CN105820229A - Duck gizzard duck's gizzard antioxidative peptide and application thereof - Google Patents
Duck gizzard duck's gizzard antioxidative peptide and application thereof Download PDFInfo
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- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
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Abstract
The invention discloses duck gizzard duck's gizzard antioxidative peptide and application thereof. The amino acid sequence of the duck gizzard duck's gizzard antioxidative peptide is as shown in Asn-Ly-Phe-Ile-Leu-Lys (a component DKFILK), and the monoisotopic peak of a parent ion of the sequence is m/z 382.2376 Da. The preparation process comprises the following steps: degreasing duck gizzard, performing enzymolysis, performing ultrafiltration on an enzymolysis product, performing gel filtration chromatography, performing reverse high performance liquid chromatography purification, and performing mass spectrum together with amino acid analysis, thereby obtaining the antioxidative peptide. The duck gizzard duck's gizzard antioxidative peptide is wide and cheap in raw material source and scientific and reasonable in process; by virtue of an enzymolysis technique and together with purification through ultrafiltration grading and chromatographic analysis, the prepared antioxidative peptide has relatively high activity; compared with an antioxidant prepared through chemical synthesis, the antioxidative peptide disclosed by the invention has the advantages of security, no toxic or side effect, good antioxidation property, easiness in digestion and absorption, and the like, and can be used as medicines, functional food and the like.
Description
Technical field
The invention belongs to technical field of polypeptide, be specifically related to a kind of duck gizzard protein sources anti-oxidation peptide and preparation method, also relate to
And the purposes of this duck gizzard protein sources anti-oxidation peptide.
Background technology
Increasing research shows, activity in vivo oxygen-derived free radicals has certain function, as immunity and signal are conducted through
Journey.But too much reactive oxygen free radical also has destruction, cause the damage of human normal cell and tissue, thus cause many
Plant disease, such as diseases such as heart disease, senile dementia, parkinson disease and tumors.Therefore, to the active oxygen that can remove internal surplus
Free radical, protection cell and mitochondrial normal configuration and function, prevent the generation of lipid peroxidation, there is induction anticancer, anti-
And the anti-ageing research of the Natural antioxidant waiting for a long time other biological activity has been increasingly becoming focus.At present, resist due to chemosynthesis
Oxidant such as BHA(tert-butyl group alkyl methoxybenzene), BHT(2,6-ditertbutylparacresol), PG(gallic acid third lipoprotein), TBHQ
(tert-butylhydroquinone) etc. owing to good other countries' food service industry the most in the world cheap, active being used widely,
But many studies have shown that, this kind of chemosynthesis antioxidant (in addition to TBHQ) is owing to also existing many side effect, as to people
The liver of body, spleen, lung all adversely affect, and can induce malignant tumor etc..Therefore, the concerned countries such as Europe, the U.S., Japan is the most right
The measure prohibitted the use of taked by this kind of chemosynthesis antioxidant.The scholar of many countries starts study hotspot turns to natural resisting
The research of oxidant, as developed Natural Antioxidant Peptides, blood pressure lowering peptide by some technology, resisted and swell from pluck and plant
Tumor peptide etc..
The anti-oxidation peptide of separate sources becomes study hotspot, such as Concha Ostreae, soybean protein, Fructus Jujubae, Corium Anas domestica, albumen etc.
