Method for preparing nutritional base material with high antioxidant activity by taking wheat gluten protein powder as raw material and application of nutritional base material

Abstract

The invention discloses a method for preparing a nutritional base material with high antioxidant activity by taking wheat gluten protein powder as a raw material and application of the nutritional base material. According to the method, pancreatin with greatly improved protein yield and alkaline protease with greatly improved activity are selected to have a synergistic effect, the advantages of the pancreatin and the alkaline protease are fully played, the solid recovery rate, the protein recovery rate and the hydrolysis degree of wheat gluten protein enzymatic hydrolysate are improved, and a high-content and high-activity antioxidant peptide component is obtained; and through the Maillard reaction after ethanol purification, the antioxidant activity of the wheat gluten protein enzymatic hydrolysate is improved, and the preparation time of the peptide with high antioxidant activity is shortened. The method is simple and convenient in process operation, low in production cost, green, environment-friendly and high in raw material utilization rate, and the obtained nutritional base material has high antioxidant activity and high safety and is particularly suitable for high-value utilization of low-value resource wheat gluten protein powder.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological enzyme hydrolysis, in particular to a method for preparing a high-antioxidant-activity nutritional base material from wheat gluten protein powder and application thereof. BACKGROUND

[0002] Wheat gluten protein powder is a by-product of wheat processing, with a protein content of more than 80%, and is a kind of plant protein with excellent quality, rich nutrition and wide sources. At present, wheat gluten protein powder is mainly used for processing traditional foods such as gluten strings, fried gluten balls and vegetarian meat, or added into flour and coarse grains as a dough strengthening agent and quality improver, and a part is used for feed production. Although the protein content in wheat gluten protein is high and the amino acid composition is balanced, the solubility is low due to the presence of a large amount of hydrophobic amino acids and uncharged amino acids, which makes it difficult to be directly used in the field of nutritional supplements.

[0003] Biological enzyme hydrolysis is a biological treatment technology based on the specific catalytic action of protease, which can cut large molecular proteins into small molecular peptides and amino acids by recognizing specific enzyme cutting sites in proteins. This method can effectively improve the digestion and absorption performance of proteins, and convert large molecular proteins that are difficult to be directly utilized by the human body into active peptide segments and amino acids with high bioavailability. In addition, enzymatic protein can also produce peptides with significant biological activity, which plays an important role in regulating human digestion and absorption and promoting health, and provides a new way for the green and efficient development of nutritional and health functional products.

[0004] Antioxidant peptides, as an important active component in enzymatic hydrolysate, exhibit good antioxidant performance. Compared with chemically synthesized antioxidants and large molecular antioxidant proteins, antioxidant peptides have the advantages of low molecular weight, high absorption efficiency and good safety. The antioxidant mechanism mainly includes scavenging free radicals, inhibiting lipid peroxidation reaction and up-regulating the activity of endogenous antioxidant enzymes. Therefore, antioxidant peptides have wide application prospects in the fields of functional foods, medicines and cosmetics, and have gradually become a hot research direction for the high-value utilization of protein resources.

[0005] Although direct enzymatic hydrolysis can obtain antioxidant peptides with certain activity, the use of a single enzyme preparation has the problems of low efficiency, limited activity of the obtained product and poor stability. The present application uses double-enzyme complex hydrolysis combined with ethanol purification and Maillard reaction, which not only effectively improves the enzymatic hydrolysis efficiency, but also further enhances the biological activity of antioxidant peptides and their stability in the gastrointestinal environment. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a method for preparing a high-antioxidant-activity nutritional base material from wheat gluten protein powder.

[0007] Another object of the present application is to provide the high antioxidant activity nutritional base prepared by the above method.

[0008] Still another object of the present application is to provide the application of the high antioxidant activity nutritional base.

