Preparation method and application of astaxanthin oil with high anti-inflammatory activity

Abstract

The invention belongs to the field of astaxanthin oil, and particularly relates to a preparation method and application of astaxanthin oil with high anti-inflammatory activity. Haematococcus pluvialis H7 and haematococcus pluvialis ENN71 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 ENN71 culture, the astaxanthin oil prepared by single haematococcus pluvialis ENN71 culture has the advantages that the astaxanthin oil content is greatly reduced, and the astaxanthin oil yield is improved. Through combined culture, the anti-inflammatory activity of the astaxanthin oil can be remarkably improved, and the neutrophil aggregation inhibition rates of zebra fish embryos are respectively improved by 76.2% and 115.2%. According to the application, the ratio of the haematococcus pluvialis H7 to the haematococcus pluvialis ENN71 in combined culture is optimized, when the mass ratio of the haematococcus pluvialis H7 to the haematococcus pluvialis ENN71 is 1: 2, the anti-inflammatory activity of the astaxanthin oil prepared through combined culture is the highest, and the zebra fish embryo neutrophile granulocyte aggregation inhibition rate reaches 77.1 + / -0.36%.

Description

Technical Field

[0001] The invention belongs to the field of astaxanthin oil, and specifically relates to a preparation method and application of astaxanthin oil with high anti-inflammatory activity. 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] Astaxanthin has anti-inflammatory effects, and its anti-inflammatory mechanism includes: Astaxanthin can inhibit the activation of the NF-κB signaling pathway, reduce the expression of inflammation-related genes, and thus reduce the production of inflammatory mediators. In addition, astaxanthin can also reduce the release of inflammatory mediators by inhibiting the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Astaxanthin has a regulatory effect on the functions of various immune cells. Astaxanthin can inhibit the activation of macrophages and reduce the release of inflammatory mediators. In addition, astaxanthin can also regulate the functions of T lymphocytes and B lymphocytes, promote the maintenance of immune balance, and thus reduce inflammatory responses. Astaxanthin is a powerful antioxidant, and its antioxidant capacity is stronger than that of common antioxidants such as vitamin C, vitamin E, and β-carotene. Astaxanthin can directly remove ROS and RNS, inhibit lipid peroxidation, and reduce the generation of oxidative stress products, thereby protecting cells and tissues from oxidative damage and reducing inflammatory responses. At present, many preclinical studies and some clinical trials have shown that astaxanthin has potential efficacy in the treatment of some inflammatory-related diseases. For example, in the treatment of arthritis, astaxanthin may reduce joint inflammation and pain and improve joint function by inhibiting inflammatory signaling pathways and reducing the production of inflammatory mediators. In the treatment of inflammatory bowel disease, astaxanthin may reduce intestinal inflammatory responses and relieve symptoms such as diarrhea and abdominal pain by regulating the function of intestinal immune cells and maintaining the integrity of the intestinal mucosal barrier.

[0004] 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.

[0005] 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.

[0006] 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 ENN71 with a preservation number of CGMCC No.5147 to prepare astaxanthin oil can significantly improve the anti-inflammatory effect of astaxanthin oil.

[0007] After searching, the prior art already has CN2020108914158 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 device for monitoring the movement state of Haematococcus pluvialis in a photobioreactor and frequency modulation (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), CN 2019100735049 A strain of Haematococcus pluvialis ENN71 with high astaxanthin production, and a cultivation method and application thereof, CN2015102369015 A strain of Haematococcus pluvialis ENN71, and a cultivation method and application thereof.

[0008] 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 ENN71, with a preservation number of CGMCC No.5147, and the extraction of astaxanthin oil are all prior arts, but the prior art does not disclose astaxanthin oil with high anti-inflammatory efficacy, nor does it disclose that astaxanthin oil prepared by the combined culture of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 has high anti-inflammatory efficacy. Summary of the invention

[0009] On one hand, the present invention provides a method for preparing astaxanthin oil with high anti-inflammatory activity, comprising using a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis ENN71, with a preservation number of CGMCC No.5147, to prepare astaxanthin oil with high anti-inflammatory activity.

