Monascus strain with high yield of monascus pigment and low yield of citrinin and application thereof

Abstract

This invention discloses a Monascus purpureus strain that produces high levels of Monascus pigment and low levels of citrinin, and its applications. The Monascus purpureus strain producing high levels of Monascus pigment and low levels of citrinin is classified as *Monascus purpureus* FAAS-M34 (…). Monascus purpureus The purple Monascus strain FAAS-M34 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2022959 and a deposit date of June 24, 2022. This strain exhibits a strong ability to produce natural red yeast rice pigment and low production of citrinin, making it suitable for preparing high-quality red yeast rice. The resulting red yeast rice product has a high color value, and the citrinin content does not exceed relevant national regulations, with a high product yield. Therefore, the purple Monascus strain FAAS-M34 can be safely used in the food processing industry. Compared with existing strains of the same type, it can produce higher-quality red yeast rice products and has high industrial application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a monascus strain with high yield of monascus pigment and low yield of citrinin and application thereof. BACKGROUND

[0002] Monascus pigment is a secondary metabolite of filamentous fungus Monascus, which is the only natural pigment of microbial origin allowed to be used in food additives. In recent years, due to the advantages of monascus pigment such as stable properties, strong heat resistance, good coloring power for protein, etc., it can replace nitrite (a primary carcinogen) and is rapidly increasing in use in meat processing and aquatic product processing fields, which has jumped to the most important application field of monascus rice and monascus pigment. Monascus and monascus pigment are one of the natural food pigments with the largest output and use in the world. Fujian Province is the birthplace and main producing area of monascus, and the output and export of Fujian monascus have been in the leading position in China for a long time. Monascus has been exported to many countries in Southeast Asia, Europe and the United States, and the output of monascus rice accounts for more than 60% of the world's total output. Fujian monascus has a high reputation at home and abroad due to its high quality and high color value, and has a broad market prospect. However, monascus mycelium often generates a toxin, citrinin, during fermentation and metabolism, which pollutes monascus products and causes safety hazards in the use of monascus pigment, seriously restricting the wide application of monascus, and has become the biggest obstacle for monascus products to enter the international market.

[0003] Citrinin is one of the main indicators of food safety national standards and an important indicator of food pollution. Some countries have established limit indicators for citrinin in food additives monascus pigment. For example, the national standards of China, i.e. "Food Additive Monascus Yellow" and "Food Additive Monascus Red" executed in 2015 and 2016, added the limit standards of citrinin in solid samples as 1 mg / Kg and 0.04 mg / Kg (both in unit of color value) respectively, which are higher than the requirements of some countries in the world, such as South Korea, Japan and the European Union, which stipulate that the limit standard of citrinin in monascus products is 0.05, 0.2 and 2 mg / Kg respectively. At present, the content of citrinin in high color value monascus products in China is still relatively high (mostly 0.23-20.65 mg / Kg), which restricts the export and use of monascus products in China, and affects the export of monascus products and the healthy development of monascus industry in Fujian Province. SUMMARY

[0004] The present application aims to provide a monascus strain with high yield of monascus pigment and low yield of citrinin and application thereof.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The present application provides a red yeast strain with high yield of monascus pigment and low yield of citrinin, which is named as Monascus purpureus FAAS-M34, has been preserved in China Center for Type Culture Collection, Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2022959, and the preservation date is June 24, 2022.

[0007] The present application provides the Monascus purpureus FAAS-M34 which can be applied to prepare high-quality red yeast rice.

[0008] The Monascus purpureus FAAS-M34 has strong ability to produce natural monascus pigment and almost no citrinin, and can be applied to prepare high-quality red yeast rice, and the obtained red yeast rice product has high color value and almost no citrinin, short culture period and high product yield. Therefore, the Monascus purpureus FAAS-M34 can be safely used in the food processing industry, and compared with the existing strains of the same type, it can produce higher quality red yeast products, and has high industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 It is a comparison chart of the ability of red yeast strain to produce pigment and citrinin in solid and liquid culture.

[0010] Figure 2 It is a colony morphology chart of the Monascus purpureus FAAS-M34.

[0011] Figure 3 It is an individual morphology chart (mycelium and conidial structure) of the Monascus purpureus FAAS-M34 under an optical microscope.

[0012] Figure 4 It is an individual morphology chart (cleistothecium and mycelium structure) of the Monascus purpureus FAAS-M34 under a scanning electron microscope.

[0013] Figure 5 It is a growth and metabolism curve chart of the Monascus purpureus FAAS-M34 in liquid culture. DETAILED DESCRIPTION

[0014] The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

[0015] Sample source

[0016] Red yeast rice or koji pool soil samples collected from various red yeast production enterprises in Fujian.

[0017] Culture medium

[0018] (1) Slant medium: malt extract 10°Bé, agar 2%, pH 6-7, after heating and dissolving, pour into test tubes, plug with cotton plug, sterilize with high pressure steam at 121°C for 20 min, take out, and place in slant to cool and form slant medium.

