feasible embodiment, each 1000 mL of culture medium contains: 40-60 g, preferably 50 g glucose, 10-20 g, preferably 15 g peptone, 1-3 g, preferably 2 g NaNO3, 0.8-1.2 g, preferably 1 g MgSO4·7H2O, 1-2 g, preferably 1.5 g KH2PO4, and the rest is water.

[0041] Preferably, the culture medium further comprises amino acid and / or organic acid additives.

[0042] Further preferably, the culture medium further comprises any one or more additives selected from the group consisting of: cysteine, histidine, glycine, tyrosine, valine, tartaric acid, ascorbic acid, citric acid and malic acid, preferably selected from the group consisting of: cysteine, histidine, glycine and tyrosine;

[0043] Preferably, the fermentation culture temperature is 25-35°C, preferably 30°C;

[0044] Preferably, the fermentation culture time is 7-15 days, preferably 8-12 days.

[0045] Optionally, the method further comprises the step of separating and purifying the monascus pigment from the fermentation culture.

[0046] Beneficial effects

[0047] Through extensive experimental screening and optimization, the present invention successfully obtained a strain of purple Monascus purpureus (Monascus purpureus) M23067Z4Fd12 with excellent fermentation performance. This strain not only produces high yields of red yeast pigment, but also, after rigorous testing, has been confirmed to not produce mycotoxins such as citrinin, showing excellent safety. It is particularly worth noting that the present invention has discovered for the first time that by adding specific types of amino acids and / or organic acids (including but not limited to amino acids such as cysteine, histidine, glycine, tyrosine, and valine, as well as organic acids such as tartaric acid, ascorbic acid, citric acid, and malic acid) to the culture medium, the secondary metabolic pathways of the strain can be significantly activated, significantly increasing the yield of red yeast pigment. This discovery provides a new technical approach for the efficient biosynthesis of red yeast pigment.

[0048] The purple Monascus M23067Z4Fd12 of the present invention exhibits excellent fermentation performance, including strong temperature adaptability, pH adaptability and alcohol tolerance, and these characteristics make it particularly suitable for industrial fermentation production.

[0049] In practical application, the strain of the present invention has broad application prospects and significant value-added effects, as shown in:

[0050] In the development of fermented foods, the purple Monascus fungus M23067Z4Fd12 is inoculated into rice, sorghum and other cereals for fermentation, which not only gives the product a natural red color but is also non-toxic and harmless, with excellent safety.

[0051] In the winemaking industry, fermentation with this strain can enrich the aroma and smooth the taste of wine. It also increases the content of functional ingredients such as red yeast rice pigment in the wine, giving the product unique health attributes.

[0052] In the field of deep processing of Chinese medicinal materials, using this strain for the biotransformation of tangerine peel can significantly increase the total flavonoids content in tangerine peel and enhance its antioxidant activity, providing a new path for the value-added development of traditional Chinese medicinal materials.

[0053] In summary, the purple Monascus M23067Z4Fd12 provided by the present invention has significant technical advantages in increasing the yield of Monascus pigment, expanding the application field, and enhancing the added value of products, providing important strain resources and technical support for the innovative development of Monascus-related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0055] Figure 1 The colony morphology of strain M23067Z4Fd12 cultured in PDA medium at 30°C for 7, 15, and 30 days is shown.

[0056] Figure 2 Shown are the results of optical microscopy observation of the M23067Z4Fd12 strain.

[0057] Figure 3 Shown is the phylogenetic tree of the M23067Z4Fd12 strain constructed based on the ITS sequencing results.

[0058] Figure 4 Shown is the phylogenetic tree of the M23067Z4Fd12 strain constructed based on the sequencing results of β-tubulin.

[0059] Figure 5 The ISSR fingerprint of strain M23067Z4Fd12 obtained after amplification with eight primers is shown; the primer names are shown above the image.

[0060] Figure 6A comparison of the appearance of red yeast rice fermented with purple Monascus sp. M23067Z4Fd12 and commercially available red yeast rice is shown.

[0061] Figure 7 A picture showing the appearance of red yeast rice fermented by purple Monascus sp. M23067Z4Fd12.

[0062] Figure 8 A picture showing the appearance of red yeast rice wine fermented by purple Monascus sp. M23067Z4Fd12.

[0063] Figure 9 A comparison of the appearance of red yeast rice peel obtained by fermentation with purple Monascus sp. M23067Z4Fd12 and that of dried tangerine peel not inoculated with the strain is shown.

[0064] Figure 10 A comparison of the appearance of the red yeast rice and tangerine peel beverage obtained by fermentation with purple Monascus sp. M23067Z4Fd12 and the tangerine peel beverage without inoculation of the strain is shown. DETAILED DESCRIPTION

[0065] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0066] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present invention can be practiced without certain specific details. In some embodiments, raw materials, components, methods, means, etc. that are well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0067] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0068] The present invention is further described in detail below through examples.

