阅读说明：本技术 一种低分量海参活性肽的提取方法 (Method for extracting low-component sea cucumber bioactive peptide ) 是由 鞠文明 孙永军 王淑倩 吴中英 李俊峰 胡炜 邱美茹 王鹏程 彭莉晶 吴学记 刘 于 2020-06-02 设计创作，主要内容包括：本发明公开了一种低分量海参活性肽的提取方法,其经过以下步骤：a.原料处理；b.原料脱色；c.酶解；d.多肽分子分级分离和纯化；e.干燥。本发明利用超临界技术脱除色素和脂肪,通过切向流过滤技术分级分离活性肽并纯化,经低温超微粉碎耦合真空干燥获得低分子量海参活性肽。本发明可以快速的对海参活性肽进行提取,提取的海参活性肽纯度高,分子量低且均一性好,生物活性高,易于被人体消化吸收,并且原料利用率高,节约资源,提取耗时较短,制作方法简单。(The invention discloses a method for extracting low-component sea cucumber bioactive peptide, which comprises the following steps: a. treating raw materials; b. decoloring raw materials; c. carrying out enzymolysis; d. separating and purifying polypeptide molecules; e. and (5) drying. The invention removes pigment and fat by using a supercritical technology, fractionally separates and purifies active peptide by using a tangential flow filtration technology, and obtains the low molecular weight sea cucumber active peptide by low-temperature ultramicro-crushing coupled vacuum drying. The method can quickly extract the sea cucumber bioactive peptide, the extracted sea cucumber bioactive peptide has high purity, low molecular weight, good uniformity, high biological activity, easy digestion and absorption by human bodies, high raw material utilization rate, resource saving, short extraction time and simple preparation method.)

1. A method for extracting low-component sea cucumber bioactive peptide is characterized by comprising the following steps: which comprises the following steps:

a. raw material treatment: selecting sea cucumbers, and crushing the sea cucumbers into strips;

b. raw material decoloration: carrying out supercritical extraction technology decoloration treatment on strip sea cucumbers;

c. enzymolysis: preparing a hydrolase solution, mixing the hydrolase solution with the decolorized sea cucumber, and carrying out enzymolysis under certain enzymolysis conditions;

d. polypeptide molecule fractionation and purification: performing polypeptide molecule fractionation and purification by adopting tangential flow ultrafiltration;

e. and (3) drying: the low-component sea cucumber bioactive peptide is prepared by adopting low-temperature superfine grinding coupled with vacuum drying.

2. The method for extracting the low-component sea cucumber bioactive peptide according to claim 1, wherein the method comprises the following steps: in the step b, the supercritical decolorizing conditions are as follows: the amount of ethanol as carrying agent is 50-60ml/500g sea cucumber raw material, the pressure is 22-25MPa, the temperature is 45-50 ℃, and the flow rate of carbon dioxide is 12-15 kg/h.

3. The method for extracting the low-component sea cucumber bioactive peptide according to claim 1, wherein the method comprises the following steps: in the step c, the hydrolase comprises alkaline protease, compound protease, flavourzyme, neutral protease and papain.

4. The method for extracting the low-component sea cucumber bioactive peptide according to claim 1, wherein the method comprises the following steps: in the step c, the enzymolysis conditions are as follows: adjusting the pH value to 7.5-9, wherein the weight ratio of the enzyme to the sea cucumber is 1: adding 150-200 alkali protease, carrying out enzymolysis for 2-4 hours at 55-62 ℃, inactivating enzyme in a reaction solution after enzymolysis in a water bath at 105 ℃ for 15 minutes, and then adjusting the pH value to 5.5-6.0; then, according to the weight ratio of the enzyme to the mixed solution of 1: 45-60, adding a compound enzyme consisting of compound protease, flavourzyme and neutral protease, carrying out enzymolysis for 2-3 hours at 45-58 ℃, inactivating enzyme in a reaction solution after enzymolysis in a water bath at 105 ℃ for 15 minutes, and finally mixing the enzyme and the mixed solution in a weight ratio of 1: adding papain to 60-80, carrying out enzymolysis for 1-3 hours at 52-55 ℃, and inactivating enzyme in a water bath at 105 ℃ for 15 minutes.

5. The method for extracting the low-component sea cucumber bioactive peptide according to claim 1, wherein the method comprises the following steps: in the step d, the tangential flow ultrafiltration adopts membranes respectively intercepting the average molecular mass of 10000u, 3000u and 1000 u.

6. The method for extracting the low-component sea cucumber bioactive peptide according to claim 1, wherein the method comprises the following steps: in the step e, the low-temperature superfine grinding coupling vacuum drying is carried out at the working temperature of the low-temperature superfine grinder ranging from-20 ℃ to-15 ℃, and the vacuum degree of a cavity of the superfine grinder ranges from 70Pa to 90 Pa.

7. The method for extracting the low-component sea cucumber bioactive peptide according to claim 4, wherein the method comprises the following steps: the compound enzyme composed of the compound protease, the flavourzyme and the neutral protease has the mass fraction of the compound protease, the flavourzyme and the neutral protease being 1:1-3: 1-2.

Technical Field

The invention relates to extraction and preparation of peptides, in particular to a method for extracting low-component sea cucumber bioactive peptides.

