Production method for improving ring opening rate of monascus milovastatin

Abstract

The invention discloses a production method for improving the ring opening rate of monascus milovastatin, and belongs to the technical field of monascus rice production. According to the method, a monascus purpureus high-yield strain is selected and inoculated into a solid indica rice matrix containing a specific additive for fermentation after spore activation, and the matrix comprises 1, 5-p-coumaroylquinic acid, sodium acetate, sodium citrate, a thermally modified soybean protein isolate polysaccharide sulfate compound and other components. Wherein sodium acetate and sodium citrate can serve as short-chain fatty acid precursors, the activity of closed-loop lactonase is inhibited through pH regulation, and generation of lactone type products is reduced; 1, 5-p-coumaroylquinic acid activates the expression of a polyketide synthase gene cluster, so that the synthesis flux of the Monacolin K with an open-loop structure is improved; nicotinamide ribose enhances the electron transfer efficiency of glycolysis and TCA circulation, provides sufficient ATP for polyketone synthesis, and promotes synthesis of Monacolin K. The compound further strengthens the synthesis flux of secondary metabolites by changing the form of hyphae and continuously releasing a nitrogen / carbon source, and promotes intracellular products to be transferred to extracellular.

Description

Technical Field

[0001] The invention belongs to the technical field of red yeast rice production, and in particular relates to a production method for improving the ring-opening rate of lovastatin in red yeast rice. Background Art

[0002] Red yeast rice is a functional product made by fermenting indica rice with Monascus spp. Its core active ingredient is lovastatin, which exists in two isomers: the open-ring acid form (Monacolin K) and the closed-ring lactone form. Studies have shown that the open-ring Monacolin K directly inhibits HMG-CoA reductase activity, competitively blocking the rate-limiting step in cholesterol biosynthesis. Its lipid-lowering efficiency is more than double that of the closed-ring form, while also significantly reducing hepatotoxicity and renal toxicity. However, in existing production processes, the closed-ring form generally accounts for over 60%-70% of lovastatin, limiting the product's efficacy. Furthermore, red yeast rice also produces citrinin, a secondary metabolite that has been shown to be nephrotoxic, causing kidney enlargement, tubular dilation, and epithelial cell necrosis, as well as teratogenicity and carcinogenesis. Furthermore, lovastatin and citrinin share the same acetyl-CoA precursor synthesis pathway, further hindering the targeted production of high-purity Monacolin K. Currently, the low ring-opening rate of lovastatin in the industrial production of red yeast rice results in low yields and high costs for Monacolin K. To achieve high yields of Monacolin K, optimization of fermentation conditions and fermentation substrates is necessary. Summary of the Invention

[0003] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a method for producing lovastatin with a high ring-opening rate from red yeast rice based on metabolic pathway regulation and mycelium morphology optimization. The purpose of increasing the ring-opening rate of lovastatin from red yeast rice is achieved by adding a composite precursor and regulating the fermentation environment.

[0004] In order to achieve the above object, the following technical solution is adopted: The present invention provides a production method for improving the ring-opening rate of red yeast rice milovastatin, comprising the following steps:

[0005] S1. Spore culture: A high-yield strain of Monascus purpureus was selected and cultured on a maltose agar slant medium for 7 days to obtain an activated spore suspension with a strain concentration of 5.0 × 10^6 CFU / g.

[0006] S2. Seed culture: Inoculate the activated spores into liquid culture medium at an inoculum size of 5%-15%, and culture at 32°C and 150-200 rpm for 48 hours to obtain seed solution;

[0007] S3, solid fermentation: the seed liquid is inoculated into a cooked indica rice substrate with a moisture content of 52-55% at an inoculum rate of 15-30%, the pH is adjusted to 3.5-5.0 using an organic acid, and the solid fermentation is carried out for 75 hours;

[0008] S4. The red yeast rice after the culture is completed is taken out and dried at 40-60° C. for 12-24 hours to obtain a red yeast rice product.

