阅读说明：本技术 一种具有抗氧化活性的鸡骨胶原蛋白肽及其应用 (Chicken bone collagen peptide with antioxidant activity and application thereof ) 是由 黄春敏 于 2021-09-30 设计创作，主要内容包括：本发明公开了一种具有抗氧化活性的鸡骨胶原蛋白肽及其应用,涉及胶原蛋白肽加工技术领域。本发明鸡骨胶原蛋白肽是经过原料的处理、提取胶原蛋白溶液,并对其进行酶解、离心脱渣、脱色脱腥、超滤、纳滤膜脱盐浓缩、离心分离、灭菌以及离心喷雾干燥后制备而成。本发明所制备的鸡骨胶原蛋白肽具有较高的抗氧化活性,对·OH、DPPH·、O2-·三种典型自由基均具有较高的清除作用,其清除效果与其浓度呈正相关。(The invention discloses chicken bone collagen peptide with antioxidant activity and application thereof, and relates to the technical field of collagen peptide processing. The chicken bone collagen peptide is prepared by treating raw materials, extracting a collagen solution, performing enzymolysis, centrifugal deslagging, decoloring and fishy smell removing, ultrafiltration, nanofiltration membrane desalination and concentration, centrifugal separation, sterilization and centrifugal spray drying. The chicken bone collagen peptide prepared by the invention has higher antioxidant activity, has higher scavenging effect on three typical free radicals of OH, DPPH and O2, and the scavenging effect is positively correlated with the concentration thereof.)

1. The chicken bone collagen peptide with antioxidant activity is characterized by comprising the following steps:

(1) treatment of raw materials: crushing and sieving the chicken bone residues;

(2) extracting a collagen solution: putting the chicken bone dregs obtained in the step (1) into a reaction kettle, washing with clear water, adding 2-4 times of water by mass, and cooking for 3-4 hours at the temperature of 115-121 ℃ and under the pressure of 0.15-0.2 MPa to obtain a collagen solution;

(3) enzymolysis: adding neutral protease, pancreatin and compound flavourzyme into the collagen solution extracted in the step (2) for carrying out step-by-step enzymolysis treatment, and carrying out enzyme deactivation treatment after the enzymolysis is finished to obtain secondary enzymolysis liquid;

(4) centrifugal deslagging: carrying out centrifugal deslagging treatment on the secondary enzymolysis liquid after enzyme deactivation and sterilization, and filtering to obtain a clear liquid;

(5) decoloring and deodorizing: maintaining the temperature of the clear liquid obtained in the step (4) at 50-60 ℃, performing decolorization and deodorization treatment, and filtering to obtain decolorized and deodorized liquid;

(6) and (3) ultrafiltration: performing ultrafiltration on the decolorized and fishy smell-removed solution by using an ultrafiltration membrane to remove impurities to obtain ultrafiltrate;

(7) and (3) nanofiltration membrane desalination and concentration: concentrating and desalting ultrafiltrate by a nanofiltration membrane to obtain concentrated solution, wherein the concentration of the concentrated solution reaches 15%, ash content is less than or equal to 1%, and the desalting rate reaches more than 95%;

(8) centrifugal separation: carrying out centrifugal separation treatment on the concentrated solution to achieve high purification of the feed liquid;

(9) and (3) sterilization: sterilizing the clear liquid obtained by centrifugal separation;

(10) centrifugal spray drying, namely performing powder spraying drying on the sterilized clear liquid by using a high-speed centrifugal spray drying granulator;

(11) and (6) packaging a finished product.

2. The chicken bone collagen peptide with antioxidant activity according to claim 1, wherein the chicken bone residue is pulverized and sieved with a 10-20 mesh sieve in the step (1).

3. The chicken bone collagen peptide with antioxidant activity according to claim 1, wherein the amount of the neutral protease added in step (3) is 0.2-0.3% by mass of the collagen solution.

4. The chicken bone collagen peptide with antioxidant activity according to claim 3, wherein the pancreatin added in step (3) is 0.2-0.3% by mass of the collagen solution.

