Preparation method of a quinoa peptide-polyphenol complex

Abstract

The present invention relates to the field of food science and technology, and particularly to a preparation method of a quinoa peptide-polyphenol complex. The present invention enhances the extraction rate and active functions of quinoa active ingredients through a three-step method of pressure cooking, enzymatic hydrolysis, and compounding, and prepares a highly active quinoa peptide-polyphenol composite functional ingredient by using intermolecular interactions. Compared with traditional functional ingredients, this complex has more diverse functions, can not only meet the needs of consumers for the diversity of functional foods, but also provide nutritional and health functions more comprehensively. Therefore, the present invention not only provides new technical support for the development of quinoa functional food ingredients, but also opens up a new way for the comprehensive utilization of quinoa, and has important practical value and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of food science and technology, and particularly to a method for preparing a quinoa peptide-polyphenol complex. Background Art

[0002] With the growing pursuit of nutritious and functional foods by people, the research on the active ingredients of quinoa has attracted extensive attention. Quinoa is rich in polyphenolic compounds, mainly composed of phenolic acids and flavonoids, which have the effects of lowering blood sugar, lowering blood lipid, antioxidant, antibacterial, and preventing cardiovascular diseases. Quinoa polyphenols have been widely used in the food, medicine and other industries. Quinoa peptides are polypeptides with specific amino acid sequences and biological activities formed by the hydrolysis of quinoa proteins after processing or digestion, and have the effects of lowering blood sugar, lowering blood pressure, and antioxidant. In the prior art, there is no relevant report on the compound preparation of quinoa peptides and quinoa polyphenols. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides a method for preparing a quinoa peptide-polyphenol complex. The present invention modifies quinoa by pressure cooking, efficiently obtains the polyphenol and protein components therein and optimizes their composition, uses a compound enzymatic hydrolysis method and molecular weight screening to prepare highly active quinoa peptides, and then spray-dries to prepare a highly active quinoa peptide-polyphenol complex, so as to improve the biological activity of the quinoa active ingredients and develop high-active functional ingredients of quinoa.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing a quinoa peptide-polyphenol complex, comprising the following steps:

[0006] 1) Mix quinoa grains with water and perform pressure cooking to obtain cooked quinoa, dry, crush and sieve the cooked quinoa to obtain quinoa powder;

[0007] The conditions for the pressure cooking are: the pressure is 0.1-0.2 MPa, the temperature is 110-120 °C, and the time is 10-20 min;

[0008] 2) Extract quinoa polyphenols from the quinoa powder obtained in step 1);

[0009] 3) Extract quinoa proteins from the quinoa powder obtained in step 1), mix the quinoa proteins with a compound enzyme and perform enzymatic hydrolysis to obtain an enzymatic hydrolysate;

[0010] The compound enzyme includes papain and alkaline protease;

[0011] 4) Pass the enzymatic hydrolysate obtained in step 3) through an ultrafiltration centrifugal tube system with a cut-off molecular weight of 3 KDa and / or 1 KDa to obtain quinoa peptides;

[0012] 5) Mix the quinoa polyphenols obtained in step 2) with the quinoa peptides and water obtained in step 4), and carry out a stirring reaction to obtain a reaction product;

[0013] 6) Dry the reaction product obtained in step 5) to obtain a quinoa peptide-polyphenol complex.

[0014] Preferably, the mass ratio of quinoa grains to water in step 1) is 10:3;

[0015] The temperature of the drying is below 60°C; the aperture of the sieve used for sieving is 80 mesh, and the material passing through the sieve is quinoa powder.

[0016] Preferably, the extraction of quinoa polyphenols in step 2) includes the following steps:

[0017] A. Mix the quinoa powder with an ethanol solution, and then carry out oscillation, ultrasonic treatment, and centrifugation in sequence to obtain a precipitate and a supernatant. Concentrate the supernatant and then dissolve it in methanol to obtain a free phenol solution;

[0018] B. Mix the precipitate obtained in step A with a sodium hydroxide solution and oscillate to obtain an oscillated product. Adjust the pH value of the oscillated product and then carry out extraction. Concentrate the obtained extract and then dissolve it in methanol to obtain a bound phenol solution;

[0019] C. Purify the free phenol solution obtained in step A and the bound phenol solution obtained in step B by macroporous resin, freeze-dry and mix them to obtain quinoa polyphenols.

[0020] Preferably, the mass ratio of quinoa powder to the volume of the ethanol solution in step A is 5 g:50 mL, and the volume percentage content of the ethanol solution is 70%;

[0021] The conditions for the oscillation include: temperature is 25°C, time is 4 h, and rotation speed is 200 rpm;

[0022] The conditions for the ultrasonic treatment include: power is 200 w, time is 15 min;

[0023] The conditions for the centrifugation include: centrifugal force is 3000 g, time is 15 min;

[0024] The temperature for the concentration is 45°C, and it is concentrated to one-third of the original volume.

[0025] Preferably, the volume ratio of the sodium hydroxide solution in step B to the ethanol solution in step A is 1:1, and the concentration of the sodium hydroxide is 4 mol / L;

[0026] The conditions for the oscillation include: temperature is 25°C, time is 4 h, and rotation speed is 200 rpm;

[0027] The pH value is 2.0, and the pH value is adjusted using a hydrochloric acid solution with a concentration of 6 mol / L.

[0028] The reagents used for extraction are ethyl acetate and diethyl ether. The volume ratio of ethyl acetate to diethyl ether is 1:1, and the number of extraction times is 3 times.

[0029] The temperature for concentration is 45 °C, and it is concentrated to one-third of the original volume.

[0030] Preferably, the macroporous resin in step C is AB-8 macroporous resin.

[0031] The conditions for purification include: the adsorption temperature is 25 °C, the adsorption time is 8 h, and after adsorption, desorption is carried out using an ethanol solution with a volume percentage of 70%.

[0032] Preferably, the extraction method of quinoa protein in step 3) includes the following steps:

[0033] a. Defat the quinoa powder to obtain defatted quinoa powder. Mix the defatted quinoa powder with water, adjust the pH value, then stir and centrifuge to obtain the supernatant.

[0034] b. Adjust the pH value of the supernatant obtained in step a and then centrifuge to obtain a precipitate.

[0035] c. Redissolve the precipitate obtained in step b, dialyze it in a dialysis bag with a molecular cut-off of 8000 - 14000 Da, adjust the pH value to neutral, and then freeze-dry to obtain quinoa protein.

