Enzymatic preparation of small molecule peptide and its immunological application

Abstract

Enzymatic Preparation and Immunological Application of Small Molecule Turtle Peptides. The method for preparing turtle peptides includes: providing turtle protein raw materials; performing a primary enzymatic hydrolysis of the turtle protein raw materials using an alkaline protease; performing a secondary enzymatic hydrolysis of the primary hydrolysis product using a complex enzyme containing flavor protease and trypsin; and inactivating, separating, and purifying the secondary hydrolysis product to obtain turtle peptides. This invention efficiently degrades large molecule turtle protein into small peptides and oligopeptides with an average molecular weight of less than 1000 Da, especially 180-800 Da, through stepwise synergistic enzymatic hydrolysis. These small peptides can be directly and completely absorbed through the intestinal peptide transport system, with an absorption rate and efficiency far exceeding that of free amino acids and whole proteins, significantly improving bioavailability. The small molecule turtle peptides prepared by this invention can significantly enhance the phagocytic capacity of macrophages and promote the proliferation and differentiation of lymphocytes, thereby enhancing the body's immune function at both the innate and adaptive immune levels.

Description

Technical Field

[0001] This invention relates to the preparation of a turtle peptide. Background Technology

[0002] Turtles, especially Chinese tortoises, have a long history of use in traditional Chinese medicine, where they are believed to have yin-nourishing, kidney-tonifying, and body-strengthening effects. Tortoise shells, plastrons, and their extracts are frequently used in traditional Chinese medicine prescriptions. Modern research shows that tortoises are rich in proteins and polypeptides, which are essential for their biological activity.

[0003] However, natural turtle protein has a large molecular weight and complex structure, making it difficult for the human gastrointestinal tract to digest and absorb after direct consumption, resulting in low bioavailability and significantly reducing its nutritional value and physiological functions. To overcome this problem, existing technologies have attempted to hydrolyze turtle protein using chemical methods or single proteases, but these methods suffer from uncontrollable hydrolysis levels, wide molecular weight distribution of products, unstable activity, and the potential generation of harmful byproducts or bitter peptides, affecting the safety and taste of the products.

[0004] Therefore, there is an urgent need to develop a technology that can efficiently and controllably degrade turtle protein into small molecule peptides that are easily absorbed and have significant immune-enhancing activity. Summary of the Invention

[0005] The purpose of this invention is to controllably degrade turtle protein raw materials into small molecule peptides that are easily absorbed and have significant immune-enhancing activity.

[0006] According to one aspect of the present invention, a method for preparing turtle peptide is provided, comprising: Provide turtle protein raw materials; The turtle protein raw material was enzymatically hydrolyzed once using alkaline protease. The product of the first enzymatic hydrolysis was subjected to a second enzymatic hydrolysis using a complex enzyme containing flavor protease and trypsin. The secondary enzymatic hydrolysis product was inactivated, separated, and purified to obtain turtle peptide.

[0007] According to a preferred embodiment of the preparation method of the present invention, the pH value of the single enzymatic hydrolysis is 7.5-9.0, the temperature is 45-55℃, and the time is 2-4 hours.

[0008] In another preferred embodiment of the preparation method according to the present invention, the pH value of the secondary enzymatic hydrolysis is 6.0-7.5, the temperature is 50-60℃, and the time is 3-5 hours.

[0009] In another preferred embodiment of the preparation method according to the present invention, purification includes: treatment with an ultrafiltration membrane with a molecular weight cutoff of 1 kD, and collection of permeate components with a molecular weight of less than 1000 Da.

[0010] The weight-average molecular weight of the turtle peptide prepared according to the present invention is preferably 180-800 Da.

[0011] According to another aspect of the present invention, a composition is also provided, comprising turtle peptide prepared according to the above method and a pharmaceutically or food-grade acceptable carrier or excipient. The composition includes, but is not limited to, functional foods, health foods, special medical foods, nutritional supplements, or pharmaceutical preparations.

