CN117186177A - Duck blood cell protein peptide with uric acid reducing activity and preparation method thereof - Google Patents
Duck blood cell protein peptide with uric acid reducing activity and preparation method thereof Download PDFInfo
- Publication number
- CN117186177A CN117186177A CN202210607233.2A CN202210607233A CN117186177A CN 117186177 A CN117186177 A CN 117186177A CN 202210607233 A CN202210607233 A CN 202210607233A CN 117186177 A CN117186177 A CN 117186177A
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- China
- Prior art keywords
- duck
- protease
- duck blood
- polypeptide
- protein peptide
- Prior art date
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a duck blood cell protein peptide with uric acid reducing activity and a preparation method thereof, belonging to the technical field of food biology. The invention provides a method for preparing duck blood protein peptide, which comprises the steps of S1) adding protease A into duck hemoglobin to obtain enzymolysis solution M1, wherein the duck blood protein peptide has 5 peptide fragments with amino acid sequences of SEQ ID No. 1-5; s2), adding protease B into the enzymolysis liquid M1 to obtain enzymolysis liquid M2; the protease A is protease derived from pineapple, and the protease B is one or two of protease derived from bacillus stearothermophilus and protease derived from pseudomonas aeruginosa. The invention provides a new choice for deep processing of poultry blood in China, enriches the variety of peptide products and has great economic and social benefits.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to a duck blood cell protein peptide with uric acid reducing activity and a preparation method thereof.
Background
Blood is a byproduct in the meat processing process, and blood resources in China are very rich. Taking ducks as an example, the number of ducks in the stock of China in 2019 is about 24.61 hundred million, and the number of ducks in the stock of China is increased year by year. About 4.42 ten thousand tons of duck blood proteins are produced annually, calculated as about 18% protein content in blood. Pathogenic bacteria or toxic metabolites may be carried in blood, and the blood is easily polluted by pathogenic microorganisms, so that the blood is inconvenient to collect and store, and only a small amount of blood is processed and utilized in the form of blood powder, blood bean curd or feed (Diab et al membranes,2020,10,257). In order to reasonably utilize blood protein resources, the deep processing and utilization of blood are of great significance. Hemoglobin is the major protein component of blood, and is present in red blood cells in an amount of 80% of the total protein content of blood. The blood cell powder is generally processed and produced by adopting a direct spray drying mode, and the cell membrane of the blood cell is limited to be broken, so that the digestion and absorption rate is not high, and the application of the blood cell powder in the food and feed industry is limited. Hemoglobin is composed of heme and globin, is reddish brown, and has a stimulating blood fishy smell. Hemoglobin is released during deep processing of the blood cells, and ferrous iron in the hemoglobin can be rapidly oxidized to ferric iron, affecting the appearance of the product (Alvarez et al LWT-Food Science and Technology,2016, 73:280-289). Because of the characteristics of duck slaughtering process and the property of easy coagulation and caking of duck blood, duck blood is more difficult to collect than pig blood, cow blood and the like. Therefore, the utilization rate of duck blood in China is low. There are no reports of duck blood proteolysis and comprehensive utilization at home and abroad, and few domestic reports are mainly focused on the aspect of preparing bioactive peptides by hydrolyzing duck blood. The acid protease, papain, alkaline protease and flavourzyme are selected by taking the degree of hydrolysis, the degree of decoloration and the yield of the product as indexes, and as a result, the acid protease can obviously degrade duck blood cell proteins, the product is milky white, the yield of duck blood cell peptides is 60.1%, and the hydrolysate has antioxidant activity (Zheng Zhaojun and the like, animal nutrition journal, 2016, 28:2521-2533). The iron ion chelating ability of duck blood cell hydrolysate is used as index, and the hydrolysis condition of alkaline protease to hydrolyze duck blood cell protein is optimized, and under the optimal condition, the iron ion chelating rate of duck blood cell hydrolysate is 65.4%, and can be used as iron supplementing agent (Yang Yan, e.g. food industry technology, 2020, 41:19).
The bioactive peptide prepared by the biological enzyme hydrolysis method is mostly derived from dietary protein, has high safety, no side effect and low induced drug resistance compared with the traditional medicine. Many bioactive peptides have been FDA approved for therapeutic use, with approximately 100 polypeptide drugs on the U.S., european and japanese markets (barbarara et al biotechnology Advances,2018, 36:415-429). Suitable proteases hydrolyze hemoglobin, not only to decolorize, but also to obtain small molecule peptides with physiological functional activity (Alvarez et al LWT-Food Science and Technology,2016, 73:280-289). The biological enzyme technology is utilized to hydrolyze duck blood cell proteins, so that proteins in blood cell are fully released, the protein utilization rate can be improved, and the small molecular peptide has a plurality of physiological regulation functions and can be used as ingredients of functional foods or health care products. The pig blood cell peptide can obviously improve average daily gain of piglets, reduce diarrhea rate and enhance immunity of the piglets after feeding the weaned piglets (Wu Yanjun, university of Yangzhou, 2010). The goose blood peptide prepared by the neutral protease, alkaline protease and papain through complex hydrolysis is fed to mice, so that the immunity of the mice is remarkably improved, and the proliferation capacity of spleen lymphocytes and the secretion amount of cytokines of the mice are remarkably improved (Wang Zheng, university of vinca industry report, 2020). To promote the application of blood peptides in the food industry, the Chinese animal husbandry society published the body standard of camel blood Polypeptides (T/CAAA 018-2019) on day 1 and 7 of 2019. The standard prescribes the related index of camel blood polypeptide prepared from camel blood, and has certain reference value for blood peptide prepared from blood of other animal sources.
Hemoglobin is present in blood cells and the blood cells need to be destroyed to release hemoglobin prior to hydrolysis. The extraction of hemoglobin mainly adopts an acid solution method or an acidic acetone method, when the pH value is lower than 3.5, ferrous ions and histidine coordination bonds are dissociated, so that heme molecules are broken, and thus hemoglobin is released, but other organic matters can be brought into the methods in the methods, a large amount of alkali liquor is needed for adjusting the pH during hydrolysis, the salt is brought in, and the refining process and cost of later desalination and impurity removal are increased (Zhong Yaoan, food science, 2004,4:66-71;Wedzicha et al, food Chemistry,1985, 17:199-207). Mechanical methods of disruption of cell walls, such as ultrasonic wall breaking, freeze thawing, homogenization, etc., have also been studied, but these methods require specialized equipment, increase production process steps and production costs (Wu Wenjin, etc., food research and development, 2021, 42:90-96). Therefore, there is a need to develop a method capable of conveniently and efficiently treating blood cells and releasing hemoglobin. The electrolyzed water is a solution obtained by electrolyzing a low-concentration salt solution in an electrolytic tank with a cation exchange membrane, the anode generates acidic electrolyzed water with strong oxidizing property and low point position, and the cathode generates alkaline electrolyzed water with strong reducing property and high potential. Electrolytic water has been used in many applications such as sterilization and bacteriostasis, food preservation, cleaners, and meat tenderization (Huang et al food Control,2008, 19:329-345). Less reports of electrolyzed water-treated proteins have been made, and the use of electrolyzed water to soak soybeans in combination with microbial fermentation has been shown to increase the ACE inhibitory activity of aqueous extracts of fermented soybeans (Li et al International Journal of Food Properties,2011, 14:145-156). The secondary structure of alkaline electrolyzed water extracted almond proteins was destroyed compared to the dissolution of extracted almond proteins with ultrapure water (Li et al Food Science and Biotechnology,2018, 28:15-23). After wheat gluten, peanut meal and walnut meal are soaked in an electrolytic tank with improved design, ACE inhibition activity of hydrolysates of three protein raw materials is remarkably improved (Zhang et al LWT-Food Science and Technology,2022,154,112864). At present, reports and patents for treating the haemoglobin by acidic electrolytic water swelling wall breaking are not yet seen.
Gout is a disease of purine nucleotide metabolic disorder that causes precipitation of sodium urate crystals at 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 material Reviews Rheumatology,2020, 16:380-390). Uric acid levels above 416. Mu. Mol/L in the serum of men (women above 357. Mu. Mol/L) are defined as hyperuricemia, which is the most important biochemical index in the pathogenesis of gout. Xanthine Oxidase (XOD) is capable of catalyzing 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 uric acid production in blood and is a key factor in causing hyperuricemia and gout. Traditional medicines for treating gout and hyperuricemia mainly comprise allopurinol and febuxostat, and the allopurinol and febuxostat all achieve the effect of reducing the uric acid concentration in serum by inhibiting the catalytic activity of XOD. Traditional uric acid lowering drugs are often associated with serious adverse effects including allergic syndromes, rashes, effects on liver metabolism, and the like (Jansen et al clinical Rheumatology,2010, 29:835-840). Therefore, the development of the bioactive peptide with the XOD inhibitory activity is used for preventing and treating hyperuricemia and gout, and has important economic and social significance.
Few bioactive peptides with XOD inhibitory activity are reported by the biological enzyme hydrolysis method. Most XOD inhibitory peptides are derived from marine fish proteins, such as neutral protease hydrolyzed bonito protein in vitro XOD IC 50 ICRK XOD IC identified from tuna hydrolysate with a value of 14.88mg/mL 50 A value of 14.18mg/mL; also present are XOD inhibiting peptides derived from other proteins, such as alkaline protease hydrolysed walnut meal having an XOD inhibition of 27.23% at a concentration of 20mg/mL (methong et al food Chemistry,2021,347:129068;Bu et al.International Conference on Energy,Environment and Bioengineering,2020,185:04062;Li et al.Food)&Functions, 2018, 9:107-116). The hydrolysates have lower activity than conventional uric acid lowering drugs (allopurinol, IC) 50 A value of 8.04. Mu.g/mL), there is a great gap between the effects, and no patent and report on the preparation of XOD inhibitory peptides from animal blood proteins has been found. In theory, the biological activity of peptides is determined by a combination of factors such as protein structure, protease type and hydrolysis conditions (Udenigwe et al journal of Food Science,2012,77: R11-R24). Thus, selection of appropriate protein substrates and proteases can enrich for XOD inhibiting peptides. The structure-activity relationship research of the XOD inhibition peptide shows that aromatic amino acid in the peptide segment can be effectively combined with the XOD catalytic active center, and plays a key role in the XOD inhibition activity. Protease PaproA is a highly active protease produced by fermentation of Pseudomonas aeruginosa (Pseudomonas aeruginosa) CAU342A (Sun Qian et al, microbiology letters, 2017, 44:86-95); protease GsProS8 is heterologously expressed and prepared by high-density fermentation The resulting Bacillus stearothermophilus (Geobacillus stearothermophilus) was protease-derived (Chang et al BMC Biotechnology,2021, 21:21). Bioinformatics combined peptide spectrum analysis shows that protease PaproA and GsProS8 can specifically hydrolyze duck hemoglobin, enrich peptide fragments containing aromatic amino acids, and are suitable for preparing XOD inhibition peptides.
Some patent reports for preparing uric acid-reducing active peptide by proteinase hydrolysis are available. The Chinese patent of invention No. ZL201310485124.9 discloses a method for preparing walnut peptide with uric acid reducing effect by hydrolyzing walnut pulp with alkaline protease and cellulase. The chinese patent application No. 202010861723.6 discloses a method for preparing kidney bean peptide having uric acid reducing activity by hydrolyzing kidney bean protein using alkaline protease. The Chinese patent application No. 202110397871.1 discloses a method for preparing cod polypeptide with xanthine oxidase inhibitory activity by hydrolyzing cod steak powder with papain, neutral protease, alkaline protease or flavourzyme. The Chinese patent application No. 20211045403.X discloses a method for preparing sturgeon peptide composite powder with uric acid reducing activity by using subtilisin, papain and alkaline protease to compound hydrolyzed sturgeon protein. The above disclosed invention patents are all methods for preparing peptides having uric acid lowering activity using protease hydrolysis technology. At present, no patent and report on preparation of uric acid reducing peptide by hydrolyzing duck blood cell protein with biological enzyme are seen.
Disclosure of Invention
The invention aims to solve the technical problems that: how to reduce uric acid and/or how to inhibit Xanthine Oxidase (XOD) activity.
To solve the technical problems. In a first aspect, the present invention provides a method of preparing a duck blood protein peptide having the following 5 peptide fragments:
p1), a polypeptide with an amino acid sequence of SEQ ID No. 1;
p2), a polypeptide with an amino acid sequence of SEQ ID No. 2;
p3), a polypeptide with an amino acid sequence of SEQ ID No. 3;
p4), polypeptide having the amino acid sequence of SEQ ID No. 4;
p5), a polypeptide with an amino acid sequence of SEQ ID No. 5;
the method comprises the steps of carrying out enzymolysis on duck hemoglobin to obtain an enzymolysis product, and extracting duck hemoglobin peptide from the enzymolysis product.
Further, in the above method, the enzymatic hydrolysis of duck hemoglobin comprises the steps of:
s1), adding protease A into duck hemoglobin to obtain enzymolysis solution M1;
s2), adding protease B into the enzymolysis liquid M1 to obtain enzymolysis liquid 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, which is a product of Shanghai-derived leaf biotechnology limited, and has a product number of: s10009;
In the present invention, the protease derived from Bacillus stearothermophilus may be named protease GsProS8. The amino acid sequence of the protease GsProS8 is shown in FIG. 1. The protease GsProS8 is obtained by introducing a 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 protease PaproA. The protease PaproA may be derived in particular from Pseudomonas aeruginosa (Pseudomonas aeruginosa) CAU342A. SDS-PAGE and zymogram of the protease PaproA are shown in FIG. 2.
Specifically, the protease GsProS8 is prepared according to a method comprising the following steps:
inoculating bacillus subtilis WB600 for expressing recombinant protease GsProS8 into LB culture medium according to an inoculum size of 1% (V/V), culturing at 37 ℃ for 12h as fermentation seed liquid, inoculating the bacillus subtilis WB600 with the inner liquid amount of 1.5L in a fermentation tank according to an inoculum size of 5% (V/V), controlling the fermentation process at 37 ℃, regulating pH to 4.0 by using ammonia water and phosphoric acid, maintaining dissolved oxygen at about 30%, starting feeding a feed medium when the residual sugar content in the culture medium is less than 1g/L, fermenting at high density for 108h, and collecting fermentation liquid, and centrifuging supernatant to obtain the protease GsProS8. Wherein, the composition of the fermentation medium is: 2g/L of ammonium sulfate, 3g/L of yeast extract powder, 1g/L of glucose, 1g/L of sodium chloride, 0.5g/L of magnesium sulfate, 0.05g/L of zinc chloride, 0.05g/L of manganese chloride, 0.1g/L of ferment, 2g/L of calcium chloride and the balance of water; the composition of the feed medium was: glucose 500g/L, peptone 37.5g/L, and water in balance. The protease activity of the protease GsProS8 enzyme solution is 3800U/mL. The method for measuring the enzyme activity of protease GsProS8 is described in GB/T23527-2009: after 1mL of enzyme solution and 1mL of casein solution are incubated at 40 ℃ for 10min, 2mL of trichloroacetic acid is added to stop the reaction, centrifugation is carried out at 10000rpm for 10min, 1mL of supernatant is taken, 5mL of sodium carbonate solution and 1mL of Fu Lin Fen reagent are added, incubation is carried out at 40 ℃ for 20min for color development, and the absorbance at 660nm is measured. The enzyme solution of trichloroacetic acid was added first to terminate the reaction. The amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute is defined as 1 enzyme activity unit (U).
The protease PaproA may be prepared according to a method comprising the steps of: pseudomonas aeruginosa (Pseudomonas aeruginosa) CAU342A is cultured in a fermentation medium at 30 ℃ for 3 days, fermentation broth is collected, the fermentation broth is centrifuged, and supernatant is collected, wherein the supernatant is protease PaproA enzyme solution. Wherein, the fermentation medium is prepared from the following raw materials: 3% of vinasse, 1.5% of yeast extract powder, 0.05% of tween-80, 0.5% of sodium chloride, 0.7% of potassium phosphate, 0.3% of dipotassium hydrogen phosphate, 0.04% of manganese sulfate and the balance of water, wherein all the percentages are in mass percent. The pH of the fermentation medium was 7.5. The protease activity of the enzyme solution for preparing the protease PaproA is 2653U/mL. The method for determining the enzyme activity of protease PaproA is described in GB/T23527-2009: after 1mL of enzyme solution and 1mL of casein solution are incubated at 40 ℃ for 10min, 2mL of trichloroacetic acid is added to stop the reaction, centrifugation is carried out at 10000rpm for 10min, 1mL of supernatant is taken, 5mL of sodium carbonate solution and 1mL of Fu Lin Fen reagent are added, incubation is carried out at 40 ℃ for 20min for color development, and the absorbance at 660nm is measured. The enzyme solution of trichloroacetic acid was added first to terminate the reaction. The amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute is defined as 1 enzyme activity unit (U).
In the invention, S1) the duck hemoglobin can be duck hemoglobin solution, and the duck hemoglobin solution can be prepared by mixing duck blood cell powder and acidic electrolyzed water, swelling and breaking walls;
in one embodiment of the invention, the duck blood cell protein solution is obtained by adding 5g of duck blood cell powder into 100ml of acidic electrolyzed water with pH of 3.0, swelling and breaking walls for 2 hours; in another embodiment of the invention, the duck blood cell protein solution is obtained by adding 1kg of duck blood cell powder into 20L of acidic electrolyzed water with pH of 3.0, swelling and breaking walls for 1 hour.
In the invention, duck blood cell powder is purchased from Handan Xinheng biotechnology limited company;
in the invention, an acidic electrolyzed water is prepared by using a laboratory self-made electrolytic tank (figure 3), electrodes are made of platinized titanium alloy, naCl or KCl is dissolved in distilled water, the mass concentration is 0.45% (w/w), then the solution is poured into the electrolytic tank, and the solution is electrolyzed for 5min under the conditions of 60V voltage, 0.5-1A current and 5-10cm electrode spacing, thus the acidic electrolyzed water is obtained by the anode electrolytic tank.
In one embodiment of the invention, S1) the protease A is added in an amount and S2) the protease B is added in an amount of 2000U/g duck blood cell powder. Further, the addition amount of the protease A is 1000U/g duck blood cell powder, and the addition amount of the protease B is 1000U/g duck blood cell powder.
Further, in the above method, the method further includes S3), where S3) is to centrifuge the enzymatic hydrolysate M2, and collect the supernatant M3 to obtain a duck blood protein peptide solution.
In the present invention, the centrifugation conditions in S3) were 8820 Xg for 10min.
Further, in the above method, the method further comprises the step of freeze-drying the supernatant M3 to obtain the duck blood protein peptide.
Further, in the above method, the method further comprises a step of separating a highly active duck blood protein peptide component from the obtained duck blood protein peptide, the step comprising B1) and B2):
b1 Dissolving the duck blood protein peptide powder with hydrochloric acid solution, centrifuging, collecting supernatant,
b2 And (3) performing gel chromatography on the supernatant obtained in the step B1), and taking an effluent liquid with the elution time of 173-180min to obtain the high-activity duck blood protein peptide component F5.
In the invention, the concentration of the hydrochloric acid solution (the solvent is water and the solute is HCL) of B1) is 10mM; the centrifugation condition is 8820 Xg for 10min;
the composition of the hydrochloric acid solution is as follows: hydrochloric acid with 36 percent: water=1:1090.
In the invention, the chromatographic conditions of the gel chromatography are as follows: AKTApurifier UPC-900 rapid protein liquid chromatograph, chromatographic column size is 1000X 10mM, column material is sephadex G-15, mobile phase is 10mM hydrochloric acid solution, flow rate is 0.8mL/min, detect: UV280nm.
Further, the high-activity duck blood protein peptide component F5 comprises a peptide fragment with an amino acid sequence shown in SEQ ID No. 1-53.
In order to solve the technical problems, in a second aspect, the invention provides the duck blood protein peptide prepared by the method.
In order to solve the technical problem, in a third aspect, the present invention provides a polypeptide, wherein the polypeptide is selected from one or N of P1) -P5), and N is less than or equal to 5:
p1), a polypeptide with an amino acid sequence of SEQ ID No. 1;
p2), a polypeptide with an amino acid sequence of SEQ ID No. 2;
p3), a polypeptide with an amino acid sequence of SEQ ID No. 3;
p4), polypeptide having the amino acid sequence of SEQ ID No. 4;
p5), the polypeptide with the amino acid sequence of SEQ ID No. 5.
In the invention, the polypeptide P1), P2) or P3) is derived from duck hemoglobin beta subunit, and the Uniprot number of the duck hemoglobin beta subunit is P01988; the polypeptide of P4) or P5) is derived from duck hemoglobin alpha subunit, and Uniprot number of the duck hemoglobin alpha subunit is P02115.
In order to solve the above technical problem, in a fourth aspect, the present invention provides the use of the duck blood protein peptide and/or the polypeptide described above, wherein the use is any one of A1) to A4):
a1 For the preparation of a product for the treatment and/or prevention of gout;
A2 Use in the preparation of uric acid-lowering products;
a3 Use in inhibiting xanthine oxidase activity);
a4 And the use thereof in the preparation of xanthine oxidase inhibitors.
In order to solve the technical problem, in a fifth aspect, the present invention provides a uric acid reducing product and/or a product for preventing and/or treating hyperuricemia, wherein the product contains the duck blood cell protein peptide and/or the polypeptide.
In the present invention, the uric acid reduction is embodied as inhibition or reduction of Xanthine Oxidase (XOD) activity.
In the present invention, xanthine Oxidase (XOD) was purchased from Sigma company under product number X4376.
In the invention, the product can be food, health food or medicine.
Furthermore, in the health food or the medicine, the duck blood protein peptide can be synthesized artificially or obtained by hydrolyzing duck blood protein.
In the invention, carrier materials can be added in the preparation of the health food or the medicine.
Such carrier materials include, but are not limited to, water soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethylcellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl ethyl cellulose, etc.). The materials can be prepared into various 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 injection and the like. Can be common preparation, slow release preparation, controlled release preparation and various microparticle administration systems. For the purpose of shaping the unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, dextrose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, and the like; disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils and the like; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets. For the purpose of formulating the unit dosage form into a pill, various carriers well known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, and the like; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. For preparing a unit dosage form into a suppository, various carriers well known in the art can be widely used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides, and the like. For preparing unit dosage forms into injectable preparations such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. may be used. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and further, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added. In addition, colorants, preservatives, flavors, flavoring agents, sweeteners, or other materials may also be added to the pharmaceutical formulation, if desired.
The dosage forms can be orally administered.
Compared with the prior art, the invention has at least the following advantages:
1. the invention firstly uses acidic electrolyzed water to swell wall broken hemocyte protein, and provides an environment-friendly, simple and efficient pretreatment method suitable for mass production for preparing hemocyte protein peptide by enzymolysis;
2. the invention utilizes one or two of bromelain and pseudomonas aeruginosa source protease PaproA or bacillus stearothermophilus source protease GsProS8 to hydrolyze duck blood cell powder step by step, enriches peptide fragments with XOD inhibitory activity by a specific enzyme cutting mode, and the prepared duck blood cell protein peptide has high uric acid reducing activity;
3. the method for preparing uric acid-reducing duck blood cell protein peptide by hydrolyzing duck blood cell powder step by step with double enzymes has the product yield of more than 62% and the molecular weight of more than 95% in the part with the molecular weight of less than 5000 Da;
4. the method for preparing uric acid-reducing duck blood cell protein peptide by hydrolyzing duck blood cell powder step by double enzymes provided by the invention has the advantages that the in vitro XOD half inhibition concentration of the prepared duck blood cell protein peptide powder is lower than 0.795mg/mL, and the duck blood cell peptide powder has good gastrointestinal digestion stability;
5. the invention provides a method for preparing uric acid-reducing duck blood cell protein peptide by hydrolyzing duck blood cell powder step by step, which separates and identifies high-activity peptide segments IVYPW, YPWTQ and LITGLW in vitro XOD IC 50 The values were 0.424mg/mL, 0.675mg/mL and 0.743mg/mL.
Drawings
FIG. 1 shows the gene, coding sequence and corresponding amino acid sequence of the Bacillus stearothermophilus protease GsProS 8.
FIG. 2 shows SDS-PAGE and an enzyme spectrum of a fermentation broth PaproA of Pseudomonas aeruginosa protease; wherein M is a low molecular weight standard protein; 1 is crude enzyme solution; 2 is a protease enzyme spectrum, in which 4 isoenzyme bands with protease activity appear. There are 2 major bands, indicating higher activity, and the corresponding proteins have molecular weights of 32kD and 50kD.
FIG. 3 is a block diagram of a laboratory self-made electrolyzer.
FIG. 4 shows the cleavage frequency of proteases PaproA and GsProS8 for hydrolyzing the carboxy-terminal amino terminus of duck blood proteins.
Fig. 5 is a gel exclusion chromatographic chart of duck blood cell protein peptide.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Pseudomonas aeruginosa (Pseudomonas aeruginosa) CAU342A in the examples below was screened, identified and saved for the present laboratory from soy sauce koji, and in literature "Sun Qian et al, fermentation conditions for protease production by Pseudomonas aeruginosa were optimized, microbiology bulletin 2017,44 (1): 86-95." the public was able to obtain the above strain from the applicant, and the obtained strain was only useful for experiments for verifying the present invention, but not for other uses;
bacillus subtilis WB600 expressing recombinant protease GsProS8 in the examples described below was kept by the present laboratory and is disclosed in the literature "Chang et al high level expression and biochemical characterization of an alkaline serine protease from Geobacillus stearothermophilus to prepare antihypertensive whey protein hydroxy ate. BMC Biotechnology,2021,21:21", which is publicly available to the applicant for the above strain, and the resulting strain was only useful for experiments to verify the present invention and not as other uses;
bromelain is a product of Shanghai source leaf biotechnology limited, with the product number: s10009;
duck blood cell powder is purchased from Handan Xinheng biotechnology limited company;
xanthine Oxidase (XOD) was purchased from Sigma under product number X4376;
The acid electrolyzed water is prepared by using a laboratory self-made electrolytic tank (figure 3), wherein an electrode is made of platinized titanium alloy, naCl or KCl is dissolved in distilled water, the mass concentration is 0.45% (w/w), then the solution is poured into the electrolytic tank, and the solution is electrolyzed for 5min under the conditions of 60V voltage, 0.5-1A current and 5-10cm electrode spacing, and the acid electrolyzed water is obtained by the anode electrolytic tank;
the following examples were run using SPSS 11.5 statistical software and the experimental results were expressed as mean ± standard deviation using One-way ANOVA test, with significant differences (P < 0.05), with very significant differences (P < 0.01), and with very significant differences (P < 0.001).
EXAMPLE 1 Single protease GsProS8 or PaproA hydrolysis of Duck blood cell proteins
Preparing duck blood cell solution with 5% substrate concentration (w/w) by using acidic electrolyzed water with pH of 2.5, stirring, swelling, breaking wall for 2h to obtain duck blood cell protein solution, and regulating pH of each substrate solution to 8.0 by using 1M NaOH solution or HCl solution. And (3) respectively adding pseudomonas aeruginosa protease PaproA and bacillus stearothermophilus protease GsProS8 for single-enzyme hydrolysis, wherein the added enzyme amounts are 2000U/g duck blood cell powder. After 10h of hydrolysis, inactivating for 10min in boiling water bath, centrifuging to obtain two duck blood cell protein hydrolysates, respectively lyophilizing the hydrolysates to obtain lyophilized products, and weighing to calculate the yield of the products. The product yield was calculated as follows:
Product yield (%) = (supernatant lyophilized powder mass/duck blood cell powder mass) ×100%;
and the lyophilized product was dissolved to 1mg/mL with distilled water to determine the XOD inhibition. The results of the product yield and XOD inhibition of the hydrolysate are shown in table 1. The peptide fragments in the two duck blood cell protein hydrolysates were identified using LC-MS/MS and the cleavage frequencies of the two proteases to hydrolyze the carboxy-terminal amino-terminus of duck hemoglobin were counted as shown in figure 4. Measurement of XOD inhibitory Activity the procedure of reference Zhong et al (Zhong et al food Chemistry,2021, 347:129068) was slightly modified as follows:
to 50. Mu.L of the hydrolysate sample, 150. Mu.L of Xanthine Oxidase (XOD) solution (0.05U/mL) was added, and after incubation at 37℃for 5min, the reaction was started by adding 150. Mu.L of xanthine solution. After 60min, 100. Mu.L of hydrochloric acid solution (1M) was added to terminate the reaction, and absorbance was measured at 290 nm. Buffer was used as negative control and allopurinol was used as positive control. The XOD inhibition rate was calculated as follows:
wherein A is 1 -sample absorbance; a is that 2 -sample blank; a is that 3 -negative control; a is that 4 -positive control.
TABLE 1 Single enzymatic hydrolysis of Duck blood cells by proteases GsProS8 and PaproA
* The hydrolysate XOD inhibitory activity was measured at a protein concentration of 1 mg/mL.
As shown in Table 1, after protease GsProS8 and PaproA are used for single enzyme hydrolysis of duck blood cell proteins, the XOD inhibition rates of the hydrolysates respectively reach 57.5% and 64.7% at the concentration of 1mg/mL, and the hydrolysates show higher XOD inhibition activities, but the yield of the hydrolyzed duck blood cell proteins is lower, and is respectively 16.4% and 28%. The cleavage sites of the two proteases on duck hemoglobin are mainly focused on hydrophobic amino acids (methionine and leucine) and aromatic amino acids (phenylalanine and tyrosine), which facilitate the release of XOD inhibitory peptide fragments.
The amino acid sequence of protease GsProS8 is shown in FIG. 1. The protease GsProS8 is obtained by introducing a coding gene of the protease GsProS8 into bacillus subtilis to obtain recombinant bacteria and fermenting for 108 hours at high density. The nucleotide sequence of the gene encoding the protease GsProS8 is shown in FIG. 1. The protease GsProS8 was prepared as follows:
inserting recombinant protease GsProS8 bacillus subtilis WB600 into LB culture medium according to 1% (V/V) inoculum size, culturing at 37deg.C for 12 hr as fermentation seed liquid, inoculating with 1.5L of fermentation tank, controlling fermentation process at 37deg.C, regulating pH to 4.0 with ammonia water and phosphoric acid, maintaining dissolved oxygen at about 30%, and keeping residual sugar content in the culture medium <And (3) at the speed of 1g/L, feeding a feed medium, fermenting at high density for 108h, collecting fermentation liquor, and centrifuging supernatant to obtain the protease GsProS8. Wherein the composition of the fermentation medium is: 2g/L of ammonium sulfate, 3g/L of yeast extract powder, 1g/L of glucose, 1g/L of sodium chloride, 0.5g/L of magnesium sulfate, 0.05g/L of zinc chloride, 0.05g/L of manganese chloride, 0.1g/L of ferment, 2g/L of calcium chloride and the balance of water; the composition of the feed medium was: glucose 500g/L, peptone 37.5g/L, and water in balance. The protease activity of the above-mentioned protease GsProS8 enzyme solution was measured in the following manner, and the result showed that the protease activity of the protease PaproA enzyme solution was 3800U/mL. The method for measuring the enzyme activity of the protease GsProS8 refers to GB/T23527-2009, and comprises the following specific steps: 1mL of protease GsProS8 enzyme solution and 1mL of casein solution (NaH with pH of 7.5 as solvent 2 PO 4 -Na 2 HPO 4 Buffer solution, solute is casein) at 40 ℃ for 10min, adding 2mL trichloroacetic acid to stop reaction, centrifuging at 10000rpm for 10min, taking 1mL supernatant, adding 5mL sodium carbonate solution and 1mL Fu Lin Fen reagent, preserving heat at 40 ℃ for 20min for color development, and measuring the absorbance at 660 nm. The enzyme solution of trichloroacetic acid was added first to terminate the reaction. The amount of enzyme required to hydrolyze casein at 40℃and pH 7.5 to produce 1. Mu.g of tyrosine per minute was defined as 1 enzyme activity unit (U).
SDS-PAGE and zymogram of the protease PaproA are shown in FIG. 2. There are 4 bands of isoenzymes with protease activity in the zymogram. There are 2 major bands, indicating higher activity, and the corresponding proteins have molecular weights of 32kD and 50kD. The protease PaproA is prepared by the following stepsThe preparation method comprises the following steps: pseudomonas aeruginosa (Pseudomonas aeruginosa) CAU342A is cultured in a fermentation medium at 30 ℃ for 3 days, fermentation broth is collected, the fermentation broth is centrifuged, and supernatant is collected, wherein the supernatant is protease PaproA enzyme solution. Wherein, the fermentation medium is prepared from the following raw materials: 3% of vinasse, 1.5% of yeast extract powder, 0.05% of tween-80,0.5% of sodium chloride, 0.7% of potassium phosphate, 0.3% of dipotassium hydrogen phosphate, 0.04% of manganese sulfate and water, wherein all percentages are by mass. The pH of the fermentation medium was 7.5. The protease activity of the enzyme solution of the protease PaproA was measured in the following manner, and the result showed that the protease activity of the enzyme solution of the protease PaproA was 2653U/mL. The method for determining the enzyme activity of protease PaproA is described in GB/T23527-2009: 1mL of protease PaproA in enzyme solution and 1mL of casein solution (NaH with pH of 7.5 as solvent 2 PO 4 -Na 2 HPO 4 Buffer solution, solute is casein) at 40 ℃ for 10min, adding 2mL trichloroacetic acid to stop reaction, centrifuging at 10000rpm for 10min, taking 1mL supernatant, adding 5mL sodium carbonate solution and 1mL Fu Lin Fen reagent, preserving heat at 40 ℃ for 20min for color development, and measuring the absorbance at 660 nm. The enzyme solution of trichloroacetic acid was added first to terminate the reaction. The amount of enzyme required to hydrolyze casein at 40℃and pH 7.5 to produce 1. Mu.g of tyrosine per minute was defined as 1 enzyme activity unit (U).
Example 2 hydrolysis condition optimization of high Activity uric acid reducing Duck blood peptide
Preparing duck blood cell powder suspension with 5% substrate concentration (liquid obtained by adding 5g duck blood cell powder into 100ml acidic electrolyzed water with pH of 3.0), swelling and breaking wall for 2h to obtain duck blood cell protein liquid. The pH of the system is adjusted to an optimal condition by using 1M sodium hydroxide or hydrochloric acid solution, and duck blood cell powder is hydrolyzed by using acid protease, bromelain, pseudomonas aeruginosa source protease PaproA and bacillus stearothermophilus protease GsProS8 in a single-enzyme or two-enzyme step hydrolysis mode, wherein the total enzyme adding amount is 2000U, and the specific hydrolysis mode is shown in Table 2. The method for determining the enzyme activity of protease PaproA is described in GB/T23527-2009: 1mL of enzyme solution and 1mL of casein solution are incubated at 40 ℃ for 10min, 2mL of trichloroacetic acid is added to stop the reaction, centrifugation is carried out at 10000rpm for 10min, and 1mL of the enzyme solution is taken The clear solution was added with 5mL of sodium carbonate solution and 1mL of Fu Lin Fen reagent, incubated at 40℃for 20min for color development, and absorbance at 660nm was measured. The enzyme solution of trichloroacetic acid was added first to terminate the reaction. The amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute is defined as 1 enzyme activity unit (U). The single enzyme hydrolysis is carried out with the enzyme quantity of 2000U/g duck blood cell powder, and the hydrolysis is carried out for 10 hours under the respective optimal conditions. In the first step of double-enzyme compound hydrolysis, the protease A is added with 1000U/g duck blood cell powder, and hydrolyzed for 6 hours under the optimal condition; and secondly, regulating the pH of the system, adding 1000U/g of protease B into duck blood cell powder, and continuously hydrolyzing for 4 hours under the optimal condition. After hydrolysis, the duck blood cell protein hydrolysate was obtained by inactivating in boiling water bath for 10min and centrifuging (8820×g for 10 min), and the supernatant was lyophilized and assayed for XOD inhibitory activity. Product yield determination method reference example 1. The hydrolysis conditions and the measurement results are shown in Table 2. XOD inhibition Activity reference example 1, 5 concentrations were selected according to the XOD inhibition activity of the hydrolysate, XOD inhibition rates at each concentration were measured (table 3), and non-linear regression was performed using SPSS software to calculate each hydrolysate IC according to the concentration and XOD inhibition rate 50 Values.
TABLE 2 hydrolysis conditions and the XOD inhibitory Activity of the respective hydrolysates
TABLE 3 XOD inhibitory Activity of the various hydrolysates at various concentrations
As an implementation mode of the embodiment, after the bromelain is compounded with the protease GsProS8 and PaproA, the product yield of duck blood cell powder is obviously improved to more than 54%, and the XOD inhibition activity of hydrolysate is improved; the XOD half-inhibitory concentrations of bromelain and duck blood cell protein peptides prepared by hydrolyzing duck blood cell powder with PaproA and GsProS8 respectively reach 0.795mg/mL and 0.744mg/mL, respectively. The acidic protease single enzyme compound protease PaproA or GsProS8 hydrolyzes duck blood cell powder, and the protein recovery rate is obviously improved, but the XOD inhibition activity of the hydrolysate is greatly reduced.
Example 3 isolation and identification of highly active peptide fragments
The duck blood cell protein peptide (bromelain complex protease GsProS8 hydrolysis) obtained in example 2 was dissolved in 10mM hydrochloric acid solution (36% concentrated hydrochloric acid: pure water=1:1090) using a 10mM hydrochloric acid solution equilibrated G15 gel column (1000X 10 mM), and 8820X G was centrifuged for 10min to obtain the supernatant for gel chromatography. Gel chromatographic separation method is adopted, and chromatographic conditions are as follows: AKTApurifier UPC-900 rapid protein liquid chromatograph; mobile phase: 10mM hydrochloric acid solution; flow rate: 0.8mL/min; and (3) detection: UV280nm. As a result, as shown in FIG. 5, 5 fractions were isolated, of which F5 fraction XOD was the most inhibitory. The peptide sequences identified in the F5 fraction are shown in Table 4, analyzed by nano LC-MS/MS. The results of the synthetic verification of 5 peptide fragments and the measurement of XOD inhibitory activity are shown in table 5. XOD inhibition activity assay reference example 2.
TABLE 4 peptide fragments identified in F5 Components
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* Numbering of parent proteins in the UniProt database
TABLE 5 XOD inhibitory Activity of synthetic peptide fragments
As an embodiment of this example, duck blood cell protein peptides are separated to five components, wherein F5 groupThe partial XOD has the highest inhibitory activity and is XOD IC in vitro 50 The value was 0.489mg/mL. The F5 component is identified to obtain 53 peptide fragments, which are mainly derived from duck hemoglobin alpha-chain (P01988) and beta-chain (P02115), and most of peptide fragment sequences contain aromatic amino acids. Synthesis verified that 5 of these peptides may have XOD inhibitory activity, with higher XOD inhibitory activity, IC 50 The values were 0.424mg/mL, 0.675mg/mL and 0.743mg/mL, respectively.
