Oil active peptide and its immobilized enzymatic preparation method

Abstract

The application discloses oil active peptides and an immobilized enzymatic preparation method thereof, and belongs to the technical field of biological catalysis and functional food. The oil active peptides disclosed by the application are antioxidant peptides or ACE inhibiting peptides; the amino acid sequences of the antioxidant peptides are ANPPHFIHL and GQTPLFPR respectively; and the amino acid sequences of the ACE inhibiting peptides are RMPVPIFF and KGWLLTL respectively. The antioxidant peptides have good scavenging capacity on ABTS, hydroxyl radicals and Fe 2+ The inhibiting rate of the ACE inhibiting peptides can be more than 90%, which provides a material basis for developing functional food and health products with definite activity.

Description

Technical Field

[0001] This invention relates to the fields of biocatalysis and functional food technology, and more specifically to active peptides from oilseeds and their immobilized enzymatic hydrolysis preparation methods. Background Technology

[0002] Oilseed proteins (such as soybean protein, peanut protein, sunflower protein, walnut protein, and flaxseed protein) are important sources of plant protein, boasting advantages such as high nutritional value and wide availability. The preparation of bioactive peptides from oilseed proteins using enzymatic hydrolysis has become a research hotspot in the functional food field. Walnut protein, in particular, has a protein content exceeding 40% in walnut meal, a byproduct of oil extraction. Rich in bioactive amino acids such as glutamic acid and arginine, it is a high-quality raw material for developing bioactive peptides with antioxidant, blood pressure-lowering, Alzheimer's disease prevention, and metabolic disorder regulation properties.

[0003] Currently, the preparation of bioactive peptides from oilseed proteins primarily employs the direct hydrolysis of free proteases. However, traditional free proteases are prone to inactivation in the reaction system, difficult to recover and reuse, and suffer from poor controllability during the hydrolysis process. This results in high enzyme usage costs, uneven product molecular weight distribution, and unstable bioactive peptide yields, severely limiting the efficiency and economics of their industrial application. Furthermore, the structures and compositions of different oilseed proteins vary significantly, and existing single-protein hydrolysis strategies often lack adaptability comparisons for different oilseed proteins, making it difficult to screen for the most promising protein raw materials. Enzyme immobilization technology is an effective strategy to improve enzyme stability and achieve recycling. Immobilizing proteases on solid supports facilitates separation and recovery, enhances operational stability, and allows for better control of the hydrolysis process. In recent years, two-dimensional nanomaterials have shown broad application prospects in the field of enzyme immobilization due to their high specific surface area and modifiability. However, existing immobilization supports generally suffer from weak binding forces and poor mass transfer efficiency.

[0004] Therefore, providing active peptides from oilseeds and their immobilized enzymatic hydrolysis preparation methods is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides an active peptide from oilseeds and a method for its immobilized enzymatic hydrolysis preparation. The magnetic carbon nitride immobilized alkaline protease has good biocompatibility, strong binding force and is easy to separate. Furthermore, a controllable enzymatic hydrolysis method suitable for various oilseed proteins is established to achieve efficient preparation and targeted screening of active peptides.

[0006] This invention overcomes the shortcomings of existing free proteases, such as poor stability, difficulty in recovery, low controllability of the hydrolysis process, and insufficient research on the adaptability of different oilseed proteins. It provides a method for preparing carbon nitride immobilized proteases, a method for preparing active peptides by hydrolyzing oilseed proteins using the immobilized enzyme, and novel antioxidant peptides and ACE inhibitory peptides isolated and identified from walnut protein hydrolysates.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A magnetic carbon nitride immobilized alkaline protease, wherein the immobilized enzyme is prepared by immobilizing the protease using aminated carbon nitride as a carrier via glutaraldehyde cross-linking; wherein the carbon nitride is non-magnetic carbon nitride or magnetic carbon nitride modified by iron salt coprecipitation to acquire magnetic properties.

[0009] Furthermore, a method for preparing magnetic carbon nitride immobilized alkaline protease involves using magnetic carbon nitride nanosheets as a carrier. The carrier surface is functionalized by introducing amino functional groups, and then the protease is cross-linked onto the carrier surface. This facilitates efficient contact between oilseed proteins and the active sites of the protease, thereby achieving efficient preparation of active peptides. Specific steps include: (1) Preparation of magnetic carbon nitride First, oxidation treatment: Weigh 0.5-2 g of graphitic carbon nitride (g-C3N4) and disperse it in 50-100 mL of nitric acid solution (6-12 mol / L). Stir at 60-70℃ for 2-4 h. After completion, adjust the pH to neutral with NaOH solution and wash several times with deionized water to obtain carbon nitride oxide. Next, magnetic modification: Sonicate the above carbon nitride oxide in 20-60 mL of water, and add 10 mL of a mixture containing 350-450 mg of ferric salt and 100-200 mg of ferrous salt dropwise, continuing sonication. Subsequently, add 10-20% ammonia solution to the mixture to adjust the pH to 9.0-12.0, and stir at 70℃ for 1-3 h to allow the magnetic particles to fully precipitate. Finally, washing and drying: Wash the precipitated particles repeatedly with deionized water until neutral, and dry overnight in an oven at 50-70℃ to obtain magnetic carbon nitride (MCN).