[0009] The object of the present application is achieved by the following technical solutions:

[0010] A method for preparing high antioxidant activity nutritional base by using wheat gluten protein powder as raw material, comprising the following steps:

[0011] (1) Dissolve the protein glutamine enzyme in water, and add the wheat gluten protein powder under high-speed stirring, and then modify by incubation and stirring, and then add trypsin and alkaline protease, and then perform enzymolysis by incubation and stirring, and then inactivate the enzyme after the enzymolysis is completed, and then centrifuge to obtain the supernatant to obtain the wheat gluten protein enzymolysis product;

[0012] (2) Add anhydrous ethanol into the wheat gluten protein enzymolysis product, mix uniformly, and then stand still, and then centrifuge to obtain the supernatant to obtain the ethanol crude separation product;

[0013] (3) Add anhydrous ethanol into the ethanol crude separation product, stand still, and then centrifuge to obtain the precipitate, and then dry to obtain the ethanol separation product;

[0014] (4) Mix the ethanol separation product, ribose and water uniformly, adjust the pH, and then perform reaction by incubation and standing still, and then cool after the reaction is completed, and then concentrate under reduced pressure to obtain the high antioxidant activity nutritional base.

[0015] The mass ratio of the wheat gluten protein powder, the protein glutamine enzyme, the trypsin and the alkaline protease in step (1) is 100:0.3-1:0.5-1:0.25-1.

[0016] The enzyme activity of the protein glutamine enzyme in step (1) is 100 U / g.

[0017] The enzyme activity of the trypsin in step (1) is 5000 U / g.

[0018] The alkaline protease in step (1) is Novozymes alkaline protease 37071.

[0019] The condition of the high-speed stirring in step (1) is 800-1000 rpm.

[0020] The condition of the modification by incubation and stirring in step (1) is 50-60℃ incubation and 30-60 rpm stirring for 3-4 h.

[0021] The condition of the enzymolysis by incubation and stirring in step (1) is 50-60℃ incubation and 30-60 rpm stirring for 12-14 h.

[0022] The centrifugation condition in step (1) is 7000-9000g centrifugation for 10-20min.

[0023] The added amount of anhydrous ethanol in step (2) is added to 35-45% of the total mass.

[0024] The standing in step (2) is standing for 1-2h.

[0025] The centrifugation condition in step (2) is 7000-9000g centrifugation for 10-20min.

[0026] The added amount of anhydrous ethanol in step (3) is added to 75-90% of the total mass.

[0027] The standing in step (3) is standing for 1-2h.

[0028] The centrifugation condition in step (3) is 7000-9000g centrifugation for 10-20min.

[0029] The mass ratio of ethanol separation product: ribose: water in step (4) is 1:1.5-2:8-12.

[0030] The pH adjustment in step (4) is adjusting the pH to 8.0-8.5.

[0031] The reaction condition of the incubation standing in step (4) is 85-90℃ reaction for 70-90min.

[0032] A high-antioxidant-activity nutritional base material is prepared by the above preparation method.

[0033] The application of the above high-antioxidant-activity nutritional base material in preparing food nutritional supplements.

[0034] The present application has the following advantages and effects relative to the prior art:

[0035] (1) The present application first disperses protein glutamine enzyme (PG enzyme) in water, then slowly adds wheat gluten protein powder in batches under high-speed stirring conditions, which can disperse the poor water-soluble wheat gluten protein powder in water, promote its full contact with protein glutamine enzyme, improve the modification efficiency of wheat gluten protein, increase the solubility of wheat gluten protein, expose more enzyme cutting sites, and improve the subsequent protease hydrolysis efficiency.

[0036] (2) The present application selects trypsin with a larger protein yield increase and alkaline protease with a larger activity increase to synergistically exert the advantages of both, so that a high-content strong-activity antioxidant peptide component is obtained, and the solid recovery rate, protein recovery rate, degree of hydrolysis and antioxidant activity of the wheat gluten protein hydrolysate are improved by controlling the temperature and time of enzymolysis, thereby shortening the preparation time of high-antioxidant-activity peptides.

[0037] (3) The present application uses ethanol fractionation precipitation technology to effectively separate and purify a component with reasonable amino acid composition and strong antioxidant activity by using the solubility difference of proteins in different solvents. This method can eliminate the adverse effects of other weakly active components and achieve sufficient enrichment of the target component, thereby significantly improving the overall antioxidant effect.

[0038] (4) The present application uses ribose to participate in the Maillard reaction, which not only significantly improves the reaction rate, but also fully exerts the physiological functions of ribose, especially the effect of protecting the heart health of middle-aged and elderly people. This invention not only achieves the purpose of nutritional supplementation, but also helps to maintain the normal physiological function of blood vessels to some extent.

