Cod skin active polypeptide powder and its use in antiviral and sterilization

Abstract

The present invention discloses a preparation method of cod skin active polypeptide powder and its use for antiviral and sterilization. Clean cod skin is soaked in water to swell, then water is added for homogenization, and then protease is added for hydrolysis. After the hydrolysis reaction is completed, the enzyme is inactivated and centrifuged, and the supernatant is freeze-dried to obtain the cod skin active polypeptide powder. The spray prepared by using the cod skin active polypeptide powder can effectively inhibit pathogenic bacteria, has a significant inhibitory effect on influenza A virus, and can be used as an antiviral, sterilization health product, medicine or disinfectant product. The present invention enriches the product form of cod skin by making the prepared cod skin polypeptide powder into a spray, is applicable to a wide range of people, avoids the waste of cod skin resources, realizes the high-value utilization of cod, and is of great significance for the development and utilization of cod skin products.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic product processing and biomedicine, and in particular to cod skin active polypeptide powder and its use in antiviral and sterilization. Background Art

[0002] Cod is a deep-sea fish of the family Gadidae in the order Gadformes. It has delicious meat, high protein and low fat, and has high economic and nutritional value. In addition, cod also has certain medicinal value, and has the effect of assisting the recovery of stomach discomfort and edema. The main species of cod include Atlantic cod, Pacific cod, Greenland cod and haddock.

[0003] Cod skin has fewer scales on the surface and is bluish-black in color. It has a soft and tough texture, low in fat, rich in collagen, and delicious taste. It also contains high DHA (docosahexaenoic acid) and other unsaturated fatty acids, which can promote fat metabolism, lower blood pressure, anti-oxidation, and enhance immunity. In addition, it also has a good effect on trauma, bleeding, and constipation due to falls, fractures, and trauma, and is an important raw material in the food and pharmaceutical industries.

[0004] Cod is mainly distributed in the Yellow Sea, Bohai Sea and the northern East China Sea in my country, with an annual output of millions of tons. Cod has many by-products, including fish heads, fish tails, fish skins, fish bones, fish fins and viscera, and the total amount of by-products accounts for more than 60% of the total amount of cod processed. In recent years, cod fillets have become the main processing method for cod. Although a few by-products (such as cod skin) have been developed as low-value products to a certain extent, most of them are still directly treated as garbage for disposal, resulting in waste of resources and pollution of the environment. With the prosperous development of my country's cod breeding industry, cod skin has become the main by-product of my country's cod processing. In recent years, the research on cod skin has mainly focused on collagen peptides, but the products developed with it as raw materials are also concentrated in the form of solid beverages and seasonings, which are mostly limited to the level of improving flavor, and the functional characteristics of cod skin protein peptides are not fully utilized, and the form of the product is not novel enough.

[0005] Influenza, or flu for short, is an acute respiratory disease caused by influenza A (A), B (B) and C (C) viruses, with clinical manifestations such as high fever, fatigue, headache, cough, and muscle aches. Among them, influenza A virus (Influenza A virus) is most susceptible to antigenic mutation and poses the greatest threat to the human body. With the invasion of the new coronavirus in the past two years, coupled with the continuous mutation of influenza viruses and the increasing resistance to anti-influenza drugs, children and the elderly, who have relatively weak resistance, have also become a high-risk group for influenza, causing serious impacts on people's health.

[0006] Since ancient times, my country has always believed that medicine and food have the same origin. Spray is a common product form that can be applied to the human nasal cavity, oral cavity, skin and other parts. It can be used as a health product or a disinfectant product. Its use method is simple and not restricted by the environment. It can be evenly absorbed after atomization without being absorbed through the stomach, reducing the irritation of the drug to the gastrointestinal tract and having no other side effects. At present, there are few reports on the extraction of peptides from fish skin for anti-influenza virus. Summary of the invention

[0007] The purpose of the present invention is to solve the above problems and provide a cod skin active polypeptide powder and its use in antiviral and sterilization.

[0008] In order to achieve its purpose, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention provides a method for preparing cod skin active polypeptide powder, comprising the following steps: taking clean cod skin and soaking it in water to swell it, then adding water to homogenize it, then adding protease to hydrolyze it, after the hydrolysis reaction is completed, inactivating the enzyme, centrifuging it, taking the supernatant and freeze-drying it to obtain the cod skin active polypeptide powder; preferably, the protease is selected from papain, trypsin and a composite protease.

[0010] In the above-mentioned technical scheme of the preparation method, the supernatant is filtered through a filter membrane to remove impurities and then freeze-dried, and preferably the filter membrane is a 0.45 μm filter membrane; preferably, the freeze-drying means that the supernatant is pre-frozen at -55°C to -45°C in a vacuum freeze dryer for 8 to 10 hours, and then the temperature is continuously raised to 35°C at uneven intervals under vacuum conditions to remove moisture and complete freeze-drying.

[0011] Preferably, the preparation method comprises the following steps: taking clean, scale-free fresh codfish skin, soaking it in hot water at 80-90°C to swell it, adding water to homogenize it according to the mass ratio of fish skin to water of 1:4-8, then adding 1-2% of the mass of the fresh codfish skin before swelling with a protease at 35-50°C for 4.5-5.5h, inactivating the enzyme after the hydrolysis reaction, centrifuging, taking the supernatant for freeze-drying, and obtaining the codfish skin active polypeptide powder; preferably, the protease is trypsin, the amount of which is 1.2-1.8% or 1.4-1.6% of the mass of the fish skin, the enzymolysis temperature is 35-38°C, and the enzymolysis is carried out under neutral conditions.

[0012] Preferably, the cod skin is derived from deep-sea cod fish, and preferably the cod fish include true cod and haddock.

[0013] The second aspect of the present invention provides a cod skin polypeptide antiviral bactericidal agent, comprising an effective amount of cod skin active polypeptide powder prepared by any of the preparation methods described above and one or more pharmaceutically acceptable excipients;

[0014] Preferably, the auxiliary materials include aromatics, sweeteners, and antioxidants; the aromatics include flavors and spices; and the antioxidants include vitamin C.

