Mussel polypeptide-zinc chelate as well as preparation method and application thereof

Abstract

The invention discloses a mussel polypeptide-zinc chelate as well as a preparation method and application thereof. The mussel polypeptide-zinc chelate is obtained by chelating mussel polypeptide of which the molecular weight is less than 3kDa and a zinc element; wherein the zinc element is derived from a water-soluble zinc salt; the mass ratio of the mussel polypeptide to the zinc element is (5.5-6.5): 1. The mussel polypeptide-zinc chelate provided by the invention can be used for wound repair. According to the preparation method disclosed by the invention, the mussel polypeptide-zinc chelate with relatively high zinc chelating rate and chelate yield can be obtained.

Description

Technical Field

[0001] The present invention relates to a mussel polypeptide-zinc chelate, a preparation method thereof and an application thereof. Background Art

[0002] As the largest organ of the human body, the skin is not only a key barrier between the body and the external environment, but also participates in various physiological processes such as immune regulation, excretion of metabolic wastes, and synthesis of vitamin D. However, due to the vulnerability of its tissue structure, the skin is easily damaged by external factors such as mechanical trauma, burns, and scalds. The healing process of skin wounds is usually slow and complex, involving multiple stages such as inflammatory response, tissue hyperplasia, and remodeling. If not intervened in time, chronic wounds may form, significantly affecting the quality of life of patients.

[0003] The purple mussel (Mytilus galloprovincialis) is a common economic shellfish along the coast of China, rich in protein, amino acids, trace elements and various bioactive components, and is known as the "Lady of the East China Sea". Traditional medicine records that the purple mussel has the effects of hemostasis, antidiarrhea, etc., and can be used to treat diseases such as asthenia and emaciation, and decline of essence and blood.

[0004] In recent years, there has been much research on polypeptide metal ion chelates. Generally, polypeptide metal ion chelates are active products produced by the coordination covalent bonding or adsorption bonding of carboxyl groups, phosphate groups, amide groups, and amino acids of small polypeptides produced by protein hydrolysis with metal ions. The chelation conditions of different polypeptides with different metal ions are different, and the properties of the obtained chelates are different.

[0005] CN110643663A discloses a preparation method of mussel polypeptide chelated calcium: mussel homogenate is enzymolyzed to obtain polypeptide powder, which is chelated with calcium chloride under low temperature and high pressure conditions, and the final product is obtained through microencapsulation treatment. The mussel polypeptide chelated calcium prepared by this preparation method reduces the stimulation to the stomach and enhances the absorption of calcium salts by the intestine.

[0006] CN114041603A discloses an application of a peptide-zinc chelate in anti-allergy products. Oyster peptide powder is chelated with zinc sulfate under certain conditions to obtain oyster peptide-zinc chelate.

[0007] CN115894666A discloses the preparation of a tuna skin collagen peptide zinc ion chelate, mainly by hydrolyzing tuna skin with a composite protease to obtain collagen active peptides, and then chelating the collagen active peptides with a zinc ion solution to obtain a tuna skin collagen peptide zinc ion chelate. It is beneficial to reduce the side effects during zinc ion absorption.

[0008] CN119350176A discloses a polypeptide metal chelate, which is formed by chelating a polypeptide compound with a metal ion compound, and the metal ion compound is a zinc ion compound. The polypeptide compound can be used in the field of cosmetics.

[0009] CN117143187A discloses a zinc ion chelated modified peptide derived from the traditional Chinese medicine Periplaneta americana, which can be used to promote wound repair or / and healing. The preparation method includes preparing a polypeptide with the sequence GHCSPPFAPRL by using the polypeptide solid-phase synthesis technique and separating and purifying it by using semi-preparative high-performance liquid chromatography to obtain the purified polypeptide; using the microwave synthesis method to chelate metal zinc ions with the three amino acid residues GHC in the purified polypeptide, and then precipitating and separating the metal zinc ion chelated modified peptide from the reaction system by the ethanol precipitation method to obtain the zinc ion chelated modified peptide derived from the traditional Chinese medicine Periplaneta americana.

[0010] So far, there has been no report on the mytilus edulis polypeptide-zinc chelate, its preparation method and its application in wound repair. Summary of the Invention

[0011] In view of this, one object of the present invention is to provide a mytilus edulis polypeptide-zinc chelate, which can be used for wound repair. Another object of the present invention is to provide a preparation method of the mytilus edulis polypeptide-zinc chelate as described above. Still another object of the present invention is to provide an application of the mytilus edulis polypeptide-zinc chelate as described above. Yet another object of the present invention is to provide a wound repair product.

[0012] The present invention adopts the following technical solutions to achieve the above objects.

[0013] On the one hand, the present invention provides a mytilus edulis polypeptide-zinc chelate, which is obtained by chelating a mytilus edulis polypeptide with a molecular weight less than 3 kDa and zinc element;

[0014] Among them, the zinc element is derived from a water-soluble zinc salt;

[0015] Among them, the mass ratio of the mytilus edulis polypeptide to the zinc element is 5.5-6.5:1.

[0016] On the other hand, the present invention also provides a preparation method of the mytilus edulis polypeptide-zinc chelate as described above, including the following steps:

[0017] 1) Dissolve the freeze-dried powder of the mytilus edulis polypeptide with a molecular weight less than 3 kDa in water to obtain a mytilus edulis polypeptide aqueous solution with a concentration of 25-120 mg / mL;

[0018] 2) Mix the mussel polypeptide aqueous solution and the water-soluble zinc salt, react at 30-60 °C for 20-80 min, and maintain the pH value of the reaction solution at 5-8 during the reaction process; wherein, the mass ratio of zinc element in the mussel polypeptide to the water-soluble zinc salt is 5.5-6.5:1;

[0019] 3) After the reaction, add alcohol for precipitation, perform solid-liquid separation, wash the separated solid and freeze-dry it to obtain the mussel polypeptide-zinc chelate.

[0020] According to the preparation method of the present invention, preferably, in step 2), the water-soluble zinc salt is zinc sulfate; in step 3), after the reaction, add absolute ethanol for precipitation, perform solid-liquid separation, wash the separated solid and freeze-dry it to obtain the mussel polypeptide-zinc chelate.

[0021] According to the preparation method of the present invention, preferably, the freeze-dried mussel polypeptide is prepared by the following steps:

[0022] (a) Remove sand and shells from mussels to obtain mussel meat; crush the mussel meat to obtain processed mussels;

[0023] (b) Calculate according to the dry weight of the processed mussels, add 10-17 times the weight of water to the processed mussels and mix, and adjust the pH value of the mixture to 7-9 to obtain a mussel homogenate;

[0024] (c) Add 1-4 wt% of trypsin to the mussel homogenate obtained in step (b), and perform enzymatic hydrolysis reaction at 45-55 °C for 2-5 h;

[0025] (d) After the enzymatic hydrolysis reaction, boil the enzymatic hydrolysis reaction product for 10-20 min, then centrifuge and collect the supernatant;

[0026] (e) Separate the supernatant through an ultrafiltration membrane, collect the filtrate of peptide segments with a molecular weight less than 3 kDa, concentrate the collected filtrate, and then freeze-dry the concentrate to obtain a freeze-dried mussel polypeptide with a molecular weight less than 3 kDa.