[0010] (2) Surface functionalization of magnetic carbon nitride 0.1-1 g of the MCN prepared in step (1) was dispersed in 100-200 mL of anhydrous ethanol containing 2-6% silane coupling agent, and the pH was adjusted to 3.0-5.0 with acetic acid. The mixture was stirred at 60-80 °C for 6-10 h, and then washed with anhydrous ethanol to obtain amino-modified MCN-NH2. The above MCN-NH2 was ultrasonically dispersed in 50-100 mL of phosphate buffer solution, a crosslinking agent was added, and the mixture was stirred at room temperature for 1-4 h. The precipitate was then washed several times with deionized water and dried to obtain surface-activated MCN-NH2.

[0011] (3) Magnetic carbon nitride immobilized protease Alkaline protease solutions with a concentration of 0.6–3 mg / mL were prepared using sodium phosphate buffer (0.05–0.1 mol / L, pH 6.0–10.0). The MCN-NH2 carrier prepared in step (2) was then ultrasonically dispersed in the alkaline protease solution at a mass-to-volume ratio of 1:(250–500) (g:mL), and incubated at 20–60 °C for 1–5 h. Subsequently, the mixture was centrifuged to collect the precipitate, lyophilized, and stored at low temperature.

[0012] Preferably, the iron salt in step (1) is FeCl3·6H2O; the ferrous salt is FeCl2·4H2O; the silane coupling agent in step (2) is 3-aminopropyltrimethoxysilane (APTMs); and the crosslinking agent is glutaraldehyde.

[0013] More preferably, the mass of FeCl3·6H2O is 380 mg; the mass of FeCl2·4H2O is 140 mg; the concentration of APTMs is 4%; and the concentration of glutaraldehyde is 4%.

[0014] Preferably, in step (3), the sodium phosphate buffer solution has a pH of 7.0, the alkaline protease concentration is 1.8 mg / mL, the immobilization temperature is 30°C, and the incubation time is 4 h.

[0015] More preferably, the protease is an alkaline protease derived from Bacillus subtilis (Xiasheng, Cangzhou, Hebei). The optimal temperature for the prepared immobilized alkaline protease is 60℃, which is higher than the 50℃ of the free enzyme; its thermal stability is significantly improved, and its half-life is 1.97 times that of the free enzyme; after being reused 5 times, it still retains 55.0% of its initial activity.

[0016] Furthermore, a method for preparing bioactive peptides by immobilizing alkaline protease to hydrolyze oilseed proteins using magnetic carbon nitride includes the following steps: Add 0.1–0.5 g of immobilized alkaline protease to 5–10 mL of 4%–10% oilseed protein solution, adjust the pH to 8.0–11.0 with sodium hydroxide solution, and enzymatically hydrolyze for 10–60 min at 40–60 °C. Then, recover the immobilized enzyme by magnetic separation. After centrifugation, take the supernatant of the oilseed protease hydrolysate and determine its biological activity.

[0017] Preferably, the oilseed protein is selected from at least one of soybean protein, peanut protein, sunflower protein, flaxseed protein, and walnut protein.

[0018] Preferably, the bioactivity is antioxidant activity (ABTS free radical scavenging rate) and ACE inhibitory activity; the ABTS free radical scavenging rate of the oilseed protein hydrolysate is 33.7%~86.5%, and the ACE inhibitory activity is 24.9%~88.6%.

[0019] More preferably, under the same enzymatic hydrolysis conditions, the antioxidant activity and ACE inhibitory activity of walnut protein hydrolysate are superior to those of other oilseed proteins, with an ABTS free radical scavenging rate of 76.25% and an ACE inhibitory activity of 88.60%.

[0020] More preferably, the molecular weight distribution of the prepared walnut protein hydrolysate is as follows: <1 kDa: 26.5%~85.3%, 1-3 kDa: 8.1%~32.2%, 3-5 kDa: 1.0%~12.3%, 5-10 kDa: 2.4%~12.7%, and above 10 kDa: 5.3%~39.5%.

[0021] Furthermore, a rapid isolation, purification, and activity screening method for bioactive peptides includes the following steps: (1) The above enzymatic hydrolysate was subjected to ultrafiltration to collect the components with a molecular weight <3 kDa; (2) The components were subjected to dextran gel G-15 chromatography, and the active elution peaks were collected; (3) Determine the antioxidant activity of each elution peak (ABTS free radical scavenging rate, hydroxyl free radical scavenging rate, Fe... 2+ Screening for highly active components based on chelation capacity or ACE inhibitory activity; (4) The highly active components were identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to obtain peptide sequences.

