A walnut peptide product with high antioxidant activity and a preparation method and application thereof

Abstract

The application discloses a walnut peptide product with high antioxidation activity, a preparation method and application thereof. The product is a negative charge enriched or positive charge enriched walnut peptide with a molecular weight of less than or equal to 3 kDa, wherein part of the acidic residues have a C-terminal distribution characteristic and are adjacent to hydrophobic residues; the preparation method comprises the steps of enzymolysis, bipolar membrane electrodialysis and molecular weight grading. The walnut peptide product provided by the application has suitable molecular size and higher interface charge expression capacity, has clear antioxidation activity structure characteristics and stronger hydroxyl radical scavenging activity, and the antioxidation activity can reach 95%; and the product shows stronger scavenging capacity in a chemical free radical system and a cell system, can be used as an antioxidant raw material of food, functional food, nutritional supplement or cosmetics, and can also be used in the field of biological medicine, such as preparation of oxidative stress treatment drugs, as a drug delivery material, maintenance of blood calcium balance and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a peptide, its preparation method, and its application, and more particularly to a walnut peptide product with high antioxidant activity, its preparation method, and its application. Background Technology

[0002] Antioxidant peptides are naturally occurring or artificially designed bioactive peptides that can reduce the levels of reactive oxygen species and other pro-oxidants, thus scavenging free radicals in the body and preventing oxidative stress and damage. Furthermore, antioxidant peptides can donate hydrogen to antioxidant enzymes and chelate metal ions, showing great potential for applications in biomedicine, food, and health care. For example, patent CN 117924418 A discloses a walnut protein octapeptide HNVADPQR with pre-protective efficacy against alcoholic hepatocellular damage, its preparation method, and its applications. This active peptide exhibits good antioxidant activity at the cellular level.

[0003] Existing walnut peptide products are mostly evaluated for their antioxidant capacity based on molecular weight, amino acid composition, or specific sequences. However, the effectiveness of antioxidant activity is closely related to whether the carboxyl groups in the sequence are in a state of exposure, dissociation, and participation in free radical reactions. In existing technologies, walnut peptides obtained by ordinary enzymatic hydrolysis have complex compositions and low contents of active components. For example, patent application CN119020444A discloses a method for preparing walnut antioxidant peptides through probiotic compound fermentation. This method involves inoculating a mixed probiotic culture into a walnut meal fermentation substrate, separating and extracting peptides of different molecular weight ranges from the fermented product using ultrafiltration, and then analyzing their antioxidant activity. However, its highest antioxidant activity is only about 70%. Therefore, there is an urgent need to develop a walnut peptide product with clearly defined antioxidant structural characteristics and high hydroxyl radical scavenging ability. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a walnut peptide product with clear antioxidant activity, high hydroxyl radical scavenging ability and cell protection effect, as well as its preparation method and application.

[0005] Technical solution: The present invention provides a walnut peptide product with high antioxidant activity, wherein the walnut peptide product is a negatively or positively charged walnut peptide with a molecular weight ≤3 kDa.

[0006] Furthermore, the walnut peptide product is a negatively charged enriched walnut peptide with a molecular weight of 1-3 kDa, or a positively charged enriched walnut peptide with a molecular weight ≤3 kDa.

[0007] Preferably, the walnut peptide exhibits a negative zeta potential or a positive zeta potential under pH conditions of 6.0-8.5.

[0008] The walnut peptide product provided by this invention has one or more of the following characteristics: the molecular weight is mainly distributed below 3kDa; it exhibits a negative or positive zeta potential under pH 6.0-8.5 conditions; the 1-3 kDa negatively charged walnut peptide has an absolute zeta potential higher than that of components with less than 1 kDa and greater than 3 kDa under low salt conditions; it has a strong hydroxyl radical scavenging ability; its antioxidant activity is significantly reduced after carboxyl neutralization or amidation; and it can reduce the level of cellular oxidative stress.

[0009] Preferably, the peptide chain terminus of the walnut peptide forms a local carboxyl group region.

[0010] Preferably, the C-terminal 1 / 3 region of the walnut peptide sequence contains aspartic acid or glutamic acid, and the adjacent amino acid of the aspartic acid or glutamic acid is a hydrophobic amino acid. Further, the last two amino acids at the C-terminus of the walnut peptide product sequence contain aspartic acid or glutamic acid, or the last amino acid at the C-terminus of the walnut peptide product sequence is aspartic acid or glutamic acid.

[0011] Preferably, the N-terminal 1 / 3 region of the walnut peptide sequence contains proline, glycine, and / or lysine. Further, the N-terminal 3-4 amino acids of the walnut peptide product sequence contain proline, glycine, and / or lysine.

