Method for screening monascus fermented corn strains
Through multiple identifications of Monascus and optimization of the fermentation process, a Monascus strain with high lovastatin production was screened out, solving the problems of mycotoxins and production costs, and achieving efficient and low-cost Monascus production.
Patent Information
- Application Number
- CN202510783203.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-12
- Publication Date
- 2025-09-12
AI Technical Summary
Existing red yeast rice strains have the problem of contaminating red yeast rice products with the fungal toxin citrinin, and the screening method of red yeast rice has failed to effectively screen out high-quality strains that produce high-yield red yeast pigments and lovastatin. In addition, red yeast rice production mainly uses rice as raw material, which is relatively expensive.
Morphological identification, ITS molecular biology identification and 18S molecular biology identification of Monascus were combined with solid-state and liquid fermentation processes to screen high-quality Monascus. By adjusting the fermentation medium composition and fermentation time, the Monascus fermentation corn strain was optimized and a Monascus strain with high lovastatin production was screened.
The high-yield lovastatin red yeast strain was efficiently screened out, which reduced production costs, improved the quality and benefits of red yeast products, and met the production needs of corn raw materials.
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Figure CN120624592A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of strain screening, and in particular relates to a method for screening red yeast rice fermentation strains. Background Art
[0002] Monascus spp., also known as Monascus purpureus, is the strain that produces red yeast rice, a traditional Chinese medicinal and edible fermentation product. Its ability to metabolize beneficial metabolites determines its application value, making the selection and breeding of high-quality Monascus particularly important. Monascus produces colorful monascus pigments (MPs), which have long been used as a food colorant. Monascus also produces the lipid-lowering ingredient monacolin K (MK, trade name lovastatin), leading to the development of high-MK content in red yeast rice as a lipid-lowering drug and health food. Monascus also produces abundant enzymes such as amylase, saccharifying enzyme, and esterifying enzyme, making it widely used in food brewing. To improve the quality of red yeast rice, isolation, identification, and selection of high-quality strains are essential. However, some strains can produce the mycotoxin citrinin, which can be used to dye red yeast rice products. Therefore, when selecting high-quality Monascus strains that produce high-quality monascus pigments, citrinin production should also be considered.
[0003] Corn is the main crop grown in Shanxi Province. Since 2004, the area and yield of corn have surpassed wheat to become the largest crop. Subsequently, the planting area and total yield have continued to increase. As of 2022, the corn planting area in Shanxi Province has reached 1.8139 million hectares. 2 , accounting for 50.2% of the total sown area of crops. Summary of the Invention
[0004] An object of the present invention is to provide a method for screening red yeast rice fermentation strains and to provide at least the advantages described below.
[0005] The present invention provides a method for screening red yeast rice fermentation strains for corn. The method comprises the following steps: collecting red yeast rice from the main red yeast rice producing areas in my country, isolating and purifying the red yeast rice, and identifying the red yeast rice using three methods: morphological identification, ITS molecular biological identification, and 18S molecular biological identification. Solid-state and liquid fermentation processes are explored for the isolated strains, and determinations of the pigment, citrinin, and lovastatin of the fermentation products are carried out, in order to screen out high-quality red yeast rice and provide bacterial germplasm resources for the production of corn red yeast rice.
