Monascus strain and use thereof

Abstract

Provided in the present application are a Monascus strain, a red yeast rice, a red yeast rice decoction piece, a preparation method for red yeast rice, a red yeast rice extract and a drug. The Latin name of the Monascus strain is Monascus purpureus, and the accession number thereof is CGMCC No. 40777. A red yeast rice product prepared by means of fermentation using the Monascus strain of the present application has a high content of lovastatin compounds and a high content of extractives, contains polyhydroxy sterol and flavonoid components and has good safety, thus improving the drug activity and quality of red yeast rice products.

Description

A Monascus strain and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311841573.2, filed on December 28, 2023, entitled “A Monascus strain and its application,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of microbial fermentation, and in particular to a Monascus strain, Monascus purpurogenum, Monascus purpurogenum slices, a preparation method of Monascus purpurogenum, a Monascus purpurogenum extract and a medicine. Background Art

[0004] Red yeast rice is a traditional fermented product in my country. Its main ingredients vary, and it's used in various fields, including medicine, food, and dyeing. The discovery of lovastatin-like compounds in red yeast rice has led to its widespread recognition as a natural medicine of choice for inhibiting cholesterol synthesis and lowering blood lipids.

[0005] There are many types of lovastatin compounds, including lovastatin (Monacolin K (MK)), lovastatin acid (acid form of Monacolin K (MKA)), lovastatin J (Monacolin J (MJ)), lovastatin J acid (acid form of Monacolin J (MJA)), lovastatin L (Monacolin J (ML)), lovastatin L acid (acid form of Monacolin L (MLA)), lovastatin M (Monacolin M (MM)), lovastatin M acid (acid form of Monacolin M (MMA)), and dehydrolovastatin (Dehydromonacolin K (DMK)). In some content determination methods, monacolin K is the sum of the contents of lovastatin and lovastatin acid, which is used as a detection indicator for red yeast rice. However, studies have shown that other lovastatin compounds also have high cholesterol synthesis inhibition and lipid-lowering activities. Therefore, while increasing the content of lovastatin and lovastatin acid, increasing the content of overall lovastatin compounds can also improve the pharmaceutical activity of red yeast rice.

[0006] Currently, red yeast rice (Red Yeast Rice) as a natural lipid-lowering agent is typically used in the form of decoction pieces. The national government, as well as some provinces and cities, have established recommended standards for the extract content and lovastatin-like compounds in red yeast rice slices to ensure their lipid-lowering efficacy. Extract content refers to the determination of soluble substances in the medicinal material and decoction pieces using water or alcoholic solvents. The extract content is used as a quality control standard. This is generally used when the active ingredient or content in the medicinal material is very low, the indicator component is unclear, or an accurate quantitative method is not available. Medicinal red yeast rice contains a variety of active ingredients with diverse pharmacological effects, such as sterols, r-aminobutyric acid, pigments, and organic acids. These ingredients have benefits such as regulating blood lipids, lowering blood sugar and cholesterol, enhancing immunity, and fighting tumors. Therefore, using extract content as a quality control measure can objectively measure the comprehensive pharmacological effects of red yeast rice products. Therefore, the extract content and lovastatin-like compound content of red yeast rice products are key to their quality.

[0007] The study found that the contents of extracts, lovastatin and lovastatin acid, citrinin (or citrinin), and aflatoxin in commercially available products varied greatly, as recorded in Table 1.

[0008] Table 1

[0009] Some commercially available medicinal slices have qualified extracts but do not contain the active ingredients lovastatin and lovastatin acid. Furthermore, the extract content is increased, and the levels of citrinin and aflatoxins, which are toxic and harmful, are increased. Alternatively, some products contain lovastatin but the extract content is unqualified.

[0010] In addition, the content of toxic components also affects the safety of red yeast rice and limits its application. Summary of the Invention

[0011] The present application provides a Monascus strain, Monascus, Monascus slices, a method for preparing Monascus, a Monascus extract and a medicine to increase the content of lovastatin compounds and the content of extracts in Monascus, Monascus slices or their products.

[0012] In a first aspect of the present application, a Monascus strain is provided. The scientific name of the Monascus strain is Monascus purpureus, and the deposit number is CGMCC No. 40777.

[0013] The second aspect of the present application provides a red yeast rice, which is obtained by fermenting the Monascus strain provided by the first aspect.

[0014] In any embodiment of the second aspect, based on the dry matter of red yeast rice, the content of lovastatin compounds in red yeast rice is 4 mg / g-40 mg / g, preferably 6 mg / g-30 mg / g, and more preferably 7.0 mg / g-22 mg / g.

[0015] In any embodiment of the second aspect, the lovastatin compound preferably includes lovastatin and lovastatin acid. Based on the dry matter of red yeast rice, the content of lovastatin in red yeast rice is preferably 3.07 mg / g-13.5 mg / g, more preferably 7.25 mg / g-13.5 mg / g, and further preferably 8.5 mg / g-13.5 mg / g; based on the dry matter of red yeast rice, the content of lovastatin acid in red yeast rice is preferably 2.0 mg / g-5.0 mg / g, more preferably 2.3 mg / g-4.25 mg / g, and further preferably 3.5 mg / g-4.23 mg / g.

[0016] In any embodiment of the second aspect, it is further preferred that the lovastatin compound further includes lovastatin L, lovastatin M acid and dehydrolovastatin. Based on the dry matter of red yeast rice, the content of lovastatin L in the red yeast rice is preferably 0.3 mg / g-1.33 mg / g, preferably 1.12 mg / g-1.33 mg / g, the content of lovastatin M acid in the red yeast rice is preferably 0.27 mg / g-0.81 mg / g, preferably 0.72 mg / g-0.81 mg / g, and the content of dehydrolovastatin in the red yeast rice is preferably 0.2 mg / g-0.78 mg / g, preferably 0.65 mg / g-0.78 mg / g.

