A method for increasing the content of astaxanthin diester in Haematococcus pluvialis culture

Abstract

The present invention provides a method for increasing the content of astaxanthin diester in Haematococcus pluvialis culture. In the first stage of culturing Haematococcus pluvialis, an organic acid magnesium salt is added to the culture medium to promote the MEP pathway and the transfer of electrons in the chloroplast, thereby enhancing the accumulation of algae cells; in the second stage of culturing, a guanidine polypeptide and a surfactant are added. On the one hand, the guanidine polypeptide participates in the metabolism of fatty acids in the algae cells, thereby promoting the large-scale accumulation of fatty acids. On the other hand, under the action of the surfactant, the catalytic action of lipase can be promoted, thereby promoting the generation of astaxanthin diester. Through the above process optimization, the content of astaxanthin diester in the total astaxanthin product is greatly improved, which is conducive to the production of commercial astaxanthin with high stability and antioxidant activity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and mainly relates to a method for increasing the content of astaxanthin diester in Haematococcus pluvialis culture. Background Art

[0002] Astaxanthin is a fat-soluble carotenoid with strong antioxidant properties. Its chemical name is 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene, and it is red or orange-yellow in appearance. Astaxanthin has excellent physiological functions, such as passing through the blood-brain barrier, protecting the brain, scavenging free radicals, protecting the skin and eyes, preventing cardiovascular and cerebrovascular aging, protecting the health of the nervous system, and relieving sports fatigue. In addition, astaxanthin itself has bright colors, which can make the feathers and skin of animals present healthy and beautiful colors. Based on the above characteristics, astaxanthin has a wide range of applications in various fields such as health care, medicine, food, cosmetics, and breeding.

[0003] The chemical structure of astaxanthin is shown in the following formula. Four isoprene units are connected in the form of conjugated double bonds, and there are two six-membered ring structures composed of isoprene units connected at both ends. Both the conjugated hydroxyl group and the unsaturated double bond can quench oxygen free radicals, making astaxanthin have a strong antioxidant function. According to research, its antioxidant activity is 550 times that of vitamin E and 10 times that of β-carotene. There are two chiral centers at both ends of the astaxanthin molecular structure. Both chiral carbon atoms can exist in the form of left-handed (S) or right-handed (R), forming three stereoisomers: 3S, 3'S, 3S, 3'R, and 3R, 3'R. According to animal experiments, its antioxidant activity is 3S, 3'S>3R, 3'R>3S, 3'R.

[0004]

[0005] At present, the mainstream methods for preparing astaxanthin include chemical synthesis, red Phaffia yeast fermentation and Haematococcus pluvialis fermentation. Among them, the chemical method prepares a mixture of 3S, 3'S: 3S, 3'R: 3R, 3'R = 1:2:1, which has the lowest antioxidant activity; more than 95% of the astaxanthin fermented by red Phaffia yeast is 3R, 3'R, and more than 95% of the astaxanthin synthesized by Haematococcus pluvialis is the most active 3S, 3'S configuration. Therefore, the development of Haematococcus pluvialis astaxanthin resources has become the focus of research for many companies.

[0006] However, the conjugated double bond structure of astaxanthin also leads to the instability of astaxanthin. Light, heat, oxides, etc. can easily destroy its structure, thus losing its antioxidant activity. The esterification protection method is a strategy to protect the antioxidant activity. Each end of the conjugated double bond has a hydroxyl group, which can form monoesters or diesters with fatty acids. For example, in the astaxanthin products of Haematococcus pluvialis, 65% exist in the form of monoesters, 30% exist in the form of diesters, and the free state accounts for about 5%. Since the diester structure of astaxanthin is more stable and not easily oxidized and denatured, increasing the content of astaxanthin diesters in Haematococcus pluvialis is beneficial to the preservation of astaxanthin. Studies have shown that in terms of fish absorption and pigment deposition, there is little difference in bioavailability between astaxanthin esters and free astaxanthin. In terms of antioxidant, compared with free astaxanthin, the activity of astaxanthin monoesters is slightly lower, and diesters have the lowest activity. However, after taking astaxanthin esters, they can be hydrolyzed into monomers by lipase in the digestive tract (Study on the digestion and absorption characteristics of astaxanthin esters from Haematococcus pluvialis [J]. Journal of Chinese Institute of Food Science and Technology, 2019, 019(004): 125-132.), which does not affect the absorption and efficacy of astaxanthin. In addition, the biological activity of astaxanthin esters is increasingly being studied. For example, studies by Rao et al. have shown that astaxanthin monoesters and diesters can prevent the occurrence of skin cancer (Effective inhibition of skin cancer, tyrosinase, and antioxidant properties by astaxanthin and astaxanthin esters from the green alga Haematococcus pluvialis. [J]. Journal of Agricultural & Food Chemistry, 2013, 61 (16): 3842-3851), and Kamath's experimental results have shown that astaxanthin esters have a relieving and therapeutic effect on gastric ulcers in rats (Ulcer preventive and antioxidative properties of astaxanthin from Haematococcus pluvialis [J]. European Journal of Pharmacology, 2008, 590 (1-3): 387-395).Kobayashi's photosensitized oxidation experiment on linoleic acid showed that the singlet oxygen quenching ability of astaxanthin esters from the green alga Haematococcus pluvialis [J]. Biotechnology Letters, 1999, 21 (4): 265-269. Therefore, increasing the content of astaxanthin diesters in the fermentation product of Haematococcus pluvialis is beneficial to protecting its antioxidant activity, and does not affect the digestion and function after taking it, which is conducive to the commercial promotion of the product.

