Haematococcus pluvialis with high content of unsaturated fatty acids and breeding method and application thereof

Abstract

The application discloses a breeding method of Haematococcus pluvialis with high content of unsaturated fatty acid, which comprises the following steps of algal strain culture, algal strain expansion culture, large-scale culture and induction. The breeding method controls the breeding conditions, so that the content of unsaturated fatty acid in the oil of the obtained Haematococcus pluvialis is increased, the obtained Haematococcus pluvialis is more suitable for developing various health products, the large-scale production can be carried out in months with low yield or stop production, the production benefit is increased, and the problem of stop production of the Haematococcus pluvialis breeding enterprise in the low-temperature season is solved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Haematococcus pluvialis with high unsaturated fatty acid content, its cultivation method, and its application. Background Technology

[0002] Haematococcus pluvialis is widely recognized as the best natural organism for producing astaxanthin. However, due to its thick spore wall, it cannot be absorbed by the human body and therefore requires extraction after cell wall disruption. Haematococcus pluvialis extract contains approximately 98.5% fat. People often only focus on the astaxanthin content (2.5-5%), neglecting other fat components. Of this fat, saturated fat accounts for about 20%, and unsaturated fat accounts for about 80%. In applications, Haematococcus pluvialis extract is often used to produce soft capsules, microcapsules, etc. When taken, other fat components are ingested along with astaxanthin. Since fat intake is closely related to human health, it is essential to provide a healthier alternative to Haematococcus pluvialis and its extract. Summary of the Invention

[0003] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for cultivating Haematococcus pluvialis with high unsaturated fatty acid content. By controlling the cultivation conditions, the content of unsaturated fatty acids in the oil of cultivated Haematococcus pluvialis is increased, making it more suitable for the development of various health products.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] A method for cultivating Haematococcus pluvialis with high unsaturated fatty acid content includes the following steps:

[0006] (1) Algal culture: Dilute the Haematococcus pluvialis culture medium I, and let it stand for a period of time, shaking it once every 2-3 hours. Culture conditions: light intensity: 1000-3000 Lux; light-dark cycle: 14h:10h; temperature: 20-25℃.

[0007] Culture medium I has the following formula: KNO3 0.1~0.2 g / L, KH2PO4 0.02~0.04 g / L, MgSO4•7H2O 0.3~0.5 g / L, NaHCO3 0.05~0.07 g / L, CaCl2 0.02~0.04 g / L, trace element stock solution I 0.3~0.5 mL / L, vitamin B1 0.05~0.2 mL / L, vitamin C 0.05~0.2 mL / L, vitamin B... 12 0.2~0.4 mL / L, biotin 0.2~0.4 mL / L, balance is water, pH 7~8.

[0008] The formula for trace element mother liquor I is as follows: EDTA-Na2 4~6g / L, FeSO4•7H2O 0.5~2g / L, MnSO4•H2O 0.5~2g / L, ZnSO4•7H2O 0.1~0.3g / L, Co(NO3)2•6H2O 0.03~0.05g / L, CuSO4 0.01~0.05g / L, H3BO3 7~11g / L, NaMoO4 0.2~0.4g / L, with the balance being water.

[0009] (2) Large-scale culture of algae: The Haematococcus pluvialis cultured in step (1) was inoculated into a flat-plate photobioreactor and cultured in medium II. For the first two days after inoculation, weak light was used with an illumination of 2000-2500 Lux, a light-dark cycle of 14h:10h, and a temperature of 20-25℃. Starting from the third day, strong light was used with an illumination of 4500-5500 Lux, a light-dark cycle of 14h:10h, and a temperature of 10-15℃. Aeration was carried out continuously, and CO2 was introduced once each in the morning and afternoon to maintain the pH at 7-8.

[0010] The formulation of culture medium II is based on culture medium I with the addition of 0.4~0.6 mL / L of trace element stock solution II, and the pH is 7~8.

[0011] The formula for trace element mother liquor II is: EDTA-Na2 4~6g / L, FeSO4•7H2O 4~6g / L, with the remainder being water.