[0036] Preferably, the conditions for defatting in step a include: mix the quinoa powder with n-hexane, then oscillate and dry to obtain defatted quinoa powder; the mass ratio of the quinoa powder to the volume of n-hexane is 1 g:4 mL; the conditions for oscillation include: the rotation speed is 200 rpm and the time is 12 h; the drying temperature is 40 °C.

[0037] The mass ratio of the defatted quinoa powder to water in step a is 1:10, the pH value is 10.0, and the pH value is adjusted using a hydrochloric acid solution with a concentration of 2 mol / L; the conditions for stirring include: the temperature is 35 °C and the time is 90 min; the conditions for centrifugation include: the temperature is 4 °C, the time is 20 min, and the centrifugal force is 6000 g.

[0038] The pH value in step b is 4.5, and the pH value is adjusted using a hydrochloric acid solution with a concentration of 0.5 mol / L; the conditions for centrifugation include: the temperature is 4 °C, the time is 20 min, and the centrifugal force is 6000 g.

[0039] The dialysis conditions in step c include: redissolving the precipitate in PBS buffer for dialysis for 24 h, and changing the buffer once every 3 h.

[0040] Preferably, the mass ratio of quinoa protein to the complex enzyme in step 3) is 100:1;

[0041] The mass ratio of papain to alkaline protease is 1-2:1-2;

[0042] The enzymatic hydrolysis conditions include: temperature of 50 °C, time of 3 h, and rotation speed of 100 rpm; the enzymatic hydrolysis is carried out under water bath conditions.

[0043] Preferably, the mass ratio of quinoa polyphenol to quinoa peptide in step 5) is 1-6:1-6;

[0044] The stirring reaction time in step 5) is 24 h;

[0045] The drying conditions in step 6) include: temperature of 80-120 °C and feed rate of 2-5 L / h.

[0046] The beneficial effects of the present invention are as follows:

[0047] (1) Efficient extraction of quinoa protein and polyphenols: By using the method of pressure cooking, the production efficiency of obtaining quinoa protein and polyphenols is improved, and the cost is reduced; the extractable content of quinoa protein and polyphenols is increased, the components are optimized, the biological activity is enhanced, and the loss of nutrients is reduced.

[0048] (2) Preparation of highly active quinoa peptides by using the complex enzyme method and molecular weight screening: To solve the problems of low efficiency and unsatisfactory activity in the traditional method of preparing bioactive peptides by single enzymatic hydrolysis, the present invention uses the complex enzyme method to prepare quinoa peptides, and screens the peptide segments with the highest activity through molecular weight screening, which can improve the enzymatic hydrolysis efficiency and screen out highly active polypeptides.

[0049] (3) Preparation of highly active quinoa peptide-polyphenol complex: By utilizing the synergistic interaction between polypeptides and polyphenols, a complex with multiple activities is prepared, which solves the limitation of the traditional functional ingredients with single function.

[0050] The present invention enables quinoa polypeptides and polyphenols to interact through environment-based non-covalent bonds, improving their physical and chemical properties through synergistic effects and complementary activities, and enhancing their biological activities. The present invention enhances the extraction rate and active functions of quinoa active ingredients through a three-step method of pressure cooking, enzymatic hydrolysis, and compounding, and prepares a highly active quinoa peptide-polyphenol composite functional ingredient by utilizing intermolecular interactions. Compared with traditional functional ingredients, this composite has more diverse functions, can not only meet the needs of consumers for the diversity of functional foods, but also provide nutritional and health functions more comprehensively. Therefore, this invention not only provides new technical support for the development of quinoa functional food ingredients, but also opens up new ways for the comprehensive utilization of quinoa, and has important practical value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0052] Figure 1 is the hypoglycemic ability (a) and antioxidant ability (b) of the quinoa protein hydrolyzate after pressure cooking;

[0053] Figure 2 is the hypoglycemic ability (a) and antioxidant ability (b) of quinoa peptides with different molecular weights;

[0054] Figure 3 is the infrared spectrum (a) and secondary structure (b) of the highly active quinoa peptide-polyphenol complex;

[0055] Figure 4 is the hypoglycemic ability (a) and antioxidant ability (b) of the highly active quinoa peptide-polyphenol complex;

[0056] Figure 5 is the bacterial growth curve of the highly active quinoa peptide-polyphenol complex Escherichia coli (a) Staphylococcus aureus (b). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0057] The present invention provides a method for preparing a quinoa peptide-polyphenol complex, comprising the following steps:

[0058] 1) Mix quinoa grains with water and subject them to pressure cooking to obtain cooked quinoa. Dry, crush, and sieve the cooked quinoa to obtain quinoa powder;

[0059] The conditions for the pressure cooking are: pressure of 0.1 - 0.2 MPa, temperature of 110 - 120 °C, and time of 10 - 20 min;

[0060] 2) Extract quinoa polyphenols from the quinoa powder obtained in step 1);

[0061] 3) Extract quinoa protein from the quinoa flour obtained in step 1), mix the quinoa protein with a complex enzyme and carry out enzymatic hydrolysis to obtain an enzymatic hydrolysate;

[0062] The complex enzyme includes papain and alkaline protease;

[0063] 4) Pass the enzymatic hydrolysate obtained in step 3) through an ultrafiltration centrifugal tube system with a cut-off molecular weight of 3 KDa and / or 1 KDa to obtain quinoa peptides;

[0064] 5) Mix the quinoa polyphenols obtained in step 2), the quinoa peptides obtained in step 4) and water, and carry out a stirring reaction to obtain a reaction product;

[0065] 6) Dry the reaction product obtained in step 5) to obtain a quinoa peptide-polyphenol complex.

[0066] In the present invention, quinoa grains are mixed with water and subjected to pressure cooking to obtain cooked quinoa, and the cooked quinoa is dried, pulverized and sieved to obtain quinoa flour; the conditions for the pressure cooking are: the pressure is 0.1-0.2 MPa, the temperature is 110-120 °C, and the time is 10-20 min. In the present invention, the mass ratio of the quinoa grains to water is preferably 10:3. In the present invention, the drying temperature is preferably below 60 °C. In the present invention, the aperture of the sieve used for sieving is preferably 80 mesh, and the material passing through the sieve is preferably quinoa flour.

[0067] Cooking is one of the common processing methods of quinoa, but ordinary cooking techniques will cause a large loss of quinoa phenolic substances and lead to a decline in antioxidant capacity. Compared with ordinary cooking and boiling, the pressure cooking technique adopted in the present invention has the advantages of fast cooking, high efficiency and low cost. It not only increases the total phenolic content of quinoa, but also improves the antioxidant capacity of quinoa polyphenols and reduces the loss of nutrients. In addition, the pressure cooking technique can play a certain role in improving the processing characteristics of quinoa protein, and has the advantages of simple operation and low cost, and is suitable for large-scale industrial processing.