[0012] According to another aspect of the present invention, the application of the turtle peptide prepared according to the above method in the preparation of an immune enhancer is also provided, the immune enhancer being used to enhance the phagocytic activity of macrophages or promote lymphocyte proliferation.

[0013] This invention utilizes the stepwise and synergistic action of a complex enzyme to efficiently degrade large-molecule turtle protein into small peptides and oligopeptides with an average molecular weight of less than 1000 Da, especially 180-800 Da. These small peptides can be directly and completely absorbed through the intestinal peptide transport system, with an absorption rate and efficiency far exceeding that of free amino acids and whole proteins, resulting in a significant improvement in bioavailability.

[0014] In vitro cell experiments have demonstrated that the small molecule turtle peptide prepared in this invention can significantly enhance the phagocytic capacity of macrophages and promote the proliferation and differentiation of lymphocytes, thereby enhancing the body's immune function from both innate and adaptive immune levels. Detailed Implementation

[0015] Example Fresh turtle meat and carapace were collected, washed, and homogenized. 100 kg of the turtle paste was added to 500 kg of purified water and stirred thoroughly. The pH was adjusted to 8.5 with 1M NaOH solution, and the temperature was raised to 50℃. 5 million units (sufficient amount) of alkaline protease (P824138-1g CAS No.: 9014-01-1, Macklin) were added, and the mixture was incubated and stirred for 3 hours for enzymatic hydrolysis. Subsequently, 1M HCl solution was added to adjust the pH to 7.0, and the temperature was raised to 55℃. Then, 3 million units (sufficient amount) of flavor protease (F768979-500g CAS No.: 9001-92-7, Macklin) and 2 million units (sufficient amount) of trypsin (T105532-100g CAS No.: 9002-07-7, Aladdin) were added, and the mixture was incubated and stirred for another 4 hours for enzymatic hydrolysis. After enzymatic hydrolysis, the solution was heated to 90°C and stirred continuously for 15 min to inactivate the enzyme. After cooling, it was centrifuged at 8000 r / min for 20 min, and the supernatant was collected. The supernatant was passed through an ultrafiltration membrane system with a molecular weight cutoff of 1 kD to remove the inactivated enzyme and unhydrolyzed large protein molecules, and the filtrate was collected. Finally, the filtrate was spray-dried at an inlet air temperature of 180°C and an outlet air temperature of 85°C to obtain approximately 15 kg of pale yellow powdered enzymatically hydrolyzed turtle peptide. The weight-average molecular weight was determined to be approximately 550 Da by high-performance gel permeation chromatography.

[0016] Macrophage phagocytic activity test RAW264.7 macrophages were treated with enzymatically hydrolyzed turtle peptide at different concentrations (0.00, 0.10, 0.20, 0.30, 0.40, 0.60, 0.80, 1.00, 1.20 mg / mL) for 48 h. Then, 10 μL of CCK-8 solution was added to each well of a 96-well plate using a CCK-8 assay kit. The plates were incubated for 3 h, and the absorbance at 450 nm was measured to determine macrophage viability. The results are shown in Table 1: the addition of 0.60 and 0.80 mg / mL of enzymatically hydrolyzed turtle peptide significantly increased the viability of RAW264.7 macrophages.

[0017]

[0018] Macrophage phagocytic capacity test for neutral red RAW264.7 macrophages were treated with enzymatically hydrolyzed turtle shell peptides at different concentrations (0.00, 0.10, 0.20, 0.30, 0.40, 0.60, 0.80, 1.00, 1.20 mg / mL) for 48 h. The culture medium was carefully discarded, and 100 μL of neutral red solution was added to each well, followed by incubation for 30 min. Cells were washed twice with 200 μL of polybutylene succinate (PBS) to remove residual neutral red. Then, 100 μL of cell lysis buffer (acetic acid and ethanol, volume ratio 1:1) was added, and the cells were gently shaken at room temperature for 2 h. The absorbance (OD) was measured at 540 nm, and the phagocytic rate of neutral red by RAW264.7 macrophages treated with different concentrations of turtle shell peptides was calculated. Phagocytic rate (%) = [(OD treated group - OD blank group) / (OD control group - OD blank group)] × 100%. A higher phagocytic rate of neutral red by macrophages indicates a stronger phagocytic capacity. The results are shown in Table 2: different concentrations of turtle peptides can significantly improve the phagocytic capacity of macrophages.

