Green directional extraction and purification method for haematococcus astaxanthin

Abstract

The invention belongs to the technical field of organic matter extraction, and particularly relates to a green directional extraction and purification method for haematococcus astaxanthin, which comprises the following steps: (1) mixing dried haematococcus powder and a composite pretreatment agent according to a mass ratio of 1: (8-15), and stirring to obtain a pretreated algae solution; (2) treating the pretreated algae liquid through ultrahigh-pressure microjet equipment to obtain wall-broken slurry; (3) mixing the wall-broken slurry and a composite extraction agent according to a volume ratio of 1: 3-1: 5, and performing extraction separation to obtain an astaxanthin extraction phase; (4) carrying out selective adsorption on the astaxanthin extraction phase through an adsorption column filled with a molecular imprinting adsorbent; (5) collecting eluent by adopting a gradient elution method; and (6) performing vacuum concentration on the eluent, and performing spray drying to obtain an astaxanthin product. According to the method, the astaxanthin extraction efficiency and the product purity are remarkably improved, meanwhile, the energy consumption is reduced to the maximum extent, toxic solvent residues are avoided, and the stability of the astaxanthin is effectively protected.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of organic matter extraction, and particularly relates to a method for green directional extraction and purification of astaxanthin from Haematococcus pluvialis. BACKGROUND

[0002] Astaxanthin is a ketone type carotenoid, which is widely distributed in nature, especially in aquatic organisms. Haematococcus pluvialis, as a kind of freshwater unicellular microalgae, is recognized as the best natural source of astaxanthin known at present, and the accumulation amount of astaxanthin can reach 3% to 5% of the dry weight of cells. This natural red pigment has very strong antioxidant capacity, and its free radical scavenging efficiency is much higher than that of common vitamin E and beta-carotene. Based on this property, astaxanthin shows important application value in multiple fields: it is used in health food to enhance the body's antioxidant defense capacity; it is used in high-end cosmetics to protect the skin from light damage; it is used as a feed additive in aquaculture to improve the color and health status of farmed aquatic products; in addition, it also shows potential research value in the field of medicine.

[0003] Efficient extraction of astaxanthin from Haematococcus pluvialis has always faced many technical difficulties. This microalgae can form a very tough cell wall under adverse conditions, which has a complex structure composed of polysaccharides and polymers such as cellulose, hemicellulose and special sporopollenin, and acts as a natural protective barrier, making it difficult for the internal astaxanthin to be released. Traditional physical crushing methods, such as ball milling or high-pressure homogenization, often require a large amount of energy and have unsatisfactory crushing effect, with the problem of incomplete cell wall breaking, and the strong mechanical action can easily cause changes in the chemical structure of astaxanthin molecules. The commonly used organic solvent extraction method, although relatively simple to operate, has the risk of toxic residue of solvents such as acetone and petroleum ether, which not only affects product safety, but also accelerates the isomerization reaction of astaxanthin during high-temperature extraction, causing it to change from the all-trans form with high biological activity to other forms with lower activity.

[0004] In view of these technical bottlenecks, researchers have proposed various improvement schemes. The supercritical fluid extraction technology uses carbon dioxide as the medium, avoiding the residue of organic solvents, but this technology has very strict requirements for the pretreatment of raw materials, and the equipment investment cost is high, which limits its popularization and application. Enzyme treatment technology has mild conditions and can specifically degrade cell wall components, but the effect of a single enzyme preparation is limited, the treatment time is long, and the cost of enzyme preparation itself is high, which is difficult to meet the demand of large-scale production. Ionic liquid pretreatment, as a new green technology, has good solubility for cellulose materials, but when used alone, the damage to the cell wall of Haematococcus pluvialis is limited, and a strong subsequent treatment is still needed. In the separation and purification stage, the conventional chromatographic filler has insufficient selective adsorption capacity for astaxanthin, resulting in a complicated purification process and low product yield.

[0005] The prior art still has obvious limitations in practical application. The synergistic effect between different pretreatment methods has not been fully studied and utilized, for example, the combined use of ionic liquids and biological enzymes lacks scientific matching basis. The balance between energy input and wall breaking efficiency in the physical breaking process is difficult to grasp, and excessive pressure not only increases energy consumption, but also may cause irreversible damage to the target product. The selection of extraction solvents often focuses on solubility performance, ignoring its influence on product stability, and lacks effective in-situ protection measures. The connection between each process link is not close enough, and most of them still use batch operation mode, which restricts the improvement of overall production efficiency. Therefore, a Haematococcus pluvialis astaxanthin green directional extraction and purification method needs to be designed. SUMMARY

[0006] In order to overcome the defects in the prior art, a Haematococcus pluvialis astaxanthin green directional extraction and purification method is provided.