Example 4 preparation of uric acid-reducing active Duck blood cell protein peptide at kilogram level and determination of molecular weight thereof
Adding 20L of acidic electrolyzed water with pH of 3.0 into an enzymolysis tank, starting a stirring paddle, slowly adding 1kg of duck blood cell powder, swelling and breaking wall for 1h to obtain duck hemoglobin solution. And adding 1M sodium hydroxide solution into the enzymolysis tank by using a peristaltic pump to adjust the pH value to 7.5, and heating to 45-55 ℃ by using a heater for heat preservation. Duck hemoglobin liquid is added with 3.5g bromelain (1×10) 6 U), after 4h of hydrolysis, the pH value of the system is adjusted to 8.0. 380mL of Pseudomonas aeruginosa-derived protease enzyme solution (1X 10) was added 6 And U) continuing to hydrolyze for 6 hours, inactivating and cooling to room temperature by using a boiling water bath after two-step hydrolysis to obtain enzymolysis liquid. And (3) carrying out plate-frame filtration on the obtained enzymolysis liquid to obtain a peptide solution, and carrying out rotary evaporation concentration to obtain a concentrated peptide solution. Spray drying the obtained concentrated peptide solution to obtain duck blood cell protein peptide powder (DHH_BP) prepared by complex hydrolysis of bromelain and pseudomonas aeruginosa source protease.
Adding 20L of acidic electrolyzed water with pH of 3.0 into an enzymolysis tank, starting a stirring paddle, slowly adding 1kg of duck blood cell powder, adjusting the pH value of the system to 3.0, and swelling and breaking the wall for 1h to obtain duck hemoglobin solution. Adding 1M sodium hydroxide solution into the enzymolysis tank by peristaltic pump to adjust pH to 7.5, heating to 45-55deg.C by heater, and maintaining the temperature. 3.5g bromelain (1X 10) was added to the duck hemoglobin suspension 6 U), after 4h of hydrolysis, the pH value of the system is adjusted to 8.5. 260mL of Bacillus stearothermophilus-derived protease enzyme solution (1X 10) 6 And U) continuing to hydrolyze for 6 hours, inactivating and cooling to room temperature by using a boiling water bath after two-step hydrolysis to obtain enzymolysis liquid. And (3) carrying out plate-frame filtration on the obtained enzymolysis liquid to obtain a peptide solution, and carrying out rotary evaporation concentration to obtain a concentrated peptide solution. Spraying the obtained concentrated peptide solutionDrying to obtain duck blood cell protein peptide powder (DHH_BG) prepared by complex hydrolysis of bromelain and bacillus stearothermophilus protease.
The peptide powder obtained by spray drying is weighed, the product yield is calculated as follows:
product yield (%) = (quality of peptide powder obtained by drying/quality of duck blood cell powder) ×100%
The molecular weight of the uric acid-reducing duck blood cell protein peptide obtained above is measured, and chromatographic conditions are measured by adopting an HPLC method: agilent high performance liquid chromatograph 1260; chromatographic column: TSKgel-G2000SWXL column (7.8X100 mm); mobile phase: acetonitrile/pure water/trifluoroacetic acid: 45/50/0.1 (v/v/v); and (3) detection: UV214nm; flow rate: 0.5mL/min; column temperature: 30 ℃. The results are shown in Table 6.
TABLE 6 molecular weight distribution of two uric acid-lowering active duck blood cell protein peptides
As an implementation mode of the example, the hydrolysis of 1kg duck blood cell protein gives 672g DHH_BG and 620g DHH_BP, the product yields are 67.2% and 62%, respectively, and the component ratios with the molecular weight less than 5000Da are 96.8% and 96.5%, respectively.
Example 5 Duck blood cell protein peptide gastrointestinal digestion stability experiment
The duck blood cell protein peptide prepared in example 4 was subjected to in vitro simulated gastrointestinal digestion with minor modifications with reference to the method of Tavares et al, as follows:
dissolving duck blood cell peptide in deionized water, regulating pH to 2.0 with dilute hydrochloric acid solution, adding pepsin (E: S2.5%, w/w), hydrolyzing at 37deg.C for 90min, regulating pH to 7.5 with 0.1M sodium hydroxide solution, adding trypsin (E: S2.5%, w/w), and hydrolyzing at 37deg.C for 2 hr. After the reaction, the mixture was kept in boiling water for 10min to inactivate enzyme, and after cooling, the mixture was centrifuged at 10000r/min for 10min, and the supernatant was lyophilized, and the inhibitory activity of the simulated digested duck blood cell peptide XOD was measured as described in example 2.
As a preferred embodiment of this example, the two duck blood cell protein peptides DHH_BG and DHH_BP of example 4, after in vitro simulated gastrointestinal digestion, were XOD IC 50 The values are improved from 0.744mg/mL and 0.795mg/mL to 0.959mg/mL and 0.892mg/mL, and the gastrointestinal digestion stability is good.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Sequence listing
<110> Chinese university of agriculture
<120> Duck blood cell protein peptide with uric acid reducing activity and preparation method thereof
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<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
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Ile Val Tyr Pro Trp
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<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
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Tyr Pro Trp Thr Gln
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<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
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Leu Ile Thr Gly Leu Trp
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<210> 4
<211> 4
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
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Tyr Pro Gln Thr
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<210> 5
<211> 5
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
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Tyr Phe Pro His Phe
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<211> 6
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
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Tyr Pro Trp Thr Gln Arg
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<210> 7
<211> 6
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
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Ile Val Tyr Pro Trp Thr
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<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
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Leu Ile Val Tyr Pro
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Leu Ile Val Tyr Pro Trp
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<210> 10
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<212> PRT
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Val Tyr Pro Trp Thr Gln
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<211> 7
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
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Leu Ile Val Tyr Pro Trp Thr
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<210> 12
<211> 4
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 12
Pro Trp Thr Gln
1
<210> 13
<211> 4
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 13
Thr Phe Ala Gln
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<210> 14
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<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 14
Thr Phe Ala Gln Leu
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<210> 15
<211> 6
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 15
Pro Trp Thr Gln Arg Phe
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<210> 16
<211> 4
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 16
Val Val Pro Trp
1
<210> 17
<211> 4
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 17
Thr Gly Leu Trp
1
<210> 18
<211> 5
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 18
Ile Thr Gly Leu Trp
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<210> 19
<211> 5
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
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Val Val Pro Trp Thr
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<211> 4