[0027] According to the preparation method of the present invention, preferably, in step (a), the mussel is Mytilus edulis; the particle size of the processed mussel is 1-5 mm; in step (b), adjust the pH value of the mixture to 7.5-8.5 with an alkali metal hydroxide solution or hydrochloric acid solution.

[0028] According to the preparation method of the present invention, preferably, in step (c), add 2-4 wt% of trypsin to the mussel homogenate obtained in step (b), and perform enzymatic hydrolysis reaction at 45-55 °C for 3.5-5 h.

[0029] According to the preparation method of the present invention, preferably, in step (d), the centrifugation speed is 4000 - 7000 rpm, and the centrifugation time is 10 - 20 min.

[0030] According to the preparation method of the present invention, preferably, in step (e), the collected filtrate is concentrated to one fifteenth to one twentieth of the original volume to obtain a concentrate; the concentrate is freeze-dried at a temperature below -35°C to obtain a freeze-dried powder of mussel polypeptide with a molecular weight less than 3 kDa.

[0031] On the other hand, the present invention also provides an application of the mussel polypeptide-zinc chelate as described above or the mussel polypeptide-zinc chelate prepared according to the preparation method as described above in the preparation of wound repair products.

[0032] On another hand, the present invention also provides a wound repair product prepared from raw materials including the mussel polypeptide-zinc chelate as described above.

[0033] The mussel polypeptide-zinc chelate of the present invention has good skin wound healing ability, and the antibacterial effect and skin wound healing rate of the chelate are significantly higher than those of the unchelated mussel polypeptide. According to the preferred technical solution of the present invention, chelating mussel polypeptide and water-soluble zinc salt under specific conditions can obtain a mussel polypeptide-zinc chelate with a high zinc chelation rate and chelate yield and good wound repair performance. The zinc chelation rate reaches more than 90%, preferably more than 96%, and the chelate yield is more than 50%. Description of the Drawings

[0034] Figure 1 It is the ultraviolet-visible spectrogram of the mussel polypeptide (MPH) prepared in Preparation Example 1 and the mussel polypeptide-zinc chelate (MPH-Zn) prepared in Example 1.

[0035] Figure 2 It is the infrared spectrogram of the mussel polypeptide (MPH) prepared in Preparation Example 1 and the mussel polypeptide-zinc chelate (MPH-Zn) prepared in Example 1.

[0036] Figure 3 It is the SEM image of the mussel polypeptide (MPH) prepared in Preparation Example 1.

[0037] Figure 4 It is the SEM image of the mussel polypeptide-zinc chelate (MPH-Zn) prepared in Example 1.

[0038] Figure 5 It is the observation result of the morphology of the constructed Caco-2 cell monolayer membrane by laser confocal microscopy.

[0039] Figure 6The effects of mussel polypeptide-zinc chelate on Escherichia coli (E. coli), Gram-positive Staphylococcus aureus (S. aureus), and methicillin-resistant Staphylococcus aureus (MRSA).

[0040] Figure 7 The results of the changes in the back wounds of mice in different groups using mussel polypeptide and mussel polypeptide-zinc chelate.

[0041] Figure 8 The wound healing rates of mice in different groups using mussel polypeptide and mussel polypeptide-zinc chelate. Specific implementation manners

[0042] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0043] As described in the background art, although there are many existing technologies for chelating polypeptides and metal ions, there is still no report on mussel polypeptide-zinc chelate and its preparation method and application so far. Moreover, since the chelating conditions of different polypeptides and zinc are different, therefore, the preparation method of the present invention using specific process parameters to obtain a relatively high zinc content (i.e., a relatively high chelation rate of zinc) and a relatively high yield of chelate does not belong to a conventional choice.

[0044] <Mussel polypeptide-zinc chelate>

[0045] A mussel polypeptide-zinc chelate provided by the present invention is obtained by chelating mussel polypeptide with a molecular weight less than 3 kDa and zinc element; wherein, the zinc element is derived from a water-soluble zinc salt; the mass ratio of mussel polypeptide to zinc element is 5.5-6.5:1. Such a mussel polypeptide-zinc chelate has good skin healing ability, and the antibacterial effect and skin healing rate of the chelated mussel polypeptide are significantly higher than those of the unchelated mussel polypeptide.

[0046] In the present invention, specifically, the mussel polypeptide-zinc chelate is obtained by chelating an aqueous solution formed by freeze-dried powder of mussel polypeptide with a molecular weight less than 3 kDa and a water-soluble zinc salt containing zinc element under specific conditions. The freeze-dried powder of mussel polypeptide with a molecular weight less than 3 kDa is preferably prepared from Mytilus edulis. The water-soluble zinc salt can be selected from at least one of zinc sulfate, zinc chloride, and zinc acetate, and is preferably zinc sulfate. The water-soluble zinc salt may contain crystal water or may not contain crystal water.

[0047] In the present invention, the mass ratio of mussel polypeptide to zinc element is 5.5-6.5:1, preferably 5.8-6.2:1, more preferably 5.9-6.1:1, for example, it can be 5.9:1, 6:1, 6.1:1. Within such a mass ratio range, the chelation rate of zinc in the obtained mussel polypeptide-zinc chelate is relatively high, the yield of chelate is relatively high, and the obtained chelate has good performance in promoting skin wound healing.

[0048] <Preparation method of mussel polypeptide-zinc chelate>

[0049] A preparation method of mussel polypeptide-zinc chelate provided by the present invention includes three steps: the preparation step of mussel polypeptide aqueous solution, the chelation step, and the post-treatment. Optionally, it also includes the preparation step of mussel polypeptide freeze-dried powder. The following is a detailed description.

[0050] Preparation steps of freeze-dried mussel polypeptides

[0051] In the present invention, the mussel polypeptide freeze-dried powder is prepared by the following steps:

[0052] (a) Remove sand and shells from mussels to obtain mussel meat; crush the mussel meat to obtain treated mussels;

[0053] (b) Calculate according to the dry weight of the treated mussels, add 10-17 times the weight of water to the treated mussels and mix, and adjust the pH value of the mixture to 7-9 to obtain a mussel homogenate;

[0054] (c) Add 1-4 wt% of trypsin to the mussel homogenate obtained in step (b), and carry out an enzymatic hydrolysis reaction at 45-55 °C for 2-5 h;

[0055] (d) After the enzymatic hydrolysis reaction is completed, boil the enzymatic hydrolysis reaction product for 10-20 min, then centrifuge, and collect the supernatant;

[0056] (e) Separate the supernatant through an ultrafiltration membrane, collect the filtrate of peptide segments with a molecular weight less than 3 kDa, concentrate the collected filtrate, and then freeze-dry the concentrate to obtain a mussel polypeptide freeze-dried powder with a molecular weight less than 3 kDa. Under such conditions, a mussel polypeptide freeze-dried powder with a molecular weight less than 3 kDa and high hydrolysis degree and polypeptide yield can be obtained.