[0012] (3) Large-scale cultivation: Clean and disinfect the culture ponds and equipment, and transfer the algae cultured in step (2) to the culture ponds. Use culture medium II for cultivation at a temperature of 10~20℃. For the first 3 days after inoculation, maintain a light intensity of 3000~5000 Lux. Starting from the 4th day, increase the light intensity to 5000~10000 Lux. At this time, add rare earth compounds to culture medium II: praseodymium sulfate Pr2(SO4)3 0.2~0.4 g / L, holmium sulfate Tb2(SO4)3•8H2O 0.1~0.3 g / L, and lutetium sulfate Lu2(SO4)3 0.2~0.4 g / L.

[0013] On day 7, add 0.05~0.5g / L of liquid food-grade lipase and stir well. Maintain the pH value below 9.0.

[0014] (4) Induction: When the color of the algal solution changes from green to yellow-green, and the diameter of the red area in the middle of the algal cell is about 1 / 3 of the cell diameter, add a mixture of EDTA-Na2 and FeSO4•7H2O to the algal solution: EDTA-Na2 10~15mg / L, FeSO4•7H2O 10~15mg / L, and the remainder is water.

[0015] When the diameter of the red area in the middle of the algal cell is about 1 / 2 of the cell diameter, add 2~3 g / L of MgSO4•7H2O to the algal solution.

[0016] After 24-48 hours, the Haematococcus pluvialis cells transform into spores. At this time, the light intensity is increased to 10,000-12,000 Lux, with a light-dark cycle of 14-16 hours: 10-8 hours. The algal solution is harvested when it turns reddish-brown and all the algal cells turn red. The algal cells are then broken down and dried to obtain Haematococcus pluvialis powder.

[0017] Preferably, in step (1), the volume ratio of Haematococcus pluvialis species to culture medium I is 1:1~5.

[0018] Preferably, in steps (2) and (3), the volume ratio of Haematococcus pluvialis species to culture medium II is 1:5~10.

[0019] Preferably, in steps (1)-(3), the pH value is adjusted by adding CO2 to the algal solution.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The conventional cultivation temperature for Haematococcus pluvialis is around 25℃; below 25℃, its growth and reproduction are inhibited. The cultivation method described in this invention utilizes low temperature and oxidative stress, and adds specific nutrients and enzymes in a specific manner, resulting in a reduction of saturated fatty acid content by more than 40%, an increase of monounsaturated fatty acid content by more than 8%, and an increase of polyunsaturated fatty acid content by more than 10% in the cultivated Haematococcus pluvialis. Unsaturated fatty acids are beneficial to cardiovascular health, lowering cholesterol and triglyceride levels in the blood and preventing cardiovascular diseases. Therefore, Haematococcus pluvialis cultivated using the method described in this invention is more suitable for developing various health products.

[0022] 2. The aquaculture method described in this invention has low aquaculture costs and is easy to operate. It significantly increases the content of unsaturated fatty acids and decreases the content of saturated fatty acids in Haematococcus pluvialis, while not affecting the astaxanthin content or yield. It can be applied to large-scale production.

[0023] 3. From November to March of the following year, temperatures are low, resulting in low yields of Haematococcus pluvialis cultivated using conventional methods, often leading to shutdowns. This leaves production equipment and workers idle, causing resource waste and economic losses for enterprises. The cultivation method described in this invention allows for large-scale cultivation of Haematococcus pluvialis with high unsaturated fatty acid content during the relatively cooler months. It fully utilizes idle production equipment and human resources during shutdowns, avoiding resource waste while increasing profits and solving the labor shortage problem for Haematococcus pluvialis cultivation enterprises during shutdowns. This method is particularly applicable in some southern regions of my country during winter, completely avoiding reduced production or shutdowns, significantly minimizing economic losses, and improving economic efficiency. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to embodiments. These embodiments are limited to providing a clearer understanding of the technical features, objectives, and effects of the present invention, and are not intended to limit the scope of the invention. Example 1

[0025] A method for cultivating Haematococcus pluvialis with high unsaturated fatty acid content includes the following steps:

[0026] I. Algal Culture

[0027] 1. Prepare culture medium I

[0028] (1) The formulation of culture medium I is as follows:

[0029]

[0030] The formula for trace element mother liquor I is as follows:

[0031]

[0032] (2) Adjust the pH of culture medium I

[0033] Add CO2 to the prepared culture medium I, adjust the pH to about 7.5, shake well, seal the bottle tightly, and set aside.

