阅读说明：本技术 一种南极磷虾小分子肽的制备方法 (Preparation method of euphausia superba small-molecule peptide ) 是由 欧阳小琨 卢雨清 杨立业 范丽红 于 2019-09-16 设计创作，主要内容包括：本发明涉及生物工程技术领域,公开了一种南极磷虾小分子肽的制备方法。包括以下步骤：1)将南极磷虾肉洗净后绞碎得肉糜,将肉糜浸入乙酸乙酯浸泡脱脂,静置,过滤分离得脱脂磷虾肉糜；2)将脱脂磷虾肉糜加入水中均匀分散,加入去氟复合颗粒,过滤分离得脱氟肉糜；3)将脱氟肉糜加入水中均匀分散,加入胰蛋白酶,加热酶解的南极磷虾酶解液；4)将南极磷虾酶解液依次经过超滤、凝胶柱层析纯化得南极磷虾小分子肽。本发明制备得到的小分子肽中氟元素含量较低,不会对人身体健康造成影响。(The invention relates to the technical field of bioengineering, and discloses a preparation method of euphausia superba small molecular peptide. The method comprises the following steps: 1) cleaning and mincing Antarctic krill meat to obtain meat paste, soaking the meat paste in ethyl acetate for degreasing, standing, filtering and separating to obtain degreased krill meat paste; 2) adding the degreased krill meat paste into water for uniform dispersion, adding defluorinated composite particles, and filtering and separating to obtain defluorinated meat paste; 3) adding the defluorinated minced meat into water for uniform dispersion, adding trypsin, and heating the enzymatic hydrolysate of the euphausia superba for enzymolysis; 4) and sequentially carrying out ultrafiltration and gel column chromatography purification on the euphausia superba enzymatic hydrolysate to obtain the euphausia superba small molecular peptide. The small molecular peptide prepared by the invention has low fluorine content, and does not affect human health.)

1. A preparation method of euphausia superba small molecule peptide is characterized by comprising the following steps:

1) cleaning and mincing Antarctic krill meat to obtain meat paste, soaking the meat paste in ethyl acetate for degreasing, standing, filtering and separating to obtain degreased krill meat paste;

2) adding the degreased krill meat paste into water for uniform dispersion, adding defluorinated composite particles, and filtering and separating to obtain defluorinated meat paste;

3) adding the defluorinated minced meat into water for uniform dispersion, adding trypsin, and heating for enzymolysis to obtain an antarctic krill enzymolysis liquid;

4) and sequentially carrying out ultrafiltration and gel column chromatography purification on the euphausia superba enzymatic hydrolysate to obtain the euphausia superba small molecular peptide.

2. The preparation method of the euphausia superba small-molecule peptide according to claim 1, wherein the mass-to-volume ratio of the meat emulsion to the ethyl acetate in the step 1) is 2-5g/100 mL.

3. The method for preparing the euphausia superba small-molecule peptide according to claim 1, wherein the trypsin is added in the step 3) in an amount of 1-3% of the mass of the defluorinated meat emulsion.

4. The method for preparing the euphausia superba small-molecule peptide according to claim 1, wherein the enzymolysis temperature in the step 3) is 40-45 ℃ and the enzymolysis time is 5-8 h.

5. The preparation method of euphausia superba small-molecule peptide according to claim 1, wherein the preparation method of the defluorinated composite particle comprises the following steps:

adding carboxymethyl chitosan into water to prepare 1-5wt% carboxymethyl chitosan water solution; adding bentonite into carboxymethyl chitosan aqueous solution, ultrasonically stirring for 4-8h, filtering to separate out bentonite, adding bentonite into sodium trimetaphosphate aqueous solution, soaking for 1-3h, filtering, separating, and drying to obtain modified bentonite; adding the modified bentonite into an ethanol solution of lanthanum nitrate, stirring for reacting for 2-5h, filtering, washing and drying to obtain the modified bentonite.

