Monascus fermentation additional culture medium and method for producing monascus based on culture medium
A Monascus fermentation culture medium with indica rice and specific liquid components enhances acid lovastatin production in Monascus rice, addressing low lovastatin content and citrinin issues, achieving high lovastatin levels and citrinin-free products.
Patent Information
- Application Number
- CN202311145607.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-06
- Publication Date
- 2024-12-13
AI Technical Summary
The lovastatin content, particularly the proportion of acid lovastatin, in solid-state fermented Monascus rice is generally low, and there is a need to address this while minimizing the production of citrinin, a nephrotoxic fungal toxin.
A Monascus fermentation additional culture medium composed of a solid matrix, preferably indica rice, and a liquid culture medium containing 50 g/L glucose, 20 g/L peptone, and 0.5 g/L MgSO4, with a 10% mass volume concentration, is used in a method involving inoculation, variable temperature fermentation, and controlled water addition to enhance lovastatin production.
The method results in Monascus rice with a high proportion of acid lovastatin and no detectable citrinin, optimizing the culture medium formula and fermentation process to improve lovastatin content.
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Figure CN119120205A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of fermentation engineering, and in particular to a Monascus fermentation supplementary culture medium and a method for producing Monascus based on the culture medium. Background Art
[0002] Lovastatin (monacolin K, MK) is the most widely used active substance in red yeast rice. Lovastatin obtained by Monascus fermentation has two forms: an acidic open-ring structure and a lactone closed-ring structure. Studies have shown that lovastatin has a pharmacological effect of lowering blood lipids because it can inhibit the synthesis of cholesterol in the human body. Among the lovastatins of the above two structures, acid lovastatin is a structural analogue of 3-carboxyl-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the cholesterol synthesis pathway, which competitively inhibits the synthesis of cholesterol. Lactone lovastatin itself is inactive and needs to be converted into acid lovastatin under the action of hydroxyesterase in the human body before it can play a cholesterol-lowering effect. Compared with Western medicine lovastatin, Chinese medicine has fewer side effects, higher safety, and better comprehensive efficacy. Therefore, increasing the lovastatin content in red yeast rice by solid-state fermentation, especially the content of acid lovastatin, has important application value.
[0003] The composition of the fermentation medium and the fermentation process have an important influence on the lovastatin content in red yeast rice. Some studies have shown that the addition of additional culture medium is beneficial to increase the lovastatin content in red yeast rice. The additional culture medium generally contains glucose, peptone, MgSO4, KH2PO4, etc. Some studies have shown that changes in process conditions such as the concentration of additional culture medium and initial pH, initial water content, culture temperature, culture time and water replenishment will affect the lovastatin content in red yeast rice.
[0004] Most species of Monascus can also produce citrinin, a nephrotoxic fungal toxin. Citrinin can cause kidney enlargement and mitochondrial enlargement, leading to large-scale cell death. According to relevant standards in my country, the limit of citrinin in liquid Monascus products is 50μg / L, and the limit of citrinin in solid products is 1mg / kg. Studies have shown that whether red yeast rice products contain citrinin (or the content of citrinin) mainly depends on the species of Monascus.
[0005] Studies have shown that the proportion of acid lovastatin in red yeast rice products is closely related to the species of Monascus. At present, there is no literature report on the research of increasing the proportion of acid lovastatin in red yeast rice products by changing the content of MgSO4 in the supplementary culture medium. Summary of the invention
[0006] The object of the present invention is to provide a Monascus fermentation additional culture medium and a method for producing Monascus based on the culture medium, so as to solve the problem that the lovastatin content in solid-state fermented Monascus rice is generally not high, especially the proportion of acid lovastatin is not high, proposed in the above-mentioned background technology.
[0007] To achieve the above object, the present invention provides the following technical solution: a Monascus fermentation additional culture medium, which is composed of a solid matrix and a liquid culture medium.
[0008] Preferably, the solid matrix is indica rice.
[0009] Preferably, the liquid culture medium is composed of 50 g / L glucose, 20 g / L peptone, 0.5 g / L MgSO4 and 1 g / L KH2PO4.
