CN111218480A - Preparation method of solid-state fermentation red yeast rice with high yield of monacolin K - Google Patents

Preparation method of solid-state fermentation red yeast rice with high yield of monacolin K Download PDF

Info

Publication number
CN111218480A
CN111218480A CN202010117381.7A CN202010117381A CN111218480A CN 111218480 A CN111218480 A CN 111218480A CN 202010117381 A CN202010117381 A CN 202010117381A CN 111218480 A CN111218480 A CN 111218480A
Authority
CN
China
Prior art keywords
monacolin
culture
follows
red yeast
fermentation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202010117381.7A
Other languages
Chinese (zh)
Inventor
张占军
王富花
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yangzhou Polytechnic College
Yangzhou Polytechnic College Yangzhou Radio and TV University
Original Assignee
Yangzhou Polytechnic College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yangzhou Polytechnic College filed Critical Yangzhou Polytechnic College
Priority to CN202010117381.7A priority Critical patent/CN111218480A/en
Publication of CN111218480A publication Critical patent/CN111218480A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a preparation method of solid state fermentation red yeast rice with high yield of monacolin K, which specifically comprises the following steps: (1) activating strains: placing Monascus into slant culture medium for activating culture, scraping slant spore into triangular flask containing sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; (2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing for 40-55 h at 28 ℃ by a shaking table at 150-200 r/min to obtain seed liquid; (3) fermenting the seed liquid: and (3) inoculating the seed solution cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 5-25%, and performing variable temperature fermentation culture for 10-20 days to obtain the fermented red yeast rice. The method can be effectively usedThe yield of monacolin K in the solid-state fermentation process of the red yeast rice is improved.