[0006] The technical solutions of the present invention are as follows: The method for screening red yeast rice fermentation strains comprises the following steps: Step 1: Strain activation Monascus strains were selected and inoculated into potato agar medium. After culturing at 30°C for 5 days, lines were drawn on potato agar plates and cultured for 5 days. Step 2 Preparation of spore suspension Add spore wash solution to the activated strain from step 1, scrape off the spores and place them in a sterile Erlenmeyer flask with glass beads. Shake to disperse the spores, filter, and count the spores under an optical microscope using a hemocytometer. Adjust the spore count to 107 cells / mL to prepare a uniform spore suspension for later use. Step 3: Preparation of seed solution The spore suspension was inoculated into the seed culture medium at a 10% inoculum rate and cultured in a constant temperature shaker at 30°C and 200 rpm for 48 h to prepare the seed culture solution. Step 4: Fermentation culture screening The seed liquid was inoculated into the fermentation medium at a volume ratio of 5%, and cultured in a constant temperature shaker at 30°C and 200 rpm for 6 days. Samples were taken for determination of reducing sugar content, pH, color value, and dry cell weight. A solid-state fermentation matrix was prepared using corn as the raw material. After being mixed evenly with an auxiliary material solution at a mass ratio of xx:xx, the culture was inoculated at a 10% v / w inoculum and cultured in a constant temperature and humidity chamber at 30°C and 80% humidity for 3 days. On the fourth day, the temperature was lowered to 25°C, and the humidity was adjusted to 60% for continued culture. During the fermentation period, the shallow trays were removed every 48 hours, turned over in a clean bench, and supplemented with pH 5.0 acetic acid solution. Samples were taken from the sixth day of fermentation, dried at 60°C, and then the lovastatin content was determined. The strain with statin content above 5% was selected as the red yeast rice fermentation corn strain.
[0007] Preferably, in the method for screening red yeast rice fermentation corn strains, the auxiliary solution is formed by crushing corn to a size of 100 mesh and adding twice the mass of water.
[0008] Preferably, in the method for screening red yeast rice fermentation strains, the method for preparing the solid-state fermentation matrix comprises: Each stainless steel shallow dish was filled with 200 g of corn and soaked overnight in acetic acid solution with a pH of 4.5; Drain the soaked corn, add soybean powder and mix thoroughly, wrap with 8 layers of gauze and place in a sterilizer to sterilize at 15℃ for 20 minutes. After sterilization, cool in a clean bench and sterilize with ultraviolet light.
[0009] The present invention has the following beneficial effects: (1) Purchase monascus powder from the market, activate it, and make monascus liquid. Then, select and catalyze it with fungi to obtain a new mother liquor. Then, use liquid chromatography to detect the statin content. Select monascus from different manufacturers, and screen it multiple times with different groups until the mother liquor contains more than 5% statin. (2) By continuously adjusting the type of koji, its ratio, and fermentation time, the optimal bacteria are selected and the formula is solidified to form a high-yielding Monascus purpurogenous bacteria.
[0010] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 This is a flow chart of an embodiment of the method for screening red yeast rice fermentation strains provided by the present invention. DETAILED DESCRIPTION
[0012] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.
[0013] It should be understood that terms such as “having,” “including,” and “comprising” used herein do not prescribe the existence or addition of one or more other elements or combinations thereof.
[0014] Existing monascus koji extracts are made from rice or glutinous rice. This invention, however, optimizes and purifies existing monascus koji extracts through fungal extraction to create a monascus koji extract specifically for corn. This allows for the reuse of corn and aged grains, providing a more valuable application. Corn is much cheaper per unit than rice, and Shanxi, Henan, Shandong, and Hebei are the main producing areas.
[0015] 1. Materials and Methods 1. Materials 1. Red yeast rice: Red yeast rice from various production areas in China 2. Culture medium: Potato agar (PDA), Czapek agar (CYA), malt extract agar (MEA), malt wort agar (Wa) and other Monascus culture media; 25% glycerol nitrate agar (G25N) is based on Czapek agar with 25% mass fraction of glycerol added.
[0016] (2) Methods 1. Isolation and purification of Monascus Take red yeast rice samples and culture them in a restricted liquid culture medium at 30°C and 200 rpm for 24 hours. Then spread them on PDA culture medium for separation and select red yeast rice with different morphologies for strain preservation. Monascus is purchased from different manufacturers on the market and purified by fungal catalysis to become a high-yield statin-like Monascus. Note: Restricted liquid culture medium 15g / L rice flour, 5g / L glucose, 3.98% volume fraction of lactic acid, 6.24% volume fraction of ethanol and 0.1g / L chloramphenicol.
[0017] 2. Morphological identification of Monascus The isolated and purified Monascus were spotted in PDA, CYA, MEA, G25N and Wa respectively and cultured at 25°C for 7 days.
