Method for preparing lysozyme and composite medical dressing by resource utilization of eggshell membrane

Abstract

The invention discloses a method for preparing lysozyme and composite medical dressing by eggshell membrane resource utilization, which comprises the following steps: mixing eggshell membrane powder and a solvent in proportion, adding an enzyme preparation for enzymolysis, and filtering after enzyme deactivation to obtain eggshell membrane enzymatic hydrolysate; replacing lysozyme in the eggshell membrane enzymatic hydrolysate with cationic resin, and performing freeze drying treatment to obtain solid lysozyme; carrying out freeze drying treatment on the eggshell membrane enzymatic hydrolysate from which the lysozyme is removed to obtain an enzymatic hydrolysate freeze-dried substance; and mixing the enzymatic hydrolysate freeze-dried substance with a chitosan solution to obtain a composite solution, and freeze-drying to obtain the composite medical dressing. The abandoned solid product is used for high-valued extraction of the lysozyme and preparation of the sponge dressing, the biomass enzymatic hydrolysate prepared by the method has remarkable antioxidant activity and shows potential application value in the fields of biomedicine, food preservation and the like, the content of the extracted lysozyme is high, and the method is suitable for industrial production. The sponge dressing has good hemostasis and oxidation resistance, can promote wound healing, and realizes utilization of zero waste biomass.

Description

Technical Field

[0001] The present invention relates to the technical field of resource utilization of eggshell membranes, and particularly relates to a method for resource utilization of eggshell membranes to prepare lysozyme and composite medical dressings. Background Art

[0002] Egg by-products (eggshells, inner eggshell membranes, egg white / yolk residues) have made significant progress in recent years in the fields of biomedicine and food preservation due to their natural antibacterial, antioxidant, and film-forming properties, mainly focusing on the extraction of active ingredients and the development of new bio-based materials.

[0003] The eggshell membrane is located between the egg white and the eggshell and is composed of two different forms of proteins. The outer eggshell membrane presents a randomly intertwined fibrous network structure, while the inner eggshell membrane shows a relatively smooth and amorphous organic colloid layer. The porous structure of the outer eggshell membrane ensures good permeability of air and moisture, facilitating gas exchange and humidity regulation during embryo development. The inner eggshell membrane has a dense structure that can effectively prevent the leakage of egg white and provides a stable internal environment for the embryo. Based on this structural feature, the eggshell membrane not only plays a key role in protecting the contents of eggs but also demonstrates its unique application potential in the field of biomaterials.

[0004] The eggshell membrane is mainly composed of proteins (accounting for 80–85%), which are distributed in the outer and inner layers, including type I, type V, and type X collagens, sialoprotein, osteopontin, and a small amount of carbohydrates. The fibrous structure of the eggshell membrane is stably connected through extensive cross-linking bonds (such as desmosine amino groups, isodesmosine amino groups, and disulfide bonds), making it highly insoluble. The protein components of the eggshell membrane include two main types: structural proteins and globular proteins. Structural proteins mainly include collagen and eggshell membrane proteins, which provide a strong framework for the eggshell membrane; while globular proteins include lysozyme, lysyl oxidase, etc., which endow the eggshell membrane with additional functional properties.

[0005] Lysozyme has received extensive attention due to its wide application and cheap source. As a natural monomeric protein, lysozyme has good antibacterial effects and a variety of types, widely existing in nature. The chemical properties of egg white lysozyme are very stable. When its pH value is in the range of 1.2–11.3, its structure hardly changes, and it also has strong thermal stability in an acidic environment. Currently, lysozyme has been widely used in the food and pharmaceutical industries. In the food industry, lysozyme is mainly used as an additive in the production of wine and beer and as an inhibitor of Clostridium growth during cheese ripening, and has been officially approved as a food preservative by many countries and organizations. Researchers have found that in addition to obtaining lysozyme from egg white, the eggshell membrane is another way to obtain lysozyme.