[0007] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions: A Haematococcus pluvialis astaxanthin green directional extraction and purification method, the method comprising the following steps: (1) Dry Haematococcus pluvialis powder is mixed with a composite pretreatment agent at a mass ratio of 1:8-15, stirred and treated at 40-48 DEG C for 1.5-2.5 hours to obtain a pretreated algae liquid; the present application first designs a composite pretreatment agent for the problem of difficult to break the cell wall of Haematococcus pluvialis. The components of the composite pretreatment agent produce a synergistic effect, the mixed ionic liquid can preliminarily swell the structural polysaccharides in the cell wall, the cellulose complex enzyme further hydrolyzes the polysaccharide molecular chain, the alkyl polysaccharide surfactant promotes the dispersion and penetration of each component, and the metal complex catalyst activates the active center of the enzyme through its unique coordination ability. This multi-level action mode makes the cell wall structure loose, but does not excessively damage the internal structure of the cell.

[0008] (2) The pretreated algae liquid is treated by an ultrahigh pressure microjet device at a pressure of 155-175 MPa for 2-3 times to obtain a broken wall slurry; the algae liquid treated by the composite pretreatment agent exhibits unique wall breaking characteristics in the ultrahigh pressure microjet device. Since the cell wall has been softened in advance, 2-3 times of treatment at a pressure of 155-175 MPa can achieve the desired crushing effect, and this pressure range is lower than directly treating the algae liquid without pretreatment. The synergistic effect of pretreatment and physical wall breaking not only reflects in the improvement of wall breaking efficiency, but more importantly, reduces the mechanical damage to astaxanthin molecules. Timely cooling measures after each treatment further protect the heat-sensitive astaxanthin, and this mild and efficient wall breaking method lays a foundation for subsequent extraction.

[0009] (3) The broken cell slurry is mixed with the composite extraction agent at a volume ratio of 1:3-1:5, and extracted at 40-45℃ and 4.5-5.5 MPa for 50-70 minutes to obtain the astaxanthin extraction phase; the broken cell slurry enters the extraction stage, and the design of the composite extraction agent fully considers the characteristics of astaxanthin. The n-butane is used as the base solvent, and 2%-4% of the co-solvent and 0.5%-1.2% of the composite antioxidant are added, wherein the co-solvent is compounded by ethyl acetate and ethyl lactate at a mass ratio of 1:1.5-2.5, and the composite antioxidant is compounded by rosemary extract and tocopherol at a mass ratio of 1:2-3. This composite design enables the extraction process to be carried out under mild conditions of 40-45℃ and 4.5-5.5 MPa, the co-solvent adjusts the polarity of the extraction system, making it more matched with the solubility characteristics of astaxanthin, and the composite antioxidant provides double protection for astaxanthin during the extraction process, effectively preventing oxidative degradation.

[0010] (4) The astaxanthin extraction phase is selectively adsorbed by an adsorption column filled with a molecular imprinting adsorbent at a flow rate of 8-15 mL / min and a temperature of 32-37℃; in the purification stage, the application of the molecular imprinting adsorbent reflects the accurate recognition of the molecular structure of astaxanthin. Using silica gel as the carrier, a surface imprinting technology is adopted, astaxanthin is used as the template molecule, dimethylaminoethyl methacrylate is used as the functional monomer, and ethylene glycol dimethacrylate is used as the crosslinking agent, and the polymerization reaction is carried out at 60-70℃ for 6-8 hours to obtain the adsorbent with specific recognition cavities after eluting the template molecule. When the astaxanthin extraction phase passes through the adsorption column, these cavities can accurately recognize and bind astaxanthin molecules, while other impurities are effectively excluded due to differences in spatial structure and chemical properties. This separation method based on molecular recognition combines with gradient elution program, first washing impurities with 30%-50% ethanol solution, and then desorbing astaxanthin with 70%-90% ethanol solution, to achieve high-efficiency purification.

[0011] (5) Gradient elution method is adopted, first washing impurities with 30%-50% ethanol solution, and then desorbing astaxanthin with 70%-90% ethanol solution, and collecting the eluent; (6) The eluent is concentrated under reduced pressure at 35-40℃, and spray-dried to obtain astaxanthin product.

[0012] The temperature control in the whole process is also carefully designed, from 40-48℃ in the pretreatment stage, to 40-45℃ in the extraction stage, to 32-37℃ in the purification stage, and finally to 35-40℃ in the concentration stage. This progressive temperature control maximizes the protection of the natural conformation of astaxanthin. The synergistic effect of each link makes the final astaxanthin product not only maintain a high purity, but also completely retain its biological activity. This comprehensive effect is difficult to achieve by single technical improvement.

[0013] The composite pretreatment agent is prepared by the following steps: mixing 1-butyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazolium chloride at a mass ratio of 2-3:1 to obtain a mixed ionic liquid, adding 0.8%-1.5% of cellulose complex enzyme, 0.3%-0.6% of alkyl polyglycoside surfactant and 0.1%-0.3% of metal complex catalyst based on the total mass of the mixed ionic liquid, then dissolving and constant volume the mixture with a phosphate buffer solution at pH 6.0-6.5 to make the mass concentration of the mixed ionic liquid in the final solution reach 10%-12%, and activating the treatment at 45-50℃ for 30-45 minutes to obtain the composite pretreatment agent.