[0012] Preferably, the N-terminal 1 / 3 region of the walnut peptide sequence contains adjacent proline and glycine.

[0013] Preferably, the walnut peptide product comprises peptide segments with sequences as shown in SEQ ID NO.1-5.

[0014] The walnut peptide product provided by this invention, after LC-MS / MS identification and statistical analysis, also has the following characteristics:

[0015] 1. Some acidic residues exhibit terminal distribution characteristics: The C-terminal and near-C-terminal regions of the peptide contain at least one aspartic acid (D) or glutamic acid (E). The carboxyl groups of the D / E side chains located at or near the C-terminus are spatially close to the α-carboxyl group at the C-terminus of the peptide, easily forming local carboxyl regions at the end of the peptide chain. Compared to D / E located in the middle of the sequence, the terminal or near-terminal acidic residues are less restricted by the surrounding residues on both sides and intramolecular interactions, and their carboxyl groups are more easily exposed to the aqueous environment and come into contact with hydroxyl radicals, metal ions, or other redox-related reaction systems.

[0016] 2. Some acidic residues tend to form terminal or near-terminal acidic-hydrophobic adjacent structures with hydrophobic residues: This invention refers to the local sequence units where D / E are directly adjacent to hydrophobic amino acids as acidic-hydrophobic adjacent sequence motifs, such as EL, LE, and VE. These structures provide ionizable carboxyl sites, and the adjacent hydrophobic residues may influence the local hydration state and membrane interface interactions, thus providing sequence-level support for the relationship between carboxyl accessibility and hydroxyl radical scavenging ability.

[0017] Furthermore, structures related to "Glu-hydrophobic amino acids" such as γ-Glu-Val and γ-Glu-Leu have been reported as active molecules of calcium ion-sensitive receptors (CaSRs), which have the function of regulating blood calcium levels. Also related to CaSR activation, γ-glutamyl peptides have been reported to have potential functions such as anti-inflammatory, hypoglycemic, and appetite-suppressing effects, and can be used for metabolic regulation and maintaining intestinal health. In addition, acidic-hydrophobic residue combinations may regulate hydration, interfacial adsorption, and assembly behavior. Therefore, this invention also has the potential to serve as an interfacial / membrane interaction and delivery material, and can be applied in the medical field.

[0018] 3. The N-terminal and near-N-terminal regions of the peptide contain one or more of proline (P), glycine (G), or lysine (K); the N-terminal and near-N-terminal regions of the peptide contain PG or GP.

[0019] Research reports that N-acetyl Pro-Gly-Pro, or N-acetyl PGP, is a tripeptide produced by the degradation of the extracellular matrix. It has a structure and function similar to CXC chemokine and can induce neutrophil chemotaxis and promote SOD production through CXC receptors.

[0020] In summary, the walnut peptide product provided by this invention possesses high antioxidant activity and specific structural characteristics, and has promising application prospects in the fields of biomedicine, food and health care.

[0021] This invention also provides a method for preparing the walnut peptide product, which involves enzymatic hydrolysis with protease, negative charge enrichment via bipolar membrane electrodialysis, and molecular weight fractionation. Under the influence of an electric field, the bipolar membrane generates H₂. + and OH - This creates a local acid-base environment, causing walnut peptides with a strong negative charge expression capacity to migrate to the acid chamber.

[0022] Preferably, the bipolar membrane electrodialysis system includes anion exchange membrane, cation exchange membrane and bipolar membrane, the method of negative charge enrichment is to collect the acid chamber enrichment solution and / or alkali chamber enrichment solution of the bipolar membrane electrodialysis, and the molecular weight fractionation is to collect peptides with a molecular weight ≤3 kDa.

[0023] In the bipolar membrane electrodialysis (BMED) separation process, peptide migration is influenced by a combination of factors, including electric field, concentration gradient, component charge, diffusion behavior, and membrane interface interactions. Therefore, peptides containing D / E at or near the C-terminus may exhibit different mass transfer behaviors than ordinary peptides under the acidified environment and applied electric field of BMED due to their higher exposure of carboxyl sites. Measurements have shown that BMED acidic and basic compartment peptides possess high hydroxyl radical scavenging activity.

[0024] Compared to fractions less than 1 kDa and fractions greater than 3 kDa, the 1-3 kDa negatively charged walnut peptide fraction exhibited stronger hydroxyl radical scavenging ability. Mechanism analysis: Fractions less than 1 kDa have relatively low active site loading due to chain length limitations; fractions greater than 3 kDa have excessively long chains, making it easier for amino acid residue side chains to interact and form stereoforms, hindering the accessibility of active sites to hydroxyl radicals in solution; while the 1-3 kDa fraction has a relatively moderate chain length, with both a large number of active sites and high accessibility, thus possessing a strong free radical scavenging ability.

