Method for preparing collagen peptide and application thereof in improving immunity

Abstract

This invention provides a method for preparing collagen peptides and their application in enhancing immunity. Furthermore, this invention screened and isolated a collagen peptide MY-4 with good antioxidant properties, which exhibits a strong protective effect against thymic lymphocytes. The peptide can effectively increase the phagocytic rate and phagocytic index of macrophages. Therefore, the collagen peptides of this invention can effectively enhance immunity and have promising application prospects.

Description

Technical Field

[0001] This application relates to the field of biology, and more specifically to methods for preparing collagen peptides and their application in enhancing immunity. Background Technology

[0002] The immune system, a vital system for maintaining human health and internal environmental balance, is closely related to various physiological functions. It is the most effective weapon against invading pathogens, capable of recognizing and eliminating antigenic foreign substances, and coordinating with other systems to maintain internal homeostasis and physiological balance. Preventing immune system disorders and improving immunity are issues of great concern and pursuit.

[0003] Collagen peptides are small peptide fragments obtained from collagen through enzymatic hydrolysis and other processes. For example, they can stimulate the proliferation of lymphocytes, which play a crucial role in the immune response. Lymphocytes, including T lymphocytes involved in cellular immunity and B lymphocytes involved in humoral immunity, are promoted by collagen peptides, which help enhance the body's immune defense capabilities. They can also enhance the phagocytic function of macrophages. Macrophages act like the body's "scavengers," engulfing foreign pathogens and some aging and damaged cells. Collagen peptides activate macrophages, enabling them to better perform their phagocytic function, thereby strengthening the body's non-specific immune response. Furthermore, collagen peptides can promote the secretion of immune-related cytokines. These cytokines coordinate communication and interactions between immune cells, helping the body to mount a more effective immune response against pathogens and maintain the balance and stability of the immune system. Collagen peptides can significantly enhance the proliferation of lymphocytes induced by concanavalin A, the number of antibody plaques formed, and the activity of natural killer cells. They can also increase the percentage of CD4+ T helper cells in the spleen of mice and the secretion of cytokines IL-2, IFN-γ, IL-5, and IL-6.

[0004] Collagen peptides are short peptides derived from the hydrolysis of collagen molecules, exhibiting good bioactivity and bioavailability. Their preparation methods mainly include hydrolysis, enzymatic hydrolysis, photochemical methods, and heating methods. Hydrolysis is a commonly used method for preparing collagen peptides. This method involves raw material preparation, enzymatic hydrolysis, cessation of enzymatic hydrolysis, and extraction of collagen peptides. Raw materials, such as animal bones, skin, or fish scales, need to be prepared. Then, the raw materials are pretreated, including removing impurities and washing. Next, the pretreated raw materials undergo enzymatic hydrolysis with suitable enzymes; commonly used enzymes include pepsin, adhesin, and lysozyme. During the enzymatic hydrolysis reaction, the degree of collagen hydrolysis can be adjusted by controlling the hydrolysis time and temperature. After the enzymatic hydrolysis reaction is complete, it needs to be stopped, usually by adding enzyme inhibitors or adjusting the reaction conditions. Collagen peptides are then extracted from the reaction solution using methods such as centrifugation and ultraviolet absorption. Enzymatic hydrolysis is another commonly used method for preparing collagen peptides. This method utilizes specific enzymes, such as pepsin, collagenase, collagenase, and collagen tripeptidase, to enzymatically hydrolyze collagen. The conditions and steps of the enzymatic hydrolysis reaction are similar to those of the hydrolysis method, but the choice of enzymes differs. Collagen peptides prepared by enzymatic hydrolysis typically have high purity and bioactivity, but the hydrolysis process is relatively long, requiring a considerable reaction time. The heating method is a simple and easy way to prepare collagen peptides. This method involves heating the collagen raw material to induce a hydrolysis reaction, thereby obtaining collagen peptides. Collagen peptides prepared by the heating method typically have lower purity and bioactivity, but the operation is simple and requires minimal equipment, therefore it is widely used in some practical applications.

