阅读说明：本技术 一种富虾青素雨生红球藻油的制备方法 (Preparation method of astaxanthin-rich haematococcus pluvialis oil ) 是由 周庆新 陈芳甜 谷彩霞 王岳琦 胡莘琳 于 2021-08-13 设计创作，主要内容包括：本发明涉及一种富虾青素雨生红球藻油的制备方法,属于化合物提取的技术领域。本发明包括以下步骤：(1)原料预处理；(2)制备酶解体系；(3)酶解处理；(4)富集过程。本发明采用酶解协同异相纯化的方法,高效的实现了雨生红球藻油中虾青素的富集,本发明最终制备得到的产品中总虾青素类物质的含量达40％以上,较好的满足了产品的应用过程中的剂量需求。(The invention relates to a preparation method of astaxanthin-rich haematococcus pluvialis oil, belonging to the technical field of compound extraction. The invention comprises the following steps: (1) pretreating raw materials; (2) preparing an enzymolysis system; (3) carrying out enzymolysis treatment; (4) and (5) an enrichment process. The method adopts the method of enzymolysis, synergetic heterogeneous purification and high-efficiency enrichment of astaxanthin in haematococcus pluvialis oil, the content of total astaxanthin substances in the finally prepared product reaches more than 40 percent, and the dosage requirement in the application process of the product is better met.)

1. The preparation method of the astaxanthin-rich haematococcus pluvialis oil is characterized by comprising the following steps: (1) pretreating raw materials; (2) preparing an enzymolysis system; (3) carrying out enzymolysis treatment; (4) and (5) an enrichment process.

2. The preparation method according to claim 1, comprising the following specific steps:

(1) feedstock and pretreatment

Taking haematococcus pluvialis oil as a raw material, adding absolute ethyl alcohol into the haematococcus pluvialis oil to enable the amount of the ethyl alcohol to be 0-30% of the mass of the haematococcus pluvialis oil, and fully stirring and uniformly mixing to obtain a haematococcus pluvialis oil pretreatment solution;

(2) preparation of an enzymatic System

Dispersing lipase and antioxidant in deionized water and/or buffer solution, and then adding the deionized water and/or buffer solution system containing the lipase and the antioxidant into the haematococcus pluvialis pretreatment solution obtained in the step (1) to obtain a haematococcus pluvialis oil enzymolysis system;

(3) enzymolysis treatment

Carrying out enzymolysis treatment on the haematococcus pluvialis oil enzymolysis system obtained in the step (2) under the condition of 35-55 ℃ water bath, wherein the enzymolysis time is 1-8 h, continuous or uninterrupted shearing emulsification is carried out in the process, the condition of the shearing emulsification is 3000-20000 rpm, nitrogen is continuously introduced in the process, and after the enzymolysis is finished, haematococcus pluvialis oil enzymolysis treatment liquid is obtained;

(4) enrichment Process

Dissolving a certain amount of urea in an ethanol solution, and heating and refluxing the urea solution to fully dissolve the urea to obtain a urea ethanol solution; rapidly mixing the haematococcus pluvialis oil enzymolysis treatment liquid obtained in the step (3) with a urea ethanol solution to ensure that the proportion of the haematococcus pluvialis oil to the urea in the total system is 1: 0.5-10, fully stirring and uniformly mixing, and then standing for 2-12 hours in an environment at the temperature of-15-25 ℃; after standing, rapidly filtering and/or centrifuging the system, collecting clear liquid, adding deionized water which is 1-10 times of the weight of haematococcus pluvialis oil into the clear liquid, adjusting the pH value of the system to 6.0-7.0, stirring and mixing, standing and layering, and collecting an organic oil phase; and adding an ethyl acetate solution with the volume 1-3 times that of the haematococcus pluvialis oil into the collected organic oil phase, uniformly mixing, washing the organic phase with deionized water for 1-3 times, and carrying out reduced pressure concentration on the organic phase to obtain the astaxanthin-rich haematococcus pluvialis oil.

3. The method according to claim 1, wherein the lipase in the step (2) is lipase powder, and the lipase powder is used as it is or after being immobilized.

4. The preparation method according to claim 1, wherein the lipase powder used in the step (2) accounts for 1-5% of the total enzymolysis system by mass.

5. The method according to claim 1, wherein the antioxidant in the step (2) is one or more selected from the group consisting of tea polyphenols, ascorbic acid, sodium edetate, erythorbic acid and its salt, and phytic acid.

6. The method according to claim 1, wherein the antioxidant is used in an amount of 0.005 to 1% by mass based on the total amount of the enzymatic hydrolysis system in the step (2).