[0007] The growth of Haematococcus pluvialis and the accumulation of astaxanthin are affected by its own and environmental factors, including the concentration of components in the culture medium, light quality and intensity, temperature, pH, etc. The growth process of Haematococcus pluvialis is divided into two stages: ① Under relatively mild and comfortable culture conditions, the algae cells are kept in the stage of rapid growth and swimming cells to obtain a higher biomass. ② By limiting stress conditions such as nitrogen sources, phosphorus sources and high light intensity, the swimming cells are induced to become immobile cells, thereby obtaining the accumulation of astaxanthin.

[0008] In summary, the existing astaxanthin preparation process has problems such as instability and easy oxidation. It is necessary to adopt appropriate process means to improve the stability of astaxanthin and promote the commercial application of Haematococcus pluvialis astaxanthin. Summary of the invention

[0009] The object of the present invention is to provide a method for increasing the content of astaxanthin diesters in Haematococcus pluvialis culture. The method is based on improving the fermentation stability and activity of astaxanthin, and adopts a novel and efficient method to increase the content of astaxanthin diesters in Haematococcus pluvialis. Through the above process, commercial production of astaxanthin in Haematococcus pluvialis can be achieved.

[0010] A method for increasing the content of astaxanthin diester in a culture of Haematococcus pluvialis comprises adding organic acid magnesium to a basal culture medium and adding a guanidine polypeptide and a surfactant to a feed culture medium.

[0011] In the above method, organic acid magnesium salt is added to the culture medium in the first growth stage to promote the MEP pathway and the transfer of electrons in the chloroplasts, thereby enhancing the accumulation of algae cells. In the second growth stage, guanidine polypeptide derivatives and surfactants are added. On the one hand, the amphiphilic polypeptide chains of the guanidine polypeptide derivatives catalyze the metabolism of fatty acids in algae cells, thereby promoting the accumulation of fatty acids in large quantities; on the other hand, the catalytic action of lipase can be promoted under the action of the surfactant, thereby promoting the production of astaxanthin diesters.

[0012] In the present invention, the culture of Haematococcus pluvialis is divided into two stages: swimming cell culture and stress culture; preferably, the culture temperature of the swimming cell culture stage is 15-25°C, and the light intensity is 50-500 μmol / (m 2 ·s), the light duration is 12-20 hours per day, and the culture time is 3-12 days; preferably, the culture temperature in the stress culture stage is 15-25°C, the light intensity is 2000-5000 μmol / (m 2 ·s), the light duration is 16-24 hours per day, and the cultivation time is 8-20 days.

[0013] In the present invention, the organic acid magnesium is one or more of magnesium citrate, magnesium lactate, magnesium glycolate, magnesium threonate, and magnesium malate; preferably, the organic acid magnesium is added in an amount of 200-1800 mg / L in the basal culture medium.

[0014] In the present invention, the guanidine polypeptide is a polypeptide consisting of 3-5 amino acids, wherein the endpoints of the polypeptide chain are respectively called the amino terminus (N terminus) and the carboxyl terminus (C terminus), and the amino group at the N terminus of the polypeptide is substituted by a guanidine group; preferably, the guanidine polypeptide structure is shown in the following formula:

[0015]

[0016] Wherein, n is an integer of 0-2; R1 is a hydrophilic group, preferably R1 is derived from one or more of the hydrophilic amino acids serine (S), cysteine ​​(C), threonine (T), and tyrosine (Y); R3 and R4 are hydrophobic groups, preferably R3 and R4 are derived from one or more of hydrophobic amino acids such as valine (V), leucine (L), isoleucine (I), and proline (P); preferably R2 is derived from one or more of lysine (K), arginine (R), histidine (H), aspartic acid (D), and glutamic acid (E), and when n is 2, the sources of R2 may be the same or different; preferably, the guanidine polypeptide derivative is added in an amount of 500-1500 mg / L in the feed medium.

[0017] In the present invention, the surfactant is one or more of alkyl glucoside, sucrose ester, potassium lauroyl glutamate, sodium cocoylaminopropionate; preferably, the surfactant is added in an amount of 200-800 mg / L in the feed medium.

[0018] In the present invention, the basic components of the basic culture medium include one or more of potassium nitrate, potassium dihydrogen phosphate, calcium chloride dihydrate, disodium EDTA, ferric chloride hexahydrate, zinc chloride, boric acid, cobalt chloride hexahydrate, copper sulfate pentahydrate, manganese chloride hexahydrate, and sodium molybdate dihydrate; preferably, the content of each basic component of the basic culture medium is: potassium nitrate 200-600 mg / L, potassium dihydrogen phosphate 10-70 mg / L, calcium chloride dihydrate 50-200 mg / L / L, disodium EDTA 1.0-10mg / L, ferric chloride hexahydrate 1.0-5.0mg / L, zinc chloride 0.0001-0.001mg / L, boric acid 0.01-0.1mg / L, cobalt chloride hexahydrate 0.0001-0.001mg / L, cupric sulfate pentahydrate 0.01-0.1mg / L, manganese chloride hexahydrate 0.0001-0.001mg / L, sodium molybdate dihydrate 0.01-0.1mg / L.

