Improved culture solution for culturing haematococcus pluvialis and preparation method thereof

Abstract

The invention belongs to the technical field of microalgae culture, and particularly provides an improved culture solution for haematococcus pluvialis culture and a preparation method thereof. The culture solution is prepared from 1.5 to 2.5 mg / L of alpha-cyclodextrin embedded sodium acetate, 0.5 to 0.5 mg / L of cobalt nitrate hexahydrate, 0.05 to 1 mg / L of copper sulfate, 0.3 to 0.9 mg / L of zinc sulfate heptahydrate, 1.5 to 2.5 mg / L of manganese chloride tetrahydrate, 3 to 7 mg / L of boric acid, 8 to 20 mg / L of sodium carbonate, 10.0 to 20.0 mg / L of disodium hydrogen phosphate dihydrate, 1200 to 1400 mg / L of sodium nitrate, 0.5 to 3 mg / L of EDTA (Ethylene Diamine Tetraacetic Acid), 20 to 50 mg / L of calcium chloride, 70 to 100 mg / L of magnesium sulfate heptahydrate, 50 to 80 mg / L of monopotassium phosphate trihydrate, 0.5 to 1.0 mg / L of ferrous sulfate heptahydrate and 0.01 to 0.03 mg / L of ammonium molybdate tetrahydrate. According to the invention, alpha-cyclodextrin is introduced to embed sodium acetate, ferrous sulfate heptahydrate and ammonium molybdate tetrahydrate are added, and a sodium carbonate-disodium hydrogen phosphate-potassium dihydrogen phosphate composite buffer system is constructed, so that the problem of easy imbalance of traditional carbon source supply is solved, and the defect of single and short elements of a traditional formula can be overcome; pH fluctuation caused by cell metabolism acid production during high-density culture can be effectively coped with, it is ensured that cells are always in a suitable acid-base environment, and nutrient absorption and metabolism pathways are stabilized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microalgae culture, and particularly relates to an improved Haematococcus pluvialis culture solution and a preparation method thereof. BACKGROUND

[0002] Haematococcus pluvialis is a kind of freshwater unicellular green algae, which has attracted much attention due to its ability to synthesize astaxanthin in large quantities under stress conditions. As a powerful antioxidant, astaxanthin has high application value in the fields of food, medicine, cosmetics and aquaculture, and the market demand continues to rise. However, the large-scale cultivation of Haematococcus pluvialis still faces many technical bottlenecks, which restricts the efficient production of astaxanthin. Currently, the cultivation of Haematococcus pluvialis mainly relies on traditional culture solutions (such as BG11 culture solution, SE culture solution, etc.), but these culture solutions have the following significant defects: 1. The carbon source in the traditional culture solution is mainly free organic carbon, and its release rate cannot match the metabolic demand of Haematococcus pluvialis under different light intensities, resulting in insufficient carbon source when light is sufficient and carbon source accumulation when light is weak, which further affects the biomass growth and astaxanthin synthesis efficiency; 2. The existing formula often lacks key trace elements such as iron and molybdenum, and iron is a core component of the photosynthetic electron transport chain, and molybdenum is a cofactor of key nitrogen metabolism enzymes (such as nitrate reductase), and the lack of elements will significantly inhibit the photosynthesis and nitrogen assimilation ability of algal cells, resulting in slow growth; 3. During the high-density cultivation of Haematococcus pluvialis, organic acids produced by metabolism will cause the pH of the culture solution to drop rapidly, and the traditional culture solution only relies on a single buffer (such as sodium carbonate) to maintain a stable acid-base environment, which further affects the absorption of nutrients and cell activity. SUMMARY

[0003] To solve the above technical problems, the application provides an improved Haematococcus pluvialis culture solution and a preparation method thereof, which can solve the technical problem of low growth and astaxanthin accumulation of Haematococcus pluvialis in traditional culture solution, and improve the astaxanthin productivity of Haematococcus pluvialis by optimizing the component ratio of the culture solution.

[0004] In a first aspect, a Haematococcus pluvialis culture solution is provided, comprising: α-cyclodextrin-embedded sodium acetate 1.5-2.5 mg / L, cobalt nitrate hexahydrate 0.05-0.5 mg / L, copper sulfate 0.05-1 mg / L, zinc sulfate heptahydrate 0.3-0.9 mg / L, manganese chloride tetrahydrate 1.5-2.5mg / L, 3-7mg / L boric acid, 8-20mg / L sodium carbonate, 10.0-20.0mg / L disodium hydrogen phosphate dihydrate, 1200-1400mg / L sodium nitrate, 0.5-3mg / L EDTA, 20-50mg / L calcium chloride, 70-100mg / L magnesium sulfate heptahydrate, 50-80mg / L potassium dihydrogen phosphate trihydrate, 0.5-1.0mg / L ferrous sulfate heptahydrate, 0.01-0.03mg / L ammonium molybdate tetrahydrate.

