CN108456243A

CN108456243A - Corn antioxidant active peptide and preparation method thereof

Info

Publication number: CN108456243A
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: 2018-08-28
Anticipated expiration: 2035-09-09
Also published as: CN108456244B; CN108456244A; CN105237622A; CN105237622B; CN108456243B

Abstract

The invention belongs to technical field of food biotechnology, and in particular to a kind of corn peptide and preparation method thereof.The present invention provides a kind of corn peptide, the amino acid sequence of the corn peptide is：ADCGWPA.The corn peptide has high anti-oxidation activity, after measured, to the IC of DPPH radicals scavengings₅₀Value is 0.23mg/mL, the IC removed to ultra-oxygen anion free radical₅₀Value is 0.19mg/mL.The present invention also provides the preparation methods of above-mentioned corn peptide, through extrusion and to remove the maize yellow-powder of starch as raw material, using proteinase synergy mode of action, obtain the mixtures of polypeptides with antioxidant activity, pass through ultrafiltration, ion-exchange chromatography, gel chromatography, reverse-phase chromatography again, the amino acid sequence for obtaining six corn peptides is sequenced finally by mass spectrum for the component of collecting high-activity and molecular weight within the scope of 600 1400Da step by step；The antioxidation activity in vitro of corn peptide is assessed after chemical synthesis, it is final to obtain corn peptide ADCGWPA.

Description

Corn antioxidant active peptide and preparation method thereof

The application be with application No. is " CN201510565561.0 ", it is entitled " corn antioxidant active peptide and its The divisional application proposed based on the Chinese patent application of preparation method ".The application full content is remembered from Mother case of the application documents It carries.

Technical field

The present invention relates to three new, active corn peptides of high anti-oxidation and preparation methods, belong to food biotechnology neck Domain.

Background technology

Oxidation reaction disease generate during play a significant role, as cancer, aging, artery sclerosis, angiocardiopathy, Diabetes, Alzheimer disease etc..The generation of these negative healths is attributed to the oxidative damage of cell component, such as cell Film, lipoprotein, enzyme and nucleic acid etc..In food, oxidation reaction can also directly affect food quality, usually with the flavor of food It is related with the change of quality.Oxidation reaction is that free radical generates caused by excess and/or the consumption of endogenous anti-oxidative ingredient.Cause This, inhibits the formation of oxidation reaction and free radical to be very important in body and food.Appropriate intake antioxidant can be with The Free Radical Level being substantially reduced in body plays a significant role keeping fit and preventing disease.Meanwhile it eating Antioxidant is mixed in product can keep quality and the nutrition of food.

According to source difference, antioxidant is divided into two types：Synthetized oxidation preventive agent and natural.Synthesize antioxygen Agent, such as BHT, BHA, TBHQ, are considered that there are security risks, seriously limit its application in food industry；And it is natural Antioxidant is due to having many advantages, such as safety, high activity, easily absorbing, without side-effects and widely distributed and be concerned.Currently, anti- It is isolated from numerous food product albumen to aoxidize peptide, such as soybean protein, rapeseed protein, rice endosperm protein, wheat Bud albumen, barley glutelin etc..

Maize yellow-powder (Corn GlutenMeal abbreviations CGM) is that corn wet produces the maximum by-product of starch process yield Object includes about 60% protein, by zeins (zein, 68%), glutelin (glutelin, 22%), ball egg (globulins, 1.2%) and albumin (albumin) composition in vain.Although maize yellow-powder is a kind of resourceful, of low cost Natural plant protein raw material, but since the content of wherein lysine and tryptophan is not high, it is clear that for a kind of non-full nutrition Albumen.In addition, the protein contained by maize yellow-powder is insoluble in water, composition is again more complicated, and mouthfeel is also very coarse, therefore, nothing By being made inquiries in field of food or in feedstuff industry all few people.Even there are many places also by it and other by-products one It rises to be not added with to recycle and just discharge naturally in vain, this is not only a kind of significant wastage to grain resource, while also to ecology Environment causes a degree of pollution.Therefore, if can carry out modifying to zein and be applied to food industry, market Value will substantially increase.

Invention content

The invention solves first technical problem be to provide three new, active corn peptides of high anti-oxidation, due to The raw materials for production of the product are maize yellow-powders, first for it is this treated coldly for a long time, outcast natural resources provide one kind has The utilization ways of effect, while further developed three and have the function of disease preventing and treating, anti-aging etc., it can be as food, medicine, change The antioxidation active peptides that the products additive such as cosmetic uses.The invention solves another technical problem to be to provide these three new , the preparation method of high anti-oxidation activity corn peptide.

