CN111534443B - High-yield MonacolinK purple monascus strain and method for producing functional red yeast rice by fermenting MonacolinK purple monascus strain - Google Patents

Abstract

The invention discloses a high-yield MonacolinK purple monascus strain and a method for producing functional monascus by fermenting the same. The invention obtains Monacolin K with higher yield and without citrinin produced during fermentation through screening and optimizing series conditions from naturally fermented red yeast rice, namely Monascus purpureus NCPS2019010, wherein the preservation number of the strain is CCTCC NO: M2019736. On the basis of the excellent strains, a polypropylene ethylene high-temperature resistant culture bag is adopted for fermentation to prepare the functional red yeast rice, water does not need to be supplemented in the fermentation process, materials do not need to be frequently turned over in the culture process, the materials are turned over only once in the third day of culture, the content of MonacolinK in the prepared functional red yeast rice reaches 0.8-1.7%, the content of open-loop MonacolinK reaches more than 80%, and no citrinin is produced.

Description

High-yield MonacolinK purple monascus strain and method for producing functional red yeast rice by fermenting MonacolinK purple monascus strain

Technical Field

The invention relates to a high-yield MonacolinK purple monascus strain and a method for producing functional red yeast rice by fermenting the MonacolinK purple monascus strain, and belongs to the technical field of microbial strain fermentation.

Background

The red yeast rice is prepared by fermenting indica rice, japonica rice, sticky rice and the like serving as raw materials through monascus, is a traditional fermented product in China, has a production and application history of more than one thousand years, and is widely applied to food coloring, wine brewing, fermented bean curd and traditional Chinese medicine. It is stated in compendium of materia medica that red yeast has the function of promoting blood circulation and removing blood stasis. In 1979, the Japanese academy of academic Long-vine chapter separated a new substance from Monascus ruber (M.ruber) fermentation product, named MonacolinK, can effectively inhibit the activity of key enzyme 3-hydroxy-methyl-glutaryl-coenzyme A reductase in cholesterol biosynthesis, block the synthesis of cholesterol, regulate abnormal blood lipid in human body, and has specific effect on reducing total serum cholesterol and low-concentration lipoprotein cholesterol which produce atherosclerosis.

In recent years, with the improvement of living standard, coronary heart disease, hyperlipidemia, cerebral thrombosis and the like caused by high cholesterol threaten human health, the demand of lipid-lowering products is increasing, and functional red yeast rice taking monacolin k (the same substance as lovastatin) as a main component gradually comes into the visual field of people. The functional red yeast rice is mainly different from common red yeast rice in that the MonacolinK content is higher. Research shows that compared with lovastatin extracted from other fungi or artificially synthesized, MonacolinK in the red yeast product has less crystallization and high solubility, so the red yeast product has more obvious curative effect and less toxic and side effect. The functional red yeast rice also contains various active substances which are beneficial to human bodies, such as gamma-aminobutyric acid, choline, ergosterol, flavone, unsaturated fatty acid and the like, so that the functional red yeast rice not only retains the traditional effects of strengthening spleen to promote digestion and promoting blood circulation to remove blood stasis, but also has the physiological activities of reducing blood cholesterol, reducing blood fat, reducing blood sugar and blood pressure, preventing cancer, improving immunity, resisting oxidation and the like, has extremely high nutritional, health-care and medicinal values, and is a natural, safe and effective health-care food and medicine raw material.

