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

Abstract

The present invention relates to the field of astaxanthin oil, specifically to a method for preparing astaxanthin oil with high anti-inflammatory activity and its application. The present invention cultivates Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71 in a racetrack photobioreactor and a pipeline photobioreactor to accumulate astaxanthin and prepare astaxanthin oil. Under the same concentration and conditions, the combined culture significantly enhances the anti-inflammatory activity of astaxanthin oil compared to astaxanthin oil prepared by culturing Haematococcus pluvialis H7 or Haematococcus pluvialis ENN71 alone, increasing the zebrafish embryo neutrophil aggregation inhibition rate by 76.2% and 115.2%, respectively. The present application optimizes the ratio of the combined culture of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71. When the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 1:2, the astaxanthin oil prepared by the combined culture has the highest anti-inflammatory activity, with a zebrafish embryo neutrophil aggregation inhibition rate of 77.1±0.36%.

Description

Technical Field

[0001] 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. Background Art

[0002] Astaxanthin's chemical structure 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 eliminate 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 properties. Its anti-inflammatory mechanisms include: astaxanthin can inhibit the activation of the NF-κB signaling pathway, reducing the expression of inflammation-related genes, and thus reducing the production of inflammatory mediators. Furthermore, astaxanthin can reduce the release of inflammatory mediators by inhibiting the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Astaxanthin modulates the functions of various immune cells. It can inhibit macrophage activation and reduce the release of inflammatory mediators. Furthermore, astaxanthin can regulate the functions of T and B lymphocytes, promoting immune balance and thus alleviating inflammatory responses. Astaxanthin is a powerful antioxidant, with antioxidant capacity greater than that of common antioxidants such as vitamin C, vitamin E, and beta-carotene. By directly scavenging ROS and RNS, astaxanthin inhibits lipid peroxidation and reduces the production of oxidative stress products, thereby protecting cells and tissues from oxidative damage and alleviating inflammatory responses. Currently, multiple preclinical studies and some clinical trials have demonstrated the potential efficacy of astaxanthin in treating several inflammatory 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] The applicant's previous research shows that (method for producing astaxanthin oil from Haematococcus pluvialis, patent number CN2020108914158) 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 Aierkang 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, the applicants 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 existing technologies include 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 High-astaxanthin-producing Haematococcus pluvialis ENN71 and its cultivation method and application, CN2015102369015 A strain of Haematococcus pluvialis ENN71 and its cultivation method and application.

[0008] It can be seen that the expansion of Haematococcus pluvialis in a racetrack 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) taking the fresh Haematococcus pluvialis algae from step (1), putting it into a dilute sulfuric acid solution with a concentration of 1-2 mol / L, stirring at 60-65°C to break the algae wall, the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15-20%, 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 92-95% of ethanol by mass, the temperature is 28-30°C, and the time is 32-35min, and the heating leaching uses 97-98% of ethanol by mass, the temperature is 60-65°C, and the time is 40-45min. n, the mass percentage of the room temperature extraction and the heated extraction liquid is 12:1-16:1; C) taking the extraction liquid and distilling it under reduced pressure to obtain crude astaxanthin oil; D) taking the crude astaxanthin oil, adding it into 70-75% ethanol by mass, with the mass ratio of crude astaxanthin to ethanol being 1:5-1:7, stirring and dispersing at 50-52° C. for 40-45 minutes, settling and separating for 3.5-4 hours to obtain the lower layer of astaxanthin oil, and desolvating it under reduced pressure to obtain the finished astaxanthin oil.

[0014] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 1:1.

[0015] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 1:2.

[0016] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 2:1.

[0017] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 2:3.

[0018] Preferably, the mass ratio of Haematococcus pluvialis H7 to Haematococcus pluvialis ENN71 is 4:3.

[0019] The astaxanthin oil with high anti-inflammatory activity is prepared by any of the aforementioned methods.

[0020] The use of the above-mentioned astaxanthin oil with high anti-inflammatory activity in the preparation of cosmetics.

[0021] The beneficial effects of the present invention include at least:

[0022] (1) Based on the previous research, the applicant surprisingly found that the combination culture of Haematococcus pluvialis H7, with the preservation number CGMCC No. 41186, and Haematococcus pluvialis ENN71 to prepare astaxanthin oil can significantly improve the anti-inflammatory activity of astaxanthin oil compared with the astaxanthin oil prepared by Haematococcus pluvialis H7 alone and the astaxanthin oil prepared by 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 the astaxanthin oil prepared by Haematococcus pluvialis H7 alone and Haematococcus pluvialis ENN71 alone, respectively.

