CN108456243B

CN108456243B - Corn antioxidant active peptide and preparation method thereof

Info

Publication number: CN108456243B
Application number: CN201810400482.8A
Authority: CN
Inventors: 刘晓兰; 郑喜群; 王晓杰; 金杜欣
Original assignee: Qiqihar University
Current assignee: Qiqihar University
Priority date: 2015-09-09
Filing date: 2015-09-09
Publication date: 2020-08-04
Anticipated expiration: 2035-09-09
Also published as: CN108456243A; CN105237622A; CN108456244A; CN108456244B; CN105237622B

Abstract

The invention belongs to the technical field of food biology, and particularly relates to a corn peptide and a preparation method thereof. The invention provides a corn peptide, wherein the amino acid sequence of the corn peptide is as follows: ADCGWPA. Said corn peptide has high antioxidant activity, and has been determined to have IC for scavenging DPPH free radicals₅₀IC for superoxide anion radical scavenging with a value of 0.23mg/m L₅₀The value is 0.19mg/m L the invention also provides a preparation method of the corn peptide, which comprises the steps of taking corn gluten meal which is extruded and bulked and has starch removed as a raw material, adopting a protease synergistic enzymolysis mode to obtain a polypeptide mixture with antioxidant activity, collecting components with high activity and molecular weight within the range of 600-1400Da step by step through ultrafiltration, ion exchange chromatography, gel chromatography and reverse phase chromatography, finally obtaining amino acid sequences of six corn peptides through mass spectrometry, evaluating the in-vitro antioxidant activity of the corn peptides after chemical synthesis, and finally obtaining the corn peptide ADCGWPA.

Description

Corn antioxidant active peptide and preparation method thereof

The application is a divisional application based on Chinese patent application with the application number of CN201510565561.0 and the invention name of corn antioxidant active peptide and a preparation method thereof. The entire contents of the present application are derived from the parent application documentation.

Technical Field

The invention relates to three novel corn peptides with high antioxidant activity and a preparation method thereof, belonging to the technical field of food biology.

Background

The oxidation reaction plays an important role in the process of disease development, such as cancer, aging, arteriosclerosis, cardiovascular diseases, diabetes, alzheimer's disease, and the like. These negative health conditions arise due to oxidative damage to cellular components such as cell membranes, lipoproteins, enzymes, and nucleic acids. In food products, oxidation reactions can also directly affect the quality of the food product, often in connection with changes in the flavor and texture of the food product. The oxidation reaction is caused by an excess of free radical production and/or consumption of endogenous antioxidant components. Therefore, it is very important to suppress oxidation reactions and the formation of free radicals in the body and food. The proper intake of the antioxidant can obviously reduce the level of free radicals in the organism, and plays an important role in keeping the health of the body and preventing diseases. Meanwhile, the quality and nutrition of the food can be maintained by adding the antioxidant into the food.

Antioxidants are divided into two types, depending on the source: synthetic antioxidants and natural antioxidants. Synthetic antioxidants, such as BHT, BHA, TBHQ, etc., are considered to present safety hazards, severely limiting their application in the food industry; natural antioxidants are receiving much attention because of their safety, high activity, easy absorption, no side effects and wide distribution. Currently, antioxidant peptides have been isolated from many food proteins, such as soy protein, rapeseed protein, rice endosperm protein, wheat germ protein, barley gluten, and the like.

Corn gluten meal (Corn GlutenMeal abbreviated as CGM) is a byproduct with the largest yield in the process of producing starch by a Corn wet method, contains about 60% of protein and consists of zein (zein, 68%), glutelin (22%), globulin (globulins, 1.2%) and albumin (albumin). The corn gluten meal is a natural vegetable protein raw material with abundant resources and low cost, but is obviously a non-total nutrient protein because the content of lysine and tryptophan is not high. In addition, the protein contained in the maize yellow powder is difficult to dissolve in water, the composition is relatively complex, and the taste is very rough, so that the maize yellow powder hardly asks for body fluid in the food field and the feed industry. Even if there are many places, the waste is naturally discharged together with other byproducts in vain without recycling, which not only wastes grain resources greatly, but also pollutes the ecological environment to a certain extent. Thus, if zein could be modified and used in the food industry, its market value would be greatly increased.

Disclosure of Invention

The first technical problem to be solved by the invention is to provide three new corn peptides with high antioxidant activity, and as the raw material for producing the product is corn gluten meal, an effective utilization way is firstly provided for the natural resource which is cooled down and abandoned for a long time, and three antioxidant active peptides which have the functions of disease prevention and treatment, aging resistance and the like and can be used as additives of products such as food, medicine, cosmetics and the like are developed. The invention also provides a preparation method of the three new corn peptides with high antioxidant activity.

