A method for rapidly screening monacolin K high-yield monascus

Abstract

The application provides a method for rapidly screening monacolin K high-yield Monascus, and belongs to the technical field of microorganism screening, and comprises the following steps: S1, preliminary screening of monacolin K high-yield Monascus; S1.1, isolation and purification of Monascus; S1.2, screening of monacolin K high-yield Monascus; S1.2.1, preparation of Neurospora crassa spore liquid; S1.2.2, preparation of a to-be-tested liquid; S1.2.3, biological identification of the to-be-tested liquid; S2, re-screening of monacolin K high-yield Monascus; S2.1, preparation of a spore suspension; S2.2, preparation of a seed liquid; S2.3, solid-state fermentation; S3, detection of monacolin K; S4, detection of citrinin; and S5, selection of optimal monacolin K high-yield Monascus. The application separates and purifies monacolin K high-yield Monascus, re-screens monacolin K high-yield Monascus through solid-state fermentation, selects optimal monacolin K high-yield Monascus by detecting the monacolin K and citrinin contents of each Monascus, and has high efficiency and low cost.

Description

Technical Field

[0001] This invention relates to the field of microbial screening technology, and in particular to a method for rapidly screening high-yielding Monakaria K red Monascus. Background Technology

[0002] Currently, there is limited research on methods for screening Monascus monazites that produce high levels of Monascus K, both domestically and internationally. The main approach involves first isolating Monascus monazites from nature and Monascus products for initial screening, then using the Monascus monazites obtained from the initial screening for fermentation, and finally detecting the Monascus K content in the fermentation products using high-performance liquid chromatography to screen for Monascus monazites that produce high levels of Monascus K. This method is not only costly but also time-consuming and labor-intensive.

[0003] To improve the efficiency of screening high-yield Monakaria K, this invention provides a method for rapidly screening high-yield Monakaria K. Summary of the Invention

[0004] The purpose of this invention is to provide a method for rapidly screening high-yield Monakolin K red Monascus, thereby solving the technical problems mentioned in the background art.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for rapidly screening high-yield Monakaria kibblea includes the following steps: S1, Initial screening of high-yield Monacoline K red yeast rice S1.1 Isolation and purification of Monascus purpureus After the red yeast rice sample was crushed and sieved, it was shaken in physiological saline, serially diluted, and cultured on PDAC plates. White fluffy single colonies were picked and repeatedly streaked twice for purification. The resulting strain was inoculated on PDA slant and cultured until it grew fully. Then, glycerol bacteria were prepared by using 20% ​​glycerol as a protectant. S1.2 Screening of Monascus purpureus for high Monascus K production S1.2.1 Preparation of *Neurospora crassa* spore liquid Culture the spores of Neurospora crassa to the early logarithmic phase and set aside; S1.2.2 Preparation of the test solution The red Monascus strain to be screened was inoculated onto a PDA slant and cultured. After ultrasonic extraction with ethyl acetate as solvent, the supernatant was collected by centrifugation and concentrated to obtain the test solution. S1.2.3, Biological identification of the test solution The inhibition zone against Neurospora crassa was detected on MEA medium using the Oxford cup method. Strains with an inhibition zone diameter greater than 1.6 cm were identified as Monascus purpureus producing high levels of Monascus K. S2, secondary screening of high-yield Monacoline K red Monascus. S2.1 Preparation of spore suspension The Monascus purpureus that produced high levels of Monascus K obtained from the initial screening was inoculated onto PDAC plates and cultured in an incubator. White colonies were picked and inoculated onto PDA slant plates and cultured. Spore suspension was prepared using sterile water as a protectant. S2.2 Preparation of Seed Liquid After diluting the spore suspension, a certain amount of the diluted spore suspension was inoculated into an Erlenmeyer flask containing seed culture medium and cultured on a shaker to obtain the seed solution. S2.3, solid-state fermentation In a clean bench, a certain amount of seed liquid is inoculated into sterilized rice substrate, thoroughly mixed and spread out, placed in an incubator for fermentation to produce red yeast rice, which is then dried, pulverized and sieved to obtain red yeast rice powder. S3, Monacoline K detection A certain amount of red yeast rice powder was placed in a centrifuge tube, ethanol was added, and the mixture was vortexed and shaken. After ultrasonic extraction and cooling, the supernatant was centrifuged and filtered through a microporous membrane. The content of monacolin K was detected by high performance liquid chromatography. S4. Detection of Citrulline A certain amount of red yeast rice powder was placed in a centrifuge tube, the extraction solution was added and vortexed to mix. After ultrasonic extraction, the supernatant was centrifuged and passed through a C18 solid phase extraction column. All eluent was collected and passed through an organic microporous membrane. The content of citrinin was detected by high performance liquid chromatography. S5. Selection of the optimal high-yield Monacoline K red yeast rice strain Based on the content of Monacolin K and Citrinin in each Monacolin K spore, the optimal Monacolin K-producing Monacolin K spore was selected.