[0025] The present invention also provides the application of the walnut peptide product in pharmaceuticals, food, nutritional supplements or cosmetic antioxidant ingredients.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0027] 1. The walnut peptide product provided by this invention has both a suitable molecular size and a high interfacial charge expression capacity, and has clear antioxidant activity structural characteristics and strong hydroxyl radical scavenging activity. At concentrations of 0.015 mg / mL and 1.25 mg / mL, the antioxidant activity of the product can reach 50% and 95%, respectively.

[0028] 2. This product not only exhibits strong scavenging ability in chemical free radical systems, but also demonstrates the effects of reducing ROS, improving antioxidant enzyme activity, and reducing lipid peroxidation levels in the HepG2 cell oxidative stress evaluation model, thus exhibiting cell protection effects.

[0029] 3. This product is derived from natural sources and prepared under mild conditions. It can be used as an antioxidant raw material in food, functional food, nutritional supplements, or cosmetics. It can also be used in the biomedical field—for the preparation of drugs for the treatment of oxidative stress, as a drug delivery material, and for maintaining blood calcium balance. Attached Figure Description

[0030] Figure 1 The IC50 values ​​represent the hydroxyl radical scavenging activity (IC50) of walnut peptides before and after separation and purification by bipolar membrane electrodialysis, compared to the control. 50 ;

[0031] Figure 2The IC50 activity of hydroxyl radical scavenging of walnut peptides of different molecular weights obtained by bipolar membrane electrodialysis is shown. 50 ;

[0032] Figure 3 These are the apparent zeta potentials of antioxidant walnut peptides of different molecular weights at different pH and salt concentrations.

[0033] Figure 4 The IC50 values ​​of antioxidant walnut peptides of different molecular weights before and after neutralizing carboxyl groups indicate their hydroxyl radical scavenging activity (IC50). 50 ;

[0034] Figure 5 The study investigated the preventive and therapeutic effects of 1-3 kDa antioxidant walnut peptide on the oxidative stress HepG2 cell model (A represents cell survival rate under different treatments, B represents cell survival rate after different treatments before and after oxidative damage, and C represents the level of oxidative indicators after different treatments).

[0035] Figure 6 These are fluorescence images and fluorescence intensity levels of HepG2 cells subjected to different treatments under oxidative stress. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0037] Example 1

[0038] This embodiment provides a walnut peptide product with high antioxidant activity, and its preparation method is as follows.

[0039] Step 1: Preparation of walnut protein hydrolysate. Walnut meal powder (after walnut oil extraction) was defatted using a supercritical fluid carbon dioxide method or an organic reagent method. Deionized water was added to prepare a 10% (w / v) dispersion, and the pH was adjusted to 7.0. Novozymes Protamex 1.6 was added for enzymatic hydrolysis at a dosage of 1200 U / g. Hydrolysis was carried out at 65℃ for 1 h, followed by enzyme inactivation at 90-100℃. After cooling, the mixture was centrifuged at 10000 × g for 10 min and filtered through a 0.22 μm filter membrane. The supernatant was collected to obtain the walnut protein hydrolysate.

[0040] Step 2: Preparation of negatively charged enriched walnut peptides. The walnut protein hydrolysate obtained in Step 1 was added to a bipolar membrane electrodialysis system. The electrodialysis membrane stack consisted of an anion exchange membrane, a cation exchange membrane, and a bipolar membrane. The concentration of walnut peptides was 5%, and the treatment system volume was 400 mL. Under the action of an electric field, the bipolar membrane generated H₂. + and OH -This creates a localized acid-base environment, causing walnut peptides with a stronger negative charge expression capacity to migrate to the acid chamber and those with a stronger positive charge expression capacity to migrate to the base chamber. The acid and base chamber solutions are collected, desalted, and freeze-dried to obtain negatively charged and positively charged walnut peptides.

[0041] Step 3: Obtaining the highly active walnut peptide product. The negatively charged enriched walnut peptide obtained in Step 2 was dissolved in water and fractionated sequentially using 3 kDa and 1 kDa ultrafiltration membranes to obtain fractions greater than 3 kDa, 1-3 kDa, and less than 1 kDa, respectively. The 1-3 kDa fraction was collected and freeze-dried to obtain the highly antioxidant active walnut peptide product. Representative peptides in the product are shown in SEQ ID NO. 1-5.

