Monascus strain with high yield of monascus pigment and application thereof

Abstract

This invention discloses a Monascus strain that produces high levels of Monascus pigment and its applications. The Monascus strain producing high levels of Monascus pigment is classified as Monascus purpureus FAAS-M3 (…). Monk purple The purple Monascus FAAS-M3 strain provided in this invention has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2022958 and deposit date of June 24, 2022. This strain exhibits a strong ability to produce natural Monascus pigments and also produces high levels of citrinin. Red yeast rice products produced using this strain have high color value and citrinin content, resulting in a high product yield. Therefore, the purple Monascus FAAS-M3 strain provided in this invention has high research value.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a Monascus strain that produces high levels of Monascus pigments and its applications. Background Technology

[0002] Monascus purpureus, a fungus unique to the East, is not only used to make various traditional foods, but it is also an important industrial fungus. Its natural pigments are of high quality and inexpensive, and its usage ranks second in the world. my country is a major producer of Monascus purpureus, with Fujian Province being the traditional birthplace and main production area. Fujian's production and exports account for more than half of the national total, maintaining a leading position both domestically and internationally for a long time. It has exported to countries such as the United States, France, Germany, Japan, and South Korea. Furthermore, Monascus purpureus produces a variety of unique bioactive metabolites, such as Monacolin K, lovastatin, γ-aminobutyric acid (GABA), and ergosterol, which have physiological effects such as lowering cholesterol, blood pressure, and lipids, and inhibiting cancer cells. Taiwan has elevated the status of Monascus purpureus and considers it the preferred industrial microbial strain for achieving local breakthroughs in bioengineering. Most regions in my country have completed the industrialization of Monascus purpureus production and the modernization of pure-culture koji-making. Only Fujian Province (the birthplace and main production area of ​​Monascus purpureus) still uses traditional koji-making techniques that have been used for nearly a thousand years, and its wild Monascus purpureus resources are abundant. Therefore, exploring the unique wild Monascus germplasm resources in Fujian Province and developing the province's unique Monascus resources to avoid the extinction of wild Monascus strains due to ecological homogenization is of great significance for the protection and development of Monascus germplasm resources in our province, and also provides a theoretical basis for the development of new Monascus processes and products. Summary of the Invention

[0003] The purpose of this invention is to provide a Monascus strain that produces high levels of Monascus pigments and its applications.

[0004] In order to achieve the above objectives, the present invention adopts the following technical solution:

[0005] This invention provides a Monascus strain that produces high levels of Monascus pigment, classified as Monascus purpureus FAAS-M3, which has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2022958 and deposit date of June 24, 2022.

[0006] The purple red yeast rice FAAS-M3 provided by this invention can be used to prepare high-color-value red yeast rice.

[0007] The purple Monascus FAAS-M3 strain provided by this invention has a strong ability to produce natural Monascus pigments and also produces high levels of citrinin. Red yeast rice products produced using this strain have high color value and citrinin content, a short cultivation cycle, and a high product yield. Therefore, the purple Monascus FAAS-M3 strain provided by this invention has high research value. Attached Figure Description

[0008] Figure 1 A graph comparing the ability of Monascus purpureus strains to produce pigments and citrinin in solid and liquid cultures.

[0009] Figure 2 This is a colony morphology diagram of Monascus purpureus FAAS-M3.

[0010] Figure 3 This is an image of the individual morphology (mycelium and conidia) of Monascus purpureus FAAS-M3 under an optical microscope.

[0011] Figure 4 The image shows the individual morphology of Monascus purpureus FAAS-M3 under a scanning electron microscope (closure shell structure and mycelial morphology).

[0012] Figure 5 This is a growth and metabolism curve of Monascus purpureus FAAS-M3 in liquid culture. Detailed Implementation

[0013] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0014] Sample source

[0015] Samples of red yeast rice or soil samples prepared from red yeast rice were collected from various red yeast rice production enterprises in Fujian.

[0016] culture medium

[0017] (1) Slant culture medium: 10°Bé malt extract, 2% agar, pH 6-7, heat to dissolve, dispense into test tubes, plug with cotton plugs, autoclave at 121℃ for 20 minutes, remove, place on slant to form slant, cool and solidify to form slant culture medium.