[0057] In step (a), preferably, the mussel is Mytilus edulis. Remove sand and shells from the mussels, drain them, and they can be used fresh or stored frozen for later use. A meat grinder can be used to crush the mussel meat to obtain a minced treated mussel. The particle size of the treated mussel is 1-5 mm, preferably 2-4 mm, and more preferably 2.5-3.5 mm. This is beneficial to the full progress of the subsequent enzymatic hydrolysis reaction and improves the hydrolysis efficiency.

[0058] In step (b), the water can be deionized water, purified water or distilled water. The amount of water is calculated according to the dry weight of the treated mussels (dry weight: after the mussel meat is minced, a part of the treated mussel meat is measured for dry weight on a moisture analyzer, in parallel three times, and the average value is taken)), and is 10 to 17 times the weight of the treated mussels, preferably 12 to 17 times, more preferably 14 to 16 times. This can ensure the fluidity of the homogenate, is beneficial to the progress of the enzymatic hydrolysis reaction, and at the same time avoids excessive dilution from affecting the efficiency of the subsequent concentration step.

[0059] In the present invention, an alkali metal hydroxide solution or a hydrochloric acid solution is used to adjust the pH value of the mixture to 7 to 9, preferably 7 to 8.5, more preferably 7.5 to 8. This is beneficial to the progress of the enzymatic hydrolysis reaction. The alkali metal hydroxide solution can be selected from one of sodium hydroxide solution and potassium hydroxide solution, preferably sodium hydroxide solution, and the concentration can be 0.05 to 0.5 mol / L, preferably 0.1 to 0.3 mol / L, more preferably 0.1 to 0.2 mol / L. The concentration of the hydrochloric acid solution can be 0.05 to 0.5 mol / L, preferably 0.1 to 0.3 mol / L, more preferably 0.1 to 0.2 mol / L.

[0060] In step (c), the protease used in the enzymatic hydrolysis reaction is preferably trypsin. The dosage of the protease is 1 to 4 wt% (based on the dry weight of the treated mussels), preferably 2 to 4 wt%, more preferably 3 to 3.5 wt%. The present invention particularly preferably uses trypsin. The preferred dosage of trypsin can ensure sufficient hydrolysis while reducing costs and reducing by-product generation, so as to obtain a better mussel polypeptide-zinc chelate with wound repair promoting performance. The temperature of the enzymatic hydrolysis reaction can be 45 to 55 °C, preferably 50 to 55 °C. The time of the enzymatic hydrolysis reaction can be 2 to 5 h, preferably 2 to 4 h, more preferably 3 to 4 h. This is beneficial to improving the degree of hydrolysis and the polypeptide yield.

[0061] In step (d), after the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysis reaction product is boiled for 10 to 20 min to inactivate the enzyme. The boiling time is preferably 10 to 15 min, more preferably 10 to 12 min. After boiling, centrifugation is carried out. The rotation speed of centrifugation can be 4000 to 7000 rpm, preferably 4500 to 6500 rpm, more preferably 5000 to 5500 rpm. The time of centrifugation can be 10 to 20 min, preferably 12 to 18 min, more preferably 15 to 17 min. The supernatant is directly taken after centrifugation, or filtration is carried out after centrifugation, and the supernatant is collected. Preferably, the process of centrifugation and filtration is repeated one to three times. Finally, the supernatant (i.e., the clear liquid) is collected.

[0062] In step (e), the molecular weight cut-off of the ultrafiltration membrane is preferably 5 kDa or more, more preferably 4 kDa or more, and even more preferably 3 kDa or more. According to a specific embodiment of the present invention, the filtrate containing peptides with a molecular weight less than 3 kDa is collected, and the collected filtrate is concentrated to one-fifteenth to one-twentieth of the original volume, and then the concentrate is freeze-dried to obtain freeze-dried mussel polypeptides with a molecular weight less than 3 kDa. The freeze-drying temperature can be less than -35 °C, preferably less than -50 °C, such as -80 °C.

[0063] Preparation steps of mussel polypeptide aqueous solution

[0064] The freeze-dried mussel polypeptides with a molecular weight less than 3 kDa are dissolved in water to obtain a mussel polypeptide aqueous solution with a concentration of 25 - 120 mg / mL.

[0065] In the mussel polypeptide aqueous solution, the concentration of the mussel polypeptide can be 25 - 120 mg / mL, preferably 40 - 120 mg / mL, more preferably 60 - 120 mg / mL, further preferably 80 - 110 mg / mL, even more preferably 90 - 110 mg / mL, for example, it can be 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL. This concentration range can ensure that the reaction system has appropriate fluidity, while providing sufficient polypeptide molecules to participate in the chelation reaction, which is beneficial to improving the chelation rate of zinc in the obtained chelate and the yield of the chelate. Under the condition of a peptide concentration lower than that of the present invention, the concentrations of the polypeptide and zinc ions are lower, and the intermolecular collision frequency decreases, resulting in a decrease in the efficiency of the chelation reaction. While under the condition of a peptide concentration higher than that of the present invention, the solubility of the polypeptide decreases, the molecular distribution is too dense, and the steric hindrance effect is enhanced, thus inhibiting the progress of the chelation reaction.

[0066] Chelation step

[0067] The mussel polypeptide aqueous solution and the water-soluble zinc salt are mixed and reacted at 30 - 60 °C for 20 - 80 min, and the pH value of the reaction solution is maintained at 5 - 8 during the reaction; wherein, the mass ratio of the mussel polypeptide to the zinc element in the water-soluble zinc salt is 5.5 - 6.5:1. This is beneficial to improving the chelation rate of zinc, and thus beneficial to obtaining a mussel polypeptide-zinc chelate with the performance of promoting wound repair.

[0068] According to an embodiment of the present invention, the water-soluble zinc salt is added to the mussel polypeptide aqueous solution and mixed. The water-soluble zinc salt can be selected from at least one of zinc sulfate, zinc chloride, and zinc acetate, preferably zinc sulfate. The water-soluble zinc salt can contain crystal water or can be anhydrous.

[0069] In the present invention, the mass ratio of zinc element in mussel polypeptide and water-soluble zinc salt (which can be abbreviated as peptide-zinc ratio) is 5.5 - 6.5:1, preferably 5.8 - 6.2:1, more preferably 5.9 - 6.1:1, and for example, it can be 5.9:1, 6:1, 6.1:1. When the peptide-zinc ratio is higher than the scope of the present invention, the polypeptide is in excess supply, and part of the polypeptide may not be able to form a stable chelate due to lack of sufficient Zn 2+ and thus exists in the reaction system in a free state and is removed during the washing process with absolute ethanol, thereby reducing the chelation rate. However, when the peptide-zinc ratio is lower than the scope of the present invention, the binding sites of the polypeptide tend to be saturated, and the excessive Zn 2+ generates insoluble precipitates, such as Zn(OH)2, etc., through competitive reactions, resulting in waste of raw materials.

[0070] In the present invention, the pH value during the chelation reaction is 5 - 8, preferably 6 - 8, more preferably 7 - 8, and still more preferably 7.5 - 8. When the solution pH is too low, hydrogen ions competitively inhibit the chelation of zinc ions; while when the pH is too high, zinc ions are prone to form precipitates with hydroxide ions, both of which are not conducive to the progress of the chelation reaction. According to a preferred embodiment of the present invention, the pH value during the chelation reaction is 7.5 - 8.