[0023] (2) This application optimizes the ratio of combined culture of Haematococcus pluvialis H7 and Haematococcus pluvialis ENN71. 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

[0024] Figure 1 Flowchart of the zebrafish embryo neutrophil aggregation inhibition assay.

[0025] Figure 2 The neutrophil measurement area in the tail of zebrafish embryos.

[0026] Figure 3 Neutrophil aggregation inhibition rate of zebrafish embryos in the model control group.

[0027] Figure 4 Neutrophil aggregation inhibition rate of zebrafish embryos in the positive control group of 10µM indomethacin.

[0028] Figure 5 Test sample group Example 1 Astaxanthin oil zebrafish embryo neutrophil aggregation inhibition rate.

[0029] Figure 6 Test sample group comparative example 1 astaxanthin oil zebrafish embryo neutrophil aggregation inhibition rate.

[0030] Figure 7 The inhibition rate of neutrophil aggregation of zebrafish embryos by astaxanthin oil in comparative example 2 of the test sample group was astaxanthin oil.

[0031] Figure 8 This is a flow chart for the expansion of Haematococcus pluvialis.

[0032] Figure 9 This is the flow chart of astaxanthin oil extraction. DETAILED DESCRIPTION

[0033] The following describes the specific embodiments of the present invention with reference to the accompanying drawings. The experimental methods used in the examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0034] Example 1

[0035] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0036] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0037] 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;

[0038] 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 mol / L, stirred at 60°C to break the algae wall, and the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15%, and calcium carbonate is added to neutralize to pH 6 .0, filter pressing to obtain a cracked Haematococcus pluvialis cake; B) taking the cracked Haematococcus pluvialis cake, putting it into alcohol, stirring and extracting, and centrifuging to obtain an extract; the extraction includes room temperature extraction and heating extraction, the room temperature extraction uses 92% ethanol by mass, the temperature is 28°C, and the time is 32 minutes, the heating extraction uses 97% ethanol by mass, the temperature is 60°C, and the time is 40 minutes, and the mass percentage of the room temperature extraction and the heating extraction liquid is both 12:1; C) taking the extract, and distilling under reduced pressure to obtain crude astaxanthin oil; D) taking crude astaxanthin oil, putting it into alcohol with a mass percentage of 75% ethanol, the mass ratio of crude astaxanthin to alcohol is 1:7, stirring and dispersing at 52°C for 45 minutes, settling and separating for 3.5 hours to obtain a lower layer of astaxanthin oil, and desolventizing to obtain a finished astaxanthin oil product.

[0039] 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.

[0040] 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. The rest is the same as Example 1.

[0041] Refer to the attached Figure 1 Flowchart 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 were tested.

[0042] Principle of anti-inflammatory activity assay:

[0043] Neutrophils in zebrafish embryos are highly similar to human neutrophils in morphology, biochemistry, and physiological function. Neutrophils are the first white blood cells to appear at sites of injury or pathogen invasion, acting to eliminate infection or harmful substances. A copper sulfate-induced model of neutrophil accumulation in the lateral line region of zebrafish embryos was used for testing. Changes in neutrophil numbers in the lateral line region of fish embryos were compared between the test substance-treated group, comparative example group 1, comparative example group 2, and the model control group. The neutrophil inhibition rate was calculated to evaluate the anti-inflammatory effect of the raw material, formula, or product.

[0044] The specific test steps are as follows:

[0045] Select healthy zebrafish embryos 3 days after fertilization. Figure 1 .

[0046] (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).

[0047] 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.

[0048] 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.

[0049] Test substance treatment group setup: 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;

[0050] 24 fish embryos were randomly selected and placed in a 3 cm culture dish, and 5 mL of oil containing 10 μM anhydrous copper sulfate and 10 μM astaxanthin from comparative example 1 was added;

[0051] 24 fish embryos were randomly selected and placed in a 3 cm culture dish, and 5 mL of oil containing 10 μM anhydrous copper sulfate and 10 μM astaxanthin from comparative example 2 was added;

[0052] (2) Place in a constant temperature box at 28℃±1℃ and incubate for 40min~45min.

[0053] (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 three times, each time for 5 minutes, and then treated with 50% ethanol for 3 minutes.

[0054] The fish embryos were stained with Sudan black staining solution at room temperature for 1 hour, then washed with 70% ethanol 4 times for 5 minutes each time, and then treated with PBST 2 times for 5 minutes each time.