[0034] 2. Vaccination

[0035] Select vigorous algal species and inoculate them with culture medium I at a volume ratio of 1:5.

[0036] 3. Cultivate

[0037] After inoculation, place the inoculated cells on a culture rack and incubate statically, shaking them every 2 hours. Light intensity: 2000 Lux; Light-dark cycle: 14h:10h; Temperature: 23℃.

[0038] II. Large-scale cultivation of algae

[0039] 1. Prepare culture medium II

[0040] Add 0.5 mL / L of trace element stock solution II to culture medium I, introduce CO2 into the prepared culture medium II, adjust the pH to about 7.5, and supplement the carbon source.

[0041] The formula for trace element mother liquor II is as follows:

[0042]

[0043] 2. Vaccination

[0044] Take the vigorous algae cultured in step one, and after microscopic examination, confirm that the Haematococcus pluvialis is growing normally and is free from contamination. It can then be used as an algae inoculation into the flat plate photobioreactor. The algae inoculate with culture medium II at a volume ratio of 1:5.

[0045] 3. Cultivate

[0046] Within 2 days after inoculation, the animals were kept under low light (2200 Lux) at 23°C with a 14h:10h light-dark cycle. From the 3rd day onwards, the animals were kept under high light (5000 Lux) at 13°C with a 14h:10h light-dark cycle. The animals were continuously aerated and stirred, and CO2 was introduced twice daily, once in the morning and once in the afternoon, to adjust the pH to 7.5.

[0047] III. Large-scale training

[0048] 1. Disinfection treatment of breeding ponds and equipment

[0049] Thoroughly clean the large tank, impeller agitator, heat exchange pipes, and the outer surface of the CO2 vent pipe with water; fill the tank with water to a depth of 20cm, and start the impeller agitator to circulate the water; add H2O2 to the water at a rate of 0.2mL / L, and stir continuously for at least 2 hours to ensure even distribution of H2O2; after disinfection for at least 12 hours, lower the water level to 15cm, add pre-dissolved anhydrous sodium sulfite at a rate of 0.4g / L, and stir continuously for at least 2 hours to ensure even distribution of anhydrous sodium sulfite; after 2 hours, the disinfection process is complete.

[0050] Disinfect the drain pipe (valve) of the flat plate reactor with ammonia water for more than 12 hours; disinfect the pipes (pipe opening and inside) used to transport algae seeds with H2O2 (27-30%) aqueous solution at 0.8 mL / L for more than 12 hours.

[0051] 2. Vaccination

[0052] Microscopic examination confirmed that the algae in the plate reactor were growing normally and were free of contamination. They were then transported to the aquaculture pond through a pipeline and cultured in culture medium II with a volume ratio of algae to culture medium II of 1:5.

[0053] 3. Cultivate

[0054] (1) Light

[0055] For 1-3 days after inoculation, when the light is strong on sunny days, culture in the shade; when it is cloudy or rainy, open the shade net to increase the light intensity, and control the light intensity at 4000 Lux.

[0056] Starting on the 4th day after vaccination, gradually open the shade netting to increase the light exposure to 7500 Lux.

[0057] At this point, add the following rare earth element compounds to the culture medium: praseodymium sulfate (Pr₂(SO₄)₃) 0.3 g / L, holmium sulfate (Tb₂(SO₄)₃·8H₂O) 0.2 g / L, and lutetium sulfate (Lu₂(SO₄)₃) 0.3 g / L. Start the impeller agitator to circulate the water and mix thoroughly.

[0058] On day 7 after inoculation, add 0.25 g / L of liquid food-grade lipase at around 20°C, at which point the enzyme activity is highest. Turn on the impeller agitator to circulate the water; continue stirring for at least 2 hours to ensure even distribution of the lipase.

[0059] (2) Temperature: 10~20℃. When the temperature is below 10℃, add warm water or geothermal water to raise the temperature according to the actual situation.

[0060] 4. Carbon source supply and pH control

[0061] When Haematococcus pluvialis cells are green, it is in the vegetative growth stage. During this stage, CO2 is introduced twice a day, once in the morning and once in the afternoon, to maintain the pH value below 9.0.