The corn peptide with high antioxidant activity is prepared by using extruded and puffed corn gluten meal without starch AS a raw material, adding a certain proportion of water to prepare a suspension with a substrate concentration of 10%, firstly inoculating alkaline protease Alcalase for enzymolysis for 75min, then inoculating flavor protease Flavourzyme for enzymolysis for 50min to obtain a polypeptide mixture with antioxidant activity, separating the mixture by adopting ultrafiltration, ion exchange chromatography, gel chromatography and reverse phase chromatography, and finally determining the amino acid sequence of the active peptide antioxidant by using Q-TOF2₅₀Values of 0.12mg/m L, 0.13mg/m L and 0.23mg/m L, respectively, and an IC for scavenging superoxide anion radicals₅₀The values were 0.39mg/m L, 0.41mg/m L and 0.19mg/m L, respectively.

The specific method for preparing the product comprises the following steps:

1. preparation of corn protein enzymolysis liquid

Taking a certain amount of corn gluten meal which is extruded and puffed and has starch removed, adding a certain proportion of water to prepare suspension with substrate concentration of 10% (m/V), and firstly carrying out enzymolysis by alkaline protease Alcalase, wherein the enzymolysis conditions are as follows: adding 2% of enzyme, performing enzymolysis at 50 deg.C and pH7.7 for 75 min; and then adding Flavourzyme Flavourzyme for synergistic enzymolysis, wherein the enzymolysis conditions are as follows: adding 5% of enzyme, performing enzymolysis at 53 deg.C and pH6.4 for 50 min. After the enzymolysis is finished, the enzymolysis liquid is heated for 10min at the temperature of 100 ℃ to passivate the activity of protease. And centrifuging the zymolyte at 4000r/min for 10min, discarding the precipitate, and obtaining supernatant which is a mixture of antioxidant active peptides.

2. Ultrafiltration of corn protein enzymolysis liquid

Ultrafiltration treatment is carried out on the corn protease hydrolysate by adopting an ultrafiltration membrane with a cut-off molecular weight of 6kDa to obtain two corn peptide components with different molecular weights of a cut-off solution with a molecular weight of more than 6kDa and a permeate with a molecular weight of less than 6 kDa. The permeate with a molecular weight of less than 6kDa is selected as a sample for further separation and purification.

3. Strong anion exchange chromatography with Q-Sepharose Fastflow

Diluting the 6kDa permeate obtained by ultrafiltration by 10 times, centrifuging at 10000r/min for 15min, collecting supernatant, filtering with 0.22 μm microporous membrane, and subjecting 50m L filtrate to strong anion exchange chromatography

Starting buffer solution, 20 mmol/L pH8.5Tris-HCl, eluent, 20 mmol/L pH8.5Tris-HCl containing 1mol NaCl, gradient elution for 15min, flow rate of 2m L/min, UV_214nmAnd (6) detecting. And (4) measuring the antioxidant activity of the collected liquid of each tube, and collecting the high-activity part for later use.

4. SephadexG-25 gel filtration chromatography

High-activity Q-Sepharose Fastflow samples were lyophilized and usedDissolving in double distilled water, and desalting by gel chromatography

Flow rate 2.0m L/min, UV_214nmAnd (6) detecting. And simultaneously carrying out molecular weight calibration, wherein standard substances for calibration are bacitracin (molecular weight of 1400Da) and reduced glutathione (molecular weight of 600 Da). Measuring the antioxidant activity of the collected liquid in each tube, and collecting the high-activity part with the molecular weight in the range of 600-1400Da for later use.

5. Reversed phase chromatography

① semi-preparative reverse phase chromatographic column for determining active region of antioxidant peptide

Weighing 20mg of active component separated from SephadexG-25, dissolving in 2% acetonitrile water solution of 1m L, filtering with 0.22 μm microporous membrane, and separating with Pronto SI L C18 (model of chromatographic column)

10 μm), flow rate of 1.5m L/min, mobile phase a as 2% acetonitrile in water containing 0.065% TFA, mobile phase B as 80% acetonitrile in water containing 0.05% TFA, gradient elution for 60min, fraction of the corn peptide at the beginning of peak-off was collected using an automatic fraction collector, 3m L was collected per tube, antioxidant activity of samples per tube was measured, and the active regions were determined to be in tubes 8, 11, and 13.

