Soybean polypeptide as well as preparation method and application thereof

Abstract

The invention discloses soybean polypeptide and a preparation method and application thereof.The preparation method comprises the steps that raw materials are pretreated, specifically, low-temperature desolventized soybean meal is smashed through a smashing machine, a product is sieved through a 60-80-mesh sieve, and soybean meal powder is obtained; acid precipitation and water washing: water is added into the soybean meal powder, the temperature is raised to 30-60 DEG C, the pH is adjusted to 3.0-6.0 with concentrated hydrochloric acid under the stirring condition, and the solid-to-liquid ratio of the soybean meal powder to the water is 1: 8-1: 12; and precipitating the soybean protein, continuously stirring for 1-2 hours, treating by adopting a centrifugal machine, and pouring out the supernatant. When the molecular weight of the prepared soybean polypeptide is 5000-10000 u, the soybean polypeptide has the highest cancer inhibition activity, and the IC50 of the soybean anti-cancer peptide is 0.47 mg / mL. After column chromatography separation, an in-vitro cell experiment result shows that the component A4 has the optimal anticancer activity, and IC50 is 0.37 mg / mL; under the concentration of 0.1 mg / mL, the IR value is 33.15% greater than 30%, and the compound has sensitive anti-cancer property; the average relative molecular mass of the soybean anticancer peptide is calculated to be 6723u through a standard curve, so that the preparation method is simple, and the purity of the soybean peptide is relatively high.

Description

Technical Field

[0001] The present invention relates to the technical field of soybean polypeptide preparation, and in particular to a soybean polypeptide and a preparation method and application thereof. Background Art

[0002] my country is a major producer of soybeans and a major consumer of soy products. Soybeans are rich in protein and are often used as a substitute for animal protein due to their reasonable amino acid ratio. Soy peptides are a class of compounds produced by the hydrolysis of soybean protein. The average peptide chain length is 2 to 10 amino acid residues, and the molecular weight distribution is usually below 3000Da. Soy peptides are not only easy to digest and absorb, but also have physiological functions such as anti-oxidation, anti-aging, lowering blood pressure, lowering blood lipids and anti-fatigue. They are a widely used functional raw material.

[0003] The preparation of soybean peptides is mainly based on soy protein isolate or soybean meal as raw materials, and is obtained by enzymatic degradation under certain temperature and pH conditions using proteases. However, due to the small number of commercial varieties and a single action site, the peptide sequences produced by catalytic degradation are often homogenized, and some functions such as anti-cancer activity are rarely reported. At present, soybean peptides are more often prepared by fermentation, that is, strains with strong protease production ability are screened to directly ferment soybean meal. On the one hand, it can reduce production costs, and on the other hand, it can also improve the flavor of the peptides. However, the peptides prepared by this method are often not of high purity and have poor efficacy. Summary of the invention

[0004] In order to solve the above technical problems, a soybean polypeptide and a preparation method and application thereof are provided. The technical solution solves the problem that the prepared polypeptides proposed in the above background technology are often of low purity and poor efficacy.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0006] A soybean polypeptide and a preparation method thereof, comprising:

[0007] S1. Raw material pretreatment: Use a pulverizer to pulverize the low-temperature desolventized soybean meal, and pass the product through a 60-80 mesh sieve to obtain soybean meal powder;

[0008] S2, acid precipitation and water washing: adding water to the soybean meal powder, raising the temperature to 30-60°C, adjusting the pH to 3.0-6.0 with concentrated hydrochloric acid under stirring conditions, wherein the solid-liquid ratio of the soybean meal powder to water is 1:8-1:12;

[0009] The soybean protein was precipitated and stirred for 1 to 2 hours, and then centrifuged and the supernatant was discarded;

[0010] Continue to add water to the precipitate according to the above solid-liquid ratio. There is no need to increase the temperature or adjust the pH value at this time.

