阅读说明：本技术 一种微藻内虾青素的快速提取方法 (Method for rapidly extracting astaxanthin in microalgae ) 是由 黄成潭 潘军 叶蕾 黄敏 于 2021-08-12 设计创作，主要内容包括：本发明涉及天然色素提取技术领域,特别涉及一种微藻内虾青素的快速提取方法。该方法包括：将含有微藻的养殖水与蛋白保护剂混合,经第一离心,得到浓缩藻液；将浓缩藻液与混合萃取液混合,对所得混合藻液进行冷藏；混合萃取液由乙醇、丙酮、正乙烷和水组成；将冷藏后的混合藻液与抗氧化剂、表面活化剂混合,进行细胞破碎,得到破碎后的藻液；将破碎后的藻液进行超声提取；经超声提取后的混合藻液进行第二离心,得到上清液；将上清液与硫酸铵、聚丙二醇混合,搅拌；将搅拌好的料液进行第三离心,将所得沉淀物进行冷冻干燥。本发明混合萃取液的萃取率高。本发明通过较为简便的方式,可将虾青素快速提取和纯化,提取成功率可达99％,且溶剂残留极低。(The invention relates to the technical field of natural pigment extraction, and particularly relates to a method for quickly extracting astaxanthin in microalgae. The method comprises the following steps: mixing culture water containing microalgae with a protein protective agent, and performing first centrifugation to obtain concentrated algae solution; mixing the concentrated algae solution with the mixed extract, and refrigerating the obtained mixed algae solution; the mixed extract consists of ethanol, acetone, n-ethane and water; mixing the refrigerated mixed algae solution with an antioxidant and a surfactant, and carrying out cell disruption to obtain a disrupted algae solution; carrying out ultrasonic extraction on the crushed algae liquid; performing second centrifugation on the mixed algae liquid subjected to ultrasonic extraction to obtain supernatant; mixing the supernatant with ammonium sulfate and polypropylene glycol, and stirring; and (4) performing third centrifugation on the stirred feed liquid, and freeze-drying the obtained precipitate. The extraction rate of the mixed extraction liquid is high. The method can rapidly extract and purify the astaxanthin in a simple and convenient mode, the extraction success rate can reach 99 percent, and the solvent residue is extremely low.)

1. A method for rapidly extracting astaxanthin in microalgae is characterized by comprising the following steps:

mixing culture water containing microalgae with a protein protective agent, and performing first centrifugation to obtain a concentrated algae solution;

mixing the concentrated algae solution with the mixed extract liquor, and refrigerating the obtained mixed algae solution; the mixed extract consists of ethanol, acetone, n-ethane and water;

mixing the refrigerated mixed algae solution with an antioxidant and a surfactant, and carrying out cell disruption to obtain a disrupted algae solution;

carrying out ultrasonic extraction on the crushed algae liquid;

performing second centrifugation on the mixed algae liquid subjected to ultrasonic extraction in the step (5) to obtain a supernatant;

mixing the supernatant with ammonium sulfate and polypropylene glycol, and stirring;

and (7) performing third centrifugation on the stirred feed liquid, and freeze-drying the obtained precipitate.

2. The extraction method according to claim 1, wherein the protein protectant is sodium azide;

in the culture water containing microalgae, the addition amount of the protein protective agent is 0.1-0.5 mg/L.

3. The extraction method according to claim 1, wherein the rotation speed of the first centrifugation is 5000-10000rpm, and the water content of the concentrated algae solution is 50-70%.

4. The extraction method according to claim 1, wherein the volume ratio of ethanol, acetone, n-ethane and water in the mixed extract is (15-25): (5-15): (10-30): (100-200);

the mass ratio of the concentrated algae liquid to the mixed extract is 1: (2-3).

5. The extraction method according to claim 1, wherein the refrigeration is performed at 0-6 ℃, and the temperature of the mixed algae solution is reduced to 4-10 ℃.

6. The extraction process according to claim 1, wherein the antioxidant is vitamin C; the surfactant is Tween 80 and Dow DF104 polyether defoaming agent;

in the mixed algae liquid after refrigeration, the addition amount of the antioxidant is 1-2mg/L, the addition amount of the Tween 80 is 1-5 mL/L, and the addition amount of the Dow DF104 polyether defoaming agent is 1-2 mL/L.