[0039] (5) The verification scheme of the present application takes into account the utilization rate of wheat gluten protein powder and the ability to improve activity, and at the same time, through a reasonable elderly gastrointestinal model, it is verified that it still maintains high activity after being destroyed by the gastrointestinal tract. This further proves the effect of Maillard reaction modification: not only has good nutritional supplement value, but also shows high biological activity.

[0040] (6) The process of the present application is simple to operate, low in production cost, green and environmentally friendly, and has high raw material utilization rate. The obtained nutritional base material has high antioxidant activity and high safety, and is particularly suitable for high-value utilization of low-value resource wheat gluten protein powder. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is a production process schematic diagram;

[0042] Figure 2 is a peptide molecular weight distribution response detection result graph of nutritional base material B and its simulated gastrointestinal digestion products;

[0043] Figure 3 is a real object graph of nutritional base material B prepared in Example 2. DETAILED DESCRIPTION

[0044] The present application will be further described in detail below in combination with examples and drawings, but the embodiments of the present application are not limited thereto.

[0045] Unless otherwise specified, the following embodiments are generally conducted according to conventional experimental conditions or according to the experimental conditions recommended by the reagent companies. If not otherwise specified, the materials, reagents, etc. used are reagents and materials obtained from commercial channels.

[0046] The experimental methods of the experimental indexes involved in the embodiments of the present application are as follows:

[0047] (1) Solid recovery rate: The solid content of the sample is determined by drying method, and the solid recovery rate of the enzyme hydrolysate is calculated according to the following formula:

[0048] Solid recovery rate = solid content of enzyme hydrolysate / total mass of enzyme hydrolysis raw material x 100%.

[0049] (2) Protein recovery rate: The total nitrogen content of the raw material and the nutrient base is determined by Kjeldahl method, and the protein recovery rate of the enzyme hydrolysate is calculated according to the following formula:

[0050] Protein recovery rate = total nitrogen content in nutrient base / total nitrogen content in raw material x 100%.

[0051] (3) Determination of degree of hydrolysis: The amino acid nitrogen content in the nutrient base is determined by formaldehyde potentiometric titration method, and the total nitrogen content of the raw material is determined by Kjeldahl method, and the calculation formula of the degree of hydrolysis of the nutrient base is as follows:

[0052] Degree of hydrolysis = amino acid nitrogen content in nutrient base / total nitrogen content of raw material mixture x 100%.

[0053] (4) In vitro gastrointestinal digestion: The Infogest standard model is used to conduct experiments according to the gastrointestinal digestion conditions of the elderly, and the formula of simulated gastric juice and simulated intestinal juice is shown in Table 1.

[0054] Specifically, 10g of the nutrient base diluent is weighed, 8g of simulated gastric juice, 1g of gastric digestion calcium chloride solution and 1g of deionized water are added, under the condition of pH = 6, 3mg of pepsin is added, and the water bath is shaken for 3h, and the shaking speed is fixed at 100rpm; After digestion, 16g of simulated intestinal juice, 4g of intestinal digestion calcium chloride solution, pH = 7 is adjusted, 8mg of trypsin and 0.0816g of ox bile salt are added, and the simulated digestion is carried out for 3h, and then the enzyme is inactivated.

[0055] In particular, the concentration of gastric digestion calcium chloride is 3mM, the concentration of intestinal digestion calcium chloride is 6mM, the enzyme activity of pepsin is 10000NFU / mg, and the enzyme activity of trypsin is 250U / mg; The enzyme inactivation condition is heating at 95-100℃ for 15min.

[0056] Table 1 Formula of simulated gastric juice and simulated intestinal juice

[0057]

[0058] (5) Antioxidant activity intensity determination:

[0059] ① DPPH radical scavenging rate: using an ultraviolet-visible spectrophotometer, the absorption capacity at 517 nm wavelength was used to determine the DPPH radical scavenging capacity of the same protein concentration sample, and the DPPH radical scavenging rate calculation formula was as follows:

[0060] DPPH radical scavenging rate = (absorbance intensity of the blank group at 517 nm - (absorbance intensity of the sample group at 517 nm - absorbance intensity of the control group at 517 nm)) / absorbance intensity of deionized water at 517 nm x 100%

[0061] In particular, the DPPH solution concentration was 0.2 mM, the sample protein concentration was 0.5 mg / mL, and the reaction time was 30 min

[0062] Specifically, 2 mL of sample solution was mixed with 2 mL of DPPH solution, and the reaction was carried out at room temperature for 30 min in the dark. The blank group was 2 mL of deionized water mixed with 2 mL of DPPH solution, and the control group was 2 mL of sample mixed with 2 mL of ethanol solution.