[0015] Preferably, the cod skin polypeptide antiviral fungicide comprises the following components in parts by weight: 0.4-0.6 parts, 0-1.25 parts of vitamin C, 0-1.2 parts of edible flavor, 0-10 parts of sweetener, 87-99.6 parts of purified water; preferably, it comprises 0.4-0.6 parts of cod skin active polypeptide powder, 0.25-1.25 parts of vitamin C, and 87-99.6 parts of purified water;

[0016] Preferably, the fish skin polypeptide antiviral bactericidal agent further comprises 6 to 10 parts of sweetener,

[0017] It is further preferred that the cod skin polypeptide antiviral fungicide further comprises 0.1 to 1.0 parts of edible flavor, wherein the edible flavor is selected from mint flavor, strawberry flavor, passion fruit flavor, and lavender flavor;

[0018] It is further preferred that the edible flavor includes 0.1-0.5 parts of mint flavor and 0.3-0.7 parts of strawberry flavor.

[0019] Preferably, the cod skin polypeptide antiviral fungicide comprises the following components in parts by weight: 0.4-0.6 parts of cod skin active polypeptide powder, 0.5-1.0 parts of vitamin C, 0.2-0.4 parts of mint flavor, 6-10 parts of sweetener, 0.4-0.6 parts of strawberry flavor, and 87.4-92.5 parts of purified water;

[0020] Preferably, the mass percentages of cod skin active polypeptide powder, vitamin C, sweetener, mint flavor, strawberry flavor and purified water are 0.5%, 0.5%, 10.0%, 0.2%, 0.5% and 88.3% respectively;

[0021] The sweetener is selected from xylitol, glucose, erythritol and steviol glycoside.

[0022] The third aspect of the present invention provides a method for preparing the cod skin polypeptide antiviral fungicide described in any one of the above items, comprising the following steps:

[0023] (1) Weigh each raw material component and add purified water to fully dissolve;

[0024] (2) Aseptic canning;

[0025] (3) sterilizing to obtain cod skin polypeptide antiviral sterilizing liquid;

[0026] Preferably, in step (1), the raw materials are dissolved by ultrasound, the ultrasound frequency is 45 khz, and the ultrasound time is 5 to 20 minutes; ultra-high temperature instantaneous sterilization is adopted, and the conditions are sterilization at a temperature of 136 to 150° C. for 5 to 10 seconds.

[0027] The fourth aspect of the present invention provides the use of cod skin active polypeptide powder prepared by any of the preparation methods described above or the cod skin polypeptide antiviral fungicide described above in the preparation of antiviral and / or antibacterial products, wherein the virus includes influenza virus, the bacteria are pathogenic fungi or pathogenic bacteria, the pathogenic fungi include Candida albicans, and the pathogenic bacteria include Escherichia coli and Staphylococcus aureus.

[0028] In the above application technology scheme, the virus is influenza A virus, including H1N1, PR8 and H3N2; the product includes medicine, disinfectant or health care product; the dosage form is a spray or solution, preferably an oral spray.

[0029] The beneficial effects of the present invention are:

[0030] The present invention treats cod skin by low-temperature enzymolysis, enzymolyzes cod skin at 37°C (enzymolysis for 4.5-5.5h), and the obtained cod skin polypeptide powder has the unique flavor of cod, and has good solubility and is convenient for long-term storage. Low-temperature freeze drying is used to better retain its quality and activity (if the spray drying temperature is too high, the obtained cod skin peptide powder is very easy to absorb moisture and is mostly destroyed by high temperature).

[0031] The deep-sea cod anti-influenza virus active peptide spray of the present invention is mainly composed of cod skin polypeptide powder, antioxidant (vitamin C) and sweetener, has good functional activity, can effectively inhibit Escherichia coli, Staphylococcus aureus and Candida albicans, has a significant inhibitory effect on influenza A viruses such as PR8 and H1N1, has a significant inhibitory effect on bacteria, fungi and influenza viruses, and can be used as antiviral, sterilization health products, medicines or disinfection products. Assisted by flavoring, it has good flavor and is easy to use, and is suitable for a wide range of people, all ages. The present invention enriches the product form of cod skin by making the obtained cod skin polypeptide powder into a spray, is suitable for a wide range of people, avoids the waste of cod skin resources, realizes the high value utilization of cod, and is of great significance for the development and utilization of cod skin products. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the determination of basic components of cod fish skin.

[0033] Figure 2 This is a schematic diagram of the effect of peptide powder obtained by different drying methods on MDCK cell viability.

[0034] Figure 3 This is a schematic diagram of the antiviral effect of peptide powder obtained by different drying methods observed under a microscope.

[0035] Figure 4This is a schematic diagram of the antiviral activity of cod skin polypeptide powder obtained by hydrolysis with different hydrolases.

[0036] Figure 5 Schematic diagram of the cytotoxicity of peptide powders prepared under neutral (pH 7.0) and alkaline (pH 8.1) conditions.

[0037] Figure 6 It is a schematic diagram of the antiviral effect of cod skin polypeptide powder prepared under the final process conditions.

[0038] Figure 7 This is the result of the antiviral activity of peptides prepared from different fish skins.

[0039] Figure 8 This is a schematic diagram of the amino acid composition of cod skin polypeptide powder.

[0040] Fig. 9 This is the molecular weight (Mw) distribution result of cod skin peptides obtained by hydrolysis with different enzymes.

[0041] Fig.10 It is the HPLC chromatogram of the molecular weight of cod skin polypeptides extracted by trypsin hydrolysis.

[0042] Fig.11 This is a graph showing the effects of additives added in different proportions on sensory quality.

[0043] Fig.12 This is a graph showing the changes in body weight of mice infected with PR8 from day 0 to day 14.

[0044] Fig.13 is the survival rate of PR8-infected mice from day 0 to day 14.

[0045] Fig.14 is the lung index of mice in each experimental group on the 4th day.

[0046] Fig.15 is the spleen index of mice in each experimental group on the 4th day.

[0047] Fig.16 This is the HE staining picture of lung tissue sections of mice in each experimental group on the 4th day.

[0048] Fig.17 is the virus titer in the lung tissue homogenate of mice in each experimental group on the 4th day.

[0049] Fig.18 is the level of inflammatory factors in the lung homogenate of mice in each experimental group on the 4th day.

[0050] Fig.19 These are the results of the evaluation of the antibacterial effect of cod skin oral spray.

[0051] Fig. 20 These are the results of the antibacterial activity of cod skin oral spray against three types of bacteria. DETAILED DESCRIPTION

[0052] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0053] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0054] The materials used in the present invention are not limited to those described in the following embodiments. Any technician in the relevant field can make appropriate changes to the brand or source of the materials required for the present invention without departing from the spirit and scope of the present invention. The cod can also be obtained from the local market.