[0014] The cellulose complex enzyme is compounded by endoglucanase, exoglucanase and beta-glucosidase at an enzyme activity unit ratio of 1:1.5-2:0.8-1.

[0015] The metal complex catalyst is zinc-phenanthroline complex or manganese-salicylaldehyde complex.

[0016] The zinc-phenanthroline complex is prepared by dissolving zinc nitrate hexahydrate and phenanthroline in anhydrous ethanol at a molar ratio of 1:1-1.2, stirring and refluxing at 60-70℃ for 2-3 hours, cooling to precipitate crystals, filtering and washing with ethanol, and vacuum drying to obtain the zinc-phenanthroline complex.

[0017] The manganese-salicylaldehyde complex is prepared by dissolving manganese acetate tetrahydrate and salicylaldehyde in methanol at a molar ratio of 1:2-2.2, adding triethylamine equivalent to 1-1.2 times the molar number of manganese salt as a basic catalyst, stirring and reacting at 50-60℃ for 3-4 hours, evaporating the solvent to obtain brown solid, and recrystallizing with diethyl ether to purify to obtain the manganese-salicylaldehyde complex.

[0018] The composite extraction agent is prepared by the following steps: taking n-butane as a basic solvent, adding 2%-4% of a cosolvent and 0.5%-1.2% of a composite antioxidant based on the volume of the basic solvent, and mixing to obtain the composite extraction agent.

[0019] The cosolvent is compounded by ethyl acetate and ethyl lactate at a mass ratio of 1:1.5-2.5; and the composite antioxidant is compounded by rosemary extract and tocopherol at a mass ratio of 1:2-3.

[0020] The preparation method of the molecular imprinting adsorbent comprises the following steps: taking silica gel as a carrier, using surface imprinting technology, taking astaxanthin as a template molecule, dimethylaminoethyl methacrylate as a functional monomer, and ethylene glycol dimethacrylate as a crosslinking agent, and polymerizing at 60-70℃ for 6-8 hours, and then eluting the template molecule to obtain an adsorbent with specific recognition cavities, which is the molecular imprinting adsorbent.

[0021] The molar ratio of the template molecule, functional monomer and crosslinking agent is 1:4-6:20-25.

[0022] Compared with the prior art, the application has the following advantages and beneficial effects: 1. The application effectively solves the problem of difficult breaking of the cell wall of Haematococcus pluvialis by using a composite pretreatment agent. The composite pretreatment agent is composed of an ionic liquid, a cellulose complex enzyme and a metal complex catalyst. The ionic liquid can preliminarily dissolve structural polysaccharides in the cell wall, the cellulose complex enzyme further hydrolyzes the polysaccharide molecular chains, and the presence of the metal complex catalyst improves the catalytic efficiency of the enzyme. The multi-component synergistic effect makes the cell wall structure loose, creating favorable conditions for subsequent physical wall breaking.

[0023] 2. In the physical wall breaking process, the pretreated algal liquid can be fully broken at a relatively low pressure in the ultrahigh pressure microjet device. This is because the mechanical strength of the cell wall has been significantly reduced after the combined action of chemicals and biological enzymes, so that the energy input required by the microjet is correspondingly reduced. This treatment method not only improves the wall breaking efficiency, but also avoids excessive damage to the astaxanthin molecular structure, which is beneficial to maintaining the natural activity of the product.

[0024] 3. The composite extraction agent of the application uses n-butane as a base solvent and adds a co-solvent and a composite antioxidant. The use of the co-solvent improves the solubility selectivity of the extraction system for astaxanthin, and the composite antioxidant provides effective protection for astaxanthin during the extraction process. This combined design enables the extraction process to be carried out under relatively mild conditions, ensuring high extraction efficiency and minimizing isomerization and oxidative degradation of astaxanthin.

[0025] 4. In the purification stage, the application uses a molecular imprinting adsorbent. This adsorbent is prepared with astaxanthin as a template molecule, and its surface has recognition sites that match the spatial structure of astaxanthin molecules. When the astaxanthin extraction phase passes through the adsorption column, these specific recognition sites can selectively bind astaxanthin molecules, thereby achieving effective separation from other impurities. This separation method based on molecular recognition has high selectivity, which helps to improve the purity of the final product. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the application.

[0027] In the DETAILED DESCRIPTION of the application, the sources of various main raw materials are briefly described as follows: Haematococcus pluvialis powder: purchased from Xi'an Tianfeng Biotechnology Co., Ltd., CAS No. 472-61-7 1-Butyl-3-methylimidazolium chloride: purchased from Wuhan Shuer Biological Technology Co., Ltd., CAS No. 79917-90-1 1-Allyl-3-methylimidazolium chloride: purchased from Hubei Xinyu Hong Biological Medicine Technology Co., Ltd., CAS No. 65039-10-3 Cellulose complex enzyme: purchased from Beijing Baiolai Biological Technology Co., Ltd., CAS No. 9012-54-8 Alkyl polyglycoside surfactant: purchased from Jining Tangyi Chemical Co., Ltd., CAS No. 68515-73-1 = 68441 Zinc nitrate hexahydrate: purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd., CAS No. 10196-18-6, analytical pure grade.