[0018] 3. Molecular biological identification of Monascus DNA from Monascus was extracted using a fungal DNA extraction kit. PCR amplification and agarose gel electrophoresis were performed to test the amplification effect. The amplified PCR product was then sent to a sequencing company for sequencing. The sequencing results were analyzed using nucleotide sequence blast analysis, and a phylogenetic tree was constructed.
[0019] 4. Determination of Monascus metabolites (1) Determination of pigments and citrinin Purified Monascus spores from PDA plates were transferred to liquid fermentation medium using physiological saline. The inoculum size was 10% by volume, and the spore suspension concentration was 1 × 10⁶ CFU / mL. The liquid fermentation medium contained 40 g / L rice flour and 4 g / L glycerol. Fermentation was performed at 30°C, 200 rpm, and 7 days. After fermentation, 0.5 mL of the fermentation broth was added to 5 mL of 75% by volume ethanol at 60°C for 120 minutes. After extraction, the supernatant was centrifuged at 4500 rpm for 15 minutes. The supernatant was filtered through a 0.45 μm filter and used as the assay for pigment and citrinin. The determination method of pigments is carried out according to the following method: Alcohol-soluble color value: Add bacteria to 70% ethanol solution, stir evenly, sonicate for 20 minutes, extract at 60°C for 2 hours, and centrifuge at 4500 rpm for 5 minutes. The resulting supernatant is appropriately diluted with 70% ethanol solution, and the absorbance at 410, 465, and 510 nm is measured using a spectrophotometer. Yellow color value = OD410nm × dilution factor, orange color value = OD465nm × dilution factor, and red color value = OD510nm × dilution factor. Total alcohol-soluble color value = alcohol-soluble yellow color value + alcohol-soluble orange color value + alcohol-soluble red color value. Determination method of citrinin: refer to the first method of the national standard "GB 5009.222-2016 Determination of citrinin in food" for the HPLC determination of citrinin in red yeast rice products.
[0020] (2) Determination of lovastatin content A. Liquid chromatography conditions Chromatographic column: C18 column, 5μm, 250 mm × 4.6 mm UV detection wavelength: 238 nm Mobile phase: V (acetonitrile): V (0.01% phosphoric acid solution) = 60:40 Injection volume: 20 μL Flow rate: 1 mL / min Column temperature: 30°C B. Lovastatin standard curve Accurately weigh 20 mg of lactone lovastatin standard, dissolve it in acetonitrile, and dilute to 10 mL. Then further dilute to 0.01, 0.02, 0.04, 0.10, 0.25, and 0.40 mg / mL. Draw a lactone lovastatin standard curve using the lactone lovastatin concentration as the horizontal axis and the corresponding HPLC peak area as the vertical axis. Take 1 mL of 2 mg / mL lactone lovastatin standard solution in a centrifuge tube, add 1 mL of 0.1 mol / L sodium hydroxide solution and mix well. Then, ultrasonicate in a water bath at 50°C for 1 h. After the reaction, cool to room temperature and dilute to 10 mL with acetonitrile. Based on the relative molecular masses of lactone lovastatin and acid lovastatin, which are 404.55 and 422.54, respectively, the concentration of acid lovastatin at this point can be calculated. A standard curve for acid lovastatin can be drawn with the acid lovastatin concentration as the horizontal axis and the corresponding HPLC peak area as the vertical axis. C. Determination of Lovastatin in Samples The optimal extraction method for red yeast rice powder was to use 55% ethanol as the solvent, extract at a liquid-to-solid ratio of 40 mL / g, a temperature of 60°C, and an ultrasonic power of 300 W for 40 min. Lovastatin was also determined in advance. Place 1 g of sample in a centrifuge tube (denoted as M) and extract according to the above method. After extraction, quickly return the sample to room temperature and dilute to the initial volume. After centrifugation, filter the supernatant through a 0.45 μm microporous membrane and analyze it with HPLC. Perform three replicates per group. Calculate the acid-type lovastatin content (C1) and lactone-type lovastatin content (C2) in the sample using the standard curve. The sum of these contents is recorded as the total lovastatin content (C3). The extraction rates of lovastatin and acid-type lovastatin can be calculated according to the following formulas:
[0021] 5. Optimization of culture medium composition for Monascus pigment production, such as Figure 1 shown.
