An active peptide with both lipid-lowering and uric acid-lowering functions, and its preparation method and application

Abstract

The present invention discloses an active peptide having both lipid-lowering and uric acid-lowering functions, and its preparation method and application, which belong to the field of biotechnology. The preparation method of the active peptide of the present invention comprises the following steps: taking quinoa powder, adding ethanol, reacting, and drying to obtain defatted quinoa powder; adding alkali solution, reacting; filtering and centrifuging the reaction solution, collecting the supernatant and adjusting, standing and then centrifuging, removing the supernatant, and retaining the protein precipitate; freeze-drying to obtain a quinoa protein crude extract; adding water to form a solution, adding protease for enzymolysis to obtain an enzymolysis solution; terminating the enzymolysis reaction, centrifuging, and collecting the supernatant. The active peptide provided by the present invention has good results in three in vitro lipid-lowering activity evaluation indicators: pancreatic lipase inhibition, sodium taurocholate binding ability, and cholesterol esterase inhibition. In addition, the xanthine oxidase inhibition effect of the quinoa protein hydrolysate shows that the quinoa protein hydrolysate under the optimal process conditions has good uric acid-lowering activity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an active peptide having both blood lipid-lowering and uric acid-lowering functions, and a preparation method and application thereof. Background Art

[0002] Quinoa is considered a functional food due to its high-quality protein. The peptides released by enzymatic hydrolysis of quinoa protein exhibit high biological activity. Currently, little research has been conducted on the lipid-lowering effects of quinoa protein and its peptides. Further research is needed to develop peptides from quinoa's high-quality protein that exhibit lipid-lowering and uric acid-lowering activities.

[0003] Hyperlipidemia is a systemic metabolic syndrome that can lead to abnormally elevated levels of triglycerides, cholesterol, and low-density lipoprotein (LDL). Hyperlipidemia patients experience inactive LDL clearance and LDL receptor expression, leading to elevated liver fat and, consequently, elevated blood cholesterol. Consequently, hyperlipidemia is increasingly endangering public health and quality of life.

[0004] Protein components in foods have been shown to modulate dyslipidemia, with their hypolipidemic effects likely derived from peptide fragments and arginine residues. The mechanism of action of lipid-lowering peptides is primarily through disruption of micellar solubility and dietary cholesterol absorption, thereby altering hepatobiliary bile acid metabolism, promoting cholesterol catabolism, and regulating lipoprotein and gene expression. The development of safe and effective lipid-lowering peptides from natural foods is imperative for fundamental regulation in preventing hyperlipidemia.

[0005] Xanthine oxidase is a key enzyme in purine metabolism in the body, catalyzing the conversion of hypoxanthine to xanthine, which is then oxidized to metabolites such as uric acid, pyrimidines, and purines. Excessive accumulation of uric acid in the body can lead to symptoms such as diarrhoea and kidney stones. Inhibiting the catalytic activity of xanthine oxidase can reduce uric acid accumulation. Therefore, uric acid-lowering activity is an effective treatment for patients with hyperuricemia. Currently, no research has been conducted on the uric acid-lowering function of quinoa protein peptides. Therefore, the development of natural uric acid-lowering active peptides has become a new research direction. Summary of the Invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing an active peptide having both lipid-lowering and uric acid-lowering functions.

[0007] Another object of the present invention is to provide an active peptide having both lipid-lowering and uric acid-lowering functions obtained by the above preparation method.

[0008] Another object of the present invention is to provide the use of the above-mentioned active peptide having both lipid-lowering and uric acid-lowering functions.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for preparing an active peptide having both lipid-lowering and uric acid-lowering functions comprises the following steps:

[0011] S1. Take quinoa powder, add ethanol, react, and dry to obtain defatted quinoa powder;

[0012] S2. Take the defatted quinoa powder obtained in step S1, add alkali solution, and react; filter and centrifuge the reaction solution, collect the supernatant and adjust the pH to 4.5±0.2, let it stand and then centrifuge, remove the supernatant, and retain the protein precipitate; freeze-dry to obtain a crude quinoa protein extract;

[0013] S3, taking the crude quinoa protein extract obtained in step S2, adding water to form a solution, adding protease for enzymatic hydrolysis, and obtaining an enzymatic hydrolyzate;

[0014] S4. Inactivate the protease in the enzymatic hydrolysis solution obtained in step S3, terminate the enzymatic hydrolysis reaction, centrifuge, and collect the supernatant to obtain the active peptide with both lipid-lowering and uric acid-lowering functions.

[0015] Preferably, the protease used in step S3 is any one or more of trypsin, papain, pepsin, alkaline protease and neutral protease, and the enzymatic hydrolysis conditions are: temperature 32-52° C., pH 1-3, enzymatic hydrolysis time 1-5 h, enzyme-substrate ratio 0.1%-0.5%, substrate concentration 1 wt%-5 wt%;

[0016] More preferably, the enzyme used is pepsin, and the enzymatic hydrolysis conditions are: temperature 37-47°C, pH 1-2, enzymatic hydrolysis time 1-1.5h, enzyme-substrate ratio 0.18-0.22%, substrate concentration 2wt%-4wt%;

[0017] Most preferably, the enzyme used is pepsin, and the enzymatic hydrolysis conditions are: temperature 42.88° C., pH 1.57, enzymatic hydrolysis time 1 h, enzyme-substrate ratio 0.2%, and substrate concentration 3.03 wt %.

[0018] Preferably, the quinoa powder in step S1 is obtained by grinding quinoa and passing it through a 10-30 mesh sieve; more preferably, it is obtained by passing it through a 20 mesh sieve.