[0010] The present invention provides a method for preparing astaxanthin oil with high anti-inflammatory activity, which specifically comprises the following steps:

[0011] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0012] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis ENN71, with a preservation number of CGMCC No.5147, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 1-4:1-3;

[0013] 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) fresh Haematococcus pluvialis algae from step (1) is put into a dilute sulfuric acid solution with a concentration of 1-2 mol / L, stirred at 60-65°C for wall breaking, the mass percentage of Haematococcus pluvialis in the algae slurry is 15-20% on a dry basis, calcium carbonate is added to neutralize to pH 6.0, and filter press to obtain wall-broken Haematococcus pluvialis cake; B) the wall-broken Haematococcus pluvialis cake is taken, put into alcohol, stirred for leaching, and centrifuged to obtain an extract; the leaching comprises room temperature leaching and heating leaching, the mass percentage of alcohol ethanol used for room temperature leaching is 92-95%, the temperature is 28-30°C, and the time is 32-35 min, the mass percentage of alcohol ethanol used for heating leaching is 97-98%, the temperature is 60-65°C, and the time is 40-45 min, and the mass percentage of the room temperature leaching and heating leaching liquid is both

[0014] 12:1-16:1; C) taking the extract and performing vacuum distillation to obtain crude astaxanthin oil; D) taking the crude astaxanthin oil and putting it into alcohol having a mass percentage of 70-75% ethanol, the mass ratio of crude astaxanthin oil to alcohol is

[0015] 1:5-1:7, stir and disperse at 50-52℃ for 40-45min, separate by sedimentation for 3.5-4h to obtain the lower layer of astaxanthin oil, and desolventize to obtain the finished astaxanthin oil.

[0016] Preferably, the mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 is 1:1.

[0017] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 1:2.

[0018] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 2:1.

[0019] Preferably, the mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 is 2:3.

[0020] Preferably, the mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 is 4:3.

[0021] The astaxanthin oil with high anti-inflammatory activity prepared by any of the aforementioned methods.

[0022] The use of the above-mentioned astaxanthin oil with high anti-inflammatory activity in preparing food.

[0023] The use of the above-mentioned high anti-inflammatory activity astaxanthin oil in the preparation of edible oil.

[0024] The use of the above-mentioned astaxanthin oil with high anti-inflammatory activity in cosmetics or medicines.

[0025] The beneficial effects of the present invention include at least:

[0026] (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 ENN71 to prepare astaxanthin oil can significantly improve the anti-inflammatory activity of astaxanthin oil compared with astaxanthin oil prepared by culturing Haematococcus pluvialis H7 alone and astaxanthin oil prepared by culturing Haematococcus pluvialis ENN71 alone under the same concentration and conditions. The inhibition rate of neutrophil aggregation in zebrafish embryos cultured in the combination culture was increased by 76.2% and 115.2% compared with Haematococcus pluvialis H7 alone and Haematococcus pluvialis ENN71 alone, respectively.

[0027] (2) The present application optimizes the ratio of the combined culture of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71, wherein when the mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 is 1:2, the astaxanthin oil prepared by the combined culture has the highest anti-inflammatory activity, and the zebrafish embryo neutrophil aggregation inhibition rate reaches 77.1±0.36%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a flow chart of the zebrafish embryo neutrophil aggregation inhibition test.

[0029] Figure 2 The neutrophil measurement area in the tail of zebrafish embryos.

[0030] Figure 3 Neutrophil aggregation inhibition rate of zebrafish embryos in the model control group.

[0031] Figure 4 Neutrophil aggregation inhibition rate of zebrafish embryos in the positive control group of 10 μM indomethacin.

[0032] Figure 5 Test sample group Example 1 Astaxanthin oil zebrafish embryo neutrophil aggregation inhibition rate.

[0033] Figure 6 Test sample group comparison example 1: Astaxanthin oil zebrafish embryo neutrophil aggregation inhibition rate.

[0034] Figure 7 Test sample group comparison example 2: astaxanthin oil zebrafish embryo neutrophil aggregation inhibition rate.

[0035] Figure 8 This is a flow chart for the expansion of Haematococcus pluvialis.

[0036] Fig. 9 This is the flow chart of astaxanthin oil extraction. DETAILED DESCRIPTION

[0037] 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.

[0038] Example 1

[0039] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0040] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0041] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis ENN71, with a preservation number of CGMCC No.5147, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 1:1;

[0042] 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 mol / L, stirring at 60°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15%, 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 ethanol with a mass percentage of 92%, and the temperature is 28°C, time is 32min, the mass percentage of ethanol used for heating extraction is 97%, the temperature is 60°C, the time is 40min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 12:1; 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 52°C for 45min, settling and separating for 3.5h to obtain the lower layer astaxanthin oil, and desolventizing to obtain the astaxanthin oil product.

[0043] 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.

[0044] Comparative Example 2: The difference from Example 1 is that the Haematococcus pluvialis in step (1) is only Haematococcus pluvialis ENN71, with a preservation number of CGMCC No.5147, and the rest is the same as Example 1. Figure 1 Flow chart of the test for inhibition of neutrophil aggregation in zebrafish embryos Neutrophil aggregation inhibition rates of test example 1, comparative example 1, and comparative example 2.

