CN109806383B

CN109806383B - Application of eel peptide in preparing food, medicine or health product for promoting immunity

Info

Publication number: CN109806383B
Application number: CN201910221495.3A
Authority: CN
Inventors: 韩庆国; 续旭; 罗文奇; 毕德成
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2019-03-22
Filing date: 2019-03-22
Publication date: 2022-08-16
Anticipated expiration: 2039-03-22
Also published as: CN109806383A

Abstract

The invention provides application of eel peptide in preparing food, medicine or health care products for promoting immunity, and relates to the technical field of biological medicine. As shown in the specific example, the Griess method detects the influence of eel peptide on the NO content produced by mouse macrophages, and the eel peptide is found to promote the release of macrophage NO. The CCK-8 method detects the influence of the eel peptide on the activity of the macrophage, and shows that the eel peptide does not influence the cell activity of the macrophage. The protein immunoblotting method is used for determining the expression conditions of iNOS protein and NF-kB signal channel, and the discovery that the eel peptide can well induce the expression of iNOS and activate the NF-kB signal channel is found. The influence of eel peptide on the TNF-alpha and IL-6 secretion amount is detected by an ELISA method, and the eel peptide is found to promote the release of cell TNF-alpha and IL-6. The eel peptide has good immunity enhancing effect, and can be developed as novel immunity promoting food, medicine or health care product.

Description

Application of eel peptide in preparing food, medicine or health product for promoting immunity

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of eel peptide in preparing foods, medicines or health-care products for promoting immunity.

Background

Immunity is a physiological function of the human body, by which the human body recognizes "self" and "non-self" components, thereby destroying and repelling antigenic substances (e.g., germs, etc.) entering the human body, or damaged cells and tumor cells, etc., which are generated from the human body itself, to maintain the health of the human body, and to resist or prevent infection by microorganisms or parasites or other undesirable states of biological invasion. Nitric Oxide (NO) and nitric oxide synthase (iNOS) are important physiological transmitters in the body and intercellular and intracellular signaling molecules involved in the regulation of physiological processes of the body and host defense and immune responses. During the immune process, when macrophages are in a stress state under external stimulation, respiratory burst can be generated, and a large amount of superoxide radicals and Reactive Oxygen Species (ROS) are generated through NADPH oxidase; meanwhile, inducible nitric oxide synthase protein (iNOS) in cells is induced to express to generate a large amount of Nitric Oxide (NO), and nitric oxide free radicals (NO) play a cytotoxic role against exogenous microorganisms by stimulating macrophages to induce inflammation and death of infected cells. NF-kB is a nuclear transcription factor which participates in the regulation of multiple functions such as immune response, inflammatory reaction, apoptosis, proliferation and the like, and has important physiological and pathological effects. The NF-kB consists of two subunits of p50 and p65, and in a resting state, the NF-kB is combined with an inhibitor protein IkB-alpha thereof to form a compound which is dissociated in cytoplasm; when activated, the p65 subunit of NF-kB and I kB-alpha are phosphorylated, NF-kB and I kB-alpha are dissociated, and then NF-kB enters the nucleus of cell and is combined with DNA to play a role in transcriptional regulation. In inflammatory response, the production of the inflammatory mediators NO and the related cytokines TNF- α and IL-6 are closely related to the activation of NF- κ B signaling pathway.

The eel is commonly called as the sea ginseng, has high nutritive value and is deeply favored by consumers. The eel breeding yield is huge in China, and the economic value is considerable. The eel polypeptide hydrolysate contains rich eel polypeptide, amino acids, minerals and other active matters, and has the main nutritious components of eel meat maintained, new health functions added to the polypeptide, and easy digestion and absorption. Therefore, the eel polypeptide hydrolysate can be used for developing functional foods; meanwhile, the seasoning is delicious and can be used for developing high-grade seasonings, and the application prospect is wide. According to the report of nursing newspaper, the nutritional ingredients of eel are much higher than weever, chicken and beef, and the contents of vitamins, minerals and trace elements are beyond the reach of land animals. Scientific research shows that the eel is one of the fishes with the highest EPA and DHA (deep sea fish oil component, DHA is docosahexaenoic acid, EPA is eicosapentaenoic acid), can reduce blood fat, resist arteriosclerosis and thrombus, and can supplement necessary nutrients for brain. DHA can promote brain development of teenagers, enhance memory, and is also helpful for the elderly to prevent brain function deterioration and senile dementia. The medical experts also find that the eel contains the beneficial components of fish oil and vegetable oil, and is an ideal food for supplementing fatty acid and amino acid which are necessary for human bodies. The zinc content, the highly unsaturated fatty acid content and the vitamin E content of the eel are all high, and the eel can assist in preventing decay and arteriosclerosis, thereby having the effects of skin care and beauty. The nutritional ingredients are rich in vitamin A and vitamin E, the content of the vitamin A and the vitamin E is 60 times and 9 times of that of common fishes respectively, and the contents of other vitamins such as vitamin B1 and vitamin B2 are also rich. In addition, eel also contains a large amount of calcium, and has certain effect of preventing osteoporosis. Eel contains a rare protein of Xihe roc in vivo, and the fishbone and meat of the eel contain rich high-quality protein and various amino acids necessary for human bodies, so how to better utilize the protein nutrient components in the eel has become the work focus of researchers.