Hydrolysate all has certain antioxidant activity, and further separates pure to these materials with antioxidant activity
Change, the anti-oxidation peptide with high anti-oxidation activity can be obtained.As utilized zymolysis technique to obtain anti-oxidation peptide from animal and internal organs,
Chinese patent CN103275180B, discloses a kind of with phosphate buffer regulation pH value, utilizes protease hydrolyzed black scraper fish
Head, obtains the antioxidation of a kind of aminoacid sequence Leu-Ser-His-Gly-Pro-Tyr by a series of separating and purifying technologies
Peptide；
Chinese patent CN103073621B discloses a kind of ground meat proteins of tuna anti-oxidation peptide and its production and use, with
Defat tuna minced meat, as raw material, add phosphate buffer, regulate pH to 5.0 ~ 7.0, obtain mixed liquor；Mixed liquor is stirred
Preheating, adds papain, obtains enzymatic hydrolysate；By enzymatic hydrolysate elder generation enzyme denaturing the most successively through desalination, ultrafiltration and chromatography, obtain ammonia
Base acid sequence is the anti-oxidation peptide of Tyr-Glu-Asn-Gly-Gly；
Chinese patent CN103467568B disclose a kind of utilization solid-liquid ratio be 1:3 defat golden cuttlefish meat solid content in add
0.02 mol/ml phosphate buffer regulates pH value, finally utilizes papain enzymolysis to obtain a kind of aminoacid sequence
For Ala-Pro-Pro-Glu-Asn-Gly-Met-Ala-Gln-Met, molecular weight is the golden cuttlefish protein antioxidant of 1045.21Da
Peptide；
Chinese patent CN103739693B discloses one and utilizes microwave-assisted enzyme process to take enzymolysis pinctada fucata meat to obtain one
Plant the pinctada fucata martensii meat anti-oxidizing peptide that sequence is Gly-Ala-Gly-Leu-Pro-Gly-Lys-Arg-Glu-Arg of peptide；In
The open equally a kind of phosphate buffer that adds in solid-to-liquid ratio is 1:15 ~ 20ml of state's patent CN103524596B regulates pH value,
Then utilizing papain to carry out enzymolysis protein to obtain the aminoacid sequence anti-oxidation peptide as Leu-Asp-Lys, these resist
Oxidation peptide can apply in the fields such as food industry, medicine and cosmetics.
Duck gizzard sweet in the mouth, property are flat, salty, have the effect of stomach invigorating.Stomachache is the clinical common a kind of disease of gastropathy, Chinese medicine usually with
This life is name of disease, is equivalent to the acute and chronic gastritis of modern medicine, gastric ulcer, stomach neurosis etc..Duck gizzard is that duck meat is processed
By-product in journey, to the utilization of duck meat by-product great majority also in the roughing stage, abundant raw material source, but due to China
Most enterprises is deficient to the deep process technology of duck gizzard, so causing some wastings of resources, therefore, utilizing cheap raw material, adopting
By certain technology, duck gizzard is developed into the higher product of added value and become livestock products enterprise problem demanding prompt solution.
ORAC(Oxgen Radical Absorbance Capacity) refer to oxygen-derived free radicals absorbability, i.e. test food
The general international standard unit of the content of polyphenoils in medicine.ORAC value is the highest, and the oxidation resistance of suppression free radical is more
By force.The method now has become as United States Department of Agriculture (USDA), U.S. sanitary institute, the major criterion of FDA evaluation food oxydating resistance ability.Europe
Continent, the food of Japan and other countries, functional food industry the most commonly used ORAC value as the important evaluation criterion of oxidation resistance,
It it is the most authoritative method of antioxidant activity in vitro of evaluating at present.
The present inventor finds in an experiment, with duck gizzard as raw material, utilizes zymolysis technique to prepare the technical study of anti-oxidation peptide
It is in the blank stage, and for free radical antioxidation isolated and purified index, duck gizzard enzymatic hydrolysate is prepared high activity with ORAC value and resist
Oxidation peptide and application thereof also have no report.
Summary of the invention
First purpose of the present invention is to provide a kind of raw material sources wide, inexpensive, safe without toxic side effect, craft science
Rationally, antioxidant activity is strong and is prone to digest and assimilate, and can remove a kind of duck gizzard egg of free radical and anti-lipid peroxidation effect
White source anti-oxidation peptide and preparation method；Second purpose is that this anti-oxidation peptide offer one can be as functional food, medicine
Purposes with food additive.
