CN105254716B - A kind of anti-oxidant hexapeptide of leek seed and the preparation method and application thereof - Google Patents
A kind of anti-oxidant hexapeptide of leek seed and the preparation method and application thereof Download PDFInfo
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- CN105254716B CN105254716B CN201510820291.3A CN201510820291A CN105254716B CN 105254716 B CN105254716 B CN 105254716B CN 201510820291 A CN201510820291 A CN 201510820291A CN 105254716 B CN105254716 B CN 105254716B
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Abstract
The invention discloses anti-oxidant hexapeptides of a kind of leek seed and the preparation method and application thereof.The anti-oxidant hexapeptide sequence is Phe-Pro-Leu-Pro-Ser-Phe(FPLPSF), experiment in vitro shows that the polypeptide can effectively remove the free radicals such as DPPH, ABTS, hydroxyl radical free radical and superoxide anion.Anti-oxidation peptide according to the present invention has the characteristics that structure is simple, anti-oxidant vigor is strong, can be used as the excellent substitution of existing artificial synthesized antioxidant, has important value with application aspect to the exploitation of novel oxidation-resistant health care product.
Description
Technical field
Application the present invention relates to a kind of leek seed natural anti-oxidation hexapeptide and preparation method thereof and in field of food belongs to
In field of food.
Background technique
China is a large agricultural country, crops it is many kinds of, but by the limited crops by processing technology condition
Processing be mostly in the junior stage, still have more large content of protein in the residue after processing, and they are mostly main
It discharges for feed industry or naturally, not only wastes resource, while also resulting in environmental pollution.The it is proposed of plant active peptides concept
And the raising of separation detection technique, make people start gradually to pay attention to the deep processing to the agricultural and sideline product rich in protein and therefrom obtains
A variety of active peptides, such as casein phosphopeptide, blood pressure lowering peptide and absorption peptide easy to digest and are opened at the research of plant origin active peptide
It sends out and plays a significant role to the added value for improving agricultural products in China deep processing.
Antioxidant at home and abroad have developed rapidly in recent years, and purposes is also increasingly wider.Artificial human body internal oxidition is studied to produce
Raw free radical is related with the aging of people and many diseases, therefore antioxidant is applied not only to the anti-oxidant of fat-containing food, and
And the exploitation as function factor for health food and cosmetics etc..There are many type of antioxidant, but some chemical syntheses
Antioxidant such as BHA, BHT etc., due to itself agreeing can have toxic side effect, national governments are one after another to its mandatory provision
ADI value controls it and is excessively added.Then people, which gradually turn to sight, extracts natural antioxygen from various plant and animal tissues
Agent.Antioxidant activity polypeptide due to low toxicity, it is efficient the features such as, antioxidant as food and body, it is considered to be artificial
The ideal replacer of synthetized oxidation preventive agent.
Anti-oxidation peptide can effectively remove the active oxygen radical of internal surplus, protect the normal configuration of cell and mitochondria
And function, prevent the generation of lipid peroxidation, and aoxidize and many diseases such as cancer of the mankind and other animals, aging, dynamic
The pathogenesis of arteries and veins hardening etc. is related.There is the substance of antioxidant activity can reduce interior free yl level for appropriate intake, prevent
Only lipid peroxidation helps body to resist disease.
Leek seed is the common edible vegetable liliaceous plant leek in ChinaAllium tuberosumRottl drying and ripening
Seed, leek seed (in pharmacopeia be frequently referred to fragrant-flowered garlic son) is that traditional Chinese medicine kind in China's first recorded in " Mingyi Bielu " is classified as middle product.
It is recorded according to pharmacopeia, leek seed has the effect of warm nourishing liver and kidney, establishing-Yang controlling nocturnal emission with astringent drugs.Leek seed is as strengthening yang and invigorating kidney with being used as medicine in China people
Between have thousands of years of history.The Ministry of Public Health, China is classified as medical and edible dual purpose plant natural products within 2002.This plantation of leek seed
Species child resource occupies first place in the world in China's very abundant, yield.At present both at home and abroad all the time to leek especially its seed
The research of leek seed is seldom.Foreign countries did not carried out the research of chemical component and effect etc. also to leek seed, and domestication studies
Research in terms of point is also and few, so far, to its active peptides or pharmacological research both at home and abroad all also almost without report.
Therefore, it is necessary to make full use of leek (leek seed) plant resources of China's very abundant, it is carried out to go deep into system research, is
It realizes the leek seed modernization of Chinese medicine and develops novel, efficient, safe healthy food and scientific basis is provided.
Summary of the invention
Simple, the anti-oxidant hexapeptide of the strong leek seed of antioxidant activity is prepared the object of the present invention is to provide a kind of, and can
The anti-oxidant hexapeptide to be applied to the development and exploitation of health food.