Monacolin k (monacolin k) is an ideal hypolipidemic agent with two structures, a lactone-type structure and an acid-type structure. Monacolin K is produced by Aspergillus terreus and Monascus ruber. Monacolin K from aspergillus terreus fermentation is a prescription drug developed by American merck company, is a main component of lipid-lowering drugs in the current market, has a lactone structure, is a prodrug, and has activity only after being hydrolyzed into an acid structure by hydroxyl esterase secreted by human liver after being taken, so as to play a role in inhibiting HMG-CoA reductase and further blocking cholesterol synthesis; however, a few of human bodies have insufficient or low activity of endocrine hydroxyesterase, so that the hydrolysis of lactone type Monacolin K is incomplete, toxic and side effects are generated, the striated muscle is dissolved and painful, and the burden of the liver and the kidney is increased. The Monacolin K in the functional red yeast rice exists in two forms at the same time, most of the Monacolin K in the functional red yeast rice obtained by natural fermentation is in an acid structure, the Monacolin K does not need hydroxyl esterase in a human body to participate in hydrolysis, the liver and kidney burden cannot be increased, the lipid-lowering effect is directly exerted, the other part of the Monacolin K in a lactone structure is the Monacolin K, the acid can be converted into the lactone structure along with the change of external moisture and temperature in the later period of fermentation, and if the conditions such as high temperature, dehydration and the like are met in the purification process, the content of the lactone Monacolin K occupies the main position.

Monacolin K produced by the fermentation method of the aspergillus terreus is of a lactone structure, generates toxic and side effects, causes dissolving and pain of striated muscles, and increases the burden of the liver and the kidney. Although Monacolin K from monascus fermentation can well solve the problem and has a plurality of other health care functions, the Monacolin K content in functional red yeast rice produced by the prior art is low, and in 1995, Blanc of French scholars detects citrin in monascus fermentation products, and the monascus fermentation products have the effects of nephrotoxicity, carcinogenicity, mutagenesis and the like, so that the application and development of monascus fermentation products are severely restricted. Therefore, obtaining the monascus purpureus strain with high acid yield and Monacolin K and low citrinin yield is a problem which needs to be solved urgently by the industry at present.

The application of functional red yeast rice in the feed has also been developed, and lovastatin has been used as a feed additive for chickens and pigs abroad to reduce the synthesis of cholesterol in the animals and reduce the cholesterol content in chicken, eggs and pork. The problem of antibiotic abuse in the aquaculture industry has long been an important factor affecting food safety and public health safety. The ministry of agricultural rural areas clearly stipulates that antibiotics are strictly prohibited to be added into daily feed of animals from 1 month and 1 day of 2020, and feed products containing the antibiotics are strictly prohibited to be sold from 7 months and 1 day, and the historical opportunity is met by 'substitute antibiotic' products. The functional red yeast contains not only MonacolinK, but also a large amount of other beneficial active substances, and has the values of improving immunity and high antioxidant activity. Therefore, the functional red yeast rice is expected to be used as a novel feed additive and has wide application space in relevant products of 'resistance reduction/resistance replacement'.

The requirement of the light industry standard of China on functional red yeast rice is that the content of MonacolinK is more than or equal to 0.4 percent. The content of MonacolinK in the common red yeast rice is extremely low, the MonacolinK can be measured after being concentrated by multiple times, the solid state fermentation process is mature and rough, the technical content is low, the fermentation is semi-open type fermentation, the mixed bacteria are easy to pollute, toxin substances are generated, the citrinin is a main toxin, the citrinin has strong nephrotoxicity and damages the metabolism of the liver, the test in vivo can be combined with serum protein, and the carcinogenicity can induce mutation. The requirements of European and American countries on the content of citrinin in food and feed are that the citrinin cannot be detected by high performance liquid chromatography. The content of the citrinin of the functional red yeast rice product in China is not higher than 0.05 mu g/g.

At present, the problems puzzling the functional red yeast rice products mainly have two aspects, namely that the large-scale fermentation technology is immature and the production cost of the products is too high; secondly, the yield of MonacolinK is low and a toxic substance citrinin is generated in the fermentation process. According to the literature reports, the laboratory fermentation level of the functional red yeast rice MonacolinK is between 2 and 10mg/g, and a few reports indicate that the fermentation level can reach 15 to 20mg/g, but the amplification of the fermentation scale is a technical bottleneck which troubles the industry because the solid fermentation of the functional red yeast rice is strictly pure. At present, most of domestic functional red yeast rice plants adopt plastic bottle fermentation production processes, the fermentation period is long, the yield is low, the labor cost is high, and although many laboratory research achievements seem to be advanced, industrialization is difficult to realize due to the production technology bottleneck. The production of citrinin in the fermentation process is another problem which troubles many developers and manufacturers.