② analytical reverse phase chromatographic column for separating antioxidant active peptide

Dissolving the active component separated by semi-preparative reverse phase chromatographic column in 2% acetonitrile water solution, and further separating and purifying with analytical column of column type XSelect CSH (Chile Sakul Seal)

3.5 μm), detection wavelength 214nm, flow rate 1m L/min, loading 20 μ L mobile phase a was 2% acetonitrile in water containing 0.065% TFA and mobile phase B was acetonitrile containing 0.05% TFA.

The tube components are separated by an analytical column to obtain eight corn peptides, the peak tip part of each peak is collected for antioxidant activity determination, and 8-II, 8-V and 8-VII are determined as main antioxidant active peptides.

The tube was fractionated to obtain five maize peptides, 11-I, 11-II, 11-III, 11-IV and 11-VI respectively. The peak part of each corn peptide is collected for the determination of antioxidant activity, and 11-III and 11-VI are determined as main antioxidant active peptides.

And (3) separating the active components of the tube by an analytical column to obtain nine peaks, and comparing the nine peaks with a solvent chromatogram to determine the corn antioxidant peptides of which 13-I, 13-III, 13-VI, 13-VII and 13-VIII are solvent peaks and 13-II, 13-IV, 13-V and 13-IX are 13 tubes. The antioxidant activity of four peak parts of the corn peptide is measured, and the 13-V is determined as the main corn antioxidant peptide.

6. Mass spectrometry sequencing:

the 8-II, 8-V, 8-VII, 11-III, 11-VI and 13-V separated by reverse phase chromatography were subjected to mass spectrometry, and the results are shown in Table 1.

TABLE 1 amino acid sequence information of six maize antioxidant peptides

Name (R)	Antioxidant active peptide sequence	Number of amino acids	Molecular weight/Da
				8-II	Lys-Ser-Cys-Ala-Pro-Leu-Ala-Ser	8	775.29
8-V	Ala-Asp-Cys-Gly-Trp-Pro-Ala	7	719
				8-VII	Ala-Leu-Thr-Ser-Pro-Ala	6	558.29
11-III	Cys-Ser-Gln-Ala-Pro-Leu-Ala	7	688.29
				11-VI	Tyr-Pro-Lys-Leu-Ala-Pro-Asn-Glu	8	930.49
13-V	Tyr-Pro-Gln-Leu-Leu-Pro-Asn-Glu	8	972.49

7. Determination of in vitro antioxidant activity of corn antioxidant peptide

After the six corn antioxidant peptides are chemically synthesized, the activity and the reducing power for removing DPPH, hydroxyl and superoxide anion free radicals are measured, and finally three new corn peptides with high antioxidant activity are obtained.

The invention has the advantages that the product has higher antioxidant activity, and the determination shows that the IC of the CSQAP L A, KSCAP L AS and the ADCGWPA can eliminate DPPH free radicals₅₀Values of 0.12mg/m L, 0.13mg/m L and 0.23mg/m L, respectively, and an IC for scavenging superoxide anion radicals₅₀The values are respectively 0.39mg/m L, 0.41mg/m L and 0.19mg/m L, the effect is equivalent to that of antioxidant vitamin C and glutathione, and the product can be used as product additive for food, medicine, cosmetics, etc。

Drawings

FIG. 1 is a flow chart of the production process of the present invention.

Detailed Description

Example 1:

1. preparation of corn protein enzymolysis liquid

Taking a certain amount of corn gluten meal which is extruded and puffed and has starch removed, adding a certain proportion of water to prepare suspension with substrate concentration of 10% (m/V), and firstly carrying out enzymolysis by alkaline protease Alcalase, wherein the enzymolysis conditions are as follows: adding 2% of enzyme, performing enzymolysis at 50 deg.C and pH7.7 for 75 min; and then adding Flavourzyme Flavourzyme for synergistic enzymolysis, wherein the enzymolysis conditions are as follows: adding enzyme 5%, performing enzymolysis at 53 deg.C and pH6.4 for 50 min. After hydrolysis, the enzymolysis liquid is heated for 10min at 100 ℃ to inactivate protease activity. And centrifuging the zymolyte at 4000r/min for 10min, discarding the precipitate, and obtaining supernatant which is a mixture of antioxidant active peptides.