[0005] Further, the preparation method of the α-cyclodextrin-embedded sodium acetate comprises: Step 1, weigh α-cyclodextrin, add deionized water, stir in a 50-70℃ constant temperature water bath until completely dissolved to form an α-cyclodextrin solution; weigh sodium acetate, add deionized water, stir to dissolve to form a sodium acetate solution; Step 2, slowly drop the sodium acetate solution into the α-cyclodextrin solution, while stirring with a magnetic stirrer at a speed of 300-600rpm, and incubate in a 50-70℃ water bath for 2 hours; Step 3, after the reaction is completed, cool the mixture to room temperature, stand to allow the inclusion compound to be analyzed, separate the precipitate, wash the precipitate with deionized water several times, and finally freeze-dry the precipitate to obtain an α-cyclodextrin-embedded sodium acetate complex.

[0006] Further, the concentration of α-cyclodextrin in the α-cyclodextrin solution in step 1 is 4-8wt%.

[0007] Further, the concentration of sodium acetate in the sodium acetate solution in step 1 is 4-8wt%; the mass ratio of α-cyclodextrin to sodium acetate is 2-3:1.

[0008] Further, the drying in step 3 is vacuum drying, and the conditions for the vacuum drying are: -30~-50℃, vacuum degree 0.1-0.5MPa.

[0009] In a second aspect, a method for culturing Haematococcus pluvialis is provided, comprising: culturing Haematococcus pluvialis in a large-scale device, and using the above-mentioned culture solution in the culturing process.

[0010] Further, the method for culturing Haematococcus pluvialis comprises: S1: Haematococcus pluvialis cultured to the stable phase by a conventional method, inoculating Haematococcus pluvialis into the culture solution for culturing; S2: a shaking table; S3: culturing for 45h; S4: inoculating the seed solution into the Haematococcus pluvialis culture solution, and continuing to culture in the shaking table for 6-15d.

[0011] Further, the pH of the culture solution in S1 is 7-8. Further, the rotation speed of the shaker in S2 is 200-300 rpm / min. Further, the culture temperature in S3 is 28 DEG C. Further, the culture temperature in S4 is 28 DEG C. Further, the rotation speed of the shaker in S4 is 200-300 rpm / min.

[0012] Further, the large-scale culture device comprises a columnar photobioreactor, a flat plate photobioreactor and a pipeline photobioreactor.

[0013] Compared with the prior art, the rain red ball algae culture solution and the preparation method thereof have the following beneficial effects: 1. The alpha-cyclodextrin-embedded sodium acetate is introduced, the carbon source release is dynamically controlled according to the light intensity, the characteristics of the rain red ball algae photosynthesis are matched, the accurate carbon source support is provided for the cell growth at different light periods, the efficient metabolism level is maintained, and the imbalance problem of the traditional carbon source supply is solved. 2. The new ferrous sulfate heptahydrate and ammonium molybdate tetrahydrate are added to perfect the trace element system, the iron element promotes the photosynthetic electron transfer, the molybdenum element assists the nitrogen metabolism enzyme activity, the algal cell growth, photosynthesis and nitrogen assimilation process are supported in all directions, the cell physiological function is improved, and the single short board of the traditional formula element is made up. 3. The "sodium carbonate - disodium hydrogen phosphate - potassium dihydrogen phosphate" composite buffer system is constructed, the pH buffer capacity of the culture solution is enhanced, the pH fluctuation caused by the cell metabolic acid during the high-density culture is effectively responded, the cell is ensured to be always in the suitable acid-base environment, the nutrient absorption and metabolic pathway are stabilized, and the culture process is ensured to be stable. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme of the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0015] The preparation method of the alpha-cyclodextrin-embedded sodium acetate used in the examples and comparative examples is as follows: Step 1, take α-cyclodextrin 5g, add 100ml deionized water, stir in 60℃ constant temperature water bath until completely dissolved, form 5%(w / v) α-cyclodextrin solution; take sodium acetate 2.5g, add 50ml deionized water, stir to dissolve, form 5%(w / v) sodium acetate solution; Step 2, slowly drop the sodium acetate solution into the α-cyclodextrin solution (dropping speed 1ml / min), while stirring with a magnetic stirrer at 300rpm, 60℃ water bath for 2 hours; Step 3, after the reaction, the mixture is cooled to room temperature, stand for 12 hours, make the inclusion complex analysis, separate the precipitate, wash the precipitate with deionized water for 3 times to remove the free sodium acetate, put the precipitate in the vacuum freeze dryer(-50℃, vacuum degree 0.1MPa) for 24 hours, get the α-cyclodextrin embedded sodium acetate complex.

