GB2590263A

GB2590263A - Peanut antioxidative peptide prepared from high pressure-assisted enzymatic hydrolysis and preparation method therefor

Info

Publication number: GB2590263A
Application number: GB2100930.3A
Authority: GB
Inventors: Dong Xinhong; Yin Feilong; Li Jing; Shan Yang
Original assignee: Guilin University of Technology
Current assignee: Guilin University of Technology
Priority date: 2019-07-08
Filing date: 2020-07-07
Publication date: 2021-06-23
Also published as: GB202100930D0; CN110272935A; WO2021004463A1; CN110272935B

Abstract

A method for preparing an antioxidative peptide from a peanut meal by employing a high pressure-assisted enzymatic hydrolysis method, comprising the extraction of heat denatured protein in the peanut meal, high-pressure preprocessing, enzymatic hydrolysis, ultrafiltration, microporous resin separation, gel filtration chromatography, antioxidative activity identification, and mass spectrometry, for producing an antioxidatively activate peptide.

Description

Descriptions

A Kind of Peanut Antioxidant Peptide Prepared by High-pressure Assisted Enzymatic Hydrolysis and its Preparation Method

Technical Field

[0001] The technical field of the present invention belongs to modification of protein, high-pressure treatment of protein, and enzymatic hydrolysis of protein, particularly relates to a kind of peanut antioxidant peptide prepared by high-pressure assisted enzymatic hydrolysis and its preparation method

Background Technology

[0002] Since Harman proposed the free radical theory in 1956, people have gradually realized that the free radicals produced by the material oxygenolysis in vivo are the key to the aging and various diseases of human beings, and intaking highly active antioxidants is an effective method to scavenge excessive free radicals in the body. Currently, chemical antioxidants are the antioxidants with largest production volume, however, in consideration of the unsafe of chemical antioxidant additives, there is restriction on the use of such antioxidants. With the increase of demand for safe food and safe antioxidants by people, the research and utilization of natural antioxidants come to the attention of the publics.

[0003] Antioxidant is a new research topic related to the studies of biological activity of peptides that the domestic and foreign scholars concern in recent years. The antioxidant peptide derived from food proteins not only has good antioxidant activity, but also has relative high safety. According to relevant researches, it has been found that the carnosine, the glutathione, and the soybean peptide have anti-oxidation effects, and gradually show the advantages in such applications as medicine, food, feedstuff, and other fields. With the constant discovery of biological active peptides, their preparation and development have gradually become a hot topic.

Summary of the Invention Technical Issues

[0004] The antioxidant peptides produced by mild hydrolysis of food proteins through protease are not only easier to digest and absorb by the human body than the protein itself, and alleviate the damage of free radicals to the body, but also improve the food quality as well as extend the food shelf-life. As a result, the application prospects of antioxidant peptides in the sectors of food and biomedicine are worthy to be expected. Currently, the content of anti-oxidant peptides prepared by the method of enzymatic hydrolysis adopted abroad is relatively high, which reaches more than 50%, meanwhile, the peptide molecular weight is very small; however, the content of hydrolysis-peptides prepared by the method adopted in China is relatively low, and the peptide molecular weight is relatively high. The reason for this issue may be that the existing methods of enzymatic hydrolysis modification are incapable of enzyme hydrolyzing proteins sufficiently, and thus, the technology of enzymatic modification of proteins needs to be further improved or perfected.

Solutions to the Issues [0005] The purpose of the present invention is to provide a kind of antioxidant peptide of high temperature peanut meal and its preparation method, wherein, the high temperature peanut meal is processed through the technology of high-pressure assisted enzymatic hydrolysis modification, and thus, modifies the denatured protein in the peanut meal, so that prepares a neotype of peanut antioxidant peptides with antioxidant activity, which can not only improve the comprehensive utilization value of peanut meal, the by-product referred to herein, but also provide a new way for the deep processing of peanut industry, which has considerable social and economic benefits.

