CN112452506A - Production method of culture medium powder - Google Patents
Production method of culture medium powder Download PDFInfo
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- CN112452506A CN112452506A CN202011227775.4A CN202011227775A CN112452506A CN 112452506 A CN112452506 A CN 112452506A CN 202011227775 A CN202011227775 A CN 202011227775A CN 112452506 A CN112452506 A CN 112452506A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B02—CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
- B02C—CRUSHING, PULVERISING, OR DISINTEGRATING IN GENERAL; MILLING GRAIN
- B02C21/00—Disintegrating plant with or without drying of the material
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B02—CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
- B02C—CRUSHING, PULVERISING, OR DISINTEGRATING IN GENERAL; MILLING GRAIN
- B02C23/00—Auxiliary methods or auxiliary devices or accessories specially adapted for crushing or disintegrating not provided for in preceding groups or not specially adapted to apparatus covered by a single preceding group
- B02C23/08—Separating or sorting of material, associated with crushing or disintegrating
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- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/14—Calcium; Ca chelators; Calcitonin
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/16—Magnesium; Mg chelators
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/76—Undefined extracts from plants
Abstract
The invention provides a production method of culture medium powder, which comprises the following specific steps: s1: putting the raw materials of the culture medium into a ball mill, and ball-milling and uniformly mixing to form a primary mixed culture medium; s2: keeping uniform powder with fineness of 50-120 meshes; s3: adding the screened primary mixed culture matrix into a hammer mill; s7: the final mixed culture medium is dissolved into the bovine serum, the bovine serum is partially replaced by the microalgae powder, so that on one hand, amino acid, vitamin, inorganic substances, lipid substances, nucleic acid derivatives and the like contained in the bovine serum are mutually supplemented with high nutritional ingredients such as protein, polysaccharide, carotenoid and trace elements contained in the microalgae, and then other small amount of nutritional ingredients are added, the mixed ingredients are few, the step procedure is simple, and the pollution during the preparation of the culture medium is effectively avoided.
Description
Technical Field
The invention mainly relates to the technical field of culture medium production, in particular to a production method of culture medium powder.
Background
The culture medium is a nutrient medium prepared from different nutrient substances for the growth and reproduction of microorganisms, plants or animals (or tissues). Generally comprises several major substances such as carbohydrate, nitrogen-containing substances, inorganic salts (including trace elements), vitamins and water. The culture medium is not only a basic substance for providing nutrition for the cells and promoting the proliferation of the cells, but also a living environment for the growth and the propagation of the cells. The culture medium has many kinds, and can be divided into natural culture medium, synthetic culture medium and semi-synthetic culture medium according to the source of the preparation raw material; can be divided into solid culture medium, liquid culture medium and semisolid culture medium according to physical state; according to the culture function, the culture medium can be divided into a basic culture medium, a selective culture medium, an enriched culture medium, an identification culture medium and the like; according to the application range, the culture medium can be divided into a bacteria culture medium, an actinomycete culture medium, a yeast culture medium, a fungus culture medium and the like. The media is typically tested and pH adjusted after preparation, and sterilized, typically by autoclaving and filtration. The culture medium is rich in nutrients and is easy to be polluted or deteriorated. It should not be placed for a long time after being prepared, and is best to be prepared for use immediately.
The culture medium has different use requirements due to different prepared raw materials, and has slightly different storage and preservation aspects, and the common culture medium is easily polluted by bacteria or decomposed and deteriorated after being heated and absorbed with moisture, so the common culture medium needs to be preserved in a damp-proof, light-proof and cool place. For some media (such as tissue culture media) that need to be sterilized strictly, storage for a longer period of time must be done in a refrigerator at 3-6 ℃. Since the liquid culture medium is not easy to be stored for a long time, it is made into powder.
The existing culture medium is cultured by using bovine serum as a main component of the culture medium, which provides basic nutrients: amino acids, vitamins, inorganic substances, lipid substances, nucleic acid derivatives and the like are essential substances for cell growth, but excessive serum contains substances which are toxic to cells, such as polyamine oxidase, complement, antibodies, bacterial toxins and the like, which affect cell growth and even cause cell death, and the serum is difficult to source and expensive.
Disclosure of Invention
The invention mainly provides a production method of culture medium powder, which is used for solving the technical problems in the background technology.