[0019] Preferably, the ethanol in step S1 is 90% to 95% ethanol; more preferably, it is 95% ethanol.

[0020] Preferably, the material-liquid ratio of quinoa powder and ethanol in step S1 is 1 g:10-30 mL; more preferably 1 g:20 mL.

[0021] Preferably, the reaction time in step S1 is 10 to 15 hours, more preferably 12 hours.

[0022] Preferably, the reaction temperature in step S1 is room temperature, which in the present invention refers to 20-30°C.

[0023] Preferably, the alkali solution in step S2 is a NaOH solution; more preferably, it is a 0.1 mol / L NaOH solution.

[0024] Preferably, the solid-to-liquid ratio of the defatted quinoa powder to the alkali solution in step S2 is 1 g:30-50 mL; more preferably 1 g:40 mL.

[0025] Preferably, the reaction conditions in step S2 are a temperature of 40-50° C. and a time of 2-4 h; more preferably, a temperature of 45° C. and a time of 3 h.

[0026] Preferably, the standing time in step S2 is 10 to 30 minutes, more preferably 20 minutes.

[0027] Preferably, the centrifugal conditions in steps S2 and S3 are both a rotation speed of 3000-5000 r / min and a time of 10-30 min; more preferably, a rotation speed of 4000 r / min and a time of 20 min.

[0028] Preferably, the reagents used to adjust the pH in steps S2 and S3 are 0.1 mol / L NaOH solution and HCl solution.

[0029] Preferably, the protease inactivation method in step S4 is to react in boiling water for 8 to 12 minutes; more preferably, the protease inactivation method is to react in boiling water for 10 minutes.

[0030] Preferably, after the active peptide with both lipid-lowering and uric acid-lowering functions is obtained in step S4, it is frozen for later use, and the freezing temperature is -20±5°C.

[0031] An active peptide with both blood lipid-lowering and uric acid-lowering functions is prepared by the above preparation method.

[0032] The use of the above active peptide having both lipid-lowering and uric acid-lowering functions in at least one of the following situations:

[0033] (1) Application in the preparation of pancreatic lipase activity inhibitors;

[0034] (2) Application in the preparation of bile salt adsorbents;

[0035] (3) Application in the preparation of cholesterol esterase activity inhibitors;

[0036] (4) Application in the preparation of lipid-lowering drugs.

[0037] The use of the above active peptide having both lipid-lowering and uric acid-lowering functions in at least one of the following situations:

[0038] (1) Application in the preparation of xanthine oxidase inhibitors;

[0039] (2) Application in the preparation of uric acid-lowering drugs;

[0040] (3) Application in the preparation of drugs for treating hyperuricemia;

[0041] (4) Application in the preparation of drugs for treating hyperuricemia and lowering blood lipids.

[0042] Preferably, in the application of preparing a pancreatic lipase activity inhibitor, the concentration of the active peptide is 10 to 500 μg / mL.

[0043] Preferably, in the application of preparing bile salt adsorbent, the concentration of the active peptide is 0.25-2 mg / mL.

[0044] Preferably, in the application of preparing cholesterol esterase activity inhibitor, the concentration of the active peptide is 0.1-2 μg / mL.

[0045] Preferably, in the application of preparing xanthine oxidase inhibitors or uric acid-lowering drugs or drugs for treating hyperuricemia, the concentration of the active peptide is 1 to 8 μg / mL.

[0046] The present invention has the following advantages and effects compared to the prior art:

[0047] The present invention uses quinoa as raw material to extract protein, and uses the pancreatic lipase inhibition ability as the determination index. The process of preparing quinoa protein hypolipidemic peptide is optimized through single factor experiment and response surface analysis. The quinoa protein hypolipidemic peptide prepared under the optimal enzymatic hydrolysis conditions is analyzed for its pancreatic lipase inhibition effect, sodium taurocholate binding effect, cholesterol pancreatic enzyme inhibition effect and xanthine oxidase inhibition effect, and the amino acid composition of the quinoa protein hypolipidemic peptide is analyzed. The results show that the optimal preparation conditions of quinoa protein hypolipidemic peptide are pepsin hydrolysis for 1h, temperature 42.88℃, pH 1.57, substrate concentration 3.03% and enzyme-substrate ratio 0.2%. At this time, the inhibition rate of the protease hydrolyzate on pancreatic lipase is 90.93%±0.10%. The activity evaluation results show that the quinoa protein hydrolyzate has a pancreatic lipase inhibition effect (IC 50 =7.49 μg / ml), sodium taurocholate binding capacity (EC 50 =0.53 mg / mL) and cholesterol esterase inhibition (IC 50=4.73mg / mL) and the results of the three in vitro lipid-lowering activity evaluation indicators were good. In addition, the xanthine oxidase inhibitory effect of quinoa protein hydrolysate (IC 50 =5.97 mg / mL), indicating that the quinoa protein hydrolysate under the optimal process conditions has excellent uric acid-lowering activity. Amino acid analysis showed that the resulting quinoa protein peptide is rich in essential amino acids (34.23%), hydrophobic amino acids (34.11%), and acidic amino acids (31.66%). This study contributes to the high-value utilization of quinoa resources and provides a theoretical basis for the development of quinoa protein lipid-lowering peptides.