[0068] The quinoa polyphenols in the quinoa flour extracted in the present invention.

[0069] In the present invention, the extraction of the quinoa polyphenols preferably includes the following steps:

[0070] A. Mix the quinoa flour with an ethanol solution, and then carry out oscillation, ultrasonic treatment and centrifugation in sequence to obtain a precipitate and a supernatant. Concentrate the supernatant and then dissolve it in methanol to obtain a free phenol solution;

[0071] B. Mix the precipitate obtained in step A with a sodium hydroxide solution and oscillate to obtain an oscillated product. Adjust the pH value of the oscillated product and then carry out extraction. Concentrate the obtained extract and then dissolve it in methanol to obtain a bound phenol solution;

[0072] C. Purify the free phenol solution obtained in step A and the conjugated phenol solution obtained in step B with macroporous resin, followed by freeze-drying and mixing to obtain quinoa polyphenols.

[0073] In the present invention, the mass ratio of the quinoa powder in step A to the volume of the ethanol solution is preferably 5 g:50 mL, and the volume percentage content of the ethanol solution is preferably 70%. In the present invention, the conditions for oscillation preferably include: temperature of 25°C, time of 4 h, and rotation speed of 200 rpm. In the present invention, the conditions for ultrasonic treatment preferably include: power of 200 w and time of 15 min. In the present invention, the conditions for centrifugation preferably include: centrifugal force of 3000 g and time of 15 min. In the present invention, the temperature for concentration is preferably 45°C, and the volume is concentrated to one-third of the original volume.

[0074] In the present invention, the volume ratio of the sodium hydroxide solution in step B to the ethanol solution in step A is preferably 1:1, and the concentration of the sodium hydroxide is preferably 4 mol / L. In the present invention, the conditions for oscillation preferably include: temperature of 25°C, time of 4 h, and rotation speed of 200 rpm. In the present invention, the pH value is preferably 2.0, and the pH value is preferably adjusted using a hydrochloric acid solution with a concentration of 6 mol / L in the present invention. In the present invention, the reagents used for extraction are preferably ethyl acetate and diethyl ether, the volume ratio of ethyl acetate to diethyl ether is preferably 1:1, and the number of extraction times is preferably 3 times. In the present invention, the temperature for concentration is preferably 45°C, and the volume is concentrated to one-third of the original volume.

[0075] In the present invention, the macroporous resin for step C is AB-8 macroporous resin. In the present invention, the purification conditions preferably include: adsorption temperature of 25°C, adsorption time of 8 h, and desorption using an ethanol solution with a volume percentage content of 70% after adsorption is completed.

[0076] The quinoa protein in the quinoa powder obtained by extraction in the present invention is mixed with a complex enzyme and enzymatically hydrolyzed to obtain a hydrolysate; the complex enzyme includes papain and alkaline protease.

[0077] In the present invention, the extraction method of the quinoa protein preferably includes the following steps:

[0078] a. Degrease the quinoa powder to obtain defatted quinoa powder, mix the defatted quinoa powder with water, adjust the pH value, stir, and centrifuge to obtain a supernatant;

[0079] b. Adjust the pH value of the supernatant obtained in step a and then centrifuge to obtain a precipitate;

[0080] c. The precipitate obtained in step b is redissolved and dialyzed in a dialysis bag with a molecular cutoff of 8000 to 14000 Da. The pH value is adjusted to neutral and then freeze-dried to obtain quinoa protein.

[0081] In the present invention, the defatting conditions in step a preferably include: mixing the quinoa powder with n-hexane, shaking and drying to obtain defatted quinoa powder. In the present invention, the mass ratio of the quinoa powder to the volume ratio of n-hexane is preferably 1g:4mL. In the present invention, the oscillation conditions preferably include: a rotation speed of 200rpm, a time of 12h; and a drying temperature of 40°C. In the present invention, the mass ratio of the defatted quinoa powder to water in step a is preferably 1:10, the pH value is 10.0, and a hydrochloric acid solution with a concentration of 2mol / L is used to adjust the pH value. In the present invention, the stirring conditions preferably include: a temperature of 35°C and a time of 90min. In the present invention, the centrifugal conditions include: a temperature of 4°C, a time of 20min, and a centrifugal force of 6000g. In the present invention, the pH value in step b is preferably 4.5, and a hydrochloric acid solution with a concentration of 0.5mol / L is used to adjust the pH value. In the present invention, the conditions for the centrifugation preferably include: a temperature of 4°C, a time of 20min, and a centrifugal force of 6000g. In the present invention, the conditions for the dialysis in step c preferably include: redissolving the precipitate in a PBS buffer for dialysis for 24h, and replacing the buffer every 3h. In the present invention, the mass ratio of the quinoa protein to the complex enzyme is preferably 100:1. In the present invention, the mass ratio of the papain to the alkaline protease is preferably 1-2:1-2. In the present invention, the conditions for the enzymolysis preferably include: a temperature of 50°C, a time of 3h, and a rotation speed of 100rpm. In the present invention, the enzymolysis is preferably carried out under water bath conditions.

[0082] Enzymatic hydrolysis is one of the most commonly used methods for preparing bioactive peptides. The prepared peptides are highly safe, easily digested and absorbed by the human body, and the production conditions are mild and easy to control. However, when a single enzyme is used to prepare bioactive peptides, the yield is low and the peptide activity is not ideal. The present invention uses an optimized composite enzyme method to prepare quinoa peptides, and screens the peptide segments with the highest activity by molecular weight, thereby overcoming the singleness of the action of a single enzyme and the disadvantages of unsatisfactory activity, and can improve the enzymatic hydrolysis efficiency and peptide activity.

[0083] The invention passes the obtained enzymolyzate through an ultrafiltration centrifuge tube system with a molecular weight cutoff of 3KDa and / or 1KDa to obtain quinoa peptide.

[0084] The present invention mixes the obtained quinoa polyphenols, the obtained quinoa peptides and water, and performs a stirring reaction to obtain a reactant. In the present invention, the mass ratio of the quinoa polyphenols to the quinoa peptides is preferably 1 to 6:1 to 6. In the present invention, the stirring reaction time is preferably 24 hours.

[0085] The obtained reactants of the present invention are dried to obtain a quinoa peptide-polyphenol complex. In the present invention, the drying conditions preferably include: a temperature of 80 to 120 °C and a feeding rate of 2 to 5 L / h.

