Preparation method of wheat oligopeptide selenium chelate

Abstract

The invention belongs to the technical field of bioactive substances, and particularly relates to a preparation method of a wheat oligopeptide selenium chelate. The preparation method comprises the following steps: firstly preparing wheat oligopeptide, then mixing the wheat oligopeptide with sodium selenite, carrying out chelation reaction under the assistance of supercritical fluid, and separating and purifying a product to obtain the wheat oligopeptide selenium chelate. The wheat oligopeptide and inorganic selenium are chelated, and compared with other protein peptide and selenium chelating, the wheat oligopeptide is higher in chelating rate and better in oxidation resistance. The chelating rate of the wheat oligopeptide selenium chelate obtained through the method is larger than or equal to 40%, and the DPPH free radical scavenging rate in an in-vitro anti-oxidation experiment is larger than or equal to 35%.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioactive substances, and particularly relates to a preparation method of a wheat oligopeptide selenium chelate. BACKGROUND

[0002] Selenium plays a crucial role in biology and is an essential trace element that has a wide impact on human health. Adequate selenium intake can significantly improve the body's antioxidant capacity and reduce the risk of chronic diseases. In recent years, peptide selenium chelates have become a research hotspot as an important form of organic selenium. By chelating selenium with peptides, not only can the bioavailability and stability of selenium be improved, but also its antioxidant and other biological activities can be enhanced.

[0003] Wheat oligopeptides are a mixture of small molecular weight peptides with biological activity derived from wheat protein through enzymatic hydrolysis. They are widely available and highly safe, and have various biological activities such as antioxidant and immune regulation. When wheat oligopeptides are chelated with selenium, the stability and bioavailability of selenium can be further improved, and synergistic effects can be produced, showing great potential in the fields of nutritional supplements, functional foods, and biological medicines. For example, in nutritional supplements, it can effectively improve the selenium deficiency of the human body and enhance the body's antioxidant defense system; in biological medicines, it is expected to develop targeted drug carriers or adjuvant drugs with specific efficacy. SUMMARY

[0004] The purpose of the present application is to break through the bottleneck of existing technology and provide a preparation process for wheat oligopeptide selenium chelate, achieving the preparation of wheat oligopeptide selenium chelate with high chelation rate and high antioxidant activity under mild conditions.

[0005] To solve the above technical problems, the technical solution adopted by the present application is as follows:

[0006] A preparation method of wheat oligopeptide selenium chelate, in which a mixed solution of wheat oligopeptide and sodium selenite is subjected to a chelation reaction with the assistance of a supercritical fluid, and the product is separated and purified to obtain wheat oligopeptide selenium chelate.

[0007] Preferably, the mass ratio of wheat oligopeptide to sodium selenite in the mixed solution is 1-3:1, preferably 2:1.

[0008] The total mass concentration of wheat oligopeptide and sodium selenite in the mixed solution is 0.2-0.4 g / mL.

[0009] The pH value of the mixed solution is controlled to be 5-7.

[0010] Further, the conditions of the supercritical fluid assisted chelation are: in a supercritical carbon dioxide reactor, under the conditions of temperature 35-55℃, pressure 8-12 MPa, and reaction time 30-90 min.

[0011] The product separation and purification is: after the reaction, the pressure of the reactor is reduced at a rate of 0.1-0.2 MPa / min to make CO2 escape, the wheat oligopeptide selenium chelate with a molecular weight cut-off of 1000-3000 Da is ultrafiltrated, and then freeze-dried to obtain the wheat oligopeptide selenium chelate.

[0012] In the above separation and purification step, the molecular weight cut-off of the ultrafiltration membrane is preferably controlled to be 2000 Da.

[0013] The wheat oligopeptide can be prepared according to the existing conventional method, and the following preparation method is provided for reference.