[0019] (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. 50 mL of liquid seed culture medium is added to a 250 mL flask and several small glass beads are added, and sterilized with high pressure steam at 121°C for 30 min.

[0020] (3) Liquid fermentation culture 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. 50 mL of liquid seed culture medium is added to a 250 mL flask and several small glass beads are added, and sterilized with high pressure steam at 121°C for 30 min.

[0021] (4) Solid state fermentation culture medium: indica rice, commercially available.

[0022] Example 1 Isolation, screening and identification of strains

[0023] 1.1 Method

[0024] 1.1.1 Strain isolation method

[0025] 1-2 g of crushed red koji rice is added to a flask containing 50 mL of sterile water, and shaken at 180 r / min for 30 min, and serial gradient dilution is performed, i.e. 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 0.1-0.2 mL of each dilution is taken and plated, and incubated at 35°C for 3-5 days, and according to the growth morphology of the colonies on the plate, colonies with fast growth, large colony diameter and dark color are picked and subcultured until a purified Monascus strain is obtained, and the strain is incubated on a slant at 35°C for 7-10 days, and then stored in a refrigerator at 4°C for preservation, and used as needed.

[0026] 1.1.2 Preparation of spore suspension

[0027] The slant inoculation of Monascus strain is cultured in a constant temperature incubator at 30-35°C for 7-10 days, and the slant is full of Monascus when it is ready. 8 mL of sterile normal saline is added to the slant seed, and the spores on the slant are scraped with an inoculation needle and dispersed in sterile water containing glass beads to prepare a uniform spore suspension. The spore concentration of the spore suspension is 1×10 7 7

[0028] 1.1.3 Preparation of liquid seed solution

[0029] The spore suspension is inoculated into the liquid seed culture medium at a 6-10% inoculation amount, and is cultured in a constant temperature shaker at 32-35°C with 160-200 rpm shaking for 2-4 days to obtain the liquid seed solution, which is taken for use.

[0030] 1.1.4 Liquid fermentation culture

[0031] The liquid seed solution is inoculated into the liquid fermentation culture medium at an 8-12% inoculation amount, and is cultured in a constant temperature shaker at 32-35°C with a rotation speed of 160-200 r / min for 4-6 days.

[0032] 1.1.5 Solid-state fermentation culture

[0033] 50-60 g of indica rice per bottle is mixed with rice soaking liquid (water) at a mass ratio of 1:1.5-2 (w / w) in a 500 mL triangular bottle, and is shaken well for about 6-8 h of soaking time. After draining, the rice is sterilized by high-pressure steam at 121°C for 30 min, and is inoculated with 6-10% of the liquid seed solution after cooling, shaken well, and cultured at 30-35°C. When the culture medium is basically covered with mycelium, 2-5 mL of vitamin complex solution is added to keep the humidity in the bottle above 85%. The culture is cultured for 10-13 days until the rice grains turn purple red. Then the Monascus rice is dried in a constant temperature oven at 60°C with ventilation until the moisture content is below 12%. The dried Monascus rice is ground into a product.

[0034] 1.1.6 Determination method

[0035] The color value of Monascus rice is determined according to the national standard GB 1886.19-2015 “Food Additive Monascus Rice”, and the citrinin is determined according to the national standard GB 5009.222-2016 “Determination of Citrinin in Food”.

[0036] 2 Results and analysis

[0037] 2.1. Isolation and screening of Monascus strain monocytes

[0038] ​​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 monaxioline and citrinin as indicators, it was found that different Monascus purpureus strains showed varying degrees of monaxioline and citrinin production capabilities. Among them, strain FAAS-M34 was a superior strain with strong monaxioline production and low citrinin production capabilities. The results are as follows: Figure 1 As shown.

[0039] 2.2 Morphological characteristics and taxonomic identification of Monascus purpureus strain FAAS-M34

[0040] 2.2.1 Colony morphology characteristics

[0041] The bacteria were cultured on malt extract agar plates for 7 days. The colony morphology, color, and elevation of the red yeast rice were observed. Initially, the mycelium was white, gradually turning red, and then purplish-red as it matured. The reverse side of the agar plate turned orange-red. Colony morphology was as follows: Figure 2 As shown, the colonies are relatively compact, with a diameter of 20-25 mm. The center of the colony is raised and has irregular radial grooves. The aerial hyphae are covered with short, fluffy hairs.

[0042] 2.2.2 Cultivation Characteristics

[0043] Culture studies revealed that strain FAAS-M34 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 is slow 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.

[0044] 2.2.3 Individual morphological characteristics

[0045] Morphology under optical microscopes and scanning electron microscopes, such as Figure 3 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. Conidia are borne at the tips of the mycelium and its branches, spherical or obpyriform, 6–9 μm in diameter, colorless, containing granules, and are solitary or in chains of 2–3. The cleistothecia are spherical, 10–20 μm in diameter, and stalked.

