CN114403455A - Cod peptide with immunoregulation activity and application thereof - Google Patents
Cod peptide with immunoregulation activity and application thereof Download PDFInfo
- Publication number
- CN114403455A CN114403455A CN202210025039.3A CN202210025039A CN114403455A CN 114403455 A CN114403455 A CN 114403455A CN 202210025039 A CN202210025039 A CN 202210025039A CN 114403455 A CN114403455 A CN 114403455A
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- cod
- peptide
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Abstract
The invention discloses cod peptide with immunoregulation activity and application thereof, and belongs to the field of biological medicines. The invention takes cod as raw material, and comprises the following steps: the cod is ground, subjected to enzymolysis, subjected to spray drying, separated and prepared, dealcoholized, desalted and freeze-dried, and the alcohol-soluble cod peptide is developed. Experiments show that the alcohol-soluble cod peptide has the capacity of promoting RAW264.7 cells to release NO under a certain dosage; the amount of NO released from 500. mu.g/mL alcohol-soluble cod peptide treated cells was 31.41% higher than that of the blank; in addition, the cod is used as a raw material, so that the safety is high. The cod is used as the raw material to develop the zymolyte mainly containing the bioactive peptide, the raw material is rich, the source is wide, the preparation process flow is simple and convenient, and the large-scale industrial production can be met; the method has important significance for developing and utilizing the cod bioactive peptide in functional food, health-care food or medicines.
Description
Technical Field
The invention relates to cod peptide with immunoregulation activity and application thereof, and belongs to the field of biological medicines.
Background
Immunity is closely related to body health, and immune homeostasis is very important to human health. Under normal physiological conditions, the immune system is responsible for maintaining immune homeostasis. An imbalance in the immune system can lead to immunodeficiency diseases, infectious diseases, hypersensitivity diseases, autoimmune diseases and malignant tumors. Adaptive immunity is highly specific for potentially dangerous foreign antigens and is classified into two types, cell-mediated immunity and antibody-mediated immunity. At present, drugs for treating immunodeficiency diseases, such as levamisole, have many side effects, so that it is very important to find a product for enhancing immunity without side effects. The RAW264.7 cell is one of the important members of immune cells in the immune system of the body, has non-negligible functions in immune defense, immune maintenance and immune monitoring, and is one of the cell types which make immune response first. Activated macrophages have phagocytic capacity and occupy a considerable position in the immune response by secreting immunologically active substances with various functions. Mouse spleen lymphocytes were isolated from spleen tissue; the spleen is the largest immune organ of the body, accounts for 25 percent of the total amount of the systemic lymphoid tissue, contains a large amount of lymphocytes and macrophages, and is the center of cellular immunity and humoral immunity of the body. Mature lymphocytes need to be stimulated by antigens to differentiate and proliferate, and then exert immune functions.
Cod is one of the highest-yielding fish species in the world today, and is produced mainly in canada, iceland, norway, russia, japan, korea, and other countries. In China, the main producing areas of the cod are distributed in the northern part of the yellow sea, the southeast part of the Shandong and relevant zones of the southeast coastal region. Cod is a deep sea fish. The nutritional value of high protein and low fat is praised as 'dietician on dining table' and 'liquid gold' in the eyes of western people who pay attention to the quality of diet, and is a precious treasure than gold. Research shows that the fish protein hydrolysate has various biological activities, such as antioxidant activity, antihypertensive activity, antidiabetic activity and antibacterial activity. The active peptides can be released from the protein sequence by an enzymatic hydrolysis process to exert a variety of biological functions or physiological effects. In recent years, researchers have found peptides having immunomodulatory activity from hydrolysates of pseudostellaria protein, zein, bovine colostrum whey protein, mytilus coruscus protein, and the like, but their industrial production and utilization are limited due to technical limitations and high process costs.
Therefore, the development of a polypeptide which has simple and convenient extraction process and potential immunoregulatory activity has important significance for the development of health-care food and medicines for enhancing immunity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of alcohol-soluble cod peptide with immunoregulation activity, so as to promote NO content released by RAW264.7 cells, promote mouse spleen cell proliferation and enhance immunoregulation function.