[0048] Figure 1 This is the effect of protease types on the pancreatic lipase inhibition rate of enzymatic hydrolysates;

[0049] Figure 2 This is the effect of enzymatic hydrolysis time on the pancreatic lipase inhibition rate of the enzymatic hydrolysate;

[0050] Figure 3 This is the effect of pH on the pancreatic lipase inhibition rate of the enzymatic hydrolysate;

[0051] Figure 4 This is the effect of enzymatic hydrolysis temperature on the pancreatic lipase inhibition rate of the enzymatic hydrolyzate;

[0052] Figure 5 This is the effect of enzyme-substrate ratio on pancreatic lipase inhibition rate of enzymatic hydrolysate;

[0053] Figure 6 This is the effect of substrate concentration on the pancreatic lipase inhibition rate of the enzymatic hydrolysate;

[0054] Figure 7 This is a graph showing the effects of temperature, pH, and substrate concentration on pancreatic lipase inhibition;

[0055] Figure 8 is a graph of pancreatic lipase inhibition at different sample concentrations;

[0056] Figure 9 is a graph of sodium taurocholate binding rates at different sample concentrations;

[0057] Figure 10 is a graph of cholesterol esterase inhibition at different sample concentrations;

[0058] Figure 11 is a graph of xanthine oxidase inhibition at different sample concentrations. DETAILED DESCRIPTION

[0059] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0060] The sources of the experimental materials in the following examples are as follows: quinoa was provided by the Key Laboratory of Cereal Processing of the Ministry of Agriculture and Rural Affairs; pepsin (3×106 U / mg), neutral protease (1×105 U / g), trypsin (2.5×105 U / g), and papain (2×105 U / g) were purchased from Nanning Pangbo Bioengineering Co., Ltd.; sodium taurocholate (STC), porcine pancreatic lipase, p-nitrophenyl laurate (pNP laurate), and p-nitrophenyl butyrate (PNPB) were purchased from Shanghai MacLean Biochemical Co., Ltd.; cholesterol esterase was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; other chemicals and reagents were of analytical grade.

[0061] Example 1

[0062] 1 Extraction of quinoa protein

[0063] Quinoa protein extraction is based on the principle of alkaline extraction and acid precipitation. After quinoa is crushed, it is passed through a 20-mesh sieve. A certain amount of quinoa powder is weighed and reacted continuously at room temperature for 12 hours with 95% ethanol at a solid-liquid ratio of 1:20. Defatted quinoa powder is then dried to obtain a certain amount of defatted quinoa powder. A 0.1 mol / L NaOH solution is added at a solid-liquid ratio of 1:40. After being water-bathed at 45°C for 3 hours, the mixture is filtered and centrifuged at 4000 r / min for 20 minutes. The supernatant is collected and the pH is adjusted to 4.5. After standing for 20 minutes, it is centrifuged at 4000 r / min for 20 minutes. After removing the supernatant, the protein precipitate is retained and freeze-dried to obtain a crude quinoa protein extract, which is then stored at -20°C.

[0064] 2 Preparation of quinoa protein peptide

[0065] A fixed amount of crude quinoa protein extract was weighed and added to an appropriate amount of distilled water to form a solution. After adjusting the pH to a specific value, a specific amount of enzyme was added. The solution was hydrolyzed in a constant-temperature water bath for 3 hours, then inactivated in boiling water for 10 minutes. After cooling to room temperature, the solution was centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected as the enzymatic solution containing the polypeptides. The pH was adjusted using 0.1 mol / L NaOH and HCl solutions.

[0066] 3 Single-factor experiments

[0067] 3.1 Screening of proteases

[0068] Trypsin, papain, pepsin, alkaline protease and neutral protease were selected to hydrolyze quinoa protein under their most suitable conditions. The optimal pH and temperature of these five enzymes were 8.0, 37℃; 6.5, 55℃; 1.5, 37℃; 9.5, 55℃ and 7.0, 45℃, respectively. Under the conditions of substrate concentration of 3% and enzyme-substrate ratio of 0.3%, enzymatic hydrolysis was carried out for 3 hours, enzyme inactivation for 10 minutes, centrifugation at 4000r / min for 20 minutes, and the supernatant hydrolyzate was taken to determine the pancreatic lipase inhibition rate to select the optimal protease.

[0069] 3.2 Effect of enzymatic hydrolysis time on the activity of enzymatic hydrolysates

[0070] Under the conditions of substrate concentration of 3%, enzyme-substrate ratio of 0.3%, pH 1.5 and 37°C, enzymatic hydrolysis was carried out with pepsin (1, 2, 3, 4, 5 h). The pancreatic lipase inhibition rate was used as an indicator, and the enzymatic hydrolysis time with the highest inhibition rate was selected as the optimal condition.

[0071] 3.3 Effect of pH on enzymatic activity

[0072] Under conditions of substrate concentration of 3%, enzyme-substrate ratio of 0.3%, and 37°C, enzymatic hydrolysis was performed with pepsin at different pH values (1.0, 1.5, 3.0, 2.5, and 3.0). The pancreatic lipase inhibition rate was used as an indicator, and the pH with the highest inhibition rate was selected as the optimal condition.

[0073] 3.4 Effect of enzymatic hydrolysis temperature on the activity of enzymatic hydrolysates

[0074] Under the conditions of substrate concentration of 3%, enzyme-substrate ratio of 0.3%, and pH 1.5, enzymatic hydrolysis temperatures (32, 37, 42, 47, 52°C) were selected, and enzymatic hydrolysis was carried out with pepsin. The pancreatic lipase inhibition rate was used as an indicator, and the enzymatic hydrolysis time with the highest inhibition rate was selected as the optimal condition.

