High-activity oat peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioactive components, and discloses a high-activity oat peptide as well as a preparation method and application thereof. The preparation method comprises the following steps: crushing the oat bran, adding water, carrying out primary enzymolysis with cellulase and xylanase, treating with L-cysteine, and carrying out ultrasonic extraction to obtain a crude extract; carrying out ammonium sulfate salting-out on the crude extract to obtain a reconstitution fluid, and treating with a composite adsorbent to obtain an enriched fluid; adding compound protease into the enriched liquid for secondary enzymolysis (containing a protective agent and inert gas) to obtain enzymatic hydrolysate; performing microfiltration and ultrafiltration on the enzymatic hydrolysate, adding glycine and glutamine transaminase for third enzymolysis, and concentrating and drying to obtain a finished product. The prepared oat peptide is high in protein dissolution rate and active peptide retention rate, excellent in ACE inhibitory activity and alpha-glucosidase inhibitory activity and good in storage stability, and can be used for preparing products for reducing blood pressure and blood sugar.

Description

Technical Field

[0001] This invention belongs to the field of bioactive ingredient technology, specifically relating to a highly active oat peptide, its preparation method, and its application. Background Technology

[0002] Oats, belonging to the genus *Avena* of the tribe Apiaceae in the family Poaceae, are annual or perennial herbaceous plants. Oats are rich in nutrients, with higher levels of protein, fat, vitamin E, dietary fiber, and minerals such as calcium, magnesium, iron, and phosphorus than other cereal crops. They possess various bioactive functions, including lowering blood lipids, cholesterol, blood pressure, and blood sugar, as well as antioxidant properties.

[0003] Oat peptides refer to small-molecule bioactive peptides extracted from oats. Oat peptides are rich in nutrients such as amino acids, minerals, vitamins, and proteins, and have effects such as moisturizing, promoting digestion, anti-aging, regulating blood sugar, and lowering cholesterol. They are widely used in health products, skincare products, pharmaceuticals, and functional foods.

[0004] Chinese invention patent application CN119020443A discloses a method for separating and purifying oat ACE inhibitory peptides prepared by enzymatic hydrolysis, comprising: preparing a mixture of oat bran and water; heating the mixture and adding amylase and cellulase under rapid flow for a first enzymatic hydrolysis; sieving the hydrolysate after the first hydrolysis, separating the residue and liquid, mixing the filter residue with water, adjusting the pH and temperature of the solution, and adding alkaline protease under rapid flow for a second enzymatic hydrolysis; sieving the hydrolysate after the second hydrolysis, separating the residue and liquid, and filtering the filtrate through a ceramic membrane for clarification; passing the filtrate filtered through the ceramic membrane through two nanofiltration membranes until the retentate concentration reaches 30%; and sterilizing the retentate by cooking and spray drying to obtain oat peptide powder. The oat protein dissolution rate in this invention is low (<80%), and the active peptides are easily oxidized and degraded during the enzymatic hydrolysis process.

[0005] Chinese invention patent application CN101709321A discloses an oat polypeptide and its preparation method and application. The preparation method of the oat polypeptide includes the following steps: 1) mixing oat bran with water, adjusting the pH to 8.5-9.5, and performing ultrasonic pretreatment at 50-60℃; 2) adding alkaline protease to the pretreated liquid and stirring at 50-60℃ for 2-5 hours; then adding flavor protease and stirring at 50-60℃ for 1-2 hours; 3) centrifuging the enzymatically hydrolyzed liquid to obtain the supernatant, adding β-glucanase, medium-temperature amylase, and saccharifying enzyme to the supernatant, reacting for 2-4 hours, and filtering to obtain a clear liquid; purifying the clear liquid using macroporous adsorption resin to obtain a solution containing oat polypeptides. The pretreatment step in this invention often uses chemical reagents, resulting in a high protein denaturation rate (>15%) and a risk of chemical residues.

[0006] Chinese invention patent application CN119530329A discloses a sea buckthorn oat peptide and its preparation and application. This invention is the first to isolate and purify three active peptides from oats, and based on these active peptides, combines broken-cell wall oat powder, sea buckthorn powder, yam powder, and three prebiotics to obtain sea buckthorn oat peptides. The active peptides prepared by this invention exhibit poor synergy with the food-medicine homologous components and do not form a targeted synergistic mechanism.