[0009] Furthermore, the solid fermentation indica rice matrix further includes components in the following concentrations: 3-5% glucose, 1-2% peptone, 0.5-1% yeast extract, 1-3% heat-modified soy protein isolate polysaccharide sulfate complex, 0.5-1% ammonium sulfate, 0.05-0.2% 1,5-p-coumarylquinic acid, 0.2-0.5% sodium acetate, 0.1-0.3% sodium citrate, 0.01-0.03% nicotinamide riboside, 0.05-0.1% ascorbic acid, 0.001-0.005% biotin, 0.001-0.01% hydrated magnesium sulfate, 0.001-0.01% hydrated zinc sulfate, and 0.005-0.01% manganese sulfate.

[0010] Among them, 1,5-p-coumarylquinic acid can scavenge free radicals produced during the fermentation process, protect the integrity of Monascus cell membrane, maintain the stability of the secondary metabolic enzyme system, activate the expression of the PKS gene cluster in Monascus, promote the extension of the polyketide chain, and increase the synthesis flux of Monacolin K.

[0011] Sodium acetate and sodium citrate can serve as short-chain fatty acid precursors, catalyzed by acetyl-CoA synthetase within the bacteria to produce acetyl-CoA, which serves as a building block for the polyketide skeleton of lovastatin. Short-chain fatty acids such as acetic acid, as precursors, are inducers of acetyl-CoA and propionyl-CoA synthetases. However, if added directly, they are easily volatile and have a certain toxicity to bacterial growth. Therefore, the present invention adds sodium acetate and sodium citrate to regulate the direction of biosynthesis and accumulation of secondary metabolites of Monascus, promoting the synthesis of Monacolin K. Furthermore, as weak acid salts, these two salts form a buffer system in the early stages of fermentation, inhibiting the activity of closed-ring lactonization enzymes, thereby reducing the production of lactone-type lovastatin.

[0012] Nicotinamide riboside is a precursor substrate of nicotinamide adenine dinucleotide (NAD+). It replenishes the intracellular NAD+ pool through the nicotinamide phosphoribosyltransferase (NAMPT) pathway, enhances the electron transfer efficiency of glycolysis and TCA cycle, provides sufficient ATP for polyketide synthesis, and promotes the synthesis of Monacolin K by utilizing acetyl-CoA produced by glycolysis.

[0013] Metal ions can serve as active ingredients of certain enzymes or as cofactors and activators of certain enzymes, and play an important role in the fermentation process. Therefore, the present invention also adds hydrated magnesium sulfate, hydrated zinc sulfate and manganese sulfate to the solid fermentation substrate.

[0014] Furthermore, the heat-modified soy protein isolate polysaccharide sulfate complex is prepared by the following steps:

[0015] The soy protein isolate was dissolved in deionized water and gently stirred at 500 r / min for 2 hours to prepare a 20 mg / mL suspension, which was heated at 95°C for 20 minutes. The suspension was centrifuged at 3000 r / min and 4°C for 10 minutes, the supernatant was collected, and the supernatant was freeze-dried to obtain heat-modified soy protein isolate, which was then configured into a heat-modified soy protein isolate solution; a dextran sulfate solution was prepared and stirred at 500 r / min for 2 hours. The dextran sulfate solution was heated at 95°C for 10 minutes and then cooled to room temperature. The dextran sulfate solution was mixed with the heat-modified soy protein isolate solution, the pH value was adjusted to 5.5 with 0.1 mol / L HCl solution, and the heat-modified soy protein isolate polysaccharide sulfate complex was obtained after freeze-drying.