The Chinese patent application No. 201710774819.7 discloses a monascus purpureus for producing high-yield acid Monacolin K and low-yield citrinin and application thereof, and provides a method for producing functional red yeast rice by fermenting the monascus purpureus and the obtained functional red yeast rice, the main contents of the monascus purpureus comprise a strain culture mode and a fermentation method, the main bright points are that the provided strain can improve the content of acid Monacolin K in the functional red yeast rice of monascus purpureus fermentation products, and simultaneously reduce the content of the citrinin, the content of the citrinin is lower than 15 mu g/kg, the content of the Monacolin K is at least 5 per thousand, and the ratio of the acid Monacolin K in the Monacolin K is at least 40%. Although the yield of Monacolin K of the strain provided by the invention is relatively high, and the content of acid Monacolin K is higher than 40%, the fermentation process still generates citrinin, the preparation process is complicated, the indica rice needs to be soaked for 4-10h in advance, then steamed by steam for about 15-30min, the rice is sterilized at 121 ℃, and the inoculation amount is large and reaches 10% -20%. In the solid culture process, the culture medium needs to be shaken and turned over once a day, and a container used for culture is a 1000ml glass shaking bottle, so that the operation not only causes high labor intensity, but also causes rapid water loss in the culture process, and according to the practical operation experience, water needs to be supplemented after 8-10 days, thereby further increasing the labor intensity. Other similar patents, such as chinese patent application No. 201810040975.5, disclose a process for producing functional red rice powder by fermenting cereal, which requires water supplement and intermittent shaking of the bottle during constant temperature cultivation, and needs shaking once every 9-15 hours, thus being very labor-intensive.

Disclosure of Invention

At present, in the solid fermentation culture of functional red yeast rice, the yield of functional substances Monacolin K is low, the content of open-loop Monacolin K (acid Monacolin K) is basically below 50%, toxic substances are produced, in the solid fermentation process, the culture medium is basically shaken every day to turn over materials, the operation not only causes high labor intensity, but also leads to fast water loss in the culture process, according to the practical operation experience, water needs to be supplemented after 8-10 days, the labor intensity is further increased, containers used for culture are all glass or plastic shake flasks, the space is wasted in the culture process, the materials are easy to accumulate and agglomerate, and the water is fast to volatilize.

Aiming at the problems, the invention provides a purple red yeast strain which has high Monacolin K yield and no citrinin production in the fermentation process, and the strain has stable performance, high growth speed and strong fermentation capacity. On the basis of excellent strains, a polypropylene ethylene high-temperature resistant culture bag is adopted for fermentation to prepare the functional red yeast rice, water does not need to be supplemented in the fermentation process, materials do not need to be frequently turned in the culture process, and the materials are only turned once in the third day of culture. The functional red yeast rice prepared by the process is fermented for 15-30 days, the MonacolinK content in the product reaches 0.8% -1.7%, the open-loop MonacolinK content reaches more than 80%, and no citrinin is produced.

The laboratory obtains a purple Monascus purpureus (Monascus purpureus) NCPS2019010 with high yield of Monacolin K and NO citrinin production in the fermentation process from naturally fermented red yeast rice after screening and series condition optimization, the bacterial strain is preserved in a China center for type culture collection (CCTCC for short, the address: China, Wuhan university) in 2019 and 23 months, and the preservation number is M2019736.

The method for producing the functional red yeast rice by fermenting the monascus purpureus NCPS2019010 strain is characterized by comprising the following steps of:

1) slant culture

Carrying out slant culture on the monascus purpureus NCPS2019010 strain at the culture temperature of 28-32 ℃ for 5-8 days;

2) seed liquid culture

Adding thallus Porphyrae into shake flask containing seed liquid culture medium, scattering thallus Porphyrae, shake-culturing with shaking table, and culturing at 28-30 deg.C for 90-100hr to obtain seed liquid;

3) fermentation culture

Filling the fermentation solid culture medium into a polypropylene high-temperature-resistant culture bag, wherein the thickness of the material is 4-5cm, scattering the material while the material is hot after sterilization, cooling to normal temperature, and inoculating 5-10ml of seed liquid according to the proportion of inoculating 100g of dry matter;

placing the inoculated culture bag in a constant-temperature culture chamber at 27-30 ℃ for culture without controlling humidity, fully and uniformly mixing after culturing for 3-4 days, continuously culturing until the monascus grows over the culture medium for 5-6 days and the materials are obviously red, transferring the culture to the condition of 22-25 ℃ for culture for 15-25 days, and after the culture is finished, sterilizing, drying and crushing to obtain the functional monascus.