[0017] In any embodiment of the second aspect, more preferably, the lovastatin compound further comprises lovastatin L acid, lovastatin J and lovastatin J acid. Based on the dry matter of red yeast rice, the content of lovastatin L acid in red yeast rice is preferably 0.29 mg / g-0.46 mg / g, preferably 0.41 mg / g-0.46 mg / g; the content of lovastatin J in red yeast rice is preferably 0.11 mg / g-0.43 mg / g, preferably 0.41 mg / g-0.43 mg / g; the content of lovastatin J acid in red yeast rice is preferably 0.16 mg / g-0.28 mg / g, preferably 0.27 mg / g-0.28 mg / g.

[0018] In any embodiment of the second aspect, based on the dry matter of red yeast rice, the content of extract in red yeast rice is preferably greater than 7%, preferably the extract content is 7.5%-30%; preferably the extract content is 8%-30%; preferably the extract content is 10%-29%; preferably the extract content is 15%-25%; preferably the extract content is 17.4%-23.6%.

[0019] The third aspect of the present application provides a red yeast rice slice, which is prepared using the red yeast rice provided by any embodiment of the second aspect.

[0020] The fourth aspect of the present application provides a method for preparing red yeast rice, which comprises using the red yeast rice enzyme strain provided in the first aspect as a strain to ferment and prepare red yeast rice.

[0021] In any embodiment of the fourth aspect, the preparation method comprises: expanding and culturing the Monascus strain in a liquid culture medium to obtain a liquid strain; inoculating the liquid strain into a solid culture medium for fermentation to obtain a fermentation product containing Monascus.

[0022] In any embodiment of the fourth aspect, the liquid culture medium comprises glucose, peptone, a phosphorus source, and trace elements.

[0023] In any embodiment of the fourth aspect, preferably, the content of glucose in the liquid culture medium is 30 g / L-150 g / L; preferably, the content of peptone in the liquid culture medium is 2 g / L-30 g / L; preferably, the content of the phosphorus source in the liquid culture medium is 0.5 g / L-7 g / L, and preferably, the phosphorus source includes NH4H2PO4, (NH4)2HPO4.

[0024] In any embodiment of the fourth aspect, preferably, the trace elements include magnesium and calcium. It is further preferred that MgSO4·7H2O is used to provide magnesium, and the content of MgSO4·7H2O in the liquid culture medium is 0.01 g / L-1.2 g / L. It is further preferred that CaCl2 is used to provide calcium, and the content of CaCl2 in the liquid culture medium is 0.01 g / L-0.8 g / L.

[0025] In any embodiment of the fourth aspect, preferably, the liquid of the liquid culture medium comprises an aqueous starch solution.

[0026] In any embodiment of the fourth aspect, the solid culture medium comprises rice, bran, peptone, glucose and water.

[0027] In any embodiment of the fourth aspect, preferably, in parts by weight, the solid culture medium comprises 100 parts of rice, 6-9 parts of bran, 2-8 parts of peptone, and 1.4-3.0 parts of glucose; preferably, the solid culture medium also comprises ethanol, and further preferably, the mass content of ethanol relative to the dry matter of the solid culture medium is 0.2%-1%, more preferably 0.35%-0.7%.

[0028] In any embodiment of the fourth aspect, preferably, the solid culture medium further comprises bean powder, further preferably, the weight portion of the bean powder is 2-8 parts, further preferably, the bean powder is selected from one or more of green bean powder, mung bean powder, black bean powder, red bean powder, and yellow bean powder.

[0029] In any embodiment of the fourth aspect, the rice is preferably steamed, boiled or hot-soaked, more preferably steamed or hot-soaked.

[0030] In any embodiment of the fourth aspect, the steaming temperature is preferably 90° C.-120° C., and the steaming time is 25 min-50 min, preferably 35 min-45 min.

[0031] In any embodiment of the fourth aspect, the ratio of water added during steaming to the mass of rice is preferably 30%-70%, more preferably 40%-55%.

[0032] In any embodiment of the fourth aspect, preferably, during hot dipping, the hot dipping temperature is 90° C.-100° C., and the hot dipping time is 6 h-18 h, more preferably 10 h-14 h.

[0033] In any embodiment of the fourth aspect, the process of inoculating the liquid strain into the solid culture medium for fermentation includes: sterilizing the solid culture medium, and then inoculating the liquid strain into the solid culture medium, preferably, the sterilization temperature is 120°C-125°C, and the time is 30min-90min; the inoculated solid culture medium is cultured in an environment of 30°C-36°C and a relative humidity of 40%-80% for 48h-120h to obtain a primary culture; and the secondary culture is cultured in an environment of 18°C-25°C and a relative humidity of 40%-80% for 7 days-30 days, preferably 7 days-20 days, to obtain a fermented system.

[0034] In any embodiment of the fourth aspect, when the primary culture lasts for 48h-120h, water is added to the solid culture medium and the solid culture medium is stirred, and the amount of water added is 5%-15% of the dry matter mass of the solid culture medium, preferably 6%-10%.

[0035] The fifth aspect of the present application provides a red yeast rice extract, which is prepared by extracting the red yeast rice provided by any embodiment of the second aspect with water or ethanol.

[0036] The sixth aspect of the present application provides a drug, including red yeast rice or red yeast rice extract, wherein the red yeast rice includes the red yeast rice provided by any embodiment of the second aspect, or the red yeast rice slices provided by any embodiment of the third aspect, and the red yeast rice extract includes the red yeast rice extract provided by any embodiment of the fifth aspect, and the drug is selected from lipid-lowering drugs, antihypertensive drugs, blood sugar-lowering drugs and / or anticancer drugs.