[0006] The specific steps of the present invention are as follows: [0007] (1) Extract peanut protein isolate from peanut meal: weigh a certain amount of peanut meal, and adjust the pH value to 10 with Imol/L of NaOH according to the solid-liquid ratio of 1:8, and then, complete the extraction process in a magnetically stirring water bath, wherein, the extraction temperature is at 60°C, and the extraction time is 60min. After stirring and extracting, centrifuge 3000g of materials at the temperature of 5°C for 10min, and then, collect the supernatant, as well as adjust the pH value to 4.5 with lmol/L of HC1, then, centrifuge 3000g of materials at the temperature of 5°C for 10min, after that, collect the sediment and dissolve it with deionized water, adjust the pH value of the solution to 7.0 with lmol/L of NaOH, and conduct freeze drying finally, and thus, obtain peanut protein isolate.

[0008] (2) High-pressure processing of peanut protein: make the peanut protein isolate (SPI) into a protein solution with a concentration of 5% (w/v) with distilled water, and then, vacuum seal the protein solution in a polyethylene bag. After that, treat it under the pressure of 200-400MPa for 18-22min. The speed to reach the target pressure is 250 MPa/min, and the releasing rate is 300 MPa/min. In the process of pressure treatment, keep the temperature at 25°C. And then, the SPI solution after pressure treatment is used to enzymatic hydrolyze after freeze drying.

[0009] (3) Enzymatic hydrolysis of peanut protein: make the peanut protein after high-pressure treatment into a solution with a concentration of 4-6% with deionized water, and then, place the solution on a thermostatic magnetic stirrer for enzymatic hydrolysis, wherein, adjust the temperature of the solution at 52-55°C, and the p1-1 value to 7-9, and then, add Alcalase (with enzyme concentration of 130-140pL/g) while slowly stirring, in the process of enzymatic hydrolysis, maintain the pH value of the reaction system by adding 1MNa0H, and the hydrolysis time is 110-130min.

[0010] (4) Ultrafiltration separation of enzymolysis products of peanut protein: take the enzymolysis products undergo enzymatic hydrolysis for 100-130min, prepare a solution with a concentration of 4-7%, and then, complete ultrafiltration separation at a rotational speed of 100-120rpm After that, further separate and purify the Fl component with the strongest antioxidant activity obtained by ultrafiltration.

[0011] (5) Separation by the method of macroporous adsorption resin: pack the pre-treated resin into the column by wet method (the column size is 20/2.0cm), and suck 20mL of the Fl component with a concentration of 18-2 lmg/mL to load sample. The flow rate of sample is 0.3-0.6BV (column bed volume)/h, then wash the chromatography column at a flow rate of 1BV (column bed volume)/h, and then, collect the eluent, and collect one tube per 15mL, when the change of water eluent A220nm is stable, conduct stepwise elution and separation at a flow rate of 1.4-1.6 BV/h with different concentrations of ethanol solution, and then, collect the eluent separated in each step, after that, concentrate them under reduced pressure and evaporate the ethanol, and then, freeze dry the concentrated solution.

[0012] (6) Gel filtration analysis: take a 1.0/80cm glass chromatography column and pack the treated Sephadex G-25 into the column. Make the sample obtained from step (5) into a solution of 3-6mg/mL after freeze drying, and the loading quantity of sample is 4mL, and then, elute the sample with distilled water at a flow rate of 3mL/10min. After that, collect one tube per 3mL, and then, use a spectrophotometer to detect and collect each elution peak at the position of 220nm, and concentrate under reduced pressure as well as freeze dry each eluted component. After that, repeat the treatment on samples, and then, combine each eluted component, and thus, evaluate the antioxidant activity. Wherein, the P3 component with stronger antioxidant activity is the antioxidant peptide of high temperature peanut meal.

