Oyster peptide and its application in improving immunity

Abstract

This invention provides an oyster peptide and its application in enhancing immunity. The oyster peptide has a novel amino acid structure. The oyster peptide can enhance the activity of immune cells, promote the proliferation of immune cells, promote the development and maturation of immune organs, regulate the in vivo immune environment, promote the secretion of immunoglobulins, and improve humoral immunity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology research and development, and specifically provides an oyster peptide and its application in improving immunity. Background Technology

[0002] Oysters, also known as oysters, are a type of shellfish widely distributed in coastal areas worldwide. They belong to the phylum Mollusca and class Bivalvia. They are not only a delicious seafood ingredient but also possess rich chemical components and various pharmacological effects. For example, the Compendium of Materia Medica records that oysters have the effects of "nourishing yin and tonifying the kidneys, consolidating essence and stopping leukorrhea." The Shennong's Classic of Materia Medica states that "oysters are salty and neutral in nature." Modern research shows that oysters are rich in diverse nutrients, including proteins, polysaccharides, minerals, amino acids, fatty acids, and oyster alkaloids (see Ren Yuyu, Lei Genping, Wang Bingyan, et al., Research on the Pharmacological Effects of Oysters, Journal of Liaoning University of Traditional Chinese Medicine, 2026, 28(04): 49-54).Amino acids are one of the main components of oysters. Oysters are rich in a variety of amino acids that the human body cannot synthesize on its own, and they play an important regulatory role in the body's metabolism (see Kakaroukas A, Abrahamse-Berkeveld M, Berrington JE, et al. An Observational Cohort Study and Nested Randomized Controlled Trial on Nutrition and Growth Outcomes in Moderate and Late Preterm Infants (FLAMINGO). Front Nutr, 2021, 8:561419). Oysters are also rich in minerals, especially zinc, selenium, iron, calcium, and magnesium. Zinc is particularly abundant in oysters, and it can regulate immune responses, enhance antioxidant capacity, promote wound healing, and play an important role in the prevention and treatment of immunodeficiency and aging (see Khalifa SAM, Elias N, Farag MA, et al. Marine Natural Products: A Source of Novel Anticancer Drugs. Mar Drugs, 2019, 17(9):). 491-522); Oyster polysaccharides are important components of oysters, mainly derived from the viscera of oysters. Oyster polysaccharides have significant immunomodulatory, antioxidant, anti-inflammatory and antitumor activities (see Wang TT, Deng JQ, Chen LZ, et al. The second member of the bacterial UDP-N-acetyl-d-glucosamine heparosan alpha-1, 4-N-acetyl-d-glucosaminyltransferase superfamily: GaKfiA from Gallibacterium anatis, Int JBiol Macromol, 2020, 147:170-176).