Product of the present invention it is new, high anti-oxidation activity corn peptide be with the maize yellow-powder through extrusion and desizing be original Material is added a certain proportion of water and prepares the suspension that concentration of substrate is 10%, first accesses alkali protease Alcalase enzymolysis 75min, then flavor protease Flavourzyme enzymolysis 50min are accessed, obtain the mixtures of polypeptides with antioxidant activity.It adopts The mixture is detached with ultrafiltration, ion-exchange chromatography, gel chromatography and RP chromatography, finally Q-TOF2 is utilized to measure The amino acid sequence of antioxidation active peptides.The antioxidation activity in vitro of corn peptide is assessed after chemical synthesis, it is final to obtain three newly , the active corn peptide of high anti-oxidation.The amino acid sequence of corn peptide is respectively：CSQAPLA, KSCAPLAS and ADCGWPA.Three IC of the person to DPPH radicals scavengings₅₀Value is respectively 0.12mg/mL, 0.13mg/mL and 0.23mg/mL；Certainly to superoxide anion The IC removed by base₅₀Value difference 0.39mg/mL, 0.41mg/mL and 0.19mg/mL.

Specific method prepared by product of the present invention：

1, the preparation of zein enzymolysis liquid

Take it is a certain amount of through extrusion and remove the maize yellow-powder of starch, a certain proportion of water be added prepare concentration of substrate and be The suspension of 10% (m/V), is first digested with alkali protease Alcalase, and enzymatic hydrolysis condition is：Enzyme concentration 2%, enzymolysis temperature 50 DEG C, pH 7.7, time 75min of degree；Then flavor protease Flavourzyme is added and carries out coordinated enzymatic hydrolysis, enzymatic hydrolysis condition is： Enzyme concentration 5%, 53 DEG C, pH 6.4, time 50min of hydrolysis temperature.Enzymolysis liquid heats under the conditions of 100 DEG C after enzymolysis 10min is to be passivated prolease activity.Zymolyte abandons precipitation in 4000r/min centrifugations 10min, and gained supernatant is antioxidant activity The mixture of peptide.

2, the ultrafiltration of zein enzymolysis liquid

Hyperfiltration treatment is carried out to zein enzymolysis liquid using the ultrafiltration membrane for blocking molecular weight 6kDa, obtains ＞ 6kDa retentions The corn peptide composition of two kinds of different molecular weights of liquid and ＜ 6kDa permeate.Select the permeate of molecular weight ＜ 6kDa as in next step The sample isolated and purified.

3, Q-Sepharose FastFlow strong anions displacement chromatography

The permeate of ＜ 6kDa obtained by ultrafiltration is diluted 10 times, 10000r/min centrifuges 15min, and supernatant is taken to cross 0.22 μm Miillpore filter, 50mL filtrates carry out strong anion displacement chromatographyStart buffer：20mmol/LpH 8.5Tris-HCl, eluent：20mmol/L pH 8.5Tris-HCl containing 1molNaCl, gradient elution 15min, flow velocity： 2mL/min, UV_214nmPlace's detection.The antioxidant activity of each pipe collection liquid is measured, collecting high-activity part is spare.

4, SephadexG-25 gel permeation chromatographies

After the high vigor sample freezing of Q-Sepharose FastFlow is drained, gel layer is carried out after being dissolved with distilled water Analyse desalinationFlow velocity 2.0mL/min, UV_214nmPlace's detection.It is carried out at the same time molecular weight calibration, the mark of calibration Quasi- substance is bacitracin (molecular weight 1400Da) and reduced glutathione (molecular weight 600Da).Measure each pipe collection liquid Antioxidant activity, molecular weight is within the scope of 600-1400Da and high activity partially is spare for collection.

5, reverse-phase chromatography

1. semi-preparative reverse-phase chromatographic column determines the active region of anti-oxidation peptide

It weighs the separating obtained active components of 20mg SephadexG-25 to be dissolved in 2% acetonitrile solution of 1mL, with 0.22 μ M filtering with microporous membrane, chromatographic column model Pronto SIL C18 (10 μm), flow velocity 1.5mL/ Min, mobile phase A are 2% acetonitrile solution containing 0.065%TFA, and Mobile phase B is 80% aqueous acetonitrile containing 0.05%TFA Liquid, gradient elution 60min.Corn peptide composition when starting appearance is collected using automatic fraction collector, often pipe collects 3mL.It surveys The antioxidant activity of fixed often pipe sample determines active region in 8,11 and 13 pipes.

