Haematococcus pluvialis culture medium and haematococcus pluvialis culture method

Abstract

The invention relates to the technical field of biological culture for producing high-added-value products from microalgae, in particular to a haematococcus pluvialis culture medium and a haematococcus pluvialis culture method. The haematococcus pluvialis culture medium is mainly based on a BG11 culture medium, an organic carbon source acetic acid for value-added growth of haematococcus pluvialis is added on the basis of the BG11 culture medium, and meanwhile, the addition amount of a chelating agent, biotin and various trace elements in the culture medium are adjusted to inhibit the growth of bacteria and promote the growth of target organism haematococcus pluvialis cells. The biomass of the haematococcus pluvialis is obviously improved, and the production economic benefits can be efficiently improved in the large-scale production of the haematococcus pluvialis.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, in particular to a Haematococcus pluvialis culture medium and a Haematococcus pluvialis culture method. Background Art

[0002] Haematococcus pluvialis, also known as lake-dwelling Haematococcus or lake-dwelling blood algae, is a ubiquitous green algae belonging to the Volvoxales order, Haematococcus family. It occurs widely in nature under favorable growth conditions. Haematococcus pluvialis is an important freshwater, single-celled green algae with a high astaxanthin content, accounting for approximately 1-7% of its dry cell weight, significantly higher than that of Phaffia rhodozyma. Astaxanthin has anti-hypertensive, anti-diabetic, and anti-cancer properties. Therefore, extracting astaxanthin from this microalgae holds great promise and has become a research hotspot for natural astaxanthin production internationally in recent years. Astaxanthin is produced by cultivating high-quality Haematococcus pluvialis strains and harvesting spores, which are then broken and dried.

[0003] Existing algae cultivation mainly uses BG11 as the basic culture medium, which can meet the needs in the algae exploration and research and development stage. However, in the large-scale cultivation of Haematococcus pluvialis, the cultivation of Haematococcus pluvialis in BG11 culture medium fails to meet production needs. The main problem is that when Haematococcus pluvialis is cultivated in BG11 culture medium, the division and growth rate are slow, and the cultivation time required is long. Prolonging the cultivation time will cause the algae species to be contaminated during the cultivation process.

[0004] Prior art CN118028111B discloses a method for culturing Haematococcus pluvialis using a modified Bold basal medium supplemented with resveratrol, 5-aminolevulinic acid, and catechol. Cultivation is carried out in an incubator and is not suitable for large-scale cultivation. Prior art CN115627237A discloses a method for increasing astaxanthin production in Haematococcus pluvialis by coupling proline with multiple stresses. This method increases astaxanthin production but is prone to cell damage under high-salt conditions and increases costs.

[0005] Therefore, developing a high-efficiency Haematococcus pluvialis culture medium that reduces culture costs and increases algal cell growth rate is a technical problem we need to solve. Summary of the Invention

[0006] The present application provides a culture medium for Haematococcus pluvialis and a culture method for Haematococcus pluvialis. The culture medium is mainly based on BG11 culture medium, to which acetic acid, an organic carbon source suitable for the growth of Haematococcus pluvialis, is added. At the same time, the amount of chelating agent added to the culture medium is adjusted to inhibit bacterial growth and promote the growth of target organism Haematococcus pluvialis cells. Secondly, multiple trace elements required for the division and growth of Haematococcus pluvialis cells are added. In combination with the provision of compressed air and carbon dioxide, sufficient artificial light required for the growth of Haematococcus pluvialis cells is supplemented under a set environment and lighting to achieve the temperature and humidity required for the growth of Haematococcus pluvialis cells, and the culture area is maintained at Class D cleanliness.

[0007] The problem of slow algal cell division and proliferation, low efficiency and inability to mass produce is solved. During the proliferation growth stage of Haematococcus pluvialis, the inoculation concentration of algae is not less than 5×10 4 The number of cells / mL can be increased to 50×10 cells / mL under the preset ventilation level environment conditions and culture time of 12 days. 4 / mL, compared with the culture conditions of BG11 medium, the cell division and proliferation advantages of Haematococcus pluvialis are very significant, which can effectively improve the production economic benefits in the large-scale production of Haematococcus pluvialis.