A kind of anti-oxidant hexapeptide of the invention, amino acid sequence Phe-Pro-Leu-Pro-Ser-Phe.Use single-letter
It is expressed as FPLPSF, i.e., by phenylalanine-proline-leucine-proline-amino acid residue structure of serine-phenylalanine 6
At.
The enzymolysis preparation of leek seed anti-oxidation peptide: it proposes the heavy method of acid using subtracting and obtains leek seed albumen, using acid egg
White enzyme is by it in optimum enzymolysis condition: pH 3.02, enzyme additive amount 1345.7U, concentration of substrate 2%(w/v), solution enzyme time 6.45h,
Boiling water bath 10min inactivation, is centrifuged 20min through 12000rpm, supernatant is taken to be freeze-dried, and as leek seed protein digestion produces
Object；
The anti-oxidant hexapeptide isolation and purification method: leek seed protein digestion product utilization Sephadex G-25 gel
Chromatography is separated, and using deionized water as eluent, 0.3 mL/min of flow velocity measures the extinction at 214 nm wavelength of elution fraction
Value；The peak with best antioxidant activity is collected, is further separated using reversed-phase high performance liquid chromatography (RP-HPLC)；RP-HPLC
Gradient be 0 ~ 5 min, 5%(V/V) acetonitrile；5 ~ 40 min, 5% ~ 40%(V/V) acetonitrile；40-50 min, 40%(V/V) second
Nitrile；Flow velocity is 2.0 mL/min, 214 nm of Detection wavelength, collects the eluting peak that elution time is 36 ~ 37min, and freeze-drying is
Obtain the anti-oxidant hexapeptide.
The present invention has the advantages that
The anti-oxidant hexapeptide have it is strongly active, have to DPPH, hydroxyl radical free radical, superoxide anion and ABTS free radical
Strong scavenging effect shows that it has important value in terms of antioxidant activity development and application.Leek seed natural anti-oxidation hexapeptide
FPLPSF has stronger removing vigor to DPPH free radical, ABTS free radical and hydroxyl radical free radical, when concentration is 0.5mg/
When mL, leek seed natural anti-oxidation hexapeptide FPLPSF is to DPPH free radical, ABTS free radical, hydroxyl radical free radical and superoxide anion
The clearance rate of free radical is respectively 56.49 ± 0.38%, 93.90 ± 2.64%, 43.41 ± 0.79% and 30.09 ± 1.80%.
Detailed description of the invention
Fig. 1: Allium tubersum rottl extract Sephedex G-25 gel filtration chromatography figure.
Fig. 2: gel chromatography eluting peak A5 reversed-phase high performance liquid chromatography figure.
Fig. 3: the mass spectrogram of anti-oxidant hexapeptide.
Fig. 4: scavenging effect of the anti-oxidant hexapeptide to ABTS free radical.
Specific embodiment
The present embodiment is implemented under the premise of the technical scheme of the present invention, gives detailed embodiment and mistake
Journey, but protection scope of the present invention is not limited only to following embodiments.
Embodiment 1
Anti-oxidant hexapeptide of the present invention is isolated and purified including Sephadex G-25 gel filtration chromatography and reversed phase high efficiency
Two steps of liquid chromatogram (RP-HPLC).
The preparation of the anti-oxidant hexapeptide enzymolysis product of leek seed: using acid protease in its optimum enzymolysis condition (pH
3.02, enzyme additive amount 1345.7U, concentration of substrate 2%(w/v)) under solve enzyme time 6.45h, boiling water bath 10min inactivation, warp
12000rpm is centrifuged 20min, takes supernatant to be freeze-dried, as leek seed protein digestion product；
Sephadex G-25 gel filtration chromatography: leek seed enzymolysis product freeze-dried powder is dissolved in deionized water,
12000 rpm are centrifuged 15 min.Take supernatant with 0.22 μm of aperture micro-filtrate membrane filtration.Sephadex G-50 gel column (1.6cm
× 100 cm) deionized water balance is used, by filtered sample upper prop.De-, 0.3 mL/min of flow velocity is washed with deionized water, in
Eluent light absorption value is detected at 214 nm wavelength, draws elution curve, as shown in Figure 1.Eluting peak peak 5 is collected, is freeze-dried ,-
80 DEG C of cryo-conservations are spare.
High performance liquid chromatography: above-mentioned 5 freeze-dried powder of peak of deionized water dissolving is further separated using RP-HPLC.Liquid chromatogram
System is LC-20A, Gemini 5 μ C18 (250mm × 10mm) reversed-phase column (Phenomenex, UK) is assembled, with water and second
Nitrile (contains 0.05%(V/V) trifluoroacetic acid) and constitute elution system carry out gradient elution.Gradient: 0 ~ 5 min, 5%(V/V) second
Nitrile；5 ~ 40 min, 5% ~ 40%(V/V) acetonitrile；40-50 min, 40%(V/V) acetonitrile；2.0 mL/min of elution flow rate detects wave
Long 214 nm, elution curve are as shown in Figure 2.Collecting elution and staying the time is 36 ~ 37 min (eluting peak 16), and freeze-drying is this
The anti-oxidant hexapeptide is invented, FF-6 is named as.