[0069] In the following examples, unless otherwise specified, all raw materials are commercially available products.

[0070] Example 1: Screening of Monascus purpurogenum M23067Z4Fd12

[0071] The strain of the present invention was obtained by screening from the Monascus library of Shenyang Agricultural University. The strains in the library were all isolated from naturally fermented red yeast rice. The purple Monascus M23067Z4Fd12 of the present invention was obtained through primary screening and secondary screening. The specific steps are as follows:

[0072] 1. Preliminary screening of bacterial strains:

[0073] The seed liquid culture method was used to preliminarily screen the red yeast rice strains with high pigment content in the fermentation broth.

[0074] The specific screening steps are as follows: take out the Monascus cake stored in the -80℃ refrigerator, dissolve it under natural conditions, and then put it into PDA culture medium and culture it at 30℃ for 7 days; pick the spores on the surface of the PDA culture medium into sterile water and beat them with sterilized glass beads to make the spore concentration reach 1×10 6 4 mL of spore suspension was added to the seed liquid culture medium and cultured at 30°C and 180 r / min for 7 days, and then the pigment content was determined.

[0075] Determination of Monascus pigment content: Take 5 mL of the fermentation broth after the above culture, add 10 mL of 70% ethanol, and place in a 60°C water bath for 30 min. After cooling to room temperature, 410nm ,OD 470nm ,OD 510nm The content of red yeast rice pigment was determined.

[0076] The results showed that 15 strains of red yeast rice had strong pigment production ability, while other strains had less pigment content or were even unable to metabolize and synthesize red yeast rice pigment.

[0077] 2. Strain rescreening:

[0078] Liquid fermentation was used to screen high-pigment red yeast rice strains.

[0079] The specific screening steps are as follows: the 15 red yeast rice strains obtained from the initial screening were fermented with seed liquid, and after constant temperature shaking at 30°C for 4 days, 4 mL of seed liquid was taken for liquid fermentation culture for 7 days to obtain fermentation liquid.

[0080] Method for determining the pigment content of Monascus: The fermentation broth was filtered and the pigment content of the obtained supernatant fermentation broth was determined (the determination method refers to the initial screening of strains).

[0081] Determination of mycelium dry weight: The precipitate obtained after filtration was rinsed with distilled water, dried, and the mycelium dry weight was determined.

[0082] Determination of citrinin content: Take 1.5 mL of fermentation broth for determination of citrinin. The specific determination method refers to GB5009.222-2016 "Determination of citrinin in foods".

[0083] The results are shown in Table 1.

[0084] Table 1 Monascus pigment content, citrinin content, and mycelial biomass of the rescreened strains

[0085]

[0086]

[0087] Table 1 shows that the fermentation broth of strain M23067Z4Fd12 has the highest content of red yeast rice pigment, which is 181.150 U / mL, and its mycelial growth is also the highest, which is 38.5 g / L, and it does not contain citrinin. In contrast, the fermentation broths of the four strains M230525Z6Ia1, M230606Z3Dc15, M230601Z3Dc16, and M230628Z3Gc23 all contain citrinin, and the red yeast rice pigment production is average; while the red yeast rice pigment production of other strains is very low.

[0088] In summary, the M23067Z4Fd12 strain grew well and had a strong pigment metabolism ability. Therefore, through primary and secondary screening, the M23067Z4Fd12 strain was finally determined to be a high-quality strain that produces high pigment and no citrinin, namely the purple Monascus of the present invention.

[0089] Example 2: Morphological identification of Monascus purpurogenum M23067Z4Fd12

[0090] 1. Observation by plate culture method: The activated M23067Z4Fd12 bacterial cake was inoculated into PDA medium and cultured at 30℃ for 7 days, 15 days and 30 days, and the colony morphology, color and growth were observed.

[0091] The results are as follows Figure 1 As shown; Figure 1 It shows that the M23067Z4Fd12 colony on the 7th day was round, with a diameter of 15-17 mm, orange mycelium and white edges; the M23067Z4Fd12 colony on the 15th day was 33-36 mm in diameter, orange-yellow mycelium with intact edges; the M23067Z4Fd12 colony on the 30th day was 45-48 mm in diameter, and red mycelium; during the entire observation period, the color of the PDA culture medium gradually changed from transparent to orange and finally to red.

[0092] 2. Observation under an optical microscope: Add an appropriate amount of distilled water to the plate and gently scrape off the hyphae and spores on the bacterial cake; aspirate 10 μL and drop it onto a glass slide, and use an optical microscope to observe the morphological characteristics of ascospores, ascocarps, etc.

[0093] The results are as follows Figure 2 As shown; Figure 2Display: The hyphae of M23067Z4Fd12 are irregularly branched, transparent, with obvious oil droplets, transverse septa, and warty crystals on the wall; the ascocarp type is cleistocarp, which is spherical or elliptical and has a stalk; there are many spherical asci scattered in the ascocarp, each containing spherical or ovoid ascospores, which disintegrate after maturity and release ascospores.