The purpose of the present invention and solve its technical problem underlying and realize by the following technical solutions: a kind of duck gizzard egg
White source anti-oxidation peptide, it is characterised in that: the aminoacid sequence of this anti-oxidation peptide is Asn-Lys-Phe-Ile-Leu-Lys (component
DKFILK), the monoisotopic peak of the parent ion of this sequence is (m/z 382.2376 Da, [M+H]+), its concrete preparation process
Comprise the following steps:
1) pretreatment of raw material: take duck gizzard and thaw, remove muscle, mince, pulverized with pulverizer；
2) petroleum ether degreasing: the duck gizzard after pulverizing adds petroleum ether and soaks 10 ~ 12h, pours out petroleum ether, by residual in fume hood
Staying petroleum ether thoroughly to volatilize, pack is preserved in refrigerator standby；
3) being homogenized, regulate pH value: be homogenized in the ratio of solid-liquid ratio 1:2 ~ 4 with water by the duck gizzard meat paste after defat, regulation pH value is extremely
6.5~7；
4) adding protease hydrolyzed: adjust the temperature to 50 ~ 55 DEG C, protease addition is: 0.4 ~ 0.6%, homogenate water bath with thermostatic control 3 ~
5h；
5) enzyme denaturing, centrifugal and lyophilization: by the duck gizzard homogenate after water-bath at 95 ~ 110 DEG C of enzyme denaturing 10 ~ 15min, after cooling,
Being centrifuged 10 ~ 20min at 4000 ~ 6000r/min, take supernatant and i.e. obtain enzymolysis solution, lyophilizing obtains zymolyte dry powder；
6) isolated and purified: above-mentioned gained enzymolysis solution is carried out respectively ultrafiltration, chromatography (including gel chromatography, anti-high performance liquid chromatography)
Deng separation purification refine process, last lyophilization obtains duck gizzard protein sources anti-oxidation peptide.
Further, in described step 4), protease is papain, and enzyme activity is 13000 IU/g.
Further, described step 6) ultrafiltration and chromatography detailed process are:
Ultrafiltration classification: by above-mentioned steps 5) to make concentration be 10 ~ 15 mg/mL solution for the zymolyte that obtains, in 0.05 ~ 0.1 MPa
Operating pressure, the revolution speed of 60 ~ 65r/min and 20 ~ 25 DEG C operating temperature under use ultrafilter membrane (molecular cut off 3
KDa) carry out hyperfiltration treatment, collect molecular weight and be less than 3 kDa parts, lyophilizing, obtain ultrafiltration zymolyte；
Chromatography process: above-mentioned ultrafiltration zymolyte (ZT1) dry powder deionized water is made into the solution of 6-15mg/ml, through gel
G15 chromatographic column (2.6cm × 60cm) separates, and is collected in each eluting peak under 214nm, lyophilizing, selects the component that ORAC value is higher to make
For anti-efficiently liquid phase purified；Zymolyte deionized water after above-mentioned gel chromatography is made into the solution of 10-20mg/ml, profit
It is purified by anti-high performance liquid chromatography (RP-HPLC), is collected in each purified components under 220nm, with the component that ORAC value is higher
As utilizing mass-spectrometric technique to identify the zymolyte of aminoacid sequence；1 high activity antioxygen is obtained according to ORAC value antioxidant activity
Change peptide sequence Asn-Lys-Phe-Ile-Leu-Lys(component DKFILK).
Further, described gel is sephadex G 15；Described anti-high performance liquid chromatography testing conditions is: sample size:
15~20ul；Chromatographic column: Lichrospher C18 post；Flow velocity: 4mL/min；Mobile phase A: 5% acetonitrile+0.1% trifluoroacetic acid；Stream
Dynamic phase B:100% acetonitrile；Detection wavelength: 220nm.
The application of above-mentioned a kind of duck gizzard protein sources anti-oxidation peptide, it is characterised in that sequence Asn-Lys-Phe-Ile-Leu-
Lys (component DKFILK) is the index using ORAC value as the activity power measuring isolated and purified anti-oxidation peptide, and cloudy to super oxygen
Ion radical (IC50 value is 14.58 mg/mL), hydroxyl radical free radical (IC50 is 0.016mg/mL), (IC50 is hydrogen peroxide
163.90 μ g/mL) there is higher scavenging capacity, the damage (IC50 value is 1.93 mg/mL) to DNA simultaneously has protective effect；
Asn-Lys-Phe-Ile-Leu-Lys (component DKFILK) has safe without toxic side effect, antioxidant activity is strong, be prone to absorption
Etc. advantage, can be as medicine, functional food application.
The present invention compared with prior art has clear advantage and beneficial effect.From above technical scheme, this
Bright raw material sources are wide, inexpensive, and craft science is reasonable, by zymolysis technique, use the purification such as ultrafiltration classification and chromatography simultaneously,
The anti-oxidation peptide prepared has higher activity；Compared with the antioxidant of chemosynthesis, the anti-oxidation peptide that the present invention prepares
Have safe without toxic side effect, antioxidant activity is strong and is prone to advantages such as digesting and assimilating, can be as medicine, functional food etc.