[0028] O-phenanthroline: purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., CAS No. 66-71-7, purity ≥ 99%.

[0029] Manganese acetate tetrahydrate: purchased from Tianjin Xinsen Biochemical Technology Co., Ltd., CAS No. 6156-78-1, purity 99%.

[0030] Salicylaldehyde: purchased from Jinan Chuangshi Chemical Co., Ltd., CAS No. 90-02-8, industrial grade.

[0031] n-Butane: purchased from Yantai Hengxin Chemical Technology Co., Ltd., high-purity solvent grade (purity ≥ 99.5%).

[0032] Ethyl acetate: purchased from Jinan Chuangshi Chemical Co., Ltd., CAS No. 141-78-6.

[0033] Ethyl lactate: purchased from Wuhan Kemik Biological Medicine Technology Co., Ltd., CAS No. 97-64-3, purity ≥ 98%.

[0034] Rosemary extract: purchased from Nuolingsheng (Lianyungang) Technology Co., Ltd., a mixture of plant-derived antioxidants containing rosemary extract.

[0035] Tocopherol: purchased from Hubei Widelai Chemical Technology Co., Ltd., CAS No. 59-02-9, food grade.

[0036] Dimethylaminoethyl methacrylate: purchased from Jinan Chuangshi Chemical Co., Ltd., CAS No. 2867-47-2.

[0037] Ethylene glycol dimethacrylate: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No. 97-90-5, crosslinking agent grade.

[0038] Silica gel: purchased from Shandong Guohua Chemical Co., Ltd., CAS No. 14808-60-7.

[0039] The technical solution of the present application is: A method for green directional extraction and purification of astaxanthin from Haematococcus pluvialis, comprising the following steps: (1) Dry Haematococcus pluvialis powder is mixed with a composite pretreatment agent at a mass ratio of 1:8-15, stirred and treated at 40-48°C for 1.5-2.5 hours to obtain pretreated algae liquid; (2) The pretreated algae liquid is treated 2-3 times by an ultrahigh-pressure microjet device at a pressure of 155-175 MPa to obtain a broken-wall slurry; (3) The broken-wall slurry is mixed with a composite extraction agent at a volume ratio of 1:3-1:5, extracted at 40-45°C and 4.5-5.5 MPa for 50-70 minutes, and astaxanthin extraction phase is separated; (4) The astaxanthin extraction phase is selectively adsorbed by an adsorption column filled with a molecular imprinting adsorbent at a flow rate of 8-15 mL / min and a temperature of 32-37°C; (5) Gradient elution is used, first 30%-50% ethanol solution is used to wash impurities, and then 70%-90% ethanol solution is used to desorb astaxanthin, and the eluate is collected; (6) The eluate is concentrated under reduced pressure at 35-40°C, and spray drying is performed to obtain astaxanthin product.

[0040] The composite pretreatment agent is prepared by the following steps: 1-butyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazolium chloride are mixed at a mass ratio of 2-3:1 to obtain a mixed ionic liquid, based on the total mass of the mixed ionic liquid, 0.8%-1.5% cellulose complex enzyme, 0.3%-0.6% alkyl polysaccharide surfactant and 0.1%-0.3% metal complex catalyst are added, then the mixture is dissolved with phosphate buffer at pH 6.0-6.5 and constant volume, so that the mass concentration of the mixed ionic liquid in the final solution reaches 10%-12%, and the mixture is activated at 45-50°C for 30-45 minutes to obtain the composite pretreatment agent.

[0041] The cellulose complex enzyme is compounded by endoglucanase, exoglucanase and beta-glucosidase at an enzyme activity unit ratio of 1:1.5-2:0.8-1.

[0042] The metal complex catalyst is zinc-orthophenanthroline complex or manganese-salicylaldehyde complex.

[0043] The preparation method of the zinc-phenanthroline complex is as follows: zinc nitrate hexahydrate and phenanthroline are dissolved in anhydrous ethanol according to a molar ratio of 1:1-1.2, stirred and refluxed at 60-70 DEG C for 2-3 hours, and then crystals are precipitated after cooling, filtered, washed with ethanol, and vacuum dried to obtain the zinc-phenanthroline complex.

[0044] The preparation method of the manganese-salicylaldehyde complex is as follows: manganese acetate tetrahydrate and salicylaldehyde are dissolved in methanol according to a molar ratio of 1:2-2.2, 1-1.2 times of triethylamine corresponding to the molar number of manganese salt is added as a basic catalyst, stirred and reacted at 50-60 DEG C for 3-4 hours, and then brown solid is obtained after evaporation of the solvent, and the manganese-salicylaldehyde complex is obtained by recrystallization with diethyl ether.