[0055] The main reagents and material sources of the embodiments of the present invention are as follows:

[0056]

[0057] Example 1 Determination of the basic components of cod skin

[0058] Experimental method: Fresh real cod purchased from the market, the cod skin was washed with deionized water to remove surface impurities, and the water content in the sample was determined by the method in "GB5009.3-2016 Determination of Water in Food"; the cod skin was washed with deionized water to remove surface impurities, and the ash content in the sample was determined by the method in "GB5009.4-2016 Determination of Ash in Food"; the cod skin was washed with deionized water to remove surface impurities, and the protein content in the sample was determined by the method in "GB5009.5-2016 Determination of Protein in Food"; the cod skin was washed with deionized water to remove surface impurities, and the fat content in the sample was determined by the method in "GB5009.6-2016 Determination of Fat in Food"; the cod skin was washed with deionized water to remove surface impurities, and the total sugar content in the sample was determined by the method in "GB / T 9695.31-2008 Determination of Total Sugar Content in Meat Products".

[0059] The results of the determination of the basic components of cod skin are as follows Figure 1 As shown in the figure, it can be seen that the highest content in cod skin is water, and the most abundant dry matter in cod skin is protein, among which collagen accounts for the majority; in addition, the fat content in cod skin is relatively small, and the sugar content is almost negligible, which is consistent with the literature reports. The above results show that cod skin is a high-quality raw material for extracting collagen peptides.

[0060] In the embodiment of the present invention, cod skin active polypeptide powder (CSP) was prepared by enzymatic extraction with different enzymes (papain, trypsin, and compound protease) and different drying methods (freeze drying and spray drying). Each process is as follows:

[0061] 1. Enzymolysis: Take fresh cod skin, cut it into square pieces (about 3×3 cm), wash it with cold water, remove residual fish scales, fish meat and a small amount of fat, etc.; then soak it in 80° C. hot water for 10 minutes to swell it, add water according to the mass ratio of fish skin to water of 1:6 and homogenize it for 2 minutes, add 1.5% of the mass of protease of fresh cod skin before swelling to hydrolyze it under the optimum temperature of enzyme for 5 hours, then kill the enzyme in a 100° C. hot water bath for 10 minutes, centrifuge the cod skin enzymolysis solution at a speed of 8000 r / min for 15 minutes, take the supernatant, and obtain the cod skin enzymolysis solution; pass the enzymolysis solution through a 0.45 μm filter membrane, remove impurities and then dry it, the powder obtained after drying is the cod skin active polypeptide powder, and seal it in a low-temperature dry environment for storage.

[0062] 2. Freeze drying: the cod skin enzymatic hydrolysis supernatant prepared by enzymatic hydrolysis is pre-frozen at -50°C in a vacuum freeze dryer for 8 hours, and then maintained in a vacuum state before freeze drying is completed. Under the vacuum state, the temperature is first increased to -10°C at a gradient of 10°C / h, and then maintained at -10°C for 4.5 hours and then increased to -5°C, and then maintained for 5.5 hours and then the temperature is increased to 0°C, and then maintained at 0°C and 5°C for 6 hours respectively, and then maintained at 10°C and 15°C for 4.5 hours respectively, and further maintained at 20°C for 6 hours before reaching 25°C, dried at 25°C for 12 hours, then heated to 35°C, and finally dried at 35°C for 1 hour to complete freeze drying, and finally the cod skin active polypeptide powder is obtained.

[0063] 3. Spray drying: Use a peristaltic pump to suck the cod skin enzymatic hydrolysis supernatant (not less than 500 mL) prepared by enzymatic hydrolysis into a spray dryer, and then the supernatant is atomized by an atomizer. The sprayed water mist combines with the 170°C high-temperature airflow during the descending process to dry and dehydrate, and finally collect the cod skin active polypeptide powder.

[0064] Example 2 Effects of peptide powders obtained by different drying methods on MDCK cell viability and antiviral activity

[0065] 1. Effects of peptide powder obtained by different drying methods on MDCK cell viability

[0066] Experimental method: Trypsin was used to hydrolyze cod skin at its optimal temperature (37°C), and dried cod skin polypeptide powder (CSP) was obtained by freeze drying and spray drying. The effects of peptide powders obtained by different drying methods on MDCK cell viability were evaluated, and the MTT method was used to determine the effects of cod skin polypeptide powder on MDCK cells. MDCK cell suspension was added to a 96-well plate at a concentration of 5×10 3 / mL, add 0.1mL to each well, and culture for 24h at 37°C and 5% CO2. Prepare a series of dilutions of cod skin polypeptide powder, replace the culture medium of monolayer cells with the dilutions, set up normal cell controls, and then culture in a 37°C, 5% CO2 incubator for 48h; add 20μL, 5mg / mL tetramethylthiazolyl salt (MTT) solution to each well, continue to culture for 4h, then discard the culture medium, add 150μL of dimethyl sulfoxide (DMSO) to each well, oscillate for 10min, and after the crystals are completely dissolved, use an enzyme marker to measure the absorbance value (OD value) at a wavelength of 490nm. Set up 3 parallels for each concentration, and use the average value to evaluate the cytotoxicity of cod skin polypeptides obtained by neutral and alkaline enzymatic hydrolysis, freeze drying and spray drying to MDCK, and the cell survival rate is calculated according to the following formula:

[0067] Where: OD0——absorbance of each well of DMSO;

[0068] OD1——absorbance value of each well in the drug treatment group;

[0069] OD2——absorbance of each well in the negative control group.

[0070] The results are as follows Figure 2 As shown, the cod skin peptides obtained by freeze drying and spray drying have no obvious cytotoxicity to MDCK in the concentration range of 1.25-5 mg / mL, but the cytotoxicity increases with the increase of concentration. When the concentration is 10 mg / mL, the activity of the peptide powder obtained by freeze drying after acting on MDCK cells is 89.51±4.26%, while the activity of the peptide powder obtained by spray drying after acting on MDCK cells is 36.81±2.54%. Therefore, the cod skin peptide obtained by freeze drying has little effect on the cell activity of MDCK. Analysis may be because the spray drying method is high-temperature drying, which causes the structure of the peptide to change or even destroy, resulting in a decrease in the nutritional value of the peptide powder, and even produces some substances that are not conducive to cell growth, while freeze drying is low-temperature drying, which has little effect on the structure and activity of the peptide.

