Preparation method of wheat peptide for enhancing immunity

Abstract

The invention relates to the technical field of active peptides, and discloses a preparation method of wheat peptides capable of enhancing immunity, which comprises the following steps: pretreatment: adding wheat vital gluten into deionized water, and violently stirring to form a suspension A; high hydrostatic pressure depolymerization treatment: putting the suspension A in a polyethylene bag, immersing the polyethylene bag in an ultrahigh pressure liquid medium, applying hydrostatic pressure at normal temperature, and then instantaneously releasing the pressure to 0 MPa to enable protein molecules to generate an oscillation depolymerization effect to obtain a suspension B; centrifuging the suspension B, and taking supernate for later use; performing enzymolysis: performing enzymolysis on the supernate by using papain and alkaline protease, performing enzyme deactivation after the enzymolysis is completed, and performing centrifugation to obtain enzymatic hydrolysate; decoloring and debitterizing; purifying and concentrating; and performing spray drying to obtain wheat peptide powder. According to the method disclosed by the invention, the structure of a protein molecule is opened and more restriction enzyme cutting sites are exposed through high hydrostatic pressure depolymerization treatment under a normal temperature condition under the condition that the activity of protein is not influenced, so that the subsequent enzymolysis efficiency is greatly improved, and the yield of the wheat peptide is improved.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to a method for preparing a wheat peptide that enhances immunity. Background Technology

[0002] Wheat gluten protein, commonly known as gluten powder or active gluten powder, is a natural protein extracted from wheat and is a byproduct of wheat starch production. The main components of wheat gluten protein are gliadin and glutenin, both of which have very similar amino acid compositions, being proteins rich in glutamic acid and proline. Besides providing the body with amino acids and energy, food proteins are also an important source of bioactive peptides. Currently, a large number of bioactive peptides have been isolated and identified from the enzymatic hydrolysates of various foods, such as antihypertensive peptides, soybean peptides, corn peptides, rice peptides, and wheat peptides. These peptides have various physiological functions, such as antioxidation, enhancing immunity, and promoting bone development and growth.

[0003] The preparation of bioactive peptides typically employs enzymatic hydrolysis, a widely used method with several applications: firstly, improving the functional properties of proteins, such as solubility, emulsification, and gelation, in the food industry; secondly, preparing hypoallergenic infant foods and nutritional products; and thirdly, preparing bioactive peptides for application in the pharmaceutical and health food fields. Therefore, existing technologies often use enzymatic hydrolysis of wheat gluten to produce wheat peptides. However, during enzymatic hydrolysis, wheat gluten exhibits low hydrolysis efficiency and incomplete reaction, resulting in low peptide yield. To address these technical problems, this invention provides a method for preparing wheat peptides. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing wheat peptides that enhance immunity. By using high hydrostatic pressure depolymerization treatment at room temperature, the structure of the protein molecules is opened up and more enzyme cleavage sites are exposed without affecting the protein activity, thereby significantly improving the efficiency of subsequent enzymatic hydrolysis and increasing the yield of wheat peptides.

[0005] To achieve the above objectives, the present invention provides a method for preparing wheat peptides that enhance immunity, comprising the following steps: Step 1, Pretreatment: Add wheat gluten powder to deionized water and stir vigorously for 20-30 minutes using a high-speed disperser to fully hydrate and disperse it, forming a uniform suspension A; Step 2, high hydrostatic pressure depolymerization treatment: Place suspension A in a polyethylene bag and immerse it in an ultra-high pressure liquid medium. Apply hydrostatic pressure at room temperature and then release the pressure instantly to 0 MPa to cause the protein molecules to undergo an oscillating depolymerization effect, thus obtaining suspension B. Step 3, heat suspension B at 8000× gCentrifuge for 10 minutes and collect the supernatant for later use; Step 4, enzymatic hydrolysis: The supernatant from step 3 is enzymatically hydrolyzed using papain and alkaline protease. After the enzymatic hydrolysis is completed, the enzymes are inactivated. The hydrolysate is obtained at 8000 rpm for 15 min. Step 5, decolorization and debittering: Adjust the pH of the enzymatic hydrolysate to 4.0-5.0, add food-grade activated carbon powder, stir at a constant temperature to allow the activated carbon to fully adsorb pigments and bitter substances, remove the activated carbon after adsorption is complete to obtain crude wheat peptide solution; Step 6, purification and concentration: use a microporous membrane to filter and remove residual impurities. Transfer the filtrate to a vacuum concentrator and concentrate it to a solid content of 25%-40% to obtain a concentrated solution. Step 7, spray drying: Spray drying of the concentrate yields wheat peptide powder.