[0011] Continue stirring for 15 to 20 minutes, wash the precipitate with water, continue centrifuging in the centrifuge, the speed of the centrifuge is 6000 to 9000 r / min, remove the supernatant and collect the precipitate;

[0012] Wash the precipitate with water according to the same operation, then discard the supernatant and collect the precipitate;

[0013] S3, enzyme hydrolysis: collecting the precipitate from the acid precipitation and water washing steps, wherein the solid-liquid ratio of the precipitate to water is 1:8 to 1:12, and mixing and stirring the precipitate with water;

[0014] The pH is adjusted to 8.0-10.0 using NaOH solution, and the temperature is raised to 35°C-60°C;

[0015] Adding enzyme solution into the soybean meal at a ratio of 40 to 100 ml of enzyme solution per kilogram of soybean meal, wherein the enzyme solution is Bacillus subtilis serine alkaline protease solution;

[0016] During the hydrolysis reaction, 0.05 mol / L NaOH was added to keep the pH value inside the system stable. When the degree of protein hydrolysis reached 20-30%, the reaction was stopped.

[0017] S4, enzyme inactivation: After the enzymatic hydrolysis is completed, the temperature is quickly raised to 90-100°C and maintained for 10-20 minutes to inactivate the enzyme;

[0018] Rapidly cool it to room temperature within 10 to 20 minutes, adjust the pH to neutral, and centrifuge it with a centrifuge at a speed of 12,000 to 15,000 r / min to separate the solid and liquid, wherein the supernatant is the soybean polypeptide stock solution;

[0019] S5, concentration: obtaining the membrane liquid from the enzyme inactivation step, and performing a reduced pressure concentration treatment on the membrane liquid to obtain a solid, wherein the content of the solid is 35-40%;

[0020] S6. Drying: After the concentration is completed, it is made into dry powder through spray drying or freeze drying.

[0021] The specific preparation process is as follows: select 20g of soybean meal powder with a protein content of 45%, mix and stir it with 200ml of water, raise the temperature to 45°C, and use concentrated hydrochloric acid to adjust the pH value of the solution to 4.2. After 2h, stop stirring, centrifuge at 9000rpm for 30min, remove the supernatant, add 200ml of water to the precipitate, continue stirring for 20min, continue centrifuging at 9000rpm for 30min, remove the supernatant, and repeat this step. Collect the precipitate, add 200ml of water, continue stirring, add 0.5mol / L NaOH, adjust the pH value to 9.5, raise the temperature to 35°C, add 1.1ml enzyme solution, and perform hydrolysis reaction. During the hydrolysis reaction, continue to maintain a temperature of 50°C and a pH of 9.5. After the degree of hydrolysis reaches 25%, stop the reaction. After the reaction is completed, quickly raise the temperature to 95°C and maintain for 10 to 15min to inactivate the enzyme. The temperature was lowered to room temperature, the pH was adjusted to 6.8, and the mixture was centrifuged at 12,000 rpm for 30 minutes. The supernatant was the crude soybean peptide liquid. The soybean peptide liquid was passed through an ultrafiltration membrane with a molecular weight of 5,000 Da to remove the macromolecular fragments. The liquid on the membrane was returned to the enzymatic hydrolysis tank and then hydrolyzed. The refined soybean peptide liquid was decompressed and concentrated to a solid content of 35-40%, and spray-dried to obtain the refined soybean peptide dry powder.

[0022] An application of soybean polypeptide, which is prepared by the above preparation method and is applied in the preparation of health food or anticancer medicine.

[0023] The experiment used the growth inhibition effect on gastric cancer cell SGC7901 as an indicator of anticancer activity. Through initial screening, it was found that the growth inhibition rate of soybean peptides on cancer cells increased with the increase of sample concentration, and the highest inhibition rates were 44.84%, 69.34%, and 50.32% respectively (see Figure 1 ). According to the definition of IC50, the IC50 values ​​of the three components with molecular weight less than 5000u, 5000-10000u, and greater than 10000u were calculated by SPSS Probit to be 1.17, 0.47, and 0.96 mg / mL, respectively. Comparing the half-lethal concentration (IC50) values ​​of each component, it can be seen that the component with a molecular weight of 5000-10000u has the best inhibitory effect on cancer cells, and the IC50 at this time is ≈ 0.47 mg / mL. Therefore, it is preliminarily determined that the molecular weight range of soybean anticancer peptides is 5000-10000u.