[0071] 2. Observation of the antiviral activity of peptide powders obtained by different drying methods under a microscope

[0072] Experimental method: Trypsin was used to hydrolyze cod skin at its optimal temperature (37°C) and optimal pH (pH 8.1), and dried cod skin polypeptide powder (CSP) was obtained by freeze drying and spray drying. The antiviral activity of peptide powders obtained by different hydrolases and drying methods was evaluated, and the inhibitory effect of peptide powder on influenza A virus H3N2 was determined by cytopathic effect (CPE) inhibition experiment. After the canine kidney cells (MDCK) in the 96-well plate grew into a monolayer, they were inoculated with H3N2 influenza A virus solution with a virus infection multiplicity (MOI) of 0.1, then incubated at 37°C for 1h, the supernatant was discarded, 200μL of cod skin polypeptide solution with a concentration of 1mg / mL was added, and then incubated at 37°C and 5% CO2 for 48h, and the cytopathic effect of MDCK was observed under a microscope.

[0073] The antiviral effects of peptide powder (1 mg / mL) obtained by different drying methods are as follows Figure 3 As shown, the normal MDCK cell control group showed a complete, regular, and plump cell structure under a microscope, while the MDCK cells infected with the H3N2 virus showed serious pathological changes such as rounding, breaking, and falling off. The pathological changes of the MDCK cells treated with the spray-dried peptide powder improved slightly, but obvious breaking and falling off were also observed. Only some of the MDCK cells treated with the freeze-dried peptide powder showed pathological changes. Therefore, the freeze-dried peptide powder has better antiviral activity than the spray-dried peptide powder.

[0074] Example 3 Antiviral activity of peptide powders obtained with different hydrolases and different pH values

[0075] 1. Antiviral activity of peptide powders obtained by different hydrolases

[0076] Experimental method: Under neutral conditions, trypsin (37°C, pH 8.1), papain (50°C, pH 6.5), and compound protease (50°C, pH 7.0) were used to hydrolyze cod skin at the optimal temperature and pH of the hydrolase, and finally the dried cod skin polypeptide powder was obtained by freeze drying. The inhibition rate of cod skin polypeptide powder on influenza A virus was determined by cytopathic effect (CPE) inhibition experiment. After the canine kidney cells (MDCK) in the 96-well plate grew into a monolayer, the PR8 influenza A virus solution with a virus infection multiplicity (MOI) of 0.1 was inoculated, and then incubated at 37°C for 1h, the supernatant was discarded, and 200μL of cod skin polypeptide solution with a concentration of 1mg / mL was added, and then incubated at 37°C and 5% CO2 for 48h. A positive drug control group was set up in the experiment, and a normal cell group and a virus control group were also set up. Finally, the cells were fixed with 4% paraformaldehyde and stained with crystal violet for 30 minutes. The absorbance of each well was measured at 570 nm using an ELISA reader. The virus inhibition rate was calculated according to the following formula:

[0077]

[0078] Among them: OD a ——Absorbance value of drug-treated group;

[0079] OD b ——Absorbance value of virus control group;

[0080] OD c ——Absorbance value of normal cell control group.

[0081] The results are as follows Figure 4 As shown, the polypeptide powder obtained by hydrolyzing cod skin with different hydrolases had a certain inhibitory effect on PR8 virus at 500 μg / mL, and the antiviral activity of the peptide powder obtained by trypsin hydrolysis was 32.16±0.84%, which was significantly higher than the antiviral activity of the peptide powder obtained by hydrolysis with combined protease (15.62±8.16%) and papain (6.91±1.50%) (p<0.05).

[0082] 2. Cytotoxicity and antiviral activity of peptide powders obtained under neutral (pH 7.0) and alkaline (pH 8.1) conditions

[0083] Furthermore, since the optimum pH of trypsin is 8.1, and the hydrolyzate in a polar environment may have a certain effect on cell viability, in order to test the cytotoxicity and antiviral activity of the peptide powder obtained by enzymatic hydrolysis under neutral (pH 7.0) and alkaline (pH 8.1) conditions, trypsin was used to enzymatically hydrolyze cod skin at the optimal temperature (37°C), neutral (pH 7.0) and alkaline (pH8.1) conditions, and finally cod skin polypeptide powder was obtained by freeze-drying. According to the method described above, the toxicity of the peptide powder to MDCK cells and the inhibitory activity to influenza A virus PR8 and H1N1 were detected by MTT method and CPE inhibition experiment, respectively.

[0084] Cytotoxicity results such as Figure 5As shown in the figure, the cytotoxicity of cod skin polypeptide powder obtained by enzymatic hydrolysis under neutral (pH 7.0) and alkaline (pH 8.1) conditions to MDCK increases with the increase of concentration. Both polypeptides have no obvious cytotoxicity to MDCK in the concentration range of 1.25-5 mg / mL, but the cytotoxicity is very obvious at 10 mg / mL, and the corresponding cell viabilities are 56.78±3.04% and 22.27±10.04%, respectively. However, it can be seen that the cell viability of the MDCK cell treatment group of the peptide powder obtained by enzymatic hydrolysis under neutral (pH 7.0) conditions is significantly higher than that of the peptide powder treated under alkaline (pH 8.1) conditions. It is speculated that the peptide powder obtained by enzymatic hydrolysis under alkaline (pH 8.1) conditions will exist in a polar form in the solution, and the cell viability is extremely sensitive to pH, with the optimal pH being about 6.8-7.6. Excessive acidity or alkalinity is not conducive to cell survival. Therefore, the optimal extraction conditions for cod skin polypeptides are: trypsin hydrolysis, enzymatic hydrolysis temperature 37°C, neutral (pH 7.0) enzymatic hydrolysis, and finally preservation by low-temperature freeze drying. The inhibitory activity against influenza A virus PR8 and H1N1 was verified according to the final conditions. The results are as follows Figure 6 As shown, the drug Ribavirin (Ribavirin, C8H 12 N4O5) was used as a positive control with a concentration of 50 μg / mL. Its inhibition rates against influenza virus PR8 and H1N1 were 75.53±2.11% and 79.99±5.09%, respectively. The inhibition rates of the cod skin peptide powder (1 mg / mL) treatment group were 57.28±3.36% and 57.74±0.22%, respectively. Therefore, the cod skin polypeptide powder prepared by the final process has an inhibitory effect on influenza A virus similar to that of drugs, and has a good antiviral effect.

[0085] 3. Verification of antiviral activity of different fish skin peptides

[0086] Furthermore, trypsin was used to enzymatically hydrolyze cod skin, sturgeon skin, sea bass skin and yellow croaker skin, and different types of fish skin polypeptides were extracted according to the optimal process determined in Example 3 above, and their anti-influenza virus activity was further analyzed. Figure 7 As shown in the results, at a concentration of 500 μg / mL, the inhibition rate of cod skin polypeptide against influenza A virus PR8 was 35.90±1.88%, significantly exceeding the activity of the other fish skin polypeptides (p<0.05). Therefore, the polypeptide extraction method of the present invention further verifies the anti-influenza virus activity of the extract from cod skin.