[0016] In a first aspect, a Haematococcus pluvialis culture solution is provided, comprising: 1.5-2.5mg / L of α-cyclodextrin embedded sodium acetate, 0.05-0.5mg / L of cobalt nitrate hexahydrate, 0.05-1mg / L of copper sulfate, 0.3-0.9mg / L of zinc sulfate heptahydrate, 1.5-2.5mg / L of potassium chloride tetrahydrate, 3-7mg / L of boric acid, 8-20mg / L of sodium carbonate, 10.0-20.0mg / L of disodium hydrogen phosphate dihydrate, 1200-1400mg / L of sodium nitrate, 0.5-3mg / L of EDTA, 20-50mg / L of calcium chloride, 70-100mg / L of magnesium sulfate heptahydrate, 50-80mg / L of potassium dihydrogen phosphate trihydrate, 0.5-1.0mg / L of ferrous sulfate heptahydrate, and 0.01-0.03mg / L of ammonium molybdate tetrahydrate.

[0017] Further, the preparation method of the α-cyclodextrin embedded sodium acetate comprises: Step 1, take α-cyclodextrin, add deionized water, stir in 50-70℃ constant temperature water bath until completely dissolved, form α-cyclodextrin solution; take sodium acetate, add deionized water, stir to dissolve, form sodium acetate solution; Step 2, slowly drop the sodium acetate solution into the α-cyclodextrin solution, while stirring with a magnetic stirrer at 300-600rpm, 50-70℃ water bath for 2 hours; Step 3, after the reaction, the mixture is cooled to room temperature, stand for 12 hours, make the inclusion complex analysis, separate the precipitate, wash the precipitate with deionized water for 3 times to remove the free sodium acetate, put the precipitate in the vacuum freeze dryer(-50℃, vacuum degree 0.1MPa) for 24 hours, get the α-cyclodextrin embedded sodium acetate complex.

[0018] Further, the concentration of the α-cyclodextrin in the α-cyclodextrin solution in step 1 is 4-8 wt%.

[0019] Further, the concentration of the sodium acetate in the sodium acetate solution in step 1 is 4-8 wt%; and the mass ratio of the α-cyclodextrin to the sodium acetate is 2-3:1.

[0020] Further, the drying in step 3 is vacuum drying, and the vacuum drying is performed under the conditions of -30 to -50℃ and a vacuum degree of 0.1-0.5 MPa.

[0021] Examples 1-3 and Comparative Example 1 The components and contents in Examples 1-3 and Comparative Example 1 are shown in Table 1. The culture solution in Examples and Comparative Example is used to culture Haematococcus pluvialis, and the cultured Haematococcus pluvialis is tested, and the results are shown in Table 2.

[0022] Table 1: Component and proportion of culture solution

[0023] Example

[0024] Culture of Haematococcus pluvialis: The Haematococcus pluvialis is purchased from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences, and the Haematococcus pluvialis strain is FACHB-877, and the culture solution is BG11. The method for culturing the Haematococcus pluvialis using the culture solution in Examples 1-3 and Comparative Example 1 is as follows: S1: An appropriate amount of algae is inoculated into each of the culture solutions (pH 7-8) in the above examples and comparative example, and the culture temperature is 25±1℃, the daily light-dark cycle is 12h:12h, the light condition is 30-40μmoL / m2·s, and the culture is static; S2: Shaking table, the shaking table speed is 200-300rpm / min; S3: The culture is performed for 45h, and the culture temperature is 28℃; S4: The seed solution is inoculated into the Haematococcus pluvialis culture solution, and the culture is continued in the shaking table for 6-15d, the shaking table speed is 200-300rpm / min, and the culture temperature is 28℃.