[0008] The first embodiment of the present application provides a culture medium for Haematococcus pluvialis, wherein the culture medium is composed of the following components: sodium nitrate: 800-1500 mg / L, magnesium sulfate heptahydrate: 75-100 mg / L, dipotassium hydrogen phosphate trihydrate: 40-100 mg / L, citric acid monohydrate: 5.5-6.6 mg / L, calcium chloride: 25-80 mg / L, ferric citrate ammonium: 6-10 mg / L, EDTA-2Na: 1 ~1.5mg / L, boric acid: 2~3mg / L, manganese sulfate: 1.5~2mg / L, zinc sulfate heptahydrate: 0.2~0.23mg / L, sodium molybdate dihydrate: 0.3~0.4mg / L, copper sulfate pentahydrate: 0.05~0.1mg / L, cobalt chloride hexahydrate: 0.04~0.5mg / L, glacial acetic acid: 40~45mL / L, sodium hydroxide: 20~25mg / L, chelating agents and trace elements.

[0009] Preferably, the trace elements include biotin, vitamin B11 and vitamin B12; and the chelating agent is EDTA-2Na.

[0010] Preferably, the added amounts of biotin, vitamin B11 and vitamin B12 are: biotin: 0.01-0.05 mg / L, vitamin B11: 0.1-0.2 mg / L, vitamin B12: 0.001-0.005 mg / L, and the added amount of EDTA-2Na is 1-1.5 mg / L.

[0011] Preferably, the culture medium is used for the cultivation and large-scale breeding of Haematococcus pluvialis.

[0012] Another aspect of the present application provides a method for culturing Haematococcus pluvialis, the method comprising preparing a culture medium for Haematococcus pluvialis, inoculating the culture medium, and amplifying the culture medium;

[0013] The steps are:

[0014] (1) The method for preparing the Haematococcus pluvialis culture medium comprises:

[0015] a. Raw material preparation and dissolution: First, a large number of elements (sodium nitrate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate trihydrate, citric acid monohydrate, calcium chloride, ferric citrate, sodium hydroxide) were dissolved in distilled water, stirred until fully dissolved and mixed to obtain a stock solution;

[0016] b. Adjust pH and volume: Then add the remaining elements and trace elements to the above solution, adjust the pH to 7.3-7.9, and dilute to 4L with distilled water to obtain Haematococcus pluvialis culture medium;

[0017] c. Sterilization: The prepared Haematococcus pluvialis culture medium is transferred into a container, sealed, and then sterilized by high-pressure steam at a sterilization temperature of 121°C for 30 min to obtain a sterilized Haematococcus pluvialis culture medium;

[0018] d. Cooling and storage: Cool the sterilized Haematococcus pluvialis culture medium to room temperature and store at 4-8°C;

[0019] (2) Inoculation and expansion culture: Under the preset culture conditions and lighting, select well-growing Haematococcus pluvialis as the seed liquid for inoculation. When inoculating, slowly pour the seed liquid into the culture vessel to ensure that the algae seeds are evenly distributed in the culture medium for expansion culture.

[0020] Preferably, the algae species is Haematococcus pluvialis in the value-added growth stage.

[0021] Preferably, the culture conditions are: temperature 23° C., humidity 45%, the culture area is a Class D clean area, and the algae are shaken twice a day; the culture lasts 5 to 20 days, and the ventilation ratio is 1% to 3% of compressed air and carbon dioxide.

[0022] Preferably, the inoculated algae concentration is not less than 5×10 4 The number of cells per mL.

[0023] Preferably, the pH in step (1) of the production method is 7.3 to 7.9, and the carbon dioxide is used to adjust the pH during culture.

[0024] Preferably, the culture illumination is as follows: 1 to 3 days after inoculation, the culture is in a weak light environment with an illumination of 800 Lux, 4 to 8 days after cultivation, the culture is in an illumination of 3200 Lux, and 9 to 12 days after cultivation, the culture is in a strong light environment with an illumination of 4800 Lux.