[0022] (1) Strain activation Inoculate into PDA slant medium, culture at 30℃ for 5 days, draw lines on PDA plates, and expand culture for 5 days.
[0023] (2) Preparation of spore suspension After culturing Monascus on plates for 5 days, add spore wash solution, gently scrape off the spores, transfer to a sterile Erlenmeyer flask with glass beads, shake to break up the spores, filter, and count the spores under an optical microscope using a hemocytometer. Adjust the spore content to 107 / mL to prepare a uniform spore suspension for later use.
[0024] (3) Preparation of seed solution The spore suspension was inoculated into the seed culture medium at a 10% inoculum rate and cultured in a constant temperature shaker at 30°C and 200 rpm for 48 h to prepare the seed culture solution.
[0025] (4) Fermentation culture The seed liquid was inoculated into the fermentation medium at a volume ratio of 5%, and cultured in a constant temperature shaker at 30°C and 200 r / min for 6 days. Samples were taken to measure various indicators.
[0026] (5) Optimization of fermentation medium composition A. Single-factor experimental design Different addition amounts of indica rice flour and corn starch were calculated with the same starch content, and these two raw materials were used to replace rice flour in the initial fermentation medium for fermentation to explore the effects of corn starch and indica rice flour on the color production ability of the tested Monascus. Fructose, glucose, sucrose, soluble starch, glycerol, maltose, or indica rice flour or corn flour were used as carbon sources to replace glucose in the basic fermentation medium; tryptone, sodium glutamate, soybean powder, yeast extract, corn steep liquor, or urea were used as nitrogen sources to replace sodium glutamate in the basic fermentation medium; sodium nitrate, magnesium sulfate heptahydrate, copper sulfate pentahydrate, zinc sulfate heptahydrate, or ferrous sulfate heptahydrate were used as metal ions to replace magnesium sulfate heptahydrate in the basic fermentation medium to explore the effects of different carbon sources, nitrogen sources, and metal ions on the color production ability of this Monascus strain; After determining the types of carbon source, nitrogen source and metal ions, conduct a single-factor test of concentration.
[0027] B. Response surface design to optimize culture medium composition Based on the results of a single-factor experiment, a Box-Behnken design was conducted using Design Expert 8.0 software to determine the optimal culture medium concentration for maltose concentration (A), monosodium glutamate concentration (B), and ferrous sulfate heptahydrate concentration (C). 1, 0, and -1 represented high, medium, and low levels of the independent variable, respectively. The total color value of alcohol-soluble soluble matter was used as the indicator. Ten milliliters of fermentation broth was centrifuged at 4500 rpm for 10 minutes to obtain the fermentation supernatant and bacterial cells for measuring various parameters. Determination of reducing sugar content in fermentation broth: The fermentation supernatant was taken to determine the reducing sugar content of the fermentation broth using the DNS colorimetric method; Determination of pH of fermentation broth: Take the fermentation supernatant to determine the pH value of the fermentation broth using a pH meter; Determination of color value of fermentation broth: alcohol-soluble color value (same as above); Determination of dry cell weight: Wash the centrifuged cells several times with distilled water, dry them in a 60°C oven until constant weight is reached, and weigh them using an analytical balance. Cell mass (g / L) = dry cell weight / fermentation broth volume.