The slant culture medium: PDA was added with wort solid medium (wort addition 4 ℃ final sugar degree).

Seed liquid culture medium (except glycerol, the mass ratio is as follows): rice flour 1.5-2.5%, corn starch 0.1-0.15%, soybean flour 1.5-2.5%, peptone 0.8-1.2%, glycerin 4-6% (V/V), glucose 1.5-2.5%, KH₂PO₄ 0.2-0.3％，NaNO₃0.3-0.8% and natural pH.

The fermentation solid culture medium comprises: comprises dry matter, inorganic salt nutrient solution and lactic acid; the addition of the inorganic salt nutrient solution is 60-70% (V/W) of the dry matter, and the addition of the lactic acid is 0.5-1.5% (V/W) of the dry matter;

the dry matter comprises the following components in parts by weight: 100 parts of polished round-grained rice or long-grained nonglutinous rice, 60-85 parts of soybean protein isolate, 10-20 parts of corn flour, 10-20 parts of bran and 8-12 parts of millet;

the inorganic salt nutrient solution comprises the following components in percentage by weight: MgSO (MgSO)₄ 0.08-0.12％，ZnSO₄0.03-0.08%, trisodium citrate 0.8-1.2%, NaNO₃ 0.3-0.8％。

The invention has the beneficial effects that:

1) the product quality is good

The fermented functional red yeast rice prepared by the process has the advantages that the Monacolin K content in the product reaches 0.8-1.7%, the open-loop Monacolin K content reaches more than 80%, no citrinin is generated, and the quality of the product is greatly higher than that of the existing product in the market.

2) Simple and convenient process

The traditional common culture bottle is too high, the space is wasted, as aerobic solid fermentation is adopted, the high-temperature-resistant polypropylene culture bag is adopted, the contact area is large, the culture medium is thin (not higher than 5cm), the mixing uniformity is good after inoculation, materials do not need to be frequently turned in the culture process, only the materials need to be turned once in the third day of culture, and the labor amount is greatly reduced. Because the material turning is reduced, the water does not need to be supplemented in the fermentation process, and the risk of infectious microbes is greatly reduced.

Drawings

FIG. 1 is a photograph of a colony of strain NCPS2019010 after being plated on a PDA for 10 days;

FIG. 2 is a photograph (front) of a colony of the strain NCPS2019010 after being plated on a PDA for 14 days;

FIG. 3 is a photograph of a colony of the strain NCPS2019010 after being plated on a PDA for 14 days (reverse side);

FIG. 4 is a photograph of 100 times stained mycelia and conidia of strain NCPS 2019010;

FIG. 5 is a 40-fold lower ascospore picture of strain NCPS 2019010;

FIG. 6 is a photograph of PCR agarose gel electrophoresis of strain NCPS 2019010; wherein, 2: NCPS2019010, M: Marker (DL 2000: 2000, 1000, 750, 500, 250, 100bp), Y: negative control;

FIG. 7 is a picture of functional red yeast rice produced by fermentation;

FIG. 8 shows the HPLC detection result of the product, in open loop form, Monacolin K.

Detailed Description

The effects of the present invention will be described below with reference to test examples.

Example 1: isolation and characterization of strains

1. Screening and optimizing monascus

Activating the plate: PDA is added with proper amount of wort (final sugar degree 4)^°)；

The formula of the liquid culture medium is as follows: 2 percent of rice flour, 2 percent of corn flour, 4 percent of soybean meal and 7 percent of glycerin(mass volume fraction), glucose 2%, KH₂PO₄ 0.25％，NaNO₃0.5％；

The solid culture medium formula comprises: 15g of rice flour, 10g of soybean meal, 2.5g of bran and 2g of corn flour, stirring and sterilizing for 20min at 121 ℃. Sterilizing, scattering while hot, adding 10mL of nutrient solution (0.1% MgSO)₄，0.05％ZnSO ₄1% trisodium citrate, 5% glycerol, 3% peptone, 0.5% NaNO₃) +0.3mL of lactic acid.