7. The extraction method according to claim 1, characterized in that the procedure of the ultrasound extraction is: carrying out ultrasonic treatment for 1-3s, and carrying out intermittent treatment for 2-5 s; ultrasonic extraction is carried out for 60-120min, and standing is carried out for 10-20 min.

8. The extraction method according to claim 1, wherein the second centrifugation is performed at 2000-3000rpm for 40-60 min.

9. The extraction method according to claim 1, wherein the amount of ammonium sulfate added to the supernatant is 10 to 15g/L, and the amount of polypropylene glycol added to the supernatant is 5 to 30 mL/L;

the stirring speed is 500-1000 rpm, and the time is 1-2 h.

10. The extraction method according to any one of claims 1 to 9, wherein the rotation speed of the third centrifugation is 5000 to 10000rpm and the time is 20 to 40 min.

Technical Field

The invention relates to the technical field of natural pigment extraction, and particularly relates to a method for quickly extracting astaxanthin in microalgae.

Background

Microalgae are baits (or baits of baits) for the lifetime or specific development stages of aquaculture animals, and are one of important bases to support the aquaculture industry to a great extent. The nannochloropsis oculata grows rapidly, has small cell particles, is rich in unsaturated fatty acids such as EPA and the like, has comprehensive nutrition and has the characteristic of thicker cell wall. Microalgae is the primary productivity of a water body ecosystem, and is a high-quality natural bait for aquatic animals because the microalgae contains rich nutrient substances such as protein, polyunsaturated fatty acid, carotenoid and vitamins; the microalgae can provide food for aquatic animals, and can absorb redundant substances such as ammonia nitrogen, phosphorus and the like in a water body through photosynthesis to play a role in stabilizing and improving water quality, so that the microalgae is widely applied to the aquaculture industry.

Astaxanthin is a red fat-soluble carotenoid, is insoluble in water, and is easily dissolved in solvents such as dimethyl sulfoxide, acetone, chloroform and the like. Research shows that astaxanthin has stronger biological activity than carotene, vitamin E and the like, and belongs to substances safe for human bodies. Astaxanthin, which has been considered to be "super vitamin E", has an antioxidant activity about 10 times higher than that of beta-carotene and about 500 times higher than that of vitamin E. Astaxanthin has been used in aquaculture, the nutritional and health care and cosmetic industries, as well as in food supplements, and it also has pharmacological effects in many diseases and is safe to humans, with broad market prospects.

Astaxanthin is divided into natural astaxanthin and artificially synthesized astaxanthin according to the source, and the synthesized astaxanthin is a racemic mixture of three isomers, and the ratio is about 1: 2: 1. natural astaxanthin is mainly derived from algae and crustaceans, and haematococcus pluvialis is the highest accumulated species of all known astaxanthin-synthesizing organisms, can accumulate more than 3% of its dry weight, and is the most used algae for producing natural astaxanthin at present. Haematococcus pluvialis is a unicellular green freshwater alga with a complex life cycle. The utilization of Haematococcus pluvialis to produce astaxanthin is gradually receiving attention from people. In recent years, the laboratory culture of haematococcus pluvialis has become a research hotspot, but due to the problems of large-scale culture and the difficulty in extraction increased by the sclerenchyma cells, the industrial production of astaxanthin is difficult to realize. Therefore, the research on the extraction method of astaxanthin in haematococcus pluvialis has certain significance for large-scale production.

At present, the extraction of astaxanthin in haematococcus pluvialis by using an organic solvent is a common method, the extraction cost is low, and the operation is simple. In astaxanthin extraction, dimethyl sulfoxide, ethanol, organic acids, acetone, chloroform, methanol, hexane, isopropanol, dichloromethane and the like are main extracting agents. Sachindra et al, which compare the extraction effects of a single organic solvent and a mixed solvent, have found that the extraction rate of the mixed solvent is higher, and these extractants are usually compounded and used in a certain proportion. The compound use of dimethyl sulfoxide and acetone is a more classical use method. The inherent toxicity of these organic solvents also adversely affects the process, and excessive solvent residue in the extract can create food safety concerns. The single organic solvent extraction method has low efficiency and long extraction time, and is usually combined with other extraction methods. How to reduce the solvent residue in the extract and maintain good extraction effect is a problem faced by the current method.