[0024] The peptide solution with a relative molecular mass of 5000 to 10000u obtained by membrane liquid was separated by Sephadex G-75 column chromatography, and the elution peaks were collected and numbered A1, A2, A3, A4, A5, A6, A7, A8, A9, A10 (see Figure 2The collected groups were divided into 0.1 mg / mL solutions for anticancer activity testing. The results are shown in Figure 3 .

[0025] Depend on Figure 3 It can be seen that among components A1 to A10, A4 has the highest anticancer activity, with a growth inhibition rate IR = 33.15%>30%, which has sensitive anticancer properties. The second is component A5, whose growth inhibition rate IR = 27.73% <30%, so it is not sensitive to the growth inhibition of cancer cells. The lowest anticancer activity is component A2, with a cancer inhibition rate of only 1.17%. The anticancer activities of each component are A4>A5>A3>A9>A1>A7>A8>A6>A10>A2. The A4 group was divided into solutions of different concentrations (0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL), and its IC50 was calculated to be 0.37 mg / mL based on the cell inhibition rate (17.58%, 20.96%, 30.59%, 35.67%, 40.2%, 46.7%, 45.5%, 44.4%, 56.3%), which was significantly better than the anticancer peptide component after membrane (IC50≈0.47 mg / mL), indicating that the purification effect was good.

[0026] Due to the complexity of peptide structure, its anti-cancer mechanism is also relatively complex. The anti-cancer activity of peptides is related to its specific amino acid composition on the one hand; on the other hand, it is closely related to its amino acid sequence. For example, Lunasin has an anti-cancer effect because it has 8 aspartic acid residues at its C-terminus. When they bind to the nuclear chromatin region (such as appearing at the centromere), they play a great role in anti-mitotic effects. As a result, the complex at the centromere cannot form normally, and the microtubules cannot be connected to the centromere, resulting in the cessation of mitosis and the ultimate death of the cells, thereby inhibiting the proliferation of cancer cells.

[0027] Determine the retention time of each known molecular weight standard according to the above column chromatography separation method, and record the retention time (min) when the maximum absorption peak of the protein standard appears. The determination results are shown in Figure 4 , with retention time as the horizontal axis and lgM as the vertical axis, draw a standard curve as shown in Figure 5 .

[0028] Depend on Figure 5 It can be seen that the molecular weight logarithm is linearly related to the retention time, and the regression equation is Y = -0.0817x + 6.4632, R 2 =0.988. In the formula, X represents the retention time, Y represents the logarithm of the molecular weight, and 0.988 is the linear correlation coefficient of the standard curve. It can be seen that the logarithm of each molecular weight has a good linear relationship with the retention time of each standard sample, and the molecular weight distribution of the anticancer peptides in the extract can be accurately calculated based on this regression equation.

[0029] Soy peptides also have the basic characteristics of being easy to digest and absorb, and having a fast absorption rate, which makes them suitable as a nutrient. Soy peptides can be used by patients in the recovery period, the elderly with impaired digestive function, and infants and young children whose digestive function is not fully mature. When using soy peptides, they can be combined with auxiliary materials to make a variety of foods, such as milk powder for the elderly. Using soy peptides as the base material, according to the nutritional requirements and physiological characteristics of the elderly, whole milk powder and honey are added, essential amino acids, namely cysteine ​​and methionine, are strengthened, and inorganic salts such as Ca, Fe and Zn are strengthened and integrated into natural fruit juice. A high-fructose, high-protein, and easily digestible milk powder for the elderly is developed, which can significantly reduce the serum cholesterol of the elderly. It is a very high-quality nutritional health food. Soy peptides have anti-antigenicity and cholesterol-lowering effects, so they can be used to produce functional health foods that have the effects of lowering blood pressure, lowering cholesterol, and preventing cardiovascular diseases.

