Soybean peptide with effect of preventing and / or treating chronic nephritis as well as preparation method and application of soybean peptide

Abstract

The invention provides a soybean peptide with an effect of preventing and / or treating chronic nephritis as well as a preparation method and application of the soybean peptide, and belongs to the technical field of bioactive peptides. According to the preparation method of the soybean peptide mixture provided by the invention, a three-step enzymolysis mode is adopted, the molecular weight of soybean peptide is controlled to be 1000 Daltons or below, micromolecular oligopeptide is used as a main component, and the prepared soybean peptide mixture is low in sodium, phosphorus and potassium content and capable of improving the nutritional status and muscle loss condition of nephritis patients. The prepared soybean peptide mixture is further separated and purified, the purified soybean peptide mixture, the single-chain polypeptide S1 and the single-chain polypeptide S2 are obtained, the content of uric acid and creatinine can be effectively reduced, inflammation can be inhibited, and the soybean peptide is expected to be developed into a novel polypeptide medicine for preventing and / or treating chronic kidney diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptides, and in particular relates to a soybean peptide having the efficacy of preventing and / or treating chronic nephritis, and a preparation method and application thereof. Background Art

[0002] Chronic kidney disease (CKD) has become a major public health issue that threatens human health worldwide. Most CKD patients have no obvious symptoms in the early stages, and are often in the middle or late stages when diagnosed, which reduces the opportunity for early intervention. The cost of treating CKD patients is high, especially after entering end-stage renal disease (ESRD). The cost of dialysis and kidney transplantation places a heavy burden on patients' families and the social medical system.

[0003] At present, the goal of drug intervention for CKD is mainly to control the progression of the disease, delay renal failure, relieve symptoms and reduce the occurrence of complications. Drug intervention is often carried out from the following perspectives: 1. Reduce urine protein and protect renal function, using angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) to reduce urine protein excretion, reduce glomerular pressure, and delay renal function deterioration. CEI / ARB is the first-line drug for patients with CKD accompanied by hypertension or proteinuria, but long-term use may cause hyperkalemia. 2. Lowering blood sugar: SGLT-2 inhibitors (such as dapagliflozin and empagliflozin) have been shown to have significant renal protective effects, can reduce proteinuria and delay the decline of renal function. SGLT-2 inhibitors have gradually become an important choice for patients with diabetes and CKD, but some patients may have poor tolerance; 3. Control hypertension: ACEI, ARB, calcium channel blockers (CCB), beta-blockers, etc. CKD patients' blood pressure needs to be controlled at <130 / 80mmHg (with proteinuria). Blood pressure control is an important measure to delay the progression of CKD. Combination medication is common, but it can easily lead to hypotension or electrolyte disorders. In addition, other complications of chronic kidney disease include: anemia, abnormal bone metabolism, hyperkalemia, abnormal acid metabolism, and other complications are often treated with different drugs. However, drug intervention often has the following limitations: 1. Drug side effects: Many drugs may cause problems such as hyperkalemia, hypotension, and gastrointestinal reactions; 2. Poor long-term compliance: Patients need to take multiple drugs for a long time, and compliance is not high; 3. Limited etiological treatment: Most existing drugs are symptomatic treatments and it is difficult to reverse the decline of renal function; 4. Insufficient individualized treatment: Drug selection needs to be adjusted according to the condition, comorbidities, and patient tolerance, but it is difficult to fully implement in clinical practice; 5. Heavy economic burden: New drugs (such as SGLT-2 inhibitors) are expensive, which limits their popularization. Therefore, in view of the limitations of drug intervention, it is necessary to combine it with non-drug means such as nutritional intervention and lifestyle adjustment to comprehensively manage the condition and quality of life of CKD patients. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a soybean peptide mixture with low sodium, phosphorus and potassium content and a preparation method thereof, which has the effect of improving the nutritional status and muscle loss of nephritis patients.

[0005] The purpose of the present invention is also to provide a purified soybean peptide mixture obtained by further separation and purification of the soybean peptide mixture, as well as single-chain polypeptide S1 and single-chain polypeptide S2, which can effectively reduce uric acid and creatinine content, inhibit inflammation, and are expected to be developed into a new polypeptide drug for the prevention and / or treatment of chronic kidney disease.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a soybean peptide mixture, comprising the following steps:

[0008] (1) adding deionized water with a pH of 9.5 to 10.5 to prepare a soy protein solution with a concentration of 4.5 to 5.5%;

[0009] (2) grinding and homogenizing the soy protein solution to obtain a soy protein homogenate having a protein particle size of 30 to 80 μm;

[0010] (3) The soy protein homogenate is kept at 53-58° C. and stirred for 0.5-1 h, the pH value is adjusted to 9.5-10.5, alkaline protease is added, and enzymolysis is performed until the pH value drops to 7.0-7.5, papain and neutral protease are added, and flavor protease is added after enzymolysis for 3.5-4.5 h, and enzymolysis is continued for 2-2.5 h to obtain an enzymolysis solution;

[0011] (4) the enzymatic hydrolyzate is heated to 108-112° C. and maintained for 3-10 min, then cooled to room temperature, filtered using a ceramic membrane, and the clear liquid filtered by the ceramic membrane is filtered using a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to obtain a nanofiltration membrane cutoff liquid;

[0012] (5) The intercepted liquid of the nanofiltration membrane is sequentially passed through a cation exchange resin and an anion exchange resin at a flow rate of 48 to 52 L / h. The material after resin adsorption is concentrated, sterilized, and dried to obtain a soybean peptide mixture.

[0013] Preferably, based on the mass of the soybean protein homogenate, the amount of alkaline protease added is 1.5 to 2.5 million U / g, the amount of papain added is 700,000 to 900,000 U / g; the amount of neutral protease added is 2.5 to 3.5 million U / g; and the amount of flavor protease added is 400,000 to 600,000 U / g.

[0014] Preferably, the soybean peptide mixture is separated and purified by preparative high performance liquid chromatography, and gradient elution is performed with mobile phase A: 0.1% trifluoroacetic acid + 99.9% ultrapure water, mobile phase B: 0.1% trifluoroacetic acid + 99.9% acetonitrile to obtain a purified soybean peptide mixture.

[0015] Preferably, the method for preparing the sample solution for separation and purification by the preparative high performance liquid chromatography comprises: dissolving the soybean peptide mixture with mobile phase A, preparing a soybean peptide solution, centrifuging, filtering the supernatant with a filter membrane, and using the filtrate as the sample solution.

[0016] Preferably, the elution procedure of the separation and purification by the preparative high performance liquid chromatography is: 0% to 5% B in 0 to 5 min, 5% B to 80% B in 5 to 20 min; the elution fraction collected in 11 to 12 min is the purified soybean peptide mixture.

[0017] The present invention also provides a soybean peptide mixture prepared by the above preparation method.

[0018] Preferably, the soybean peptide mixture comprises polypeptide S1 and polypeptide S2.

[0019] The present invention also provides polypeptide S1, which is a single-chain polypeptide with a structural sequence of YDPSTGIY and a peptide chain molecular weight of 914.95 Da.

[0020] The present invention also provides polypeptide S2, which is a single-chain polypeptide with a structural sequence of VNEPAPGLK and a peptide chain molecular weight of 924.05 Da.

[0021] The present invention also provides the use of the soybean peptide mixture or the polypeptide S1 or the polypeptide S2 in preparing a drug for preventing and / or treating chronic nephritis.