[0006] Furthermore, in step S1.1, the shaking temperature is 30°C, the shaking speed is 180 r / min, and the shaking time is 1 h. Furthermore, in step S1.1, the coating culture temperature is 30°C and the coating culture time is 2 days. Further, in step S1.2.1, *Neurospora crassa* is inoculated onto MEA plates and cultured in an incubator at 28°C. Single colonies are picked and inoculated into Erlenmeyer flasks containing PD medium and cultured on a shaker for 24-72 hours to establish a functional relationship between the concentration of Monacolin K and the diameter of the inhibition zone.

[0007] Furthermore, in step S1.2.2, the culture temperature is 30℃ and the culture time is 10 days. Furthermore, in step S1.2.2, the ultrasonic extraction time is 1 h; the centrifugation speed is 5000 r / min and the centrifugation time is 2 min; the concentration temperature is 30℃ and the concentration time is 15 min.

[0008] Furthermore, in step S2.1, the temperature of the incubator is 30°C. Furthermore, in step S2.2, the concentration of the diluted spores is 10⁶~10⁷ CFU / mL; the inoculation amount of the spore suspension is 2% (v / v).

[0009] Furthermore, in step S2.2, the temperature of the shaker culture is 30°C, the rotation speed of the shaker culture is 180 r / min, and the shaker culture time is 12 h.

[0010] Furthermore, in step S2.3, the inoculation amount of the seed liquid is 8% (v / g); the fermentation temperature is 30℃; and the drying temperature is 60℃.

[0011] Furthermore, in step S2.3, the fermentation process involves turning the koji once after 72 hours of fermentation and continuing fermentation for another 14 days.

[0012] The present invention, by adopting the above-described technical solution, has the following beneficial effects: 1. This invention utilizes the antifungal activity of Monacolin K and the sensitivity of Neurospora crassa to the β-hydroxy acid of Monacolin K, using Neurospora crassa as an indicator bacterium to screen Monascus purpureus that produce high levels of Monacolin K, thereby improving the efficiency of screening Monascus purpureus that produce high levels of Monacolin K.

[0013] 2. This invention isolates and purifies Monascus purpureus that produce high levels of Monascus K, and then re-screens Monascus purpureus that produce high levels of Monascus K through solid-state fermentation. By detecting the content of Monascus K and citrinin in each Monascus purpureus strain, the optimal Monascus K-producing Monascus purpureus is selected, which is highly efficient and low in cost. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the correlation between MKA concentration and inhibition zone diameter in this invention. Figure 2 This is a schematic diagram of the inhibition zone of the high-yield Monacoline K red Monascus of this invention; Figure 3 This is a chromatogram of the MKL and MKA standards of this invention; Figure 4 This is a schematic diagram of the standard curve of the MKL of this invention; Figure 5 This is a schematic diagram of the standard curve of the MKA of this invention; Figure 6 This is a schematic diagram of the standard curve of CIT in this invention; Figure 7 This is the chromatogram of the CIT standard of this invention; Figure 8 This is a schematic diagram illustrating the ability of different Monascus purpureus strains to produce MK and CIT according to the present invention. Detailed Implementation To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are merely to provide the reader with a thorough understanding of one or more aspects of the present invention, and these aspects of the invention can be implemented even without these specific details.

