Method for improving stable activity and antioxidant activity of chicken peptide ADH as well as polypeptide and application thereof

Abstract

The invention discloses a method for improving the stable activity and antioxidant activity of chicken peptide ADH, and a polypeptide and application thereof. The ADH stable activity and the antioxidant activity of the chicken peptide can be improved by performing the plastein reaction on the chicken peptide, and it is proved for the first time that the ADH stable activity and the antioxidant activity of the chicken peptide can be further remarkably improved by adding exogenous amino acid in the plastein reaction; compared with the unreacted chicken peptide and the chicken peptide subjected to plastein reaction, the ADH stable activity and the oxidation resistance of the prepared product are remarkably improved by at least 105%; wherein under the plastein reaction action of alkaline protease, cysteine, leucine, tryptophan and lysine, the ADH stable activity and antioxidant activity of the chicken peptide are remarkably improved. The method provided by the invention is simple, convenient and rapid, the prepared polypeptide product has a remarkable effect, and more product raw material sources with better effects are provided.

Description

Technical Field

[0001] This invention relates to the fields of peptide preparation and biomedicine, and more specifically, to a method for improving the ADH stabilizing and antioxidant activities of chicken peptides, as well as the peptides and their applications. Background Technology

[0002] The plastein reaction, also known as the protein-like reaction, is a protein hydrolysis and resynthesis reaction catalyzed by proteases. Essentially, it is a reverse hydrolysis process of enzymes. Under specific high-concentration (usually >20%) substrate conditions, proteolytic enzymes catalyze the relinking of fragments in a broken peptide chain, forming new, larger, or structurally altered polypeptide products, thereby changing the structure and function of the polypeptide. The plastein reaction is mainly used for (1) enhancing / imparting bioactivity: enhancing the antioxidant, ACE inhibitory, antibacterial, and mineral binding activities of peptides by recombination or the introduction of specific groups; (2) improving nutritional and physical properties: eliminating unpleasant flavors and bitterness; improving solubility, emulsification, and foam stability; and improving the safety of allergenic proteins; (3) upgrading and utilizing protein resources: converting low-value proteins (such as fish processing by-products and oilseed meal) into high-value, easily digestible, and flavorful functional protein ingredients; (4) synthesizing customized structural peptides: using them as biocatalytic tools to synthesize peptides with specific sequences or structures under mild conditions for use in scientific research or pharmaceutical precursors; (5) using them in drug delivery systems: utilizing their ability to form hydrophobic aggregates to encapsulate hydrophobic active substances (such as vitamins, polyphenols, and drugs) to improve stability and bioavailability.

[0003] Due to the limitations of traditional Plastein reactions, such as high randomness, weak targeting, and unpredictable products, existing research typically employs chemical modification methods (e.g., PEGylation) to improve stability and half-life by chemically linking modifying groups. However, these methods involve harsh reaction conditions that may destroy activity and pose a risk of reagent toxicity. Current research has explored introducing amino acids to alter peptide structure and enhance activity. For example, existing technology discloses that adding cysteine ​​to the Plastein reaction can increase the DPPH radical scavenging rate of chicken lung peptides by 51.85% and the OH radical scavenging rate by 31.30%, thereby improving its thermal stability and raising the denaturation temperature of chicken lung peptides from 122.06℃ to 136.15℃. However, there are currently few Plastein reaction methods and reports for enhancing ADH (alcohol dehydrogenase) activity. Therefore, this invention application is filed to further enhance the ADH stability and antioxidant activity of chicken-derived peptides. Summary of the Invention

[0004] The technical problem to be solved by this invention is to improve the ADH stabilizing activity and antioxidant activity of existing chicken-derived peptides. This invention provides a method for improving the ADH stabilizing activity and antioxidant activity of chicken peptides, as well as the peptides and their applications.

[0005] The first objective of this invention is to provide a method for improving the stabilizing and antioxidant activities of chicken peptide ADH.

[0006] A second objective of this invention is to provide an application of the above-described method.

[0007] A third objective of this invention is to provide a chicken peptide.

[0008] A fourth objective of this invention is to provide applications of the aforementioned chicken peptides.