[0006] After extracting lysozyme, the eggshell membrane hydrolysate still contains abundant bioactive substances such as collagen and has the potential for further high-value utilization. Collagen has the characteristics of low immunogenicity, good biocompatibility and biodegradability. Based on collagen, biological dressings can promote the proliferation, differentiation and secretion of various cells in the skin, accelerate the formation and remodeling of the extracellular matrix, and thus promote wound healing. In the context of the rapid development of modern society, injuries caused by factors such as traffic accidents, high-altitude falls, mechanical extrusion and natural disasters occur frequently. Such traumas are often accompanied by complex wound surfaces, and the loss of skin integrity may lead to serious disabilities or even death. Therefore, the development of new materials with both biocompatibility and high-efficiency hemostatic performance has important clinical value for promoting rapid wound healing. The eggshell membrane hydrolysate is rich in bioactive substances such as collagen. The eggshell membrane hydrolysate after extracting lysozyme can be used to prepare medical dressings, realizing zero waste and multi-level comprehensive utilization of the eggshell membrane and increasing the added value of the eggshell membrane.

[0007] At present, there is no reported method for efficiently extracting lysozyme from eggshell membrane and comprehensively utilizing the eggshell membrane hydrolysate. Efficiently extracting lysozyme from waste eggshell membrane and simultaneously preparing medical dressings that can significantly improve the blood coagulation and healing speed of wounds is of great significance for realizing the reduction, zero waste treatment and high-value resource utilization of agricultural waste. Summary of the Invention

[0008] The present invention provides a method for resource utilization of eggshell membrane to prepare lysozyme and composite medical dressings to solve the above problems.

[0009] To achieve the above object, the technical solution of the present invention is:

[0010] A method for resource utilization of eggshell membrane to prepare lysozyme and composite medical dressings, comprising the following steps:

[0011] S1: Collect eggshell membrane, wash, dry, powder and sieve it to obtain eggshell membrane powder for standby;

[0012] S2: Mix the eggshell membrane powder with a solvent in a certain proportion, add an enzyme preparation for enzymatic hydrolysis, inactivate the enzyme, and then filter to obtain eggshell membrane hydrolysate;

[0013] S3: Use cation resin to displace lysozyme in the eggshell membrane hydrolysate, and freeze-dry the solution containing lysozyme to obtain solid lysozyme;

[0014] S4: Freeze-dry the eggshell membrane hydrolysate after removing lysozyme to obtain a freeze-dried product of the hydrolysate;

[0015] S5: Mix the freeze-dried product of the hydrolysate with a 1-3% chitosan solution to obtain a composite solution;

[0016] S6: Inject the composite solution into a mold. After freeze-drying, a composite medical dressing is prepared.

[0017] Further, in S2, the enzyme preparation is alkaline protease, and the enzyme addition amount is 8–10 U / mg substrate.

[0018] The enzymatic hydrolysis conditions are: enzymatic hydrolysis for 3–5 h at 45–55 °C.

[0019] Further, in S1, the solvent is deionized water, and the mass ratio of eggshell membrane powder to deionized water is 1:2–25.

[0020] Further, in S3, cation exchange resin is used for replacement, including adsorption and desorption steps. When adsorbing, the mass-to-volume ratio of cation exchange resin to eggshell membrane enzymatic hydrolysate is 1:6 (g:mL).

[0021] When desorbing, the mass ratio of cation exchange resin to sodium chloride is 1:4.

[0022] Further, in S5, the chitosan is medical-grade chitosan with a deacetylation degree ≥85% and a viscosity of 100–200 mPa·s. The chitosan and the freeze-dried product of the enzymatic hydrolysate are dissolved together in a 1–2% acetic acid solution and then freeze-dried to prepare a composite medical dressing.

[0023] Further, in S6, the freeze-drying conditions are: pre-freezing at -80 °C for 4–8 h, then transferring to a freeze dryer, and performing sublimation drying at a vacuum degree ≤15 Pa for 24–48 h.

[0024] Further, in S2, the enzyme inactivation treatment uses a double inactivation method of heating at 90–95 °C for 10–15 min and adjusting the pH to 3.5–4.5. After enzyme inactivation, the enzymatic hydrolysate is microfiltered through a 0.45 μm filter.

[0025] Further, the composite medical dressing comprises 1–3 wt% of the freeze-dried product of the enzymatic hydrolysate and 1–3 wt% of chitosan.

[0026] Further, the antioxidant peptides and collagen in the eggshell membrane enzymatic hydrolysate after removing lysozyme have a molecular weight of 5–50 kDa.