Disclosure of Invention

The invention provides the application of eel peptide in preparing food, medicine or health care product for promoting immunity in order to overcome the defect of insufficient application and development of the prior eel active ingredient, the eel peptide has no cytotoxicity, can induce the improvement of cell activity, and can be used as a preparation raw material of the food, medicine or health care product for promoting immunity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of eel peptide in preparing food, medicine or health care product for promoting immunity.

Preferably, the eel peptide is prepared by a method comprising the following steps:

(1) cooking eel bone, pulverizing, performing enzymolysis with protease, performing solid-liquid separation, and collecting liquid to obtain enzymolysis solution;

(2) sequentially filtering, decoloring and filtering with a plate frame to obtain a refined solution;

(3) concentrating the refined solution, performing precision filtration, spray drying the filtrate obtained by precision filtration, and performing magnetic separation to obtain eel peptide coarse powder;

(4) purifying the eel peptide coarse powder by an ion exchange column and a molecular sieve in sequence, and drying to obtain the eel peptide.

Preferably, the average molecular weight of the eel peptide is 485-1800 Da.

Preferably, the enhancing immunity comprises enhancing iNOS protein expression, activating NF-kB signal path, and inducing TNF-alpha and IL-6 secretion.

Preferably, the dosage form of the food, the medicine or the health care product comprises a tablet, an oral liquid, a powder or a paste.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides application of eel peptide in preparing food, medicine or health care product for promoting immunity. As shown in experiments of specific embodiments of the invention, the influence of eel peptide on the NO content of mouse macrophage (RAW264.7 cell) is detected by a Griess method, and the eel peptide is found to promote the release of macrophage NO and has dose dependence. The CCK-8 method is used for detecting the influence of the eel peptide on the activity of the macrophage, and the result shows that the eel peptide does not influence the cell activity of the macrophage and has no cytotoxic effect. Furthermore, the expression conditions of RAW264.7 cell nitric oxide synthase (iNOS) protein and NF-kB signal channel are measured by a protein immunoblotting method, and the eel peptide is found to well induce the expression of iNOS and activate the NF-kB signal channel. Finally, the influence of eel peptide on the TNF-alpha and IL-6 secretion amount of RAW264.7 cells is detected by an ELISA method, and the eel peptide is found to promote the release of the TNF-alpha and IL-6 of the RAW264.7 cells and has dose dependence. Therefore, the eel peptide has good immunity enhancing effect, which indicates that the eel peptide can be developed as a novel immunity promoting food, a medicament or a health product.

Drawings

FIG. 1 Effect of Anguillar Japonica peptide on NO production by RAW264.7 cells;

FIG. 2 cell viability effect of eel peptides on RAW264.7 cells;

FIG. 3 influence of eel peptide on the expression level of iNOS protein in RAW264.7 cells;

FIG. 4 Effect of Anguillar Japonica peptide on NF-. kappa.B signaling pathway in RAW264.7 cells;

FIG. 5 Effect of Anguillar Japonica peptide on TNF-alpha secretion from RAW264.7 cells;

FIG. 6 influence of Anguillar Japonica peptide on IL-6 secretion amount of RAW264.7 cells.

Detailed Description

The invention provides application of eel peptide in preparing food, medicine or health product for promoting immunity. In the present invention, the average molecular weight of the eel peptide is 485-1800 Da.

The eel peptide can be sourced from commercial products or prepared by self. In the present invention, the eel peptide is preferably prepared by a method comprising the following steps:

(2) sequentially filtering, decoloring and filtering by a plate frame to obtain a refined solution;

The method comprises the steps of stewing eel bones, crushing, carrying out protease enzymolysis, carrying out solid-liquid separation, and taking liquid parts to obtain an enzymolysis liquid;

in the invention, the eel bones are selected as raw materials to prepare the eel peptide, mainly because the eel bones are worthless byproducts of eel food processing, which is beneficial to recycling of waste. In the present invention, before the eel bones are cooked, it is preferable to rinse the eel bones with water for removing impurities, internal organs, and the like from the eel bones. In the present invention, the rinsing water is preferably pure water. In the invention, the rinsing temperature is preferably 0-20 ℃ in ice water.