[0007] Therefore, there is an urgent need to develop a low-damage, high-retention, green and environmentally friendly oat peptide extraction process to solve the problems of activity loss and pollution in existing technologies. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a method for preparing highly active oat peptides and the resulting product.

[0009] To achieve the above-mentioned objectives of this invention, the specific technical solution adopted by this invention is as follows: A method for preparing highly active oat peptides includes the following steps: (1) Crush oat bran, add water, add cellulase and xylanase for the first enzymatic hydrolysis, then add L-cysteine, and extract by ultrasonication to obtain crude oat protein extract; (2) Add ammonium sulfate to the crude oat protein extract, let stand, centrifuge, collect the precipitate, add water, and obtain protein complex solution; (3) Add composite adsorbent to protein complex solution for adsorption treatment, stir, centrifuge, collect supernatant to obtain protein enrichment solution; (4) Add a complex protease to the protein enrichment solution for a second enzymatic hydrolysis, while adding a protective agent and passing in an inert gas to obtain the hydrolysate; (5) The enzymatic hydrolysate is subjected to microfiltration and ultrafiltration in sequence. The ultrafiltration permeate is collected, and glycine and glutamine transaminase are added for a third enzymatic hydrolysis. The solution is then concentrated and dried to obtain the highly active oat peptide.

[0010] Preferably, in step (1), the protein content of the oat bran is ≥18%, and it is pulverized to 80-100 mesh. The amount of water added is 5-7 times the mass of the oat bran. The amounts of cellulase and xylanase added are 0.4%-0.8% and 0.2%-0.5% of the mass of the oat bran, respectively. The pH of the first enzymatic hydrolysis is 5.5-6.0, the temperature of the first enzymatic hydrolysis is 50-55℃, and the time of the first enzymatic hydrolysis is 60-90 min.

[0011] Preferably, in step (1), the amount of L-cysteine ​​added is 0.3%-0.6% of the weight of oat bran. After adding L-cysteine, the pH is adjusted to 8.0-8.5. The ultrasonic power is 300-350W, the ultrasonic frequency is 30-40kHz, and the ultrasonic time is 15-25min.

[0012] Preferably, the final concentration of ammonium sulfate in step (2) is 2.0-2.5 mol / L, the standing time is 7-9 h, the centrifugation speed is 5000-7000 r / min, the centrifugation time is 15-25 min, and the amount of water added is 30%-40% of the volume of the crude oat protein extract.

[0013] Preferably, the composite adsorbent in step (3) is a mixture of chitosan and activated carbon, wherein the mass ratio of chitosan to activated carbon is 3:2; the amount of composite adsorbent added is 0.8-1.2% of the mass of the protein complex solution; the pH of the adsorption treatment is 6.0-6.5; the temperature is 30-35℃; the stirring speed is 70-90 r / min; the stirring time is 50-70 min; the centrifugation speed is 4000-6000 r / min; and the centrifugation time is 10-20 min.

[0014] Preferably, the complex protease in step (4) is composed of alkaline protease, flavor protease and bromelain, the mass ratio of alkaline protease, flavor protease and bromelain is 2-3:1-2:1, the amount of complex protease is 2.5-3.5% of the protein mass, the temperature of the second enzymatic hydrolysis is 50-55℃, and the time of the second enzymatic hydrolysis is 2-3h.

[0015] Preferably, the protective agent in step (4) is tea polyphenols and vitamin E, and the amount added is 0.5-0.9% and 0.3-0.6% of the protein content, respectively, and the inert gas is nitrogen or argon.

[0016] Preferably, in step (5), the microfiltration uses a 0.1-0.3 μm ceramic membrane and the microfiltration operating pressure is 0.3-0.5 MPa; the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 2-3 kDa and the ultrafiltration operating pressure is 0.6-0.8 MPa; the amount of glycine and glutamine transaminase added is 0.6%-1.0% and 0.2%-0.4% of the permeate mass, respectively; the pH of the third enzymatic hydrolysis is 6.2-6.8; the temperature of the third enzymatic hydrolysis is 37-41℃; the time of the third enzymatic hydrolysis is 25-35 min; and the drying is freeze drying.

[0017] This invention also relates to highly active oat peptides prepared by the above-described method.