[0071] In the present invention, an alkali metal hydroxide solution or a hydrochloric acid solution can be used to maintain the pH value within a specific range. The alkali metal hydroxide solution can be one of sodium hydroxide solution and potassium hydroxide solution, preferably sodium hydroxide solution, and the concentration can be 0.05 - 0.5 mol / L, preferably 0.1 - 0.3 mol / L, more preferably 0.1 - 0.2 mol / L. The concentration of the hydrochloric acid solution can be 0.05 - 0.5 mol / L, preferably 0.1 - 0.3 mol / L, more preferably 0.1 - 0.2 mol / L. This is conducive to maintaining the constancy of the pH value of the reaction solution, thus conducive to improving the zinc chelation rate of the obtained chelate.

[0072] The temperature of the chelation reaction can be 30 - 60 °C, preferably 40 - 60 °C, more preferably 45 - 55 °C, and still more preferably 50 - 55 °C. The time of the chelation reaction can be 20 - 80 min, preferably 30 - 70 min, more preferably 40 - 65 min, and still more preferably 55 - 60 min. Such a temperature range and reaction time are conducive to improving the zinc chelation rate.

[0073] Post-treatment step

[0074] After the reaction is completed, alcohol is added for precipitation, solid-liquid separation is carried out, and the separated solid is washed and freeze-dried to obtain the mussel polypeptide-zinc chelate. This is conducive to improving the chelate yield and conducive to obtaining a mussel polypeptide-zinc chelate with better performance.

[0075] According to an embodiment of the present invention, after the chelation reaction is completed, precipitation is carried out using ethanol. The amount of ethanol used is 4 to 6 times the volume of the chelation reaction solution after the chelation reaction, preferably 4.5 to 5.5 times, and more preferably 5 to 5.5 times. Ethanol is preferably anhydrous ethanol. When the amount of ethanol used is within the above range, it is beneficial to improve the chelation rate of zinc and the yield of the chelate. If the amount of ethanol is too small, the purity of the chelate is low; if the amount of ethanol is too large, the yield of the chelate is low. After precipitation, it can be refrigerated and left to stand. The refrigeration and standing time is not specifically limited, as long as the precipitation is fully deposited, such as refrigerating and standing for 5 to 20 h, preferably 10 to 14 h, and more preferably 11 to 13 h. Sufficient standing time is beneficial to the formation and full precipitation of chelate crystals.

[0076] Solid-liquid separation can be carried out by centrifugation or filtration. When performing solid-liquid separation, the solid precipitate can be washed with a solvent. The washing method can be washing with anhydrous ethanol, and the amount of anhydrous ethanol used is not specifically limited, as long as various reagents on the surface of the precipitate can be washed away.

[0077] Using the preparation method of the present invention, the chelation rate of zinc can reach more than 90%, and can reach more than 96% in the preferred technical solution; the yield of the chelate can reach more than 50%, and can reach more than 51% in the preferred technical solution. The preparation method of the present invention can take into account both the chelation rate of zinc and the yield of the chelate.

[0078] <Application of Mytilus edulis Polypeptide-Zinc Chelate>

[0079] The present invention also provides an application of the Mytilus edulis polypeptide-zinc chelate as described above or the Mytilus edulis polypeptide-zinc chelate prepared by the above preparation method in the preparation of a wound repair product.

[0080] <Wound Repair Product>

[0081] A wound repair product provided by the present invention is prepared from raw materials including the Mytilus edulis polypeptide-zinc chelate as described above. That is, it can include the Mytilus edulis polypeptide-zinc chelate and other raw materials. The wound repair product is a skin external use product, and the specific form is not limited. For example, it can be an ointment, a gel, a wound dressing, etc.

[0082] The following uses specific examples and experimental examples to illustrate the embodiments and technical effects of the present invention, but does not constitute a limitation on the protection scope of the present invention.

[0083] <Analysis Method>

[0084] Ultraviolet-visible absorption spectrum: A ultra-micro high-precision ultraviolet / visible light photometer is used, manufactured by DeNovix in the United States, model DS-11FX.

[0085] Infrared absorption spectrum: A Fourier transform infrared spectrometer from Bruker is used.

[0086] SEM test: The EVO LS15 scanning electron microscope energy spectrometer of Carl Zeiss AG in Germany was used.

[0087] Amino acid content analysis: The fully automatic amino acid analyzer of SECOM in Germany, model S-433D, was used.

[0088] Definition and test of degree of hydrolysis: The o-phthalaldehyde (OPA) method was used to determine the degree of hydrolysis (DH) of proteins. The principle is that OPA reacts specifically with the free α-amino groups in the hydrolysis products, and the content of peptides and amino acids is indirectly quantified through absorbance. This application was optimized based on the methods of Church and Nielsen, and a UV-visible spectrophotometer was used to analyze the MPH solution. 400 μL of the diluted sample solution was added to a test tube containing 3 mL of OPA reagent (7.620 g of sodium tetraborate, 200 mg of sodium dodecyl sulfate, 16 mL of OPA, 176 mg of dithiothreitol, and 200 mL of deionized water), and this moment was marked as time point 0. After the mixture was stirred thoroughly for 5 seconds, it was left standing for 2 minutes, and then the absorbance was immediately measured at 340 nm. Deionized water or buffer was used as the blank to correct the absorbance. Glycine was used as the standard product to calculate the free amino group content (μmol NH2 / mL) in each sample. The experiment was set up with two parallel runs in each group, for a total of two parallel experiments. The degree of hydrolysis of each sample at each digestion time point was calculated as follows:

[0089]

[0090] The degree of hydrolysis (DH%) is defined as the percentage of peptide bond cleavage during protein hydrolysis. In this calculation, NH 2t represents the concentration of free α-amino groups (mg / mL) at a specific digestion time point, NH2 t0 represents the initial concentration of amino groups (mg / mL) before digestion, and Total NH2 refers to the total amino group concentration (mg / mL) calculated based on the total number of amino groups in the amino acid molecule.

[0091] Testing the amount of zinc element in the obtained chelate: The method of "GB 5009.14-2017 Ethylenediaminetetraacetic acid disodium (EDTA) titration method" was adopted with some modifications. Accurately weigh 0.5 - 1.0 g of the MPH-Zn sample and place it in a 250 mL conical flask, and moisten it with a small amount of ultrapure water. Add 5 mL of HCl solution (V / V = 1:4) and 50 mL of ultrapure water, and dissolve it by ultrasonic wave. Add 10 mL of ammonium fluoride solution (100 g / L), 10 mL of thiourea solution (50 g / L) and 0.2 g of ascorbic acid in sequence, and shake until completely dissolved. Add 3 drops of xylenol orange indicator, and titrate with 0.05 M EDTA standard solution until the solution changes from purplish red to bright yellow, which is the titration end point, and record the titration volume V1. At the same time, conduct a blank test and record the titration volume V0. The zinc content (X, in mass percentage) in the chelate sample is calculated according to the following formula:

[0092]

[0093] In the formula: V1 is the volume of EDTA consumed for titrating the sample solution (mL), V0 is the volume of EDTA consumed for titrating the blank solution (mL), C is the actual concentration of EDTA (mol / L), m is the sample mass (g), and 0.06539 is a constant.