In the invention, the stewing temperature of the eel bone is preferably 90-95 ℃; the cooking time is preferably 40-120 min, and more preferably 60 min. In the present invention, the pulverized particle size is preferably 60 to 120 mesh, and more preferably 80 mesh. In the present invention, the protease includes, but is not limited to, trypsin, flavourzyme. In the invention, the enzymolysis time is preferably 2-4 h, and more preferably 3 h. In the invention, the enzymolysis temperature is preferably 50-70 ℃, and more preferably 60 ℃. The solid-liquid separation method in the present invention is not particularly limited, and a method of solid-liquid separation known in the art, for example, filtration, may be employed.

After the enzymolysis liquid is obtained, the enzymolysis liquid is sequentially filtered, decolored and filtered by a plate frame to obtain a refined liquid.

In the present invention, the filtration is preferably performed by using a cloth bag, and the material of the cloth bag includes but is not limited to PP (polypropylene), PE (polyester), and PTFE (polytetrafluoroethylene). In the present invention, the decolorization is preferably performed using activated carbon. In the invention, in the plate-frame filtration, the aperture of a plate-frame filter membrane is preferably 80-150 meshes, and more preferably 120 meshes.

After the refined liquid is obtained, the refined liquid is concentrated and then is subjected to precise filtration, the filtrate obtained by the precise filtration is subjected to spray drying and magnetic separation, and the eel peptide coarse powder is obtained.

In the present invention, the concentration is preferably performed at a low temperature of 10 ℃ or lower. In the invention, the microfiltration and nanofiltration can remove bacteria and impurities in the peptide liquid by using a 0.45-micron filter membrane. In the invention, the grain size of the eel peptide coarse powder obtained after spray drying is in the range of 4-80 meshes.

In the invention, the metal debris in the peptide powder can be removed by magnetic separation.

After eel peptide coarse powder is obtained, the eel peptide coarse powder is purified and dried by an ion exchange column and a molecular sieve in sequence to obtain the eel peptide.

In the present invention, the specification of the ion exchange column is preferably DEAE dextran a25 gel separation. The molecular sieve specification is preferably P6 column separation to obtain active eel peptide.

As shown in the specific embodiment of the invention, the immunity promoting effect of the eel peptide comprises the functions of promoting iNOS protein expression of RAW264.7 cells, activating NF-kB signal path, and inducing TNF-alpha and IL-6 secretion, namely, the eel peptide has the effect of improving the immunity of the cells.

In the present invention, the dosage form of the food, drug or health product includes, but is not limited to, tablets, oral liquids, powders or pastes.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1 preparation of Anguillar Japonica peptides

The eel bones were rinsed with ice water to obtain a crude product. Pulverizing the crude product, steaming at 90 deg.C for 60min, and performing enzymolysis with trypsin and flavourzyme at 60 deg.C for 2 hr to obtain eel peptidase hydrolysate. Filtering eel peptidase hydrolyzed solution with cloth bag, decolorizing with active carbon, and filtering with plate frame to obtain eel peptide refined extractive solution. And (3) carrying out low-temperature nanofiltration concentration on the eel peptide refined extract, cooling, and carrying out spray drying to obtain eel peptide powder. Separating eel peptide powder with ion exchange column DEAE dextran A25 gel, and separating with P6 column molecular sieve to obtain eel active peptide.

The eel bone peptide powder is soluble in water, is white, has no taste, and has peptide content of 98%.

Example 2

Experimental raw materials and experimental methods:

1. experimental materials and related reagents

The eel peptide of the experiment is from Shanguan Weiming Biotech limited (prepared according to the method shown in example 1); high-glucose DMEM medium, penicillin, streptomycin were purchased from Hyclone, usa; fetal bovine serum was purchased from BI corporation; the NO detection kit and the CCK-8 kit are purchased from Shanghai Bintian biological technology research institute; cell lysates were purchased from Shanghai Bocai Biotech; iNOS, p-IkB, p-p65, p65 monoclonal antibody, goat rabbit secondary antibody purchased from CST company, Germany; ECL luminophores were purchased from Thermo Fisher Scientific; LPS and other common biochemicals were purchased from Sigma; mouse TNF-alpha and IL-6 ELISA kits were purchased from Shenzhen Xinbo Sheng Biotech Co.