[0006] Furthermore, in step 1, the concentration of suspension A is 5-8%.

[0007] Furthermore, in step 2, the static pressure control is 120-150 MPa, and the processing time is 5 minutes.

[0008] Furthermore, in step 4, the specific enzymatic hydrolysis operations are as follows: ① Adjust the substrate concentration to 6%, add 2% papain, and hydrolyze for 90 minutes at 45℃ and pH 6.5; ② Raise the temperature to 50-55℃, add 1% alkaline protease, adjust the pH to 9, and hydrolyze for 60 minutes.

[0009] Furthermore, in step 5, the amount of food-grade activated carbon powder added is 1%-3%, the temperature is 50-60℃, and the time is 30-45 minutes.

[0010] Furthermore, in step 6, the pore size of the microporous filter membrane is 0.22μm-0.45μm, the temperature of the vacuum concentrator is set to 50-60℃, and the vacuum degree is -0.08MPa to -0.09MPa.

[0011] Furthermore, in step 7, the spray drying conditions are: inlet temperature 160-180℃, outlet temperature 80-90℃, and atomizer speed 15000-30000 rpm.

[0012] To achieve the above objectives, the present invention also provides an immune-enhancing wheat peptide, which is prepared by the above preparation method.

[0013] To achieve the above objectives, the present invention also provides the application of the aforementioned immune-enhancing wheat peptide in the preparation of immune-enhancing products.

[0014] Furthermore, the products include food and pharmaceuticals.

[0015] The advantages and positive effects of the method for preparing immune-enhancing wheat peptides described in this invention are as follows: 1. In this invention, a high hydrostatic pressure depolymerization treatment is performed at room temperature before enzymatic hydrolysis, which opens up the protein molecular structure, exposes more enzyme cleavage sites, and makes subsequent enzymatic hydrolysis more complete and efficient, making it easier to generate a large number of specific short peptides with immunomodulatory activity, thereby improving the yield of wheat peptides.

[0016] 2. This invention first uses papain to perform preliminary cleavage under mild conditions, breaking down large protein molecules into medium-sized peptides. Then, alkaline protease is used for further deep hydrolysis to generate the target small molecule active peptides. This stepwise enzymatic hydrolysis of "mild first, then strong" avoids the limitations of single enzymatic hydrolysis, significantly improves the conversion rate and yield of active peptides, and ensures that the final product contains a high concentration of functional components.

[0017] 3. The invention employs a dual approach of two-step enzymatic hydrolysis and activated carbon treatment, which greatly improves the solubility of wheat peptides and eliminates bitterness, making wheat peptides easier to apply in various food and beverage systems, resulting in better taste and higher consumer acceptance. 4. This invention uses activated carbon decolorization and microporous membrane filtration to effectively remove pigments, unhydrolyzed protein particles and tiny impurities, resulting in a light-colored, clear and transparent peptide solution, which significantly improves the purity and sensory quality of wheat peptides.

[0018] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0019] The technical solution of the present invention will be further described below through embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0023] Papain (500-2000 U / g) and alkaline protease (200000 U / g) were purchased from Sigma.