[0075] 3.5 Effect of enzyme-substrate ratio on hydrolysate activity

[0076] Enzymatic hydrolysis was carried out with pepsin under the conditions of substrate concentration 3%, pH 1.5, 37°C, and enzymatic hydrolysis for 3 h. The pancreatic lipase inhibition rate was used as an indicator under different enzyme-substrate ratios (0.1%, 0.2%, 0.3%, 0.4%, and 0.5%), and the pH with the highest inhibition rate was selected as the optimal condition.

[0077] 3.6 Effect of substrate concentration on enzymatic activity

[0078] Under the conditions of enzyme-substrate ratio of 0.3%, pH 1.5, and 37°C, pepsin was used for enzymatic hydrolysis for 3 h. The substrate concentrations (1%, 2%, 3%, 4%, and 5%) were examined. The pancreatic lipase inhibition rate was used as an indicator, and the substrate concentration with the highest inhibition rate was selected as the optimal condition.

[0079] 4 Response surface optimization experiment

[0080] In the single-factor experiment, the optimal conditions of enzymatic hydrolysis time, pH, enzymatic hydrolysis temperature, enzyme-substrate ratio, and substrate concentration were selected as independent variables. A three-factor, three-level response surface experiment was designed with pancreatic lipase inhibition as the response value. The response surface experiment design is shown in Table 1.

[0081] Table 1 Response surface factors and level design

[0082]

[0083]

[0084] 5. Determination of pancreatic lipase inhibition rate

[0085] Type II porcine pancreatic lipase is prepared into 5 mg / mL with first-grade water. A certain amount of reaction substrate p-nitrophenyl laurate (pNP laurate) is dissolved in a sodium acetate aqueous solution containing 1% Triton X-100 to prepare a 0.1% (w / v) pNP laurate solution. Heat in a water bath to accelerate dissolution, cool to room temperature or cool for later use. Add 250 μL of reaction substrate, 100 μL of enzymatic solution and 200 μL of reaction buffer to a centrifuge tube, and finally add 150 μL of lipase solution to start the reaction. React at 37°C for 2 hours, centrifuge at 10,000 rpm for 1 minute, and measure the absorbance of the supernatant at a wavelength of 420 nm. For the blank control, replace the sample with buffer solution, and for the sample control, replace the lipase solution with reaction buffer. Calculate the lipase inhibition rate and calculate the IC 50 The value is calculated according to the following formula:

[0086]

[0087] Where: A1 is the absorbance of the sample supernatant; A2 is the absorbance of the sample control supernatant; A0 is the absorbance of the blank supernatant.

[0088] Analysis of the lipid-lowering activity of quinoa protein peptides

[0089] 6.1 Inhibitory effect of quinoa protein peptide on pancreatic lipase

[0090] The enzymatic hydrolysate obtained under the optimal process conditions was freeze-dried and then prepared into sample solutions of different concentrations (10, 50, 100, 200, 500 μg / mL) with phosphate buffer solution. The pancreatic lipase inhibition rate was determined and the IC 50 The value is the polypeptide concentration when the pancreatic lipase inhibition rate reaches 50%.

[0091] 6.2 Adsorption capacity of quinoa protein peptides for bile salts

[0092] Transfer 1 mL of the sample to be tested to a centrifuge tube. Add 1 mL of 0.01 mol / L HCl solution to each tube to simulate the acidic environment of the human stomach. After incubation at 37°C in a shaking waterbath for 1 hour, adjust the pH of the solution to 6.3 with 0.1 mol / L NaOH, then add 5 mL of a prepared sodium taurocholate (STC) standard solution (1 mmol / mL). Add 5 mL of phosphate buffer to a blank tube. After constant shaking at 37°C for 1 hour, centrifuge at 4000 rpm for 20 minutes. Transfer 2.5 mL of the supernatant to a new centrifuge tube, add 7.5 mL of 60% sulfuric acid solution, incubate at 70°C for 30 minutes, and cool to room temperature. Finally, measure the absorbance of each sample at 387 nm. Each sample is analyzed in triplicate, and the values are determined using a bile salt standard curve. The standard curve is generated by adding 0.5, 1.0, 1.5, 2.0, and 2.5 mL of sodium taurocholate solution at a concentration of 1 mmol / L. The bile salt binding capacity was calculated using the following formula:

[0093]

[0094] Where: C0 is the concentration of bile salt blank (without peptide); C1 is the concentration of bile salt in the supernatant after adding peptide.

[0095] 6.3 Inhibitory effect of quinoa protein peptide on cholesterol esterase

[0096] Porcine pancreatic cholesterol esterase was prepared at 4.2 μg / mL using first-grade water. All reactions were carried out in sodium phosphate buffer (0.1 mol / L, pH 7.0) containing NaCl (0.1 mol / L), p-nitrophenylbutyrate PNPB (0.2 mmol / L), and sodium taurocholate STC (5.16 mmol / L). 10 μL of reaction substrate PNPB, 25 μL of sample solution, and 50 μL of buffer were added to a centrifuge tube. Finally, 50 μL of cholesterol esterase solution was added to start the reaction. The reaction was carried out at 25°C for 5 minutes, and the absorbance was measured at 405 nm. The blank tube used first-grade water instead of the sample solution, the blank control tube used first-grade water instead of the enzyme solution and sample solution, and the background control tube used first-grade water instead of the enzyme solution. Calculate the cholesterol esterase inhibitory activity and calculate the IC 50 The value is calculated as follows:

[0097]

[0098] Where: A is the absorbance of the supernatant in the blank tube; B is the absorbance of the supernatant in the blank control tube; C is the absorbance of the supernatant in the sample tube; and D is the absorbance of the supernatant in the sample control tube.

