Scapharca broughtonii source small molecule active peptide and application thereof

Abstract

The invention provides a scapharca broughtonii source small molecule active peptide and application thereof, and belongs to the field of biological medicine. The amino acid sequence of the scapharca broughtonii source polypeptide is as follows: TVGMGVPAV. The scapharca broughtonii source small molecule active peptide can directly coat the surface of any transparent material to endow a base material with an excellent and stable anti-fog function, also has the activities of resisting oxidation, promoting fibroblast cell proliferation and the like, improves the culture income of scapharca broughtonii, and has a certain application value.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a small molecule active peptide derived from quail eggs and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Transparent materials such as glass, quartz, and plastic are widely used in daily life, industry, agriculture, and medical treatment, such as various medical endoscope lenses, automobile windshields, and agricultural plastic films. However, when these materials are in an environment with high humidity or a large temperature difference between the inside and outside, the surface of the material is prone to fogging, and the fog droplets will cause incident light scattering, further resulting in a significant reduction in the light transmittance of the material, and the actual application is limited. It is worth noting that when these transparent materials are used for goggles, food wrap, windshields, masks, etc., the fogging of the material surface may not only cause serious medical and traffic accidents, but also affect the purchase of vegetables and fruits.

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

[0005] The ark shellfish, commonly known as red shellfish, blood shellfish, and large hairy clam, is a large marine benthic economic shellfish, widely distributed in the Sea of ​​Japan, the Yellow Sea, the Bohai Sea, and the East China Sea in the northwest Pacific Ocean. The adult shellfish is large and fat, with delicious meat and high economic value. In recent years, the scale of ark shellfish seed production and breeding has been continuously expanded, with significant economic and social benefits. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a novel active peptide from clams with medicinal value and its preparation. The present invention extracts small molecule active peptides from clams and develops its potential applications in the fields of anti-fog and wound repair, which can further enhance its production and breeding value.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a small molecule active peptide from kuiguan source, the amino acid sequence of which is: TVGMGVPAV.

[0009] The second aspect of the present invention provides a method for extracting active peptides from small fractions of clams, comprising:

[0010] Remove the shell of the cockle, take out the meat, wash it, add PBS solution, homogenize it, separate the solid and liquid, and take the supernatant;

[0011] adding ammonium sulfate to the supernatant to react, and after the reaction is completed, performing solid-liquid separation and collecting the precipitate;

[0012] Adding Tris buffer to the precipitate, dissolving the precipitate, dialyzing I, and then freeze-drying;

[0013] The freeze-dried sample is separated by column chromatography, and the collected liquid is dialyzed II and freeze-dried to obtain the small molecule active peptide from Kuihan source.

[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 volume ratio of ammonium sulfate to supernatant is 50-70 g:100 mL.

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

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

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

[0019] More specifically, they include:

[0020] (1) Remove the shell of the cockle, remove the meat, wash with ultrapure water, add three times the volume of PBS solution with pH 8.0, homogenize with a tissue homogenizer, centrifuge for 30 minutes, and collect the supernatant;

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

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

[0023] (4) The freeze-dried samples were separated by Deae and Sephadex G100 column chromatography, and the collected liquid was dialyzed by dialysis bag and freeze-dried to obtain the small molecule active peptide from Kuiquan source.

[0024] The second aspect of the present invention provides a multifunctional antifogging agent, which is composed of the following raw materials in parts by weight: 1-5 parts of small molecule active peptide from Kuiguan source, 0.02-0.10 parts of CGS19 antimicrobial peptide, 1×10 3 -1.5×10 3 share;

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

[0026] The third aspect of the present invention provides the use of the above-mentioned small molecule active peptide from kuiguan source in the preparation of antifogging agents, products with antifogging function, antibacterial agents or antioxidants.

[0027] Preferably, the products with anti-fog function include: glasses, swimming goggles, goggles, endoscopes, food preservative films, windshields and their coatings, which have good biological safety, anti-fog, food preservation and other functions.

[0028] The fourth aspect of the present invention provides the use of the above-mentioned small molecule active peptide derived from clams in the preparation of drugs or dressings for promoting wound repair, wherein the clams polypeptide can significantly promote the proliferation of fibroblasts.