Application.
Accompanying drawing explanation
Fig. 1 is ultrafiltration zymolyte each component ORAC value figure of the present invention；
Fig. 2 is sephadex G 15 chromatography analysis figure of the present invention；
Fig. 3 is that G15 of the present invention separates four components ZTT1, the ORAC activity value figures of ZTT2, ZTT3, ZTT4；
Fig. 4 A is the RP-HPLC analysis chart that sephadex G 15 of the present invention prepares zymolyte (ZTT3)；
Fig. 4 B is that separated each component is to ORAC oxygen-derived free radicals activity figure；
Fig. 5 a is the MS mass spectrum of i.e. sequence Asn-Lys-Phe-Ile-Leu-Lys of separation component V of the present invention (component DKFILK)
Figure；
Fig. 5 b is MS/MS mass spectrum；
Fig. 6 is the ultra-oxygen anion free radical scavenging capacity figure of anti-oxidation peptide of the present invention；
Fig. 7 is the Scavenging activity on hydroxyl free radical figure of anti-oxidation peptide of the present invention；
Fig. 8 is the hydrogen peroxide scavenging capacity figure of anti-oxidation peptide of the present invention；
Fig. 9 is the DNA damage protective effect figure of anti-oxidation peptide of the present invention.
Detailed description of the invention
Below in conjunction with accompanying drawing and preferred embodiment, to a kind of duck gizzard protein sources anti-oxidation peptide proposed according to the present invention and
Application detailed description of the invention, feature and effect thereof, after describing in detail such as.
See Fig. 1-9, a kind of duck gizzard protein sources anti-oxidation peptide, it is characterised in that the aminoacid sequence of this anti-oxidation peptide is
Asn-Lys-Phe-Ile-Leu-Lys (component DKFILK), the monoisotopic peak of the parent ion of this sequence is (m/z
382.2376 Da, [M+H]+), its concrete preparation process comprises the following steps:
1) pretreatment of raw material: take duck gizzard and thaw, remove muscle, mince, pulverized with pulverizer；
2) petroleum ether degreasing: the duck gizzard after pulverizing adds petroleum ether and soaks 10 ~ 12h, pours out petroleum ether, by residual in fume hood
Staying petroleum ether thoroughly to volatilize, pack is preserved in refrigerator standby；
3) being homogenized, regulate pH value: be homogenized in the ratio of solid-liquid ratio 1:2 ~ 4 with water by the duck gizzard meat paste after defat, regulation pH value is extremely
6.5~7；
4) adding protease hydrolyzed: adjust the temperature to 50 ~ 55 DEG C, protease addition is: 0.4 ~ 0.6%, homogenate water bath with thermostatic control 3 ~
5h；
5) enzyme denaturing, centrifugal and lyophilization: by the duck gizzard homogenate after water-bath at 95 ~ 110 DEG C of enzyme denaturing 10 ~ 15min, after cooling,
Being centrifuged 10 ~ 20min at 4000 ~ 6000r/min, take supernatant and i.e. obtain enzymolysis solution, lyophilizing obtains zymolyte dry powder；
6) isolated and purified: above-mentioned gained enzymolysis solution is carried out respectively ultrafiltration, chromatography (including gel chromatography, anti-high performance liquid chromatography)
Deng separation purification refine process, last lyophilization obtains duck gizzard protein sources anti-oxidation peptide.
Further, in described step 4), protease is papain, and enzyme activity is 13000 IU/g.
Further, described step 6) ultrafiltration and chromatography detailed process are:
Ultrafiltration classification: by above-mentioned steps 5) to make concentration be 10 ~ 15mg/mL solution for the zymolyte that obtains, in 0.05 ~ 0.1MPa's
Ultrafilter membrane (molecular cut off 3kDa) is used to enter under the operating temperature of operating pressure, the revolution speed of 60 ~ 65r/min and 20 ~ 25 DEG C
Row hyperfiltration treatment, collects molecular weight and is less than 3kDa part, lyophilizing, obtain ultrafiltration zymolyte；
Chromatography process: above-mentioned ultrafiltration zymolyte (ZT1) dry powder deionized water is made into the solution of 6-15mg/ml, through gel
G15 chromatographic column (2.6cm × 60cm) separates, and is collected in each eluting peak under 214nm, lyophilizing, selects the component that ORAC value is higher to make
For anti-efficiently liquid phase purified；Zymolyte deionized water after above-mentioned gel chromatography is made into the solution of 10-20mg/ml, profit
It is purified by anti-high performance liquid chromatography (RP-HPLC), is collected in each purified components under 220nm, with the component that ORAC value is higher
As utilizing mass-spectrometric technique to identify the zymolyte of aminoacid sequence；1 high activity antioxygen is obtained according to ORAC value antioxidant activity
Change peptide sequence Asn-Lys-Phe-Ile-Leu-Lys(component DKFILK).