[0018] This invention also relates to the application of the highly active oat peptides prepared by the above-mentioned method in the preparation of products for lowering blood pressure and blood sugar.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention adopts a synergistic technology of “compound enzymatic hydrolysis + L-cysteine ​​bond breaking + ultrasonic extraction”. Cellulase and xylanase break the dietary fiber network, L-cysteine ​​breaks the protein disulfide bonds, and ultrasonic mass transfer is enhanced, so that the protein dissolution rate is increased to more than 90% and the protein denaturation rate is ≤1.6%, which significantly reduces the activity loss during the raw material processing.

[0020] (2) This invention effectively prevents the oxidative inactivation of active peptides by synergistic enzymatic hydrolysis of alkaline protease, flavor protease and bromelain, combined with tea polyphenol-vitamin E complex protective agent system and inert gas protection; the specific enzyme combination and optimized process conditions enable the product to have both high ACE inhibition rate (≥89%) and high α-glucosidase inhibition rate (≥83%), and the active peptide retention rate reaches more than 85%.

[0021] (3) In this invention, the third enzymatic hydrolysis (glutamine transaminase + glycine modification) optimizes the peptide structure through cross-linking reaction, improves intestinal absorption efficiency and storage stability, and the activity retention rate is ≥90% after 6 months of storage at 4℃, which solves the problem of easy degradation of traditional oat peptides. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0023] Cellulase (biotechnology grade, 400 U / mg), xylanase (BR, 300,000 U / g), alkaline protease (BR, 200 U / mg), flavor protease (BR, 20 U / mg), bromelain (biotechnology grade, 600 U / mg), transglutaminase (BR, 200 U / g), and amylase (biotechnology grade, ~50 U / mg) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0024] Chitosan, purchased from Shandong Quanhang Marine Biotechnology Co., Ltd., specification: food grade; activated carbon, purchased from Hunan Changtai Biotechnology Co., Ltd., specification: fine powder.

[0025] Example 1 A green extraction process for highly active oat peptides, comprising the following steps: (1) Take oat bran (protein content 19.2%), grind it to 100 mesh, add 6 times the weight of deionized water, adjust the pH to 5.8, add 0.6% of the weight of oat bran cellulase and 0.35% of the weight of oat bran xylanase, stir at 52℃ for 75 min for enzymatic hydrolysis; add 0.45% of the weight of oat bran L-cysteine, adjust the pH to 8.2, stir at 48℃ for 30 min; use 320W, 35kHz ultrasonic extraction for 20 min, centrifuge at 6000r / min for 20 min, collect the supernatant to obtain crude oat protein extract (extraction rate 92.7%).

[0026] (2) Add ammonium sulfate to the crude extract to a final concentration of 2.2 mol / L, let stand at 23°C for 8 h, centrifuge at 6000 r / min for 20 min, collect the precipitate; add 35% of the volume of deionized water of the crude extract to reconstitute and obtain the protein reconstituted solution.

[0027] (3) Add 1.0% of the composite adsorbent (chitosan: activated carbon = 3:2) to the compound solution, adjust the pH to 6.2, stir at 32℃ and 80r / min for 60min, centrifuge at 5000r / min for 15min, collect the supernatant, and obtain an enriched solution with a protein purity of 91.8%.

[0028] (4) Adjust the pH of the enrichment solution to 7.8, add a complex protease (alkaline protease: flavor protease: bromelain = 3:2:1), the total amount of enzyme added is 3.0% of the protein in the enrichment solution, and add 0.7% of the protein in tea polyphenols and 0.45% of vitamin E; introduce argon gas (oxygen content ≤0.5%), incubate at 52℃ for 2.5h for enzymatic hydrolysis; heat to 90℃ and incubate for 15min for inactivation, cool down to obtain the enzymatic hydrolysate.

[0029] (5) The enzymatic hydrolysate was microfiltered through a 0.2 μm ceramic membrane (operating pressure 0.4 MPa), and the permeate was collected; the permeate was ultrafiltered through a 2.5 kDa ultrafiltration membrane (operating pressure 0.7 MPa), and the ultrafiltration permeate was collected; the pH of the permeate was adjusted to 6.5, and 0.8% glycine and 0.3% glutamine transaminase by mass of the permeate were added. The mixture was stirred at 39 °C for 30 min; the reaction was terminated by incubation at 85 °C for 10 min. The product was concentrated under vacuum at 0.08 MPa and 48°C to one-third of its original volume, and then freeze-dried at -40°C and 15 Pa to obtain the highly active oat peptide product.

