阅读说明：本技术 一种南极磷虾蛋白的提取方法 (Extraction method of euphausia superba protein ) 是由 欧阳小琨 卢雨清 杨立业 范丽红 于 2019-09-04 设计创作，主要内容包括：本发明涉及蛋白提取技术领域,公开了一种南极磷虾蛋白的提取方法。包括以下步骤：1)将南极磷虾冷冻干燥,粉碎过筛,加入蒸馏水中混合匀浆得浆液；2)向浆液中加入氢氧化钠溶液调节pH至9-10,再加入蛋白酶进行酶解得酶解液；3)将酶解液进行超声处理后进行蛋白酶灭活,离心获取蛋白上清液；4)向蛋白上清液中加入盐酸溶液进行酸沉,然后加入海泡石进行吸附处理；再加入氢氧化钠溶液调节pH至10-13,离心,取上清液；5)向上清液中加入盐酸溶液进行二次酸沉,过滤分离,洗涤,冷冻干燥,即得。本发明南极磷虾蛋白提取过程中加入海泡石对磷虾蛋白进行脱腥处理,且海泡石不会吸附磷虾蛋白,提高磷虾蛋白的提取率。(The invention relates to the technical field of protein extraction, and discloses an extraction method of euphausia superba protein. The method comprises the following steps: 1) freeze-drying Euphausia superba, pulverizing, sieving, adding distilled water, mixing and homogenizing to obtain a slurry; 2) adding sodium hydroxide solution into the slurry to adjust the pH to 9-10, and then adding protease for enzymolysis to obtain an enzymolysis solution; 3) carrying out ultrasonic treatment on the enzymolysis liquid, then carrying out protease inactivation, and centrifuging to obtain a protein supernatant; 4) adding a hydrochloric acid solution into the protein supernatant for acid precipitation, and then adding sepiolite for adsorption treatment; adding sodium hydroxide solution to adjust pH to 10-13, centrifuging, and collecting supernatant; 5) adding hydrochloric acid solution into the supernatant, performing secondary acid precipitation, filtering, separating, washing, and freeze drying. In the extraction process of the euphausia superba protein, the sepiolite is added to carry out deodorization treatment on the euphausia superba protein, and the sepiolite cannot adsorb the euphausia superba protein, so that the extraction rate of the euphausia superba protein is improved.)

1. The extraction method of the euphausia superba protein is characterized by comprising the following steps:

1) freeze-drying Euphausia superba, pulverizing, sieving, adding distilled water, mixing and homogenizing to obtain a slurry;

2) adding sodium hydroxide solution into the slurry to adjust the pH to 9-10, and then adding protease for enzymolysis to obtain an enzymolysis solution;

3) carrying out ultrasonic treatment on the enzymolysis liquid, then carrying out protease inactivation, and centrifuging to obtain a protein supernatant;

4) adding a hydrochloric acid solution into the protein supernatant for acid precipitation, and then adding sepiolite for adsorption treatment; adding sodium hydroxide solution to adjust pH to 10-13, centrifuging, and collecting supernatant;

5) adding hydrochloric acid solution into the supernatant, performing secondary acid precipitation, filtering, separating, washing, and freeze drying.

2. The extraction method for Euphausia superba protein according to claim 1, wherein the concentration of the sodium hydroxide solution is 5-10% by mass.

3. The extraction method for Euphausia superba protein according to claim 1, wherein the concentration of the hydrochloric acid solution is 10-15% by mass.

4. The extraction method of euphausia superba protein according to claim 1, wherein the pH of the acid precipitation step in step 4) is adjusted to 4-5 by adding hydrochloric acid solution.

5. The extraction method for Euphausia superba protein according to claim 1, wherein sepiolite is added in an amount of 2-6wt% of the supernatant in step 4).

6. The extraction method of euphausia superba protein according to claim 1, wherein the pH of the solution obtained by the second acid precipitation step in step 5) is adjusted to 4-5 by adding hydrochloric acid solution.

7. The extraction method of euphausia superba protein according to claim 1, wherein the sepiolite is subjected to modification treatment, and the modification treatment method comprises the following steps:

adding sepiolite into an ethanol water solution, stirring and dispersing uniformly to obtain a suspension, adding a silane coupling agent into the suspension, heating in a water bath to 40-50 ℃, stirring and reacting for 1-2h, filtering, and drying to obtain alkylated sepiolite; adding the alkylated sepiolite into a 0.5-3wt% polyethyleneimine water solution, heating to 60-65 ℃, and reacting for 3-5h under heat preservation to obtain the final product.

8. The extraction method for Euphausia superba protein according to claim 7, wherein the mass-to-volume ratio of the alkylated sepiolite to the polyethyleneimine water solution is 1-3g/50 mL.

Technical Field

The invention relates to the technical field of protein extraction, in particular to an extraction method of euphausia superba protein.