[0016] The heat-modified soy protein isolate polysaccharide sulfate complex is a composite of heat-modified soy protein isolate and glucan sulfate with sulfate ester bonds. This provides additional nitrogen and carbon sources, and in a low pH environment, the hydrophobic groups of the complex (hydrophobic amino acid residues of soy protein) are exposed, allowing for stronger interfacial adsorption with the hydrophobic surface of Monascus mycelium through hydrophobic interactions. This facilitates stable attachment of the mycelium to the solid substrate, improves the mycelium's nutrient absorption efficiency, and accelerates the synthesis of secondary metabolites. The complex also forms micelles or aggregates at low pH, slowly degrading and continuously releasing monosaccharides produced by the decomposition of soy protein hydrolysates and glucan sulfate. This avoids the metabolic imbalance caused by the rapid consumption of traditional nitrogen sources (such as peptone) and maintains the stable synthesis of secondary metabolites in the middle and late stages of fermentation. The sulfur-containing groups released from the complex can also participate in the sulfur metabolism pathway, promoting the regeneration of coenzyme A, thereby enhancing the conversion of acetyl-CoA to monacolin K synthesis. In addition, the addition of heat-modified soy protein isolate polysaccharide sulfate complex also changed the mycelial morphology, increasing the degree of mycelial wrinkling, increasing the specific surface area, and strengthening the permeability of the cell membrane, thereby transferring lovastatin from the cell to the extracellular space and increasing lovastatin production.

[0017] Furthermore, the slant culture medium comprises the following components in the following concentrations: 8-12% maltose juice, 1.5-2.5% agar, 0.05-0.1% potassium dihydrogen phosphate, 0.01-0.03% hydrated magnesium sulfate, and 0.001-0.005% vitamin B1.

[0018] Furthermore, the seed culture medium includes components with the following concentrations: 5-8% glucose, 0.5-1.5% peptone, 0.05-0.5% sodium nitrate, 0.2-0.6% yeast extract, 0.1-0.2% potassium dihydrogen phosphate, and 0.005-0.01% hydrated magnesium sulfate. Furthermore, the concentration of the heat-modified soy protein isolate solution is 10-30 mg / mL.

[0019] Furthermore, the concentration of the dextran sulfate solution is 5-15 mg / mL, and the degree of sulfation is 0.8-1.2.

[0020] Furthermore, the mass ratio of the hot dextran sulfate solution to the heat-modified soy protein isolate solution is 1:3-6.

[0021] Furthermore, the solid fermentation comprises the following steps:

[0022] 0-24h: Temperature 30-32℃, RH 85%, turn the compost twice;

[0023] 24-72h: Raise the temperature to 38-42℃;

[0024] After 72h: cool to 30-32℃, RH70%.

[0025] Furthermore, the dissolved oxygen in the solid fermentation process is maintained at 3-5 mg / L.

[0026] Furthermore, the organic acid is one of citric acid, malic acid and lactic acid.

[0027] The beneficial effects of the present invention are:

[0028] (1) The present invention adds 1,5-p-coumarylquinic acid to scavenge free radicals and activate the expression of the polyketide synthase gene cluster, combines a weak acid buffer system of sodium acetate and sodium citrate to inhibit the activity of closed-ring lactonase, blocks the formation of closed-ring lovastatin from the metabolic pathway, and significantly increases the proportion of open-ring structure. The introduction of heat-modified soybean protein isolate polysaccharide sulfate complex further enhances the synthesis flux of secondary metabolites by changing the mycelial morphology and continuously releasing nitrogen / carbon sources, while promoting the transfer of intracellular products to the extracellular space;

[0029] (2) By optimizing the culture medium composition and fermentation conditions, the distribution pathway of acetyl-CoA is balanced, the accumulation of precursor substances required for citrinin synthesis is reduced, and the sulfur-containing groups released from the complex participate in sulfur metabolism, promote the regeneration of Coenzyme A, and further guide the metabolic flow toward the synthesis of Monacolin K, thereby reducing the risk of citrinin contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1These are morphological analysis images of Monascus fermentation mycelium in Example 3 of the present invention and Comparative Examples 1-3.

[0031] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and the experimental materials used in the following examples, unless otherwise specified, are all purchased from commercial channels.