[0094] Example 3: Molecular Identification of Monascus purpurogenum M23067Z4Fd12

[0095] DNA extraction: The mycelia of strain M23067Z4Fd12 were placed in PD culture medium, cultured on a constant temperature shaker at 32°C and 120 rpm for 4 days, filtered, and dried at 60°C. 0.1 g of mycelia were taken and the strain DNA was extracted using a fungal genomic DNA kit (Shenyang Wanlei Company) according to its instructions.

[0096] The strain identification used ITS sequence and β-tubulin sequence for analysis. The specific operation is as follows:

[0097] 1. ITS phylogenetic tree analysis:

[0098] ITS sequence amplification: PCR amplification was performed using the primer pair ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3', SEQ ID NO: 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO: 2) and the above-extracted strain DNA as a template.

[0099] Sequencing and identification: The PCR product was purified and sent to Shanghai Bioengineering Company for sequencing. The sequence obtained (i.e., the ITS sequence of strain M23067Z4Fd12) is shown in SEQ ID NO: 3 below:

[0100] .

[0101] The phylogenetic tree of strain M23067Z4Fd12 was constructed based on the ITS sequencing results. Figure 3 As shown; Figure 3 The results showed that the homology between strain M23067Z4Fd12 and Monascus purpureus MN 156545.1 was 99%, thus confirming that strain M23067Z4Fd12 belonged to the genus Monascus.

[0102] 2. β-tubulin phylogenetic tree analysis:

[0103] β-tubulin sequence amplification:

[0104] The Ben A region was amplified by PCR using the primer pair Bt2a (5'-GGTAACCAAATCGGTGCTGCTTTC-3', SEQ ID NO: 4) and Bt2b (5'-ACCCTCAGTGTAGTGACCCTTGGC-3', SEQ ID NO: 5) and the above-extracted strain DNA as a template.

[0105] Sequencing and identification: The PCR product was purified and sent to Shanghai Bioengineering Company for sequencing. The sequence obtained (i.e., the β-tubulin sequence of M23067Z4Fd12) is shown below in SEQ ID NO: 6:

[0106] .

[0107] The phylogenetic tree of strain M23067Z4Fd12 was constructed based on the β-tubulin sequencing results. Figure 4 As shown; Figure 4 The results showed that the homology between strain M23067Z4Fd12 and Monascus purpureus MN 229577.1 was 99%, thus confirming that strain M23067Z4Fd12 was Monascus purpureus.

[0108] Example 4: ISSR fingerprint of Monascus purpurogenum M23067Z4Fd12

[0109] ISSR is suitable for marking differences within microbial populations. In this example, eight ISSR primers were used to construct a fingerprint of the strain M23067Z4Fd12 of the present invention.

[0110] Primer selection: The eight primers selected were all from the 100 ISSR primers published by Columbia University, numbered 816, 817, 836, 855, 857, 888, 889, and 890. The specific sequences are shown in Table 2.

[0111] Table 2 ISSR primers tested

[0112]

[0113] Note: Among them: Y=(C,T), B=(C,G,T), D=(A,G,T), H=(A,C,T), V=(A,C,G)

[0114] The PCR reaction system (total volume 25 μL) was as follows: 1.5 μL DNA, 1.5 μL primers, 9.5 μL dd H2O, and 12.5 μL 2× TaqMaster Mix DNA polymerase.

[0115] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, optimal annealing temperature for 45 s for a total of 35 cycles, and extension at 72°C for 7 min.

[0116] The ISSR map construction process is as follows: prepare a 1.5% agarose gel, take 5 μL of the above PCR product and load it for electrophoresis at a voltage of 100 V. Stop the electrophoresis when the bromophenol blue dye band in the gel moves close to the end of the gel. Observe on a gel imaging system, take pictures and record. The resulting map is the DNA fingerprint map of the PCR product.

[0117] The ISSR fingerprints of the strain obtained after amplification with 8 primers are shown in Figure 5 .

[0118] Figure 5The results show that the amplified pattern of primer 816 has 7 obvious bands, 4 of which are distributed in the range of 1000-2500bp, 1 in the range of 750bp, and 2 in the range of 500bp; the amplified pattern of primer 817 has 8 obvious bands, 1 in the range of 2500bp, 5 in the range of 750-2000bp, and 2 in the range of 500-750bp; the amplified pattern of primer 836 has more bands, with a total of 8 clear bands, 3 in the range of 1000-2000bp, and 5 concentrated in the range of 250-750bp; the amplified pattern of primer 855 has 1 clear band, concentrated in the range of 1000-2000bp. 0bp interval; Primer 857 has 4 clear bands, 3 of which are concentrated in the 500-1000bp interval, and 1 is distributed in the 2000bp interval; Primer 888 has 5 clear bands, 1 is distributed in the 2000bp interval, 2 are distributed in the 1000-2000bp interval, and 2 are distributed in the 500-750bp interval; Primer 889 has 2 clear bands, one of which is distributed in the 1000bp interval with the most prominent band brightness, and the other is distributed in the 250bp interval; Primer 890 has 2 clear bands, one in the 1000bp interval with prominent brightness, and the other is distributed in the 1000-2000bp interval.