[0018] (2) Liquid seed culture medium: 3-6% soluble starch, 3-6% glucose, 1-2% peptone, 0.1-0.2% NaNO3, 0.1-0.2% KH2PO4, 0.05%-0.15% MgSO4, 0.05-0.115% K2HPO4, pH adjusted to 4.6-6.0. Add 50 mL of liquid seed culture medium and several small glass beads to a 250 mL Erlenmeyer flask, and autoclave at 121℃ for 30 min.

[0019] (3) Liquid fermentation medium: 3-6% soluble starch, 3-6% glucose, 1-2% peptone, 0.1-0.2% NaNO3, 0.05-0.15% NH4NO3, 0.1-0.2% KH2PO4, 0.05%-0.15% MgSO4, 0.10%-0.20% ZnSO4, 0.05%-0.15% FeSO4, 0.05-0.15% K2HPO4, pH adjusted to 4.6-6.0. Add 50 mL of liquid seed culture medium and several small glass beads to a 250 mL Erlenmeyer flask, and autoclave at 121℃ for 30 min.

[0020] (4) Solid fermentation culture medium: Indica rice, commercially available.

[0021] Example 1: Isolation, Screening, and Identification of Strains

[0022] 1.1 Method

[0023] 1.1.1 Methods for strain isolation

[0024] Add 1-2g of crushed red yeast rice to an Erlenmeyer flask containing 50mL of sterile water, shake at 180r / min for 30min, and perform serial dilutions, i.e., 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 For each dilution, take 0.1-0.2 mL and spread it on a plate. Incubate at 35°C for 3-5 days. Based on the morphological characteristics of the colonies on the plate, select those with fast growth, large diameter, and dark color for transfer culture until a purified Monascus strain is obtained. Incubate on a slant at 35°C for 7-10 days. After the hyphae have covered the slant, store in a refrigerator at 4°C for later use.

[0025] 1.1.2 Preparation of spore suspension

[0026] Inoculate Monascus purpureus strains onto slant agar plates and incubate at 30-35℃ for 7-10 days, or until the slant plates are fully covered with Monascus purpureus. Add 8 mL of sterile physiological saline to the slant agar seeds, scrape the slant agar spores with an inoculation needle, and disperse them in sterile water containing glass beads to prepare a homogeneous spore suspension. Count the spores using a hemocytometer; the spore concentration of the suspension should be 1 × 10⁻⁶. 7 2.5 × 10⁻² / mL 7 per mL.

[0027] 1.1.3 Preparation of Liquid Seed Solution

[0028] Inoculate the spore suspension into the liquid seed culture medium at an inoculation rate of 6-10%, place it in a constant temperature shaker at 32-35℃, and shake at 160-200 rpm for 2-4 days to obtain the liquid seed solution, which can then be used.

[0029] 1.1.4 Liquid fermentation culture

[0030] Inoculate the liquid seed culture into the liquid fermentation medium at an inoculation rate of 8-12%, and culture it in a constant temperature shaker at 32-35℃ with a rotation speed of 160-200 r / min for 4-6 days.

[0031] 1.1.5 Solid-state fermentation culture

[0032] Mix 50-60g of indica rice with soaking liquid (water) at a mass ratio of 1:1.5-2 (w / w) in a 500mL Erlenmeyer flask, shake well, and soak for approximately 6-8 hours. After draining, autoclave at 121℃ for 30 minutes. After cooling, inoculate with 6-10% liquid seed culture, shake well, and incubate at 30-35℃. Once the culture medium is basically covered with mycelia, add 2-5mL of vitamin complex solution to maintain the humidity inside the flask above 85%. Incubate for 10-13 days, until the rice grains turn purplish-red. Then, dry the red yeast rice in a constant temperature incubator at 60℃ with ventilation until the moisture content is below 12%. Grind the dried red yeast rice into the finished product.

[0033] 1.1.6 Determination Method

[0034] The color value of red yeast rice was determined according to the national standard GB 1886.19-2015 "Food Additives - Red Yeast Rice", and the citrinin content was determined according to the national standard GB 5009.222-2016 "Determination of Citrinin in Food".