[0094] In the process of preparing freeze-dried mussel polypeptides and mussel polypeptide-zinc chelates, unless otherwise specified, all experiments were independently repeated three times, and the data were expressed as mean ± standard deviation (x ± s.d.), and the significance level was set as P < 0.05. Data processing was completed by origin 2024 software, and charts were drawn according to the experimental results.

[0095] The following introduces the sources of some raw materials:

[0096] The mussel, with the English name Mytilus galloprovincialis, was purchased from Fanghuayuan Market in Yantai, China. It was a live individual with a body length of 5 - 8 cm and a weight of 20 - 30 g per mussel. Before use, it was frozen and stored at -80 °C after shelling, cleaning and draining.

[0097] Trypsin, pepsin, neutral protease, and alkaline protease were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. MEM basal medium: purchased from Wuhan Procell Life Science Co., Ltd. Hank's balanced salt solution: purchased from Wuhan Procell Life Science Co., Ltd. The moisture analyzer was purchased from Shanghai Youke Instrument Co., Ltd., model DSH-10A.

[0098] Preparation Example 1

[0099] The mussel meat was ground into minced meat (particle size 1 - 5 mm) with a meat grinder to obtain the processed mussel.

[0100] Add 15 times the weight of deionized water to the processed mussels (based on the dry weight of the processed mussels; method for dry weight: after the mussel meat is minced, take a portion of the processed mussels and measure the dry weight on a moisture analyzer, repeat three times in parallel, and take the average), and mix thoroughly. Then, adjust the pH value of the mixture to 8 using 0.1 mol / L sodium hydroxide or 0.1 mol / L hydrochloric acid solution to obtain a mussel homogenate.

[0101] Add 3 wt% trypsin (based on the dry weight of the processed mussels) to the mussel homogenate, and carry out an enzymatic hydrolysis reaction on the mussel homogenate at 50 °C for 4 h.

[0102] After the enzymatic hydrolysis reaction is completed, boil the enzymatic hydrolysis reaction product for 10 min to inactivate the enzyme, and then quickly cool it in an ice bath to obtain an inactivated product. Then, centrifuge the inactivated product at 5000 rpm for 15 min, filter it by suction, and repeat the steps of centrifugation and suction filtration of the filtrate twice. Collect the supernatant (i.e., the clear liquid).

[0103] Separate the supernatant through an ultrafiltration membrane, collect the filtrate of the peptide segments with a molecular weight less than 3 kDa, concentrate the collected filtrate to 1 / 20 of the original volume, and freeze-dry the concentrate at -80 °C to obtain a freeze-dried powder of mussel polypeptide with a molecular weight less than 3 kDa. Denote it as MPH.

[0104] Preparation Examples 2-12 and Comparative Preparation Examples 1-3

[0105] In Preparation Examples 2 to 12 and Comparative Preparation Examples 1 to 3, some process parameters refer to Table 1 below, and the remaining parameters and operating steps are the same as those in Preparation Example 1.

[0106] Table 1

[0107]

[0108] Example 1

[0109] Dissolve the freeze-dried powder of mussel polypeptide with a molecular weight less than 3 kDa prepared in Preparation Example 1 in water to obtain an aqueous solution of mussel polypeptide with a concentration of 100 mg / mL.

[0110] Mix the aqueous solution of mussel polypeptide and zinc sulfate, react at 50 °C for 60 min, and maintain the pH value of the reaction solution at 7.5 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution during the reaction process; wherein, the mass ratio of the mussel polypeptide in the aqueous solution of mussel polypeptide to the zinc element in zinc sulfate is 6:1.

[0111] After the reaction was completed, 5 volumes of absolute ethanol (the amount of absolute ethanol was based on the volume of the reaction solution after the chelation reaction) were added for precipitation, and the mixture was allowed to stand at 4 °C for 12 h for precipitation. Then, solid-liquid separation was carried out. The separated solid precipitate was washed with absolute ethanol and then freeze-dried at -80 °C to obtain the mussel polypeptide-zinc chelate, denoted as MPH-Zn.

[0112] Comparative Examples 1-6

[0113] In Comparative Examples 1 to 6, some process parameters were referred to Table 2 below, and the remaining parameters and operation steps were the same as those in Example 1.

[0114] Table 2

[0115]

[0116] Note: In Table 2, the chelation rate of zinc = (mass of zinc element in the chelate / mass of zinc element in the raw materials used) × 100%; the yield of the chelate = (mass of the obtained chelate / sum of the masses of the raw mussel polypeptide freeze-dried powder and zinc sulfate) × 100%; the chelate refers to the mussel polypeptide-zinc chelate. The peptide-zinc ratio refers to the mass ratio of mussel polypeptide to zinc element in zinc sulfate. The definitions in the following table are the same.

[0117] It can be seen from the comparison of Comparative Examples 1 to 6 that, under other unchanged conditions, as the peptide concentration increases, the chelation rate of zinc first increases and then decreases. It can be seen from the comparison of Comparative Example 1 and Example 1 that although the yield of the chelate in Comparative Example 1 is relatively high, the chelation rate of zinc in its chelate is relatively low, that is, the zinc content in the chelate is relatively low.

[0118] Example 2 and Comparative Examples 7-8

[0119] In Example 2 and Comparative Examples 7 and 8, some process parameters were referred to Table 3 below, and the remaining parameters and operation steps were the same as those in Example 1.

[0120] Table 3

[0121]

[0122] It can be seen from Table 3 that when the peptide-zinc ratio is 6 - 7, the chelation rate of zinc is relatively high, especially when the peptide-zinc ratio is 6, the chelation rate of zinc is even higher.

[0123] Comparative Examples 9-18

[0124] In Comparative Examples 9 - 18, some process parameters were referred to Table 4 below, and the remaining parameters and operation steps were the same as those in Example 1.

[0125] Table 4

[0126]

[0127] It can be seen from Comparative Examples 9 to 12 that as the pH value increases, the chelation rate of zinc first increases and then decreases. It can be seen from Comparative Examples 15 to 18 that as the amount of absolute ethanol increases, the chelation rate of zinc increases, but the yield of the chelate first increases and then decreases. The chelation rates of zinc in the chelates are generally low, all lower than 90%.

[0128] Experimental Example 1

[0129] (I). Ultraviolet absorption spectrum determination

[0130] The MPH obtained in Preparation Example 1 and the MPH-Zn obtained in Example 1 were subjected to ultraviolet absorption spectrum determination. Appropriate amounts of MPH and MPH-Zn were weighed and diluted to a concentration of 0.2 mg / mL with deionized water respectively. The measurement was carried out using an ultra-micro high-precision ultraviolet / visible light photometer, and the measurement range was between 190 - 400 nm. The instrument was calibrated with deionized water for blank.