[0063] ② ABTS radical scavenging rate: using an ultraviolet-visible spectrophotometer, the absorption capacity at 734 nm wavelength was used to determine the ABTS radical scavenging capacity of the same protein concentration sample, and the ABTS radical scavenging rate calculation formula was as follows:

[0064] ABTS radical scavenging rate = (absorbance intensity of the blank group at 734 nm - absorbance intensity of the sample group at 517 nm) / absorbance intensity of the blank group at 517 nm x 100%

[0065] In particular, the ABTS solution concentration was 1.3 mg / mL, the sample protein concentration was 3 mg / mL, and the reaction time was 15 min

[0066] Specifically, 0.1 mL of sample solution was mixed with 5 mL of ABTS solution, and the reaction was carried out at room temperature for 15 min in the dark. The blank group was 0.1 mL of deionized water mixed with 5 mL of ABTS solution.

[0067] (1) Peptide molecular weight determination: high performance liquid chromatography was used, phosphate buffer system (38 g NaH2PO4·2H2O + 5.04 g Na2HPO4·12H2O + 1 ml trifluoroacetic acid + deionized water to 1 L → water phase membrane twice → ultrasonic degassing for 20 min) was selected, the flow rate was 1 mL / min, the sample concentration was 2.5 mg / ml, the injection volume was 10 μL, and the detection wavelength was 220 nm

[0068] Specifically, Agilent 1260 high performance liquid chromatograph is selected, and TSKgel G2000SWXL (7.8*300) chromatographic column is selected.

[0069] The enzymes used in the embodiment of the application include: protein glutaminase (PG enzyme) with a specification of 100 U / g; pancreatin with a specification of 5000 U / g; Novozymes alkaline protease 37071 with a nominal activity of >=4AU-A / g.

[0070] Example 1

[0071] A method for preparing a high-antioxidant-activity nutritional base material from wheat gluten protein, comprising the following steps:

[0072] (1) 0.3 g of protein glutaminase (PG enzyme) is added to an enzymolysis tank to be dissolved in 125 g of water, 50 g of wheat gluten protein powder is slowly added in steps under high-speed stirring at 1000 rpm, the temperature is raised to 53 DEG C, and stirring is carried out at 60 rpm for 4 h to obtain a wheat gluten protein modified product;

[0073] (2) The pH of the wheat gluten protein modified product is adjusted to 8.0 by using 4 mol / L hydrochloric acid solution and 4 mol / L sodium hydroxide solution, 0.16 g of pancreatin and 0.8 g of Novozymes alkaline protease 37071 are added, and after 12 h of stirring and temperature maintaining at 60 rpm, the temperature is raised to 95 DEG C for 15 min for enzyme inactivation, a tubular centrifuge is used for centrifugation at 8000* g for 15 min, and the supernatant is taken to obtain a wheat gluten protein enzymolysis product A;

[0074] (3) 26.92 g of anhydrous ethanol is added to 50 g of the wheat gluten protein enzymolysis product A, stirring is uniformly carried out to make the ethanol concentration reach 35%, the mixture is left to stand for 1.0 h, then centrifugation is carried out, and the supernatant is taken; anhydrous ethanol is continuously added to the supernatant, stirring is uniformly carried out to make the ethanol solution concentration reach 75% (mass concentration), the mixture is left to stand for 1.0 h, then a tubular centrifuge is used for centrifugation at 8000* g for 15 min, the precipitate is taken, and drying is carried out in a vacuum drying box to obtain an ethanol separation product A;

[0075] (4) 5 g of the ethanol separation product A and 5 g of ribose are uniformly mixed with 40 g of water, 4 mol / L hydrochloric acid solution and 4 mol / L sodium hydroxide solution are used to adjust the pH to 8.0, the temperature is raised to 85 DEG C, the mixture is left to stand for 70 min, then rapid cooling is carried out to room temperature, under a vacuum degree of 0.09 MPa, 50 DEG C, the mixture is concentrated under reduced pressure to 50% of solid content. Nutritional base material A is obtained.