[0099] 6.4 Inhibitory effect of quinoa protein peptide on xanthine oxidase

[0100] Prepare sample solutions of varying concentrations (0.5, 1, 2, 4, and 8 mg / mL) using buffer solution. Add 50 μL of the sample, 50 μL of 0.04 U / mL xanthine oxidase solution, and 50 μL of 0.48 mmol / L xanthine solution to a 96-well plate. Mix by oscillation at room temperature. After 30 minutes, measure the absorbance at 290 nm. Calculate the xanthine oxidase inhibition rate using the following formula:

[0101]

[0102] Where: A1 is the absorbance of the supernatant in the sample tube; A2 is the absorbance of the supernatant in the sample control tube without enzyme; A3 is the absorbance of the supernatant in the blank tube; A4 is the absorbance of the supernatant in the blank tube without enzyme.

[0103] 7 Amino acid composition determination

[0104] The amino acid composition of the optimal enzymatic hydrolysate was determined using GB 5009.124-2016 “Determination of amino acids in foods” (National Health and Family Planning Commission of the People’s Republic of China, China Food and Drug Administration. National Food Safety Standard Determination of amino acids in foods).

[0105] 8 Statistical analysis

[0106] All experiments were repeated at least three times. Data were analyzed using SPSS 21.0 software, and the results are expressed as mean ± standard deviation (SD). Response surface optimization experiments were designed and analyzed using Design Expert 8.0.6.1 software. One-way analysis of variance (ANOVA) was used to analyze the significance of differences between samples (P < 0.05).

[0107] 8.1 Single-factor experiment

[0108] 8.1.1 Screening of proteases

[0109] The substrate specificity of proteases determines the site of action of the enzymatic hydrolysis reaction, resulting in different compositions and properties of the hydrolysis products under the action of different proteases. Using the pancreatic lipase inhibition rate as the activity index of protease enzymatic hydrolysis of quinoa protein, five different proteases were selected to prepare quinoa protein hydrolysates. The results are as follows Figure 1 As shown, the pancreatic lipase inhibition rate of quinoa protein peptide obtained by pepsin was higher than that of the other four enzymes, reaching 81.64% ± 0.458%, indicating that pepsin has a strong ability to inhibit pancreatic lipase against quinoa protein. Pepsin will be used in subsequent enzymatic hydrolysis process optimization experiments.

[0110] 8.1.2 Effect of enzymatic hydrolysis time on the activity of enzymatic hydrolysates

[0111] like Figure 2 As shown, the pancreatic lipase inhibition rate of the hydrolyzate decreased with increasing hydrolysis time, reaching a maximum of 85.61% ± 0.58% (P < 0.05) after 1 hour of hydrolysis. After 1 hour of reaction, the enzyme activity gradually decreased, and the degree of inhibition gradually stabilized. This may be because the enzymatic activity is higher in a shorter hydrolysis time, and the quinoa protein has more cleavage sites, resulting in the best inhibitory effect on lipase by the obtained peptide. However, longer hydrolysis time will gradually destroy the active structure of the peptide and hydrolyze it into free amino acids, reducing the inhibitory activity of lipase. Therefore, a hydrolysis time of 1 hour was selected for the next step of enzymatic process optimization experiment.

[0112] 8.1.3 Effect of pH on Enzyme Hydrolysate Activity

[0113] like Figure 3 As shown, the inhibitory effects of peptides obtained by enzymatic hydrolysis under different pH conditions showed significant differences. The pancreatic lipase inhibition rate of the enzymatic peptides first increased and then decreased with increasing pH, reaching a maximum of 81.29% ± 2% (P < 0.05) at pH 1.5. The optimal enzymatic activity of pepsin lies between 1.0 and 2.0. pH values below or above this range alter the enzyme's spatial structure, reducing enzymatic activity and leading to decreased pancreatic lipase inhibition. Therefore, pH values of 1, 1.5, and 2 were selected for subsequent response surface optimization.

[0114] 8.1.4 Effect of enzymatic hydrolysis temperature on the activity of enzymatic hydrolysates

[0115] like Figure 4 As shown, the inhibitory activity of the peptide hydrolysate against pancreatic lipase first increased and then decreased with temperature. At 42°C, the pancreatic lipase inhibition rate of the hydrolysate reached a maximum of 87.77% ± 1.73% (P < 0.05). Pepsin exerts its maximum activity at the optimal temperature, accelerating molecular thermal motion and enabling solvent penetration and solute diffusion. This results in sufficient enzymatic degradation of quinoa protein, resulting in more active peptides in the hydrolysate. Furthermore, above the optimal temperature range, the protease may lose its enzymatic activity due to denaturation, resulting in a decrease in the number of peptides obtained, leading to a decrease in the pancreatic lipase inhibition rate of the hydrolysate. We will select 37°C, 42°C, and 47°C for the next step of response surface optimization.

[0116] 8.1.5 Effect of Enzyme-Substrate Ratio on Hydrolysate Activity

[0117] like Figure 5As shown, as the enzyme-substrate ratio increases, the level of pancreatic lipase inhibition by the hydrolyzate first increases and then decreases, reaching a maximum inhibition rate of 85.85% ± 2.89% at an enzyme-substrate ratio of 0.2%. When the enzyme-substrate ratio is less than 0.2%, the enzymatic hydrolysis reaction is not saturated, and the quinoa protein is not fully hydrolyzed. When the enzyme-substrate ratio exceeds 0.2%, the hydrolysis reaction of pepsin and quinoa protein is oversaturated, no longer promoting protein hydrolysis, resulting in enzyme waste. Therefore, an enzyme-substrate ratio of 0.2% was selected for subsequent enzymatic process optimization experiments.