[0042] Example 2

[0043] This example is the sequencing identification and statistical analysis of the walnut peptide product in Example 1.

[0044] Samples before bipolar membrane electrodialysis and samples from the acid chamber were identified by LC-MS / MS, and weighted statistical analysis was performed based on peptide abundance. Peptides containing at least one aspartic acid (D) or glutamic acid (E) moiety were defined as acidic peptides, and peptides with a D / E ratio ≥ 2 were defined as polyacidic peptides (D / E represents aspartic acid or glutamic acid).

[0045] The results showed that the total amount of acidic residues in the acid chamber samples did not increase, but rather the sequence distribution abundance was reconstructed.

[0046] Table 1. Amino acid composition and sequence position distribution of walnut peptides before and after BMED treatment.

[0047] index BMED pre-sample acid chamber sample Change characteristics D / E abundance weighting 12.38% 2.18% It did not show an increase in total volume. Abundance percentage of polyacidic peptides 26.85% 0.51% The proportion of polyacidic peptides decreased D / E is located in the 1 / 3 area of ​​C. 10.18% 87.36% Increased by approximately 8.58 times The ratio of D / E within the two amino acids at the C-terminus 20.66% 92.31% Terminal distribution enhancement D / E is precisely located at the C end ratio 11.13% 52.17% Precise terminal acid residue increase N-end G / P / K percentage 0.01% 43.61% Significantly increased N-end GP / PG ratio 0.46% 53.23% Increased by approximately 114.8 times

[0048] The above results indicate that the main sequence characteristic of the acid chamber samples after BMED treatment is that the D / E groups are significantly concentrated in the C-terminal and near-C-terminal regions. The C-terminus of the peptide contains a free α-carboxyl group, and the D / E side chains also contain ionizable carboxyl groups. When the D / E is located at or near the C-terminus, the side chain carboxyl group is spatially close to the terminal α-carboxyl group, easily forming a local carboxyl region at the peptide chain end. Compared to D / E located in the middle of the sequence, the terminal or near-terminal acidic residues are less restricted by the surrounding residues and intramolecular interactions, making their carboxyl groups more easily exposed to the aqueous environment and in contact with hydroxyl radicals, metal ions, or other redox-related reaction systems.

[0049] From the BMED separation process, peptide migration is influenced by a combination of factors, including electric field, concentration gradient, component charge, diffusion behavior, and membrane interface interactions. Therefore, peptides containing D / E at or near the C-terminus may exhibit different mass transfer behaviors than ordinary peptides under the acidified environment of BMED and an applied electric field due to their higher exposure of carboxyl sites.

[0050] Further analysis revealed that some representative peptides in the acid chamber samples exhibited local sequence features where the D / E position is adjacent to a hydrophobic amino acid. These local sequence units where the D / E position is directly adjacent to a hydrophobic amino acid are termed acidic-hydrophobic adjacent sequence motifs, such as EL, LE, and VE. This terminology is used to describe the local sequence features observed in this invention and is not limited to known universal antioxidant functional motifs.

[0051] Table 2. Representative peptides with C-terminal or near-C-terminal acidic characteristics in acid chamber samples.

[0052] Representative peptides Main sequence features illustrate GPKTKLEL Includes EL / LE, E near C end Acidic-hydrophobic adjacent sequence motif GPKTLKEL Contains EL, E near C end Acidic-hydrophobic adjacent sequence motif PLASLVEL Contains VE / EL, E near the C-terminus Acidic-hydrophobic adjacent sequence motif PGKLPPTVVEL Contains VE / EL, E near the C-terminus Acidic-hydrophobic adjacent sequence motif GVVEL Contains VE / EL, E near the C-terminus acidic characteristics of short peptide ends PPLE Includes LE, C-end E Precise terminal acidity characteristics ACCSD C-end D Precise terminal acidity characteristics

[0053] Among the total abundance of acidic peptides, peptides containing terminal D / EH or HD / E structures accounted for 54.90%, higher than the 45.54% in pre-BMED acidic peptides; where H represents hydrophobic amino acids. This result suggests that some acidic residues in the acid compartment not only exhibit terminal distribution characteristics but also tend to form terminal or near-terminal acidic-hydrophobic adjacent structures with hydrophobic residues. These structures provide ionizable carboxyl sites, and the adjacent hydrophobic residues may influence local hydration states and membrane interface interactions, thus providing sequence-level support for the relationship between carboxyl accessibility and hydroxyl radical scavenging ability.

[0054] Example 3

[0055] In this embodiment, the salicylic acid-Fenton system was used to evaluate the hydroxyl radical scavenging ability of each component of walnut peptide in Example 1.