[0013] (7) Use liquid chromatography-mass spectrometry technology to identify the structure of the antioxidant peptide contained in the P3 component with stronger antioxidant activity obtained after separation in step (6), [0014] (8) PSI-MS/MS identification: the sample flow analyzed by RP-HPLC enters the mass spectrometer (BrukerDaltonik GmbH, Bremen, Germany) pass through the interface of electrospray at a speed of 10ORL/min, and the positive ion scanning mode is adopted. In addition, the atomizing gas and the drying gas are high-purity nitrogen, and the scanning range is at the nucleo-cytoplasmic ratio of 200-2000(m/z), [0015] The sequence analysis of MS/MS peptide uses BioTools (Version3.0, Bruker Daltonics DataAnalysis 3.3) software combined with manual calculation, and thus, identify the amino acid sequence and molecular weight of the functional peptide factors contained in the P3 component of the Fl component.

The Beneficial Effects of the Invention [0016] The antioxidant peptide prepared by the present invention is a kind of tetrapeptide structure of four amino acid sequences, wherein, the molecular weight is 471.1 Da, and has certain antioxidant activity. Antioxidant peptide can improve the activity of superoxide dismutase, catalase, and glutathione peroxidase in cells, and have protective effects on DNA damage induced by hydroxyl free radicals, and thus, can be served as functional active components or antioxidants, and to be used in fields of health care products, food, nutritional supplements, animal feedstuff, cosmetics, and daily chemical products, etc.

Brief Description of the Drawings

[0017] For the purpose of clarifying the objectives, technical schemes, and advantages of the present invention, the text below will further describe the present invention in conjunction with the accompanying drawings, wherein: [0018] Figure 1 shows the impact of high-pressure treatment on enzymolysis effects of peanut protein; [0019] Figure 2 shows the results of ultrafiltration separation of enzymolysis products of peanut protein; (wherein, A: the impact of different enzymatic hydrolysis time on membrane flux; B: the impact of rotational speed of peristaltic pump on membrane flux; C: the impact of the concentration of enzymolysis products on membrane flux); [0020] Figure 3 shows the antioxidant activity analysis of different ultrafiltration components; [0021] Figure 4 shows the gel filtration chromatography analysis of PPIH<3K components contained in the products of enzymatic hydrolysis; [0022] Figure 5 shows the mass spectrum identification results of antioxidant peptides.

Embodiments of the Invention [0023] Embodiment 1 [0024] Take a certain amount of peanut meal, and extract peanut protein isolate by the method of alkali-extraction and acid-precipitation. Seal the peanut protein isolate with a concentration of 3% in a polyethylene bag, and then conduct freeze drying after being subjected to high-pressure treatment at the pressure of 200 N4Pa for 18min. After freeze drying, the sample is made into a solution with a concentration of 3% and place it on a thermostatic magnetic stirrer, and then, adjust the temperature of the solution at 52°C, and the pH value to 7, after that, add Alcalase (with enzyme concentration of 130 pL/g) for enzymatic hydrolysis for 110min. Then, separate the products of enzymatic hydrolysis with a concentration of 3%, and control the rotational speed of constant flow pump at 100rpm. After that, use the method of macroporous adsorption resin to separate and purify the Fl component with strong antioxidant activity after ultrafiltration separation, and suck 20mL of sample solution (with a concentration of 18mg/mL) and load the sample, control the flow rate at 0.3BV (column bed volume)/h, and then, wash the chromatography column at a flow rate of 1BV (column bed volume)/h, then, collect the eluent, and collect one tube per 15mL, when the change of water eluent A220nm is stable, conduct stepwise elution and separation at a flow rate of 1.4 BV/h with different concentrations of ethanol solution, and then, collect the eluent separated in each step, after that, concentrate them under reduced pressure and evaporate the ethanol, and then, freeze dry the concentrated solution. After that, complete gel filtration analysis on the sample after freeze drying, and make the sample after freeze drying into a solution of 3mg/mL, and the loading quantity of sample is 4m1L, and then, elute the sample with distilled water at a flow rate of 3mL/10min. After that, collect one tube per 3mL, and then, use a spectrophotometer to detect and collect each elution peak at the position of 220nm, and concentrate under reduced pressure as well as freeze dry each eluted component. After that, repeat the treatment on samples, and then, combine each eluted component, and thus, evaluate the antioxidant activity. Wherein, the P3 component with stronger antioxidant activity is the antioxidant peptide of high temperature peanut meal.