[0086] Currently, there is no product containing highly active quinoa peptide-polyphenol functional components on the market. The preparation of a highly active quinoa peptide-polyphenol complex by spray drying can fill the gap in highly active quinoa peptide-polyphenol functional ingredients on the market, greatly enhance the active functions of products, and overcome the limitation of the single function of traditional functional ingredients.

[0087] Pressure cooking significantly increases the free phenols, bound phenols and total phenol contents of quinoa. The antioxidant, antibacterial properties and in vitro digestibility of its polyphenols are all significantly improved, and the content of polyphenols is closely related to antioxidant and antibacterial activities. At the same time, pressure cooking also enhances the emulsifying stability and thermal stability of quinoa protein.

[0088] The enzymatic hydrolysate of quinoa protein has high hypoglycemic, antioxidant and antibacterial abilities, and the effect of combined enzymatic hydrolysis is better; and highly active quinoa peptides are screened out by molecular weight screening, greatly improving the enzymatic hydrolysis efficiency and polypeptide activity of quinoa protein.

[0089] The hypoglycemic, antioxidant and antibacterial abilities of the highly active quinoa peptide-polyphenol complex are improved compared with those of polyphenol and polypeptide monomers, and there is a synergistic activity enhancement between polyphenols and polypeptides; it fills the gap in highly active quinoa peptide-polyphenol functional ingredients on the market and provides technical support for the development of functional foods and the comprehensive utilization of quinoa.

[0090] The materials, instruments, etc. used in the following examples are as follows:

[0091] Materials and reagents:

[0092] Quinoa was purchased from Qinghai Haixi Organic Farm.

[0093] DPPH, ABTS, TPTZ, Trolox, Folin-Ciocalteu phenol reagent, methanol, glacial acetic acid, absolute ethanol, ethyl acetate, sodium hydroxide, acetone, potassium persulfate, hydrochloric acid, ferric chloride hexahydrate are all of analytical grade; yeast extract, tryptone, agar are all of biological reagent purity; α-amylase and α-glucosidase were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; papain and alkaline protease were purchased from Tianjin Taijin Technology Co., Ltd.; the polyphenol standard product is of chromatographic purity and was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0094] Instruments and equipment:

[0095] Spray dryer (YC-015), Marin Christ, Germany; High performance liquid chromatograph (1260 infinity), Agilent Technologies, Inc.; Scanning electron microscope (SU-1510), Hitachi, Japan; Thermogravimetric analyzer (TGA-Q50), TA Instruments, USA; Desktop high-speed refrigerated centrifuge (Neofuge 13R), Heal Force Development Ltd; Pulverizer (30b), Changzhou Panfeng Drying Equipment Co., Ltd.; Multifunctional microplate reader (Epoch2), ThermoFisher Scientific, USA; Fourier transform infrared spectrometer (IS50), ThermoFisher Scientific, USA; Mass spectrometer (Q Exactive), ThermoFisher Scientific, USA.

[0096] To further illustrate the present invention, the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0097] Example 1

[0098] Method for extracting quinoa polyphenols by pressure cooking treatment of quinoa, the extraction method is as follows:

[0099] (1) Put 100 g of quinoa seeds and 30 mL of distilled water into a glassware, shake for 2 h to evenly distribute the moisture on the surface of the quinoa, and then pressure cook in an autoclave at 0.1 MPa and 120 °C for 20 min. Then, collect the pretreated quinoa and dry it at 60 °C. Crush the quinoa through an 80-mesh sieve and store it at 4 °C until use.

[0100] (2) Mix 5.0 g of quinoa flour with 50 mL of 70% (v / v) ethanol, shake at a speed of 200 r / min at 25 °C for 4 h, then sonicate for 15 min, centrifuge at 3000×g for 15 min, collect the supernatant. Repeat the extraction of the precipitate with 70% ethanol twice, combine the supernatants, evaporate and concentrate under vacuum using a rotary evaporator at 45 °C. The concentrated solution is redissolved in 10 mL of methanol as the free phenol fraction and stored at -20 °C until further analysis. In the remaining residue above, add 50 mL of 4 mol / L NaOH, shake at a speed of 200 r / min at 25 °C for 4 h. Adjust the pH of the mixture to 2.0 with 6 mol / L HCl, extract with ethyl acetate / ether (1:1, v / v) three times, then evaporate and concentrate under vacuum at 45 °C, and finally reconstitute to 10 mL with methanol as the bound phenol fraction and store at -20 °C until further analysis. Purify the free phenol and bound phenol using AB-8 macroporous resin, with an adsorption temperature of 25 °C and an adsorption time of 8 h. After adsorption, desorb with 70% ethanol as the eluent. Use a vacuum rotary evaporator to remove ethanol at 45 °C and freeze-dry to obtain the quinoa polyphenol sample.

[0101] Example 2

[0102] Example 2 is similar to Example 1, except that it is pressure-cooked in an autoclave at 0.2 MPa and 120 °C for 20 min.

[0103] Example 3

[0104] Example 3 is similar to Example 1, except that it is pressure-cooked in an autoclave at 0.1 MPa and 110 °C for 20 min.

[0105] Example 4

[0106] Example 4 is similar to Example 1, except that it is pressure-cooked in an autoclave at 0.1 MPa and 120 °C for 10 min.

[0107] Example 5

[0108] (1) Appropriately take the quinoa flour prepared in Example 1, remove the fat by shaking with n-hexane (material-liquid ratio 1:4) at 200 r / min for 12 h, dry the defatted sample at 40 °C, add distilled water at a ratio of 1:10, adjust the pH of the solution to 10.0 with 2 mol / L NaOH, stir magnetically at 35 °C for 90 min, then centrifuge at 6000×g and 4 °C for 20 min, collect the supernatant, adjust the pH to 4.5 with 0.5 mol / L HCl, and then centrifuge at 6000×g and 4 °C for 20 min to collect the precipitate. Redissolve it with PBS buffer solution at pH 7.0, put it into a dialysis bag with a molecular cut-off of 8000 - 14000 Da, and dialyze at low temperature in PBS buffer solution for 24 h, changing the buffer solution every 3 h. Finally, adjust the pH value to 7.0 and freeze-dry it under vacuum to obtain the quinoa protein sample.