[0035] The strain number of the Monascus purpureus is CGMCC3.15544, which was purchased from the China General Microbiological Culture Collection Center.

[0036] Example 1. A method for increasing the ring-opening rate of red yeast rice milovastatin, comprising the following steps:

[0037] S1. Spore culture: A high-yield strain of Monascus purpureus was selected and cultured on a maltose agar slant medium for 7 days to obtain an activated spore suspension with a strain concentration of 5.0 × 10^6 CFU / g.

[0038] S2. Seed culture: Inoculate the activated spores into liquid culture medium at a 5% inoculum size and culture at 32°C and 150 rpm for 48 h to obtain seed solution.

[0039] S3, solid fermentation: the seed liquid was inoculated into a cooked indica rice substrate with a moisture content of 52% at an inoculum rate of 15%, the pH was adjusted to 3.5 with citric acid, and the solid fermentation was carried out for 75 hours;

[0040] S4. The red yeast rice after the culture is completed is taken out and dried at 40° C. for 12 h to obtain a red yeast rice product.

[0041] The solid fermentation indica rice matrix further includes components having the following concentrations: 3% glucose, 1% peptone, 0.5% yeast extract, 1% heat-modified soy protein isolate polysaccharide sulfate complex, 0.5% ammonium sulfate, 0.05% 1,5-p-coumarylquinic acid, 0.2% sodium acetate, 0.1% sodium citrate, 0.01% nicotinamide riboside, 0.05% ascorbic acid, 0.001% biotin, 0.001% hydrated magnesium sulfate, 0.001% hydrated zinc sulfate, and 0.005% manganese sulfate.

[0042] The heat-modified soy protein isolate polysaccharide sulfate complex is prepared by the following steps:

[0043] The soy protein isolate was dissolved in deionized water and gently stirred at 500 r / min for 2 hours to prepare a 20 mg / mL suspension, which was heated at 95°C for 20 minutes. The suspension was centrifuged at 3000 r / min and 4°C for 10 minutes, the supernatant was collected, and the supernatant was freeze-dried to obtain heat-modified soy protein isolate, which was then prepared into a 10 mg / mL heat-modified soy protein isolate solution; a 5 mg / mL dextran sulfate solution (sulfation degree 0.8) was prepared and stirred at 500 r / min for 2 hours. The dextran sulfate solution was heated at 95°C for 10 minutes, cooled to room temperature, and mixed with the heat-modified soy protein isolate solution in a mass ratio of 1:3. The pH value was adjusted to 5.5 with 0.1 mol / L HCl solution, and the complex was obtained after freeze-drying.

[0044] The slant culture medium comprises the following components in the following concentrations: 8% maltose extract, 1.5% agar, 0.05% potassium dihydrogen phosphate, 0.01% hydrated magnesium sulfate, and 0.001% vitamin B1.

[0045] The seed culture medium includes the following components in the following concentrations: 5% glucose, 0.5% peptone, 0.05% sodium nitrate, 0.2% yeast extract, 0.1% potassium dihydrogen phosphate, and 0.005% hydrated magnesium sulfate.

[0046] The solid fermentation comprises the following steps:

[0047] 0-24h: Temperature 30℃, RH 85%, turn the compost twice;

[0048] 24-72h: Raise the temperature to 38°C;

[0049] After 72h: cool to 30℃, RH 70%.

[0050] The dissolved oxygen in the solid fermentation process was maintained at 3 mg / L.

[0051] Example 2. A method for increasing the ring-opening rate of red yeast rice milovastatin, comprising the following steps:

[0052] S1. Spore culture: A high-yield strain of Monascus purpureus was selected and cultured on a maltose agar slant medium for 7 days to obtain an activated spore suspension with a strain concentration of 5.0 × 10^6 CFU / g.