5. The chicken bone collagen peptide with antioxidant activity according to claim 4, wherein the compound flavor protease in the step (3) is a protease prepared by fermenting and refining Aspergillus oryzae strains, and comprises two components of polypeptide exo-active enzyme and endo-active enzyme, and the adding amount of the compound flavor protease is 0.05-0.15% of the mass of the collagen solution.

6. The chicken bone collagen peptide with antioxidant activity according to claim 5, wherein the step-by-step enzymolysis in step (3) is to perform enzymolysis on a collagen solution by using neutral protease and pancreatin, and then add compound flavourzyme for enzymolysis; or firstly carrying out enzymolysis by using neutral protease, then adding pancreatin for enzymolysis, and finally adding compound flavor protease for enzymolysis; or firstly carrying out enzymolysis by using neutral protease, and then adding pancreatin and compound flavor protease for mixed enzymolysis.

7. The chicken bone collagen peptide with antioxidant activity according to claim 1, wherein the decolorization and deodorization treatment in step (5) is carried out by using activated carbon fiber membrane equipment.

8. The chicken bone collagen peptide with antioxidant activity according to claim 1, wherein the rotation speed of the centrifugal treatment in step (8) is 16000r/min, and the separation factor is: 15050 r.c.f.

9. The chicken bone collagen peptide with antioxidant activity according to claim 1, wherein the sterilization treatment in step (9) is carried out by using a tubular high temperature instantaneous sterilization apparatus provided with a preheating device, a sterilization device, a heat recovery device and a cooling device.

10. Use of the chicken bone collagen peptide with antioxidant activity according to any one of claims 1 to 9 as an antioxidant.

Technical Field

The invention belongs to the technical field of collagen peptide processing, and particularly relates to chicken bone collagen peptide with antioxidant activity and application thereof.

Background

Free radical induced Reactive Oxygen Species (ROS), oxidative stress and lipid peroxidation have attracted widespread attention. A large number of active oxygen particles and free radicals are easy to react with other components in the organism in metabolism, thereby accelerating the aging of the organism and playing a leading role in a series of disease deterioration, such as cardiovascular diseases, cancers and the like. The antioxidant active peptide can reduce free radicals and improve the antioxidant function of organisms. The food-derived antioxidant peptide gradually becomes a safe and effective antioxidant because of having the characteristics of small molecular weight, simple structure, easy absorption by human bodies, high antioxidant activity, strong stability under different environmental conditions and the like.

Animal bones contain a large amount of collagen, and researches show that animals with shorter growth periods have richer collagen content, the collagen is easier to dissolve out after being heated, and the collagen dissolution rate of aged animals is low after being heated. The slaughtering period of the chicken is 60 days, the slaughtering period of the pig is 70 days, the growing period of the cattle is more than one year, and obviously, the extraction of the collagen in the chicken bones has superiority and operability.

At present, the processing of the chicken bones is mainly based on products with low added values, such as crude bone meal, bone paste, bone oil and the like, and the chicken bones have heavy fishy smell and obvious granular sensation after being crushed, which is a technical problem that the processing of bone foods is difficult to avoid. The chicken bone resources are mostly used for animal feed and waste except for part of processing and eating. The chicken bone contains rich nutrient substances, particularly rich protein, and more than 90% of bone protein is collagen. Therefore, the technical problem to be solved by the industry at present is to obtain the chicken bone collagen peptide with antioxidant activity from chicken bones.

Disclosure of Invention

Based on the above, the invention provides the chicken bone collagen peptide with the antioxidant activity, which takes chicken bones as raw materials to obtain the collagen peptide with excellent antioxidant performance.