[0087] Example 4 Composition Analysis of Cod Skin Active Peptide Powder

[0088] 1. Determination of amino acid composition of cod skin polypeptide powder

[0089] The amino acid composition and content of cod skin polypeptide powder prepared under the optimal extraction conditions were determined by the method in "GB5009.124-2016 Determination of Amino Acids in Food". The results are as follows: Figure 8 As shown in the figure, in the cod skin polypeptide powder, the higher contents are Gly, Glu and Pro, accounting for 9.57±0.27g / 100g, 4.62±0.11g / 100g and 4.49±0.36g / 100g respectively. Gly and Pro are the main chains constituting collagen, which corresponds to the higher content of collagen in cod skin. Glu is a flavor amino acid. The higher content of Glu is the main reason why the cod skin polypeptide tastes delicious.

[0090] 2. Analysis of the components of cod skin peptides

[0091] The cod skin polypeptide powder was dissolved in ultrapure water to prepare an aqueous solution with a concentration of 1 mg / mL. The solution was filtered through a 0.22 μm water filter to obtain a polypeptide solution to be tested. The molecular weight distribution (Mw) of the cod skin polypeptide was determined using a high performance liquid chromatograph. The chromatographic column was TSK gel G2000 SWXL, the injection volume was set to 10 μL, the elution buffer was a 30% acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid), the flow rate was set to 0.5 mL / min, and the molecular weight distribution results of the cod skin polypeptide were analyzed after continuous detection for 30 minutes.

[0092] Fig. 9 The molecular weight (Mw) distribution results of cod skin peptides obtained by different enzyme hydrolysis are shown, among which the composite protease is composed of trypsin, compound protease and papain in a mass ratio of 1:1:1. The results show that the content of Mw < 2000Da in the cod skin peptides extracted by trypsin is 97.94%, of which the content of peptides with Mw < 500Da accounts for 52.96%, the highest content among all enzyme-extracted peptides; while the content of peptides with Mw < 2000Da in the peptides extracted by papain accounts for 51.75%, of which the content of peptides less than 500Da accounts for only 18.86%, the least among all enzyme-extracted peptides, and the large molecular peptides with Mw > 10000Da account for 6.16%; according to the analysis of the results, it is believed that small molecule peptides have a stronger effect on influenza virus. Therefore, the peptides obtained by papain hydrolysis of cod skin have a large molecular weight, which is not conducive to the interaction with the protein on the surface of influenza virus. However, small molecule peptides that significantly inhibit influenza A virus can be obtained by trypsin hydrolysis, and their molecular weight chromatograms are shown in the figure below. Fig.10 shown.

[0093] Example 5 Effects of different additive ratios on the sensory quality of cod skin polypeptide spray

[0094] 1. Single Factor Experiment

[0095] Sensory evaluation: Invite 9 sensory evaluators who have received sensory training to conduct sensory evaluation of the product in the food sensory evaluation room and record the sensory scores. The team members conducted sensory evaluation on the spray from four aspects: color, texture, smell and taste according to the sensory scoring rules (see Table 1). The sensory evaluation was conducted on the semi-finished spray product, and the average value was taken as the sensory scoring result.

[0096] Table 1 Sensory scoring details

[0097]

[0098] Experimental method: Since cod skin polypeptide powder has basically no cytotoxicity to MDCK cells within the concentration range of 0-5 mg / mL, its addition ratio is set to 0.5% and remains unchanged. According to the addition ratios of mint flavor, xylitol, and strawberry flavor of 0.3%, 8%, and 0.5%, respectively, 0.25%, 0.5%, 0.75%, 1.0%, and 1.25% vitamin C are added, and finally supplemented with pure water, mixed and sterilized by bus, and canned into vials in a sterile environment to obtain the semi-finished cod skin oral spray. Explore the effects of different addition ratios of vitamin C on the sensory quality of cod skin oral spray and determine the optimal addition amount of vitamin C.

[0099] Furthermore, the addition ratios of vitamin C, xylitol and strawberry flavor were kept at 0.75%, 8% and 0.5% respectively, and mint flavor was added at 0.1%, 0.2%, 0.3%, 0.4% and 0.5% respectively. After supplementing with water, a cod skin oral spray semi-finished product was prepared to explore the effects of mint flavor in different addition ratios on the sensory quality of the spray and determine the optimal addition amount of mint flavor.

[0100] Furthermore, the addition ratios of vitamin C, mint flavor and strawberry flavor were kept at 0.75%, 0.3% and 0.5% respectively, and 4%, 6%, 8%, 10% and 12% of xylitol were added respectively. After supplementing with water, a cod skin oral spray semi-finished product was prepared to explore the effects of different addition ratios of xylitol on the sensory quality of the spray and determine the optimal addition amount of xylitol.

[0101] Furthermore, the addition ratios of vitamin C, mint flavor and xylitol were kept at 0.75%, 0.3%, and 8%, respectively, and 0.3%, 0.4%, 0.5%, 0.6% and 0.7% of strawberry flavor were added, respectively. After supplementing with water, a cod skin oral spray semi-finished product was prepared to explore the effects of strawberry flavor in different addition ratios on the sensory quality and to determine the optimal addition amount of strawberry flavor.

[0102] The addition ratio of the above components is the mass percentage of each component in the total mass of the spray.

[0103] The results are as follows Fig.11 As shown in the figure, additives with different addition ratios have a more obvious effect on the sensory quality of the spray. When the addition amount of vitamin C is 0.75%, the score is the highest, and then the score decreases with the increase of the addition amount of vitamin C. This is because the pH of vitamin C in aqueous solution is acidic, and a higher addition ratio of vitamin C will give people a strong sense of sourness, thereby reducing the sensory score of the spray; when the addition amount of mint flavor is 0.3%, the sensory score is the highest, and then the score decreases with the increase of the addition amount of mint flavor. This is because excessive mint flavor makes the taste bitter and the smell pungent, resulting in a decrease in sensory quality; the addition ratio of xylitol for the most suitable taste is 8%, and as the addition amount of xylitol continues to increase, the color will decrease and the taste will be bitter, resulting in a decrease in sensory quality; strawberry flavor has little effect on the sensory quality of the spray, but excessive strawberry flavor will cause the smell to be too strong and the taste to be overly sweet. Therefore, combined with the results of the single factor experiment, the added amounts of vitamin C, mint flavor, xylitol and strawberry flavor were kept within the ranges of 0.5-1.0%, 0.2-0.4%, 6-10% and 0.4-0.6%, respectively, which could improve the sensory quality of the spray.