[0029] Beneficial Effects of the Invention

[0030] (1) The small molecule active peptide from Kuihan source disclosed in the present invention can be directly coated onto the surface of different transparent materials through a one-step deposition method, thereby imparting an anti-fog function to the substrate and improving the biological safety of the substrate; compared with the existing commercial 3M anti-fog goggles and commercial anti-fog agents, after the multifunctional anti-fog agent prepared by the present invention is coated onto the surface of the goggles, not only does the lens maintain a high light transmittance after being treated with 85°C water vapor for 6 hours, but it also exhibits excellent antibacterial activity against Escherichia coli and Staphylococcus aureus.

[0031] (2) The small molecule active peptide from Kuiquan source disclosed in the present invention has excellent functions of scavenging DPPH free radicals and promoting fibroblast proliferation, and can be applied to the fields of cosmetics, medical beauty, health care, food, etc., and has great application value.

[0032] (3) The kuian source disclosed in the present invention is a peptide with a MW < 3 kDa, which can be used as a stable antioxidant in medicines, foods, and cosmetics. At the same time, compared with other longer peptides, short peptides have the advantages of low production cost and simple quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 This is a scanning electron micrograph of the small molecule active peptide from Kuihanyuan;

[0035] Figure 2 It is the small molecule active peptide from Kuihan source that determines the cell survival rate;

[0036] Figure 3 This is the antioxidant activity diagram of the small molecule active peptide from Kuihan source;

[0037] Figure 4 This is the water contact angle diagram of Kuihanyuan small molecule active peptide;

[0038] Figure 5 This is the anti-fog effect picture of Kuihanyuan small molecule active peptide;

[0039] Figure 6 It is to use the small molecule peptide of Kuiquan source to prepare the multifunctional antifogging agent antifogging photos;

[0040] Figure 7 It is a photo of using small molecule peptides from Kuihan source to prepare multifunctional antifogging agent and antibacterial agent;

[0041] Figure 8 It is to use the small molecule peptide of Kuiquan source to prepare multifunctional antifogging agent food preservation photos;

[0042] Fig. 9 This is the mass spectrum of the small molecule active peptide from Kuihanyuan. DETAILED DESCRIPTION

[0043] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0044] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0045] Example 1: Preparation of small molecule active peptides from clams

[0046] The fresh clams were shelled and the meat was taken out, which was washed with ultrapure water. Three times of PBS solution with pH 8.0 was added according to the volume-to-mass ratio, and the mixture was homogenized with a tissue homogenizer and centrifuged for 30 minutes to obtain the supernatant. 60 g of ammonium sulfate was slowly added to each 100 mL of the supernatant. After the ammonium sulfate was completely dissolved, the mixture was stirred for 60 minutes, and then centrifuged for 30 minutes to collect the precipitate. The collected precipitate was dissolved with Tris buffer (pH 8.0), and then dialyzed with ultrapure water for 3 days, and freeze-dried to obtain a crude product. The crude product was separated by Deae and Sephadex G100 column chromatography (the separation liquid was PBS solution with pH 8.0 and rinsed at 4°C), and the collected liquid was dialyzed with a dialysis bag with ultrapure water at 4°C, and freeze-dried to obtain small molecule active peptides from clams.

[0047] After mass spectrometry identification, the amino acid sequence of the active peptide is: TVGMGVPAV, such as Fig. 9 shown.

[0048] (Threonine-Valine-Glycine-Methionine-Glycine-Valine-Proline-Alanine-Valine)

[0049] The specific feature of the present 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 As shown, the scanning electron micrograph of the small molecule active peptide from Kuiquan source, the active peptide exists in the form of nanoparticles in aqueous solution.

[0051] Example 2: Cell proliferation experiment of small molecule active peptides from Kuihan source

[0052] L929 fibroblast cell line was co-incubated with small molecule active peptides from Kuihan source, and CCK-8 kit was used to detect blood cell survival rate. First, L929 fibroblasts were dispersed in a 96-well plate at a density of 10,000 cells per well. After incubation for 24 hours, a certain volume of PBS solution (concentration of small molecule active peptides from Kuihan source: 0 μg / mL) and small molecule active peptide solution from Kuihan source were added to the above-mentioned well plate in sequence, and co-culture was continued in a constant temperature cell culture incubator at 37°C and 5% CO2, wherein the concentrations of small molecule active peptides from Kuihan source were 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL and 100 μg / mL, respectively. After 48 hours, 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 to each well in sequence, and placed in a 37°C incubator for further incubation for 2 hours. Finally, the absorbance of the cells at 480 nm was measured using an enzyme-labeled instrument to calculate the cell survival rate.