The chicken bone collagen peptide with antioxidant activity of the invention is prepared by the following steps:

(1) treatment of raw materials: crushing and sieving the chicken bone residues;

(2) extracting a collagen solution: putting the chicken bone dregs obtained in the step (1) into a reaction kettle, washing with clear water, adding 2-4 times of water by mass, and cooking for 3-4 hours at the temperature of 115-121 ℃ and under the pressure of 0.15-0.2 MPa to obtain a collagen solution;

(3) enzymolysis: adding neutral protease, pancreatin and compound flavourzyme into the collagen solution extracted in the step (2) for carrying out step-by-step enzymolysis treatment, and carrying out enzyme deactivation treatment after the enzymolysis is finished to obtain secondary enzymolysis liquid;

(4) centrifugal deslagging: carrying out centrifugal deslagging treatment on the secondary enzymolysis liquid after enzyme deactivation and sterilization, and filtering to obtain a clear liquid;

(5) decoloring and deodorizing: maintaining the temperature of the clear liquid obtained in the step (4) at 50-60 ℃, performing decolorization and deodorization treatment, and filtering to obtain decolorized and deodorized liquid;

(6) and (3) ultrafiltration: performing ultrafiltration on the decolorized and fishy smell-removed solution by using an ultrafiltration membrane to remove impurities to obtain ultrafiltrate;

(7) and (3) nanofiltration membrane desalination and concentration: concentrating and desalting ultrafiltrate by a nanofiltration membrane to obtain concentrated solution, wherein the concentration of the concentrated solution reaches 15%, ash content is less than or equal to 1%, and the desalting rate reaches more than 95%;

(8) centrifugal separation: carrying out centrifugal separation treatment on the concentrated solution to achieve high purification of the feed liquid;

(9) and (3) sterilization: sterilizing the clear liquid obtained by centrifugal separation;

(10) centrifugal spray drying, namely performing powder spraying drying on the sterilized clear liquid by using a high-speed centrifugal spray drying granulator;

(11) and (6) packaging a finished product.

Preferably, in the step (1), the chicken bone residues are crushed and sieved by a 10-20-mesh sieve.

Preferably, the neutral protease in the step (3) is produced by bioscience, ltd, changhua, south ning, and the addition amount of the neutral protease accounts for 0.2-0.3% of the mass of the collagen solution.

More preferably, the pancreatin added in the step (3) accounts for 0.2 to 0.3 percent of the mass of the collagen solution.

More preferably, the compound flavor protease in the step (3) is a protease prepared by fermenting and refining aspergillus oryzae strains, comprises two components of polypeptide exo-active enzyme and endo-active enzyme, and is provided by southern ningdong Hengchengdao biotechnology limited company, and the adding amount of the compound flavor protease accounts for 0.05-0.15% of the mass of the collagen solution.

Particularly preferably, the step-by-step enzymolysis in the step (3) is to perform enzymolysis on a collagen solution by using neutral protease and pancreatin, and then add the compound flavourzyme for performing enzymolysis; or firstly carrying out enzymolysis by using neutral protease, then adding pancreatin for enzymolysis, and finally adding compound flavor protease for enzymolysis; or firstly carrying out enzymolysis by using neutral protease, and then adding pancreatin and compound flavor protease for mixed enzymolysis.

Preferably, activated carbon fiber membrane equipment is adopted in the step (5) for decolorization and deodorization treatment.

Preferably, the rotation speed of the centrifugal treatment in the step (8) is 16000r/min, and the separation factor is as follows: 15050R.C.F

Preferably, the step (9) adopts a tubular high-temperature instantaneous sterilization device for sterilization treatment, and the sterilization device is provided with a preheating device, a sterilization device, a heat recovery device and a cooling device.

The chicken bone collagen peptide obtained by the invention has higher antioxidant performance and can be used as an antioxidant.

Compared with the prior art, the invention has the following beneficial effects:

the molecular weight of the chicken bone collagen peptide prepared by the invention is relatively centralized and is less than 10000Da, through a PGAR sequence obtained by LC-MS/MS polypeptide sequence analysis, through three typical free radical system experiments of OH, DPPH, O2 and iron ion reduction capability experiments, PGAR (chicken bone collagen peptide) has a scavenging effect on three free radicals, and the scavenging effect is positively correlated with the concentration of the chicken bone collagen peptide. When the concentration of PGAR reaches 20mg/mL, the DPPH & clearance rate is 80.24%; while at a concentration of 9.0mg/mL, the OH clearance reaches 92.50%; while at a concentration of 20mg/mL, the scavenging effect of O2-was 59.80%. It can be seen that PGAR has significant antioxidant activity in the pathway of blocking free radical reactions. PGAR shows stronger reducing capability in the experiment of reducing iron ions, which also indicates that PGAR has antioxidant activity through a way of complexing metal ions.