7. The method according to claim 1, wherein the amount of deionized water and/or buffer used in step (2) is 30 to 80% of the total enzymatic hydrolysis system.

8. The preparation method according to claim 1, wherein in the urea ethanol solution in the step (4), the mass volume fraction of urea in the ethanol solution is 7-14%, and the mass volume fraction of the ethanol solution is not less than 90% of the ethanol aqueous solution.

Technical Field

The invention belongs to the technical field of compound extraction, and particularly relates to a preparation method of astaxanthin-rich haematococcus pluvialis oil.

Background

Astaxanthin (Astaxanthin, 3,3 '-dihydroxy-4, 4-diketo-beta, beta' -carotene), a red ketocarotenoid, is found in some aquatic animals. Astaxanthin is reported to be more than 10 times more antioxidant than beta-carotene and more than 500 times more antioxidant than vitamin E, and is called "super vitamin E". The astaxanthin can be used as a colorant for valuable aquatic products and poultry cultivation to increase the additional commercial value, and in addition, the astaxanthin can be selectively accumulated by a human body, so that the astaxanthin has the effects of resisting oxidation, aging and tumors, preventing cardiovascular and cerebrovascular diseases and the like. And thus has received much attention in recent years.

Astaxanthin derived from natural algae and its extract are widely used in developed countries such as Europe and America, Japan, and southeast Asia, and the people in China who know astaxanthin are few, and China is currently known only by a few people in the scientific and fashionable beauty circles. Astaxanthin (Astaxanthin), a natural carotenoid component, has the potential to be novel (anti-oxidant/anti-inflammatory agents) and is expected to lift the surge of third prophylactic drugs after statins and antiplatelet drugs, as reported by the harvard investigator Preston. China is a large country for astaxanthin raw material resources, most of the astaxanthin raw materials and primary products are exported at present, and the resource advantages are not converted into product advantages and technical advantages. Particularly, the content of astaxanthin in haematococcus pluvialis oil in the current market is only 5-10 percent mostly, so that the application of the haematococcus pluvialis oil in high-end products is severely limited. Therefore, the development of a technology for enriching astaxanthin from Haematococcus pluvialis oil, which is suitable for industrial production, is urgently needed.

Disclosure of Invention

Aiming at the problem of low astaxanthin content in haematococcus pluvialis oil in the current market, the invention provides a preparation method of the astaxanthin-rich haematococcus pluvialis oil, and the preparation method is used for solving the problem.

The technical scheme of the invention is as follows: a preparation method of astaxanthin-rich haematococcus pluvialis oil comprises the following steps: (1) pretreating raw materials; (2) preparing an enzymolysis system; (3) carrying out enzymolysis treatment; (4) and (5) an enrichment process.

The preparation method comprises the following specific steps:

(1) feedstock and pretreatment

The haematococcus pluvialis oil pretreatment solution is prepared by taking haematococcus pluvialis oil as a raw material, adding absolute ethyl alcohol into the haematococcus pluvialis oil, enabling the amount of the ethyl alcohol to be 0-30% of the mass of the haematococcus pluvialis oil, and fully stirring and uniformly mixing.

(2) Preparation of an enzymatic System

And (2) dispersing lipase and antioxidant in deionized water and/or buffer solution, and then adding the deionized water and/or buffer solution system containing the lipase and the antioxidant into the haematococcus pluvialis pretreatment solution obtained in the step (1) to obtain the haematococcus pluvialis oil enzymolysis system.

(3) Enzymolysis treatment

And (3) carrying out enzymolysis treatment on the haematococcus pluvialis oil enzymolysis system obtained in the step 2) under the condition of 35-55 ℃ water bath, wherein the enzymolysis time is 1-8 h, continuous or uninterrupted shearing emulsification is carried out in the process, the shearing emulsification condition is 3000-20000 rpm, nitrogen is uninterruptedly introduced in the process, and the haematococcus pluvialis oil enzymolysis treatment liquid is obtained after the enzymolysis is finished.

(4) Enrichment Process

Dissolving a certain amount of urea in an ethanol solution, and heating and refluxing the urea solution to fully dissolve the urea to obtain a urea ethanol solution; rapidly mixing the haematococcus pluvialis oil enzymolysis treatment liquid obtained in the step (3) with a urea ethanol solution to ensure that the proportion of the haematococcus pluvialis oil to the urea in the total system is 1: 0.5-10, fully stirring and uniformly mixing, and then standing for 2-12 hours in an environment at the temperature of-15-25 ℃; after standing, rapidly filtering and/or centrifuging the system, collecting clear liquid, adding deionized water which is 1-10 times of the weight of haematococcus pluvialis oil into the clear liquid, adjusting the pH value of the system to 6.0-7.0, stirring and mixing, standing and layering, and collecting an organic oil phase; and adding an ethyl acetate solution with the volume 1-3 times that of the haematococcus pluvialis oil into the collected organic oil phase, uniformly mixing, washing the organic phase with deionized water for 1-3 times, and carrying out reduced pressure concentration on the organic phase to obtain the astaxanthin-rich haematococcus pluvialis oil.