The invention provides cod peptide powder which is prepared according to the following method:
s1, grinding the cod: adding water into the cod, and grinding to obtain cod mixed solution;
s2, enzymolysis: adding alkali into the cod mixed liquor obtained in the step S1, adjusting the pH value to 8.0-8.5, heating to 55-58 ℃, adding alkaline protease according to the proportion of 0.19-0.21%, uniformly stirring, performing enzymolysis for 1.8-2.2 h, heating to inactivate enzyme for 10-15 min, cooling to below 65 ℃, performing filter pressing, centrifuging through a vibrating screen, performing membrane filtration separation, and concentrating to obtain cod enzymatic hydrolysate;
s3, spray drying: spray drying the cod enzymolysis liquid obtained in the step S2 to obtain cod peptide powder;
s4, separation preparation: adding the cod peptide powder obtained in the step S3 into 75-85% of ethanol according to the mass ratio of (1: 18) - (1: 22), stirring for 1-2 h at 20-30 ℃, centrifuging for 20-30 min at the rotating speed of 7500-8500 g, and taking supernatant to obtain alcohol-soluble cod peptide;
s5, dealcoholization: removing ethanol from the alcohol-soluble cod peptide obtained in the step S4 by using a rotary evaporator to 3-5% of the original volume to obtain alcohol-removed alcohol-soluble cod peptide;
s6, desalting: and (4) adding deionized water into the dealcoholized alcohol-soluble cod peptide obtained in the step (S5) according to the volume ratio of 1 (5-7), respectively performing osmotic desalting treatment, removing salt to obtain the cod peptide, and freeze-drying and storing the cod peptide.
In one embodiment of the present invention, step S1 is: adding water into the cod according to the mass ratio of 1 (1.5-2.0) and grinding to obtain a cod mixed solution;
in one embodiment of the present invention, the enzyme deactivation temperature in step S2 is 85 ℃ or higher.
In one embodiment of the present invention, in step S2, the vibrating screen has a size of 120 mesh.
In one embodiment of the invention, the molecular weight of the alcohol-soluble cod peptide is distributed between 200Da and 3000Da, and the nitrogen content is 80-90%.
In one embodiment of the present invention, the temperature of the rotary evaporation in step S5 is set to 50 to 60 ℃, and the rotation speed is set to 60 to 80 rpm.
In one embodiment of the invention, the osmotic desalination method in the step S6 comprises pouring the dealcoholized alcohol-soluble cod peptides into a dialysis bag of 100-200 Da, and dialyzing at 2-8 ℃ for 24-48 h.
The invention provides a product, which contains the cod peptide.
In one embodiment of the invention, the product is a food, pharmaceutical or nutraceutical product.
In one embodiment of the invention, the food is protein powder, candy or beverage containing the cod peptide.
In one embodiment of the invention, the pharmaceutical product comprises the cod peptide described above, a pharmaceutical carrier and/or a pharmaceutical excipient.
The invention provides application of the cod peptide in preparation of a product with immunoregulatory activity.
In one embodiment of the invention, the product is a pharmaceutical or nutraceutical product.
In one embodiment of the invention, the pharmaceutical product comprises the cod peptide described above, a pharmaceutical carrier and/or a pharmaceutical excipient.
The invention provides application of the cod peptide in preparation of a product for promoting NO release of RAW264.7 cells.
In one embodiment of the invention, the product is a pharmaceutical or nutraceutical product.
In one embodiment of the invention, the pharmaceutical product comprises the cod peptide described above, a pharmaceutical carrier and/or a pharmaceutical excipient.
The invention provides application of the cod peptide in preparation of a product for promoting mouse spleen lymphocyte proliferation.
In one embodiment of the invention, the product is a pharmaceutical or nutraceutical product.
In one embodiment of the invention, the pharmaceutical product comprises the cod peptide described above, a pharmaceutical carrier and/or a pharmaceutical excipient.
Advantageous effects
1. The invention develops alcohol-soluble cod peptide capable of promoting NO release of RAW264.7 cells and promoting mouse spleen lymphocyte proliferation by taking cod as a RAW material, and develops the application of marine organisms in development of health care products and medicines for enhancing immunity.
2. Compared with the prior art, the method for obtaining the cod peptide is simpler, and the obtained cod peptide has stronger immunoregulation activity.
3. Experiments show that the alcohol-soluble cod peptide has the capacity of promoting RAW264.7 cells to release NO under a certain dosage; the amount of NO released by the 500 mu g/mL alcohol-soluble cod peptide treated cells is 31.41% higher than that of the blank group, and has a significant difference (p is less than 0.05) compared with the blank group.
4. The invention develops an extraction process of the codfish immunoregulation active peptide by adopting an alcohol-soluble extraction method for the first time, and the codfish immunoregulation active peptide has equivalent immunoregulation activity effect and selectable method.
5. The cod is used as the raw material to develop the zymolyte mainly containing the bioactive peptide, the raw material is rich, the source is wide, the preparation process flow is simple and convenient, and the large-scale industrial production can be met; the method has important significance for developing and utilizing the cod bioactive peptide in functional food, health-care food or medicines.
Drawings
FIG. 1: molecular weight distribution of common cod peptide, alcohol soluble cod peptide, and alcohol insoluble cod peptide.
FIG. 2: graph of the effect of regular cod peptides, alcohol-soluble cod peptides, and alcohol-insoluble cod peptides on RAW264.7 cell viability.
FIG. 3: graph showing the effect of regular cod peptides, alcohol-soluble cod peptides, and alcohol-insoluble cod peptides on the amount of NO released from RAW264.7 cells.
FIG. 4: graph of the effect of regular cod peptides, alcohol-soluble cod peptides, and alcohol-insoluble cod peptides on the 24h proliferation of splenic lymphocytes in mice.
