CN117230140A

CN117230140A - Preparation method and application of high antioxidant peptide

Info

Publication number: CN117230140A
Application number: CN202311192956.1A
Authority: CN
Inventors: 单良; 单琪; 丁占生; 周敏; 李妍
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2023-09-15
Filing date: 2023-09-15
Publication date: 2023-12-15

Abstract

The application discloses a preparation method and application of high antioxidant peptide, which successfully prepares polypeptide with high antioxidant activity from sea buckthorn leaves by preferably selecting protease to compound and jointly enzymolyze sea buckthorn leaf proteins with metal ions, preferably selecting proper metal ions to activate the activity of compound protease, and synergistically improving enzymolysis efficiency by influencing enzyme conformation and catalytic activity.

Description

Preparation method and application of high antioxidant peptide

Technical Field

The application belongs to the technical field of active peptide extraction, and particularly relates to a preparation method and application of high antioxidant peptide.

Background

Sea buckthorn is widely planted in northeast, northwest and southwest areas of China, and the sea buckthorn leaves are rich in resources. Hippophae rhamnoides has effects of relieving cough and asthma, resisting oxidation, and reducing blood lipid. The flavonoid compounds contained in the sea buckthorn leaves can also play an antioxidant role, and some skin aging resisting medicines contain the flavonoid compounds of the sea buckthorn leaves. Meanwhile, the flavonoid compound can well regulate blood viscosity, so that the flavonoid compound can play a role in reducing blood fat, plays a good role in preventing cardiovascular and cerebrovascular diseases, and is suitable for people with hyperglycemia and hypertension. The protein content in the sea buckthorn leaves is about 15% -20%, wherein the content of essential amino acids in human body is higher, especially the content of methionine, lysine and tryptophan is higher.

The polypeptide produced by the hydrolysis of the sea buckthorn leaf protein has the functions of resisting oxidation, resisting inflammation, reducing blood sugar, reducing blood fat and the like, and can be used for the production of various functional foods. The active peptide with antioxidant function can reduce free radical generation, inhibit lipid peroxidation, and relieve cell injury. Several reports of developing active peptide by utilizing sea buckthorn byproduct protein at present take sea buckthorn seed protein as raw material, and reports of preparing antioxidant peptide by utilizing sea buckthorn leaf protein are fresh. The sea buckthorn leaves are rich in nutrition and multiple in physiological active ingredients, and have good medical care effect. Therefore, the method has high development and utilization value. According to the previous research and practice, the industries which can be expected to develop with the seabuckthorn leaves are pharmaceutical industry, food industry, beverage industry, cosmetic industry, feed industry and the like, and the development and utilization of the seabuckthorn leaves are important components of the seabuckthorn industry.

At present, research shows that the sea-buckthorn leaf extract has an antioxidation effect, the sea-buckthorn leaf extract can be used for preventing and treating oxidization, animal experiments prove that the sea-buckthorn leaf extract has the antioxidation effect, but specific effects and research are still lacking, and how to efficiently extract peptides with high antioxidation activity from sea-buckthorn leaves is a problem to be solved.

Disclosure of Invention

This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.

The present application has been made in view of the above and/or problems occurring in the prior art.

Therefore, the application aims to overcome the defects in the prior art and provide a preparation method of high antioxidant peptide.

In order to solve the technical problems, the application provides the following technical scheme: comprising the steps of (a) a step of,

dissolving sea buckthorn leaf protein powder in deionized water to obtain sea buckthorn leaf protein solution, adding composite proteinase and metal ion into the solution for enzymolysis, centrifuging to obtain polypeptide liquid, concentrating and drying to obtain sea buckthorn leaf high antioxidant peptide.

As a preferable scheme of the preparation method of the high antioxidant peptide, the preparation method comprises the following steps: the ratio of the sea buckthorn protein powder to deionized water in the sea buckthorn leaf protein solution is 1:35-1:55.

As a preferable scheme of the preparation method of the high antioxidant peptide, the preparation method comprises the following steps: the compound protease comprises two or more of neutral protease, alkaline protease, trypsin, pepsin or papain.

As a preferable scheme of the preparation method of the high antioxidant peptide, the preparation method comprises the following steps: the compound protease is neutral protease and trypsin, wherein the compound ratio of the neutral protease to the trypsin is 1:0.5-3.