[0131] The ultraviolet absorption spectra of MPH and MPH-Zn are as Figure 1 shown. MPH and MPH-Zn have a maximum absorption peak at 195 nm, which belongs to the characteristic absorption peak range of peptide bonds. The absorption peak at 195 nm is usually related to the carbonyl group (C=O) and amide bond (-CONH-) in the peptide chain, mainly caused by π-π* transition and n-π* transition, and is a typical absorption characteristic of MPH in the far ultraviolet region. The absorption peak between 250 - 280 nm is the characteristic absorption peak of aromatic amino acids containing benzene rings (such as phenylalanine, tyrosine and tryptophan). For both absorption peaks, the absorption intensity of MPH-Zn is lower than that of MPH, which should be because after Zn 2+ forms coordination bonds with the N and O atoms in the peptide molecule, resulting in changes in the intramolecular chromophore groups (such as -C=O, -COOH) and the spatial structure of their internal atoms, leading to a decrease in the absorption intensity in the ultraviolet absorption spectrum. These changes indicate that the chelate is different from the original peptide and is a new molecular structure.

[0132] (II). Infrared absorption spectrum determination

[0133] The MPH obtained in Preparation Example 1 and the MPH-Zn obtained in Example 1 were analyzed by Fourier transform infrared spectroscopy. The spectral data were collected at room temperature in the wavenumber range of 4000 cm -1 to 400 cm -1 . When preparing the sample, an appropriate amount of MPH or MPH-Zn was mixed with anhydrous potassium bromide, ground into a fine powder, and pressed into a transparent thin film for spectral analysis.

[0134] The results are as Figure 2 shown. From Figure 2It can be seen that the coordination of zinc ions affects the formation of intramolecular hydrogen bonds, which is reflected by the change in the position of the absorption peak and the appearance of new peaks. In the MPH-Zn complex, the N-H stretching vibration peak shifts from 3424.61 cm -1 to 3421.67 cm -1 . This should be because the electron-withdrawing effect of Zn 2+ enhances the electron density around the N-H bond. The N-terminus and N-H groups of lysine (Lys) and arginine (Arg) in MPH may form coordination bonds with Zn 2+ , thus forming N-Zn bonds. The absorption band of MPH at 1633.79 cm -1 corresponds to the amide I band (1600 - 1700 cm -1 ), which is mainly caused by the stretching of the C-O bond and the bending vibration of the N-H bond. After zinc chelation, the absorption peak in MPH-Zn shifts to 1625.33 cm -1 , and the vibration intensity weakens, indicating that the C-O group forms a coordination bond with Zn 2+ .

[0135] In MPH, the infrared absorption peak of the amide II band appears at 1516.97 cm -1 . After chelation, the amide group forms a new chemical environment with zinc ions, thus affecting its vibration characteristics and resulting in the disappearance of this characteristic peak. Before and after chelation, the wave number of -COO - changes from 1405.54 cm -1 to 1406.61 cm -1 . Although the infrared absorption change is small, the increase in frequency indicates that -COO - may coordinate with zinc ions to form a stronger chemical bond. In addition, the peak of MPH at 1043.45 cm -1 shifts to 1126.02 cm -1 in MPH-Zn. Due to the stretching vibration of C-O, it indicates the formation of the C-O-Zn bond. The changes in the FTIR spectrum show that after the combination of metal ions and organic ligand groups, the characteristic absorption peaks of peptides change significantly. The strong interaction between the -CO, -NH2, and -COOH groups in MPH and Zn 2+ ions ultimately leads to the formation of MPH-Zn.

[0136] (III) Determination of Amino Acid Composition

[0137] The amino acid compositions of MPH in Preparation Example 1 and MPH-Zn in Example 1 were analyzed using an automatic amino acid analyzer. Before analysis, the MPH and MPH-Zn samples were processed to remove proteins, lipids, and pigments, and ammonia was removed if necessary. The samples were separated using ion exchange chromatography.

[0138] Amino acid sequence and composition are key factors affecting the interaction between polypeptides and metal ions. The amino acid compositions of MPH and MPH-Zn are shown in Table 5. 17 kinds of amino acids are detected in both MPH and MPH-Zn, and the content of glutamic acid is the highest, reaching 17.39%. The significant feature of MPH is that it contains a large amount of Asp (9.51%), Glu (13.40%), Lys (9.06%), His (2.00%) and Arg (8.25%). The inventors believe that these residues can provide a large number of reaction sites for binding zinc ions through electrostatic or coordination interactions. After conjugation with zinc ions, the contents of Asp, Glu, Lys, His and Arg in MPH-Zn increase significantly compared with MPH. Their contents increase to 12.67%, 17.39%, 12.07%, 3.08% and 11.20% respectively. The contents of acidic amino acids and basic amino acids are 22.91% and 30.06% respectively. In contrast, the contents of MPH-Zn increase significantly to 19.31% and 26.35%. Research shows that acidic amino acids and basic amino acids become the key sites for the formation of mussel polypeptide-zinc chelates due to the presence of free carboxyl groups and amino groups; Cys has sulfhydryl and hydroxyl residues and plays a significant role in the formation of mussel polypeptide-zinc chelates; Pro forms a more stable cyclic or large chelate structure by inducing the formation of a bent conformation in the internal structure of the peptide chain. In addition, the essential amino acid contents of MPH and MPH-Zn are 41.28% and 34.16% respectively, and the contents of Glu, Asp, Gly, Pro, etc. are rich, which conforms to the characteristics of collagen peptides.

[0139] Table 5

[0140]

[0141] Note: In the table, % refers to weight percentage.

[0142] (IV) SEM test

[0143] The microscopic morphology results of MPH in Preparation Example 1 and MPH-Zn in Example 1 are shown in Figure 3 、 Figure 4 . It can be seen from Figure 3 、 Figure 4 that the surface structure of MPH is mainly composed of large and unevenly distributed irregular particles. In contrast, MPH-Zn shows significantly different characteristics. Chelation makes its surface structure denser and forms a crystalline cluster structure.

[0144] In summary, the observation of chemical characteristics before and after chelation by UV-Vis and FTIR indicates the influence of chelation on molecular electronic transitions and functional groups. The UV spectral results show that chelation leads to changes in the internal electronic transitions of the molecule, manifested as lower absorption intensities of MPH-Zn than MPH in the ranges of 190 nm and 250 - 280 nm. The FTIR spectrum shows that functional groups such as the carbonyl group and amino group of MPH form coordination bonds with zinc ions, thus causing obvious shifts and intensity changes in the characteristic absorption peaks of MPH-Zn. For example, the amide I band shifts from 1633.79 cm -1 to 1625.33 cm -1 , and the N-H stretching vibration peak shifts from 3424.61 cm -1 to 3421.67 cm -1 . The microscopic morphology results show that the average particle size of MPH-Zn decreases significantly, indicating that the chelate is evenly dispersed and the system is concentrated. Amino acid composition analysis shows that in MPH-Zn, the contents of acidic and basic amino acids increase significantly, and Asp, Glu, Lys, His, and Arg, both acidic and basic amino acids, are the main sites for the formation of mussel polypeptide-zinc chelate.

