阅读说明：本技术 一种从南极磷虾中提取虾青素的方法 (Method for extracting astaxanthin from antarctic krill ) 是由 杨立业 范丽红 欧阳小琨 卢雨清 于 2019-10-15 设计创作，主要内容包括：本发明涉及虾青素的提取技术领域,针对酶解法提取虾青素时间长会使虾青素变性的问题,提供一种从南极磷虾中提取虾青素的方法,包括以下步骤：1)将干的南极磷虾破碎,加入由蛋白酶和几丁质酶组成的复合酶,在水溶液中酶解,得到酶解液、残渣和色素沉淀,旋转蒸发后离心取沉淀物；2)离心后的沉淀物加入乙酸乙酯和甲醇的混合溶剂,搅拌提取过滤,收集滤液,旋转蒸发去除混合溶剂,浓缩滤液；3)滤液先通过大孔吸附树脂吸附游离的虾青素,后经硅胶柱进一步分离纯化,最后冷冻干燥得到虾青素粉末。本发明通过复合抗氧化剂各成分的协同作用降低酶解过程中对类胡萝卜素蛋白的破坏,虾青素提取率高。(The invention relates to the technical field of astaxanthin extraction, and provides a method for extracting astaxanthin from antarctic krill aiming at the problem that astaxanthin is denatured due to long time of astaxanthin extraction by an enzymatic hydrolysis method, which comprises the following steps: 1) crushing dried antarctic krill, adding a complex enzyme consisting of protease and chitinase, carrying out enzymolysis in an aqueous solution to obtain an enzymolysis solution, residues and pigment precipitate, carrying out rotary evaporation, and centrifuging to obtain a precipitate; 2) adding the precipitate after centrifugation into a mixed solvent of ethyl acetate and methanol, stirring, extracting and filtering, collecting filtrate, removing the mixed solvent by rotary evaporation, and concentrating the filtrate; 3) adsorbing free astaxanthin by the filtrate through macroporous adsorption resin, further separating and purifying through a silica gel column, and finally freeze-drying to obtain astaxanthin powder. The method reduces the damage to carotenoid protein in the enzymolysis process through the synergistic effect of the components of the composite antioxidant, and has high extraction rate of astaxanthin.)

1. A method for extracting astaxanthin from antarctic krill is characterized by comprising the following steps:

1) taking dried euphausia superba as a raw material, crushing, adding a compound enzyme which is 1-5% of the mass of the euphausia superba and consists of protease and chitinase, carrying out enzymolysis in an aqueous solution to obtain an enzymolysis liquid, residues and pigment precipitate, and centrifuging after rotary evaporation to obtain a precipitate;

2) adding mixed solvent of ethyl acetate and methanol into the centrifuged precipitate according to the feed-liquid ratio of 0.2-0.5 g/mL, stirring, extracting and filtering, collecting filtrate, removing the mixed solvent by rotary evaporation, and concentrating the filtrate;

3) adsorbing free astaxanthin by the filtrate through macroporous adsorption resin, further separating and purifying through a silica gel column, and finally freeze-drying to obtain astaxanthin powder.

2. The method for extracting astaxanthin from antarctic krill according to claim 1, wherein the step 1) is to add 0.6-3% of compound antioxidant by mass of antarctic krill in the enzymolysis process, wherein the compound antioxidant comprises tert-butyl hydroquinone, one antioxidant selected from citric acid, vitamin A, vitamin E and vitamin C, and mesoporous molecular sieve.

3. The method for extracting astaxanthin from antarctic krill as claimed in claim 2, wherein the preparation method of the compound antioxidant comprises the following steps: firstly, mixing tert-butyl hydroquinone and a mesoporous molecular sieve according to a mass ratio (0.03-0.1):1 to obtain a first mixture, mixing an antioxidant selected from citric acid, vitamin A, vitamin E and vitamin C and the mesoporous molecular sieve according to a mass ratio (0.02-0.06):1 to obtain a second mixture, and mixing the first mixture and the second mixture according to a mass ratio (2.2-2.5): 1.