2. Ultrafiltration of corn protein enzymolysis liquid

3. Strong anion exchange chromatography with Q-Sepharose Fastflow

Diluting the 6kDa permeate obtained by ultrafiltration by 10 times, centrifuging at 10000r/min for 15min, collecting supernatant, filtering with 0.22 μm microporous membrane, and subjecting the filtrate to strong anion exchange chromatography

4. SephadexG-25 gel filtration chromatography

High-activity Q-Sepharose Fastflow samples were lyophilized and then reconstituted with bis-SepharoseDissolving in distilled water, and desalting by gel chromatography

5. Reversed phase chromatography

10 μm), the loading was 500 μ L, the flow rate was 1.5m L/min, the detection wavelength was 214nm, mobile phase a was 2% acetonitrile in water containing 0.065% TFA, mobile phase B was 80% acetonitrile in water containing 0.05% TFA, gradient elution was performed for 60min, the peptide fraction of corn at the beginning of peak emergence was collected using an automatic fraction collector, 3m L was collected for each tube, the antioxidant activity of the samples for each tube was determined, the active region was determined at 8, 11 and 13 tubes, and the samples were collected repeatedly and freeze-dried for use.

And separating the tube components by an analytical column to obtain eight corn peptides, collecting the peak tip part of each peak to perform antioxidant activity determination, and determining 8-II, 8-V and 8-VII as main antioxidant active peptides. The three fractions were collected repeatedly, freeze-dried and left for mass spectrometric sequencing.

The tube components obtain five corn peptides which are respectively 11-I, 11-II, 11-III, 11-IV and 11-VI. And collecting the peak part of each corn peptide for measuring the antioxidant activity, and determining 11-III and 11-VI as main antioxidant active peptides. The two peptides were collected repeatedly, freeze-dried and left for mass spectrometric sequencing.

The active components of the tube are separated by an analytical column to obtain nine peaks, and the corn antioxidant peptides with 13-I, 13-III, 13-VI, 13-VII and 13-VIII as solvent peaks and 13-II, 13-IV, 13-V and 13-IX as 13 tubes are determined by comparing with a solvent chromatogram. And determining 13-V as the main corn antioxidant peptide by measuring the antioxidant activity of the peak parts of the four corn peptides. Collecting the components for several times, freezing, vacuum drying, sealing, and storing.

6. Mass spectrometry sequencing:

and (3) carrying out mass spectrum sequencing on 8-II, 8-V, 8-VII, 11-III, 11-VI and 13-V separated by the reverse phase chromatography to obtain six amino acid sequences.

7. Determination of in vitro antioxidant activity of corn antioxidant peptide

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of ziqi hall

<120> antioxidant active peptide of corn and preparation method thereof

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>7

<212>PRT

<213>Zea mays

<400>1

Cys Ser Gln Ala Pro Leu Ala

1 5

<210>2

<211>8

<212>PRT

<213>Zea mays

<400>2

Lys Ser Cys Ala Pro Leu Ala Ser

1 5

<210>3

<211>7

<212>PRT

<213>Zea mays

<400>3

Ala Asp Cys Gly Trp Pro Ala

1 5

Claims

1. A maize peptide having the amino acid sequence: ADCGWPA.

2. The method for preparing the corn peptide as claimed in claim 1, wherein the corn peptide is prepared by using corn gluten meal which is extruded and puffed and has starch removed as a raw material, adopting a protease synergistic enzymolysis mode to obtain a polypeptide mixture with antioxidant activity, sequentially carrying out ultrafiltration, ion exchange chromatography, gel chromatography and reverse phase chromatography, gradually collecting components with high activity and molecular weight within the range of 600-1400Da, and finally carrying out mass spectrometry to obtain amino acid sequences of six corn peptides; evaluation of in vitro antioxidant Activity of maize peptides after chemical Synthesis to finally obtain IC for DPPH free radical scavenging₅₀IC for superoxide anion radical scavenging with a value of 0.23mg/m L₅₀The value of the corn peptide ADCGWPA is 0.19mg/m L, and the protease synergistic enzymolysis specifically comprises taking a certain amount, extruding and puffing, and removing starchAdding a certain proportion of water into the corn gluten meal to prepare suspension with a substrate concentration of 10% by mass-volume ratio, and firstly carrying out enzymolysis by using alkaline protease Alcalase, wherein the enzymolysis conditions are as follows: adding 2% of enzyme, performing enzymolysis at 50 deg.C and pH7.7 for 75 min; then adding Flavourzyme Flavourzyme into the alkaline protease enzymolysis liquid for synergistic enzymolysis, wherein the enzymolysis conditions are as follows: adding 5% of enzyme, performing enzymolysis at 53 deg.C and pH6.4 for 50 min; after the enzymolysis is finished, heating the enzymolysis liquid for 10min at the temperature of 100 ℃ in cooperation with the enzymolysis liquid to passivate the activity of protease; centrifuging the obtained zymolyte, removing the precipitate, and obtaining supernatant as a mixture of antioxidant active peptides.