[0014] The wheat oligopeptide is prepared by dissolving the wheat gluten in deionized water to form an aqueous solution, adjusting the pH to 8.5, adding alkaline protease for enzymatic reaction for 50-70 min, boiling for 15 min and cooling, then adjusting the pH to 6.5, adding flavor protease for enzymatic reaction for 90-100 min, boiling for 15 min, centrifuging to obtain the supernatant, filtering the supernatant through an ultrafiltration membrane, and freeze-drying to obtain the wheat oligopeptide.

[0015] Preferably, the addition amount of the alkaline protease is 1.0% of the mass of the wheat gluten, the enzymatic reaction temperature is controlled to be 55℃, and the enzymatic reaction time is controlled to be 60 min; the addition amount of the flavor protease is 2.0% of the mass of the wheat gluten, the enzymatic reaction temperature is controlled to be 50℃, and the enzymatic reaction time is controlled to be 100 min.

[0016] In the process of filtering the supernatant through an ultrafiltration membrane and freeze-drying to obtain the wheat oligopeptide, the molecular weight cut-off of the ultrafiltration membrane is 5000 Da, and the centrifugation conditions are 8000 r / min and 10 min.

[0017] Specifically, a preparation method of a wheat oligopeptide selenium chelate includes the following steps:

[0018] (1) Preparation of wheat oligopeptide: dissolving the wheat gluten in deionized water to form an aqueous solution, adjusting the pH to 8.5, adding alkaline protease for enzymatic reaction for 50-70 min, boiling for 15 min and cooling, then adjusting the pH to 6.5, adding flavor protease for enzymatic reaction for 90-100 min, boiling for 15 min, centrifuging to obtain the supernatant, filtering the supernatant through an ultrafiltration membrane, and freeze-drying to obtain the wheat oligopeptide;

[0019] (2) wheat oligopeptide and sodium selenite with a mass ratio of 2:1 are configured into a solution with a mass concentration of 0.2 g / mL by using deionized water; the solution is injected into a supercritical CO2 reactor, and the reaction is carried out at a temperature of 35-55 DEG C and a pressure of 8-12 MPa for 30-90 min;

[0020] (3) product separation and purification: after the reaction is completed, the pressure of the reactor is reduced at a rate of 0.1-0.2 MPa / min to make CO2 escape, ultrafiltration is used to separate the wheat oligopeptide selenium chelate with a molecular weight cut-off of 1000-3000 Da, and then freeze-drying is performed to obtain the wheat oligopeptide selenium chelate.

[0021] Compared with other protein peptides and selenium chelation, the chelation rate of the wheat oligopeptide is higher, the antioxidant property is better, and the Se 4+ The structure of the wheat oligopeptide is not significantly affected. The chelation rate of the wheat oligopeptide selenium chelate obtained by the preparation method is greater than or equal to 40%, and the DPPH free radical scavenging rate in the in-vitro antioxidant experiment is greater than or equal to 35%. Compared with existing chelation methods, the method is simpler and greener, the chelation rate and in-vitro antioxidant capacity are higher, and the method is more suitable for the preparation of the wheat oligopeptide selenium chelate.

[0022] The obtained wheat oligopeptide selenium chelate has good application in the preparation of functional food.

[0023] Compared with the prior art, the present application has the following advantages:

[0024] (1) Compared with the traditional method, the supercritical fluid assisted process is particularly selected according to the wheat oligopeptide selenium chelate to be prepared, so that the chelation rate of the wheat oligopeptide and selenium is increased by 10%, and the chelation rate is detected to be greater than or equal to 45%, which greatly improves the raw material utilization rate and product yield;

[0025] (2) The reaction is carried out under relatively mild temperature and pressure conditions, which effectively avoids the damage of high temperature and strong alkali to the structure and activity of the wheat oligopeptide, and ensures that the final product can completely retain the biological activity of the wheat oligopeptide and the effective function of selenium, and the product quality is stable and reliable;

[0026] (3) The supercritical CO2 has the functions of reaction medium and separation medium, which omits many complicated intermediate separation steps in the traditional process, such as multiple precipitation and complex acid-base adjustment, significantly shortens the process flow, reduces equipment investment and labor cost, improves production efficiency, and is suitable for large-scale industrial production;

[0027] (4) CO2 is used as the supercritical fluid, which avoids the use of a large amount of chemical reagents, reduces environmental pollution from the source, conforms to the current green chemistry and sustainable development concept, and has good ecological and social benefits. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The infrared scanning spectrum of the wheat oligopeptide and the wheat oligopeptide selenium chelate is shown in Figure 1.