Description

Preparation method of solid-state fermentation red yeast rice with high yield of monacolin K
Technical Field
The invention relates to the fields of microbiology and solid state fermentation, in particular to a preparation method of solid state fermentation red yeast rice with high yield of monacolin K.
Background
Red yeast rice is also called red rice, red yeast and good rice, is a substance used as both medicine and food and is prepared by inoculating monascus fungi on steamed rice for solid state fermentation, has a history of production and application for thousands of years in China, and is generally applied to the aspects of wine brewing, red fermented bean curd manufacturing, vinegar production, fermented beverages, meat products, blood pressure and lipid lowering medicines and the like. In 1979, the Japanese academy of academic Long Teng chapter separated from the secondary metabolite of Monascus purpureus to obtain an active substance capable of effectively inhibiting cholesterol synthesis, which is named Monacolin K. Lovastatin (lovastatin), an active substance that also has cholesterol-inhibiting properties, was discovered in A.terreus by the American scientist Alberts [9] in 1980. The red yeast is a natural solid fermentation product, and does not need any purification, so the red yeast is safer and more reliable than the lovastatin which is a prescription medicine in the aspect of safety. Besides monacolin K, the fermentation product also contains a series of lovastatin structural analogs, Y-aminobutyric acid and other physiologically active substances, and the related components are combined with the monacolin K, so that the effect of synergistically regulating blood fat is achieved, and the side effect of pure lovastatin is greatly reduced. With the increasing demand of people on red yeast products such as health-care food and health-care medicine developed by monascus, the method for effectively improving the content of monacolin K in the solid state fermentation of red yeast has profound significance on the further development and application of the red yeast products.
Therefore, it is necessary to develop a method for preparing high-yield Monacolin K by solid state fermentation red yeast rice; the ecological benefit, the social benefit and the enterprise economic benefit are better unified.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of high-yield monascus by solid state fermentation.
In order to solve the technical problems, the invention adopts the technical scheme that the preparation method of the high-yield monascus by solid state fermentation of monacolin K specifically comprises the following steps:
(1) activating strains: putting the screened monascus into a slant culture medium for activated culture, wherein the culture temperature is as follows: and (3) culturing at 28-30 ℃ for: 16-24 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mLAnd is ready for use;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 0.5-3%; the culture conditions are as follows: shaking and culturing the seeds for 40-55 h at 26-30 ℃ by a shaking table at 150-200 r/min to obtain seed liquid;
(3) fermenting the seed liquid: and (3) inoculating the seed solution cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 5-25%, and performing variable temperature fermentation culture for 10-20 days to obtain the fermented red yeast rice.
By adopting the technical scheme, the activated strain is inoculated to the seed culture medium to obtain the seed solution, then the seed solution is inoculated to the solid fermentation culture medium for fermentation, the appropriate inoculation amount is crucial to the normal growth and metabolism of monascus, when the inoculation amount is small, the yield of monacolin K is increased, until the peak value is reached, the yield of monacolin K is reduced along with the increase of the inoculation amount, namely, the subsequent continuous increase of the inoculation amount has an inhibiting effect on the production of monacolin K. The reason is probably that the larger inoculation amount is favorable for the monascus to produce more metabolites, but if the inoculation amount is too large, the thallus grows too fast to be beneficial to the formation of the metabolites, and the accumulation of the metabolites has a feedback inhibition effect on the fermentation process. On the other hand, the continuous formation of metabolites can also increase the viscosity of a fermentation medium, so that the mass transfer process is hindered, and the growth and metabolism of thalli are finally influenced; the method can effectively improve the yield of monacolin K in the solid-state fermentation process of red yeast rice.
As a preferred technical scheme of the invention, in the step (3), the solid state fermentation culture medium is optimized firstly: the yield of monacolin K in the red yeast rice is optimized and improved by investigating factors of inoculation amount, charging amount, corn flour addition amount and peptone addition amount.
By adopting the technical scheme, the activated strain is inoculated to the seed culture medium to obtain the seed solution, then the seed solution is inoculated to the solid fermentation culture medium for fermentation, the appropriate inoculation amount is crucial to the normal growth and metabolism of monascus, when the inoculation amount is small, the yield of monacolin K is increased, until the peak value is reached, the yield of monacolin K is reduced along with the increase of the inoculation amount, namely, the subsequent continuous increase of the inoculation amount has an inhibiting effect on the production of monacolin K. The reason is probably that the larger inoculation amount is favorable for the monascus to produce more metabolites, but if the inoculation amount is too large, the thallus grows too fast to be beneficial to the formation of the metabolites, and the accumulation of the metabolites has a feedback inhibition effect on the fermentation process. On the other hand, the continuous formation of metabolites can also increase the viscosity of a fermentation medium, so that the mass transfer process is hindered, and the growth and metabolism of thalli are finally influenced; the method can effectively improve the yield of monacolin K in the solid-state fermentation process of red yeast rice.