[0104] 2. Orthogonal test

[0105] Based on the results of the single factor experiment, the sensory score was used as an indicator to design L9(3 4 ) Orthogonal experiment was used to further determine the addition ratio of vitamin C, xylitol, mint flavor and strawberry flavor. The experimental factor design is shown in Table 2.

[0106] Table 2 Orthogonal experimental factor design

[0107]

[0108] The results of the orthogonal test are shown in Table 3. It can be seen that the sensory results of the spray are more obviously affected by vitamin C. The order of factors affecting the sensory perception of the spray is vitamin C>xylitol>mint flavor>strawberry flavor. Through K value analysis, it can be seen that the optimal addition ratio combination of vitamin C, mint flavor, xylitol and strawberry flavor is A1B1C3D2, that is, the optimal addition ratios of vitamin C, mint flavor, xylitol and strawberry flavor are 0.5%, 0.2%, 10.0% and 0.5% respectively, and finally supplemented with pure water. At this time, the sensory evaluation effect is the best.

[0109] Table 3 Effects of different addition ratios of each formula on product sensory

[0110]

[0111] 3. Best preparation method for cod skin polypeptide oral spray

[0112] (1) Mixing: Weigh the required weight according to the order of cod skin polypeptide powder, vitamin C, and sweetener according to the proportion. Mint essence and strawberry essence can be added in the required amount using a micro-pipette. The addition ratios are 0.5%, 0.5%, 10.0%, 0.2%, and 0.5%, respectively. Finally, add 88.3% of pure water to make up the system. Ultrasonic dissolution is uniform, the ultrasonic frequency is 45 khz, and the ultrasonic time is 10 min.

[0113] (2) Packaging: The mixed spray concentrate is packaged in a spray bottle, and finally ultra-high temperature instantaneous sterilization is performed at 136-150° C. for 5-10 seconds to obtain the cod skin polypeptide oral spray.

[0114] Example 6 In vivo experiment of cod skin spray against influenza virus

[0115] 1. Animal Culture

[0116] 70 SPF female BALB / c mice (14-16 g) were selected and weighed one by one. The mice were randomly divided into 7 groups according to their weight (the specific experimental grouping is shown in Table 4), with 10 mice in each group. The experiment was started after 1 day of adaptation. The feeding and research of mice strictly followed the protocol approved by the Animal Care and Use Committee of Ocean University of China (OUCYY-2016003). After the mice were anesthetized with 30% ulose, the mice were breathing rapidly at this time, and each mouse was quickly infected with PR8 virus (2LD 50 / mice); cod skin polypeptide (CSP) and cod skin oral spray (Antiflupep, ATEP) were used as experimental groups, and the normal control group and placebo control group were given the same volume of normal saline as control. At the same time, recombinant human interferon α2b (Interferon, ITF) commercially available anti-influenza product was used as a positive control. The day of infection was counted as day 0. Administration began 4 hours after virus infection, once a day, for a total of 3 days. On the 4th day, the mice were killed, and the lung tissue was homogenized and then the relevant indicators were measured. In the survival experiment, the weight changes and deaths of the mice were recorded every day for 14 consecutive days. During this period, the supply of feed and drinking water for the mice was guaranteed, but not too much.

[0117] Table 4 Animal experiment groups

[0118]

[0119] 2. Index determination

[0120] Mouse survival rate and weight change: After inoculation with the virus, the death and weight of mice were recorded every day for 14 consecutive days, and the weight recovery after administration was compared (when the weight of the mouse decreased by more than 25% relative to the weight on the day of inoculation, it was considered dead), the survival rate of the mice was calculated, and a survival curve was drawn. The survival rate and weight change were calculated according to the following formulas,

[0121]

[0122] The results are as follows Fig.12 As shown, the proportion of initial body weight of mice in the placebo control group was significantly reduced (55.56%), and the proportion of initial body weight of mice in the recombinant interferon α2b (10000U / day) treatment group was 99.69±4.66%, which shows that the antiviral product has a significant effect on inhibiting the weight loss of mice infected with the virus. The results of the low-dose group (5mg / kg / day) and high-dose group (20mg / kg / day) of cod skin polypeptide were consistent with those of the antiviral product (87.41±4.38% and 96.53±3.08%), and the proportion of initial body weight of mice in the low-dose group (5mg / kg / day) and high-dose group (20mg / kg / day) of cod skin oral spray was 99.63±3.99% and 101.44±4.51%, respectively, which exceeded the effect of the antiviral product interferon α2b (10000U / day) treatment group. Fig.13 The results show the survival rate of mice after drug administration. The final survival rate of mice in the placebo group was 14.29%, which is an extremely low survival rate. The survival rate of mice in the interferon α2b (10000U / day) treatment group was 42.86%. It can be seen that the current broad-spectrum antiviral products have a certain effect on improving the survival rate of mice infected with the virus. The survival rates of mice in the cod skin polypeptide (5mg / kg / day) and cod skin spray (5mg / kg / day) treatment groups were 57.14 and 71.43%, respectively. The survival rates of the cod skin polypeptide (20mg / kg / day) and cod skin spray (20mg / kg / day) treatment groups were both 85.71%. It can be seen that cod skin polypeptide and cod skin oral spray can improve the survival of mice infected with the virus. 3. Mouse lung, spleen index and lung pathology analysis

[0123] Four days after the mice were infected, three mice were killed by cervical dislocation in each group and then dissected. The complete lung and spleen tissues were taken out and weighed. The lung index and spleen index of each group of mice were calculated based on the weight of the mice. A small part of the lung tissue (3 parts / group) was taken for formaldehyde fixation and paraffin embedding, and then HE staining analysis was performed to observe the lung lesions. The lung index was calculated according to the following formula:

[0124]

[0125] The lung and spleen tissues of mice were swollen due to virus infection, which led to a significant increase in the proportion of lung and spleen tissue in the body. Fig.14As shown, the lung index of the placebo control group was 1.19±0.15%, significantly higher than that of the normal control group (0.63±0.02%, p<0.05), while the lung index of the interferon α2b (10000U / day) treatment group was 0.78±0.14%, significantly lower than that of the placebo control group (p<0.05), and the lung index of the low-dose cod skin polypeptide (5mg / kg / day) treatment group was 0.98±0.10%, although there was a certain decrease, but the effect was not significant. The lung indexes of the cod skin polypeptide (20 mg / kg / day), cod skin oral spray (5 mg / kg / day) and cod skin oral spray (20 mg / kg / day) treatment groups were 0.69±0.04%, 0.82±0.02% and 0.65±0.04%, respectively. Compared with the placebo control group, the lung index was significantly reduced (p<0.05), indicating that the therapeutic effect of cod skin oral spray is comparable to that of the commercially available antiviral product interferon α2b (10000U / day).

