阅读说明：本技术 一种低致敏性脱腥南极磷虾肽的制备方法 (Preparation method of low-sensitization fishy smell-removed Antarctic krill peptide ) 是由 于建伟 魏星 邹鹏飞 蒋一博 唐文金 卢伟超 杜可欣 于 2019-11-22 设计创作，主要内容包括：本发明涉及一种低致敏性脱腥南极磷虾肽的制备方法,按制备顺序依次包括酶解、超滤、闪蒸、膜分级；所述膜分级为使用半透膜装置将闪蒸获得的浓缩酶解液分离成多个分子量不同的组分,分别检测除分子量最小的组分之外的每个组分的过敏原含量,将过敏原含量最高的组分与分子量最小的组分抛弃,并将其他组分合并。本发明通过多层定向透析技术有效脱除南极磷虾肽中的致敏物质,同时采用超滤闪蒸串联的一步浓缩法,获得的南极磷虾肽液,具有低致敏性、无腥味等特点。本发明制造过程效率高,易于产业化,肽活性高,功效明显,同时使提取液可以广泛应用于营养品、护肤品、药品等领域。(The invention relates to a preparation method of low-sensitization fishy smell-removed Antarctic krill peptide, which sequentially comprises enzymolysis, ultrafiltration, flash evaporation and membrane classification according to the preparation sequence; the membrane fractionation is to separate the concentrated enzymatic hydrolysate obtained by flash evaporation into a plurality of components with different molecular weights by using a semi-permeable membrane device, detect the allergen content of each component except the component with the minimum molecular weight, discard the component with the highest allergen content and the component with the minimum molecular weight, and combine the other components. The method effectively removes the sensitizing substances in the Antarctic krill peptide by a multilayer directional dialysis technology, and simultaneously adopts a one-step concentration method of ultrafiltration flash evaporation series connection to obtain the Antarctic krill peptide liquid, which has the characteristics of low sensitization, no fishy smell and the like. The invention has high efficiency of the manufacturing process, easy industrialization, high peptide activity and obvious effect, and simultaneously, the extracting solution can be widely applied to the fields of nourishment, skin care products, medicines and the like.)

1. A preparation method of low-sensitization fishy smell-removed Antarctic krill peptide is characterized by sequentially comprising enzymolysis, ultrafiltration, flash evaporation and membrane classification according to a preparation sequence;

the membrane fractionation is to separate the concentrated enzymatic hydrolysate obtained by flash evaporation into a plurality of components with different molecular weights by using a semi-permeable membrane device, detect the allergen content of each component except the component with the minimum molecular weight, discard the component with the highest allergen content and the component with the minimum molecular weight, and collect and combine other components.

2. The method according to claim 1, wherein the concentrated enzymatic hydrolysate is separated into 3 to 5 fractions having different molecular weights by using a semi-permeable membrane device.

3. The method according to claim 2, wherein the concentrated enzymatic hydrolysate is separated into 5 fractions having different molecular weights by using 4 semipermeable membrane units, and the cut-off molecular weights of the 4 semipermeable membrane units are 800 to 2500Da,500 to 1800Da, 300 to 1200Da, and 100 to 800Da, respectively.

4. The method of claim 1, wherein the semipermeable membrane device is a dialysis bag.

5. The method of claim 4, wherein the semipermeable membrane device comprises a plurality of dialysis bags with different molecular weight cut-off from inside to outside.

6. The preparation method according to claim 5, wherein the dialysis bags are 4, and the cut-off molecular weights of the dialysis bags are 800-2500 Da, 500-1800 Da, 300-1200 Da and 100-800 Da from inside to outside in sequence; the dialysis temperature is 4-15 ℃, and the dialysis time is 12-48 h.

7. The preparation method according to claim 1, wherein the enzymatic hydrolysis is carried out under the following specific working conditions: crushing Antarctic krill, and mixing with water according to a mass ratio of 1: 2-1: 8, uniformly mixing, adjusting the pH value to 2.0-6.0, adding 1.0-6.0 wt% of pepsin based on the mass of a reaction system, performing enzymolysis for 0.5-2 h at the temperature of 30-50 ℃, heating to 85-95 ℃, and keeping for 5-20 min to inactivate enzyme; cooling the reaction system to below 30 ℃, centrifuging, collecting clear liquid after centrifugation, and removing residues to obtain crude enzymolysis liquid.