[0053] S2. Seed culture: Inoculate the activated spores into liquid culture medium at an inoculum size of 15%, and culture at 32°C and 200 rpm for 48 h to obtain seed solution;

[0054] S3, solid fermentation: the seed liquid was inoculated into a cooked indica rice substrate with a moisture content of 55% at an inoculum rate of 30%, malic acid was used to adjust the pH to 5.0, and solid fermentation was carried out for 75 hours;

[0055] S4. The red yeast rice after the culture is completed is taken out and dried at 60° C. for 24 h to obtain a red yeast rice product.

[0056] The solid fermentation indica rice matrix further includes components having the following concentrations: 5% glucose, 2% peptone, 1% yeast extract, 3% heat-modified soy protein isolate polysaccharide sulfate complex, 1% ammonium sulfate, 0.2% 1,5-p-coumarylquinic acid, 0.5% sodium acetate, 0.3% sodium citrate, 0.03% nicotinamide riboside, 0.1% ascorbic acid, 0.005% biotin, 0.01% hydrated magnesium sulfate, 0.01% hydrated zinc sulfate, and 0.01% manganese sulfate.

[0057] The heat-modified soy protein isolate polysaccharide sulfate complex is prepared by the following steps:

[0058] The soy protein isolate was dissolved in deionized water and gently stirred at 500 r / min for 2 hours to prepare a 20 mg / mL suspension, which was heated at 95°C for 20 minutes. The suspension was centrifuged at 3000 r / min and 4°C for 10 minutes, the supernatant was collected, and the supernatant was freeze-dried to obtain heat-modified soy protein isolate, which was then prepared into a 30 mg / mL heat-modified soy protein isolate solution; a 15 mg / mL dextran sulfate solution (sulfation degree 1.2) was prepared and stirred at 500 r / min for 2 hours. The dextran sulfate solution was heated at 95°C for 10 minutes, cooled to room temperature, and mixed with the heat-modified soy protein isolate solution in a mass ratio of 1:6. The pH value was adjusted to 5.5 with 0.1 mol / L HCl solution, and the complex was freeze-dried to obtain.

[0059] The slant culture medium comprises the following components in the following concentrations: 12% maltose extract, 2.5% agar, 0.1% potassium dihydrogen phosphate, 0.03% hydrated magnesium sulfate, and 0.005% vitamin B1.

[0060] The seed culture medium includes the following components in the following concentrations: 8% glucose, 1.5% peptone, 0.5% sodium nitrate, 0.6% yeast extract, 0.2% potassium dihydrogen phosphate, and 0.01% hydrated magnesium sulfate.

[0061] The solid fermentation comprises the following steps:

[0062] 0-24h: Temperature 32℃, RH 85%, turn the compost twice;

[0063] 24-72h: Raise the temperature to 42°C;

[0064] After 72 hours: cool to 32°C, RH 70%.

[0065] The dissolved oxygen in the solid fermentation process was maintained at 5 mg / L.

[0066] Example 3. A method for increasing the ring-opening rate of red yeast rice milovastatin, comprising the following steps:

[0067] S1. Spore culture: A high-yield strain of Monascus purpureus was selected and cultured on a maltose agar slant medium for 7 days to obtain an activated spore suspension with a strain concentration of 5.0 × 10^6 CFU / g.

[0068] S2. Seed culture: Inoculate the activated spores into liquid culture medium at a 10% inoculum size and culture at 32°C and 175 rpm for 48 h to obtain seed solution.

[0069] S3, solid fermentation: the seed liquid was inoculated into a cooked indica rice substrate with a moisture content of 53% at an inoculum size of 22%, the pH was adjusted to 4.2 using lactic acid, and the solid fermentation was carried out for 75 hours;

[0070] S4. The red yeast rice after the culture is completed is taken out and dried at 50° C. for 18 h to obtain a red yeast rice product.