[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. A blank control was used without inoculation. The tubes were incubated at 30°C for 7-14 days. Observation revealed indentations in the gelatin curd beneath the colonies, indicating that this strain hydrolyzes the gelatin.

[0049] Table 1 Physiological and Biochemical Characteristics

[0050] Glucose Maltose Fructose Sucrose Lactose Sorbitol 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'-TCCGTAGGTGAACCTGC GG-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-M34 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-M34 can be identified as Monascus purpureus FAAS-M34, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2022959 and deposit date of June 24, 2022.

[0053] Table 2. ITS rDNA sequencing results of strain FAAS-M34

[0054]

[0055]

[0056] 2.2.6 Assessment of growth and metabolic capacity

[0057] The *Monascus purpureus* strain FAAS-M34 was cultured in liquid form, and the mycelial weight, color production, and citrinin production at different growth stages were measured. The biomass reached 2.2 ± 0.1 g / 100 mL, the color value was as high as 126.10 ± 17.82 U / mL, and the citrinin production was as low as 0.19 ± 0.52 mg / L. Its growth and metabolism were as follows: Figure 5 As shown.

[0058] Example 2: Application of Monascus purpureus FAAS-M34 in the preparation of red yeast rice

[0059] The process for preparing red yeast rice using Monascus purpureus FAAS-M34 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-M34 *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 shake well. 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. The nutrient enzyme accelerator contains 0.1-0.2g NH4SO4, 0.05-0.15g MgSO4, 0.01-0.02g MnSO4, 0.01-0.02g ZnSO4, 0.01-0.02g FeSO4, 0.1-0.2g KH2PO4, and 0.05-0.115g K2HPO4 per 1mL. The pH of the rice soaking solution is adjusted to 3-4 with acetic acid, and then sterilized at 121℃ 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 pigment. 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-M34, 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 can be seen from Table 3, Treatment 1, which uses rice soaking liquid and sterile water for replenishment as the control of the conventional process, and Treatments 2, 3, and 4, which add different concentrations of external nutrient enzymes during rice soaking, are significantly better than Treatment 1 in all aspects. The color value of the three treatments increased significantly by 50%-96%, while the content of citrinin also increased slightly by 21-39%; the product yield increased by 18%-29%.

[0072] When adding vitamin complex solution of different concentrations during the replenishment, the color value of red yeast rice cultured in treatments 5, 6, and 7 increased slightly by 6%-51%, the product yield increased slightly by 11%-25%, and the production of citrinin was effectively inhibited, with the citrinin content reduced by 22%-42%.

[0073] Simultaneous addition of different concentrations of nutrient enzyme catalysts and vitamin complex solution for treatments 8, 9, and 10 significantly improved the color value of the product, which was 1.97-2.64 times that of the control, and the inhibition effect of citrinin was significant, reducing it by 66%-82%. Red yeast rice prepared using the strain and process of this invention has a cultivation period of 10-13 days; after testing, the color value of the red yeast rice product reached 6600-7500 μ / g, the citrinin content in the finished product did not exceed the relevant national regulations, and the product yield reached 39%.

Claims

1. The application of a Monascus purpureus strain with high production of monaxioline and low production of citrinin in the preparation of red yeast rice, characterized in that, The Monascus strain mentioned is Monascus purpureus (purple Monascus). Monascus purpureus FAAS-M34 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2022959 and deposit date of June 24, 2022. The preparation process of the red yeast rice is as follows: 1) Inoculate the slant with purple Monascus FAAS-M34 and incubate in a constant temperature incubator at 30-35℃ for 7-10 days until the slant is covered with Monascus ferruginea. Add sterile physiological saline to the slant seeds, scrape the slant spores with an inoculation needle, and disperse them in sterile water containing glass beads to make a spore suspension. 2) 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 for 2-4 days to obtain the liquid seed solution; 3) Mix the indica rice and soaking liquid in an Erlenmeyer flask at a mass ratio of 1:1.5-2, shake well, soak for 6-8 hours, drain, sterilize, cool, and then inoculate with 6-10% of the liquid seed solution obtained in step 2). Shake well and incubate at 30-35℃. When the culture medium is covered with mycelium, add vitamin complex solution to maintain the humidity in the flask above 85%. Incubate for 10-13 days, then dry and grind the obtained red yeast rice to obtain the finished product. The rice soaking solution contains 1-3% by weight of a nutrient enzyme accelerator added 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 3-4. The vitamin complex solution contains 0.005-0.01g pyridoxine, 0.001-0.005g folic acid, 0.02-0.03g tocopherol and 0.03-0.04g biotin per 100mL, with the addition amount being 1-2% of the dry weight of the material. The pH of the vitamin complex solution is 3.0-3.5.

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