[0126] Depend on Fig.15 It can be seen that the spleen index of the interferon α2b (10000U / day) treatment group was 0.34±0.02%, and the spleen index of the cod skin polypeptide and cod skin oral spray in the high-dose treatment group (20mg / kg / day) was 0.34±0.11% and 0.24±0.06%, respectively. In particular, the effect of the cod skin oral spray on reducing the spleen index exceeded that of the antiviral product interferon α2b (10000U / day) treatment group, but there was no significant difference. There was no significant difference between the spleen index of the placebo control group (0.46±0.05%) and the spleen index of the normal control group (0.29±0.02%). It can be seen that the influenza virus has less infection of the spleen, and mainly causes lung lesions by infecting the lung tissue. In short, the cod skin oral spray has a certain protective effect on the lung tissue and spleen tissue of mice infected with influenza virus.

[0127] H&E staining results of mouse lung tissue are shown in Fig.16 As shown in the figure, compared with the normal control group, the lung tissue of mice infected with PR8 virus showed severe pulmonary edema, alveolar concavity and alveolar wall thickening. Treatment with recombinant interferon α2b (10000U / day) can reduce lung tissue lesions. The low-dose group of cod skin polypeptide (5mg / kg / day) and cod skin oral spray (5mg / kg / day) had less inhibitory effect on lung lesions, and some inflammatory cells still existed. However, the high-dose group of cod skin polypeptide (20mg / kg / day) and cod skin oral spray (20mg / kg / day) significantly reduced the infiltration of inflammatory cells in the alveolar wall after treatment, and the lung bronchial epithelium and other tissues were relatively intact.

[0128] 4. Mouse lung virus proliferation assay

[0129] Most of the lung tissue was removed for preparation of lung tissue homogenate, and the NA titer method was used to evaluate the reduction of virus titer in the lung homogenate. The results are as follows Fig.17 shown.

[0130] The results of lung viral NA titer are as follows Fig.17 As shown, taking the NA titer in the lung tissue homogenate of the placebo control group as 100%, the virus titer of the interferon α2b (10000U / day) treatment group was 59.62±4.31%. It can be seen that the recombinant interferon α2b (10000U / day) in the commercially available antiviral products can significantly reduce the virus titer in the lung (p<0.05). The virus titers of the cod skin polypeptide low-dose group (5mg / kg / day) and high-dose group (20mg / kg / day) were 84.49±2.83% and 41.20±5.09%, respectively. It can be seen that the cod skin polypeptide powder has a significant effect on reducing the virus titer in the lungs of mice infected with the virus (p<0.05). The lung virus titers in the low-dose group (5 mg / kg / day) and high-dose group (20 mg / kg / day) of cod skin oral spray also showed a significant decrease (p < 0.05), which were 61.61 ± 5.31% and 33.89 ± 5.31%, respectively. The effect of the cod skin oral spray in the high-dose group (20 mg / kg / day) even significantly exceeded that of the current antiviral product recombinant interferon α2b (10000 U / day) treatment group (p < 0.05).

[0131] 5. Determination of inflammatory factors

[0132] After homogenizing the mouse lung tissue, the changes of inflammatory factors such as CXCL1, RANTES, IL-6 and TNF-α in the mouse lung tissue homogenate were determined according to the method in the instructions of the inflammatory factor ELISA kit. The results are as follows Fig.18 shown.

[0133] After mice are infected with the virus, the level of inflammatory factors in their lungs will rise sharply. Therefore, the concentration of inflammatory factors in the lungs is measured to detect the regulatory mechanism of cod skin oral spray on the secretion level of cytokines in the lung tissue of mice induced by PR8 influenza virus, so as to verify the ultimate anti-influenza effect of cod skin oral spray. Fig.18It can be seen that the cod skin peptides in the low-dose group (5mg / kg / day) and the high-dose group (20mg / kg / day) can reduce the level of inflammatory factors, but the significance is low. The cod skin oral spray in the low-dose group (5mg / kg / day) and the high-dose group (20mg / kg / day) also has a reducing effect on inflammatory factors, and the effect of the high-dose group (20mg / kg / day) is the most significant. Compared with the placebo control group, the CXCL1 level of the cod skin oral spray (20mg / kg / day) treatment group was significantly reduced from 327.82±24.31pg / mL to 49.91±11.55pg / mL (p<0.05), which is also significantly higher than the antiviral product recombinant interferon α2b The treatment effect of (10000U / day) was significantly reduced (p<0.05), RANTES level was significantly reduced from 1508.04±48.45pg / mL to 1185.51±38.14pg / mL (p<0.05), IL-6 level was significantly reduced from 178.06±3.43pg / mL to 139.05±4.34pg / mL (p<0.05), TNF-α level was significantly reduced from 211.12±20.18pg / mL to 145.03±5.51pg / mL (p<0.05). The above results fully demonstrate that downregulating the level of cellular inflammatory factors is an important way for cod skin oral spray to inhibit influenza virus infection in the lungs of mice.

[0134] Example 7 Antibacterial activity of cod skin oral spray

[0135] 1. Observation of the antibacterial effect of cod skin oral spray by plate coating method

[0136] Experimental method: Gram-positive bacteria Escherichia coli, Gram-negative bacteria Staphylococcus aureus and fungi Candida albicans were selected as experimental strains. After the strains were cultured to the logarithmic growth phase, the bacterial solution was diluted 1000 times with liquid culture medium in a sterile 5mL test tube, and then mixed with cod skin oral spray 1:1 (v / v). After shaking for 2 hours in an incubator at the optimal culture temperature for the strain, 20μL of the mixed solution was fully applied to the solid culture medium, and the culture medium was inverted and cultured for 24 hours. After the culture was completed, the growth of the colony was observed. Among them, ampicillin (50μg / mL) was used as a positive control for the inhibition experiment of Escherichia coli and Staphylococcus aureus, and amphotericin B (50μg / mL) was used as a positive control for the inhibition experiment of Candida albicans. Sterile water was used as a negative control for the antibacterial experiment.