[0003] Among the many derivatives of oysters, oyster peptides have a wide range of biological activities. They exert multifunctional effects through different molecular mechanisms, such as free radical scavenging, signaling pathway regulation, gut microbiota regulation, and cell function protection, including antioxidant, anti-hyperglycemic, antihypertensive, anti-inflammatory, and anti-cancer effects. Oyster peptides exert their functions through multiple physiological pathways, including: First, oyster peptides are rich in amino acids such as aspartic acid, glutamic acid, cysteine, leucine, lysine, and arginine. These amino acids can work synergistically to achieve antioxidant effects by directly scavenging free radicals, inhibiting lipid peroxidation, and enhancing the activity of endogenous antioxidant enzymes (including superoxide dismutase and catalase). Compared with antioxidants from other sources, oyster peptides have low toxicity and good bioavailability. Their complex composition enables them to maintain stable antioxidant activity under various physiological conditions (see Yang X, Wang S, Liu H, et al. A dual absorption pathway of novel oyster-derived peptide-zinc complex enhances zinc bioavailability and restores mitochondrial function. Journal of Advanced Research, 2025, 78: 47–62.). Second, it has been reported that oyster peptides can improve glucose metabolism disorders in mice fed a high-fat and high-sugar diet, thereby activating the insulin signaling pathway and upregulating glucose transporter 2 (GLUT2) in the liver and glucose transporter 4 (GLUT4) in skeletal muscle, thereby enhancing glucose uptake and utilization (see Chen Z, Su X, Cao W, et al. The discovery and characterization of a potent DPP-IVinhibitory peptide from oysters for the treatment of type 2 diabetes based on computational and experimental studies. Marine Drugs, 2024, 22(8): 361).Third, oyster peptides exhibit anti-inflammatory activity due to their unique amino acid composition and structural characteristics. For example, they effectively alleviate inflammation by inhibiting the activation of the nuclear factor-κB (NF-κB) signaling pathway and downregulating the expression of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) (see Guo H, Xie W, Ji Z, et al. Oysterpeptides ameliorate dextran sulfate sodium-induced ulcerative colitis via modulating the gut microbiota and inhibiting the TLR4 / NF-κB pathway. Nutrients, 2024, 16(11): 1591). This anti-inflammatory effect is beneficial for alleviating various inflammation-related diseases such as cardiovascular disease, asthma, diabetes, Alzheimer's disease, lung disease, and autoimmune diseases (see Rivera-Jim'enez, J, Berraquero-García C, Pérez-Gálvez R et al. Peptides and protein hydrolysates exhibiting anti-inflammatory activity: Sources, structural features and modulation mechanisms. Food&Function, 2022, 13(24), 12510–12540).

[0004] Despite experimental demonstrations of functional effects in cell and animal models, the practical application of oyster-derived peptides faces several challenges. These include difficulties in peptide isolation and structural characterization, low in vivo stability and bioavailability, and unclear mechanisms of action and target identification. This invention discovers a novel oyster peptide with a stable and well-defined amino acid structure that can effectively enhance the body's immune function, providing a research foundation for the development of related health foods or drugs. Summary of the Invention

[0005] To address the shortcomings of the prior art, the first aspect of the present invention provides an oyster peptide that improves immunity, wherein the amino acid sequences of the active peptide are shown in SEQ ID NO: 1-5.

[0006] A second aspect of the present invention provides the use of the oyster peptide in the preparation of products that enhance immunity.

[0007] Furthermore, the oyster peptides are prepared by solid-phase synthesis or enzymatic hydrolysis.

[0008] Furthermore, the oyster peptides are prepared using an enzymatic hydrolysis method, the specific steps of which include: (1) Prepare a compound enzyme solution, wherein the compound enzyme includes at least one of trypsin, proteinase K, papain, pepsin, bromelain, neutral protease and flavor protease; (2) Take fresh oysters and homogenize them under low temperature conditions; (3) Add the compound enzyme solution and enzymatically hydrolyze at 37℃ for 5-10 h; (4) The oyster peptides were separated and obtained by chromatography.

[0009] Furthermore, the complex enzyme is composed of trypsin, papain, and flavor protease in a ratio of 2:1:3.

[0010] A third aspect of the present invention provides a product for improving immunity, the product comprising the oyster peptide.

[0011] Furthermore, the product in question is a health supplement.

[0012] Furthermore, the product also includes nutritionally acceptable excipients.

[0013] Furthermore, the product is a pharmaceutical product.

[0014] Furthermore, the product also includes pharmaceutically acceptable excipients.

[0015] Beneficial effects This invention provides an oyster peptide and its application in enhancing immunity, as detailed below: 1. The oyster peptides mentioned above can enhance the activity of immune cells and promote their proliferation; 2. The oyster peptides mentioned above can promote the development and maturation of immune organs and regulate the immune environment in the body; 3. The oyster peptides mentioned above can promote the secretion of immunoglobulins and improve humoral immunity. Attached Figure Description

[0016] Figure 1 Oyster peptides promote NK cell proliferation. Figure 2 Mouse serum hemolysin levels Figure 3 IgG levels in mouse serum Detailed Implementation Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents, biological materials, and detection kits are all commercially available.