[0022] Preferably, the component with the highest antioxidant activity is identified as an antioxidant peptide with amino acid sequences ANPPHFIHL (SEQ ID NO.1) and GQTPLFPR (SEQ ID NO.2); the component with the highest ACE inhibitory activity is identified as an ACE inhibitory peptide with amino acid sequences RMPVPIFF (SEQ ID NO.3) and KGWLLTL (SEQ ID NO.4).

[0023] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses an active peptide from oilseeds and a method for its immobilized enzymatic hydrolysis preparation, which has the following beneficial effects: (1) This invention uses magnetic carbon nitride as a carrier and covalently immobilizes alkaline protease on the carrier surface through amylation modification and glutaraldehyde crosslinking, which significantly enhances enzyme stability and substrate accessibility and improves hydrolysis efficiency. The optimal temperature of the immobilized enzyme (60℃) is higher than that of the free enzyme (50℃), and the thermal stability is significantly improved (the half-life is 1.97 times that of the free enzyme). After being reused 5 times, it still retains 55.0% of the initial activity, which has good prospects for industrial application.

[0024] (2) The present invention uses iron salt coprecipitation to impart magnetic properties to carbon nitride, which facilitates the rapid separation and recovery of immobilized enzymes, overcomes the defects of poor controllability and difficulty in reusing free enzymes, reduces production costs, and improves process controllability.

[0025] (3) In this invention, immobilized alkaline protease was used to hydrolyze various oilseed proteins such as soybean, peanut, sunflower, flax, and walnut. The antioxidant activity and ACE inhibitory activity of different oilseed protein hydrolysates were systematically compared, and walnut protein was selected as the optimal raw material. Furthermore, through the synergistic regulation of two factors (pH and temperature), the molecular weight of walnut hydrolysate was controllable (the proportion of small molecule peptides of 100~3000 Da can reach more than 89.5%).

[0026] (4) This invention isolated and identified two novel antioxidant peptides (ANPPHFIHL and GQTPLFPR) and two ACE inhibitory peptides (RMPVPIFF and KGWLLTL) from walnut protein hydrolysate. In vitro activity verification showed that the antioxidant peptides inhibited ABTS, hydroxyl radicals, and Fe. 2+ All of them have good scavenging ability, and the inhibition rate of ACE inhibitory peptides can reach more than 90%, providing a material basis for the development of functional foods and health products with clear activity. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 The images are scanning electron microscope (SEM) images of CN, MCN-NH2, and Ap@MCN-NH2 in Example 1; where A: CN; B: MCN-NH2; C: Ap@MCN-NH2.

[0029] Figure 2 The images show (a) VSM plots and (b) FT-IR plots of MCN, MCN-NH2, and Ap@MCN-NH2 in Example 1.

[0030] Figure 3 The enzyme activity of free alkaline protease (Ap) and Ap@MCN-NH2 in Example 2 at 40-80℃.

[0031] Figure 4 The half-life of free alkaline protease (Ap) and Ap@MCN-NH2 in Example 3 after incubation at 60°C.

[0032] Figure 5 This demonstrates the reusability of the immobilized enzyme in Example 4.

[0033] Figure 6 The ABTS free radical scavenging rate of oilseed protein hydrolysates.

[0034] Figure 7 The ACE inhibition rate of oilseed protein hydrolysates.

[0035] Figure 8 The molecular weight distribution of walnut protein hydrolyzed by immobilized enzymes is shown in the figure. (a) Enzymatic hydrolysis reaction for 10 min under different pH and temperature conditions; (b) Enzymatic hydrolysis reaction for 30 min under different pH and temperature conditions; (c) Enzymatic hydrolysis reaction for 60 min under different pH and temperature conditions; (d) Enzymatic hydrolysis reaction for 90 min under different pH and temperature conditions.

[0036] Figure 9 The molecular weight distribution of walnut protein hydrolyzed by free enzymes is shown in the figure; (a) different hydrolysis temperatures; (b) different hydrolysis times.

[0037] Figure 10 The DPPH (a), ABTS (b), ·OH radical scavenging rate (c), and Fe of ultrafiltration components WPH-I and WPH-II in Example 17 are shown. 2+ Chelation capacity (d) graph.

[0038] Figure 11 ABTS (a), ·OH radical scavenging rate (b), and Fe were measured for gel chromatography fractions WPH-II-F1, WPH-II-F2, WPH-II-F3, and WPH-II-F4 in Example 17. 2+ Chelation capacity (c) diagram.

[0039] Figure 12 The mass spectra of two antioxidant peptides are shown, including (a) GQTPLFPR mass spectrum and (b) ANPPHFIHL mass spectrum.