[0024] Example 1 A method for preparing wheat peptides that enhance immunity includes the following steps: Step 1, Pretreatment: Add wheat gluten powder to deionized water and stir vigorously for 20-30 minutes using a high-speed disperser to fully hydrate and disperse it, forming a uniform suspension A with a concentration of 8%; Step 2, high hydrostatic pressure depolymerization treatment: Place suspension A in a polyethylene bag and immerse it in an ultra-high pressure liquid medium (water). Apply a hydrostatic pressure of 150 MPa at room temperature for 5 minutes, and then instantly release the pressure to 0 MPa to cause the protein molecules to undergo an oscillating depolymerization effect, thus obtaining suspension B. Step 3, heat suspension B at 8000× g Centrifuge for 10 minutes and collect the supernatant for later use; Step 4, enzymatic hydrolysis: First, adjust the substrate concentration in the supernatant to 6%, add 2% papain, and hydrolyze for 90 min at 45℃ and pH 6.5; then raise the temperature to 50℃, add 1% alkaline protease, adjust the pH to 9, and hydrolyze for 60 min. After the enzymatic hydrolysis is completed, inactivate the enzyme, and obtain the hydrolysate at 8000 rpm for 15 min. Step 5, decolorization and debittering: Adjust the pH of the enzymatic hydrolysate to 4.0-5.0, add 2% food-grade activated carbon powder, stir at 50-60℃ for 30 minutes to allow the activated carbon to fully adsorb pigments and bitter substances. After adsorption is complete, remove the activated carbon to obtain crude wheat peptide solution. Step 6, purification and concentration: Filter using a 0.22μm-0.45μm microporous membrane to remove residual impurities, transfer the filtrate to a vacuum concentrator, and concentrate it to a solid content of 30% under the conditions of 50-60℃ and a vacuum degree of -0.08MPa to -0.09MPa to obtain a concentrated solution; Step 7, spray drying: Spray drying of the concentrate yields wheat peptide powder.

[0025] Example 2 A method for preparing wheat peptides that enhance immunity includes the following steps: Step 1, Pretreatment: Add wheat gluten powder to deionized water and stir vigorously for 20-30 minutes using a high-speed disperser to fully hydrate and disperse it, forming a uniform suspension A with a concentration of 5%; Step 2, high hydrostatic pressure depolymerization treatment: Place suspension A in a polyethylene bag and immerse it in an ultra-high pressure liquid medium (water). Apply a hydrostatic pressure of 120 MPa at room temperature for 5 minutes, and then instantly release the pressure to 0 MPa to cause the protein molecules to undergo an oscillating depolymerization effect, thus obtaining suspension B. Step 3, heat suspension B at 8000× g Centrifuge for 10 minutes and collect the supernatant for later use; Step 4, enzymatic hydrolysis: First, adjust the substrate concentration in the supernatant to 6%, add 2% papain, and hydrolyze for 90 min at 45℃ and pH 6.5; then raise the temperature to 50℃, add 1% alkaline protease, adjust the pH to 9, and hydrolyze for 60 min. After the enzymatic hydrolysis is completed, inactivate the enzyme, and obtain the hydrolysate at 8000 rpm for 15 min. Step 5, decolorization and debittering: Adjust the pH of the enzymatic hydrolysate to 4.0-5.0, add 2% food-grade activated carbon powder, stir at 50-60℃ for 30 minutes to allow the activated carbon to fully adsorb pigments and bitter substances. After adsorption is complete, remove the activated carbon to obtain crude wheat peptide solution. Step 6, purification and concentration: Filter using a 0.22μm-0.45μm microporous membrane to remove residual impurities, transfer the filtrate to a vacuum concentrator, and concentrate it to a solid content of 30% under the conditions of 50-60℃ and a vacuum degree of -0.08MPa to -0.09MPa to obtain a concentrated solution; Step 7, spray drying: Spray drying of the concentrate yields wheat peptide powder.

[0026] Example 3 A method for preparing wheat peptides that enhance immunity includes the following steps: Step 1, Pretreatment: Add wheat gluten powder to deionized water and stir vigorously for 20-30 minutes using a high-speed disperser to fully hydrate and disperse it, forming a uniform suspension A with a concentration of 7%; Step 2, high hydrostatic pressure depolymerization treatment: Place suspension A in a polyethylene bag and immerse it in an ultra-high pressure liquid medium (water). Apply a hydrostatic pressure of 130 MPa at room temperature for 5 minutes, and then instantly release the pressure to 0 MPa to cause the protein molecules to undergo an oscillating depolymerization effect, thus obtaining suspension B. Step 3, heat suspension B at 8000× g Centrifuge for 10 minutes and collect the supernatant for later use; Step 4, enzymatic hydrolysis: First, adjust the substrate concentration in the supernatant to 6%, add 2% papain, and hydrolyze for 90 min at 45℃ and pH 6.5; then raise the temperature to 50℃, add 1% alkaline protease, adjust the pH to 9, and hydrolyze for 60 min. After the enzymatic hydrolysis is completed, inactivate the enzyme, and obtain the hydrolysate at 8000 rpm for 15 min. Step 5, decolorization and debittering: Adjust the pH of the enzymatic hydrolysate to 4.0-5.0, add 2% food-grade activated carbon powder, stir at 50-60℃ for 30 minutes to allow the activated carbon to fully adsorb pigments and bitter substances. After adsorption is complete, remove the activated carbon to obtain crude wheat peptide solution. Step 6, purification and concentration: Filter using a 0.22μm-0.45μm microporous membrane to remove residual impurities, transfer the filtrate to a vacuum concentrator, and concentrate it to a solid content of 30% under the conditions of 50-60℃ and a vacuum degree of -0.08MPa to -0.09MPa to obtain a concentrated solution; Step 7, spray drying: Spray drying of the concentrate yields wheat peptide powder.

[0027] Comparative Example 1 A method for preparing wheat peptides that enhance immunity includes the following steps: Step 1, Pretreatment: Add wheat gluten powder to deionized water and stir vigorously for 20-30 minutes using a high-speed disperser to fully hydrate and disperse it, forming a uniform suspension A with a concentration of 8%; Step 2, place suspension A at 8000× g Centrifuge for 10 minutes and collect the supernatant for later use; Step 3, enzymatic hydrolysis: First, adjust the substrate concentration in the supernatant to 6%, add 2% papain, and hydrolyze for 90 min at 45℃ and pH 6.5; then raise the temperature to 50℃, add 1% alkaline protease, adjust the pH to 9, and hydrolyze for 60 min. After the enzymatic hydrolysis is completed, inactivate the enzyme, and obtain the hydrolysate at 8000 rpm for 15 min. Step 4, Decolorization and Debittering: Adjust the pH of the enzymatic hydrolysate to 4.0-5.0, add 2% food-grade activated carbon powder, and stir at a constant temperature of 50-60℃ for 30 minutes to allow the activated carbon to fully adsorb pigments and bitter substances. After adsorption is complete, remove the activated carbon to obtain crude wheat peptide solution. Step 5, purification and concentration: filter using a 0.22μm-0.45μm microporous membrane to remove residual impurities, transfer the filtrate to a vacuum concentrator, and concentrate it to a solid content of 30% under the conditions of 50-60℃ and a vacuum degree of -0.08MPa to -0.09MPa to obtain a concentrated solution; Step 6, spray drying: Spray drying of the concentrate yields wheat peptide powder.