2. analytic type reverse-phase chromatography post separation antioxidation active peptides

The separating obtained active component of reverse-phase chromatographic column will partly be prepared to be dissolved in 2% acetonitrile solution, further use analytical column Isolated and purified, the column type of analytical column be Xselect CSH (3.5 μm), Detection wavelength 214nm, stream Speed is 1mL/min, and applied sample amount is 20 μ L.Mobile phase A be 2% acetonitrile solution containing 0.065%TFA, Mobile phase B be containing The acetonitrile of 0.05%TFA.

Pipe component after post separation, obtains eight corn peptides by analysis, and the peak nose part progress for collecting each peak is anti-oxidant Determination of activity determines that 8-II, 8-V and 8-VII are main antioxidation active peptides.

Pipe component obtains five corn peptides, respectively 11-I, 11-II, 11-III, 11-IV and 11-VI.It collects each beautiful The peak nose part of rice peptide carries out the measurement of antioxidant activity, determines that 11-III and 11-VI is main antioxidation active peptides.

Pipe active component determines 13-I, 13- through analyzing nine peaks of post separation acquisition through making comparisons with solvent chromatogram III, 13-VI, 13-VII, 13-VIII are solvent peak, and 13-II, 13-IV, 13-V, 13-IX are the corn antioxidant peptide of 13 pipes. By measuring the antioxidant activity of four corn peptide peak nose parts, determine that 13-V is main corn antioxidant peptide.

6, mass spectrum is sequenced：

8-II, 8-V, 8-VII, 11-III, 11-VI and 13-V that reversed phase chromatography separation is come out carry out mass spectrum sequencing, knot Fruit is as shown in table 1.

The amino acid sequence information of 1 six kinds of corn antioxidant peptides of table

Title	Antioxidant activity peptide sequence	Amino acid number	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, the measurement of corn antioxidant peptide antioxidation activity in vitro

After six corn antioxidant peptides of acquisition are carried out chemical synthesis, measure to DPPH, hydroxyl and superoxide anion certainly It is final to obtain three new, active corn peptides of high anti-oxidation by the scavenging capacity and reducing power of base.

The beneficial effects of the invention are as follows：Product of the present invention have higher antioxidant activity, after measured CSQAPLA, IC of the KSCAPLAS and ADCGWPA threes to DPPH radicals scavengings₅₀Value be respectively 0.12mg/mL, 0.13mg/mL and 0.23mg/mL；The IC that ultra-oxygen anion free radical is removed₅₀Value difference 0.39mg/mL, 0.41mg/mL and 0.19mg/mL, effect Fruit is suitable with antioxidant vitamin C and glutathione, can be used as the use of the products additive such as food, medicine, cosmetics.

Description of the drawings

Fig. 1 is the production technological process of the present invention.

Specific implementation mode

Embodiment 1：

1, the preparation of zein enzymolysis liquid

Take it is a certain amount of through extrusion and remove the maize yellow-powder of starch, a certain proportion of water be added prepare concentration of substrate and be The suspension of 10% (m/V), is first digested with alkali protease Alcalase, and enzymatic hydrolysis condition is：Enzyme concentration 2%, enzymolysis temperature 50 DEG C, pH7.7, time 75min of degree；Then flavor protease Flavourzyme is added and carries out coordinated enzymatic hydrolysis, enzymatic hydrolysis condition is： Enzyme concentration 5%, 53 DEG C, pH6.4, time 50min of hydrolysis temperature.Enzymolysis liquid heats 10min under the conditions of 100 DEG C after hydrolysis To be passivated prolease activity.Zymolyte abandons precipitation in 4000r/min centrifugations 10min, and gained supernatant is antioxidation active peptides Mixture.

2, the ultrafiltration of zein enzymolysis liquid

3, Q-Sepharose FastFlow strong anions displacement chromatography

The permeate of ＜ 6kDa obtained by ultrafiltration is diluted 10 times, 10000r/min centrifuges 15min, and supernatant is taken to cross 0.22 μm Miillpore filter, filtrate carry out strong anion displacement chromatographyStart buffer：20mmol/LpH 8.5Tris-HCl, eluent：20mmol/L pH 8.5Tris-HCl containing 1molNaCl, gradient elution 15min, flow velocity： 2mL/min, UV_214nmPlace's detection.The antioxidant activity of each pipe collection liquid is measured, collecting high-activity part is spare.