[0010] Preferably, the amount of liquid culture medium added is 10% of the mass volume concentration of the solid matrix.
[0011] A method for producing red yeast rice based on a Monascus fermentation supplementary medium, the method for producing red yeast rice based on a Monascus fermentation supplementary medium comprising the following steps:
[0012] S1: Take a loop of Monascus mycelium with an inoculation loop and culture it in seed culture medium for 72 hours;
[0013] S2: 50 g of indica rice in a 250 mL triangular bottle was soaked in a pH 5.5 lactic acid aqueous solution for 12 hours, the water was drained to 40% of the initial water content of the medium, liquid culture medium was added, and steam sterilized at 115 °C for 30 minutes;
[0014] S3: After the substrate is cooled naturally, 10% of HNU01 spore solution is inoculated and the solid-state fermentation is carried out at variable temperature for 18 days.
[0015] Preferably, the seed culture medium is composed of glucose, soy peptone, NaNO3, MgSO4 and KH2PO3.
[0016] Preferably, in step S3, the temperature of the variable temperature solid-state fermentation is 30° C. for the first 3 days and 24° C. for the next 15 days.
[0017] Preferably, in the step S3, the koji is stirred and turned over on the 4th day of the variable temperature solid-state fermentation, and 10% of water is added, and the koji is stirred and turned over once every two days thereafter.
[0018] Compared with the prior art, the beneficial effects of the present invention are: the Monascus fermentation additional culture medium and the method for producing Monascus based on the culture medium produce Monascus rice containing a high proportion of acid lovastatin by optimizing the culture medium formula and fermentation process. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is a simplified process flow diagram for producing red yeast rice by solid-state fermentation.
[0020] Figure 2 This is a graph showing the effects of culture medium composition and fermentation process conditions on the content of lovastatin in red yeast rice.
[0021] Figure 3 This is a graph showing the effect of MgSO4 concentration on the proportion of acid lovastatin in red yeast rice.
[0022] Figure 4 The figure shows the HPLC spectrum and standard curve of the acid MK standard.
[0023] Figure 5 The figure shows the HPLC spectrum and standard curve of the lactone MK standard product.
[0024] Figure 6 This is the HPLC spectrum and standard curve of citrinin standard product.
[0025] Figure 7 The HPLC spectrum of MK mixed standard and the HPLC spectrum of MK in the fermentation products under different initial water content conditions.
[0026] Figure 8 This is the HPLC spectrum of citrinin in red yeast rice under optimized conditions in various single-factor experiments. DETAILED DESCRIPTION
[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0028] The invention provides a Monascus fermentation additional culture medium and a method for producing Monascus based on the culture medium. Monascus rice containing a high proportion of acid lovastatin is produced by optimizing the culture medium formula and the fermentation process. The Monascus fermentation additional culture medium consists of a solid matrix and a liquid culture medium. The liquid culture medium consists of 50 g / L of glucose, 20 g / L of peptone, 0.5 g / L of MgSO4 and 1 g / L of KH2PO4. The amount of liquid culture medium added is 10% of the mass volume concentration of the solid matrix.
[0029] The strain used in this application is Monascus purpureus (NHU01);
[0030] Reagents:
[0031] Yeast extraction and tryptone: Oxoid Company, UK;
[0032] Agarose: Sangon Biotechnology (Shanghai) Co., Ltd.;
[0033] Glucose, agar powder: Sinopharm Chemical Reagent Co., Ltd.;
[0034] Common reagents and culture medium formula:
[0035] Potato dextrose agar medium (PDA medium): 200 g potato, 20 g glucose, 20 g agar, add distilled water to make up to 1000 mL;
[0036] Shake flask seed culture medium: glucose, soy peptone, NaNO3, MgSO4·7H2O, 0.1% KH2PO3, acetic acid to adjust pH to 5.0;
[0037] Additional culture medium (dissolved in a small amount of water and added to the drained rice): glucose, soy peptone, KH2PO4, MgSO4·7H2O, and the solid-state fermentation matrix is southern indica rice.