The antioxidant composition being collected into is freeze-dried, obtained compositional purity is examined using high performance liquid chromatography.Through
Detection, the anti-oxidation peptide compositional purity reach 95%, can measure its amino acid sequence.
(LC-MS/MS) method measurement amino acid sequence (Fig. 3) is used in conjunction with liquid chromatogram and mass spectrum, obtains the amino of FF-6
Acid sequence FPLPSF.
Embodiment 2
Natural anti-oxidation hexapeptide activity obtained in embodiment 1 is studied:
DPPH radicals scavenging effect: the sample of 0.4 mL various concentration and isometric 4%(W/V) DPPH dehydrated alcohol
Solution is sufficiently mixed, and is protected from light stands 30 min at room temperature, measure light absorption value under 517 nm.With deionized water and reduced form paddy Guang
Sweet peptide replaces sample to make blank control and positive control respectively.The DPPH radicals scavenging vigor of sample is based on following equation (1)
It calculates:
In formula, A0: blank control group light absorption value；As: sample sets light absorption value
ABTS radicals scavenging effect: the ABTS of 7 mmol/L is prepared with distilled water+The over cure of solution and 2.45 mmol/L
Sour potassium solution (must first place 16 h at room temperature before), respectively by ABTS+It is mixed by 1: 1 volume ratio with potassium persulfate solution
It closes, mixed liquor is diluted to light absorption value A with the phosphate buffer solution of 7.4,5 mmol/L of Ph before use734For 0.70 ±
0.02.After taking the ABTS free-atom aqueous solution mixing of the sample and 0.5 mL of 0.5 mL various concentration to stand 10 min under 734 nm
Survey its light absorption value.Sample is replaced to make blank control and positive control respectively with deionized water and reduced glutathione.ABTS is certainly
Vigor is removed by base to calculate by following equation (2):
(2)
In formula, A0: blank control group light absorption value；As: sample sets light absorption value.
Ultra-oxygen anion free radical scavenging effect: take 0.4 mL sample solution that the Tris-HCl buffering of 50 mmol/L is added
0.4 mL of solution (pH 8.3) replaces sample as blank control pipe with distilled water.Concussion mixes, and keeps the temperature 10 in 25 DEG C of water-baths
ML(25 DEG C of water-bath preheating of pyrogallol hydrochloric acid solution 0.1 of 1.5 mmol/L is added after min), 5 min of reaction are mixed rapidly,
Absorbance value A is measured at 320 nm every 30 s320.Sample is replaced to make sky respectively with deionized water and reduced glutathione
White control and positive control.The regression equation changed over time as light absorption value, the slope of curve are mouse thymus cells rate, sample
Product are calculated the removing vigor of superoxide anion by following equation (3):
(3)
In formula, Δ A0/ min: the blank control group light absorption value slope of curve；ΔAs/ min: the sample sets light absorption value slope of curve.
Hydroxyl radical free radical scavenging capacity: 100uL sample and 100uL 6.0mmol/L ferrous sulfate, 100uL are taken
It is protected from light at room temperature after the mixing of 6.0mmol/L hydrogen peroxide and is incubated for 10min, the salicylism reaction of 10uL 6.0mmol/L is added
30min measures the light absorption value of reactant under 510nm wavelength.Blank control is done with distilled water.The scavenging capacity of hydroxyl radical free radical
It is calculated by following equation (4):
In formula,A s: the light absorption value of experimental group；A 0: the light absorption value of the experimental group without hydrogen peroxide；A b: blank control group
Light absorption value
Chelated iron activity: 100uL sample is taken to mix with 10uL 2.0mmol/L frerrous chloride and 270uL deionized water
It is incubated at room temperature 3 minutes, the luxuriant and rich with fragrance Lip river piperazine of 20uL 5.0mmol/L is added, measure reaction under 562nm wavelength again after reacting 10min
The light absorption value of object.Blank control is done with distilled water.Chelated iron activity is calculated with following formula (5):
In formula,A b: control group light absorption value；A s: experimental group light absorption value.
Measured through this embodiment, leek seed natural anti-oxidation hexapeptide FPLPSF to DPPH free radical, ABTS free radical and
Hydroxyl radical free radical all has stronger removing vigor, when concentration is 0.5mg/mL, leek seed natural anti-oxidation hexapeptide FPLPSF
Clearance rate to DPPH free radical, ABTS free radical, hydroxyl radical free radical and ultra-oxygen anion free radical is respectively 56.49 ±
0.38%, 93.90 ± 2.64%, 43.41 ± 0.79% and 30.09 ± 1.80%(table 1).