4. The method for extracting astaxanthin from antarctic krill as claimed in claim 1, 2 or 3, wherein the conditions of the enzymolysis in the step 1) are as follows: the pH value is 5-8, the temperature is 30-50 ℃, and the time is 4-7 h.

5. The method for extracting astaxanthin from antarctic krill according to claim 1, wherein the mass ratio of the protease to the chitinase in the step 1) is (2-4): 1.

6. The method for extracting astaxanthin from Antarctic krill according to claim 1, wherein the volume ratio of ethyl acetate to methanol in step 2) is (1-3):3, the extraction temperature is 30-50 ℃, and the rotation speed is 500-1500 r/min.

7. The method for extracting astaxanthin from antarctic krill as claimed in claim 1, wherein the conditions of freeze-drying in step 3) are as follows: freezing at-50 deg.C to-10 deg.C to solid, and drying in vacuum under atmospheric pressure of 17 Pa for 6-8 hr.

Technical Field

The invention relates to the technical field of astaxanthin extraction, in particular to a method for extracting astaxanthin from antarctic krill.

Background

Antarctic krill is an invertebrate of the marine soft-shelled turtle class, is the species with the largest biomass energy in the world, and is the fishery resource which is the most abundant and urgent to be developed and utilized on the earth. The antarctic krill is rich in carotenoid substances such as astaxanthin, astaxanthin ester is the main component of pigment in the carapace of the antarctic krill, and therefore the antarctic krill is natural astaxanthinOne of the excellent sources. Natural astaxanthin is a polyene carotenoid with active hydroxy ketone group and molecular formula C₄₀H₅₂O₄. Astaxanthin is one of the most important natural carotenoids, is a natural molecule with the strongest oxidation resistance in the nature, and has various beneficial physiological functions such as a coloring function, an immunity enhancing function, cancer prevention, ultraviolet resistance and the like, so the natural astaxanthin has multi-directional research values and has wide development and utilization prospects in the health care product and cosmetic manufacturing industries. Natural astaxanthin is mostly protein-bound and exists in the form of carotenoid protein conjugates. The enzymatic hydrolysis method is applied to extracting astaxanthin because of mild extraction conditions and high extraction rate, but the continuous proteolysis can reduce the protection value of carotenoid protein, and once astaxanthin is free and unstable, molecules are easily oxidized into astaxanthin due to the influence of conditions such as light, heat, alkali, acid, oxidant, ultraviolet light and the like, so that an ideal astaxanthin extraction method is needed.

Disclosure of Invention

In order to overcome the problem that the astaxanthin is denatured due to the fact that the astaxanthin is extracted by an enzymolysis method for too long time, the method for extracting the astaxanthin from the antarctic krill is provided, damage to carotenoid protein in the enzymolysis process is reduced through the synergistic effect of all components of the composite antioxidant, and the extraction rate of the astaxanthin is high.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for extracting astaxanthin from antarctic krill is characterized by comprising the following steps:

1) taking dried euphausia superba as a raw material, crushing, adding a compound enzyme which is 1-5% of the mass of the euphausia superba and consists of protease and chitinase, carrying out enzymolysis in an aqueous solution to obtain an enzymolysis liquid, residues and pigment precipitate, and centrifuging after rotary evaporation to obtain a precipitate;

2) adding mixed solvent of ethyl acetate and methanol into the centrifuged precipitate according to the feed-liquid ratio of 0.2-0.5 g/mL, stirring, extracting and filtering, collecting filtrate, removing the mixed solvent by rotary evaporation, and concentrating the filtrate;

3) adsorbing free astaxanthin by the filtrate through macroporous adsorption resin, further separating and purifying through a silica gel column, and finally freeze-drying to obtain astaxanthin powder.