Further, described gel is sephadex G 15；Described anti-high performance liquid chromatography testing conditions is: sample size:
15~20ul；Chromatographic column: Lichrospher C18 post；Flow velocity: 4mL/min；Mobile phase A: 5% acetonitrile+0.1% trifluoroacetic acid；Stream
Dynamic phase B:100% acetonitrile；Detection wavelength: 220nm.
The application of above-mentioned a kind of duck gizzard protein sources anti-oxidation peptide, it is characterised in that sequence Asn-Lys-Phe-Ile-Leu-
Lys (component DKFILK) is the index using ORAC value as the activity power measuring isolated and purified anti-oxidation peptide, and cloudy to super oxygen
Ion radical (IC50 value is 14.58 mg/mL), hydroxyl radical free radical (IC50 is 0.016mg/mL), (IC50 is hydrogen peroxide
163.90 μ g/mL) there is higher scavenging capacity, the damage (IC50 value is 1.93 mg/mL) to DNA simultaneously has protective effect；
Asn-Lys-Phe-Ile-Leu-Lys (component DKFILK) has safe without toxic side effect, antioxidant activity is strong, be prone to absorption
Etc. advantage, can be as medicine, functional food application.
In the present invention, (Oxygen radical absorbance capacity, oxygen-derived free radicals absorbs energy to use ORAC
Power) activity value evaluates the activity of anti-oxidation peptide.
Concrete ORAC reactions steps is: enter under the NaH2PO4-Na2HPO4 buffered environment of 75 mmol/L pH=7.4
OK, with NaH2PO4-Na2HPO4 buffer, enzymolysis solution protein concentration is diluted to 0.04 mg/mL before reaction.At 96 hole fluorescent screens
Micropore adds testing sample 20 L, simultaneously with NaH2PO4-Na2HPO4 buffer Trolox is made into 10,20,40,60,
80,100 mol/L(do blank with 20 L NaH2PO4-Na2HPO4 buffer) do mark song；120 are added with multichannel pipettor
L 116.7 nmol/L(final concentration 70 nmol/L) fluorescein (FL), after being incubated 15 min at 37 DEG C, use multichannel pipettor
60 L 40 mmol/L(final concentration 12 mmol/L are added rapidly in each hole) AAPH starts reaction.AAPH pH 7.4
75 mmol/L NaH2PO4-Na2HPO4 delay liquid and prepare 38.25 mM, the freshest preparation of AAPH of use.Microwell plate is placed in
With excitation wavelength 485 nm at 37 DEG C in microplate reader, launch wavelength 520 nm and start clock reaction reading (f0) every 1min survey
Measure the most each hole fluorescence intensity (f1, f2, f3f100), METHOD FOR CONTINUOUS DETERMINATION 100 times, each reading is linked to be curve for fixed 1 time.Each
Sample arranges 3 multiple holes.The fluorescence intensity data of each micropore reaction of experiment gained is input in Excel enter one by software
Step processes, and the absolute fluorescence intensity data of each micropore different time points, compared with the blank fluorescence intensity of-AAPH, obtain relatively
Fluorescence intensity f0, uses approximate integration to calculate the area (AUC) under fluorescence decay curve, fluorescence decay with relative intensity of fluorescence
Area under a curve can approximate regards each trapezoidal area sum as, and AUC represents area under a curve.Formula is expressed as follows: AUC=
0.5 (f0+fn)+(f1+f2++ fi ++fn-1)
Net AUC= AUCsample-AUCblank
Absolute fluorescence intensity when wherein fi represents i-th measuring point, t is the time interval between adjacent two measuring points,
In this test ORAC assay method, t is set as 1 min.The area under fluorescence decay curve under antioxidant action and nonreactive
The decay part area of the difference of the area under the fluorescence decay curve of Free Radical in the presence of oxidant, i.e. fluorescence quenching curve
Net AUC, for the protected area of antioxidant.ORAC, is the protected area by fluorescence decay curve and mark
The protected area of quasi-antioxidant is compared and is obtained.