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

[0054] Example 3: Antioxidant activity experiment of small molecule active peptides from kui kan source

[0055] Dissolve the small molecule active peptide from Kuihan source in ultrapure water and prepare solutions of 1, 5, 10, 15 and 20 mg / mL of the small molecule active peptide from Kuihan source. Prepare 0.2 mM DPPH ethanol solution. Pipette 2 mL of DPPH ethanol solution and Kuihan source small molecule active peptide solution respectively, mix well, and place at room temperature away from light. After 30 minutes, use a UV spectrophotometer to measure the absorbance of the solution at 517 nm. The DPPH free radical scavenging rate is calculated according to the following formula:

[0056]

[0057] Among them, A S is the absorbance of the sample reaction solution, A c 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 the small molecule active peptide from Kuiquan source, its DPPH scavenging rate gradually increased, and the highest DPPH scavenging rate was about 35%, showing a certain antioxidant activity.

[0059] Example 4: Wettability experiment of small molecule active peptides from Kuihan source

[0060] A 10 mg / mL aqueous solution of the small molecule active peptide from the kui kan source was prepared, 20 μL of the solution was transferred and coated on the surface of a polycarbonate substrate, and dried at room temperature. The surface wettability was measured using a water contact angle meter.

[0061] The results are shown in Figure 4. After the small molecule active peptide from Kuiquan source was coated on the surface of the polycarbonate substrate, the contact angle of the polycarbonate substrate surface was greatly reduced, showing super hydrophilicity, and the water contact angle was 6.4°.

[0062] Example 5: Anti-fog experiment of small molecule active peptide from Kuihan source

[0063] 10 mg / mL of the small molecule active peptide from the source of kui kan was prepared, and 50 μL / single goggles were coated on the surface of the goggles, and dried at room temperature to obtain anti-fog goggles coated with the small molecule active peptide from the source of kui kan. The goggles coated with the small molecule active peptide from the source of kui kan were then placed in a hot steam environment at a temperature of ~85°C for 2 hours, and anti-fog photos were obtained at different time intervals to evaluate the anti-fog performance of the small molecule active peptide from the source of kui kan.

[0064] The results are as follows Figure 5 As shown, the goggles coated with the small molecule active peptide from Kuihanyuan were treated with hot steam at 85°C for 2 hours. The fonts on the back of the goggles were clearly visible, and no fogging was observed on the surface, showing excellent anti-fog performance.

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

[0066] In order to further broaden the application of the small molecule active peptide from the source of the kui kan, it was cross-linked with polyvinyl alcohol (PVA) rich in hydroxyl groups and sea cucumber active peptide (GS19) with excellent antibacterial activity to obtain a multifunctional antifogging agent coating with excellent antibacterial, self-repairing, self-cleaning and other properties. The specific experimental steps are as follows: 1 mg of the small molecule active peptide from the source of the kui kan and 20 μg of the CGS19 antibacterial peptide (disclosed in the existing patent) were dissolved in 1 mL of PVA aqueous solution (1wt%), stirred at room temperature to obtain a uniform solution. Then, according to the coating area of ​​the goggles, 240 μL was applied to the goggles, dried at room temperature, and the goggles coated with the coating were obtained. Then the goggles (PSG-3), the goggles coated with commercial antifogging agents (agents) and the commercial antifogging goggles (3M) were placed in a hot steam environment at a temperature of ~85 ° C for 6 hours, and antifogging photos at different time intervals were obtained to evaluate the antifogging performance and stability of the functional coating.

[0067] The results are as follows Figure 6 As shown in the figure, the goggles coated with commercial antifog agents and commercial antifog goggles have excellent antifog performance in the first 5 minutes, but the antifog effect gradually disappears as time goes on. Among them, the antifog performance of commercial antifog goggles disappears after being treated with 85℃ steam for 30 minutes, a large amount of fogging forms on the surface of the goggles, and it is difficult to observe the fonts behind the goggles through the lens; the antifog performance of goggles coated with antifog agents is better than that of commercial antifog goggles, but the antifog performance disappears after being treated with 85℃ steam for 2 hours, and the surface of the lens fogs up, and the antifog performance and stability are limited. Compared with the goggles coated with commercial antifog agents and commercial antifog goggles, the antifog agent coated with the coating shows excellent antifog performance and stability. After being treated with 85℃ steam for 6 hours, no fogging is observed on the surface, and the fonts behind the goggles are clearly visible, showing excellent antifog performance and stability.