[0029] Figure 2 The infrared scanning spectrum of different peptides and peptide selenium chelates is shown in Figure 1. DETAILED DESCRIPTION

[0030] The technical solutions of the present application are described below with specific examples, but the protection scope of the present application is not limited thereto:

[0031] In the supercritical CO2 reaction, the material of the reaction kettle is 316L stainless steel, the volume is 1000 mL, and the reaction kettle is equipped with a high-precision temperature sensor (accuracy ±0.1℃) and a pressure sensor (accuracy ±0.05MPa).

[0032] Example 1

[0033] The wheat oligopeptide selenium chelate is prepared according to the following steps:

[0034] (1) Preparation of wheat oligopeptide: 1 kg of gluten meal (crude protein content 50%) is dissolved in 2 L of deionized water to prepare a water solution with a protein concentration of 25%, the pH is adjusted to 8.5, and 1.0% of alkaline protease based on the mass of the gluten meal is added. After 60 min of enzymatic reaction at 55℃, it is boiled for 15 min and cooled; then the pH is adjusted to 6.5, and 2.0% of flavor protease based on the mass of the gluten meal is added. After 100 min of enzymatic reaction at 50℃, it is boiled for 15 min to obtain a wheat powder enzymatic solution; the obtained enzymatic solution is subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 5000 Da, and the permeate is collected to obtain a wheat oligopeptide solution; the obtained wheat oligopeptide solution is freeze-dried to obtain wheat oligopeptide.

[0035] (2) Chelation reaction: the obtained wheat oligopeptide and sodium selenite are configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution is 2:1, and the pH of the solution is adjusted to 7; the mixed solution is injected into a supercritical CO2 reaction kettle, and reacted at a temperature of 35℃ and a pressure of 10 MPa for 30 min.

[0036] (3) Product separation and purification: the reaction solution of the previous step is subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain a wheat oligopeptide selenium chelate.

[0037] Example 2

[0038] The wheat oligopeptide selenium chelate is prepared according to the following steps:

[0039] (1) Preparation of wheat oligopeptide: same as Example 1;

[0040] (2) Chelation reaction: the obtained wheat oligopeptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, and the pH of the solution was adjusted to 7; the mixed solution was injected into a supercritical CO2 reactor, and reacted at a temperature of 45°C and a pressure of 10 MPa for 30 min;

[0041] (3) Product separation and purification: the reaction solution of the last step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain a wheat oligopeptide selenium chelate.

[0042] Example 3

[0043] A wheat oligopeptide selenium chelate was prepared according to the following steps:

[0044] (1) Preparation of wheat oligopeptide: same as Example 1;

[0045] (2) Chelation reaction: the obtained wheat oligopeptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, and the pH of the solution was adjusted to 7; the mixed solution was injected into a supercritical CO2 reactor, and reacted at a temperature of 55°C and a pressure of 10 MPa for 30 min;

[0046] (3) Product separation and purification: the reaction solution of the last step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain a wheat oligopeptide selenium chelate.

[0047] Example 4

[0048] A wheat oligopeptide selenium chelate was prepared according to the following steps:

[0049] (1) Preparation of wheat oligopeptide: same as Example 1;

[0050] (2) Chelation reaction: the obtained wheat oligopeptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, and the pH of the solution was adjusted to 7; the mixed solution was injected into a supercritical CO2 reactor, and reacted at a temperature of 45°C and a pressure of 8 MPa for 30 min;

[0051] (3) Product separation and purification: the reaction solution of the last step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain a wheat oligopeptide selenium chelate.