[0055] Treat the fish embryos with bleach solution for 10 minutes (if treating in a test tube, keep the lid open). Then treat with 70% ethanol solution for 5 minutes, PBST for 1 minute, Clearing Solution 1 for 15 minutes, Clearing Solution 2 for 10 minutes, and PBST for 3 minutes.

[0056] (4) Microscopic analysis of the sample: Place the fish embryo on its side. Then, place it under a stereo microscope and take a picture of the tail of the fish embryo.

[0057] (5) Data and result calculation: Count the lateral line area of ​​each fish embryo from the anus to the tail (see Figure 2 ) of neutrophils.

[0058] Calculate the neutrophil aggregation inhibition rate:

[0059]

[0060] (1) Where:

[0061] S—the average number of neutrophils in fish embryos in the test substance treatment group, in granules per tail (granules / tail);

[0062] M—mean number of neutrophils in fish embryos of the model control group, in granules per tail (granules / tail);

[0063] B—The average number of neutrophils in fish embryos of the blank control group, the unit is granules per tail (granules / tail);

[0064] The mean and standard error (SD) of each test group were calculated, and statistical results were expressed as mean ± SD. Data were analyzed using statistical software for analysis of variance. Two-tailed T-tests were performed to determine the p-value for the differences in neutrophil counts between the test group and the model control group. A p < 0.05 indicated a significant difference.

[0065] The specific test results are shown in Table 1 below.

[0066] Table 1

[0067] Neutrophil aggregation inhibition rate in zebrafish embryos Model control ( Figure 3 ) 0% Positive control group: 10µM indomethacin ( Figure 4 ) 28.7±0.19% Test sample group: Example 1 Astaxanthin oil ( Figure 5 ) 74.9±0.33% Test sample group: Comparative Example 1 Astaxanthin Oil ( Figure 6 ) 42.5±0.24% Test sample group: Comparative Example 2 Astaxanthin Oil ( Figure 7 ) 34.8±0.27%

[0068] 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.

[0069] Example 2

[0070] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0071] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0072] 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;

[0073] 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 mol / L, stirred at 60°C to break the algae wall, and the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15%, and calcium carbonate is added to neutralize to pH 6 .0, filter pressing to obtain a cracked Haematococcus pluvialis cake; B) taking the cracked Haematococcus pluvialis cake, putting it into alcohol, stirring and extracting, and centrifuging to obtain an extract; the extraction includes room temperature extraction and heating extraction, the room temperature extraction uses 92% ethanol by mass, the temperature is 28°C, and the time is 32 minutes, the heating extraction uses 97% ethanol by mass, the temperature is 60°C, and the time is 40 minutes, and the mass percentage of the room temperature extraction and the heating extraction liquid is both 12:1; C) taking the extract, and distilling under reduced pressure to obtain crude astaxanthin oil; D) taking crude astaxanthin oil, putting it into alcohol with a mass percentage of 75% ethanol, the mass ratio of crude astaxanthin to alcohol is 1:7, stirring and dispersing at 52°C for 45 minutes, settling and separating for 3.5 hours to obtain a lower layer of astaxanthin oil, and desolventizing to obtain a finished astaxanthin oil product.

[0074] 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%.

[0075] Example 3

[0076] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0077] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0078] 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;

[0079] 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 mol / L, stirred at 60°C to break the algae wall, and the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15%, and calcium carbonate is added to neutralize to pH 6 .0, filter pressing to obtain a cracked Haematococcus pluvialis cake; B) taking the cracked Haematococcus pluvialis cake, putting it into alcohol, stirring and extracting, and centrifuging to obtain an extract; the extraction includes room temperature extraction and heating extraction, the room temperature extraction uses 92% ethanol by mass, the temperature is 28°C, and the time is 32 minutes, the heating extraction uses 97% ethanol by mass, the temperature is 60°C, and the time is 40 minutes, and the mass percentage of the room temperature extraction and the heating extraction liquid is both 12:1; C) taking the extract, and distilling under reduced pressure to obtain crude astaxanthin oil; D) taking crude astaxanthin oil, putting it into alcohol with a mass percentage of 75% ethanol, the mass ratio of crude astaxanthin to alcohol is 1:7, stirring and dispersing at 52°C for 45 minutes, settling and separating for 3.5 hours to obtain a lower layer of astaxanthin oil, and desolventizing to obtain a finished astaxanthin oil product.

[0080] 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%.