[0019] In the present invention, the guanidine polypeptide and the surfactant are added to the photobioreactor in the stress culture stage by means of fed-batch feeding; preferably, the flow rate of the guanidine polypeptide and the surfactant is 0.5-5 mL / h.

[0020] In the present invention, the astaxanthin ester obtained by the method is calculated by converting the molecular weight into astaxanthin, and the total astaxanthin accounts for 3-10% of the wet weight of the algae; preferably, the free astaxanthin in the astaxanthin accounts for 2%-20%, the astaxanthin diester accounts for 60%-70%, and the astaxanthin monoester accounts for 20%-28%, based on the total mass of astaxanthin.

[0021] In one embodiment, an extractant is added to the algae, and the algae are broken by an ultrasonic cell disruptor, centrifuged, and the extractant is collected. The extractant is then added to the algae precipitate, and the above extraction-breaking-centrifugation-collecting extractant steps are repeated until the algae residue is white, and the collected extractant supernatants are combined, and the content of free astaxanthin and astaxanthin esters therein is detected by high performance liquid chromatography-mass spectrometry. Among them, the extractant can be selected from acetone, cyclohexanone, ethyl acetate, methyl tert-butyl ether, etc., and the mass added each time is 3-10 times the wet weight of the algae.

[0022] Another object of the present invention is to provide an astaxanthin product.

[0023] An astaxanthin is prepared by the above method, wherein the free astaxanthin accounts for 2%-20%, the astaxanthin diester accounts for 60%-70%, and the astaxanthin monoester accounts for 20%-28%, based on the total mass of the astaxanthin.

[0024] Compared with the prior art, the positive effects of the present invention are:

[0025] (1) In the existing Haematococcus pluvialis cultivation method, the astaxanthin content is 1.5% to 3% on average. The astaxanthin content in the embodiment of the present invention can be increased to 3% to 10%, which can increase the astaxanthin yield and reduce production costs.

[0026] (2) The existing content of astaxanthin diester in Haematococcus pluvialis is only about 30%. The present invention can effectively increase the proportion of astaxanthin diester in astaxanthin to 60% to 70%, protect the hydroxyl group in the form of ester bonds, greatly reduce the oxidation of astaxanthin during production and storage, make the astaxanthin in Haematococcus pluvialis more stable, and facilitate commercial promotion. DETAILED DESCRIPTION

[0027] In order to further illustrate the method for increasing the astaxanthin ester content provided by the present invention, the present invention is described in detail below with reference to the examples, but it does not constitute a limitation of the present invention.

[0028] Haematococcus pluvialis, strain model H3, purchased from Shanghai Guangyu Biotechnology Co., Ltd.;

[0029] Guanidine peptide derivatives were purchased from Jier Biochemical (Shanghai) Co., Ltd.;

[0030] Magnesium citrate, magnesium lactate, magnesium glycolate, magnesium threonate, magnesium malate, etc. were purchased from Aladdin Company and the specifications were analytical grade;

[0031] Alkyl glucoside, sucrose ester, potassium lauroyl glutamate, sodium cocoylaminopropionate were purchased from SIGMA Company, and the specifications were analytical grade;

[0032] All inorganic salt reagents were commercially available and of analytical grade.

[0033] Analytical test methods:

[0034] The liquid chromatography-mass spectrometry detection method of astaxanthin and esters is as follows: the detection instrument is an Agilent high performance liquid chromatography-mass spectrometer, equipped with a Phenomenx C18 reverse phase column 5μm (4.6mm×250mm). Mobile phase A is a mixture of water and methanol (volume ratio of 3:1), mobile phase B is acetonitrile (100%), flow rate is 0.5ml / min, and the elution program is shown in Table 1. Ultraviolet detection is adopted, and the detection wavelength is 476nm. The mass spectrometer uses point spray ionization as the ion source, point spray ionization negative ion mode, capillary voltage 4000V, sprayer gas flow rate and pressure are 5L / min and 40psi respectively, nitrogen air temperature is 325℃, and injection volume is 10μL. Astaxanthin, astaxanthin monoester and diester in the extract are separated and identified, and their contents are counted.

[0035] Table 1 Gradient elution program

[0036]

[0037]

[0038] Example 1

[0039] (1) Cultivation of swimming cells of Haematococcus pluvialis

[0040] The culture medium used for the cultivation of Haematococcus pluvialis is MCM culture medium, and its formula is as follows: 870 mg / L of magnesium lactate, 200 mg / L of potassium nitrate, 34.6 mg / L of potassium dihydrogen phosphate, 73.8 mg / L of calcium chloride dihydrate, 4.7 mg / L of disodium EDTA, 1.9 mg / L of ferric chloride hexahydrate, 0.0001 mg / L of zinc chloride, 0.09 mg / L of boric acid, 0.0008 mg / L of cobalt chloride hexahydrate, 0.015 mg / L of copper sulfate pentahydrate, 0.0006 mg / L of manganese chloride hexahydrate, and 0.05 mg / L of sodium molybdate dihydrate. After 3L of culture medium solution is prepared according to the above formula, it is sterilized at 121°C for 20 minutes, placed to cool to 25°C, and inoculated with Haematococcus pluvialis seed liquid, and the inoculation amount is 10% of the volume of the culture medium. A self-dried photobioreactor with a built-in light source is added to start the cultivation of Haematococcus pluvialis swimming cells to expand the algae. The culture temperature at this stage was 19°C and the light intensity was 150 μmol / (m 2 ·s), the light duration was 17 hours per day, and the culture time was 3 days.

