CN117186177B - Duck blood cell protein peptide with uric acid reducing activity and preparation method thereof - Google Patents

Abstract

The present invention discloses a duck blood globulin peptide with uric acid lowering activity and a preparation method thereof, and belongs to the field of food biotechnology. The present invention provides a method for preparing duck blood protein peptide, the duck blood protein peptide has 5 peptide segments whose amino acid sequence is SEQ ID No.1-5, and the method includes steps S1), adding protease A to duck hemoglobin to obtain enzymolysis solution M1; S2), adding protease B to enzymolysis solution M1 to obtain enzymolysis solution M2; the protease A is a protease derived from pineapple, and the protease B is selected from one or two of the protease derived from thermophilic bacillus and the protease derived from Pseudomonas aeruginosa. The present invention provides a new choice for the deep processing of poultry blood in my country, enriches the types of peptide products, and has greater economic and social benefits.

Description

A duck hemoglobin peptide with uric acid-lowering activity and preparation method thereof

Technical Field

The invention belongs to the field of food biotechnology, and in particular relates to a duck hemoglobin peptide with uric acid-lowering activity and a preparation method thereof.

Background Art

Blood is a byproduct of meat processing, and my country has abundant blood resources. Taking ducks as an example, the number of ducks in my country in 2019 was about 2.461 billion, and it has been increasing year by year. According to the protein content of about 18% in the blood, about 44,200 tons of duck blood protein are produced each year. Blood may carry pathogenic bacteria or toxic metabolites and is easily contaminated by pathogenic microorganisms, which makes blood collection and storage inconvenient. Only a small amount of blood is processed and utilized in the form of blood meal, blood tofu or feed (Diab et al. Membranes, 2020, 10, 257). In order to rationally utilize blood protein resources, deep processing and utilization of blood is of great significance. Hemoglobin is the main protein component of blood, present in red blood cells, accounting for 80% of the total protein content of blood. Blood cell powder is generally processed and produced by direct spray drying. The rupture of the blood cell membrane is limited, resulting in low digestion and absorption rate, which limits its application in the food and feed industry. Hemoglobin is composed of heme and globin, is reddish brown, and has a pungent bloody smell. Hemoglobin is released during the deep processing of blood cells, and the divalent iron in hemoglobin can be quickly oxidized to trivalent iron, affecting the appearance of the product (Alvarez et al. LWT-Food Science and Technology, 2016, 73: 280-289). Due to the characteristics of the duck slaughtering process and the nature of duck blood that is easy to coagulate and clump, duck blood collection is more difficult than pig blood and cow blood. Therefore, the utilization rate of duck blood in my country is low. There are no reports on the hydrolysis and comprehensive utilization of duck blood protein abroad, and there are few domestic reports, mainly focusing on the preparation of bioactive peptides by hydrolyzing duck blood. Acidic protease, papain, alkaline protease and flavor protease were screened with hydrolysis degree, decolorization degree and product yield as indicators. The results showed that acidic protease can significantly degrade duck blood globulin, the product is milky white, the yield of duck blood globulin is 60.1%, and the hydrolyzate has antioxidant activity (Zheng Zhaojun et al., Journal of Animal Nutrition, 2016, 28: 2521-2533). Taking the iron ion chelation ability of duck blood globulin hydrolysate as an indicator, the hydrolysis conditions of duck blood globulin by alkaline protease were optimized. Under the optimal conditions, the iron ion chelation rate of duck blood globulin hydrolysate was 65.4%, which can be used as an iron supplement (Yang Yanru, Food Industry Science and Technology, 2020, 41:19).

Most of the bioactive peptides prepared by bioenzymatic hydrolysis are derived from dietary proteins, with high safety, no side effects, and low induction of drug resistance compared to traditional drugs. Many bioactive peptides have been approved by the FDA for use in therapeutic drugs, and there are about 100 peptide drugs on the market in the United States, Europe and Japan (Barbara et al. Biotechnology Advances, 2018, 36: 415-429). Suitable protease hydrolysis of hemoglobin can not only decolorize, but also obtain small molecule peptides with physiological functional activity (Alvarez et al. LWT-Food Science and Technology, 2016, 73: 280-289). Using bioenzyme technology to hydrolyze duck hemoglobin and fully release the protein in the blood cells can not only improve the protein utilization rate, but also the small molecule peptides have many physiological regulation functions and can be used as ingredients for functional foods or health products. Feeding pig hemoglobin peptides to weaned piglets can significantly increase the average daily weight gain of piglets, reduce the diarrhea rate, and enhance the immunity of piglets (Wu Yanjun, Journal of Yangzhou University, 2010). Feeding mice with wild goose blood peptides prepared by hydrolysis of neutral protease, alkaline protease and papain significantly improved the immunity of mice, and significantly increased the proliferation capacity of spleen lymphocytes and cytokine secretion of mice (Wang Zheng, Journal of Changchun University of Technology, 2020). In order to promote the application of blood peptides in the food industry, the China Animal Husbandry Association issued the "Camel Blood Peptide" group standard (T/CAAA 018-2019) on January 7, 2019. This standard specifies the relevant indicators of camel blood peptides prepared from camel blood, which has a certain reference value for blood peptides prepared from blood of other animal sources.

