CN106520877A - Method for preparing pig cerebral protein antioxidative peptide - Google Patents
Method for preparing pig cerebral protein antioxidative peptide Download PDFInfo
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- CN106520877A CN106520877A CN201611048652.8A CN201611048652A CN106520877A CN 106520877 A CN106520877 A CN 106520877A CN 201611048652 A CN201611048652 A CN 201611048652A CN 106520877 A CN106520877 A CN 106520877A
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- sus domestica
- medulla sus
- polypeptide
- enzymolysis
- defat
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
Abstract
The invention discloses a method for preparing pig cerebral protein antioxidative peptide. The method comprises the following steps of 1, pretreatment, wherein fresh pig brain is taken to be prepared into degreased cerebral protein powder for use; 2, ultrasonic treatment of pig cerebral protein powder, wherein the degreased cerebral protein powder is added into deionized water according to the volume proportion of 1:200, the cerebral protein powder is completely dissolved; countercurrent pulse ultrasonic treatment is carried out, the cerebral protein powder cooled to room temperature is placed in a countercurrent pulse ultrasonic treating device to be treated, the ultrasonic frequency is 20 kHz, the power ranges from 60 W to 100 W, ultrasonic treatment is carried out for 4 min to 6 min, ultrasonic time is 2 s, and intermittent time is 2 s; 3, enzymolysis to extract crude pig brain polypeptide, wherein pig brain polypeptide crude extracting liquid is prepared; 4, separation, wherein an ultrafiltration membrane with the molecular cut off of 3-5 kDa is used for fractionation; 5, purifying, wherein a DEAE-52 fiber resin layer is utilized for carrying for secondary purification on the pig brain peptide prepared in the step 4, and the pig cerebral protein antioxidative peptide is prepared.
Description
Technical field
The present invention relates to a kind of protein preparation method.It is more particularly related to a kind of Medulla sus domestica protein antioxidant peptide
Preparation method.
Background technology
Each position of pig all has important nutritive value.Especially not only meat is fine and smooth for Medulla sus domestica, fresh and tender good to eat, is rich in
Cephalin and protein, and the content of calcium, phosphorus, ferrum is higher than Carnis Sus domestica, can increase the immunity of body, enhance metabolism and be good for
Brain function.
Cerebral tissue is nutritious, and containing rich in protein, in recent years, researcher separates albumen from animal brain
Matter, enzyme process digest into the zymolyte containing free aminoacid and low molecular peptide, with its disease in terms for the treatment of brain.Austria according to than
The cerebrolysin vial that prestige pharmaceutical factory (Ebewe) manufactures is the widest brain protein zymolyte product of clinical practice, and it is by Medulla sus domestica
Albumen Jing enzyme process digests pharmaceutical preparation obtained in laggard onestep extraction, consisting of 75% free amino acid and little point of 25%
Sub- peptide, research show that the biological activity of cerebrolysin is closely related with the small-molecule peptide which contains, at present to Medulla sus domestica albumen and many
The correlational study of peptide is less, therefore studies the preparation method of animal brain's zymolyte small molecular peptide, isolates and purifies and its raw
Thing activity has great importance to social development.
The content of the invention
It is an object of the invention to solve at least the above, and provide the advantage that at least will be described later.
It is a still further object of the present invention to provide a kind of preparation method of Medulla sus domestica protein antioxidant peptide, which utilizes ultrasonic wave added
Enzyme alkali carries, quickly and efficiently extraction prepare Medulla sus domestica protein antioxidant peptide；Which is simple to operate, and process time is short, and albumen yield is high.