[0005] Collagen peptides prepared by different methods have different sequences and functions. Therefore, developing collagen peptides with better anti-immune effects is an important research direction. Summary of the Invention

[0006] This invention overcomes the deficiencies of the prior art and provides an isolated collagen peptide, wherein the collagen peptide is MY-4 and the amino acid sequence is shown in SEQ ID NO: 1.

[0007] Specifically, the polypeptides of the present invention can be modified or have amino acids replaced, while still maintaining the corresponding polypeptide activity.

[0008] Furthermore, the polypeptide may also have minor amino acid substitutions, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1 to 3 amino acids.

[0009] Examples of conserved substitutions include substitutions within the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific reactivity are known in the art and can be found, for example, in H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. The most common exchanges are alanine to serine, valine to isoleucine, aspartic acid to glutamic acid, threonine to serine, alanine to glycine, alanine to threonine, serine to asparagine, alanine to valine, serine to glycine, tyrosine to phenylalanine, alanine to proline, lysine to arginine, aspartic acid to asparagine, leucine to isoleucine, leucine to valine, alanine to glutamic acid, and aspartic acid to glycine.

[0010] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6- / V-methyllysine, 2-aminoisobutyric acid, isovaleine, and α-methylserine) may substitute amino acid residues in wild-type polypeptides. A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, and non-natural amino acids may substitute amino acid residues. "Non-natural amino acids" are modified after protein synthesis and / or have chemical structures in their side chains that differ from those of standard amino acids. Non-natural amino acids are chemically synthesized and preferably commercially available, and include piperidinic acid, thiazolidinic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

[0011] The present invention further provides a composition for enhancing immunity, characterized in that it contains the collagen peptide of the present invention, which is MY-4, and the amino acid sequence is shown in SEQ ID NO: 1.

[0012] The compositions provided herein may contain inorganic or organic buffers (e.g., sodium or potassium salts of phosphates, carbonates, acetates, or citrates) and pH adjusters (e.g., hydrochloric acid, sodium or potassium hydroxide, citrates or acetates, amino acids and their salts), antioxidants (e.g., ascorbic acid, α-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), solution and / or low-temperature / lyophilization stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic conditioners (e.g., salts or sugars), antimicrobial agents (e.g., benzoic acid, phenol, gentamicin), defoamers (e.g., polydimethylsiloxane), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymer stabilizers and viscosity modifiers (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethyl cellulose), and cosolvents (e.g., glycerol, polyethylene glycol, ethanol).

[0013] The compositions of this invention may further include one or more additional components selected from a wide variety of excipients known in the pharmaceutical formulation field. Any number of components may be selected, used alone or in combination, depending on the desired properties of the tablets or capsules, the selection being based on their known use in the preparation of the compositions of this invention. Such components include, but are not limited to, water; non-aqueous solvents (e.g., ethanol); coating agents; capsule shells; colorants; waxes; gelling agents; flavoring agents; preservatives (e.g., methyl benzoate, sodium benzoate, and potassium benzoate); antioxidants [e.g., butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E and vitamin E esters such as tocopheryl acetate]; flavor enhancers; sweeteners (e.g., aspartame and saccharin); concentrating agents; surfactants, etc.

[0014] One embodiment of the invention is a pharmaceutical composition suitable for parenteral administration, comprising a compound of formula (I) and a surfactant system comprising a polymer composition that allows the compound of formula (I) to be released over a period of one to three months. Suitable combinations of polymers are, for example, polysorbate 20 and polyethylene glycol (PEG) 3350.