As a preferable scheme of the preparation method of the high antioxidant peptide, the preparation method comprises the following steps: the metal ions include Cu ²⁺ 、Zn ²⁺ 、Ni ²⁺ Or Ca ²⁺ One of them.

As a preferable scheme of the preparation method of the high antioxidant peptide, the preparation method comprises the following steps: the metal ion is Zn ²⁺ And Ca ²⁺ One of them.

As a preferable scheme of the preparation method of the high antioxidant peptide, the preparation method comprises the following steps: the enzymolysis time is 0.2-1 h, and the enzymolysis temperature is 40-50 ℃.

As a preferable scheme of the preparation method of the high antioxidant peptide, the preparation method comprises the following steps: the enzyme adding amount of the compound protease is 5000U/g-20000U/g.

As a preferable scheme of the preparation method of the high antioxidant peptide, the preparation method comprises the following steps: the concentration of the metal ions is 0.1-1.0 mg/mL.

It is a further object of the present application to provide the use of a highly antioxidant peptide for the preparation of a product for the treatment or prevention of aging.

The application has the beneficial effects that:

the application successfully prepares the polypeptide with high antioxidant activity from the sea buckthorn leaves by preferentially selecting protease to combine with metal ions to jointly hydrolyze sea buckthorn leaf proteins, preferably selecting proper metal ions to activate the activity of compound protease, and synergistically improving enzymolysis efficiency by influencing the conformation and catalytic activity of the enzyme.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:

FIG. 1 is a graph showing the effect of DPPH radical scavenging rate of antioxidant peptide obtained by different ratios of feed to water in example 3 of the present application.

FIG. 2 is a graph showing the effect of DPPH radical scavenging rate of antioxidant peptide obtained by different enzyme addition amounts in example 4 of the present application.

FIG. 3 is a graph showing the effect of DPPH radical scavenging rate of antioxidant peptides obtained by adding different amounts of metal ions in example 5 of the present application.

FIG. 4 is a graph showing the effect of the compound ratios of the different complex enzymes of example 6 on DPPH radical scavenging rate of antioxidant peptide.

Detailed Description

In order that the above-recited objects, features and advantages of the present application will become more apparent, a more particular description of the application will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present application is not limited to the specific embodiments disclosed below.

Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the application. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The sea buckthorn leaf protein powder used in the application is sea buckthorn leaf crude protein powder, and the extraction method comprises the following steps:

pouring the sea buckthorn leaf fragments into a powdering machine, picking most sea buckthorn branches, crushing the sea buckthorn branches to 60 meshes, preparing the sea buckthorn branches by an alkali dissolution and acid precipitation method according to a feed liquid ratio of 1:30, regulating the pH to 11 by using a 1mol/L sodium hydroxide solution at a rotating speed of 850r/min, and timing for 90min. Then the stirrer is turned off, the sea buckthorn leaf solution is poured into a 500mL centrifugal bottle, the sea buckthorn leaf solution is placed into a centrifugal machine after being balanced, the sea buckthorn leaf solution is centrifuged for 20min at 4000r/min, and the supernatant is poured into a 5000mL beaker. Stirring the supernatant with a stirrer at 850r/min, adjusting pH to 3.5 with 1mol/L hydrochloric acid, turning off the stirrer, and standing for 30min. And pouring most of supernatant liquid to leave sediment, pouring the sediment into a 500mL centrifugal bottle, balancing, putting the sediment into a centrifugal machine, centrifuging for 20min at the rotating speed of 4000r/min, and taking the sediment to obtain the sea buckthorn leaf protein powder.