[0037] The red yeast rice product prepared by fermenting the Monascus strain provided in the present application has a high content of lovastatin compounds and extracts, and contains polyhydroxy sterols and flavonoids, and has good safety, thereby improving the pharmaceutical activity and quality of the red yeast rice product.

[0038] Biomaterial deposit information

[0039] The Monascus strain provided by the present invention was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on August 4, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and the deposit number is CGMCC No. 40777. The culture name is Monascus purpureus, and the Latin name is Monascus purpureus. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments of the present application. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without creative work.

[0041] FIG1 shows the lethality rates at different chemical mutagenesis times during the Monascus strain screening process of the present application.

[0042] FIG2 shows the lethality rates at different UV mutagenesis times during the Monascus strain screening process of the present application.

[0043] FIG3 shows the morphology and microscopic characteristics of the Monascus colony of the present application.

[0044] Figure 4 shows the fingerprints of the red yeast rice slices obtained at 15 days and 26 days in Example 5 of the present application.

[0045] FIG5 shows a liquid chromatogram of aflatoxin in the red yeast rice slices obtained on day 26 in Example 5 of the present application.

[0046] FIG6 shows a liquid chromatogram of citrinin in the red yeast rice slices obtained on day 26 in Example 5 of the present application. DETAILED DESCRIPTION

[0047] The following detailed description of the embodiments of the present application is provided in conjunction with the accompanying drawings and examples. The detailed description of the following examples is used to illustrate the principles of the present application, but is not intended to limit the scope of the present application, i.e., the present application is not limited to the described embodiments.

[0048] As analyzed in the background technology of this application, the content of lovastatin compounds and the content of extracts in the red yeast rice products of the prior art cannot be increased at the same time. In order to solve this problem, this application provides a Monascus strain, red yeast rice, red yeast rice slices and a method for preparing red yeast rice.

[0049] In a first embodiment of the present application, a Monascus strain is provided. The scientific name of the Monascus strain is Monascus purpureus, and the deposit number is CGMCC No.40777.

[0050] The red yeast rice product prepared by fermenting the Monascus strain provided in the present application has a high content of lovastatin compounds and extracts, and contains polyhydroxy sterols and flavonoids, and has good safety, thereby improving the pharmaceutical activity and quality of the red yeast rice product.

[0051] In some embodiments, the sequence of the 18S rRNA of the above-mentioned Monascus strain is shown in SEQ ID No. 1, including the 18S rRNA fragment, the full sequence of ITS 1, 5.8S rRNA, ITS2 and the 28S rRNA region sequence fragment:

[0052] In a second embodiment of the present application, a red yeast rice is provided, which is fermented using the Monascus strain provided in the first embodiment. This red yeast rice contains high levels of lovastatin compounds and extracts. Furthermore, research conducted by the present invention has found that even when the extract content is increased, no toxic or harmful components, such as citrinin and aflatoxins, are detected.

[0053] In addition, the red yeast rice can be used as a raw material for functional red yeast rice or medicinal red yeast rice. The red yeast rice can be used as red yeast rice medicinal materials, decoction pieces, and various raw materials, and can be used directly or after being processed.

[0054] In some embodiments, based on the dry matter of red yeast rice, the content of lovastatin compounds in red yeast rice is 4 mg / g-40 mg / g, preferably 6 mg / g-30 mg / g, and more preferably 7.0 mg / g-22 mg / g.

[0055] In some embodiments, the lovastatin compound preferably includes lovastatin and lovastatin acid. Based on the dry matter of red yeast rice, the content of lovastatin in red yeast rice is preferably 3.07 mg / g-13.5 mg / g, more preferably 7.25 mg / g-13.5 mg / g, and further preferably 8.5 mg / g-13.5 mg / g; the content of lovastatin acid in red yeast rice is preferably 2.0 mg / g-5.0 mg / g, more preferably 2.3 mg / g-4.25 mg / g, and further preferably 3.5 mg / g-4.23 mg / g.

[0056] In some embodiments, it is further preferred that the lovastatin compound further includes lovastatin L, lovastatin M acid and dehydrolovastatin. Based on the dry matter of red yeast rice, the content of lovastatin L in the red yeast rice is preferably 0.3 mg / g-1.33 mg / g, preferably 1.12 mg / g-1.33 mg / g, the content of lovastatin M acid in the red yeast rice is preferably 0.27 mg / g-0.81 mg / g, preferably 0.72 mg / g-0.81 mg / g, and the content of dehydrolovastatin in the red yeast rice is preferably 0.2 mg / g-0.78 mg / g, preferably 0.65 mg / g. -0.78mg / g; more preferably, the lovastatin compounds also include lovastatin L acid, lovastatin J and lovastatin J acid, preferably the content of lovastatin L acid in red yeast rice is 0.29mg / g-0.46mg / g, preferably 0.41mg / g-0.46mg / g, preferably the content of lovastatin J in red yeast rice is 0.11mg / g-0.43mg / g, preferably 0.41mg / g-0.43mg / g, preferably the content of lovastatin J acid in red yeast rice is 0.16mg / g-0.28mg / g, preferably 0.27mg / g-0.28mg / g.

[0057] Many red yeast rice products do not contain or contain only trace amounts of lovastatin J acid that cannot be detected. The red yeast rice of the present invention contains a certain amount of this component.