[0068] Example 7 Antibacterial activity of the multifunctional antifogging agent prepared using small molecule active peptides from the source of kui kan

[0069] The small molecule active peptide (Glu) from Kuiquan source and PBS solution were used as the control group, and the antibacterial activity of the multifunctional coating was detected using Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). The specific experimental steps are as follows: 1 mg of small molecule active peptide from Kuiquan source and 20 μg of CGS19 antibacterial peptide were dissolved in 1 mL of PVA aqueous solution (1wt%), stirred at room temperature to obtain a uniform solution, and freeze-dried to obtain PSG-3 powder; similarly, PSG-1 and PSG-2 were obtained according to the experimental method of PSG-3, where the mass of CGS19 antibacterial peptide in PSG-1 and PSG-2 was 5 μg and 10 μg, respectively). The small molecule active peptide from Kuiquan source, PBS buffer, PSG-1, PSG-2 and PSG-3 were mixed with 1 mL of bacterial suspension (bacterial concentration was 10 6 CFU / mL) for co-culture. After 6 hours, the bacterial suspension was diluted 100,000 times with sterile PBS solution, inoculated on the culture medium, and placed in a 37°C incubator for further culture. After 24 hours, the number of colony units was counted by plate counting method to analyze the antibacterial activity.

[0070] The results are as follows Figure 7 As shown, the multifunctional coating exhibited excellent antibacterial activity against E. coli and S. aureus, with an antibacterial rate of more than 99.9%.

[0071] Example 8: Application of small molecule active peptides from kui kan source in food insurance

[0072] 200 μL of the antifogging agent solution in Example 6 was applied to the surface of the food packaging box, and the untreated food packaging box was used as a blank control. The food packaging box was placed at room temperature to observe the deterioration of the strawberries. In addition, the food packaging box was placed in a 4°C environment to observe the fogging on the surface of the fresh-keeping box.

[0073] The results are as follows Figure 8 As shown, the surface of the food preservation box without any treatment is fogged, and the strawberries behind the coated preservation box are clearly visible. The strawberries in the coated preservation box placed at room temperature have no obvious changes after being placed at room temperature for a period of time, while the strawberries in the food preservation box without any post-treatment quickly deteriorate.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

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

2. A method for extracting active peptides from small fractions of clams, characterized in that: include: Remove the shell of the cockle, take out the meat, wash it, add PBS solution, homogenize it, separate the solid and liquid, and take the supernatant; adding ammonium sulfate to the supernatant to react, and after the reaction is completed, performing solid-liquid separation and collecting the precipitate; Adding Tris buffer to the precipitate, dissolving the precipitate, dialyzing I, and then freeze-drying; The freeze-dried sample is separated by column chromatography, and the collected liquid is dialyzed II and freeze-dried to obtain the small molecule active peptide from Kuihan source.

3. The method for extracting small molecule active peptides from clams as claimed in claim 2, characterized in that: The volume of the PBS solution is 3-6 times that of the clam meat.

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

5. The method for extracting small molecule active peptides from clams as claimed in claim 2, characterized in that: The reaction time is 60-90 min.

6. The method for extracting small molecule active peptides from clams as claimed in claim 2, characterized in that: The column chromatography method uses Deae and Sephadex G100 column chromatography for separation respectively.

7. A multifunctional antifogging agent, characterized in that: The raw materials are as follows: 1-5 parts of small molecule active peptide from Kuihan source, 0.02-0.10 parts of CGS19 antimicrobial peptide, 1×10 3 -1.5×10 3 share; Wherein, the concentration of the PVA aqueous solution is 1-2wt%.

8. Use of the small molecule active peptide from the source of kuiguanyin according to claim 1 in the preparation of antifogging agents, products with antifogging function, antibacterial agents or antioxidants.

9. Use of the small molecule active peptide from Kuiquan source as claimed in claim 8 in the preparation of an antifogging agent or a product or coating with an antifogging function, characterized in that: The products with anti-fog function include: glasses, swimming goggles, goggles, endoscopes, food wrap, windshields and their coatings.

10. Use of the small molecule active peptide from kuiguan source as claimed in claim 1 in the preparation of drugs or dressings that promote wound repair.

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