[0052] Example 5

[0053] Wheat oligopeptide selenium chelate was prepared according to the following steps:

[0054] (1) Preparation of wheat oligopeptide: same as Example 1;

[0055] (2) Chelation reaction: the obtained wheat oligopeptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, and the pH of the solution was adjusted to 7; the mixed solution was injected into a supercritical CO2 reactor, and reacted for 30 min under the conditions of a temperature of 45 ℃ and a pressure of 12 MPa;

[0056] (3) Product separation and purification: the reaction solution of the last step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain the wheat oligopeptide selenium chelate.

[0057] Example 6

[0058] Wheat oligopeptide selenium chelate was prepared according to the following steps:

[0059] (1) Preparation of wheat oligopeptide: same as Example 1;

[0060] (2) Chelation reaction: the obtained wheat oligopeptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, and the pH of the solution was adjusted to 7; the mixed solution was injected into a supercritical CO2 reactor, and reacted for 60 min under the conditions of a temperature of 45 ℃ and a pressure of 10 MPa;

[0061] (3) Product separation and purification: the reaction solution of the last step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain the wheat oligopeptide selenium chelate.

[0062] Example 7

[0063] Wheat oligopeptide selenium chelate was prepared according to the following steps:

[0064] (1) Preparation of wheat oligopeptide: same as Example 1;

[0065] (2) Chelation reaction: the obtained wheat oligopeptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, and the pH of the solution was adjusted to 7; the mixed solution was injected into a supercritical CO2 reactor, and reacted for 90 min under the conditions of a temperature of 45 ℃ and a pressure of 10 MPa;

[0066] (3) Product separation and purification: the reaction solution of the last step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain the wheat oligopeptide selenium chelate.

[0067] Comparative Example 1

[0068] A wheat oligopeptide was prepared according to the method of Step (1) of Example 1.

[0069] Comparative Example 2

[0070] Preparation of a wheat oligopeptide selenium chelate:

[0071] (1) Preparation of a wheat oligopeptide: according to Example 1;

[0072] (2) Chelation reaction: the obtained wheat oligopeptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, the pH of the solution was adjusted to 7, and stirring was performed at 55°C for 40 min;

[0073] (3) Product separation and purification: the reaction solution of the previous step was subjected to ultrafiltration using an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-drying was performed to obtain a wheat oligopeptide selenium chelate.

[0074] Comparative Example 3

[0075] Preparation of a wheat oligopeptide selenium chelate:

[0076] (1) Preparation of a wheat oligopeptide: according to Example 1;

[0077] (2) Chelation reaction: the obtained wheat oligopeptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, the pH of the solution was adjusted to 7, and microwave treatment was performed for 3 min at a microwave power of 500 W, and stirring was performed at 90°C for 30 min;

[0078] (3) Product separation and purification: the reaction solution of the previous step was subjected to ultrafiltration using an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-drying was performed to obtain a wheat oligopeptide selenium chelate.

[0079] Comparative Example 4

[0080] Preparation of a wheat oligopeptide selenium chelate:

[0081] (1) Preparation of a wheat oligopeptide: according to Example 1;

[0082] (2) Chelation reaction: the obtained wheat oligopeptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, the pH of the solution was adjusted to 7, and ultrasonic cavitation treatment was performed at 50°C for 30 min at an ultrasonic power of 300 W;

[0083] (3) Product separation and purification: the reaction solution of the previous step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain the selenium chelate of wheat oligopeptide.

[0084] Comparative Example 5

[0085] Preparation of a selenium chelate of corn oligopeptide:

[0086] (1) Chelation reaction: corn peptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, the solution pH was adjusted to 7, and the solution was injected into a supercritical CO2 reactor, and reacted at a temperature of 45℃ and a pressure of 10 MPa for 90 min;

[0087] (2) Product separation and purification: the reaction solution of the previous step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain the selenium chelate of corn oligopeptide.

[0088] Comparative Example 6

[0089] Preparation of a selenium chelate of soybean oligopeptide:

[0090] (1) Chelation reaction: corn peptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, the solution pH was adjusted to 7, and the solution was injected into a supercritical CO2 reactor, and reacted at a temperature of 45℃ and a pressure of 10 MPa for 90 min;

[0091] (2) Product separation and purification: the reaction solution of the previous step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain the selenium chelate of corn oligopeptide.