The method for detecting the antioxidant activity in the application comprises the following steps:

0.00197g of DPPH powder is weighed, added into 40mL of absolute ethyl alcohol for dissolution, and the solution is prepared into 0.5mmol/LDPPH solution by constant volume into a 100mL volumetric flask;

during detection, 1mL of 0.5mmol/LDPPH solution is accurately taken out by a liquid-transfering gun and added into 9mL of absolute ethyl alcohol, the solution is diluted to 0.05mmol/L, 1mL of the sea buckthorn leaf crude protein solution after enzymolysis is accurately taken out by the liquid-transfering gun and added into 9mL of deionized water, the solution is diluted to 10 times, and then 4mL of the sea buckthorn leaf crude protein solution after enzymolysis which is diluted by 10 times is accurately taken out by the liquid-transfering gun and added into 4mL of deionized water, and a sample is diluted to 20;

the blank group was also diluted to 20-fold in the same manner;

the group A is to accurately absorb 0.5mL of sea buckthorn leaf crude protein solution diluted 20 times by a liquid-transfering gun, add the solution into a 5mL small centrifuge tube, and then add 3.5mL of 0.05mmol/LDPPH solution;

the group B is to accurately absorb 0.5mL of sea buckthorn leaf crude protein solution diluted 20 times by a liquid-transferring gun, add the solution into a 5mL small centrifuge tube, and then add 3.5mL of absolute ethyl alcohol;

group C was 4mL of a 0.05mmol/LDPPH solution and was set as a control group.

Incubation was carried out in a constant temperature bath at 37℃for 30min in the absence of light, and after removal, the absorbance was measured at a wavelength of 517nm and the antioxidant activity was calculated.

The other raw materials used in the application are common and commercially available in the field without special description, and in particular, the enzyme activities of the protease used in the application are respectively as follows: pepsin (15000U/mg), trypsin (250U/mg), papain (800U/mg), alkaline protease (200U/mg), neutral protease (100U/mg).

Example 1

The embodiment provides a preparation method of high antioxidant peptide, which specifically comprises the following steps:

dissolving Hippophae rhamnoides powder in deionized water according to a ratio of 1:45 to obtain Hippophae rhamnoides leaf protein solution, adding 10000U/g of compound protease (neutral protease and trypsin are compounded according to 1:2) and 0.5mg/ml Ca into the solution ²⁺ Regulating pH of enzymolysis solution to 7.5, performing enzymolysis at 45deg.C for 0.6 hr, heating and inactivating enzyme in water bath at 90deg.C, centrifuging at high speed for 20min, collecting supernatant to obtain antioxidant active peptide liquid, concentrating and drying to obtain Hippophae rhamnoides leaf high antioxidant peptide, and determining that the antioxidant active peptide DPPH free radical clearance is 95.23%

Example 2

This example differs from example 1 in that Ca was adjusted ²⁺ Is Zn ²⁺ The method specifically comprises the following steps:

dissolving Hippophae rhamnoides powder in deionized water according to a ratio of 1:45 to obtain Hippophae rhamnoides leaf protein solution, adding 10000U/g of compound protease (neutral protease and trypsin are compounded according to 1:2) and 0.5mg/ml Zn into the solution ²⁺ Regulating pH of enzymolysis solution to 7.5, performing enzymolysis at 45deg.C for 0.6 hr, heating and inactivating enzyme in water bath at 90deg.C, centrifuging at high speed for 20min, collecting supernatant to obtain antioxidant active peptide liquid, concentrating and drying to obtain Hippophae rhamnoides leaf high antioxidant peptide, and determining that the antioxidant active peptide DPPH free radical clearance obtained in this example is 91.37%

Example 3

The influence of different ratios of water to the obtained antioxidant active peptide liquid in the sea buckthorn protein liquid is examined, and the difference of the method is that the method is different from the method in the embodiment 1 in that the ratio of feed liquid is adjusted to be 1:35, 1:40, 1:45, 1:50 and 1:55 respectively, the rest of the process steps are the same as the embodiment 1, the antioxidant active peptide liquid in the embodiment under different ratios of water is obtained, and the DPPH free radical clearance is measured, and the result is shown in figure 1.

As can be seen from fig. 1, the solution-to-solution ratio has a significant effect on the DPPH radical scavenging rate of the oxidation-resistant active peptide solution, and the radical scavenging rate is highest at a solution-to-solution ratio of 1:45, indicating that the enzyme concentration and the substrate concentration reach equilibrium at this ratio, so that the enzymolysis reaction rate is maximized.

Example 4

The present example was used to investigate the effect of the enzyme addition amounts of different compound proteases on the effect of scavenging free radicals in the obtained antioxidant active peptide liquid, and was different from example 1 in that the enzyme addition amounts of the compound proteases were adjusted to 5000, 10000, 15000 and 20000U/g, respectively, and the other process steps were the same as in example 1, to obtain antioxidant active peptide liquid under different enzyme addition amounts of the compound proteases of this example, and the scavenging free radicals of DPPH was measured, and the results are shown in fig. 2.