[0109] Hydrolyze quinoa protein by the combined enzyme method, and the enzymatic hydrolysis conditions are as follows:

[0110] Prepare the extracted quinoa protein into a solution with a concentration of 3% (w / v) with distilled water. Under the condition of enzyme:substrate = 1:100 (w / w), carry out enzymatic hydrolysis by the combined action of two enzymes (papain: alkaline protease, mass ratio 1:1), adjust the pH of the suspension to 7.0 with 0.5 mol / L NaOH, carry out enzymatic hydrolysis in a 50 °C water bath at a speed of 100 r / min for 3 h, and boil for 10 min to terminate the enzyme reaction. Then centrifuge at 8000×g and 4 °C for 10 min to collect the supernatant to obtain the enzymatic hydrolysis product.

[0111] Example 6

[0112] Example 6 is similar to Example 5, except that the ratio of papain: alkaline protease is 2:1.

[0113] Example 7

[0114] Example 7 is similar to Example 5, except that the ratio of papain: alkaline protease is 1:2.

[0115] Example 8

[0116] Ultrafilter the quinoa protein hydrolysis product to screen out quinoa peptides with high activity. The screening method is as follows:

[0117] Pass the enzymatic hydrolysis product through a Millipore ultrafiltration centrifuge tube (15 mL) with a molecular weight cut-off of 1 kDa to obtain quinoa peptides after ultrafiltration <1 kDa, and carry out vacuum freeze-drying for later use.

[0118] Example 9

[0119] Example 9 is similar to Example 8, except that the remaining enzymatic hydrolysate of Example 8 is passed through a Millipore ultrafiltration centrifuge tube (15 mL) with a molecular weight cut-off of 3 KDa to obtain quinoa peptides after ultrafiltration of (1-3 KDa), which are then vacuum freeze-dried for later use.

[0120] Example 10

[0121] Example 10 is similar to Example 8, except that the remaining enzymatic hydrolysate of Example 9 is passed through a Millipore ultrafiltration centrifuge tube (15 mL) with a molecular weight cut-off of 10 KDa to obtain quinoa peptides after ultrafiltration of (3-10 KDa) and (>10 KDa), which are then vacuum freeze-dried for later use.

[0122] Example 11

[0123] A preparation method of a high-activity quinoa peptide-polyphenol complex is as follows:

[0124] Disperse quinoa polyphenols in an appropriate amount of methanol, and then adjust to 100 mL with distilled water. Adjust the pH of the polyphenol solution to 9.0 with 0.1 mol / L NaOH, and stir magnetically for 12 h to completely disperse the polyphenol solution to obtain a polyphenol solution. Then dissolve the quinoa peptides obtained in Example 9 in the above polyphenol solution, and the ratio of polypeptide / polyphenol is 6:1 (w / w). After adding the polypeptide of Example 9, continue to stir the mixed solution magnetically at room temperature for 24 h to ensure complete reaction of the polypeptide and polyphenol. Spray-dry the solution at 120 °C and 5 L / h to obtain a high-activity quinoa peptide-polyphenol complex.

[0125] Example 12

[0126] Example 12 is similar to Example 11, except that the spray-drying conditions are 60 °C and 2 L / h.

[0127] Example 13

[0128] Example 13 is similar to Example 11, except that the spray-drying conditions are 80 °C and 3 L / h.

[0129] Example 14

[0130] Example 14 is similar to Example 11, except that the ratio of polypeptide / polyphenol is 2:1 (w / w).

[0131] Example 15

[0132] Example 15 is similar to Example 11, except that the ratio of polypeptide / polyphenol is 4:1 (w / w).

[0133] Example 16

[0134] Example 16 is similar to Example 11, except that the ratio of polypeptide / polyphenol is 1:2 (w / w).

[0135] Example 17

[0136] Example 17 is similar to Example 11, except that the ratio of polypeptide / polyphenol is 1:4 (w / w).

[0137] Example 18

[0138] Example 17 is similar to Example 11, except that the ratio of polypeptide / polyphenol is 1:6 (w / w).

[0139] Comparative Example 1

[0140] The difference between Comparative Example 1 and Example 1 is only that it is steamed by the ordinary steaming method (120 °C) for 20 min.

[0141] Comparative Example 2

[0142] The difference between Comparative Example 2 and Example 5 is only that single enzyme (papain / alkaline protease) is used to enzymatically hydrolyze quinoa protein.

[0143] Comparative Example 3

[0144] The difference between Comparative Example 3 and Example 8 is only that ultrafiltration centrifugal tubes are not used for sieving.

[0145] Comparative Example 4

[0146] The difference between Comparative Example 4 and Example 10 is only that the freeze-drying method is adopted, that is, the solution is frozen at -80 °C for 12 hours and then freeze-dried for 24 hours to obtain the complex.

[0147] Comparative Example 5

[0148] The difference between Comparative Example 5 and Example 11 is only that the ratio of polypeptide / polyphenol is 1:1 (w / w).

[0149] α-Amylase inhibition rate: Prepare 1% starch solution and 1 U / mL α-amylase solution with 0.1 mol / L, pH = 6.8 PBS buffer solution. Accurately pipette 200 μL of the sample solution and 200 μL of the α-amylase solution, mix them and place them in a reaction at 37 °C for 5 min, add 500 μL of the starch solution to start the reaction, keep the mixture at a constant temperature of 37 °C for 10 min, after the reaction, add 200 μL of DNS reagent to it, heat the reaction solution in a water bath at 100 °C for 15 min to terminate the reaction, then place it in ice water to cool down rapidly, and then add 2 mL of distilled water, and measure the absorbance at 540 nm. The inhibition rate of α-amylase is calculated according to the following formula:

[0150]

[0151] A: Control group, absorbance value with PBS buffer replacing the sample; B: Blank group, absorbance value with PBS buffer replacing the enzyme; C: Sample group, absorbance value of the sample supernatant.

[0152] α - Glucosidase inhibition rate: Prepare a 4 mmol / L 4 - nitrophenyl - β - D - glucopyranoside (pNPG) solution and a 0.2 U / mL α - glucosidase solution with 0.1 M PBS at pH = 6.8. Accurately pipette 100 μL of the sample solution, add 100 μL of PBS and 100 μL of pNPG, and react at 37 °C for 5 min to mix well. Then add 50 μL of the α - glucosidase solution to initiate the reaction and react fully at 37 °C for 30 min. Finally, add 80 μL of 1 mol / L Na 2 CO 3 to terminate the reaction, and measure its absorbance value at 405 nm, record the content of 4 - nitrophenol (PNP) released and record. The α - glucosidase inhibition rate is calculated according to the following formula:

[0153]

[0154] A: Control group, absorbance value after the enzyme reacts with the substrate; B: Blank group, absorbance value with PBS buffer replacing the sample to be measured and the α - glucosidase solution; C: Sample group, absorbance value of the sample supernatant; D: Sample control group, absorbance value with an equal volume of PBS buffer replacing the α - glucosidase solution.