[0058] In some embodiments, based on the dry matter of red yeast rice, the extract content in the red yeast rice is preferably greater than 7%, preferably the extract content is 7.5%-30%; preferably the extract content is 8%-30%; preferably the extract content is 10%-29%; preferably the extract content is 15%-25%; preferably the extract content is 17.4%-23.6%.

[0059] In a third embodiment of the present application, a red yeast rice slice is provided, which is prepared using any red yeast rice provided in the second embodiment.

[0060] In a fourth embodiment of the present application, a method for preparing red yeast rice is provided. The method comprises using the red yeast rice enzyme strain provided in the first embodiment as a strain to ferment and prepare red yeast rice.

[0061] The red yeast rice product prepared by fermenting the Monascus strain provided in the present application has a high content of lovastatin compounds and an extract content, thereby improving the pharmaceutical activity of the red yeast rice product.

[0062] In some embodiments of the present application, the above-mentioned preparation method includes: using liquid culture medium to expand the Monascus strain to obtain liquid strains; inoculating the liquid strains into solid culture medium for fermentation to obtain a fermentation product containing Monascus.

[0063] The Monascus strain is first expanded and cultured, and then fermented, thereby improving the fermentation efficiency.

[0064] When the Monascus strain is expanded, the composition of the liquid culture medium used can refer to the composition of the liquid culture medium for the expansion culture of the conventional Monascus strain. In some embodiments, the liquid culture medium includes glucose, peptone, a phosphorus source and trace elements; to improve the efficiency of the expansion culture. In some embodiments, preferably, the content of glucose in the liquid culture medium is 30g / L-150g / L; preferably, the content of peptone in the liquid culture medium is 2g / L-30g / L; preferably, the content of the phosphorus source in the liquid culture medium is 0.5g / L-7g / L, preferably, the phosphorus source includes NH4H2PO4, (NH4)2HPO4; preferably, the trace elements include magnesium and calcium, further preferably using MgSO4·7H2O to provide magnesium, the content of MgSO4·7H2O in the liquid culture medium is 0.01g / L-1.2g / L, further preferably using CaCl2 to provide calcium, the content of CaCl2 in the liquid culture medium is 0.01g / L-0.8g / L; to improve the utilization rate of the liquid culture medium. Preferably, the liquid of the liquid culture medium comprises an aqueous starch solution, such as potato boil water or an aqueous solution of potato starch.

[0065] When the liquid strain is fermented and cultured, the composition of the solid culture medium can also refer to the solid culture medium used for conventional Monascus fermentation culture. In some embodiments, the solid culture medium includes rice, bran, peptone, glucose and water. In order to optimize the nutrient composition of the solid culture medium and further increase the yield and extract content of lovastatin compounds, in some embodiments, preferably, in parts by weight, the solid culture medium includes 100 parts of rice, 6-9 parts of bran, 2-8 parts of peptone, and 1.4-3.0 parts of glucose.

[0066] In some embodiments, the solid culture medium further comprises ethanol, and preferably the ethanol content relative to the dry matter of the solid culture medium is 0.2%-1%, preferably 0.35%-0.7%, wherein the ethanol is used to inhibit the effects of miscellaneous bacteria and trace alcohol metabolic factors.

[0067] In some embodiments of the present application, the solid culture medium further comprises bean powder, preferably the bean powder is in an amount of 2 to 8 parts by weight, and further preferably the bean powder is selected from one or more of green bean powder, mung bean powder, black bean powder, red bean powder, and yellow soybean powder, so as to further increase the content of lovastatin compounds and the content of extracts.

[0068] The rice can be rice flour or rice grains. In some embodiments, to improve the fermentation efficiency of the rice, the rice is preferably steamed, boiled, hot-soaked, or cold-soaked, with steaming or hot-soaking being more preferred. These rice forms are more readily utilized by Monascus after maturation, thereby further improving fermentation efficiency.

[0069] For example, rice and water are mixed at a mass ratio of (30-120):100 (the mass percentage of rice in the solid culture medium is based on the dry weight of rice), and then steamed, boiled, hot-soaked, or cold-soaked. After boiling, hot-soaking, or cold-soaking, the water is drained before use.

[0070] In some embodiments, when the rice is steamed, boiled, or hot-soaked, the weight ratio of water to rice is (30-70):100. For example, when steaming, the weight ratio of water to rice is 30%-70%.

[0071] In some embodiments, the steaming temperature is 90°C-120°C (e.g., 90°C, 100°C, 110°C, 120°C), the mass ratio of water added to rice is 30%-70%, more preferably 40%-55% (e.g., 40%, 45%, 50% or 55%), and the steaming time is 25min-50min (e.g., 25min, 30min, 35min, 40min, 45min or 50min), preferably 35min-45min.

[0072] In some embodiments, the preferred hot dip temperature is 50-100°C (such as 60, 70, 80, 85, 90, 95°C), the preferred hot dip temperature is 90-100°C, and the hot dip time is 6-18 hours, preferably 10-14 hours.

[0073] In some embodiments, the process of inoculating the liquid strain into the solid culture medium for fermentation includes: sterilizing the solid culture medium, inoculating the liquid strain into the solid culture medium, preferably, the sterilization temperature is 120 ° C to 125 ° C, and the time is 30 min to 90 min; the inoculated solid culture medium is cultured in an environment of 30 ° C to 36 ° C and a relative humidity of 40% to 80% for 48 h to 120 h to obtain a primary culture; the primary culture is cultured in an environment of 18 ° C to 25 ° C and a relative humidity of 40% to 80% for 7 days to 30 days, preferably 7 days to 20 days, more preferably 10 days to 20 days to obtain a fermented system. After the above fermentation process, the content of lovastatin compounds and extracts can be fully increased, and the output of citrinin and aflatoxin B1 can be reduced, thereby improving the overall quality of red yeast rice.