FIG. 5: graph of the effect of regular cod peptides, alcohol-soluble cod peptides, and alcohol-insoluble cod peptides on the proliferation of spleen lymphocytes of mice for 48 h.
FIG. 6: graph of the effect of regular cod peptides, alcohol-soluble cod peptides, and alcohol-insoluble cod peptides on the proliferation of mouse spleen lymphocytes for 72 h.
FIG. 7: graph of the effect of alcohol-soluble cod peptide on the proliferation of spleen lymphocytes in mice for 48 h.
FIG. 8: graph of the effect of alcohol-soluble cod peptide mouse T/B lymphocyte proliferation for 48 h.
Detailed Description
The present invention is described below with reference to the results of cell assay data, and the reagents used in the examples are all conventional commercial products or reagents prepared according to conventional methods, and the methods in the examples are all conventional experimental methods, unless otherwise specified.
The alkaline proteases referred to in the examples below were purchased from Novoxin (China) Biotechnology Ltd. Examples the following examples are high-glucose DMEM complete medium, RPMI-1640 medium, purchased from Gibco.
The mice referred to in the following examples were purchased from Liaoning Biotechnology Ltd; RAW264.7 cells were purchased from shanghai cell bank, china academy of sciences.
The media involved in the following examples are as follows:
high-glucose DMEM complete medium: high-glucose DMEM medium was supplemented with 10% Fetal Bovine Serum (FBS) and 1% diabody (streptomycin mixed solution).
RPMI-1640 complete medium: RPMI-1640 medium was supplemented with 10% Fetal Bovine Serum (FBS) and 1% double antibody (penicillin streptomycin mixture).
The detection methods referred to in the following examples are as follows:
protein content determination: kjeldahl method
The assay was carried out with minor modifications with reference to the national standard GB 5009.5-2016. 0.2g of ordinary cod peptide powder (blank group without sample), 0.2g of copper sulfate pentahydrate, 6g of potassium sulfate and 15mL of concentrated sulfuric acid were added into a digestion tube, and gradient digestion was performed using a graphite digestion furnace (220 ℃ C. for 30min, 320 ℃ C. for 30min, 420 ℃ C. for 60 min). And (3) automatically adding alkali, diluting, distilling and titrating by using an SKD-1000 full-automatic Kjeldahl azotometer. Protein content was calculated according to the following formula:
in the formula:
x-protein content in the sample, in units%;
V1-the volume of standard hydrochloric acid consumed by the test solution in mL;
V2-the blank consumes a volume of standard hydrochloric acid in mL;
c-standard hydrochloric acid concentration, unit mol/L;
m is the mass of the sample in g.
And (3) total sugar content determination: phenol-sulfuric acid process
Making a standard curve: accurately weighing 20mg of standard glucose into a 500mL volumetric flask, adding water to the scale, respectively sucking 0.4 mL, 0.6 mL, 0.8 mL, 1.0mL, 1.2 mL, 1.4 mL, 1.6 mL and 1.8mL, respectively supplementing distilled water to 2.0mL, then adding 1.0mL of 6% phenol and 5.0mL of concentrated sulfuric acid, shaking uniformly and cooling, placing in a dark place at room temperature for 20min, measuring the optical density at 490nm, using 2.0mL of water as a blank according to the same color development operation, taking the abscissa as the microgrammes of polysaccharide and the ordinate as the optical density value, and obtaining a standard curve. And (3) determining the total sugar content of the sample: accurately weighing 0.1g of common cod peptide powder in a 50mL volumetric flask, adding water to a scale, after completely dissolving, taking 2.0mL of common cod peptide powder solution in a test tube, then adding 1.0mL of 6% phenol and 5.0mL of concentrated sulfuric acid, shaking up and cooling, standing at room temperature in a dark place for 20min, measuring the optical density at 490nm, adding 2.0mL of common cod peptide powder solution as a control group by using 6.0mL of water instead of phenol and concentrated sulfuric acid, and using 2.0mL of water as a blank according to the same color development operation. Calculating the total sugar content of the common cod peptide powder: and respectively subtracting the optical density values of the control group and the blank group from the optical density value of the experimental group, and substituting the obtained optical density values into a standard curve to obtain the total sugar content of the common cod peptide powder solution.