6. The preparation method of the euphausia superba small-molecule peptide according to claim 5, wherein the mass-to-volume ratio of the bentonite to the carboxymethyl chitosan aqueous solution is 1-3g/80 mL.

7. The method for preparing the euphausia superba small-molecule peptide according to claim 5, wherein the mass concentration of the aqueous solution of sodium trimetaphosphate is 3-6%.

Technical Field

The invention relates to the technical field of bioengineering, in particular to a preparation method of euphausia superba small molecular peptide.

Background

With the continuous progress of fishery equipment and technology, a brand new type euphausia superba industry which is supported by an ecological efficient fishing technology, driven by products with high added values and integrated with marine fishing and shipborne processing is formed, but the fishing and processing technology is not mature in China due to late start, and the euphausia superba industry in China is still in the initial stage of development at present. Active peptides are a class of peptides having specific physiological activities. Active peptides have various molecular structures ranging from simple dipeptides to cyclic macromolecular polypeptides, and these polypeptides can be modified by phosphorylation, glycosylation or acylation. Active peptides can be roughly classified into physiologically active peptides (such as antihypertensive peptides, antibacterial peptides), flavor-improving peptides, antioxidant peptides, and nutrient peptides, etc., according to their functions. The Antarctic krill peptide is rich in various amino acids, active peptides, organic acids and the like, wherein the protein content of the Antarctic krill small molecular peptide reaches more than 80%, and the Antarctic krill small molecular peptide has the characteristics of small molecular weight, high water solubility and high absorption rate. However, the application of antarctic krill is limited by the high fluorine content of antarctic krill, so that the low-fluorine antarctic krill uniform liquid should be prepared by using the biological defluorinating agent. The euphausia superba small molecular peptide has small molecular weight and good water solubility, can be directly absorbed and utilized without passing through a digestive tract, and can also directly enter muscle cells through a cell barrier to provide a protein source for muscle proliferation. Gel chromatography, also known as gel filtration, is a chromatographic method for separating substances by molecular weight. Therefore, the euphausia superba small molecular peptide can be obtained by utilizing gel chromatography purification, and a foundation is laid for developing functional low-fluorine euphausia superba small molecular peptide.

Chinese patent publication No. CN109880871 discloses a method for extracting small molecule peptides by using euphausia superba powder as a raw material, which comprises the steps of extracting by hot water, inactivating enzyme hydrolase to obtain an enzyme shearing liquid, precipitating by calcium salt, filtering, passing through a composite tubular membrane, removing small molecule impurities and hyphae, passing through a nanofiltration membrane, concentrating under reduced pressure to a certain concentration, and then carrying out centrifugal spray drying to obtain the small molecule peptides. Because the krill contains more fluorine elements, the small fraction titanium prepared by the conventional method contains high-content fluorine, and is not beneficial to the health of human bodies.

Disclosure of Invention

The invention aims to overcome the technical problems and provides a preparation method of euphausia superba small molecular peptide. The small molecular peptide prepared by the invention has low fluorine content, and does not affect human health.

In order to achieve the purpose, the invention adopts the following technical scheme: a preparation method of euphausia superba small molecule peptide comprises the following steps:

1) cleaning and mincing Antarctic krill meat to obtain meat paste, soaking the meat paste in ethyl acetate for degreasing, standing, filtering and separating to obtain degreased krill meat paste;

2) adding the degreased krill meat paste into water for uniform dispersion, adding defluorinated composite particles, and filtering and separating to obtain defluorinated meat paste;

3) adding the defluorinated minced meat into water for uniform dispersion, adding trypsin, and heating the enzymatic hydrolysate of the euphausia superba for enzymolysis;

4) and sequentially carrying out ultrafiltration and gel column chromatography purification on the euphausia superba enzymatic hydrolysate to obtain the euphausia superba small molecular peptide.