[0074] After testing, in some embodiments, water is added to the solid culture medium during the first stage of culture for 72 hours to 120 hours to replenish the fermentation process moisture. The amount of water added is 5% to 15% of the dry matter weight of the solid culture medium. Adding water during or at the end of high-temperature culture further increases the content of lovastatins and extracts in the red yeast rice.

[0075] In some embodiments, after fermentation, the above preparation method further includes a process of drying the fermentation product. This process is not only a process of removing water, but also a balance process in which some acid components, such as lovastatin acid, are converted into lactone lovastatin. If the conditions are severe, that is, the drying temperature is too high, the acid and lactone types may degrade or be converted into irreversible dehydrated lovastatin, which will affect the content. Other paired statin components change similarly. Therefore, in some embodiments, the drying temperature is 50°C-100°C, preferably 60-70°C, and the drying time is 6h-15h.

[0076] In some embodiments, the above-mentioned red yeast rice slices are prepared using the preparation method provided in the second embodiment above.

[0077] The fifth embodiment of the present application provides a red yeast rice extract, which is prepared by extracting any one of the red yeast rices provided in the second embodiment with water or ethanol.

[0078] The sixth embodiment of the present application provides a medicine, including red yeast rice or red yeast rice extract, wherein the red yeast rice includes the red yeast rice provided in the second embodiment, or the red yeast rice slices provided in the third embodiment, and the red yeast rice extract includes the red yeast rice extract provided in the fifth embodiment, and the medicine is selected from lipid-lowering drugs, antihypertensive drugs, blood sugar-lowering drugs and / or anticancer drugs.

[0079] Since the red yeast rice of the present application has high contents of extract and lovastatin, it has high lipid-lowering, blood sugar-lowering and / or blood pressure-lowering effects.

[0080] The beneficial effects of the present application will be further illustrated below with reference to examples, but the scope of the present invention is not limited to these examples.

[0081] Experimental instruments and equipment: ZHWY-2102 constant temperature culture oscillator: Shanghai Zhicheng Co., Ltd.; LRH-250-HSE constant temperature and humidity incubator: Zhujiang Taihongjun Instrument; BXM-110VE vertical pressure steam sterilizer: Boxun; WGL-230B electric heating blast drying oven: Tianjin Test Instrument Co., Ltd.; FW80 high-speed universal grinder: Tianjin Test Instrument Co., Ltd.

[0082] Liquid chromatograph: LC20AT, Shimadzu Corporation, Japan, UV detector. Chromatographic column: octadecylsilane bonded silica gel (Waters Symmetry C18, 3.9 × 150 mm, 5 μm). Electronic balance: XS205DU, METTLER; ultrapure water analyzer: PALL Cascada IX; ultrasonic cleaning machine: KH-500DB, Kunshan Hechuang Ultrasonic Instrument Co., Ltd.; rapid moisture analyzer: MA50, Sartorius.

[0083] Materials and reagents: Acetonitrile: chromatographic grade, Fisher (Lot 211921); Trifluoroacetic acid: chromatographic grade, Fisher (Lot 145624); 95% ethanol: AR, Sinopharm Chemical Reagent Company; Purified water: homemade; Methanol: chromatographic grade, Fisher (Lot 157751). Reference substance: lovastatin (Monacolin K, China National Institute for Food and Drug Control, purity >99%, Lot 100600). 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG): Sigma.

[0084] Monascus strain screening process

[0085] Preparation of bacterial suspension: The initial strain (number WP03) was inoculated on PDA medium and cultured at 29°C for 7 days. Mycelia and spores were scraped off with an inoculating loop and transferred to a triangular flask containing 20 mL of sterile water. The bottom of the triangular flask was covered with glass beads and shaken at 180 rpm for 20 min. The suspension was then filtered to prepare a spore suspension. The suspension was diluted to a spore concentration of 10 6 / mL of bacterial suspension.

[0086] Chemical mutagenesis: Preparation of MNNG solution: weigh 50 mg of MNNG, place it in a volumetric flask, add acetone to 5 ml, and prepare a stock solution with a concentration of 1%. 6 To a spore suspension of 0.9 and 0.8 mL of spores / mL, 0.1 mL and 0.2 mL of 1% MNNG stock solution (concentrations of 0.1% and 0.2%, respectively) were added. After treatment for 10, 30, 60, and 90 minutes, the suspension was diluted in a gradient manner, and 0.1 mL of the suspension was plated. Spores were incubated at 29°C for 3-5 days, and the spore count was calculated using the untreated control (lethality rate shown in Figure 1). The strain treated with 0.2% MNNG for 90 minutes, which achieved the optimal lethality, was used for the subsequent UV mutagenesis.

[0087] Lethality rate / %=(number of colonies grown on the control plate-number of colonies grown on the mutagenesis plate) / number of colonies on the control plate.

[0088] UV mutagenesis: Prepare 10 MNNG mutagenesis strains according to the above method. 65 mL of spore suspension was taken and placed at 20 cm under a 20 W UV lamp for 0 s, 30 s, 60 s, 90 s, 120 s, and 180 s, respectively. The plates were coated with the same method as above, and the lethality was calculated using the unirradiated control as the control (see Figure 2 for lethality). The optimal UV irradiation time was 120 s.

[0089] The results of the above chemical UV mutagenesis were finally obtained. The Monascus mutant strain was deposited in the China General Microbiology Center (CGMCC) under the deposit number CGMCC No. 40777.

[0090] The sequence of the 18S rRNA of the above-mentioned Monascus strain is shown in SEQ ID No. 1, including the 18S rRNA fragment, the full sequences of ITS 1, 5.8S rRNA, ITS2, and the 28S rRNA region sequence fragment:

[0091] The morphology and microscopic characteristics of the Monascus colony are shown in FIG3 .