[0025] Test method for total yield of astaxanthin: After the culture is completed, the content of astaxanthin in the Haematococcus pluvialis and the dry weight of the Haematococcus pluvialis cells are determined; the content of astaxanthin is determined according to the HPLC method; and the pH is determined using a pH detector.

[0026] Table 2: Content of astaxanthin in Haematococcus pluvialis and dry weight of Haematococcus pluvialis cells

[0027]

[0028] The results show that the dry weight of Haematococcus pluvialis and the content of astaxanthin in Haematococcus pluvialis of the embodiment are higher than those of the comparative examples, and therefore the Haematococcus pluvialis culture solution of the embodiment can improve the dry weight of Haematococcus pluvialis and the content of astaxanthin in Haematococcus pluvialis.

[0029] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A modified culture medium for cultivating Haematococcus pluvialis, characterized in that, include: α-Cyclodextrin encapsulated sodium acetate 1.5-2.5 mg / L, cobalt nitrate hexahydrate 0.05-0.5 mg / L, copper sulfate 0.05-1 mg / L, zinc sulfate heptahydrate 0.3-0.9 mg / L, manganese chloride tetrahydrate 1.5-2.5 mg / L, boric acid 3-7 mg / L, sodium carbonate 8-20 mg / L, disodium hydrogen phosphate dihydrate 10.0-20.0 mg / L, sodium nitrate 1200-1400 mg / L, EDTA 0.5-3 mg / L, calcium chloride 20-50 mg / L, magnesium sulfate heptahydrate 70-100 mg / L, potassium dihydrogen phosphate trihydrate 50-80 mg / L, ferrous sulfate heptahydrate 0.5-1.0 mg / L, ammonium molybdate tetrahydrate 0.01-0.03 mg / L.

2. The improved Haematococcus pluvialis culture medium according to claim 1, characterized in that, The method for preparing the α-cyclodextrin-encapsulated sodium acetate includes: Step 1: Weigh α-cyclodextrin, add deionized water, and stir in a constant temperature water bath at 50-70℃ until completely dissolved to form an α-cyclodextrin solution; weigh sodium acetate, add deionized water, and stir to dissolve to form a sodium acetate solution. Step 2: Slowly add sodium acetate solution dropwise into α-cyclodextrin solution while stirring with a magnetic stirrer at 300-600 rpm and keep the mixture in a water bath at 50-70℃ for 2 hours. Step 3: After the reaction is complete, cool the mixture to room temperature, let it stand to allow the inclusion complex to fully precipitate, separate the precipitate, wash the precipitate several times with deionized water, and finally freeze-dry the precipitate to obtain the α-cyclodextrin-encapsulated sodium acetate complex.

3. The improved culture medium for Haematococcus pluvialis cultivation according to claim 2, characterized in that, The concentration of α-cyclodextrin in the α-cyclodextrin solution described in step 1 is 4-8 wt%.

4. The improved culture medium for Haematococcus pluvialis cultivation according to claim 2, characterized in that, The concentration of sodium acetate in the sodium acetate solution in step 1 is 4-8 wt%; the mass ratio of α-cyclodextrin to sodium acetate is 2-3:

1.

5. The improved culture medium for Haematococcus pluvialis cultivation according to claim 2, characterized in that, The drying described in step 3 is vacuum drying, and the conditions for vacuum drying are: -30~-50℃, vacuum degree 0.1-0.5MPa.

6. A method for culturing Haematococcus pluvialis, characterized in that, include: Haematococcus pluvialis is cultured in a large-scale device, using the culture medium described in any one of claims 1-5 during the culture process.

7. The method for culturing Haematococcus pluvialis according to claim 6, characterized in that, The specific steps are as follows: S1: Haematococcus pluvialis cultured to the stable phase using conventional methods, Haematococcus pluvialis inoculated into culture medium and cultured; S2: Shaking bed; S3: Incubate for 45 hours; S4: Inoculate the seed culture into the Haematococcus pluvialis culture medium and continue to culture on a shaker for 6-15 days.

8. The method for culturing Haematococcus pluvialis according to claim 7, characterized in that, The pH of the culture medium in S1 is 7-8; the shaking speed in S2 is 200-300 rpm / min; the culture temperature in S3 is 28℃; the culture temperature in S4 is 28℃; and the shaking speed in S4 is 200-300 rpm / min.

9. The method for culturing Haematococcus pluvialis according to claim 6, characterized in that, The large-scale cultivation device includes a column photobioreactor, a flat-plate photobioreactor, and a tubular photobioreactor.

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