[0041] (2) Astaxanthin production by Haematococcus pluvialis

[0042] After the end of the Haematococcus pluvialis swimming cell culture period, the light intensity was increased to 3600 μmol / (m 2 ·s), the illumination time is 24 hours per day. And feed medium is introduced into the reactor, there are two components in the medium, the structure of the guanidine polypeptide derivative is: guanidine-CRVP, the addition amount is 500mg / L, and the content of potassium lauroyl glutamate is 450mg / L. The above feed medium is also sterilized at 121°C for 20min, placed to cool down to below 25°C, and then introduced into the photobioreactor by feeding method, and the flow rate is 5.0mL / h. Astaxanthin is generated and accumulated under this stress condition. The culture time at this stage is 19 days.

[0043] (3) Collection of algae and detection of astaxanthin ester content

[0044] After the culture is completed, 10 mL of the culture solution of Haematococcus pluvialis is taken and placed in a 15 mL centrifuge tube that has been weighed (the mass is recorded as M1). The sum of the mass of the algae solution and the centrifuge tube is recorded as M2. After centrifugation at 6000 rpm for 10 minutes, the upper supernatant is discarded and the sum of the mass of the lower algae and the centrifuge tube is recorded as M3. The wet weight of the algae in the solution is (M2-M3) / (M2-M1). After testing, the wet weight of the algae is 10%.

[0045] Acetone is added to the algae as an extractant, and the algae are broken by an ultrasonic cell disruptor, centrifuged, and the extractant is collected. The extractant is then added to the algae precipitate, and the extraction-breaking-centrifugation-collecting extractant steps are repeated, and the amount of the extractant added each time is 9.3 times the wet weight of the algae. After repeating three times, the extractant is extracted until the algae residue appears white, and the collected extractant supernatants are combined, and the content of free astaxanthin and astaxanthin esters therein is detected by high performance liquid chromatography-mass spectrometry, wherein astaxanthin esters are calculated by converting astaxanthin into astaxanthin according to molecular weight, and the total astaxanthin accounts for 7% of the wet weight of the algae, of which free astaxanthin accounts for 5.1%, astaxanthin diester accounts for 80%, and astaxanthin monoester accounts for 14.9%.

[0046] Example 2

[0047] (1) Cultivation of swimming cells of Haematococcus pluvialis

[0048] The culture medium used for the cultivation of Haematococcus pluvialis is MCM culture medium, and its formula is as follows: 200 mg / L magnesium threonate, 500 mg / L potassium nitrate, 59 mg / L potassium dihydrogen phosphate, 50 mg / L calcium chloride dihydrate, 10 mg / L disodium EDTA, 5.0 mg / L ferric chloride hexahydrate, 0.001 mg / L zinc chloride, 0.06 mg / L boric acid, 0.0007 mg / L cobalt chloride hexahydrate, 0.03 mg / L copper sulfate pentahydrate, 0.0009 mg / L manganese chloride hexahydrate, 0.02 mg / L sodium molybdate dihydrate. After preparing 3L of culture medium solution according to the above formula, sterilize at 121°C for 20 minutes, place it to cool down to below 25°C, and inoculate Haematococcus pluvialis seed liquid, with an inoculation amount of 10% of the culture medium volume. Add a self-dried photobioreactor with a built-in light source to start the cultivation of Haematococcus pluvialis swimming cells to expand the algae. The culture temperature at this stage was 21°C and the light intensity was 400 μmol / (m 2 ·s), the light duration was 16 hours per day, and the culture time was 6 days.

[0049] (2) Astaxanthin production by Haematococcus pluvialis

[0050] After the end of the Haematococcus pluvialis swimming cell culture period, the light intensity was increased to 4700 μmol / (m 2·s), the illumination time is 18 hours per day. And feed medium is introduced into the reactor, there are two components in the medium, the structure of the guanidine polypeptide derivative is: guanidine-SIL, the addition amount is 500 mg / L, and the sucrose ester content is 500 mg / L. The above feed medium is also sterilized at 121°C for 20 minutes, placed to cool down to below 25°C, and then introduced into the photobioreactor by feeding, and the flow rate is 5.0 mL / h. Astaxanthin is generated and accumulated under this stress condition. The culture time at this stage is 8 days.

[0051] (3) Collection of algae and detection of astaxanthin ester content

[0052] After the culture is completed, 10 mL of the culture solution of Haematococcus pluvialis is taken and placed in a 15 mL centrifuge tube that has been weighed (the mass is recorded as M1). The sum of the mass of the algae solution and the centrifuge tube is recorded as M2. After centrifugation at 6000 rpm for 10 minutes, the upper supernatant is discarded and the sum of the mass of the lower algae and the centrifuge tube is recorded as M3. The wet weight of the algae in the solution is (M2-M3) / (M2-M1). After testing, the wet weight of the algae is about 7%.