[0056] See results Figure 1 , Figure 2 Both acid-cell and alkali-cell walnut peptides exhibit high hydroxyl radical scavenging activity. In acid-cell walnut peptides, the IC50 of the 1-3 kDa fraction is... 50 The lower than 1 kDa component and the higher than 3 kDa component indicate that it has a stronger hydroxyl radical scavenging ability.

[0057] Example 4

[0058] This embodiment evaluates the zeta potential of each walnut peptide component in Example 1. Each walnut peptide component was dissolved in buffer solutions at pH 6.0, 7.4, and 8.5, with different NaCl concentrations.

[0059] See results Figure 3 The 1-3 kDa fraction exhibits a higher absolute zeta potential under most conditions, with a more pronounced negative charge expression, especially under low ionic strength and high pH conditions, indicating stronger carboxyl accessibility and interfacial negative charge expression.

[0060] Example 5

[0061] This embodiment verifies the neutralization of carboxyl groups in the walnut peptides from Example 1. An EDC / NHS activation system was used, with ethanolamine as the nucleophile, to neutralize or amidate the carboxyl groups of the walnut peptides.

[0062] See results Figure 4 The hydroxyl radical scavenging ability of the 1-3 kDa component decreased significantly after neutralization by the carboxyl group, indicating that accessible carboxyl groups and negative charge expression are important sources of its high antioxidant activity.

[0063] Example 6

[0064] This embodiment evaluates the cellular antioxidant effects of the walnut peptides from Example 1. A hydrogen peroxide-induced oxidative stress evaluation model was established using HepG2 cells. Walnut peptides enriched with a 1-3 kDa negative charge did not exhibit significant cytotoxicity; at a concentration of 200 μg / mL, cell viability was as high as 109.6%. Walnut peptides enriched with a 1-3 kDa negative charge were used to prevent and treat H2O2-induced oxidative damage.

[0065] See results Figure 5 , Figure 6 A walnut peptide fraction of 50 μg / mL improved cell viability (approximately 98.9%), reduced intracellular ROS levels, improved the activity of antioxidant enzymes such as CAT, SOD, and GSH-Px, and significantly reduced MDA levels. Figure 5 In step C, the MDA value was first standardized using the control group as a benchmark, and then reversed to make its trend consistent with that of CAT, SOD, and GSH. This way, the higher the value of the four indicators in the graph, the better the antioxidant status.

[0066] In summary, the walnut peptide product provided in Example 1 has both a suitable molecular size and a high interfacial charge expression capacity, with clear antioxidant activity structural characteristics, strong hydroxyl radical scavenging activity and good cell protection effect, and has broad application prospects.

Claims

1. A walnut peptide product with high antioxidant activity, characterized in that, The walnut peptide product is a negatively or positively charged walnut peptide with a molecular weight ≤3 kDa.

2. The walnut peptide product according to claim 1, characterized in that, The walnut peptide exhibits a negative or positive zeta potential under pH conditions of 6.0-8.

5.

3. The walnut peptide product according to claim 1, characterized in that, The walnut peptide forms a local carboxyl region at the end of its peptide chain.

4. The walnut peptide product according to claim 3, characterized in that, The walnut peptide sequence contains aspartic acid or glutamic acid in the C-terminal 1 / 3 region, and the adjacent amino acids of the aspartic acid or glutamic acid are hydrophobic amino acids.

5. The walnut peptide product according to claim 1, characterized in that, The walnut peptide sequence contains proline, glycine, and / or lysine in the N-terminal 1 / 3 region.

6. The walnut peptide product according to claim 5, characterized in that, The walnut peptide sequence contains adjacent proline and glycine in the N-terminal 1 / 3 region.

7. The walnut peptide product according to any one of claims 1-6, characterized in that, The walnut peptide product contains peptides with sequences as shown in SEQ ID NO.1-5.

8. A method for preparing the walnut peptide product according to any one of claims 1-7, characterized in that, It was obtained through enzymatic hydrolysis with proteases, charge enrichment by bipolar membrane electrodialysis, and molecular weight fractionation.

9. The preparation method according to claim 8, characterized in that, The bipolar membrane electrodialysis system includes anion exchange membrane, cation exchange membrane and bipolar membrane, the charge enrichment method is to collect the acid chamber enrichment solution and / or alkali chamber enrichment solution of the bipolar membrane electrodialysis, and the molecular weight fractionation is to collect peptides with a molecular weight ≤3 kDa.

10. The use of the walnut peptide product according to any one of claims 1-6 in pharmaceuticals, food, nutritional supplements or cosmetic antioxidant ingredients.

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