In order to realize these purposes of the invention and further advantage, there is provided a kind of system of Medulla sus domestica protein antioxidant peptide
Preparation Method, which comprises the following steps：
Step one, pretreatment：
Take fresh Medulla sus domestica to rinse, chopping, through defat, drying and pulverize, defat brain egg albumen powder is obtained standby；
Step 2, ultrasonic Treatment pig brain protein powder：
It is 1 that defat brain egg albumen powder obtained in step one is taken according to volume ratio:200 ratio adds deionized water, magnetic force to stir
20-40min is mixed, is placed in 40-60 DEG C of water-bath and is incubated 0.5-1.5h, be completely dissolved brain egg albumen powder；
The process of adverse current pulse ultrasonic wave：The brain egg albumen powder that will be cooled to room temperature is placed in place in adverse current pulse ultrasonic wave processor
Reason, wherein, supersonic frequency is 20kHz, and power is 60-100W, ultrasonic 4-6min, period ultrasonic time 2s, intermittent time 2s；
Step 3, the thick Medulla sus domestica polypeptide of enzymolysis and extraction：Medulla sus domestica protein solution after ultrasound adds alkaline protease to be digested,
Wherein enzyme bottom ratio is 1000-3000U/g；PH value is 8.0-9.0, and hydrolysis temperature is 30-50 DEG C, and enzymolysis time is 80-100min；
Immediately enzymolysis solution is placed in boiling water bath after enzymolysis and stands 5-15min；Room temperature is cooled to, 10- is centrifuged in 8000-12000rpm
20min, takes supernatant, and Medulla sus domestica polypeptide crude extract is obtained；
Step 4, separates：
The Medulla sus domestica polypeptide crude extract that ultrasonic wave added enzymolysis is obtained, adopting molecular cut off is carried out for the ultrafilter membrane of 3-5kDa
Fractionated, is obtained Medulla sus domestica polypeptide after collecting products therefrom lyophilization, wherein, control ultrafilter membrane outlet pressure is less than 5Bar；
Step 5, purification：
Medulla sus domestica polypeptide prepared by step 4 is carried out using DEAE-52 fibre resins chromatography post secondarily purified；Prepared Medulla sus domestica
Protein antioxidant peptide.
Preferably, described in the pretreatment, defat is specifically included：Fresh Medulla sus domestica after by chopping adds its volume 4-6
5-15min is boiled in boiling water again, defat 20- at the ethanol of the 75-90% of 2-4 times of volume, 60-80 DEG C is added after filtration
40min, is cooled to room temperature, after 3000-6000rmp be centrifuged 10-20min, collect precipitation；The drying is referred specifically to, in baking
It is dried to constant weight at 50-70 DEG C in case.
Preferably, adjust described with 0.1-0.5mol/L NaOH solutions during the ultrasonic Treatment of the step 2
The pH value of Medulla sus domestica protein solution is constant for 8.0-9.0.
Preferably, the purification of the step 5 is specially：The Medulla sus domestica polypeptide 50-100mg of step 4 preparation is weighed, is dissolved in
The 0.02mol/L sodium-acetate buffers of 5-10mL pH 7.5-8.5, are centrifuged 5-10min in 3000-5000rpm, take supernatant work
To enter sample liquid；
Sample introduction drop is added in DEAE-52 fibre resins chromatography post, successively with the 0.02mol/L acetic acid of pH 7.5-8.5
Sodium buffer, pH 8.0Tris-HCl buffer, carry out eluting, using DHL-A constant flow pumps, coutroi velocity 0.8-1.0mL/min,
Collected with automatic fraction collector, collection liquid adopts molecular cut off carries out desalination for the polypeptide bag filter of 300Da；It is cold after desalination
The dry prepared Medulla sus domestica protein antioxidant peptide of lyophilizing.
Preferably, the pH 8.0Tris-HCl buffer contains the NaCl of 0.0-2.0mol/L.
Preferably, the preparation method of the Medulla sus domestica protein antioxidant peptide, comprises the following steps：
Step one, pretreatment：
Take fresh Medulla sus domestica to rinse, chopping, through defat, drying and pulverize, defat brain egg albumen powder is obtained standby；It is described
Defat is specifically included：Fresh Medulla sus domestica after by chopping boils 5-15min in adding the boiling water of its volume 4-6 times, after filtration again plus
Enter defat 20-40min at the ethanol of the 75-90% of 2-4 times of volume, 60-80 DEG C, be cooled to room temperature, after 3000-
6000rmp is centrifuged 10-20min, collects precipitation；The drying is referred specifically to, 50-70 DEG C in the baking oven at be dried to constant weight；
Step 2, ultrasonic Treatment pig brain protein powder：
It is 1 that defat brain egg albumen powder obtained in step one is taken according to volume ratio:200 ratio adds deionized water, magnetic force to stir
20-40min is mixed, is placed in 40-60 DEG C of water-bath and is incubated 0.5-1.5h, be completely dissolved brain egg albumen powder；Use 0.1-0.5mol/L
The pH value that NaOH solution adjusts the Medulla sus domestica protein solution is constant for 8.0-9.0；