[0015] Example Experimental strains and samples 1. Neurospora crassa: provided by China General Microbiological Culture Collection Center (AGMCC).

[0016] 2. Red yeast rice fermentation substrate: commercially available Akita Komachi pearl rice.

[0017] 3. Information on the red yeast rice products preserved in this experiment is shown in Table 1 below.

[0018] Table 1. Relevant information on red yeast rice products Preparation of culture media and solutions 1. PD medium: 6.0 g potato extract powder, 20.0 g glucose, dissolved in deionized water and brought to a final volume of 1000 mL, then autoclaved at 121°C for 20 min.

[0019] 2. PDA medium: 6.0 g potato extract powder, 20.0 g glucose, 20 g agar, dissolved in deionized water by heating and brought to a final volume of 1000 mL, then autoclaved at 121°C for 20 min.

[0020] 3. PDAC medium: 46.1 g of potato dextrose agar (containing chloramphenicol) medium was dissolved in deionized water by heating and then brought to a final volume of 1000 mL. The mixture was then autoclaved at 121°C for 20 min.

[0021] 4. Sterile physiological saline: Dissolve 0.90 g of sodium chloride in deionized water and bring the volume to 100 mL. Sterilize at 121°C under high temperature and pressure for 20 min.

[0022] 5. 20% Glycerin: 20.0 g glycerin, 80 mL deionized water, autoclaved at 121°C for 20 min.

[0023] 6. Preparation of MEA medium: 20.0 g malt extract, 8.0 g peptone, 20.0 g glucose, 6.0 g yeast extract, 13.0 g agar, dissolved in deionized water by heating and brought to a final volume of 1000 mL. Sterilize at 121℃ for 20 min.

[0024] 7. Red Monascus seed culture medium: 50.0 g glucose, 15.0 g peptone, 10.0 g yeast extract, 3.0 g NaNO3, 0.5 g MgSO4·7H2O, 1.0 g KH2PO4, dissolved in deionized water and brought to a final volume of 1000 mL. Sterilize at 121℃ under high temperature and pressure for 20 min.

[0025] 8. Solid-state fermentation substrate: Weigh 25.0 g of rice into a 90 mm petri dish, soak in water overnight, drain the water, and then autoclave at 121℃ for 20 min.

[0026] 9. 75% ethanol solution: Mix 75 mL of anhydrous ethanol with 25 mL of deionized water.

[0027] 10. Monacoline K standard stock solution (lactone): Accurately weigh 20.0 mg of monacoline K into a 10 mL beaker, dissolve it in a small amount of acetonitrile, transfer it to a 10 mL volumetric flask, wash the beaker 2-3 times with acetonitrile, transfer the washing solution to the volumetric flask, and then add acetonitrile to make up to volume.

[0028] 11. 0.2 mol / L sodium hydroxide solution: Weigh 0.80 g of sodium hydroxide into a 200 mL beaker, add 100 mL of deionized water, stir with a glass rod to dissolve, and then pour into a 100 mL sample bottle.

[0029] 12. 0.1% phosphoric acid solution: Transfer 0.6 mL of phosphoric acid (density 1.69 g / mL) into a 1000 mL beaker, add 1000 mL of deionized water and mix well. Filter through a 0.45 μm microporous membrane before use. Prepare fresh before use.

[0030] 13. Citrulline extract: Prepared at a volume ratio of acetonitrile:isopropanol:ulpure water = 7:2:11, and adjusted to pH 1.5 with phosphoric acid.