[0025] Embodiment 2 [0026] Take a certain amount of peanut meal, and extract peanut protein isolate by the method of alkali-extraction and acid-precipitation. Seal the peanut protein isolate with a concentration of 4% in a polyethylene bag, and then conduct freeze drying after being subjected to high-pressure treatment at the pressure of 300 IVIPa for 18min. After freeze drying, the sample is made into a solution with a concentration of 4% and place it on a thermostatic magnetic stirrer, and then, adjust the temperature of the solution at 53°C, and the pH value to 7, after that, add Alcalase (with enzyme concentration of 140 htL/g) for enzymatic hydrolysis for 100min. Then, separate the products of enzymatic hydrolysis with a concentration of 4%, and control the rotational speed of constant flow pump at 100rpm. After that, use the method of macroporous adsorption resin to separate and purify the F] component with strong antioxidant activity after ultrafiltration separation, and suck 20mL of sample solution (with a concentration of 18mg/mL) and load the sample, control the flow rate at 0.6BV (column bed volume)/h, and then, wash the chromatography column at a flow rate of 1BV (column bed volume)/h, then, collect the eluent, and collect one tube per 15mL, when the change of water eluent A220nm is stable, conduct stepwise elution and separation at a flow rate of 1.6 BV/h with different concentrations of ethanol solution, and then, collect the eluent separated in each step, after that, concentrate them under reduced pressure and evaporate the ethanol, and then, freeze dry the concentrated solution. After that, complete gel filtration analysis on the sample after freeze drying, and make the sample after freeze drying into a solution of 3mg/mL, and the loading quantity of sample is 4mL, and then, elute the sample with distilled water at a flow rate of 3mL/10min. After that, collect one tube per 3mL, and then, use a spectrophotometer to detect and collect each elution peak at the position of 220nm, and concentrate under reduced pressure as well as freeze dry each eluted component. After that, repeat the treatment on samples, and then, combine each eluted component, and thus, evaluate the antioxidant activity. Wherein, the P3 component with stronger antioxidant activity is the antioxidant peptide of high temperature peanut meal.

[0027] Embodiment 3 [0028] Take a certain amount of peanut meal, and extract peanut protein isolate by the method of alkali-extraction and acid-precipitation. Seal the peanut protein isolate with a concentration of 4% in a polyethylene bag, and then conduct freeze drying after being subjected to high-pressure treatment at the pressure of 400 AIN for 20min After freeze drying, the sample is made into a solution with a concentration of 4% and place it on a thermostatic magnetic stirrer, and then, adjust the temperature of the solution at 54°C, and :5 the pH value to 9, after that, add Alcalase (with enzyme concentration of 130 hiL/g) for enzymatic hydrolysis for 120min. Then, separate the products of enzymatic hydrolysis with a concentration of 4%, and control the rotational speed of constant flow pump at 100rpm. After that, use the method of macroporous adsorption resin to separate and purify the F] component with strong antioxidant activity after ultrafiltration separation, and suck 20mL of sample solution (with a concentration of 18mg/mL) and load the sample, control the flow rate at 0.5BV (column bed volume)/h, and then, wash the chromatography column at a flow rate of 1BV (column bed volume)/h, then, collect the eluent, and collect one tube per 15mL, when the change of water eluent A220nm is stable, conduct stepwise elution and separation at a flow rate of 1.4 BV/h with different concentrations of ethanol solution, and then, collect the eluent separated in each step, after that, concentrate them under reduced pressure and evaporate the ethanol, and then, freeze dry the concentrated solution. After that, complete gel filtration analysis on the sample after freeze drying, and make the sample after freeze drying into a solution of 3mg/mL, and the loading quantity of sample is 4mL, and then, elute the sample with distilled water at a flow rate of 3mL/10min. After that, collect one tube per 3mL, and then, use a spectrophotometer to detect and collect each elution peak at the position of 220nm, and concentrate under reduced pressure as well as freeze dry each eluted component. After that, repeat the treatment on samples, and then, combine each eluted component, and thus, evaluate the antioxidant activity. Wherein, the P3 component with stronger antioxidant activity is the antioxidant peptide of high temperature peanut meal.