Hemoglobin is present in blood cells, and blood cells need to be destroyed before hydrolysis to release hemoglobin. Hemoglobin extraction mainly uses acid solution method or acid acetone method. When the pH value is lower than 3.5, divalent iron ions dissociate from histidine coordination bonds, resulting in the breakage of heme molecules, thereby releasing hemoglobin, but these methods will bring in other organic matter, and a large amount of alkali solution needs to be used to adjust the pH during hydrolysis, bringing in salt, and increasing the refining process and cost of later desalination and impurity removal (Zhong Yaoguang, Food Science, 2004, 4: 66-71; Wedzicha et al., Food Chemistry, 1985, 17: 199-207). There are also studies that use mechanical methods to destroy cell walls, such as ultrasonic wall breaking, freeze-thaw treatment, homogenization treatment, etc., but these methods require professional equipment, increase production process steps and production costs (Wu Wenjin et al., Food Research and Development, 2021, 42: 90-96). Therefore, it is necessary to develop a method that can conveniently and efficiently process blood cells and release hemoglobin. Electrolyzed water is a solution obtained by electrolyzing a low-concentration salt solution in an electrolytic cell with a cation exchange membrane. The anode produces acidic electrolyzed water with strong oxidizing properties and low points, and the cathode produces alkaline electrolyzed water with strong reducing properties and high potential. At present, electrolyzed water has been applied to many fields, such as bactericidal and antibacterial, food preservation, detergents and meat tenderization (Huang et al. Food Control, 2008, 19: 329-345). There are few reports on electrolyzed water protein treatment. Soaking soybeans in electrolyzed water combined with microbial fermentation can improve the ACE inhibitory activity of fermented soybean water extract (Li et al., International Journal of Food Properties, 2011, 14: 145-156). Compared with almond protein extracted by ultrapure water dissolution, the secondary structure of almond protein extracted by alkaline electrolyzed water is destroyed (Li et al., Food Science and Biotechnology, 2018, 28: 15-23). After wheat gluten, peanut meal and walnut meal were treated with an improved electrolytic cell, the ACE inhibitory activity of the three protein raw material hydrolysates was significantly improved (Zhang et al. LWT-Food Science and Technology, 2022, 154, 112864). At present, there are no reports or patents on the swelling and wall breaking of hemoglobin by acidic electrolyzed water.

Gout is a purine nucleotide metabolic disorder that causes sodium urate crystals to precipitate in the joints, accompanied by acute pain. The prevalence and incidence of gout are 6.8‰ and 2.89‰, respectively, and are increasing year by year worldwide (Dehlin et al. Mature Reviews Rheumatology, 2020, 16: 380-390). Male serum uric acid levels above 416 μmol/L (female levels above 357 μmol/L) are defined as hyperuricemia, which is the most important biochemical indicator in the pathogenesis of gout. Xanthine oxidase (XOD) can catalyze the oxidation of hypoxanthine to xanthine and xanthine to uric acid, and is a key enzyme in the human uric acid regulation system. XOD increases the production of uric acid in the blood and is a key factor in causing hyperuricemia and gout. Traditional drugs for the treatment of gout and hyperuricemia are mainly allopurinol and febuxostat, both of which achieve the effect of reducing serum uric acid concentration by inhibiting the catalytic activity of XOD. Traditional uric acid-lowering drugs are often accompanied by serious adverse reactions, including allergic syndrome, rash, and effects on liver metabolism (Jansen et al. Clinical Rheumatology, 2010, 29: 835-840). Therefore, the development of bioactive peptides with XOD inhibitory activity for the prevention and treatment of hyperuricemia and gout has important economic and social significance.

There are few reports on the preparation of bioactive peptides with XOD inhibitory activity by bioenzymatic hydrolysis. Most XOD inhibitory peptides are derived from marine fish proteins, such as the in vitro XOD IC ₅₀ value of bonito protein hydrolyzed by neutral protease is 14.88 mg/mL, and the XOD IC ₅₀ value of ICRK identified from bluefin tuna hydrolysate is 14.18 mg/mL; there are also XOD inhibitory peptides derived from other proteins, such as walnut meal hydrolyzed by alkaline protease, with an XOD inhibition rate of 27.23% at a concentration of 20 mg/mL (Zhong et al. Food Chemistry, 2021, 347: 129068; Bu et al. International Conference on Energy, Environment and Bioengineering, 2020, 185: 04062; Li et al. Food & Function, 2018, 9: 107-116). The activity of these hydrolyzates is low, and there is a big gap compared to the effect of traditional uric acid-lowering drugs (allopurinol, IC ₅₀ value is 8.04 μg/mL). There are no patents or reports on the preparation of XOD inhibitory peptides using animal blood proteins. In theory, the biological activity of peptides is determined by multiple factors such as protein structure, protease type and hydrolysis conditions (Udenigwe et al. Journal of Food Science, 2012, 77: R11-R24). Therefore, selecting suitable protein substrates and proteases can enrich XOD inhibitory peptides. The structure-activity relationship study of XOD inhibitory peptides shows that the aromatic amino acids in the peptide segment can effectively bind to the XOD catalytic active center, which plays a key role in the XOD inhibitory activity. Protease PaproA is a highly active protease produced by fermentation of Pseudomonas aeruginosa CAU342A (Sun Qian et al., Bulletin of Microbiology, 2017, 44: 86-95); protease GsProS8 is a protease derived from Geobacillus stearothermophilus produced by heterologous expression and high-density fermentation (Chang et al. BMC Biotechnology, 2021, 21: 21). Bioinformatics combined with peptide mapping analysis found that proteases PaproA and GsProS8 can specifically hydrolyze duck hemoglobin and enrich peptides containing aromatic amino acids, which are suitable for the preparation of XOD inhibitory peptides.

There have been some patent reports on the preparation of uric acid-lowering active peptides by protease hydrolysis. The Chinese invention patent with patent number ZL201310485124.9 discloses a method for preparing walnut peptides with uric acid-lowering efficacy by hydrolyzing walnut meal using alkaline protease and cellulase. The Chinese invention patent with application number 202010861723.6 discloses a method for preparing kidney bean peptides with uric acid-lowering activity by hydrolyzing kidney bean protein using alkaline protease. The Chinese invention patent with application number 202110397871.1 discloses a method for preparing codfish peptides with xanthine oxidase inhibitory activity by hydrolyzing cod fillet powder using papain, neutral protease, alkaline protease or flavor protease. The Chinese invention patent with application number 20211045403.X discloses a method for preparing sturgeon peptide composite powder with uric acid-lowering activity by using subtilisin, papain and alkaline protease to hydrolyze sturgeon protein. The above-disclosed invention patents are all methods for preparing peptides with uric acid lowering activity using protease hydrolysis technology. At present, there are no patents or reports on the preparation of uric acid lowering peptides by bioenzymatic hydrolysis of duck hemoglobin.

Summary of the invention

The technical problem to be solved by the present invention is: how to reduce uric acid and/or how to inhibit the activity of xanthine oxidase (XOD).

In order to solve the above technical problems, in the first aspect, the present invention provides a method for preparing duck blood protein peptide, wherein the duck blood protein peptide has the following five peptide segments:

P1), a polypeptide having an amino acid sequence of SEQ ID No. 1;

P2), a polypeptide having an amino acid sequence of SEQ ID No. 2;

P3), a polypeptide having an amino acid sequence of SEQ ID No.3;

P4), a polypeptide having an amino acid sequence of SEQ ID No.4;

P5), a polypeptide having an amino acid sequence of SEQ ID No.5;

The method comprises enzymatically hydrolyzing duck hemoglobin to obtain an enzymatic hydrolysate, and extracting duck blood protein peptides from the enzymatic hydrolysate.