Preferably, the lipase in the step (2) is lipase powder, and the lipase powder is directly used or used after being immobilized.

Preferably, the amount of the lipase powder in the step (2) accounts for 1-5% of the total enzymolysis system by mass.

Preferably, the antioxidant in step (2) is one or more of tea polyphenol, ascorbic acid, sodium edetate, isoascorbic acid and salts thereof, and phytic acid.

Preferably, the amount of the antioxidant in the step (2) is 0.005-1% by mass of the total enzymolysis system.

Preferably, the amount of the deionized water and/or the buffer solution in the step (2) accounts for 30-80% of the total enzymolysis system.

Preferably, in the urea ethanol solution in the step (4), the mass volume fraction of urea in the ethanol solution is 7-14%, and the mass volume fraction of the ethanol solution is not less than 90%.

The invention has the beneficial effects that:

(1) the method for purifying astaxanthin in haematococcus pluvialis oil through enzymolysis synergistic heterogeneous phase is adopted, the astaxanthin enrichment in the haematococcus pluvialis oil is efficiently realized, the content of total astaxanthin substances in the finally prepared product reaches more than 40%, and the dosage requirement in the application process of the product is well met.

(2) The method uses the combined action of the antioxidant and the nitrogen in the preparation process, and avoids the generation of byproducts in the enzymolysis and enrichment processes of the astaxanthin.

(3) The preparation method is simple and easy to operate, low in cost, mild in condition, free of chemical change in the process, free of change in the physiological activity of the astaxanthin and suitable for large-scale industrial popularization. The astaxanthin-rich haematococcus pluvialis oil obtained by the method can be widely applied to the industries of chemical products, daily chemical products, medicines, foods, health-care products and the like, and has a wide market prospect.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a process flow diagram of the present invention.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment 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.

Example 1

A preparation method of astaxanthin-rich haematococcus pluvialis oil comprises the following specific preparation steps:

(1) feedstock and pretreatment

Taking 10g haematococcus pluvialis oil, adding 3g of absolute ethyl alcohol into the haematococcus pluvialis oil, and fully stirring and uniformly mixing to obtain the haematococcus pluvialis oil pretreatment solution.

(2) Preparation of an enzymatic System

Dispersing 4g of lipase powder and 0.8g of ascorbic acid in 65g of deionized water, and then adding a deionized water solution containing lipase and ascorbic acid into the haematococcus pluvialis pretreatment solution obtained in the step (1) to obtain the haematococcus pluvialis oil enzymolysis system.

(3) Enzymolysis treatment

And (3) carrying out enzymolysis treatment on the haematococcus pluvialis oil enzymolysis system obtained in the step (2) under the water bath condition of 45 ℃, wherein the enzymolysis time is 5h, continuous or uninterrupted shearing emulsification is carried out in the process, the shearing emulsification condition is 8000rpm, nitrogen is continuously introduced in the process, and the haematococcus pluvialis oil enzymolysis treatment solution is obtained after the enzymolysis is finished.

(4) Enrichment Process

Dissolving 10g of urea in 90ml of 95% ethanol solution, and heating and refluxing to fully dissolve the urea to obtain urea ethanol solution; rapidly mixing the haematococcus pluvialis oil enzymolysis treatment liquid obtained in the step (3) with a urea ethanol solution, fully stirring and uniformly mixing, and standing for 5 hours at the temperature of 0 ℃; after standing, rapidly filtering the system, collecting clear liquid, adding 50ml of deionized water into the clear liquid, adjusting the pH value of the system to 6-7, stirring and mixing, standing for layering, and collecting an organic oil phase; adding 30ml of ethyl acetate into the collected organic oil phase, uniformly mixing, washing the organic phase for 3 times by using 30ml of deionized water multiplied by 3, and concentrating the organic phase under reduced pressure to obtain 5.76g of astaxanthin-rich haematococcus pluvialis oil with the yield of 57.6%.