[0119] Example 5: Effects of different exogenous additives on the pigment production ability of Monascus purpurogenum M23067Z4Fd12

[0120] Ten culture solutions were prepared, with no addition (CK) and nine different exogenous additives added. The nine exogenous additives were cysteine, histidine, glycine, tyrosine, valine, tartaric acid, ascorbic acid, citric acid, and malic acid. Seed solution of Monascus purpurogenum M23067Z4Fd12 was added to ten culture solutions (culture solution: 50 g glucose, 15 g peptone, 2 g NaNO₃, 1 g MgSO₄·7H₂O, 1.5 g KH₂PO₄) and cultured at 30°C and 180 rpm for 7 days. Five milliliters of the fermented broth were added with 10 mL of 70% ethanol, incubated at 60°C in a water bath for 30 minutes, and cooled to room temperature. The monascus pigment content and citrinin content were then determined using the same method as in Example 1.

[0121] The results are shown in Table 3.

[0122] Table 3 Effects of different exogenous additives on monascus pigment and citrinin in Monascus purpurogenum M23067Z4Fd12 strain

[0123]

[0124]

[0125] Table 3 shows that after adding cysteine ​​or histidine, the increase in the pigment content of red yeast rice was the most significant, with the total pigment content being 439.24 U / mL and 434.25 U / mL, which was 4 times the pigment content of the CK group (181.150 U / mL), and no citrinin was produced; the addition of glycine, tyrosine, valine, tartaric acid, ascorbic acid, citric acid, and malic acid can promote the increase in the total pigment content, which is 2-3 times the pigment content of the CK group (181.150 U / mL), and no citrinin was produced; in addition, it can be concluded from Table 3 that exogenous additives of amino acids can significantly promote the production of extracellular pigments, while exogenous additives of organic acids can significantly increase the production of intracellular pigments.

[0126] Example 6: Temperature adaptability determination of Monascus purpurogenum M23067Z4Fd12

[0127] An appropriate amount of spore suspension of Monascus purpurogenum M23067Z4Fd12 was inoculated into PD medium and PDA plate medium, respectively. Then, the PD medium inoculated with the strain spores was placed on a constant temperature shaker at 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C, and cultured at 180 r / min. The PDA plate medium inoculated with the strain spores was placed on a constant temperature shaker at 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C, and the mycelial growth and colony diameter were measured.

[0128] The results are shown in Table 4.

[0129] Table 4 Effect of temperature on the growth of fermented purple Monascus M23067Z4Fd12

[0130]

[0131]

[0132] Note: “-” means no growth; “±” means slight growth; “+” means normal growth; “++” means good growth; “+++” means vigorous growth; the volume of the sample for growth measurement is 30 mL.

[0133] As shown in Table 4, the M23067Z4Fd12 strain can grow at temperatures between 15°C and 45°C, indicating that it has very strong temperature adaptability.

[0134] Example 7: pH adaptability determination of Monascus purpurogenum M23067Z4Fd12

[0135] An appropriate amount of spore suspension of Monascus purpurogenum M23067Z4Fd12 was inoculated into PD culture medium with pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively. The culture was carried out at a constant temperature of 30°C and 180 rpm, and the mycelial growth under different conditions was measured.

[0136] The results are shown in Table 5.

[0137] Table 5 Effect of pH value on the growth of fermented purple Monascus M23067Z4Fd12

[0138]

[0139] Note: “-” means no growth; “±” means slight growth; “+” means normal growth; “++” means good growth; “+++” means vigorous growth; the volume of the sample for growth measurement is 30 mL.

[0140] As shown in Table 5, Monascus purpurogenum M23067Z4Fd12 can grow in both strong acid and weak alkaline fermentation environments, indicating that it has strong adaptability to pH values.

[0141] Example 8: Alcohol tolerance determination of Monascus purpurogenum M23067Z4Fd12

[0142] An appropriate amount of spore suspension of Monascus purpurogenum M23067Z4Fd12 was inoculated into PD culture medium and PDA plate culture medium with alcohol concentrations of 8%, 10%, 12%, 15%, 18% and 20%, respectively. Then, the PD culture medium inoculated with the strain spores was placed at a constant temperature of 30°C and 180r / min for culture, and the PDA plate culture medium inoculated with the strain spores was placed at a constant temperature of 30°C for static culture, and the colony diameter and growth amount were measured.

[0143] The results are shown in Table 6.

[0144] Table 6 Effect of alcohol concentration on the growth of fermented purple Monascus M23067Z4Fd12

[0145]

[0146]

[0147] Note: “-” means no growth; “±” means slight growth; “+” means normal growth; “++” means good growth; “+++” means vigorous growth; the volume of the sample for growth measurement is 30 mL.