[0009] The fifth objective of this invention is to provide a product.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a method for improving the stabilizing and antioxidant activities of chicken peptide ADH, comprising the following steps: S1. Preparation of chicken peptides: Chop chicken breast meat, add 0.8 to 10 times the volume of distilled water, adjust the pH value, add 0.01 to 10% alkaline protease, hydrolyze for 0.5 to 72 hours, centrifuge after enzyme inactivation, collect the supernatant, freeze dry to obtain chicken peptides; S2. Add 0.01-10% protease to chicken peptides with a concentration of 15-50%, adjust the pH to 6-11, and hydrolyze with shaking at 15-70℃ for 0.5-72 hours. Or S2. Add 0.01-10% protease and 1-10% amino acids to chicken peptides at a concentration of 15-50%, adjust the pH to 6-11, and hydrolyze with shaking at 15-70℃ for 0.5-72 hours. S3. After completing the Plastein reaction, inactivate the enzyme, centrifuge, collect the supernatant, and freeze-dry or spray-dry to obtain polypeptide powder.

[0011] The chicken peptide CP prepared based on previous research of this invention possesses ADH stabilizing activity and antioxidant properties (ADH stabilizing activity of chicken peptide CP is 68.68±0.71%, ABTS free radical scavenging activity is 7982.44±349.86 μmol TE / g sample, and DPPH free radical scavenging activity is 15.5±0.27%), and can be used to alleviate alcoholic liver damage. This invention, through the plastein reaction, can improve the ADH stabilizing activity and antioxidant activity of chicken peptide (specifically, for ADH stabilizing activity, the N2 group showed the highest increase to 113.71±0.51% of CP; for ABTS free radical scavenging activity, the P2 group showed the highest increase to 123.87±2.41% of CP; for DPPH free radical scavenging activity, the A2 group showed the highest increase to 142.36±3.05% of CP). This invention is the first to demonstrate that the simultaneous addition of exogenous amino acids during the plastein reaction of chicken peptides can significantly enhance the ADH stabilizing and antioxidant activities of CP. Products prepared by linking cysteine, leucine, tryptophan, lysine, proline, or alanine showed significantly improved ADH stabilizing and antioxidant activities compared to unreacted chicken peptides and those undergoing only the plastein reaction (specifically, for ADH stabilizing activity, the A2-C5 group showed the highest increase to 135.91±2.23% of CP; for ABTS radical scavenging activity, the A2-W5 group showed the highest increase to 241.09±2.95% of CP; and for DPPH radical scavenging activity, the A2-C5 group showed the highest increase to 211.89±3.10% of CP). The method provided by this invention is simple, rapid, and uses mild and environmentally friendly reaction conditions, while also exhibiting high safety. The prepared peptide products show significantly improved efficacy and effectively avoid the effects of chemical modification methods, providing more and better-performing chicken peptide sources for alleviating alcoholic liver damage.

[0012] Preferably, 0.01-5% alkaline protease is used in S1.

[0013] Preferably, the protease in S2 is selected from alkaline protease, papain, or neutral protease.

[0014] More preferably, the protease is selected from alkaline proteases or neutral proteases.

[0015] More preferably, the protease is an alkaline protease.

[0016] Preferably, the concentration of the protease in S2 is 0.1-5%.

[0017] Preferably, the amino acid in S2 is selected from cysteine, leucine, tryptophan, lysine, proline, or alanine.

[0018] More preferably, the amino acid is selected from cysteine, leucine, tryptophan, or lysine.

[0019] More preferably, the amino acid is selected from cysteine ​​or tryptophan.

[0020] Preferably, the concentration of the chicken peptide CP is 25-35%.

[0021] More preferably, the concentration of the chicken peptide CP is 30%.

[0022] Preferably, the concentration of tryptophan is 3-8%.

[0023] More preferably, the concentration of tryptophan is 5%.

[0024] Preferably, the pH of S2 is adjusted to 6-9, and the mixture is hydrolyzed by shaking at 40-60°C for 1-12 hours.

[0025] This invention provides the application of the above method in the preparation of peptide products with high ADH stabilizing activity and antioxidant activity.

[0026] This invention provides a chicken peptide, which is prepared by the above method.

[0027] This invention provides the application of the above-mentioned chicken peptide in the preparation of ADH-activated products and / or antioxidant products.

[0028] The present invention also provides the application of the above-mentioned chicken peptide in the preparation of products that alleviate alcoholic liver damage.

[0029] Specifically, based on existing research reports that chicken peptide CP (Xiao Chuqiao. Preparation of ADH-activating peptide from chicken protein and its protective mechanism against alcoholic liver injury in mice [D]. South China University of Technology, 2020. DOI:10.27151 / d.cnki.ghnlu.2020.000069.) possesses ADH-stabilizing and antioxidant activities, and can be used to alleviate alcoholic liver injury and prepare products that alleviate alcoholic liver injury, those skilled in the art can expect that the polypeptide product prepared by the present invention based on chicken peptide CP will have better ADH-stabilizing and antioxidant activities, and can also be used to alleviate alcoholic liver injury, with a better expected effect. Therefore, the present invention also provides the application of the polypeptide product prepared by the above method in the preparation of products that alleviate alcoholic liver injury.

[0030] In addition, the present invention provides an ADH-activating product and / or an antioxidant product containing the above-mentioned chicken peptides.

[0031] The present invention has the following beneficial effects: This invention enhances the ADH stabilizing and antioxidant activities of chicken peptides through a Plastein reaction. The highest ADH stabilizing activity was increased to 113.71±0.51% of the CP, the highest ABTS radical scavenging activity was increased to 123.87±2.41% of the CP, and the highest DPPH radical scavenging activity was increased to 142.36±3.05% of the CP. Furthermore, it is the first time that the simultaneous addition of exogenous amino acids during the Plastein reaction of chicken peptides can further significantly enhance the ADH stabilizing and antioxidant activities of the CP, with the highest ADH stabilizing activity increased to 135.91±2.23% of the CP, the highest ABTS radical scavenging activity increased to 241.09±2.95% of the CP, and the highest DPPH radical scavenging activity increased to 211.89±3.10% of the CP. Products prepared by linking cysteine, leucine, tryptophan, lysine, proline, or alanine show significantly enhanced ADH stability and antioxidant activity compared to unreacted chicken peptides and chicken peptides undergoing only the plastein reaction. The method provided by this invention is simple, rapid, and uses mild and environmentally friendly reaction conditions, while also exhibiting high safety. The prepared peptide products show significantly improved efficacy and effectively avoid the negative effects of chemical modification methods, providing more and better-performing chicken peptide sources for alleviating alcoholic liver damage. Attached Figure Description

[0032] Figure 1 The results show the determination of ADH stability and antioxidant activity of different proteases and amino acids in the Plastein reaction.

[0033] Figure 2 Results of Plastein reaction ADH stabilization activity and antioxidant activity determination for different amino acid addition amounts.

[0034] Note: In the diagram, CP represents chicken peptides; A2, P2, and N2 represent Plastein reaction products hydrolyzed by alkaline protease, papain, and neutral protease, respectively; A2-C2, A2-L2, A2-W2, A2-K2, A2-P2, and A2-A2 represent Plastein reaction products hydrolyzed by alkaline protease with the addition of cysteine ​​(C), leucine (L), tryptophan (W), lysine (K), proline (P), and alanine (A), respectively; P2-C2, P2-L2, P2-W2, P2-K2, and P2... -P2 and P2-A2 represent the Plastein reaction products obtained by adding cysteine ​​(C), leucine (L), tryptophan (W), lysine (K), proline (P), and alanine (A) to papain, respectively; N2-C2, N2-L2, N2-W2, N2-K2, N2-P2, and N2-A2 represent the Plastein reaction products obtained by adding cysteine ​​(C), leucine (L), tryptophan (W), lysine (K), proline (P), and alanine (A) to neutral protease, respectively. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] Example 1: Preparation of Chicken Peptides The chicken peptides used in this embodiment (enzymatic hydrolysis products of chicken breast) were prepared according to the methods disclosed in existing research techniques (Xiao Chuqiao. Preparation of chicken protein source ADH activating peptides and study on their protective mechanism against alcoholic liver injury in mice [D]. South China University of Technology, 2020. DOI:10.27151 / d.cnki.ghnlu.2020.000069.).