Furthermore, in the above method, the enzymatic hydrolysis of duck hemoglobin comprises the following steps:

S1), adding protease A to duck hemoglobin to obtain enzymatic solution M1;

S2), adding protease B to the enzymatic hydrolysate M1 to obtain enzymatic hydrolysate M2;

The protease A may be a protease derived from pineapple, and the protease B may be a protease derived from Bacillus stearothermophilus or a protease derived from Pseudomonas aeruginosa.

In the present invention, the pineapple-derived protease may be bromelain, and the bromelain is a product of Shanghai Yuanye Biotechnology Co., Ltd., and its product number is: S10009;

In the present invention, the protease derived from Bacillus stearothermophilus can be named protease GsProS8. The amino acid sequence of the protease GsProS8 is shown in Figure 1. The protease GsProS8 is a protease obtained by introducing the coding gene of the protease GsProS8 into Bacillus subtilis for expression. The nucleotide sequence of the coding gene of the protease GsProS8 is shown in Figure 1.

In the present invention, the protease derived from Pseudomonas aeruginosa may be named as protease PaproA. The protease PaproA may be derived from Pseudomonas aeruginosa CAU342A. The SDS-PAGE and enzyme spectrum of the protease PaproA are shown in FIG2 .

Specifically, the protease GsProS8 is prepared according to a method comprising the following steps:

Bacillus subtilis WB600 expressing the recombinant protease GsProS8 was inoculated into LB culture medium at an inoculum size of 1% (V/V), and cultured at 37°C for 12 hours as a fermentation seed liquid. The fermenter was filled with 1.5 L of liquid and inoculated at an inoculum size of 5% (V/V). The fermentation process was controlled at 37°C, the pH was adjusted to 4.0 using ammonia water and phosphoric acid, and the dissolved oxygen was maintained at about 30%. When the residual sugar content in the culture medium was less than 1 g/L, the feed culture medium was started. After 108 hours of high-density fermentation, the fermentation liquid was collected, and the centrifuged supernatant was the protease GsProS8. The composition of the fermentation medium is: 2g/L ammonium sulfate, 3g/L yeast extract, 1g/L glucose, 1g/L sodium chloride, 0.5g/L magnesium sulfate, 0.05g/L zinc chloride, 0.05g/L manganese chloride, 0.1g/L enzyme, 2g/L calcium chloride, and the rest is water; the composition of the feed medium is: 500g/L glucose, 37.5g/L peptone, and the rest is water. The protease activity of the protease GsProS8 enzyme solution is 3800U/mL. The method for determining the enzyme activity of protease GsProS8 refers to GB/T 23527-2009: 1mL enzyme solution and 1mL casein solution are incubated at 40℃ for 10min, then 2mL trichloroacetic acid is added to terminate the reaction, centrifuged at 10000rpm for 10min, 1mL supernatant is added with 5mL sodium carbonate solution and 1mL Folin phenol reagent, incubated at 40℃ for 20min to develop color, and the absorbance is measured at 660nm. The enzyme solution to which trichloroacetic acid is added to terminate the reaction is used as a control. The amount of enzyme required to hydrolyze casein to produce 1μg tyrosine per minute is defined as 1 enzyme activity unit (U).

The protease PaproA can be prepared according to a method comprising the following steps: Pseudomonas aeruginosa CAU342A is cultured at 30°C in a fermentation medium for 3 days, the fermentation broth is collected, the fermentation broth is centrifuged, and the supernatant is collected, which is the enzyme solution of the protease PaproA. The fermentation medium is made of the following raw materials: 3% lees, 1.5% yeast extract powder, 0.05% Tween-80, 0.5% sodium chloride, 0.7% potassium phosphate, 0.3% dipotassium hydrogen phosphate, 0.04% manganese sulfate, and the rest is water, and all percentages are mass percentages. The pH of the fermentation medium is 7.5. The protease activity of the enzyme solution of the protease PaproA prepared is 2653U/mL. The enzyme activity determination method of protease PaproA refers to GB/T 23527-2009: 1mL enzyme solution and 1mL casein solution are incubated at 40℃ for 10min, then 2mL trichloroacetic acid is added to terminate the reaction, centrifuged at 10000rpm for 10min, 1mL supernatant is added with 5mL sodium carbonate solution and 1mL Folin phenol reagent, incubated at 40℃ for 20min to develop color, and the absorbance is measured at 660nm. The enzyme solution to which trichloroacetic acid is added to terminate the reaction is used as a control. The amount of enzyme required to hydrolyze casein to produce 1μg tyrosine per minute is defined as 1 enzyme activity unit (U).

In the present invention, the duck hemoglobin described in S1) can be a duck hemoglobin solution, and the duck hemoglobin solution can be prepared by mixing duck hemoglobin powder and acidic electrolyzed water to swell and break the wall;

In one embodiment of the present invention, the duck blood globulin solution is prepared by adding 5 g of duck blood cell powder to 100 ml of acidic electrolyzed water with a pH value of 3.0, and swelling and breaking the cell wall for 2 hours to obtain duck blood globulin liquid; in another embodiment of the present invention, the duck blood globulin solution is prepared by adding 1 kg of duck blood cell powder to 20 L of acidic electrolyzed water with a pH value of 3.0, and swelling and breaking the cell wall for 1 hour to obtain duck blood globulin liquid.

In the present invention, duck blood cell powder was purchased from Handan Xinheng Biotechnology Co., Ltd.

In the present invention, acidic electrolyzed water is prepared using a laboratory-made electrolytic cell (Figure 3), the electrode is made of platinum-plated titanium alloy, NaCl or KCl is dissolved in distilled water with a mass concentration of 0.45% (w/w), and then poured into the electrolytic cell, and electrolyzed for 5 minutes under the conditions of voltage 60V, current 0.5-1A, and electrode spacing of 5-10cm, and the anode electrolytic cell obtains acidic electrolyzed water.

In one embodiment of the present invention, the total amount of protease A added in S1) and protease B added in S2) is 2000U/g duck blood powder. Further, the amount of protease A added in S1) is 1000U/g duck blood powder, and the amount of protease B added in S2) is 1000U/g duck blood powder.