[0045] The composite extraction agent is prepared by the following steps: n-butane is used as a basic solvent, 2%-4% of a cosolvent and 0.5%-1.2% of a composite antioxidant are added to the basic solvent, and then the composite extraction agent is obtained by mixing.

[0046] The cosolvent is compounded by ethyl acetate and ethyl lactate according to a mass ratio of 1:1.5-2.5, and the composite antioxidant is compounded by rosemary extract and tocopherol according to a mass ratio of 1:2-3.

[0047] The preparation method of the molecular imprinting adsorbent comprises the following steps: silica gel is used as a carrier, a surface imprinting technology is adopted, astaxanthin is used as a template molecule, dimethylaminoethyl methacrylate is used as a functional monomer, ethylene glycol dimethacrylate is used as a crosslinking agent, polymerization reaction is carried out at 60-70 DEG C for 6-8 hours, and then the adsorbent with specific recognition cavities, i.e., the molecular imprinting adsorbent, is obtained after elution of the template molecule.

[0048] The molar ratio of the template molecule, the functional monomer and the crosslinking agent is 1:4-6:20-25.

[0049] The series of synergistic innovations of the composite pretreatment agent, the ultrahigh-pressure microjet wall breaking, the composite extraction agent containing an antioxidant and the molecular imprinting adsorption purification can significantly improve the astaxanthin extraction efficiency and product purity, maximize the reduction of energy consumption, avoid the residue of toxic solvents, and effectively protect the stability of astaxanthin.

[0050] The present application is described in detail through the following examples and comparative examples, but the protection scope of the present application is not limited to these examples. The chemical reagents and raw materials used in the following examples and comparative examples are all conventional commercially available products, unless otherwise specified. Example 1

[0051] Take 150 grams of dried Haematococcus pluvialis powder and mix with 1200 milliliters of a composite pretreatment agent at a material mass ratio of 1:8. The composite pretreatment agent contains a mixed ionic liquid, which is formed by mixing 1-butyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazolium chloride at a mass ratio of 3:1. Based on the total mass of the mixed ionic liquid, add 1.5% of a cellulose composite enzyme, which is formed by mixing endoglucanase, exoglucanase, and beta-glucosidase at an enzyme activity unit ratio of 1:2:1; at the same time, add 0.6% of an alkyl polyglycoside surfactant and 0.3% of a zinc-phenanthroline complex catalyst. Dissolve the above mixture in a phosphate buffer solution at pH 6.5 and make up to volume, so that the mass concentration of the mixed ionic liquid in the final solution reaches 10%, and then activate the treatment at 50°C for 30 minutes to prepare the composite pretreatment agent. Pretreatment is carried out at 48°C for 2.5 hours with stirring.

[0052] Subsequently, the pretreated algae solution is treated by an ultra-high pressure microjet device at a pressure of 175 MPa for 3 times to obtain a broken cell slurry. The broken cell slurry is mixed with a composite extraction agent at a volume ratio of 1:5 for extraction. The composite extraction agent is prepared by using n-butane as a base solvent, adding a co-solvent at a volume of 4%, and adding a composite antioxidant at a volume of 1.2%; wherein the co-solvent is formed by mixing ethyl acetate and ethyl lactate at a mass ratio of 1:2.5, and the composite antioxidant is formed by mixing rosemary extract and tocopherol at a mass ratio of 1:3. Extraction is carried out at 45°C and 5.5 MPa for 70 minutes to separate astaxanthin extraction phase. The extraction phase is purified by passing through an adsorption column packed with a molecular imprinting adsorbent, which is prepared by using a molar ratio of template molecule, functional monomer dimethylaminoethyl methacrylate, and crosslinking agent ethylene glycol dimethacrylate of 1:6:25. The operating flow rate of the adsorption column is 15 mL / min, and the column temperature is 37°C. Gradient elution is used, first using 50% ethanol solution to wash impurities, and then using 90% ethanol solution to desorb astaxanthin, and collecting the eluate.

[0053] Finally, the eluate is concentrated under reduced pressure at 40°C, and spray dried to obtain the final astaxanthin product. Example 2

[0054] In this embodiment, the same as in Example 1 will not be repeated, and the differences are as follows: Take 150 grams of dried Haematococcus pluvialis powder and mix it with 2250 milliliters of a composite pretreatment agent at a material mass ratio of 1:15. In the composite pretreatment agent, the mixed ionic liquid is formed by mixing 1-butyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazolium chloride at a mass ratio of 2:1, and the mass concentration is 12%. Based on the total mass of the mixed ionic liquid, 0.8% of a cellulose composite enzyme is added, and the enzyme is compounded by endoglucanase, exoglucanase, and β-glucosidase at an enzyme activity unit ratio of 1:1.5:0.8; at the same time, 0.3% of an alkyl polyglycoside surfactant and 0.1% of a manganese-salicylaldehyde complex catalyst are added.

[0055] The above mixture is dissolved and constant volume with a phosphate buffer solution at pH 6.0, and activated at 45°C for 45 minutes. The pretreatment is stirred at 40°C for 1.5 hours. The physical wall breaking is carried out twice in an ultrahigh pressure microjet device at a pressure of 155 MPa.