[0038] The specific preparation method is as follows: Chicken breast is chopped and mixed with 5 times its volume of distilled water (mass / volume), then the pH is adjusted to 8.0. An alkaline protease is added at a concentration equivalent to 0.5% of the protein content, and hydrolysis is carried out in a 55°C constant-temperature shaker for 8 hours. Afterward, the enzyme is inactivated in boiling water for 15 minutes. After cooling to room temperature, the hydrolysate is centrifuged at 8000g for 15 minutes at 4°C. The supernatant is collected and freeze-dried to prepare chicken peptide CP.

[0039] Example 2 Plastein reaction method 1. Plastein reaction of different proteases Chicken peptide (CP) at a concentration of 30% w / v was treated with 1% of the following proteases: alkaline protease A2 (pH 8, 55℃), papain P2 (pH 7, 55℃), and neutral protease N2 (pH 7, 50℃). The pH was adjusted, and the mixture was hydrolyzed in a constant temperature shaker for 2 hours. The sample was then incubated in a boiling water bath for 10 minutes to inactivate the enzymes. After centrifugation at 4℃ and 5000g for 5 minutes, the supernatant was collected to obtain the Plastein reaction products, which were named A2, P2, and N2, respectively, using different proteases.

[0040] 2. Adding different amino acids to carry out the Plastein reaction Based on the above Plastein reaction, 30% CP was taken, and 1% alkaline protease, papain, and neutral protease were added respectively. At the same time, 2% of cysteine ​​(C), leucine (L), tryptophan (W), lysine (K), proline (P), and alanine (A) were added to CP. The Plastein reaction was carried out for 2 hours at the optimal pH and temperature of the enzyme. After that, the sample was placed in a boiling water bath for 10 minutes to inactivate the enzyme. It was then centrifuged at 4℃ and 5000g for 5 minutes. The supernatant was collected to obtain the Plastein reaction products. These Plastein reaction products of chicken peptide CP with different proteases and amino acids were named A2-C~A, P2-C~A, and N2-C~A respectively.

[0041] Example 3: Determination of ADH stabilizing activity and antioxidant properties (1) Relative ADH stability activity assay: 50 μL of sample solution (0.1 mg / mL of each product and CP from Example 2) was mixed with 50 μL of ADH (0.2 U / mL in 10 mM PBS, pH 7.4) and incubated at 37°C for 60 minutes. Then, 150 μL of detection reagent (containing 22.4 mM sodium pyrophosphate, 3.3% ethanol, and 7.8 mM NAD) was added. + The absorbance at 340 nm was recorded every minute for 10 minutes. Ultrapure water was used as a control. The initial reaction rate of unincubated ADH was defined as V0, while the reaction rate of ADH after 60 minutes of incubation was defined as Vs.

[0042] The relative ADH stable activity is calculated using the following formula: ADH stable activity = (Vs / V0) × 100%.

[0043] (2) ABTS free radical scavenging activity assay: A 7 mM ABTS stock solution was prepared using 2.45 mM potassium persulfate and stored at 4°C for 16 hours. Before use, the ABTS solution was diluted with 50 mM PBS (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. In the experiment, 20 μL of sample (0.1 mg / mL of each group of products and CP from Example 2) was mixed with 200 μL of ABTS solution, incubated in the dark for 6 minutes, and the absorbance was measured at 734 nm. PBS and Trolox were used as blank control and positive control, respectively.

[0044] The free radical scavenging rate is calculated using the following formula: ABTS scavenging rate (%) = (absorbance of control group - absorbance of sample) / absorbance of control group × 100%.

[0045] (3) DPPH radical scavenging activity: 30 μL of sample solution (10 mg / mL of each group of products and CP from Example 2) was mixed with 150 μL of ethanol, followed by the addition of 37.5 μL of DPPH ethanol solution (0.02% w / v). After mixing, the reaction was carried out in the dark for 60 minutes, and the absorbance was measured at 517 nm. The control group was prepared by replacing the sample with an equal volume of water.

