Preparation method of phaseolus calcaratus polypeptide, compound beverage and application thereof

Abstract

The invention discloses a preparation method of phaseolus calcaratus polypeptide, a compound beverage of the phaseolus calcaratus polypeptide and application of the phaseolus calcaratus polypeptide, and aims to solve the technical problems that high-value utilization of phaseolus calcaratus is difficult to realize at present, the phaseolus calcaratus polypeptide is single in purpose, and functional effects of Alkaline protease is used for preparing the phaseolus calcaratus polypeptide, the enzymolysis conditions are optimized, and the polypeptide content can reach 133.89 mg / g. Meanwhile, the formula of the compound beverage containing the phaseolus calcaratus polypeptide is optimized, so that the compound beverage has relatively good antioxidant activity on the premise that the taste is proper. The requirement of people for high-speed growth of functional food can be met, the health level of people is improved, deep processing of low-added-value agricultural products is facilitated, then the agricultural structure is improved, and the income of farmers is increased.

Description

Technical Field

[0001] The present invention relates to the field of food processing technology, and specifically to a method for preparing adzuki bean polypeptide, a composite beverage thereof and applications thereof. Background Art

[0002] Adzuki beans, as a widely planted herb seed, are also known as red beans, red beans, and red beans. They are harvested in summer and autumn when the pods mature. The dried seeds are not only edible, but also have important medicinal value. In traditional medicine, adzuki beans are regarded as a good product for promoting blood circulation, strengthening the spleen and removing dampness, and promoting diuresis and reducing swelling. Its rich nutrients and medicinal properties make it a hot topic in the field of health foods.

[0003] With the development of modern biotechnology, the research on adzuki beans has gradually deepened to its molecular level. Alkaline protease, an enzyme that exhibits efficient proteolysis ability in an alkaline environment, is favored by the food and pharmaceutical industries because it can break down proteins into small molecular peptides that are easy to absorb under suitable conditions. These peptides not only retain some of the biological activity of proteins, but also enhance their absorption efficiency and utilization rate in the human body due to their low molecular weight characteristics, further broadening the application potential of natural ingredients such as adzuki beans.

[0004] Appropriate intake of antioxidants by the human body (especially through food) is an effective way to reduce the level of free radicals in the body, prevent lipid peroxidation, and resist diseases. Compared with the possible side effects of drugs, food-derived antioxidants have become a more popular choice due to their safety and sustainability. Therefore, in-depth research on natural antioxidant foods such as adzuki beans is conducive to the development of new drugs and functional foods, and can also provide new functional base materials and additives for the food and pharmaceutical industries, and promote the innovative development of the health industry. However, current research on adzuki beans mainly focuses on the preparation and extraction of polyphenol components, while there is less research on other important nutrients (such as peptides, etc.), which undoubtedly greatly limits its application areas and application value. At present, it is urgent to explore the deeper nutritional value and application potential of adzuki beans, expand its application methods, and explore its application in health care products such as compound beverages.

[0005] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0006] In view of at least one of the above technical problems, the present disclosure provides, on the one hand, a method for preparing adzuki bean polypeptide with a simple process flow and low cost, aiming to solve the current technical problem that adzuki bean is difficult to achieve high-value utilization; on the other hand, it provides a compound beverage containing adzuki bean polypeptide, aiming to solve the technical problem that adzuki bean polypeptide has a single use and its functional effect is difficult to fully exert.

[0007] According to one aspect of the present disclosure, a method for preparing adzuki bean polypeptide is provided, comprising the following steps: (1) Grinding and sieving adzuki beans to obtain adzuki bean powder; (2) adding the adzuki bean powder to water to form a bean powder suspension with a concentration of 2-3%; (3) adjusting the pH value of the soybean powder suspension to 8-8.5 using acid or alkali; (4) Then, 3800-4200 U / mg alkaline protease was added thereto, and the mixture was subjected to enzymatic hydrolysis at 54-56°C for 2.5-3.5 h; (5) After enzymatic hydrolysis is completed, sterilize in boiling water for 12-17 minutes. After cooling to room temperature, adjust the pH value of the enzymatic hydrolysis product to 7-7.5, and then centrifuge at 3500-4000 r / min for 15-20 minutes. Take the supernatant.