Placing purchased multiple red yeast rice into sterile water, shaking for about 2 hr, diluting in series with dilution degree of 10²、10³、10⁴Coating PDA on the suspension, adding appropriate amount of solid plate (containing 0.01% chloramphenicol), culturing at 28-30 deg.C for 4-5 days, selecting single colony with good growth condition to slant culture medium according to colony appearance, culturing for 6-7 days, diluting, purifying, and slant culturing the single colony.

Detecting the growth activity of a single colony: the liquid culture medium is subpackaged in 500mL triangular flasks, each flask is filled with 100mL, and sterilized at 121 ℃ for later use. The slant digging block is connected to the liquid culture medium, the liquid culture medium is cultured for 96 hours at 28 ℃ and 180rpm by shaking in a shaking table, the growth vigor is observed in the culture process, liquid with vigorous growth vigor and dark color is selected to inoculate the solid fermentation culture medium according to the inoculation amount of 10 percent, after the culture is finished, the solid fermentation culture medium is dried for 2 hours at the temperature of 60 ℃, the MK content is determined, 10 strains with the highest MK content are selected to carry out continuous three-time solid fermentation, the MK content and the citrinin content are determined, and the results are as follows.

TABLE 1 screening results of Red Rice strains

The 010- # strain is fermented to produce the citrinin with the highest MK content and no toxin substance, the performance of continuous three-time solid fermentation is stable, and multiple repeated experiments show that the strain is very good in stability, so that the 010- # strain is selected as a final strain to perform strain identification and is named as NCPS 2019010.

2. Characterization and characterization of the strains

1) And (3) observing colony morphology: morphological identification of Monascus purpureus No. 010 strain selected was spotted on PDA plates, cultured at 30 ℃ for 10-14 days, and the color, morphology, size and texture changes of the strain growth were observed and recorded. The shapes of hypha, conidiophore chain, cyst closure shell, ascospore and the like are observed by adopting a slide culture method, and the pictures are observed by a microscope.

And (3) colony morphology characteristics: the strain activated by the inclined plane is spotted on a PDA flat plate, and observed after being cultured in a constant temperature incubator at 32 ℃ for 10 days, the colony is round, the edge is neat, long villus hyphae are arranged, and the villus is white (figure 1); after 14 days of culture, the colony is round, the edge is neat, the villus is shortened, the colony is orange red, the edge is light yellow in a circle, and the back surface is orange red (figures 2-3).

2) And (3) microscopic observation: individual morphological characteristics

Hypha diameter 2-7 μm, with transverse septa and irregular branches (FIG. 4); conidia are unicellular, colorless and transparent, and are single-grown or chain-formed, and are inverted pear-shaped or spherical, and are grown on the top of hyphae and branches thereof (fig. 4). The cyst-closing shell is elliptical, has a diameter of 30-50 mu m, has a handle, and is filled with densely arranged spores, and the ascospores are oval or spherical (figure 5);

through observing colony morphological characteristics and microscopic structural characteristics such as hypha, spores and the like, the NCPS2019010 fungus can be preliminarily identified as the monascus fungus according to a fungus identification manual and a Chinese fungistatic book, but the species is difficult to identify only through morphological characteristics, and further identification needs to be carried out by combining a molecular biology means.