As a preferred technical scheme of the invention, the formula of the slant culture in the step (1) is as follows: 15-25 g of potatoes, 1.5-2.5 g of glucose, 1.0-2.0 g of agar, 90-110 mL of distilled water and pH of 7.0-7.2.
As a preferred technical solution of the present invention, the formulation of the seed culture medium in the step (2) is: 15-25 g of peptone, 15-25 g of corn flour, 5-15 g of yeast extract, 0.3-0.8 g of magnesium sulfate, 0.5-1.5 g of dipotassium hydrogen phosphate, 0.05-0.15 g of ferrous sulfate, 900-1100 mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 110-130 ℃ for 15-25 min.
As a preferred embodiment of the present invention, the formula of the solid state fermentation medium in the step (3) is: the method comprises the steps of soaking indica rice for 4-6 h, steaming without half-cooked, placing 20-40 g of the indica rice into a 200-300 mL sterilization triangular flask, adding corn flour and peptone, wherein the addition amount of the corn flour is 0.5-10%, the addition amount of the peptone is 0.5-10%, and sterilizing for 15-25 min at 110-130 ℃ and 0.1 MPa. The solid culture medium is important for culturing the high monacolin K, wherein the charging amount and the adding amounts of the corn flour and the peptone influence the monacolin K of the red yeast to different degrees; wherein the amount of the charged materials affects the heat dissipation and oxygen supply in the growth and metabolism process of the monascus so that the amount of the charged materials has an important relation with the generation of monacolin K; corn flour is used as a carbon source in the fermentation process of monascus and is superior to other carbon sources such as glucose and soluble starch. The corn flour is used as an organic carbon source, so that the nutrition is rich and comprehensive, and the rapid growth of monascus at the early stage is facilitated; the peptone is the best nitrogen source for Monacolin K production by monascus and is beneficial to the growth of monascus. The invention optimizes the solid fermentation culture medium before solid fermentation culture: the yield of monacolin K in the red yeast rice is optimized and improved by observing factors of inoculation amount, charging amount, corn flour addition amount and peptone addition amount, so that the optimized solid state fermentation culture medium is obtained.
In a preferred embodiment of the present invention, the temperature-variable fermentation culture in the step (3) is performed under the conditions of 30 ℃ for the first 3d and 26 ℃ for the second 4-20 d. The temperature-variable fermentation condition is adopted, so that the fermentation of monascus can be further facilitated, and high monacolin K can be obtained.
In a preferred embodiment of the present invention, in the step (3), the amount of the seed solution cultured in the step (2) inoculated into the optimized solid fermentation medium is 15%. A large number of experiments show that the optimal inoculation amount of the seed liquid to be inoculated to the solid culture medium is 15%.
As a preferred embodiment of the present invention, the formulation of the solid fermentation medium in the step (3): immersing indica rice for 5h, steaming without half-cooked, placing 30g into a 250mL sterilized triangular flask, adding corn flour and peptone, wherein the addition amount of the corn flour is 3%, the addition amount of the peptone is 2%, and sterilizing at 121 ℃ and 0.1MPa for 20 min. A large number of experiments show that the optimal charging amount is 30g/250mL, the addition amount of the corn flour is 3%, and the addition amount of the peptone is 2%.
Drawings
FIG. 1 is a graph showing the effect of Monascus purpureus went on the inoculation of Monascus purpureus went in the preparation of high Monacolin K yield by solid state fermentation;
FIG. 2 is a graph showing the effect of Monascus purpureus went on the amount of monascus charged in the solid medium in the preparation of a solid fermentation red yeast for high yield of Monacolin K;
FIG. 3 is a graph showing the effect of Monascus purpureus went on the production of Monacolin K by monascus in the amount of corn flour added to the solid medium in the preparation method of the solid fermentation red yeast rice;
FIG. 4 is a graph showing the effect of Monascus purpureus K production by monascus on the amount of peptone added to the solid medium in the preparation method of solid fermentation red yeast rice with high Monacolin K production;
FIG. 5 shows high yield monacolin K monascus strains in a method for preparing high yield monacolin K solid state fermentation monascus;
FIG. 6 shows a solid state fermentation red yeast rice with high monacolin K yield in the preparation method of the solid state fermentation red yeast rice with high monacolin K yield.
Detailed Description
The reagents used in the following examples were:
monacolin K standard: sigma, USA; potato, corn flour, long-shaped rice: is sold on the market; peptone, yeast extract, glucose, magnesium sulfate, agar, dipotassium hydrogen phosphate, ferrous sulfate: chengdu Kelong chemical reagent factory. Methanol, acetonitrile, phosphoric acid: chemical reagents of the national drug group, ltd, is chromatographically pure.
The apparatus used in the following examples was: DHG-9030A type electric heating constant-temperature air-blast drying oven: shanghai-constant technology, Inc.; SW-CJ-2G superclean bench: suzhou clarification plant, Inc.; DS-1 high speed tissue masher: shanghai Biao Ben model plant; model 1260 high performance liquid chromatograph: agilent technologies, Inc.; SB-5200DTD ultrasonic cleaning machine: ningbo Xinzhi Biotechnology GmbH; LDZX-KBS pressure steam sterilizer: shanghai Shenan medical instruments factory.