[0071] The solid fermentation indica rice matrix also includes the following components: glucose 4%, peptone

[0072] 1.5%, yeast extract 0.8%, heat-modified soy protein isolate polysaccharide sulfate complex

[0073] 2%, ammonium sulfate 0.8%, 1,5-p-coumarylquinic acid 0.12%, sodium acetate

[0074] 0.35%, sodium citrate 0.2%, nicotinamide riboside 0.02%, ascorbic acid

[0075] 0.08%, Biotin 0.003%, Hydrated Magnesium Sulfate 0.005%, Hydrated Zinc Sulfate

[0076] 0.005%, manganese sulfate 0.007%.

[0077] The heat-modified soy protein isolate polysaccharide sulfate complex is prepared by the following steps:

[0078] Soy protein isolate was dissolved in deionized water and stirred gently at 500 r / min for 2 h to prepare

[0079] The 20 mg / mL suspension was heated at 95°C for 20 min, the suspension was centrifuged at 3000 r / min and 4°C for 10 min, the supernatant was collected, and the supernatant was lyophilized to obtain heat-modified soy protein isolate, which was then prepared into a 20 mg / mL heat-modified soy protein isolate solution; a 10 mg / mL dextran sulfate solution (sulfation degree

[0080] 1.0) and stirred at 500 r / min for 2 h. The dextran sulfate solution was heated at 95° C. for 10 min, cooled to room temperature, and mixed with the heat-modified soy protein isolate solution at a mass ratio of 1:4.5. The pH value was adjusted to 5.5 with 0.1 mol / L HCl solution, and the complex was obtained after lyophilization.

[0081] The slant culture medium comprises the following components in the following concentrations: 10% maltose extract, 2.0% agar, 0.08% potassium dihydrogen phosphate, 0.02% hydrated magnesium sulfate, and 0.003% vitamin B1.

[0082] The seed culture medium includes components with the following concentrations: 6% glucose, 1.0% peptone, 0.3% sodium nitrate, 0.4% yeast extract, 0.15% potassium dihydrogen phosphate, and 0.007% hydrated magnesium sulfate.

[0083] The solid fermentation comprises the following steps:

[0084] 0-24h: Temperature 31℃, RH 85%, turn the compost twice;

[0085] 24-72h: Raise the temperature to 40℃;

[0086] After 72h: cool to 31°C, RH 70%.

[0087] The dissolved oxygen in the solid fermentation process was maintained at 4 mg / L.

[0088] Comparative Example 1

[0089] Compared with Example 3, 1,5-p-coumaroylquinic acid was removed from the substrate of the solid fermentation, and other components and process parameters remained unchanged.

[0090] Comparative Example 2

[0091] Compared with Example 3, the heat-modified soy protein isolate polysaccharide sulfate complex was replaced with an equal amount of ordinary soy protein isolate, and the other components and process parameters remained unchanged.

[0092] Comparative Example 3

[0093] Compared with Example 3, the substrate for solid fermentation lacks nicotinamide riboside, and other components and process parameters remain unchanged.

[0094] Result Analysis

[0095] Test Example 1. Analysis of Monacolin K Yield, Ring Opening Rate, and Citrinin Content

[0096] The monacolin K yield, ring-opening rate, and citrinin content in each group of red yeast rice prepared in each example and comparative example were determined by HPLC. Each group was tested 10 times. The results are shown in Table 1. HPLC conditions were as follows: a C18 reverse-phase column (ZORBAX SBC18), dimensions 150-250 mm × 4.6 mm × 5 μm, a mobile phase consisting of acetonitrile and 0.1% aqueous phosphoric acid (65:35 volume ratio), a flow rate of 1.0 mL / min, and a column temperature of 25°C; injection volume: 20 μL, detection wavelength: 238 nm. Red yeast rice samples were ultrasonically extracted with 75% methanol or ethanol (30-50 min), centrifuged, and filtered through a 0.45 μm filter. Qualitative analysis was performed using the elution time of the monacolin K and citrinin standards, and quantitative analysis was performed using the external standard peak area method.