[0029] Embodiment 4 [0030] Take a certain amount of peanut meal, and extract peanut protein isolate by the method of alkali-extraction and acid-precipitation. Seal the peanut protein isolate with a concentration of 5% in a polyethylene bag, and then conduct freeze drying after being subjected to high-pressure treatment at the pressure of 300 MIPa for 22min. After freeze drying, the sample is made into a solution with a concentration of 4% and place it on a thermostatic magnetic stirrer, and then, adjust the temperature of the solution at 53°C, and the pH value to 7, after that, add Alcalase (with enzyme concentration of 130 pL/g) for enzymatic hydrolysis for 120min. Then, separate the products of enzymatic hydrolysis with a concentration of 5%, and control the rotational speed of constant flow pump at 100rpm. After that, use the method of macroporous adsorption resin to separate and purify the Fl component with strong antioxidant activity after ultrafiltration separation, and suck 20mL of sample solution (with a concentration of 20mg/mL) and load the sample, control the flow rate at 0.5BV (column bed volume)/h, and then, wash the chromatography column at a flow rate of 1BV (column bed volume)/h, then, collect the eluent, and collect one tube per 15mL, when the change of water eluent A220nm is stable, conduct stepwise elution and separation at a flow rate of 1.5 BV/h with different concentrations of ethanol solution, and then, collect the eluent separated in each step, after that, concentrate them under reduced pressure and evaporate the ethanol, and then, freeze dry the concentrated solution. After that, complete gel filtration analysis on the sample after freeze drying, and make the sample after freeze drying into a solution of 5mg/mL, and the loading quantity of sample is 4mL, and then, elute the sample with distilled water at a flow rate of 3mL/10min. After that, collect one tube per 3mL, and then, use a spectrophotometer to detect and collect each elution peak at the position of 220nm, and concentrate under reduced pressure as well as freeze dry each eluted component. After that, repeat the treatment on samples, and then, combine each eluted component, and thus, evaluate the antioxidant activity. Wherein, the P3 component with stronger antioxidant activity is the antioxidant peptide of high temperature peanut meal.