[0056] The broken wall slurry is mixed with a composite extraction agent at a volume ratio of 1:3 for extraction. The composite extraction agent is prepared by adding 2% of a cosolvent and 0.5% of a composite antioxidant to n-butane as a base solvent; the cosolvent is compounded by ethyl acetate and ethyl lactate at a mass ratio of 1:1.5, and the composite antioxidant is compounded by rosemary extract and tocopherol at a mass ratio of 1:2. The extraction conditions are 40°C, 4.5 MPa, and a duration of 50 minutes. When adsorbing and purifying, the molar ratio of the template molecule, the functional monomer dimethylaminoethyl methacrylate, and the crosslinking agent ethylene glycol dimethacrylate of the molecular imprinting adsorbent used is 1:4:20, the adsorption column operating flow rate is 8 mL / min, the temperature is 32°C, and gradient elution is performed with 30% and 70% ethanol solutions in sequence. The eluent is concentrated under reduced pressure at 35°C and then spray dried to obtain the final astaxanthin product. Example 3

[0057] In this embodiment, the same as in Example 1 will not be repeated, and the differences are as follows: Take 150 grams of dried Haematococcus pluvialis powder and mix it with 2250 milliliters of a composite pretreatment agent at a material mass ratio of 1:15. In the composite pretreatment agent, the mixed ionic liquid is formed by mixing 1-butyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazolium chloride at a mass ratio of 2:1, and the mass concentration is 12%. Based on the total mass of the mixed ionic liquid, 0.8% of a cellulose composite enzyme is added, and the enzyme is compounded by endoglucanase, exoglucanase, and β-glucosidase at an enzyme activity unit ratio of 1:1.5:0.8; at the same time, 0.3% of an alkyl polyglycoside surfactant and 0.1% of a manganese-salicylaldehyde complex catalyst are added.

[0058] Based on the total mass of the mixed ionic liquid, 1.1% of a cellulose composite enzyme is added, and the enzyme is compounded by endoglucanase, exoglucanase, and β-glucosidase at an enzyme activity unit ratio of 1:1.7:0.9; at the same time, 0.45% of an alkyl polyglycoside surfactant and 0.19% of a zinc-orthophenanthroline complex catalyst are added.

[0059] The mixture was dissolved with phosphate buffer solution at pH 6.3 and constant volume, and activated at 47℃ for 37 minutes. The pretreatment was stirred at 44℃ for 2 hours. The physical wall breaking was carried out twice in an ultrahigh pressure microfluidization device at a pressure of 165 MPa. The wall broken slurry was mixed with the composite extractant at a volume ratio of 1:4 for extraction. The composite extractant was prepared by taking n-butane as the base solvent, adding a co-solvent at a volume of 3% and a composite antioxidant at 0.85%; the co-solvent was compounded by ethyl acetate and ethyl lactate at a mass ratio of 1:2, and the composite antioxidant was compounded by rosemary extract and tocopherol at a mass ratio of 1:2.5.

[0060] The extraction conditions were 42℃, 5.0 MPa, and lasted for 60 minutes. When adsorbing and purifying, the molar ratio of the template molecule, the functional monomer dimethylaminoethyl methacrylate, and the crosslinking agent ethylene glycol dimethacrylate of the molecular imprinting adsorbent used was 1:5:22, the operating flow rate of the adsorption column was 12 mL / min, the temperature was 35℃, and gradient elution was carried out with 40% and 80% ethanol solutions in sequence. After the eluent was concentrated under reduced pressure at 37℃, spray drying was carried out to obtain the final astaxanthin product.

[0061] Comparative Example 1 Comparative Example 1 is basically the same as Example 3, and the core difference lies in that no cellulose complex enzyme is added when preparing the composite pretreatment agent. The pretreatment agent only contains mixed ionic liquid, alkyl polyglycoside surfactant and zinc-phenanthroline complex catalyst, and the proportions and preparation steps of other components are the same as those of Example 3.

[0062] Comparative Example 2 Comparative Example 2 is basically the same as Example 3, and the core difference lies in that no metal complex catalyst, i.e. zinc-phenanthroline complex, is added when preparing the composite pretreatment agent. The pretreatment agent only contains mixed ionic liquid, cellulose complex enzyme and alkyl polyglycoside surfactant, and the proportions and preparation steps of other components are the same as those of Example 3.

[0063] Comparative Example 3 Comparative Example 3 is basically the same as Example 3, and the core difference lies in that the ultrahigh pressure microfluidization device is not used in the wall breaking step, but a conventional high-speed shearing disperser is used at 10,000 rpm for 20 minutes. The remaining step parameters are consistent with those of Example 3.

[0064] Comparative Example 4 Comparative Example 4 is basically the same as Example 3, and the core difference lies in that no composite antioxidant, i.e. rosemary extract and tocopherol, is added when preparing the composite extractant. The extractant only consists of n-butane and the co-solvent ethyl acetate and ethyl lactate. The remaining step parameters are consistent with those of Example 3.