2. Experimental method

2.1 cell culture

Mouse macrophage RAW264.7 was purchased from American type culture Collection, cultured in complete medium consisting of 90% high-glucose DMEM medium, 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin, and placed in a cell culture chamber at 37 ℃ in 5% CO 2.

2.2 detection of NO

Mouse macrophage RAW264.7 at 1 × 10 ⁵ Inoculating each well in a 96-well plate, culturing at 37 deg.C for 4h, removing supernatant, adding 90 μ L serum-free high-sugar DMEM culture medium and 10 μ L eel peptide (160 μ g/mL, 800 μ g/mL, 1600 μ g/mL) at different concentrations into each well of the experimental group, adding 90 μ L serum-free high-sugar DMEM culture medium and 10 μ L LPS (100ng/mL) into each well of the positive control group, and adding 100 μ L serum-free high-sugar DMEM culture medium and 10 μ L LPS (100ng/mL) into the blank control groupThe culture was continued for 24 hours at 37 ℃ in DMEM medium with sugar.

And then sucking out the culture supernatant to be detected, sequentially placing the culture supernatant into a new 96-well plate, respectively adding 50 mu L of Sulfanilamide solution into the standard well and the experimental well, incubating for 5-10 min at room temperature in a dark place, respectively adding 50 mu L of ED solution, and incubating for 5-10 min at room temperature in a dark place. Reading simultaneously with A550nm and A630nm dual wavelengths within 30min, and processing the result with A ₅₅₀ The reading of A630nm was subtracted from the nm reading. According to the standard curve, the cellular NO content is calculated.

2.3 Western blotting

The mouse macrophage RAW264.7 cells (2 multiplied by 106 cells/hole) are inoculated in a 6-hole plate, after the cells are attached to the wall, the original culture medium is discarded, and the culture medium containing 16 mu g/mL, 80 mu g/mL and 160 mu g/mL of eel peptide is added for co-culture for 24 h. Then, the cells were washed 3 times with precooled PBS, RIPA lysate containing protease inhibitor was added, the cells were lysed at 4 ℃ for 30min, and the cell lysate was collected. Then, the total protein concentration in the cell lysate was detected by BCA protein quantification kit. Mixing cell lysate solution containing equal amount of protein with 6 × loading buffer at a ratio of 1:5 to obtain protein sample solution, boiling in boiling water bath for 10min, centrifuging instantaneously, and separating by 10% SDS-polyacrylamide gel electrophoresis under conditions of 80V 20min and 120V 100 min. Then, the protein was transferred to the PVDF membrane by electric transfer at a current of 100mA for 100 min. Blocking with 5% skimmed milk powder in blocking solution at room temperature for 2h, and hybridizing with primary antibody (1:1000) at 4 deg.C overnight. The membrane was then washed 3 times with TBST, a secondary antibody (1:10000) was added, hybridization was carried out at room temperature for 2 hours, and after washing the membrane 3 times with TBST, development was carried out.

2.4 cell viability assay

Mouse macrophage RAW264.7 cells were seeded in 96-well plates at 1X 10 cells per well ⁵ Carrying out adherent culture for 2-6h, discarding the supernatant, adding 100 mu L of culture medium containing 16 mu g/mL, 80 mu g/mL and 160 mu g/mL of eel peptide respectively, co-culturing for 24h, then adding 10 mu L of CCK-8 reagent into each hole, incubating for 1h, and detecting the light absorption value under the wavelength of 450nm by using an enzyme labeling instrument.

2.5 cytokine detection

RAW264.7Cells or primary macrophages were seeded in 96-well plates at 2X 10 cells per well ⁵ Performing adherent culture for 2-6h, discarding supernatant, adding 100 μ L of culture medium containing Anguillar Japonica peptide 16 μ g/mL, 80 μ g/mL, and 160 μ g/mL, and co-culturing for 24 h. The content of each cytokine in the cell supernatant was measured by ELISA. And carrying out experimental operation according to the specification, and finally detecting the light absorption value under the wavelength of 450nm by using an enzyme-labeling instrument.

3. The experimental results are as follows:

3.1 Effect of Anguillar Japonica peptide on NO production by RAW264.7 cells

Macrophages produce NO through a reactive nitrogen intermediate action system, thereby exerting a killing function. As shown in figure 1, after the RAW264.7 cells are cultured by using complete culture media containing different concentrations of eel peptides for 24 hours, the capability of the cells for generating NO is measured, and the result shows that the eel peptides can effectively promote the RAW264.7 cells to generate NO and the effect of the eel peptides is dose-dependent.