The antioxidant activity of one, FF-6 of table
The sample concentration of all tests is 0.5mg/mL;
a : DPPH radicals scavenging vigor;
b : ABTS+Radicals scavenging vigor;
c : hydroxyl radical free radical removes vigor;
d : superoxide radical removes vigor;
e : chelating vigor.
SEQUENCE LISTING
<110>University of Fuzhou
<120>anti-oxidant hexapeptide of a kind of leek seed and the preparation method and application thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 6
<212> PRT
<213>amino acid sequence
<400> 1
Phe Pro Leu Pro Ser Phe
1 5
Claims (3)
1. a kind of anti-oxidant hexapeptide of leek seed, it is characterised in that: the anti-oxidant hexapeptide amino acid sequence is Phe-Pro-Leu-
Pro-Ser-Phe。
2. a kind of preparation method of the anti-oxidant hexapeptide of leek seed as described in claim 1, it is characterised in that: from natural leek seed
In isolate and purify or artificial synthesis obtains；Specific steps include the following:
(1) preparation of leek seed protein hydrolysate: obtaining leek seed albumen using alkali extraction and acid precipitation, will using acid protease
It is digested under optimum enzymolysis condition: pH 3.02, enzyme additive amount 1345.7U, concentration of substrate 2%(w/v), solution enzyme time 6.45h,
Boiling water bath 10min inactivation, is centrifuged 20min through 12000rpm, supernatant is taken to be freeze-dried, and as leek seed protein digestion produces
Object；
(2) enzymolysis product of leek seed albumen is separated using Sephadex G-25 gel chromatography, is elution with deionized water
Liquid, 0.3 mL/min of flow velocity measure the light absorption value at 214 nm wavelength of elution fraction；Collecting has best antioxidant activity
Peak is further separated using reversed-phase high performance liquid chromatography (RP-HPLC)；The gradient of RP-HPLC is 0 ~ 5 min, 5%(V/V)
Acetonitrile；5 ~ 40 min, 5% ~ 40%(V/V) acetonitrile；40-50 min, 40%(V/V) acetonitrile；Flow velocity is 2.0 mL/min, detects wave
Long 214 nm collect the eluting peak that elution time is 36 ~ 37min, are freeze-dried up to the anti-oxidant hexapeptide.
3. a kind of anti-oxidant hexapeptide of leek seed as described in claim 1 is preparing the application in antioxidant health-care product.
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| CN105601707B (en) * | 2016-02-05 | 2019-03-12 | 福州大学 | A kind of anti-oxidant tetrapeptide of dandelion seed and preparation method thereof |
| CN106478770B (en) * | 2016-10-13 | 2019-12-17 | 福州大学 | Perilla seed antioxidant dipeptide and preparation method and application thereof |
| CN108191958B (en) * | 2018-01-09 | 2020-10-09 | 福州大学 | Linseed meal antioxidant hexapeptide and preparation method and application thereof |
| CN109055463A (en) * | 2018-09-17 | 2018-12-21 | 河南城建学院 | A kind of preparation method of fragrant-flowered garlic seed polypeptide |
| CN111978370B (en) * | 2020-08-31 | 2021-11-30 | 福州大学 | Chia seed antioxidant peptide and preparation method and application thereof |
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| CN102197856A (en) * | 2011-06-22 | 2011-09-28 | 山西大学 | Preparation method of pumpkin seed antioxidant peptide |
| CN103864891A (en) * | 2014-03-25 | 2014-06-18 | 福州大学 | Chinese chive seed antioxidative tripeptide, and preparation and application thereof |
| CN103936827A (en) * | 2014-04-22 | 2014-07-23 | 福州大学 | Chinese chive seed antibacterial tripeptide and preparation method and use thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102197856A (en) * | 2011-06-22 | 2011-09-28 | 山西大学 | Preparation method of pumpkin seed antioxidant peptide |
| CN103864891A (en) * | 2014-03-25 | 2014-06-18 | 福州大学 | Chinese chive seed antioxidative tripeptide, and preparation and application thereof |
| CN103936827A (en) * | 2014-04-22 | 2014-07-23 | 福州大学 | Chinese chive seed antibacterial tripeptide and preparation method and use thereof |
Non-Patent Citations (2)
| Title |
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| Purification and characterization of anantioxidant peptide (GSQ) from Chinese leek(Allium tuberosum Rottler) seeds;Jing Hong等;《Journal of functional foods》;20140621;第10卷;第145页右侧第1段、第2.2-2.5部分、第4部分结论 |
| 抗氧化肽的酶解制备及其构效关系的研究进展;程云辉 等;《食品与机械》;20091130;第25卷(第6期);174-178 |
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