Drawings

FIG. 1 is a process flow diagram of the chicken bone collagen peptide of the present invention;

FIG. 2 is a total ion chromatogram of chicken bone collagen peptide of example 1;

FIG. 3 is a second-order mass spectrum of PGAR peptide fragment of example 1 of the present invention;

FIG. 4 is a total ion chromatogram of chicken bone collagen peptide of example 2;

FIG. 5 is a second-order mass spectrum of PGAR peptide fragment of example 2 of the present invention;

FIG. 6 is a total ion chromatogram of chicken bone collagen peptide of example 3;

FIG. 7 is a second-order mass spectrum of PGAR peptide fragment of example 3 of the present invention;

FIG. 8 shows the DPPH free radical scavenging effect of chicken bone collagen Peptide (PGAR) and Vc according to the present invention;

FIG. 9 shows the effect of chicken bone collagen Peptide (PGAR) and Vc on the removal of hydroxyl radicals in accordance with the present invention;

FIG. 10 shows the scavenging effect of chicken bone collagen Peptide (PGAR) and Vc on superoxide anion radical according to the present invention;

FIG. 11 shows the reduction performance test of chicken bone collagen Peptide (PGAR) and Vc according to the present invention.

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1

A preparation method of chicken bone collagen peptide with antioxidant activity comprises the following steps:

(1) the raw materials are preferably as follows: taking a certain amount of crushed chicken bone residues qualified by inspection and quarantine, wherein the mesh number of the chicken bone residues is 10-20 meshes;

(2) extracting a collagen solution: putting the chicken bone dregs obtained in the step (1) into a reaction kettle, washing with clear water, adding 2 times of water by mass, and cooking for 4 hours at 120 ℃ under 0.2 MPa;

(3) step-by-step enzymolysis: adding neutral protease accounting for 0.3 percent of the mass of the collagen liquid and pancreatin accounting for 0.2 percent of the mass of the collagen liquid into the collagen liquid extracted in the step (2), carrying out enzymolysis for 3 hours at the temperature of 52 ℃ and the pH value of 7, adding compound flavor protease accounting for 0.1 percent of the mass of the collagen liquid into the obtained enzymolysis liquid, carrying out enzymolysis for 0.5 hour at the temperature of 55 ℃ and the pH value of 7, and carrying out enzyme deactivation;

(4) centrifugal deslagging: removing residues of the enzyme-inactivated and sterilized enzymolysis solution by a horizontal spiral sedimentation centrifuge, and completely separating suspended powder and liquid in the enzymolysis solution;

(5) decoloring and deodorizing: cooling the filtered clear liquid obtained in the step (4) to 50 ℃, and then decoloring and deodorizing by using activated carbon fiber membrane equipment;

(6) membrane separation: ultrafiltering the decolorized fishy smell removing solution by a multifunctional inorganic ceramic membrane separation device, efficiently removing impurities, and filtering the filtrate to a clear solution storage tank at the filtering temperature of 60 ℃ and the filtering pressure of 0.2 MPa;

(7) and (3) nanofiltration membrane desalination and concentration: concentrating and desalting the filtrate by using a nanofiltration membrane device, wherein the concentration of the polypeptide solution is controlled to be 5% before concentration, the concentration of the concentrated material reaches 15%, the operating pressure is 2MPa, and the operating temperature is 40 ℃;

(8) high-speed tubular separation: the concentrated solution is separated by a high-speed tubular separator, so that the feed liquid is highly purified and has no impurities. The rotating speed of the separating cylinder is 16000r/min, and the separating factor is as follows: 15050 R.C.F;