[0017] Example 1: Screening and preparation of oyster peptides 1.1 Preparation of Oyster Peptides A complex enzyme solution was prepared using trypsin, papain, and flavor protease in a 2:1:3 ratio, with a total enzyme concentration of 3000 U / g. Fresh oysters were weighed and a suitable amount of ice-cold PBS solution was added. The mixture was homogenized at 4°C, and deionized water was added at a liquid-to-solid ratio of 6:1. The mixture was stirred thoroughly, and the pH was precisely adjusted to the optimal pH range for the protease using 1 mol / L NaOH or HCl solution. The complex enzyme solution was added, and the mixture was incubated at 37°C for 5-10 h. After hydrolysis, the enzyme activity was immediately inactivated by boiling in a water bath for 15 min, thus terminating the reaction. The hydrolysate was centrifuged at 4°C (10,000 r / min, 15 min), and the supernatant was collected and concentrated.

[0018] The peptides were secreted using Sephadex G-25 gel filtration chromatography. The gel suspension was poured into the chromatography column, avoiding air bubbles, and allowed to settle. The column was equilibrated with 2-3 column volumes of phosphate buffer. The enzymatic digest was concentrated and loaded onto the column, with the loading volume not exceeding 5% of the column volume. Elution was performed using phosphate buffer at a flow rate of 0.5-1.0 mL / min. The eluent was collected in separate tubes, and the protein absorption peaks were monitored using a UV detector (280 nm or 214 nm). The peptides were then collected separately, and their amino acid sequences were further identified. As shown in Table 1, five novel oyster bioactive peptides were identified.

[0019] Table 1 Oyster bioactive peptides 1.2 Preliminary identification of the immunomodulatory capacity of oyster bioactive peptides Resuscitate and culture NK92 cells. When cell confluence reaches 80% or higher, passage the cells for a total of 3 passages to fully activate them. Take NK92 cells in the logarithmic growth phase, adjust the cell density, and add 2 × 10⁻⁶ cells. 5 Cells were seeded in 96-well plates and cultured at 37°C for 4 hours. They were then randomly divided into 6 groups: experimental group (containing the five active peptides mentioned above at a concentration of 50 μg / mL); control group (containing an equal volume of cell culture medium); and blank control group (containing only cell culture medium). Cells were incubated at 37°C and 5% CO2 for 24 hours. 10 μL of MTT solution (5 mg / mL, final concentration 0.5 mg / mL) was added to each well, and incubation continued for another 4 hours. The culture was terminated, and the cells were centrifuged at 1000 rpm for 5 minutes to precipitate the cells. The supernatant was carefully removed, and 150 μL of DMSO was added to each well. The cells were shaken slowly on a shaker for 10 minutes to fully dissolve the crystals. The absorbance at 570 nm was measured using a microplate reader. Cell proliferation rate (%) was calculated as follows: (ODg = ...)g 对照 -OD 阴性对照) / (OD 实验 -OD 阴性对照 ) × 100%.

[0020] It has been reported that marine bioactive peptides can promote the proliferation of immune cells. This invention verified the proliferative capacity of the bioactive peptides on NK92 cells, and the results are as follows: Figure 1 As shown, different bioactive peptides exhibited different cell proliferation-promoting abilities. Among them, 1B5, 3D5, and 3F6 showed strong proliferation-promoting abilities, with 3D5 exhibiting the strongest activity. Subsequent experiments in this application were based on the 3D5 oyster peptide.

[0021] Example 2: Animal Experiment 2.1 Administration Forty healthy male Balb / C mice were randomly divided into four groups of 10 mice each after 5 days of acclimatization: low-dose group (500 mg / kg oyster peptide daily by gavage); medium-dose group (1000 mg / kg oyster peptide daily by gavage); high-dose group (2000 mg / kg oyster peptide daily by gavage); and control group (equal volume of physiological saline daily by gavage). The administration was carried out for a total of 3 weeks.