[0030] Example 2 A green extraction process for highly active oat peptides, comprising the following steps: (1) Take oat bran (protein content 18.2%), grind it to 80 mesh, add 5 times the weight of deionized water, adjust the pH to 5.5, add 0.4% of oat bran weight of cellulase (enzyme activity 10000U / g) and 0.2% of oat bran weight of xylanase (enzyme activity 8000U / g), stir at 50℃ for 90 min for enzymatic hydrolysis; then add 0.3% of oat bran weight of L-cysteine, adjust the pH to 8.0, stir at 45℃ for 30 min; use 300W, 30kHz ultrasonic extraction for 25 min, centrifuge at 5000r / min for 15 min, collect the supernatant to obtain crude oat protein extract (extraction rate 90.1%).

[0031] (2) Add ammonium sulfate powder slowly to the crude extract while stirring until the final concentration of ammonium sulfate reaches 2.0 mol / L. Let it stand at 20℃ for 7 h, centrifuge at 5000 r / min for 25 min, and collect the precipitate. Add 30% of the volume of deionized water of the crude extract to reconstitute and obtain the protein reconstituted solution.

[0032] (3) Add 0.8% of the composite adsorbent (chitosan: activated carbon = 3:2) to the compound solution, adjust the pH to 6.0, stir at 30℃ and 70r / min for 70min, centrifuge at 4000r / min for 20min, collect the supernatant, and obtain an enriched solution with a protein purity of 90.3%.

[0033] (4) Adjust the pH of the enrichment solution to 7.5, add a complex protease (alkaline protease: flavor protease: bromelain = 2:1:1, total enzyme activity 40000U / g), the total amount of enzyme added is 2.5% of the protein in the enrichment solution, and at the same time add 0.5% of the protein in tea polyphenols and 0.3% of vitamin E; purge with nitrogen (oxygen content ≤0.5%), incubate at 50℃ for 3h for enzymatic hydrolysis, raise the temperature to 88℃ and incubate for 15min for inactivation, cool down, and obtain the enzymatic hydrolysate.

[0034] (5) The enzymatic hydrolysate was microfiltered through a 0.2 μm ceramic membrane (operating pressure 0.3 MPa), and the permeate was collected; the permeate was ultrafiltered through a 2 kDa ultrafiltration membrane (operating pressure 0.6 MPa), and the ultrafiltration permeate was collected; the pH of the permeate was adjusted to 6.2, and 0.6% glycine and 0.2% glutamine transaminase (enzyme activity 100 U / g) by mass of the permeate were added. The mixture was stirred at 37°C for 35 min, and then the reaction was terminated by incubation at 85°C for 10 min. The product was concentrated under vacuum at 0.07 MPa and 45°C to 1 / 4 of its original volume, and then freeze-dried at -40°C and 10 Pa to obtain the highly active oat peptide product.

[0035] Example 3 A green extraction process for highly active oat peptides, comprising the following steps: (1) Take oat bran (protein content 20.1%), grind it to 100 mesh, add 7 times the weight of deionized water, adjust the pH to 6.0, add 0.8% of the weight of oat bran cellulase and 0.5% of the weight of oat bran xylanase, stir at 55℃ for 60 min for enzymatic hydrolysis; add 0.6% of the weight of oat bran L-cysteine, adjust the pH to 8.5, stir at 50℃ for 30 min; use 350W, 40kHz ultrasonic extraction for 15 min, centrifuge at 7000r / min for 15 min, collect the supernatant to obtain crude oat protein extract (extraction rate 93.5%).

[0036] (2) Add ammonium sulfate to the crude extract to a final concentration of 2.5 mol / L, let stand at 25°C for 9 h, centrifuge at 7000 r / min for 15 min, collect the precipitate; add 40% of the volume of deionized water of the crude extract to reconstitute and obtain the protein reconstituted solution.

[0037] (3) Add 1.2% of the composite adsorbent (chitosan: activated carbon = 3:2) to the compound solution, adjust the pH to 6.5, stir at 35℃ and 90r / min for 50min, centrifuge at 6000r / min for 10min, collect the supernatant, and obtain an enriched solution with a protein purity of 92.5%.

[0038] (4) Adjust the pH of the enrichment solution to 8.0, add a complex protease (alkaline protease: flavor protease: bromelain = 3:1:1), the total amount of enzyme added is 3.5% of the protein content in the enrichment solution, and add 0.9% of tea polyphenols and 0.6% of vitamin E of protein content at the same time; introduce nitrogen gas (oxygen content ≤0.5%), incubate at 55℃ for 2h for enzymatic hydrolysis; heat to 92℃ and incubate for 15min for inactivation, cool down, and obtain the enzymatic hydrolysate.

