Preparation method and application of astaxanthin oil with high oxidation resistance

Abstract

The invention belongs to the field of astaxanthin oil, and particularly relates to a preparation method and application of astaxanthin oil with high oxidation resistance. Haematococcus pluvialis H7 and haematococcus pluvialis JNU35 are cultured in a runway type photobioreactor and a pipeline type photobioreactor to accumulate astaxanthin, the astaxanthin oil is prepared, and under the same concentration and conditions, compared with astaxanthin oil prepared by single haematococcus pluvialis H7 or single haematococcus pluvialis JNU35 culture, the astaxanthin oil prepared by single haematococcus pluvialis H7 or single haematococcus pluvialis JNU35 culture has the advantages that the astaxanthin oil content is increased, and the astaxanthin oil yield is increased. Through combined culture, the oxidation resistance of the astaxanthin oil can be obviously improved, and the ROS clearance rates are respectively improved by 65.1% and 171.3%. According to the application, the ratio of the haematococcus pluvialis H7 to the haematococcus pluvialis JNU35 for combined culture is optimized, and when the mass ratio of the haematococcus pluvialis H7 to the haematococcus pluvialis JNU35 is 4: 3, the astaxanthin oil prepared by combined culture has the highest oxidation resistance, and the ROS clearance rate reaches 32.8 + / -0.34%.

Description

Technical Field

[0001] The invention belongs to the field of astaxanthin oil, and specifically relates to a preparation method and application of high-antioxidant astaxanthin oil. Background Art

[0002] The chemical structure of astaxanthin gives it antioxidant properties. Astaxanthin itself is a fat-soluble carotenoid with a long conjugated double bond system in its molecular structure. This conjugated double bond can capture and remove free radicals. The conjugated double bonds of astaxanthin can provide electrons to neutralize free radicals, thereby preventing the chain reaction of free radicals.

[0003] It is known from the applicant's preliminary research (Method for preparing astaxanthin oil from Haematococcus pluvialis, patent number CN2020108914158) that astaxanthin in Haematococcus pluvialis exists in an esterified state and coexists with Haematococcus pluvialis algae oil. Usually, the fat-soluble extract of Haematococcus pluvialis is a mixture of astaxanthin esters and algae oil, and is called astaxanthin oil because it is rich in astaxanthin.

[0004] One of the main sources of astaxanthin oil is Haematococcus pluvialis. Under specific growth conditions, Haematococcus pluvialis can synthesize a large amount of astaxanthin. Through a series of biotechnologies and extraction processes, such as cell wall breaking and extraction, astaxanthin oil is extracted from algae cells to make astaxanthin oil.

[0005] The applicants of this application, "Yunnan Aierfa Biotechnology Co., Ltd.", "Yunnan Aiercon Biotechnology Co., Ltd." and "Aierfa Biotechnology (Jiaxing) Co., Ltd.", have previously applied for patent research on Haematococcus pluvialis tunnel photobioreactor, pipeline photobioreactor, Haematococcus pluvialis H7 with a preservation number of CGMCC No.41186, and a method for producing astaxanthin oil from Haematococcus pluvialis. Based on the previous research, this applicant surprisingly found that the combination culture of Haematococcus pluvialis H7 with a preservation number of CGMCC No.41186 and Haematococcus pluvialis JNU35 with a preservation number of CGMCC No.16438 to prepare astaxanthin oil can significantly improve the antioxidant property of astaxanthin oil.

[0006] After searching, the prior art already includes CN2020108914158, a method for preparing astaxanthin oil from Haematococcus pluvialis (the applicant's prior application), CN202410747172, a high temperature and alkali-resistant Haematococcus pluvialis and screening method and application (the applicant's prior application), CN2024110855601, a movement state monitoring and frequency modulation device for Haematococcus pluvialis in a photobioreactor (the applicant's prior application), CN2021104699161, a method for preparing high-purity astaxanthin esters from Haematococcus pluvialis (the applicant's prior application), CN202322590980.2, a racetrack photobioreactor for accelerating the cultivation of Haematococcus pluvialis to accumulate astaxanthin (the applicant's prior application), and CN 2019100735049, a high-astaxanthin-producing Haematococcus pluvialis JNU35 and its cultivation method and application.