As can be seen from FIG. 2, the enzyme addition has a significant effect on DPPH radical scavenging rate against the oxidation-active peptide solution, and the radical scavenging rate is highest at an enzyme addition of 10000U/g.

Example 5

The influence of the addition amounts of different metal ions on the effect of the free radical scavenging rate of the obtained antioxidant active peptide liquid was examined in this example, and the difference from example 1 was that the addition amounts of the metal ions were adjusted to 0.25, 0.5, 0.75, 1.0 and 2.0mg, respectively, and the other steps were the same as in example 1, to obtain antioxidant active peptide liquid at the addition amounts of different metal ions in this example, and the DPPH free radical scavenging rate was measured, and the result is shown in FIG. 3.

As can be seen from fig. 3, the addition amount of the metal ion has a remarkable influence on the DPPH radical scavenging rate of the oxidation-active peptide solution, the radical scavenging rate is highest at an enzyme addition amount of 0.5mg, and too much addition amount of the metal ion has an inhibitory effect on the enzyme activity, so that it is necessary to reasonably control the addition amount of the metal ion.

Example 6

The present example was used to investigate the effect of the compound ratio of neutral protease to trypsin in different compound proteases on the effect of scavenging free radicals in the obtained antioxidant active peptide liquid, and was different from example 1 in that the neutral protease and trypsin were adjusted to be 1:1, 1:2, and 1:3, respectively, and the remaining process steps were the same as example 1, to obtain antioxidant active peptide liquid in different compound ratios in this example, and the result of measuring the scavenging free radicals of DPPH was shown in fig. 4.

As can be seen from FIG. 4, the better effect can be obtained under the combination ratio of the neutral protease and the trypsin, and the best effect is achieved when the ratio of the neutral protease to the trypsin is 1:2.

Comparative example 1

The comparative example was different from example 1 in that the complex protease was adjusted to be neutral protease only, and the other steps were the same as in example 1 to obtain an antioxidant active peptide solution of the comparative example, and the DPPH radical scavenging rate was measured.

Comparative example 2

The comparative example was different from example 1 in that the complex protease was adjusted to be alkaline protease only, and the other steps were the same as in example 1 to obtain an antioxidant active peptide solution of the comparative example, and the DPPH radical scavenging rate was measured.

Comparative example 3

The comparative example was different from example 1 in that trypsin was used as the only compound protease, and the other steps were the same as those in example 1 to obtain an antioxidant active peptide solution of the comparative example, and DPPH radical scavenging rate was measured.

Comparative example 4

The comparative example was different from example 1 in that pepsin alone was used as the modified complex protease, and the other steps were the same as in example 1 to obtain an antioxidant active peptide solution of the comparative example, and the DPPH radical scavenging rate was measured.

Comparative example 5

The comparative example was different from example 1 in that the compound protease was adjusted to be papain only, and the other steps were the same as in example 1 to obtain an antioxidant active peptide solution of the comparative example, and the DPPH radical scavenging rate was measured.

Comparative example 6

The comparative example is different from example 1 in that the compound protease is regulated to be neutral protease and alkaline protease to be compounded according to the ratio of 1:2, and the rest process steps are the same as those of example 1, so that the antioxidant active peptide liquid of the comparative example is obtained, and the DPPH free radical clearance is measured.

Comparative example 7

The comparative example is different from example 1 in that the compound protease is prepared by compounding pepsin and papain according to a ratio of 1:2, and the rest process steps are the same as those of example 1, so that the antioxidant active peptide liquid of the comparative example is obtained, and the DPPH free radical clearance is measured.

The DPPH radical scavenging rates of the antioxidant active peptide solutions of comparative example 1 and comparative examples 1 to 7 are shown in Table 1.