[0030] Soybean peptides also have good moisture retention and hygroscopicity, which can soften products and improve taste. Therefore, they can be used to produce various soy products with excellent flavor and quality, rich nutrition and easy digestion and absorption. When used in high-protein products such as meat and fish, they can highlight the meat flavor of the products, enhance the freshness of food, and help optimize taste and quality. When used in baked foods, they can increase the aroma and flavor of food and ensure that the food texture is loose. For example, when added to bread dough, it can increase viscoelasticity, reduce water loss in bread, ensure that bread has a softer texture, increase volume, and increase aroma. When used in the production of cakes, candies, and chocolates, it can reduce the sweetness of products, reduce costs, significantly increase the content of amino acids in protein, help improve taste quality, and extend shelf life. Because soybean peptides have high water solubility, they are easy to cause low viscosity, are not easy to precipitate under acidic conditions, and also have a certain foaming effect, so they can be used in the production of acidic beverages, soda, nutritional drinks, instant milk powder and other foods. High fructose and high protein, easy to digest elderly milk powder can significantly reduce the elderly.

[0031] Compared with the prior art, the present invention provides a soybean polypeptide and a preparation method and application thereof, which have the following beneficial effects:

[0032] The soybean polypeptide prepared by the present invention has the highest anti-cancer activity when the molecular weight is 5000-10000u, and the IC50 of the soybean anti-cancer peptide is 0.47mg / mL. After column chromatography separation, the results of in vitro cell experiments show that component A4 has the best anti-cancer activity, with an IC50 of 0.37mg / mL; at a concentration of 0.1mg / mL, its IR value is 33.15%>30%, and has sensitive anti-cancer properties; the average relative molecular mass of the soybean anti-cancer peptide calculated by the standard curve is 6723u, so the preparation method is relatively simple, the soybean polypeptide has a high purity, and the efficacy is more prominent. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The cancer cell inhibition rate and IC50 value of each component after membrane in the present invention;

[0034] Figure 2 This is the Sephadex G-75 gel elution spectrum of the soybean anticancer peptide in the present invention;

[0035] Figure 3 is the OD value and cancer cell inhibition rate of each eluted component in the present invention;

[0036] Figure 4 It is the Sephadex G-75 gel elution spectrum of the protein standard in the present invention;

[0037] Figure 5 It is the molecular weight standard curve diagram of the present invention;

[0038] Figure 6 It is a schematic diagram of the preparation method of soybean polypeptide in the present invention. DETAILED DESCRIPTION

[0039] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0040] Please refer to Figure 6 As shown, a soybean polypeptide and a preparation method thereof, comprising:

[0041] S1. Raw material pretreatment: Use a pulverizer to pulverize the low-temperature desolventized soybean meal, and pass the product through a 60-80 mesh sieve to obtain soybean meal powder;

[0042] S2, acid precipitation and water washing: add water to the soybean meal powder, raise the temperature to 30-60°C, and adjust the pH to 3.0-6.0 with concentrated hydrochloric acid under stirring;

[0043] The soybean protein was precipitated and stirred for 1 to 2 hours, and then centrifuged and the supernatant was discarded;

[0044] Continue to add water to the precipitate according to the above solid-liquid ratio. There is no need to increase the temperature or adjust the pH value at this time.

[0045] Continue stirring for 15 to 20 minutes, wash the precipitate with water, continue centrifuging, remove the supernatant and collect the precipitate;

[0046] Wash the precipitate with water according to the same operation, then discard the supernatant and collect the precipitate;

[0047] S3, enzyme hydrolysis: collecting the precipitate from the acid precipitation and water washing steps, mixing and stirring it with water;

[0048] The pH is adjusted to 8.0-10.0 using NaOH solution, and the temperature is raised to 35°C-60°C;

[0049] Add enzyme solution to the soybean meal at a ratio of 40 to 100 ml of enzyme solution per kilogram of soybean meal;

[0050] During the hydrolysis reaction, 0.05 mol / L NaOH was added to keep the pH value inside the system stable. When the degree of protein hydrolysis reached 20-30%, the reaction was stopped.