[0008] According to the second aspect of the present disclosure, the adzuki bean polypeptide prepared by the above method is used in the preparation of an antioxidant biological agent.

[0009] According to the third aspect of the present disclosure, the adzuki bean polypeptide prepared by the above method is used in the preparation of a biological agent for scavenging hydroxyl radicals and / or DPPH radicals.

[0010] According to a fourth aspect of the present disclosure, a composite beverage is provided, which is made of the following components in percentage by mass: 8-10% honey, 23-25% adzuki bean polypeptide prepared by the above method, 0.4-0.5% citric acid, 1.8-2.2% seabuckthorn concentrated juice, and 63-65% purified water.

[0011] The seabuckthorn among the above raw materials is a unique plant that can be used as both medicine and food. Its fruit is rich in a variety of biologically active substances that are beneficial to the human body, such as flavonoids, superoxide dismutase and ω-6 polyunsaturated fatty acids. When combined with adzuki bean polypeptides, it has multiple health functions such as enhanced anti-aging, anti-oxidation, anti-radiation, enhanced immunity and anti-tumor.

[0012] In some embodiments of the present disclosure, the compound beverage is made of the following raw materials in percentage by mass: Honey 10%, adzuki bean polypeptide prepared by the above method 24%, citric acid 0.4%, sea buckthorn concentrated juice 2%, and purified water 63.6%.

[0013] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: 1. This application uses alkaline protease to hydrolyze adzuki beans. It is an enzyme that can show efficient protein hydrolysis ability in an alkaline environment. The obtained polypeptide not only greatly retains the biological activity of the protein, but also improves its absorption efficiency and utilization rate in the human body due to its low molecular weight characteristics, further broadening the application methods and application value of natural ingredients such as adzuki beans.

[0014] 2. The preparation method of the present application is simple, and alkaline protease is used to prepare adzuki bean polypeptide, and the optimal process control parameters for preparing adzuki bean polypeptide are optimized, that is, when the enzymatic hydrolysis temperature is 55°C, the enzymatic hydrolysis time is 3h, and the enzymatic hydrolysis pH value is 8.4, the adzuki bean polypeptide content can reach 133.89 mg / g, and its hydroxyl radical scavenging rate can reach 71%, and the DPPH radical scavenging rate can reach 83%.

[0015] 3. In the compound application research, it was found that the adzuki bean polypeptide and sea buckthorn compound beverage had the best taste when the honey addition amount was 10%, the adzuki bean polypeptide addition amount was 24%, and the citric acid addition amount was 0.40%. The various ingredients synergized with each other, and the scavenging rates of hydroxyl free radicals and DPPH free radicals were 78% and 89.67% respectively, showing good antioxidant activity.

[0016] 4. Processing adzuki beans into high value-added antioxidant peptides through biotechnology can not only meet people's rapidly growing demand for functional foods and improve people's health level, but also be conducive to the deep processing of low value-added agricultural products, improve agricultural structure, increase farmers' income and develop rural economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the effect of different enzyme sources on the polypeptide content in one embodiment of the present application.

[0018] Figure 2 This is the effect of enzymatic hydrolysis temperature on polypeptide content in one embodiment of the present application.

[0019] Figure 3 This is the effect of enzymatic hydrolysis pH on polypeptide content in one embodiment of the present application.

[0020] Figure 4 This is the effect of substrate concentration on polypeptide content in one embodiment of the present application.

[0021] Figure 5 This is the effect of the amount of enzyme added on the polypeptide content in one embodiment of the present application.

[0022] Figure 6 This is the effect of enzymatic hydrolysis time on polypeptide content in one embodiment of the present application.

[0023] Figure 7 This is the effect of the interaction between enzymatic hydrolysis time and pH on the polypeptide content in one embodiment of the present application.

[0024] Figure 8 This is the effect of the interaction between enzymatic hydrolysis temperature and pH on the polypeptide content in one embodiment of the present application.

[0025] Fig. 9 This is the effect of the interaction between enzymatic hydrolysis time and temperature on the polypeptide content in one embodiment of the present application.