The process of adverse current pulse ultrasonic wave：The brain egg albumen powder that will be cooled to room temperature is placed in place in adverse current pulse ultrasonic wave processor
Reason, wherein, supersonic frequency is 20kHz, and power is 60-100W, ultrasonic 4-6min, period ultrasonic time 2s, intermittent time 2s；
Step 3, the thick Medulla sus domestica polypeptide of enzymolysis and extraction：Medulla sus domestica protein solution after ultrasound adds alkaline protease to be digested,
Wherein enzyme bottom ratio is 1000-3000U/g；PH value is 8.0-9.0, and hydrolysis temperature is 30-50 DEG C, and enzymolysis time is 80-100min；
Immediately enzymolysis solution is placed in boiling water bath after enzymolysis and stands 5-15min；Room temperature is cooled to, 10- is centrifuged in 8000-12000rpm
20min, takes supernatant, and Medulla sus domestica polypeptide crude extract is obtained；
Step 4, separates：
The Medulla sus domestica polypeptide crude extract that ultrasonic wave added enzymolysis is obtained, adopting molecular cut off is carried out for the ultrafilter membrane of 3-5kDa
Fractionated, is obtained Medulla sus domestica polypeptide after collecting products therefrom lyophilization, wherein, control ultrafilter membrane outlet pressure is less than 5Bar；
Step 5, purification：
Medulla sus domestica polypeptide prepared by step 4 is carried out using DEAE-52 fibre resins chromatography post secondarily purified；Prepared Medulla sus domestica
Protein antioxidant peptide；The purification is specially：The Medulla sus domestica polypeptide 50-100mg of step 4 preparation is weighed, 5-10mL pH are dissolved in
The 0.02mol/L sodium-acetate buffers of 7.5-8.5, are centrifuged 5-10min in 3000-5000rpm, take supernatant as entering sample liquid；
Sample introduction drop is added in DEAE-52 fibre resins chromatography post, successively with the 0.02mol/L acetic acid of pH 7.5-8.5
Sodium buffer, pH 8.0Tris-HCl buffer, carry out eluting, using DHL-A constant flow pumps, coutroi velocity 0.8-1.0mL/min,
Afterwards by the liquid collected be added drop-wise to again DEAE-52 fibre resins chromatography post in carry out it is secondarily purified after collection liquid, receive
Liquid collecting adopts molecular cut off carries out desalination for the polypeptide bag filter of 300Da；Lyophilization after desalination is obtained Medulla sus domestica albumen antioxygen
Change peptide；The pH 8.0Tris-HCl buffer contains the NaCl of 0.0-2.0mol/L.
The present invention at least includes following beneficial effect：The preparation method of Medulla sus domestica protein antioxidant peptide of the present invention, by pig
Brain egg albumen powder solution before enzymolysis using adverse current pulse ultrasonic wave process, Medulla sus domestica albumen Jing after super process sound, more brain eggs
Peptide bond in white is exposed, so as to be conducive to protease to play the zymolysis of albumen.By the mode of action compared with routine, this
Bright middle proteolysiss degree improves 30.6%.Part is passed through description below body by the further advantage of the present invention, target and feature
Existing, part will be also understood by the person skilled in the art by the research to the present invention and practice.
Description of the drawings
Fig. 1 be Medulla sus domestica protein antioxidant peptide of the present invention preparation method in hydrolysis curves figure；
Fig. 2 is the concentration curve of polypeptide after the preparation method of Medulla sus domestica protein antioxidant peptide of the present invention is digested；
Fig. 3 be Medulla sus domestica protein antioxidant peptide of the present invention preparation method in variable concentrations UPCHPs remove ABTS+ abilities
Curve chart；
Fig. 4 be Medulla sus domestica protein antioxidant peptide of the present invention preparation method in UPCHPs ultrafiltration components (1mg/mL) to ABTS from
By the Scavenging activity curve chart of base；
Fig. 5 be Medulla sus domestica protein antioxidant peptide of the present invention preparation method in UPCHPs-III DEAE-52 elution curves；
Fig. 6 be Medulla sus domestica protein antioxidant peptide of the present invention preparation method in UPCHPs-F3 Tricine-SDS-PAGE it is electric
Swimming figure；
Fig. 7 be Medulla sus domestica protein antioxidant peptide of the present invention preparation method in UPCHPs F3 Jing Tricine-SDS-PAGE point
From Scavenging activity curve chart of the purified components to ABTS+；
Fig. 8 is the preparation method Ultra MALDI-TOF/TOF-MS identification aminoacid of Medulla sus domestica protein antioxidant peptide of the present invention
Sequence chart；
Fig. 9 is the preparation method Ultra MALDI-TOF/TOF-MS identification aminoacid of Medulla sus domestica protein antioxidant peptide of the present invention
Sequence chart.
Specific embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to description text
Word can be implemented according to this.