Trolox concentration is directly proportional to NetAUC, is substituted into by sample NetAUC, and conversion obtains ORAC value, and i.e. every gram enzymolysis produces
Thing is equivalent to the amount (molTrolox equivalents/g) of Torlox.ORAC value is with Trolox equivalent TE (molTrolox
Equivalent Per g Protein) represent.
ORAC value is the highest, and the oxidation resistance of its product is the strongest.Duck gizzard protein sources anti-oxidation peptide of the present invention is at zymolyte
Carry out ORAC value in the separation purge process such as ultrafiltration and chromatography and be shown in Table 1.
Embodiment:
1) take Sansui duck duck gizzard to thaw, remove muscle, mince, pulverized with pulverizer.
2) the duck gizzard meat paste after pulverizing adds petroleum ether and carries out defat 10 ~ 12h, pour out petroleum ether, in fume hood
Thoroughly being volatilized by unrecovered oil ether, collect solid content, pack is preserved in refrigerator standby.
3) being homogenized in the ratio of solid-liquid ratio 1:2 ~ 4 with water by the duck gizzard meat paste after defat, regulation pH value is to 6.5 ~ 7.
4) water-bath temperature is regulated to 50 ~ 55 DEG C, in defat meat paste liquid, add the papain of 0.4 ~ 0.6%, then
By homogenate water bath with thermostatic control 3 ~ 5h.
5) by the duck gizzard homogenate after water-bath at 95 ~ 110 DEG C of enzyme denaturing 10 ~ 15min, after cooling, at 4000 ~ 6000r/min
Centrifugal 10 ~ 20min, takes supernatant and i.e. obtains enzymolysis solution, lyophilizing, obtain zymolyte lyophilized powder.
6) above-mentioned gained zymolyte is carried out respectively ultrafiltration, chromatography (including gel chromatography, anti-high performance liquid chromatography) isochrome
Spectrum purge process, lyophilization obtains duck gizzard protein sources anti-oxidation peptide, utilizes Q Exactive quadrupole rod-electrostatic field track trap high score
Distinguishing mass-spectrometric technique its structure of binding amino acid sequence Analysis and Identification, detailed process is:
(a) ultrafiltration classification: it is 10 ~ 15 mg/mL solution that the zymolyte obtained is made concentration, in the work of 0.05 ~ 0.1 MPa
Ultrafilter membrane (molecular cut off 3kDa) is used to surpass under the operating temperature of pressure, the revolution speed of 60 ~ 65r/min and 20 ~ 25 DEG C
Filter processes, and collects molecular weight and is less than 3kDa part, and lyophilizing, the component that wherein ORAC value is the highest is ultrafiltration zymolyte (ZT1).
B () gel chromatography chromatographs: above-mentioned ultrafiltration zymolyte (ZT1) dry powder deionized water is made into the molten of 6-15mg/ml
Liquid, separates through gel G15 chromatographic column (2.6cm × 60cm), is collected in each eluting peak under 214nm, lyophilizing, wherein ORAC value
The highest component is that zymolyte (ZTT3) prepared by gel.
(c) RP-HPLC chromatogram purification: the zymolyte deionized water after above-mentioned gel chromatography is made into 10-20mg/ml's
Solution, utilizes anti-high performance liquid chromatography (RP-HPLC) to be purified (testing conditions: sample size: 15 ~ 20ul；Chromatographic column:
Lichrospher C18 post；Flow velocity: 4mL/min；Mobile phase A: 5% acetonitrile+0.1% trifluoroacetic acid；Mobile phase B: 100% acetonitrile；
Detection wavelength: 220nm), obtain 1 high activity anti-oxidation peptide (component V) according to the ORAC value the highest component of activity.
D () Q Exactive quadrupole rod-electrostatic field track trap high resolution mass spectrum binding amino acid sequence identifies structure: RP-
The high activity anti-oxidation peptide (component V) that HPLC detection is collected is simple spike, component V is carried out first mass spectrometric and second order ms divides
Analysis, resolves by pFind software, and the aminoacid sequence identifying component V is Asn-Lys-Phe-Ile-Leu-Lys(component
DKFILK), the monoisotopic peak of the parent ion of its sequence is m/z 382.2376 Da.