[0027] Further, the eggshell membrane is an eggshell membrane, a duck eggshell membrane, a quail eggshell membrane, or a goose eggshell membrane.

[0028] The beneficial effects of the present invention are:

[0029] First, a method for the resource utilization of eggshell membrane to prepare lysozyme and composite medical dressings disclosed in the present invention. The present invention aims at the solid products that are often ignored and discarded in the process of biomass production and treatment, and proposes to highly utilize them in the extraction of lysozyme and the preparation of sponge dressings. It not only realizes the efficient utilization and reduction treatment of agricultural biomass waste, but also the solid product of biomass enzymatic hydrolysis shows potential application value in the fields of biomedicine, food preservation, etc. due to its significant collagen content and antioxidant activity, effectively promoting the realization of the zero-waste biomass utilization method;

[0030] Second, the present invention obtains eggshell membrane lysozyme protein and heteroprotein through the enzymatic hydrolysis treatment of biomass solid products. The heteroprotein is compounded with a hydrophilic polymer to prepare a sponge dressing with liquid absorption performance and blood coagulation promotion. The dressing maintains good hemostasis and antioxidant properties. At the same time, through the actual application verification of mouse in vivo wound healing, the dressing effectively promotes the wound healing period of mice, fully demonstrating its application prospect as a functional additive in the field of medical sponge dressings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is a graph showing the influence results of different enzyme preparations on the lysozyme activity and the DPPH scavenging rate of the enzymatic hydrolysate disclosed in Example 2 of the present invention;

[0033] Figure 2 It is a graph showing the influence results of different enzyme preparations on the lysozyme activity and the DPPH scavenging rate of the enzymatic hydrolysate disclosed in Example 3 of the present invention;

[0034] Figure 3 It is a graph showing the influence results of different enzymatic hydrolysis temperatures on the lysozyme activity and the DPPH scavenging rate of the enzymatic hydrolysate disclosed in Example 4 of the present invention;

[0035] Figure 4 It is a graph showing the influence results of different enzymatic hydrolysis times on the lysozyme activity and the DPPH scavenging rate of the enzymatic hydrolysate disclosed in Example 5 of the present invention;

[0036] Figure 5 It is the characterization result of the eggshell membrane enzymatic hydrolysate prepared in Example 1 of the present invention, where a is the determination result of the lysozyme activity before and after resin adsorption; b is the infrared spectrum of the prepared lysozyme; c is the SDS-PAGE gel electrophoresis of lysozyme;

[0037] Figure 6 These are the characterization results of the composite medical sponge dressing prepared in Example 1 of the present invention. Among them, a is the thermogravimetric analysis result graph, b is the differential thermogravimetric analysis result graph, c is the infrared spectrum graph, d is the liquid absorption result, e is the in vitro coagulation index result, f is the in vitro enzymatic hydrolysis result, g is the hemolytic result, and h is the cytotoxicity result;

[0038] Figure 7 This is the HE staining result graph of epithelial cells after the composite medical dressing prepared in Example 1 of the present invention is applied to the wound. Detailed implementation manners

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0040] Example 1:

[0041] A method for the resource utilization of eggshell membrane to prepare lysozyme and a composite medical dressing, comprising the following steps:

[0042] S1: Raw material pretreatment: Collect eggshell membranes, wash them three times with deionized water, dry them at 40–50 °C, and sieve them through a 40-mesh sieve to obtain eggshell membrane powder;

[0043] S2: Preparation of eggshell membrane enzymatic hydrolysate:

[0044] Add 5 g of eggshell membrane powder, 100 g of deionized water, and 9 U / mg of alkaline protease into a beaker, immerse it in a preheated water bath at 50 °C, react for 4 h, then take the beaker out of the water bath and immediately immerse it in a boiling water bath to quench the reaction. After inactivating the enzyme, filter it through a 0.45-μm microfiltration membrane to obtain eggshell membrane enzymatic hydrolysate;

[0045] S3: Preparation of lysozyme:

[0046] Use cation resin to displace the lysozyme in the eggshell membrane enzymatic hydrolysate. Add 17.5 g of d152 macroporous acrylic weak acidic cation exchange resin to the eggshell membrane enzymatic hydrolysate for adsorption for 4 h, then add 70 g of sodium chloride for desorption for at least 30 min. Freeze the solution containing lysozyme at -80 °C for 6 h, then transfer it to a freeze dryer, and freeze-dry it under a vacuum degree of ≤15 Pa for 48 h to obtain solid lysozyme;

[0047] S4: Obtain freeze-dried enzymatic hydrolysate:

[0048] The eggshell membrane enzymolysis solution after removing lysozyme was pre-frozen at -80°C for 6 h and then transferred to a freeze dryer for freeze-drying treatment under a vacuum degree of ≤15 Pa for 36 h to obtain the freeze-dried product of the enzymolysis solution.

[0049] S5: Preparation of composite solution:

[0050] The freeze-dried product of the enzymolysis solution was mixed with a 1-3% chitosan solution and dissolved in a 1–2% acetic acid solution to obtain a composite solution.

[0051] S6: Preparation of composite medical dressing:

[0052] The composite solution was injected into a mold, pre-frozen at -80°C for 6 h and then transferred to a freeze dryer for freeze-drying treatment under a vacuum degree of ≤15 Pa for 48 h to obtain a porous sponge-like dressing, and thus the composite medical dressing was prepared.

[0053] Example 2: Experiment on the effects of different enzyme preparations on the lysozyme activity and DPPH scavenging rate of the enzymolysis solution

[0054] According to the method in Example 1, 4 groups of eggshell membrane powders were taken, and neutral protease, pepsin, papain, and trypsin were used to enzymolyze the eggshell membrane powders respectively, and compared with Example 1. The activity of lysozyme in the obtained eggshell membrane enzymolysis solution was detected by turbidimetry, and the antioxidant performance of the eggshell membrane enzymolysis solution was detected by DPPH·.

[0055] The results are as Figure 1 shown. It can be seen from the figure that the lysozyme activity and DPPH scavenging rate in the eggshell membrane enzymolysis solution prepared with alkaline protease are significantly higher than those in the groups using neutral protease, pepsin, papain, and trypsin. Therefore, the best enzyme preparation for preparing lysozyme is alkaline protease.

[0056] Example 3: Experiment on the effects of adding different doses of enzyme preparation on the lysozyme activity and DPPH scavenging rate of the enzymolysis solution

[0057] According to the method in Example 1, 4 groups of eggshell membrane powders were taken, and alkaline protease with 3 U / mg, 6 U / mg, 12 U / mg, and 15 U / mg was used to prepare the eggshell membrane enzymolysis solution respectively, and compared with Example 1. The activity of lysozyme in the obtained eggshell membrane enzymolysis solution was detected by turbidimetry, and the antioxidant performance of the eggshell membrane enzymolysis solution was detected by DPPH·.

[0058] The results are as Figure 2As shown in the figure, it can be seen that when the addition amount of alkaline protease is 9 U / mg, the activity of lysozyme and the DPPH scavenging rate in the eggshell membrane enzymatic hydrolysate are significantly higher than those when the addition amounts are 3 U / mg and 6 U / mg, and the lysozyme activity is higher than that in the group with an enzyme preparation addition amount of 12 U / mg. When the addition amount of alkaline protease is increased to 15 U / mg, the activity of lysozyme and the DPPH scavenging rate do not increase significantly. Therefore, considering economic applicability, the optimal addition amount of alkaline protease is 9 U / mg.

[0059] Example 4: Experiment on the effect of different enzymatic hydrolysis temperatures on the activity of lysozyme and the DPPH scavenging rate of the enzymatic hydrolysate

[0060] According to the method in Example 1, take 4 groups of eggshell membrane powders, and use enzymatic hydrolysis temperatures of 30 °C, 40 °C, 60 °C, and 70 °C respectively to carry out enzymatic hydrolysis on the eggshell membrane powders, and compare with Example 1. Use the turbidimetry method to detect the activity of lysozyme in the obtained eggshell membrane enzymatic hydrolysate, and use DPPH· to detect the antioxidant performance of the eggshell membrane enzymatic hydrolysate.