[0076] The flowchart of the embodiment is as shown in Figure 1The solid recovery rate, degree of hydrolysis and protein recovery rate of the wheat gluten protease product A are shown in Table 2, and the DPPH radical scavenging rate and ABTS radical scavenging rate of the nutritional base A are shown in Table 3.

[0077] Example 2

[0078] A method for preparing a high-antioxidant-activity nutritional base from wheat gluten protein, comprising the following steps:

[0079] (1) 0.4 g of protein glutaminase (PG enzyme) is added to an enzyme hydrolysis tank dissolved in 100 g of water, high-speed stirring is started, and 50 g of wheat gluten protein powder is slowly added in steps; the temperature is raised to 55℃, and low-speed stirring is maintained for 3.5 h to obtain a wheat gluten protein modified product;

[0080] (2) The pH of the wheat gluten protein modified product is adjusted to 8.0 with 4 mol / L hydrochloric acid solution and 4 mol / L sodium hydroxide solution, 0.2 g of trypsin and 0.1 g of Novozymes alkaline protease 37071 are added, and after 13 h of incubation and stirring, the temperature is raised to 95℃ for 15 min for enzyme inactivation; the product is centrifuged at 8000 x g for 15 min using a tubular centrifuge, and the supernatant is taken to obtain a wheat gluten protease product B;

[0081] (3) 33.33 g of anhydrous ethanol is added to 50 g of the wheat gluten protease product B, and stirred to make the ethanol concentration 40%, and then left to stand for 1.0 h, followed by centrifugation, and the supernatant is taken; anhydrous ethanol is continuously added to the supernatant, and stirred to make the ethanol solution concentration 80%, and then left to stand for 1.0 h, followed by centrifugation at 8000 x g for 15 min using a tubular centrifuge, and the precipitate is taken and dried in a vacuum drying oven to obtain an ethanol separation product B;

[0082] (4) 5 g of the ethanol separation product B and 5 g of ribose are mixed with 50 g of water, the pH is adjusted to 8.0 with 4 mol / L hydrochloric acid solution and 4 mol / L sodium hydroxide solution, the temperature is raised to 90℃, and left to stand for 80 min, then quickly cooled to room temperature, and concentrated to a solid content of 50% at 50℃ under a vacuum of 0.09 MPa to obtain a nutritional base B.

[0083] The solid recovery rate, degree of hydrolysis and protein recovery rate of the wheat gluten protease product B are shown in Table 2.

[0084] The actual product diagram of the nutritional base B prepared in this example is shown in Figure 3 Table 3 shows the DPPH radical scavenging rate and ABTS radical scavenging rate of the original solution and the gastrointestinal digestion product, and Table 4 shows the molecular weight distribution of the nutritional base B and the simulated digestion product peptides.

[0085] Example 3

[0086] A method for preparing a high antioxidant activity nutritional base material from wheat gluten protein, comprising the following steps:

[0087] (1) 0.5 g of protein glutaminase (PG enzyme) is added to an enzymolysis tank dissolved in 100 g of water, high-speed stirring is started, and 50 g of wheat gluten protein powder is slowly added in steps; the temperature is raised to 57℃, and low-speed stirring is maintained for 3 h to obtain a wheat gluten protein modification product;

[0088] (2) The pH of the wheat gluten protein modification product is adjusted to 8.5 using 4 mol / L hydrochloric acid solution and 4 mol / L sodium hydroxide solution, 0.179 g of trypsin and 0.071 g of Novozymes alkaline protease 37071 are added, and after 12 h of incubation and stirring, the temperature is raised to 95℃ for 15 min for enzyme inactivation; the product is centrifuged at 8000 x g for 15 min using a tubular centrifuge, and the supernatant is obtained to obtain a wheat gluten protein enzymolysis product C;

[0089] (3) 40.91 g of anhydrous ethanol is added to 50 g of the wheat gluten protein enzymolysis product A, and stirred to make the ethanol concentration reach 45%, and then left to stand for 1.0 h, followed by centrifugation, and the supernatant is taken; anhydrous ethanol is continuously added to the supernatant, and stirred to make the ethanol solution concentration reach 80%, and then left to stand for 1.5 h, followed by centrifugation at 8000 x g for 15 min using a tubular centrifuge, and the precipitate is taken and dried in a vacuum drying oven to obtain an ethanol separation product C;

[0090] (4) 5 g of the ethanol separation product C and 5 g of ribose are mixed with 60 g of water, and the pH is adjusted to 8.5 using 4 mol / L hydrochloric acid solution and 4 mol / L sodium hydroxide solution, and the temperature is raised to 90℃, and then left to stand for 90 min, followed by rapid cooling to room temperature, and then concentrated to a solid content of 50% at 50℃ under a vacuum of 0.09 MPa to obtain a nutritional base material C.