[0015] A suitable combination of polymers, namely wetting agents and stabilizers, is required to produce stable suspensions. Wetting agents can be selected from nonionic and ionic surfactant classes. Representative examples of wetting agents include: polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters (polysorbates), dehydrated sorbitan esters of fatty acids (SPAN), poloxamer, TM such as LUTROL F68, F108 and F127, which are block copolymers of ethylene oxide and propylene oxide, sodium dodecyl sulfate and sodium dodecyl sulfonate.

[0016] Representative stabilizers include, but are not limited to: polyethylene glycol, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxymethylpropyl cellulose, polysaccharides, hyaluronic acid, polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP).

[0017] Examples of combinations of polymers include: polysorbates, such as polysorbate 20 or polysorbate 60 as a wetting agent, and polyethylene glycol (PEG) as a stabilizer, such as PEG 3350, PEG 4000 or PEG 8000.

[0018] In one embodiment, the pharmaceutically acceptable wetting agent or surfactant is present in an amount of about 0.01% to about 5.0% (weight / volume), preferably about 0.05% to about 2.0% (weight / volume), more preferably about 0.1% to about 1.0% (weight / volume), for example about 0.1%, 0.2%, 0.5%, 1.0% (weight / volume).

[0019] Examples of suitable pharmaceutically acceptable isotonic modifiers include, but are not limited to, D-mannitol, glucose, glycerol, sodium chloride, potassium chloride, calcium chloride, and magnesium chloride, or any combination thereof. Various nitrates, citrates, acetates, or mixtures thereof may also be used. In one embodiment, the pharmaceutically acceptable isotonic modifier is present in an amount of about 0.1% to about 5.0% (weight / volume), preferably about 1% to about 3% (weight / volume).

[0020] Examples of suitable pharmaceutically acceptable preservatives include, but are not limited to, benzalkonium chloride, benzyl chloride and cetylpyridine chloride, benzyl bromide, benzyl alcohol, disodium EDTA, phenylmercuric nitrate, phenylmercuric acetate, sodium ethylmercuric thiosalicylate, thimerosal, acetate and borate phenylmercuric acid, polymyxin B sulfate, chlorhexidine, methylparaben and propylparaben, phenethyl alcohol, quaternary ammonium chloride, sodium benzoate, sodium propionate, stabilized oxychloro complex, sorbic acid, or mixtures thereof. Preferred pharmaceutically acceptable preservatives include disodium EDTA (ethylenediaminetetraacetic acid disodium salt) and benzalkonium chloride, or mixtures thereof. In one embodiment, the pharmaceutically acceptable preservative is present in an amount from about 0.01% to about 2.0% (weight / volume), preferably from about 0.05% to about 1% (weight / volume). Examples of suitable pharmaceutically acceptable buffers include, but are not limited to, sodium chloride, glucose, lactose and phosphate-buffered saline (PBS), or any combination thereof. Other suitable pharmaceutically acceptable buffers include, but are not limited to, disodium succinate hexahydrate, borate, citrate, phosphate, acetate, physiological saline, tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), HEPES, sodium phosphate, sodium borate, physiological saline, citrate, carbonate, phosphate and / or mixtures thereof, to obtain the desired osmolarity. In one embodiment, the pharmaceutically acceptable buffer is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume).

[0021] Typically, the pharmaceutical compositions of the present invention comprise 0.1-50% by weight of a polypeptide. Typically, the pharmaceutical compositions of the present invention comprise 0.1-5% polysorbate 20 as a surfactant and 0.1-5% polyethylene glycol. The pharmaceutical compositions of the present invention may comprise 0.1-10% polysorbate 20 as a surfactant and 0.1-10% polyethylene glycol.