[0040] Figure 13 The mass spectra of two ACE inhibitory peptides are shown, including (a) RMPVPIFF mass spectrum and (b) KGWLLTL mass spectrum. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1 A method for preparing magnetically immobilized alkaline protease (Ap@MCN-NH2) using carbon nitride includes the following steps: (1) Preparation of magnetic carbon nitride (MCN): 0.5 g of carbon nitride (CN) was weighed and added to 50 mL of 6 mol / L nitric acid solution. The mixture was magnetically stirred at 70 °C for 4 h for oxidation. After the reaction, the pH was adjusted to neutral with NaOH solution, and the mixture was washed at least three times with deionized water to obtain carbon nitride oxide. The carbon nitride oxide was ultrasonically dispersed in 50 mL of water, and 10 mL of a mixed solution containing 380 mg FeCl3·6H2O and 140 mg FeCl2·4H2O was added dropwise. The mixture was ultrasonically treated for 10 min. Subsequently, 20% ammonia solution was added dropwise to the mixture to adjust the pH to 10.0, and the mixture was stirred at 70 °C for 1 h to allow the magnetic particles to fully precipitate. Finally, the precipitated particles were repeatedly washed with deionized water and dried overnight in an oven at 60 °C to obtain magnetic carbon nitride (MCN).

[0043] (2) Surface functionalization of magnetic carbon nitride: 0.5 g of MCN was dispersed in 100 mL of anhydrous ethanol containing 4% APTMs, and the pH was adjusted to 4.5 with acetic acid. The mixture was stirred at 70 °C for 6 h. After the reaction was complete, the mixture was washed with anhydrous ethanol to obtain amino-modified MCN-NH2. The prepared MCN-NH2 was then ultrasonically dispersed in 50 mL of phosphate buffer solution (0.05 mol / L, pH 8.0), and 4% (v / v) glutaraldehyde was added. The mixture was stirred at room temperature for 2 h. After the reaction was complete, the precipitate was collected by centrifugation at 8000 rpm for 5 min, washed repeatedly with deionized water, and dried to obtain surface-activated MCN-NH2.

[0044] (3) Preparation of alkaline protease immobilized by magnetic carbon nitride: A 1.8 mg / mL alkaline protease solution was prepared using sodium phosphate buffer (0.05 mol / L, pH 8.0) and set aside. Then, 20 mg of the prepared MCN-NH2 carrier (surface-activated MCN-NH2) was ultrasonically dispersed in 5 mL of the alkaline protease solution and incubated at 30 °C for 4 h. After the reaction, the precipitate was collected by centrifugation, lyophilized, and stored at low temperature to obtain the magnetically immobilized carbon nitride alkaline protease (Ap@MCN-NH2). The enzyme loading was determined to be 109.7 mg / g.

[0045] Scanning electron microscope images of CN, MCN-NH2, and Ap@MCN-NH2 are shown below. Figure 1 .like Figure 1 As shown in AC, CN exhibits a relatively smooth sheet-like structure. After magnetization treatment, the surface of MCN-NH2 becomes significantly rougher, with a large number of magnetic particles aggregated, indicating that the magnetic modification of this material was successful.

[0046] The (a) VSM plot and (b) FT-IR plot of MCN, MCN-NH2, and Ap@MCN-NH2 are shown below. Figure 2 Compared to MCN, MCN-NH2 at 2920 cm⁻¹ 1 The CH symmetric and antisymmetric stretching vibration peaks appear at 1079 cm⁻¹. 1 The presence of Si-O-Si characteristic peaks at 3100–3600 cm⁻¹ confirms the successful grafting of APTMS onto the MCN surface. 1 The stretching of NH in free AP amides can be observed, and the peak signal in AP@MCN-NH2 is significantly increased compared to MCN-NH2, thus confirming the immobilization of AP.

[0047] Example 2 The optimal temperature for immobilizing alkaline protease and free enzyme (Ap) is determined through the following steps: The reaction temperature was set at 40–80 °C. The free and immobilized enzymes were incubated in a constant temperature water bath at 40–80 °C for 5 min. Casein solution was added, and the reaction was allowed to proceed for 10 min. The reaction was then immediately terminated, and the optimal reaction temperatures for the free and immobilized enzymes were determined by measuring enzyme activity. The protease activity was determined using the Folin-phenol method: 5 mg of magnetic carbon nitride-immobilized alkaline protease was dispersed in 1 mL of PBS buffer (50 mol / L, pH 10) and incubated at 50 °C for 5 min. Then, 1 mL of 20 mg / mL casein was added, and the reaction was allowed to proceed for 10 min. The reaction was then terminated by adding 2 mL of trichloroacetic acid (0.4 mol / L), and the mixture was centrifuged for 1 min to obtain the supernatant. 1.0 mL of the supernatant was taken, and 5 mL of Na₂CO₃ solution (0.4 mol / L) and 1.0 mL of Folin-phenol reagent were added sequentially. After mixing, the mixture was incubated at 40 °C for 20 min, and the absorbance at 680 nm was measured. The tyrosine standard curve was used (Y = 0.0105X + 0.0108, R0). 2 =0.9995) to calculate the amount of tyrosine released. One unit of enzyme activity (U) is defined as the amount of protease required to generate 1.0 μg of tyrosine in 1 min at pH 10 and 50°C. The formula for calculating enzyme activity is as follows:

[0048] In the formula: U: enzyme activity (U / g); C: The concentration of tyrosine calculated based on the standard curve; V: Total volume of the enzymatic hydrolysis reaction system (mL); N: The multiple of the measured reaction solution to the total reaction solution; n: the dilution factor of the enzyme solution; t: reaction time (min); m: Mass (g) of immobilized alkaline protease used to determine enzyme activity.