[0046] DPPH free radical scavenging activity is calculated using the following formula: DPPH free radical scavenging activity (%) = (control absorbance) (Sample absorbance) × 100 / control absorbance.

[0047] The results are as follows Figure 1 As shown, compared to chicken peptide CP without Plastein reaction, both the ADH stabilizing activity and antioxidant activity of chicken peptide CP were significantly improved by performing a protein-like reaction on CP alone (with the addition of different proteases) and by performing a protein-like reaction with the simultaneous addition of six exogenous amino acids.

[0048] Regarding ADH stabilizing activity and antioxidant activity (ABTS and DPPH free radical scavenging effects), after 2 hours of hydrolysis, the reaction product introduced by the alkaline protease-promoted protein-like reaction, which introduced exogenous amino acids, was more beneficial in enhancing the ADH stabilizing activity of chicken peptide CP. Furthermore, the addition of 2% C, L, W, and K was superior to P and A. Simultaneously, under the action of alkaline protease, the addition of C, L, W, and K amino acids was also significantly more effective than the product that underwent the Plastein reaction without the addition of exogenous amino acids.

[0049] Example 4: Effect of different amounts of added amino acids on Based on the results of Example 3, alkaline protease and four exogenous amino acids (C, L, W, and K) were selected to study the effects of Plastein reaction products on ADH stabilization activity and antioxidant capacity under different amounts of exogenous amino acids. The Plastein reaction method was the same as in Example 3, except that the amount of amino acid added was set to 5%. After obtaining the reaction products, their relative ADH stabilization activity and antioxidant capacity were measured using the same method as in Example 3.

[0050] The results are as follows Figure 2 As shown, adding 5% C and W in the Plastein reaction is more beneficial to improving ADH stabilizing activity and its antioxidant activity, which is also better than that of chicken peptides and Plastein reaction products without added exogenous amino acids. Among them, the improvement effect of adding 5% W is more comprehensive and better, with the following specific effects: ADH stabilizing activity increased to 134.68±0.64% of CP, ABTS free radical scavenging activity increased to 241.10±2.95% of CP, and DPPH free radical scavenging activity increased to 209.64±2.20% of CP.

[0051] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for improving the stabilizing and antioxidant activities of chicken peptide ADH, characterized in that, Includes the following steps: S1. Preparation of chicken peptides: Chop chicken breast meat, add 0.8 to 10 times the volume of distilled water, adjust the pH value, add 0.01 to 10% alkaline protease, hydrolyze for 0.5 to 72 hours, centrifuge after enzyme inactivation, collect the supernatant, freeze dry to obtain chicken peptides; S2. Add 0.01-10% protease to chicken peptides with a concentration of 15-50%, adjust the pH to 6-11, and hydrolyze with shaking at 15-70℃ for 0.5-72 hours. Or S2. Add 0.01-10% protease and 1-10% amino acids to chicken peptides at a concentration of 15-50%, adjust the pH to 6-11, and hydrolyze with shaking at 15-70℃ for 0.5-72 hours. S3. After completing the Plastein reaction, inactivate the enzyme, centrifuge, collect the supernatant, and freeze-dry or spray-dry to obtain polypeptide powder.

2. The method according to claim 1, characterized in that, The protease described in S2 is selected from alkaline protease, papain, or neutral protease.

3. The method according to claim 1, characterized in that, The amino acid described in S2 is selected from cysteine, leucine, tryptophan, lysine, proline, or alanine.

4. The method according to claim 1, characterized in that, The concentration of chicken peptides mentioned in S2 is 25-35%.

5. The method according to claim 1, characterized in that, The concentration of the amino acid described in S2 is 3-8%.

6. The use of the method according to any one of claims 1 to 5 in the preparation of polypeptide products with high ADH stabilizing activity and antioxidant activity.

7. A chicken peptide, characterized in that, It is prepared by the method described in any one of claims 1 to 6.

8. The use of the chicken peptide of claim 7 in the preparation of ADH-activated products and / or antioxidant products.

9. The use of the chicken peptide of claim 7 in the preparation of a product for alleviating alcoholic liver damage.

10. An ADH activating product and / or an antioxidant product, characterized in that, Contains the chicken peptide as described in claim 7.

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