[0155] Polyphenol content: The polyphenol content in quinoa was determined by the Folin - Ciocalteu method. Take 50 μL of the above - mentioned polyphenol extract, add 950 μL of distilled water and mix well. Add 5 mL of distilled water, 500 μL of Folin - Ciocalteu solution, and 250 μL of Na 2 CO 3 solution (20%, w / v), measure its absorbance value at 760 nm after reacting in the dark for 20 min. Use the gallic acid standard curve (y = 0.0055x + 0.0381, R 2 = 0.9969) to calculate the polyphenol content, and the result is expressed as mg gallic acid equivalent (mg GAE) per gram of dry weight (DW).

[0156] In vitro digestibility: The in vitro digestibility of quinoa polyphenols was determined using the previously reported in vitro gastrointestinal digestion method with minor modifications. Simulated gastric juice was prepared with 0.625 g of pepsin, 0.25 g of sodium malate, 0.25 g of sodium citrate, 250 μL of acetic acid, 210 μL of lactic acid, and distilled water, and adjusted to pH = 2.0 with 0.5 mol / L HCl, finally obtaining 500 mL of simulated gastric juice. 5.0 g of quinoa flour was mixed with 100 mL of simulated gastric juice and reacted with shaking in a water bath at 37 °C and 100 r / min for 1 h. Then, the pH of the mixture was adjusted to 7.0 to stop digestion. Subsequently, 87.0 mg of bile salts and 25 mg of pancreatin were added to the mixture. After 2 h, the mixture was centrifuged at 3000×g for 10 min, and the supernatant was collected. The in vitro digestibility of polyphenols was determined as the percentage of phenolic content in the supernatant relative to the total phenolic content. 3 The pH of the mixture was adjusted to 7.0 to stop digestion. Subsequently, 87.0 mg of bile salts and 25 mg of pancreatin were added to the mixture. After 2 h, the mixture was centrifuged at 3000×g for 10 min, and the supernatant was collected. The in vitro digestibility of polyphenols was determined as the percentage of phenolic content in the supernatant relative to the total phenolic content.

[0157] DPPH radical scavenging ability: DPPH standard curve: 1.0 mg of Trolox was prepared as a 400 μmol / L solution with absolute ethanol and diluted with distilled water to concentration gradients of 80, 160, 240, 320, and 400 μmol / L. 50 μL of Trolox solutions with different concentrations were taken respectively, 200 μL of DPPH solution and 250 μL of absolute ethanol were added thereto, and the absorbance was measured at 517 nm after reacting in the dark for 30 min. The standard curve was obtained: y = -0.0011x + 0.7048, R 2 = 0.9994. Sample determination: The phenolphthalein complex was prepared as a 1 mg / mL solution, and the determination method was the same as above. The DPPH radical scavenging ability of the complex was calculated according to the standard curve.

[0158] ABTS radical scavenging ability: ABTS standard curve: 2.5 mg of Trolox was prepared as a 1000 μmol / L solution with absolute ethanol and diluted with distilled water to concentration gradients of 200, 400, 600, 800, and 1000 μmol / L. ABTS working solution: 7 mmol / L ABTS solution and 2.45 mmol / L K 2 S 2 O 8 solution were prepared with distilled water respectively. The ABTS solution and K 2 S 2 O 8The solutions were mixed at a ratio of 2:1 (v:v), and after mixing, the mixture was placed in a dark environment and reacted for 16 h to obtain the ABTS radical stock solution. The ABTS radical stock solution was diluted with absolute ethanol to an absorbance value of 0.70 ± 0.02 at 734 nm and 30 °C before use to obtain the ABTS radical working solution, which was prepared and used immediately. 20 μL of Trolox solutions with different concentrations were taken respectively, and 500 μL of the ABTS working solution was added thereto. After reacting for 10 min under dark conditions, the absorbance value was measured at 734 nm. The standard curve was obtained: y = -0.0007x + 0.6632, R 2 = 0.9997. Sample determination: The quinoa peptide-polyphenol complex was prepared into a 1 mg / mL solution, and the determination method was the same as above. The ABTS radical scavenging ability of the complex was calculated according to the standard curve.

[0159] Ferric reducing ability: FRAP standard curve: 5.0 mg of Trolox was prepared into a 2000 μmol / L solution with absolute ethanol and diluted with distilled water to concentration gradients of 400, 800, 1200, 1600, and 2000 μmol / L. FRAP working solution: Prepare a 300 mmol / L sodium acetate solution with pH = 3.6, a 10 mmol / L TPTZ solution with 40 mmol / L hydrochloric acid as the solvent, and a 20 mmol / L FeCl 3 ·6H 2 O solution. The three were mixed at a volume ratio of 10:1:1 to obtain the FRAP working solution, which was prepared and used immediately. 10 μL of Trolox solutions with different concentration gradients were taken respectively and mixed with 990 μL of the FRAP working solution. Then, the mixture was reacted in a 37 °C water bath for 30 min, and the absorbance value was measured at 593 nm. The standard curve was obtained: y = 0.0002x + 0.1252, R 2 = 0.9993. Sample determination: The phenolphthalein complex was prepared into a 1 mg / mL solution, and the determination method was the same as above. The ferric reducing ability of the complex was calculated according to the standard curve.

[0160] Determination of antibacterial ability: Escherichia coli and Staphylococcus aureus were selected as the test strains in this experiment to determine the antibacterial activity of the phenolphthalein complex. Activation of bacteria: Under sterile conditions, the test strains were picked with an inoculation loop and inoculated onto LB solid medium by three-zone streaking. Then the petri dishes were sealed and inverted and placed in a constant temperature incubator at 37 °C for 24 h, and repeatedly cultured and activated for 2 - 3 generations. Preparation of bacterial suspension: The activated test strains were inoculated into LB liquid medium, and then placed in an air bath shaker (37 °C, 180 r / min) for 6 - 8 h. Then the absorbance value was measured at 600 nm, and a bacterial suspension with a concentration of 1×106 CFU / mL was selected, that is, the bacterial suspension when the absorbance value reached 0.78 ± 0.02, and stored at 4 °C for use. Minimum inhibitory concentration (MIC): A complex solution with a concentration of 15 mg / mL was prepared as the initial concentration. 150 μL of the sample solution and 150 μL of LB liquid medium were added to a sterile 96-well plate and mixed well. The sample solution was serially diluted to form different concentration gradients by the two-fold dilution method. Finally, 5 μL of the diluted bacterial suspension was added to the wells of the sample solution and the liquid medium. In addition, only 150 μL of LB liquid medium was added to the wells of the negative control, and 150 μL of LB liquid medium and 5 μL of bacterial suspension were added to the wells of the positive control. After adding the samples, the 96-well plate was sealed and incubated at 37 °C for 24 h. The sample concentration with no obvious bacterial growth was the MIC.