The technical scheme adopted by the invention for solving the technical problems is as follows: a production method of culture medium powder comprises the following specific steps:
s1: according to the formula of the culture medium, putting the raw materials of the culture medium into a ball mill, and carrying out ball milling and mixing uniformly to form a primary mixed culture medium;
s2: sieving the primary mixed culture matrix through a first sieve and a second sieve in sequence after uniform mixing, removing super-large particles and super-small particles in the primary mixed culture matrix, and reserving uniform powder with the fineness of 50-120 meshes;
s3: adding the screened primary mixed culture matrix into a hammer mill, adjusting the rotating speed of the hammer mill to 3000-12000 r/min, and controlling the temperature of the materials to be 5-30 ℃ in the process of milling, wherein the milling time is 20-30 min;
s4: cooling for 20-30 minutes, sieving again through a third screen, reserving uniform powder with the fineness of 80-100 meshes, and finally mixing the crushed raw materials for 20-40 minutes to obtain a powder cell culture medium;
s5: crushing the sterilized microalgae solid by a hammer mill, screening to form microalgae powder, and uniformly mixing;
s6: uniformly mixing the microalgae powder and the powder type cell culture medium to obtain a final mixed culture medium;
s7: taking a dried and sterilized beaker, measuring 50ml of bovine serum, dissolving the final mixed culture medium into the bovine serum, adding distilled water during the period, stirring, and adjusting the pH to 4.5-4.9 by adopting HCl or NaOH to finish the preparation of the culture medium liquid;
s8: and after the culture medium liquid is prepared, subpackaging the culture medium liquid into sterile containers, sealing, performing steam sterilization, subpackaging and cooling to obtain the solid culture medium.
The culture medium formula comprises inorganic salt components, vitamin components and carbon and nitrogen source components, wherein the inorganic salt components are one or more of sodium chloride, calcium chloride, magnesium sulfate, calcium nitrate tetrahydrate, anhydrous sodium dihydrogen phosphate, potassium chloride and anhydrous potassium dihydrogen phosphate.
The vitamin component is one or more of vitamin B1, vitamin B2, vitamin A, vitamin B12, vitamin H, folic acid, D-calcium pantothenate, glutathione, choline chloride, pyridoxal hydrochloride, linoleic acid and vitamin C.
The carbon and nitrogen source component is one or more of anhydrous glucose, pyruvate and plant protein hydrolysate.
In step S5, the sterilization method of the microalgae fixture is irradiation sterilization, including ultraviolet, chi-ray or gamma-ray irradiation.
In the step S6, mixing the microalgae powder and the powder cell culture medium for 20-35min to obtain a final mixed culture medium, and cooling for 3 h.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the technical scheme, the bovine serum is partially replaced by the microalgae powder, so that on one hand, amino acids, vitamins, inorganic substances, lipid substances, nucleic acid derivatives and the like contained in the bovine serum are mutually supplemented with high nutritional ingredients such as proteins, polysaccharides, carotenoids, trace elements and the like contained in the microalgae, and then other small amount of nutritional ingredients are added, the mixed ingredients are few, the step procedure is simple, and the pollution during the preparation of the culture medium is effectively avoided.
2. The microalgae adopted in the method is low-grade aquatic plants which are extremely wide in distribution and rich in nutrition, the preparation method is simple, the raw materials are easy to obtain, the method is safe and reliable, the cost is low, the survival rate and the cell activity of the organisms to be cultured can be obviously improved, the content of bovine serum is reduced, and the cost is saved.
3. Through the technical scheme, the method can produce stable and uniform cell culture media, has small occupied area, can realize continuous production, has large yield, small batch difference and stable product properties, and can save production cost, production time and investment capital.