(9) tubular high-temperature instantaneous sterilization: and (3) sterilizing the separated clear liquid by adopting a tube array type high-temperature instantaneous sterilization device, wherein the sterilization temperature is as follows: the discharging temperature is 50 ℃ at 120 ℃;

(10) high-speed centrifugal spray drying: carrying out powder spraying drying on the concentrated solution reaching the sterilization effect by a high-speed centrifugal spray drying granulator, wherein the air inlet temperature is 200 ℃, and the air outlet temperature is 98 ℃;

(11) determination of amino acid sequence: the LC-MS/MS-based polypeptide complete sequence analysis is a very key technology for analyzing biological medicines, and the combination of liquid chromatography and mass spectrometry can be used for determining not only amino acid sequences, but also comprehensive biomolecule information such as mutation, posttranslational modification and the like. Four short peptide sequences of AGPK, VGPT, PGAR and AGPR are obtained by the method, and secondary mass spectrum scores are respectively as follows: 65.3, 64.9, 151.3, 85.4, as shown in table 1.

TABLE 1 EXAMPLE 1 identification of peptide fragments of chicken bone collagen peptide

Example 2

A preparation method of chicken bone collagen peptide with antioxidant activity comprises the following steps:

(1) the raw materials are preferably as follows: taking a certain amount of crushed chicken bone residues qualified by inspection and quarantine, wherein the mesh number of the chicken bone residues is 10-20 meshes;

(2) extracting a collagen solution: putting the chicken bone dregs obtained in the step (1) into a reaction kettle, washing with clear water, adding 2.5 times of water by mass, and cooking for 4 hours at 120 ℃ under 0.2 MPa;

(3) step-by-step enzymolysis: adding neutral protease accounting for 0.2% of the mass of the collagen liquid into the collagen liquid extracted in the step (2), carrying out enzymolysis for 2 hours at the temperature of 52 ℃ and the pH of 7, adding pancreatin accounting for 0.3% of the mass of the collagen liquid and compound flavor protease accounting for 0.1% of the mass of the collagen liquid into the obtained enzymolysis liquid, carrying out enzymolysis for 1.5 hours at the temperature of 55 ℃ and the pH of 7, and carrying out enzyme deactivation;

(4) centrifugal deslagging: removing residues of the enzyme-inactivated and sterilized enzymolysis solution by a horizontal spiral sedimentation centrifuge, and completely separating suspended powder and liquid in the enzymolysis solution;

(5) decoloring and deodorizing: cooling the filtered clear liquid obtained in the step (4) to 50 ℃, and then decoloring and deodorizing by using activated carbon fiber membrane equipment;

(6) membrane separation: ultrafiltering the decolorized fishy smell removing solution by a multifunctional inorganic ceramic membrane separation device, efficiently removing impurities, and filtering the filtrate to a clear solution storage tank at the filtering temperature of 60 ℃ and the filtering pressure of 0.2 MPa;

(7) and (3) nanofiltration membrane desalination and concentration: concentrating the filtrate with nanofiltration membrane equipment, desalting, controlling the concentration of the polypeptide solution to be 5% before concentration, controlling the concentration of the concentrated material to be 15%, and controlling the operation pressure to be 2.5MPa and the operation temperature to be 43 ℃;

(8) high-speed tubular separation: the concentrated solution is separated by a high-speed tubular separator, so that the feed liquid is highly purified and has no impurities. The rotating speed of the separating cylinder is 16000r/min, and the separating factor is as follows: 15050 R.C.F;

(9) tubular high-temperature instantaneous sterilization: and (3) sterilizing the separated clear liquid by adopting a tube array type high-temperature instantaneous sterilization device, wherein the sterilization temperature is as follows: 115 ℃ and the discharging temperature is 54 ℃;

(10) high-speed centrifugal spray drying: carrying out powder spraying drying on the concentrated solution reaching the sterilization effect by a high-speed centrifugal spray drying granulator, wherein the air inlet temperature is 200 ℃, and the air outlet temperature is 102 ℃;

(11) determination of amino acid sequence: the LC-MS/MS-based polypeptide complete sequence analysis is a very key technology for analyzing biological medicines, and the combination of liquid chromatography and mass spectrometry can be used for determining not only amino acid sequences, but also comprehensive biomolecule information such as mutation, posttranslational modification and the like. Four short peptide sequences of AGPK, VGPT, PGAR and AGPR are obtained by the method, and secondary mass spectrum scores are respectively as follows: 22.2, 33.8, 133.1, 45.4, as shown in table 2.