[0049] Relative enzyme activity calculation: The highest enzyme activity measured is taken as 100%, and the remaining enzyme activities are compared with it to calculate the relative enzyme activity (%).

[0050] Figure 3 The results showed that the optimal temperatures for the free enzyme and the immobilized enzyme (AP@MCN-NH2) were 50℃ and 60℃, respectively, with comparable enzyme activities. When the temperature exceeded 70℃, the relative enzyme activity of the free enzyme dropped directly to 14.78%, while AP@MCN-NH2 retained 81.55% until the temperature rose to 80℃, at which point its relative enzyme activity decreased to 8.44%.

[0051] Example 3 The thermal stability of immobilized alkaline protease and free enzyme is determined through the following steps: Free and immobilized enzymes were incubated at 60°C for different times, and enzyme activity was measured every 30 minutes. Enzyme activity was defined as 100% when incubated for 5 minutes under optimal conditions, and the residual activity of the protease was used for evaluation.

[0052] Figure 4 The results showed that after incubation at 60℃ for 60 min, the AP@MCN-NH2 retained approximately 83.6% of its initial activity, while the free enzyme retained only 49.6% of its initial activity. The half-life of the immobilized enzyme was 133.7 min, while that of the free enzyme was 67.9 min, the former being 1.97 times longer than the latter. Therefore, the carbon nitride immobilized enzyme can maintain good catalytic activity for a longer period at higher temperatures, improving its applicability and controllability in high-temperature industrial applications.

[0053] Example 4 The reusability of immobilized alkaline protease is determined through the following steps: The immobilized enzyme was incubated at 60°C for 5 min and then used to enzymatically hydrolyze casein solution for 10 min. The enzyme activity was measured using the Folin-phenol method. The immobilized enzyme was then recovered and used for the next enzymatic hydrolysis, and the process was repeated 5 times.

[0054] Figure 5 The results showed that the immobilized enzyme retained 55.21% of its initial enzyme activity after being reused five times, indicating that the immobilized enzyme has high reusability potential and is suitable for industrial production.

[0055] Example 5 The preparation of bioactive peptides from soybean protein by enzymatic hydrolysis using immobilized alkaline protease (Ap@MCN-NH2) includes the following steps: 0.1 g of immobilized alkaline protease was added to 10 mL of soybean protein solution (40 mg / mL), and the mixture was enzymatically hydrolyzed for 60 min at 50 °C and pH 10.0. The ABTS free radical scavenging rate and ACE inhibition rate of the hydrolysate were then measured.

[0056] Method for determining ABTS free radical scavenging rate: An equal volume of 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution was mixed and incubated at room temperature in the dark for 16 h to obtain an ABTS stock solution. Before use, the solution was diluted with deionized water to a absorbance of 0.70 ± 0.02 at 734 nm to obtain the ABTS preparation solution. 50 μL of the sample solution and 150 μL of the ABTS preparation solution were added to a 96-well plate and mixed thoroughly. After incubation at room temperature for 6 min, the absorbance was measured at 734 nm. The calculation method is as follows:

[0057] In the formula: A0: Absorbance of blank control (deionized water + DPPH solution); A1: Absorbance of the sample group (sample + DPPH solution); A2: Absorbance of sample blank (sample + anhydrous ethanol).

[0058] ACE inhibition rate assay: First, prepare the sample solution: Dissolve the sample in 100 mmol / L sodium borate buffer (pH 8.3) containing 300 mmol / L NaCl. Separately, dissolve hippuryl histidine leucine (HHL) in the same buffer to prepare a 5 mmol / L HHL substrate solution. Mix 50 μL of the sample solution with 100 μL of HHL (5 mmol / L), incubate at 37°C for 10 min, then add 20 μL of ACE enzyme (0.1 U / mL), and continue the reaction at 37°C for 30 min. Finally, add 200 μL of HCl (0.05 mol / L) to terminate the reaction. Extract the released hippuric acid with 1.5 mL of ethyl acetate, shake thoroughly, and allow to separate into layers. Take the ethyl acetate layer, evaporate it by rotary evaporation at 50°C, and redissolve the residue with 1 mL of NaCl solution (1 mol / L). Measure the absorbance at 228 nm. The ACE inhibition rate formula is as follows:

[0059] In the formula: A0: Absorbance of the control group (using buffer solution instead of the sample); A1: Absorbance of the experimental group (sample + ACE + HHL); A2: Absorbance of the blank sample group (using buffer instead of ACE).