3.2 Effect of Anguillar Japonica peptide on RAW264.7 cell viability

The CCK-8 kit is used for detecting the influence of the eel peptide on the cell viability of RAW264.7 cells, and the result is shown in figure 2, after the eel peptide with different concentrations (16 mug/mL, 80 mug/mL and 160 mug/mL) and the RAW264.7 cells are co-cultured for 24 hours, the cell viability of an experimental group is not reduced compared with that of a control group, which indicates that the eel peptide has no toxic effect on the RAW264.7 cells.

3.3 Effect of Anguillar Japonica peptide on expression level of iNOS protein in RAW264.7 cells

In immune cells, the nitric oxide synthase iNOS is immunoregulatory by the induction of the intracellular messenger NO. As shown in FIG. 3, the eel peptide promotes iNOS protein expression in RAW264.7 cells and is dose-dependent.

3.4 Effect of Anguillar Japonica peptide on NF- κ B Signal pathway of RAW264.7 cells

NF-kB is a nuclear transcription factor which participates in the regulation of multiple functions such as immune response, inflammatory reaction, apoptosis, proliferation and the like, and has important physiological and pathological effects. As shown in FIG. 4, the eel peptide can promote the expression of kinase (p-I kappa B, p-p65) in NF-kappa B signaling pathway of RAW264.7 cells and has dose dependence.

3.5. Influence of Anguillar Japonica peptide on TNF-alpha, IL-6 secretion amount of RAW264.7 cell

TNF-alpha is a cytokine which can directly kill tumor cells without obvious toxicity to normal cells, and participates in important physiological and pathological activities such as immune response regulation, inflammatory response regulation and the like. As shown in FIG. 5, the eel peptide can promote the secretion of TNF-alpha from RAW264.7 cells and has dose dependence.

IL-6 is a cytokine that stimulates the proliferation and differentiation of cells involved in immune responses and enhances their function, and has important physiological and pathological roles. As shown in FIG. 6, eel peptide promotes the secretion of IL-6 cytokine from RAW264.7 cells, and is dose-dependent.

4. Conclusion

In conclusion, the eel peptide which is a polypeptide product obtained by rinsing, crushing, enzymolysis, purification and separation of eel bones can effectively induce the enhancement of the immunological activity of RAW264.7 cells. The following conclusions can be drawn from the study:

firstly: eel peptide can effectively induce RAW264.7 cells to generate NO, and the effect of the eel peptide is dose-dependent;

secondly, the method comprises the following steps: eel peptide has no cytotoxicity on RAW264.7 cell activity;

thirdly, the method comprises the following steps: eel peptide can effectively induce the expression of iNOS protein of RAW264.7 cells, and the action of the eel peptide is dose-dependent;

fourthly: the eel peptide can effectively activate NF-kB signal channels of RAW264.7 cells, and the effect of the eel peptide is dose-dependent;

fifth: eel peptide can effectively induce TNF-alpha and IL-6 secretion of RAW264.7 cells, and its effect is dose-dependent

In conclusion, the research discovers for the first time that the eel peptide has the effects of inducing RAW264.7 cells to generate NO, inducing iNOS expression, activating NF-kB signal channels and promoting TNF-alpha and IL-6 secretion, proves that the eel peptide has good immunoregulation activity, and has wide market application prospect when being used as a novel immunity promoting medicine and a novel health product.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The application of the eel peptide in preparing foods, medicines or health products for promoting immunity, wherein the promotion of immunity is the promotion of the release of macrophage NO, the promotion of iNOS protein expression, the activation of NF-kB signal path, and the induction of TNF-alpha and IL-6 secretion;

the macrophage is RAW 264.7;

the average molecular weight of the eel peptide is 485-1800 Da;

the eel peptide powder is white;

the eel peptide is prepared by the following steps:

the cooking temperature is 90-95 ℃, and the cooking time is 60 min;

the crushing granularity is 80 meshes;

the protease is trypsin and flavourzyme;

the enzymolysis time is 3 hours; the enzymolysis temperature is 60 ℃;

(2) sequentially performing cloth bag filtration, activated carbon decolorization and plate-and-frame filtration on the enzymolysis solution to obtain refined solution;

the aperture of the plate frame filtration is 120 meshes;

the concentration is low-temperature concentration; the low-temperature concentration temperature is below 10 ℃;

(4) purifying the eel peptide coarse powder by an ion exchange column and a molecular sieve in sequence, and drying to obtain eel peptide;

the ion exchange column is DEAE dextran A25 gel; the molecular sieve is a P6 column molecular sieve.

2. The use according to claim 1, wherein the food, pharmaceutical or nutraceutical formulation comprises a tablet, oral liquid, powder or paste.