[0025] The beneficial effects of the embodiments of the present application are:

[0026] (1) During the growth stage of Haematococcus pluvialis, the inoculation concentration of algae should be no less than 5×10 4 The number of cells / mL can be increased to 50×10 cells / mL under the preset ventilation level environment conditions and culture time of 12 days. 4 / mL, compared with the culture conditions of BG11 medium, the cell division and proliferation advantages of Haematococcus pluvialis are very significant, which can effectively improve the production economic benefits in the large-scale production of Haematococcus pluvialis.

[0027] (2) Organic carbon source Acetic acid provides a carbon source in the culture of Haematococcus pluvialis. Haematococcus pluvialis needs a carbon source to synthesize organic substances in the cell, such as carbohydrates, proteins, fats, etc. during its growth. As an organic carbon source, acetic acid can be absorbed and utilized by Haematococcus pluvialis cells, providing the necessary carbon skeleton for its growth and metabolism, thereby promoting the proliferation of algal cells and the increase of biomass, and promoting the synthesis and accumulation of astaxanthin; adding acetic acid can affect the expression of pigment synthesis-related genes in algal cells and increase the activity of enzymes related to astaxanthin synthesis, so that Haematococcus pluvialis accumulates more astaxanthin during its growth, improving the growth of shrimp. Acetic acid can affect the physiological state of Haematococcus pluvialis cells and help maintain the normal metabolism and function of the cells. It can regulate the pH value in the cells, keep the intracellular environment stable, and provide suitable conditions for various enzymatic reactions in the cells. At the same time, it also participates in the energy metabolism process in the cells and provides energy for the growth and activity of the cells. Acetic acid can enhance the resistance of Haematococcus pluvialis to these environmental stresses, regulate the osmotic pressure in the cells, stabilize the cell membrane structure, reduce the damage to the cells caused by stress conditions, and enable Haematococcus pluvialis to maintain a good growth state in adverse environments, thereby improving its survival and adaptability.

[0028] (3) Biotin participates in the regulation of the activity of some photosynthesis-related enzymes in Haematococcus pluvialis cells, which helps to optimize the photosynthesis process. It can affect the synthesis and stability of photosynthetic pigments, enabling algal cells to absorb light energy more effectively and convert it into chemical energy, providing sufficient energy for cell growth and metabolism, thereby improving photosynthesis efficiency and promoting the growth and material accumulation of Haematococcus pluvialis. It participates in the signal transduction process within cells, can regulate physiological activities such as cell growth, differentiation and reproduction, and enable Haematococcus pluvialis cells to maintain a good physiological state, which is beneficial to their growth and the synthesis and accumulation of astaxanthin, and improves stress resistance.

[0029] (4) Vitamin B11 and vitamin B112 have a synergistic effect. Vitamin B11 provides one-carbon units for DNA synthesis, participates in the synthesis of purines and pyrimidines, and is the key material basis for DNA replication during cell division. Vitamin B12 participates in intracellular methyl transfer reactions, ensuring the normal progress of some important methylation reactions in the cell, which is crucial for maintaining the normal physiological function of the cell and promoting cell division. For example, vitamin B12 can transfer methyl groups from methyltetrahydrofolate to homocysteine to generate methionine and tetrahydrofolate. Tetrahydrofolate is an important form of vitamin B11. In this way, vitamin B12 ensures the normal metabolic cycle of one-carbon units involved in vitamin B11. The two synergistically promote the cell division and growth of Haematococcus pluvialis, increasing the number and biomass of algal cells.

[0030] (5) Vitamin B11 participates in the metabolism of various amino acids, such as the mutual conversion of serine and glycine, providing raw materials for protein synthesis. Vitamin B12 plays a role in fatty acid metabolism, participates in propionic acid metabolism to generate succinyl coenzyme A, enters the tricarboxylic acid cycle to provide energy for cells, and also provides a material basis for lipid synthesis. The synergistic effect of the two makes the protein and lipid metabolism in Haematococcus pluvialis cells more coordinated, providing sufficient substances and energy for physiological processes such as cell growth and astaxanthin synthesis, and optimizing the overall metabolic function of the cells.

[0031] (6) Vitamin B11 participates in the synthesis of enzymes and proteins related to photosynthesis, such as chlorophyll-binding protein, which is crucial for the synthesis of chlorophyll and the stability of the photosynthetic system. Vitamin B12 affects the synthesis and stability of photosynthetic pigments, participates in regulating the synthesis and modification of photosynthesis-related proteins, and helps to improve the efficiency of the photosynthetic system. The two work together to improve the photosynthetic efficiency of Haematococcus pluvialis from different aspects, enabling algal cells to use light energy more effectively, converting carbon dioxide and water into organic matter and oxygen, and providing sufficient carbon source and energy for cell growth and astaxanthin synthesis; Vitamin B11 is involved in regulating the antioxidant system in cells, enhancing Haematococcus pluvialis's resistance to oxidative stress and reducing the damage to cells by free radicals. Vitamin B12 helps maintain the osmotic pressure balance in cells and helps cells maintain normal morphology and function under stress conditions such as high salt and drought. When Haematococcus pluvialis faces various environmental stresses, vitamin B11 and vitamin B12 can enhance the stress resistance of algal cells from multiple angles such as anti-oxidation and maintaining cell osmotic pressure, allowing Haematococcus pluvialis to maintain a good growth and metabolic state in adverse environments, which is beneficial to the synthesis and accumulation of astaxanthin. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram of a Haematococcus pluvialis culture medium after sterilization according to the present invention;

[0033] Figure 2 Schematic diagram comparing the culture results of a Haematococcus pluvialis culture medium of the present invention and BG11;

[0034] Figure 3 This is a schematic diagram of the daily growth curve of algal cell concentration of the present invention;

[0035] Figure 4 This is a graph showing the antibacterial results of adding a chelating agent and potassium persulfate to the present invention;

[0036] Figure 5 Schematic diagram of the diameter of the inhibition zone of the chelating agent and potassium persulfate at different concentrations of the present invention;

[0037] Figure 6 This is a graph showing the antibacterial results of adding chelating agents and trace elements to the present invention;

[0038] Figure 7 This is a schematic diagram of the diameter of the inhibition zone when adding different concentrations of chelating agents and trace elements in the present invention. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0040] For a better understanding of the present invention and not to limit the scope of the present invention, all numbers expressing amounts, percentages, and other numerical values used in this application should be understood as being modified by the word "about" in all cases, and each numerical parameter should at least be considered to be obtained in light of the number of reported significant digits and by ordinary rounding techniques.

[0041] All components used in the present invention can be purchased commercially.

[0042] Comparative Example: Preparation of BG11 Medium

[0043] (1) The composition is shown in Table 1:

[0044] Table 1BG11 liquid culture medium formula

[0045]

[0046]

[0047] (2) Preparation method, the steps are:

[0048] a. Raw material preparation and dissolution: First, sodium nitrate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate trihydrate, citric acid monohydrate, calcium chloride, ferric citrate, EDTA-2Na, boric acid, manganese sulfate, and sodium carbonate were dissolved in distilled water, stirred with a glass rod to dissolve quickly and fully, and mixed to obtain a stock solution;

[0049] b. Adjust pH and fix volume: Next, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt chloride are added to the above stock solution, stirred evenly, and the pH is adjusted to 7.3-7.9. The volume is fixed to 5 L with distilled water and mixed evenly to obtain BG11 medium.