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 20
Leu Ile Val Tyr
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<210> 21
<211> 8
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 21
Leu Ile Val Tyr Pro Trp Thr Gln
1 5
<210> 22
<211> 4
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 22
Ile Val Tyr Pro
1
<210> 23
<211> 7
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 23
Val Asp Pro Glu Asn Phe Arg
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<210> 24
<211> 7
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 24
Leu Leu Ile Val Tyr Pro Trp
1 5
<210> 25
<211> 10
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 25
Leu His Val Asp Pro Glu Asn Phe Arg Leu
1 5 10
<210> 26
<211> 9
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 26
Leu Leu Ile Val Tyr Pro Trp Thr Gln
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<210> 27
<211> 9
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 27
His Val Asp Pro Glu Asn Phe Arg Leu
1 5
<210> 28
<211> 6
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 28
Ala Ala Trp Gln Lys Leu
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<210> 29
<211> 7
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 29
Ala Trp Gln Lys Leu Val Arg
1 5
<210> 30
<211> 11
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 30
Thr Ala Glu Glu Lys Gln Leu Ile Thr Gly Leu
1 5 10
<210> 31
<211> 6
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 31
Trp Gln Lys Leu Val Arg
1 5
<210> 32
<211> 21
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 32
Ala Asp Cys Gly Ala Glu Ala Leu Ala Arg Leu Leu Ile Val Tyr Pro
1 5 10 15
Trp Thr Gln Arg Phe
20
<210> 33
<211> 10
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 33
Val His Trp Thr Ala Glu Glu Lys Gln Leu
1 5 10
<210> 34
<211> 10
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 34
Ile Val Tyr Pro Trp Thr Gln Arg Phe Phe
1 5 10
<210> 35
<211> 6
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 35
Ala Ser Leu Asp Lys Phe
1 5
<210> 36
<211> 4
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 36
Glu Tyr Gly Ala
1
<210> 37
<211> 6
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 37
Thr Tyr Phe Pro His Phe
1 5
<210> 38
<211> 5
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 38
Phe Pro His Phe Asp
1 5
<210> 39
<211> 4
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 39
Phe His Pro Phe
1
<210> 40
<211> 5
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 40
Thr Tyr Phe Pro His
1 5
<210> 41
<211> 11
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 41
Thr Pro Glu Val His Ala Ser Leu Asp Lys Phe
1 5 10
<210> 42
<211> 14
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 42
Phe Ile Ala Tyr Pro Gln Thr Lys Thr Tyr Phe Pro His Phe
1 5 10
<210> 43
<211> 10
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 43
Val Gly Ala Val Leu Thr Ala Lys Tyr Arg
1 5 10
<210> 44
<211> 7
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 44
Lys Thr Tyr Phe Pro His Phe
1 5
<210> 45
<211> 12
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 45
Asp Leu Ser His Gly Ser Ala Gln Ile Lys Ala His
1 5 10
<210> 46
<211> 12
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 46
Ile His His Pro Ala Ala Leu Thr Pro Glu Val His
1 5 10
<210> 47
<211> 7
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 47
Tyr Phe Pro His Phe Asp Leu
1 5
<210> 48
<211> 15
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 48
Ala Val Asn His Ile Asp Asp Ile Ala Gly Ala Leu Ser Lys Leu
1 5 10 15
<210> 49
<211> 8
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 49
Ala Glu Thr Leu Glu Arg Met Phe
1 5
<210> 50
<211> 8
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 50
Thr Tyr Phe Pro His Phe Asp Leu
1 5
<210> 51
<211> 4
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 51
Ala Leu Val Glu
1
<210> 52
<211> 18
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 52
Leu Val Val Val Ala Ile His His Pro Ala Ala Leu Thr Pro Glu Val
1 5 10 15
His Ala
<210> 53
<211> 12
<212> PRT
<213> green-head duck subspecies (Anas platyrhynchos platyrhynchos)
<400> 53
Asp Lys Leu His Val Asp Pro Glu Asn Phe Arg Leu
1 5 10
Claims (10)
1. A method for preparing duck blood protein peptide, which is characterized in that: the duck blood protein peptide has the following 5 peptide fragments:
p1), a polypeptide with an amino acid sequence of SEQ ID No. 1;
p2), a polypeptide with an amino acid sequence of SEQ ID No. 2;
p3), a polypeptide with an amino acid sequence of SEQ ID No. 3;
p4), polypeptide having the amino acid sequence of SEQ ID No. 4;
p5), a polypeptide with an amino acid sequence of SEQ ID No. 5;
the method comprises the steps of carrying out enzymolysis on duck hemoglobin to obtain an enzymolysis product, and extracting duck blood protein peptide from the enzymolysis product.
2. The method according to claim 1, characterized in that: the enzymatic hydrolysis duck hemoglobin comprises the following steps:
s1), adding protease A into duck hemoglobin to obtain enzymolysis solution M1;
s2), adding protease B into the enzymolysis liquid M1 to obtain enzymolysis liquid M2;
the protease A is protease derived from pineapple, and the protease B is protease derived from bacillus stearothermophilus or protease derived from pseudomonas aeruginosa.
3. The method according to claim 1 or 2, characterized in that: the method further comprises S3), wherein S3) is to centrifuge the enzymolysis liquid M2, collect supernatant M3 and obtain duck blood protein peptide solution.
4. A method according to any one of claims 1-3, characterized in that: the method further comprises the step of freeze-drying the supernatant M3 to obtain the duck blood protein peptide with xanthine oxidase activity inhibition.
5. The method according to claim 4, wherein: the method further comprises the step of isolating a highly active duck blood protein peptide from the duck blood protein peptide having xanthine oxidase inhibitory activity, the step comprising B1) and B2):
b1 Dissolving the duck blood protein peptide powder with hydrochloric acid solution, centrifuging, and taking supernatant;
b2 Gel chromatography is carried out on the supernatant obtained in the step B1), effluent liquid with the elution time of 173-180min is taken, the duck blood protein peptide with the name of high-activity duck blood protein peptide component F5 is obtained,
the chromatographic conditions of the gel chromatography are as follows: akta purifier UPC-900 flash protein liquid chromatograph, column size: 1000X 10mm, column material: sephadex G-15, mobile phase: 10mM hydrochloric acid solution, flow rate: 0.8mL/min, detection: UV280 nm.
6. The method according to claim 5, wherein: the high-activity duck blood protein peptide component F5 has a peptide segment with an amino acid sequence of SEQ ID No. 1-53.
7. A duck blood protein peptide prepared by the method of any one of claims 1-6.
8. A polypeptide, characterized in that: the polypeptide is selected from one or N of P1) -P5), and N is less than or equal to 5:
p1), a polypeptide with an amino acid sequence of SEQ ID No. 1;
p2), a polypeptide with an amino acid sequence of SEQ ID No. 2;
p3), a polypeptide with an amino acid sequence of SEQ ID No. 3;
p4), polypeptide having the amino acid sequence of SEQ ID No. 4;
p5), the polypeptide with the amino acid sequence of SEQ ID No. 5.
9. Use of a duck blood protein peptide as claimed in claim 7 and/or a polypeptide as claimed in claim 8, characterized in that: the application is any one of A1) -A4):
a1 For the preparation of a product for the treatment and/or prevention of gout;
a2 Use in the preparation of uric acid-lowering products;
a3 Use in inhibiting xanthine oxidase activity);
a4 And the use thereof in the preparation of xanthine oxidase inhibitors.
10. A uric acid lowering product and/or a product for preventing and/or treating hyperuricemia, characterized in that: the product comprises the duck blood cell protein peptide of claim 7 and/or the polypeptide of claim 8.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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CN202210607233.2A CN117186177B (en) | 2022-05-31 | 2022-05-31 | Duck blood cell protein peptide with uric acid reducing activity and preparation method thereof |
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