[0062] IV. Spore Induction

[0063] 1. Induction

[0064] When the color of the algal solution changes from green to yellowish-green, and the diameter of the red area in the middle of the algal cells is about 1 / 3 of the cell diameter when examined under a microscope, a mixture of EDTA-Na2 and FeSO4•7H2O is added to the algal solution: EDTA-Na2 12.5 mg / L, FeSO4•7H2O 12.5 mg / L, with the remainder being water.

[0065] After 24 to 48 hours, microscopic examination was performed. When the diameter of the red area in the middle of the algal cell was about 1 / 2 of the cell diameter, MgSO4•7H2O 2.5g / L was added to the algal solution.

[0066] After 24 to 48 hours, the Haematococcus pluvialis cells transform into spores.

[0067] 2. Light control

[0068] After the Haematococcus pluvialis cells transformed into spores, the shading net was fully opened, and the light intensity was increased to 10,000 Lux, with a light-dark cycle of 15h:9h.

[0069] 3. pH adjustment

[0070] After the Haematococcus pluvialis cells transform into spores, CO2 supply is stopped, allowing the pH of the algal solution to gradually increase. When the macroscopic state of the algal solution in the culture pond turns reddish-brown, random samples are taken for microscopic examination while the algal solution is circulated and stirred evenly. Under high magnification, 20-30 algal cells are randomly observed. Once all the cells turn red, they can be harvested. After cell wall disruption and drying, Haematococcus pluvialis powder is obtained.

[0071] In the above steps, natural light is usually used as the light source. When natural light is insufficient, artificial light can be used to supplement the light. Example 2

[0072] A method for cultivating Haematococcus pluvialis with high unsaturated fatty acid content includes the following steps:

[0073] I. Algal Culture

[0074] 1. Prepare culture medium I

[0075] (1) The formulation of culture medium I is as follows:

[0076]

[0077] The formula for trace element mother liquor I is as follows:

[0078]

[0079] (2) Adjust the pH of culture medium I

[0080] Add CO2 to the prepared culture medium I, adjust the pH to about 7, shake well, seal the bottle tightly, and set aside.

[0081] 2. Vaccination

[0082] Select vigorous algal species and inoculate them with culture medium I at a volume ratio of 1:3.

[0083] 3. Cultivate

[0084] After inoculation, the cells were incubated statically, shaken every 3 hours. Light intensity: 1000 Lux; light-dark cycle: 14h:10h. Temperature: 20℃.

[0085] II. Large-scale cultivation of algae

[0086] 1. Prepare culture medium II

[0087] Based on culture medium I, 0.6 mL / L of trace element stock solution II was added, CO2 was introduced into the prepared culture medium II, the pH was adjusted to about 7, and a carbon source was added at the same time.

[0088] The formula for trace element mother liquor II is as follows:

[0089]

[0090] 2. Vaccination

[0091] Take the vigorous algae cultured in step one, and after microscopic examination, confirm that the Haematococcus pluvialis is growing normally and is free from contamination. It can then be used as an algae inoculation into the flat plate photobioreactor. The algae inoculate with culture medium II at a volume ratio of 1:8.

[0092] 3. Cultivate

[0093] Within 2 days after inoculation, the animals were exposed to low light (2000 Lux) at 20°C with a 14h:10h light-dark cycle. Starting from the 3rd day, the animals were exposed to high light (4500 Lux) at 10°C with a 14h:10h light-dark cycle. The animals were continuously aerated and stirred, and CO2 was introduced twice daily, once in the morning and once in the afternoon, to adjust the pH to 7.

[0094] III. Large-scale training

[0095] 1. Disinfection treatment of breeding ponds and equipment

[0096] Thoroughly clean the large tank, impeller agitator, heat exchange pipes, and the outer surface of the CO2 vent pipe with water; fill the tank with water to a depth of 20cm, and start the impeller agitator to circulate the water; add H2O2 to the water at a rate of 0.2mL / L, and stir continuously for at least 2 hours to ensure even distribution of H2O2; after disinfection for at least 12 hours, lower the water level to 15cm, add pre-dissolved anhydrous sodium sulfite at a rate of 0.4g / L, and stir continuously for at least 2 hours to ensure even distribution of anhydrous sodium sulfite; after 2 hours, the disinfection process is complete.