[0065] Comparative Example 5 Comparative Example 5 is basically the same as Example 3, with the core difference being that the purification step does not use a specially designed molecular imprinting adsorbent, but uses ordinary specifications of silica gel with a particle size of 100-200 mesh as the filler of the adsorption column. The elution solvent system and operating conditions are the same as those of Example 3.

[0066] Performance test results and analysis In order to scientifically evaluate the comprehensive performance of the method of the present application, a series of tests were conducted on the astaxanthin products prepared in the above three examples and five comparative examples, and the test results are shown in Table 1. The test indicators and methods include: 1. Breakage rate: determined by using a hemocytometer and trypan blue staining method. The specific method is to count the number of intact cells and total cells under a microscope, and calculate the breakage rate.

[0067] 2. Astaxanthin extraction rate: determined by UV-visible spectrophotometry. After the final product is appropriately dissolved with dimethyl sulfoxide, the absorbance is measured near 492 nm wavelength, the astaxanthin content is calculated through the standard curve, and converted into milligrams of astaxanthin per gram of dry algal powder.

[0068] 3. Product purity and all-trans astaxanthin proportion: determined by high performance liquid chromatography. A C18 chromatographic column is used, and a mixture of methanol and water is used as the mobile phase for gradient elution, and the detection wavelength is usually selected near 478 nm or 480 nm, the total purity of astaxanthin is calculated by area normalization method, and the proportion of all-trans configuration is calculated by comparing with the standard.

[0069] 4. Antioxidant activity retention rate: determined by DPPH free radical scavenging method. The half-inhibitory concentration of the sample is determined, and compared with the half-inhibitory concentration of the high-purity all-trans astaxanthin standard, and the activity retention rate is calculated.

[0070] Table 1 Analysis test results

[0071] As can be seen from Table 1, the three examples all use the complete composite pretreatment agent containing ionic liquid, cellulose complex enzyme and metal complex catalyst, and the cell wall breaking rate is higher than 97.5% and the astaxanthin extraction rate is more than 37.9 mg / g. In contrast, the comparative example 1 lacks cellulose complex enzyme, and its cell wall breaking rate and extraction rate are significantly reduced to 85.3% and 28.1 mg / g. This shows that although ionic liquid can initially dissolve the structural polysaccharides of the cell wall, the lack of specific hydrolysis of polysaccharide molecular chains by cellulose complex enzyme makes it difficult to effectively break down the strong network structure of the cell wall. The comparative example 2 lacks a metal complex catalyst, and its cell wall breaking rate and extraction rate are also significantly lower than the examples, indicating that the metal complex plays an important role in maintaining or enhancing the catalytic efficiency of the cellulose complex enzyme in the pretreatment environment. The synergistic effect of the three can effectively reduce the mechanical strength of the cell wall and create the best conditions for subsequent physical cell wall breaking.

[0072] In the physical cell wall breaking step, examples 1-3 only need to be subjected to ultra-high pressure microjet treatment at a pressure of 155-175 MPa after composite pretreatment to achieve a very high cell wall breaking rate. In contrast, comparative example 3, which has not been subjected to effective composite pretreatment and directly uses high-speed shearing to break the cell wall, has the worst breaking effect, with a breaking rate of only 78.2%, and the purity and all-trans configuration of astaxanthin in the extract are also the lowest. This shows that strong mechanical shear force can not only break the cell wall incompletely, but also can damage astaxanthin molecules. The combination of ultra-high pressure microjet technology and effective pretreatment can better protect the natural activity of astaxanthin while ensuring the breaking effect.

[0073] Examples 1 to 3 add a composite antioxidant composed of rosemary extract and tocopherol to the composite extraction agent, which effectively inhibits the oxidation and isomerization of astaxanthin during the extraction process, so the proportion of all-trans astaxanthin in the product is high, and the retention rate of antioxidant activity is also high. Comparative example 4 does not add a composite antioxidant, although its cell wall breaking rate and extraction rate are similar to those of the examples, but the proportion of all-trans astaxanthin and the retention rate of antioxidant activity are significantly reduced, indicating that in the extraction process, the lack of in-situ protection, astaxanthin has undergone significant oxidative degradation and isomerization reaction. The use of a composite solvent of ethyl acetate and ethyl lactate helps to improve the solubility selectivity of n-butane for astaxanthin.

[0074] Examples 1 to 3 use a specially designed molecular imprint adsorbent, the surface of which has recognition sites matching the spatial structure of astaxanthin molecules, so the purity of the final product is higher than 91.8%. While comparative example 5 uses ordinary silica gel adsorbent, the purity of the product is significantly reduced to 82.3%, which clearly shows that the specific recognition cavities produced by molecular imprinting technology can selectively bind astaxanthin molecules, thereby achieving efficient separation from impurities. This separation method based on molecular recognition has a selectivity that ordinary adsorbents cannot match.