[0007] It can be seen that the expansion of Haematococcus pluvialis in a raceway photobioreactor and a pipeline photobioreactor to accumulate astaxanthin, Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, Haematococcus pluvialis JNU35, with a preservation number of CGMCC No.16438, and the extraction of astaxanthin oil are all prior arts, but the prior art does not disclose highly antioxidant astaxanthin oil, nor does it disclose that the astaxanthin oil prepared by the combined culture of Haematococcus pluvialis H7 and Haematococcus pluvialis JNU35 has high antioxidant properties. Summary of the invention

[0008] On one hand, the present invention provides a method for preparing high antioxidant astaxanthin oil, comprising using a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis JNU35, with a preservation number of CGMCC No.16438, to prepare high antioxidant astaxanthin oil.

[0009] The present invention provides a method for preparing high antioxidant astaxanthin oil, which specifically comprises the following steps:

[0010] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0011] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis JNU35, with a preservation number of CGMCC No.16438;

[0012] (2) Extraction of astaxanthin oil, specifically comprising the following steps: A) taking fresh Haematococcus pluvialis from step (1), placing it into a dilute sulfuric acid solution with a concentration of 1.5-2 mol / L, stirring at 65-70° C. to break the algae wall, wherein the mass percentage of Haematococcus pluvialis in the algae slurry is 20-25% on a dry basis, adding calcium carbonate to neutralize to a pH of 6.0-6.5, and filtering to obtain a cracked Haematococcus pluvialis cake; B) taking the cracked Haematococcus pluvialis cake, placing it into alcohol, stirring and leaching, and centrifuging to obtain an extract; the leaching comprises room temperature leaching and heating leaching, wherein the room temperature leaching uses 90%-93% alcohol by mass, the temperature is 25-28° C., and the time is The method comprises the following steps: heating the extract for 30-35 minutes, heating the extract for 45-50 minutes, the alcohol mass percentage of the alcohol used is 95%-98%, the temperature is 55-58°C, the time is 45-50 minutes, and the mass percentage of the room temperature extract and the heated extract is 15:1-17:1; C) taking the extract, and distilling under reduced pressure to obtain crude astaxanthin oil; D) taking the crude astaxanthin oil, putting it into alcohol with a mass percentage of 70%-75% ethanol, the mass ratio of the crude astaxanthin oil to the alcohol is 1:5-1:7, stirring and dispersing at 50-55°C for 40-45 minutes, settling and separating for 3-3.5 hours to obtain the lower layer of astaxanthin oil, and desolventizing to obtain the finished astaxanthin oil.

[0013] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 1-4:1-3.

[0014] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 1:1.

[0015] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 1:2.

[0016] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 2:1.

[0017] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 2:3.

[0018] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 4:3.

[0019] The high antioxidant astaxanthin oil prepared by any of the aforementioned methods.

[0020] The use of the above-mentioned high antioxidant astaxanthin oil in the preparation of food.

[0021] The use of the above-mentioned high antioxidant astaxanthin oil in the preparation of edible oil.

[0022] Application of the above-mentioned high antioxidant astaxanthin oil in cosmetics or medicines.

[0023] The beneficial effects of the present invention include at least:

[0024] (1) Based on previous research, the applicant surprisingly found that the combination culture of Haematococcus pluvialis H7, with a preservation number of CGMCC No. 41186, and Haematococcus pluvialis JNU35 to prepare astaxanthin oil can significantly improve the antioxidant activity of astaxanthin oil compared with astaxanthin oil prepared by culturing Haematococcus pluvialis H7 alone and astaxanthin oil prepared by culturing Haematococcus pluvialis JNU35 alone under the same concentration and conditions. The ROS scavenging rate of the combination culture is increased by 65.1% and 171.3% compared with that of Haematococcus pluvialis H7 alone and Haematococcus pluvialis JNU35 alone, respectively.

[0025] (2) The present application optimizes the ratio of the combined culture of Haematococcus pluvialis H7 and Haematococcus pluvialis JNU35, wherein when the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 4:3, the astaxanthin oil prepared by the combined culture has the highest antioxidant activity, and the ROS scavenging rate reaches 32.8±0.34%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a flow chart of the zebrafish embryo reactive oxygen species (ROS) scavenging test.

[0027] Figure 2 This is the region where reactive oxygen species (ROS) are measured in zebrafish embryos.