[0026] Fig.10 This is the effect of the amount of honey added on the sensory score in one embodiment of the present application.

[0027] Fig.11 This is the effect of the amount of adzuki bean polypeptide liquid added on the sensory score in one embodiment of the present application.

[0028] Fig.12 This is the effect of the amount of citric acid added on the sensory score in one embodiment of the present application.

[0029] Fig.13 This is the effect of the amount of seabuckthorn concentrated juice added on the sensory score in one embodiment of the present application.

[0030] Fig.14 This is the effect of the interaction between the amount of honey added and the amount of adzuki bean polypeptide liquid added on the sensory score in one embodiment of the present application.

[0031] Fig.15 This is the effect of the interaction between the amount of honey added and the amount of sea buckthorn concentrated juice added on the sensory score in one embodiment of the present application.

[0032] Fig.16 This is the effect of the interaction between the addition amount of adzuki bean polypeptide liquid and the addition amount of seabuckthorn concentrated juice on the sensory score in one embodiment of the present application. DETAILED DESCRIPTION

[0033] The specific implementation methods of the present invention are described below in conjunction with examples, but the following examples are only used to illustrate the present application in detail and do not limit the scope of the present application in any way.

[0034] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; unless otherwise specified, the reagents involved are all commercially available conventional reagents; unless otherwise specified, the preparation or detection methods involved are all conventional methods.

[0035] Example 1. Enzymatic preparation of adzuki bean polypeptide and optimization of enzymatic conditions 1. Enzymatic preparation of adzuki bean peptides Grind the adzuki beans and pass them through a 50-mesh sieve to form adzuki bean powder. Weigh a certain mass of adzuki bean powder and add it to 100 ml of deionized water to prepare a bean powder suspension of a certain concentration. Adjust its pH value with 10% NaOH solution and 10% hydrochloric acid, then add protease, and hydrolyze it in a water bath for a certain time. After enzymolysis, inactivate the enzyme in boiling water for 15 minutes, cool to room temperature, adjust the pH value to 7, and then centrifuge at 4000r / min for 20 minutes.

[0036] Based on the above method, a single factor experiment was conducted on the preparation of adzuki bean polypeptide, using alkaline protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd.), papain and trypsin as single enzyme sources. Figure 1 As shown in the figure, the enzymatic hydrolysis effect of alkaline protease is significantly better than that of papain and trypsin. Therefore, alkaline protease is used to prepare adzuki bean polypeptide.

[0037] 2. Determination of adzuki bean polypeptide content (1) Preparation of relevant reagents Bovine serum albumin standard solution: Accurately weigh 100 mg of bovine serum albumin standard sample (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.), dissolve it in a small amount of distilled water, stir with a glass rod until completely dissolved, and then dilute it to 10 mL in a volumetric flask to obtain a standard protein solution (10 mg / mL).

[0038] (2) Preparation of standard curve First, take 6 test tubes and add 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the prepared bovine serum albumin standard solution into the test tubes respectively. Add distilled water to each test tube to make the total volume of the solution reach 1.0 mL; then add 4.0 mL of biuret reagent to each test tube, mix thoroughly and let stand at room temperature for 30 minutes; next, use a UV spectrophotometer to measure the absorbance at a wavelength of 540 nm (use distilled water for the blank group, and repeat the test three times to ensure data accuracy); finally, use protein concentration as the horizontal axis and absorbance value as the vertical axis to draw a protein concentration standard curve.

[0039] (3) Determination of absorbance of adzuki bean polypeptide After centrifugation, take 2 ml of the supernatant into a centrifuge tube, and use a pipette to draw an equal volume of 2 mL of 10% trichloroacetic acid into the centrifuge tube, let it stand for 20 minutes, and then centrifuge (4000 r / min, 20 minutes). Use a pipette to draw 1 mL of the supernatant after centrifugation into a test tube, and add 4 mL of biuret reagent, shake well, let it stand at room temperature for 30 minutes, and then measure the absorbance at a wavelength of 540 nm using a UV spectrophotometer.