[0092] The tested strain grew rapidly on malt extract agar medium. After culturing at 25°C in the dark for 10 days, the colony diameter was 40-45 mm, with luxuriant aerial hyphae. The color was light in the early stage (as shown in the upper left picture of Figure 3) and orange-red in the later stage. The back of the colony was dark orange, and the pigment was dissolved in the culture medium.

[0093] Conidia are produced in large quantities, and the conidiophores are not obviously specialized, straight or slightly curved. The conidia are spherical, colorless, with slightly rough walls, truncate bases, and arranged in strings, 5.5 to 10.1 m (as shown in the upper right, lower left, and lower right figures of Figure 3).

[0094] Study on strain stability

[0095] After the strain was passaged 20 times, the contents of lovastatin and lovastatin acid in the obtained red yeast rice slices remained basically unchanged, as shown in Table 2, indicating that the strain of the invention has good stability (using the fermentation conditions of Example 5, with a culture period of 23 days).

[0096] Table 2

[0097] Example 1

[0098] A Monascus strain (CGMCC No. 40777, hereinafter referred to as Monascus strain 1) and an initial strain (WP03) were inoculated at a ratio of 5% (volume of the culture slant suspension / volume of the liquid culture medium) into a liquid fermentation medium containing 85 g / L glucose, 15 g / L peptone, 1.5 g / L NH4H2PO4, 0.8 g / L MgSO4·7H2O, and 0.2 g / L CaCl2. The fermentation medium was prepared with distilled water. The fermentation temperature was 30°C, and the fermentation time was 40-48 hours. The culture was shaken at 30°C for 2 days.

[0099] Absorb the liquid seeds into the solid culture medium and stir to mix. Solid culture medium: rice, soak at room temperature (20℃-25℃) (14 hours), the mass ratio of water to rice is 120:100, drain the water, add nutrients in proportion (the composition is 100 parts of rice (before adding water), 8 parts of bran, 2 parts of peptone, 2 parts of glucose) and stir evenly; carry out primary culture for 5 days in an environment with a fermentation temperature of 30℃-36℃ and a relative humidity of 40%-80%, and carry out secondary culture from the 6th day in an environment of 18℃-25℃ and a relative humidity of 40%-80% for 25 days to obtain a fermentation product. Dry at 70℃ to obtain red yeast rice. The determination of lovastatin content refers to the 2020 edition of the Pharmacopoeia.

[0100] The extract determination method is the hot soak method under the alcohol-soluble extract determination method (General Chapter 2201 of the Chinese Pharmacopoeia 2020 Edition), using 70% ethanol as the solvent.

[0101] Citrinin detection (detection limit 5 ppb) refers to the first method (immunoaffinity column cleanup-high performance liquid chromatography) in the national standard - GB 5009.222-2016 "Determination of citrinin in food" to detect citrinin (same as "citrinin") content.

[0102] The method for detecting aflatoxin refers to the Pharmacopoeia Aflatoxin Determination Method (Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 2351-Liquid Chromatography / Photochemical Derivatization Method).

[0103] The statin content was determined by high-performance liquid chromatography (HPLC) using the following methods: HPLC conditions: acetonitrile as mobile phase A, 0.1% (volume fraction) trifluoroacetic acid (TFA) in water as mobile phase B, gradient elution (0-28 min: 35% mobile phase A and 65% mobile phase B; 28-30 min: linear gradient: mobile phase A decreased from 75% to 35% and mobile phase B increased from 25% to 65%; 30-35 min: return to the initial conditions of 35% mobile phase A and 65% mobile phase B). Flow rate: 1.0 mL / min, column temperature: 30°C, injection: 20 μL, PDA settings: 200-350°C, detection wavelength: 237 nm.

[0104] The test results are recorded in Table 3.

[0105] Table 3

[0106] The lovastatin content of the strain of the invention is about 2.14 times that of the initial strain, and does not contain citrinin and aflatoxin.

[0107] The following fermentation was carried out using Monascus strain 1 to investigate the rice processing method used in the solid culture medium, the solid culture medium prescription, and the culture process.

[0108] Example 2

[0109] The rice in solid culture medium are as follows:

[0110] Hot soaking rice: Mix rice with high temperature water (80℃) and soak for 4h, 12h and 18h ​​respectively, maintaining the temperature above 70℃, and the mass ratio of water to rice is 120:100.

[0111] Cooking rice: Mix rice with 100℃ hot water and continue heating, keeping the temperature at 90℃-100℃ for 2 minutes. The mass ratio of water to rice is 120:100.

[0112] Low-temperature soaking: Add water at 20-25°C and soak for 14 hours. The mass ratio of water to rice is 120:100.

[0113] Fermentation was carried out under the fermentation conditions of Example 1, and the extract and lovastatin content in the red yeast rice after fermentation were compared. See Table 4 for details.

[0114] Table 4

[0115] It can be seen that hot soaking and boiling rice have better effects and can significantly increase the content of lovastatin. In particular, the content of lovastatin in the red yeast rice obtained from the solid culture medium containing boiled rice under the same fermentation conditions is 3.33 times that of the rice soaked at room temperature, and the content of the extract is increased by 4.5 times. However, the experiment found that the rice was relatively sticky during the cooking process, agglomerated during fermentation, and the fermentation was uneven. Therefore, the main processing methods of rice are boiling water soaking or steaming.