Disclosure of Invention

In view of the above, the invention provides a method for rapidly extracting astaxanthin from microalgae. The method has the advantages of high speed, high extraction rate, high purification, and low solvent residue.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for rapidly extracting astaxanthin in microalgae, which comprises the following steps:

mixing culture water containing microalgae with a protein protective agent, and performing first centrifugation to obtain a concentrated algae solution;

mixing the concentrated algae solution with the mixed extract liquor, and refrigerating the obtained mixed algae solution; the mixed extract consists of ethanol, acetone, n-ethane and water;

mixing the refrigerated mixed algae solution with an antioxidant and a surfactant, and carrying out cell disruption to obtain a disrupted algae solution;

carrying out ultrasonic extraction on the crushed algae liquid;

performing second centrifugation on the mixed algae liquid subjected to ultrasonic extraction in the step (5) to obtain a supernatant;

mixing the supernatant with ammonium sulfate and polypropylene glycol, and stirring;

and (7) performing third centrifugation on the stirred feed liquid, and freeze-drying the obtained precipitate.

Preferably, the protein protectant is sodium azide;

preferably, the addition amount of the protein protective agent in the culture water containing microalgae is 0.1-0.5 mg/L.

In the specific embodiment provided by the invention, the adding amount of the protein protective agent in the culture water containing microalgae is 0.5 mg/L.

Preferably, the rotation speed of the first centrifugation is 5000-10000rpm, and the water content of the concentrated algae solution is 50-70%.

In the specific embodiment provided by the invention, the rotation speed of the first centrifugation is 10000rpm, and the water content of the concentrated algae liquid is 70%.

Preferably, in the mixed extract, the volume ratio of ethanol, acetone, n-ethane and water is (15-25): (5-15): (10-30): (100-200);

preferably, the volume ratio of the ethanol, the acetone, the n-ethane and the water in the mixed extract is (18-20): 9-11): 13-17): 100-120.

In the specific embodiment provided by the invention, the volume ratio of the ethanol, the acetone, the n-ethane and the water in the mixed extract is 19:10:15: 101.

Preferably, the mass ratio of the concentrated algae solution to the mixed extract is 1: (2-3).

In the specific embodiment provided by the invention, the mass ratio of the concentrated algae liquid to the mixed extract is 1: 3.

preferably, the refrigeration is performed at 0-6 ℃, and the temperature of the mixed algae liquid is reduced to 4-10 ℃.

In the specific embodiment provided by the invention, the refrigeration is performed at 4 ℃, and the temperature of the mixed algae liquid is reduced to 10 ℃.

Preferably, the antioxidant is vitamin C.

Preferably, the surfactants are tween 80 and dow DF104 polyether defoamer.

Preferably, in the mixed algae liquid after refrigeration, the addition amount of the antioxidant is 1-2mg/L, the addition amount of the Tween 80 is 1-5 mL/L, and the addition amount of the Dow DF104 polyether defoamer is 1-2 mL/L.

In the specific embodiment provided by the invention, in the mixed algae liquid after refrigeration, the addition amount of the antioxidant is 2mg/L, the addition amount of the Tween 80 is 5mL/L, and the addition amount of the Dow DF104 polyether defoamer is 2 mL/L.

Preferably, the procedure of ultrasound extraction is: carrying out ultrasonic treatment for 1-3s, and carrying out intermittent treatment for 2-5 s; ultrasonic extraction is carried out for 60-120min, and standing is carried out for 10-20 min.

In the specific embodiment provided by the present invention, the procedure of ultrasound extraction is: ultrasonic for 2s, and intermittent for 5 s; ultrasonic extracting for 120min, and standing for 20 min.

Preferably, the rotation speed of the second centrifugation is 2000-3000rpm, and the time is 40-60 min.