[0137] The results are as follows Fig.19As shown in the figure, the total colony counts of the three bacteria in the plates after coating with the cod skin oral spray (Antiflupep) tended to decrease compared with the negative control group, among which the inhibitory effect on Candida albicans was the most obvious. It can be seen that the cod skin oral spray has a certain inhibitory effect on the three bacteria, but the specific inhibition rate of the cod skin oral spray on the three bacteria cannot be obtained by the total colony counts.

[0138] 2. Determination of antibacterial activity of cod skin oral spray by microdilution method

[0139] Experimental method: In order to further obtain the antibacterial rate of cod skin oral spray, the antibacterial activity of cod skin oral spray was determined by microdilution method. First, the strain was cultured in culture medium to the logarithmic growth phase, 50 μL of bacterial solution was taken in a 96-well plate, and an equal volume of cod skin oral spray was added and shaken in an incubator at the optimal culture temperature of the strain for 16 to 24 hours, and finally the absorbance of each well was measured at a wavelength of 600 nm by an ELISA instrument. Among them, ampicillin (50 μg / mL) was used as a positive control for the inhibition experiment on Escherichia coli and Staphylococcus aureus, and amphotericin B (50 μg / mL) was used as a positive control for the inhibition experiment on Candida albicans. Sterile water was used as a negative control for the antibacterial experiment. Before the experiment, the cod skin oral spray was filtered and sterilized with a 0.22 μm sterile filter membrane. The antibacterial rate was calculated according to the following formula:

[0140]

[0141] Where: OD0——absorbance value of each well in blank group (without inoculation of bacterial solution);

[0142] OD1——absorbance value of each well in the negative control group (sterile water);

[0143] OD2—absorbance value of each well in the experimental group (cod skin oral spray).

[0144] Antibacterial activity of cod skin spray against three bacteria Fig. 20 As shown, the cod skin oral spray has a high inhibitory activity against Staphylococcus aureus and Escherichia coli, with inhibition rates of 81.06±3.62% and 73.46±8.00% respectively, and also has a certain inhibitory activity against Candida albicans, with an inhibition rate of 53.21±3.98%. It can be seen that the cod skin oral spray has a certain antibacterial activity against Escherichia coli, Staphylococcus aureus and Candida albicans.

[0145] Lung infection is the main result of severe influenza, so preventing influenza virus from infecting the lungs is an important way to prevent influenza. Neuraminidase (NA) is an important glycoprotein of influenza virus, which plays an important role in the spread of virus between cells. Influenza virus enters the lungs through the respiratory tract and spreads in the lungs through neuraminidase (NA), which increases the level of lung inflammatory factors, thereby causing lung inflammation and lesions. In this experiment, cod skin oral spray can significantly reduce the NA titer in the lungs, prevent the virus from spreading further in the body, and significantly reduce the level of lung inflammatory factors induced by the virus, preventing lung lesions caused by increased inflammatory factors, and then inhibiting various influenza symptoms caused by lung lesions. In addition, the experiment also confirmed that cod skin oral spray has a high inhibitory activity against Escherichia coli, Staphylococcus aureus and Candida albicans, and can effectively avoid various diseases caused by bacterial and fungal infections.

[0146] The cod skin active polypeptide and spray of the present invention make full use of cod skin, improve the high-value utilization of cod, reduce resource waste, protect the environment, and make up for the deficiencies of current anti-influenza virus products, providing a theoretical basis and technical support for the research of anti-influenza virus drugs. The product can be in the form of an oral spray, which is easy to use, non-toxic and harmless to the human body, can effectively prevent influenza virus infection and bacterial invasion, has good sensory quality, and is suitable for various groups of people.

Claims

1. Use of cod skin active polypeptide powder or an antiviral fungicide containing the cod skin active polypeptide powder in the preparation of antiviral and / or antibacterial products, characterized in that: The virus is influenza A virus PR8, the bacteria is a pathogenic fungus or a pathogenic bacterium, the pathogenic fungus is Candida albicans, and the pathogenic bacterium is Escherichia coli or Staphylococcus aureus; The antiviral fungicide contains an effective amount of cod skin active polypeptide powder and one or more pharmaceutically acceptable excipients; The preparation method of the cod skin active polypeptide powder comprises the following steps: taking clean fresh cod skin without fish scales, soaking it in 80-90°C hot water to swell it, then adding water in a mass ratio of fish skin to water of 1:4-8 to homogenize it, then adding 1-2% of the mass of the fresh cod skin before swelling, trypsin, and hydrolyzing it at 35-50°C under neutral conditions for 4.5-5.5 hours, inactivating the enzyme after the hydrolysis reaction, centrifuging, filtering the supernatant to remove impurities, and then freeze-drying it to obtain the cod skin active polypeptide powder.

2. The use according to claim 1, characterized in that: The supernatant is filtered through a filter membrane to remove impurities and then freeze-dried, wherein the filter membrane is a 0.45 μm filter membrane; the freeze-drying means that the supernatant is pre-frozen at -55°C to -45°C for 8 to 10 hours in a vacuum freeze dryer, and then the temperature is continuously raised to 35°C at uneven intervals under vacuum conditions to remove moisture and complete freeze-drying.

3. The use according to claim 1, characterized in that: The amount of trypsin added is 1.2~1.8% of the fish skin mass, and the enzymatic hydrolysis temperature is 35~38 ℃.

4. The use according to claim 1, characterized in that: The auxiliary materials include aromatics, sweeteners, and antioxidants; the aromatics include essences and spices; and the antioxidants include vitamin C.

5. The use according to claim 4, characterized in that: The antiviral fungicide comprises the following components in parts by weight: 0.4-0.6 parts of cod skin active polypeptide powder, 0.25-1.25 parts of vitamin C, 0.1-1.0 parts of edible flavor, 6-10 parts of sweetener, and 87-99.6 parts of purified water; The edible flavor is selected from mint flavor, strawberry flavor, passion fruit flavor and lavender flavor.

6. The use according to claim 5, characterized in that: The antiviral fungicide comprises the following components in parts by weight: 0.4-0.6 parts of cod skin active polypeptide powder, 0.5-1.0 parts of vitamin C, 0.2-0.4 parts of mint essence, 6-10 parts of sweetener, 0.4-0.6 parts of strawberry essence, and 87.4-92.5 parts of purified water; The sweetener is selected from xylitol, glucose, erythritol and steviol glycoside.

7. The use according to claim 1, characterized in that: The products include medicines and disinfectants; the dosage forms are sprays or solutions.

8. The use according to claim 7, characterized in that: The dosage form is an oral spray.

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