[0053] Ethyl acetate is added to the algae as an extractant, and the algae are broken by an ultrasonic cell disruptor, centrifuged, and the extractant is collected. The extractant is then added to the algae precipitate, and the extraction-breaking-centrifugation-collecting extractant steps are repeated, and the amount of the extractant added each time is 10 times the wet weight of the algae. After repeating three times, the extractant is extracted until the algae residue appears white, and the collected extractant supernatants are combined, and the content of free astaxanthin and astaxanthin esters therein is detected by high performance liquid chromatography-mass spectrometry, wherein astaxanthin esters are calculated by converting astaxanthin into astaxanthin according to molecular weight, and the total astaxanthin accounts for 5% of the wet weight of the algae, of which free astaxanthin accounts for 7%, astaxanthin diester accounts for 68.1%, and astaxanthin monoester accounts for 24.8%.

[0054] Example 3

[0055] (1) Cultivation of swimming cells of Haematococcus pluvialis

[0056] The culture medium used for the cultivation of Haematococcus pluvialis is MCM culture medium, and its formula is as follows: 680mg / L of magnesium citrate, 530mg / L of potassium nitrate, 70mg / L of potassium dihydrogen phosphate, 90.9mg / L of calcium chloride dihydrate, 3.2mg / L of disodium EDTA, 4.1mg / L of ferric chloride hexahydrate, 0.0003mg / L of zinc chloride, 0.01mg / L of boric acid, 0.0001mg / L of cobalt chloride hexahydrate, 0.1mg / L of copper sulfate pentahydrate, 0.0001mg / L of manganese chloride hexahydrate, and 0.01mg / L of sodium molybdate dihydrate. After 3L of culture medium solution is prepared according to the above formula, it is sterilized at 121°C for 20min, placed to cool down to below 25°C, and inoculated with Haematococcus pluvialis seed liquid, and the inoculation amount is 10% of the culture medium volume. A self-dried photobioreactor with a built-in light source is added to start the cultivation of Haematococcus pluvialis swimming cells to expand the algae. The culture temperature at this stage was 25°C and the light intensity was 500 μmol / (m 2 ·s), the light duration was 12 hours per day, and the culture time was 10 days.

[0057] (2) Astaxanthin production by Haematococcus pluvialis

[0058] After the end of the Haematococcus pluvialis swimming cell culture period, the light intensity was increased to 2000 μmol / (m 2 ·s), the illumination time is 21 hours per day. And feed medium is introduced into the reactor, there are two components in the medium, the structure of the guanidine polypeptide derivative is: guanidine-YEDLI, the addition amount is 1500mg / L, and the alkyl glycoside content is 750mg / L. The above feed medium is also sterilized at 121°C for 20min, placed to cool down to below 25°C, and then introduced into the photobioreactor by feeding method, and the flow rate is 0.5mL / h. Astaxanthin is generated and accumulated under this stress condition. The culture time at this stage is 20 days.

[0059] (3) Collection of algae and detection of astaxanthin ester content

[0060] After the culture is completed, 10 mL of the culture solution of Haematococcus pluvialis is taken and placed in a 15 mL centrifuge tube that has been weighed (the mass is recorded as M1). The sum of the mass of the algae solution and the centrifuge tube is recorded as M2. After centrifugation at 6000 rpm for 10 minutes, the upper supernatant is discarded and the sum of the mass of the lower algae and the centrifuge tube is recorded as M3. The wet weight of the algae in the solution is (M2-M3) / (M2-M1). After testing, the wet weight of the algae is about 15%.

[0061] Methyl tert-butyl ether is added to the algae as an extractant, and the algae are broken by an ultrasonic cell disruptor, centrifuged, and the extractant is collected. The extractant is then added to the algae precipitate, and the above extraction-breaking-centrifugation-collecting extractant steps are repeated, and the amount of the extractant added each time is 4 times the wet weight of the algae. The extractant extraction is repeated three times until the algae residue appears white, and the collected extractant supernatants are combined, and the content of free astaxanthin and astaxanthin esters therein is detected by high performance liquid chromatography-mass spectrometry, wherein astaxanthin esters are calculated by converting astaxanthin into astaxanthin according to molecular weight, and the total astaxanthin accounts for 6% of the wet weight of the algae, of which free astaxanthin accounts for 5.5%, astaxanthin diester accounts for 75.1%, and astaxanthin monoester accounts for 19.5%.

[0062] Example 4

[0063] (1) Cultivation of swimming cells of Haematococcus pluvialis

[0064] The culture medium used for culturing Haematococcus pluvialis is MCM culture medium, and its formula is as follows: 1800mg / L of magnesium malate, 350mg / L of potassium nitrate, 31.3mg / L of potassium dihydrogen phosphate, 171.6mg / L of calcium chloride dihydrate, 8.0mg / L of disodium EDTA, 3.7mg / L of ferric chloride hexahydrate, 0.0002mg / L of zinc chloride, 0.05mg / L of boric acid, 0.0004mg / L of cobalt chloride hexahydrate, 0.08mg / L of copper sulfate pentahydrate, 0.0008mg / L of manganese chloride hexahydrate, and 0.07mg / L of sodium molybdate dihydrate. After 3L of culture medium solution is prepared according to the above formula, it is sterilized at 121℃ for 20min, placed to cool down to below 25℃, and inoculated with Haematococcus pluvialis seed liquid, and the inoculation amount is 10% of the volume of the culture medium. A self-dried photobioreactor with a built-in light source is added to start the culture of Haematococcus pluvialis swimming cells to expand the algae. The culture temperature at this stage was 21°C and the light intensity was 225 μmol / (m 2 ·s), the light duration was 15 hours per day, and the culture time was 12 days.