According to the method, firstly, the glycosidic bond of the chitin in the antarctic krill is broken through enzymolysis, the stable astaxanthin-protein combination is obtained, and then the mixed organic solvent is used for extraction, so that the dosage of the organic solvent is reduced compared with that of direct extraction.

Preferably, in the step 1), a compound antioxidant with the mass of 0.6-3% of that of the Antarctic krill is added in the enzymolysis process, wherein the compound antioxidant comprises tert-butyl hydroquinone, one antioxidant selected from citric acid, vitamin A, vitamin E and vitamin C, and a mesoporous molecular sieve. Although the enzymatic hydrolysis method has mild extraction conditions and a high extraction rate, continuous proteolysis lowers the protective value of carotenoid protein and denatures astaxanthin, so that an antioxidant needs to be added. Because the enzymolysis reaction is very complex and the antioxidant mechanisms of different antioxidants are different, the antioxidant activity achieved by using the antioxidant with a single component is relatively single, the oxidative stress reaction in organisms cannot be effectively inhibited, and the antioxidant effect is limited. In the experimental process, a plurality of known antioxidants are selected and combined, and finally the antioxidant formed by compounding the components is screened out, wherein tert-butylhydroquinone is matched with one of citric acid, vitamin A, vitamin E and vitamin C, so that the comprehensive antioxidant capacity is better. After the compound antioxidant is used, the extraction rate of the astaxanthin can be improved by properly prolonging the enzymolysis time without worrying about the reduction of the protection value of the carotenoid protein. In addition, citric acid and vitamin C can also play roles in decalcification and softening.

Preferably, the preparation method of the compound antioxidant comprises the following steps: firstly, mixing tert-butyl hydroquinone and a mesoporous molecular sieve according to a mass ratio (0.03-0.1):1 to obtain a first mixture, mixing an antioxidant selected from citric acid, vitamin A, vitamin E and vitamin C and the mesoporous molecular sieve according to a mass ratio (0.02-0.06):1 to obtain a second mixture, and mixing the first mixture and the second mixture according to a mass ratio (2.2-2.5): 1. The antioxidant is loaded in the mesoporous molecular sieve, so that the dispersion degree of particles and the size of the particles can be controlled by utilizing the characteristics of the pore channels of the molecular sieve, and the activity of the antioxidant is improved.

Preferably, the conditions of the enzymolysis in the step 1) are as follows: the pH value is 5-8, the temperature is 30-50 ℃, and the time is 4-7 h.

Preferably, the mass ratio of the protease to the chitinase in the step 1) is (2-4): 1. The protease is papain, subtilisin, etc.

Preferably, the volume ratio of the ethyl acetate to the methanol in the step 2) is (1-3):3, the extraction temperature is 30-50 ℃, and the rotation speed is 500-1500 r/min.

Preferably, the conditions for freeze-drying are: freezing at-50 deg.C to-10 deg.C to solid, and drying in vacuum under atmospheric pressure of 17 Pa for 6-8 hr. The freeze drying can prevent astaxanthin denaturation caused by high temperature during drying.

Therefore, the invention has the following beneficial effects: 1) according to the method, firstly, the glycosidic bond of chitin in the antarctic krill is broken through enzymolysis, a stable astaxanthin-protein combination is obtained, and then the mixed organic solvent is used for extraction, so that the dosage of the organic solvent is reduced compared with that of direct extraction; 2) adding antioxidant during enzymolysis to prevent astaxanthin denaturation caused by reduction of the protective value of carotenoid protein; 3) the tert-butyl hydroquinone is matched with one of citric acid, vitamin A, vitamin E and vitamin C, so that the comprehensive oxidation resistance is better; 4) the antioxidant is loaded in the mesoporous molecular sieve, so that the dispersion degree of particles and the size of the particles can be controlled by utilizing the characteristics of the pore channels of the molecular sieve, and the activity of the antioxidant is improved.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.

In the present invention, unless otherwise specified, all the raw materials and equipment used are commercially available or commonly used in the art, and the methods in the examples are conventional in the art unless otherwise specified.