[0092] Comparative Example 7

[0093] Preparation of a selenium chelate of pea oligopeptide:

[0094] (1) Chelation reaction: corn peptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, the solution pH was adjusted to 7, and the solution was injected into a supercritical CO2 reactor, and reacted at a temperature of 45℃ and a pressure of 10 MPa for 90 min;

[0095] (2) Product separation and purification: the reaction solution of the previous step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cut-off of 2000 Da, and then freeze-dried to obtain the selenium chelate of corn oligopeptide.

[0096] Comparative Example 8

[0097] Preparation of a selenium chelate of rice peptide:

[0098] (1)Chelation reaction: rice peptide and sodium selenite were configured into a solution with a mass concentration of 0.2 g / mL using deionized water, the mass ratio of the two in the solution was 2:1, the pH of the solution was adjusted to 7, and the solution was injected into a supercritical CO2 reactor, and reacted at a temperature of 45°C and a pressure of 10 MPa for 90 min;

[0099] (2) Product separation and purification: the reaction solution of the above step was subjected to ultrafiltration through an ultrafiltration membrane device with a molecular weight cutoff of 2000 Da, and then freeze-dried to obtain a rice peptide selenium chelate.

[0100] Index determination method and results:

[0101] 1. Chelation rate determination

[0102] Prepare a 1 mg / kg selenium standard solution, accurately take 0.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mL of the selenium standard solution, add deionized water to 40 mL, adjust the pH to 2-3 with hydrochloric acid, add 4 mL of 0.2 M EDTA-2Na to mask interfering ions, then add 2 mL of 0.5% DAB, shake well, and place in a 60°C water bath for 20 min (in a dimly lit environment), remove, adjust the pH to 7-7.5 with NaOH, accurately transfer 4.0 mL of toluene, shake vigorously for 1 min, stand for 3-4 min to separate the layers, then filter the organic layer through a filter paper to a 1 cm cuvette, and measure the absorbance of the solution at 420 nm (toluene as blank).

[0103] The products obtained in Examples 1-7 and Comparative Examples 1-8 were subjected to the following operation: first, take about 0.1 g of the sample and place it in a 25 mL K flask, add 5 mL of digestion solution (10 g of sodium molybdate, dissolved in 150 mL of distilled water, slowly add 150 mL of concentrated sulfuric acid, cool, add 200 mL of 70%-72% perchloric acid, mix well), and digest at low temperature until the sample solution is colorless and transparent. After cooling, the digestion solution is transferred to a 100 mL beaker, and the residual digestion sample solution in the K flask is washed with distilled water and combined into the beaker. The above digestion solution is adjusted to a pH of about 7.0 with 40% NaOH, and finally the solution is diluted to 50 mL.

[0104] Take 5 mL of the digested sample and repeat the standard solution determination operation to calculate the chelation rate.

[0105] Chelation rate (%) = (m1 / m2) x 100 (m1 is the mass of selenium in the chelate, and m2 is the mass of selenium added)

[0106] The results are shown in Table 1 below:

[0107] Table 1

[0108] Chelation rate / % Example 1 41.75 Example 2 47.94 Example 3 45.31 Example 4 45.05 Example 5 47.28 Example 6 48.53 Example 7 49.22 Comparative Example 1 0 Comparative Example 2 36.74 Comparative Example 3 31.64 Comparative Example 4 42.51 Comparative Example 5 47.47 Comparative Example 6 43.59 Comparative Example 7 38.75 Comparative Example 8 39.06

[0109] From the above table, it can be seen that the chelation rates of Examples 1-7 are all between 40% and 50%, and the chelation rate is the highest when the supercritical parameters are reaction temperature 45°C, pressure 10 MPa, and reaction time 90 min.

[0110] The chelation rate of Comparative Example 1 is 0, further proving that no peptide selenium chelate is generated when no chelation reaction is performed, eliminating the interference of selenium contained in the wheat oligopeptide itself on the experimental results, and ensuring the accuracy of the chelation rate data. The chelation rate of Comparative Example 2 (traditional process) is 36.74%, the chelation rate of Comparative Example 3 (microwave-assisted chelation process) is only 31.64%, and the chelation rate of Comparative Example 4 (ultrasonic cavitation chelation process) is 42.51%. The chelation rate of the supercritical process is increased by about 7%-15% compared to the traditional process, the microwave-assisted chelation process, and the ultrasonic cavitation chelation process, fully verifying the core advantages of the supercritical fluid-assisted process in improving raw material utilization and product yield.

[0111] Meanwhile, from Examples 7 and Comparative Examples 5-8, it can be seen that the chelation rate of the peptide selenium chelate is related to the type of peptide. The chelation rate of the wheat oligopeptide selenium chelate is slightly higher than that of the corn oligopeptide selenium chelate.

[0112] 2. DPPH free radical scavenging rate determination

[0113] Prepare a 0.2 mmol / L DPPH free radical solution. Mix 2 mL of 0.5 mg / mL sample solution and 2 mL of DPPH solution, mix thoroughly at room temperature, and avoid light reaction for 30 min. Then use a UV spectrophotometer to measure the absorbance of the solution at 517 nm as A sample. Under the same reaction conditions, replace the above DPPH solution with anhydrous methanol solution to measure the absorbance as A blank, and replace the sample with pure water to measure the absorbance as A control.

[0114] DPPH free radical scavenging capacity (%) = [1-(A sample-A blank) / A control] x 100%

[0115] The following Table 2 lists the free radical scavenging rate determination results of the samples of the examples and comparative examples.

[0116] Table 2

[0117]

[0118]

[0119] As can be seen from Table 2, the DPPH radical scavenging rate of Examples 1-7 (supercritical process wheat oligopeptide selenium chelate) is between 36% and 43%, and the rate of Comparative Example 1 (pure wheat oligopeptide) is only 32.53%. When the supercritical parameters are a reaction temperature of 45°C, a pressure of 10 MPa, and a reaction time of 90 min, the DPPH radical scavenging rate is the highest, which is basically consistent with the parameter influence trend of the chelation rate, indicating that there is a certain positive correlation between the chelation rate and the antioxidant property, and the more sufficient the chelation, the higher the antioxidant activity of the product. It is shown that after the wheat oligopeptide is chelated with selenium, the in vitro antioxidant capacity of the product is significantly improved through the synergistic effect of the antioxidant activity of selenium and the activity of the peptide chain itself. At the same time, as can be seen from Example 7 and Comparative Examples 5-8, the antioxidant capacity of the peptide selenium chelate is related to the type of the peptide.

[0120] 3. Determination of peptide selenium chelate structure by Fourier infrared spectroscopy

[0121] In the agate mortar, 1.0 mg of sample (product of Example 7) was placed, and then 10.0 mg of dried KBr was added and ground uniformly. The sample was placed in a tablet press device, and a pressure of 20 MPa was applied. After 1 min, the tablet was taken out and was in a translucent state. Subsequently, qualitative analysis was performed on the tablet by using a Fourier infrared spectrometer, and an infrared spectrum graph with a wavelength of 400-4000 cm -1 was obtained.

[0122] Infrared spectroscopy can effectively distinguish the structural differences between two substances, and therefore it is widely used in reactions between organic groups and metal ions. Figure 1 The results show that Se 4+ is mainly chelated with the amino group (-NH2) and the carboxyl group (-COOH) through covalent coordination. The 1455 cm -1 absorption band of the amide I band does not change after the chelation reaction, which indicates that the carbonyl group (C=O) in the peptide chain does not participate in the coordination of Se 4+ . The characteristic absorption peak of the carboxyl group shifts from 1554 cm -1 to 1523 cm -1 , which indicates that the oxygen atom of the carboxyl group is an important binding site of Se 4+ . Figure 2 The results show that there are differences in the intensity and peak position of the infrared spectrum of the peptide and the peptide selenium chelate. By comparing the intensity and area of the peaks between the wheat oligopeptide and the rice peptide, the soybean oligopeptide, the corn oligopeptide, and the lentil oligopeptide, it can be seen that the relative content of the amino group and the amino functional group that binds with Se 4+ in the wheat oligopeptide is relatively low, and the change in the peak intensity of the wheat oligopeptide after binding with Se 4+ is relatively small, which indicates that compared with the rice peptide, the soybean oligopeptide, the corn oligopeptide, and the lentil oligopeptide, the Se 4+The wheat oligopeptide selenium chelate has good chelation rate while the structure of the wheat oligopeptide is not significantly affected.

[0123] By analyzing the secondary structure of the infrared spectrum (results shown in Table 3) (corresponding to the wheat oligopeptide and wheat oligopeptide selenium chelate of Example 7), the proportion of beta-sheet structure in the secondary structure of the wheat oligopeptide is increased and the proportion of beta-turn structure is reduced by supercritical CO2 assisted chelation, compared with the traditional chelation method, in the process of supercritical CO2 assisted chelation, the change of mass transfer and reaction rate in the reaction system makes the peptide chain in the formation of secondary structure be subjected to different kinetic limitations, thereby leading to the final secondary structure being different from the wheat oligopeptide.

[0124] Table 3 Secondary structure of wheat oligopeptide and wheat oligopeptide selenium chelate

[0125] Sample Beta-sheet Random coil Alpha-helix Beta-turn Wheat oligopeptide 18.03％ 0 41.64％ 40.33％ Wheat oligopeptide selenium chelate 20.83％ 0 41.76％ 37.41％

[0126] In summary, the wheat oligopeptide selenium chelate prepared by the present application has high selenium chelation rate and strong antioxidant activity, the selenium chelation rate is as high as 49.22%, and the DPPH free radical scavenging rate is as high as 40.57%. The high diffusivity of supercritical CO2 enables selenium ions to quickly approach the wheat oligopeptide molecules, accelerating the chelation reaction rate. The low viscosity of supercritical CO2 provides a good environment for the free movement of microparticles, which is beneficial to the mass transfer and the probability of intermolecular collision in the reaction process. It is fully proved that the supercritical fluid assisted process has double advantages in improving the chelation rate and ensuring the antioxidant activity, breaking through the technical bottleneck of the traditional process, and having significant innovation and advancement.

Claims

1. A method for preparing wheat oligopeptide selenium chelates, characterized in that, A chelation reaction was carried out in a mixed solution of wheat oligopeptides and sodium selenite with the assistance of supercritical fluid. The product was then separated and purified to obtain wheat oligopeptide selenium chelate.

2. The method for preparing wheat oligopeptide selenium chelate as described in claim 1, characterized in that, The conditions for supercritical fluid-assisted chelation are as follows: reaction in a supercritical carbon dioxide reactor at a temperature of 35~55℃ and a pressure of 8~12MPa for 30~90 min.

3. The method for preparing wheat oligopeptide selenium chelate as described in claim 1, characterized in that, The mass ratio of wheat oligopeptides to sodium selenite is 1~3:

1.

4. The method for preparing wheat oligopeptide selenium chelate as described in claim 3, characterized in that, The total mass concentration of wheat oligopeptides and sodium selenite in a mixed solution is 0.2~0.4 g / mL.

5. The method for preparing wheat oligopeptide selenium chelate as described in claim 1, characterized in that, The pH of the mixed solution should be controlled between 5 and 7.

6. The method for preparing wheat oligopeptide selenium chelate as described in claim 1, characterized in that, The product separation and purification process is as follows: after the reaction is completed, the pressure of the reactor is reduced at a rate of 0.1~0.2 MPa / min to allow CO2 to escape, and wheat oligopeptide selenium chelate with a molecular weight cutoff of 1000~3000 Da is retained by ultrafiltration, and then the wheat oligopeptide selenium chelate is obtained by freeze drying.

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