[0097] Disinfect the drain pipe (valve) of the flat plate reactor with ammonia water for more than 12 hours; disinfect the pipes (pipe opening and inside) used to transport algae seeds with H2O2 (27-30%) aqueous solution at 0.8 mL / L for more than 12 hours.

[0098] 2. Vaccination

[0099] Microscopic examination confirmed that the algae in the plate reactor were growing normally and were free of contamination. They were then transported to the aquaculture pond through a pipeline and cultured in culture medium II with a volume ratio of algae to culture medium II of 1:8.

[0100] 3. Cultivate

[0101] (1) Light

[0102] Natural light.

[0103] For 1-3 days after inoculation, when the light is strong on sunny days, culture in the shade; when it is cloudy or rainy, open the shade net to increase the light, and control the light intensity to 3000 Lux.

[0104] Starting on the 4th day after vaccination, gradually open the shade netting to increase the light exposure to 5000 Lux.

[0105] At this point, add the following rare earth element compounds to the culture medium: praseodymium sulfate (Pr₂(SO₄)₃) 0.4 g / L, holmium sulfate (Tb₂(SO₄)₃·8H₂O) 0.1 g / L, and lutetium sulfate (Lu₂(SO₄)₃) 0.2 g / L. Start the impeller agitator to circulate the water and mix thoroughly.

[0106] On day 7 after inoculation, add 0.05 g / L of liquid food-grade lipase at around 20°C, at which point the enzyme activity is highest. Turn on the impeller agitator to circulate the water; continue stirring for at least 2 hours to ensure the lipase is evenly distributed.

[0107] (2) Temperature: 10~20℃. When the temperature is below 10℃, add warm water or geothermal water to raise the temperature according to the actual situation.

[0108] 4. Carbon source supply and pH control

[0109] When Haematococcus pluvialis cells are green, it is in the vegetative growth stage. During this stage, CO2 is introduced twice a day, once in the morning and once in the afternoon, to maintain the pH value below 9.0.

[0110] IV. Spore Induction

[0111] 1. Induction

[0112] When the color of the algal solution changes from green to yellowish-green, and the diameter of the red area in the middle of the algal cells is about 1 / 3 of the cell diameter when examined under a microscope, a mixture of EDTA-Na2 and FeSO4•7H2O is added to the algal solution: EDTA-Na2 10mg / L, FeSO4•7H2O 10mg / L, with the remainder being water.

[0113] After 24 to 48 hours, under a microscope, when the diameter of the red area in the middle of the algal cell is about 1 / 2 of the cell diameter, MgSO4•7H2O 3g / L is added to the algal solution.

[0114] After 24 to 48 hours, the Haematococcus pluvialis cells transform into spores.

[0115] 2. Light control

[0116] After the Haematococcus pluvialis cells transformed into spores, the shading net was fully opened, and the light intensity was increased to 11,000 Lux, with a light-dark cycle of 16h:8h.

[0117] 3. pH adjustment

[0118] After the Haematococcus pluvialis cells transform into spores, CO2 supply is stopped, allowing the pH of the algal solution to gradually increase. When the macroscopic state of the algal solution in the culture pond turns reddish-brown, random samples are taken for microscopic examination while the algal solution is circulated and stirred evenly. Under high magnification, 20-30 algal cells are randomly observed. Once all the cells turn red, they can be harvested. After cell wall disruption and drying, Haematococcus pluvialis powder is obtained. Example 3

[0119] A method for cultivating Haematococcus pluvialis with high unsaturated fatty acid content includes the following steps:

[0120] I. Algal Culture

[0121] 1. Prepare culture medium I

[0122] (1) The formulation of culture medium I is as follows:

[0123]

[0124] The formula for trace element mother liquor I is as follows:

[0125]

[0126] (2) Add CO2 to the prepared culture medium I, adjust the pH value to about 8, shake well, seal the bottle mouth tightly, and set aside.

[0127] 2. Select a vigorous algal species and inoculate it with culture medium I at a volume ratio of 1:1 between the algal species and culture medium I.

[0128] 3. After inoculation, allow the mixture to stand still, shaking it every 2.5 hours. Light intensity: 3000 Lux; light-dark cycle: 14h:10h. Temperature: 25℃.

[0129] II. Large-scale cultivation of algae

[0130] 1. Prepare culture medium II

[0131] Based on culture medium I, 0.4 mL / L of trace element stock solution II was added, CO2 was introduced into the prepared culture medium II, the pH was adjusted to about 8, and a carbon source was added at the same time.

[0132] The formula for trace element mother liquor II is as follows:

[0133]

[0134] 2. Take the vigorous algae cultured in step one, and after microscopic examination, confirm that the Haematococcus pluvialis is growing normally and is free from contamination. It can then be used as the algae inoculated into the flat plate photobioreactor. The algae inoculates the culture medium II at a volume ratio of 1:10.

[0135] 3. Within 2 days after inoculation, use low-light illumination (2500 Lux), 25℃, and a light-dark cycle of 14h:10h. Starting from the 3rd day, use high-light illumination (5500 Lux), a light-dark cycle of 14h:10h, and 15℃. Continuously aerate and agitate, and add CO2 twice a day, once in the morning and once in the afternoon, to adjust the pH to 8.

[0136] III. Large-scale training

[0137] 1. Clean and disinfect the breeding ponds and equipment.

[0138] 2. Take normally growing algae from the plate reactor and transfer it to the aquaculture pond. Cultivate it using culture medium II, with a volume ratio of algae to culture medium II of 1:10. The temperature should be 10-20℃.

[0139] 3. For 1-3 days after inoculation, maintain a light intensity of 5000 Lux. Starting from day 4 after inoculation, increase the light intensity to 10000 Lux. At this time, add the following rare earth element compounds to the culture medium: praseodymium sulfate (Pr₂(SO₄)₃) 0.2 g / L, holmium sulfate (Tb₂(SO₄)₃·8H₂O) 0.3 g / L, and lutetium sulfate (Lu₂(SO₄)₃) 0.4 g / L. Mix thoroughly.

[0140] On day 7 after inoculation, add 0.5 g / L of liquid food-grade lipase and stir well.

[0141] 4. When the Haematococcus pluvialis cells are green, it is in the vegetative growth stage. During this stage, CO2 should be introduced once in the morning and once in the afternoon each day to maintain the pH value below 9.0.

[0142] IV. Spore Induction

[0143] 1. When the color of the algal solution changes from green to yellowish-green, and the diameter of the red area in the middle of the algal cells is about 1 / 3 of the cell diameter, add a mixture of EDTA-Na2 and FeSO4•7H2O to the algal solution: EDTA-Na2 15mg / L, FeSO4•7H2O 15mg / L, with the remainder being water.

[0144] After 24 to 48 hours, when the diameter of the red area in the middle of the algal cell is about 1 / 2 of the cell diameter, add 2 g / L of MgSO4•7H2O to the algal solution.

[0145] After 24 to 48 hours, the Haematococcus pluvialis cells transform into spores.

[0146] 2. Light control

[0147] After Haematococcus pluvialis cells transformed into spores, the light intensity was increased to 12000 Lux, with a light-dark cycle of 14h:10h.

[0148] 3. pH adjustment

[0149] After the Haematococcus pluvialis cells transform into spores, CO2 supply is stopped, allowing the pH of the algal solution to gradually increase. When the algal solution in the culture pond turns reddish-brown and all the cells turn red, it can be harvested. After cell wall breaking and drying, Haematococcus pluvialis powder is obtained. Example 4

[0150] This embodiment follows conventional methods for cultivating Haematococcus pluvialis. The Haematococcus pluvialis cultivated in this embodiment, as well as in Examples 1, 2, and 3, were all analyzed for fat content according to the method specified in GB 5009.6-2016 National Food Safety Standard - Determination of Fat in Food. Astaxanthin content was determined according to GB / T31520-2015 Determination of Astaxanthin in Haematococcus pluvialis by Liquid Chromatography. The results are shown in Table 1.

[0151]

[0152] As shown in Table 1, compared with Haematococcus pluvialis cultured by conventional methods, the Haematococcus pluvialis cultured by the method described in this invention exhibits a saturated fatty acid content that decreases by more than 40%, a monounsaturated fatty acid content that increases by more than 8%, and a polyunsaturated fatty acid content that increases by more than 10%. The astaxanthin content remains unaffected.