[0050] Example 1: Haematococcus pluvialis culture medium

[0051] (1) The composition is shown in Table 2:

[0052] Table 2 Haematococcus pluvialis culture medium formula

[0053]

[0054]

[0055] (2) Preparation method, the steps are:

[0056] a. Raw material preparation and dissolution: First, sodium nitrate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate trihydrate, citric acid monohydrate, calcium chloride, ferric citrate, and sodium hydroxide were dissolved in distilled water, stirred until fully dissolved to obtain a stock solution;

[0057] b. Adjust pH and volume: Then add the remaining elements and trace elements to the above solution, adjust the pH to 7.3-7.9, and dilute to 4L with distilled water to obtain Haematococcus pluvialis culture medium;

[0058] c. Sterilization: Transfer the prepared Haematococcus pluvialis culture medium into a container, seal it, and sterilize it with high-pressure steam at a temperature of 121°C for 30 minutes to obtain the sterilized Haematococcus pluvialis culture medium. Figure 1 As shown;

[0059] d. Cooling and storage: Cool the sterilized Haematococcus pluvialis culture medium to room temperature and store at 4-8°C;

[0060] Test Example 1: Light Adjustment Comparison

[0061] (1) Experimental materials: BG11 culture medium of comparative example, Haematococcus pluvialis culture medium of Example 1;

[0062] (2) Experimental methods:

[0063] a. Inoculation culture: Select the value-added growth stage of Haematococcus pluvialis for inoculation, and inoculate them into the comparative example BG11 medium and the Haematococcus pluvialis medium of Example 1; initial algal cell density: 5×10 4 / mL, the culture volume is 5L, the ventilation is compressed air and the ratio of carbon dioxide is 1% to 3%, and the pH range is 7.3 to 7.9, which is controlled by the amount of added carbon dioxide;

[0064] b. Environment: Cultivate under low light conditions of 800 Lux for 1-3 days after inoculation, 3200 Lux for 4-8 days, and strong light conditions of 4800 Lux for 9-12 days. Maintain an ambient temperature of 23°C and a humidity of 45% in a Class D cleanroom. Shake the algae twice daily.

[0065] (3) Experimental data: The comparison of light regulation between BG11 culture medium and Haematococcus pluvialis culture medium is shown in Table 3:

[0066] Table 3 Comparison of light regulation between BG11 culture medium and Haematococcus pluvialis culture medium

[0067]

[0068] (4) Experimental results: It was verified in long-term large-scale cultivation that algae species cultured in Haematococcus pluvialis culture medium can adapt to strong light sources more quickly when cultured in a clean area; compared with algae species cultured in traditional BG11 culture medium, the light resistance of algae species cultured in Haematococcus pluvialis culture medium is increased by about one-fold; algae species cultured under high light intensity in a clean area can better adapt to the outdoor strong light environment in the next stage of outdoor cultivation.

[0069] Test Example 2: Comparison of algal cell division and proliferation

[0070] (1) Experimental materials: BG11 culture medium of comparative example, Haematococcus pluvialis culture medium of Example 1;

[0071] (2) Experimental methods:

[0072] a. Inoculation culture: Select the value-added growth stage of Haematococcus pluvialis for inoculation, and inoculate them into the comparative example BG11 medium and the Haematococcus pluvialis medium of Example 1; initial algal cell density: 5×10 4 / mL, the culture volume is 5L, the ventilation is compressed air and the ratio of carbon dioxide is 1% to 3%, and the pH range is 7.3 to 7.9, which is controlled by the amount of added carbon dioxide;

[0073] b. Environment: Cultivate under low light conditions of 800 Lux for 1-3 days after inoculation, 3200 Lux for 4-8 days, and strong light conditions of 4800 Lux for 9-12 days. Maintain an ambient temperature of 23°C and a humidity of 45% in a Class D cleanroom. Shake the algae twice daily.

[0074] (3) Experimental data: The number of algae cells is shown in Table 4, the appearance comparison is shown in Figure 2, and the daily growth curve of algae cell concentration is shown in Figure 3 As shown:

[0075] Table 4 Algal cell concentrations of BG11 and Haematococcus pluvialis culture media under the same conditions

[0076]

[0077] (4) Experimental results: Table 4 shows that under the same environment and the same initial algal cell concentration, the algal cell concentration of BG11 and Haematococcus pluvialis culture medium was greater than that of BG11 culture medium when cultured for 12 days. Figure 2 The different culture media shown were cultured for 12 days at the same inoculation concentration and environment. The algae liquid color of the traditional BG11 culture medium was green, while the algae liquid color of the culture medium for Haematococcus pluvialis was dark green. The comparison results showed that the concentration of Haematococcus pluvialis algae cells cultured in the Haematococcus pluvialis culture medium was significantly higher than that of algae cells cultured in the traditional BG11 culture medium. Figure 3 As shown, the daily growth rate curve of the algal cell concentration cultured in Haematococcus pluvialis culture medium is significantly higher than that of the algal cell concentration cultured in BG11 culture medium.