4, SephadexG-25 gel permeation chromatographies

5, reverse-phase chromatography

It weighs the separating obtained active components of 20mg SephadexG-25 to be dissolved in 2% acetonitrile solution of 1mL, with 0.22 μ M filtering with microporous membrane, chromatographic column model Pronto SIL C18 (10 μm), applied sample amount is 500 μ L, Flow velocity is 1.5mL/min, and Detection wavelength 214nm, mobile phase A is 2% acetonitrile solution containing 0.065%TFA, Mobile phase B For 80% acetonitrile solution containing 0.05%TFA, gradient elution 60min.When collecting beginning appearance using automatic fraction collector Corn peptide composition, often pipe collect 3mL.Measure the antioxidant activity of often pipe sample.Determine active region in 8,11 and 13 pipes.Instead Again collect sample after freezing drain it is spare.

The separating obtained active component of reverse-phase chromatographic column will partly be prepared to be dissolved in 2% acetonitrile solution, further use analytical column Isolated and purified, the column type of analytical column be Xselect CSH (3.5 μm), Detection wavelength 214nm, Flow velocity is 1mL/min, and applied sample amount is 20 μ L.Mobile phase A be 2% acetonitrile solution containing 0.065%TFA, Mobile phase B be containing The acetonitrile of 0.05%TFA.

Pipe component after post separation, obtains eight corn peptides by analysis, and the peak nose part progress for collecting each peak is anti-oxidant Determination of activity determines that 8- II, 8- V and 8- VII are main antioxidation active peptides.These three components are collected repeatedly, and freezing is drained, and is stayed Wait for that mass spectrum is sequenced.

Pipe component obtains five corn peptides, respectively 11-I, 11- II, 11- III, 11- IV and 11- VI.Collect each corn The peak nose part of peptide carries out the measurement of antioxidant activity, determines that 11- III and 11- VI is main antioxidation active peptides.It collects repeatedly The two peptides, freezing are drained, and mass spectrum sequencing is remained.

Pipe active component obtains nine peaks through analyzing post separation, through making comparisons with solvent chromatogram, determine 13- I, 13- III, 13- VI, 13- VII, 13- VIII are solvent peak, 13- II, 13- IV, 13- V, the corn antioxidant peptide that 13- Ⅸ is 13 pipes.Pass through survey The antioxidant activity of fixed four corn peptide peak nose parts determines that 13- V is main corn antioxidant peptide.The group is received several times Collection, freezing are drained, are sealed spare.

6, mass spectrum is sequenced：

8- II, 8- V, 8- VII, 11- III, 11- VI and the 13- V that reversed phase chromatography separation is come out carry out mass spectrum sequencing, obtain Obtain six amino acid sequences.

7, the measurement of corn antioxidant peptide antioxidation activity in vitro

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Qiqihar University

<120>Corn antioxidant active peptide 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 kind of corn peptide, which is characterized in that the amino acid sequence of the corn peptide is：ADCGWPA.

2. the preparation method of corn peptide described in claim 1, which is characterized in that the corn peptide is through extrusion and to remove The maize yellow-powder of starch is raw material, using proteinase synergy mode of action, obtains the mixtures of polypeptides with antioxidant activity, then Ultrafiltration, ion-exchange chromatography, gel chromatography, reverse-phase chromatography are passed sequentially through, step by step collecting high-activity and molecular weight is in 600- The amino acid sequence for obtaining six corn peptides is sequenced finally by mass spectrum for component within the scope of 1400Da；It is assessed after chemical synthesis The antioxidation activity in vitro of corn peptide, the final IC obtained to DPPH radicals scavengings₅₀Value be 0.23mg/mL, to super oxygen the moon from The IC of sub- radicals scavenging₅₀Value is the corn peptide ADCGWPA of 0.19mg/mL.

3. preparation method according to claim 2, which is characterized in that the proteinase synergy, which digests, is specially：It takes certain The maize yellow-powder for measuring through extrusion and removing starch, it is mass volume ratio 10% that a certain proportion of water, which is added, and prepares concentration of substrate Suspension, first digested with alkali protease Alcalase, enzymatic hydrolysis condition is：Enzyme concentration 2%, 50 DEG C of hydrolysis temperature, pH 7.7, time 75min；Then flavor protease Flavourzyme is added into alkali protease enzymolysis liquid and carries out coordinated enzymatic hydrolysis, Enzymatic hydrolysis condition is：Enzyme concentration 5%, 53 DEG C, pH 6.4, time 50min of hydrolysis temperature；Coordinated enzymatic hydrolysis liquid is 100 after enzymolysis 10min is heated under the conditions of DEG C to be passivated prolease activity；Precipitation is abandoned in obtained zymolyte centrifugation, and gained supernatant is anti-oxidant work The mixture of property peptide.