Claims

Claims 1. A kind of peanut antioxidant peptide prepared by high-pressure assisted enzymatic hydrolysis, characterized in that the said peanut antioxidant peptide is a kind of tetrapeptide structure of four amino acid sequence, wherein, the molecular weight is 471.1 Da, and has certain antioxidant activity.
2. A method for preparing the said peanut antioxidant peptide as described above, characterized in that the specific steps are: (1) Weigh a certain amount of peanut meal, and adjust the pH value to 10 with Imol/L of NaOH according to the solid-liquid ratio of 1:8, and then, complete the extraction process in a magnetically stirring water bath, wherein, the extraction temperature is at 60°C, and the extraction time is 60min. After stirring and extracting, centrifuge 3000g of materials at the temperature of 5°C for 10min, and then, collect the supernatant, as well as adjust the pH value to 4.5 with Imol/L of HC1, then, centrifuge 3000g of materials at the temperature of 5°C for 10min, after that, collect the sediment and dissolve it with deionized water, adjust the pH value of the solution to 7.0 with lmol/L of NaOH, and conduct freeze drying finally, and thus, obtain peanut protein isolate.(2) Make the peanut protein isolate (SPI) obtained in step (1) into a protein solution with a concentration of 3-5% (w/v) with distilled water, and then, vacuum seal the protein solution in a polyethylene bag. After that, treat it under the pressure of 200-400MPa for 18-22min. The speed to reach the target pressure is 250 MPaimin, and the releasing rate is 300 MPaimin. In the process of pressure treatment, keep the temperature at 25°C. And then, the SPI solution after pressure treatment is used to enzymatic hydrolyze after freeze drying.(3) Make the peanut protein obtained in step (2) into a solution with a concentration of 4-6% with deionized water, and then, place the solution on a thermostatic magnetic stirrer for enzymatic hydrolysis, wherein, adjust the temperature of the solution at 52-55°C, and the pH value to 7-9, and then, add Alcalase while slowly stirring, and adjust the enzyme concentration to 130-14041g, in the process of enzymatic hydrolysis, maintain the pH value of the reaction system by adding IMNa0H, and the hydrolysis time is 110-130min.(4) Separate the enzymolysis products of peanut protein obtained in step (3) by ultrafiltration, and the condition of ultrafiltration separation is to obtain the enzymolysis products undergo enzymatic hydrolysis for 100-130min, prepare a solution with a concentration of 4-7%, and the rotational speed at 100-120rpm. After that, further separate and purify the Fl component with the strongest antioxidant activity obtained by ultrafiltration.(5) Purify the Ft component obtained from process of ultrafiltration separation in step (4) by the method of macroporous adsorption resin, pack the pre-treated resin into the column by wet method, and the size of chromatography column is 20x2.0cm, and suck 20mL of the Ft component with a concentration of 18-21mg/mL to load sample. The flow rate of sample is 0.3-0.6BV (column bed volume)/h, then, wash the chromatography column at a flow rate of 1BV (column bed volume)/h, and then, collect the eluent, and collect one tube per 15mL, when the change of water eluent A220nm is stable, conduct stepwise elution and separation at a flow rate of 1.4-1.6 BV/h with different concentrations of ethanol solution, and then, collect the eluent separated in each step, after that, concentrate them under reduced pressure and evaporate the ethanol, and then, freeze dry the concentrated solution.(6) Conduct gel filtration analysis on the component obtained after freeze drying in step (5), take a 1.0/80cm glass chromatography column and pack the treated Sephadex G-25 into the column. Make the sample obtained from step (5) into a solution of 3-6mg/mL after freeze drying, and the loading quantity of sample is 4mL, and then, elute the sample with distilled water at a flow rate of 3mL/10min. After that, collect one tube per 3mL, and then, use a spectrophotometer to detect and collect each elution peak at the position of 220nm, and concentrate under reduced pressure as well as freeze-dry each eluted component. After that, repeat the treatment on samples, and then, combine eluted components, and thus, evaluate the antioxidant activity. Wherein, the P3 component with stronger antioxidant activity is the antioxidant peptide of high temperature peanut meal.(7) Use liquid chromatography-mass spectrometry technology to identify the structure of the antioxidant peptide contained in the P3 component with stronger antioxidant activity obtained after separation in step (6); (8) EST-MS/MS identification: the sample flow analyzed by RP-HPLC enters the mass spectrometer (BmkerDaltonik GmbH, Bremen, Germany) pass through the interface of electrospray at a speed of 1001.1L/min, and the positive ion scanning mode is adopted. In addition, the atomizing gas and the drying gas are high-purity nitrogen, and the scanning range is at the nucleo-cytoplasmic ratio of 200--2000(m/z), and the sequence analysis of MS/MS peptide uses BioTools (Version3.0, Bruker Daltonics DataAnalysis 3.3) software combined with manual calculation, and thus, identify the amino acid sequence and molecular weight of the functional peptide factors contained in the P3 component of the Fl component.