阅读说明：本技术 小公鱼免疫活性肽及其制备方法和用途 (Gorgon fish immune active peptide and preparation method and application thereof ) 是由 杨最素 叶蕾 唐云平 徐宝贵 黄芳芳 余方苗 于 2020-05-22 设计创作，主要内容包括：本发明公开了小公鱼免疫活性肽及其制备方法和用途,属于生物技术领域,该小公鱼免疫活性肽具有氨基酸序列Tyr–Val–Met-Arg-Phe或Ser–Arg–Gln–Met–Ser。本发明小公鱼免疫活性肽能促进巨噬细胞系RAW 264.7细胞的增殖,可用于制备提高免疫细胞增殖药品或保健品,或者制备治疗免疫疾病的药物。(The invention discloses a small male fish immune active peptide, a preparation method and application thereof, belonging to the technical field of biology. The Erichopus japonicus immune active peptide can promote the proliferation of macrophage cell RAW264.7, and can be used for preparing medicines or health products for improving the proliferation of immune cells or preparing medicines for treating immune diseases.)

1. The small male fish immunoactive peptide has an amino acid sequence of Tyr-Val-Met-Arg-Phe or Ser-Arg-Gln-Met-Ser.

2. The bioactive peptide of small fish of claim 1, wherein: the red carp immune active peptide has the function of activating immune cells.

3. The rapid hemostatic hydrogel according to claim 2, wherein: the immune cells include macrophages.

4. A method for preparing an immunoactive peptide of a small fish of any one of claims 1 to 3, comprising:

pretreatment: desalting and degreasing the small fish, washing the small fish with pure water to be neutral, crushing, draining, and freezing at-20 ℃ to obtain processed small fish meat for later use;

enzymolysis: adding protease into the pretreated small fish meat for enzymolysis, boiling an enzymolysis product after the enzymolysis is finished, and centrifuging to obtain supernatant enzymolysis liquid;

and (3) ultrafiltration: carrying out ultrafiltration treatment on the enzymolysis liquid to obtain ultrafiltrate containing the small carp immunoactive peptide;

separation and purification: separating and purifying the ultrafiltrate by DEAE Sepharose Fast Flow and HPLC to obtain the bioactive peptide of the small male fish.

5. The method for preparing an immunoactive peptide of small fish of claim 4, wherein: the protease is selected from pepsin, neutral protease, alkaline protease, trypsin or papain.

6. The method for preparing an immunoactive peptide of small fish of claim 4, wherein: the ultrafiltration is carried out as follows: and (3) performing ultrafiltration on the enzymolysis liquid by using a 1KDa ultrafiltration membrane, and collecting ultrafiltrate with a molecular weight section of <1 KDa.

7. The method for preparing an immunoactive peptide of small fish of claim 4, wherein: the relative proliferation rate of the ultrafiltrate with the molecular weight range of <1KDa to the macrophage is up to 70.03%.

8. Use of the immunoactive peptide of claim 1 to 3 for the preparation of a pharmaceutical or nutraceutical agent for enhancing immune cell proliferation.

9. Use according to claim 8, characterized in that: the immune cells include macrophages.

10. Use of the immunoactive peptide of claim 1 to 3 for the manufacture of a medicament for the treatment of immune disorders.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a small male fish immunoactive peptide, and a preparation method and application thereof.

Background

The immune system consists of immune organs, immune cells, immune active substances and other components, has the functions of immune defense, immune monitoring, immune regulation and the like, and is related to etiological mechanisms of various diseases. Once the immune system of a human body is dysfunctional or impaired, it is easy to cause infection or induce various diseases, and therefore, it is of great significance to develop compounds that enhance the immunoregulatory activity of the human body. The immunomodulatory peptides are reported to have the most potential in improving the low immune function of human bodies, can participate in the immune regulation of the bodies, and find new immunomodulatory peptides from food proteins to provide advantages for dietary therapy. The preparation of immunoactive peptides from marine organisms by the enzymatic hydrolysis method has received extensive attention from researchers due to its characteristics of good effect, high safety, low cost, etc.

Chinese small fish (Stolephoruschinginensis), commonly known as the Goldfish, belongs to the order Clupeiformes, Engraulidae, genus Erigocephalus, and is distributed along the coast of south China sea and east China sea. The analysis result of the nutritional composition shows that the Chinese small male fish is rich in 18 common amino acids, various mineral elements such as zinc, strontium, calcium and the like and various unsaturated fatty acids, has high crude protein content, water content of 59.54 percent and crude fat content of 3.02 percent, has ash content of 10.35 grams per 100 grams, and is a high-protein low-fat economical and practical fish. At present, the research on the Chinese small male fish at home and abroad is mainly the relation between the distribution characteristics and the environment or the storage mode, but the research on the extraction and activity of the small male fish polypeptide is less reported.

Disclosure of Invention

The invention aims to provide an immunoactive peptide of small male fish, which can promote the proliferation of macrophage system RAW264.7 cells.