[0022] The compositions of the present invention can also be in the form of an emulsion. An emulsion is a two-phase system prepared by mixing two immiscible liquid carriers, wherein one carrier is uniformly dispersed in the other and consists of microspheres with a diameter equal to or greater than the maximum particle diameter. The microsphere size is critical and must allow the system to achieve maximum stability. Typically, separation of the two phases will not occur unless a third substance, i.e., an emulsifier, is incorporated. Thus, a basic emulsion contains at least three components: two immiscible liquid carriers and an emulsifier, and an active ingredient. Most emulsions involve mixing an aqueous phase into a non-aqueous phase (or vice versa). However, it is possible to prepare substantially non-aqueous emulsions, for example, anionic and cationic surfactants in a non-aqueous immiscible system of glycerol and olive oil. Therefore, the compositions of the present invention can be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin, or a mixture thereof. Suitable emulsifiers can be naturally occurring gums, such as gum arabic or astragalus gum; naturally occurring phospholipids, such as soybean phospholipids and lecithin; and esters or metaesters obtained from fatty acids and hexitanic anhydrides, such as sorbitan monooleate; and condensation products of metaesters and ethylene oxide, such as polyoxyethylene sorbitan monooleate.

[0023] According to some other embodiments, the therapeutic amount of the collagen peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 100 mg / kg body weight. According to another embodiment, the therapeutic amount of the collagen peptide in the pharmaceutical composition is from about 0.00001 mg / kg body weight to about 100 mg / kg body weight. According to another embodiment, the therapeutic amount of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 2 mg / kg body weight to about 10 mg / kg body weight. According to another embodiment, the therapeutic amount of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 3 mg / kg body weight to about 10 mg / kg body weight. According to another embodiment, the therapeutic amount of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 4 mg / kg body weight to about 10 mg / kg body weight. According to another embodiment, the therapeutic amount of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 5 mg / kg body weight to about 10 mg / kg body weight. According to another embodiment, the therapeutic amount of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 60 mg / kg body weight to about 100 mg / kg body weight. According to another embodiment, the therapeutic dose of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 70 mg / kg body weight to about 100 mg / kg body weight. According to another embodiment, the therapeutic dose of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 80 mg / kg body weight to about 100 mg / kg body weight. According to another embodiment, the therapeutic dose of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 90 mg / kg body weight to about 100 mg / kg body weight. According to another embodiment, the therapeutic dose of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 90 mg / kg body weight. According to another embodiment, the therapeutic dose of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 80 mg / kg body weight. According to another embodiment, the therapeutic dose of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 70 mg / kg body weight. According to another embodiment, the therapeutic dose of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 60 mg / kg body weight. According to another embodiment, the therapeutic dose of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 50 mg / kg body weight. According to another embodiment, the therapeutic dose of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 40 mg / kg body weight. According to another embodiment, the therapeutic dose of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 30 mg / kg body weight.According to another embodiment, the therapeutic amount of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 20 mg / kg body weight. According to another embodiment, the therapeutic amount of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 10 mg / kg body weight. According to another embodiment, the therapeutic amount of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 1 mg / kg body weight. According to another embodiment, the therapeutic amount of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 0.1 mg / kg body weight. According to another embodiment, the therapeutic amount of the therapeutic inhibitory peptide in the pharmaceutical composition is from about 0.000001 mg / kg body weight to about 0.01 mg / kg body weight.

[0024] Furthermore, the pharmaceutical composition of the present invention may also include a second therapeutic agent.

[0025] The second therapeutic agent can be any suitable second drug that enhances the body's immunity. The second drug can be ginseng extract.

[0026] Beneficial effects This invention provides a method for preparing collagen peptides and their application in enhancing immunity. Furthermore, this invention screened and isolated a collagen peptide MY-4 with good antioxidant properties, which exhibits a strong protective effect against thymic lymphocytes. The peptide can effectively increase the phagocytic rate and phagocytic index of macrophages. Therefore, the collagen peptides of this invention can effectively enhance immunity and have promising application prospects. Attached Figure Description