By above-mentioned gained duck gizzard protein sources anti-oxidation peptide Asn-Lys-Phe-Ile-Leu-Lys(component
DKFILK) antioxidation in vitro free radical scavenging test is carried out.Test result indicate that: Asn-Lys-Phe-Ile-Leu-Lys(component
DKFILK) to ultra-oxygen anion free radical (IC50 value is 14.58 mg/mL), hydroxyl radical free radical (IC50 is 0.016mg/mL),
Hydrogen peroxide (IC50 is 163.90 μ g/mL) has a higher scavenging capacity, and to the damage of DNA, (IC50 value is 1.93 mg/ simultaneously
ML) there is protective effect.
The above, be only presently preferred embodiments of the present invention, and the present invention not makees any pro forma restriction, appoints
What is without departing from technical solution of the present invention content, any is simply repaiied according to what above example made by the technical spirit of the present invention
Change, equivalent variations and modification, all still fall within the range of technical solution of the present invention.
Claims (6)
1. a duck gizzard protein sources anti-oxidation peptide, it is characterised in that: the aminoacid sequence of this anti-oxidation peptide is Asn-Lys-Phe-
Ile-Leu-Lys, component is DKFILK, and the monoisotopic peak of the parent ion of its sequence is m/z 382.2376 Da, [M+H]+；
Its preparation method comprises the following steps:
(1) pretreatment of raw material: take duck gizzard and thaw, remove muscle, mince, pulverized with pulverizer；
(2) petroleum ether degreasing: the duck gizzard after pulverizing adds petroleum ether and soaks 10 ~ 12h, pours out petroleum ether, will in fume hood
Unrecovered oil ether thoroughly volatilizees, and pack is preserved in refrigerator standby；
(3) being homogenized, regulate pH value: be homogenized in the ratio of solid-liquid ratio 1:2 ~ 4 with water by the duck gizzard meat paste after defat, regulation pH value is extremely
6.5~7；
(4) adding protease hydrolyzed: regulation temperature 50 ~ 55 DEG C, protease addition is: 0.4 ~ 0.6%, homogenate water bath with thermostatic control 3 ~
5h；
(5) enzyme denaturing, centrifugal and lyophilization: by the duck gizzard homogenate after water-bath at 95 ~ 110 DEG C of enzyme denaturing 10 ~ 15min, after cooling,
Being centrifuged 10 ~ 20min at 4000 ~ 6000r/min, take supernatant and i.e. obtain enzymolysis solution, lyophilizing obtains zymolyte dry powder；
(6) isolated and purified: above-mentioned gained zymolyte is carried out respectively ultrafiltration purification and gel chromatography chromatography, anti-high performance liquid chromatography
Chromatography purification refine process, last lyophilization obtains duck gizzard protein sources anti-oxidation peptide.
2. a kind of duck gizzard protein sources anti-oxidation peptide as claimed in claim 1, it is characterised in that: described step (4) protease is
Papain, enzyme activity is 13000IU/g.
3. a kind of duck gizzard protein sources anti-oxidation peptide as claimed in claim 1, it is characterised in that: ultrafiltration purification mistake in step (6)
Cheng Wei: using hollow cellulose membrane ultrafiltration to separate, retaining molecular weight MWCO is 3kDa, revolution speed: 60-65 r/min, work
Make temperature: 20 ~ 25 DEG C, outlet pressure is to carry out classification under 0.05 ~ 0.1MPa, collect gained molecular cut off less than 3 kDa groups
Divide ZT1 ultrafiltrate, lyophilization, obtain ultrafiltration zymolyte；Choose ORAC activity value the highest, molecular cut off is less than 3 kDa
Component ZT1 separates as chromatography zymolyte.
