A small molecule bioactive peptide derived from cockle and its application

Abstract

This invention provides a small molecule bioactive peptide derived from cockle and its applications, belonging to the field of biomedicine. The amino acid sequence of this cockle-derived peptide is: TVGMGVPAV. The small molecule bioactive peptide from cockle of this application can not only be directly coated onto the surface of any transparent material, imparting excellent and stable anti-fogging properties to the substrate, but also possesses antioxidant activities and promotes fibroblast cell proliferation, thereby improving the profitability of cockle farming and demonstrating certain application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a small molecule bioactive peptide derived from cockle and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Transparent materials such as glass, quartz, and plastics are widely used in daily life, industry, agriculture, and medicine, for example, in various medical endoscope lenses, automobile windshields, and agricultural plastic films. However, when these materials are in environments with high humidity or large temperature differences, their surfaces are prone to fogging. These fog droplets scatter incident light, further reducing the material's light transmittance and limiting its practical applications. It is worth noting that when these transparent materials are used in goggles, food preservation films, windshields, face masks, etc., surface fogging can not only lead to serious medical and traffic accidents but also affect the purchase of vegetables and fruits.

[0004] The main methods to solve the fogging of material surfaces include: (1) Cleaning: Use a clean, soft cloth or sponge dipped in water or detergent to gently wipe away the fog caused by surface contamination; (2) Drying: In environments with high humidity, a heater or drying oven can be used to fully dry the surface; (3) Adding anti-fogging agents: Adding anti-fogging agents is a common method. Anti-fogging agents can be added to plastic materials during the production process to improve the application performance of the materials; (4) Using anti-fogging coatings: Apply an anti-fogging coating to the surface of plastic products using electrostatic spraying, vacuum coating, dip coating, etc., to inhibit the condensation and accumulation of water vapor, thereby achieving the anti-fogging effect. Compared with "cleaning" and "drying", "adding anti-fogging agents" and "using anti-fogging coatings" usually involve coating the material surface with materials that can prevent fogging in the early or later stages to prevent fogging of transparent materials. The methods are simpler. However, existing anti-fogging agents have poor adhesion and are easily washed away by water when the air humidity is high or when the environment is in a high humidity environment for a long time, which limits their practical application. Therefore, there is an urgent need to develop new, long-lasting, and stable anti-fogging agents.

[0005] The giant ark clam, also known as the red clam, blood clam, or large hairy clam, is a large marine benthic economic shellfish widely distributed along the coasts of the Sea of ​​Japan, Yellow Sea, Bohai Sea, and East China Sea in the northwestern Pacific Ocean. Adult ark clams are large and plump, with delicious meat, and have high economic value. In recent years, the production and aquaculture of giant ark clam seedlings have been continuously expanding, resulting in significant economic and social benefits. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a novel bioactive peptide derived from cockle and its preparation. This invention extracts small-molecule bioactive peptides from cockle and explores their potential applications in areas such as anti-fogging and wound healing, which can further enhance its production and aquaculture value.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a small molecule bioactive peptide derived from cockle, with the amino acid sequence: TVGMGVPAV.

[0009] A second aspect of the present invention provides a method for extracting small bioactive peptides from cockle shells, comprising:

[0010] Remove the shells from the cockles, wash them, add PBS solution, homogenize, separate the solids and liquids, and collect the supernatant.

[0011] Ammonium sulfate was added to the supernatant to carry out the reaction. After the reaction was completed, the solid and liquid were separated and the precipitate was collected.

[0012] Add Tris buffer to the precipitate to dissolve it, dialyze through I, and then freeze-dry.

[0013] The lyophilized sample was separated by column chromatography. The collected liquid was dialyzed II and lyophilized to obtain small molecule active peptides derived from cockle.

[0014] In some embodiments, the volume of the PBS solution is 3-6 times that of the cockle meat.

[0015] In some embodiments, the mass-to-volume ratio of ammonium sulfate to supernatant is 50-70 g: 100 mL.

[0016] In some embodiments, the reaction time is 60-90 minutes.