[0028] Figure 3 Blank control group: ROS clearance rate.

[0029] Figure 4 Positive control group: glutathione ROS clearance rate.

[0030] Figure 5 Test sample group: ROS clearance rate of astaxanthin oil in Example 1.

[0031] Figure 6 Test sample group: Comparative Example 1 Astaxanthin oil ROS clearance rate.

[0032] Figure 7 Test sample group: ROS clearance rate of astaxanthin oil in comparative example 2.

[0033] Figure 8 This is a flow chart for the expansion of Haematococcus pluvialis.

[0034] Fig. 9 This is the flow chart of astaxanthin oil extraction. DETAILED DESCRIPTION

[0035] The specific implementation of the present invention is described below with reference to the accompanying drawings. The experimental methods used in the implementation examples are all conventional methods unless otherwise specified; the materials and reagents used are all commercially available unless otherwise specified.

[0036] Example 1

[0037] A method for preparing high antioxidant astaxanthin oil comprises the following steps:

[0038] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0039] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis JNU35, with a preservation number of CGMCC No.16438, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 1:1;

[0040] Referring to the scheme of the applicant's prior patent CN 111875528 A, astaxanthin oil is extracted, and the specific steps are as follows: A) taking the fresh Haematococcus pluvialis algae of step (1), putting it into a dilute sulfuric acid solution with a concentration of 1.5 mol / L, stirring at 65°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry is 20% on a dry basis, adding calcium carbonate to neutralize to pH 6.0, and filtering to obtain a broken Haematococcus pluvialis algae cake; B) taking the broken Haematococcus pluvialis algae cake, putting it into alcohol, stirring and leaching, and centrifuging to obtain an extract; the leaching includes room temperature leaching and heating leaching, the room temperature leaching uses 90% ethanol by mass, and the heating leaching uses 90% ethanol by mass. The temperature is 25°C, the time is 30 min, the mass percentage of ethanol used for heating extraction is 95%, the temperature is 55°C, the time is 45 min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 15:1; C) taking the extract, distilling under reduced pressure to obtain crude astaxanthin oil; D) taking the crude astaxanthin oil, putting it into alcohol with a mass percentage of 70% ethanol, the mass ratio of crude astaxanthin oil to alcohol is 1:5, stirring and dispersing at 50°C for 40 min, settling and separating for 3 h to obtain the lower layer of astaxanthin oil, and desolventizing to obtain the astaxanthin oil product.

[0041] Comparative Example 1: The difference from Example 1 is that the Haematococcus pluvialis in step (1) is only Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and the rest is the same as Example 1.

[0042] Comparative Example 2: The difference from Example 1 is that the Haematococcus pluvialis in step (1) is only Haematococcus pluvialis JNU35, with a preservation number of CGMCC No.16438, and the rest is the same as Example 1.

[0043] Refer to the attached Figure 1 Zebrafish embryo reactive oxygen species (ROS) scavenging test flow chart Test Example 1, Comparative Example 1, Comparative Example 2 Reactive oxygen species (ROS) scavenging rate;

[0044] Principle of Antioxidant Activity Assay:

[0045] Reactive oxygen species (ROS) in living cells of the human body can oxidize proteins, nucleic acids, lipids or other molecules, and change the expression of downstream genes through Keap1-Nrf2 signaling in physiological and pathological diseases. The Keap1-Nrf2 signaling in zebrafish is the same as that in humans, and certain fluorescent probes, such as H2DCFDA, can monitor ROS in zebrafish embryos through live staining. Zebrafish embryos are small, which is convenient for taking pictures under a microscope, and software analysis is used to quantitatively read the green fluorescence signal in zebrafish embryos that increases with the increase of ROS. The changes in ROS levels in fish embryos in the treatment group and the blank control group are tested and compared, and the ROS clearance rate is calculated to evaluate the antioxidant effect of the raw materials or products.

[0046] The specific test steps are as follows:

[0047] Pick healthy 2-day-post-fertilization (DPF) zebrafish embryos.

[0048] (1) Test grouping: The test needs to set up a blank control group (fish embryo culture solution), a positive control group (glutathione working solution) and a test substance group (Example 1 group, Comparative Example 1 group, Comparative Example 2 group). If an organic solvent is used for dissolution, the solvent concentration in each group of solutions must be the same.