[0028] Comparative Example 2 A method for preparing wheat peptides that enhance immunity includes the following steps: Step 1, Pretreatment: Add wheat gluten powder to deionized water and stir vigorously for 20-30 minutes using a high-speed disperser to fully hydrate and disperse it, forming a uniform suspension A with a concentration of 5%; Step 2, high-pressure homogenization: Transfer suspension A into a high-pressure homogenizer, set the pressure to 120 MPa, the time to 5 min, and turn on the cooling system to obtain suspension B; Step 3, heat suspension B at 8000× g Centrifuge for 10 minutes and collect the supernatant for later use; Step 4, enzymatic hydrolysis: First, adjust the substrate concentration in the supernatant to 6%, add 2% papain, and hydrolyze for 90 min at 45℃ and pH 6.5; then raise the temperature to 50℃, add 1% alkaline protease, adjust the pH to 9, and hydrolyze for 60 min. After the enzymatic hydrolysis is completed, inactivate the enzyme, and obtain the hydrolysate at 8000 rpm for 15 min. Step 5, decolorization and debittering: Adjust the pH of the enzymatic hydrolysate to 4.0-5.0, add 2% food-grade activated carbon powder, stir at 50-60℃ for 30 minutes to allow the activated carbon to fully adsorb pigments and bitter substances. After adsorption is complete, remove the activated carbon to obtain crude wheat peptide solution. Step 6, purification and concentration: Filter using a 0.22μm-0.45μm microporous membrane to remove residual impurities, transfer the filtrate to a vacuum concentrator, and concentrate it to a solid content of 30% under the conditions of 50-60℃ and a vacuum degree of -0.08MPa to -0.09MPa to obtain a concentrated solution; Step 7, spray drying: Spray drying of the concentrate yields wheat peptide powder.

[0029] Effect test The peptide yield of wheat peptides prepared in Examples 1-3 and Comparative Examples 1-2 was determined. The peptide yield was calculated using the following formula: Peptide yield (%) = (Peptide content in the supernatant of the enzymatic hydrolysate / Total protein content in the raw material) × 100%. The results are shown in Table 1. Table 1 Peptide yield

[0030] As shown in Table 1, the peptide yields of wheat peptides prepared in Examples 1-3 were all above 80%, with a maximum of 86%. In contrast, the peptide yields of wheat peptides prepared in Comparative Examples 1-2 were all below 70%, significantly lower than those in Examples 1-3. Comparative Example 1 did not undergo high hydrostatic depolymerization treatment, resulting in a compact protein molecular structure and lower efficiency during enzymatic hydrolysis, leading to a low peptide yield. Comparative Example 2 underwent high-pressure homogenization, which opened up the protein molecular structure. However, the high-pressure homogenization process significantly increased the temperature, causing localized overheating, protein denaturation, and inactivation, thus affecting the peptide yield.

[0031] Immunity enhancement assay: 2g of wheat peptide was dissolved in 10mL of distilled water to prepare a wheat peptide solution. The experimental subjects were inbred female mice, weighing 18-22g, with 10 mice in each group, divided into 4 groups. The grouping and drug administration are shown in Table 2.

[0032] Table 2 Grouping and Dosing Information

[0033] Thirty days later, mice in each group underwent an experiment on the phagocytosis of chicken red blood cells by mouse peritoneal macrophages.

[0034] Experimental steps: ① Activation of mouse macrophages: Four days before the experiment, each mouse was injected intraperitoneally with 0.2 mL of 2% hematocrit sheep red blood cells. Mice were euthanized by cervical dislocation, and each mouse was injected intraperitoneally with 4 mL of Hank's solution containing fetal bovine serum. The abdomen was gently massaged 20 times to thoroughly wash out peritoneal macrophages. Then, a small incision was made in the abdominal wall, and 2 mL of peritoneal lavage fluid was drawn into a test tube using a pipette.

[0035] ② Using a 1mL pipette, add 0.5mL of peritoneal lavage fluid to a test tube containing 0.5mL of 1% chicken blood erythrocyte suspension and mix well. Using a syringe (with a large needle), add 0.5mL of the mixture to an agar ring on a glass slide. Incubate at 37℃ for 15-20 minutes. After incubation, quickly rinse away any non-adherent cells with physiological saline, fix in methanol for 1 minute, and stain with Giemsa solution for 15 minutes. Rinse thoroughly with distilled water, air dry, and count the phagocytic rate and phagocytic index using a 40× microscope. The phagocytic rate is the percentage of macrophages that phagocytose chicken erythrocytes per 100 macrophages; the phagocytic index is the average number of chicken erythrocytes phagocytosed by each macrophage. The experimental results are shown in Table 3.