The technical scheme adopted by the invention for realizing the purpose is as follows:

an immunoactive peptide of small male fish has amino acid sequence Tyr-Val-Met-Arg-Phe or Ser-Arg-Gln-Met-Ser. Through MTT experiments, the herein-disclosed small fish immunoactive peptide can promote proliferation of macrophage cell line RAW264.7, and has a high Relative Proliferation Rate (RPR) to RAW264.7 cells, so that the small fish immunoactive peptide can be developed into marine functional food by preparing the herein-disclosed small fish immunoactive peptide.

Preferably, the small fish immunoactive peptide has the function of activating immune cells.

More preferably, the immune cells comprise macrophages.

The invention also aims to provide a preparation method of the small carp immune active peptide, which can prepare the small carp enzymatic hydrolysis polypeptide with the immune regulation activity on macrophage RAW264.7 and has high yield.

The technical scheme adopted by the invention for realizing the purpose is as follows:

the preparation method of the small fish immunoactive peptide comprises the following steps:

pretreatment: desalting and degreasing the small fish, washing the small fish with pure water to be neutral, crushing, draining, and freezing at-20 ℃ to obtain processed small fish meat for later use;

enzymolysis: adding protease into the pretreated small fish meat for enzymolysis, boiling an enzymolysis product after the enzymolysis is finished, and centrifuging to obtain supernatant enzymolysis liquid;

and (3) ultrafiltration: carrying out ultrafiltration treatment on the enzymolysis liquid to obtain ultrafiltrate containing the small carp immunoactive peptide;

separation and purification: separating and purifying the ultrafiltrate by DEAE Sepharose Fast Flow and HPLC to obtain the bioactive peptide of the small male fish.

The preparation method takes the Chinese small male fish (hereinafter referred to as small male fish) as a RAW material, and prepares the small male fish enzymolysis polypeptide with immunoregulatory activity on macrophage RAW264.7 by process optimization and a separation and purification technology of modern marine bioactive peptide, thereby providing experimental basis and theoretical guidance for improving the biological added value of the small male fish and developing immunoregulator functional food; the preparation method of the invention ensures that the amino acid sequence is Tyr-Val-Met-Arg-Phe or Ser-Arg-Gln-Met-Ser small male fish immunoactive peptide, and the yield is higher.

Preferably, the protease is selected from pepsin, neutral protease, alkaline protease, trypsin or papain.

More preferably, the enzymatic conditions are: the protease is pepsin, the temperature is 30-45 ℃, the pH is 1.0-2.5, the material-liquid ratio is 1:2-5(g/mL), the time is 4-7h, and the enzyme adding amount is 1500-.

Preferably, the ultrafiltration is carried out as follows: and (3) performing ultrafiltration on the enzymolysis liquid by using a 1KDa ultrafiltration membrane, and collecting ultrafiltrate with a molecular weight section of <1 KDa.

Preferably, the relative rate of macrophage proliferation of ultrafiltrate in the <1kDa molecular weight range is as high as 70.03%.

The invention also aims to provide the application of the small male fish immune active peptide in preparing medicines or health-care products for improving immune cell proliferation.

Preferably, the immune cells comprise macrophages.

The invention also aims to provide the application of the small male fish immunoactive peptide in preparing the medicines for treating the immune diseases. Preferably, the disease of an immune disease comprises a condition of hypoimmunity.

The invention has the beneficial effects that: the small male fish immunocompetence peptide can promote the proliferation of macrophage cell RAW 264.7; the preparation method of the invention prepares the barracuda enzymolysis polypeptide with immunoregulation activity on macrophage RAW264.7, which provides experimental basis and theoretical guidance for improving the biological additional value of the barracuda and developing immunoregulator functional food; the preparation method of the invention ensures that the amino acid sequence is Tyr-Val-Met-Arg-Phe or Ser-Arg-Gln-Met-Ser small male fish immunoactive peptide, and the yield is higher. Therefore, the invention is the small male fish immunoactive peptide which can promote the proliferation of macrophage cell RAW 264.7.

Drawings

FIG. 1 shows the effect of different proteases on the proliferative effect of RAW 264.7;

FIG. 2 is a graph showing the effect of pH on the proliferation rate of RAW264.7 cells;

FIG. 3 is a graph of the effect of temperature on the proliferation rate of RAW264.7 cells;

FIG. 4 is a graph of the effect of time on the proliferation rate of RAW264.7 cells;

FIG. 5 is a graph of the effect of feed liquid ratio on the proliferation rate of RAW264.7 cells;

FIG. 6 is a graph showing the effect of enzyme addition on the proliferation rate of RAW264.7 cells;

FIG. 7 is a graph of the effect of various ultrafiltration components on RAW264.7 cell proliferation;

FIG. 8 shows elution peaks of anionic DEAE Sepharose Fast Flow;

FIG. 9 shows the magnitude of the RPR value of each elution peak;

FIG. 10 shows the elution peaks of the RP-HPLC column;

FIG. 11 is a graph showing the effect of peak II-IV components on the proliferative capacity of RAW264.7 cells.

Detailed Description

The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings: