A method for separating and purifying high antioxidant activity peptide by bipolar membrane electrodialysis

Abstract

The application discloses a method for separating and purifying high-antioxidant-activity peptides by using a bipolar membrane electrodialysis, which is based on the structure of the antioxidant-activity peptides, and the peptides with different charge expression characteristics are subjected to directional migration, fractional collection and activity screening by the bipolar membrane electrodialysis, and combined with molecular weight grading, so that the obtained active peptides have a significantly enhanced hydroxyl radical scavenging capacity and high antioxidant capacity. The application is suitable for the preparation of high-antioxidant-activity peptides, and provides an effective technical path for the separation and purification of charge-responsive antioxidant peptides from different protein sources, and can be used for the preparation of antioxidant raw materials for medicines, foods, nutritional supplements or cosmetics.

Description

Technical Field

[0001] This invention relates to a method for separating and purifying peptides with high antioxidant activity, and more particularly to a method for separating and purifying peptides with high antioxidant activity using bipolar membrane electrodialysis. 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 strong application potential in biomedicine, food, and health care.

[0003] Patent CN 111777664 A discloses a walnut meal antioxidant peptide YFW, its preparation method, and its application. This antioxidant peptide is obtained through alkaline protease hydrolysis, ultrafiltration separation, and dextran gel chromatography purification, exhibiting high antioxidant activity. Patent application CN 117486971 A discloses an antioxidant peptide, walnut peptide, its preparation method, and its application. This method uses supercritical fluid extraction technology to defatted walnut meal, and sequentially adds alkaline and acidic solutions to adjust the pH, separate different components, and finally obtains an antioxidant peptide with strong antioxidant activity and anti-fatigue effects through enzymatic hydrolysis, filtration, concentration, and drying.

[0004] Most of these methods use molecular weight or polarity as screening criteria to separate and purify peptides, but peptides with specific molecular weights or polarities do not necessarily have high antioxidant activity. Therefore, there is currently a lack of methods capable of separating and purifying peptides with high antioxidant activity. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for separating and purifying peptides with high antioxidant activity using bipolar membrane electrodialysis (BMED).

[0006] Technical solution: The present invention provides a method for separating and purifying peptides with high antioxidant activity, the method comprising the step of enriching negatively charged peptides and / or positively charged peptides.

[0007] Technological Motivation: From the structural perspective of antioxidant activity, the negative carboxyl potential provided by acidic amino acid residues such as aspartic acid D and glutamic acid E is one of the important factors affecting the scavenging of peptide hydroxyl radicals and antioxidant activity. Therefore, targeted migration, fractional collection, and activity screening of peptides with different charge expression characteristics can yield peptides with high antioxidant activity.

[0008] The step of enriching negatively charged peptides and / or positively charged peptides is preferably to treat the peptide solution with bipolar membrane electrodialysis and collect acidic and / or basic peptides.

[0009] Bipolar membrane electrodialysis is an electro-membrane separation technique that combines selective migration of ion-exchange membranes with water dissociation of bipolar membranes. This technique drives the migration of charged components under electrode potential differences. Therefore, applying bipolar membrane electrodialysis to peptide separation can achieve the targeted enrichment of negatively or positively charged antioxidant peptides. Based on the structural basis of the antioxidant activity of antioxidant peptides, the inventors' initial experimental objective was to enrich negatively charged peptides through bipolar membrane electrodialysis. After comparing the antioxidant activity of BMED three-compartment (acid compartment, base compartment, and sample compartment) peptides, the inventors also found that not only acid compartment peptides have high antioxidant activity, but base compartment peptides also have high antioxidant activity.

[0010] Conventional electrodialysis primarily relies on ion-exchange membranes and electric fields to achieve desalination or concentration of charged components, making it difficult to collect peptides with different charge characteristics separately. Charged peptides tend to migrate along with inorganic salts or other small charged molecules to the concentration compartment, hindering the separation of acidic, basic, and relatively neutral peptides. In contrast, bipolar membrane electrodialysis utilizes the H2O generated by the hydrolysis of the bipolar membrane. + and OH - In-situ acidification and alkalization environments are created in different compartments of the membrane stack. Combined with the selective mass transfer of anion and cation exchange membranes, the charge expression and migration direction of peptides under different pH conditions are regulated, thereby achieving the enrichment and collection of peptides with different charge characteristics in different compartments. Among them, peptides with strong negative or positive charge expression capabilities undergo directional migration under the action of an electric field and are enriched in the acid or alkali compartments respectively. Therefore, the bipolar membrane electrodialysis in this invention is not a simple desalination step, but a targeted enrichment technology for active peptides based on peptide charge differences and acid-base environment regulation, providing a more targeted separation and purification pathway for obtaining highly antioxidant active peptides.

[0011] The preferred bipolar membrane electrodialysis system includes anion exchange membrane, cation exchange membrane, and bipolar membrane.

[0012] The membrane stack of the bipolar membrane electrodialysis is preferably composed of multiple sets of membrane units.

[0013] The peptide solution is preferably obtained by enzymatic hydrolysis of proteins.

[0014] The protein sources include plants.

[0015] After the enzymatic hydrolysis is completed, it is preferable to first heat to inactivate the enzyme, then cool it and centrifuge to collect the supernatant, and filter it to obtain the peptide solution.

[0016] Preferably, the acidic and / or basic peptides are desalted and dried to obtain peptide powder with high antioxidant activity.

[0017] After desalting, it is preferable to first grade the obtained peptides by molecular weight fractionation and then perform drying treatment.

[0018] The molecular weight fractionation preferably collects peptides ≤3 kDa. Specifically, for base-compartment peptides, the molecular weight fractionation preferably collects peptides less than 1 kDa. For acid-compartment peptides, the molecular weight fractionation preferably collects peptides between 1 and 3 kDa. Compared to fractions greater than 3 kDa, peptides less than 1 kDa and those between 1 and 3 kDa exhibit stronger hydroxyl radical scavenging capabilities. Mechanism analysis: 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; fractions less than 1 kDa have shorter chains, less steric hindrance, and greater exposure of active sites; the 1-3 kDa chain length is relatively moderate, with more active sites and greater accessibility, thus possessing a strong free radical scavenging ability.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0020] 1. Unlike active peptide separation methods that rely on ultrafiltration, chromatography, or ordinary electrodialysis for coarse separation, this invention provides a method for preparing antioxidant active peptides by enriching negatively charged or positively charged peptides based on the relationship between peptide hydroxyl radical scavenging activity and the negative potential point of the D / E carboxyl group of acidic amino acids. The resulting antioxidant peptides have high antioxidant activity and can be used to prepare therapeutic drugs for oxidative stress-related diseases, or as antioxidant raw materials for food, nutritional supplements, or cosmetics, with broad application prospects.

[0021] 2. This invention uses bipolar membrane electrodialysis to target and enrich peptides with strong negative charge expression ability and carboxyl accessibility or peptides with strong positive charge expression ability. Compared with ordinary electrodialysis, bipolar membrane electrodialysis can free charged peptides from salt interference and achieve efficient separation and recovery of peptides with different charge characteristics.

[0022] 3. The acid and alkali chamber enriched components obtained by this invention both exhibit significantly enhanced hydroxyl radical scavenging ability; compared with the original protein hydrolysate, the apparent antioxidant activity of the acid and alkali chamber products after bipolar membrane electrodialysis treatment is significantly improved, indicating that the process not only has desalting or concentration effects, but also can reshape the charge composition and functional phenotype of peptides.

[0023] 4. This invention combines bipolar membrane electrodialysis charge enrichment with molecular weight fractionation to identify the ≤3 kDa fraction as the high-activity window. This fraction has both a suitable molecular size and a strong ability to express negative or positive charges. It exhibits a low IC50 and a high absolute ζ potential in the hydroxyl radical scavenging system, indicating that there is a more favorable match between its molecular size and accessible active sites.