It should be appreciated that it is used herein such as " have ", "comprising" and " including " term are not precluded from one or many
The presence or addition of individual other elements or its combination.
The present invention provides a kind of preparation method of Medulla sus domestica protein antioxidant peptide, and which comprises the following steps：
Step one, pretreatment：
Take fresh Medulla sus domestica to rinse, chopping, through defat, drying and pulverize, defat brain egg albumen powder is obtained standby；
Step 2, ultrasonic Treatment pig brain protein powder：
It is 1 that defat brain egg albumen powder obtained in step one is taken according to volume ratio:200 ratio adds deionized water, magnetic force to stir
20-40min is mixed, is placed in 40-60 DEG C of water-bath and is incubated 0.5-1.5h, be completely dissolved brain egg albumen powder；
The process of adverse current pulse ultrasonic wave：The brain egg albumen powder that will be cooled to room temperature is placed in place in adverse current pulse ultrasonic wave processor
Reason, wherein, supersonic frequency is 20kHz, and power is 60-100W, ultrasonic 4-6min, period ultrasonic time 2s, intermittent time 2s；
Step 3, the thick Medulla sus domestica polypeptide of enzymolysis and extraction：Medulla sus domestica protein solution after ultrasound adds alkaline protease to be digested,
Wherein enzyme bottom ratio is 1000-3000U/g；PH value is 8.0-9.0, and hydrolysis temperature is 30-50 DEG C, and enzymolysis time is 80-100min；
Immediately enzymolysis solution is placed in boiling water bath after enzymolysis and stands 5-15min；Room temperature is cooled to, 10- is centrifuged in 8000-12000rpm
20min, takes supernatant, and Medulla sus domestica polypeptide crude extract is obtained；
Step 4, separates：
The Medulla sus domestica polypeptide crude extract that ultrasonic wave added enzymolysis is obtained, adopting molecular cut off is carried out for the ultrafilter membrane of 3-5kDa
Fractionated, is obtained Medulla sus domestica polypeptide after collecting products therefrom lyophilization, wherein, control ultrafilter membrane outlet pressure is less than 5Bar；
Step 5, purification：
Medulla sus domestica polypeptide prepared by step 4 is carried out using DEAE-52 fibre resins chromatography post secondarily purified；Prepared Medulla sus domestica
Protein antioxidant peptide.
Wherein in one embodiment, defat described in the pretreatment is specifically included：Fresh Medulla sus domestica after by chopping adds
5-15min is boiled in the boiling water for entering its volume 4-6 times, the ethanol of the 75-90% of 2-4 times of volume after filtration, is added, 60-80 DEG C
Lower defat 20-40min, is cooled to room temperature, after 3000-6000rmp be centrifuged 10-20min, collect precipitation；The dry tool
Body refers to, 50-70 DEG C in the baking oven at be dried to constant weight.
It is wherein in one embodiment, molten with 0.1-0.5mol/L NaOH during the ultrasonic Treatment of the step 2
The pH value that liquid adjusts the Medulla sus domestica protein solution is constant for 8.0-9.0.
Wherein in one embodiment, the purification of the step 5 is specially：Weigh the Medulla sus domestica polypeptide 50- of step 4 preparation
100mg, is dissolved in the 0.02mol/L sodium-acetate buffers of 5-10mL pH 7.5-8.5, and 5-10min is centrifuged in 3000-5000rpm,
Supernatant is taken as entering sample liquid；
Sample introduction drop is added in DEAE-52 fibre resins chromatography post, successively with the 0.02mol/L acetic acid of pH 7.5-8.5
Sodium buffer, pH 8.0Tris-HCl buffer, carry out eluting, using DHL-A constant flow pumps, coutroi velocity 0.8-1.0mL/min,
Collected with automatic fraction collector, collection liquid adopts molecular cut off carries out desalination for the polypeptide bag filter of 300Da；It is cold after desalination
The dry prepared Medulla sus domestica protein antioxidant peptide of lyophilizing.
Wherein in one embodiment, the pH 8.0Tris-HCl buffer contains the NaCl of 0.0-2.0mol/L.
Embodiment 1
The preparation method of the Medulla sus domestica protein antioxidant peptide is comprised the following steps：
(1) preparation of defat pig brain protein powder
Fresh Medulla sus domestica：Purchased from Zhenjiang market, clot and meninges (pia mater encephali and arachnoidea) are removed, with normal saline flushing,
Chopping, adds 4 times of boiling water, boils 5min, the ethanol of the 75-90% of 2 times of volumes, defat at 60 DEG C are added after filtration
20min, after being cooled to room temperature, 3000-6000rmp centrifugation 10-20min, after collecting precipitation, 50 DEG C in the baking oven at be dried to perseverance
Weight, grinding are preserved in drying basin, standby.
(2) ultrasonic wave added enzymolysis prepares Medulla sus domestica polypeptide
Defat brain egg albumen powder 1g is placed in 500mL conical flasks, the deionized water of addition 200mL, magnetic agitation 20-40min,
It is placed in 40 DEG C of water-baths and is incubated 0.5h, is completely dissolved brain egg albumen powder, is cooled to room temperature.Adverse current pulse ultrasonic wave processor bar
Part：Supersonic frequency is 20kHz, and power is 60-100W, ultrasonic 4-6min (ultrasonic time 2s, intermittent time 2s).Use 0.1-
The pH value that 0.5mol/L NaOH adjust solution is constant for 8.0-9.0.
After ultrasound, Medulla sus domestica protein solution adds alkaline protease 0.1g (enzyme bottom ratio, 1000-3000U/g), in pH 8.0-
9.0,30-50 DEG C of hydrolysis temperature is digested, and after enzymolysis time 80min, enzymolysis solution is placed in boiling water bath stands 5min immediately；
Room temperature is cooled to, 10-20min is centrifuged in 8000-12000rpm, supernatant is taken standby.
(3) ultra-filtration and separation
The Medulla sus domestica polypeptide (UPCHPs) that the extra coarse sound assistance enzymolysis for obtaining are obtained, using molecular cut off (MWCO) 5kDa
Ultrafilter membrane carry out fractionated to UPCHPs, control ultrafilter membrane outlet pressure is less than 5Bar.Collect obtained component UPCHPs-
III (3-5kDa), and by each component freeze-dried back.
(4) DEAE-52 cellulose columns are isolated and purified
The polypeptide 50mg after lyophilization is weighed, 5mL pH 7.5-8.5 sodium-acetate buffers (0.02mol/L) is dissolved in, is filled
Divide 3000-5000g centrifugation 5-10min after dissolving, supernatant is taken as sample liquid is entered, it is standby.
Sample introduction drop is added in DEAE-52 chromatographic columns, successively with pH 7.5-8.5 sodium-acetate buffer (0.02mol/
L), pH 8.0Tris-HCl buffer (NaCl containing 0.0-2.0mol/L), carry out eluting, using DHL-A constant flow pumps, control
Flow velocity 0.8-1.0mL/min, is collected with automatic fraction collector.Merge each eluting peak eluent concentration after, by DEAE-52 from
Sub- post separation obtains antioxidant activity highest component and adopts molecular cut off carrying out desalination for the polypeptide bag filter of 300Da, collects
Obtain final product after sample lyophilizing.
Embodiment 2
The preparation method of the Medulla sus domestica protein antioxidant peptide is comprised the following steps：
(1) preparation of defat pig brain protein powder
Fresh Medulla sus domestica：Purchased from Zhenjiang market, clot and meninges (pia mater encephali and arachnoidea) are removed, with normal saline flushing,
Chopping, adds 6 times of boiling water, boils 15min, the ethanol of the 75-90% of 4 times of volumes is added after filtration, defat 20- at 80 DEG C
40min, after being cooled to room temperature, 3000-6000rmp centrifugation 10-20min, after collecting precipitation, 50-70 DEG C in the baking oven at be dried
To constant weight, grinding, preserve in drying basin, it is standby.
(2) ultrasonic wave added enzymolysis prepares Medulla sus domestica polypeptide
Defat brain egg albumen powder 2g is placed in 500mL conical flasks, adds the deionized water of 400mL, magnetic agitation 40min to put
1.5h is incubated in 40-60 DEG C of water-bath, is completely dissolved brain egg albumen powder, is cooled to room temperature.Adverse current pulse ultrasonic wave processor bar
Part：Supersonic frequency is 20kHz, and power is 60-100W, ultrasonic 4-6min (ultrasonic time 2s, intermittent time 2s).Use 0.1-
The pH value that 0.5mol/L NaOH adjust solution is constant for 8.0-9.0.
After ultrasound, Medulla sus domestica protein solution adds alkaline protease 0.1-0.2g (enzyme bottom ratio, 1000-3000U/g), in pH
8.0-9.0,30-50 DEG C of hydrolysis temperature are digested, and after enzymolysis time 80-100min, immediately enzymolysis solution are placed in boiling water bath
Stand 5-15min；Room temperature is cooled to, 20min is centrifuged in 8000-12000rpm, supernatant is taken standby.