[0061] The results are as Figure 3 shown. It can be seen from the figure that when the enzymatic hydrolysis temperature is 50 °C, the activity of lysozyme and the DPPH scavenging rate in the eggshell membrane enzymatic hydrolysate are significantly higher than those of other groups. Therefore, the optimal enzymatic hydrolysis temperature is 50 °C.

[0062] Example 5: Experiment on the effect of different enzymatic hydrolysis times on the activity of lysozyme and the DPPH scavenging rate of the enzymatic hydrolysate

[0063] According to the method in Example 1, take 4 groups of eggshell membrane powders, and use enzymatic hydrolysis times of 2 h, 6 h, 8 h, and 10 h respectively to carry out enzymatic hydrolysis on the eggshell membrane powders, and compare with Example 1. Use the turbidimetry method to detect the activity of lysozyme in the obtained eggshell membrane enzymatic hydrolysate, and use DPPH· to detect the antioxidant performance of the eggshell membrane enzymatic hydrolysate.

[0064] The results are as Figure 4 shown. It can be seen from the figure that when the enzymatic hydrolysis time is 4 h, the activity of lysozyme in the eggshell membrane enzymatic hydrolysate is higher than that in the groups of 6 h, 8 h, and 10 h. Its lysozyme activity is basically equivalent to that in the 2 h group, but its DPPH scavenging rate is significantly higher than that in the 2 h group. Therefore, the optimal enzymatic hydrolysis time is 4 h.

[0065] Therefore, the optimal conditions for preparing lysozyme are the preparation conditions in Example 1, that is, using 9 U / mg of alkaline protease, in a water bath at 50 °C, reacting for 4 h;

[0066] The lysozyme activity of the enzymolysis eggshell membrane (E-ESM) prepared in Example 1 was determined by turbidimetry, and its lysozyme activity reached 2.52 U / mL. In terms of antioxidant performance, the DPPH· method was used to determine the antioxidant activity of the enzymolysis eggshell membrane solution. After diluting the enzymolysis eggshell membrane solution ten times, its DPPH radical scavenging rate reached 38.23%.

[0067] The lysozyme solution of eggshell membrane was obtained through D152 macroporous acrylic weak acidic cation exchange resin, and the purified eggshell membrane lysozyme (PE-ESM) was obtained by ultrafiltration desalting and freeze-drying. The lysozyme activity changed before and after resin exchange ( Figure 5 a), proving that it was successfully replaced.

[0068] The main functional groups of the lysozyme standard product (such as –OH, –NH, C–H, C=O, and C–O) all exist in PE-ESM (such as Figure 5 b), and a broad peak appears at around 3400 cm – -1. Although the shape is slightly different, it still indicates that there are still a large number of hydrogen bonds, but PE-ESM may be affected by other functional groups. At around 1650 cm – -1, both the lysozyme standard product and PE-ESM show obvious C=O stretching vibration peaks, but the peak position of PE-ESM is slightly different because there are different amide bonds due to the presence of heteroproteins.

[0069] The purified product (purified eggshell membrane lysozyme PE-ESM) was characterized by SDS-PAGE gel electrophoresis technology, and the results showed that Figure 5 c there was a protein band, and there was a clear band between 10–15 kDa, indicating that the PE-ESM lysozyme was purified into a monomer protein and was consistent with the molecular weight of the standard product.

[0070] It was determined that 80 mg of lysozyme was obtained per gram of eggshell membrane powder.

[0071] The freeze-dried product of the heteroprotein after removing lysozyme was compounded with chitosan to prepare a medical sponge dressing (DeleteEnzymolysis eggshell membrane, DE-ESM / CS), and the following characterizations were carried out:

[0072] In terms of thermogravimetric analysis, DE-ESM / CS was thermally stable and only lost water in the first stage, and began to decompose at 280℃ in the second stage ( Figure 6 a, 6b). It was not easily thermally decomposed at room temperature, indicating that this composite medical dressing had good thermal stability and had advantages in application and storage.

[0073] Figure 6 c. The absorption peak between 1000 cm-1 and 1200 cm-1 in the infrared spectrum is related to the C–O stretching vibration. The absorption peaks of DE-ESM / CS are more obvious or broader at certain wave numbers because the vibration modes of some chemical bonds have changed after adding DE-ESM. This shows the similarity in chemical structure between CS and DE-ESM / CS and also reveals the influence of the introduction of DE-ESM on the structure of CS.