[0038] Solid-state fermentation:
[0039] Select 250mL triangular flask, put 50g rice into each bottle, soak it in lactic acid solution of pH5.5 for 12h, wash the rice milk, drain it with gauze to 40% water content (initial water content, relative dry basis), add 5mL additional culture medium, sterilize at 115℃ for 30min, cool naturally, inoculate with 5mL red yeast spore suspension fermented in 3d shake flask, stir evenly. Cultivate at 30℃ constant temperature for the first 3 days, replenish water (10%) and turn over on the 4th day. Continue to cultivate for 15 days at 24℃ afterwards. The fermented product is dried in 45℃ vacuum for 6h, to be tested.
[0040] High performance liquid chromatography (HPLC) analysis of MK and citrinin content:
[0041] The MK content was determined using a LC-20AT high performance liquid chromatograph, Shim-pack C18 chromatographic column and SPD-20A ultraviolet detector from Shimadzu Corporation of Japan, and lovastatin standard product was purchased from Shanghai McLean Biochemical Technology Co., Ltd. Accurately weigh 0.8 g of the sample, fully grind it in a mortar and pass it through a 100-mesh sieve, weigh 0.5 g of rice flour, add 10 mL of 75% ethanol solution (volume fraction: 75%) of the extractant, ultrasonicate for 50 min, centrifuge at 4000 r / min for 10 min, and take the supernatant to make the volume 10 mL. Take the supernatant solution and filter it through a 0.22 μm microporous membrane, the filtrate is set aside, and the MK content is measured by HPLC under 237 nm ultraviolet light.
[0042] MK content t (mg / g) = 0.05c (mg / L), t (mg / g) represents the mass of total MK (including acid and lactone forms) in each gram of dry red yeast rice product, and c represents the MK concentration in the extract (mg / L).
[0043] The content of citrinin was determined using a LC-20AT high performance liquid chromatograph and RF-20A fluorescence detector from Shimadzu Corporation of Japan, and the citrinin standard product was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Take a 50mL beaker, accurately weigh 0.8g of the sample, fully grind it in a mortar and pass it through a 100-mesh sieve to obtain 0.5g of rice flour, add an extractant V (toluene): V (ethyl acetate): V (formic acid) = 7:3:1, 10mL, ultrasonicate for 5min, centrifuge at 4000r / min for 5min, take the supernatant and pour it into a beaker, add the extractant to the residue to make up to 10mL, ultrasonicate for 5min, centrifuge at 4000r / min for 5min; take the supernatant and pour it into a beaker again, add the extractant to the residue to make up to 10mL, ultrasonicate for 5min, centrifuge at 4000r / min for 5min, take the upper clear night and pour it into a beaker. The supernatants obtained from the three times were mixed evenly, and the supernatant solution was filtered through a 0.22 μm microporous filter membrane. The filtrate was set aside for use. The concentration of citrinin was measured by HPLC at a fluorescence detection excitation wavelength of 331 nm and an emission wavelength of 500 nm.
[0044] Example 1
[0045] Potato dextrose agar medium (PDA medium): 200 g potato, 20 g glucose, 20 g agar, add distilled water to make up to 1000 mL.
[0046] Shake flask seed culture medium: 6% glucose, 2.5% soy peptone, 0.2% NaNO3, 0.1% MgSO4·7H2O, 0.1% KH2PO3, pH adjusted to 5.0 with acetic acid.
[0047] 250mL triangular flasks were selected, 50g of rice was put into each flask, and the flask was soaked in pH 6.5 lactic acid solution for 12 hours. Additional culture medium (dissolved in a small amount of water and added to the drained rice): 5% glucose, 2% soy peptone, 0.1% KH2PO4, 0.05% MgSO4·7H2O. The solid-state fermentation substrate was southern indica rice.
[0048] Initial pH value 6.5, initial water content 40%, glucose 50g / L, peptone 20g / L, MgSO40.5g / L, KH2PO41g / L, variable temperature fermentation (30°C for the first 3 days, 24°C for the next 15 days), total fermentation time 18 days. On the 4th day, 10% water was added, and the koji was stirred and turned over, and thereafter, the koji was stirred and turned over once every two days.
[0049] The fermentation product was vacuum dried at 45°C for 6 h, and the MK and citrinin contents were analyzed by high performance liquid chromatography (HPLC).