[0040] (4) Calculation of peptide content The measured absorbance value was compared with the standard curve to calculate the peptide content according to the following formula: ; Where: c—protein concentration / (mg / mL); V—volume of supernatant after centrifugation / mL; m—mass of adzuki beans in 1 mL of supernatant / g.

[0041] 3. Single factor experiment of preparing adzuki bean peptides by alkaline protease hydrolysis In this case, a single factor experiment (enzyme hydrolysis temperature, enzyme hydrolysis pH, substrate concentration, enzyme addition amount, and enzyme hydrolysis time) was conducted on the basis of the above method to prepare red bean peptides using alkaline protease.

[0042] (1) Effect of enzymatic hydrolysis temperature on the preparation of adzuki bean peptides by alkaline protease hydrolysis Prepare adzuki bean polypeptide according to the above method, crush adzuki bean and pass through a 50-mesh sieve, weigh 2g of adzuki bean powder and add it to 100ml of deionized water to make a 2% bean powder suspension, adjust the pH value to 8.5, add 4000U / mg of alkaline protease, enzymolysis in a water bath for 3h, adjust the enzymolysis temperature to 45, 50, 55, 60, 65℃, inactivate the enzyme in boiling water for 15min after enzymolysis, adjust the pH value to 7 after cooling to room temperature, and then centrifuge at 4000r / min for 20min. Then take the supernatant to measure the absorbance of adzuki bean polypeptide and calculate the polypeptide content. The results are as follows Figure 2 As shown in the figure, at 45℃~55℃, the polypeptide content increases with the increase of temperature, and the polypeptide content decreases after exceeding 55℃, which may be because the enzyme structure is destroyed and the enzyme activity decreases.

[0043] (2) Effect of enzymatic hydrolysis pH on the preparation of adzuki bean peptides by alkaline protease hydrolysis Grind the adzuki beans and pass through a 50-mesh sieve, weigh 2g of adzuki bean powder and add it to 100ml of deionized water to make a 2% bean powder suspension, adjust the pH to 7.5, 8.0, 8.5, 9.0, 9.5, add 4000U / mg of alkaline protease, continue to maintain the temperature at 55℃ in a water bath for 3h, inactivate the enzyme in boiling water for 15min after enzymolysis, adjust the pH to 7 after cooling to room temperature, and then centrifuge at 4000r / min for 20min. Then take the supernatant to measure the absorbance of adzuki bean polypeptide and calculate the polypeptide content. The results are as follows: Figure 3 As shown in the figure, the polypeptide content increases first and then decreases with the increase of pH value. This is because the catalytic activity of the enzyme as a catalyst is extremely sensitive to pH value. Too high or too low pH environment will have an adverse effect on the conformation and function of the enzyme, thereby inhibiting its catalytic efficiency and affecting the progress of the enzymatic reaction.

[0044] (3) Effect of substrate concentration on the preparation of adzuki bean peptides by alkaline protease hydrolysis Grind the adzuki beans and pass through a 50-mesh sieve. Weigh 2 g of adzuki bean powder and add it to deionized water to make bean powder suspensions with concentrations of 1%, 2%, 3%, 4%, and 5%, respectively. Adjust the pH to 8.5, add 4000 U / mg of alkaline protease, and continue to hydrolyze in a water bath at 55°C for 3 hours. After hydrolysis, boil water to inactivate the enzyme for 15 minutes, cool to room temperature, adjust the pH to 7, and centrifuge at 4000 r / min for 20 minutes. Then take the supernatant to measure the absorbance of adzuki bean polypeptide and calculate the polypeptide content. The results are as follows: Figure 4 As shown in the figure, when the substrate concentration increases from 1% to 2%, the peptide content increases significantly. However, once the concentration threshold of 2% is exceeded, the peptide content shows a downward trend. Further in-depth research found that this is because within the lower substrate concentration range, increasing the substrate concentration can effectively promote the reaction rate, but after reaching 2%, excessively high substrate concentrations may instead trigger a substrate inhibition effect, that is, the substrate molecules begin to compete with the active site of the enzyme for binding, thereby hindering the further progress of the forward reaction.