[0118] 8.1.6 Effect of substrate concentration on enzymatic activity

[0119] like Figure 6 As shown, the pancreatic lipase inhibition rate increases with increasing substrate concentration, reaching a maximum of 81.53% ± 1.53% at 3%, then gradually decreasing and reaching equilibrium at 4%. The enzymatic reaction rate gradually increases with increasing substrate concentration. When the substrate concentration exceeds the optimal range, the peptide's reaction sites may be blocked, and interactions between substrates may occur, reducing the inhibitory activity. Furthermore, since the substrate and pepsin have already reacted completely, the enzymatic reaction rate will not increase with increasing substrate concentration. Therefore, substrate concentrations of 2%, 3%, and 4% will be selected for response surface optimization.

[0120] 8.2 Response surface optimization experiment

[0121] Based on a single-factor experiment, a three-factor, three-level experiment was designed and conducted with temperature (A), pH (B), and substrate concentration (C) as independent variables and pancreatic lipase inhibition rate as the activity indicator. The results of the response surface experiment are shown in Table 2.

[0122] Table 2 Response surface design scheme and results

[0123]

[0124] Table 3 Significance test of regression model

[0125]

[0126]

[0127] Note: P<0.05, significant, marked with *; P<0.01, extremely significant, marked with **; P>0.05, not significant.

[0128] According to the data in Table 2, multiple regression fitting was performed using Design Expert 8.0.6.1 software to obtain the quadratic polynomial regression equation between the three influencing factors and the response value pancreatic lipase inhibition rate (Y):

[0129] Y=90.2+1.23×A+1.63×B+0.41×C+1.31×A×B-0.16×A×C-0.98×B×C-4×A 2 -6.13×B 2 -4.66×C 2

[0130] The F-values and P-values in Table 3 reflect the correlation and significance between pancreatic lipase inhibition and each factor. The high or low F-values reflect the influence of each factor on the experimental model. The regression model and equation were validated for significance. The model's F-value was 25.65, and the P-value was 0.0002 < 0.01, indicating that the established regression model exhibited highly significant differences, high reliability, and the experimental method was feasible. In the regression model, F(A) = 6.97, F(B) = 12.39, and F(C) = 0.77. Based on this, it is speculated that the order of influence of factors on pancreatic lipase inhibition is pH > temperature > substrate concentration. The F-value for the lack-of-fit term was 3.38, and the P-value was 0.1353 > 0.05, indicating that the established regression model was minimally affected by random factors such as experimental manipulation. The coefficient of variation (CV) was 1.58% < 10%, indicating that the experimental results are reliable. In summary, the experiment was stable and the model had a high degree of fit. It can be used to observe the changes in quinoa protein hydrolysate with experimental conditions and to analyze and predict the optimal process conditions when the hydrolysate has the highest pancreatic lipase inhibitory ability.

[0131] The corresponding contour map and surface map can reflect the interaction effect between two factors among A, B or C. Figure 7 As shown in the table, the interactive effects of temperature, pH, and substrate concentration on pancreatic lipase inhibition all show an initial upward and then downward trend, with a maximum value within a certain range. At a certain substrate concentration, the steepness of the response surface fluctuated significantly with changes in pH and temperature, indicating that the experimental model for pancreatic lipase inhibition by quinoa protein hydrolysate is primarily influenced by pH and temperature, with their effects being greater than those of substrate concentration, consistent with the variance analysis results in Table 3.

[0132] Taking into account the effects of temperature, pH, and substrate concentration on pancreatic lipase inhibition, a response surface optimization method was used to determine the optimal preparation process for quinoa lipid-lowering peptide: a temperature of 42.88°C, a pH of 1.57, a substrate concentration of 3.03, a reaction time of 1 hour, and an enzyme-substrate ratio of 0.2%. Under these conditions, the quinoa peptide exhibited an inhibition rate of 90.43% against pancreatic lipase. Experimental verification of these predicted optimal conditions revealed an actual inhibition rate of 90.93% ± 0.10% for quinoa lipid-lowering peptide against pancreatic lipase, which was very close to the predicted value.

[0133] 8.3 Analysis of the lipid-lowering activity of quinoa protein peptides

[0134] 8.3.1 Inhibitory Effect of Quinoa Protein Peptides on Pancreatic Lipase

[0135] When the body consumes high-fat foods, fat cannot be absorbed directly. Instead, it is hydrolyzed by pancreatic lipase to produce fatty acids and 3-monoacylglycerides, which are then mixed with bile salts and enter the lipid cells and epithelial cell membranes of the intestine in the form of colloids. Inhibiting the activity of lipase can reduce the absorption of fat by the intestine. The ability of quinoa protein peptide to inhibit pancreatic lipase is as follows: Figure 8 As shown in the figure, with the increase of quinoa protein peptide concentration, the inhibitory effect on pancreatic lipase showed an upward trend. When the concentration was 500 μg / mL, the maximum value was 62.53% ± 0.11% (P < 0.05). At 10 μg / mL, the pancreatic lipase inhibition rate exceeded 50%, and the IC50 for inhibiting pancreatic lipase activity was calculated by the software. 50 It is 7.49 μg / ml (Table 4). It can be seen that quinoa protein peptide has a significant effect on inhibiting the activity of pancreatic lipase. The reason may be that quinoa protein peptide affects the electron distribution of pancreatic lipase, inhibits the binding of substrate and pancreatic lipase, affects the catalytic action of pancreatic lipase, inhibits the activity of pancreatic lipase, and achieves the effect of lowering blood lipids.