[0050] The MK content was 6.5 mg / g, and citrinin was not detected.
[0051] Example 2
[0052] Potato dextrose agar medium (PDA medium): 200 g potato, 20 g glucose, 20 g agar, add distilled water to make up to 1000 mL.
[0053] Shake flask seed culture medium: 6% glucose, 2.5% soy peptone, 0.2% NaNO3, 0.1% MgSO4·7H2O, 0.1% KH2PO3, pH adjusted to 5.0 with acetic acid.
[0054] Select 250mL triangular flasks, put 50g of rice into each flask, and soak it in pH5.5 lactic acid solution for 12 hours. Add additional culture medium (dissolved in a small amount of water and added to the drained rice): 5% glucose, 2% soy peptone, 0.1% KH2PO4, 0.05% MgSO4·7H2O. The solid-state fermentation substrate is southern indica rice.
[0055] Initial pH value 5.5, initial water content 50%, glucose 50g / L, peptone 20g / L, MgSO40.5g / L, KH2PO41g / L, variable temperature fermentation (30°C for the first 3 days, 24°C for the next 15 days), total fermentation time 18 days. On the 4th day, 10% water was added, and the koji was stirred and turned over, and thereafter, the koji was stirred and turned over once every two days.
[0056] The fermentation product was vacuum dried at 45°C for 6 h, and the MK and citrinin contents were analyzed by high performance liquid chromatography (HPLC).
[0057] The MK content was 7.1 mg / g, and citrinin was not detected.
[0058] Example 3
[0059] Potato dextrose agar medium (PDA medium): 200 g potato, 20 g glucose, 20 g agar, add distilled water to make up to 1000 mL.
[0060] Shake flask seed culture medium: 6% glucose, 2.5% soy peptone, 0.2% NaNO3, 0.1% MgSO4·7H2O, 0.1% KH2PO3, pH adjusted to 5.0 with acetic acid.
[0061] Select 250mL triangular flasks, put 50g of rice into each flask, and soak it in pH5.5 lactic acid solution for 12 hours. Add additional culture medium (dissolved in a small amount of water and added to the drained rice): 5% glucose, 2% soy peptone, 0.1% KH2PO4, 0.05% MgSO4·7H2O. The solid-state fermentation substrate is southern indica rice.
[0062] Initial pH value 5.5, initial water content 40%, glucose 40g / L, peptone 20g / L, MgSO40.5g / L, KH2PO41g / L, variable temperature fermentation (30°C for the first 3 days, 24°C for the next 15 days), total fermentation time 18 days. On the 4th day, 10% water was added, and the koji was stirred and turned over, and thereafter, the koji was stirred and turned over once every two days.
[0063] The fermentation product was vacuum dried at 45°C for 6 h, and the MK and citrinin contents were analyzed by high performance liquid chromatography (HPLC).
[0064] The MK content was 7.7 mg / g, and citrinin was not detected.
[0065] Example 4
[0066] Potato dextrose agar medium (PDA medium): 200 g potato, 20 g glucose, 20 g agar, add distilled water to make up to 1000 mL.
[0067] Shake flask seed culture medium: 6% glucose, 2.5% soy peptone, 0.2% NaNO3, 0.1% MgSO4·7H2O, 0.1% KH2PO3, pH adjusted to 5.0 with acetic acid.
[0068] Select 250mL triangular flasks, put 50g of rice into each flask, and soak it in pH5.5 lactic acid solution for 12 hours. Add additional culture medium (dissolved in a small amount of water and added to the drained rice): 5% glucose, 2% soy peptone, 0.1% KH2PO4, 0.05% MgSO4·7H2O. The solid-state fermentation substrate is southern indica rice.
[0069] Initial pH value 5.5, initial water content 40%, glucose 50g / L, peptone 20g / L, MgSO40.5g / L, KH2PO41g / L, variable temperature fermentation (30°C for the first 3 days, 24°C for the next 13 days), total fermentation time 16 days. On the 4th day, 10% water was added, and the koji was stirred and turned over, and thereafter, the koji was stirred and turned over once every two days.