3) Molecular biological identification of monascus

Performing PCR amplification by using ITS identification primers (ITS1/ITS4) and 18S identification primers (NS1/NS8), detecting PCR products by agarose gel electrophoresis, respectively detecting the sizes of the products to be about 300-700 bp and 1800bp (shown in figure 6), performing DNA sequencing, performing homology comparison with known sequences in GenBank, judging the type of the fungus, and classifying the fungus into genus or species. The ITS sequence of NCPS2019010 is determined, and the result is shown in (SEQ-1):

CTAGTGTAGCATTTATACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCTTACTACATGGATACCTGTGGTAATTCTAGAGCTAATACATGCTAAAAACCCCGACTTCGGAAGGGGTGTATTTATTAGATAAAAAACCAACGCCCTTCGGGGCTCCTTGGTGAATCATAATAACTAAACGAATCGCATGGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATAGTGGCCTACCATGGTGGCAACGGGTAACGGGGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATACTGATACGGGGCTCTTTCGGGTCTCGTAATCGGAATGAGAACGACCTAAATAACCTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAACCTTGGGTCTGGCTGGCCGGTCCGCCTCATCGCGAGTACTGGTCCGGCCGGACCTTTCCTTCTGGGGAACCTCATGGCCTTCACTGGCTGTGGGGGGAACCAGGACTTTTACTGTGAAAAAATTAGAGTGTTCAAAGCAGGCCTTTGCTCGAATACATTAGCATGGAATAATAGAATAGGACGTGCGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGATAGTCGGGGGCGTCAGTATTCAGCTGTCAGAGGTGAAATTCTTGGATTTGCTGAAGACTAACTACTGCGAAAGCATTCGCCAAGGATGTTTTCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGGGTTTCTATGATGACCCGTTCGGCACCTTACGAGAAATCAAAGTTTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGGGCACCACAAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAAAATAAGGATTGACAGATTGAGAGCTCTTTCTTGATCTTTTGGATGGTGGTGCATGGCCGTTCCTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAGACCTCGGCCCTTAAATAGCCCGGTCCGCATTTGCGGGCCGCTGGCTTCTTAAGGGGACTATCGGCTCAAGCCGATGGAAGTGCGCGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGACAGGGCCAGCGAGTACATCACCTTGGCCGAGAGGCCTGGGTAATCTTGTTAAACCCTGTCGTGCTGGGGATAGAGCATTGCAATTATTGCTCTTCAACGAGGAATGCCTAGTAGGCACGAGTCATCAGCTCGTGCCGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTCCGGACTGGCCCAGGGAGGTTGGCAACGACCCCCCCGGGCCGGAAAGCTGGTCAAACTCGGTCA。

through molecular biological identification, the strain NCPS2019010 is Monascus purpureus (Monascus purpureus), and the strain is preserved in a China center for type culture Collection (CCTCC for short, the address: China, Wuhan university) in 23 months in 2019, and the preservation number is CCTCC NO: M2019736.

Example 2: sterile pure-breed fermentation

Slant culture medium: PDA was added with wort solid medium (wort addition 4 ℃ final sugar degree).

Seed liquid culture medium: 2% of rice flour, 0.12% of corn steep liquor powder, 2% of soybean flour, 1.0% of peptone, 5% (V/V) of glycerol, 2% of glucose and KH₂PO₄ 0.25％，NaNO₃0.5% and natural pH.

Fermentation solid medium: 100g of polished round-grained rice or long-grained nonglutinous rice, 60-85g of soybean protein isolate, 10-20g of corn flour, 10-20g of bran, 8-12g of millet and inorganic salt nutrient solution (0.1% MgSO (MgSO)) as nutrient solution₄，0.05％ZnSO ₄1% trisodium citrate, 0.5% NaNO₃) The addition amount is 66% (V/W) of the dry matter, and the addition amount of the lactic acid is 1% (V/W) of the dry matter.

The specific method comprises the following steps:

1) slant culture

Carrying out slant culture on the monascus purpureus NCPS2019010 strain at 28-32 ℃ for 5-8 days, and storing the monascus purpureus NCPS2019010 strain in a refrigerator at 4 ℃ for use when the slant is full of mycelia and the culture medium turns red;

2) seed liquid culture

The seed liquid culture medium is subpackaged into 500mL triangular flasks, each flask is filled with 100mL, sterilized for 30min at 121 ℃, and taken out for later use after cooling;

the inoculation method comprises the following steps: performing aseptic operation, digging 2-3 pieces of thallus Porphyrae with soybean granule size on the inclined plane, adding into shake flask, scattering thallus Porphyrae as much as possible, shake culturing at 28-30 deg.C and 180rpm for 90-100hr to obtain seed solution;