3. The method of claim 2, wherein the step of separating by reverse phase chromatography comprises:

Weighing active components separated by SephadexG-25, dissolving the active components in 2% acetonitrile water solution, filtering by a 0.22 mu m microporous membrane, wherein the specification of a chromatographic column is phi 10 × mm, a filler is ProntoSI L C18 with the particle size of 10 mu m, the flow rate is 1.5m L/min, a mobile phase A is 2% acetonitrile water solution containing 0.065% TFA, and a mobile phase B is 80% acetonitrile water solution containing 0.05% TFA;

Dissolving the active component separated by the semi-preparative reverse phase chromatographic column in 2% acetonitrile aqueous solution, and further separating and purifying by using an analytical column, wherein the column shape of the analytical column is phi 4.6 × 150mm, the XSelectCSH is 3.5 mu m, the detection wavelength is 214nm, the flow rate is 1m L/min, and the sample loading amount is 20 mu L;

separating the No. 8 tube group by an analytical column to obtain eight corn peptides, collecting the peak tip part of each peak to perform antioxidant activity determination, and determining 8-II, 8-V and 8-VII as main antioxidant active peptides;

grouping No. 11 tubes to obtain five corn peptides which are respectively 11-I, 11-II, 11-III, 11-IV and 11-VI; collecting peak part of each corn peptide for measuring antioxidant activity, and determining 11-III and 11-VI as main antioxidant active peptides;

separating the active component of No. 13 tube by an analytical column to obtain nine peaks, and comparing with a solvent chromatogram map to determine corn antioxidant peptides of No. 13-I, 13-III, 13-VI, 13-VII and 13-VIII as solvent peaks and No. 13-II, 13-IV, 13-V and 13-IX as No. 13 tubes; the antioxidant activity of four peak parts of the corn peptide is measured, and the 13-V is determined as the main corn antioxidant peptide.

4. The method according to claim 2, wherein the ultrafiltration comprises the following steps: performing ultrafiltration treatment on the polypeptide mixture with the antioxidant activity by adopting an ultrafiltration membrane with the cut-off molecular weight of 6kDa to obtain a corn peptide component of a trapped fluid with the molecular weight of more than 6kDa and a permeate with the molecular weight of less than 6 kDa; the permeate with a molecular weight of less than 6kDa is selected as a sample for further separation and purification.

5. The method according to claim 2, wherein the ion exchange chromatography comprises the steps of diluting the permeate having a molecular weight of less than 6kDa obtained by ultrafiltration with Q-Sepharose Fast Flow by 10 times, centrifuging at 10000r/min for 15min, passing the supernatant through a 0.22 μm microfiltration membrane, subjecting the 50m L filtrate to strong anion exchange chromatography with a column having a diameter of 1.6 × 30cm, starting with a buffer solution of 20 mmol/L and pH8.5Tris-HCl, eluting with a buffer solution of 20 mmol/L pH8.5Tris-HCl containing 1mol of NaCl, and performing UV-based ion exchange chromatography at a Flow rate of 2m L/min_214nmDetecting; and (4) measuring the antioxidant activity of the collected liquid of each tube, and collecting the high-activity part for later use.

6. The preparation method according to claim 2, wherein the gel chromatography uses SephadexG-25 with a chromatographic column specification of phi 1.6 × 90cm, after a high-activity sample of Q-Sepharose Fast Flow is freeze-dried, the sample is dissolved by double distilled water, gel chromatography desalination is carried out while molecular weight calibration is carried out, standard substances used for calibration are bacitracin with a molecular weight of 1400Da and reduced glutathione with a molecular weight of 600Da, the antioxidant activity of the collected liquid in each tube is measured, and the high-activity part with a molecular weight in the range of 600-1400Da is collected for standby.

7. The method of claim 2, wherein the mass spectrometry sequencing is: performing mass spectrometry on 8-II, 8-V, 8-VII, 11-III, 11-VI and 13-V separated by reversed phase chromatography to obtain six amino acid sequences; chemically synthesizing the obtained six corn antioxidant peptides, measuring the scavenging activity and reducing power of DPPH, hydroxyl and superoxide anion free radicals, and finally obtaining the IC for scavenging DPPH free radicals₅₀IC for superoxide anion radical scavenging with a value of 0.23mg/m L₅₀The value was 0.19mg/m L maize peptide ADCGWPA.