[0136] 8.3.2 Adsorption Capacity of Quinoa Protein Peptides for Bile Salt

[0137] Cholesterol is metabolized into bile salts in the body. A decrease in the bile salt content in the liver and intestines can promote the metabolism of cholesterol, thereby reducing the intestinal absorption of cholesterol and achieving the effect of lowering blood lipids.

[0138] like Figure 9 As shown in the results, within the range of 0.1-2 mg / mL, the binding rate of sodium taurocholate showed an increasing trend with the increase of quinoa protein peptide concentration. When the concentration of quinoa protein peptide was 2 mg / mL, the binding rate of sodium taurocholate reached a maximum of 76.95% ± 0.37% (P < 0.05). The EC 50 The value is 0.53mg / mL. The ability of quinoa protein peptide to bind bile salts is significant, revealing the possibility that quinoa protein peptide can play a lipid-lowering role in the intestine. By inhibiting the intestinal absorption of bile salts and excreting them from the body, cholesterol is metabolized in the body and converted into bile acids, thereby achieving a lipid-lowering mechanism.

[0139] 8.3.3 Inhibitory Effect of Quinoa Protein Peptides on Cholesterol Esterase

[0140] Cholesterol esters are hydrolyzed into cholesterol and fatty acids under the catalysis of cholesterol esterase. Cholesterol can only be absorbed by the human body after it is dissolved in cholesterol micelles. Therefore, inhibiting the activity of cholesterol esterase is an effective way to reduce the absorption of cholesterol. Figure 10As shown in the results, within a certain concentration range, quinoa protein peptide showed a significant upward trend in inhibiting cholesterol esterase. At 2 μg / mL, the cholesterol esterase inhibition rate reached the highest value of 41.18% ± 0.20% (P < 0.05). SPSS software analysis showed that IC 50 The result was 4.73 mg / mL. This indicates that quinoa protein peptides effectively inhibit the activity of cholesterol esterase. It is speculated that the acidic peptides obtained by the action of pepsin on quinoa protein bind to cholesterol esterase, thereby reducing the catalytic effect on the substrate. In summary, quinoa protein peptides are natural active peptides with lipid-lowering functions.

[0141] Table 4 Evaluation of quinoa protein peptides' lipid-lowering activity

[0142]

[0143] 8.3.4 Xanthine Oxidase Inhibitory Activity Analysis of Quinoa Protein Hydrolysates

[0144] Xanthine oxidase is a key enzyme in purine metabolism in the body. It catalyzes the conversion of hypoxanthine to xanthine, which is then oxidized to produce metabolites such as uric acid, pyrimidine, and purine. Excessive accumulation of uric acid in the body can lead to symptoms such as diarrhoea or kidney stones. Therefore, inhibiting the catalytic activity of xanthine oxidase can reduce uric acid accumulation.

[0145] like Figure 11 As shown in the results, the inhibitory effect of xanthine oxidase increased with the concentration of quinoa protein hydrolysate. When the concentration of quinoa protein hydrolysate was 8 mg / mL, the xanthine oxidase inhibitory effect was as high as 67.96% ± 0.90% (P < 0.05). IC 50 The result was 5.97 mg / mL, which was higher than that of bonito synthetic peptide (ACECD) (IC 50 =7.23mg / mL) has a better inhibitory effect (Enzymatic preparation and functional activity evaluation of bonito xanthine oxidase inhibitory peptide). In addition, Zhan Suhong et al. ultrafiltrated Far Eastern sardine protein to obtain an enzymatic hydrolyzate component with a molecular weight of less than 1000kDa, and its IC 50 The concentration of quinoa lipid-lowering peptides reached 15.89 g / L (Preparation of Xanthine Oxidase Inhibitory Peptides from Far Eastern Sardinella and Study on Their Uric Acid-Lowering Activity). The experimental results showed that under the optimal enzymatic hydrolysis conditions, quinoa lipid-lowering peptides also had good uric acid-lowering activity.

[0146] 8.4 Amino Acid Composition Analysis

[0147] The amino acid composition of quinoa protein peptide is shown in Table 5. It primarily consists of a peptide mixture containing 17 amino acids, with glutamic acid being the most abundant, accounting for 19.85% of the total amino acid content, followed by arginine and aspartic acid, at 10.80% and 9.58%, respectively. Studies have shown that arginine promotes nitric oxide production in the body, increasing blood flow and thereby lowering low-density lipoprotein cholesterol levels. Essential amino acids account for 34.23% of the total amino acid content, close to the WHO / FAO recommended value of 36%. A higher proportion of essential amino acids indicates a higher nutritional value for quinoa protein. The proportion of hydrophobic amino acids is 34.11%, while the proportion of acidic amino acids is 31.66%. Studies have shown that a high content of hydrophobic amino acids plays a significant role in bile acid binding. Hydrophobic amino acids bind to bile acids through hydrophobic interactions, forming insoluble complexes that are excreted. Furthermore, studies have shown that a higher content of hydrophobic amino acids promotes their incorporation into lipid micelles. Furthermore, acidic amino acids have a positive effect on lipid-lowering activity. In summary, the active peptides from quinoa protein hydrolysate in this study have potential lipid-lowering effects, and their specific peptide composition and amino acid sequence analysis need further study.

[0148] Table 5 Amino acid composition of quinoa protein hydrolysate

[0149]

[0150]

[0151] Note: *EAA: essential amino acids (Thr, Val, Met, Ile, Leu, Phe, Lys, His);

[0152] #HAA: hydrophobic amino acids (Ala, Val, Ile, Leu, Phe, Trp, Pro, Met);

[0153] AAA: acidic amino acids (Asp, Glu).