[0035] 2 Results and Analysis

[0036] 2.1. Isolation and screening of single cells of Monascus purpureus strains

[0037] Forty-five Monascus purpureus strains, numbered M1-M45, were isolated and purified from red yeast rice and soil samples. After shake-flask screening, 14 strains exhibiting typical purple Monascus purpureus colony characteristics were selected. These strains were then cultured in both liquid and solid-state fermentation processes. Using the production of monadin and citrinin as indicators, it was found that different Monascus purpureus strains showed varying degrees of monadin and citrinin production. Among them, strain FAAS-M3 exhibited a strong ability to produce both monadin and citrinin, as shown in the results below. Figure 1 As shown.

[0038] 2.2 Morphological characteristics and taxonomic identification of Monascus purpureus strain FAAS-M3

[0039] 2.2.1 Colony morphology characteristics

[0040] The colonies were incubated on malt extract agar plates for 7 days, and the morphology, color, and elevation of the red yeast rice colonies were observed. The mycelium was red, and the reverse side of the agar plate appeared dark red. The colony morphology was as follows. Figure 2 As shown, the colonies are relatively dense, felt-like and flat, expanding and growing with smooth edges. The colony diameter is 35-50 mm, and the colonies have irregular radial grooves. The aerial hyphae are covered with short, fluffy hairs.

[0041] 2.2.2 Cultivation Characteristics

[0042] Culture studies revealed that the FAAS-M3 strain can be cultured in both solid and liquid media at temperatures ranging from 25 to 40°C, with the optimal growth temperature being 32 to 35°C. Growth slows above 37°C and below 30°C, and almost ceases above 40°C. The most suitable growth medium is malt extract agar, which produces the most stable colony morphology.

[0043] 2.2.3 Individual morphological characteristics

[0044] Morphology under optical microscopes and scanning electron microscopes, such as Figure 3-4 As shown. The mycelium has numerous irregular branches, with transverse septa and multiple nuclei. The mycelium is 3-5 μm in diameter and contains granules. The conidia are borne at the tips of the mycelium and its branches. They are spherical, 6-9 μm in diameter, colorless, contain granules, and are solitary or in chains of 5-8. The cleistothecia are spherical with raised dots on the surface and are 10-20 μm in diameter.

[0045] 2.2.4 Biochemical characteristics

[0046] 2.2.4 Biochemical characteristics

[0047] Add 2% glucose, maltose, fructose, sucrose, lactose, and sorbose to sugar-free Czapek's medium, respectively, and autoclave at 121℃ for 20 min before use. Inoculate with Monascus purpureus strain FAAS-M34 and incubate at 30℃ for 4 days. The carbon source utilization results are shown in Table 1, indicating that strain FAAS-M34 can grow in glucose, maltose, fructose, sucrose, and lactose media, but can hardly utilize sorbose.

[0048] The gelatin culture medium was dispensed into test tubes and autoclaved at 121°C for 20 minutes, then cooled for later use. Spores of *Monascus purpureus* strain FAAS-M34 were picked and inoculated into the center of the test tube using the puncture method. An uninoculated control was used. The tubes were incubated at 30°C for 7-14 days. Observe the gelatin curd beneath the colonies; a depression will appear, indicating that this strain can hydrolyze gelatin.

[0049] Table 1 Physiological and Biochemical Characteristics

[0050] glucose maltose fructose sucrose lactose sorbose Gelatin hydrolysis + + + + + - +

[0051] 2.2.5 Molecular biological identification

[0052] DNA was extracted from Monascus purpureus mycelium using a novel plant genomic DNA extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.). PCR amplification was performed using universal primers ITS-1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS-4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR amplification conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 55℃ annealing for 1 min, 72℃ extension for 1 min, 30 cycles, followed by a final extension at 72℃ for 10 min. The PCR products were verified by agarose gel electrophoresis and then directly sequenced bidirectionally using an ABI-PRISM3730 automated DNA sequencer. The sequences were compared for similarity in the GenBank database using BLAST software. Table 2 shows that the amplified fragments from strain FAAS-M3 using primers ITS1 / ITS4 showed 100% similarity to those from Monascus purpureus. Based on the comprehensive morphological and physiological and biochemical identification results, strain FAAS-M3 can be identified as Monascus purpureus FAAS-M3, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2022958 and deposit date of June 24, 2022.