[0060] The results showed that the ABTS free radical scavenging rate of soybean protein hydrolysate was ( Figure 6 ) and ACE inhibition rate ( Figure 7 The figures were 33.68% and 64.28%, respectively.

[0061] Example 6 Bioactive peptides were prepared by enzymatic hydrolysis of peanut protein with immobilized alkaline protease (Ap@MCN-NH2), following the same procedure as in Example 5.

[0062] The results showed that the ABTS free radical scavenging rate of peanut protein hydrolysate was ( Figure 6 ) and ACE inhibition rate ( Figure 7 The percentages were 54.23% and 84.38%, respectively.

[0063] Example 7 Active peptides were prepared by enzymatic hydrolysis of sunflower protein with immobilized alkaline protease (Ap@MCN-NH2), following the same procedure as in Example 5.

[0064] The results showed that the ABTS free radical scavenging rate of sunflower protein hydrolysate was ( Figure 6 ) and ACE inhibition rate ( Figure 7 The figures were 86.52% and 24.90%, respectively.

[0065] Example 8 The immobilized alkaline protease (Ap@MCN-NH2) was used to enzymatically hydrolyze flaxseed protein to prepare bioactive peptides, following the same procedure as in Example 5.

[0066] The results showed that the ABTS free radical scavenging rate of linolenic acid hydrolysate was ( Figure 6 ) and ACE inhibition rate ( Figure 7 The percentages were 61.25% and 53.68%, respectively.

[0067] Example 9 Immobilized alkaline protease (Ap@MCN-NH2) was used to enzymatically hydrolyze walnut protein to prepare bioactive peptides, following the same procedure as in Example 5.

[0068] The results showed that the ABTS free radical scavenging rate of walnut protein hydrolysate was ( Figure 6 ) and ACE inhibition rate ( Figure 7 The figures were 76.25% and 88.60%, respectively.

[0069] Example 10 0.1 g of immobilized alkaline protease (Ap@MCN-NH2) was used to enzymatically hydrolyze walnut protein at pH 7 and temperature (T) 30℃ for 10, 30, 60, and 90 min, and the proportion of different molecular weight components was determined.

[0070] Figure 8 The results showed that macromolecular components >10 kDa accounted for 15.4%–39.5%, medium molecular weight components (3–10 kDa) accounted for 18.1%–25.0%, and small molecular weight components <3 kDa accounted for 42.4%–61.7%. Overall, macromolecules accounted for a relatively high proportion, while the enrichment of small molecules was limited.

[0071] Example 11 0.1 g of immobilized alkaline protease (Ap@MCN-NH2) was used to hydrolyze walnut protein at pH 9 and T 30℃ for 10, 30, 60, and 90 min, and the proportion of different molecular weight components was determined.

[0072] Figure 8The results showed that the proportion of >10 kDa components decreased to 11.3%–21.8%, the proportion of 3–10 kDa components remained stable at 18.2%–22.4%, and the proportion of <3 kDa components increased to 56.1%–70.5%. Compared with pH 7, alkaline pH was more conducive to the degradation of large molecular weight peptides into small molecular weight peptides, while the proportion of medium molecular weight components remained basically stable.

[0073] Example 12 0.1 g of immobilized alkaline protease (Ap@MCN-NH2) was used to hydrolyze walnut protein at pH 7 and T 50℃ for 10, 30, 60, and 90 min, and the proportion of different molecular weight components was determined.

[0074] Figure 8 The results showed that the >10 kDa fraction accounted for 3.2%–18.6%, the 3–10 kDa fraction accounted for 3.4%–15.8%, and the <3 kDa fraction accounted for 65.6%–93.4%. Heating to 50℃ effectively promoted the degradation of macromolecules and improved the enrichment efficiency of small peptides.

[0075] Example 13 0.1 g of immobilized alkaline protease (Ap@MCN-NH2) was used to hydrolyze walnut protein at pH 9 and T 50℃ for 10, 30, 60, and 90 min, and the proportion of different molecular weight components was determined.

[0076] Figure 8 The results showed that the >10 kDa component accounted for 5.3%–25.7%, the 3–10 kDa component accounted for 5.2%–23.1%, and the <3 kDa component accounted for 51.2%–89.5%.

[0077] Example 14 0.1 g of immobilized alkaline protease (Ap@MCN-NH2) was used to hydrolyze walnut protein at pH 9 and T 50℃ for 30 min, and the proportion of different molecular weight components was determined.

[0078] Figure 8 The results showed that the >10 kDa fraction was 5.3%, the 3-10 kDa fraction was 5.2%, and the <3 kDa fraction was 89.5%. Under these conditions, the immobilized alkaline protease exhibited the strongest enzymatic activity, which can efficiently enrich active peptides, shorten reaction time, and improve production efficiency, making it the optimal combination of conditions for enriching small molecule peptides.

[0079] Example 15 0.05 g of free alkaline protease was hydrolyzed at pH 9 for 30 min, and the hydrolysis temperatures were set at 20, 30, 40, 50, and 60 °C. The proportion of components with different molecular weights was determined.