[0045] (4) Effect of enzyme dosage on the preparation of adzuki bean peptides by alkaline protease hydrolysis Grind the adzuki beans and pass through a 50-mesh sieve, weigh 2g of adzuki bean powder and add it to deionized water to make a 2% bean powder suspension, adjust the pH value to 8.5, adjust the amount of enzyme (alkaline protease) to 2000, 2500, 3000, 3500, 4000, 4500U / mg, continue to maintain the temperature at 55℃ in a water bath for 3h, inactivate the enzyme in boiling water for 15min after enzymolysis, adjust the pH value to 7 after cooling to room temperature, and then centrifuge at 4000r / min for 20min. Then take the supernatant to measure the absorbance of adzuki bean polypeptide and calculate the polypeptide content. The results are as follows: Figure 5 As shown in the figure, increasing the amount of enzyme can significantly improve the binding efficiency of enzyme and substrate, thereby promoting the generation and accumulation of red bean polypeptides. However, with the continuous increase of enzyme amount, when the enzyme amount increases to 4000 U / mg, it will cause excessive hydrolysis of polypeptides due to the relative lack of substrate, that is, while the enzyme catalyzes the generation of polypeptides, it also begins to decompose the generated polypeptides, resulting in a decrease in the final polypeptide content.

[0046] (5) Effect of enzymatic hydrolysis time on the preparation of adzuki bean peptides by alkaline protease hydrolysis Grind the adzuki beans and pass through a 50-mesh sieve. Weigh 2 g of adzuki bean powder and add it to deionized water to make a 2% bean powder suspension. Adjust the pH value to 8.5, add 4000 U / mg of alkaline protease, and perform enzymolysis in a water bath at 55°C. Adjust the enzymolysis time to 2.0, 2.5, 3.0, 3.5, and 4.0 h. After enzymolysis, inactivate the enzyme in boiling water for 15 minutes. After cooling to room temperature, adjust the pH value to 7, and centrifuge at 4000 r / min for 20 minutes. Then take the supernatant to measure the absorbance of adzuki bean polypeptide and calculate the polypeptide content. The results are as follows: Figure 6 As shown, alkaline protease, as a highly specific enzyme, has specific peptide bond action sites. During protein hydrolysis, as the specific peptide bonds of the substrate decrease, the reaction rate decreases accordingly. In addition, as the reaction proceeds, the enzyme activity gradually weakens, thereby limiting the further hydrolysis of the protein by the protease.

[0047] 4. Optimization of the preparation process of adzuki bean peptide Based on the above single factor experiment, with enzymatic hydrolysis temperature, enzymatic hydrolysis pH value and enzymatic hydrolysis time as investigation factors and adzuki bean polypeptide content as investigation index, according to the Box-Behnken experimental design principle, a three-factor three-level response surface optimization experiment of adzuki bean polypeptide enzymatic hydrolysis process was designed. The response surface experimental factors and levels, response surface experimental design scheme and results, and regression model variance analysis are shown in Table 1, Table 2, and Table 3, respectively.

[0048] Table 1 Factors and levels of response surface experiment .

[0049] Table 2 Response surface experimental design scheme and results .

[0050] Table 3 Analysis of variance of regression model Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).

[0051] As can be seen from Table 3, the F value of each factor is ranked as C>A>B, that is, temperature>pH>time. The regression of the model is significant (P<0.0001), the lack of fit term is not significant, and the model R2=0.9993, Radj2 =0.9984, and the model fit is high. The data were fitted and analyzed using Design-Expert 13 software, and the quadratic polynomial regression equation was obtained: Y=133.58-2.21A-1.48B+4.12C-1.31AB-0.6625AC-0.585BC-8.44A2-13.8B2-14.96C2.