[0065] (2) Astaxanthin production by Haematococcus pluvialis

[0066] After the end of the Haematococcus pluvialis swimming cell culture period, the light intensity was increased to 5000 μmol / (m 2·s), the illumination time is 22 hours per day. And feed medium is introduced into the reactor, there are two components in the medium, the structure of the guanidine polypeptide derivative is: guanidine-TDHPV, the addition amount is 736mg / L, and the sucrose ester content is 350mg / L. The above feed medium is also sterilized at 121°C for 20min, placed to cool down to below 25°C, and then introduced into the photobioreactor by feeding method, and the flow rate is 4.3mL / h. Astaxanthin is generated and accumulated under this stress condition. The culture time at this stage is 16 days.

[0067] (3) Collection of algae and detection of astaxanthin ester content

[0068] After the culture is completed, 10 mL of the culture solution of Haematococcus pluvialis is taken and placed in a 15 mL centrifuge tube that has been weighed (the mass is recorded as M1). The sum of the mass of the algae solution and the centrifuge tube is recorded as M2. After centrifugation at 6000 rpm for 10 minutes, the upper supernatant is discarded and the sum of the mass of the lower algae and the centrifuge tube is recorded as M3. The wet weight of the algae in the solution is (M2-M3) / (M2-M1). After testing, the wet weight of the algae is about 8%.

[0069] Acetone is added to the algae as an extractant, and the algae are broken by an ultrasonic cell disruptor, centrifuged, and the extractant is collected. The extractant is then added to the algae precipitate, and the extraction-breaking-centrifugation-collecting extractant steps are repeated, and the amount of the extractant added each time is 5.7 times the wet weight of the algae. The extractant extraction is repeated three times until the algae residue appears white, and the collected extractant supernatants are combined, and the content of free astaxanthin and astaxanthin esters therein is detected by high performance liquid chromatography-mass spectrometry, wherein astaxanthin esters are calculated by converting astaxanthin into astaxanthin according to molecular weight, and the total astaxanthin accounts for 3% of the wet weight of the algae, of which free astaxanthin accounts for 2%, astaxanthin diester accounts for 78%, and astaxanthin monoester accounts for 20%.

[0070] Example 5

[0071] (1) Cultivation of swimming cells of Haematococcus pluvialis

[0072] The culture medium used for the cultivation of Haematococcus pluvialis is MCM culture medium, and its formula is as follows: 1200mg / L magnesium threonate, 600mg / L potassium nitrate, 10mg / L potassium dihydrogen phosphate, 200mg / L calcium chloride dihydrate, 1.0mg / L disodium EDTA, 1.0mg / L ferric chloride hexahydrate, 0.0009mg / L zinc chloride, 0.1mg / L boric acid, 0.001mg / L cobalt chloride hexahydrate, 0.07mg / L copper sulfate pentahydrate, 0.001mg / L manganese chloride hexahydrate, 0.1mg / L sodium molybdate dihydrate. After 3L of the culture medium solution is prepared according to the above formula, it is sterilized at 121°C for 20min, placed to cool down to below 25°C, and inoculated with Haematococcus pluvialis seed liquid, and the inoculation amount is 10% of the culture medium volume. A self-dried photobioreactor with a built-in light source is added to start the cultivation of Haematococcus pluvialis swimming cells to expand the algae. The culture temperature at this stage was 15°C and the light intensity was 50 μmol / (m 2 ·s), the light duration was 20 hours per day, and the culture time was 8 days.

[0073] (2) Astaxanthin production by Haematococcus pluvialis

[0074] After the end of the Haematococcus pluvialis swimming cell culture period, the light intensity was increased to 2500 μmol / (m 2 ·s), the illumination time is 16 hours per day. And feed medium is introduced into the reactor, there are two components in the medium, the structure of the guanidine polypeptide derivative is: guanidine-CKVL, the addition amount is 828mg / L, and the content of sodium cocoylaminopropionate is 200mg / L. The above feed medium is also sterilized at 121°C for 20min, placed to cool down to below 25°C, and then introduced into the photobioreactor by feeding method, and the flow rate is 1.9mL / h. Astaxanthin is generated and accumulated under this stress condition. The culture time at this stage is 14 days.

[0075] (3) Collection of algae and detection of astaxanthin ester content

[0076] After the culture is completed, 10 mL of the culture solution of Haematococcus pluvialis is taken and placed in a 15 mL centrifuge tube that has been weighed (the mass is recorded as M1). The sum of the mass of the algae solution and the centrifuge tube is recorded as M2. After centrifugation at 6000 rpm for 10 minutes, the upper supernatant is discarded and the sum of the mass of the lower algae and the centrifuge tube is recorded as M3. The wet weight of the algae in the solution is (M2-M3) / (M2-M1). After testing, the wet weight of the algae is about 5%.