4. preparation method according to claim 2, which is characterized in that the specific process of the ultrafiltration is as follows：Using blocking The ultrafiltration membrane of molecular weight 6kDa carries out hyperfiltration treatment to the mixtures of polypeptides with antioxidant activity, obtains molecular weight ＞ The corn peptide composition of 6kDa trapped fluids and molecular weight ＜ 6kDa permeate；Select the permeate of molecular weight ＜ 6kDa as in next step The sample isolated and purified.

5. preparation method according to claim 2, which is characterized in that the specific process of the ion-exchange chromatography is as follows： Used strong anion exchanger is Q-Sepharose Fast Flow, by the permeate of molecular weight ＜ 6kDa obtained by ultrafiltration 10 times of dilution, 10000r/min centrifuge 15min, and supernatant is taken to cross 0.22 μm of miillpore filter, and 50mL filtrates carry out strong anion The chromatographic column specification of displacement chromatography, the strong anion displacement chromatography isStart buffer：20mmol/LpH 8.5Tris-HCl, eluent：8.5 Tris-HCl of 20mmol/LpH of the NaCl containing 1mol, flow velocity：2mL/min, UV_214nmPlace Detection；The antioxidant activity of each pipe collection liquid is measured, collecting high-activity part is spare.

6. preparation method according to claim 2, which is characterized in that the gel chromatography use chromatographic column specification forSephadexG-25, after the freezing of the high vigor sample of Q-Sepharose Fast Flow is drained, with double It carries out carrying out molecular weight calibration while gel chromatography desalination after steaming water dissolution, the standard substance of calibration is molecular weight 1400Da Bacitracin and molecular weight be 600Da reduced glutathione；The antioxidant activity of each pipe collection liquid is measured, molecular weight is collected Within the scope of 600-1400Da and high activity partially is spare.

7. preparation method according to claim 2, which is characterized in that the separating step of the reverse-phase chromatography includes：

The separating obtained active components of SephadexG-25 are weighed to be dissolved in 2% acetonitrile solution, with 0.22 μm of filtering with microporous membrane, The specification of chromatographic column isFiller is ProntoSILC18, the flow velocity 1.5mL/min of 10 μm of grain size, flowing Phase A is 2% acetonitrile solution containing 0.065%TFA, and Mobile phase B is 80% acetonitrile solution containing 0.05%TFA；Using certainly Dynamic fraction collector collects corn peptide composition when starting appearance, and often pipe collects 3mL；Measure the antioxidant activity of often pipe sample； Determine that active region is managed at No. 8, No. 11 and No. 13；

The separating obtained active component of reverse-phase chromatographic column will partly be prepared to be dissolved in 2% acetonitrile solution, further carried out with analytical column It isolates and purifies, the column type of analytical column is3.5 μm of XselectCSH, Detection wavelength 214nm, flow velocity are 1mL/min, applied sample amount are 20 μ L；Mobile phase A is 2% acetonitrile solution containing 0.065%TFA, and Mobile phase B is containing 0.05% The acetonitrile of TFA；

No. 8 pipe components after post separation, obtain eight corn peptides by analysis, and the peak nose part for collecting each peak carries out anti-oxidant work Property measure, determine 8-II, 8-V and 8-VII be main antioxidation active peptides；

No. 11 pipe components obtain five corn peptides, respectively 11-I, 11-II, 11-III, 11-IV and 11-VI；It collects each beautiful The peak nose part of rice peptide carries out the measurement of antioxidant activity, determines that 11-III and 11-VI is main antioxidation active peptides；

No. 13 pipe active components determine 13-I, 13- through analyzing nine peaks of post separation acquisition through making comparisons with solvent chromatogram III, 13-VI, 13-VII, 13-VIII are solvent peak, and 13-II, 13-IV, 13-V, 13-IX are the corn antioxidant of No. 13 pipes Peptide；By measuring the antioxidant activity of four corn peptide peak nose parts, determine that 13-V is main corn antioxidant peptide.

8. preparation method according to claim 7, which is characterized in that the mass spectrum, which is sequenced, is：Reversed phase chromatography separation is gone out 8-II, 8-V, 8-VII, 11-III, 11-VI and the 13-V come carries out mass spectrum sequencing, obtains six amino acid sequences；By acquisition After six corn antioxidant peptides carry out chemical syntheses, measure to the scavenging capacity of DPPH, hydroxyl and ultra-oxygen anion free radical and Reducing power, the final IC obtained to DPPH radicals scavengings₅₀Value is 0.23mg/mL, is removed to ultra-oxygen anion free radical IC₅₀Value is 0.19mg/mL corn peptides ADCGWPA.