Table 2 example 2 identification of peptide fragments of chicken bone collagen peptide

Example 3

A preparation method of chicken bone collagen peptide with antioxidant activity comprises the following steps:

(1) the raw materials are preferably as follows: taking a certain amount of crushed chicken bone residues qualified by inspection and quarantine, wherein the mesh number of the chicken bone residues is 10-20 meshes;

(2) extracting a collagen solution: putting the chicken bone dregs obtained in the step (1) into a reaction kettle, washing with clear water, adding 2.2 times of water by mass, and cooking for 4 hours at 120 ℃ under 0.2 MPa;

(3) step-by-step enzymolysis: adding neutral protease accounting for 0.3% of the mass of the collagen liquid into the collagen liquid extracted in the step (2), carrying out enzymolysis for 2 hours at the temperature of 52 ℃ and the pH value of 7, adding pancreatin accounting for 0.25% of the mass of the collagen liquid into the obtained enzymolysis liquid, carrying out enzymolysis for 1 hour at the temperature of 52 ℃ and the pH value of 7, finally adding composite flavor protease accounting for 0.05% of the mass of the collagen liquid, carrying out enzymolysis for 0.5 hour at the temperature of 55 ℃ and the pH value of 7, and inactivating enzymes;

(4) centrifugal deslagging: removing residues of the enzyme-inactivated and sterilized enzymolysis solution by a horizontal spiral sedimentation centrifuge, and completely separating suspended powder and liquid in the enzymolysis solution;

(5) decoloring and deodorizing: cooling the filtered clear liquid obtained in the step (4) to 50 ℃, and then decoloring and deodorizing by using activated carbon fiber membrane equipment;

(6) membrane separation: ultrafiltering the decolorized fishy smell removing solution by a multifunctional inorganic ceramic membrane separation device, efficiently removing impurities, and filtering the filtrate to a clear solution storage tank at the filtering temperature of 60 ℃ and the filtering pressure of 0.2 MPa;

(7) and (3) nanofiltration membrane desalination and concentration: concentrating the filtrate with nanofiltration membrane equipment, desalting, controlling the concentration of the polypeptide solution to be 5% before concentration, and controlling the concentration of the concentrated material to be 15%, the operating pressure to be 2.2MPa, and the operating temperature to be 42 ℃;

(8) high-speed tubular separation: the concentrated solution is separated by a high-speed tubular separator, so that the feed liquid is highly purified and has no impurities. The rotating speed of the separating cylinder is 16000r/min, and the separating factor is as follows: 15050 R.C.F;

(9) tubular high-temperature instantaneous sterilization: and (3) sterilizing the separated clear liquid by adopting a tube array type high-temperature instantaneous sterilization device, wherein the sterilization temperature is as follows: the discharging temperature is 55 ℃ at 120 ℃;

(10) high-speed centrifugal spray drying: carrying out powder spraying drying on the concentrated solution reaching the sterilization effect by a high-speed centrifugal spray drying granulator, wherein the air inlet temperature is 210 ℃, and the air outlet temperature is 100 ℃;

(11) determination of amino acid sequence: the LC-MS/MS-based polypeptide complete sequence analysis is a very key technology for analyzing biological medicines, and the combination of liquid chromatography and mass spectrometry can be used for determining not only amino acid sequences, but also comprehensive biomolecule information such as mutation, posttranslational modification and the like. Four short peptide sequences of AGPK, VGPT, PGAR and AGPR are obtained by the method, and secondary mass spectrum scores are respectively as follows: 160.7, 21.9, 144.4, 151.9, as shown in table 3.