[0075] The test result shows that by the processes of composite pretreatment agent, super high pressure micro jet breaking wall, composite extraction agent containing antioxidant and molecular imprint adsorption purification, the application significantly improves the astaxanthin extraction efficiency and product purity, maximally reduces energy consumption, avoids toxic solvent residue and effectively protects the stability of astaxanthin (especially high bioactivity of all-trans configuration).

[0076] The above is the preferred embodiment of the application, it should be noted that for those skilled in the art, without departing from the principles of the application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the application.

Claims

1. A method for green directional extraction and purification of astaxanthin from Haematococcus sp., characterized by, The method comprises the following steps: (1) mixing dry Haematococcus pluvialis powder with a composite pretreatment agent at a mass ratio of 1:8-15, stirring and treating at 40-48 DEG C for 1.5-2.5 hours to obtain a pretreated algae solution; (2) treating the pretreated algae solution by an ultrahigh pressure microjet device at a pressure of 155-175 MPa for 2-3 times to obtain a broken cell slurry; (3) mixing the broken cell slurry with a composite extraction agent at a volume ratio of 1:3-1:5, extracting at 40-45 DEG C and 4.5-5.5 MPa for 50-70 minutes, and separating to obtain an astaxanthin extraction phase; (4) selectively adsorbing the astaxanthin extraction phase by an adsorption column filled with a molecular imprinting adsorbent at a flow rate of 8-15 mL / min and a temperature of 32-37 DEG C; (5) using a gradient elution method, first washing impurities with 30%-50% ethanol solution, then desorbing astaxanthin with 70%-90% ethanol solution, and collecting the eluate; (6) concentrating the eluate at 35-40 DEG C under reduced pressure, and spray drying to obtain astaxanthin product.

2. The method according to claim 1, wherein the method is characterized by, The composite pretreatment agent is prepared by the following steps: mixing 1-butyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazolium chloride at a mass ratio of 2-3:1 to obtain a mixed ionic liquid, adding 0.8%-1.5% cellulose complex enzyme, 0.3%-0.6% alkyl polyglycoside surfactant and 0.1%-0.3% metal complex catalyst based on the total mass of the mixed ionic liquid, then dissolving and constant volume the mixture with a phosphate buffer solution at pH 6.0-6.5, so that the mass concentration of the mixed ionic liquid in the final solution reaches 10%-12%, and activating at 45-50 DEG C for 30-45 minutes to obtain the composite pretreatment agent.

3. The method according to claim 2, wherein the method is characterized by, The cellulose complex enzyme is compounded by endoglucanase, exoglucanase and beta-glucosidase at an enzyme activity unit ratio of 1:1.5-2:0.8-1.

4. The method according to claim 2, wherein the method is characterized by, The metal complex catalyst is zinc-phenanthroline complex or manganese-salicylaldehyde complex.

5. The method according to claim 4, wherein the method is characterized by, The zinc-phenanthroline complex is prepared by the following method: dissolving zinc nitrate hexahydrate and phenanthroline in anhydrous ethanol at a molar ratio of 1:1-1.2, stirring and refluxing at 60-70 DEG C for 2-3 hours, cooling to precipitate crystals, filtering and washing with ethanol, and vacuum drying to obtain zinc-phenanthroline complex.

6. The method according to claim 4, wherein the method is characterized by, The manganese-salicylaldehyde complex is prepared by the following method: dissolving manganese acetate tetrahydrate and salicylaldehyde in methanol at a molar ratio of 1:2-2.2, adding triethylamine equivalent to 1-1.2 times the molar number of manganese salt as base catalyst, stirring at 50-60 DEG C for 3-4 hours, evaporating the solvent to obtain brown solid, and recrystallizing with diethyl ether to purify to obtain manganese-salicylaldehyde complex.

7. The method according to claim 1, wherein the method is characterized by, The composite extraction agent is prepared by the following steps: taking n-butane as a base solvent, adding 2%-4% cosolvent and 0.5%-1.2% composite antioxidant based on the volume of the base solvent, and mixing to obtain a composite extraction agent.

8. The method according to claim 7, wherein the method is characterized by, The co-solvent is compounded by ethyl acetate and ethyl lactate at a mass ratio of 1:1.5-2.5; the composite antioxidant is compounded by rosemary extract and tocopherol at a mass ratio of 1:2-3.

9. The method according to claim 1, wherein the method is characterized by, The preparation method of the molecular imprinting adsorbent comprises the following steps: taking silica gel as a carrier, adopting a surface imprinting technology, taking astaxanthin as a template molecule, taking dimethylaminoethyl methacrylate as a functional monomer, taking ethylene glycol dimethacrylate as a crosslinking agent, performing polymerization reaction at 60-70 DEG C for 6-8 hours, and then eluting the template molecule to obtain the adsorbent with specific recognition cavities, namely the molecular imprinting adsorbent.

10. The method according to claim 9, wherein the method is characterized by, The molar ratio of the template molecule, the functional monomer and the crosslinking agent is 1:4-6:20-25.

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