[0024] 5. This invention further demonstrates, through zeta potential characterization, ion strength shielding, carboxyl neutralization, and cellular oxidative stress evaluation, that effective negative charge expression and accessible acidic sites are important factors affecting the antioxidant activity of peptides. This invention is applicable to the preparation of peptides with high antioxidant activity and provides an effective technical route for the isolation and purification of charge-responsive antioxidant peptides from different protein sources.

[0025] 6. Mass spectrometry analysis of bipolar membrane electrodialysis acid chamber products showed that acid chamber peptides have the following characteristics: (1) acidic residues have C-terminal distribution characteristics and are adjacent to hydrophobic residues, such as γ-Glu-Val, γ-Glu-Leu, etc.; (2) the near-N-terminal region contains one or more of proline, glycine or lysine, such as PG, GP, PGP, etc.

[0026] Regarding the above-mentioned feature (1), the 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 receptor (CaSR) and have the function of regulating blood calcium content; similarly related to CaSR activation, γ-glutamyl peptide has been reported to have potential functions such as anti-inflammatory, hypoglycemic, and appetite suppression, and can be used for metabolic regulation and maintenance of intestinal health; in addition, the combination of acidic-hydrophobic residues may regulate hydration, interfacial adsorption and assembly behavior, so the active peptide obtained in this invention also has the potential to be used as an interfacial / membrane interaction and delivery material and can be applied in the medical field.

[0027] Regarding the above feature (2), studies have reported 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.

[0028] In summary, this invention can efficiently prepare peptides with high antioxidant activity, and has good application prospects in the fields of biomedicine, food and health care. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the separation and purification of walnut peptides using bipolar membrane electrodialysis;

[0030] Figure 2 The negative charge density of <1, 1-3 and >3 kDa walnut peptides as determined by zeta potential;

[0031] Figure 3 This is a comparison of the hydroxyl radical scavenging activity of walnut peptides after electrodialysis. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0033] Example 1

[0034] This embodiment provides a method for separating and purifying peptides with high antioxidant activity using bipolar membrane electrodialysis. The specific steps are as follows.

[0035] Step 1: Preparation of Walnut Peptide Stock Solution. Weigh defatted walnut meal powder (obtained after walnut oil extraction by defatting methods such as supercritical fluid carbon dioxide method or organic reagent method), add deionized water, and prepare a 10% (w / v) substrate dispersion. Adjust the pH to 7.0, add Novozymes complex protease (Protamex 1.6) at a dosage of 1200 U / g substrate, and hydrolyze at 55℃ for 1 h. After hydrolysis, inactivate the enzyme by heating at 90-100℃. After cooling, centrifuge at 10000×g for 10 min, collect the supernatant, and filter through a 0.22 μm filter membrane to obtain the walnut peptide stock solution.

[0036] Step 2: Enrichment of negatively or positively charged peptides using bipolar membrane electrodialysis. The walnut peptide stock solution obtained in Step 1 is added to a bipolar membrane electrodialysis system. The system includes anion exchange membranes, cation exchange membranes, and a bipolar membrane, with the membrane stack consisting of multiple membrane units. The concentration of the walnut peptide stock solution is 5%, and the processing volume is 400 mL. The system operates under an electric field, with the preferred operating voltage being 35 V. Hydrolysis of the bipolar membrane generates H₂. + and OH − This creates localized acidification and alkalization environments in different compartments. Walnut peptides with a stronger negative charge expression capacity migrate into the acidic compartment, while those with a stronger positive charge expression capacity migrate into the alkali compartment (e.g.,...). Figure 1 (As shown). The acid and alkali solutions were collected to obtain negatively charged enriched walnut peptide solutions and positively charged enriched walnut peptide solutions.

[0037] Step 3: Desalting and Drying. The solutions obtained in Step 2 were subjected to desalting treatment to remove small molecule salts and other impurities. After desalting, they were freeze-dried to obtain negatively charged enriched walnut peptide powder and positively charged enriched walnut peptide powder.

[0038] Step 4: Molecular weight fractionation. The negatively charged enriched walnut peptide powder obtained in Step 3 was redissolved in water and fractionated sequentially using 3 kDa and 1 kDa ultrafiltration membranes to obtain walnut peptide fractions with values ​​greater than 3 kDa, 1-3 kDa, and less than 1 kDa, respectively. The 1-3 kDa fraction was collected, freeze-dried, and the highly antioxidant walnut peptide was obtained.

[0039] Example 2

[0040] In this embodiment, the salicylic acid-Fenton system and the iron-free UV / H2O2-TPA system were used to evaluate the hydroxyl radical scavenging ability of the walnut peptide stock solution obtained in step 1 of Example 1, the three-chambered walnut peptide obtained in step 2, and the three walnut peptide components obtained in step 4.

[0041] The results showed that, after enrichment by bipolar membrane electrodialysis, acid- and base-chambered walnut peptides with significantly enhanced hydroxyl radical scavenging activity were obtained (see...). Figure 3 Among them, the 1-3 kDa component exhibited the lowest IC50, indicating that this component is the main highly active component.

[0042] Example 3

[0043] In this embodiment, zeta potential analysis was used to evaluate the charge expression ability of the three walnut peptide components obtained in step 4 of Example 1 under different NaCl concentrations.

[0044] The results showed that the 1-3 kDa fraction had a high absolute zeta potential under low ionic strength conditions, indicating that its acidic sites had good accessibility and negative charge expression ability.

[0045] Example 4

[0046] This embodiment verifies the accessibility of carboxyl groups by using EDC / NHS-mediated amidation reaction between ethanolamine and the carboxyl groups of walnut peptides to achieve carboxyl group blocking or charge shielding.

[0047] The results showed that the hydroxyl radical scavenging ability of the 1-3 kDa fraction decreased significantly after the carboxyl groups were neutralized, indicating that the highly active walnut peptides enriched by bipolar membrane electrodialysis are closely related to their accessibility of carboxyl groups and their ability to express negative charges.

[0048] In summary, Examples 1-4, using walnut peptide as an example, demonstrate that the present invention can effectively prepare peptides with high antioxidant activity by directionally enriching negatively charged or positively charged peptides through bipolar membrane electrodialysis, providing an effective technical path for the separation and purification of charge-responsive antioxidant peptides from different protein sources.

Claims

1. A method for separating and purifying peptides with high antioxidant activity, characterized in that, The method includes the step of enriching negatively charged peptides and / or positively charged peptides.

2. The method according to claim 1, characterized in that, The step of enriching negatively charged peptides and / or positively charged peptides involves treating the peptide solution with bipolar membrane electrodialysis and collecting acidic and / or basic peptides.

3. The method according to claim 2, characterized in that, The bipolar membrane electrodialysis system includes anion exchange membranes, cation exchange membranes, and bipolar membranes.

4. The method according to claim 2, characterized in that, The bipolar membrane electrodialysis membrane stack consists of multiple sets of membrane units.

5. The method according to claim 2, characterized in that, The peptide solution is obtained by enzymatic hydrolysis of proteins.

6. The method according to claim 5, characterized in that, The protein sources include plants.

7. The method according to claim 5, characterized in that, After the enzymatic hydrolysis is completed, the enzyme is first inactivated by heating, then cooled and centrifuged to collect the supernatant, and then filtered to obtain the peptide solution.

8. The method according to claim 2, characterized in that, The acidic and / or basic peptides are desalted and dried to obtain a peptide powder with high antioxidant activity.

9. The method according to claim 8, characterized in that, After desalting, the obtained peptides are first graded by molecular weight and then dried.

10. The method according to claim 8, characterized in that, The molecular weight classification is for collecting peptides ≤3 kDa.

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