Detailed Description
While the present invention will be described more fully hereinafter for purposes of facilitating an understanding thereof, the present invention may be embodied in many different forms and is not limited to the embodiments described herein, but rather, these embodiments are provided so that this disclosure will be thorough and complete.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may be present, and when an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present, as the terms "vertical", "horizontal", "left", "right" and the like are used herein for descriptive purposes only.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the knowledge of the terms used herein in the specification of the present invention is for the purpose of describing particular embodiments and is not intended to limit the present invention, and the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The first embodiment is as follows:
a production method of culture medium powder comprises the following specific steps:
s1: according to the formula of the culture medium, putting the raw materials of the culture medium into a ball mill, and carrying out ball milling and mixing uniformly to form a primary mixed culture medium;
s2: sieving the primary mixed culture matrix through a first sieve and a second sieve in sequence after uniform mixing, removing super-large particles and super-small particles in the primary mixed culture matrix, and reserving uniform powder with the fineness of 50-120 meshes;
s3: adding the screened primary mixed culture matrix into a hammer mill, adjusting the rotating speed of the hammer mill to 3000-12000 r/min, and controlling the temperature of the materials to be 5-30 ℃ in the process of milling, wherein the milling time is 20-30 min;
s4: cooling for 20-30 minutes, sieving again through a third screen, reserving uniform powder with the fineness of 80-100 meshes, and finally mixing the crushed raw materials for 20-40 minutes to obtain a powder cell culture medium;
s5: crushing the sterilized microalgae solid by a hammer mill, screening to form microalgae powder, and uniformly mixing;
s6: uniformly mixing the microalgae powder and the powder type cell culture medium to obtain a final mixed culture medium;
s7: taking a dried and sterilized beaker, measuring 50ml of bovine serum, dissolving the final mixed culture medium into the bovine serum, adding distilled water during the period, stirring, and adjusting the pH to 4.5-4.9 by adopting HCl or NaOH to finish the preparation of the culture medium liquid;
s8: and after the culture medium liquid is prepared, subpackaging the culture medium liquid into sterile containers, sealing, performing steam sterilization, subpackaging and cooling to obtain the solid culture medium.
The culture medium formula comprises inorganic salt components, vitamin components and carbon and nitrogen source components, wherein the inorganic salt components are one or more of sodium chloride, calcium chloride, magnesium sulfate, calcium nitrate tetrahydrate, anhydrous sodium dihydrogen phosphate, potassium chloride and anhydrous potassium dihydrogen phosphate.
The vitamin component is one or more of vitamin B1, vitamin B2, vitamin A, vitamin B12, vitamin H, folic acid, D-calcium pantothenate, glutathione, choline chloride, pyridoxal hydrochloride, linoleic acid and vitamin C.
The carbon nitrogen source component is one or more of anhydrous glucose, pyruvate and plant protein hydrolysate.
In step S5, the sterilization method of the microalgae fixture is irradiation sterilization, including ultraviolet, chi-ray or gamma-ray irradiation.
In step S6, mixing microalgae powder and powdered cell culture medium for 20-35min to obtain final mixed culture medium, and cooling for 3 hr.
Example two:
a production method of culture medium powder comprises the following specific steps:
s1: selecting calcium nitrate tetrahydrate, anhydrous sodium dihydrogen phosphate and potassium chloride in inorganic salt components, selecting glutathione, choline chloride and pyridoxal hydrochloride in vitamin components, selecting anhydrous glucose, pyruvate and plant protein hydrolysate in carbon and nitrogen source components, and putting the anhydrous glucose, pyruvate and plant protein hydrolysate in a ball mill for ball milling and uniformly mixing to form a primary mixed culture matrix;
s2: sieving the primary mixed culture matrix through a first sieve and a second sieve in sequence after uniform mixing, removing super-large particles and super-small particles in the primary mixed culture matrix, and reserving uniform powder with the fineness of 50-120 meshes;
s3: adding the screened primary mixed culture matrix into a hammer mill, adjusting the rotating speed of the hammer mill to 8000r/min, and controlling the temperature of the material to be 15 ℃ in the process of milling, wherein the milling time is 30 min;
s4: cooling for 30 minutes, sieving again through a third screen, reserving uniform powder with the fineness of 80-100 meshes, and finally mixing the crushed raw materials for 20-40 min to obtain a powder cell culture medium;
s5: crushing the sterilized microalgae solid by a hammer mill, screening to form microalgae powder, and uniformly mixing;
s6: uniformly mixing the microalgae powder and the powder type cell culture medium to obtain a final mixed culture medium;
s7: taking a dried and sterilized beaker, measuring 50ml of bovine serum, dissolving the final mixed culture medium into the bovine serum, adding distilled water during the period, stirring, and adjusting the pH to 4.9 by adopting HCl or NaOH to finish the preparation of the culture medium liquid;
s8: and after the culture medium liquid is prepared, subpackaging the culture medium liquid into sterile containers, sealing, performing steam sterilization, subpackaging and cooling to obtain the solid culture medium.