Example 1: the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 5%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150 mm. times.4.6 mm. times.5 μm), acetonitrile, water, 0.5% phosphoric acid (60: 35: 5), column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 2: the difference from the example 1 is that the seed solution in the step (3) is different in the inoculation amount, and the inoculation amount is 10%; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH value of 6.0, 7.0-7.2, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150 mm. times.4.6 mm. times.5 μm), acetonitrile, water, 0.5% phosphoric acid (60: 35: 5), column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 3: the difference from the example 1 is that the seed solution in the step (3) has different inoculation amount, and the inoculation amount is 15%; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to 15% of inoculation amount, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 4: the difference from the example 1 is that the seed solution in the step (3) has different inoculation amount, and the inoculation amount is 20%; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows:20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 20%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 5: the difference from the example 1 is that the seed solution in the step (3) has different inoculation amount, and the inoculation amount is 25%; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spores into a triangular flask filled with sterile water, shaking uniformly, filtering with sterile absorbent cotton,diluting to a concentration of 106～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 25%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
The results of the effect of different inoculum sizes of examples 1-5 on monacolin K production by Monascus are shown in FIG. 1.
The appropriate amount of inoculation is critical for the normal growth and metabolism of monascus; as can be seen from FIG. 1, the yield of monacolin K increased with the increase of the inoculum size until the inoculum size reached 15%, and the yield of monacolin K was the highest at 9.25mg/g when the inoculum size reached 15%. Continuing to increase the inoculum size thereafter inhibited the production of monacolin K. The reason is probably that the larger inoculation amount is favorable for the monascus to produce more metabolites, but if the inoculation amount is too large, the thallus grows too fast to be beneficial to the formation of the metabolites, and the accumulation of the metabolites has a feedback inhibition effect on the fermentation process. On the other hand, the continuous formation of metabolites can also increase the viscosity of a fermentation medium, so that the mass transfer process is hindered, and the growth and metabolism of thalli are finally influenced. Therefore, the optimal inoculum size was selected to be 15%.
Example 6: the difference from example 2 is that the solid medium in the step (3) is charged in an amount of 20g/250ml, which is different from the amount charged; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 20g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 7: the difference from example 2 is that the solid medium in the step (3) is charged in an amount of 25g/250ml, which is different from the charged amount; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 25g into a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the addition amount of the corn flour is 1%, the addition amount of the peptone is 1%, and sterilizing at 121 ℃ and 0.1MPa for 20 min;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 8: the difference from example 2 is that the solid medium in the step (3) is charged in an amount of 35g/250ml, which is different from the amount charged; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 35g into a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the addition amount of the corn flour is 1%, the addition amount of the peptone is 1%, and sterilizing at 121 ℃ and 0.1MPa for 20 min;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 9: the difference from example 2 is that the solid medium in the step (3) is charged in an amount of 40g/250ml, which is different from the amount charged; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 40g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
The results of the effect of different loading levels in examples 2 and 6-9 on monacolin K production by Monascus are shown in FIG. 2.
As can be seen from FIG. 2, the yield of monacolin K reached a maximum of 7.26mg/g at a charge of 30g/250 mL. After which the charge was increased and the yield of monacolin K began to decline. The larger charging amount plays a role in inhibiting the generation of monacolin K, which may be caused by the fact that the viscosity of a fermentation substrate is increased due to the growth metabolic activity of monascus in the fermentation process, the permeability is reduced, the heat dissipation process is influenced, the internal temperature of the material is increased, and the generation of metabolites is further influenced. Therefore, the optimum charge was selected to be 30g/250 mL.
Example 10: the difference from the example 2 is that the solid culture medium in the step (3) is added with different corn flour, and the addition amount of the corn flour is 0.5%; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: placing Monascus purpureus into the inclined planePerforming activation culture in a culture medium, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 11: the difference from the example 2 is that the solid culture medium in the step (3) is added with different corn flour, and the addition amount of the corn flour is 3 percent; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the addition amount of the corn flour is 3%, the addition amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 12: the difference from the example 2 is that the solid culture medium in the step (3) is added with corn flour in an amount of 5%; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the addition amount of the corn flour is 5%, the addition amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 13: the difference from the example 2 is that the solid culture medium in the step (3) is added with corn flour in an amount of 7%; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the addition amount of the corn flour is 7%, the addition amount of the peptone is 1%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