[0074] In terms of liquid absorption, as Figure 6 shown in d, compared with chitosan (CS), DE-ESM / CS has a higher absorption rate, up to 1700%, which is very beneficial for the rapid absorption of wound tissue fluid exudation.

[0075] As Figure 6 shown in e, the in vitro coagulation index of DE-ESM / CS is lower than that of the commercial hemostatic agent Celox and shows positive procoagulant performance.

[0076] As Figure 6 shown in f, all samples degraded to about 40% after 1 h of in vitro enzymatic hydrolysis by 20 mg / L lysozyme and were almost completely degraded after 2 h. The faster degradation rate of this composite medical dressing confirmed its good degradation ability.

[0077] The in vitro hemolytic property of the composite medical dressing was evaluated by measuring the degree of hemolysis caused when the material was in contact with blood. The results are as Figure 6 shown in g. The hemolysis percentage after 4 h of contact with the sample at 37°C was lower than 5% of the hemolysis standard.

[0078] In terms of cytotoxicity, CCK-8 cells were co-incubated with DE-ESM / CS and CS for 48 hours respectively, and the control group without adding the medical dressing was used to measure the CCK-8 cell proliferation rate. The results are as Figure 6 shown in h. The cells of DE-ESM / CS all showed good viability, and the cell proliferation rate exceeded 90%. This result indicates that the DE-ESM / CS antibacterial film has good biocompatibility and is safe for use in wound healing.

[0079] DE-ESM / CS and CS dressings were respectively applied to the wounds of mice. The control group did not use the dressing. The wounds and the surrounding tissues were taken, and through HE staining, the growth, development, metabolism, and other processes of cells and tissues in the mouse wound sections were observed, as well as various physiological and pathological changes, as Figure 7The staining results (Column A) showed that on the 3rd day, there was an obvious inflammatory response in the wound area of the control group mice, with a large number of inflammatory cells and irregular cell arrangement. The inflammatory response in the CS group was relatively mild, and the cell arrangement was relatively orderly. The inflammatory response in the DE-ESM / CS group (Delete Enzymolysis eggshell membrane, DE-ESM) was the mildest, and the cell arrangement was the most orderly. On the 7th day, the inflammatory response in the control group was still obvious, with irregular cell arrangement. The inflammatory response in the CS group was somewhat reduced, and the inflammatory response in the DE-ESM / CS group was further reduced, with more orderly cell arrangement. On the 13th day, the inflammatory response in the control group still existed but was somewhat reduced, and the cell arrangement was still irregular. The inflammatory response in the CS group was significantly reduced, and the cell arrangement was relatively orderly. The inflammatory response in the DE-ESM / CS group almost disappeared, the cell arrangement was very orderly, and the tissue structure was close to normal.

[0080] Figure 7 The Masson staining results (Column B) showed that on the 3rd day, there were fewer collagen fibers in the control group, mainly inflammatory cells stained red. Collagen fibers began to form in the CS group, and the blue-stained area increased. There were more collagen fibers in the DE-ESM / CS group, and the blue-stained area increased significantly. On the 7th day, the collagen fibers in the control group increased slightly, but still mainly stained red. The cutin layer was not fully formed, there was less neovascularization, and the hair follicles were more immature. The collagen fibers in the CS group further increased, and the blue-stained area increased. The collagen fibers in the DE-ESM / CS group increased significantly, and the blue-stained area increased significantly. In addition, hair follicles, sebaceous glands, and neovascularization also existed. On the 13th day, the collagen fibers in the control group continued to increase, but still mainly stained red. The collagen fibers in the CS group increased significantly, and the blue-stained area increased. The collagen fibers in the DE-ESM / CS group were the most, and the blue-stained area increased significantly, and the tissue structure was close to normal.