[0078] Test Example 3 Antibacterial Experiment - Potassium Persulfate

[0079] (1) Experimental materials: nutrient agar medium, drug-sensitive paper, chelating agent (disodium ethylenediaminetetraacetic acid), potassium persulfate (K2S2O8);

[0080] (2) Experimental method: The nutrient agar medium was inoculated with the liquid of Haematococcus pluvialis cultured outdoors, and the experiment was conducted using drug-sensitive paper strips soaked in chelating agents and antibacterial agents. The addition amount of disodium EDTA was 0.002 g / L, and the addition amount of potassium persulfate was 50 ppm, 100 ppm, 150 ppm, and 200 ppm, respectively. Four groups of experiments were repeated.

[0081] (3) Experimental data: The diameter of the inhibition zone and the antibacterial effect are shown in Table 5. The experimental results are shown in Figure 5. Figure 4 As shown, the schematic diagram of the inhibition zone diameter data size is as follows Figure 5 As shown;

[0082] Table 5 Antibacterial zone diameter data and antibacterial effect

[0083]

[0084] (4) Experimental results: From Table 5, Figure 4 and Figure 5 The diameter of the inhibition zone shown in the figure shows that the experimental results of different addition amounts of potassium persulfate (K2S2O8) are all drug-resistant.

[0085] Test Example 4 Antibacterial Experiment-Biotin

[0086] (1) Experimental materials: nutrient agar medium, drug-sensitive paper, chelating agent (disodium ethylenediaminetetraacetic acid), biotin (C 10 H 16 N2O3S);

[0087] (2) Experimental method: The nutrient agar medium was inoculated with the liquid of Haematococcus pluvialis cultured outdoors, and the experiment was conducted using drug-sensitive paper sheets soaked in chelating agents and antibacterial agents. The addition amount of disodium ethylenediaminetetraacetic acid was 0.002 g / L, and biotin (C 10 H 16 The addition amounts of N2O3S) were 50ppm, 100ppm, 150ppm and 200ppm respectively, and 4 groups of experiments were repeated.

[0088] (3) Experimental data: The diameter of the inhibition zone and the antibacterial effect are shown in Table 6. The experimental results are shown in Figure 6. Figure 6 As shown, the schematic diagram of the inhibition zone diameter data size is as follows Figure 7 As shown;

[0089] Table 6 Antibacterial zone diameter data and antibacterial effect

[0090]

[0091] (4) Experimental results: According to Table 6, Figure 6 and Figure 7 As shown, the diameter of the inhibition zone when the biotin content is 200 ppm is 32 mm. Compared with 50 ppm, 100 ppm, and 150 ppm, the diameter of the inhibition zone when the addition amount is 200 ppm is the largest, and the antibacterial effect is sensitive. Comparing the results of Test Example 3 with those of Test Example 4, the antibacterial effect of adding 200 ppm of biotin is significantly better than that of adding potassium persulfate, so the antibacterial effect of the Haematococcus pluvialis culture medium of the present invention is better.

[0092] In summary, the present invention provides a culture medium and a method for cultivating Haematococcus pluvialis, wherein the inoculated algae concentration is not less than 5×10 4 After 12 days of culture, the cell number can increase to 50×10 4 / mL or more, compared with the BG11 culture medium under the same culture conditions, the cell proliferation is significantly better than the traditional BG11 culture medium, the advantage of Haematococcus pluvialis cell division and proliferation is very significant, which can effectively improve the production economic benefits in the large-scale production of Haematococcus pluvialis.