[0017] In some implementations, the duration of dialysis I is 3-4 days.

[0018] In some embodiments, the column chromatography method employs Deae and Sephadex G100 column chromatography for separation, respectively.

[0019] More specifically, including:

[0020] (1) Remove the shells from the cockles and take the meat. Wash with ultrapure water, add three times the volume of pH 8.0 PBS solution, homogenize with a tissue homogenizer, centrifuge for 30 min, and take the supernatant.

[0021] (2) Add 50-70g of ammonium sulfate to every 100mL of supernatant, stir for 60min, and then centrifuge for 30min to collect the precipitate;

[0022] (3) Add Tris buffer (pH 8.0) to the precipitate to dissolve it, dialyze it through a dialysis bag for 3 days, and then freeze-dry it.

[0023] (4) The freeze-dried sample was separated by Deae and Sephadex G100 column chromatography respectively. The collected liquid was dialyzed using a dialysis bag and freeze-dried to obtain small molecule active peptides from cockle.

[0024] A second aspect of the present invention provides a multifunctional antifogging agent, comprising the following raw materials in parts by weight: 1-5 parts of small molecule active peptides derived from cockle clams, 0.02-0.10 parts of CGS19 antimicrobial peptides, and 1×10⁻⁶ PVA aqueous solution. 3 -1.5×10 3 share;

[0025] The concentration of the PVA aqueous solution is 1-2 wt%.

[0026] A third aspect of the present invention provides the use of the above-mentioned small molecule active peptides derived from cockle in the preparation of antifogging agents, products with antifogging function, antibacterial agents, or antioxidants.

[0027] Preferably, the products with anti-fog function include: goggles, swimming goggles, safety goggles, endoscopes, food preservation films, windshields and their coatings, providing good biocompatibility, anti-fog, and food preservation functions.

[0028] A fourth aspect of the present invention provides the application of the above-mentioned small molecule active peptides derived from cockle shells in the preparation of wound-healing drugs or dressings. Specifically, the cockle shell polypeptides can significantly promote fibroblast proliferation.

[0029] Beneficial effects of the present invention

[0030] (1) The small molecule active peptides of cockle source disclosed in this invention can be directly coated onto the surface of different transparent materials by a one-step deposition method, giving the substrate anti-fog function and improving the biosafety of the substrate. Compared with existing commercial 3M anti-fog goggles and commercial anti-fog agents, the multifunctional anti-fog agent prepared by it, after being coated onto the surface of the goggles, not only maintains high light transmittance after being treated with 85°C water vapor for 6 hours, but also shows excellent antibacterial activity against Escherichia coli and Staphylococcus aureus.

[0031] (2) The small molecule active peptides of cockle source disclosed in this invention have excellent functions of scavenging DPPH free radicals and promoting fibroblast proliferation. They can be applied in cosmetics, medical aesthetics, health care, food and other fields, and have great application value.

[0032] (3) The clams disclosed in this invention are peptides with MW < 3kDa, which can be used as stable antioxidants in pharmaceuticals, food and cosmetics. At the same time, compared with other longer peptides, short peptides have the advantages of low production cost and simple quality control. Attached Figure Description

[0033] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0034] Figure 1 This is a scanning electron microscope image of small molecule active peptides derived from cockle;

[0035] Figure 2 Only small molecule active peptides derived from cockle can improve cell survival rate;

[0036] Figure 3 This is a graph showing the antioxidant activity of small molecule bioactive peptides from cockle source;

[0037] Figure 4 This is a diagram of the water contact angle of small molecule active peptides from cockle source;

[0038] Figure 5 This is an image showing the anti-fogging effect of Kuihanyuan small molecule active peptides;

[0039] Figure 6 This is a photograph of a multifunctional antifogging agent prepared using small molecule peptides derived from cockle shells.