[0031] I. Initial screening of high-yield Monakaria koraiensis 1. Isolation, purification and preservation of Monascus purpureus The laboratory-preserved red yeast rice sample was pulverized using a high-speed blender and passed through a 40-mesh sieve to obtain red yeast rice powder. An appropriate amount of sample was placed in a straight bottle containing 10 mL of sterile physiological saline and shaken on a shaker at 180 r / min for 1 h at 30℃. The mixture was then serially diluted (10... -2 10 -4 10 -5 10 -6), respectively take 100 μL of the dilution and spread it on a PDAC plate, culture it in an incubator at 30°C for 2 days. After white villous hyphae grow out, pick a single colony and transfer it to another PDAC plate for culture. After repeated isolation and purification for 2 times, a strain with the basic characteristics of Monascus is obtained. Then inoculate the strain on a PDA slant, culture until the 6th day. After the thalli grow fully, add an appropriate amount of 20% glycerol to the slant, gently scrape off the mycelia with a sterile inoculation shovel to prepare glycerol bacteria, pipette 1.5 mL of the glycerol bacteria into a 2 mL centrifuge tube, classify and number it, and store it at -20°C.

[0032] 2. Screening of Monascus with high Monacolin K yield It was found through preliminary experiments that among 5 strains with larger inhibition zone diameters, after 14 days of solid-state fermentation, for strains whose Monacolin K yield is greater than 4 mg / g, their corresponding inhibition zone diameters are greater than 1.6 cm. Therefore, the agar diffusion method is used to screen Monascus with high Monacolin K yield, and strains with an inhibition zone diameter greater than 1.6 cm are considered as Monascus with high Monacolin K yield.

[0033] 2.1 Preparation of Neurospora crassa spore suspension Take out Neurospora crassa preserved in a -20°C refrigerator and let it thaw naturally, then streak and inoculate it on an MEA plate, place it in a 28°C incubator for culture for 24-72 h, and use this bacterial suspension as the indicator strain. With agar at a concentration of 1.3% (g / mL), a culture volume of 20 mL and a loading volume of 120 μL of Monacolin K, the functional relationship between Monacolin K concentration and inhibition zone diameter is established, as Figure 1 shown.

[0034] 2.2 Preparation of test solution After activating the Monascus to be screened, pick a single colony and inoculate it on a PDA slant, culture it at 30°C for 10 days, add 3 mL of ethyl acetate into the test tube to immerse the thalli, perform ultrasonic extraction for 1 h, take 1.5 mL of the extract into a 2 mL centrifuge tube, centrifuge at 5000 r / min for 2 min, take 1 mL of the supernatant into another new centrifuge tube, place it on a vacuum concentrator to concentrate at 30°C for 15 min until about 300 μL of the extract remains, and place it in a 4°C refrigerator for storage and testing.

[0035] 2.3 Bioassay of test solution The agar concentration of MEA medium is 1.3 g / 100mL, the volume of the medium is 20 mL, and the loading volume is 120 μL. To improve screening efficiency, pour the MEA medium into a petri dish containing Oxford cups arranged in a "pin" shape, resulting in the formation of 3 wells.

[0036] Preliminary screening results: Forty suspected Monascus strains were isolated from the Monascus samples preserved in our laboratory. The production of Monacolin K by these 40 strains and eight strains (R1, R2, R3, R4, M1, M2, M3, M4) preserved in our laboratory was investigated. If a strain produces high levels of Monacolin K, its extract will diffuse within the pores, inhibiting the growth of surrounding Neurospora crassa and forming an inhibition zone with a diameter greater than 1.6 cm. The experimental phenomena are as follows: Figure 2 As shown in Table 2, the diameters of the two perpendicular inhibition zones were measured using calipers. The results show that the diameters of the inhibition zones corresponding to the extracts of nine suspected Monascus strains were greater than 1.6 cm. These nine suspected Monascus strains were considered to be high producers of Monascus K, namely strains FG1, FG3, FG4, JHS1, KM1, M2, M4, MS1, and SST1.