Example 2

A preparation method of astaxanthin-rich haematococcus pluvialis oil comprises the following specific preparation steps:

(1) feedstock and pretreatment

Taking 10g haematococcus pluvialis oil, adding 2g of absolute ethyl alcohol into the haematococcus pluvialis oil, and fully stirring and uniformly mixing to obtain the haematococcus pluvialis oil pretreatment solution.

(2) Preparation of an enzymatic System

3g of lipase powder and 0.5g of sodium ethylene diamine tetracetate are dispersed in 60g of deionized water, and then the deionized water solution containing the lipase and the sodium ethylene diamine tetracetate is added into the haematococcus pluvialis pretreatment solution obtained in the step (1), so that the haematococcus pluvialis oil enzymolysis system is obtained.

(3) Enzymolysis treatment

And (3) carrying out enzymolysis treatment on the haematococcus pluvialis oil enzymolysis system obtained in the step (2) under the condition of 50 ℃ water bath, wherein the enzymolysis time is 7h, continuous or uninterrupted shearing and emulsification are carried out in the process, the shearing and emulsification condition is 10000rpm, nitrogen is continuously introduced in the process, and the haematococcus pluvialis oil enzymolysis treatment liquid is obtained after the enzymolysis is finished.

(4) Enrichment Process

Dissolving 10g of urea in 90ml of 95% ethanol solution, and heating and refluxing to fully dissolve the urea to obtain urea ethanol solution; rapidly mixing the haematococcus pluvialis oil enzymolysis treatment liquid obtained in the step (3) with a urea ethanol solution, fully stirring and uniformly mixing, and standing for 5 hours at the temperature of 0 ℃; after standing, rapidly filtering the system, collecting clear liquid, adding 50ml of deionized water into the clear liquid, adjusting the pH value of the system to 6-7, stirring and mixing, standing for layering, and collecting an organic oil phase; adding 30ml of ethyl acetate into the collected organic oil phase, uniformly mixing, washing the organic phase for 3 times by using 30ml of deionized water multiplied by 3, and concentrating the organic phase under reduced pressure to obtain 5.23g of astaxanthin-rich haematococcus pluvialis oil with the yield of 52.3%.

Example 3

A preparation method of astaxanthin-rich haematococcus pluvialis oil comprises the following specific preparation steps:

(1) feedstock and pretreatment

Taking 10g haematococcus pluvialis oil, adding 3g of absolute ethyl alcohol into the haematococcus pluvialis oil, and fully stirring and uniformly mixing to obtain the haematococcus pluvialis oil pretreatment solution.

(2) Preparation of an enzymatic System

Dispersing 4g of lipase powder and 0.8g of sodium ethylene diamine tetracetate into 65g of deionized water, and then adding the deionized water solution containing the lipase and the sodium ethylene diamine tetracetate into the haematococcus pluvialis pretreatment solution obtained in the step (1) to obtain the haematococcus pluvialis oil enzymolysis system.

(3) Enzymolysis treatment

And (3) carrying out enzymolysis treatment on the haematococcus pluvialis oil enzymolysis system obtained in the step (2) under the water bath condition of 45 ℃, wherein the enzymolysis time is 5h, continuous or uninterrupted shearing emulsification is carried out in the process, the shearing emulsification condition is 8000rpm, nitrogen is continuously introduced in the process, and the haematococcus pluvialis oil enzymolysis treatment solution is obtained after the enzymolysis is finished.

(4) Enrichment Process

Dissolving 10g of urea in 90ml of 95% ethanol solution, and heating and refluxing to fully dissolve the urea to obtain urea ethanol solution; rapidly mixing the haematococcus pluvialis oil enzymolysis treatment liquid obtained in the step (3) with a urea ethanol solution, fully stirring and uniformly mixing, and standing for 5 hours at the temperature of 0 ℃; after standing, rapidly filtering the system, collecting clear liquid, adding 50ml of deionized water into the clear liquid, adjusting the pH value of the system to 6-7, stirring and mixing, standing for layering, and collecting an organic oil phase; adding 30ml of ethyl acetate into the collected organic oil phase, uniformly mixing, washing the organic phase for 3 times by using 30ml of deionized water multiplied by 3, and concentrating the organic phase under reduced pressure to obtain 5.52g of astaxanthin-rich haematococcus pluvialis oil with the yield of 55.2%.

The astaxanthin-rich haematococcus pluvialis oil prepared in the examples 1-3 is subjected to content detection, and the specific detection results are shown in the following table 1:

TABLE 1 results of the measurements

As can be seen from the detection results in Table 1, the haematococcus pluvialis oil with the total astaxanthin content of more than 40% can be obtained by the preparation method of the invention, and is obviously superior to the commercial products.

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.