[0040] Figure 7 Photographs show the preparation of multifunctional antifogging agents and antibacterial agents using small molecule peptides derived from cockle shells;

[0041] Figure 8 Photo of a multifunctional anti-fogging agent for food preservation prepared using small molecule peptides derived from cockle shells;

[0042] Figure 9 This is the mass spectrum of small molecule bioactive peptides derived from cockle. Detailed Implementation

[0043] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0044] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0045] Example 1: Preparation of small molecule bioactive peptides derived from cockle

[0046] Fresh cockles were shelled and the meat extracted. The meat was washed with ultrapure water and then homogenized with three times the volume of pH 8.0 PBS solution using a tissue homogenizer. After centrifugation for 30 min, the supernatant was collected. 60 g of ammonium sulfate was slowly added to every 100 mL of supernatant. After the ammonium sulfate was completely dissolved, stirring was continued for 60 min, followed by centrifugation for 30 min. The precipitate was collected and dissolved in Tris buffer (pH 8.0), then dialyzed with ultrapure water for 3 days. The crude product was then lyophilized. The crude product was separated using Deae and Sephadex G100 column chromatography (with PBS solution at pH 8.0 and washing at 4°C as the separation buffer). The collected liquid was dialyzed with ultrapure water at 4°C using a dialysis bag and then lyophilized to obtain small molecule bioactive peptides derived from cockles.

[0047] Mass spectrometry analysis revealed the amino acid sequence of the active peptide to be: TVGMGVPAV. Figure 9 As shown.

[0048] (Threonine-valine-glycine-methionine-glycine-valine-proline-alanine-valine)

[0049] Another specific feature of this invention is that the active peptide contains 9 amino acid residues, has a molecular weight of 845.4316 Da, and an isoelectric point of 5.18.

[0050] like Figure 1 The image shows a scanning electron microscope image of a small molecule active peptide derived from cockle. This active peptide exists in aqueous solution in the form of nanoparticles.

[0051] Example 2: Cell proliferation experiment of small molecule bioactive peptides derived from cockle

[0052] L929 fibroblast cell line was co-incubated with small molecule bioactive peptides derived from cockle shells, and the viability of blood cells was detected using a CCK-8 assay kit. First, L929 fibroblasts were dispersed in 96-well plates at a density of 10,000 cells per well. After 24 h of incubation, specific volumes of PBS solution (co-derived small molecule bioactive peptide concentration: 0 μg / mL) and co-derived small molecule bioactive peptide solution were added sequentially to the wells. Co-culture continued at 37°C and 5% CO2 in an incubator, with the concentrations of co-derived small molecule bioactive peptides being 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL, respectively. After 48 h, each group of cells was washed three times with fresh culture medium, and then 100 μL of fresh culture medium and 10 μL of CCK-8 solution were added sequentially to each well. The cells were then incubated at 37°C for another 2 h. Finally, the absorbance of the cells at 480 nm was measured using an ELISA reader, and the cell viability was calculated.

[0053] The results are as follows Figure 2As shown, cell survival first increased and then decreased with the increase of the concentration of small molecule active peptides from cockle source. The cell survival rate was the highest, about 140%, when the concentration of small molecule active peptides from cockle source was 80 μg / mL.

[0054] Example 3: Antioxidant Activity Experiment of Small Molecule Active Peptides from Clams

[0055] Small molecule bioactive peptides derived from cockle were dissolved in ultrapure water to prepare solutions with concentrations of 1, 5, 10, 15, and 20 mg / mL. A 0.2 mM DPPH ethanol solution was also prepared. 2 mL of each solution was transferred, mixed thoroughly, and incubated at room temperature in the dark. After 30 min, the absorbance of the solution at 517 nm was measured using a UV spectrophotometer. The DPPH free radical scavenging rate was calculated using the following formula:

[0056]

[0057] Among them, A S It is the absorbance of the sample reaction solution, A c A is the absorbance of 2 mL of ethanol and 2 mL of sample solution, and A is the absorbance of 2 mL of DPPH and 2 mL of ethanol.

[0058] The results are as follows Figure 3 As shown, with the increase of the concentration of small molecule active peptides from cockle source, its DPPH scavenging rate gradually increases, with the highest DPPH scavenging rate being about 35%, exhibiting certain antioxidant activity.