[0037] Table 2. Screening results of high-yield Monakaria kibblea (Monakaria purpureus) Note: "-" indicates no inhibition zone or an indistinct inhibition zone.

[0038] II. Secondary screening of high-yield Monacoline K-producing Monascus purpureus 1. Preparation of spore suspension The suspected high-yield Monakolin K-producing Monascus purpureus obtained from the initial screening was taken out of the freezer at -20℃, thawed at room temperature, and shaken well. It was then streaked onto a PDA plate using a sterile inoculation loop and cultured in an incubator at 30℃ until white single colonies grew. A single colony was picked up with a sterile inoculation loop and inoculated onto a PDA slant. After culturing at 30℃ for 5 days, the bacterial cells turned reddish-brown. 4 mL of sterile water was added to the test tube, and the hyphae and spores were gently scraped off with a sterile inoculation spatula to prepare a spore suspension.

[0039] 2. Preparation of Seed Liquid The spore suspension was diluted and counted using a hemocytometer to achieve a spore concentration of 10⁶–10⁷ CFU / mL. The diluted spore suspension was then inoculated at a rate of 2% (v / v) into an Erlenmeyer flask containing 50 mL of Monascus seed culture medium. The flask was incubated at 30°C and 180 rpm for 12 h on a shaker to obtain the seed culture.

[0040] 3. Solid-state fermentation Inside a clean bench, seed liquid was inoculated into sterilized rice substrate at an inoculation rate of 8% (v / g). The mixture was thoroughly dispersed and spread using a sterile inoculation spatula, ensuring that the seed liquid was evenly distributed on the solid substrate. The substrate was then placed in an incubator at 30°C for 72 hours of fermentation. Small water droplets were observed to condense on the inside of the petri dish lid. The substrate was then turned over to ensure thorough mixing before fermentation continued until the fermentation ended on the 14th day. The resulting red yeast rice was then dried in an oven at 60°C, pulverized, and passed through a 40-mesh sieve for testing.

[0041] III. Detection of Monacoline K 1. Testing conditions The content of monacolin K in fermentation products was determined by high performance liquid chromatography (HPLC), referring to the detection method in "QB / T 2847-2023 Functional Red Yeast Rice (Powder)". The chromatographic column was LaChrom C18 (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile: 0.1% phosphoric acid aqueous solution (v / v) = 65:35; the flow rate was 1.0 mL / min; the UV detection wavelength was 238 nm; the injection volume was 20 μL; the column temperature was 30℃; the detector was a UV detector; and external standard quantification was used.

[0042] 2. Qualitative judgment Monacoline K (MKL) can be converted to monacoline K (MKA) under alkaline conditions. 1 mL of 2 mg / mL monacoline K was mixed with 1 mL of 0.2 mol / L sodium hydroxide solution and sonicated for 1 h. The solution was then diluted to 10 mL with acetonitrile to obtain a standard solution of monacoline K. Since the relative molecular masses of monacoline K and monacoline K are 404.55 and 422.57 respectively, the concentration of monacoline K is 208.9 μg / mL. The 2 mg / mL monacoline K standard solution was diluted to 200 μg / mL, and the two standard solutions were mixed at a volume ratio of 1:1 to obtain a standard mixture of MKA and MKL. After filtering through a 0.22 μm microporous membrane, the mixture was analyzed using high-performance liquid chromatography (HPLC). Figure 3 The chromatogram shows that the peak time of MKA is 8.500 min and the peak time of MKL is 13.707 min, indicating a good separation effect.