And (3) measuring the content of crude fat: soxhlet extraction method
The assay was carried out with slight modification with reference to GB 5009.6-2016. Cleaning a fat receiving bottle, drying in an oven at 105 ℃ until the weight is constant (the difference between the front and the back is not more than 2mg), accurately putting about 2g of common cod peptide powder into a filter paper cylinder which is plugged into cotton from top to bottom, and sealing the upper opening and the lower opening of the filter paper cylinder to prevent a sample from leaking. Putting the filter paper cylinder with the sample into a clean Soxhlet extractor, connecting the filter paper cylinder with a constant-weight fat receiving bottle, adding anhydrous ether which is about two thirds of the volume of the fat receiving bottle, then putting the filter paper cylinder on a crude fat determinator, adjusting the water bath to 65 ℃, opening a condensed water switch, and heating to enable the filter paper cylinder to continuously reflux and extract for about 6 hours. After extraction, the fat receiving bottle is taken down, residual diethyl ether is volatilized on a water bath at 65 ℃, and after the diethyl ether is basically completely volatilized, the fat receiving bottle is continuously dried in an oven at 105 ℃ until the weight is constant (the difference between the front and the back is not more than 2 mg). The crude fat content was calculated according to the following formula:
in the formula:
x-the fat content in the sample, in units%;
m1-the total mass of the fat receiving flask and fat after constant weight, in g;
m0-mass of the fat receiving bottle after constant weight, in g;
m2mass of sample, unit g.
Determination of molecular weight distribution
The molecular weight distribution of cod peptides was determined using an Agilent 1260 HPLC system equipped with a TSKgel G2000SWXL (7.8X 300mm) chromatography column. Setting HPLC parameters: the column temperature was 25 deg.C, the flow rate was 0.5mL/min, and the elution was isocratic for 30min (H)2ACN: TFA: 55:45:0.1, v/v/v), loading 10 μ L, and detection was performed at 214nm with an ultraviolet detector. A standard curve was prepared using cytochrome C (12384Da), aprotinin (6511Da), octapeptide (1072Da), vitamin E (431Da), and L-lysine (146Da) as standards. The molecular weight range is divided into 5 parts (more than or equal to 3000Da, 1000-3000Da, 600-1000Da, 300-600Da and less than or equal to 300Da) according to the standard curve.
Determination of amino acid content
The amino acid content of the cod peptide was analyzed using a Hitachi LA8080 amino acid analyzer using a Hitachi high performance cation exchange column. Setting parameters: the temperature of the separation column is 57 ℃, the temperature of the derivatization column is 135 ℃, and the sample loading quantity is 20 mu L. The cod peptide powder was hydrolyzed in 6M HCl at 110 ℃ for 22 h. After rotary evaporation, the same volume of 0.02M HCl is taken for redissolving, and a sample passes through a 0.22 mu M water system filter membrane and is diluted and then loaded.
Example 1: preparation of common cod peptide powder and characterization of performance thereof
The method comprises the following specific steps:
1. preparation method of common cod peptide powder
(1) Grinding the cod: cutting the cod fish with fish scales, fish fins, fish heads and internal organs removed into small blocks of about 5cm in size, wherein the mass ratio of the small blocks is 1: 1.5 adding water and grinding to obtain a cod fish mixed solution;
(2) enzymolysis: adding alkali into the cod meat mixed liquor obtained in the step (1), adjusting the pH value to 8.0, heating to 58 ℃, adding alkaline protease according to the mass ratio of 0.20%, uniformly stirring, performing enzymolysis for 2h, heating to 85-100 ℃, performing enzyme deactivation for 15min, cooling to 64 ℃, performing pressure filtration, centrifuging through a 120-mesh vibrating screen, performing membrane filtration separation, and finally concentrating to obtain cod enzymatic hydrolysate;
(3) spray drying: and (3) carrying out spray drying on the cod enzymatic hydrolysate in the step (2) to obtain the common cod peptide powder.
2. Characterization of cod peptide powder
The protein content, total sugar content and crude fat content of the cod peptide powder were measured, respectively, and the results are shown in table 1.
Table 1: basic content of cod peptide powder
The results showed that the cod peptide powder obtained by the measurement of the cod peptide powder had a protein content of 84.92%, a total sugar content of 0.68%, and a crude fat content of 1.46%, and thus it could be confirmed that the cod peptide powder prepared according to step 1 had a high protein content.
Example 2: preparation of cod peptides and characterization of their properties
The method comprises the following specific steps:
1. preparation of cod peptide
(1) Separation preparation: the common cod peptide powder obtained in example 1 is prepared by mixing the following raw materials in a mass ratio of 1: 20, adding the mixture into 80 mass percent ethanol, stirring for 1h at 25 ℃, centrifuging for 20min at 8000g of rotation speed, and taking supernatant to obtain alcohol-soluble cod peptide;
collecting the precipitate to obtain alcohol-insoluble cod peptide;
(2) dealcoholizing: removing ethanol from the alcohol-soluble cod peptide obtained in the step (1) by using a rotary evaporator to 3% of the original volume to obtain dealcoholized alcohol-soluble cod peptide;
(3) desalting: mixing the ordinary cod peptide powder obtained in example 1, the alcohol-insoluble cod peptide obtained in the step (1), and the dealcoholized alcohol-soluble cod peptide obtained in the step (2) in a mass ratio of 1: 5, adding deionized water according to the proportion, and respectively performing osmotic desalination treatment, wherein the osmotic desalination method comprises the following steps: putting the cod peptide into a 100Da dialysis bag, changing dialysis water every 6h at 4 deg.C, and dialyzing for 24 h.