[0116] Example 3

[0117] Using the hot-soaked rice soaked for 18 hours in Example 2 as the rice for the solid culture medium, the effect of alcohol in the solid culture medium and the addition of water during the 5-day primary culture process were investigated. Table 5 shows the peptone and ethanol contents, as well as the amount of water added (% water added is the amount of water added relative to the dry matter mass of the solid culture medium).

[0118] Fermentation was carried out under the fermentation conditions of Example 1, and the extract and lovastatin content in the red yeast rice after fermentation were compared. See Table 5 for details.

[0119] Table 5

[0120] The results showed that the contents of lovastatin, lovastatin acid and extracts could be increased when ethanol was used or water was added during the primary culture process.

[0121] Example 4

[0122] The solid culture medium contained 0.7% ethanol (relative to the weight content of dry matter), and the experiment was conducted on soaked rice and steamed rice using a non-watering process.

[0123] Among them, the soaking conditions for soaking rice are: soaking temperature 90°C, adding 1.2 times the amount of water, and time 18 hours.

[0124] Conditions for steaming rice:

[0125] Condition 1: Temperature 110°C, water content 45% of rice weight, time 40 minutes;

[0126] Condition 2: 90°C, 45% of the rice weight in water, 50 minutes.

[0127] Condition 3: Temperature 120°C, water 45% of the rice weight, 25 minutes;

[0128] Condition 4: Temperature 110°C, water 30% of the rice weight, 40 minutes;

[0129] Condition 5: Temperature 110°C, water addition amount 70% of rice weight, time 40 minutes.

[0130] In addition, the initial strain and condition 1 were used for steaming rice.

[0131] Fermentation was carried out under the fermentation conditions of Example 1, and the extract and lovastatin content in the red yeast rice after fermentation were compared. See Table 6 for details.

[0132] Table 6

[0133] The results showed that the steaming method was more stable than the soaking method and had a higher lovastatin content.

[0134] In addition, the content of statins in the red yeast rice obtained by fermentation under test conditions 4, condition 5 and the initial strain is recorded in the following Table 7.

[0135] Table 7

[0136] Example 5

[0137] The rice in the solid culture medium was treated according to Condition 1 of Example 4. Fermentation was carried out under the same conditions as in Example 1, with an 8% water supplementation process during the primary culture. The culture duration was investigated, and starting from day 15, the rice's appearance, content, extract, citrinin, and aflatoxin B1 all met the strictest provincial regulations for processing. The extract and lovastatin content in the red yeast rice after fermentation were compared. The results are reported in Table 8.

[0138] Table 8

[0139] It can be seen that the red yeast rice strain 1 of the present application can significantly shorten the fermentation cycle to 15 days. The content of ingredients such as lovastatin is already relatively high. If red yeast rice is used for different purposes, the fermentation days can be even shorter.

[0140] In addition, the fingerprints of the red yeast rice slices obtained on the 15th and 26th days are recorded in Figure 4 and detected using the red yeast rice fingerprint determination method of the Pharmacopoeia 2020. The study found that the red yeast rice of the present invention contains many components, including flavonoids, sterols, amides, pigments, and other components in addition to statins.

[0141] Moreover, Figures 5 and 6 show the liquid chromatograms of aflatoxins and citrinin of the red yeast rice slices obtained 26 days ago. The red yeast test solution is the test solution of the red yeast rice slices to be tested. There are no peaks corresponding to the positions of aflatoxins and citrinin, proving that their respective contents are 0.

[0142] Example 6

[0143] The rice in the solid culture medium was treated according to Condition 1 of Example 4. Fermentation was performed according to the fermentation conditions of Example 1, and the primary culture was supplemented with 8% water (sterile water was added to the culture vessel with stirring). The culture time was 30 days. The amounts of peptone, green pea flour, or mung bean flour added to the solid culture medium were adjusted (see Table 9 for details).

[0144] Comparison of extracts and lovastatin content in fermented red yeast rice slices. See Table 9 for details.

[0145] Table 9

[0146] The results showed that when the solid culture medium contained 5 parts of green bean flour and 5 parts of peptone, the lovastatin content was 3.19 times that of peptone alone, and the lovastatin acid content was 1.53 times that of peptone alone. Black beans, red beans, and yellow beans can also be used in combination or individually.

[0147] In addition, the contents of 9 kinds of lovastatin in the red yeast rice slices obtained in Group 3 and Group 6 in Table 9 are recorded in Table 10.

[0148] Table 10

[0149] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A Monascus strain, characterized in that, The Latin name of the Monascus strain is Monascus purpureus, and the preservation number is CGMCC No. 40777.

2. A Monascus, characterized in that, It is obtained by fermenting with the Monascus strain described in claim 1.

3. The monascus according to claim 2, characterized in that, Based on the dry matter of the red yeast rice, the content of lovastatin compounds in the red yeast rice is 4 mg / g - 40 mg / g, preferably 6 mg / g - 30 mg / g, more preferably 7.0 mg / g - 22 mg / g; Preferably, the lovastatin compounds include lovastatin and lovastatin acid. Based on the dry matter of the red yeast rice, preferably the content of lovastatin in the red yeast rice is 3.07 mg / g - 13.5 mg / g, further preferably 7.25 mg / g - 13.5 mg / g, and further preferably 8.5 mg / g - 13.5 mg / g; Based on the dry matter of the red yeast rice, preferably the content of lovastatin acid in the red yeast rice is 2.0 mg / g - 5.0 mg / g, further preferably 2.3 mg / g - 4.25 mg / g, and further preferably 3.5 mg / g - 4.23 mg / g; More preferably, the lovastatin compounds further include lovastatin L, lovastatin M acid, and dehydro-lovastatin. Based on the dry matter of the red yeast rice, preferably the content of lovastatin L in the red yeast rice is 0.3 mg / g - 1.33 mg / g, preferably 1.12 mg / g - 1.33 mg / g, preferably the content of lovastatin M acid in the red yeast rice is 0.27 mg / g - 0.81 mg / g, preferably 0.72 mg / g - 0.81 mg / g, preferably the content of dehydro-lovastatin in the red yeast rice is 0.2 mg / g - 0.78 mg / g, preferably 0.65 mg / g - 0.78 mg / g; Even more preferably, the lovastatin compounds further include lovastatin L acid, lovastatin J, and lovastatin J acid. Based on the dry matter of the red yeast rice, preferably the content of lovastatin L acid in the red yeast rice is 0.29 mg / g - 0.46 mg / g, preferably 0.41 mg / g - 0.46 mg / g; preferably the content of lovastatin J in the red yeast rice is 0.11 mg / g - 0.43 mg / g, preferably 0.41 mg / g - 0.43 mg / g; preferably the content of lovastatin J acid in the red yeast rice is 0.16 mg / g - 0.28 mg / g, preferably 0.27 mg / g - 0.28 mg / g.