The results of the effect of different amounts of corn flour added in the above examples 2 and 10-13 on monacolin K production by monascus are shown in fig. 3.
As can be seen from fig. 3, the yield of monacolin K gradually increased with increasing amounts of added corn meal. When the addition amount of the corn flour reaches 3%, the yield of the monacolin K reaches the maximum 8.66 mg/g. While as the corn meal addition continued to increase, the yield of monacolin K decreased, indicating that too much addition was not conducive to increased yields of monacolin K. The reason is that the corn flour has high viscosity, and the ventilation quantity is reduced when the addition amount is larger, so that the accumulation of biomass is reduced, and the growth of monascus and the generation of metabolites are inhibited to a certain extent. Therefore, the optimum amount of corn meal is selected to be 3%.
Example 14: the difference from example 2 is that the solid medium in step (3) is added with peptone in an amount of 0.5%; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 0.5%, and sterilizing at 121 ℃ and 0.1MPa for 20 min;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 15: the difference from example 2 is that the solid medium in step (3) is added with peptone in an amount of 2% different from the solid medium; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 2%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 16: the difference from example 2 is that the solid medium in step (3) is added with peptone in an amount of 3% different from the solid medium; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the formula of the slant culture in the step (1) is as follows: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 3%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
Example 17: the difference from example 2 is that the solid medium in step (3) is added with peptone in an amount of 4% different from the solid medium; specifically, the method comprises the following steps:
the preparation method of the solid state fermentation red yeast rice with high yield of monacolin K specifically comprises the following steps:
(1) activating strains: putting monascus into a slant culture medium for activation culture, wherein the culture temperature is as follows: 29 ℃, incubation time: 20 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby; the preparation of the slant culture of the step (1)The method comprises the following steps: 20g of potatoes, 2.0g of glucose, 1.5g of agar, 100mL of distilled water and pH of 7.0-7.2;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 2%; the culture conditions are as follows: shaking and culturing at 28 deg.C with 160r/min shaking table for 48h to obtain seed solution; the formula of the seed culture medium in the step (2) is as follows: 20g of peptone, 20g of corn flour, 10g of yeast extract, 0.5g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 0.1g of ferrous sulfate, 1000mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 20 min;
(3) fermenting the seed liquid: inoculating the seed liquid cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 10%, and performing variable temperature fermentation culture for 15d to obtain fermented red yeast rice; wherein the front 3d is 30 ℃ and the rear 12d is 26 ℃; the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g of the indica rice in a 250mL sterilization triangular flask, adding corn flour and peptone, wherein the adding amount of the corn flour is 1%, the adding amount of the peptone is 4%, and sterilizing for 20min at 121 ℃ and 0.1 MPa;
(4) drying the fermented red yeast rice obtained in the step (3) at 50 ℃ and crushing to 40 meshes, weighing 0.5g to 50mL volumetric flask, adding methanol for ultrasonic extraction for 1h, centrifuging at 4000r/min for 10min, filtering the supernatant with a 0.45 mu m filter membrane, detecting by HPLC, and calculating the content of Monacolin K according to a Monacolin K standard curve; chromatographic conditions are as follows: ZORBAX300SB-C18(150mm X4.6 mm X5 μm), acetonitrile, water, 0.5% phosphoric acid 60:35:5 as mobile phase, column temperature 30 deg.C, detection wavelength of 238nm, flow rate of 1.0mL/min, and sample volume of 20 μ L.
The results of the effect of the addition of peptone to monacolin K produced by Monascus purpureus in examples 2 and 14-17 are shown in FIG. 4.
As can be seen from FIG. 4, the production of monacolin K gradually increased with the increase in the amount of peptone added. When the addition amount of peptone reached 2%, the yield of monacolin K reached the maximum of 7.91 mg/g. Therefore, the optimal addition amount of peptone is selected to be 2%.
To sum up: through single factor and response surface tests, under the condition of variable temperature fermentation for 15d of fermentation time, the process conditions for high yield of monacolin K in the process of monascus solid state fermentation are optimized, and the process conditions for obtaining the monacolin K through monascus solid state fermentation high yield are 17.50% of the inoculation amount. In addition, when the inoculation amount is 15%, under the condition that the charging amount is 32.95g/250mL, the adding amount of corn flour is 3.80% and the adding amount of peptone is 2.37%, the yield of monacolin K can reach 11.25 mg/g.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (8)