[0043] 3. Plotting the standard curve of lactone-type monacolin K (MKL) Accurately weigh 20 mg of monacolin K lactone into a 10 mL beaker. Dissolve the dissolved monacolin in an appropriate amount of acetonitrile and transfer the solution to a 10 mL volumetric flask. Wash the beaker 2-3 times with acetonitrile, transferring the washings to the volumetric flask. After bringing the solution to volume with acetonitrile, continue diluting with acetonitrile to prepare standard solutions of 400, 200, 100, 50, 25, 5, and 0.5 μg / mL, respectively. These are MKL standard solutions. Filter the solutions through a 0.22 μm microporous membrane and analyze them using high-performance liquid chromatography (HPLC). Measure the corresponding peak areas. Plot a standard curve of MKL with the concentration of MKL on the x-axis and the peak area on the y-axis. Figure 4 As shown, the linear regression equation is 62036x + 26418, R² = 0.9999, indicating that there is a good linear relationship between peak area and MKL concentration, and the concentration of MKL can be calculated based on the peak area.

[0044] 4. Plot the standard curve of monacolin acid K (MKA). Mix 1 mL of 2 mg / mL monacolin K lactone with 1 mL of 0.1 mol / L sodium hydroxide solution, sonicate for 1 h, cool to room temperature, and then bring the volume to 10 mL with acetonitrile (concentration 208.9 μg / mL). Further dilution with acetonitrile to prepare standard solutions of MKA with concentrations of 156.7, 104.4, 52.4, 20.9, and 4.2 μg / mL, respectively. Filter the solutions through a 0.22 μm microporous membrane and analyze the peak areas using high-performance liquid chromatography (HPLC). Plot a standard curve for MKA with MKA concentration on the x-axis and peak area on the y-axis. Figure 5 As shown, the linear regression equation is y = 58188x - 36942, R² = 0.9997, indicating that there is a good linear relationship between peak area and MKA concentration, and the concentration of MKA can be calculated based on the peak area.

[0045] 5. Monacoline K is calculated according to formula (1): ...(1) X: The content of monacolin K in the sample, in milligrams per gram (mg / g). V: Volume of sample extract, in milliliters (mL); m: Sample weight, in grams (g). X1: The concentration of lactone-type monacolin K in the sample calculated from the above MKL standard curve, in micrograms per milliliter (μg / mL). X2: The concentration of monacolin K in the sample calculated from the above MKA standard curve, in micrograms per milliliter (μg / mL).

[0046] 1000: Conversion factor for units of mass.

[0047] 6. Sample pretreatment Take 500 mg of red yeast rice powder into a 50 mL centrifuge tube, add 40 mL of 75% ethanol, vortex and shake well, sonicate at room temperature for 1 h, cool to room temperature, centrifuge at 3500 r / min for 10 min, take the supernatant and filter it through a 0.22 μm microporous membrane, and analyze it using high performance liquid chromatography.

[0048] IV. Detection of Citrulline 1. Testing conditions The content of citrinin in red yeast rice was determined by high performance liquid chromatography (HPLC). The method of purification using a C18 solid-phase extraction column-HPLC was followed according to GB 5009.222-2016 "National Food Safety Standard - Determination of Citrinin in Food". The detection conditions were as follows: detector: fluorescence detector; column: Agilent Zorbax SB-C18 column (4.6 mm × 250 mm, 5 µm); mobile phase: acetonitrile: 0.1% phosphoric acid aqueous solution (v / v) = 60:40; flow rate: 1.0 mL / min; fluorescence detection wavelength: λ. ex =331 nm, λ em =500 nm; injection volume: 10 μL; column temperature: 28℃; external standard quantification.

[0049] 2. Citrulline stock solution Accurately weigh 1 mg of citrinin standard, dissolve it in 2 mL of methanol to obtain a concentration of 500 μg / mL, and store it in a refrigerator at 4°C.