[0093] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A culture medium for Haematococcus pluvialis, characterized in that: The culture medium consists of the following components: Sodium nitrate: 800-1500 mg / L, magnesium sulfate heptahydrate: 75-100 mg / L, dipotassium hydrogen phosphate trihydrate: 40-100 mg / L, citric acid monohydrate: 5.5-6.6 mg / L, calcium chloride: 25-80 mg / L, ferric citrate ammonium: 6-10 mg / L, boric acid: 2-3 mg / L, manganese sulfate: 1.5-2 mg / L, zinc sulfate heptahydrate: 0.2-0.23 mg / L, sodium molybdate dihydrate: 0.3-0.4 mg / L, copper sulfate pentahydrate: 0.05-0.1 mg / L, cobalt chloride hexahydrate: 0.04-0.5 mg / L, glacial acetic acid: 40-45 mL / L, sodium hydroxide: 20-25 mg / L, chelating agents and trace elements.

2. A culture medium for Haematococcus pluvialis according to claim 1, characterized in that The trace elements include biotin, vitamin B11 and vitamin B12; and the chelating agent is EDTA-2Na.

3. A culture medium for Haematococcus pluvialis as claimed in claim 2, characterized in that: The added amounts of biotin, vitamin B11 and vitamin B12 are: biotin: 0.01-0.05 mg / L, vitamin B11: 0.1-0.2 mg / L, vitamin B12: 0.001-0.005 mg / L, and the added amount of EDTA-2Na is 1-1.5 mg / L.

4. A culture medium for Haematococcus pluvialis according to claim 1, characterized in that The culture medium is used for the cultivation and large-scale breeding of Haematococcus pluvialis.

5. A method for cultivating Haematococcus pluvialis, characterized in that: The method comprises the following steps: (1) The method for preparing the Haematococcus pluvialis culture medium comprises: a. Raw material preparation and dissolution: First, a large number of elements (sodium nitrate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate trihydrate, citric acid monohydrate, calcium chloride, ferric citrate ammonium, sodium hydroxide) were dissolved in distilled water, stirred until fully dissolved and then mixed to obtain a stock solution; b. Adjust pH and volume: Then add the remaining elements and trace elements to the above solution, adjust the pH, and dilute to 4L with distilled water to obtain Haematococcus pluvialis culture medium; c. Sterilization: The prepared Haematococcus pluvialis culture medium is transferred into a container, sealed, and then sterilized by high-pressure steam at a sterilization temperature of 121°C for 30 min to obtain a sterilized Haematococcus pluvialis culture medium; d. Cooling and storage: Cool the sterilized Haematococcus pluvialis culture medium to room temperature and store at 4-8°C; (2) Inoculation and expansion culture: Under the preset culture conditions and lighting, select well-growing Haematococcus pluvialis as the seed liquid for inoculation. When inoculating, slowly pour the seed liquid into the culture vessel to ensure that the algae seeds are evenly distributed in the culture medium for expansion culture.

6. A method for culturing Haematococcus pluvialis according to claim 5, characterized in that: The algae species is Haematococcus pluvialis in the value-added growth stage.

7. A method for culturing Haematococcus pluvialis according to claim 5, characterized in that: The culture conditions are as follows: temperature of 23° C., humidity of 45%, culture for 5 to 20 days, and ventilation with a ratio of compressed air to carbon dioxide of 1% to 3%.

8. The method for culturing Haematococcus pluvialis according to claim 5, wherein: The inoculated algae concentration is not less than 5×10 4 The number of cells per mL.

9. A method for culturing Haematococcus pluvialis according to claim 5, characterized in that: The pH is 7.3 to 7.

9.

10. The method for culturing Haematococcus pluvialis according to claim 5, wherein step (2) is characterized in that: The culture illumination is as follows: 1 to 3 days after inoculation, the culture is in a weak light environment with an illumination of 800 Lux, 4 to 8 days after cultivation, the culture is in a 3200 Lux illumination, and 9 to 12 days after cultivation, the culture is in a strong light environment with an illumination of 4800 Lux.

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