[0027] Figure 1 Figure 1 shows the scavenging rate of DPPH free radicals by different concentrations of MY-4 peptide. Figure 2 Phagocytosis rate results for each group Detailed Implementation Specific embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0028] Example 1: Preparation of Collagen Peptides Take sea bream skin, crush it, soak it in 6M HCl for 2 hours, rinse it with distilled water, and sterilize it by moist heat at 121℃ for 20 minutes. Then, enzymatically hydrolyze it with papain at pH 5, temperature 50℃, and enzyme dosage of 1000u / g for 6 hours. After enzyme inactivation, enzymatically hydrolyze it with alkaline protease at pH 8.5, temperature 45℃, and enzyme dosage of 1000u / g for 24 hours. After enzyme inactivation at 90℃ for 15 minutes, centrifuge it at 8500r / min at 4℃ for 15 minutes and collect the supernatant.

[0029] The supernatant was subjected to ultrafiltration to remove components with a molecular weight cutoff of less than 3 kDa. The components were then separated by column chromatography using a DEAE Sepharose FF anion exchange column. The component with the strongest scavenging ability was further separated by reversed-phase high-performance liquid chromatography using the DPPH free radical scavenging ability assay, and the main peak with the strongest antioxidant ability was obtained. The amino acid sequence of this main peak was identified by sequencing, as shown in SEQ ID NO: 1, and named MY-4.

[0030] Example 2: Identification of the antioxidant capacity of MY-4 peptide Take 2 mL of sample solution and 1×10 －4 Add 2 mL of mol / L DPPH solution to a stoppered test tube and shake well. Incubate at room temperature in the dark for 30 min. Using pure solvent as a reference, measure the absorbance at a wavelength of 517 nm. Calculate the clearance rate using the following formula: Clearance rate = [1 - (A)] S -A SB ) / A C [×100%. Where: A] S A represents the absorbance of the DPPH solution after adding the sample solution. SB A represents the absorbance of the sample solution. C The absorbance of the DPPH solution without sample solution is shown. The scavenging rate of the antioxidant Vitamin C on DPPH free radicals was measured for comparison; specific concentrations and results are shown below. Figure 1 As shown.

[0031] from Figure 1 It can be seen that as the concentration of the MY-4 peptide gradually increases from 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, to 2.0 mg / mL, the DPPH free radical scavenging ability also increases. The DPPH free radical scavenging ability of the 1 mg / mL peptide group was (79.32±2.86)%, compared with that of the positive control group (45.83±1.06)%. This fully demonstrates that the peptide of the present invention has good antioxidant properties.

[0032] Example 3: Effects of MY-4 peptide on immune cells in vitro Mouse thymic lymphocytes (catalog number: CP-M197, Wuhan Pronosei Life Sciences Co., Ltd.) were cultured and their concentration was adjusted to 2×10⁻⁶. 6 Cells were cultured at a density of 1 ml / well, and the cell suspension was divided into 5 groups. Each group was seeded in a 24-well plate at 1 ml per well, with 5 replicates per group. RPMI-1640 was added as a negative control (CK), and MY-4 peptide was added at different concentrations (0.1%, 0.2%, and 0.5%). A positive control group (levamisole hydrochloride 0.2%) was also included. Cells were incubated for 12 hours at an incubation temperature of 3.0 × 10⁻⁶. -5 J / cm 2 After irradiation with ultraviolet light, 100 μL of MTT solution was added to each well of each group, and the cells were incubated at 37°C for 4 hours before the culture was terminated. The cells were centrifuged to remove the nutrient solution, collected, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The cells were shaken for 10 minutes, and the absorbance of each well was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The results (OD values) were recorded, and the OD values ​​were used to reflect the protective effect of the peptide on thymic lymphocytes. The results are shown in Table 1.

[0033] Table 1. Protective effects of MY-4 peptide on immune cells. As shown in Table 1, the use of peptides and positive controls significantly increased the OD value of immune cells after radiation irradiation compared to the negative control, with a significant difference compared to the negative control. # (P<0.05); This indicates that the MY-4 peptide of the present invention has a good protective effect on immune cells.