[0049] Blank control group setting: 24 fish embryos were randomly selected and placed in a 96-well plate, with each well containing one fish embryo and 0.2 mL of fish embryo culture medium.

[0050] Positive control group setting: 24 fish embryos were randomly selected and placed in a 96-well plate, with each well containing one fish embryo and 0.2 mL of glutathione working solution.

[0051] Test substance treatment: 24 fish embryos were randomly selected and placed in a 96-well plate, with each well containing one fish embryo and 0.2 mL of astaxanthin oil from Example 1;

[0052] Randomly select 24 fish embryos and place them in a 96-well plate, with each well containing one fish embryo and 0.2 mL of astaxanthin oil from Comparative Example 1;

[0053] Randomly select 24 fish embryos and place them in a 96-well plate, with each well containing one fish embryo and 0.2 mL of astaxanthin oil from Comparative Example 2;

[0054] (2) Place in a 28℃±1℃ incubator for 24h±1h. Transfer to a 24-well plate, with each well containing 12 fish embryos and 2mL of reactive oxygen species (ROS) test solution, and place in a 28℃±1℃ incubator for 2h±0.2h.

[0055] (3) Microscopic analysis of samples: The fish embryos were placed sideways and photographed under a fluorescent stereo microscope using uniform photographic parameters.

[0056] (4) Data and result calculation: Use analysis software such as Image J to open the photos and calculate the yolk area of ​​each fish embryo (see Appendix Figure 2 ) to mark, then select "Measure average intensity" in the "Measurement" column, and select the "Average signal intensity" in the test results as the ROS signal intensity.

[0057] Calculation of reactive oxygen species (ROS) clearance rate:

[0058]

[0059] (1) Where:

[0060] S—the average value of the “average signal intensity” of ROS in the fish embryos of the test substance treatment group;

[0061] B—The average value of “average signal intensity” of ROS in fish embryos of the blank control group;

[0062] The mean and standard error of each test group were calculated, and the statistical results were expressed as mean ± standard error. The data were analyzed by variance analysis using statistical software, and the average signal intensity of ROS between the test group and the blank control group (or solvent control group) was tested by two-tailed T test to obtain the p value. p<0.05 indicated a significant difference.

[0063] The specific test results are shown in the following table 1

[0064] Table 1

[0065] ROS clearance rate Blank control group ( Figure 3 ) 0％ Positive control group: glutathione ( Figure 4 ) 15.3±0.26％ Test sample group: Example 1 Astaxanthin oil ( Figure 5 ) 27.4±0.31％ Test sample group: Comparative Example 1 Astaxanthin oil ( Figure 6 ) 16.6±0.23％ Test sample group: Comparative Example 2 Astaxanthin Oil ( Figure 7 ) 10.1±0.18％

[0066] From the attached Figure 3-7 As can be seen from Table 1, at the same amount and concentration, the tested sample groups: Example 1 Astaxanthin Oil ( Figure 5 ), that is, astaxanthin oil prepared by the combined culture of Haematococcus pluvialis H7 and Haematococcus pluvialis JNU35, can significantly improve the antioxidant activity of astaxanthin oil.

[0067] Example 2

[0068] A method for preparing high antioxidant astaxanthin oil comprises the following steps:

[0069] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0070] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis JNU35, with a preservation number of CGMCC No.16438, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 2:1;

[0071] Referring to the scheme of the applicant's prior patent CN 111875528 A, astaxanthin oil is extracted, and the specific steps are as follows: A) taking the fresh Haematococcus pluvialis algae of step (1), putting it into a dilute sulfuric acid solution with a concentration of 1.5 mol / L, stirring at 65°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry is 20% on a dry basis, adding calcium carbonate to neutralize to pH 6.0, and filtering to obtain a broken Haematococcus pluvialis algae cake; B) taking the broken Haematococcus pluvialis algae cake, putting it into alcohol, stirring and leaching, and centrifuging to obtain an extract; the leaching includes room temperature leaching and heating leaching, the room temperature leaching uses 90% ethanol by mass, and the heating leaching uses 90% ethanol by mass. The temperature is 25°C, the time is 30 min, the mass percentage of ethanol used for heating extraction is 95%, the temperature is 55°C, the time is 45 min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 15:1; C) taking the extract, distilling under reduced pressure to obtain crude astaxanthin oil; D) taking the crude astaxanthin oil, putting it into alcohol with a mass percentage of 70% ethanol, the mass ratio of crude astaxanthin oil to alcohol is 1:5, stirring and dispersing at 50°C for 40 min, settling and separating for 3 h to obtain the lower layer of astaxanthin oil, and desolventizing to obtain the astaxanthin oil product.

[0072] Using the method of Example 1, the ROS scavenging rate of the astaxanthin oil of Example 2 was determined to be 26.5±0.32%.

[0073] Example 3

[0074] A method for preparing high antioxidant astaxanthin oil comprises the following steps:

[0075] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0076] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis JNU35, with a preservation number of CGMCC No.16438, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 1:2;

[0077] Referring to the scheme of the applicant's prior patent CN 111875528 A, astaxanthin oil is extracted, and the specific steps are as follows: A) taking the fresh Haematococcus pluvialis algae of step (1), putting it into a dilute sulfuric acid solution with a concentration of 1.5 mol / L, stirring at 65°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry is 20% on a dry basis, adding calcium carbonate to neutralize to pH 6.0, and filtering to obtain a broken Haematococcus pluvialis algae cake; B) taking the broken Haematococcus pluvialis algae cake, putting it into alcohol, stirring and leaching, and centrifuging to obtain an extract; the leaching includes room temperature leaching and heating leaching, the room temperature leaching uses 90% ethanol by mass, and the heating leaching uses 90% ethanol by mass. The temperature is 25°C, the time is 30 min, the mass percentage of ethanol used for heating extraction is 95%, the temperature is 55°C, the time is 45 min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 15:1; C) taking the extract, distilling under reduced pressure to obtain crude astaxanthin oil; D) taking the crude astaxanthin oil, putting it into alcohol with a mass percentage of 70% ethanol, the mass ratio of crude astaxanthin oil to alcohol is 1:5, stirring and dispersing at 50°C for 40 min, settling and separating for 3 h to obtain the lower layer of astaxanthin oil, and desolventizing to obtain the astaxanthin oil product.

[0078] Using the method of Example 1, the ROS scavenging rate of the astaxanthin oil of Example 3 was determined to be 25.8±0.28%.

[0079] Example 4

[0080] A method for preparing high antioxidant astaxanthin oil comprises the following steps:

[0081] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0082] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis JNU35, with a preservation number of CGMCC No.16438, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 2:3;

[0083] Referring to the scheme of the applicant's prior patent CN 111875528 A, astaxanthin oil is extracted, and the specific steps are as follows: A) taking the fresh Haematococcus pluvialis algae of step (1), putting it into a dilute sulfuric acid solution with a concentration of 1.5 mol / L, stirring at 65°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry is 20% on a dry basis, adding calcium carbonate to neutralize to pH 6.0, and filtering to obtain a broken Haematococcus pluvialis algae cake; B) taking the broken Haematococcus pluvialis algae cake, putting it into alcohol, stirring and leaching, and centrifuging to obtain an extract; the leaching includes room temperature leaching and heating leaching, the room temperature leaching uses 90% ethanol by mass, and the heating leaching uses 90% ethanol by mass. The temperature is 25°C, the time is 30 min, the mass percentage of ethanol used for heating extraction is 95%, the temperature is 55°C, the time is 45 min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 15:1; C) taking the extract, distilling under reduced pressure to obtain crude astaxanthin oil; D) taking the crude astaxanthin oil, putting it into alcohol with a mass percentage of 70% ethanol, the mass ratio of crude astaxanthin oil to alcohol is 1:5, stirring and dispersing at 50°C for 40 min, settling and separating for 3 h to obtain the lower layer of astaxanthin oil, and desolventizing to obtain the astaxanthin oil product.

[0084] Using the method of Example 1, the ROS scavenging rate of the astaxanthin oil of Example 4 was determined to be 29.7±0.36%.