[0145] Experimental Example 2

[0146] (I). Experimental methods

[0147] 1. Cell activation and resuscitation

[0148] Before the experiment, turn on the ultraviolet lamp to sterilize the operation room and the laminar flow hood, and wipe the tabletop and the surface of the instruments with 75% alcohol. Put the frozen Caco-2 cells into a 37 °C water bath for rapid thawing, with the time controlled within 1 minute. After thawing, transfer the cells from the cryopreservation tube to a centrifuge tube under sterile conditions using a pipette, and add 1 mL of MEM basal medium. Subsequently, centrifuge the centrifuge tube at a speed of 1000 rpm for 5 minutes. After centrifugation, discard the supernatant, add 2 mL of complete medium, and gently pipette 20 - 30 times to fully mix the cells. Take 2 mL of the cell suspension, mix it with an equal volume of medium, inject it into a culture flask, gently shake to evenly distribute the cells, and finally place the culture flask in an incubator at 37 °C and 5% CO2 for culture.

[0149] 2. Cell culture and subculture

[0150] When the growth rate of Caco-2 cells reaches 80%, rinse the cells with 2 mL of PBS buffer, add 1 mL of trypsin, digest for 1 minute and then aspirate, and continue to digest for 4 minutes. After digestion, add 2 mL of complete medium to terminate the digestion reaction, and gently pipette 20 - 30 times to evenly disperse the cells. Subculture at a ratio of 1:2.

[0151] 3. Cell cryopreservation

[0152] Digest and collect the cells, and centrifuge them at 1500 rpm for 5 minutes. Discard the supernatant, resuspend with 1 mL of cryopreservation solution (culture medium:DMSO = 9:1), aliquot into cryotubes, store at -80 °C overnight and then transfer to liquid nitrogen for storage.

[0153] 4. Cytotoxicity assay (MTT method)

[0154] Inoculate 180 μL of Caco-2 cell suspension (at a density of 5×10 4 cells / mL) into a 96-well plate and incubate in a 37 °C incubator for 24 h. Subsequently, replace with fresh medium containing different concentrations of MPH-Zn (0.1, 0.25, 0.5, 1 mg / mL) and culture for 30, 60, and 120 minutes respectively. After incubation, aspirate the medium, add 100 μL of MTT solution to each well, and continue to incubate at 37 °C for 4 h. Then, discard the MTT solution, add 150 μL of DMSO to each well to dissolve the formazan crystals, and measure the absorbance at a wavelength of 490 nm using an enzyme-linked immunosorbent assay reader. The cell survival rate is calculated according to the following formula:

[0155] Cell survival rate (%) = (OD of the drug-treated group - blank / OD of the normal group - blank) × 100%.

[0156] 5. Establishment of Caco-2 monolayer model

[0157] Cells of passages 15 - 20 are inoculated into a Transwell plate at 1.5×10 5 cells / well. After culturing for several days, evaluate the integrity of the monolayer cells by detecting the transepithelial electrical resistance (TEER), alkaline phosphatase (AKP) activity, and observation with a confocal laser scanning microscope.

[0158] Caco-2 cell transepithelial electrical resistance (TEER) = (Ω - Ω0) × 1.12.

[0159] 6. Caco-2 cell monolayer membrane absorption experiment

[0160] Wash the Caco-2 cell monolayer membrane with Hank's buffer. Add 0.5 mL of zinc sulfate solution and solutions of mussel polypeptide-zinc chelate at different concentrations (0.1, 0.05, 0.025, 0.01 mg / mL) to the apical (AP) side, and add 1.5 mL of Hank's buffer to the basolateral (BL) side. Continue to culture the cells in the incubator, and collect samples from the AP and BL sides at 30 minutes, 60 minutes, and 120 minutes respectively, and determine the zinc ion concentration using ICP-MS technology.

[0161] 7. Skin wound healing experiment

[0162] 7.1 In vitro antibacterial experiment

[0163] Bacterial culture: Take cryopreserved E. coli, S. aureus, and MRSA. After thawing in a 37°C water bath, inoculate them into LB liquid medium and culture them in a shaker at 37°C and 250 rpm for 12 h. Take 100 μL of the bacterial solution and add it to 5 mL of LB medium, and continue to culture for 6 h until the logarithmic growth phase. Determine the bacterial solution concentration by the spread plate method and dilute it to 10 3 -10 9 CFU / mL gradient, take 0.1 mL and spread it on a plate for culturing and counting.

[0164] OD600 antibacterial experiment: Add 100 μL of mussel polypeptide-zinc chelate (0.1 - 1 mg / mL) to a 96-well plate, add 190 μL of nutrient broth and 10 μL of bacterial suspension (1×10 6 CFU / mL) to each well, and culture at 37°C for 16 h. Measure the absorbance at 600 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the antibacterial rate:

[0165] Antibacterial rate (%) = 1 - [(ODcontrol group - ODblank group) / (ODdrug administration group - ODblank group)] × 100%.

[0166] 7.2 Mouse epidermal wound healing experiment

[0167] Experimental grouping: Use 15 Kunming mice (female, 35 - 40 g), randomly divide them into a blank group, a mussel polypeptide group ( Figure 7 - Figure 8 medium, denoted as the polypeptide group), and a mussel polypeptide-zinc chelate group ( Figure 7 - Figure 8 medium, denoted as the chelate group), with 5 mice in each group.

[0168] Wound model establishment: The mice are fasted for 8 h before the operation and intraperitoneally injected with 10% anesthetic. After depilation on the back, excise the full-thickness skin with a diameter of 7 mm to establish a wound model.

[0169] Wound closure rate determination: Take pictures of the wounds on days 0, 3, 6, 9, 12, and 15, measure the wound area with ImageJ software, and calculate the closure rate:

[0170] Wound closure rate (%) = (initial area - area on day n) / initial area × 100%.

[0171] Histological analysis: The mice are sacrificed on day 15, and the tissues around the wounds and the main organs (heart, liver, spleen, lung, kidney) are taken. After the tissues are fixed, paraffin-embedded, H&E staining, Masson staining, and immunohistochemistry (TNF-α, IL-6) analysis are performed. The stained sections are deparaffinized, hydrated, stained, dehydrated, cleared, and sealed, and observed and photographed under a microscope.

[0172] (II). Experimental results

[0173] 1. Cytotoxicity experiment

[0174] The experimental results showed that low concentrations and short-term treatments had no significant effect on the viability of Caco-2 cells. When treated with 0.5 mg / mL MPH-Zn, the apoptosis rate of the cells decreased significantly to below 80%. MPH-Zn at a concentration ≤ 0.25 mg / mL did not exhibit toxic effects on Caco-2 cells. Therefore, 0.25 mg / mL was selected as the highest concentration for subsequent zinc transport and absorption experiments.

[0175] 2. TEER value of the Caco-2 cell monolayer membrane

[0176] The TEER value is particularly important for evaluating the tightness and integrity of the Caco-2 cell monolayer membrane. The larger the TEER value, the better the tightness and integrity of the Caco-2 cell monolayer membrane. The results showed that the TEER value stabilized after 300 Ω·cm 2 on the 15th day, indicating that the Caco-2 cells had completed differentiation and formed a monolayer membrane structure, which could meet the requirements of subsequent experiments.