[0148] As shown in Table 6, the purple Monascus sp. M23067Z4Fd12 can grow in an alcohol content range of ≤18%, indicating that it has strong tolerance to alcohol. In addition, the alcohol content of commercially available red yeast rice wine generally does not exceed 18%. Therefore, this strain can not only adapt well to the fermentation environment, but also has the potential for winemaking.

[0149] Example 9: Application of purple Monascus M23067Z4Fd12 in the preparation of high-pigment red yeast rice

[0150] Preparation of fermentation agent of Monascus purpurogenum M23067Z4Fd12

[0151] 4 mL of the spore suspension of the purple Monascus M23067Z4Fd12 strain was inoculated into a liquid culture medium (culture medium composition reference Example 5), and cultured at 30° C. and 180 r / min for 4 days to obtain a fermentation agent of the purple Monascus M23067Z4Fd12 strain.

[0152] Preparation of red yeast rice

[0153] 20g of high-quality indica rice is weighed and placed in a fermentation tank. The rice is then washed with clean water 3-4 times until the water is no longer turbid. 40mL of water is added to the fermentation tank and the rice is soaked for 24-28 hours until the rice has fully absorbed the water. The soaked rice is then washed and drained, and sterilized at 115°C for 20 minutes. After cooling, a purple Monascus M23067Z4Fd12 fermentation inoculum is added to the fermentation tank at a volume-to-mass ratio of 10% (i.e., volume (mL) of fermentation inoculum / weight (g) of rice) and stirred thoroughly to evenly distribute the seed liquid over the rice grains. The fermentation tank is sealed and cultured in a 30°C constant temperature incubator for 7 days to obtain red yeast rice.

[0154] Determination of related indicators of red yeast rice

[0155] The red yeast rice was ground into powder, and the monascus pigment content and citrinin content were determined. The specific determination method was referred to Example 1. The determination method of monascus pigment and citrinin content was referred to the national standards GB 1886.19-2015 "Food Additive Red Yeast Rice" and GB 5009.222-2016 "Determination of Citrinin in Foods".

[0156] Measurement results

[0157] 1) The red yeast rice fermented with purple Monascus sp. M23067Z4Fd12 had a color value of 4230.47 U / g and produced no citrinin. In comparison, the color value of commercially available red yeast rice is generally 2004.7 U / g. That is, the red yeast rice fermented with purple Monascus sp. M23067Z4Fd12 of the present invention has a color value far higher than that of commercially available red yeast rice.

[0158] 2) The appearance of red yeast rice obtained by fermentation of purple Monascus M23067Z4Fd12 is compared with that of commercially available red yeast rice. Figure 6 shown; according to Figure 6 The sensory evaluation of the red yeast rice fermented by purple Monascus M23067Z4Fd12 found that the red yeast rice fermented by purple Monascus M23067Z4Fd12 was uniformly dark red in color, with complete rice grains, crisp texture and no mold, red cross-section, and the inherent aroma of red yeast.

[0159] Example 10: Application of purple Monascus M23067Z4Fd12 in the preparation of high-pigment red yeast sorghum

[0160] Preparation of red yeast rice

[0161] Weigh 20g of sorghum rice and wash it with clean water 3-4 times until the water is no longer turbid. Add 40mL of water to a fermentation tank and soak for 24-28h until the sorghum rice fully absorbs water; wash and drain the soaked sorghum and sterilize it at 115°C for 20min; after cooling, absorb a purple Monascus M23067Z4Fd12 fermentation agent (prepared as in Example 9) at a volume-to-mass ratio of 10% (i.e., volume (mL) of fermentation agent / weight (g) of rice) and place it in a fermentation tank. Place the fermentation agent in a constant temperature incubator at 30°C for 7d to obtain red yeast rice.

[0162] Determination of Related Indexes of Red Yeast Sorghum Rice

[0163] The red yeast rice sorghum was ground into powder, and the red yeast rice pigment content and citrinin content were determined. The specific determination method is shown in Example 1.

[0164] Measurement results

[0165] 1) The red yeast rice sorghum fermented by purple Monascus sp. M23067Z4Fd12 had a color value of 4015.67 U / g and produced no citrinin.

[0166] 2) The appearance of red yeast rice sorghum obtained by fermentation with purple Monascus M23067Z4Fd12 is as follows Figure 7 shown; according to Figure 7 The sensory evaluation of the red yeast rice sorghum obtained by fermenting purple Monascus M23067Z4Fd12 found that the red yeast rice sorghum obtained by fermenting purple Monascus M23067Z4Fd12 was bright red in appearance, had no obvious impurities, was in the form of complete particles, had a red cross-section, and had a fermented yeast aroma.