[0053] Table 2. ITS rDNA sequencing results of strain FAAS-M3

[0054]

[0055]

[0056] 2.2.6 Assessment of growth and metabolic capacity

[0057] The purple Monascus faecium strain FAAS-M3 was cultured in liquid form, and the mycelial weight, color production, and citrinin production at different growth stages were measured. The biomass reached 1.9±0.2 g / 100 mL, the color value was as high as 147.82±19.19 U / mL, and the citrinin production was as high as 22.56±2.11 mg / L. Its growth and metabolism were as follows: Figure 5 As shown.

[0058] Example 2: Application of Monascus purpureus FAAS-M3 in the preparation of red yeast rice

[0059] The process for preparing red yeast rice using Monascus purpureus FAAS-M3 is as follows:

[0060] Slant culture → Spore suspension → Liquid seed liquid → Indica rice → Washing → Soaking (with nutrient enzymes) → Draining → Sterilization → Cooling → Inoculation → Culture → Liquid replenishment (vitamin complex solution) → Drying → Finished product

[0061] 1) Inoculate FAAS-M3 *Monascus purpureus* onto slant agar and incubate at 30-35℃ for 7-10 days until the slant is fully covered with *Monascus purpureus*. Add 8 mL of sterile physiological saline to the slant agar seeds, scrape the slant spores with an inoculation needle, and disperse them in sterile water containing glass beads to prepare a homogeneous spore suspension. Count the spores using a hemocytometer; the spore concentration of the suspension should be 1 × 10⁻⁶. 7 2.5 × 10⁻² / mL 7 per mL.

[0062] 2) Preparation of liquid seed solution

[0063] Inoculate the spore suspension into the liquid seed culture medium at an inoculation rate of 6-10%, place it in a constant temperature shaker at 32-35℃, and shake at 160-200 rpm for 2-4 days to obtain the liquid seed solution.

[0064] 3) Solid-state fermentation culture

[0065] Mix indica rice and soaking liquid at a mass ratio of 1:1.5-2 (w / w) in a 500mL Erlenmeyer flask (50-60g of rice per flask) and soak for 6-8 hours. After draining, autoclave at 121℃ for 30 minutes. After cooling, inoculate with 6-10% of the liquid seed solution obtained in step 2, shake well, and incubate at 30-35℃. Once the culture medium is basically covered with mycelia, supplement with vitamin complex solution to maintain the humidity inside the flask above 85%. Incubate for 10-13 days, until the rice grains turn purplish-red. Then, dry the red yeast rice in a constant temperature incubator at 60℃ with ventilation until the moisture content is below 12%. Grind the dried red yeast rice into the finished product.

[0066] In this invention, the rice soaking solution is prepared by adding 1-3% (by weight of water) of a nutrient enzyme accelerator to the water. Each 1 mL of the nutrient enzyme accelerator contains 0.1-0.2 g NH4SO4, 0.05-0.15 g MgSO4, 0.01-0.02 g MnSO4, 0.01-0.02 g ZnSO4, 0.01-0.02 g FeSO4, 0.1-0.2 g KH2PO4, and 0.05-0.115 g K2HPO4. The pH of the rice soaking solution is adjusted to 3-4 with acetic acid, and then sterilized at 121°C for 30 minutes. During the red yeast rice fermentation process, Fe... 2+ and Zn 2+ Various metal ions participate in enzymatic reactions, promoting mycelial growth and metabolizing into red yeast pigments. Therefore, supplementing rice with external nutrient enzymes during soaking can promote the early growth of Monascus purpureus.

[0067] In the above process, after the culture medium is basically covered with mycelia, 3-5 mL of vitamin complex solution is added. The vitamin complex solution contains 0.005-0.01 g pyridoxine, 0.001-0.005 g folic acid, 0.02-0.03 g tocopherol, and 0.03-0.04 g biotin per 100 mL, with an addition amount of 1-2% of the dry weight of the material. The pH of the vitamin complex solution is adjusted to 3.0-3.5 with acetic acid. Aseptic operation is used; the vitamin complex solution is prepared with sterile water, and the pH of the replenishment solution is adjusted to acidic with acetic acid. Sterilization is not required. When the culture medium is covered with Monascus purpureus mycelia and Monascus purpureus is the dominant organism, the vitamin complex solution is added as a replenishment solution, which can greatly avoid the loss of vitamin complex solution due to high-temperature sterilization.