[0091] The solid recovery rate, degree of hydrolysis, and protein recovery rate of the wheat gluten protein enzymolysis product C are shown in Table 2.

[0092] The DPPH free radical scavenging rate and ABTS free radical scavenging rate of the nutritional base material C before and after gastrointestinal digestion are shown in Table 3.

[0093] Comparative Example 1

[0094] Prepared according to the method of Reference Example 2, except that the amount of protein glutaminase added in step (2) is adjusted to 0.1 g to obtain a wheat gluten protein enzymolysis product 1 and a nutritional base material 1. The solid recovery rate, degree of hydrolysis, and protein recovery rate of the wheat gluten protein enzymolysis product 1 are shown in Table 2.

[0095] The DPPH radical scavenging rate and ABTS radical scavenging rate of the nutritional base 1 are shown in Table 3.

[0096] Comparative Example 2

[0097] Prepared according to the method of Example 2, except that no Novozyme alkaline protease 37071 was added in step (2), only trypsin was used for enzymatic hydrolysis treatment, and the amount of trypsin added was adjusted to 0.3 g, to obtain wheat gluten protein hydrolysate 2 and nutritional base 2.

[0098] The solid recovery rate, degree of hydrolysis and protein recovery rate of the wheat gluten protein hydrolysate 2 are shown in Table 2.

[0099] The DPPH radical scavenging rate and ABTS radical scavenging rate of the nutritional base 2 before and after gastrointestinal digestion are shown in Table 3.

[0100] Comparative Example 3

[0101] Prepared according to the method of Example 2, except that the ribose added in step (4) was replaced with glucose for reaction, to obtain wheat gluten protein hydrolysate 3 and nutritional base 3.

[0102] The solid recovery rate, degree of hydrolysis and protein recovery rate of the wheat gluten protein hydrolysate 3 are shown in Table 2.

[0103] The DPPH radical scavenging rate and ABTS radical scavenging rate of the nutritional base 3 before and after gastrointestinal digestion are shown in Table 3.

[0104] Comparative Example 4

[0105] Prepared according to the method of Example 2, except that the temperature of the Maillard reaction in step (4) was set to 60°C, to obtain wheat gluten protein hydrolysate 4 and nutritional base 4.

[0106] The solid recovery rate, degree of hydrolysis and protein recovery rate of the wheat gluten protein hydrolysate 4 are shown in Table 2.

[0107] The DPPH radical scavenging rate and ABTS radical scavenging rate of the nutritional base 4 before and after gastrointestinal digestion are shown in Table 3.

[0108] Results and discussion of Example 4

[0109] The detection results of the products prepared in the above examples and comparative examples are shown in Figure 2 and Tables 2-4.

[0110] Table 2 Enzymatic efficiency of wheat gluten protein hydrolysate

[0111]

[0112] Table 3 Antioxidant activity of nutritional bases

[0113]

[0114] Table 4 Peptide molecular weight distribution of nutritional base B and its simulated gastrointestinal digestion products

[0115]

[0116] As can be seen from Table 2, the enzymatic hydrolysis of wheat gluten protein powder using the method of the present application can achieve a high enzymatic hydrolysis efficiency. Specifically, the solid yield of wheat gluten protein hydrolysates A-C is 83.01-85.62%, the protein recovery rate is 75.07-79.13%, and the degree of hydrolysis is 15.06-16.98%. In addition, the nutritional bases prepared by the method of the present application have high antioxidant activity (as shown in Table 3), wherein the DPPH radical scavenging rate can reach 75.77-89.31%, and the simulated gastrointestinal digestion products still have a scavenging rate of 39.31-46.47%; the ABTS radical scavenging rate can reach 50.35-52.17%, and the simulated gastrointestinal digestion products have a scavenging rate of 41.59-44.11%. These results show that the method of the present application not only can achieve a high enzymatic hydrolysis efficiency, but also can impart good antioxidant effect and gastrointestinal digestion stability to the product.