[0167] Example 11: Application of purple Monascus M23067Z4Fd12 in the preparation of high-pigment red yeast rice wine

[0168] Preparation of red yeast rice wine

[0169] 20g of high-quality indica rice was weighed and placed in a conical flask, and washed with clean water for 3-4 times until the rice washing water was no longer turbid. 40mL of water was subsequently added and the rice was soaked for 24-48h until the rice fully absorbed water. The soaked rice was washed and drained, and sterilized at 115°C for 20min. After cooling to room temperature, 40mL of sterile distilled water, 10% of the red yeast rice prepared in Example 9, and 0.72% of mesophilic saccharifying enzyme were added. The mixture was sealed and placed in a 30°C constant temperature culture for 7d, during which the wine was shaken every 12h. The fermented wine was squeezed and filtered, and the wine was decocted in an 80°C constant temperature water bath for 15min. After complete sedimentation, the supernatant was transferred to a sterile liquid storage bottle, sealed, and stored at 4°C.

[0170] Determination of relevant indicators of red yeast rice wine

[0171] An appropriate amount of the prepared red yeast rice wine was collected and assayed for red yeast rice pigment, citrinin, reducing sugar, and total acid content, and the wine was then subjected to sensory evaluation. These indicators were determined in accordance with national standards GB 1886.19-2015, "Food Additive Red Yeast Rice," GB 5009.222-2016, "Determination of Citrinin in Foods," QB 5334-2019, "Red Yeast Rice," and GB 13662-2018, "Yellow Rice Wine."

[0172] Measurement results

[0173] 1) The red yeast rice wine obtained by fermentation of purple Monascus sp. M23067Z4Fd12 has a pigment content of 7.139 U / mL, a reducing sugar content of 32.84 g / L, a total acid content of 4.5 g / L, an alcohol content of 9.8%, and is citrinin-free.

[0174] 2) The appearance of the red yeast rice wine obtained by fermentation with purple Monascus sp. M23067Z4Fd12 is as follows: Figure 8 shown; according to Figure 8 The sensory evaluation of the red yeast rice wine obtained by fermenting purple Monascus sp. M23067Z4Fd12 found that the red yeast rice wine obtained by fermenting purple Monascus sp. M23067Z4Fd12 was orange-red in color, with a uniform and natural color; the red yeast rice wine contained the aroma of rice, wine and the unique aroma of red yeast rice, with rich layers of aroma; the red yeast rice wine had a smooth taste, was slightly sweet and mellow, and had no alcohol stimulation.

[0175] Example 12: Fermentation of dried tangerine peel using Monascus purpurogenum M23067Z4Fd12

[0176] Tangerine peel is a traditional Chinese medicinal ingredient, commonly used in traditional Chinese medicine and traditional diet therapy. It has benefits such as regulating qi and strengthening the spleen, relieving cough and reducing phlegm, and providing antioxidant and anti-inflammatory properties. Monascus, a fungus that can be used as both medicine and food, has cholesterol-lowering and blood sugar-lowering, as well as antioxidant properties. Fermenting tangerine peel with Monascus not only significantly increases the content of its functional ingredients but also imparts the functional properties of Monascus to the tangerine peel.

[0177] In this embodiment, the purple Monascus M23067Z4Fd12 of the present invention was used to ferment tangerine peel to test its application effect in tangerine peel fermentation, as follows.

[0178] Method of fermenting tangerine peel

[0179] Take 5g dried tangerine peel and put it in fermentation tank, rinse with clear water 3-4 time, drain subsequently, and sterilize 20min under 115 ℃ of conditions, after being cooled to room temperature, according to 10% volume mass ratio (that is, the quality (g) of volume (mL) / meter of fermentation bacteria agent), add purple Monascus M23067Z4Fd12 fermentation bacteria agent (its preparation method is with Example 9) and be placed in fermentation tank, seal and preserve, cultivate 7d in 30 ℃ of constant temperature incubators, during this period, shake once every 12h, obtain red yeast rice dried tangerine peel.

[0180] Determination of related indices of red yeast rice and tangerine peel

[0181] An appropriate amount of the prepared red yeast rice tangerine peel was taken to measure the red yeast rice pigment, citrinin, total flavonoids, antioxidant activity and related indicators, and a sensory evaluation was performed. The specific operation method is as follows:

[0182] 1. Total flavonoids assay: Pulverize and sieve a sample of red yeast rice and dried tangerine peel. Weigh approximately 0.1g of the powder, add 5mL of 70% ethanol, incubate in a water bath for 1 hour, and centrifuge at 8000r / min for 5 minutes. Collect the supernatant. Measure 0.5mL of the test solution, add 1mL of 5% NaNO₂, shake well, and let stand for 6 minutes. Add 1mL of 10% Al(NO₃)₃, shake well, and let stand for 6 minutes. Finally, add 5mL of 4% NaOH, make up to volume with 70% ethanol, shake well, and let stand for 15 minutes. Measure the absorbance at 510nm and calculate the total flavonoid content in mg / g.