[0052] The response surface optimization experiment uses a multivariate quadratic regression equation to fit the functional relationship between factors and the evaluation indicators, and expresses it simply and intuitively with a three-dimensional stereogram and a contour map, and then analyzes the optimal enzymatic hydrolysis process parameters. The closer the contour map is to an ellipse, the stronger the interaction between the two factors. The effects of the interaction between enzymatic hydrolysis time and enzymatic hydrolysis pH on the polypeptide content, the interaction between enzymatic hydrolysis temperature and enzymatic hydrolysis pH on the polypeptide content, and the interaction between enzymatic hydrolysis time and enzymatic hydrolysis temperature on the polypeptide content are shown in the figure below. Figure 7 , Figure 8 , Fig. 9 As shown in the figure, it can be seen that the interaction between enzymatic hydrolysis time and enzymatic hydrolysis pH is significant, the interaction between enzymatic hydrolysis temperature and enzymatic hydrolysis pH is significant, but the interaction between enzymatic hydrolysis time and enzymatic hydrolysis temperature is not significant.

[0053] Taking into account the effects of the interaction among temperature, time and pH on the content of adzuki bean polypeptide, the adzuki bean polypeptide content was taken as the optimization target. According to the results of Design-Expert 13 software, the optimal enzymatic hydrolysis conditions of adzuki bean polypeptide were temperature: 55.69 ℃, time: 2.97 h, pH: 8.43. Under these conditions, the adzuki bean polypeptide content predicted by the model was 134.11 mg / g.

[0054] To ensure the optimal enzymatic hydrolysis conditions for alkaline protease, this example, based on the results predicted by the response surface software model and considering the feasibility of actual operation, optimized enzymatic hydrolysis temperature of 55°C, enzymatic hydrolysis time of 3h, enzymatic hydrolysis pH of 8.4, substrate concentration of 2%, and enzyme addition amount of 4000U / mg were selected. The experiment was repeated three times according to the optimal enzymatic hydrolysis conditions, and the average polypeptide content was 133.89 mg / g, which was close to the result predicted by the model. This shows that the enzymatic hydrolysis optimization based on the response surface model in this example was successful.

[0055] Example 2: Optimization of formula of red bean polypeptide and sea buckthorn composite beverage and detection of antioxidant activity Mix adzuki bean polypeptide, sea buckthorn concentrated juice, honey and citric acid in distilled water in proportion (honey added in an amount of 9%, adzuki bean polypeptide liquid added in an amount of 25%, sea buckthorn concentrated juice added in an amount of 2%, citric acid added in an amount of 0.40%), stir with a glass rod until the mixture is evenly mixed, filter twice with gauze, and then sterilize by pasteurization, keep at 65°C for 30 minutes, and refrigerate after cooling to room temperature.

[0056] The sensory evaluation team was composed of 10 students majoring in food. They rinsed their mouths with pure water before conducting the sensory evaluation. The taste, color, flavor, texture and mouthfeel were scored. Each sample was evaluated three times and the average was taken. The total score was 100 points, of which taste, texture, color, flavor and mouthfeel each accounted for 20 points. The sensory evaluation table is shown in Table 4.

[0057] Table 4 Sensory evaluation table .

[0058] 1. Optimization of the formula of red bean peptide and sea buckthorn compound beverage (1) Single factor experiment on adzuki bean peptide and sea buckthorn compound beverage This example uses the above formula as the basic blending condition, and conducts a single factor experiment with the added amount of honey (7%, 8%, 9%, 10%, 11%), adzuki bean polypeptide liquid (15%, 20%, 25%, 30%, 35%), sea buckthorn concentrated juice (1%, 2%, 3%, 4%, 5%), and citric acid (0.30%, 0.35%, 0.40%, 0.45%, 0.50%) as variables to study the effects of various factors on the sensory quality of adzuki bean polypeptide and sea buckthorn compound beverage. The sensory score was used as an indicator to determine the formula content range of different ingredients of the beverage. The results are as follows: Fig.10 , Fig.11 , Fig.12 , Fig.13 As shown: When the honey content reaches 9%, the taste is best and the sweetness and sourness are appropriate; if the honey content is greater than 9%, the drink will be too sweet; and if the content is less than 9%, the taste is sour. When the adzuki bean polypeptide liquid is added at 25%, the drink does not have the bitterness of the bean taste, while retaining the taste of the adzuki bean itself; when the adzuki bean polypeptide liquid is added at less than 25%, the bean taste in the drink is not obvious; and when it exceeds 25%, it has a significant bean bitterness. When the amount of citric acid added to the drink is 0.40%, the drink tastes moderately sweet and sour; when it is less than 0.40%, the taste is bland; when it is greater than 0.40%, the taste is sour and astringent. When the amount of sea buckthorn concentrated juice added is 2%, the drink tastes best; when it is less than 2%, the taste of the drink is not prominent; when it is greater than 2%, the taste is sour and astringent.