Claims

1. A method for cultivating Haematococcus pluvialis with high unsaturated fatty acid content, characterized in that, Includes the following steps: (1) Culture of algae: The Haematococcus pluvialis algae was diluted and cultured in medium I. Culture conditions: light intensity: 1000~3000 Lux; light-dark cycle: 14h:10h; Temperature: 20~25℃; Culture medium I has the following formula: KNO3 0.1~0.2 g / L, KH2PO4 0.02~0.04 g / L, MgSO4•7H2O 0.3~0.5 g / L, NaHCO3 0.05~0.07 g / L, CaCl2 0.02~0.04 g / L, trace element stock solution I 0.3~0.5 mL / L, vitamin B1 0.05~0.2 mL / L, vitamin C 0.05~0.2 mL / L, vitamin B... 12 0.2~0.4 mL / L, biotin 0.2~0.4 mL / L, balance water, pH 7~8; The formula for trace element mother liquor I is as follows: EDTA-Na2 4~6g / L, FeSO4•7H2O 0.5~2g / L, MnSO4•H2O 0.5~2g / L, ZnSO4•7H2O 0.1~0.3g / L, Co(NO3)2•6H2O 0.03~0.05g / L, CuSO4 0.01~0.05g / L, H3BO3 7~11g / L, Na2MoO4 0.2~0.4g / L, with the balance being water; (2) Large-scale culture of algae: The Haematococcus pluvialis cultured in step (1) was inoculated into a flat plate photobioreactor and cultured in medium II. Within 2 days after inoculation, weak light was used with 2000~2500 Lux, light-dark cycle of 14h:10h, and temperature of 20~25℃. From the 3rd day onwards, strong light was used with 4500~5500 Lux, light-dark cycle of 14h:10h, and temperature of 10~15℃. The algae were continuously aerated and stirred. CO2 was added once in the morning and once in the afternoon each day to maintain pH at 7~8. The formulation of culture medium II is based on culture medium I with the addition of 0.4~0.6 mL / L of trace element stock solution II, and the pH is 7~8; The formula for trace element mother liquor II is: EDTA-Na2 4~6g / L, FeSO4•7H2O 4~6g / L, with the remainder being water; (3) Large-scale cultivation: Clean and disinfect the culture pond and culture equipment, transport the algae cultured in step (2) to the culture pond, and culture it using culture medium II at a temperature of 10~20℃; for the first 3 days after inoculation, maintain a light intensity of 3000~5000 Lux; starting from the 4th day, increase the light intensity to 5000~10000 Lux; at this time, add rare earth compounds to culture medium II: praseodymium sulfate Pr2(SO4)3 0.2~0.4 g / L, holmium sulfate Ho2(SO4)3•8H2O 0.1~0.3 g / L, lutetium sulfate Lu2(SO4)3 0.2~0.4 g / L; On day 7, add 0.05~0.5g / L of liquid food-grade lipase and stir well; maintain the pH value below 9.

0. (4) Induction: When the color of the algal solution changes from green to yellow-green, and the diameter of the red area in the middle of the algal cell accounts for 1 / 3 of the cell diameter, add a mixture of EDTA-Na2 and FeSO4•7H2O to the algal solution: EDTA-Na2 10~15mg / L, FeSO4•7H2O 10~15mg / L, and the remainder is water; When the diameter of the red area in the middle of the algal cell is 1 / 2 of the cell diameter, add 2~3 g / L of MgSO4•7H2O to the algal solution; After 24 to 48 hours, Haematococcus pluvialis cells transform into spores. At this time, increase the light intensity to 10,000 to 12,000 Lux and the light-dark cycle to 14 to 16 hours: 8 to 10 hours. When the algal solution turns reddish-brown and all the algal cells turn red, it is ready for harvesting.

2. The aquaculture method according to claim 1, characterized in that, In step (1), the volume ratio of Haematococcus pluvialis species to culture medium I is 1:1~5.

3. The aquaculture method according to claim 1, characterized in that, In steps (2) and (3), the volume ratio of Haematococcus pluvialis species to culture medium II is 1:5~10.

4. The aquaculture method according to claim 1, characterized in that, The pH value was adjusted by adding CO2 to the algal solution.

5. Haematococcus pluvialis cultured by the cultivation method according to any one of claims 1-4.

6. The application of Haematococcus pluvialis as described in claim 5, characterized in that, The Haematococcus pluvialis can be used to prepare health products.

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