[0154] 9 Conclusion

[0155] This study used quinoa protein as the research object, with pancreatic lipase inhibition rate as the main indicator, and obtained the optimal process conditions for preparing quinoa protein lipid-lowering peptides through single factor and response surface experiments. The results showed that the enzymatic hydrolysate obtained by pepsin hydrolysis of quinoa protein had the best activity. Under the conditions of enzymatic hydrolysis time of 1h, temperature of 42.88℃, pH 1.57, substrate concentration of 3.03% and enzyme-substrate ratio of 0.2%, its pancreatic lipase inhibition rate was 90.93%±0.10%, which was close to the software predicted value (90.43%), indicating that the model can accurately predict the optimal process conditions for quinoa protein. Using pancreatic lipase inhibition rate, sodium taurocholate binding rate, cholesterol esterase inhibition rate and xanthine oxidase inhibition rate as activity evaluation indicators, the results showed that quinoa protein hydrolysate had the best pancreatic lipase inhibition effect (IC 50 =7.49 μg / ml), sodium taurocholate binding capacity (EC 50 =0.53 mg / mL), cholesterol esterase inhibition (IC 50 =4.73 mg / mL) xanthine oxidase inhibition rate (IC 50 =5.97 mg / mL), indicating that quinoa protein hydrolysate has an effect on lowering blood lipids and that the hydrolysate has good uric acid-lowering activity. In addition, amino acid composition analysis of quinoa protein peptides was performed. Quinoa protein lipid-lowering peptides are rich in essential amino acids (34.23%), hydrophobic amino acids (34.11%), and acidic amino acids (31.66%), playing an important role in lowering blood lipids and uric acid.

[0156] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of an active peptide having both lipid-lowering and uric acid-lowering functions in at least one of the following situations: (1) Application in the preparation of uric acid-lowering drugs; (2) Application in the preparation of drugs for treating hyperuricemia; (3) Application in the preparation of drugs for treating hyperuricemia and lowering blood lipids; The active peptide is prepared by a method comprising the following steps: S1. Take quinoa powder, add ethanol, react, and dry to obtain defatted quinoa powder; S2. Take the defatted quinoa powder obtained in step S1, add alkali solution, and react; filter and centrifuge the reaction solution, collect the supernatant and adjust the pH to 4.5±0.2, let it stand and then centrifuge, remove the supernatant, and retain the protein precipitate; freeze-dry to obtain a crude quinoa protein extract; S3. The crude quinoa protein extract obtained in step S2 is added with water to form a solution, and a protease is added for enzymolysis to obtain an enzymatic solution; the protease used is pepsin, and the enzymatic hydrolysis conditions are: temperature 37-47° C., pH 1.5-2, enzymatic hydrolysis time 1-1.5 h, enzyme-substrate ratio 0.18-0.22%, and substrate concentration 2 wt %-4 wt %; S4. Inactivate the protease in the enzymatic hydrolysis solution obtained in step S3, terminate the enzymatic hydrolysis reaction, centrifuge, and collect the supernatant to obtain the active peptide with both lipid-lowering and uric acid-lowering functions.

2. The use according to claim 1, characterized in that: The drug reduces uric acid accumulation by inhibiting the catalytic action of xanthine oxidase.

3. The use according to claim 1, characterized in that: The enzyme used in step S3 is pepsin, and the enzymatic hydrolysis conditions are: temperature 42.88° C., pH 1.57, enzymatic hydrolysis time 1 h, enzyme-substrate ratio 0.2%, and substrate concentration 3.03 wt %.

4. The use according to any one of claims 1 to 3, characterized in that: The quinoa powder in step S1 is obtained by grinding quinoa and passing it through a 10-30 mesh sieve; The ethanol described in step S1 is 90% to 95% ethanol; The material-liquid ratio of quinoa powder and ethanol in step S1 is 1 g: 10-30 mL; The reaction time in step S1 is 10 to 15 h; The alkali solution described in step S2 is NaOH solution; The solid-to-liquid ratio of the defatted quinoa powder to the alkali solution in step S2 is 1 g: 30-50 mL; The reaction conditions in step S2 are a temperature of 40-50° C. and a time of 2-4 h; The standing time in step S2 is 10 to 30 minutes; The centrifugation conditions in steps S2 and S4 are both 3000-5000 r / min and 10-30 min; The reagents used for adjusting pH in steps S2 and S3 are 0.1 mol / mL NaOH solution and HCl solution; The method of inactivating the protease in step S4 is to react in boiling water for 8 to 12 minutes.

5. The use according to claim 4, characterized in that: The quinoa powder in step S1 is obtained by grinding quinoa and passing it through a 20-mesh sieve; The ethanol described in step S1 is 95% ethanol; The material-liquid ratio of quinoa powder and ethanol in step S1 is 1 g:20 mL; The reaction time in step S1 is 12 h; The alkali solution in step S2 is a 0.1 mol / L NaOH solution; The solid-to-liquid ratio of the defatted quinoa powder to the alkali solution in step S2 is 1 g:40 mL; The reaction conditions in step S2 are temperature 45°C and time 3 h; The standing time described in step S2 is 20 min; The centrifugation conditions in steps S2 and S4 are both 4000 r / min and 20 min; The protease inactivation method in step S4 is to react in boiling water for 10 minutes.

6. An active peptide with both lipid-lowering and uric acid-lowering functions, characterized by: It is prepared by the method described in any one of claims 1 to 5.

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