[0039] (5) The enzymatic hydrolysate was microfiltered through a 0.3 μm ceramic membrane (operating pressure 0.5 MPa), and the permeate was collected; the permeate was ultrafiltered through a 3 kDa ultrafiltration membrane (operating pressure 0.8 MPa), and the ultrafiltration permeate was collected; the pH of the permeate was adjusted to 6.8, and 1.0% glycine and 0.4% glutamine transaminase by mass of the permeate were added. The mixture was stirred at 41 °C for 25 min; the reaction was terminated by incubation at 85 °C for 10 min. The above product was concentrated under vacuum at 0.09 MPa and 50°C to 1 / 3 of its original volume, and then freeze-dried at -40°C and 20 Pa to obtain the high-activity oat peptide product.

[0040] Comparative Example 1 The only difference between this comparative example and Example 1 is that L-cysteine ​​was not added in step (1), while the other steps and parameters are completely consistent with those of Example 1.

[0041] The specific steps (1) are as follows: Take oat bran (protein content of 19.2%), grind it to 100 mesh, add 6 times the weight of oat bran deionized water, adjust the pH to 5.8, add 0.6% of oat bran weight of cellulase and 0.35% of oat bran weight of xylanase, stir at 52℃ for 75 min for enzymatic hydrolysis; directly adjust the pH to 8.2, stir at 48℃ for 30 min; use 320W, 35kHz ultrasonic extraction for 20 min, centrifuge at 6000r / min for 20 min, collect the supernatant, and obtain crude oat protein extract.

[0042] Comparative Example 2 The only difference between this comparative example and Example 1 is that in step (1), xylanase was replaced with amylase (enzyme activity 8000U / g). The remaining steps and parameters are completely consistent with those of Example 1.

[0043] The specific steps (1) are as follows: Take oat bran (protein content of 19.2%), grind it to 100 mesh, add 6 times the weight of deionized water, adjust the pH to 5.8, add 0.6% of cellulase and 0.35% of amylase by weight of oat bran, stir at 52℃ for 75 min for enzymatic hydrolysis; add 0.45% of L-cysteine ​​by weight of oat bran, adjust the pH to 8.2, stir at 48℃ for 30 min; use 320W, 35kHz ultrasonic extraction for 20 min, centrifuge at 6000r / min for 20 min, collect the supernatant to obtain crude oat protein extract.

[0044] Comparative Example 3 The only difference between this comparative example and Example 1 is that ammonium sulfate is replaced with ethanol (final concentration 40%, v / v) in step (2). The remaining steps and parameters are completely consistent with those of Example 1.

[0045] The specific steps (2) are as follows: slowly add anhydrous ethanol to the crude extract while stirring, so that the final concentration of ethanol reaches 40% (v / v), stand at 23℃ for 8 hours, centrifuge at 6000r / min for 20 minutes, and collect the precipitate; add 35% of the volume of deionized water of the crude extract to reconstitute and obtain the protein reconstituted solution.

[0046] Comparative Example 4 The only difference between this comparative example and Example 1 is that in step (3), only chitosan was added as an adsorbent (addition amount 1.0%), and activated carbon was not added. The remaining steps and parameters are completely consistent with Example 1.

[0047] Comparative Example 5 The only difference between this comparative example and Example 1 is that in step (4), only alkaline protease (3.0% enzyme amount) was added, and flavor protease and bromelain were not added. The remaining steps and parameters are completely consistent with Example 1.

[0048] Comparative Example 6 The only difference between this comparative example and Example 1 is that the complex protease in step (4) is: flavor protease: bromelain = 2:1 (total enzyme addition 3.0%), without alkaline protease. The remaining steps and parameters are completely consistent with Example 1.

[0049] Comparative Example 7 The only difference between this comparative example and Example 1 is that in step (4), the complex protease is: alkaline protease: flavor protease = 3:2 (total enzyme amount 3.0%), and does not contain bromelain. The remaining steps and parameters are completely consistent with Example 1.

[0050] Comparative Example 8 The only difference between this comparative example and Example 1 is that the complex protease in step (4) is: alkaline protease: bromelain = 3:1 (total enzyme amount 3.0%), without flavor protease. The remaining steps and parameters are completely consistent with Example 1.