[0034] Example 4: In vivo experiments of MY-4 peptide Half of the purchased clean-grade Kunming mice were male and half female, weighing 18–24 g. The experimental animals were randomly divided into four groups of 20 mice each: a normal control group (saline group), three MY-4 peptide groups (low-dose group [10 mg / (kg·bw)], medium-dose group [50 mg / (kg·bw)], and high-dose group [200 mg / (kg·bw)]), and a positive control group (levamisole hydrochloride [50 mg / (kg·bw)]). 0.3 mL of the MY-4 peptide was administered by gavage twice daily for two weeks. Twelve hours after the last administration, the mice were weighed. After weighing, the mice were euthanized by cervical dislocation. The animals were dissected, and the thymus and spleen were removed. Blood was blotted dry with filter paper, and the mice were immediately weighed using an electronic balance. The data were recorded, and the thymus index and spleen index were calculated using the following formulas: Thymus index = thymus weight (mg) / body weight (g) × 100%; Spleen index = spleen weight (mg) / body weight (g) × 100%.

[0035] Table 2. Effects of MY-4 peptide on immune organ indices in mice (x ± s) The spleen and thymus are important immune organs in animals, and their quality can reflect the strength of their immune function to a certain extent. Table 2 shows that after the addition of polypeptides, the spleen and thymus indices increased with increasing concentration compared to the normal control group, indicating that the addition can enhance immune function.

[0036] The same administration method for mice was used as described above. Subsequently, 1 mL of 20% chicken erythrocyte suspension was injected intraperitoneally into each mouse. After 30 minutes, the mice were euthanized by cervical dislocation and fixed supine on a wax plate. The abdominal skin was cut open from the midline, and 2 mL of physiological saline was injected into the peritoneal cavity. The plate was rotated for 1 minute to ensure thorough mixing of the saline and peritoneal fluid. Then, 1 mL of peritoneal fluid was aspirated and evenly distributed onto two glass slides, placed in an enamel box lined with damp gauze, and incubated at 37°C for 30 minutes. After incubation, the slides were removed and rinsed in physiological saline to remove attached cells. After drying, the slides were fixed with a 1:1 acetone-methanol solution for 20 minutes, stained with Giemsa stain for 3 minutes, rinsed with distilled water, and dried. Macrophages were counted under a microscope. The phagocytic rate was calculated using the formula: Phagocytic rate (%) = Macrophages phagocytosed chicken erythrocytes / Counted macrophages × 100%. The results are as follows: Figure 2 As shown.

[0037] Figure 2 The results showed that the low-dose group, medium-dose group, high-dose group, and positive control group all significantly increased the macrophage phagocytic rate compared with the normal control group (P<0.05); in particular, the phagocytic rate of the high-dose group reached (79.6±2.8)%, which was significantly higher than that of the positive control group (62.8±2.5)%.

[0038] When implementing or testing embodiments of the present invention, optional methods and materials similar to or equivalent to those described in this specification may be used, although preferred methods, apparatus, and materials are described in this specification. However, before describing the materials and methods of the present invention, it should be understood that the specific sizes, shapes, dimensions, materials, methods, means, etc., described in this specification can be modified according to conventional experimental methods and for optimization purposes; therefore, the present invention is not limited to these. Furthermore, it should be understood that the technical terms used in this specification are only used to describe specific types or embodiments and are not intended to limit the scope of the present invention, which is limited only by the scope of the appended claims.

Claims

1. An isolated collagen peptide, characterized in that... The collagen peptide is MY-4, and its amino acid sequence is shown in SEQ ID NO:

1.

2. A pharmaceutical composition for enhancing immunity, characterized in that... It contains the collagen peptides as described in claim 1.

3. Use of the collagen peptide as described in claim 1 in the preparation of a drug for enhancing immunity.

4. The use as described in claim 3, wherein the medicament comprises a suitable carrier or excipient.

5. The use as described in claim 3, wherein the drug is further supplemented with a suitable second agent to enhance immunity.

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