Furthermore, in the above method, the method also includes S3), wherein S3) is centrifuging the enzymatic hydrolyzate M2 and collecting the supernatant M3 to obtain a duck blood protein peptide solution.

In the present invention, the centrifugation condition in S3) is 8820×g for 10 min.

Furthermore, in the above method, the method also includes the step of freeze-drying the supernatant M3 to obtain duck blood protein peptide.

Furthermore, in the above method, the method also includes the step of separating the duck blood protein peptide from the obtained duck blood protein peptide to obtain a high-activity duck blood protein peptide component, and the steps include B1) and B2):

B1), dissolving the duck blood protein peptide powder with hydrochloric acid solution, centrifuging, and taking the supernatant,

B2) The supernatant obtained in B1) is subjected to gel chromatography, and the effluent with an elution time of 173-180 min is taken to obtain a highly active duck blood protein peptide component F5.

In the present invention, the concentration of the hydrochloric acid solution (solvent is water, solute is HCL) in B1) is 10 mM; the centrifugation condition is 8820×g for 10 min;

The composition of the hydrochloric acid solution is: hydrochloric acid with a content of 36%: water = 1:1090.

In the present invention, the chromatographic conditions of the gel chromatography are: AKTApurifier UPC-900 fast protein liquid chromatograph, the chromatographic column size is 1000×10 mm, the column material is Sephadex G-15, the mobile phase is 10 mM hydrochloric acid solution, the flow rate is 0.8 mL/min, and the detection is UV280 nm.

Furthermore, the highly active duck blood protein peptide component F5 includes a peptide segment having an amino acid sequence shown as SEQ ID No. 1-53.

In order to solve the above technical problems, in a second aspect, the present invention provides duck blood protein peptide prepared by the above method.

To solve the above technical problems, in a third aspect, the present invention provides a polypeptide, wherein the polypeptide is selected from one or N of P1)-P5), N≤5:

P1), a polypeptide having an amino acid sequence of SEQ ID No. 1;

P2), a polypeptide having an amino acid sequence of SEQ ID No. 2;

P3), a polypeptide having an amino acid sequence of SEQ ID No.3;

P4), a polypeptide having an amino acid sequence of SEQ ID No.4;

P5), a polypeptide having an amino acid sequence of SEQ ID No.5.

In the present invention, the polypeptide described in P1), P2) or P3) is derived from duck hemoglobin β subunit, and the Uniprot number of the duck hemoglobin β subunit is P01988; the polypeptide described in P4) or P5) is derived from duck hemoglobin α subunit, and the Uniprot number of the duck hemoglobin α subunit is P02115.

In order to solve the above technical problems, in a fourth aspect, the present invention provides the application of the above duck blood protein peptide and/or the above polypeptide, wherein the application is any one of A1)-A4):

A1) Use in the preparation of products for the treatment and/or prevention of gout;

A2) Application in the preparation of products for reducing uric acid;

A3) Application in inhibiting xanthine oxidase activity;

A4), application in the preparation of xanthine oxidase inhibitors.

To solve the above technical problems, in a fifth aspect, the present invention provides a product for lowering uric acid and/or a product for preventing and/or treating hyperuricemia, wherein the product contains the above-mentioned duck hemoglobin peptide and/or the above-mentioned polypeptide.

In the present invention, the uric acid lowering is manifested as inhibiting or reducing the activity of xanthine oxidase (XOD).

In the present invention, xanthine oxidase (XOD) was purchased from Sigma Company with product number X4376.

In the present invention, the product can be food, health food or medicine.

Furthermore, in the above-mentioned health food or medicine, the duck blood protein peptide can be artificially synthesized or obtained by hydrolysis of duck blood protein.

In the present invention, a carrier material may be added when preparing the above health food or medicine.

The carrier material includes, but is not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare the unit dosage form into a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare the unit dosage form into an injectable preparation, such as a solution, emulsion, freeze-dried powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare isotonic injections, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparations, and conventional cosolvents, buffers, pH adjusters, etc. may also be added. In addition, colorants, preservatives, spices, flavoring agents, sweeteners or other materials may also be added to the pharmaceutical preparations as needed.

The above-mentioned dosage forms can be used for oral administration.

Compared with the prior art, the present invention has at least the following advantages:

1. The present invention uses acidic electrolyzed water to swell and break the wall of hemoglobin for the first time, providing an environmentally friendly, simple, efficient and suitable pretreatment method for large-scale production for preparing hemoglobin peptides by enzymatic hydrolysis;

2. The present invention uses bromelain and one or both of the protease PaproA from Pseudomonas aeruginosa or the protease GsProS8 from Bacillus stearothermophilus to hydrolyze duck blood cell powder in steps, and enriches peptides with XOD inhibitory activity by specific enzyme cleavage. The prepared duck blood globulin peptide has high uric acid lowering activity;

3. The method for preparing uric acid-lowering duck hemoglobin peptide by double-enzyme step-by-step hydrolysis of duck hemoglobin powder provided by the present invention has a product yield greater than 62%, and the fraction with a molecular weight less than 5000Da is greater than 95%;

4. The method for preparing uric acid-lowering duck hemoglobulin peptide by double-enzyme stepwise hydrolysis of duck hemoglobulin powder provided by the present invention, wherein the prepared duck hemoglobulin peptide powder has an in vitro XOD half-inhibitory concentration of less than 0.795 mg/mL and has good gastrointestinal digestion stability;

5. The method for preparing uric acid-lowering duck hemoglobin peptides by step-by-step hydrolysis of duck hemoglobin powder provided by the present invention, the highly active peptide segments IVYPW, YPWTQ and LITGLW separated and identified had in vitro XOD _IC50 values of 0.424 mg/mL, 0.675 mg/mL and 0.743 mg/mL.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the gene, coding sequence and corresponding amino acid sequence of Bacillus stearothermophilus protease GsProS8.

Figure 2 is the SDS-PAGE and zymogram of the fermentation broth of Pseudomonas aeruginosa protease PaproA; M is a low molecular weight standard protein; 1 is a crude enzyme solution; 2 is a protease zymogram, in which 4 isozyme bands with protease activity appear. There are 2 main bands, indicating that the activity is relatively high, and the corresponding protein molecular weights are 32kD and 50kD.

Figure 3 is a diagram of the structure of a laboratory-made electrolytic cell.