Example three:
a production method of culture medium powder comprises the following specific steps:
s1: selecting sodium chloride, calcium chloride and magnesium sulfate in inorganic salt components, selecting vitamin B1, vitamin B2, vitamin A, vitamin B12, vitamin H, folic acid and D-calcium pantothenate in vitamin components, selecting anhydrous glucose, pyruvate and plant hydrolyzed protein in carbon-nitrogen source components, putting the anhydrous glucose, pyruvate and plant hydrolyzed protein in a ball mill, and uniformly mixing to form a primary mixed culture substrate;
s2: sieving the primary mixed culture matrix through a first sieve and a second sieve in sequence after uniform mixing, removing super-large particles and super-small particles in the primary mixed culture matrix, and reserving uniform powder with the fineness of 50-120 meshes;
s3: adding the screened primary mixed culture matrix into a hammer mill, adjusting the rotation speed of the hammer mill to 10000r/min, and controlling the temperature of the material to be 20 ℃ in the process of milling, wherein the milling time is 20 min;
s4: cooling for 30 minutes, sieving again through a third screen, reserving uniform powder with the fineness of 80-100 meshes, and finally mixing the crushed raw materials for 20-40 min to obtain a powder cell culture medium;
s5: crushing the sterilized microalgae solid by a hammer mill, screening to form microalgae powder, and uniformly mixing;
s6: uniformly mixing the microalgae powder and the powder type cell culture medium to obtain a final mixed culture medium;
s7: taking a dried and sterilized beaker, measuring 50ml of bovine serum, dissolving the final mixed culture medium into the bovine serum, adding distilled water during the period, stirring, and adjusting the pH to 4.9 by adopting HCl or NaOH to finish the preparation of the culture medium liquid;
s8: and after the culture medium liquid is prepared, subpackaging the culture medium liquid into sterile containers, sealing, performing steam sterilization, subpackaging and cooling to obtain the solid culture medium.
The invention has been described in an illustrative manner, and it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description, since such modifications are intended to be included within the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. A production method of culture medium powder is characterized by comprising the following specific steps:
s1: according to the formula of the culture medium, putting the raw materials of the culture medium into a ball mill, and carrying out ball milling and mixing uniformly to form a primary mixed culture medium;
s2: sieving the primary mixed culture matrix through a first sieve and a second sieve in sequence after uniform mixing, removing super-large particles and super-small particles in the primary mixed culture matrix, and reserving uniform powder with the fineness of 50-120 meshes;
s3: adding the screened primary mixed culture matrix into a hammer mill, adjusting the rotating speed of the hammer mill to 3000-12000 r/min, and controlling the temperature of the materials to be 5-30 ℃ in the process of milling, wherein the milling time is 20-30 min;
s4: cooling for 20-30 minutes, sieving again through a third screen, reserving uniform powder with the fineness of 80-100 meshes, and finally mixing the crushed raw materials for 20-40 minutes to obtain a powder cell culture medium;
s5: crushing the sterilized microalgae solid by a hammer mill, screening to form microalgae powder, and uniformly mixing;
s6: uniformly mixing the microalgae powder and the powder type cell culture medium to obtain a final mixed culture medium;
s7: taking a dried and sterilized beaker, measuring 50ml of bovine serum, dissolving the final mixed culture medium into the bovine serum, adding distilled water during the period, stirring, and adjusting the pH to 4.5-4.9 by adopting HCl or NaOH to finish the preparation of the culture medium liquid;
s8: and after the culture medium liquid is prepared, subpackaging the culture medium liquid into sterile containers, sealing, performing steam sterilization, subpackaging and cooling to obtain the solid culture medium.
2. The method for producing a culture medium powder according to claim 1, wherein the culture medium formulation comprises inorganic salt components, vitamin components and carbon-nitrogen source components, and the inorganic salt components comprise one or more of sodium chloride, calcium chloride, magnesium sulfate, calcium nitrate tetrahydrate, anhydrous sodium dihydrogen phosphate, potassium chloride and anhydrous potassium dihydrogen phosphate.
3. The method for producing a medium powder according to claim 2, wherein the vitamin component is one or a combination of more than one of vitamin B1, vitamin B2, vitamin A, vitamin B12, vitamin H, folic acid, calcium D-pantothenate, glutathione, choline chloride, pyridoxal hydrochloride, linoleic acid, and vitamin C.
4. The method for producing a culture medium powder according to claim 1, wherein the carbon and nitrogen source component is one or a combination of more than one of anhydrous glucose, pyruvate and plant protein hydrolysate.
5. The method for producing a culture medium powder as claimed in claim 1, wherein the sterilization method of the microalgae-fixing object in step S5 is irradiation sterilization method, including ultraviolet, chi-ray or gamma-ray irradiation.
6. The method for producing culture medium powder as claimed in claim 1, wherein in step S6, the microalgae powder and the powdered cell culture medium are mixed for 20-35min to obtain a final mixed culture medium, and cooled for 3 h.
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