In the specific embodiment provided by the invention, the rotation speed of the second centrifugation is 3000rpm, and the time is 40 min.

Preferably, in the supernatant, the adding amount of ammonium sulfate is 10-15g/L, and the adding amount of polypropylene glycol is 5-30 mL/L;

in the specific embodiment provided by the invention, the adding amount of ammonium sulfate in the supernatant is 15g/L, and the adding amount of polypropylene glycol is 5 mL/L.

Preferably, the stirring speed is 500-1000 rpm, and the time is 1-2 h.

In the specific embodiment provided by the invention, the stirring speed is 1000rpm, and the time is 1 h.

Preferably, the rotation speed of the third centrifugation is 5000-10000rpm, and the time is 20-40 min.

In the specific embodiment provided by the invention, the rotating speed of the third centrifugation is 5000rpm, and the time is 30 min.

In the present invention, the microalgae are selected from algae of the phylum cyanophyta, chlorophyta, chrysophyta or rhodophyta. Such as haematococcus pluvialis, chlorella, nannochloropsis, etc.

The invention provides a method for rapidly extracting astaxanthin in microalgae, which comprises the following steps: mixing culture water containing microalgae with a protein protective agent, and performing first centrifugation to obtain concentrated algae solution; mixing the concentrated algae solution with the mixed extract, and refrigerating the obtained mixed algae solution; the mixed extract consists of ethanol, acetone, n-ethane and water; mixing the refrigerated mixed algae solution with an antioxidant and a surfactant, and carrying out cell disruption to obtain a disrupted algae solution; carrying out ultrasonic extraction on the crushed algae liquid; performing second centrifugation on the mixed algae liquid subjected to ultrasonic extraction to obtain supernatant; mixing the supernatant with ammonium sulfate and polypropylene glycol, and stirring; and (4) performing third centrifugation on the stirred feed liquid, and freeze-drying the obtained precipitate. The invention has the technical effects that:

compared with the extraction effect of a single extraction liquid and a composite extraction liquid, the extraction efficiency of the mixed solvent consisting of ethanol, acetone, n-ethane and water is higher.

In the invention, sodium azide is used as a protein protective agent, so that protein denaturation and decomposition in a centrifugation process are avoided, and impurities are formed. The vitamin C has antioxidant effect, and can prevent astaxanthin from oxidative decomposition; tween 80 and Dow DF104 polyether defoamer are used as surface active agents, and astaxanthin can be separated out from the supernatant by combined use. The combination of ammonium sulfate and polypropylene glycol can precipitate astaxanthin dissolved in supernatant to form granule precipitate.

The method can rapidly extract and purify the astaxanthin by a simple and convenient mode, the yield reaches 90-95%, the purity can reach 99%, and the solvent residue is extremely low (< 0.01%).

Detailed Description

The invention discloses a method for quickly extracting astaxanthin in microalgae, which can be realized by appropriately improving process parameters by a person skilled in the art with reference to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The method for rapidly extracting the astaxanthin comprises the following specific steps:

1. adding sodium azide into cultured haematococcus pluvialis at a ratio of 0.1-0.5 mg/L. And then, carrying out centrifugal separation on the microalgae liquid by using a disc type centrifuge, and extracting algae somatic cells to form concentrated algae liquid. The rotation speed of the centrifuge is 5000 plus 10000rpm, and the water content of the separated algae liquid is 50-70 percent.

2. Preparing a mixed extraction liquid: 95% ethanol, 100% acetone, 100% n-ethane and purified water are mixed according to the volume ratio of (20-25) to (5-10) to (15-30) to (100-

Adding the separated concentrated algae solution into the mixed extract liquor, wherein the addition amount of the extract liquor is 2-3 times (mass ratio) of the concentrated algae, stirring uniformly, placing in a refrigerator at 4 ℃ for cold storage, and cooling the mixed algae solution to 4-10 ℃.

4. Adding vitamin C (1-2 mg/L), Tween 80 (1-5 ml/L) and Dow DF104 polyether defoaming agent (1-2ml/L) into the refrigerated mixed algae solution, mixing, and placing into a homogenizing crusher for cell crushing.