4. The monascus according to claim 2 or 3, characterized in that, Based on the dry matter of the red yeast rice, preferably the content of the extract in the red yeast rice is greater than 7%, preferably the extract content is 7.5% - 30%; preferably the extract content is 8% - 30%; preferably the extract content is 10% - 29%; preferably the extract content is 15% - 25%; preferably the extract content is 17.4% - 23.6%.

5. A Monascus prepared slice, characterized in that, It is prepared from the red yeast rice described in any one of claims 2 to 4.

6. A method for preparing red yeast rice, the preparation method includes using the Monascus enzyme strain described in claim 1 as a strain for fermentation to prepare red yeast rice.

7. The preparation method according to claim 6, characterized in that, The preparation method includes: The Monascus strain is expanded and cultured using a liquid medium to obtain a liquid strain; The liquid strain is inoculated into a solid medium for fermentation to obtain a fermentation product containing Monascus.

8. The preparation method according to claim 7, characterized in that, The liquid medium includes glucose, peptone, a phosphorus source, and trace elements; Preferably, the content of glucose in the liquid medium is 30 g / L - 150 g / L; preferably, the content of peptone in the liquid medium is 2 g / L - 30 g / L; preferably, the content of the phosphorus source in the liquid medium is 0.5 g / L - 7 g / L, and preferably, the phosphorus source includes NH4H2PO4 and (NH4)2HPO4; Preferably, the trace elements include magnesium and calcium. More preferably, MgSO4·7H2O is used to provide magnesium, and the content of MgSO4·7H2O in the liquid medium is 0.01 g / L - 1.2 g / L. More preferably, CaCl2 is used to provide calcium, and the content of CaCl2 in the liquid medium is 0.01 g / L - 0.8 g / L; Preferably, the liquid of the liquid medium includes a starch aqueous solution.

9. The preparation method according to claim 7 or 8, characterized in that, The solid medium includes rice, bran, peptone, glucose, and water; Preferably, by weight, the solid medium includes 100 parts of rice, 6 - 9 parts of bran, 2 - 8 parts of peptone, and 1.4 - 3.0 parts of glucose; preferably, the solid medium further includes ethanol, and more preferably, the mass content of ethanol relative to the dry matter of the solid medium is 0.2% - 1%, and more preferably 0.35% - 0.7%; Preferably, the solid medium further includes soybean powder, and more preferably, the weight of the soybean powder is 2 - 8 parts, and more preferably, the soybean powder is selected from one or more of green soybean powder, mung bean powder, black soybean powder, red bean powder, and yellow soybean powder; Preferably, the rice is steamed, boiled, or hot - soaked, and more preferably steamed or hot - soaked; Preferably, the steaming temperature is 90°C - 120°C, and the steaming time is 25 min - 50 min, preferably 35 min - 45 min; Preferably, the mass addition ratio of water added during steaming relative to rice is 30% - 70%, and more preferably 40% - 55%; Preferably, during hot - soaking, the hot - soaking temperature is 90°C - 100°C, and the hot - soaking time is 6 h - 18 h, more preferably 10 h - 14 h.

10. The preparation method according to any one of claims 7 to 9, characterized in that, The process of inoculating the liquid strain into the solid medium for fermentation includes: After the solid medium is sterilized, the liquid strain is inoculated into the solid medium. Preferably, the temperature of the sterilization treatment is 120°C - 125°C, and the time is 30 min - 90 min; The inoculated solid medium is subjected to primary culture for 48 h - 120 h in an environment of 30°C - 36°C and relative humidity of 40% - 80% to obtain a primary culture; The secondary culture is subjected to secondary culture for 7 days - 30 days, preferably 7 days - 20 days, in an environment of 18°C - 25°C and relative humidity of 40% - 80% to obtain a post - fermentation system.

11. The preparation method according to claim 10, wherein When the primary culture lasts for 48 h - 120 h, water is added to the solid medium and the solid medium is subjected to stirring treatment. The addition amount of the water is 5% - 15% of the dry matter mass of the solid medium, preferably 6% - 10%.

12. A monascus extract, characterized in that, It is prepared by extracting the Monascus purpureus Went described in any one of claims 2 to 4 with water or ethanol.

13. A drug, comprising red yeast rice or red yeast rice extract, characterized in that, The Monascus purpureus Went includes the Monascus purpureus Went described in any one of claims 2 to 4, or the Monascus purpureus Went decoction pieces described in claim 5. The Monascus purpureus Went extract includes the Monascus purpureus Went extract described in claim 12. The drug is selected from lipid-lowering drugs, antihypertensive drugs, hypoglycemic drugs, and / or anti-cancer drugs.

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