After the salt is removed, obtaining desalted common cod peptide powder, desalted alcohol-soluble cod peptide and desalted alcohol-insoluble cod peptide;
(4) and (3) freeze drying: and (4) freeze-drying the desalted common cod peptide powder, the desalted alcohol-soluble cod peptide and the desalted alcohol-insoluble cod peptide obtained in the step (3) to respectively obtain three kinds of cod peptides.
2. Characterization of cod peptides
Respectively detecting the molecular weight distribution and the amino acid content of the three cod peptides obtained in the step 1; the results are shown in tables 2 to 3 and FIG. 1.
Table 2: molecular weight distribution of plain cod peptide powder, alcohol-soluble cod peptide and alcohol-insoluble cod peptide
Table 3: amino acid content and hydrophobic amino acid content of common cod peptide, alcohol-soluble cod peptide and alcohol-insoluble cod peptide
"#" is a hydrophobic amino acid
(1) Molecular weight distribution analysis:
as can be seen from FIG. 1 and Table 2, the alcohol-soluble cod peptides after alcohol separation have a lower molecular weight, 94.28% of peptides with a molecular weight below 1000Da, wherein 12.08% of peptides with a molecular weight between 600 and 1000Da, 29.22% of peptides with a molecular weight between 300 and 600Da, and 52.97% of peptides with a molecular weight below 300 Da.
The separated ordinary cod peptide powder contains 83.42% peptides with molecular weight below 1000 Da.
The other part of alcohol-insoluble cod peptides after alcohol separation had a molecular weight below 1000Da represented 71.41%.
The above results show that the alcohol-soluble cod peptide obtained by alcohol separation contains more small-molecular-weight peptides and is more likely to have biological activity.
(2) Analysis of amino acid content
The most common residues of the immunomodulatory peptides are hydrophobic amino acids, as shown in table 3, with 17 amino acids detected in total, of which:
the content of hydrophobic amino acid in the common cod peptide accounts for 28.80% of the total amino acid content;
the hydrophobic amino acid content of the alcohol-soluble cod peptide accounts for 38.80 percent of the total amino acid content;
the hydrophobic amino acid content of the alcohol-insoluble cod peptides was 20.71% of the total amino acid content.
The results show that the alcohol-soluble cod peptide obtained by alcohol separation has higher hydrophobic amino acid content and is obviously higher than common cod peptide and alcohol-insoluble cod peptide, so that the alcohol separation method can effectively enrich the peptide segment containing hydrophobic amino acid polypeptide, and the peptide segment containing hydrophobic amino acid polypeptide has immunoregulatory activity more easily.
Example 3: effect of alcohol-soluble cod peptide on NO content released from RAW264.7 cells
The method comprises the following specific steps:
1. culture of RAW264.7 cells
RAW264.7 cells were cultured in complete high-glucose DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% diabody (mixed solution of streptomycin) and the cells were stored at 37 ℃ in the presence of 5% CO2In a moist incubator.
2. Cytotoxicity assays
The RAW264.7 cytotoxicity test is determined by adopting an MTT method, and comprises the following specific steps:
(1) preparing samples with different concentrations:
the desalted plain cod peptide powder, desalted alcohol-soluble cod peptide and desalted alcohol-insoluble cod peptide prepared in example 2 were prepared into cod peptide solutions having concentrations of 100. mu.g/mL, 200. mu.g/mL and 500. mu.g/mL, respectively, using high-glucose DMEM complete medium.
(2) mu.L of RAW264.7 cells per well (cell density 5X 10)5one/mL) were seeded in 96-well plates at 37 ℃ with 5% CO2And (2) incubating for 24 hours, and then incubating for 24 hours by using different samples prepared in the step (1).
mu.L of MTT at a concentration of 5mg/mL was added to each well, incubated under the same conditions for an additional 4h, the supernatant was discarded, and 150. mu.L of dimethyl sulfoxide (DMSO) was added to measure the absorbance at 490 nm. The results are shown in FIG. 2, with an equal amount of medium being used as a blank.
The results show that treatment of RAW264.7 cells with three cod peptides at concentrations of 100 μ g/mL, 200 μ g/mL, 500 μ g/mL, respectively, for 24h, showed no significant difference compared to the blank group (p < 0.05);
among them, the cell survival rates of the common cod peptide powder, the alcohol-soluble cod peptide and the alcohol-insoluble cod peptide are 95.97-108.38%, so that the cod peptide, the alcohol-soluble cod peptide and the alcohol-insoluble cod peptide are considered to have no toxicity to RAW264.7 cells at concentrations of 100. mu.g/mL, 200. mu.g/mL and 500. mu.g/mL.