[0059] (2) Response surface optimization experiment of red bean peptide and sea buckthorn compound beverage According to the design principle of Box-Behnken central composite experiment, on the basis of single factor experimental results, three more significant factors (adzuki bean peptide addition amount, honey addition amount, sea buckthorn concentrated juice addition amount) were selected as independent variables, and the sensory score of adzuki bean peptide and sea buckthorn compound beverage was used as the response value. A three-factor three-level response surface analysis experiment was conducted. The response surface experimental factors and levels, response surface experimental design scheme and results, and regression model variance analysis are shown in Table 5, Table 6, and Table 7, respectively.

[0060] Table 5 Response surface experimental factors and levels .

[0061] Table 6 Response surface experimental design scheme and results .

[0062] Table 7 Analysis of variance of regression model Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).

[0063] As can be seen from the table above, the F value of each factor is ranked as E>D>G, that is, the amount of red bean polypeptide liquid added>the amount of honey added>the amount of concentrated sea buckthorn juice added. The regression of the model is significant (P<0.0001), the lack of fit term is not significant, and the model R2=0.9951, Radj2=0.9888, and the model fit is high. The data was fitted and analyzed using Design-Expert13 software, and the quadratic multinomial regression equation was obtained: Y=93.6+D-1.25E-0.5G-0.25DE+0.75DG-0.75EG-2.68D2-4.18E2-8.17G2.

[0064] The results of the interaction between the amount of adzuki bean peptide, the amount of honey, and the amount of seabuckthorn concentrated juice added are as follows: Fig.14 , Fig.15 , Fig.16 As shown, the results showed that the interaction between the amount of honey added and the amount of red bean polypeptide liquid added was not significant, the interaction between the amount of honey added and the amount of sea buckthorn concentrated juice added was significant, and the interaction between the amount of red bean polypeptide liquid added and the amount of sea buckthorn concentrated juice added was significant.

[0065] Taking into account the effects of the interactions among the amount of honey added, the amount of adzuki bean polypeptide liquid added, and the amount of sea buckthorn concentrated juice added on the sensory score, the score was taken as the optimization target. According to the results of the Design-Expert 13 software operation, the optimal score condition was 10% honey addition, 24.09% adzuki bean polypeptide liquid addition, and 2.02% sea buckthorn concentrated juice addition. Under this condition, the model predicted a score of 92.06 points.

[0066] According to the prediction results of the response surface software and combined with the feasibility of the actual process conditions, the optimal scoring conditions were selected as 10% honey addition, 24% red bean polypeptide liquid addition, and 2% sea buckthorn concentrated juice addition. The experiment was repeated three times under this condition, and the average score was 93.5 points, which was close to the model prediction result, indicating that the method for optimizing the formula of red bean polypeptide and sea buckthorn compound beverage based on the response surface model is feasible. Therefore, in this example, the honey addition amount in the red bean polypeptide and sea buckthorn compound beverage is preferably 10%, the red bean polypeptide liquid addition amount is preferably 24%, the sea buckthorn concentrated juice addition amount is preferably 2%, and the citric acid addition amount is preferably 0.4%.

[0067] 2. Antioxidant activity test of adzuki bean peptide and sea buckthorn compound beverage (1) Hydroxyl radical scavenging rate First, add 1.0 mL of the liquid to be tested into a test tube; then, add 0.2 mL of hydrogen peroxide solution, 0.2 mL of ferrous sulfate solution, and 0.2 mL of salicylic acid-ethanol solution into the test tube in order; finally, add 3.4 mL of distilled water. After the mixture in the test tube is shaken, place it in a constant temperature water bath at 37°C for 30 minutes. After that, take out the test tube and use a UV spectrophotometer to measure its absorbance value A at a wavelength of 510 nm. F .