[0080] Figure 9 (a) The results showed that the >2 kDa component accounted for 13.0–23.1%, the 1.2–2 kDa component for 13.4–15.5%, the 0.2–1.2 kDa component for 25.9–29.1%, and the <0.2 kDa component for 36.9–44.8%. Under these conditions, changing the enzymatic hydrolysis temperature did not significantly alter the molecular weight of the walnut protein hydrolysate, making molecular weight control difficult.

[0081] Example 16 Free alkaline protease was used to hydrolyze walnut protein at pH 9 and T 50℃ for 20, 30, 40, 50 and 60 min, and the proportion of different molecular weight components was determined.

[0082] Figure 9 (b) The results showed that the >2 kDa fraction was 5.4–7.6%, the 1.2–2 kDa fraction was 16.3–20.0%, the 0.2–1.2 kDa fraction was 25.9–27.8%, and the <0.2 kDa fraction was 44.6–52.1%. Under these conditions, changing the enzymatic hydrolysis time did not significantly alter the molecular weight of the walnut protein hydrolysate, indicating that controlling the molecular weight was difficult.

[0083] Example 17 The isolation and purification of antioxidant peptides from walnut protein hydrolysates specifically includes the following steps: (1) The walnut protein hydrolysate prepared in Example 14 was filtered through a 0.45 μm membrane to remove impurities, and then separated using an ultrafiltration tube with a molecular weight cutoff of 3 kDa to obtain component WPH-I (>3 kDa) and component WPH-II (<3 kDa). After lyophilization, DPPH, ABTS, ·OH free radical scavenging rate and Fe were measured. 2+ Chelating ability.

[0084] (2) The ultrafiltration fraction powder was further separated, and a 50 mg / mL solution was prepared and loaded onto a Sephadex G-15 dextran gel column. The solution was eluted with sodium phosphate buffer (50 mmol / L, pH 7) at a flow rate of 1 mL / min. The collected fraction was then monitored at 220 nm and its antioxidant activity was measured after lyophilization.

[0085] The results showed that the WPH-II component had better antioxidant activity. Figure 10 After further separation using dextran gel G-15, four components, WPH-II-F1, WPH-II-F2, WPH-II-F3, and WPH-II-F4, were obtained. The antioxidant activities of the four components were compared. Figure 11 F3 was selected for subsequent mass spectrometry identification.

[0086] Example 18 The mass spectrometry identification and analysis of component WPH-II-F3 specifically includes the following steps: (1) Peptides in component F3 were identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0087] (2) Bioinformatics prediction of peptides identified by mass spectrometry was performed using websites such as PeptideRanker, ExPASy, ToxinPred, BIOPEP, AnOxPePred, and AutoDock.

[0088] The results showed that mass spectrometry identified 160 different peptides with a molecular weight range of 357.55–1093.44 Da, mainly heptapeptides, octapeptides, nonapeptides, decapeptides, and peptides with more than ten peptides. Bioinformatics analysis predicted that two peptides, ANPPHFIHL (SEQ ID NO.1) and GQTPLFPR (SEQ ID NO.2), possess antioxidant activity. The mass spectra of the two antioxidant peptides are shown below. Figure 12 .

[0089] Example 19 The isolation and purification of the ACE inhibitory peptide from the walnut protein hydrolysate were performed in the same manner as in Example 17.

[0090] The results showed that the ACE-inhibiting peptides of WPH-I and WPH-II fractions were comparable. After further separation by dextran gel G-15, the ACE inhibition rate of WPH-II-F6 fraction was the best, and it was selected for subsequent mass spectrometry identification.

[0091] Example 20 The mass spectrometry identification and analysis of component WPH-II-F6 were performed in the same manner as in Example 18.

[0092] The results showed that mass spectrometry identified nine different peptides with a molecular weight range of 829.50–2906.40 Da. Bioinformatics analysis predicted that two of these peptides, RMPVPIFF (SEQ ID NO.3) and KGWLLTL (SEQ ID NO.4), possessed ACE inhibitory activity. The mass spectra of the two ACE-inhibiting peptides are shown below. Figure 13 .

[0093] Example 21 The solid-phase synthesis verification of antioxidant peptides follows these steps: To verify the accuracy of the predicted antioxidant peptides, two peptides, ANPPHFIHL and GQTPLFPR, were synthesized using solid-phase synthesis. Their ABTS and ·OH radical inhibition rates and Fe were then evaluated. 2+ The inhibition rate of chelation at different concentrations was measured, and the half-maximal inhibitory concentration (IC50) was determined. 50 )value.

[0094] The results show that ANPPHFIHL's IC 50 The values ​​were: ABTS free radical scavenging rate 0.061 mg / mL, hydroxyl free radical scavenging rate 1.01 mg / mL, Fe... 2+ Chelating capacity 0.72 μg / mL; IC50 of GQTPLFPR 50 The values ​​were 0.111 mg / mL, 1.28 mg / mL, and 0.75 μg / mL, respectively.