[0117] Correspondingly, the difference between Comparative Example 1 and Example 2 is that Comparative Example 2 uses a lower addition level of protein glutaminase (PGase) to modify the wheat gluten protein powder, resulting in a poor enzymatic hydrolysis efficiency of trypsin and alkaline protease on the wheat gluten protein, with a solid recovery rate, protein recovery rate, and degree of hydrolysis of only 52.45%, 52.64%, and 6.48%, respectively. In addition, the antioxidant activity of the nutritional base 1 of Comparative Example 1 (DPPH scavenging rate 69.86%, ABTS scavenging rate 45.77%) is also much lower than that of the nutritional base B of Example 2 (DPPH scavenging rate 84.42%, ABTS scavenging rate 51.76%). This difference is attributed to the modification effect of PGase: this enzyme can specifically catalyze the deamidation of glutamine residues in wheat gluten protein to generate glutamic acid residues, thereby enhancing its hydrophilicity and exposing more enzyme cleavage sites. This not only improves the enzymatic hydrolysis efficiency, but also promotes the release of more peptides with antioxidant activity. At a lower addition level, the contact of PGase with wheat gluten protein is limited, and the catalytic effect is limited by the amount of enzyme added, failing to achieve the modification effect.

[0118] The enzymatic hydrolysis efficiency (solid recovery rate 67.41%, protein recovery rate 63.88%, degree of hydrolysis 13.37%) of the wheat gluten protein modification product hydrolyzed by pancreatin only in Comparative Example 2 is obviously lower than that of the enzymatic hydrolysis efficiency of pancreatin and alkaline protease in Example 2. The difference is mainly due to the fact that the alkaline protease can specifically cut the amino terminal of aromatic amino acid residues and the carboxyl terminal of hydrophobic amino acids, and the endo-acting and exo-acting enzyme activity of pancreatin on basic amino acids such as lysine and arginine synergize, thereby significantly improving the overall enzymatic hydrolysis efficiency. In addition, as can be seen from the results in Table 3, the antioxidant activity (DPPH clearance rate 75.66%, ABTS clearance rate 48.24%) of the nutritional base 2 prepared in Comparative Example 2 is also lower than that of the nutritional base B in Example 2. This is because the double-enzyme synergistic enzymolysis can release more small peptides containing hydrophobic amino acids, and such peptides usually have high antioxidant activity.

[0119] In Comparative Example 3, the same optimum conditions as in the examples were used in the enzymolysis stage, but glucose was selected as the reactant precursor for the Maillard reaction. As can be seen from the results in Table 3, the antioxidant effect of the product (nutritional base 3) obtained by using glucose for the Maillard reaction is far inferior to that of the product obtained by using ribose for the Maillard reaction, and the DPPH free radical clearance rate is only 17.61%, and the ABTS free radical clearance rate is 48.24%, both of which are lower than those of the nutritional bases A-C. This is mainly because glucose is a six-carbon sugar, and its Maillard reaction activity is lower than that of ribose, a five-carbon sugar. The cyclic structure of ribose is less stable and is more likely to open to form active intermediates (such as open-chain structures), thereby being more likely to undergo Maillard reaction with active peptides to generate more products with antioxidant capacity.

[0120] In Comparative Example 4, the same optimum conditions as in the examples were used in the enzymolysis stage, but the temperature for the Maillard reaction was selected as 60°C. The antioxidant activity of the obtained nutritional base 2 is significantly lower than that of the nutritional bases A-C subjected to the Maillard reaction at 80-90°C, and the DPPH and ABTS free radical clearance rates are 36.06% and 36.35%, respectively. This is mainly because the Maillard reaction needs to reach a certain temperature, and as the temperature increases, the reaction rate and the degree of reaction also increase. At a suitable temperature, a large number of active intermediates (such as various glycopeptides, small molecule compounds and Amadori rearrangement products) can be generated, and components with certain antioxidant capacity can also be formed at the end of the reaction, which together contribute to the free radical clearance capacity. Therefore, it is necessary to reach a temperature at which the Maillard reaction can occur to promote the normal progress of the Maillard reaction. On the basis of enzymolysis and purification, the introduction of the Maillard reaction can further effectively improve the antioxidant activity of the product.