3) fermentation culture

Filling the fermentation solid culture medium into a polypropylene high-temperature-resistant culture bag, wherein the thickness of the material is 4-5cm, sterilizing at 121 ℃ for 90 minutes, scattering the material while the material is hot, cooling to normal temperature, and inoculating 5-10ml of seed liquid according to the proportion of inoculating every 100g of dry material;

placing the inoculated culture bag in a constant-temperature culture chamber at 27-30 ℃ for culture without controlling humidity, fully and uniformly mixing after culturing for 3-4 days, continuously culturing until the monascus is overgrown with the culture medium for 5-6 days and the material is obviously red, transferring the culture to a condition of 22-25 ℃ for culture for 15-25 days (20 days in the embodiment), finally culturing to obtain the functional monascus monacolin K (MonacolinK) with the content of more than 0.8% (1.0-1.5% in the embodiment), drying the water at 60 ℃ to 8-12% after carrying out moist heat sterilization at 121 ℃ for 15 minutes, and preparing a picture of the monascus as shown in figure 7, wherein the picture of the monascus can be further crushed according to needs.

HPLC detection analysis of MonacolinK

Chromatograph: agilent 1260;

a chromatographic column: ZORBAX XDB-C184.6 mm, 5 μm;

mobile phase: acetonitrile, 0.2% aqueous phosphoric acid solution 60: 40;

detection wavelength: 237 nm;

column temperature: 30 ℃;

the test treatment was carried out in QB/T2847-2007, and the results are shown in FIG. 8 (RT of open-loop Monacolin K is 10.785, RT of lactone-type Monacolin K is 18.507), as shown in the figure: the content of the open-loop Monacolin K accounts for more than 95 percent of the total content of the Monacolin K.

SEQUENCE LISTING

<110> institute of agricultural products of academy of agricultural sciences of Shandong province

<120> high-yield MonacolinK purple monascus strain and method for producing functional red yeast rice by fermenting MonacolinK purple monascus strain