Background

Antarctic krill is the most abundant fishery resource on the earth and needs to be developed and utilized urgently, and can be used as a potential food source for human beings due to high nutritional value. As an abundant strategic resource, the development and utilization of the euphausia superba is an effective utilization of global resources, and has important strategic significance for China. As a polar marine organism with huge resource amount and high nutritional value, the protein content is high, the amino acid composition meets the high-quality protein standard specified by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) of the United nations, and the risk of cholesterol intake is reduced while sufficient nutrition is provided. The protein is a high-quality protein with good development and utilization potential, is widely concerned, and is expected to be developed into functional marine food. Chinese patent publication No. CN103583789 discloses an ultrasonic-assisted extraction method of euphausia superba protein isolate, which sequentially comprises alkaline leaching, ultrasonic extraction, centrifugation, secondary ultrasonic extraction, acid precipitation, alcohol washing and water washing; on the basis of extracting the protein isolate by alkali extraction and acid precipitation, an ultrasonic-assisted method is adopted to optimize the extraction process of the protein isolate from the Antarctic krill, but the obtained krill protein has more impurities and heavier fishy smell; chinese patent publication No. CN106798073 discloses a method for extracting protein from antarctic krill, which uses antarctic krill as a raw material, utilizes endogenous protease of the antarctic krill to carry out staged autolysis at different temperatures, enables the best effect of different endogenous proteases to be exerted to achieve the maximum separation of the protein in the antarctic krill, uses activated carbon to carry out decoloration and deodorization treatment on the krill protein, removes bitter amino acid and fishy trimethylamine nitrogen, but the adsorption effect of the activated carbon can adsorb the protein after the enzymolysis of the antarctic krill at the same time, so that the yield of the krill protein is reduced.

Disclosure of Invention

The invention aims to overcome the technical problems and provides an extraction method of euphausia superba protein.

In order to achieve the purpose, the invention adopts the following technical scheme: an extraction method of euphausia superba protein comprises the following steps:

1) freeze-drying Euphausia superba, pulverizing, sieving, adding distilled water, mixing and homogenizing to obtain a slurry;

2) adding sodium hydroxide solution into the slurry to adjust the pH to 9-10, and then adding protease for enzymolysis to obtain an enzymolysis solution;

3) carrying out ultrasonic treatment on the enzymolysis liquid, then carrying out protease inactivation, and centrifuging to obtain a protein supernatant;

4) adding a hydrochloric acid solution into the protein supernatant for acid precipitation, and then adding sepiolite for adsorption treatment; adding sodium hydroxide solution to adjust pH to 10-13, centrifuging, and collecting supernatant;

5) adding hydrochloric acid solution into the supernatant, performing secondary acid precipitation, filtering, separating, washing, and freeze drying.

The method comprises the steps of firstly carrying out freeze drying and crushing treatment on krill, adding distilled water to mix into slurry, and then carrying out enzymolysis on the krill slurry by using protease under an alkaline condition to obtain krill protein; krill protein is precipitated under acidic condition, then adsorbent is added for treatment, and the adsorbent is suspended and dispersed in the solution to adsorb bitter amino acid, fishy trimethylamine nitrogen and small particle impurities; and adjusting the pH value of the solution to be alkaline, dispersing the precipitated krill protein in the solution, performing centrifugal treatment to precipitate sepiolite suspended in the solution, collecting supernatant, and performing secondary acid precipitation to separate the krill protein. According to the method, the sepiolite is used for adsorbing and removing the fishy smell in the krill protein extraction process, so that the obtained krill protein is pure and has no fishy smell; the krill protein precipitates in the process of adsorbing and removing the fishy smell, and the sepiolite suspended and dispersed in the solution cannot adsorb the precipitated krill protein, so that the extraction amount of the krill protein is increased.

Preferably, the mass concentration of the sodium hydroxide solution is 5 to 10%.

Preferably, the mass concentration of the hydrochloric acid solution is 10-15%.

Preferably, the pH value is adjusted to 4-5 by adding hydrochloric acid solution in the step of adjusting acid precipitation in the step 4).

Preferably, the sepiolite is added in the step 4) in an amount of 2-6wt% of the supernatant.

Preferably, hydrochloric acid solution is added in the secondary acid precipitation step in the step 5) to adjust the pH value to 4-5.

Preferably, the sepiolite is subjected to modification treatment, and the modification treatment method comprises the following steps:

adding sepiolite into an ethanol water solution, stirring and dispersing uniformly to obtain a suspension, adding a silane coupling agent into the suspension, heating in a water bath to 40-50 ℃, stirring and reacting for 1-2h, filtering, and drying to obtain alkylated sepiolite; adding the alkylated sepiolite into a 0.5-3wt% polyethyleneimine water solution, heating to 60-65 ℃, and reacting for 3-5h under heat preservation to obtain the final product.

The invention is to improve the adsorption capacity of sepiolite to bitter amino acid and fishy trimethylamine nitrogen, modify sepiolite, graft silane coupling agent to sepiolite to alkylate sepiolite, then bond macromolecule polyethyleneimine and silane coupling agent, thus connect polyethyleneimine to sepiolite surface, polyethyleneimine has more amino in the molecule, the amino has adsorption to bitter amino acid and fishy trimethylamine nitrogen, the invention combines the physical adsorption of sepiolite with the chemical adsorption of amino, greatly improve the removal effect of fishy smell.

Preferably, the mass-volume ratio of the alkylated sepiolite to the polyethyleneimine water solution is 1-3g/50 mL.

Therefore, the invention has the following beneficial effects: (1) according to the method, the sepiolite is used for adsorbing and removing the fishy smell in the krill protein extraction process, so that the obtained krill protein is pure and has no fishy smell; the krill protein precipitate is adsorbed and removed in the fishy smell removing process, and the sepiolite suspended and dispersed in the solution cannot adsorb the precipitated krill protein, so that the extraction amount of the krill protein is increased; (2) the method comprises the steps of modifying the sepiolite, grafting a silane coupling agent onto the sepiolite to alkylate the sepiolite, and bonding macromolecular polyethyleneimine with the silane coupling agent, so that the polyethyleneimine is connected to the surface of the sepiolite, the polyethyleneimine molecules have more amino groups, and the amino groups have adsorption effects on bitter amino acids and fishy trimethylamine nitrogen.

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.