[0068] Using Monascus purpureus FAAS-M3, solid-cultured red yeast rice was treated with different soaking solutions and vitamin compound solutions. The color value, citrinin content, and product yield of the red yeast rice were used as evaluation indicators. The results are shown in Table 3.

[0069] Table 3 Color value, citrinin content and yield of red yeast rice products

[0070]

[0071] As shown in Table 3, compared with treatment 1, which used rice soaking liquid and sterile water for replenishment as the conventional process control, treatments 2, 3, and 4, which added different concentrations of external nutrient enzymes during rice soaking, were significantly better than treatment 1 in all aspects. The color value of the three treatments increased by 8%-48%; the yield of citrinin decreased slightly by 10%-28%; and the product yield increased by 11%-26%.

[0072] Treatments 5, 6, and 7, which involved adding different concentrations of vitamin complex solution during rehydration, showed a slight increase in red yeast rice beige value of 5%-9%, while effectively inhibiting the production of citrinin, reducing citrinin levels by 44%-62%.

[0073] Treatments 8, 9, and 10, which involved adding different concentrations of nutrient enzyme catalysts and vitamin complex solutions, showed an increase in color value of 36%-68%, a significant inhibition of citrinin, reducing it by 64%-73%, and an increase in product yield of 19%-41%. Red yeast rice prepared using the strain and process of this invention, with a cultivation period of 10-13 days, exhibited a color value as high as 8200 μ / g and a product yield exceeding 35% after testing.

Claims

1. The use of a Monascus strain with high yield of Monascus pigment in the preparation of high color value Monascus rice, characterized in that , and the Monascus strain is Monascus purpureus Monascus purpureus ) FAAS-M3, which has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2022958, and the preservation date is June 24, 2022; and the preparation process of the red koji rice is as follows: 1) Inclination surface inoculates purple red mold FAAS-M3, is placed in 30-35 ℃ constant temperature incubator and is cultivated 7-10 d, until the inclination surface is full of red mold, takes 8 mL sterile normal saline to add to the inclination surface seed, is scraped with inoculation needle to the inclination surface spore, is scattered in the sterile water with glass bead, is made into the uniform spore suspension, is counted through the blood cell counting board, and the spore concentration of spore suspension is 1×10 7 To 2.5×10 7 Individuals / mL; 2) Preparation of liquid seed solution The spore suspension is inoculated into the liquid seed culture medium at an inoculation amount of 6-10%, and is placed in a constant temperature shaker at 32-35°C, and is shaken at 160-200 rpm for 2-4 days to obtain the liquid seed solution; 3) Solid state fermentation culture The indica rice and the rice soaking solution are mixed in a 500 mL triangular flask at a mass ratio of 1:1.5-2 (w / w), 50-60 g of indica rice per flask, soaked for 6-8 hours, drained, and then autoclaved at 121°C for 30 minutes. After cooling, 6-10% of the liquid seed solution obtained in step 2 is inoculated, shaken, and cultured at 30-35°C. When the culture medium is basically covered with mycelium, a vitamin complex solution is added to maintain the humidity in the bottle at more than 85%, and the culture is continued for 10-13 days until the rice grains turn purple red. Then the red koji rice is dried at 60°C in a constant temperature oven until the moisture content is less than 12%, and the dried red koji rice is ground into a product; The rice soaking solution is prepared by adding 1-3% of a nutritional enzyme activator to water, wherein the nutritional enzyme activator contains 0.1-0.2 g of NH4SO4, 0.05-0.15 g of MgSO4, 0.01-0.02 g of MnSO4, 0.01-0.02 g of ZnSO4, 0.01-0.02 g of FeSO4, 0.1-0.2 g of KH2PO4, and 0.05-0.115 g of K2HPO4 per 1 mL, and the pH of the rice soaking solution is adjusted to 3-4 with acetic acid, and is autoclaved at 121°C for 30 minutes; In the above process, when the culture medium is basically covered with mycelium, 3-5 mL of a vitamin complex solution is added, wherein the vitamin complex solution contains 0.005-0.01 g of pyridoxine, 0.001-0.005 g of folic acid, 0.02-0.03 g of tocopherol, and 0.03-0.04 g of biotin per 100 mL, and the pH of the vitamin complex solution is adjusted to 3.0-3.5 with acetic acid.

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