[0051] Comparative Example 9 The only difference between this comparative example and Example 1 is that in step (5), after ultrafiltration, the sample is directly concentrated and dried without adding glycine and glutamine transaminase for the reaction. The remaining steps and parameters are completely consistent with those of Example 1.

[0052] Effect test 1. Determination of protein dissolution rate: Referring to the Kjeldahl method in GB 5009.5-2016 National Food Safety Standard for Determination of Protein in Food, the total protein content of the raw material and the protein content of the crude extract were determined, and the dissolution rate was calculated. Protein dissolution rate = (protein content in crude extract / total protein content of raw material) × 100%.

[0053] 2. Determination of protein purity: High-performance liquid chromatography (HPLC) was used for detection. A C18 column (4.6 mm × 250 mm, 5 μm) was used. The mobile phase was acetonitrile-0.1% trifluoroacetic acid aqueous solution (gradient elution: 0-10 min, acetonitrile 10%-20%; 10-30 min, acetonitrile 20%-40%; 30-40 min, acetonitrile 40%-80%). The flow rate was 1.0 mL / min, the column temperature was 30℃, and the detection wavelength was 220 nm. Protein purity was calculated using a bovine serum albumin standard curve.

[0054] 3. Determination of protein denaturation rate: The change in thermal stability was measured using differential scanning calorimetry (DSC). The instrument used was a TA Q2000 differential scanning calorimeter, with a scanning range of 20-120℃, a heating rate of 10℃ / min, and a nitrogen atmosphere (flow rate 50mL / min). The enthalpy value (ΔH) of the sample denaturation endothermic peak was recorded. 样品 ), and the denaturation enthalpy (ΔH) of natural oat protein standards. 天然Protein denaturation rate (%) = [1 - (ΔH)] 样品 / ΔH 天然 )]×100%.

[0055] 4. Determination of active peptide retention rate: Trichloroacetic acid (TCA) precipitation combined with the Folin-phenol method was used. The enzymatic hydrolysate and the final product solution were mixed with an equal volume of 15% TCA solution, centrifuged, and the supernatant was collected. The absorbance was measured at 750 nm using Folin-phenol reagent for color development. The peptide content was calculated using a tyrosine standard curve. Active peptide retention rate = (peptide content in the finished product / peptide content in the enzymatic hydrolysate) × 100%.

[0056] 5. Assay for ACE inhibition activity: Using hippuric acid (HHL) as the substrate, the reaction system consisted of 80 μL of 5 mmol / L HHL solution (pH 8.3, 0.05 mol / L Tris-HCl buffer), 40 μL of sample solution (1.0 mg / mL), and 10 μL of 0.1 U / mL ACE solution. The mixture was incubated at 37°C for 30 min. The reaction was terminated by adding 200 μL of 1 mol / L HCl. Hippuric acid was extracted with 1.2 mL of ethyl acetate, centrifuged at 1000 r / min for 10 min, and the organic phase was dried at 80°C. The phase was reconstituted with 2 mL of deionized water, and the absorbance was measured at 228 nm. ACE inhibition rate (%) = [1 - (ODa - ODc) / (ODb - ODc)] × 100%, where ODa is the absorbance of sample + ACE + substrate, ODb is the absorbance of blank (buffer solution + ACE + substrate), and ODc is the absorbance of control (sample + buffer solution + substrate).

[0057] 6. Determination of α-glucosidase inhibition rate: Using p-nitrobenzene-α-D-glucopyranoside (pNPG) as the substrate, the reaction system included 50 μL of 0.1 U / mL α-glucosidase solution, 50 μL of sample solution (1.0 mg / mL), and 50 μL of 5 mmol / L pNPG solution (pH 6.8, 0.05 mol / L phosphate buffer). The reaction was carried out in a water bath at 37℃ for 20 min. The reaction was terminated by adding 100 μL of 0.5 mol / L Na2CO3 solution, and the absorbance was measured at 405 nm.

[0058] α-glucosidase inhibition rate (%) = [1-(OD 样品 -OD 样品空白 ) / (OD 对照 -OD 对照空白 )]×100%, where OD 样品空白 Sample + buffer + substrate (enzyme-free), OD 对照空白 The solution consists of buffer, enzyme, and substrate (without sample).

[0059] The test results are shown in Table 1.

[0060]

[0061] 7. Storage stability test: The finished product was sealed and stored at 4℃ for 6 months. The ACE inhibition rate after storage was measured, and the activity retention rate was calculated as (inhibition rate after storage / initial inhibition rate) × 100%. The test results are shown in Table 2.