[0068] In order to ensure the accuracy of the experimental results, a control group needs to be set up. First, take 4.4 mL of distilled water and add it to the test tube; then, add 0.2 mL of hydrogen peroxide solution, 0.2 mL of ferrous sulfate solution, and 0.2 mL of salicylic acid-ethanol solution to the test tube in order. After shaking the mixture in the test tube, place it in a constant temperature water bath at 37°C for 30 minutes. After that, take out the test tube and use a UV spectrophotometer to measure its absorbance value A at a wavelength of 510 nm. E .

[0069] At the same time, a blank group was set up, which was measured only with distilled water. To ensure the reliability of the data, the experiment was repeated three times. Finally, the hydroxyl radical scavenging rate was calculated using the following formula: .

[0070] (2) DPPH free radical scavenging rate First, inject 2.0 mL of adzuki bean polypeptide sample solution into the test tube, and then add 2.0 mL of DPPH solution as the test sample A. B In addition, 2.0 mL of red bean polypeptide sample solution was added with 2.0 mL of 95% ethanol as control sample A. C , take 2.0 mL of DPPH solution and add it to 2.0 mL of 95% ethanol as blank sample A D . All test tubes were kept at 25°C in the dark for 30 minutes. Subsequently, the absorbance was measured using a spectrophotometer with a wavelength of 517 nm. To ensure the accuracy of the results, the experiment was repeated twice. Finally, the DPPH free radical scavenging rate was calculated according to the following formula: .

[0071] The results of the antioxidant activity measurement of the adzuki bean polypeptide and sea buckthorn compound beverage are shown in Table 8. From Table 8, it can be seen that the hydroxyl radical scavenging rate of the adzuki bean polypeptide is 71%, and the DPPH radical scavenging rate is 83%. Compared with the hydroxyl radical scavenging rate of 58.86% of the adzuki bean polypeptide prepared by Liang Yingyue in the literature, and the DPPH radical scavenging rate of 59.1% of the germinated chickpea polypeptide prepared by Liang Xuerong et al., the adzuki bean polypeptide prepared by alkaline protease in this application has good antioxidant properties. The DPPH radical scavenging rate and hydroxyl radical scavenging rate of the adzuki bean polypeptide and sea buckthorn compound beverage prepared by blending and sterilization reached 78% and 89.67% respectively, indicating that the compound beverage prepared with the prepared adzuki bean polypeptide has good antioxidant properties.

[0072] Table 8 Antioxidant activity of adzuki bean peptide and sea buckthorn compound beverage .

[0073] Although some preferred embodiments of the present application have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

[0074] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A method for preparing adzuki bean polypeptide, characterized in that: The following steps are involved: (1) Grinding and sieving adzuki beans to obtain adzuki bean powder; (2) adding the adzuki bean powder to water to form a bean powder suspension with a concentration of 2-3%; (3) adjusting the pH value of the soybean powder suspension to 8-8.5 using acid or alkali; (4) Then, 3800-4200 U / mg alkaline protease was added thereto, and the mixture was subjected to enzymatic hydrolysis at 54-56°C for 2.5-3.5 h; (5) After enzymatic hydrolysis is completed, sterilize in boiling water for 12-17 minutes. After cooling to room temperature, adjust the pH value of the enzymatic hydrolysis product to 7-7.5, and then centrifuge at 3500-4000 r / min for 15-20 minutes. Take the supernatant.

2. Use of the adzuki bean polypeptide obtained according to claim 1 in the preparation of an antioxidant biological agent.

3. Use of the adzuki bean polypeptide obtained according to claim 1 in the preparation of biological preparations for scavenging hydroxyl radicals and / or DPPH radicals.

4. A compound beverage, characterized in that: Made of the following raw materials in percentage by mass: 8-10% honey, 23-25% adzuki bean polypeptide prepared according to claim 1, 0.4-0.5% citric acid, 1.8-2.2% seabuckthorn concentrated juice, and 63-65% purified water.

5. The compound beverage according to claim 4, characterized in that Made of the following raw materials in percentage by mass: 10% honey, 24% adzuki bean polypeptide prepared according to claim 1, 0.4% citric acid, 2% seabuckthorn concentrated juice, and 63.6% purified water.

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