阅读说明：本技术 一种应用双水相体系分离制备虾青素的方法 (Method for preparing astaxanthin by applying aqueous two-phase system separation ) 是由 冯金华 王岳鸿 李利 王小瑞 于 2019-10-12 设计创作，主要内容包括：本发明公开了一种应用双水相体系分离制备虾青素的方法,具体涉及生物工程技术领域,包括含有虾青素的生物原料、亲水性低分子有机溶剂、无机盐溶液和浓度为5％的HCl。本发明通过利用双水相体系从含虾青素的物料中分离制备虾青素沉淀,不仅有利于对含虾青素的生物物料的开发利用,开创一种新的虾青素提取分离方法,而且降低虾青素的生产成本,解决虾青素产业化的一些分离制备的难题,具有在环境温度下进行制备、无需加热,分离快,不需要大型设备,而且蛋白质、多糖、重金属等水溶性杂质及一些脂溶性杂质减少,制备的虾青素含量高,杂质少,对虾青素的破坏少,有利于后续虾青素的收集及后续的纯化和利用。(The invention discloses a method for separating and preparing astaxanthin by using a double aqueous phase system, and particularly relates to the technical field of biological engineering, wherein the method comprises a biological raw material containing astaxanthin, a hydrophilic low-molecular organic solvent, an inorganic salt solution and HCl with the concentration of 5%. The method disclosed by the invention has the advantages that the astaxanthin sediment is separated and prepared from the astaxanthin-containing material by using the aqueous two-phase system, the development and utilization of the astaxanthin-containing biological material are facilitated, a novel astaxanthin extraction and separation method is created, the production cost of the astaxanthin is reduced, the problems of separation and preparation of astaxanthin in industrialization are solved, the method is capable of being prepared at an ambient temperature, heating is not needed, the separation is fast, large-scale equipment is not needed, water-soluble impurities such as protein, polysaccharide and heavy metal and fat-soluble impurities are reduced, the content of the prepared astaxanthin is high, the impurities are few, the damage to the astaxanthin is few, and the subsequent collection, purification and utilization of the astaxanthin are facilitated.)

1. A method for preparing astaxanthin by using an aqueous two-phase system to separate comprises a biological raw material containing astaxanthin, a hydrophilic low-molecular organic solvent, an inorganic salt solution and HCl with the concentration of 5 percent, and is characterized in that: the specific operation steps are as follows:

the method comprises the following steps: collecting astaxanthin-containing biological raw materials such as dried shrimp and crab shells, fresh products, haematococcus pluvialis, phaffia rhodozyma and the like and waste materials such as shrimp and crab shells and the like produced by astaxanthin-containing aquatic products such as shrimps and crabs and the like, and washing out surface impurities;

step two: drying the raw materials collected in the step one, soaking the raw materials in HCl with the concentration of 5% for 1 hour for decalcification, repeatedly washing the raw materials with distilled water to be neutral after soaking, drying the raw materials again, and crushing dry products by using a crusher for later use after drying;

step three: dissolving 20g of inorganic salt in water, and fixing the volume to 100mL of inorganic salt solution;

step four: adding a hydrophilic low-molecular organic solvent into the powdery raw materials prepared in the second step according to the proportion, uniformly stirring, standing in the dark for 0.5-2h, dissolving astaxanthin, and centrifuging the extracting solution to remove insoluble substances to obtain a low-molecular organic solution containing astaxanthin;

step five: adding the filtrate into the inorganic salt solution prepared in the third step according to a certain proportion, uniformly mixing to form a certain double aqueous phase, uniformly stirring, standing for phase separation for 0.5-2h, allowing astaxanthin to form precipitates on the interface of the two phases, quickly removing the upper phase, centrifuging the middle phase and the lower phase in a refrigerated centrifuge at 12000r/min for 20min, removing the supernatant after centrifugation, and keeping the precipitates;

step six: injecting distilled water into the centrifuge tube, shaking, centrifuging at 12000r/min in a refrigerated centrifuge for 20min, collecting centrifuged astaxanthin, drying at low temperature, and storing.

2. The method for preparing astaxanthin by using the aqueous two-phase system for separation according to claim 1, which is characterized in that: and in the second step, the particle size of the powdery raw material is 50-100 meshes.