[0050] 3. Preparation of the citrinin standard curve The stock solution of citrinin was diluted with methanol to prepare standard solutions of citrinin at concentrations of 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, and 1.5625 ng / mL. After filtering through a 0.22 μm microporous membrane, the solutions were injected sequentially from lowest to highest concentration, and the corresponding peak areas were measured. A standard curve of citrinin was plotted with the concentration of citrinin on the x-axis and the peak area on the y-axis. Figure 6 As shown, the linear regression equation is y = 12778x + 3257.6, R0 2 = 0.9992, indicating a good linear relationship between peak area and citrinin concentration. The concentration of citrinin can be calculated based on the peak area. The chromatogram of citrinin is shown below. Figure 7 As shown.

[0051] 4. Citrulline is calculated according to formula (2). ...(2) X: The content of citrinin in the sample, in micrograms per gram (μg / kg); : Concentration of citrinin in the eluent, in nanograms per milliliter (ng / mL). 10: Volume of eluent, in milliliters (mL); 30: Volume of sample passing through the elution column, in milliliters (mL); V: Extraction volume of the sample, in milliliters (mL); m: Sample weight, in grams (g).

[0052] 5. Sample pretreatment Weigh 1 g of red yeast rice powder into a 50 mL centrifuge tube, add 40 mL of extraction solution (acetonitrile + isopropanol + ultrapure water), vortex to mix, and then sonicate for 30 min. Centrifuge at 3500 r / min for 10 min, take 30 mL of supernatant and pass it through a pre-washed C18 solid-phase extraction column. Then wash the column with 5 mL of ultrapure water. After all the eluent has flowed out of the column, dry under reduced pressure for 3 min, and then elute with 10 mL of methanol. Collect all the eluent, filter it through a 0.22 μm organic microporous membrane, and analyze it using high performance liquid chromatography.

[0053] Results: Nine suspected high-yield Monakaria kibble strains (FG1, FG3, FG4, JHS1, KM1, M2, M4, MS1, and SST1) obtained from the initial screening were inoculated into rice substrate. After 14 days of fermentation, red yeast rice was produced. The contents of MK and CIT in the fermentation product were detected by HPLC. The results are as follows: Figure 8 As shown, the MK content produced by Monascus purpureus strains FG1, FG4, JHS1, KM1, M2, M4, MS1, and SST1 were all above 4 mg / g, specifically 4.005 mg / g, 6.059 mg / g, 10.184 mg / g, 8.064 mg / g, 5.861 mg / g, 9.034 mg / g, 7.875 mg / g, and 8.009 mg / g, respectively. Among these, Monascus purpureus strain M4 produced a significantly lower CIT content than the other strains (P<0.05), at 102 μg / kg. Monascus purpureus strains FG1, FG3, FG4, and M2 showed relatively weaker MK production capacity compared to other strains, while producing lower CIT content. The content of CIT produced by these strains was significantly higher than that produced by other Monascus purpureus strains. Combined with the antibacterial experiment results in the screening of high-yielding Monascus purpureus K, the reason why the diameter of the inhibition zone of these strains was larger may be because CIT also has a certain antibacterial effect on Neurospora crassa. In conclusion, strain M4 is the best strain.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapidly screening high-yield Monakaria kibblea, characterized in that: Includes the following steps: S1, Initial screening of high-yield Monacoline K red yeast rice S1.1 Isolation and purification of Monascus purpureus After the red yeast rice sample was crushed and sieved, it was shaken in physiological saline, serially diluted, and cultured on PDAC plates. White fluffy single colonies were picked and repeatedly streaked twice for purification. The resulting strain was inoculated on PDA slant and cultured until it grew fully. Then, glycerol bacteria were prepared by using 20% ​​glycerol as a protectant. S1.2 Screening of Monascus purpureus for high Monascus K production S1.2.1 Preparation of *Neurospora crassa* spore liquid Culture the spores of Neurospora crassa to the early logarithmic phase and set aside; S1.2.2 Preparation of the test solution The red Monascus strain to be screened was inoculated onto a PDA slant and cultured. After ultrasonic extraction with ethyl acetate as solvent, the supernatant was collected by centrifugation and concentrated to obtain the test solution. S1.2.3, Biological identification of the test solution The inhibition zone against Neurospora crassa was detected on MEA medium using the Oxford cup method. Strains with an inhibition zone diameter greater than 1.6 cm were identified as Monascus purpureus producing high levels of Monascus K. S2, secondary screening of high-yield Monacoline K red Monascus. S2.1 Preparation of spore suspension The Monascus purpureus that produced high levels of Monascus K obtained from the initial screening was inoculated onto PDAC plates and cultured in an incubator. White colonies were picked and inoculated onto PDA slant plates and cultured. Spore suspension was prepared using sterile water as a protectant. S2.2 Preparation of Seed Liquid After diluting the spore suspension, a certain amount of the diluted spore suspension was inoculated into an Erlenmeyer flask containing seed culture medium and cultured on a shaker to obtain the seed solution. S2.3, solid-state fermentation In a clean bench, a certain amount of seed liquid is inoculated into sterilized rice substrate, thoroughly mixed and spread out, placed in an incubator for fermentation to produce red yeast rice, which is then dried, pulverized and sieved to obtain red yeast rice powder. S3, Monacoline K detection A certain amount of red yeast rice powder was placed in a centrifuge tube, ethanol was added, and the mixture was vortexed and shaken. After ultrasonic extraction and cooling, the supernatant was centrifuged and filtered through a microporous membrane. The content of monacolin K was detected by high performance liquid chromatography. S4. Detection of Citrulline A certain amount of red yeast rice powder was placed in a centrifuge tube, the extraction solution was added and vortexed to mix. After ultrasonic extraction, the supernatant was centrifuged and passed through a C18 solid phase extraction column. All eluent was collected and passed through an organic microporous membrane. The content of citrinin was detected by high performance liquid chromatography. S5. Selection of the optimal high-yield Monacoline K red yeast rice strain Based on the content of Monacolin K and Citrinin in each Monacolin K spore, the optimal Monacolin K-producing Monacolin K spore was selected.