[0036] Table 3. Phagocytosis rate and phagocytosis index (mean)

[0037] As shown in Table 3, compared with the blank control group, the phagocytic rate and phagocytic index of mice in Examples 1-3 were significantly increased, indicating that the phagocytic capacity of the phagocytes in the mice in Examples 1-3 was strong and their immunity was significantly improved. Therefore, the wheat peptides prepared in Examples 1-3 can improve the immunity of mice, thus demonstrating that wheat peptides can be used to prepare related products that enhance immunity.

[0038] Therefore, the present invention adopts the above-mentioned method for preparing wheat peptides that enhance immunity. Through high hydrostatic pressure depolymerization treatment at room temperature, the structure of the protein molecules is opened up and more enzyme cleavage sites are exposed without affecting the protein activity, thereby greatly improving the efficiency of subsequent enzymatic hydrolysis and increasing the yield of wheat peptides.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing wheat peptides that enhance immunity, characterized in that, Includes the following steps: Step 1, Pretreatment: Add wheat gluten powder to deionized water and stir vigorously for 20-30 minutes using a high-speed disperser to fully hydrate and disperse it, forming a uniform suspension A; Step 2, high hydrostatic pressure depolymerization treatment: Place suspension A in a polyethylene bag and immerse it in an ultra-high pressure liquid medium. Apply hydrostatic pressure at room temperature and then release the pressure instantly to 0 MPa to cause the protein molecules to undergo an oscillating depolymerization effect, thus obtaining suspension B. Step 3, heat suspension B at 8000× g Centrifuge for 10 minutes and collect the supernatant for later use; Step 4, enzymatic hydrolysis: The supernatant from step 3 was enzymatically hydrolyzed using papain and alkaline protease. After the enzymatic hydrolysis was completed, the enzymes were inactivated. The hydrolysate was obtained at 8000 rpm for 15 min. Step 5, decolorization and debittering: Adjust the pH of the enzymatic hydrolysate to 4.0-5.0, add food-grade activated carbon powder, stir at a constant temperature to allow the activated carbon to fully adsorb pigments and bitter substances, remove the activated carbon after adsorption is complete to obtain crude wheat peptide solution; Step 6, purification and concentration: use a microporous membrane to filter and remove residual impurities. Transfer the filtrate to a vacuum concentrator and concentrate it to a solid content of 25%-40% to obtain a concentrated solution. Step 7, spray drying: Spray drying of the concentrate yields wheat peptide powder.

2. The method for preparing an immune-enhancing wheat peptide according to claim 1, characterized in that: In step 1, the concentration of suspension A is 5-8%.

3. The method for preparing an immune-enhancing wheat peptide according to claim 1, characterized in that: In step 2, the static pressure control is 120-150 MPa, and the processing time is 5 minutes.

4. The method for preparing an immune-enhancing wheat peptide according to claim 1, characterized in that, In step 4, the specific enzymatic hydrolysis operations are as follows: ① Adjust the substrate concentration to 6%, add 2% papain, and hydrolyze for 90 minutes at 45℃ and pH 6.5; ② Raise the temperature to 50-55℃, add 1% alkaline protease, adjust the pH to 9, and hydrolyze for 60 minutes.

5. The method for preparing an immune-enhancing wheat peptide according to claim 1, characterized in that: In step 5, the amount of food-grade activated carbon powder added is 1%-3%, the temperature is 50-60℃, and the time is 30-45 minutes.

6. The method for preparing an immune-enhancing wheat peptide according to claim 1, characterized in that: In step 6, the pore size of the microporous filter membrane is 0.22μm-0.45μm, the temperature of the vacuum concentrator is set to 50-60℃, and the vacuum degree is -0.08MPa to -0.09MPa.

7. The method for preparing an immune-enhancing wheat peptide according to claim 1, characterized in that: In step 7, the spray drying conditions are: inlet temperature 160-180℃, outlet temperature 80-90℃, and atomizer speed 15000-30000 rpm.

8. A wheat peptide that enhances immunity, characterized in that: It is prepared by the preparation method according to any one of claims 1-7.

9. The use of the immune-enhancing wheat peptide according to claim 8 in the preparation of immune-enhancing products.

10. The application according to claim 9, characterized in that: The products include food and pharmaceuticals.

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