[0045] Principle of anti-inflammatory activity assay:

[0046] Neutrophils in zebrafish embryos are highly similar to human neutrophils in morphology, biochemistry and physiological functions. Neutrophils are the first white blood cells to appear at the site of injury or pathogen invasion, and they act to remove infection or harmful substances. A model in which copper sulfate was used to induce damage to neuromast cells in the lateral line region of zebrafish embryos and cause neutrophil aggregation was tested. The changes in the number of neutrophils in the lateral line region of fish embryos in the test substance treatment group, comparative example group 1, comparative example group 2 and model control group were compared, and the neutrophil inhibition rate was calculated to evaluate the anti-inflammatory effect of raw materials, formulas or products.

[0047] The specific test steps are as follows:

[0048] Select healthy zebrafish embryos that are 3 days old after fertilization. Figure 1 .

[0049] (1) Test groups: The test needs to set up a model control group (copper sulfate working solution), a positive control group (copper sulfate working solution + indomethacin working solution) and a test substance group (Example 1 group, Comparative Example 1 group, Comparative Example 2 group).

[0050] Model control group setting: 24 fish embryos were randomly selected and placed in a 3 cm culture dish, and 5 mL of fish embryo culture medium containing 10 μM anhydrous copper sulfate was added.

[0051] Positive control group setting: 24 fish embryos were randomly selected and placed in a 3 cm culture dish, and 5 mL of fish embryo culture medium containing 10 μM anhydrous copper sulfate and 10 μM indomethacin was added.

[0052] The test substance treatment group was set up as follows: 24 fish embryos were randomly selected and placed in a 3 cm culture dish, and 5 mL of 10 μM anhydrous copper sulfate and 10 μM astaxanthin oil from Example 1 were added;

[0053] 24 fish embryos were randomly selected and placed in a 3 cm culture dish, and 5 mL of 10 μM anhydrous copper sulfate and 10 μM astaxanthin oil of comparative example 1 were added;

[0054] 24 fish embryos were randomly selected and placed in a 3 cm culture dish, and 5 mL of 10 μM anhydrous copper sulfate and 10 μM comparative example 2 astaxanthin oil were added;

[0055] (2) Place in a constant temperature incubator at 28°C ± 1°C and incubate for 40 min to 45 min.

[0056] (3) Fish embryo fixation and staining: After each test group of fish embryos were fixed in paraformaldehyde for at least 1 hour, the fish embryos were treated with PBST for 3 times, each time for 5 minutes, and then treated with 50% ethanol for 3 minutes.

[0057] After the fish embryos were stained with Sudan black staining solution at room temperature for 1 hour, they were washed with 70% ethanol 4 times, each time for 5 minutes, and then treated with PBST twice, each time for 5 minutes.

[0058] Treat the fish embryos with bleach solution for 10 minutes. If treated in a test tube, keep the lid open. Then treat with 70% ethanol solution for 5 minutes, PBST for 1 minute, clear solution 1 for 15 minutes, clear solution 2 for 10 minutes, and PBST for 3 minutes.

[0059] (4) Microscopic analysis of samples: Place the fish embryo on its side and then place it under a stereo microscope to take a picture of the fish embryo's tail.

[0060] (5) Data and result calculation: Count the lateral line area of ​​each fish embryo from three quarters of the tail starting from the anus (see Figure 2 ) of neutrophils.

[0061] Calculate the neutrophil aggregation inhibition rate:

[0062]

[0063] (1) Where:

[0064] S—mean value of neutrophil number in fish embryos of the test substance treatment group, unit is granule per tail (granule / tail);

[0065] M—mean value of neutrophil number in fish embryos of the model control group, unit is granule per tail (granule / tail);

[0066] B—The average number of neutrophils in fish embryos of the blank control group, in granules per tail (granules / tail);

[0067] 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 number of neutrophils between the test group and the model control group was tested by two-tailed T test to obtain the p value. p < 0.05 indicated a significant difference.

[0068] The specific test results are shown in the following table 1

[0069] Table 1

[0070]

[0071] From the attached Figure 3-7 As can be seen from Table 1, at the same amount and concentration, the test sample group: Example 1 astaxanthin oil, i.e., astaxanthin oil prepared by combined culture of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71, can significantly improve the anti-inflammatory activity of astaxanthin oil.

[0072] Example 2

[0073] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0074] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0075] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis ENN71, with a preservation number of CGMCC No.5147, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 2:1;

[0076] 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 mol / L, stirring at 60°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15%, 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 ethanol with a mass percentage of 92%, and the temperature is 28°C, time is 32min, the mass percentage of ethanol used for heating extraction is 97%, the temperature is 60°C, the time is 40min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 12:1; 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 52°C for 45min, settling and separating for 3.5h to obtain the lower layer astaxanthin oil, and desolventizing to obtain the astaxanthin oil product.