[0097] Table 1. Analysis results of Monacolin K and ring-opening rate

[0098]

[0099]

[0100] As can be seen from Table 1, the total yield of Monacolin K in Examples 1-3 is significantly higher than that in the comparative example, and the total yield in Example 3 is the highest, which is 54.4%, 50.0% and 69.4% higher than that in Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively. This shows that the synergistic effect of the composite precursor (1,5-p-coumarylquinic acid, nicotinamide riboside) and the thermally modified soy protein isolate polysaccharide sulfate complex significantly enhances the flux of the polyketide synthesis pathway. The ring-opening rate of lovastatin in Example 3 reached 85.6%, while the ring-opening rates of Comparative Examples 1-3 were only 62.1%, 65.4% and 60.7%, respectively. This confirms that 1,5-p-coumarylquinic acid blocks the production of closed-ring lovastatin by activating the expression of the PKS gene cluster, sodium acetate / sodium citrate inhibits the activity of closed-ring lactonase, and the complex optimizes the mycelial metabolic environment. The citrinin content of Examples 1-3 is all less than 0.5 μg / g, which is much lower than that of Comparative Examples 1-3. This is because the metabolic pathway regulation causes acetyl-CoA to preferentially flow to Monacolin K synthesis, reducing the competition for citrinin precursors. At the same time, the sulfur-containing groups in the complex promote the regeneration of Coenzyme A, further guiding the metabolic flow toward the target product.

[0101] Test Example 2. Morphological Analysis of Monascus Fermentation Mycelium

[0102] The morphological differences of the mycelia at the end of solid fermentation between Example 3 and Comparative Examples 1-3 were detected by scanning electron microscopy. Figure 1 .

[0103] from Figure 1 It can be seen that the mycelial surface in Example 3 is uneven, the specific surface area is significantly increased, and the cell membrane permeability is enhanced. This morphological change is conducive to the adsorption of nutrients in the solid matrix by the bacteria through hydrophobic interaction, and accelerates the transfer of intracellular metabolites to the extracellular space, reducing product feedback inhibition. However, the mycelial surface of each example is smooth, and the absorption of nutrients is limited, which easily leads to insufficient product accumulation and reduces the yield of lovastatin.

[0104] Test Example 3. Transcriptional Level Analysis of Genes Related to Monacolin K Synthesis

[0105] The transcription levels of monacolin K synthesis-related genes in the fermentation strains of Example 3 and Comparative Examples 1-2 were detected by fluorescent quantitative PCR technology, that is, the relative expression levels of monacolin K synthesis-related genes mokA-mokI were detected at 24, 36, and 72 hours of solid fermentation. The results are shown in Table 2.

[0106] Table 2. Transcriptional level analysis results of genes related to Monacolin K synthesis

[0107]

[0108] As can be seen from Table 2, the peaks of the related genes mokA-mokH in Example 3 are earlier and more intense, indicating that the present invention comprehensively regulates the genes related to monacolin K synthesis by adding exogenous additives during the solid fermentation process, thereby affecting the synthesis of monacolin K and improving the monacolin K yield.

[0109] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

[0110] The present invention and its embodiments are described above. Such description is not restrictive. The drawings show only one embodiment of the present invention, and actual applications are not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the present invention, any method and embodiment similar to the technical solution without creative design shall fall within the scope of protection of the present invention.