[0019]

[0020] Lymphocyte proliferation test After one week of acclimatization feeding, the weight of each SD rat was recorded. Twelve rats (half male, half female) were then randomly selected as the control group and administered an equal volume of 0.9% sodium chloride solution by gavage for 14 consecutive days. The remaining rats were intraperitoneally injected with cyclophosphamide solution for 3 consecutive days. Subsequently, the rats were randomly divided into a model group and a drug group [low-dose turtle peptide group (low-dose group), medium-dose turtle peptide group (medium-dose group), high-dose turtle peptide group (high-dose group), and a positive control group], with 12 rats in each group (half male, half female). On the fourth day at 9:00 AM, the model group and the drug group were intraperitoneally injected with cyclophosphamide. At 3:00 PM, except for the model group, the drug groups were administered the corresponding turtle peptide and pidotimod solutions by gavage, respectively. The model group was administered an equal volume of 0.9% sodium chloride solution by gavage. This administration was continued for 10 consecutive days at a gavage volume of 1 mL / 100 g. Blood was collected from the abdominal aorta and stored in anticoagulant tubes. Flow cytometry was used to detect changes in peripheral blood T lymphocytes in each group. Prepare Trucount tubes, add 10 μL each of anti-PECD3 monoclonal antibody and anti-APC-CD4 monoclonal antibody to each tube, add 50 μL of anticoagulated whole blood to the Trucount tubes, vortex to mix, and incubate at room temperature in the dark for 20 min. Add 450 μL of LACS lysis buffer, vortex to mix, and incubate at room temperature in the dark for 10 min. Flow cytometry was used for detection, and cells were collected and analyzed simultaneously using MultiSet software to obtain the absolute counts of CD3+ and CD4+ T lymphocytes. The results are shown in Table 3: Different doses of turtle peptide improved lymphocyte proliferation.

[0021]

[0022] This invention employs a bio-enzymatic hydrolysis method with mild reaction conditions, avoiding the pollution and nutrient loss that may occur with chemical methods. The step-by-step use of proteases with different properties makes the hydrolysis process more precise and controllable, ensuring the stability and consistency of the product's activity. Through the effective action of flavor proteases, the content of bitter peptides in the hydrolysate is significantly reduced, improving the product's taste and flavor, making it more acceptable to consumers. The entire production process involves no harmful chemical reagents, making it safe and non-toxic.

Claims

1. A method for preparing turtle peptide, comprising: Provide turtle protein raw materials; The turtle protein raw material was enzymatically hydrolyzed once using alkaline protease. The product of the first enzymatic hydrolysis was subjected to a second enzymatic hydrolysis using a complex enzyme containing flavor protease and trypsin. The secondary enzymatic hydrolysis product was inactivated, separated, and purified to obtain turtle peptide.

2. The preparation method according to claim 1, wherein the pH value of the first enzymatic hydrolysis is 7.5-9.0, the temperature is 45-55℃, and the time is 2-4 hours.

3. The preparation method according to claim 1, wherein the pH value of the secondary enzymatic hydrolysis is 6.0-7.5, the temperature is 50-60℃, and the time is 3-5 hours.

4. The preparation method according to claim 1, wherein purification includes: The permeate was processed using an ultrafiltration membrane with a molecular weight cutoff of 1 kD, and components with a molecular weight less than 1000 Da were collected.

5. The preparation method according to claim 1, wherein the weight-average molecular weight of the obtained turtle peptide is 180-800 Da.

6. A composition comprising turtle peptide prepared by the method according to any one of claims 1-5 and a pharmaceutically or food-grade acceptable carrier or excipient.

7. The use of a turtle peptide prepared according to any one of claims 1-5 in the preparation of an immune enhancer, wherein the immune enhancer is used to enhance the phagocytic activity of macrophages or promote lymphocyte proliferation.

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