[0059] Example 4: Infiltration experiment of small molecule active peptides derived from cockle

[0060] A 10 mg / mL aqueous solution of small molecule active peptides derived from cockle was prepared. 20 μL of this solution was transferred and coated onto the surface of a polycarbonate substrate, then allowed to dry at room temperature. The surface wettability was determined using a water contact angle meter.

[0061] As shown in Figure 4, after coating the small molecule active peptides of cockle source onto the surface of polycarbonate substrate, the contact angle of the polycarbonate substrate surface was significantly reduced, exhibiting superhydrophilicity with a water contact angle of 6.4°.

[0062] Example 5: Anti-fogging experiment of small molecule active peptides derived from cockle

[0063] A 10 mg / mL solution of small molecule active peptides from cockle shells was prepared and coated onto the surface of each goggle lens at a rate of 50 μL. The coating was then dried at room temperature to obtain anti-fog goggles coated with the small molecule active peptides from cockle shells. The goggles coated with these peptides were then placed in a hot steam environment at ~85℃ for 2 hours, and anti-fog photographs were obtained at different time intervals to evaluate the anti-fog performance of the small molecule active peptides from cockle shells.

[0064] The results are as follows Figure 5 As shown, the goggles coated with small molecule active peptides from kuaiyanyuan, after being treated with 85℃ hot steam for 2 hours, still showed clear lettering on the back of the goggles, with no fogging on the surface, demonstrating excellent anti-fog performance.

[0065] Example 6: Application of small molecule active peptides derived from cockle in multifunctional antifogging agents

[0066] To further broaden the application of small molecule active peptides derived from cockle shells, they were crosslinked with hydroxyl-rich polyvinyl alcohol (PVA) and sea cucumber active peptides (GS19) with excellent antibacterial activity to obtain a multifunctional anti-fogging coating with excellent antibacterial, self-repairing, and self-cleaning properties. The experimental steps were as follows: 1 mg of small molecule active peptides derived from cockle shells and 20 μg of CGS19 antibacterial peptide (disclosed in existing patents) were dissolved in 1 mL of PVA aqueous solution (1 wt%) and stirred at room temperature to obtain a homogeneous solution. Then, based on the coating area of ​​the goggle lens, 240 μL was coated onto the goggle lens and dried at room temperature to obtain coated goggles. These goggles (PSG-3), commercially available anti-fogging coated goggles (Agents), and commercially available anti-fogging goggles (3M) were then placed in a hot steam environment at ~85℃ for 6 hours, and anti-fogging photographs were obtained at different time intervals to evaluate the anti-fogging performance and stability of the functional coating.

[0067] The results are as follows Figure 6 As shown, both commercially available anti-fog goggles coated with anti-fog agent and commercially available anti-fog goggles exhibited excellent anti-fog performance in the first 5 minutes, but the anti-fog effect gradually disappeared over time. Specifically, the commercially available anti-fog goggles lost their anti-fog performance after 30 minutes of treatment with 85℃ water vapor, with extensive fogging on the surface, making it difficult to see the text behind the lenses. The anti-fog agent-coated goggles showed better anti-fog performance than the commercially available goggles, but their anti-fog performance still disappeared after 2 hours of treatment with 85℃ water vapor, with fogging on the lens surface, indicating limited anti-fog performance and stability. Compared to the commercially available anti-fog agent-coated goggles and commercially available anti-fog goggles, the coating-coated anti-fog agent exhibited excellent anti-fog performance and stability. After 6 hours of treatment with 85℃ water vapor, no fogging was observed on the surface, and the text behind the lenses was clearly visible, demonstrating excellent anti-fog performance and stability.

[0068] Example 7: Antibacterial activity of a multifunctional antifogging agent prepared using small molecule active peptides derived from cockle shells.