[0028] 6. Solid-state fermentation (1) Soak corn: Place 200 g of corn in each stainless steel shallow tray and soak overnight in acetic acid solution with pH 4.5; (2) Preparation of solid-state fermentation matrix: After draining the soaked corn, add soybean powder and mix thoroughly. Wrap it with 8 layers of gauze and place it in a sterilizer at 115℃ for 20 minutes. After sterilization, cool it in a clean bench and sterilize it with ultraviolet light. The auxiliary material solution is also sterilized in a sterilizer at 115℃ for 20 minutes and then allowed to cool. (3) Inoculation: Stir the cooled auxiliary material solution and solid fermentation matrix evenly, then inoculate the cultured seed solution at a 10% (v / w) inoculation rate (i.e., 10 mL of seed solution per 100 g of dry corn) and stir evenly; (4) Solid-state fermentation: The inoculated shallow tray was placed in a constant temperature and humidity chamber at 30°C and 80% humidity for 3 days. On the 4th day, the temperature was changed to 25°C and the humidity was adjusted to 60% for further culturing. During the fermentation period, the shallow tray was removed every 48 hours, turned over in a clean bench, and acetic acid solution with pH 5.0 was added. Samples were taken from the 6th day of fermentation and dried at 60°C for determination of lovastatin content.
[0029] Corn is crushed to a size of 100 mesh, and twice the weight of water is added to form a solution. The solution is then heated to 35°C, and the selected strain of bacteria is added. Ferment at this constant temperature for 15-20 days. The statin content of the solution is measured. The optimal strain is selected by adjusting the koji type, ratio, and fermentation time. The formula is then solidified to produce a high-statin-producing Monascus strain.
[0030] Statin testing follows the national standard method, and the fermentation method is the same for both solid and liquid methods. The difference lies in the red yeast rice strain. Current red yeast rice extracts are produced from rice, but corn has a low yield. Our invention involves collecting and cultivating red yeast rice extracts from various origins. Through fungal stimulation, we optimize and purify them, then re-stimulate, optimize, and purify them, transforming them to better adapt to corn starch. Each time we optimize and purify the mother liquor, we test it using the national standard method. Through countless repeated comparative tests, we find the red yeast rice that is most compatible with corn. We then dry-powder this extract for easier storage and transportation.
[0031] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.
Claims
1. A method for screening red yeast rice fermentation strains, characterized in that: The following steps are involved: Step 1: Strain activation: Select Monascus strains and inoculate them into potato agar medium. After culturing at 30°C for 5 days, draw lines on potato agar plates and expand the culture for 5 days. Step 2: Preparation of spore suspension: Add spore washing solution to the activated strain in step 1, scrape the spores into a sterile triangular flask with glass beads, shake to break up the spores, filter, count the spores under an optical microscope using a hemocytometer, and adjust the spore content to 10 7 / mL, and make a uniform spore suspension for later use; Step 3: Preparation of seed solution: Inoculate 10% of the spore suspension into the seed culture medium and incubate in a constant temperature shaker at 30°C and 200 rpm for 48 h to prepare the seed culture solution. Step 4: Fermentation culture screening The seed liquid was inoculated into the fermentation medium at a volume ratio of 5%, and cultured in a constant temperature shaker at 30°C and 200 rpm for 6 days. Samples were taken for determination of reducing sugar content, pH, color value, and dry cell weight. A solid-state fermentation substrate was prepared from corn. After being mixed evenly with an auxiliary material solution at a mass ratio of 10:1, the substrate was inoculated at a 10% v / w inoculum and cultured in a constant temperature and humidity chamber at 30°C and 80% humidity for 3 days. On the fourth day, the temperature was lowered to 25°C, and the humidity was adjusted to 60% for continued incubation. During fermentation, the trays were removed every 48 hours, turned over in a clean bench, and supplemented with pH 5.0 acetic acid solution. Samples were collected from the sixth day of fermentation and dried at 60°C before determination of lovastatin content. The strain with statin content above 5% was selected as the red yeast rice fermentation corn strain.
2. The method for screening red yeast rice fermented corn strains as claimed in claim 1, wherein: The auxiliary solvent is formed by crushing corn to 100 mesh and adding twice the mass of water.
3. The method for screening red yeast rice fermented corn strains as claimed in claim 2, wherein: The preparation method of the solid-state fermentation matrix comprises: Each stainless steel shallow dish was filled with 200 g of corn and soaked overnight in acetic acid solution with a pH of 4.5; Drain the soaked corn, add soybean powder and mix thoroughly, wrap with 8 layers of gauze and place in a sterilizer to sterilize at 15℃ for 20 minutes. After sterilization, cool in a clean bench and sterilize with ultraviolet light.