[0022] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0023] The present invention adopts a three-step enzymatic hydrolysis method to prepare a soybean peptide mixture with low sodium, phosphorus and potassium contents, and controls the molecular weight of the soybean peptide to be below 1000 Daltons, accounting for more than 96%, and mainly consisting of small molecule oligopeptides. The sodium content of the obtained soybean peptide mixture is controlled at about 26 mg / 100 g, and the potassium content is controlled at about 0.12 mg / 100 g, and it contains almost no phosphorus. By controlling sodium, phosphorus and potassium, the kidney burden of patients with chronic nephritis is reduced, and the nutritional status and muscle loss of patients with chronic nephritis are improved through the intake of soybean peptides.

[0024] The present invention further separates and purifies the soybean peptide mixture to obtain a separated and purified soybean peptide mixture, and structurally identifies the separated and purified soybean peptide mixture to obtain a single-chain polypeptide S1 with a structure of YDPSTGIY and a single-chain polypeptide S2 with a structure of VNEPAPGLK. Polypeptide S1 and polypeptide S2 are not degraded by digestive proteases, can effectively reduce uric acid and creatinine content, and inhibit inflammation, and are expected to be developed into a new polypeptide drug for preventing and / or treating chronic kidney disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 :Standard curve for creatinine detection;

[0026] Figure 2 : Chromatogram of the soybean peptide mixture prepared in Example 1;

[0027] Figure 3 : Chromatogram of component 12 obtained in Example 2;

[0028] Figure 4 : MS / MS spectrum of single-chain peptide S1;

[0029] Figure 5 : MS / MS spectrum of single-chain peptide S2;

[0030] Figure 6 : Typical fluorescence images of zebrafish nephritis cells in each group;

[0031] Figure 7 :Results of histopathological examination of the liver of experimental mice (HE staining, 20×);

[0032] Figure 8 : Results of histopathological examination of kidneys of experimental mice (HE staining, 20×). DETAILED DESCRIPTION

[0033] The present invention provides a method for preparing a soybean peptide mixture, comprising the following steps:

[0034] (1) adding deionized water with a pH of 9.5 to 10.5 to prepare a soy protein solution with a concentration of 4.5 to 5.5%;

[0035] (2) grinding and homogenizing the soy protein solution to obtain a soy protein homogenate having a protein particle size of 30 to 80 μm;

[0036] (3) The soy protein homogenate is kept at 53-58° C. and stirred for 0.5-1 h, the pH value is adjusted to 9.5-10.5, alkaline protease is added, and enzymolysis is performed until the pH value drops to 7.0-7.5, papain and neutral protease are added, and flavor protease is added after enzymolysis for 3.5-4.5 h, and enzymolysis is continued for 2-2.5 h to obtain an enzymolysis solution;

[0037] (4) the enzymatic hydrolyzate is heated to 108-112° C. and maintained for 3-10 min, then cooled to room temperature, filtered using a ceramic membrane, and the clear liquid filtered by the ceramic membrane is filtered using a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to obtain a nanofiltration membrane cutoff liquid;

[0038] (5) The intercepted liquid of the nanofiltration membrane is sequentially passed through a cation exchange resin and an anion exchange resin at a flow rate of 48 to 52 L / h. The material after resin adsorption is concentrated, sterilized, and dried to obtain a soybean peptide mixture.

[0039] In step (1) of the present invention, the soy protein is commercially available soy protein; the deionized water with a pH of 9.5 to 10.5 is obtained by adjusting deionized water with 30% NaOH, and the pH value of the deionized water is preferably 10; the water temperature of the deionized water is 45 to 55° C., preferably 50° C.; and the concentration of the soy protein solution is preferably 5%.

[0040] In step (2) of the present invention, the grinding is preferably performed by a colloid mill, and the grinding time is 1 to 2 hours, preferably 1.5 hours; the homogenization is preferably performed by a high-pressure homogenizer, and the pressure is 45 to 55 MPa, preferably 50 MPa.

[0041] In step (3) of the present invention, the soy protein homogenate is preferably kept warm at 55° C., and the pH value is preferably adjusted to 10; the pH is adjusted using 30% NaOH; based on the mass of the soy protein homogenate, the amount of alkaline protease added is 1.5 to 2.5 million U / g, preferably 2 million U / g, the amount of papain added is 700,000 to 900,000 U / g, preferably 800,000 U / g, the amount of neutral protease added is 2.5 to 3.5 million U / g, preferably 3 million U / g, and the amount of flavor protease added is 400,000 to 600,000 U / g, preferably 500,000 U / g.

[0042] In step (4) of the present invention, the enzymatic hydrolyzate is preferably heated to 110° C. and the holding time is preferably 5 min; the pore size of the ceramic membrane is 45 to 55 nm, preferably 50 nm. The present invention can filter out macromolecular proteins that are not completely hydrolyzed through the ceramic membrane, and filter out small molecular substances such as free amino acids and sodium chloride in the enzymatic hydrolyzate through the nanofiltration membrane.

[0043] In step (5) of the present invention, cation exchange resin and anion exchange resin are used to adsorb heavy metals in the enzymatic hydrolyzate. The nanofiltration membrane intercepted liquid preferably passes through the cation exchange resin and the anion exchange resin in sequence at a flow rate of 50L / h; as an optional embodiment, the cation exchange resin is pretreated before use, and the pretreatment step includes: passing 4 times the volume of resin through the resin column with a 4% sodium hydroxide solution at a flow rate of 1 to 2 times the volume of resin per hour, and then washing the resin with pure water, and stopping washing when the outlet pH is neutral; then passing 4 times the volume of resin through the resin column with a 4% hydrochloric acid solution at a flow rate of 1 to 2 times the volume of resin per hour, and then washing the resin with pure water, and stopping washing when the outlet pH is neutral, and standby. As an optional embodiment, the anion exchange resin is pretreated before use, and the pretreatment step includes: passing 3 times the volume of resin through the resin column with a 4% sodium hydroxide solution at a flow rate of 2 times the volume of the resin per hour, and then washing the resin with pure water until the outlet pH is <9, and then passing 3 times the volume of resin through the resin column with a 4% hydrochloric acid solution at a flow rate of 2 times the volume of the resin per hour, and then washing the resin with pure water until the outlet pH is >5 for standby use.

[0044] In step (5) of the present invention, the concentration is preferably carried out by double-effect falling film concentration to increase the material concentration to 35% to 40%; the sterilization is preferably carried out by a high-temperature sterilizer, and the sterilization conditions are 115°C and 3s; the drying is preferably spray drying, and a spray drying tower is used for treatment, and the drying conditions are: inlet air temperature 180-190°C, exhaust air temperature 70-80°C, and atomization pressure 150-160Bar.

[0045] The soybean peptide mixture prepared by the present invention has high protein and peptide contents and low sodium, phosphorus and potassium contents, can effectively reduce the uric acid content and creatinine content of the zebrafish nephritis model, and has the effect of assisting in improving nephritis.

[0046] The soybean peptide mixture of the present invention is separated and purified by a preparative high performance liquid chromatograph, and the separation and purification is performed with mobile phase A: 0.1% trifluoroacetic acid + 99.9% ultrapure water, mobile phase B: 0.1% trifluoroacetic acid + 99.9% acetonitrile for gradient elution to obtain a purified soybean peptide mixture. The preparation of the sample solution for separation and purification of the present invention includes: dissolving the soybean peptide mixture with mobile phase A, preparing a soybean peptide solution, centrifuging, filtering the supernatant with a filter membrane, and the filtrate is the sample solution; the concentration of the soybean peptide solution is preferably 8 to 12 mg / mL, more preferably 10 mg / mL; the centrifugation conditions are preferably 9000 to 11000 r / min for 3 to 8 min, more preferably 10000 r / min for 5 min; the filter membrane is preferably a 0.22 μm filter membrane. The elution procedure of the present invention is: 0% to 5% B in 0 to 5 minutes, 5% B to 80% B in 5 to 20 minutes; the elution fraction of 11 to 12 minutes is collected as the purified soybean peptide mixture; the sample loading volume during the elution is preferably 15 mL.

[0047] The soybean peptide mixture obtained by separation and purification by a preparative high performance liquid chromatograph can effectively reduce the uric acid content and creatinine content of a zebrafish nephritis model, and has an auxiliary effect of improving nephritis.

[0048] The soybean peptide mixture prepared by the present invention is structurally identified by nanoliter liquid chromatography-Q EXACTIVE mass spectrometry, and two polypeptides are identified, including polypeptide S1 and polypeptide S2.

[0049] The polypeptide S1 of the present invention is a single-chain polypeptide, the structural sequence is YDPSTGIY (H-Tyr-Asp-Pro-Ser-Thr-Gly-Ile-Tyr-OH), and the molecular weight of the peptide chain is 914.95Da. The polypeptide S2 is a single-chain polypeptide, the structural sequence is VNEPAPGLK (H-Val-Asn-Glu-Pro-Ala-Pro-Gly-Leu-Lys-OH), and the molecular weight of the peptide chain is 924.05Da. The polypeptide S1 and polypeptide S2 prepared by the present invention have significant effects of lowering uric acid, lowering creatinine, and inhibiting inflammation, and have good antioxidant activity, and the effect of polypeptide S1 is better than that of polypeptide S2, and has obvious kidney protection effect.

[0050] The present invention also provides the use of the soybean peptide mixture or the polypeptide S1 or the polypeptide S2 in the preparation of a drug for preventing and / or treating chronic nephritis, which is expected to be developed into a new polypeptide drug for improving or treating chronic kidney disease.

[0051] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] In the specific embodiments and comparative examples of the present invention, soy protein was purchased from Shandong Yuwang Ecological Food Co., Ltd.; alkaline protease was purchased from Novozymes (China) Biotechnology Co., Ltd.; papain was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.; neutral protease was purchased from Novozymes (China) Biotechnology Co., Ltd.; flavor protease was purchased from Novozymes (China) Biotechnology Co., Ltd.; potassium oxonate (PO) was purchased from Beijing Solebold Technology Co., Ltd., with a purity of ≥98%; allopurinol tablets were purchased from Hefei Jiulian Pharmaceutical Co., Ltd.; sodium carboxymethyl cellulose (CMC-Na) was purchased from Tianjin Komio Chemical Reagent Co., Ltd.; uric acid, xanthine oxidase, and adenosine deaminase kits were purchased from Nanjing Jiancheng Bioengineering Institute; urea nitrogen and creatinine kits were purchased from Ningbo Meikang Biotechnology Co., Ltd., and other reagents were all analytically pure.

[0053] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0055] Example 1

[0056] Preparation of soy peptide mixture:

[0057] 1. Prepare 1000 L of deionized water (conductivity less than 20 microsiemens per centimeter (us / cm)) and heat it to 50° C. Use 30% NaOH to adjust the pH of the deionized water to 10.0, take 50 kg of soy protein for slurry preparation, and obtain a soy protein solution with a concentration of 5%.

[0058] 2. Stir the soybean protein solution evenly, treat it with a colloid mill for 1.5 hours, and then use a high-pressure homogenizer to treat it at a pressure of 50 MPa for 2 hours to obtain a homogenate. The particle size of the protein particles in the homogenate is 30 to 80 μm.

[0059] 3. Heat the homogenate to 55°C and stir for 0.5 to 1 hour.

[0060] 4. Adjust the pH value of the heat-insulated homogenate to 10.0 with 30% NaOH solution and keep the temperature constant.

[0061] 5. Add alkaline protease (2 million U / g) to the homogenate in step 4, and continuously monitor the pH during enzymolysis. When the pH drops to 7.0-7.5, add papain (800,000 U / g) and neutral protease (3 million U / g). Continue enzymolysis for 4 hours, then add flavor protease (500,000 U / g) and continue enzymolysis for 2-2.5 hours.

[0062] 6. The enzymatic hydrolysate obtained in step 5 was heated to 110°C and maintained for 5 min to inactivate the protease.

[0063] 7. After the enzyme is inactivated, the enzymatic hydrolyzate is cooled to room temperature and then passed through a 50 nm pore size ceramic membrane.

[0064] 8. The clear liquid filtered by the ceramic membrane is collected and filtered using a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to obtain a nanofiltration membrane cutoff liquid.

[0065] 9. Use anion exchange resin and cation exchange resin to adsorb heavy metals in the enzymatic hydrolyzate.

[0066] 9.1. Cation exchange resin pretreatment

[0067] (1) Resin column loading: Weigh 50 kg of cation exchange resin (D113 model from Langfang Jinnan Resin Co., Ltd.) using an electronic scale, compact it, and transfer it to the resin column with deionized water, leaving 3 to 5 cm of water on the top of the resin.

[0068] (2) Resin pretreatment: Pass 4 times the volume of resin through the resin column with a 4% sodium hydroxide solution at a flow rate of 1 to 2 times the volume of resin per hour. After the resin is washed with pure water, stop washing when the outlet pH is neutral. Then pass 4 times the volume of resin through the resin column with a 4% hydrochloric acid solution at a flow rate of 1 to 2 times the volume of resin per hour. After the resin is washed with deionized water, stop washing when the outlet pH is neutral and set aside.

[0069] 9.2. Anion exchange resin pretreatment

[0070] (1) Resin column loading: 50 kg of anion exchange resin (D301 model from Langfang Jinnan Resin Co., Ltd.) was weighed using an electronic scale, compacted, and transferred to the resin column with deionized water, leaving 3 to 5 cm of water above the resin.

[0071] (2) Resin pretreatment: Pass 3 times the volume of resin through the resin column with a 4% sodium hydroxide solution at a flow rate of 2 times the volume of resin per hour. After the end, wash the resin with pure water until the outlet pH is <9. Then pass 3 times the volume of resin through the resin column with a 4% hydrochloric acid solution at a flow rate of 2 times the volume of resin per hour. After the end, wash the resin with deionized water until the outlet pH is >5 and set aside.

[0072] 9.3. Column passing: The feed liquid to be treated (the nanofiltration membrane interception liquid obtained in step 8) is passed through the resin column at a flow rate of 50 L / h. The feed liquid first passes through the cation exchange resin and then passes through the anion exchange resin. The conductivity of the outlet effluent is detected. When the feed liquid concentration is 5%, the conductivity is higher than 500 micro Siemens per centimeter (us / cm). If it does not meet the requirements, stop passing the feed liquid.

[0073] 9.4. Water washing: Use 1 to 2 times the volume of resin to replace the residual liquid in the resin column.

[0074] 10. The material treated by resin adsorption is concentrated by double-effect falling film to increase the material concentration to 35% to 40%.

[0075] 11. The concentrated material is sterilized in a high-temperature sterilizer at 115°C for 3 seconds.

[0076] 12. After sterilization, the material is dried in a spray drying tower. The drying conditions are: inlet air temperature 180-190°C, exhaust air temperature 70-80°C, atomization pressure 150-160Bar, and finally 25kg of soybean peptide mixture is obtained after spray drying.

[0077] The basic physical and chemical properties of the soybean peptide mixture were determined according to the national standard GB / T 22492-2008 for soybean peptides. The results are shown in Table 1.

[0078] Table 1 Basic physicochemical properties of the soybean peptide mixture prepared in Example 1

[0079] Protein content (%) Moisture content (%) Ash content (%) 95.6 4.2 0.11 Carbohydrate content (%) Fat content (%) Peptide content (%) 0.05 0.04 96.3 Sodium (mg / 100g) Potassium (mg / 100g) Phosphorus (mg / 100g) 26.2 0.12 0

[0080] As can be seen from Table 1, the present invention prepares a soybean peptide mixture low in sodium, phosphorus and potassium.

[0081] Comparative Example 1

[0082] Preparation of soy peptide mixture:

[0083] Steps 1 to 8 are the same as in Example 1.

[0084] 9. The nanofiltration membrane interception liquid obtained in step 8 is concentrated by double-effect falling film to increase the material concentration to 35% to 40%.

[0085] 10. The concentrated material is sterilized in a high-temperature sterilizer at 115°C for 3 seconds.

[0086] 11. After sterilization, the material is dried in a spray drying tower. The drying conditions are: inlet air temperature 180-190°C, exhaust air temperature 70-80°C, atomization pressure 150-160Bar, and finally 25kg of soybean peptide mixture is obtained after spray drying.

[0087] The basic physical and chemical properties of the soybean peptide mixture were determined according to the national standard GB / T 22492-2008 for soybean peptides. The results are shown in Table 2.

[0088] Table 2 Basic physicochemical properties of the soybean peptide mixture obtained in Comparative Example 1

[0089] Protein content (%) Moisture content (%) Ash content (%) 89.79 4.1 5.9 Carbohydrate content (%) Fat content (%) Peptide content (%) 0.12 0.09 83.9 Sodium (mg / 100g) Potassium (mg / 100g) Phosphorus (mg / 100g) 2126.1 365.3 86.9

[0090] It can be seen from Table 2 that the soybean peptide mixture prepared in Comparative Example 1 has higher sodium, phosphorus, potassium and ash contents, and slightly lower peptide and protein contents.

[0091] Test Example 1

[0092] The efficacy of the soybean peptide mixture prepared in Example 1 and Comparative Example 1 in assisting the improvement of nephritis in zebrafish was evaluated:

[0093] 5dpf wild-type AB strain zebrafish were randomly selected in a six-well plate, and 30 zebrafish were treated in each well, with a capacity of 3mL per well. The experimental group, positive control group and model control group used potassium oxonate combined with aristolochic acid to induce zebrafish to establish a nephritis model. The experimental group was given a water-soluble soybean peptide mixture (prepared in Example 1 and Comparative Example 1) at concentrations of 50, 250, and 1250μg / mL, respectively, and the positive control group was given allopurinol at a concentration of 136μg / mL. The normal control group used fish farming water to treat zebrafish, and the model control group used fish farming water to treat zebrafish. After each group was treated for 1d, the uric acid content and creatinine content in the zebrafish were detected.

[0094] 1. Sample preparation: Collect zebrafish from each treatment group, rinse them thoroughly with ultrapure water, transfer them into 2 mL centrifuge tubes, absorb as much water as possible, then add 500 μL of deionized water to each tube, grind them evenly using a tissue grinder under ice bath conditions to prepare homogenate, centrifuge the homogenate at 10,000 rpm for 10 min, and take the supernatant for later use.

[0095] 2. Detection of uric acid content: The supernatant obtained in step 1 was detected by high performance liquid chromatography. Chromatographic conditions: detector wavelength: γ = 288nm, chromatographic column: Shimadzu C18 (5μm 4.6×250mm); mobile phase: water (0.2% acetic acid): methanol (V:V) = 94:6; flow rate: 1.0mL / min; column temperature: 30°C; injection volume: 10μL. The statistical analysis results of uric acid content were used to evaluate the efficacy of soybean peptide components in assisting the improvement of nephritic zebrafish. The statistical processing results are expressed as mean±SE. The calculation formula for the uric acid lowering effect is as follows:

[0096]

[0097] Statistical analysis was performed using analysis of variance and Dunnett's T-test, with P < 0.05 indicating significant differences. The results are shown in Table 3.

[0098] Table 3 Experimental results of uric acid reduction after soybean peptide treatment

[0099]

[0100] The uric acid content in zebrafish treated with soybean peptide mixture was compared with that in the model control group, *P<0.05; compared with the normal control group, #P<0.05, ##P<0.01.

[0101] The results showed that the uric acid content of zebrafish in the model control group was compared with that in the normal control group, P<0.01, indicating that the model was successfully established. The uric acid content of zebrafish in the positive control group was compared with that in the model control group, P<0.05, and its uric acid-reducing effect on zebrafish was 21%, indicating that allopurinol has a reducing effect on zebrafish uric acid. Compared with the model control group, the soybean peptide mixture group in Example 1 had a P<0.05 in the 250μg / mL and 1250μg / mL concentration groups, indicating that soybean peptides have a reducing effect on zebrafish uric acid under the conditions of 250μg / mL and 1250μg / mL concentrations. Compared with the normal control group, the uric acid content of different groups in the comparative example 1 soybean peptide mixture group had significant differences. Compared with the model control group, the uric acid reducing effects of the 250μg / mL and 1250μg / mL concentration groups were 5.3% and 6.3%, respectively. The results show that the uric acid-lowering effect of the soybean peptide mixture in Example 1 is better than that of the soybean peptide mixture in comparative example 1.

[0102] 3. Creatinine content detection: The change of creatinine content is mainly determined by the filtration capacity of the kidney. When the kidney function is impaired, creatinine accumulates in the body and the content increases. The creatinine detection kit was used to detect the creatinine content in different groups of zebrafish.

[0103] Standard curve establishment: Prepare creatinine standard solutions of different concentrations (range: 0, 10, 20, 50, 100, 200 μmol / L), add reagents to each standard solution according to the kit operation procedure, and after the reaction is completed, measure the absorbance value at 570 nm with an enzyme marker, record the absorbance value of each concentration standard solution, and use the standard curve formula y=kx+b to establish the relationship between concentration and absorbance. The standard curve is as follows Figure 1 shown.

[0104] Sample detection: Take the supernatant obtained in step 1, add reagents and react according to the instructions of the kit. After the reaction is completed, use an enzyme marker to measure the absorbance value at 570nm to calculate the creatinine content. The results are shown in Table 4.

[0105] Table 4 Results of the experiment on creatinine reduction after soybean peptide treatment

[0106]

[0107]

[0108] The creatinine content of zebrafish treated with soybean peptide mixture was compared with that of model control group, *P<0.05; compared with normal control group, #P<0.05, ##P<0.01.

[0109] The results showed that the zebrafish creatinine content in the model control group was compared with that in the normal control group, P<0.01, indicating that the model was successfully established. The zebrafish creatinine content in the positive control group was compared with that in the model control group, P<0.01, indicating that allopurinol has a reducing effect on zebrafish creatinine. The zebrafish creatinine content in the soybean peptide mixture group of Example 1 was compared with that in the model control group, and the 250μg / mL and 1250μg / mL concentration groups were P<0.01, indicating that soybean peptides have a reducing effect on zebrafish creatinine under the conditions of 250μg / mL and 1250μg / mL concentrations. Compared with the normal control group, the uric acid content of different groups in the soybean peptide mixture group of Comparative Example 1 showed significant differences. Compared with the model control group, the uric acid reduction effects of the 250μg / mL and 1250μg / mL concentration groups were 7.4% and 9.5%, respectively. The results showed that the uric acid-lowering effect of the soybean peptide mixture in Example 1 was better than that of the soybean peptide mixture in Comparative Example 1.

[0110] Example 2

[0111] The soybean peptide mixture prepared in Example 1 was separated and purified using a preparative high performance liquid chromatograph:

[0112] The separation and purification parameters of preparative HPLC are as follows:

[0113] Mobile phase A: 0.1% trifluoroacetic acid + 99.9% ultrapure water; Mobile phase B: 0.1% trifluoroacetic acid + 99.9% acetonitrile;

[0114] Detection wavelength: 214nm; detection time: 30min; column temperature: 21℃~25℃.

[0115] The mobile phase A was used at 10 ml / min for equilibration for 6 column volumes until the baseline was stable.

[0116] The obtained soybean peptide mixture was dissolved in mobile phase A to prepare a soybean peptide concentration of 10 mg / mL, centrifuged at 10000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane. The sample volume was 15 mL. A mixture of mobile phase A and mobile phase B was used for gradient elution, and the elution gradient is shown in Table 5.

[0117] Table 5 Analytical liquid phase mobile phase gradient

[0118]

[0119]

[0120] From 0 to 5 min, the concentration of mobile phase B increased from 0% to 5% by gradient; from 5 to 20 min, the concentration of mobile phase B increased from 5% to 80% by gradient; from 20 to 25 min, the concentration of mobile phase B decreased from 80% to 5% by gradient; from 25 to 30 min, the concentration of mobile phase B was maintained at 5% for elution for 5 min.

[0121] Different components were collected every 1 min, and 16 components were collected. The peak times of the 16 components are shown in Table 6.

[0122] Table 6 Peak time of 16 components

[0123] Components Peak time Components Peak time 1 0~1min 9 8~9min 2 1~2min 10 9-10 minutes 3 2~3min 11 10~11min 4 3~4min 12 11~12min 5 4-5 minutes 13 12~13min 6 5~6min 14 13~14min 7 6~7min 15 14-15 minutes 8 7~8min 16 15~16min

[0124] The chromatogram of the soybean peptide mixture prepared in Example 1 is as follows: Figure 2 As shown, the separation and purification chromatogram of component 12 is as follows Figure 3 As shown. Figure 2 It can be seen that the chromatographic peak area of ​​component 12 in the soybean peptide mixture is relatively large, indicating that component 12 accounts for a relatively high proportion in the soybean peptide mixture. Figure 3 It can be seen that the purity of the component 12 obtained by separation and purification is relatively high. The purity and structure of the obtained component 12 were further identified using a nanoliter liquid chromatography-Q EXACTIVE mass spectrometry system. Detection conditions:

[0125] Mobile phase: Phase A: 100% pure water + 0.1% formic acid; Phase B: 100% acetonitrile + 0.1% formic acid;

[0126] Mobile phase flow rate: 300 nL / min; injection volume: 1 μL supernatant; mobile phase gradient program is shown in Table 7.

[0127] Table 7 Mobile phase gradient program

[0128] Time (min) 0 2.0 36.0 38.0 41.0 42.0 45.0 A(％) 97 97 63 10 10 97 97 B(％) 3 3 37 90 90 3 3

[0129] The structure of component 12 was identified by nanoliter liquid chromatography-Q EXACTIVE mass spectrometry. Two polypeptides were identified. The amino acid composition of S1 was YDPSTGIY (H-Tyr-Asp-Pro-Ser-Thr-Gly-Ile-Tyr-OH) with a molecular weight of 914.95 Da. The secondary mass spectrum is shown in Figure 4As shown; the amino acid composition of S2 is VNEPAPGLK (H-Val-Asn-Glu-Pro-Ala-Pro-Gly-Leu-Lys-OH), its molecular weight is 924.05Da, and the secondary mass spectrum is as shown Figure 5 The mass percentages of the two single-chain polypeptides in soybean peptides were further determined by liquid chromatography-mass spectrometry and were 1.26% and 0.35% respectively.

[0130] Test Example 2

[0131] The efficacy of the soybean peptide mixture components 1 to 16 purified from Example 2 was evaluated for assisting in improving nephritis in zebrafish:

[0132] 5dpf wild-type AB strain zebrafish were randomly selected in a six-well plate, 30 zebrafish were treated in each well, and the capacity of each well was 3mL. The experimental group and the model control group used potassium oxonate combined with aristolochic acid to induce zebrafish to establish a nephritis model. The experimental group was given water-soluble soybean peptide mixture components 1 to 16 at a concentration of 50μg / mL. The normal control group used fish farming water to treat zebrafish, and the model control group used fish farming water to treat zebrafish. After 1d of treatment in each group, the uric acid content and creatinine content in the zebrafish were detected. The detection method is the same as that in Experimental Example 1, and the test results are shown in Table 8.

[0133] Table 8 Experimental results of creatinine and uric acid reduction after treatment with soybean peptide mixture components 1 to 16

[0134]

[0135] The results showed that the soybean peptide mixture component 12 had a significantly higher effect on reducing creatinine and uric acid than other components.

[0136] Test Example 3

[0137] In vitro digestion test was performed on peptide S1 and peptide S2:

[0138] Preparation of artificial gastric juice: Take 240 mL of HCl solution (pH = 2), 1.8 g of pepsin (3500 U / g), shake well, dilute to 300 mL with water, and place in a 4°C refrigerator for later use.

[0139] Prepare artificial small intestinal fluid: take 2 g of potassium dihydrogen phosphate, add 150 mL of water to dissolve it, adjust the pH value to 6.8 with 0.1 mol / L NaOH solution, take 3 g of pancreatic enzyme (3000 U / g), add appropriate amount of water to dissolve it, mix the above prepared solutions, dilute with water to 300 mL, and place in a 4°C refrigerator for use.

[0140] Artificial gastric juice and intestinal juice were used as solvents, and polypeptide S1 and polypeptide S2 were added respectively, and the concentration was controlled at 4 mg / mL. Take 1 mg of sample, add 20 mL of artificial gastric juice, shake at 37 ° C, 100 r / min for 2 hours, adjust the pH to 6.8, transfer 20 mL of artificial small intestinal juice and shake well, shake at 37 ° C, 100 r / min for 3 hours, and finally inactivate the enzyme at 85 ° C for 10 minutes, cool to room temperature, perform ultrafiltration centrifugation, and retain the filtrate. The filtrate was detected by mass spectrometry, and it was identified that the molecular weight of polypeptide S1 and polypeptide S2 did not change, indicating that polypeptide S1 and polypeptide S2 were not degraded by digestive proteases.

[0141] Test Example 4

[0142] Based on the structures of polypeptide S1 and polypeptide S2 obtained in Example 2, Shanghai Newpu Biotechnology Co., Ltd. was commissioned to synthesize two single-chain polypeptides and evaluate their efficacy in assisting the improvement of nephritis in zebrafish:

[0143] 5dpf wild-type AB strain zebrafish were randomly selected in a six-well plate, and 30 zebrafish were treated in each well, and the volume of each well was 3mL. The experimental group, positive control group and model control group were induced with potassium oxonate combined with aristolochic acid to establish a nephritis model in zebrafish. The experimental group was given water-soluble soybean peptide S1 and soybean peptide S2 at concentrations of 5, 25, and 125μg / mL. The positive control group was given allopurinol at a concentration of 136μg / mL. The normal control group used fish farming water to treat zebrafish, and the model control group used fish farming water to treat zebrafish.

[0144] 1. After each group was treated for 1 day, the uric acid content in zebrafish was detected using the same detection method as in Experimental Example 1. The results are shown in Table 9.

[0145] Table 9 Experimental results of uric acid lowering effect after treatment with soybean peptides S1 and S2

[0146]

[0147] The uric acid content in zebrafish treated with soybean peptide was compared with that in the model control group, *P<0.05; compared with the normal control group, #P<0.05, ##P<0.01.

[0148] The results showed that the uric acid content of zebrafish in the model control group was compared with that in the normal control group, P<0.01, indicating that the model was successfully established. The uric acid content of zebrafish in the positive control group was compared with that in the model control group, P<0.01, and its uric acid reduction effect on zebrafish was 28.2%, indicating that allopurinol has a reducing effect on zebrafish uric acid. Compared with the model control group, the soybean peptide S1 group had a uric acid reduction effect of 11.4% and 14.5% in the 5μg / mL and 25μg / mL concentration groups, P<0.05, and a uric acid reduction effect of 24.7% in the 125μg / mL concentration group, indicating that soybean peptide S1 has a reducing effect on zebrafish uric acid. Compared with the model control group, the soybean peptide S2 group had a uric acid reduction effect of 11.3% and 19.3% in the 25μg / mL and 125μg / mL concentration groups, P<0.05, respectively. The results showed that soybean peptide S1 had a better uric acid reduction effect than soybean peptide S2.

[0149] 2. After each group was treated for 1 day, the creatinine content in zebrafish was detected using the same detection method as in Experimental Example 1. The results are shown in Table 10.

[0150] Table 10 Experimental results of creatinine reduction after treatment with soybean peptides S1 and S2

[0151]

[0152] The creatinine content of zebrafish treated with soybean peptide was compared with that of the model control group, *P<0.05; compared with the normal control group, #P<0.05, ##P<0.01.

[0153] The results showed that the creatinine content of zebrafish in the model control group was compared with that in the normal control group, P<0.01, indicating that the model was successfully established. The creatinine content of zebrafish in the positive control group was compared with that in the model control group, P<0.01, and its creatinine reduction effect on zebrafish was 18.5%, indicating that allopurinol has a reducing effect on zebrafish creatinine. Compared with the model control group, the soybean peptide S1 group had a creatinine reduction effect of 11.7% and 20.1% in the 25μg / mL and 125μg / mL concentration groups, P<0.01, respectively, indicating that soybean peptide S1 has a reducing effect on zebrafish creatinine under 25μg / mL and 125μg / mL concentration conditions. Compared with the normal control group, the uric acid content of the soybean peptide S2 group was significantly different in different groups. Compared with the model control group, the uric acid reduction effect of the 25μg / mL and 125μg / mL concentration groups was 7.7% and 14.6%, respectively. The results showed that the creatinine reduction effect of soybean peptide S1 was better than that of soybean peptide S2.

[0154] 3. Kidney inflammation detection: After each group was treated for 18 hours, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope. The data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the fluorescence intensity (H) of zebrafish kidney inflammation cells. The statistical analysis results of this indicator were used to evaluate the inhibitory effect of the sample on kidney inflammation. The statistical processing results were expressed as mean ± SE. The formula for calculating the inhibitory effect of the sample on kidney inflammation is as follows:

[0155]

[0156] The results are as follows Figure 6 As shown in Table 11. Figure 6 In the middle, red fluorescence indicates zebrafish nephritis cells.

[0157] Table 11 Experimental results of each group's inhibition of renal inflammatory cell efficacy evaluation (n=10)

[0158]

[0159] Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0160] The results showed that the fluorescence intensity of zebrafish kidney inflammatory cells in the 5μg / mL concentration group of soybean peptide S1 was 295055 pixels, which was P<0.01 compared with the model control group, indicating that soybean peptide S1 had a significant inhibitory effect on the growth of zebrafish kidney inflammation under this concentration condition. The fluorescence intensity of zebrafish kidney inflammatory cells in the 25μg / mL and 125μg / mL concentration groups of soybean peptide S1 was 250933 and 219110 pixels, respectively, compared with the model control group P<0.001, and the inflammation inhibition effect was 29.7% and 38.61%, respectively. This shows that soybean peptide S1 has an inhibitory effect on zebrafish kidney inflammation. The fluorescence intensity of zebrafish kidney inflammatory cells in the 5μg / mL and 125μg / mL concentration groups of soybean peptide S2 was 302772 pixels and 278807 pixels, respectively, compared with the model control group P<0.01, indicating that soybean peptide S2 has a significant inhibitory effect on zebrafish kidney inflammation under this concentration condition. The fluorescence intensity of inflammatory cells in zebrafish kidneys in the soybean peptide S2 125 μg / mL concentration group was 253213 pixels, and compared with the model control group, P<0.001, the inflammation inhibition effect was 29.06%, indicating that soybean peptide S2 also has the effect of inhibiting inflammation in zebrafish kidneys.

[0161] 4. The sample preparation steps in Experimental Example 1 were used to obtain supernatant, and MDA content, CAT activity and GSH content were detected using MDA, CAT and GSH kits. The statistical results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and P < 0.05 indicated that the difference was statistically significant. The results are shown in Table 12.

[0162] Table 12 Evaluation results of MDA content, CAT activity and GSH content in each group (n=3)

[0163]

[0164]

[0165] Compared with the model control group, **P<0.01, ***P<0.001.

[0166] The results showed that the normal control group was compared with the model control group zebrafish, the MDA content was P<0.001, the CAT activity was P<0.01, and the GSH content was P<0.01, indicating that the model was successfully established. The MDA content, CAT activity and GSH content of zebrafish in the positive control group were compared with those in the model control group, which were P<0.01, indicating that allopurinol had good antioxidant activity. Compared with the model control group, the soybean peptide S1 group had a 25μg / mL concentration group with a MDA content of P<0.01, a CAT activity of P<0.01, and a GSH content of P<0.01, and a 125μg / mL concentration group with a MDA content of P<0.001, a CAT activity of P<0.001, and a GSH content of P<0.01. Compared with the model control group, the MDA content of the soybean peptide S2 group at 25 μg / mL concentration was P<0.01, the CAT activity was P<0.01, and the GSH content was P<0.01; the MDA content of the 125 μg / mL concentration was P<0.01, the CAT activity was P<0.001, and the GSH content was P<0.01, indicating that soybean peptides S1 and S2 have good antioxidant activity at concentrations of 25 μg / mL and 125 μg / mL, which is specifically manifested in reducing the MDA content, increasing the GSH content of zebrafish and increasing the CAT activity.

[0167] Test Example 5

[0168] According to the structure of the polypeptide S1 obtained in Example 2, Shanghai Newpu Biotechnology Co., Ltd. was commissioned to synthesize it and evaluate its efficacy in assisting the improvement of nephritis in rats:

[0169] Experimental animals: 90 SPF-grade 5-week-old male C57BL / 6J mice weighing (20.0±2) g were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The experimental animal production license number is SCXK (Beijing) 2021-0006. The experimental unit use license number is SYXK (Lu) 20190005. The animals were kept in an animal room with a constant temperature (22±2)℃ and constant humidity (55±5)% and a light-dark alternation cycle of 12h / 12h. Ordinary feed was purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., and free food and drinking water. According to the principle of balanced weight of mice, the mice were randomly divided into 9 groups according to the experimental items, with 10 mice in each group.

[0170] Test substance dosage setting: The recommended intake of soybean peptide for human body (the soybean peptide mixture prepared in Example 1) of the test sample is 10g / 60kg.bw per person per day, which is equivalent to 0.1667mg / g.bw per day. The low, medium and high doses of mice are determined according to 5 times, 10 times and 30 times the recommended amount for human body, that is, the daily intake of mice in each group of sample group is 0.8335mg / g.bw, 1.667mg / g.bw and 5mg / g.bw respectively. Soy peptide S1 is 1g / 60kg.bw per person per day, which is equivalent to 0.01667mg / g.bw per day. The low, medium and high doses of mice are determined according to 5 times, 10 times and 30 times the recommended amount for human body, that is, the daily intake of mice in each group of sample group is 0.08335mg / g.bw, 0.1667mg / g.bw and 0.5mg / g.bw respectively. The recommended dose of allopurinol tablets for the positive control group is 0.2g / 60kg.bw, equivalent to 3.3mg / kg.bw. According to the equivalent dose ratio between humans and animals calculated by body surface area in the "Appendix to Pharmacological Experimental Methodology", the daily intake for mice is 46.2mg / kg.bw.

[0171] Experimental groups: After the mice were fed maintenance feed under the barrier system and observed for 7 days, they were randomly divided into 9 groups according to body weight, with 10 mice in each group, namely normal control group, model control group, positive control group, low-dose soy peptide group, medium-dose soy peptide group, high-dose soy peptide group, low-dose soy peptide S1 group, medium-dose soy peptide S1 group, and high-dose soy peptide S1 group.

[0172] Construction of experimental model and sample intervention: At 9:00 am after the adaptation period, except for the normal control group, which was gavaged with 0.50% CMC-Na solution, the other groups were gavaged with 250 mg / kg PO (suspended in 0.50% CMC-Na solution) for model construction, and the gavage volume was 0.1 mL / 10 g.bw. At 2:00 pm, mice were gavaged with the test substance. The concentrations of the test substance aqueous solution in the low, medium and high dose groups of soybean peptide samples were 83.35 mg / mL, 166.7 mg / mL and 500 mg / mL, respectively, and the concentrations of the test substance aqueous solution in the low, medium and high dose groups of soybean peptide S1 samples were 8.335 mg / mL, 16.67 mg / mL and 50 mg / mL, respectively, and the positive control group was 4.62 mg / mL. The normal control group and the model control group were gavaged with an equal volume of deionized water instead of the test substance solution, and the gavage volume was 0.1 mL / 10 g.bw. On the 33rd day, urine was collected for 24 hours using a metabolic cage. On the 35th day, 30 minutes after gavage, each group's eyeballs were removed for blood collection, and the liver and kidney tissues were dissected to measure various indicators of auxiliary uric acid-lowering function.

[0173] 1. Determination of mouse body weight and organ index: During the feeding process, the experimental animals were weighed weekly. After removing the eyeballs and drawing blood, the experimental animals were dissected, and the liver and kidneys were taken and weighed together for wet weight, and the organ index was calculated. Organ index (%) = organ weight (g) / experimental animal body weight (g) × 100%. The results are shown in Table 13.

[0174] Table 13 Body weight and organ index of mice in each group

[0175] Group Number of animals Body weight (g) Liver index Renal Index Normal control group 10 49.05±3.21 4.51±0.36 1.45±0.19 Model control group 10 46.12±5.35 4.01±0.41 1.25±0.35 Positive control group 10 48.91±4.12 4.39±0.61 1.41±0.13 Soy peptide low dose group 10 47.32±4.20 4.44±0.83 1.26±0.10 Soy peptide medium dose group 10 47.85±3.99 4.21±0.65 1.29±0.23 Soy peptide high dose group 10 48.59±4.30 4.49±0.72 1.35±0.31 Soy peptide S1 low dose group 10 46.55±4.33 4.19±0.58 1.26±0.15 Soy peptide S1 medium dose group 10 47.61±3.12 4.34±0.66 1.25±0.22 Soy peptide S1 high dose group 10 47.72±4.68 4.39±0.28 1.33±0.15

[0176] *Compared with the model control group, P<0.05; **Compared with the model control group, P<0.01.

[0177] The results showed that compared with the normal control group, the body weight of the model group decreased, but there was no significant difference in the final body weight (P>0.05). Compared with the model control group, there was no statistically significant difference in body weight, liver index, and kidney index among the groups (P>0.05).

[0178] 2. Determination of blood uric acid, blood urea nitrogen and blood creatinine: After blood collection, let it stand at room temperature for 1 hour, centrifuge it at 3000r / min for 10 minutes, take serum, and determine SUA, BUN and SCr contents according to the kit. The results are shown in Table 14.

[0179] Table 14 Blood uric acid, blood urea nitrogen and blood creatinine levels in each group of mice

[0180]

[0181]

[0182] *Compared with the model control group, P<0.05; **Compared with the model control group, P<0.01.

[0183] Compared with the normal control group, the blood uric acid level in the model control group was significantly increased (P<0.01), indicating that the nephritis mouse model was successfully established; the blood urea nitrogen and blood creatinine levels in the model control group were significantly increased (P<0.01), indicating that the renal function injury model was successfully established. Compared with the model control group, the positive control group and each dose group of soybean peptide S1 were reduced (P<0.05, P<0.01). Compared with the positive control group and the model control group, the levels of uric acid, creatinine and urea nitrogen in the positive control group were significantly reduced, indicating that the positive drug has a good renal function protective effect, close to the normal control group. The levels of uric acid, creatinine and urea nitrogen in the soybean peptide group showed a dose-dependent gradient downward trend. Among them, the medium and high dose groups of soybean peptide had significant and extremely significant differences (P<0.05, P<0.01). The effect of reducing urea nitrogen in the high dose group was close to that of the positive control group, indicating that soybean peptide has a strong renal function protective effect. The various dose groups of soybean peptide S1 were superior to the common soybean peptide group in reducing uric acid, improving creatinine and urea nitrogen levels, especially the high-dose group, which had a significant improvement effect, and was close to the positive drug group in reducing uric acid, improving creatinine and urea nitrogen. The creatinine and urea nitrogen levels of the high-dose soybean peptide S1 group were close to those of the positive control group, showing its excellent renal function protection effect.

[0184] 3. Determination of xanthine oxidase (XOD) and adenosine deaminase (ADA) activity in liver tissue: liver tissue was mixed with pre-cooled physiological saline in an ice water bath at a ratio of 1 g: 9 mL, and then made into 10% homogenate using a tissue homogenizer, centrifuged at 2500 r / min for 10 min, the lipid layer was carefully removed, and the supernatant was taken to determine the XOD and ADA activities according to the kit. The results are shown in Table 15.

[0185] Table 15 XOD and ADA values ​​of mice in each group

[0186] Group Number of animals XOD(U / g) ADA(U / g) Normal control group 10 <![CDATA[12.56±2.67 ** ]]> <![CDATA[0.63±0.04 ** ]]> Model control group 10 21.71±3.31 1.41±0.14 Positive control group 10 <![CDATA[14.06±2.16 ** ]]> <![CDATA[0.81±0.09 ** ]]> Soy peptide low dose group 10 19.41±3.72 1.31±0.13 Soy peptide medium dose group 10 <![CDATA[17.02±2.61 * ]]> <![CDATA[1.03±0.10 * ]]> Soy peptide high dose group 10 <![CDATA[15.34±2.81 ** ]]> <![CDATA[0.93±0.12 ** ]]> Soy peptide S1 low dose group 10 18.79±3.13 1.23±0.16 Soybean peptide S1 medium dose group 10 <![CDATA[16.41±2.69 ** ]]> <![CDATA[1.13±0.08 * ]]> Soy peptide S1 high dose group 10 <![CDATA[14.73±1.65 ** ]]> <![CDATA[0.94±0.10 ** ]]>

[0187] *Compared with the model control group, P<0.05; **Compared with the model control group, P<0.01.