[0183] 2. Determination of DPPH free radical scavenging ability: After centrifugation of the fermentation broth, the supernatant was taken and diluted 10 times with distilled water. 0.3 mL was added to 3.7 mL of 1.0×10 -4 mol / L DPPH solution, after thorough mixing, react at room temperature in the dark for 20 minutes, centrifuge at 8000r / min for 5 minutes, take the supernatant and measure its absorbance at a wavelength of 517nm, record it as A1, replace the supernatant with distilled water and measure its absorbance at a wavelength of 517nm, record it as A0, replace the DPPH solution with anhydrous ethanol and measure its absorbance at a wavelength of 517nm, record it as A2, and calculate the DPPH free radical scavenging rate to reflect its antioxidant activity. The calculation formula of DPPH free radical scavenging rate is as follows:

[0184] DPPH free radical scavenging rate % = [A0-(A1-A2) / A0] × 100

[0185] 3. The determination methods of monascus pigment and citrinin refer to the national standards GB 1886.19-2015 "Food Additive Red Yeast Rice" and GB 5009.222-2016 "Determination of citrinin in Foods".

[0186] Measurement results

[0187] 1) The results of the determination of pigment content, total flavonoids content and antioxidant activity of red yeast rice peel obtained by fermentation of purple Monascus sp. M23067Z4Fd12 are shown in Table 7.

[0188] Table 7 Comparison of pigment content, total flavonoids and antioxidant activity between fermented and unfermented tangerine peel

[0189]

[0190] As can be seen from Table 7, the color value of red yeast rice peel fermented with purple Monascus M23067Z4Fd12 was 1422.6 μ / g, and the total flavonoid content was 11.91 mg / g, which is 1.8 times the total flavonoid content of uninoculated tangerine peel (6.31 mg / g), and there was no citrinin. This result shows that tangerine peel fermented with Monascus M23067Z4Fd12 not only contains red yeast pigment, but also significantly increases the total flavonoid content in tangerine peel.

[0191] 2) Furthermore, after inoculation with Monascus purpurogenum M23067Z4Fd12, the DPPH free radical scavenging rate in dried tangerine peel reached 82.08%, twice that of uninoculated dried tangerine peel (45.62%). This result indicates that fermentation with Monascus purpurogenum M23067Z4Fd12 significantly enhances the antioxidant capacity of dried tangerine peel.

[0192] 3) The appearance of red yeast rice peel obtained by fermentation of purple Monascus M23067Z4Fd12 is shown in Figure 9 ;Depend on Figure 9 The sensory evaluation of red yeast rice tangerine peel fermented by purple Monascus M23067Z4Fd12 found that the red yeast rice tangerine peel fermented by purple Monascus M23067Z4Fd12 was dark brown in color, crisp in texture, and had the aroma of fermentation and the fragrance of tangerine peel.

[0193] Example 13: Application of purple Monascus M23067Z4Fd12 in the preparation of high-pigment red yeast rice and tangerine peel beverage

[0194] Preparation and determination of red yeast rice fermented tangerine peel beverage

[0195] Get the red yeast rice tangerine peel prepared in 1.5g embodiment 12, with the unfermented tangerine peel of same weight as contrast.Add 13.5mL normal temperature pure water respectively and brew, after brewing 10min, respectively get 1mL fermented tangerine peel and the tangerine peel beverage of uninoculated bacterial strain, contrast beverage color, total flavonoids content and antioxidant activity.The relevant index determination concrete operation of red yeast rice tangerine peel beverage is shown in embodiment 12.

[0196] Measurement results

[0197] 1) The pigment content, total flavonoids content and antioxidant activity of the red yeast rice and tangerine peel beverage obtained by fermentation of purple Monascus M23067Z4Fd12 are shown in Table 8.

[0198] Table 8 Comparison of pigment content, total flavonoids and antioxidant activity between fermented tangerine peel and unfermented tangerine peel beverages

[0199]

[0200] As can be seen in Table 8, the red yeast rice and tangerine peel beverage fermented with purple Monascus purpurogenum M23067Z4Fd12 had a pigment content of 3.47 U / mL and a total flavonoid content (6.81 mg / g) that was 1.8 times that of the uninoculated beverage (3.67 mg / g), with no citrinin production. These results indicate that the beverage fermented with Monascus purpurogenum M23067Z4Fd12 not only contains red yeast rice pigment but also significantly increases its total flavonoid content.

[0201] 2) Furthermore, the DPPH free radical scavenging rate of the red yeast rice and tangerine peel beverage fermented with Monascus purpurogenum M23067Z4Fd12 was 66.01%, 1.6 times that of the uninoculated beverage (39.21%). This result indicates that fermentation with Monascus purpurogenum M23067Z4Fd12 significantly enhances the antioxidant capacity of the red yeast rice and tangerine peel beverage.