[0069] Using glutathione-derived small molecule active peptide (Glu) and PBS solution as control groups, the antibacterial activity of the multifunctional coating was detected by testing Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). The specific experimental steps are as follows: 1 mg of glutathione-derived small molecule active peptide and 20 μg of CGS19 antimicrobial peptide were dissolved in 1 mL of PVA aqueous solution (1 wt%), stirred at room temperature to obtain a homogeneous solution, and lyophilized to obtain PSG-3 powder; similarly, PSG-1 and PSG-2 were obtained according to the experimental method for PSG-3, wherein the mass of CGS19 antimicrobial peptide in PSG-1 and PSG-2 were 5 μg and 10 μg, respectively. Gluathione-derived small molecule active peptide, PBS buffer, PSG-1, PSG-2, and PSG-3 were added to 1 mL of bacterial suspension (bacterial concentration 10). 6 The bacteria were co-cultured with CFU / mL. After 6 hours, the bacterial suspension was diluted 100,000 times with sterile PBS solution, inoculated onto the culture medium, and incubated at 37°C. After 24 hours, the number of colony units was counted using the plate count method to analyze the antibacterial activity.

[0070] The results are as follows Figure 7 As shown, this multifunctional coating exhibits excellent antibacterial activity against E. coli and S. aureus, with an antibacterial rate exceeding 99.9%.

[0071] Example 8: Application of small molecule bioactive peptides derived from cockle shells in food preservation

[0072] The anti-fogging agent solution from Example 6 was applied to the surface of a food packaging box at a rate of 200 μL, with an untreated food packaging box serving as a blank control. The strawberries were observed to spoil at room temperature. Additionally, the boxes were placed in an environment of 4°C to observe the fogging condition on their surface.

[0073] The results are as follows Figure 8 As shown, the surface of the untreated food storage container is heavily fogged up, while the strawberries behind the coated food storage container are clearly visible. Strawberries in the coated food storage container, left at room temperature for a period of time, show no significant changes, while the strawberries in the untreated food storage container spoil rapidly.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A small molecule bioactive peptide derived from cockle, characterized in that, The amino acid sequence is: TVGMGVPAV.

2. A method for extracting small bioactive peptides from cockle shells as described in claim 1, characterized in that, include: Remove the shells from the cockles, wash them, add PBS solution, homogenize, separate the solids and liquids, and collect the supernatant. Ammonium sulfate was added to the supernatant to carry out the reaction. After the reaction was completed, the solid and liquid were separated and the precipitate was collected. Add Tris buffer to the precipitate to dissolve it, dialyze through I, and then freeze-dry. The lyophilized sample was separated by column chromatography. The collected liquid was dialyzed II and lyophilized to obtain small molecule active peptides derived from cockle.

3. The extraction method of small molecule active peptides from cockle as described in claim 2, characterized in that, The volume of the PBS solution is 3-6 times that of the cockle meat.

4. The extraction method of small molecule active peptides from cockle as described in claim 2, characterized in that, The mass-to-volume ratio of ammonium sulfate to supernatant is 50-70 g: 100 mL.

5. The method for extracting small molecule active peptides from cockle shells as described in claim 2, characterized in that, The reaction time is 60-90 minutes.

6. The method for extracting small molecule active peptides from cockle shells as described in claim 2, characterized in that, The column chromatography method used Deae and Sephadex G100 column chromatography for separation.

7. A multifunctional anti-fogging agent, characterized in that, It is composed of the following raw materials in parts by weight: 1-5 parts of the small molecule active peptide from cockle as described in claim 1, 0.02-0.10 parts of CGS19 antimicrobial peptide, and 1×10⁻⁶ PVA aqueous solution. 3 -1.5×10 3 share; The concentration of the PVA aqueous solution is 1-2 wt%.

8. The use of the small molecule active peptide derived from cockle as described in claim 1 in the preparation of products with anti-fogging function, antibacterial agents or antioxidants.

9. The application of the small molecule active peptide derived from cockle as described in claim 8 in the preparation of products with anti-fogging function, antibacterial agents, or antioxidants, characterized in that, The products with anti-fog function include: eyeglasses, swimming goggles, safety goggles, endoscopes, food preservation film, windshields and their coatings.

10. The use of the small molecule active peptide derived from cockle as described in claim 1 in the preparation of wound-healing drugs or dressings.

11. The use of the small molecule active peptide derived from cockle as described in claim 1 in the preparation of antifogging agents.

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