[0077] Cyclohexanone is added to the algae as an extractant, and the algae are broken by an ultrasonic cell disruptor, centrifuged, and the extractant is collected. The extractant is then added to the algae precipitate, and the extraction-breaking-centrifugation-collecting extractant steps are repeated, and the amount of the extractant added each time is 3 times the wet weight of the algae. The extractant extraction is repeated three times until the algae residue appears white, and the collected extractant supernatants are combined, and the content of free astaxanthin and astaxanthin esters therein is detected by high performance liquid chromatography-mass spectrometry, wherein astaxanthin esters are calculated by converting astaxanthin into astaxanthin according to molecular weight, and the total astaxanthin accounts for 10% of the wet weight of the algae, of which free astaxanthin accounts for 10%, astaxanthin diester accounts for 60%, and astaxanthin monoester accounts for 30%.

[0078] Example 6

[0079] (1) Cultivation of swimming cells of Haematococcus pluvialis

[0080] The culture medium used for the cultivation of Haematococcus pluvialis is MCM culture medium, and its formula is as follows: 1500mg / L of magnesium glycolate, 430mg / L of potassium nitrate, 43.5mg / L of potassium dihydrogen phosphate, 115.9mg / L of calcium chloride dihydrate, 3.7mg / L of disodium EDTA, 4.3mg / L of ferric chloride hexahydrate, 0.0005mg / L of zinc chloride, 0.08mg / L of boric acid, 0.0009mg / L of cobalt chloride hexahydrate, 0.01mg / L of copper sulfate pentahydrate, 0.0001mg / L of manganese chloride hexahydrate, and 0.08mg / L of sodium molybdate dihydrate. After 3L of the culture medium solution is prepared according to the above formula, it is sterilized at 121°C for 20min, placed to cool down to below 25°C, and inoculated with Haematococcus pluvialis seed liquid, and the inoculation amount is 10% of the culture medium volume. A self-dried photobioreactor with a built-in light source is added to start the cultivation of Haematococcus pluvialis swimming cells to expand the algae. The culture temperature at this stage was 16°C and the light intensity was 380 μmol / (m 2 ·s), the light duration was 18 hours per day, and the culture time was 9 days.

[0081] (2) Astaxanthin production by Haematococcus pluvialis

[0082] After the end of the Haematococcus pluvialis swimming cell culture period, the light intensity was increased to 3500 μmol / (m 2 ·s), the illumination time is 20 hours per day. And feed medium is introduced into the reactor, there are two components in the medium, the structure of the guanidine polypeptide derivative is: guanidine-YHIP, the addition amount is 1074mg / L, and the alkyl glycoside content is 800mg / L. The above feed medium is also sterilized at 121°C for 20min, placed to cool down to below 25°C, and then introduced into the photobioreactor by feeding method, and the flow rate is 3.3mL / h. Astaxanthin is generated and accumulated under this stress condition. The culture time at this stage is 15 days.

[0083] (3) Collection of algae and detection of astaxanthin ester content

[0084] After the culture is completed, 10 mL of the culture solution of Haematococcus pluvialis is taken and placed in a 15 mL centrifuge tube that has been weighed (the mass is recorded as M1). The sum of the mass of the algae solution and the centrifuge tube is recorded as M2. After centrifugation at 6000 rpm for 10 minutes, the upper supernatant is discarded and the sum of the mass of the lower algae and the centrifuge tube is recorded as M3. The wet weight of the algae in the solution is (M2-M3) / (M2-M1). After testing, the wet weight of the algae is about 12%.

[0085] Ethyl acetate is added to the algae as an extractant, and the algae are broken by an ultrasonic cell disruptor, centrifuged, and the extractant is collected. The extractant is then added to the algae precipitate, and the extraction-breaking-centrifugation-collecting extractant steps are repeated, and the amount of the extractant added each time is 7.4 times the wet weight of the algae. The extractant extraction is repeated three times until the algae residue appears white, and the collected extractant supernatants are combined, and the content of free astaxanthin and astaxanthin esters therein is detected by high performance liquid chromatography-mass spectrometry, wherein astaxanthin esters are calculated by converting astaxanthin into astaxanthin according to molecular weight, and the total astaxanthin accounts for 9% of the wet weight of the algae, of which free astaxanthin accounts for 10%, astaxanthin diester accounts for 80%, and astaxanthin monoester accounts for 10%.

[0086] Comparative Example 1

[0087] The method of Example 1 was followed, except that in step (2), 500 mg of isoleucine was added instead of the guanidine polypeptide derivative, and the other operations and parameters were the same.

[0088] After testing, the total astaxanthin in step (3) accounted for 3% of the wet weight of the algae, of which free astaxanthin accounted for 32%, astaxanthin diester accounted for 23%, and astaxanthin monoester accounted for 45%.

[0089] Comparative Example 2

[0090] The method of Example 6 was followed, except that in step (1), magnesium sulfate heptahydrate was added instead of magnesium glycolate at a concentration of 1500 mg, and other operations and parameters were the same.

[0091] After testing, the total astaxanthin in step (3) accounted for 4% of the wet weight of the algae, of which free astaxanthin accounted for 27%, astaxanthin diester accounted for 25%, and astaxanthin monoester accounted for 48%.