[0095] Example 22 The solid-phase synthesis verification of ACE inhibitory peptides is performed using the following steps: To verify the accuracy of the predicted ACE-inhibiting peptides, two peptides, RMPVPIFF and KGWLLTL, were synthesized using solid-phase synthesis. Their ACE inhibition rates at different concentrations were then evaluated, and the half-maximal inhibitory concentration (IC50) was determined. 50 )value.

[0096] The results show that the ICs of RMPVPIFF and KGWLLTL 50 The values ​​were 6.89 μg / mL and 24.80 μg / mL, respectively. This demonstrates that the ACE inhibitory activity of these two peptides is accurately predicted and exhibits strong ACE inhibitory activity.

[0097] The physicochemical properties of the four bioactive peptides are shown in Table 1.

[0098] Table 1. Physicochemical properties of the four bioactive peptides

[0099] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An active peptide from oilseeds, characterized in that, It is an antioxidant peptide or an ACE inhibitory peptide; The amino acid sequences of the antioxidant peptides are ANPPHFIHL and GQTPLFPR, respectively. The amino acid sequences of the ACE inhibitory peptides are RMPVPIFF and KGWLLTL, respectively.

2. A method for immobilized enzymatic hydrolysis preparation of oilseed bioactive peptides, characterized in that, The protein was prepared by immobilizing alkaline protease with magnetic carbon nitride to hydrolyze oilseed proteins. The specific method is as follows: Add 0.1–0.5 g of magnetically immobilized alkaline protease to 5–10 mL of 4%–10% oilseed protein solution, adjust the pH to 8.0–11.0 with sodium hydroxide solution, and enzymatically hydrolyze for 10–60 min at 40–60 °C. Then, recover the immobilized enzyme by magnetic separation. After centrifugation, take the supernatant of the oilseed protein hydrolysate and determine its biological activity.

3. The method for immobilized enzymatic hydrolysis preparation of oilseed active peptides according to claim 2, characterized in that, The method for preparing the magnetic carbon nitride immobilized alkaline protease is as follows: (1) Preparation of magnetic carbon nitride Weigh 0.5-2 g of graphitic carbon nitride and disperse it in 50-100 mL of 6-12 mol / L nitric acid solution. Stir at 60-70℃ for 2-4 h. After completion, adjust the pH to neutral with NaOH solution and wash with deionized water several times to obtain carbon nitride oxide. The carbon nitride was ultrasonically dispersed in 20-60 mL of water, and 10 mL of a mixture containing 350-450 mg of iron salt and 100-200 mg of ferrous salt was added dropwise, followed by ultrasonic treatment. Subsequently, 10-20% ammonia solution was added dropwise to the mixture to adjust the pH to 9.0-12.0, and the mixture was stirred at 70℃ for 1-3 h to allow the magnetic particles to fully precipitate. Finally, the precipitated particles were repeatedly washed with deionized water until neutral, and dried overnight in an oven at 50-70℃ to obtain magnetic carbon nitride MCN. (2) Surface functionalization of magnetic carbon nitride Disperse 0.1-1 g of the MCN prepared in step (1) in 100-200 mL of anhydrous ethanol containing 2-6% silane coupling agent, adjust the pH to 3.0-5.0 with acetic acid, stir at 60-80℃ for 6-10 h, and then wash with anhydrous ethanol to obtain amino-modified MCN-NH2; ultrasonically disperse the MCN-NH2 in 50-100 mL of phosphate buffer solution, add crosslinking agent, stir at room temperature for 1-4 h, wash the precipitate multiple times with deionized water and dry to obtain surface-activated MCN-NH2; (3) Magnetic carbon nitride immobilized protease Prepare an alkaline protease solution with a concentration of 0.6-3 mg / mL using a sodium phosphate buffer solution with a concentration of 0.05-0.1 mol / L and a pH of 6.0-10.0; ultrasonically disperse the surface-activated MCN-NH2 prepared in step (2) in the alkaline protease solution, with a mass-to-volume ratio of surface-activated MCN-NH2 to alkaline protease solution of 1:(250-500), and incubate at 20-60℃ for 1-5 h; centrifuge the mixture to collect the precipitate, freeze-dry it, and store it at low temperature.

4. The method for immobilized enzymatic hydrolysis preparation of oilseed active peptides according to claim 3, characterized in that, The iron salt mentioned in step (1) is FeCl3·6H2O; the ferrous salt is FeCl2·4H2O; the silane coupling agent mentioned in step (2) is 3-aminopropyltrimethoxysilane (APTMs); the crosslinking agent is glutaraldehyde; and the concentration of glutaraldehyde is 4%.

5. The method for immobilized enzymatic hydrolysis preparation of oilseed active peptides according to claim 2, characterized in that, The oilseed protein is selected from at least one of soybean protein, peanut protein, sunflower protein, flaxseed protein, and walnut protein.

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