Table 3 example 3 identification of peptide fragments of chicken bone collagen peptide

Combining the short peptide sequences obtained in the embodiments 1, 2 and 3, the PGAR with the successfully identified short peptide sequences and higher scores is synthesized by adopting Fmoc solid phase synthesis technology to carry out PGAR polypeptide sequence synthesis. The synthesized PGAR polypeptide is subjected to in vitro oxidation resistance research, and DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, superoxide anion free radical scavenging capacity and reducing capacity are measured.

Determination of DPPH radical scavenging Capacity: 1.5mL of the sample solution and 1.5mL of the 0.1mmol/L DPPH solution are respectively added into a 5mL test tube with a plug, shaken up, and after reaction for 30min in a dark place, the absorbance Ai of the solution is measured at 517nm, and the absorbance A0 of a mixed solution of 1.5mL of the 0.1mmol/L DPPH solution and 1.5mL of 95% ethanol and the absorbance Aj of a mixed solution of 1.5mL of the sample solution and 1.5mL of 95% ethanol are simultaneously measured. As shown in FIG. 8, the DPPH radical scavenging rate of PGAR increased in the selected concentration range, with an IC50 value of about 6.4mg/mL, indicating that PGAR has a strong DPPH radical scavenging ability. VC as a high-efficiency antioxidant has extremely strong DPPH free radical scavenging capacity and has an IC50 value of about 4.0 mg/mL.

Measurement of hydroxyl radical scavenging ability: by adopting a Sminoff sodium salicylate method, 1mL of 2.3mmol/L FeSO4 solution, 1mL of 2.3mmol/L sodium salicylate-ethanol solution and 0.5mL of sample solution are respectively added into a 10mL test tube with a plug, and finally 1mL of 2.2mmol/L H2O2 solution is added to start reaction, the reaction is carried out for 1h at 37 ℃, and the light absorption value is measured at 510 nm. As shown in FIG. 9, the scavenging action of PGAR on OH was enhanced, the ascending trend was reduced at high concentration, and the clearance reached 92.50% at 9 mg/mL. The IC50 values for PGAR and Vc were approximately 4.5mg/mL and 0.68mg/mL, respectively.

Superoxide anion radical scavenging capacity determination: adopts a pyrogallol autoxidation method. A50 mmol/LTris-HCl buffer solution (pH 8.2)4.5mL and deionized water 4.1mL were added to 10mL stoppered tubes, and after incubation in a water bath at 25 ℃ for 20min, 0.1mL of the sample solution and 0.3mL of 3mmol/L pyrogallol preheated at 25 ℃ were immediately added, and absorbance was measured at 325nm every 30 seconds. As shown in FIG. 10, PGAR significantly decreased the rate of pyrogallol autoxidation in a dose-dependent manner, with increasing concentrations increasing the clearance rate, but was relatively weak at scavenging O2-compared to VC.

And (3) measuring the reducing capability: by adopting the Oyaizu method, 2mL of sample solution, 2mL of 0.2mol/L phosphate buffer solution (pH 6.6) and 2mL of potassium ferricyanide with the mass fraction of 1 percent are respectively added into a 10mL test tube with a plug, and 2mL of trichloroacetic acid with the mass fraction of 10 percent is added after heat preservation in a water bath at 50 ℃ for 20 min. 2mL of deionized water and 0.4mL of 0.1% FeCl3 solution by mass fraction were added to 2mL of the reaction solution, and after reaction for 10min, the absorbance was measured at 700 nm. As shown in FIG. 11, in a certain concentration range, the reduction capacities of both PGAR and VC are gradually increased with the increase of the concentration, and the reduction capacity of PGAR has a certain dose-effect relationship.

It should be noted that the above-mentioned embodiments are merely examples of the present invention, and it is obvious that the present invention is not limited to the above-mentioned embodiments, and other modifications are possible. All modifications directly or indirectly derivable by a person skilled in the art from the present disclosure are to be considered within the scope of the present invention.