Figure 4 shows the enzymatic cleavage frequency of the carboxyl and amino termini of duck blood protein hydrolyzed by proteases PaproA and GsProS8.

FIG. 5 is a graph showing gel exclusion chromatography analysis of duck hemoglobin peptide.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

The Pseudomonas aeruginosa CAU342A in the following examples was screened, identified and preserved by our laboratory from soy sauce koji, and is disclosed in the document "Sun Qian et al., Optimization of fermentation conditions for protease production by Pseudomonas aeruginosa, Microbiology Bulletin, 2017, 44(1): 86-95." The public can obtain the above strain from the applicant, and the obtained strain can only be used for experiments to verify the present invention and cannot be used for other purposes;

The Bacillus subtilis WB600 expressing the recombinant protease GsProS8 in the following examples is preserved by this laboratory and is disclosed in the document "Chang et al. High level expression and biochemical characterization of an alkaline serine protease from Geobacillus stearothermophilus to prepare antihypertensive whey protein hydrolysate. BMC Biotechnology, 2021, 21: 21". The public can obtain the above strain from the applicant, and the obtained strain can only be used for experiments to verify the present invention and cannot be used for other purposes;

Bromelain is a product of Shanghai Yuanye Biotechnology Co., Ltd., and its product number is: S10009;

Duck blood cell powder was purchased from Handan Xinheng Biotechnology Co., Ltd.;

Xanthine oxidase (XOD) was purchased from Sigma, product number X4376;

Acidic electrolyzed water was prepared using a laboratory-made electrolytic cell (Figure 3). The electrode was made of platinum-plated titanium alloy. NaCl or KCl was dissolved in distilled water with a mass concentration of 0.45% (w/w). Then it was poured into the electrolytic cell and electrolyzed for 5 minutes at a voltage of 60V, a current of 0.5-1A, and an electrode spacing of 5-10cm. The anode electrolytic cell obtained acidic electrolyzed water.

The following examples were processed using SPSS 11.5 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. * indicates a significant difference (P < 0.05), ** indicates a very significant difference (P < 0.01), and *** indicates a very significant difference (P < 0.001).

Example 1: Hydrolysis of duck hemoglobin by single protease GsProS8 or PaproA

Use acidic electrolyzed water with pH 2.5 to prepare a duck blood globulin solution with a substrate concentration of 5% (w/w), stir and swell and break the wall for 2 hours to obtain a duck blood globulin solution, and use 1M NaOH solution or HCl solution to adjust the pH of each substrate solution to 8.0. Add Pseudomonas aeruginosa protease PaproA and Bacillus stearothermophilus protease GsProS8 single enzymes for hydrolysis, and the enzyme addition amount is 2000U/g duck blood globulin powder. After hydrolysis for 10 hours, inactivate in a boiling water bath for 10 minutes, centrifuge to obtain two duck blood globulin hydrolyzates, freeze-dry the hydrolyzates, obtain freeze-dried products, and weigh to calculate the product yield. The product yield calculation formula is as follows:

Product yield (%) = (mass of supernatant freeze-dried powder/mass of duck blood cell powder) × 100%;

The lyophilized product was dissolved in distilled water to 1 mg/mL to determine the XOD inhibition rate. The product yield and the XOD inhibition rate of the hydrolyzate are shown in Table 1. The peptides in the two duck hemoglobin hydrolysates were identified by LC-MS/MS, and the enzymatic cleavage frequencies of the two proteases hydrolyzing the carboxyl and amino ends of duck hemoglobin were counted, as shown in Figure 4. The XOD inhibitory activity determination was slightly modified according to the method of Zhong et al. (Zhong et al. Food Chemistry, 2021, 347: 129068), and the specific method is as follows:

Add 150 μL of xanthine oxidase (XOD) solution (0.05 U/mL) to 50 μL of hydrolyzate sample, keep at 37°C for 5 minutes, and then add 150 μL of xanthine solution to start the reaction. After 60 minutes, add 100 μL of hydrochloric acid solution (1M) to end the reaction, and measure the absorbance at 290 nm. Buffer solution is used as a negative control, and allopurinol is used as a positive control. The XOD inhibition rate is calculated as follows:

Where _A1 is sample absorbance; _A2 is sample blank; _A3 is negative control; _A4 is positive control.

Table 1. Hydrolysis results of duck hemoglobin by protease GsProS8 and PaproA

^* XOD inhibitory activity of hydrolysate was measured at 1 mg/mL protein concentration.

The results are shown in Table 1. After the protease GsProS8 and PaproA hydrolyzed duck hemoglobin, the XOD inhibition rates of the hydrolyzates at a concentration of 1 mg/mL reached 57.5% and 64.7%, respectively, showing high XOD inhibition activity, but the yields of the duck hemoglobin hydrolyzed products were low, 16.4% and 28%, respectively. The cleavage sites of the two proteases on duck hemoglobin are mainly concentrated in hydrophobic amino acids (methionine and leucine) and aromatic amino acids (phenylalanine and tyrosine), which is conducive to the release of XOD inhibitory peptide fragments.

The amino acid sequence of protease GsProS8 is shown in Figure 1. Protease GsProS8 is a protease obtained by introducing the coding gene of protease GsProS8 into Bacillus subtilis to obtain a recombinant bacterium and fermenting it at high density for 108 hours. The nucleotide sequence of the coding gene of protease GsProS8 is shown in Figure 1. Protease GsProS8 is prepared according to the following steps:

The recombinant protease GsProS8 Bacillus subtilis WB600 was introduced into LB culture medium at an inoculation rate of 1% (V/V) and cultured at 37°C for 12 hours as fermentation seed liquid. The liquid volume in the fermenter was 1.5L and inoculated at an inoculation rate of 5% (V/V). The fermentation process was controlled at 37°C, the pH was adjusted to 4.0 using ammonia water and phosphoric acid, and the dissolved oxygen was maintained at about 30%. When the residual sugar content in the culture medium was <1g/L, the feed culture medium was started. After 108 hours of high-density fermentation, the fermentation liquid was collected, and the centrifuged supernatant was the protease GsProS8. The composition of the fermentation medium is: 2g/L ammonium sulfate, 3g/L yeast extract, 1g/L glucose, 1g/L sodium chloride, 0.5g/L magnesium sulfate, 0.05g/L zinc chloride, 0.05g/L manganese chloride, 0.1g/L enzyme, 2g/L calcium chloride, and the rest is water; the composition of the feed medium is: 500g/L glucose, 37.5g/L peptone, and the rest is water. The protease activity of the enzyme solution of the protease GsProS8 was determined according to the following method, and the results showed that the protease activity of the enzyme solution of the protease PaproA was 3800U/mL. The method for determining the enzyme activity of protease GsProS8 refers to GB/T 23527-2009, and the specific steps are as follows: 1 mL of protease GsProS8 enzyme solution and 1 mL of casein solution (the solvent is NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer solution with a pH of 7.5, and the solute is casein) are kept at 40°C for 10 minutes, and then 2 mL of trichloroacetic acid is added to terminate the reaction, and the reaction is centrifuged at 10000 rpm for 10 minutes. 1 mL of supernatant is added with 5 mL of sodium carbonate solution and 1 mL of Folin phenol reagent, and the reaction is kept at 40°C for 20 minutes for color development, and the absorbance is measured at 660 nm. The enzyme solution in which trichloroacetic acid is first added to terminate the reaction is used as a control. The amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute at 40°C and pH 7.5 is defined as 1 enzyme activity unit (U).

The SDS-PAGE and zymogram of the protease PaproA are shown in Figure 2. Four isozyme bands with protease activity appear in the zymogram. Among them, there are two main bands, indicating that its activity is relatively high, and the molecular weight of the corresponding protein is 32kD and 50kD. The protease PaproA is prepared according to a method comprising the following steps: Pseudomonas aeruginosa CAU342A is cultured in a fermentation medium at 30°C for 3 days, the fermentation broth is collected, the fermentation broth is centrifuged, and the supernatant is collected, which is the enzyme solution of the protease PaproA. Among them, the fermentation medium is made of the following raw materials: 3% lees, 1.5% yeast extract powder, 0.05% Tween-80, 0.5% sodium chloride, 0.7% potassium phosphate, 0.3% dipotassium hydrogen phosphate, 0.04% manganese sulfate and water, and all percentages are mass percentages. The pH of the fermentation medium is 7.5. The enzyme activity of the enzyme solution of the protease PaproA was determined according to the following method. The results showed that the enzyme activity of the enzyme solution of the protease PaproA was 2653U/mL. The enzyme activity determination method of the protease PaproA refers to GB/T 23527-2009: 1mL of the enzyme solution of the protease PaproA and 1mL of the casein solution (the solvent is a NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer solution with a pH of 7.5, and the solute is casein) are incubated at 40°C for 10 minutes, and then 2mL of trichloroacetic acid is added to terminate the reaction. The reaction is centrifuged at 10000rpm for 10 minutes, and 1mL of the supernatant is added with 5mL of sodium carbonate solution and 1mL of Folin phenol reagent. The color is developed by incubating at 40°C for 20 minutes, and the absorbance is measured at 660nm. The enzyme solution in which the reaction is terminated by adding trichloroacetic acid first is used as a control. The amount of enzyme required to hydrolyze casein to produce 1μg of tyrosine per minute at 40°C and pH 7.5 is defined as 1 enzyme activity unit (U).

Example 2: Optimization of hydrolysis conditions of highly active uric acid-lowering duck blood peptide

Prepare a duck blood cell powder suspension with a substrate concentration of 5% (liquid obtained by adding 5g duck blood cell powder to 100ml acidic electrolyzed water with pH 3.0), and swell and break the wall for 2h to obtain duck blood globulin liquid. Use 1M sodium hydroxide or hydrochloric acid solution to adjust the system pH to the optimal condition, use acid protease, bromelain, Pseudomonas aeruginosa source protease PaproA and thermophilic Bacillus stearothermophilus protease GsProS8, hydrolyze duck blood cell powder in a single enzyme or two enzyme step-by-step hydrolysis mode, the total enzyme addition amount is 2000U, and the specific hydrolysis method is shown in Table 2. The enzyme activity determination method of protease PaproA refers to GB/T 23527-2009: 1mL enzyme solution and 1mL casein solution are kept at 40℃ for 10min, then 2mL trichloroacetic acid is added to terminate the reaction, centrifuged at 10000rpm for 10min, 1mL supernatant is added with 5mL sodium carbonate solution and 1mL Folin phenol reagent, kept at 40℃ for 20min to develop color, and the absorbance is measured at 660nm. The enzyme solution to which trichloroacetic acid is added to terminate the reaction is used as a control. The amount of enzyme required to hydrolyze casein to produce 1μg tyrosine per minute is defined as 1 enzyme activity unit (U). The amount of enzyme added for single enzyme hydrolysis is 2000U/g duck blood cell powder, and the hydrolysis is carried out for 10h under the respective optimal conditions. In the first step of the double enzyme compound hydrolysis, the amount of protease A added was 1000U/g duck blood powder, and the hydrolysis was carried out under its optimal conditions for 6h; in the second step, the pH of the system was adjusted, and the amount of protease B added was 1000U/g duck blood powder, and the hydrolysis was continued for 4h under its optimal conditions. After the hydrolysis was completed, the duck blood globulin hydrolyzate was inactivated in a boiling water bath for 10min, centrifuged (8820×g centrifugation for 10min) to obtain the duck blood globulin hydrolyzate, and the supernatant was freeze-dried and the XOD inhibitory activity was determined. The product yield determination method refers to Example 1. The hydrolysis conditions and the determination results are shown in Table 2. XOD inhibitory activity refers to Example 1, and 5 concentrations are selected according to the XOD inhibitory activity of the hydrolyzate, and the XOD inhibition rate of each concentration is determined respectively (Table 3). According to the concentration and XOD inhibition rate, the IC ₅₀ value of each hydrolyzate is calculated using SPSS software nonlinear regression.

Table 2. Hydrolysis conditions and XOD inhibitory activity of each hydrolyzate

Table 3. XOD inhibitory activity of hydrolysates at different concentrations

As an implementation method of this embodiment, after bromelain is used in combination with proteases GsProS8 and PaproA, the product yield of duck blood cell powder is significantly improved, reaching more than 54%, and the XOD inhibitory activity of the hydrolyzate is improved; the XOD half-inhibition concentration of duck blood globulin peptides prepared by hydrolyzing duck blood cell powder with bromelain, PaproA and GsProS8 respectively reaches 0.795 mg/mL and 0.744 mg/mL. Although the acid protease single enzyme combined with protease PaproA or GsProS8 to hydrolyze duck blood cell powder significantly improves the protein recovery rate, the XOD inhibitory activity of the hydrolyzate is greatly reduced.