[0077] Using the method of Example 1, the zebrafish embryo neutrophil aggregation inhibition rate of the astaxanthin oil of Example 2 was determined to be 75.4±0.31%.

[0078] Example 3

[0079] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0080] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0081] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis ENN71, with a preservation number of CGMCC No.5147, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 1:2;

[0082] 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 mol / L, stirring at 60°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15%, 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 ethanol with a mass percentage of 92%, and the temperature is 28°C, time is 32min, the mass percentage of ethanol used for heating extraction is 97%, the temperature is 60°C, the time is 40min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 12:1; 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 52°C for 45min, settling and separating for 3.5h to obtain the lower layer astaxanthin oil, and desolventizing to obtain the astaxanthin oil product.

[0083] Using the method of Example 1, the zebrafish embryo neutrophil aggregation inhibition rate of the astaxanthin oil of Example 3 was determined to be 77.1±0.36%.

[0084] Example 4

[0085] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0086] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0087] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis ENN71, with a preservation number of CGMCC No.5147, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 2:3;

[0088] 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 mol / L, stirring at 60°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15%, 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 ethanol with a mass percentage of 92%, and the temperature is 28°C, time is 32min, the mass percentage of ethanol used for heating extraction is 97%, the temperature is 60°C, the time is 40min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 12:1; 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 52°C for 45min, settling and separating for 3.5h to obtain the lower layer astaxanthin oil, and desolventizing to obtain the astaxanthin oil product.

[0089] Using the method of Example 1, the zebrafish embryo neutrophil aggregation inhibition rate of the astaxanthin oil of Example 4 was determined to be 74.2±0.27%.

[0090] Example 5

[0091] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0092] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0093] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis ENN71, with a preservation number of CGMCC No.5147, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 4:3;

[0094] 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 mol / L, stirring at 60°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15%, 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 ethanol with a mass percentage of 92%, and the temperature is 28°C, time is 32min, the mass percentage of ethanol used for heating extraction is 97%, the temperature is 60°C, the time is 40min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 12:1; 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 52°C for 45min, settling and separating for 3.5h to obtain the lower layer astaxanthin oil, and desolventizing to obtain the astaxanthin oil product.

[0095] Using the method of Example 1, the zebrafish embryo neutrophil aggregation inhibition rate of the astaxanthin oil of Example 5 was determined to be 73.3±0.25%.

[0096] Example 6

[0097] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0098] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0099] The Haematococcus pluvialis is a mixture of Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186, and Haematococcus pluvialis ENN71, with a preservation number of CGMCC No.5147, and the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 1:2;

[0100] 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 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 95% ethanol by mass, and the temperature is 100°C. The temperature is 30°C, the time is 35min, the mass percentage of ethanol used for heating extraction is 98%, the temperature is 65°C, the time is 45min, and the mass percentage of the room temperature extraction and the heating extraction liquid is 16:1; 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 70% ethanol, the mass ratio of crude astaxanthin oil to alcohol is 1:5, stirring and dispersing at 50°C for 40min, settling and separating for 4h to obtain the lower layer astaxanthin oil, and vacuum desolventizing to obtain the astaxanthin oil product.

[0101] Using the method of Example 1, the zebrafish embryo neutrophil aggregation inhibition rate of the astaxanthin oil of Example 6 was determined to be 76.6±0.28%.

[0102] 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 astaxanthin oil with high anti-inflammatory activity, characterized in that The invention comprises using a mixture of Haematococcus pluvialis H7 with a preservation number of CGMCC No.41186 and Haematococcus pluvialis ENN71 with a preservation number of CGMCC No.5147 to prepare astaxanthin oil with high anti-inflammatory activity.

2. A method for preparing astaxanthin oil with high anti-inflammatory activity according to claim 1, characterized in that The mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 is 1-4:1-3.

3. A method for preparing astaxanthin oil with high anti-inflammatory activity according to claim 1, characterized in that The mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 is 1:

1.

4. A method for preparing astaxanthin oil with high anti-inflammatory activity according to claim 1, characterized in that The mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 is 1:

2.

5. A method for preparing astaxanthin oil with high anti-inflammatory activity according to claim 1, characterized in that The mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 is 2:

1.

6. A method for preparing astaxanthin oil with high anti-inflammatory activity according to claim 1, characterized in that The mass ratio of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 is 2:

3.

7. Astaxanthin oil with high anti-inflammatory activity prepared by the method according to any one of claims 1 to 6.

8. Use of the astaxanthin oil with high anti-inflammatory activity according to claim 7 in preparing food.

9. Use of the astaxanthin oil with high anti-inflammatory activity according to claim 7 in the preparation of edible oil.

10. Use of the astaxanthin oil with high anti-inflammatory activity according to claim 7 in cosmetics or medicines.

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