4. a kind of duck gizzard protein sources anti-oxidation peptide as claimed in claim 1, it is characterised in that: gel chromatography layer in step (6)
Analysis, anti-high performance liquid chromatography chromatograph and are: above-mentioned ultrafiltration zymolyte ZT1 dry powder deionized water is made into the solution of 6-15mg/ml,
Separate with 2.6cm × 60cm G15 chromatographic column, be collected in each eluting peak under 214nm, lyophilizing, select the component that ORAC value is higher to make
For anti-efficiently liquid phase purified；Zymolyte deionized water after above-mentioned gel chromatography being chromatographed is made into the molten of 10-20mg/ml
Liquid, utilizes anti-high performance liquid chromatography RP-HPLC to carry out chromatography purification, is collected in each purified components under 220nm, higher with ORAC value
Component as utilizing mass-spectrometric technique to identify the zymolyte of aminoacid sequence；1 high work is obtained according to ORAC value antioxidant activity
Property anti-oxidation peptide sequence: Asn-Lys-Phe-Ile-Leu-Lys, component is DKFILK.
5. a kind of duck gizzard protein sources anti-oxidation peptide, it is characterised in that: described gel is polydextran gel
G15；Described anti-high performance liquid chromatography testing conditions is: sample size: 15 ~ 20ul；Chromatographic column: Lichrospher C18 post；Stream
Speed: 4mL/min；Mobile phase A: 5% acetonitrile+0.1% trifluoroacetic acid；Mobile phase B: 100% acetonitrile；Detection wavelength: 220nm.
6. the application of a kind of duck gizzard protein sources anti-oxidation peptide as described in one of claim 1-5, it is characterised in that: can be as sky
So application on functional food, medicine prepared by polyphenoils.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107099573A (en) * | 2017-06-13 | 2017-08-29 | 江苏省农业科学院 | The method that optimization degreasing mode combining ultrasonic assistance enzymolysis duck liver prepares anti-oxidation peptide |
| CN109111503A (en) * | 2018-07-23 | 2019-01-01 | 华南理工大学 | A kind of synthesis polypeptide with anti-oxidation efficacy and gene and preparation method and the application for encoding the polypeptide |
| CN111004309A (en) * | 2019-12-31 | 2020-04-14 | 福建省水产研究所（福建水产病害防治中心） | ACE inhibitory peptide prepared from Takifugu flavidus fish skin and preparation method thereof |
| CN113307844A (en) * | 2021-05-25 | 2021-08-27 | 青岛新万福食品有限公司 | Polypeptide separated from pig liver and having strong oxidation resistance and promoting lipid metabolism, preparation method and application |
| CN114525321A (en) * | 2022-03-22 | 2022-05-24 | 江西煌上煌集团食品股份有限公司 | Antioxidant peptide derived from duck viscera and preparation method thereof |
| CN115947787A (en) * | 2022-09-06 | 2023-04-11 | 宁波大学 | Duck liver protein source antioxidant functional peptide and preparation method and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107099573A (en) * | 2017-06-13 | 2017-08-29 | 江苏省农业科学院 | The method that optimization degreasing mode combining ultrasonic assistance enzymolysis duck liver prepares anti-oxidation peptide |
| CN109111503A (en) * | 2018-07-23 | 2019-01-01 | 华南理工大学 | A kind of synthesis polypeptide with anti-oxidation efficacy and gene and preparation method and the application for encoding the polypeptide |
| CN109111503B (en) * | 2018-07-23 | 2021-08-10 | 华南理工大学 | Synthetic polypeptide with antioxidant effect, gene for encoding polypeptide, preparation method and application thereof |
| CN111004309A (en) * | 2019-12-31 | 2020-04-14 | 福建省水产研究所（福建水产病害防治中心） | ACE inhibitory peptide prepared from Takifugu flavidus fish skin and preparation method thereof |
| CN113307844A (en) * | 2021-05-25 | 2021-08-27 | 青岛新万福食品有限公司 | Polypeptide separated from pig liver and having strong oxidation resistance and promoting lipid metabolism, preparation method and application |
| CN113307844B (en) * | 2021-05-25 | 2022-03-01 | 青岛新万福食品有限公司 | Polypeptide separated from pig liver and having strong oxidation resistance and promoting lipid metabolism, preparation method and application |
| CN114525321A (en) * | 2022-03-22 | 2022-05-24 | 江西煌上煌集团食品股份有限公司 | Antioxidant peptide derived from duck viscera and preparation method thereof |
| CN115947787A (en) * | 2022-09-06 | 2023-04-11 | 宁波大学 | Duck liver protein source antioxidant functional peptide and preparation method and application thereof |
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