Background

Sea cucumbers are distributed in all oceans in the world, the most varieties are in the Indian-Western Pacific area, and most of the edible sea cucumbers are distributed in tropical coral reefs. Only one edible sea cucumber, namely stichopus japonicus, is produced in the north of China, and more than ten edible sea cucumbers are produced in the south of the sea and the west sand islands. Most of the edible sea cucumbers live in intertidal zones or shallow seas. Sea cucumbers are narrow-salt animals and are rare in brackish or low-salt seawater. The sea cucumber culture medium is sensitive to water pollution, and the sea cucumber is difficult to survive in polluted seawater.

The polypeptide is a compound formed by connecting α -amino acids together by peptide bonds, is an intermediate product of protein hydrolysis, is a compound formed by dehydrating and condensing two or more amino acid molecules, is generally a compound formed by dehydrating and condensing 10-100 amino acid molecules, is called polypeptide, has promotion effects on growth, development, immunoregulation and metabolism of a human body, but the low-molecular-weight sea cucumber peptide is easier to absorb and has the most obvious activity.

Disclosure of Invention

In order to overcome the defects of low extraction efficiency and complex operation in the existing preparation of the low-molecular-weight sea cucumber peptide, the invention aims to provide the extraction method of the low-component sea cucumber bioactive peptide, which has high extraction efficiency and simple and convenient operation. Aiming at sea cucumber resources, the invention removes pigment and fat by using a supercritical technology, fractionally separates and purifies active peptide by using a tangential flow filtration technology, and obtains the low molecular weight sea cucumber active peptide by low-temperature ultramicro crushing and vacuum drying.

The technical scheme adopted by the invention for solving the technical problems is as follows: a method for extracting low-component sea cucumber bioactive peptide is characterized by comprising the following steps: the method comprises the following process steps:

a. raw material treatment: selecting sea cucumbers, and crushing the sea cucumbers into strips;

b. raw material decoloration: carrying out supercritical extraction technology decoloration treatment on strip sea cucumbers;

c. enzymolysis: preparing a hydrolase solution, mixing the hydrolase solution with the decolorized sea cucumber, and carrying out enzymolysis under certain enzymolysis conditions;

d. polypeptide molecule fractionation and purification: performing polypeptide molecule fractionation and purification by adopting tangential flow ultrafiltration;

e. and (3) drying: the low-component sea cucumber bioactive peptide is prepared by adopting low-temperature superfine grinding coupled with vacuum drying.

Preferably, in step b, the supercritical decolorizing conditions are as follows: the amount of ethanol as carrying agent is 50-60ml/500g sea cucumber raw material, the pressure is 22-25MPa, the temperature is 45-50 ℃, and the flow rate of carbon dioxide is 12-15 kg/h.

Preferably, in step c, the hydrolase includes alkaline protease, complex protease, flavourzyme, neutral protease and papain.

Preferably, in step c, the enzymolysis conditions are as follows: adjusting the pH value to 7.5-9, wherein the weight ratio of the enzyme to the sea cucumber is 1: adding 150-200 alkali protease, carrying out enzymolysis for 2-4 hours at 55-62 ℃, inactivating enzyme in a reaction solution after enzymolysis in a water bath at 105 ℃ for 15 minutes, and then adjusting the pH value to 5.5-6.0; then, according to the weight ratio of the enzyme to the mixed solution of 1: 45-60, adding a compound enzyme consisting of compound protease, flavourzyme and neutral protease, carrying out enzymolysis for 2-3 hours at 45-58 ℃, inactivating enzyme in a reaction solution after enzymolysis in a water bath at 105 ℃ for 15 minutes, and finally mixing the enzyme and the mixed solution in a weight ratio of 1: adding papain to 60-80, carrying out enzymolysis for 1-3 hours at 52-55 ℃, and inactivating enzyme in a water bath at 105 ℃ for 15 minutes.

Preferably, in step d, the tangential flow ultrafiltration is performed using membranes having a cut-off of mean molecular mass of 10000u, 3000u and 1000u, respectively.

Preferably, in the step e, the low-temperature ultrafine grinding and vacuum drying are carried out at the working temperature of-20 to-15 ℃ in the low-temperature ultrafine grinder, and the vacuum degree of the cavity of the ultrafine grinder is 70 to 90 Pa.

As a preferable method, the compound enzyme composed of the compound protease, the flavourzyme and the neutral protease has the ratio of 1:1-3:1-2 according to the mass fraction of the compound protease, the flavourzyme and the neutral protease.

The invention has the beneficial effects that:

(1) the supercritical technology is used for preprocessing the sea cucumber raw material to remove pigment, so that the purity of the product is improved, and most importantly, the supercritical technology is a nondestructive processing technology, so that the quality of the raw material is maintained;

(2) the multi-enzyme compound enzymolysis method utilizes the sea cucumber raw material to the maximum extent, ensures the effectiveness of enzymolysis products, and obtains polypeptide molecules with higher biological activity;

(3) the tangential flow ultrafiltration can be divided into different molecular weight grades according to the property and the requirement of the active peptide, which is beneficial to the fractional utilization of the active peptide, and more importantly, the peptide with a specific function can be positioned, thereby reducing the waste of peptide resources;

(4) the method is suitable for low-temperature treatment process, ensures the biological activity of the product, treats ice into micron-sized raw materials at low temperature by utilizing ultramicro crushing, realizes instant drying through vacuum gasification and achieves the effect of vacuum freeze drying. The method has the advantages of higher extraction efficiency, higher yield and simple and convenient operation, and is suitable for industrial production.

Detailed Description

The invention is described in detail below with reference to specific embodiments, which are intended to facilitate the understanding and implementation of the invention and are not intended to limit the invention. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.