[0092] Comparative Example 3

[0093] The method of Example 2 was followed, except that in step (2), no sucrose ester surfactant was added, and other operations and parameters were the same.

[0094] After testing, the total astaxanthin in step (3) accounted for 4% of the wet weight of the algae, of which free astaxanthin accounted for 24%, astaxanthin diester accounted for 11%, and astaxanthin monoester accounted for 65%.

[0095] Those skilled in the art will appreciate that, based on the teachings of this specification, some modifications or adjustments may be made to the present invention, and these modifications or adjustments should also be within the scope defined by the claims of the present invention.

Claims

1. A method for increasing the content of astaxanthin diester in a culture of Haematococcus pluvialis, characterized in that: The method adds organic acid magnesium to the basal medium and adds guanidine polypeptide and surfactant to the feed medium; The culture of Haematococcus pluvialis is divided into two stages: swimming cell culture and stress culture; the guanidine polypeptide and surfactant are added in the stress culture stage; The guanidine polypeptide is a polypeptide composed of 3-5 amino acids, wherein the endpoints of the polypeptide chain are the amino terminus (N terminus) and the carboxyl terminus (C terminus), and the amino group at the N terminus of the polypeptide is replaced by a guanidine group; the guanidine polypeptide structure is shown in the following formula: Wherein, n is an integer of 0-2; R1 is a hydrophilic group; R3 and R4 are hydrophobic groups; Wherein, the surfactant is one or more of alkyl glucoside, sucrose ester, potassium lauroyl glutamate, and sodium cocoyl aminopropionate.

2. The method according to claim 1, characterized in that The culture temperature of the motile cell culture stage is 15-25°C, and the light intensity is 50-500 μmol / (m 2 ·s), the light duration is 12-20 hours per day, and the cultivation time is 3-12 days; The culture temperature during the stress culture stage is 15-25°C, and the light intensity is 2000-5000 μmol / (m 2 ·s), the light duration is 16-24 hours per day, and the cultivation time is 8-20 days.

3. The method according to claim 1 or 2, characterized in that The organic acid magnesium is one or more of magnesium citrate, magnesium lactate, magnesium glycolate, magnesium threonate and magnesium malate.

4. The method according to claim 3, characterized in that The organic acid magnesium is added in an amount of 200-1800 mg / L in the basic culture medium.

5. The method according to claim 1 or 2, characterized in that: The amount of the guanidine polypeptide added to the feed medium is 500-1500 mg / L.

6. The method according to claim 1, characterized in that In the guanidine polypeptide structure, R1 is derived from one of the hydrophilic amino acids serine, cysteine, threonine, and tyrosine; R3 and R4 are derived from one of the hydrophobic amino acids valine, leucine, isoleucine, and proline; R2 is derived from one or more of lysine, arginine, histidine, aspartic acid, and glutamic acid. When n is 2, the sources of R2 may be the same or different.

7. The method according to claim 1 or 2, characterized in that: The surfactant is added in an amount of 200-800 mg / L in the feed medium.

8. The method according to claim 1 or 2, characterized in that: The basic components of the basic culture medium include one or more of potassium nitrate, potassium dihydrogen phosphate, calcium chloride dihydrate, disodium EDTA, ferric chloride hexahydrate, zinc chloride, boric acid, cobalt chloride hexahydrate, copper sulfate pentahydrate, manganese chloride hexahydrate, and sodium molybdate dihydrate.

9. The method according to claim 8, characterized in that The contents of the basic components of the basic culture medium are as follows: 200-600 mg / L potassium nitrate, 10-70 mg / L potassium dihydrogen phosphate, 50-200 mg / L calcium chloride dihydrate, 1.0-10 mg / L disodium EDTA, 1.0-5.0 mg / L ferric chloride hexahydrate, 0.0001-0.001 mg / L zinc chloride, 0.01-0.1 mg / L boric acid, 0.0001-0.001 mg / L cobalt chloride hexahydrate, 0.01-0.1 mg / L copper sulfate pentahydrate, 0.0001-0.001 mg / L manganese chloride hexahydrate, and 0.01-0.1 mg / L sodium molybdate dihydrate.

10. The method according to claim 1 or 2, characterized in that: The guanidine polypeptide and the surfactant are added into the photobioreactor in the stress culture stage by means of feeding.

11. The method according to claim 10, characterized in that The flow rate of the guanidine polypeptide and surfactant is 0.5-5 mL / h.

12. The method according to claim 1 or 2, characterized in that: According to the astaxanthin ester obtained by the method, the astaxanthin accounts for 3-10% of the wet weight of the algae when converted into astaxanthin based on the molecular weight.

13. The method according to claim 12, characterized in that In the astaxanthin, free astaxanthin accounts for 2%-20%, astaxanthin diester accounts for 60%-70%, and astaxanthin monoester accounts for 20%-28%, calculated based on the total mass of astaxanthin.

14. An astaxanthin prepared by the method according to any one of claims 1 to 13, characterized in that: Astaxanthin esters are calculated by converting astaxanthin into astaxanthin according to the molecular weight, and astaxanthin accounts for 3-10% of the wet weight of the algae, of which free astaxanthin accounts for 2%-20%, astaxanthin diester accounts for 60%-70%, and astaxanthin monoester accounts for 20%-28%, based on the total mass of astaxanthin.

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