1. A preparation method of solid state fermentation red yeast rice with high yield of monacolin K is characterized by comprising the following steps:
(1) activating strains: putting the screened monascus into a slant culture medium for activated culture, wherein the culture temperature is as follows: and (3) culturing at 28-30 ℃ for: 16-24 h; scraping the slant spore into a triangular flask filled with sterile water, shaking, filtering with sterile absorbent cotton, and diluting to 10% concentration6～107Per mL for standby;
(2) preparing a seed solution: inoculating the strain treated in the step (1) into a seed culture medium for culture in an inoculation amount of 0.5-3%; the culture conditions are as follows: shaking and culturing the seeds for 40-55 h at 26-30 ℃ by a shaking table at 150-200 r/min to obtain seed liquid;
(3) fermenting the seed liquid: and (3) inoculating the seed solution cultured in the step (2) to the optimized solid fermentation culture medium according to the inoculation amount of 5-25%, and performing variable temperature fermentation culture for 10-20 days to obtain the fermented red yeast rice.
2. The method for preparing high-yield monascus by solid state fermentation of monacolin K according to claim 1, wherein in the step (3), a solid state fermentation culture medium is optimized: the yield of monacolin K in the red yeast rice is optimized and improved by investigating factors of inoculation amount, charging amount, corn flour addition amount and peptone addition amount.
3. The method for preparing the solid state fermentation red yeast rice with high monacolin K yield according to claim 1, wherein the formula of the slant culture in the step (1) is as follows: 15-25 g of potatoes, 1.5-2.5 g of glucose, 1.0-2.0 g of agar, 90-110 mL of distilled water and pH of 7.0-7.2.
4. The method for preparing high-yield monacolin K solid state fermentation red yeast rice according to claim 1, wherein the formula of the seed culture medium in the step (2) is as follows: 15-25 g of peptone, 15-25 g of corn flour, 5-15 g of yeast extract, 0.3-0.8 g of magnesium sulfate, 0.5-1.5 g of dipotassium hydrogen phosphate, 0.05-0.15 g of ferrous sulfate, 900-1100 mL of distilled water with the pH of 6.0, 7.0-7.2 of pH, and sterilizing at 110-130 ℃ for 15-25 min.
5. The method for preparing high-yield monacolin K solid state fermentation red yeast rice according to claim 1, wherein the formula of the solid state fermentation culture medium in the step (3) is as follows: the method comprises the steps of soaking indica rice for 4-6 h, steaming without half-cooked, placing 20-40 g of the indica rice into a 200-300 mL sterilization triangular flask, adding corn flour and peptone, wherein the addition amount of the corn flour is 0.5-10%, the addition amount of the peptone is 0.5-10%, and sterilizing for 15-25 min at 110-130 ℃ and 0.1 MPa.
6. The method for preparing high-yield monacolin K by solid state fermentation of red yeast rice according to claim 5, wherein the temperature-variable fermentation culture in the step (3) is a fermentation culture at 30 ℃ for the first 3 days and 26 ℃ for the second 4-20 days.
7. The method for preparing high-yield monascus by solid state fermentation of monacolin K according to claim 5, wherein the inoculation amount of the seed solution cultured in the step (2) to the optimized solid state fermentation medium in the step (3) is 15%.
8. The method for preparing high-yield monascus by solid state fermentation of monacolin K according to claim 5, wherein the formula of the solid fermentation medium in the step (3) is as follows: immersing indica rice for 5h, steaming without half-cooked, placing 30g into a 250mL sterilized triangular flask, adding corn flour and peptone, wherein the addition amount of the corn flour is 3%, the addition amount of the peptone is 2%, and sterilizing at 121 ℃ and 0.1MPa for 20 min.
CN202010117381.7A 2020-02-25 2020-02-25 Preparation method of solid-state fermentation red yeast rice with high yield of monacolin K Pending CN111218480A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010117381.7A CN111218480A (en) 2020-02-25 2020-02-25 Preparation method of solid-state fermentation red yeast rice with high yield of monacolin K