3. Determination of content of NO released by RAW264.7 cells
mu.L of RAW264.7 cells per well (cell density 5X 10)5one/mL) of cells were seeded in a 96-well plate at 37 ℃ with 5% CO2And (4) incubating for 24 h. The supernatant was discarded and 100. mu.L of each of the different samples prepared in the above step (1) (three kinds of cod peptides having concentrations of 100. mu.g/mL, 200. mu.g/mL, and 500. mu.g/mL, respectively) were added and incubated for 24 hours. After the culture was completed, the cell supernatant was collected.
Meanwhile, Lipopolysaccharide (LPS) is prepared to have a final concentration: 2 μ g/mL lipopolysaccharide solution, as a positive control, and the same amount of medium as a blank control, as described above.
The NO content in the supernatant was measured by using NO assay kits, and the results are shown in table 4 and fig. 3.
Table 4: effect of different concentrations of different cod peptides on NO content released from RAW264.7 cells
The results show that after the RAW264.7 cells are respectively treated by three cod peptides with the concentrations of 100 mu g/mL, 200 mu g/mL and 500 mu g/mL for 24 hours, the common cod peptide can not promote the RAW264.7 cells to release NO, the alcohol-soluble cod peptide can promote the RAW264.7 cells to release NO, and the amount of the released NO is gradually increased along with the increase of the sample concentration, so that the concentration dependence is realized.
The 500 μ g/mL alcohol-soluble cod peptide treated cells were 31.41% higher than the blank, with a significant difference compared to the blank (p < 0.05). The effect of the alcohol-insoluble cod peptides on NO release from RAW264.7 cells was not significant and was not regular, so it was considered that the ability of the alcohol-insoluble cod peptides to promote NO release from RAW264.7 cells was weak.
Therefore, of the three cod peptides, the cod peptide with better capability of promoting the RAW264.7 cells to release NO has alcohol solubility.
Example 4: effect of cod peptide on splenic lymphocytes of mice
The method comprises the following specific steps:
(1) extracting and culturing splenic lymphocytes of mice: the eyeballs of the mice were bled and sacrificed. Taking a mouse spleen under a sterile environment, grinding the spleen by using a syringe piston to prepare a single cell suspension, filtering by using a 70-mesh screen, filtering by using a 100-mesh screen again, rotating at 1000rpm, centrifuging for 5min, removing a supernatant, adding 5mL of erythrocyte lysate (purchased from Solibao), blowing off cells, lysing for 3min, centrifuging for 5min at 1000rpm, removing the supernatant, washing twice with PBS (phosphate buffer solution) for precipitation, and centrifuging for 5min at 1000rpm each time.
The mouse spleen lymphocytes obtained by the above method were cultured in RPMI-1640 complete medium.
(2) The desalted plain cod peptide powder, desalted alcohol-soluble cod peptide and desalted alcohol-insoluble cod peptide prepared in example 2 were prepared into solutions of 1.1mg/mL, 2.2mg/mL and 5.5mg/mL, respectively, using RPMI-1640 complete medium.
The mouse spleen lymphocytes obtained in the step (1) are added into the culture medium according to the proportion that each hole is 100 mu L of the mouse spleen lymphocytes (the cell density is 1 multiplied by 10)6one/mL) was inoculated on a 96-well plate, 10. mu.L of each of the samples (three kinds of cod peptides) prepared as described above at different concentrations were added to give final concentrations of 100. mu.g/mL, 200. mu.g/mL, and 500. mu.g/mL; then the reaction system is processed at 37 ℃ and 5% CO2Respectively incubating for 24h, 48h and 72h under the environment of (1).
After the incubation is finished, adding 10 mu L of CKK-8 into each hole, and incubating for 3h under the same conditions; detecting a light absorption value at 450nm after the incubation is finished; making a blank control by using an equivalent culture medium; the results are shown in Table 5 and FIGS. 4 to 6.
Table 5: effect of various concentrations of cod peptide on mouse splenic lymphocyte proliferation
The result shows that after three kinds of cod peptides are respectively treated for 24 hours, the common cod peptide has no capacity of promoting the mouse spleen lymphocyte proliferation, the alcohol-soluble cod peptide has the capacity of obviously promoting the mouse spleen lymphocyte proliferation, and has a significant difference (p is less than 0.05) compared with a blank group, and the alcohol-insoluble cod peptide has a certain capacity of promoting the mouse spleen lymphocyte proliferation but has no concentration dependence.
When the cell is cultured for 48 hours, the common cod peptide shows certain capacity of promoting the mouse spleen lymphocyte proliferation activity, the cell treated by the alcohol-soluble cod peptide increases along with the culture time, the alcohol-soluble cod peptide shows strong proliferation promoting activity, the proliferation rate of the alcohol-soluble cod peptide gradually increases, the mouse spleen lymphocyte proliferation activity can be improved by 34.79% at 500 mu g/mL, the cell proliferation activity is obviously higher than that of a blank group, and the alcohol-insoluble cod peptide also has the capacity of promoting the mouse spleen lymphocyte proliferation activity but has no strong effect of the alcohol-soluble cod peptide.