[0121] As shown in Table 3, after simulated gastrointestinal digestion, the antioxidant activity of the nutritional base of the examples and the comparative examples all decreased to a certain extent. The DPPH free radical scavenging rate and the ABTS free radical scavenging rate of the nutritional base A-C after digestion were still maintained at 39.31%-46.47% and 41.59%-44.11%, respectively; while the DPPH and ABTS scavenging rates of the comparative nutritional base 1-4 were 13.52%-38.88% and 26.35%-41.10%, respectively. The difference shows that the active peptide obtained by complex enzyme hydrolysis has strong digestion stability, and the Maillard reaction further enhances the gastrointestinal digestion stability of the antioxidant peptide, so it can still retain high antioxidant activity after gastrointestinal digestion.

[0122] As shown in Table 4, the small molecular components less than 1000 Da in the nutritional base 2 accounted for nearly 50%. It shows that the nutritional base contains rich small molecular active components. The simulated digestion experiment data shows that only a small amount of peptide segments in the range of 200-500 Da are lost, which shows that the active peptide in this molecular weight range has high stability in the gastrointestinal environment, can better maintain its activity, and is more conducive to human absorption and biological utilization, and is a nutritional base with good utilization.

[0123] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and all shall be included in the protection scope of the present application.

Claims

1. A method for preparing a high antioxidant activity nutritional base material from wheat gluten meal, characterized in that It comprises the following steps: (1) Dissolve the protein glutamine enzyme in water, add wheat gluten protein powder under high-speed stirring, modify by incubation and stirring, then add trypsin and alkaline protease, and carry out enzymolysis under incubation and stirring. After the enzymolysis is completed, the enzyme is inactivated, the supernatant is obtained by centrifugation, and a wheat gluten protein enzymolysis product is obtained; (2) Add anhydrous ethanol to the wheat gluten protein enzymolysis product, mix, stand, centrifuge, and obtain the supernatant to obtain an ethanol crude separation product; (3) Add anhydrous ethanol to the ethanol crude separation product, stand, centrifuge, and obtain the precipitate, which is dried to obtain an ethanol separation product; (4) Mix the ethanol separation product, ribose, and water, adjust the pH, incubate and stand, cool after the reaction is completed, and concentrate under reduced pressure to obtain a high-antioxidant-activity nutritional base material.

2. The method according to claim 1, wherein: in step (1), the mass ratio of the wheat gluten protein powder, the protein glutamine enzyme, the trypsin, and the alkaline protease is 100:0.3-1:0.5-1:0.25-1; in step (1), the enzyme activity of the protein glutamine enzyme is 100 U / g; in step (1), the enzyme activity of the trypsin is 5000 U / g; and in step (1), the alkaline protease is Novozymes alkaline protease 37071.

3. The method according to claim 1, wherein: in step (1), the modification by incubation and stirring is carried out at 50-60°C for 3-4 h under stirring at 30-60 rpm; and in step (1), the enzymolysis by incubation and stirring is carried out at 50-60°C for 12-14 h under stirring at 30-60 rpm.

4. The method according to claim 1, wherein: in step (1), the high-speed stirring is carried out at 800-1000 rpm; and in step (1), the centrifugation is carried out at 7000-9000 g for 10-20 min.

5. The method according to claim 1, wherein: in step (2), the amount of anhydrous ethanol added is 35-45% of the total mass; in step (2), the standing is for 1-2 h; and in step (2), the centrifugation is carried out at 7000-9000 g for 10-20 min.

6. The method according to claim 1, wherein: in step (3), the amount of anhydrous ethanol added is 75-90% of the total mass; in step (3), the standing is for 1-2 h; and in step (3), the centrifugation is carried out at 7000-9000 g for 10-20 min.

7. The method according to claim 1, wherein: ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The mass ratio of ethanol separation product: ribose: water is 1: 1.5-2: 8-12. The pH adjustment in step (4) is to adjust the pH to 8.0-8.

5.

8. The method according to claim 1, wherein the high-antioxidant-activity nutritional base is prepared from wheat gluten protein powder. The reaction condition of the heat preservation and standing in step (4) is 85-90℃ for 70-90min.

9. A high-antioxidant-activity nutritional base, wherein: The high-antioxidant-activity nutritional base is prepared by the method according to any one of claims 1-8.

10. The use of the high-antioxidant-activity nutritional base according to claim 9 in the preparation of food nutritional supplements.

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