According to the method, firstly, ethyl acetate is used for soaking the euphausia superba meat emulsion to remove lipid in the euphausia superba meat emulsion, then, fluorine-removing particles are added to the euphausia superba meat emulsion to remove fluorine ions and micromolecular impurities in the euphausia superba meat emulsion through a physical adsorption method, pure euphausia superba meat emulsion is obtained after adsorption treatment, the euphausia superba meat emulsion is subjected to enzymolysis treatment through trypsin, euphausia superba proteolysis is subjected to enzymolysis to micromolecular polytitanium, and pure euphausia superba micromolecular peptide is obtained after ultrafiltration and gel column chromatography purification in sequence. The invention aims to prevent small molecular peptides obtained by particle adsorption and enzymolysis from being removed and improve the yield of krill small molecular peptides by adsorbing and hydrolyzing krill meat paste before enzymolysis. According to the invention, fluorine elements in the euphausia superba are removed by using fluorine-removing particles, then the euphausia superba is subjected to enzymolysis by using trypsin to obtain small molecular peptides, and the small molecular peptides of the euphausia superba are further purified by using ultrafiltration and gel chromatography. The obtained antarctic krill active peptide has low fluorine content, and improves the safety and nutritive value of the product.

Preferably, the mass-to-volume ratio of the meat emulsion to the ethyl acetate in the step 1) is 2-5g/100 mL.

Preferably, the adding amount of the trypsin in the step 3) is 1-3% of the mass of the defluorinated meat paste.

Preferably, the enzymolysis temperature in the step 3) is 40-45 ℃, and the enzymolysis time is 5-8 h.

Preferably, the preparation method of the defluorinated composite particle comprises the following steps:

adding carboxymethyl chitosan into water to prepare 1-5wt% carboxymethyl chitosan water solution; adding bentonite into carboxymethyl chitosan aqueous solution, ultrasonically stirring for 4-8h, filtering to separate out bentonite, adding bentonite into sodium trimetaphosphate aqueous solution, soaking for 1-3h, filtering, separating, and drying to obtain modified bentonite; adding the modified bentonite into an ethanol solution of lanthanum nitrate, stirring for reacting for 2-5h, filtering, washing and drying to obtain the modified bentonite.

According to the method, fluorine ions are firstly adsorbed by utilizing the adsorption effect of bentonite, and then the lanthanum ions in the bentonite layer are used for fixing the fluorine, so that the desorption of the fluorine ions is prevented, and the fluorine content of the obtained krill protein is low. The preparation method of the defluorination composite particle comprises the steps of firstly immersing bentonite into an aqueous solution of carboxymethyl chitosan, dispersing carboxymethyl chitosan molecules in a layered structure of the bentonite through ultrasonic oscillation, adding a cross-linking agent, enabling the carboxymethyl chitosan molecules in a bentonite interlayer to be cross-linked through a suspension polymerization method to form a three-dimensional network polymer, adding the three-dimensional network polymer into an ethanol solution of lanthanum nitrate, combining active sites on the three-dimensional network polymer with lanthanum ions in the lanthanum nitrate, and thus fixing and dispersing the lanthanum ions in the three-dimensional network polymer, wherein the three-dimensional network polymer can increase the dispersion uniformity of the lanthanum ions among the bentonite, and further improve the fixation effect of the lanthanum on the fluoride ions.

Preferably, the mass-volume ratio of the bentonite to the carboxymethyl chitosan aqueous solution is 1-3g/80 mL.

Preferably, the mass concentration of the sodium trimetaphosphate aqueous solution is 3-6%.

Therefore, the invention has the following beneficial effects: (1) according to the invention, fluorine elements in the euphausia superba are removed by using fluorine-removing particles, then the euphausia superba is subjected to enzymolysis by using trypsin to obtain small molecular peptides, and the small molecular peptides of the euphausia superba are further purified by using ultrafiltration and gel chromatography, so that the obtained active peptide of the euphausia superba has low fluorine content, and the safety and the nutritional value of the product are improved; (2) according to the method, fluorine ions are firstly adsorbed by utilizing the adsorption effect of bentonite, and then the lanthanum ions in the bentonite layer are used for fixing the fluorine, so that the desorption of the fluorine ions is prevented, and the fluorine content of the obtained krill protein is low.

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.