5. And (3) carrying out ultrasonic extraction on the crushed mixed algae liquid by using an ultrasonic instrument for 1-3s and 2-5s at intervals for 60-120min, and standing for 10-20 min.

6. And centrifuging the mixed algae liquid after ultrasonic treatment at a low rotation speed of 2000-3000rpm for 40-60 min by using a disc centrifuge, collecting supernatant, and removing precipitates.

7. Adding ammonium sulfate solid (the adding amount is 10-15g/L) and polypropylene glycol (the adding amount is 5-30ml/L) into the collected supernatant, and stirring at 500-1000 rpm for 1-2 h.

8. And (4) centrifuging the stirred material liquid at a high rotating speed of 5000 plus 10000rpm for 20-40 min by using a disc centrifuge, and collecting the precipitate.

9. And (5) freeze-drying the precipitate to obtain astaxanthin freeze-dried powder.

The reagents or apparatus used in the present invention are commercially available.

The invention is further illustrated by the following examples:

example 1

1. Adding sodium azide into the cultured haematococcus pluvialis at the amount of 0.5 mg/L. And then, carrying out centrifugal separation on the microalgae liquid by using a disc type centrifuge, and extracting algae somatic cells to form concentrated algae liquid. The rotation speed of the centrifuge is 10000rpm, and the separated algae liquid contains 70 percent of water.

2. Preparing a mixed extraction liquid: 95% ethanol, 100% acetone, 100% n-ethane, purified water, and mixed at a volume ratio of 20:10:15:100 (ethanol: acetone: n-ethane: water: 19:10:15:101) to form a mixed extract.

3. Adding the separated concentrated algae solution into the mixed extract liquor, wherein the addition amount of the extract liquor is 3 times (mass ratio) of the concentrated algae, uniformly stirring, placing in a refrigerator at 4 ℃ for cold storage, and reducing the temperature of the mixed algae solution to 10 ℃.

4. Adding vitamin C (added amount is 2mg/L), tween 80 (added amount is 5mL/L) and Dow DF104 polyether defoaming agent (added amount is 2mL/L) into the refrigerated mixed algae solution, mixing uniformly, and placing into a homogenizing crusher for cell crushing.

5. And (3) carrying out ultrasonic extraction on the crushed mixed algae liquid by using an ultrasonic instrument for 2s and 5s at an ultrasonic interval for 120min, and standing for 20 min.

6. Centrifuging the mixed algae solution after ultrasonic treatment at 3000rpm for 40min by a disc centrifuge, collecting supernatant, and removing precipitate.

7. To the collected supernatant, ammonium sulfate solid (added amount: 15g/L) and polypropylene glycol (added amount: 5mL/L) were added, and the mixture was stirred at 1000rpm for 1 hour.

8. And (3) centrifuging the stirred feed liquid at a high rotation speed of 5000rpm for 30min by using a disc centrifuge, and collecting the precipitate.

9. And (5) freeze-drying the precipitate to obtain astaxanthin freeze-dried powder.

Detection shows that the yield of the astaxanthin dry powder after freeze-drying is 93 percent, and the purity is 99.2 percent.

Comparative example 1

The extractant used in this comparative example was replaced with the mixed extract in step (2) of example 1, and the other operation steps were the same.

TABLE 1

Extracting agent	Yield (%)	Purity (%)
			Methanol	65	73
Ethanol	68	76
			Acetone (II)	60	70
Ethyl acetate	40	56
			Chloroform	48	65
N-hexane	75	78
			Butane	75	80
Dichloromethane-methanol (v: v ═ 1:3)	72	85
			Methanol-acetone (v: v ═ 1:1)	69	73
N-butanol-acetone (v: v ═ 5:1)	72	70
			Ethyl acetate-ethanol (v: v ═ 1:1)	58	61
Ethanol-ethyl acetate (v: v ═ 1:6)	62	65
			Methanol-petroleum ether-dichloromethane (v: v ═ 5:13:5)	67	79
Dichloromethane-n-hexane (v: v ═ 3:1)	57	66
			N-hexane-ethanol (v: v ═ 1:1)	62	80
N-hexane-ethyl acetate (v: v ═ 1:1)	70	65

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.