2. The method for rapid screening of high-yield Monakaria K red Monascus according to claim 1, characterized in that: In step S1.1, the shaking temperature is 30℃, the shaking speed is 180 r / min, and the shaking time is 1h.

3. The method for rapid screening of high-yield Monakaria K red Monascus according to claim 1, characterized in that: In step S1.1, the temperature for coating culture is 30℃, and the coating culture time is 2 days.

4. The method for rapid screening of high-yield Monakaria K red mold according to claim 1, characterized in that: In step S1.2.2, the culture temperature is 30℃ and the culture time is 10 days.

5. The method for rapid screening of high-yield Monakaria K red Monascus according to claim 1, characterized in that: In step S1.2.2, the ultrasonic extraction time is 1 h; the centrifugation speed is 5000 r / min and the centrifugation time is 2 min; the concentration temperature is 30℃ and the concentration time is 15 min.

6. The method for rapid screening of high-yield Monakaria K red Monascus according to claim 1, characterized in that: In step S2.1, the temperature of the incubator is 30°C.

7. The method for rapid screening of high-yield Monakaria K red Monascus according to claim 1, characterized in that: In step S2.2, the concentration of the diluted spores is 106~107 CFU / mL; the inoculation amount of the spore suspension is 2% (v / v).

8. The method for rapid screening of high-yield Monakaria K red Monascus according to claim 1, characterized in that: In step S2.2, the temperature of the shaker culture is 30℃, the rotation speed of the shaker culture is 180 r / min, and the shaker culture time is 12 h.

9. The method for rapid screening of high-yield Monakaria K red mold according to claim 1, characterized in that: In step S2.3, the inoculation amount of seed liquid is 8% (v / g); the fermentation temperature is 30℃; and the drying temperature is 60℃.

10. The method for rapid screening of high-yield Monakaria K red Monascus according to claim 1, characterized in that: In step S2.3, the fermentation process involves turning the koji once after 72 hours of fermentation and continuing fermentation for another 14 days.

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