8. The preparation method according to claim 1, wherein the ultrafiltration is carried out under the following specific working conditions: and (3) introducing the crude enzymolysis liquid obtained after enzymolysis into an ultrafiltration device, wherein the membrane aperture is 10-5000 nm.

9. The preparation method according to claim 8, wherein the flux of the ultrafiltration device is 50-100L/h, and the operating pressure difference is 50-500 kPa.

10. The preparation method according to claim 1, wherein the flash evaporation is carried out under the following specific working conditions: and (3) carrying out flash evaporation on the enzymolysis liquid obtained by ultrafiltration for 10-100 s at the temperature of 100-200 ℃.

Technical Field

The invention particularly relates to a preparation method of low-sensitization fishy smell-removed Antarctic krill peptide.

Background

Euphausia superba (Eucheuma superba) belongs to the family Euphausiaceae, the phylum Arthropoda, and is widely distributed in Antarctic oceanic regions, and the resource storage capacity of Euphausia superba is huge and is estimated to be between 6.5 and 10 hundred million tons. Ecological scientists consider it a potential food source for human beings because of its large number and rich nutrition. Antarctic krill protein contains 8 essential amino acids for human body, and the content and score of the essential amino acids are higher than those of the ideal protein model recommended by FAO/WHO. Research shows that the Antarctic krill peptide has biological activities of resisting oxidation, lowering blood pressure, lowering cholesterol, resisting bacteria and the like, and has great application potential.

However, krill belongs to crustaceans, one of the eight major groups of sensitized foods proposed by the food and agriculture organization of the united nations. The clinical symptoms of the crustacean after allergy are skin reaction and digestive tract reaction, and serious allergy can be shock or even death. Enzymolysis is a common means for protein desensitization, part of allergens can be removed after krill protein is subjected to enzymolysis, still considerable allergic substances exist, the number of residual allergic substances is multiple, the system is complex, and the removal of the residual allergic substances is difficult through a common purification method. In addition, the fishy smell characteristic of marine peptides also affects their applications.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a preparation and purification method of low-sensitization deodorization Antarctic krill peptide, which has the advantages of simple process, easy operation, low cost and high desensitization efficiency.

The specific technical scheme is as follows:

a preparation method of low-sensitization deodorization Antarctic krill peptide comprises sequentially performing enzymolysis, ultrafiltration, flash evaporation and membrane classification according to a preparation sequence;

the membrane is classified into a plurality of components with different molecular weights by separating the concentrated enzymolysis liquid obtained by flash evaporation through a semi-permeable membrane device, respectively detecting the allergen content of each component except the component with the minimum molecular weight, discarding the component with the highest allergen content and the interval component with the minimum molecular weight, and collecting other components. The collected components can be used separately or combined.

The concentrated enzymolysis liquid obtained by flash evaporation is separated into a plurality of components with different molecular weights, namely the enzymolysis liquid is divided into a plurality of molecular weight regions by utilizing a semipermeable membrane.

The inventor unexpectedly finds that the anaphylactic substances have a certain degree of molecular weight aggregation in the enzymolysis products;

in addition, the small molecular components have more allergic substances, and the small molecular components are removed, so that the small molecular components also have the effects of desalting and removing free amino acids.

The detection method is a thiazole blue method, namely an MTT method for short, wherein thiazole blue can permeate cell membranes to enter cells, amber dehydrogenase in mitochondria of living cells can reduce exogenous MTT into blue-purple Formazan (Formazan) crystals which are difficult to dissolve in water and deposit the crystals in the cells, the crystals can be dissolved by acid isopropyl alcohol, an enzyme linked immunosorbent assay detector is used for measuring the light absorption value at the wavelength of 570nm, the number of the living cells can be indirectly reflected, and a test object ET50 can be detected by an MTT cell activity detection method. Preferably, the concentrated enzymatic hydrolysate is separated into 3 to 5 components having different molecular weights by using a semipermeable membrane device.

The amount of the components is obtained by comprehensively considering desensitization effect, yield and process cost.

Preferably, 4 semipermeable membrane devices are used for separating the concentrated enzymolysis liquid into 5 components with different molecular weights, and the cut-off molecular weights of the 4 semipermeable membrane devices are 800-2500 Da, 500-1800 Da, 300-1200 Da and 100-800 Da respectively.