[0202] 3) The appearance of the red yeast rice and tangerine peel beverage obtained by fermentation of purple Monascus M23067Z4Fd12 is shown in FIG. Figure 10 ;Depend on Figure 10 The sensory evaluation of the red yeast rice and tangerine peel beverage obtained by fermentation of purple Monascus M23067Z4Fd12 found that the red yeast rice and tangerine peel beverage of purple Monascus M23067Z4Fd12 was brown-yellow in color, natural in color, and had the aroma of tangerine peel and unique yeast fragrance.

[0203] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A purple Monascus strain M23067Z4Fd12, whose taxonomic name is Monascus purpureus, was deposited in the China Center for Type Culture Collection (CCTCC) on September 25, 2024, with the deposit number CCTCC NO: M20242078.

2. A leavening agent, characterized in that The fermentation agent comprises the Monascus purpurogenum M23067Z4Fd12 or a metabolite thereof according to claim 1.

3. The leavening agent according to claim 2, characterized in that The fermentation agent is a fermentation culture of Monascus purpurogenum M23067Z4Fd12; Preferably, the starter culture is a fermentation culture of Monascus purpurogenum M23067Z4Fd12 in a culture medium containing glucose, peptone, NaNO3, MgSO4, and KH2PO4; further preferably, the culture medium further contains amino acid and / or organic acid additives; even more preferably, the culture medium further contains any one or more additives selected from the following: cysteine, histidine, glycine, tyrosine, valine, tartaric acid, ascorbic acid, citric acid, and malic acid, preferably selected from the following: cysteine, histidine, glycine, and tyrosine; And / or, the fermentation agent is a liquid or solid preparation; optionally, the dosage form of the fermentation agent is selected from: solution, suspension, emulsion, powder, granule, pill, tablet, gel.

4. The method for preparing a leavening agent according to claim 2 or 3, wherein: The preparation method comprises: fermenting and culturing the purple Monascus M23067Z4Fd12 according to claim 1 in a culture medium; Preferably, the culture medium comprises glucose, peptone, NaNO3, MgSO4, and KH2PO4; Further preferably, the culture medium further comprises amino acid and / or organic acid additives; even more preferably, the culture medium further comprises any one or more additives selected from the group consisting of cysteine, histidine, glycine, tyrosine, valine, tartaric acid, ascorbic acid, citric acid and malic acid, preferably selected from the group consisting of cysteine, histidine, glycine and tyrosine; Preferably, the fermentation culture temperature is 25-35°C, preferably 30°C; Preferably, the fermentation culture time is 7-15 days, preferably 8-12 days.

5. A composition, characterized in that The composition comprises the Monascus purpurogenum M23067Z4Fd12 according to claim 1, or comprises the fermentation agent according to claim 2 or 3.

6. The composition according to claim 5, characterized in that The composition is a food or a pharmaceutical composition, which further comprises a food or a pharmaceutically acceptable carrier.

7. Use of the Monascus purpurogenum M23067Z4Fd12 according to claim 1, the leavening agent according to claim 2 or 3, and / or the composition according to claim 5 or 6 in preparing fermented foods.

8. The use according to claim 7, characterized in that The fermented food is made from cereals, beans or Chinese medicinal materials; Preferably, the raw material of the fermented food is rice, sorghum or tangerine peel; Further preferably, the fermented food comprises: red yeast rice, red yeast sorghum, red yeast wine, red yeast tangerine peel, and red yeast tangerine peel beverage.

9. A method for preparing a fermented food, characterized in that: The method comprises: using the purple Monascus M23067Z4Fd12 according to claim 1, the starter according to claim 2 or 3 and / or the composition according to claim 5 or 6, and fermenting cereals, beans or Chinese medicinal materials as raw materials to obtain the product.

10. The method according to claim 9, characterized in that The raw materials are selected from: rice, sorghum or tangerine peel; And / or, the fermented food includes: red yeast rice, red yeast sorghum, red yeast wine, red yeast tangerine peel, red yeast tangerine peel beverage; and / or, the fermentation temperature is 25-35°C, preferably 30°C; And / or, the fermentation time is 5-10 days, preferably 6-8 days.

11. A method for producing monascus pigment, characterized in that: The method comprises: fermenting the purple Monascus sp. M23067Z4Fd12 according to claim 1 in a culture medium containing glucose, peptone, NaNO3, MgSO4, and KH2PO4 to produce red yeast rice pigment; Optionally, the method further comprises the step of separating and purifying the monascus pigment from the fermentation culture; Preferably, the culture medium further comprises amino acid and / or organic acid additives; Further preferably, the culture medium further comprises any one or more additives selected from the group consisting of: cysteine, histidine, glycine, tyrosine, valine, tartaric acid, ascorbic acid, citric acid and malic acid, preferably selected from the group consisting of: cysteine, histidine, glycine and tyrosine; Preferably, the fermentation culture temperature is 25-35°C, preferably 30°C; Preferably, the fermentation culture time is 7-15 days, preferably 8-12 days.

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