When the culture time reaches 72h, the cell proliferation rate is slightly reduced compared with 48h, which is probably because the cell density is higher, the contact inhibition is partially generated, but the proliferation activity is still significantly different from that of the blank group (p < 0.05). Therefore, the alcohol-soluble cod peptide can promote the mouse spleen lymphocyte proliferation and is dose-dependent.
In conclusion, the alcohol-soluble cod peptide can promote NO release of RAW264.7 cells and spleen lymphocyte proliferation of mice, and has a certain immunoregulation activity. The alcohol-soluble cod peptide can be applied to development of health-care food and medicines related to diseases such as immunoregulation and the like. The invention develops the application of the cod peptide, lays a certain foundation for the application of the cod active peptide in the field of functional foods and the like
Example 5: effect of alcohol-soluble cod peptide on splenic lymphocytes of mice
The method comprises the following specific steps:
(1) extracting and culturing splenic lymphocytes of mice: the eyeballs of the mice were bled and sacrificed. Taking a mouse spleen under a sterile environment, grinding the spleen by using a syringe piston to prepare a single cell suspension, filtering by using a 70-mesh screen, filtering by using a 100-mesh screen again, rotating at 1000rpm, centrifuging for 5min, removing a supernatant, adding 5mL of erythrocyte lysate (purchased from Solibao), blowing off cells, lysing for 3min, centrifuging for 5min at 1000rpm, removing the supernatant, washing twice with PBS (phosphate buffer solution) for precipitation, and centrifuging for 5min at 1000rpm each time.
The mouse spleen lymphocytes obtained by the above method were cultured in RPMI-1640 complete medium.
(2) The desalted alcohol-soluble cod peptide prepared in example 2 was prepared into solutions of 0.11mg/mL, 0.55mg/mL, 1.10mg/mL, 2.20mg/mL, 5.50mg/mL, 7.70mg/mL using RPMI-1640 complete medium.
The mouse spleen lymphocyte proliferation index determination method comprises the following steps: the mouse spleen lymphocytes obtained in the step (1) are added into the culture medium according to the proportion that each hole is 100 mu L of the mouse spleen lymphocytes (the cell density is 1 multiplied by 10)6one/mL) was inoculated into a 96-well plate, and 10. mu.L of each of the samples prepared in step (1) and having different concentrations were added to the plate so as to give final concentrations of 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 500. mu.g/mL, and 700. mu.g/mL, respectively. Then 5% CO at 37 ℃2Respectively incubating for 48h under the environment of (1).
The mouse T lymphocyte proliferation index determination method comprises the following steps: inducing the mouse spleen lymphocytes obtained in the step (1) by using ConA with the final concentration of 1 mu g/mL to induce the mouse spleen lymphocytes into T lymphocytes. mu.L of mouse T spleen lymphocytes per well (cell density 1X 10)6one/mL) was inoculated into a 96-well plate, and 10. mu.L of each of the samples prepared in step (1) and having different concentrations were added to the plate so as to give final concentrations of 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 500. mu.g/mL, and 700. mu.g/mL. Then 5% CO at 37 ℃2Respectively incubating for 48h under the environment of (1).
The mouse B lymphocyte proliferation index determination method comprises the following steps: inducing the mouse spleen lymphocytes obtained in the step (1) into B lymphocytes by using LPS with the final concentration of 10 mug/mL. mu.L of mouse B spleen lymphocytes per well (cell density 1X 10)6one/mL) was inoculated into a 96-well plate, and 10. mu.L of each of the samples prepared in step (1) and having different concentrations were added to the plate so as to give final concentrations of 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 500. mu.g/mL, and 700. mu.g/mL. Then 5% CO at 37 ℃2Respectively incubating for 48h under the environment of (1).
After the incubation is finished, adding 10 mu L of CKK-8 into each hole, and incubating for 3h under the same conditions; detecting a light absorption value at 450nm after the end; making a blank control by using an equivalent culture medium; the results are shown in Table 6 and FIGS. 7 to 8.
Table 6: effect of alcohol-soluble cod peptide of different concentrations on mouse spleen lymphocyte, T lymphocyte and B lymphocyte proliferation
The result shows that the alcohol-soluble cod peptide can promote the mouse spleen lymphocyte proliferation, the stimulation index is increased along with the increase of the concentration of the alcohol-soluble cod peptide, and the stimulation index is 1.18 when the alcohol-soluble cod peptide is cultured for 48 hours at 700 mu g/mL.
ConA can induce T lymphocyte mitosis, LPS can induce B lymphocyte mitogen, promote cell growth and immunoglobulin release; as can be seen from FIG. 8, alcohol-soluble cod peptide promoted ConA-induced T cell proliferation, and 500. mu.g/mL of alcohol-soluble cod peptide had a T cell proliferation index of 1.11, which is higher than that of the other groups.