3. The method for preparing astaxanthin by using the aqueous two-phase system for separation according to claim 1, which is characterized in that: the inorganic salt in the third step is one or a combination of ammonium sulfate, potassium phosphate, sodium chloride, sodium sulfate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

4. The method for preparing astaxanthin by using the aqueous two-phase system for separation according to claim 1, which is characterized in that: the hydrophilic low molecular organic matter is one or a combination of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, ethylene glycol and acetone.

5. The method for preparing astaxanthin by using the aqueous two-phase system for separation according to claim 1, which is characterized in that: the ratio of the powdery raw materials to the hydrophilic low-molecular organic solvent in the fourth step is set as 1: 5-50 ℃ and the extraction temperature is set to be 15-50 ℃.

6. The method for preparing astaxanthin by using the aqueous two-phase system for separation according to claim 1, which is characterized in that: and in the fifth step, the ratio of the low-molecular-weight organic solution containing astaxanthin to the inorganic salt solution is set to be 3: 4-5.

7. The method for preparing astaxanthin by using the aqueous two-phase system for separation according to claim 1, which is characterized in that: in the whole aqueous two-phase system, the mass percent of the added inorganic salt is 10-20%, and the mass fraction of the added hydrophilic low-molecular organic matter in the whole aqueous two-phase system is 30-70%.

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for separating and preparing astaxanthin by using a double aqueous phase system.

Background

Astaxanthin (Astaxanthin), 3, 3 ' -dihydroxy-4, 4 ' -diketo-beta, beta ' -carotene, with molecular formula C40H52O4 and relative molecular mass 596.86, is a carotenoid. Astaxanthin is widely found in nature, for example in most crustaceans and salmonids, in certain fungi and fungi, in the leaves, flowers, fruits of plants, and in the feathers of flamingos. Astaxanthin is currently the strongest natural antioxidant known to man. Astaxanthin also has immunity enhancing and anticancer effects, and can promote growth and reproduction of some aquatic products. The astaxanthin with the characteristics has wide prospect in the fields of beauty and health care, agricultural feed and even medical treatment. The research on the preparation of astaxanthin has great practical significance.

The preparation of astaxanthin mainly comprises two methods, namely a natural extraction method and an artificial synthesis method. The chemically synthesized astaxanthin and natural astaxanthin have obvious difference in structure, function, application, safety and other aspects, and the stability and the antioxidant activity and the coloring property are obviously lower than those of the natural astaxanthin, so the current trend is to adopt a natural extraction method for production. At present, the international mainstream is to culture rhodococcus pluvialis or phaffia rhodozyma and extract astaxanthin from the rhodococcus pluvialis or the phaffia rhodozyma, because of problems of strain culture and the like, the total yield of astaxanthin is always low, and in addition, the waste products of shrimps and crabs are also the main distribution points of astaxanthin in the nature. The current main methods for extracting astaxanthin include: alkali extraction method, oil dissolution method, organic solvent method, and supercritical CO₂Extraction method, enzymatic hydrolysis method, etc. However, the extraction method of astaxanthin removes supercritical CO₂In addition to extraction, astaxanthin is prepared by separating it from the extraction solution. However, there is no effective method for separating astaxanthin from the solution, except for evaporation under reduced pressure. However, due to the sensitivity of astaxanthin to temperature, the structural damage of common evaporation methods to astaxanthin is large, and supercritical CO is adopted₂Although the extraction technology can be used for efficiently preparing the astaxanthin solid, the requirement on equipment is high, the cost is high, and the application range is relatively small. Therefore, a method for separating and preparing astaxanthin from the solution quickly and effectively for protecting the stability of astaxanthin and being beneficial to industrialization is found, and the method has very high value on research and development of astaxanthin.

The application of aqueous two-phase systems now involves the separation and purification of various active ingredients such as enzymes, nucleic acids, growth hormones, viruses, etc. Because of its advantages of less activity loss, less separation steps, mild operation conditions, no organic solvent residue, etc., the method is undoubtedly promising in the aspect of extracting effective components in natural products. However, the application of the existing aqueous two-phase system mainly relates to the extraction capacity of the aqueous two-phase system. There is no report on the research of using the hydrophilic low-molecular organic solvent/inorganic salt system to precipitate astaxanthin from the organic solvent and simultaneously using the system to remove water-soluble components and some fat-soluble components in the astaxanthin low-molecular inorganic solvent extract.