[0081] Masson staining can reflect the inflammatory condition of cell tissue and the generation of collagen fibers at the wound of mice. The DE-ESM / CS group showed the mildest inflammatory response and the most orderly cell arrangement at all time points. The CS group was the second, with a relatively mild inflammatory response and relatively orderly cell arrangement. The control group had the heaviest inflammatory response and irregular cell arrangement. The DE-ESM / CS group showed the most collagen fiber generation and a significant increase in the blue-stained area at all time points. The CS group was the second, with more collagen fiber generation and an increase in the blue-stained area. The control group had the least collagen fiber generation, mainly inflammatory cells stained red.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing lysozyme and a composite medical dressing by resource utilization of eggshell membrane, characterized in that It includes the following steps: S1: Collect eggshell membrane, after cleaning and drying, powder it and sieve it to obtain eggshell membrane powder for standby; S2: After mixing the eggshell membrane powder with a solvent in proportion, add an enzyme preparation for enzymatic hydrolysis, inactivate the enzyme, and then filter to obtain an eggshell membrane enzymatic hydrolysate; S3: Use a cation exchange resin to displace the lysozyme in the eggshell membrane enzymatic hydrolysate, and freeze-dry the solution containing lysozyme to obtain solid lysozyme; S4: Freeze-dry the eggshell membrane enzymatic hydrolysate after removing lysozyme to obtain a freeze-dried product of the enzymatic hydrolysate; S5: Mix the freeze-dried product of the enzymatic hydrolysate with a 1–3% chitosan solution to obtain a composite solution; S6: Inject the composite solution into a mold, and after freeze-drying, prepare a composite medical dressing.

2. The method for preparing lysozyme and composite medical dressings by resource utilization of eggshell membranes according to claim 1, characterized in that, In S2, the enzyme preparation is alkaline protease, and the enzyme addition amount is 8–10 U / mg substrate; The enzymatic hydrolysis conditions are: enzymatic hydrolysis for 3–5 h at 45–55 °C.

3. The method for preparing lysozyme and composite medical dressing by resource utilization of eggshell membrane according to claim 1, characterized in that, In S1, the solvent is deionized water, and the mass ratio of the eggshell membrane powder to deionized water is 1:2–25.

4. The method for preparing lysozyme and composite medical dressings by resource utilization of eggshell membrane according to claim 1, characterized in that In S3, the displacement with a cation exchange resin includes adsorption and desorption steps. During adsorption, the mass-to-volume ratio of the cation exchange resin to the eggshell membrane enzymatic hydrolysate is 1:6 (g:mL); During desorption, the mass ratio of the cation exchange resin to the sodium chloride solution is 1:

4.

5. The method for preparing lysozyme and a composite medical dressing by resource utilization of eggshell membrane according to claim 1, characterized in that, In S5, the chitosan is a medical-grade chitosan with a deacetylation degree ≥ 85% and a viscosity of 100–200 mPa·s. The chitosan and the freeze-dried product of the enzymatic hydrolysate are dissolved in a 1–2% acetic acid solution and then freeze-dried to prepare a composite medical dressing.

6. The method for preparing lysozyme and a composite medical dressing by resource utilization of eggshell membrane according to claim 1, wherein, In S6, the freeze-drying conditions are: pre-freeze at -80 °C for 4–8 h, then transfer to a freeze dryer, and perform sublimation drying for 24–48 h under a vacuum degree ≤ 15 Pa.

7. The method for preparing lysozyme and a composite medical dressing by resource utilization of eggshell membranes according to claim 1, characterized in that, In S2, the enzyme inactivation treatment adopts a double inactivation method of heating at 90–95 °C for 10–15 min and adjusting the pH to 3.5–4.

5. After enzyme inactivation, the enzymatic hydrolysate is filtered through a 0.45 μm microporous membrane.

8. The method for preparing lysozyme and composite medical dressings by resource utilization of eggshell membranes according to claim 1, characterized in that, The composite medical dressing includes 1–3 wt% of the freeze-dried product of the enzymatic hydrolysate and 1–3 wt% of chitosan.

9. The method for preparing lysozyme and composite medical dressings by resource utilization of eggshell membrane according to claim 1, characterized in that, The antioxidant peptides and collagen in the eggshell membrane enzymatic hydrolysate after removing lysozyme have a molecular weight of 5–50 kDa.

10. The method for preparing lysozyme and composite medical dressings by resource utilization of eggshell membranes according to claim 1, wherein, The eggshell membrane is an eggshell membrane, a duck eggshell membrane, a quail eggshell membrane, or a goose eggshell membrane.

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