Example 3: Isolation and identification of highly active peptides

A G15 gel column (1000×10 mm) was equilibrated with a 10 mM hydrochloric acid solution, and the duck hemoglobin peptide (bromelain compound protease GsProS8 hydrolysis) obtained in Example 2 was dissolved with a 10 mM hydrochloric acid solution (36% concentrated hydrochloric acid: pure water = 1:1090), centrifuged at 8820×g for 10 min, and the supernatant was taken for gel chromatography. The gel chromatography separation method was adopted, and the chromatographic conditions were: AKTApurifier UPC-900 fast protein liquid chromatograph; mobile phase: 10 mM hydrochloric acid solution; flow rate: 0.8 mL/min; detection: UV280 nm. As shown in FIG5 , 5 components were separated, among which the F5 component had the highest XOD inhibitory activity. According to nanoLC-MS/MS analysis, the peptide sequence identified in the F5 component is shown in Table 4. Five of the peptides were artificially synthesized and verified, and the XOD inhibitory activity was measured, and the results are shown in Table 5. The XOD inhibitory activity determination method refers to Example 2.

Table 4. Peptides identified in fraction F5

^* The parent protein number in the UniProt database

Table 5. XOD inhibitory activity of synthetic peptides

As an implementation method of this embodiment, duck hemoglobin peptides were separated to obtain five components, among which the F5 component had the highest XOD inhibitory activity, and its in vitro XOD IC ₅₀ value was 0.489 mg/mL. A total of 53 peptides were identified in the F5 component, mainly derived from duck hemoglobin α-chain (P01988) and β-chain (P02115), and most of the peptide sequences contained aromatic amino acids. Synthesis verified that 5 of the peptides may have XOD inhibitory activity, among which IVYPW, YPWTQ and LITGLW had higher XOD inhibitory activity, with IC ₅₀ values of 0.424 mg/mL, 0.675 mg/mL and 0.743 mg/mL, respectively.

Example 4: Preparation of kilogram-scale duck hemoglobin peptide with uric acid-lowering activity and determination of its molecular weight

Add 20L of acidic electrolyzed water with pH 3.0 to the enzymolysis tank, start the stirring paddle, slowly add 1kg of duck blood globulin powder, swell and break the wall for 1h to obtain duck hemoglobin solution. Use a peristaltic pump to add 1M sodium hydroxide solution to the enzymolysis tank to adjust the pH value to 7.5, and heat the heater to 45-55℃ for insulation. Add 3.5g bromelain (1×10 ⁶ U) to the duck hemoglobin solution, and after hydrolysis for 4h, adjust the system pH to 8.0. Add 380mL of Pseudomonas aeruginosa-derived protease enzyme solution (1×10 ⁶ U) and continue hydrolysis for 6h. After two-step hydrolysis, inactivate in a boiling water bath and cool to room temperature to obtain an enzymolysis solution. The obtained enzymolysis solution is plate-and-frame filtered to obtain a peptide solution, and concentrated by rotary evaporation to obtain a concentrated peptide solution. The obtained concentrated peptide solution is spray-dried to obtain duck hemoglobin peptide powder (DHH_BP) prepared by composite hydrolysis of bromelain and Pseudomonas aeruginosa-derived protease.

Add 20L of acidic electrolyzed water with pH 3.0 to the enzymolysis tank, start the stirring paddle, slowly add 1kg of duck blood ball powder, adjust the pH value of the system to 3.0, and swell and break the wall for 1h to obtain duck hemoglobin solution. Use a peristaltic pump to add 1M sodium hydroxide solution to the enzymolysis tank to adjust the pH value to 7.5, and heat the heater to 45-55℃ for insulation. Add 3.5g bromelain (1×10 ⁶ U) to the duck hemoglobin suspension, and after hydrolysis for 4h, adjust the pH value of the system to 8.5. Add 260mL of thermophilic Bacillus stearothermophilus protease enzyme solution (1×10 ⁶ U) and continue hydrolysis for 6h. After two-step hydrolysis, inactivate in boiling water bath and cool to room temperature to obtain enzymolysis solution. The obtained enzymolysis solution is plate-frame filtered to obtain a peptide solution, and concentrated by rotary evaporation to obtain a concentrated peptide solution. The obtained concentrated peptide solution is spray-dried to obtain duck hemoglobin peptide powder (DHH_BG) prepared by composite hydrolysis of bromelain and thermophilic Bacillus stearothermophilus protease.

Weigh the peptide powder obtained by spray drying and calculate the product yield as follows:

Product yield (%) = (mass of peptide powder obtained by drying/mass of duck blood cell powder) × 100%

The molecular weight of the uric acid-lowering duck hemoglobulin peptide obtained above was determined by HPLC method, and the chromatographic conditions were: Agilent high performance liquid chromatograph 1260; chromatographic column: TSKgel-G2000SWXL column (7.8×300mm); mobile phase: acetonitrile/pure water/trifluoroacetic acid: 45/50/0.1 (v/v/v); detection: UV214nm; flow rate: 0.5mL/min; column temperature: 30°C. The results are shown in Table 6.

Table 6. Molecular weight distribution of two duck hemoglobin peptides with uric acid lowering activity

As an implementation method of this example, the hydrolysis of 1 kg of duck hemoglobulin obtained a total of 672 g DHH_BG and 620 g DHH_BP, with product yields of 67.2% and 62% respectively, and components with a molecular weight less than 5000 Da accounted for 96.8% and 96.5% respectively.

Example 5: Gastrointestinal digestion stability test of duck hemoglobin peptide

The duck hemoglobulin peptide prepared in Example 4 was subjected to simulated gastrointestinal digestion in vitro, with reference to the method of Tavares et al. with slight modifications. The specific method is as follows:

The duck blood globulin peptide was dissolved in deionized water, the pH value of the peptide solution was adjusted to 2.0 with a dilute hydrochloric acid solution, pepsin (E: S 2.5%, w/w) was added, and the solution was placed in a constant temperature shaker at 37°C for hydrolysis for 90 min, and then the pH value of the peptide solution was adjusted to 7.5 with a 0.1M sodium hydroxide solution, trypsin (E: S 2.5%, w/w) was added, and the solution was placed in a constant temperature shaker at 37°C for hydrolysis for 2 h. After the reaction was completed, the enzyme was incubated in boiling water for 10 min to inactivate the enzyme, cooled and centrifuged at 10000 r/min for 10 min, the supernatant was freeze-dried, and the XOD inhibitory activity of the duck blood globulin peptide after simulated digestion was determined, and the specific determination method was as described in Example 2.