However, alcohol-soluble cod peptides at different concentrations had no significant proliferative effect on LPS-induced B cells. These results indicate that alcohol-soluble cod peptide can promote mouse spleen lymphocyte proliferation, T lymphocyte proliferation, but not B lymphocyte proliferation.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The cod peptide powder is characterized by being prepared according to the following method:
s1, grinding the cod: adding water into the cod, and grinding to obtain cod mixed solution;
s2, enzymolysis: adding alkali into the cod mixed liquor obtained in the step S1, adjusting the pH value to 8.0-8.5, heating to 55-58 ℃, adding alkaline protease according to the proportion of 0.19-0.21%, uniformly stirring, performing enzymolysis for 1.8-2.2 h, heating again to inactivate enzyme for 10-15 min, and obtaining cod enzymatic hydrolysate;
s3, spray drying: spray drying the cod enzymolysis liquid obtained in the step S2 to obtain cod peptide powder;
s4, separation preparation: adding the cod peptide powder obtained in the step S3 into 75-85% of ethanol according to the mass ratio of (1: 18) - (1: 22), stirring for 1-2 h at 20-30 ℃, centrifuging for 20-30 min at the rotating speed of 7500-8500 g, and taking supernatant to obtain alcohol-soluble cod peptide;
s5, dealcoholization: removing ethanol from the alcohol-soluble cod peptide obtained in the step S4 by using a rotary evaporator to 3-5% of the original volume to obtain alcohol-removed alcohol-soluble cod peptide;
s6, desalting: and (4) adding deionized water into the dealcoholized alcohol-soluble cod peptide obtained in the step (S5) according to the volume ratio of 1 (5-7), respectively performing osmotic desalting treatment, removing salt to obtain the cod peptide, and freeze-drying and storing the cod peptide.
2. A product comprising the cod peptide of claim 1.
3. The product of claim 2, wherein the product is a food, pharmaceutical or nutraceutical product.
4. A product as claimed in claim 2 or claim 3 wherein said food is a protein powder, a confectionery, a drink containing said cod peptides.
5. A product according to claim 4, wherein the product comprises cod peptide according to claim 1, a pharmaceutical carrier and/or a pharmaceutical excipient.
6. Use of cod peptides according to claim 1 for the preparation of a product with immunomodulatory activity.
7. The use of claim 6, wherein the product is a pharmaceutical or nutraceutical product.
8. Use according to claim 7, wherein the medicament comprises cod peptide according to claim 1, a pharmaceutical carrier and/or a pharmaceutical excipient.
9. Use of cod peptide according to claim 1 in the manufacture of a product for promoting NO release from RAW264.7 cells.
10. Use of cod peptide according to claim 1 in the manufacture of a product for promoting proliferation of splenic lymphocytes in mice.
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| JP2010001262A (en) * | 2008-06-23 | 2010-01-07 | Kita Nippon Kagaku Kk | Functional composition and health maintenance food |
| CN107151686A (en) * | 2017-05-15 | 2017-09-12 | 中国海洋大学 | A kind of cod albumen of highly dissoluble |
| CN109806383A (en) * | 2019-03-22 | 2019-05-28 | 深圳大学 | A kind of sea eel peptide promotes the application in immune food, drug or health care product in preparation |
| CN110521849A (en) * | 2019-08-26 | 2019-12-03 | 大连工业大学 | A kind of preparation method and applications with the high gadus protein peptides for digesting and assimilating characteristic |
| CN111763243A (en) * | 2020-05-22 | 2020-10-13 | 浙江海洋大学 | Gorgon fish immune active peptide and preparation method and application thereof |
| CN112877390A (en) * | 2021-01-28 | 2021-06-01 | 江苏大学 | Preparation method of functional alcohol-soluble sturgeon cartilage preparation |
| CN113789361A (en) * | 2021-09-24 | 2021-12-14 | 王朝辉 | Cod peptide and preparation method and application thereof |
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Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2010001262A (en) * | 2008-06-23 | 2010-01-07 | Kita Nippon Kagaku Kk | Functional composition and health maintenance food |
| CN107151686A (en) * | 2017-05-15 | 2017-09-12 | 中国海洋大学 | A kind of cod albumen of highly dissoluble |
| CN109806383A (en) * | 2019-03-22 | 2019-05-28 | 深圳大学 | A kind of sea eel peptide promotes the application in immune food, drug or health care product in preparation |
| CN110521849A (en) * | 2019-08-26 | 2019-12-03 | 大连工业大学 | A kind of preparation method and applications with the high gadus protein peptides for digesting and assimilating characteristic |
| CN111763243A (en) * | 2020-05-22 | 2020-10-13 | 浙江海洋大学 | Gorgon fish immune active peptide and preparation method and application thereof |
| CN112877390A (en) * | 2021-01-28 | 2021-06-01 | 江苏大学 | Preparation method of functional alcohol-soluble sturgeon cartilage preparation |
| CN113789361A (en) * | 2021-09-24 | 2021-12-14 | 王朝辉 | Cod peptide and preparation method and application thereof |
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