<130> 0

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1677

<212> DNA

<213> ITS sequence of Monascus purpureus (Monascus purpureus) NCPS2019010

<400> 1

ctagtgtagc atttatactg tgaaactgcg aatggctcat taaatcagtt atcgtttatt 60

tgatagtacc ttactacatg gatacctgtg gtaattctag agctaataca tgctaaaaac 120

cccgacttcg gaaggggtgt atttattaga taaaaaacca acgcccttcg gggctccttg 180

gtgaatcata ataactaaac gaatcgcatg gccttgcgcc ggcgatggtt cattcaaatt 240

tctgccctat caactttcga tggtaggata gtggcctacc atggtggcaa cgggtaacgg 300

ggaattaggg ttcgattccg gagagggagc ctgagaaacg gctaccacat ccaaggaagg 360

cagcaggcgc gcaaattacc caatcccgac acggggaggt agtgacaata aatactgata 420

cggggctctt tcgggtctcg taatcggaat gagaacgacc taaataacct aacgaggaac 480

aattggaggg caagtctggt gccagcagcc gcggtaattc cagctccaat agcgtatatt 540

aaagttgttg cagttaaaaa gctcgtagtt gaaccttggg tctggctggc cggtccgcct 600

catcgcgagt actggtccgg ccggaccttt ccttctgggg aacctcatgg ccttcactgg 660

ctgtgggggg aaccaggact tttactgtga aaaaattaga gtgttcaaag caggcctttg 720

ctcgaataca ttagcatgga ataatagaat aggacgtgcg gttctatttt gttggtttct 780

aggaccgccg taatgattaa tagggatagt cgggggcgtc agtattcagc tgtcagaggt 840

gaaattcttg gatttgctga agactaacta ctgcgaaagc attcgccaag gatgttttca 900

ttaatcaggg aacgaaagtt aggggatcga agacgatcag ataccgtcgt agtcttaacc 960

ataaactatg ccgactaggg atcggacggg tttctatgat gacccgttcg gcaccttacg 1020

agaaatcaaa gtttttgggt tctgggggga gtatggtcgc aaggctgaaa cttaaagaaa 1080

ttgacggaag ggcaccacaa ggcgtggagc ctgcggctta atttgactca acacggggaa 1140

actcaccagg tccagacaaa ataaggattg acagattgag agctctttct tgatcttttg 1200

gatggtggtg catggccgtt cctagttggt ggagtgattt gtctgcttaa ttgcgataac 1260

gaacgagacc tcggccctta aatagcccgg tccgcatttg cgggccgctg gcttcttaag 1320

gggactatcg gctcaagccg atggaagtgc gcggcaataa caggtctgtg atgcccttag 1380

atgttctggg ccgcacgcgc gctacactga cagggccagc gagtacatca ccttggccga 1440

gaggcctggg taatcttgtt aaaccctgtc gtgctgggga tagagcattg caattattgc 1500

tcttcaacga ggaatgccta gtaggcacga gtcatcagct cgtgccgatt acgtccctgc 1560

cctttgtaca caccgcccgt cgctactacc gattgaatgg ctcagtgagg cctccggact 1620

ggcccaggga ggttggcaac gacccccccg ggccggaaag ctggtcaaac tcggtca 1677

Claims (3)

1. A method for producing functional red yeast rice by fermentation is characterized by comprising the following steps:

1) slant culture

The purple monascus with the preservation number of CCTCC NO: M2019736 (A)Monascus purpureus) Carrying out slant culture on the NCPS2019010 strain at the culture temperature of 28-32 ℃ for 5-8 days;

2) seed liquid culture

Adding thallus Porphyrae into shake flask containing seed liquid culture medium, scattering thallus Porphyrae, shake-culturing with shaking table, and culturing at 28-30 deg.C for 90-100hr to obtain seed liquid;

3) fermentation culture

Filling the fermentation solid culture medium into a polypropylene high-temperature-resistant culture bag, wherein the thickness of the material is 4-5cm, sterilizing, scattering, and inoculating 5-10ml of seed solution per 100g of dry matter of the fermentation solid culture medium;

placing the inoculated culture bag in a constant-temperature culture chamber at 27-30 ℃ for culture without controlling humidity, fully and uniformly mixing after culturing for 3-4 days, continuously culturing until the monascus grows over the culture medium for 5-6 days and the material is obviously red, transferring the culture to the condition of 22-25 ℃ for culture for 15-25 days, and after the culture is finished, sterilizing and drying to obtain the functional red yeast rice; the content of the open-loop MonacolinK in the functional red yeast rice reaches more than 80 percent, and no citrinin is generated;

the fermentation solid culture medium comprises: dry matter, inorganic salt nutrient solution and lactic acid; according to the volume mass ratio, the addition of the inorganic salt nutrient solution is 60-70% of the dry matter, and the addition of the lactic acid is 0.5-1.5% of the dry matter;

the dry matter comprises the following components in parts by weight: 100 parts of polished round-grained rice or long-grained nonglutinous rice, 60-85 parts of soybean protein isolate, 10-20 parts of corn flour, 10-20 parts of bran and 8-12 parts of millet;

the inorganic salt nutrient solution comprises the following components in percentage by weight: MgSO (MgSO)₄ 0.08-0.12%， ZnSO₄0.03-0.08%, trisodium citrate 0.8-1.2%, NaNO₃ 0.3-0.8%。

2. The method for producing functional red yeast rice by fermentation as claimed in claim 1, wherein said slant culture medium used for slant culture is PDA plus wort solid medium, and the amount of wort added is 4 ° at the final sugar degree.

3. The method for producing functional red yeast rice by fermentation according to claim 1, wherein the seed liquid culture medium is: rice flour 1.5-2.5%, corn starch 0.1-0.15%, soybean flour 1.5-2.5%, peptone 0.8-1.2%, glycerin 4-6%, glucose 1.5-2.5%, KH₂PO₄ 0.2-0.3%，NaNO₃ 0.3-0.8%, pH is natural; wherein the proportion of the glycerol is volume ratio, and the rest is mass ratio.

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