[0188] The results showed that the XOD and ADA activities in the model control group were significantly higher than those in the normal control group, indicating that the mice in the model group experienced oxidative stress and abnormal adenosine metabolism, suggesting that the model was successfully established. The XOD and ADA activities in the positive control group were significantly reduced, especially the reduction of XOD was more obvious, which was significantly different from the model control group (P<0.01), indicating that the positive control drug had good antioxidant and anti-inflammatory effects. Soy peptide low-dose group: The reduction in XOD and ADA activities was small and did not reach a significant difference (P>0.05). The XOD and ADA activities in the medium-dose group of soybean peptides decreased significantly, especially the effect of the medium-dose group was more significant (P<0.05). The XOD and ADA activities in the high-dose group of soybean peptides decreased significantly, with a larger decrease, close to the level of the positive control group, with a significant difference (P<0.01), indicating that the high-dose group has stronger antioxidant and anti-inflammatory effects. The XOD and ADA activities in the low-dose group of soybean peptide S1 decreased, but the effect was slightly weaker than that of the low-dose group of ordinary soybean peptides. Soy peptide S1 medium-dose group: XOD and ADA activities decreased significantly, and the effect was weaker than that of ordinary soy peptide medium-dose group. Soy peptide S1 high-dose group: OD and ADA activities decreased significantly, close to the positive control group, and the decrease was close to that of ordinary soy peptide high-dose group, showing extremely significant differences (P<0.01).

[0189] 4. Pathological observation of renal tissue: The same part of liver tissue and kidney tissue was fixed in 4% neutral formaldehyde fixative, dehydrated and embedded to make paraffin sections, and HE staining was performed and observed under a microscope.

[0190] Histopathological examination of the liver of experimental mice (HE staining, 20×) Figure 7 The results showed that the liver tissue cells in each group had normal morphology and were arranged neatly and orderly.

[0191] The histopathological examination of the kidneys of experimental mice (HE staining, 20×) was as follows Figure 8 The results showed that compared with the normal control group, the renal tissue of the model group had obvious lesions, manifested as cell atrophy, tubular dilatation, inflammatory cell infiltration, etc. The test sample group was improved compared with the model group.

[0192] 5. Uric acid content determination: fresh urine at 3000r·min -1 After centrifugation for 15 minutes, the sediment was removed, and the supernatant was taken to determine the UUA content according to the kit. The results are shown in Table 16.

[0193] Table 16 Uric acid content in urine of mice in each experimental group

[0194] Group Number of animals Urine uric acid (mg / L) Normal control group 10 60.10±16.03 Model control group 10 169.50±25.81 Positive control group 10 <![CDATA[86.29±31.26 ** ]]> Soy peptide low dose group 10 139.51±27.92 Soy peptide medium dose group 10 <![CDATA[105.08±28.30 * ]]> Soy peptide high dose group 10 <![CDATA[96.21±20.38 ** ]]> Soy peptide S1 low dose group 10 <![CDATA[126.82±22.13 * ]]> Soybean peptide S1 medium dose group 10 <![CDATA[95.31±23.63 ** ]]> Soy peptide S1 high dose group 10 <![CDATA[86.07±29.06 ** ]]>

[0195] *Compared with the model control group, P<0.05; **Compared with the model control group, P<0.01.

[0196] The results showed that the uric acid level in the model control group was significantly higher than that in the normal control group (about 1.6 times), indicating that the model successfully induced a hyperuric acid state. The uric acid level in the positive control group was significantly reduced, with a very significant difference compared with the model control group (P<0.01), indicating that the positive drug has a good uric acid-lowering effect, close to the level of the normal control group. The uric acid level in the low-dose group of soybean peptide was not significantly reduced, and there was no significant difference from the model control group (P>0.05). The uric acid level in the medium-dose group of soybean peptide was significantly reduced (P<0.05), showing a significant uric acid-lowering effect. The uric acid level in the high-dose group of soybean peptide was further reduced, close to the level of the positive control group, with a very significant difference (P<0.01). The uric acid level in the low-dose group of soybean peptide S1 was slightly better than that in the low-dose group of ordinary soybean peptide, with a significant difference. The uric acid level in the medium-dose group of soybean peptide S1 was significantly reduced, and the uric acid-lowering effect was better than that in the medium-dose group of ordinary soybean peptide, indicating that soybean peptide S1 has higher biological activity at the same dose. The uric acid level in the high-dose soybean peptide S1 group was close to that in the positive control group, and further decreased compared with the high-dose soybean peptide group, showing a very significant difference (P<0.01).

[0197] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a soybean peptide mixture, characterized in that: The following steps are involved: (1) adding deionized water with a pH of 9.5 to 10.5 to prepare a soy protein solution with a concentration of 4.5 to 5.5%; (2) grinding and homogenizing the soy protein solution to obtain a soy protein homogenate having a protein particle size of 30 to 80 μm; (3) The soy protein homogenate is kept at 53-58° C. and stirred for 0.5-1 h, the pH value is adjusted to 9.5-10.5, alkaline protease is added, and enzymolysis is performed until the pH value drops to 7.0-7.5, papain and neutral protease are added, and flavor protease is added after enzymolysis for 3.5-4.5 h, and enzymolysis is continued for 2-2.5 h to obtain an enzymolysis solution; (4) the enzymatic hydrolyzate is heated to 108-112° C. and maintained for 3-10 min, then cooled to room temperature, filtered using a ceramic membrane, and the clear liquid filtered by the ceramic membrane is filtered using a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to obtain a nanofiltration membrane cutoff liquid; (5) The intercepted liquid of the nanofiltration membrane is sequentially passed through a cation exchange resin and an anion exchange resin at a flow rate of 48 to 52 L / h. The material after resin adsorption is concentrated, sterilized, and dried to obtain a soybean peptide mixture.

2. The preparation method according to claim 1, characterized in that: Based on the mass of soybean protein homogenate, the addition amount of alkaline protease is 1.5-2.5 million U / g, the addition amount of papain is 700,000-900,000 U / g; the addition amount of neutral protease is 2.5-3.5 million U / g; and the addition amount of flavor protease is 400,000-600,000 U / g.

3. The preparation method according to claim 1, characterized in that: The soybean peptide mixture was separated and purified by preparative high performance liquid chromatography, and the purified soybean peptide mixture was obtained by gradient elution with mobile phase A: 0.1% trifluoroacetic acid + 99.9% ultrapure water and mobile phase B: 0.1% trifluoroacetic acid + 99.9% acetonitrile.

4. The preparation method according to claim 3, characterized in that: The method for preparing the sample solution separated and purified by the preparative high performance liquid chromatography comprises: dissolving the soybean peptide mixture with mobile phase A, preparing a soybean peptide solution, centrifuging, filtering the supernatant with a filter membrane, and using the filtrate as the sample solution.

5. The preparation method according to claim 3, characterized in that: The elution procedure of the separation and purification by the preparative high performance liquid chromatography is: 0% to 5% B for 0 to 5 minutes, 5% B to 80% B for 5 to 20 minutes; the elution fraction collected for 11 to 12 minutes is the purified soybean peptide mixture.

6. The soybean peptide mixture prepared by the preparation method according to any one of claims 1 to 5.

7. The soybean peptide mixture according to claim 6, characterized in that The soybean peptide mixture comprises polypeptide S1 and polypeptide S2.

8. Polypeptide S1, characterized in that The polypeptide S1 is a single-chain polypeptide with a structural sequence of YDPSTGIY and a peptide chain molecular weight of 914.95 Da.

9. Polypeptide S2, characterized in that The polypeptide S2 is a single-chain polypeptide with a structural sequence of VNEPAPGLK and a peptide chain molecular weight of 924.05 Da.

10. Use of the soybean peptide mixture according to any one of claims 6 to 7, the polypeptide S1 according to claim 8, or the polypeptide S2 according to claim 9 in the preparation of a medicament for preventing and / or treating chronic nephritis.

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