Disclosure of Invention

In order to overcome the above-mentioned drawbacks of the prior art, embodiments of the present invention provide a method for preparing astaxanthin using aqueous two-phase system separation, the astaxanthin sediment is separated and prepared from the astaxanthin-containing material by using the aqueous two-phase system, which is not only beneficial to the development and utilization of the astaxanthin-containing biological material, but also creates a new method for extracting and separating the astaxanthin, but also reduces the production cost of the astaxanthin, solves the problems of separation and preparation of the astaxanthin in industrialization, has the advantages of preparation at ambient temperature, no need of heating, quick separation, no need of large-scale equipment, but also reduces water-soluble impurities such as protein, polysaccharide, heavy metal and the like and some fat-soluble impurities, the content of the prepared astaxanthin is high, the impurities are few, the method has less damage to the astaxanthin, is beneficial to the subsequent collection and the subsequent purification and utilization of the astaxanthin and solves the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a method for separating and preparing astaxanthin by using an aqueous two-phase system comprises a biological raw material containing astaxanthin, a hydrophilic low-molecular organic solvent, an inorganic salt solution and HCl with the concentration of 5 percent, and comprises the following specific operation steps:

the method comprises the following steps: collecting astaxanthin-containing biological raw materials such as dried shrimp and crab shells, fresh products, haematococcus pluvialis, phaffia rhodozyma and the like and waste materials such as shrimp and crab shells and the like produced by astaxanthin-containing aquatic products such as shrimps and crabs and the like, and washing out surface impurities;

step two: drying the raw materials collected in the step one, soaking the raw materials in HCl with the concentration of 5% for 1 hour for decalcification, repeatedly washing the raw materials with distilled water to be neutral after soaking, drying the raw materials again, and crushing dry products by using a crusher for later use after drying;

step three: dissolving 20g of inorganic salt in water, and fixing the volume to 100mL of inorganic salt solution;

step four: adding a hydrophilic low-molecular organic solvent into the powdery raw materials prepared in the second step according to the proportion, uniformly stirring, standing in the dark for 0.5-2h, dissolving astaxanthin, and centrifuging the extracting solution to remove insoluble substances to obtain a low-molecular organic solution containing astaxanthin;

step five: adding the filtrate into the inorganic salt solution prepared in the third step according to a certain proportion, uniformly mixing to form a certain double aqueous phase, uniformly stirring, standing for phase separation for 0.5-2h, allowing astaxanthin to form precipitates on the interface of the two phases, quickly removing the upper phase, centrifuging the middle phase and the lower phase in a refrigerated centrifuge at 12000r/min for 20min, removing the supernatant after centrifugation, and keeping the precipitates;

step six: injecting distilled water into the centrifuge tube, shaking, centrifuging at 12000r/min in a refrigerated centrifuge for 20min, collecting centrifuged astaxanthin, drying at low temperature, and storing.

Preferably, the particle size of the powdery raw material in the second step is 50-100 meshes.

Preferably, the inorganic salt in the step three is one or a combination of more of ammonium sulfate, potassium phosphate, sodium chloride, sodium sulfate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

Preferably, the hydrophilic low molecular organic substance is one or a combination of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, ethylene glycol and acetone.

Preferably, the ratio of the powdery raw material to the hydrophilic low-molecular organic solvent in the fourth step is set to be 1: 5-50 ℃ and the extraction temperature is set to be 15-50 ℃.

Preferably, the ratio of the low-molecular-weight organic solution containing astaxanthin to the inorganic salt solution in the fifth step is set to be 3: 4-5.

Preferably, in the whole aqueous two-phase system, the mass percentage of the added inorganic salt is 10-20%, and the mass fraction of the added hydrophilic low-molecular organic matter in the whole aqueous two-phase system is 30-70%.

The invention has the technical effects and advantages that:

1. the method has the advantages that the astaxanthin sediment is separated and prepared from the astaxanthin-containing material by using a double-aqueous phase system, development and utilization of the astaxanthin-containing biological material are facilitated, a novel astaxanthin extraction and separation method is created, the production cost of astaxanthin is reduced, the difficult problems of separation and preparation of astaxanthin industrialization are solved, preparation at an ambient temperature is realized, heating is not needed, separation is fast, large-scale equipment is not needed, water-soluble impurities such as protein, polysaccharide and heavy metal and fat-soluble impurities are reduced, the content of the prepared astaxanthin is high, the impurities are few, the damage to astaxanthin is few, and subsequent collection, subsequent purification and utilization of astaxanthin are facilitated.

Detailed Description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.