[0081] Example 4

[0082] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0083] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0084] 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;

[0085] 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 mol / L, stirred at 60°C to break the algae wall, and the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15%, and calcium carbonate is added to neutralize to pH 6 .0, filter pressing to obtain a cracked Haematococcus pluvialis cake; B) taking the cracked Haematococcus pluvialis cake, putting it into alcohol, stirring and extracting, and centrifuging to obtain an extract; the extraction includes room temperature extraction and heating extraction, the room temperature extraction uses 92% ethanol by mass, the temperature is 28°C, and the time is 32 minutes, the heating extraction uses 97% ethanol by mass, the temperature is 60°C, and the time is 40 minutes, and the mass percentage of the room temperature extraction and the heating extraction liquid is both 12:1; C) taking the extract, and distilling under reduced pressure to obtain crude astaxanthin oil; D) taking crude astaxanthin oil, putting it into alcohol with a mass percentage of 75% ethanol, the mass ratio of crude astaxanthin to alcohol is 1:7, stirring and dispersing at 52°C for 45 minutes, settling and separating for 3.5 hours to obtain a lower layer of astaxanthin oil, and desolventizing to obtain a finished astaxanthin oil product.

[0086] 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%.

[0087] Example 5

[0088] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0089] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0090] 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;

[0091] 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 mol / L, stirred at 60°C to break the algae wall, and the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 15%, and calcium carbonate is added to neutralize to pH 6 .0, filter pressing to obtain a cracked Haematococcus pluvialis cake; B) taking the cracked Haematococcus pluvialis cake, putting it into alcohol, stirring and extracting, and centrifuging to obtain an extract; the extraction includes room temperature extraction and heating extraction, the room temperature extraction uses 92% ethanol by mass, the temperature is 28°C, and the time is 32 minutes, the heating extraction uses 97% ethanol by mass, the temperature is 60°C, and the time is 40 minutes, and the mass percentage of the room temperature extraction and the heating extraction liquid is both 12:1; C) taking the extract, and distilling under reduced pressure to obtain crude astaxanthin oil; D) taking crude astaxanthin oil, putting it into alcohol with a mass percentage of 75% ethanol, the mass ratio of crude astaxanthin to alcohol is 1:7, stirring and dispersing at 52°C for 45 minutes, settling and separating for 3.5 hours to obtain a lower layer of astaxanthin oil, and desolventizing to obtain a finished astaxanthin oil product.

[0092] 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%.

[0093] Example 6

[0094] A method for preparing astaxanthin oil with high anti-inflammatory activity comprises the following steps:

[0095] (1) Haematococcus pluvialis was cultured in a raceway photobioreactor and a pipeline photobioreactor for 15 days to allow it to accumulate astaxanthin;

[0096] 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;

[0097] Referring to the scheme of the applicant's prior patent CN 111875528 A, the astaxanthin oil is extracted, and the specific steps are as follows: A) taking the fresh Haematococcus pluvialis algae from 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, and the mass percentage of Haematococcus pluvialis in the algae slurry on a dry basis is 20%, and adding calcium carbonate to neutralize to pH 6 .0, filter pressing to obtain a cracked Haematococcus pluvialis cake; B) taking the cracked Haematococcus pluvialis cake, putting it into alcohol, stirring and extracting, and centrifuging to obtain an extract; the extraction includes room temperature extraction and heating extraction, the room temperature extraction uses 95% ethanol by mass, the temperature is 30°C, and the time is 35 minutes, the heating extraction uses 98% ethanol by mass, the temperature is 65°C, and the time is 45 minutes, and the mass percentage of the room temperature extraction and the heating extraction liquid is 16:1; C) taking the extract, and distilling under reduced pressure to obtain crude astaxanthin oil; D) taking crude astaxanthin oil, putting it into alcohol with a mass percentage of 70% ethanol, the mass ratio of crude astaxanthin to alcohol is 1:5, stirring and dispersing at 50°C for 40 minutes, settling and separating for 4 hours to obtain a lower layer of astaxanthin oil, and desolventizing to obtain a finished astaxanthin oil product.

[0098] 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%.

[0099] Although the present invention has been disclosed above in terms of preferred embodiments, 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, which has a preservation number of CGMCC No. 41186, and Haematococcus pluvialis ENN71, which has a preservation number of CGMCC No. 5147, to prepare astaxanthin oil with high anti-inflammatory activity.

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

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

1.

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

2.

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

1.

6. The method for preparing astaxanthin oil with high anti-inflammatory activity according to claim 1, wherein 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 the preparation of cosmetics.

Images