[0161] Table 1 Effects of pressure cooking on the content, in vitro digestibility and antioxidant capacity of quinoa polyphenols

[0162]

[0163]

[0164] Analysis of the effects of pressure cooking on the content, in vitro digestibility and antioxidant capacity of quinoa polyphenols: The effects of pressure cooking on the content, in vitro digestibility and antioxidant capacity of quinoa polyphenols are shown in Table 1. Compared with Comparative Example 1, pressure cooking significantly increased the content of quinoa polyphenols (P < 0.05), and the total phenol content in Example 1 was the highest. At the same time, it can be observed that pressure cooking significantly increased the in vitro digestibility of quinoa polyphenols (P < 0.05), which was 18.46% higher than that in Comparative Example 1. After pressure cooking, the antioxidant capacity of quinoa polyphenols increased significantly. Pressure cooking may have broken the covalent bonds between polyphenols and cell walls, resulting in more phenolic hydroxyl groups being exposed and enhancing its antioxidant capacity.

[0165] Table 2 Effects of pressure cooking on the antibacterial property of quinoa polyphenols

[0166]

[0167] Analysis of the antibacterial properties of quinoa polyphenols by pressure cooking: Compared with Comparative Example 1, pressure cooking significantly improved the antibacterial properties of quinoa polyphenols, which may be due to the increase in the content of quinoa polyphenols after pressure cooking treatment, resulting in stronger antibacterial ability. Among them, Example 1 had the best antibacterial property.

[0168] Table 3 Effects of complex enzymatic hydrolysis on the hypoglycemic ability, antioxidant ability and antibacterial properties of quinoa protein hydrolysates

[0169]

[0170] Analysis of the hypoglycemic ability and antioxidant ability of quinoa protein hydrolysates by complex enzymatic hydrolysis: Table 3 shows the effects of complex enzymatic hydrolysis on the hypoglycemic ability and antioxidant ability of quinoa protein hydrolysates. Among them, the hypoglycemic ability of the complex enzyme hydrolysis group was significantly stronger than that of Comparative Example 2, and Example 5 had the strongest hypoglycemic ability. At the same time, complex enzymatic hydrolysis also significantly improved the antioxidant ability of quinoa protein hydrolysates.

[0171] Analysis of the antibacterial properties of quinoa protein hydrolysates by complex enzymatic hydrolysis: Compared with Comparative Example 2, complex enzymatic hydrolysis significantly improved the antibacterial properties of quinoa protein hydrolysates. Among them, Example 5 had the best antibacterial property, with a MIC of 1.89 g / L against Escherichia coli and 0.94 g / L against Staphylococcus aureus.

[0172] Table 4 Hypoglycemic ability, antioxidant ability and antibacterial properties of quinoa peptides with different molecular weights

[0173]

[0174]

[0175] Analysis of the hypoglycemic ability and antioxidant ability of quinoa peptides with different molecular weights: As shown in Table 4, the inhibition rates of α-amylase and α-glucosidase in Example 10 and Comparative Example 3 were significantly lower than those of the quinoa peptides in Example 8 and Example 9 (P<0.05). At the same time, there were significant differences in the DPPH, ABTS radical scavenging ability and iron reducing ability of quinoa peptide components with different molecular weights (P<0.05). The antioxidant ability of the quinoa peptides in Example 8 and Example 9 was significantly higher than that of the quinoa peptides in Example 10 and Comparative Example 3. The results showed that the antioxidant ability of low molecular weight quinoa peptides was higher than that of high molecular weight quinoa peptides.

[0176] Analysis of the antibacterial properties of quinoa peptides with different molecular weights: Quinoa peptides with different molecular weights had strong antibacterial effects on both Escherichia coli and Staphylococcus aureus, and the quinoa peptides in Example 9 and Example 10 (3-10KDa) were higher than those in Example 10 (10KDa) and Example 8. In summary, the quinoa peptides in Example 9 had the strongest comprehensive activity.

[0177] Table 5 Effects of Different Spray-Drying Conditions on Hypoglycemic Ability, Antioxidant Ability and Bacteriostasis of High-Activity Quinoa Peptide-Polyphenol Complex

[0178]

[0179] Analysis of the Effects of Different Spray-Drying Conditions on High-Activity Quinoa Peptide-Polyphenol Complex: Table 5 shows the effects of different spray-drying conditions on high-activity quinoa peptide-polyphenol complex. The hypoglycemic ability and antioxidant ability of the spray-drying group were significantly improved compared with the freeze-drying group, and the effect was better. Among them, the conditions of Example 11 were the best, and the prepared complex had the strongest activity. The growth of Escherichia coli and Staphylococcus aureus intervened by the complex was effectively inhibited within 48 h, and the bacteriostatic effect of the complex prepared under the conditions of Example 11 was the best.

[0180] Table 6 Effects of Different Polypeptide / Polyphenol (w / w) Ratios on Hypoglycemic Ability, Antioxidant Ability and Bacteriostasis of High-Activity Quinoa Peptide-Polyphenol Complex

[0181]

[0182] Analysis of the Effects of Different Polypeptide / Polyphenol (w / w) Ratios on High-Activity Quinoa Peptide-Polyphenol Complex: Table 6 shows the effects of different polypeptide / polyphenol (w / w) ratios on high-activity quinoa peptide-polyphenol complex. Different polypeptide / polyphenol (w / w) ratios had a significant effect on the activity of high-activity quinoa peptide-polyphenol complex. Among them, the conditions of Example 11 were the best, and the prepared complex had the strongest activity. The growth of Escherichia coli and Staphylococcus aureus intervened by the complex was effectively inhibited within 48 h, and the bacteriostatic effect of the complex prepared under the conditions of Example 11 was the best.

[0183] Table 7 Minimum Inhibitory Concentration of Quinoa Protein Hydrolysate after Pressure Cooking

[0184]

[0185]

[0186] Note: Comparative Example 2(1), Comparative Example 2(2), and Example 5 are the enzymolysis products after enzymolysis by papain, alkaline protease, and double enzyme combination, respectively.