[0062]

[0063] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing high activity oat peptides, characterized by, The method comprises the following steps: (1) crushing oat bran, adding water, adding cellulase and xylanase for first enzymolysis, then adding L-cysteine, ultrasonic extraction, to obtain oat protein crude extract; (2) adding ammonium sulfate to the oat protein crude extract, standing, centrifugation, collecting the precipitate, adding water, to obtain a protein resuspension solution; (3) adding a composite adsorbent to the protein resuspension solution for adsorption treatment, stirring, centrifugation, collecting the supernatant, to obtain a protein enrichment solution; (4) adding a complex protease to the protein enrichment solution for second enzymolysis, adding a protective agent and introducing an inert gas at the same time, to obtain an enzymolysis solution; (5) the enzymolysis solution is sequentially subjected to microfiltration and ultrafiltration, the ultrafiltration permeate is collected, glycine and transglutaminase are added for third enzymolysis, concentration, drying, to obtain the high-activity oat peptide.

2. The production method according to claim 1, characterized by, In step (1), the protein mass percentage of the oat bran is ≥18%, the oat bran is crushed to 80-100 mesh, the water is added in an amount of 5-7 times the mass of the oat bran, the cellulase and the xylanase are added in amounts of 0.4%-0.8% and 0.2%-0.5% of the mass of the oat bran respectively, the pH of the first enzymolysis is 5.5-6.0, the temperature of the first enzymolysis is 50-55℃, and the time of the first enzymolysis is 60-90 min.

3. The preparation method according to claim 1, characterized in that, In step (1), the L-cysteine is added in an amount of 0.3%-0.6% of the mass of the oat bran, the pH is adjusted to 8.0-8.5 after the L-cysteine is added, the power of the ultrasonic is 300-350 W, the frequency of the ultrasonic is 30-40 kHz, and the time of the ultrasonic is 15-25 min.

4. The method of claim 1, wherein, In step (2), the final concentration of the ammonium sulfate is 2.0-2.5 mol / L, the standing time is 7-9 h, the centrifugation speed is 5000-7000 r / min, the centrifugation time is 15-25 min, and the water is added in an amount of 30%-40% of the volume of the oat protein crude extract.

5. The preparation method according to claim 1, characterized in that, In step (3), the composite adsorbent is a mixture of chitosan and activated carbon, the mass ratio of the chitosan to the activated carbon is 3:2, the composite adsorbent is added in an amount of 0.8-1.2% of the mass of the protein resuspension solution, the pH of the adsorption treatment is 6.0-6.5, the temperature is 30-35℃, the stirring speed is 70-90 r / min, the stirring time is 50-70 min, the centrifugation speed is 4000-6000 r / min, and the centrifugation time is 10-20 min.

6. The method of claim 1, wherein, In step (4), the complex protease is composed of alkaline protease, flavor protease and bromelain, the mass ratio of the alkaline protease, the flavor protease and the bromelain is 2-3:1-2:1, the amount of the complex protease is 2.5-3.5% of the mass of the protein, the temperature of the second enzymolysis is 50-55℃, and the time of the second enzymolysis is 2-3 h.

7. The preparation method according to claim 1, characterized in that, In step (4), the protective agent is tea polyphenol and vitamin E, which are added in amounts of 0.5-0.9% and 0.3-0.6% of the mass of the protein respectively, and the inert gas is nitrogen or argon.

8. The method of claim 1, wherein, The microfiltration in step (5) uses a 0.1-0.3 μm ceramic membrane, and the operation pressure of the microfiltration is 0.3-0.5 MPa; the ultrafiltration uses an ultrafiltration membrane with a molecular weight cut-off of 2-3 kDa, and the operation pressure of the ultrafiltration is 0.6-0.8 MPa; the addition amount of the glycine and glutamine transaminase is 0.6%-1.0% and 0.2%-0.4% of the permeate, respectively; the pH of the third enzymatic hydrolysis is 6.2-6.8, the temperature of the third enzymatic hydrolysis is 37-41 °C, the time of the third enzymatic hydrolysis is 25-35 min, and the drying is freeze drying.

9. A high-activity oat peptide prepared by the preparation method of any one of claims 1-8.

10. Use of a high-activity oat peptide prepared by the preparation method of any one of claims 1-8 in the preparation of a blood pressure-lowering and blood sugar-lowering product.

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