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010117381.7A CN111218480A (en) 2020-02-25 2020-02-25 Preparation method of solid-state fermentation red yeast rice with high yield of monacolin K

Publications (1)

Publication Number Publication Date
CN111218480A true CN111218480A (en) 2020-06-02

Family

ID=70807397

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010117381.7A Pending CN111218480A (en) 2020-02-25 2020-02-25 Preparation method of solid-state fermentation red yeast rice with high yield of monacolin K

Country Status (1)

Country Link
CN (1) CN111218480A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108707555A (en) * 2018-06-05 2018-10-26 成都医学院 Monascus strain and its application of the high yield rich in Monacolin K
CN116083252A (en) * 2023-04-12 2023-05-09 北京一品堂医药科技有限公司 Red yeast rice fermentation preparation method of high-yield lipid-lowering natural Mo Lake forest K

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102429928A (en) * 2011-11-17 2012-05-02 成都格蓝洋生物医药科技有限公司 Preparation method for coix seed red yeast rice
CN104109700A (en) * 2014-07-09 2014-10-22 江南大学 Method of producing monacolin K functional red yeast rice by solid-liquid co-fermentation
CN110607332A (en) * 2019-08-14 2019-12-24 南京工业大学 Culture medium for improving content of functional red yeast rice Monacolin K and fermentation method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102429928A (en) * 2011-11-17 2012-05-02 成都格蓝洋生物医药科技有限公司 Preparation method for coix seed red yeast rice
CN104109700A (en) * 2014-07-09 2014-10-22 江南大学 Method of producing monacolin K functional red yeast rice by solid-liquid co-fermentation
CN110607332A (en) * 2019-08-14 2019-12-24 南京工业大学 Culture medium for improving content of functional red yeast rice Monacolin K and fermentation method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KWON,HYUNG-JIN: "Some Monascus purpureus genomes lack the monacolin K biosynthesis locus", 《JOURNAL OF APPLIED BIOLOGICAL CHEMISTRY》 *
李同乐等: "功能性红曲发酵条件的优化", 《中国酿造》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108707555A (en) * 2018-06-05 2018-10-26 成都医学院 Monascus strain and its application of the high yield rich in Monacolin K
CN116083252A (en) * 2023-04-12 2023-05-09 北京一品堂医药科技有限公司 Red yeast rice fermentation preparation method of high-yield lipid-lowering natural Mo Lake forest K

Similar Documents

Publication Publication Date Title
CN113186121B (en) Caproic acid bacteria capable of utilizing various substrates and application thereof
CN112760271B (en) Process for producing clostridium butyricum through high-density fermentation under negative pressure condition and application
CN111218480A (en) Preparation method of solid-state fermentation red yeast rice with high yield of monacolin K
CN111172059A (en) Fermentation method for increasing number of bacillus coagulans spores
CN104987316A (en) Marine fungus-derived polyketone compound and application thereof in treatment of type 2 diabetes
CN103276018A (en) Method for producing lycopene by improving fermentation of Blakeslea trispora
CN113046253B (en) Culture method for improving heat resistance of kluyveromyces marxianus
CN104277989B (en) One plant of Saccharomyces cerevisiae and its application in fermenting and producing DPN
CN101805760A (en) Method for producing microbial oil comprising arachidonic acid
CN110551636B (en) Monascus purpureus MY-21 strain and application thereof
CN103880826A (en) Isobenzofuranone compounds as well as preparation method and application thereof
CN102648757A (en) Preparation method of fermented soybeans with alpha-glucosaccharase inhibitory activity
CN111205987A (en) Monascus, method for preparing strong aromatic Chinese spirit esterification liquid by using same and application thereof
CN105192592A (en) Laba beans with function of reducing blood glucose and processing method thereof
CN104480152A (en) Method for increasing content of docosahexaenoic acid in schizochytrium limacinum grease
CN104404103B (en) Zymotic fluid prepared by a kind of method and this method for improving Polysaccharide from Portulaca oleracea
CN107929331B (en) Application of paecilomyces coralloides extract in antidiabetic
CN102286565A (en) Preparation method of theaflavin monomer
CN106591155B (en) Fusarium oxysporum strain and its 3 β, 7 α are being prepared, the application in 15 α-trihydroxyandrost -5- alkene -17- ketone
CN114875104B (en) Mulberry leaf ferment stock solution and preparation method and application thereof
CN115521949B (en) Method for producing butyric acid by clostridium cellulosum fermentation and method for measuring content of fermentable sugar in wheat bran
CN110016489B (en) Natamycin fermentation process based on cell immobilization technology
CN104561135B (en) Method for producing aromatic substance by trichoderma reesei
CN114940951B (en) Sack-coating film yeast and application thereof in Xiaoqu fen-flavor wine base
CN112779302A (en) Method for preparing Antrodin C and Antrodin A by submerged culture of antrodia camphorata liquid

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20200602