[0051] S4, enzyme inactivation: After the enzymatic hydrolysis is completed, the temperature is quickly raised to 90-100°C and maintained for 10-20 minutes to inactivate the enzyme;

[0052] Rapidly cool it to room temperature within 10 to 20 minutes, adjust the pH to neutral, centrifuge it, separate the solid and liquid, and the supernatant is the soybean polypeptide stock solution;

[0053] S5, concentration: obtaining the membrane liquid from the enzyme inactivation step, and performing a reduced pressure concentration treatment on the membrane liquid to obtain a solid;

[0054] S6. Drying: After the concentration is completed, it is made into dry powder through spray drying or freeze drying.

[0055] During the acid precipitation and water washing process, the solid-liquid ratio of soybean meal powder and water is 1:8 to 1:12.

[0056] During the acid precipitation and water washing process, the centrifuge speed is 6000-9000r / min.

[0057] During the enzymatic hydrolysis process, the solid-to-liquid ratio of precipitate to water is 1:8 to 1:12.

[0058] In the enzymatic hydrolysis process, the enzyme solution is Bacillus subtilisin serine alkaline protease solution.

[0059] During the enzyme inactivation process, the centrifuge speed is 12000-15000r / min.

[0060] During the concentration process, the solid content is 35-40%.

[0061] The specific preparation process is as follows: select 20g of soybean meal powder with a protein content of 45%, mix and stir it with 200ml of water, raise the temperature to 45°C, and use concentrated hydrochloric acid to adjust the pH value of the solution to 4.2. After 2h, stop stirring, centrifuge at 9000rpm for 30min, remove the supernatant, add 200ml of water to the precipitate, continue stirring for 20min, continue centrifuging at 9000rpm for 30min, remove the supernatant, and repeat this step. Collect the precipitate, add 200ml of water, continue stirring, add 0.5mol / L NaOH, adjust the pH value to 9.5, raise the temperature to 35°C, add 1.1ml enzyme solution, and perform hydrolysis reaction. During the hydrolysis reaction, continue to maintain a temperature of 50°C and a pH of 9.5. After the degree of hydrolysis reaches 25%, stop the reaction. After the reaction is completed, quickly raise the temperature to 95°C and maintain for 10 to 15min to inactivate the enzyme. The temperature was lowered to room temperature, the pH was adjusted to 6.8, and the mixture was centrifuged at 12,000 rpm for 30 minutes. The supernatant was the crude soybean peptide liquid. The soybean peptide liquid was passed through an ultrafiltration membrane with a molecular weight of 5,000 Da to remove the macromolecular fragments. The liquid on the membrane was returned to the enzymatic hydrolysis tank and then hydrolyzed. The refined soybean peptide liquid was decompressed and concentrated to a solid content of 35-40%, and spray-dried to obtain the refined soybean peptide dry powder.

[0062] An application of soybean polypeptide, which is prepared by the above preparation method and is applied in the preparation of health food or anticancer medicine.

[0063] The experiment used the growth inhibition effect on gastric cancer cell SGC7901 as an indicator of anticancer activity. Through initial screening, it was found that the growth inhibition rate of soybean peptides on cancer cells increased with the increase of sample concentration, and the highest inhibition rates were 44.84%, 69.34%, and 50.32% respectively (see Figure 1 ). According to the definition of IC50, the IC50 values ​​of the three components with molecular weight less than 5000u, 5000-10000u, and greater than 10000u were calculated by SPSS Probit to be 1.17, 0.47, and 0.96 mg / mL, respectively. Comparing the half-lethal concentration (IC50) values ​​of each component, it can be seen that the component with a molecular weight of 5000-10000u has the best inhibitory effect on cancer cells, and the IC50 at this time is ≈ 0.47 mg / mL. Therefore, it is preliminarily determined that the molecular weight range of soybean anticancer peptides is 5000-10000u.