[0187] Table 8 Minimum Inhibitory Concentration of Quinoa Peptides with Different Molecular Weights

[0188]

[0189] Note: Example 8, Example 9, Example 10(1), and Example 10(2) are quinoa peptides with <1KDa, 1-3KDa, 3-10KDa, and >10KDa in the double enzyme combined enzymolysis product of quinoa protein after pressure cooking, respectively.

[0190] Table 9 Antibacterial ability of high-activity quinoa peptide-polyphenol complex

[0191]

[0192] Note: QPD-III and CQPD-III are quinoa peptides of 1-3KDa in the products of double-enzyme combined hydrolysis of quinoa protein before and after pressure cooking, respectively

[0193] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention

Claims

1. A method for preparing a quinoa peptide-polyphenol complex, characterized in that: The following steps are involved: 1) mixing quinoa seeds with water and pressure cooking to obtain cooked quinoa, and drying, crushing and sieving the cooked quinoa to obtain quinoa flour; The conditions of the pressure cooking are: pressure of 0.1-0.2 MPa, temperature of 110-120° C., and time of 10-20 min; 2) extracting quinoa polyphenols from the quinoa powder obtained in step 1); 3) extracting quinoa protein from the quinoa powder obtained in step 1), mixing the quinoa protein with a composite enzyme, and performing enzymolysis to obtain an enzymolysate; The complex enzyme comprises papain and alkaline protease; 4) passing the enzymatic hydrolysate obtained in step 3) through an ultrafiltration centrifuge tube system with a molecular weight cutoff of 3KDa and / or 1KDa to obtain quinoa peptide; 5) mixing the quinoa polyphenol obtained in step 2) with the quinoa peptide obtained in step 4) and water, and stirring to react to obtain a reactant; 6) drying the reactant obtained in step 5) to obtain a quinoa peptide-polyphenol complex; The step 2) of extracting quinoa polyphenols comprises the following steps: A. mixing the quinoa powder and the ethanol solution, and then shaking, ultrasonicating, and centrifuging in sequence to obtain a precipitate and a supernatant, concentrating the supernatant, and then dissolving the supernatant in methanol to obtain a free phenol solution; B. mixing the precipitate obtained in step A with a sodium hydroxide solution, shaking, to obtain an oscillated product, adjusting the pH value of the oscillated product, extracting, concentrating the obtained extract, and then dissolving it in methanol to obtain a combined phenol solution; C. Purifying the free phenol solution obtained in step A and the bound phenol solution obtained in step B through a macroporous resin, freeze-drying and mixing to obtain quinoa polyphenols; The step 3) of extracting quinoa protein comprises the following steps: a. defatting the quinoa powder to obtain defatted quinoa powder, mixing the defatted quinoa powder with water, adjusting the pH value, stirring, and centrifuging to obtain a supernatant; b. adjusting the pH value of the supernatant obtained in step a and centrifuging to obtain a precipitate; c. The precipitate obtained in step b is redissolved and dialyzed in a dialysis bag with a molecular cutoff of 8000 to 14000 Da. The pH value is adjusted to neutral and then freeze-dried to obtain quinoa protein.

2. The preparation method according to claim 1, characterized in that: In the step 1), the mass ratio of quinoa grains to water is 10:3; The drying temperature is below 60° C.; the sieve used for sieving has an aperture of 80 meshes, and the material under the sieve is quinoa flour.

3. The preparation method according to claim 1, characterized in that: The volume ratio of the quinoa powder in step A to the ethanol solution is 5 g:50 mL, and the volume percentage of the ethanol solution is 70%; The oscillation conditions include: temperature of 25°C, time of 4h, and rotation speed of 200rpm; The ultrasonic conditions include: power of 200w, time of 15min; The centrifugal conditions include: centrifugal force of 3000g, time of 15min; The concentration temperature is 45° C., and the concentration is performed to one third of the original volume.

4. The preparation method according to claim 1, characterized in that: The volume ratio of the sodium hydroxide solution in step B to the ethanol solution in step A is 1:1, and the concentration of the sodium hydroxide is 4 mol / L; The oscillation conditions include: temperature of 25°C, time of 4h, and rotation speed of 200rpm; The pH value is 2.0, and the pH value is adjusted using a hydrochloric acid solution with a concentration of 6 mol / L; The extraction reagents used are ethyl acetate and diethyl ether, the volume ratio of ethyl acetate to diethyl ether is 1:1, and the extraction times are 3 times; The concentration temperature is 45° C., and the concentration is performed to one third of the original volume.

5. The preparation method according to claim 1, characterized in that: The macroporous resin in step C is AB-8 macroporous resin; The purification conditions include: adsorption temperature of 25° C., adsorption time of 8 hours, and desorption using an ethanol solution with a volume percentage of 70% after adsorption.

6. The preparation method according to claim 1, characterized in that: The defatting conditions in step a include: mixing the quinoa powder with n-hexane, shaking and drying to obtain defatted quinoa powder; the mass ratio of the quinoa powder to the volume of n-hexane is 1 g:4 mL; the oscillation conditions include: a rotation speed of 200 rpm and a time of 12 h; the drying temperature is 40° C.; The mass ratio of defatted quinoa powder to water in step a is 1:10, the pH value is 10.0, and a hydrochloric acid solution with a concentration of 2 mol / L is used to adjust the pH value; the stirring conditions include: temperature of 35° C., time of 90 min; the centrifugal conditions include: temperature of 4° C., time of 20 min, and centrifugal force of 6000 g; The pH value in step b is 4.5, and a hydrochloric acid solution with a concentration of 0.5 mol / L is used to adjust the pH value; the centrifugal conditions include: temperature of 4°C, time of 20 min, and centrifugal force of 6000 g; The dialysis conditions in step c include: redissolving the precipitate in PBS buffer and dialyzing for 24 hours, and replacing the buffer every 3 hours.

7. The preparation method according to claim 1, characterized in that: In step 3), the mass ratio of quinoa protein to complex enzyme is 100:1; The mass ratio of papain to alkaline protease is 1-2:1-2; The conditions for the enzymatic hydrolysis include: a temperature of 50° C., a time of 3 hours, and a rotation speed of 100 rpm; and the enzymatic hydrolysis is carried out in a water bath.

8. The preparation method according to claim 1, characterized in that: In step 5), the mass ratio of quinoa polyphenols to quinoa peptides is 1-6:1-6; The stirring reaction time in step 5) is 24 hours; The drying conditions in step 6) include: a temperature of 80 to 120° C. and a feed rate of 2 to 5 L / h.

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