[0070] The fermentation product was vacuum dried at 45°C for 6 h, and the MK and citrinin contents were analyzed by high performance liquid chromatography (HPLC).
[0071] The MK content was 7.4 mg / g, and citrinin was not detected.
[0072] Example 5
[0073] Potato dextrose agar medium (PDA medium): 200 g potato, 20 g glucose, 20 g agar, add distilled water to make up to 1000 mL.
[0074] Shake flask seed culture medium: 6% glucose, 2.5% soy peptone, 0.2% NaNO3, 0.1% MgSO4·7H2O, 0.1% KH2PO3, pH adjusted to 5.0 with acetic acid.
[0075] Select 250mL triangular flasks, put 50g of rice into each flask, and soak it in pH5.5 lactic acid solution for 12 hours. Add additional culture medium (dissolved in a small amount of water and added to the drained rice): 5% glucose, 2% soy peptone, 0.1% KH2PO4, 0.05% MgSO4·7H2O. The solid-state fermentation substrate is southern indica rice.
[0076] Initial pH value 5.5, initial water content 40%, glucose 50g / L, peptone 20g / L, MgSO40.5g / L, KH2PO41g / L, variable temperature fermentation (30°C for the first 3 days, 24°C for the next 15 days), total fermentation time 18 days. On the 4th day, 10% water was added, and the koji was stirred and turned over, and thereafter, the koji was stirred and turned over once every two days.
[0077] The fermentation product was vacuum dried at 45°C for 6 h, and the MK and citrinin contents were analyzed by high performance liquid chromatography (HPLC).
[0078] The MK content was 9.5 mg / g, of which acid MK accounted for 90%. No citrinin was detected.
[0079] Although the present invention has been described above with reference to the embodiments, various modifications may be made thereto and parts thereof may be replaced with equivalents without departing from the scope of the present invention. In particular, as long as there is no structural conflict, the various features in the embodiments disclosed by the present invention may be used in combination with each other in any manner, and the fact that these combinations are not exhaustively described in this specification is only for the sake of omitting space and saving resources. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.
Claims
1. A Monascus fermentation supplementary medium, characterized in that: The Monascus fermentation additional culture medium consists of a solid matrix and a liquid culture medium.
2. The additional culture medium for Monascus fermentation according to claim 1, characterized in that: The solid matrix is indica rice.
3. The additional culture medium for Monascus fermentation according to claim 2, characterized in that: The liquid culture medium is composed of 50 g / L glucose, 20 g / L peptone, 0.5 g / L MgSO4 and 1 g / L KH2PO4.
4. The additional culture medium for Monascus fermentation according to claim 3, characterized in that: The amount of liquid culture medium added is 10% of the mass volume concentration of the solid matrix.
5. A method for producing red yeast rice based on Monascus fermentation supplementary medium as claimed in claim 4, characterized in that: The method for producing red yeast rice based on the additional culture medium of Monascus fermentation comprises the following steps: S1: Take a loop of Monascus mycelium with an inoculation loop and culture it in seed culture medium for 72 hours; S2: 50 g of indica rice in a 250 mL triangular bottle was soaked in a pH 5.5 lactic acid aqueous solution for 12 hours, the water was drained to 40% of the initial water content of the medium, liquid culture medium was added, and steam sterilized at 115 °C for 30 minutes; S3: After the substrate is cooled naturally, 10% of HNU01 spore solution is inoculated and the solid-state fermentation is carried out at variable temperature for 18 days.
6. The method for producing red yeast rice based on Monascus fermentation supplementary medium according to claim 5, characterized in that: The seed culture medium is composed of glucose, soy peptone, NaNO3, MgSO4 and KH2PO3.
7. The method for producing red yeast rice based on Monascus fermentation supplementary medium according to claim 6, characterized in that: In step S3, the temperature of the solid-state fermentation is 30° C. for the first 3 days and 24° C. for the next 15 days.
8. The method for producing red yeast rice based on Monascus fermentation supplementary medium according to claim 7, characterized in that: In the step S3, the koji is stirred and turned over on the 4th day of the variable temperature solid-state fermentation, and 10% of water is added. Thereafter, the koji is stirred and turned over once every two days.