[0085] Example 5

[0086] A method for preparing high antioxidant astaxanthin oil comprises the following steps:

[0087] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0088] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis JNU35, with a preservation number of CGMCC No.16438, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 4:3;

[0089] Referring to the scheme of the applicant's prior patent CN 111875528 A, astaxanthin oil is extracted, and the specific steps are as follows: A) taking the fresh Haematococcus pluvialis algae of step (1), putting it into a dilute sulfuric acid solution with a concentration of 1.5 mol / L, stirring at 65°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry is 20% on a dry basis, adding calcium carbonate to neutralize to pH 6.0, and filtering to obtain a broken Haematococcus pluvialis algae cake; B) taking the broken Haematococcus pluvialis algae cake, putting it into alcohol, stirring and leaching, and centrifuging to obtain an extract; the leaching includes room temperature leaching and heating leaching, the room temperature leaching uses 90% ethanol by mass, and the heating leaching uses 90% ethanol by mass. The temperature is 25°C, the time is 30 min, the mass percentage of ethanol used for heating extraction is 95%, the temperature is 55°C, the time is 45 min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 15:1; C) taking the extract, distilling under reduced pressure to obtain crude astaxanthin oil; D) taking the crude astaxanthin oil, putting it into alcohol with a mass percentage of 70% ethanol, the mass ratio of crude astaxanthin oil to alcohol is 1:5, stirring and dispersing at 50°C for 40 min, settling and separating for 3 h to obtain the lower layer of astaxanthin oil, and desolventizing to obtain the astaxanthin oil product.

[0090] Using the method of Example 1, the ROS scavenging rate of the astaxanthin oil of Example 5 was determined to be 32.8±0.34%.

[0091] Example 6

[0092] A method for preparing high antioxidant astaxanthin oil comprises the following steps:

[0093] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 20 days to allow it to accumulate astaxanthin;

[0094] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis JNU35, with a preservation number of CGMCC No.16438, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 4:3;

[0095] Referring to the scheme of the applicant's prior patent CN 111875528 A, astaxanthin oil is extracted, and the specific steps are as follows: A) taking the fresh Haematococcus pluvialis algae of step (1), putting it into a dilute sulfuric acid solution with a concentration of 2 mol / L, stirring at 70°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 25%, adding calcium carbonate to neutralize to pH 6.5, and filtering to obtain a broken Haematococcus pluvialis algae cake; B) taking the broken Haematococcus pluvialis algae cake, putting it into alcohol, stirring and leaching, and centrifuging to obtain an extract; the leaching includes room temperature leaching and heating leaching, the room temperature leaching uses ethanol with a mass percentage of 93%, and the temperature is 28°C, time is 35min, the mass percentage of ethanol used for heating extraction is 98%, the temperature is 58°C, the time is 50min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 17:1; C) taking the extract, vacuum distilling to obtain crude astaxanthin oil; D) taking the crude astaxanthin oil, putting it into alcohol with a mass percentage of 75% ethanol, the mass ratio of crude astaxanthin oil to alcohol is 1:7, stirring and dispersing at 55°C for 45min, settling and separating for 3.5h to obtain the lower layer astaxanthin oil, and vacuum desolventizing to obtain the astaxanthin oil product.

[0096] Using the method of Example 1, the ROS scavenging rate of the astaxanthin oil of Example 6 was determined to be 33.7±0.33%.

[0097] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing high antioxidant astaxanthin oil, characterized in that The invention comprises using a mixture of Haematococcus pluvialis H7 with a preservation number of CGMCC No.41186 and Haematococcus pluvialis JNU35 with a preservation number of CGMCC No.16438 to prepare high antioxidant astaxanthin oil.

2. The method for preparing a high antioxidant astaxanthin oil according to claim 1, characterized in that The mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis JNU35 is 1-2:1-3.

3. The method for preparing a high antioxidant astaxanthin oil according to claim 1, characterized in that The mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis JNU35 is 1:

1.

4. The method for preparing a high antioxidant astaxanthin oil according to claim 1, characterized in that The mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis JNU35 is 1:

2.

5. The method for preparing a high antioxidant astaxanthin oil according to claim 1, characterized in that The mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis JNU35 is 2:

1.

6. The method for preparing a high antioxidant astaxanthin oil according to claim 1, characterized in that The mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis JNU35 is 2:

3.

7. High antioxidant astaxanthin oil prepared by the method according to any one of claims 1 to 6.

8. Use of the high antioxidant astaxanthin oil according to claim 7 in preparing food.

9. Use of the high antioxidant astaxanthin oil according to claim 7 in the preparation of edible oil.

10. Use of the high antioxidant astaxanthin oil according to claim 7 in cosmetics or medicines.

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