As a preferred embodiment of this embodiment, the XOD IC ₅₀ values of the two duck hemoglobulin peptides DHH_BG and DHH_BP described in Example 4 increased from 0.744 mg/mL and 0.795 mg/mL to 0.959 mg/mL and 0.892 mg/mL after simulated gastrointestinal digestion in vitro, and had good gastrointestinal digestion stability.

The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Sequence Listing

<110> China Agricultural University

<120> A duck hemoglobin peptide with uric acid lowering activity and its preparation method

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<141> 2022-05-31

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Leu Leu Ile Val Tyr Pro Trp

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Ala Trp Gln Lys Leu Val Arg

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Ile Val Tyr Pro Trp Thr Gln Arg Phe Phe

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Ala Ser Leu Asp Lys Phe

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<213> Mallard duck nominate subspecies (Anas platyrhynchos platyrhynchos )

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Met Arg Gly Lys Lys Val Trp Ile Ser Leu Leu Phe Ala Leu Ala Leu

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Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Pro Ala Gln Ala Ala Gly

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Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gly Phe Lys Gln Thr Met

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Ser Thr Met Ser Ala Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly

50 55 60

Gly Lys Val Gln Lys Gln Phe Lys Tyr Val Asp Ala Ala Ser Ala Thr

65 70 75 80

Leu Asn Glu Lys Ala Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala

85 90 95

Tyr Val Glu Glu Asp His Val Ala Gln Ala Tyr Ala Gln Ser Val Pro

100 105 110

Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Phe

115 120 125

Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser

130 135 140

Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala Ser Met Val Pro Ser

145 150 155 160

Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His Gly Thr His Val Ala

165 170 175

Gly Thr Val Ala Ala Leu Asn Asn Ser Val Gly Val Leu Gly Val Ala

180 185 190

Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser

195 200 205

Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Tyr

210 215 220

Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala

225 230 235 240

Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Ile Val Val

245 250 255

Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Ser Ser Ser Thr Val

260 265 270

Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala Val Gly Ala Val Asn

275 280 285

Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val Gly Ser Glu Leu Asp

290 295 300

Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr Leu Pro Gly Asn Lys

305 310 315 320

Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly

325 330 335

Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln

340 345 350

Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys Leu Gly Asp Ala Phe

355 360 365

Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln

370 375 380

Claims (7)

1. A method for preparing duck blood protein peptide, characterized in that: the duck blood protein peptide has the following five peptide segments: P1), a polypeptide having an amino acid sequence of SEQ ID No. 1; P2), a polypeptide having an amino acid sequence of SEQ ID No. 2; P3), a polypeptide having an amino acid sequence of SEQ ID No.3; P4), a polypeptide having an amino acid sequence of SEQ ID No.4; P5), a polypeptide having an amino acid sequence of SEQ ID No.5; The method comprises the steps of enzymolyzing duck hemoglobin to obtain an enzymolyzate, and extracting duck blood protein peptides from the enzymolyzate. The enzymolyzing duck hemoglobin comprises the following steps: S1), adding protease A to duck hemoglobin to obtain enzymatic solution M1; S2), adding protease B to the enzymatic hydrolysate M1 to obtain enzymatic hydrolysate M2; S3), centrifuging the enzymatic hydrolyzate M2 and collecting the supernatant M3; S4), freeze-drying the supernatant M3 to obtain duck blood protein peptides with xanthine oxidase inhibitory activity, The protease A is bromelain, The protease B is protease GsProS8, and the amino acid sequence of the protease GsProS8 is SEQ ID No.54. 2. The method according to claim 1, characterized in that: the method further comprises the step of separating the duck blood protein peptide with high activity from the duck blood protein peptide having the activity of inhibiting xanthine oxidase, and the steps include B1) and B2): B1), dissolving the duck blood protein peptide powder with hydrochloric acid solution, centrifuging and taking the supernatant; B2), the supernatant obtained in B1) is subjected to gel chromatography, and the effluent with an elution time of 173-180 min is taken to obtain a duck blood protein peptide named as a highly active duck blood protein peptide component F5, The chromatographic conditions of the gel chromatography are: AKTApurifier UPC-900 fast protein liquid chromatograph, chromatographic column size: 1000×10 mm, column material: Sephadex G-15, mobile phase: 10 mM hydrochloric acid solution, flow rate: 0.8 mL/min, detection: UV280 nm. 3. The method according to claim 2, characterized in that the highly active duck blood protein peptide component F5 has an amino acid sequence of a peptide segment of SEQ ID No. 1-53. 4. The duck blood protein peptide prepared by the method according to any one of claims 1 to 3, wherein the duck blood protein peptide has the following five peptide segments: P1), a polypeptide having an amino acid sequence of SEQ ID No. 1; P2), a polypeptide having an amino acid sequence of SEQ ID No. 2; P3), a polypeptide having an amino acid sequence of SEQ ID No.3; P4), a polypeptide having an amino acid sequence of SEQ ID No.4; P5), a polypeptide having an amino acid sequence of SEQ ID No.5. 5. A polypeptide, characterized in that: the polypeptide is a polypeptide with an amino acid sequence of SEQ ID No.3 or a polypeptide with an amino acid sequence of SEQ ID No.5. 6. The composition is characterized in that the composition is any one of the following: B1) is composed of a polypeptide having an amino acid sequence of SEQ ID No. 3 and any one, two, three or four of the polypeptides shown in SEQ ID NOs. 1-2 and 4-5; B2) It is composed of a polypeptide having an amino acid sequence of SEQ ID No. 5 and any one, two or three of the polypeptides shown in SEQ ID NOs. 1-2 and 4. 7. Use of the duck blood protein peptide according to claim 4, the polypeptide according to claim 5 and/or the composition according to claim 6 in any of the following: A1) Application in inhibiting xanthine oxidase activity, wherein the application is non-disease diagnosis and treatment; A2) Application in the preparation of xanthine oxidase inhibitors.