[0177] 3. AKP activity of the Caco-2 cell monolayer membrane

[0178] AKP is a key marker enzyme for the polar differentiation of small intestinal epithelial brush border cells. Caco-2 cells were seeded in 24-well Transwell culture plates, and the permeability and polar differentiation characteristics of the cell monolayer were verified by measuring the AKP activities of the AP and BL sides. The results showed that the AKP activities of the Caco-2 cell monolayer membrane were 5.74 ± 0.18 U / L and 3.36 ± 0.11 U / L, respectively. The alkaline phosphatase activity on the AP side was significantly higher than that on the BL side (P < 0.0001), indicating obvious polar differentiation on the AP and BL sides of the Caco-2 cell monolayer membrane.

[0179] 4. Morphology of the Caco-2 cell monolayer membrane

[0180] During the construction of the Caco-2 cell monolayer membrane, a laser confocal microscope was used to observe the cell morphology to evaluate the continuity and integrity of the monolayer membrane. The serosal proteins of the Caco-2 cell monolayer membrane were stained green with FITC-Phalloidin, and the cell nuclei were stained blue with DAPI, as shown Figure 5 (see A and B in Figure 5 respectively). The Merge (merge) is shown in Figure 5 C in. The results showed that the cell monolayer membrane structure was clear, presenting a typical "paving stone-like" arrangement, with tight cell junctions and uniform distribution of cell nuclei, indicating the successful construction of a complete Caco-2 cell monolayer membrane model.

[0181] 5. Results of antibacterial and skin wound experiments

[0182] The mussel polypeptide-zinc chelate (MPH-Zn) prepared in Example 1 was subjected to antibacterial and mouse skin wound experiments, and compared with the unchelated mussel polypeptide (MPH) obtained in Preparation Example 1, and the blank group was used as a reference to the skin healing ability of mussel polypeptide and mussel polypeptide-zinc chelate. The results are as follows: Figure 6 , Figure 7 and Figure 8 shown.

[0183] Figure 6 The effect of mussel polypeptide-zinc chelate on Escherichia coli (E. coli), Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA). Figure 6 In the figure, different letters of the same color indicate significant differences among the groups (P<0.05).Mussel polypeptide (MPH) did not show antibacterial effect at the same concentration and under the same experimental conditions as mussel polypeptide-zinc chelate (MPH-Zn).

[0184] Figure 7 The results of the changes in back wounds of mice in different groups treated with mussel polypeptides and mussel polypeptide-zinc chelate.

[0185] Figure 8 The wound healing rates of mice in different groups treated with mussel polypeptides and mussel polypeptide-zinc chelate. Figure 8 In the table, *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001.

[0186] Figure 7 , Figure 8 In the present invention, the chelate group refers to the mussel polypeptide-zinc chelate group, and the polypeptide group refers to the mussel polypeptide group.

[0187] The above experiments show that the mussel polypeptide-zinc chelate of the present invention has good skin healing ability as a whole, and the antibacterial effect and skin healing rate of the chelated mussel polypeptide-zinc chelate are significantly higher than those of the unchelated mussel polypeptide.

[0188] The present invention is not limited to the above-mentioned embodiments. Without departing from the essential content of the present invention, any deformation, improvement and substitution that can be conceived by those skilled in the art shall fall within the scope of the present invention.

Claims

1. A mussel polypeptide-zinc chelate, characterized in that, It is obtained by chelating mussel polypeptides with a molecular weight less than 3 kDa and zinc element; Among them, the zinc element is derived from a water-soluble zinc salt; Among them, the mass ratio of the mussel polypeptide to the zinc element is 5.5 - 6.5:

1.

2. The preparation method of the mussel polypeptide-zinc chelate according to claim 1, characterized in that, It includes the following steps: 1) Dissolve the freeze-dried powder of mussel polypeptides with a molecular weight less than 3 kDa in water to obtain a mussel polypeptide aqueous solution with a concentration of 25 - 120 mg / mL; 2) Mix the mussel polypeptide aqueous solution and the water-soluble zinc salt, and react at 30 - 60 °C for 20 - 80 min, and maintain the pH value of the reaction solution at 5 - 8 during the reaction process; among them, the mass ratio of the mussel polypeptide to the zinc element in the water-soluble zinc salt is 5.5 - 6.5:1; 3) After the reaction, add alcohol for precipitation, perform solid-liquid separation, wash the separated solid and freeze-dry it to obtain the mussel polypeptide-zinc chelate.

3. The preparation method according to claim 2, wherein In step 2), the water-soluble zinc salt is zinc sulfate; in step 3), after the reaction, add absolute ethanol for precipitation, perform solid-liquid separation, wash the separated solid and freeze-dry it to obtain the mussel polypeptide-zinc chelate.

4. The preparation method according to claim 2, characterized in that, The freeze-dried powder of mussel polypeptides is prepared by the following steps: (a) Remove sand and shells from mussels to obtain mussel meat; crush the mussel meat to obtain processed mussels; (b) Calculate according to the dry weight of the processed mussels, add 10 - 17 times the weight of water to the processed mussels and mix, and adjust the pH value of the mixture to 7 - 9 to obtain a mussel homogenate; (c) Add 1 - 4 wt% of trypsin to the mussel homogenate obtained in step (b), and perform enzymatic hydrolysis reaction at 45 - 55 °C for 2 - 5 h; (d) After the enzymatic hydrolysis reaction, boil the enzymatic hydrolysis reaction product for 10 - 20 min, and then centrifuge to collect the supernatant; (e) Separate the supernatant through an ultrafiltration membrane, collect the filtrate of the peptide segments with a molecular weight less than 3 kDa, concentrate the collected filtrate, and then freeze-dry the concentrate to obtain the freeze-dried powder of mussel polypeptides with a molecular weight less than 3 kDa.

5. The preparation method according to claim 4, characterized in that, In step (a), the mussel is Mytilus edulis; the particle size of the processed mussel is 1 - 5 mm; in step (b), use an alkali metal hydroxide solution or hydrochloric acid solution to adjust the pH value of the mixture to 7.5 - 8.

5.

6. The preparation method according to claim 4, characterized in that, In step (c), add 2 - 4 wt% of trypsin to the mussel homogenate obtained in step (b), and perform enzymatic hydrolysis reaction at 45 - 55 °C for 3.5 - 5 h.

7. The preparation method according to claim 4, characterized in that, In step (d), the centrifugation speed is 4000 - 7000 rpm, and the centrifugation time is 10 - 20 min.

8. The preparation method according to claim 4, characterized in that, In step (e), concentrate the collected filtrate to one-fifteenth to one-twentieth of the original volume to obtain a concentrate; freeze-dry the concentrate at a temperature below -35 °C to obtain the freeze-dried powder of mussel polypeptides with a molecular weight less than 3 kDa.

9. The application of the mussel polypeptide-zinc chelate according to claim 1 or the mussel polypeptide-zinc chelate prepared by the preparation method according to any one of claims 2 - 8 in the preparation of wound repair products.

10. A wound repair product, characterized in that, It is prepared from raw materials including the mussel polypeptide-zinc chelate according to claim 1.

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