[0064] The peptide solution with a relative molecular mass of 5000 to 10000u obtained by membrane liquid was separated by Sephadex G-75 column chromatography, and the elution peaks were collected and numbered A1, A2, A3, A4, A5, A6, A7, A8, A9, A10 (see Figure 2 The collected groups were divided into 0.1 mg / mL solutions for anticancer activity testing. The results are shown in Figure 3 .

[0065] Depend on Figure 3 It can be seen that among components A1 to A10, A4 has the highest anticancer activity, with a growth inhibition rate IR = 33.15%>30%, which has sensitive anticancer properties. The second is component A5, whose growth inhibition rate IR = 27.73% <30%, so it is not sensitive to the growth inhibition of cancer cells. The lowest anticancer activity is component A2, with a cancer inhibition rate of only 1.17%. The anticancer activities of each component are A4>A5>A3>A9>A1>A7>A8>A6>A10>A2. The A4 group was divided into solutions of different concentrations (0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL), and its IC50 was calculated to be 0.37 mg / mL based on the cell inhibition rate (17.58%, 20.96%, 30.59%, 35.67%, 40.2%, 46.7%, 45.5%, 44.4%, 56.3%), which was significantly better than the anticancer peptide component after membrane (IC50≈0.47 mg / mL), indicating that the purification effect was good.

[0066] Due to the complexity of peptide structure, its anti-cancer mechanism is also relatively complex. The anti-cancer activity of peptides is related to its specific amino acid composition on the one hand; on the other hand, it is closely related to its amino acid sequence. For example, Lunasin has an anti-cancer effect because it has 8 aspartic acid residues at its C-terminus. When they bind to the nuclear chromatin region (such as appearing at the centromere), they play a great role in anti-mitotic effects. As a result, the complex at the centromere cannot form normally, and the microtubules cannot be connected to the centromere, resulting in the cessation of mitosis and the ultimate death of the cells, thereby inhibiting the proliferation of cancer cells.

[0067] Determine the retention time of each known molecular weight standard according to the above column chromatography separation method, and record the retention time (min) when the maximum absorption peak of the protein standard appears. The determination results are shown in Figure 4 , with retention time as the horizontal axis and lgM as the vertical axis, draw a standard curve as shown in Figure 5 .

[0068] Depend on Figure 5 It can be seen that the molecular weight logarithm is linearly related to the retention time, and the regression equation is Y = -0.0817x + 6.4632, R 2=0.988. In the formula, X represents the retention time, Y represents the logarithm of the molecular weight, and 0.988 is the linear correlation coefficient of the standard curve. It can be seen that the logarithm of each molecular weight has a good linear relationship with the retention time of each standard sample, and the molecular weight distribution of the anticancer peptides in the extract can be accurately calculated based on this regression equation.

[0069] Soy peptides also have the basic characteristics of being easy to digest and absorb, and having a fast absorption rate, which makes them suitable as a nutrient. Soy peptides can be used by patients in the recovery period, the elderly with impaired digestive function, and infants and young children whose digestive function is not fully mature. When using soy peptides, they can be combined with auxiliary materials to make a variety of foods, such as milk powder for the elderly. Using soy peptides as the base material, according to the nutritional requirements and physiological characteristics of the elderly, whole milk powder and honey are added, essential amino acids, namely cysteine ​​and methionine, are strengthened, and inorganic salts such as Ca, Fe and Zn are strengthened and integrated into natural fruit juice. A high-fructose, high-protein, and easily digestible milk powder for the elderly is developed, which can significantly reduce the serum cholesterol of the elderly. It is a very high-quality nutritional health food. Soy peptides have anti-antigenicity and cholesterol-lowering effects, so they can be used to produce functional health foods that have the effects of lowering blood pressure, lowering cholesterol, and preventing cardiovascular diseases.