[0022] 2.2 Organ coefficient determination: spleen, thymus After administration, the mice were weighed, and the thymus and spleen were weighed to calculate the thymus and spleen indices. Organ index = organ weight / mouse body weight × 100%. The results are shown in Table 2. After administration of oyster peptide, the immune-related organ indices of each group increased to varying degrees in a dose-dependent manner, indicating that oyster peptide can stimulate the growth of immune organs and promote the improvement of the body's immune capacity.

[0023] Table 2. Organ Index of Mice 2.3 Serum hemolysin levels After administration, serum hemolysin levels were detected using conventional methods, as described in the literature (Fu Shiqing, Yang Xu, Wang Jiaoyan, et al., Improvement effect of East China Sea Cucumber Peptide on Cyclophosphamide-Induced Immunological Insufficiency in Mice, Modern Food Science and Technology, 2026, 42(1): 9-16). Specific steps included injecting diluted sheep red blood cells into mice, collecting and diluting serum, then adding complement obtained by diluting serum and SA at a ratio of 1:8 and 10% (V / V) SRBC, and incubating with 1% (V / V) SRBC as a control tube, followed by calculation of the half-hemolysis value (HC50). HC50 = A sample / A control × 100%, where A sample is the absorbance of the sample tube at 540 nm, and A2 is the absorbance of the control tube at 540 nm.

[0024] The results are as follows Figure 2As shown, the oyster peptide described in this application has a certain stimulating effect on the serum hemolysin level of mice at high, medium and low doses, with the high dose group showing the most significant effect. This indicates that the oyster peptide can effectively activate the humoral immune system and improve the body's immune function.

[0025] 2.4 Immunoglobulin levels After drug administration, blood was collected from the eyeballs of mice in each group, allowed to stand at room temperature for 30-60 minutes, and then centrifuged at 3000 rpm for 15 minutes at 4°C to collect mouse serum. The serum immunoglobulin IgG content was then measured using an ELISA kit (purchased from R&D Systems).

[0026] Immunoglobulins in the blood are important carriers of the body's immune function and can participate in various immunomodulatory effects such as antibacterial, antiviral, and antioxidant activity. The results are as follows... Figure 3 As shown, the oyster peptide provided in this application can effectively increase the level of immunoglobulins in mouse serum, indicating that the oyster peptide can effectively improve the body's immune function.

[0027] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An oyster peptide that improves immunity, characterized in that, The amino acid sequences of the oyster peptides are shown in SEQ ID NO: 1-5.

2. The use of the oyster peptide as described in claim 1 in the preparation of products that enhance immunity.

3. In the application according to claim 2, the oyster peptide is prepared by solid-phase synthesis or enzymatic hydrolysis.

4. The application according to claim 3, wherein the oyster peptide is prepared by enzymatic hydrolysis, and the specific steps include: (1) Prepare a compound enzyme solution, wherein the compound enzyme includes at least one of trypsin, proteinase K, papain, pepsin, bromelain, neutral protease and flavor protease; (2) Take fresh oysters and homogenize them under low temperature conditions; (3) Add the compound enzyme solution and enzymatically hydrolyze at 37℃ for 5-10 h; (4) The oyster peptides were separated and obtained by chromatography.

5. In the application according to claim 4, the composite enzyme is selected from trypsin, papain and flavor protease in a ratio of 2:1:

3.

6. A product for improving immunity, characterized in that, The product includes the oyster peptide described in claim 1.

7. The product according to claim 6, wherein the product is a health product.

8. The product according to claim 7, wherein the product further comprises nutritionally acceptable excipients.

9. The product according to claim 6, wherein the product is a pharmaceutical product.

10. The product according to claim 9, further comprising pharmaceutically acceptable excipients.

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