Preferably, the semipermeable membrane device is a dialysis bag.

Dialysis bags with different cut-off molecular weights can be selected for dialysis in sequence from large to small. Specifically, the enzymolysis liquid is dialyzed by using a dialysis bag with the largest molecular weight cut-off, the liquid in the bag is reserved, and the dialysis effluent (namely the residual liquid) is dialyzed by using a dialysis bag with the next first molecular weight cut-off, and so on.

Further preferably, the semipermeable membrane device comprises a plurality of dialysis bags which are sleeved from inside to outside in sequence according to the molecular weight cut-off from large to small.

The dialysis bags are preferably 4, and the cut-off molecular weights of the dialysis bags are 800-2500 Da, 500-1800 Da, 300-1200 Da and 100-800 Da from inside to outside in sequence; the dialysis temperature is 4-15 ℃, and the dialysis time is 12-48 h.

Among them, the most preferable are those having a molecular weight cut-off of 1000Da, 800Da, 500Da, 200Da in order from the inside to the outside.

A plurality of dialysis bags which are sleeved from inside to outside in sequence according to the molecular weight cut-off from large to small are used, so that components with a plurality of molecular weights can be obtained simultaneously, multilayer directional dialysis is achieved, and the production process is simplified.

The invention also designs a multilayer directional dialysis device which comprises a dialysis container and a plurality of dialysis bags which are arranged in the container and sleeved from inside to outside in sequence according to the molecular weight cut-off, wherein the diameters of the sections of the dialysis bags are increased from inside to outside in sequence. The multi-layer directional dialysis device can also comprise a plurality of sleeve skeletons corresponding to the shapes and the sizes of the dialysis bags, and the dialysis bags are adhered to the sleeve skeletons so as to fix the shapes of the dialysis bags. When the device is used, concentrated enzymatic hydrolysate obtained by ultrafiltration is poured into the dialysis bag at the innermost layer, a proper amount of water or buffer solution is poured into the container, and the concentrated enzymatic hydrolysate is divided into a plurality of components with different molecular weights due to the continuous molecular weight cutoff difference among the dialysis bags.

Preferably, the specific working conditions of the enzymolysis are as follows:

crushing Antarctic krill, and mixing with water according to a mass ratio of 1: 2-1: 8, uniformly mixing, adjusting the pH value to 2.0-6.0, adding 1.0-6.0 wt% of pepsin based on the mass of the total reaction system, performing enzymolysis at the temperature of 30-50 ℃ for 0.5-2 h, heating to 85-95 ℃, and keeping for 5-20 min to inactivate enzyme; and cooling the reaction system to room temperature, centrifuging, collecting clear liquid, and removing residues to obtain a crude enzymolysis liquid.

Preferably, the specific working conditions of the ultrafiltration are as follows: and (3) introducing the crude enzymolysis liquid obtained after enzymolysis into an ultrafiltration device, wherein the membrane aperture is 10-5000 nm.

Preferably, a high-flux ultrafiltration device is used, the flux is preferably 50-100L/h, and the operating pressure difference is 50-500 kPa.

Preferably, the flash distillation has the following specific working conditions: and (3) carrying out flash evaporation on the enzymolysis liquid obtained by ultrafiltration for 10-100 s at the temperature of 100-200 ℃.

The ultrafiltration device can be connected with the ultrahigh-temperature flash evaporation device in series, so that the working efficiency is improved. And by adopting a one-step concentration method of ultrafiltration flash evaporation series connection, the effective deodorization of krill enzymolysis liquid can be realized while the concentration efficiency is improved.

The invention has the following beneficial effects:

the method effectively removes the sensitizing substances in the Antarctic krill peptide by a multilayer directional dialysis technology, and simultaneously adopts a one-step concentration method of ultrafiltration flash evaporation series connection to obtain the Antarctic krill peptide liquid, which has the characteristics of low sensitization, no fishy smell and the like. The invention has high efficiency of the manufacturing process, easy industrialization, high peptide activity and obvious effect, and simultaneously, the extracting solution can be widely applied to the fields of nourishment, skin care products, medicines and the like.

Drawings

FIG. 1 is a schematic view of a multi-layered directional dialysis apparatus according to the present invention;

in the figure: 1. a dialysis container; 2. a dialysis bag.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The pepsin used in the example was purchased from a drugstore organism, and the enzyme activity was 10000 u/mg.