[0070] Soybean peptides also have good moisture retention and hygroscopicity, which can soften products and improve taste. Therefore, they can be used to produce various soy products with excellent flavor and quality, rich nutrition and easy digestion and absorption. When used in high-protein products such as meat and fish, they can highlight the meat flavor of the products, enhance the freshness of food, and help optimize taste and quality. When used in baked foods, they can increase the aroma and flavor of food and ensure that the food texture is loose. For example, when added to bread dough, it can increase viscoelasticity, reduce water loss in bread, ensure that bread has a softer texture, increase volume, and increase aroma. When used in the production of cakes, candies, and chocolates, it can reduce the sweetness of products, reduce costs, significantly increase the content of amino acids in protein, help improve taste quality, and extend shelf life. Because soybean peptides have high water solubility, they are easy to cause low viscosity, are not easy to precipitate under acidic conditions, and also have a certain foaming effect, so they can be used in the production of acidic beverages, soda, nutritional drinks, instant milk powder and other foods. High fructose and high protein, easy to digest elderly milk powder can significantly reduce the elderly.

[0071] In summary:

[0072] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A soybean polypeptide and a preparation method thereof, characterized in that: include: S1. Raw material pretreatment: Use a pulverizer to pulverize the low-temperature desolventized soybean meal, and pass the product through a 60-80 mesh sieve to obtain soybean meal powder; S2, acid precipitation and water washing: add water to the soybean meal powder, raise the temperature to 30-60°C, and adjust the pH to 3.0-6.0 with concentrated hydrochloric acid under stirring; The soybean protein was precipitated and stirred for 1 to 2 hours, and then centrifuged and the supernatant was discarded; Continue to add water to the precipitate according to the above solid-liquid ratio. There is no need to increase the temperature or adjust the pH value at this time. Continue stirring for 15 to 20 minutes, wash the precipitate with water, continue centrifuging, remove the supernatant and collect the precipitate; Wash the precipitate with water according to the same operation, then discard the supernatant and collect the precipitate; S3, enzyme hydrolysis: collecting the precipitate from the acid precipitation and water washing steps, mixing and stirring it with water; The pH is adjusted to 8.0-10.0 using NaOH solution, and the temperature is raised to 35°C-60°C; Add enzyme solution to the soybean meal at a ratio of 40 to 100 ml of enzyme solution per kilogram of soybean meal; During the hydrolysis reaction, 0.05 mol / L NaOH was added to keep the pH value inside the system stable. When the degree of protein hydrolysis reached 20-30%, the reaction was stopped. S4, enzyme inactivation: After the enzymatic hydrolysis is completed, the temperature is quickly raised to 90-100°C and maintained for 10-20 minutes to inactivate the enzyme; Rapidly cool it to room temperature within 10 to 20 minutes, adjust the pH to neutral, centrifuge it, separate the solid and liquid, and the supernatant is the soybean polypeptide stock solution; S5, concentration: obtaining the membrane liquid from the enzyme inactivation step, and performing a reduced pressure concentration treatment on the membrane liquid to obtain a solid; S6. Drying: After the concentration is completed, it is made into dry powder through spray drying or freeze drying.

2. A soybean polypeptide and a preparation method thereof according to claim 1, characterized in that: During the acid precipitation and water washing process, the solid-to-liquid ratio of the soybean meal powder to water is 1:8 to 1:

12.

3. A soybean polypeptide and a preparation method thereof according to claim 1, characterized in that: During the acid precipitation and water washing process, the rotation speed of the centrifuge is 6000-9000r / min.

4. A soybean polypeptide and a preparation method thereof according to claim 1, characterized in that: During the enzymatic hydrolysis process, the solid-to-liquid ratio of the precipitate to water is 1:8 to 1:

12.

5. A soybean polypeptide and a preparation method thereof according to claim 1, characterized in that: In the enzymatic hydrolysis process, the enzyme solution is Bacillus subtilisin serine alkaline protease solution.

6. A soybean polypeptide and a preparation method thereof according to claim 1, characterized in that: During the enzyme inactivation process, the rotation speed of the centrifuge is 12000-15000 r / min.

7. A soybean polypeptide and a preparation method thereof according to claim 1, characterized in that: During the concentration process, the solid content is 35-40%.

8. An application of soybean polypeptide, characterized in that: The product is prepared by the preparation method according to claims 1 to 7 and is used in the preparation of health food or anticancer drugs.

Images