TABLE 1

From table 1, it can be seen that when the enzyme is selected as the complex enzyme of neutral proteinase and trypsin, the obtained antioxidant peptide has highest free radical clearance, which is superior to the single use of the two, indicating that the two have synergistic effect, and can effectively identify the structure of sea buckthorn leaf protein so as to improve the enzymolysis effect; in addition, pepsin exhibits the best enzymatic hydrolysis effect when different kinds of enzymes are used alone, but when it is compounded with trypsin, the effect is rather inferior to that of neutral protease, which may be due to the similar substrate binding site between pepsin and trypsin, thereby producing an inhibitory effect, and conversely reducing the enzymatic hydrolysis effect.

Comparative example 8

This comparative example is different from example 1 in that the metal ion species was adjusted to Cu ²⁺ The other steps were the same as in example 1 to obtain an antioxidant active peptide solution of this comparative example, and the DPPH radical scavenging rate was measured.

Comparative example 9

This comparative example is different from example 1 in that the metal ion species was adjusted to Ni ²⁺ The other steps were the same as in example 1 to obtain an antioxidant active peptide solution of this comparative example, and the DPPH radical scavenging rate was measured.

Comparative example 10

This comparative example is different from example 1 in that the metal ion species was adjusted to Fe ²⁺ The other steps were the same as in example 1 to obtain an antioxidant active peptide solution of this comparative example, and the DPPH radical scavenging rate was measured.

Comparative example 11

This comparative example is different from example 1 in that the metal ion species was adjusted to Co ²⁺ The other steps were the same as in example 1 to obtain an antioxidant active peptide solution of this comparative example, and the DPPH radical scavenging rate was measured.

TABLE 2

As can be seen from Table 2, the different kinds of metal ions have a significant effect on the radical scavenging rate of the product, since the metal ions can bind to the enzyme to improve the enzymatic hydrolysis efficiency by changing the conformation and activity of the enzyme, ca ²⁺ Ion and Zn ²⁺ The addition of ions can be better combined with trypsin and neutral protease, so that the active site of the enzyme is more easily combined with a substrate, the catalytic effect of the enzyme is improved, and in addition, the two metal ions can stabilize the structure of the enzyme and improve the heat resistance and stability of the enzyme. And Fe (Fe) ²⁺ Ion and Co ²⁺ Ions may not bind to the trypsin of the application with neutral protease sufficiently stably, even inhibiting enzyme activity, resulting in reduced free radical clearance of the product.

In conclusion, the application successfully prepares the polypeptide with high antioxidant activity from the sea buckthorn leaves by preferentially selecting protease to compound and jointly enzymolyze the sea buckthorn leaf protein together with metal ions, preferably selecting proper metal ions to activate the activity of compound protease, and synergistically improving the enzymolysis efficiency by influencing the conformation and the catalytic activity of the enzyme.

It should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered in the scope of the claims of the present application.

Claims

1. A preparation method of a high antioxidant peptide is characterized by comprising the following steps: comprising the steps of (a) a step of,

2. The method for producing a highly antioxidant peptide according to claim 1, wherein: the ratio of the sea buckthorn protein powder to deionized water in the sea buckthorn leaf protein solution is 1:35-1:55.

3. The method for producing a highly antioxidant peptide according to claim 1, wherein: the compound protease comprises two or more of neutral protease, alkaline protease, trypsin, pepsin or papain.

4. The method for producing a highly antioxidative peptide according to claim 3, wherein: the compound protease is neutral protease and trypsin, wherein the compound ratio of the neutral protease to the trypsin is 1:0.5-3.

5. The method for producing a highly antioxidant peptide according to claim 1, wherein: the metal ions include Cu ²⁺ 、Zn ²⁺ 、Ni ²⁺ Or Ca ²⁺ One of them.

6. The method for producing a highly antioxidative peptide according to claim 5, wherein: the metal ion is Zn ²⁺ And Ca ²⁺ One of them.

7. The method for producing a highly antioxidant peptide according to claim 1, wherein: the enzymolysis time is 0.2-1 h, and the enzymolysis temperature is 40-50 ℃.

8. The method for producing a highly antioxidant peptide according to claim 1, wherein: the enzyme adding amount of the compound protease is 5000U/g-20000U/g.

9. The method for producing a highly antioxidant peptide according to claim 1, wherein: the concentration of the metal ions is 0.1-1.0 mg/mL.

10. Use of a highly antioxidant peptide prepared by the preparation method according to any one of claims 1 to 9 for the preparation of a product for the treatment or prevention of aging.