Claims

1. A production method for improving the ring-opening rate of red yeast rice milovastatin, characterized in that: The following steps are involved: S1. Spore culture: A high-yield strain of Monascus purpureus was selected and cultured on a maltose agar slant medium for 7 days to obtain an activated spore suspension with a strain concentration of 5.0 × 10^6 CFU / g. S2. Seed culture: Inoculate the activated spores into liquid culture medium at an inoculum size of 5%-15%, and culture at 32°C and 150-200 rpm for 48 hours to obtain seed solution; S3, solid fermentation: the seed liquid is inoculated into a cooked indica rice substrate with a moisture content of 52-55% at an inoculum rate of 15-30%, the pH is adjusted to 3.5-5.0 using an organic acid, and the solid fermentation is carried out for 75 hours; S4, taking out the red yeast rice after the culture is completed, and drying it at 40-60 ° C for 12-24 hours to obtain a red yeast rice product; The solid fermentation indica rice matrix further comprises the following components in concentrations: 3-5% glucose, 1-2% peptone, 0.5-1% yeast extract, 1-3% heat-modified soy protein isolate polysaccharide sulfate complex, 0.5-1% ammonium sulfate, 0.05-0.2% 1,5-p-coumarylquinic acid, 0.2-0.5% sodium acetate, 0.1-0.3% sodium citrate, 0.01-0.03% nicotinamide riboside, 0.05-0.1% ascorbic acid, 0.001-0.005% biotin, 0.001-0.01% hydrated magnesium sulfate, 0.001-0.01% hydrated zinc sulfate, and 0.005-0.01% manganese sulfate; The heat-modified soy protein isolate polysaccharide sulfate complex is prepared by the following steps: The soy protein isolate was dissolved in deionized water and gently stirred at 500 r / min for 2 hours to prepare a 20 mg / mL suspension, which was heated at 95°C for 20 minutes. The suspension was centrifuged at 3000 r / min and 4°C for 10 minutes, the supernatant was collected, and the supernatant was freeze-dried to obtain heat-modified soy protein isolate, which was then configured into a heat-modified soy protein isolate solution; a dextran sulfate solution was prepared and stirred at 500 r / min for 2 hours. The dextran sulfate solution was heated at 95°C for 10 minutes and then cooled to room temperature. The dextran sulfate solution was mixed with the heat-modified soy protein isolate solution, the pH value was adjusted to 5.5 with 0.1 mol / L HCl solution, and the heat-modified soy protein isolate polysaccharide sulfate complex was obtained after freeze-drying.

2. The method for increasing the ring-opening rate of red yeast rice milovastatin according to claim 1, wherein: The slant culture medium comprises the following components in the following concentrations: 8-12% of maltose juice, 1.5-2.5% of agar, 0.05-0.1% of potassium dihydrogen phosphate, 0.01-0.03% of hydrated magnesium sulfate, and 0.001-0.005% of vitamin B1.

3. The method for increasing the ring-opening rate of red yeast rice milovastatin according to claim 1, wherein: The seed culture medium comprises the following components in the following concentrations: 5-8% glucose, 0.5-1.5% peptone, 0.05-0.5% sodium nitrate, 0.2-0.6% yeast extract, 0.1-0.2% potassium dihydrogen phosphate, and 0.005-0.01% hydrated magnesium sulfate.

4. The method for increasing the ring-opening rate of red yeast rice milovastatin according to claim 1, wherein: The concentration of the heat-modified soy protein isolate solution is 10-30 mg / mL.

5. The method for increasing the ring-opening rate of red yeast rice milovastatin according to claim 1, wherein: The concentration of the dextran sulfate solution is 5-15 mg / mL, and the degree of sulfation is 0.8-1.

2.

6. The method for increasing the ring-opening rate of red yeast rice milovastatin according to claim 1, wherein: The mass ratio of the hot dextran sulfate solution to the heat-modified soy protein isolate solution is 1:3-6.

7. The method for increasing the ring-opening rate of red yeast rice milovastatin according to claim 1, wherein: The solid fermentation comprises the following steps: 0-24h: Temperature 30-32℃, RH 85%, turn the compost twice; 24-72h: Raise the temperature to 38-42℃; After 72h: cool to 30-32℃, RH70%.

8. The method for increasing the ring-opening rate of red yeast rice milovastatin according to claim 1, wherein: The dissolved oxygen in the solid fermentation process is maintained at 3-5 mg / L.

9. The method for increasing the ring-opening rate of red yeast rice milovastatin according to claim 1, wherein: The organic acid is one of citric acid, malic acid and lactic acid.

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