(3) ultra-filtration and separation
The Medulla sus domestica polypeptide (UPCHPs) that the extra coarse sound assistance enzymolysis for obtaining are obtained, using molecular cut off (MWCO) 3kDa
Ultrafilter membrane carry out fractionated to UPCHPs, control ultrafilter membrane outlet pressure is less than 5Bar.Collect obtained component UPCHPs- IV
(1-3kDa), and by component freeze-dried back.
(4) DEAE-52 cellulose columns are isolated and purified
The polypeptide 100mg after lyophilization is weighed, 5-10mL pH 7.5-8.5 sodium-acetate buffer (0.02mol/ are dissolved in
L), after fully dissolving, 3000-5000g centrifugations 5-10min, takes supernatant as sample liquid is entered, standby.
Sample introduction drop is added in DEAE-52 chromatographic columns, successively with pH 7.5-8.5 sodium-acetate buffer (0.02mol/
L), pH 8.0Tris-HCl buffer (NaCl containing 0.0-2.0mol/L), carry out eluting, using DHL-A constant flow pumps, control
Flow velocity 0.8-1.0mL/min, is collected with automatic fraction collector.Merge each eluting peak eluent concentration after, by DEAE-52 from
Sub- post separation obtains antioxidant activity highest component and adopts molecular cut off carrying out desalination for the polypeptide bag filter of 300Da, collects
After sample lyophilizing.
Embodiment 3
The preparation method of the Medulla sus domestica protein antioxidant peptide is comprised the following steps：
(1) preparation of defat pig brain protein powder
Fresh Medulla sus domestica：Purchased from Zhenjiang market, clot and meninges (pia mater encephali and arachnoidea) are removed, with normal saline flushing,
Chopping, adds 5 times of boiling water, boils 10min, the ethanol of the 75-90% of 2-4 times of volume, defat at 70 DEG C are added after filtration
20-40min, after being cooled to room temperature, 3000-6000rmp centrifugation 15min, after collecting precipitation, 50-70 DEG C in the baking oven at be dried
To constant weight, grinding, preserve in drying basin, it is standby.
(2) ultrasonic wave added enzymolysis prepares Medulla sus domestica polypeptide
Defat brain egg albumen powder 1.5g is placed in 500mL conical flasks, adds the deionized water of 300mL, magnetic agitation 20-
40min, is placed in 50 DEG C of water-baths and is incubated 1.0h, is completely dissolved brain egg albumen powder, is cooled to room temperature.The process of adverse current pulse ultrasonic wave
Device condition：Supersonic frequency is 20kHz, and power is 60-100W, ultrasonic 4-6min (ultrasonic time 2s, intermittent time 2s).Use 0.1-
The pH value that 0.5mol/L NaOH adjust solution is constant for 8.0-9.0.
After ultrasound, Medulla sus domestica protein solution adds alkaline protease 0.1-0.2g (enzyme bottom ratio, 1000-3000U/g), in pH
8.0-9.0,30-50 DEG C of hydrolysis temperature are digested, and after enzymolysis time 80-100min, immediately enzymolysis solution are placed in boiling water bath
Stand 5-15min；Room temperature is cooled to, 10-20min is centrifuged in 8000-12000rpm, supernatant is taken standby.
(3) ultra-filtration and separation
The Medulla sus domestica polypeptide (UPCHPs) that the extra coarse sound assistance enzymolysis for obtaining are obtained, using molecular cut off (MWCO) 1kDa
Ultrafilter membrane carry out fractionated to UPCHPs, control ultrafilter membrane outlet pressure is less than 5Bar.Collect obtained component UPCHPs- V
(<1kDa) and by each component freeze-dried back.
(4) DEAE-52 cellulose columns are isolated and purified
The polypeptide 80mg after lyophilization is weighed, 8mL pH 7.5-8.5 sodium-acetate buffers (0.02mol/L) is dissolved in, is filled
Divide 3000-5000g centrifugation 5-10min after dissolving, supernatant is taken as sample liquid is entered, it is standby.
Sample introduction drop is added in DEAE-52 chromatographic columns, successively with pH 7.5-8.5 sodium-acetate buffer (0.02mol/
L), pH 8.0Tris-HCl buffer (NaCl containing 0.0-2.0mol/L), carry out eluting, using DHL-A constant flow pumps, control
Flow velocity 0.8-1.0mL/min, is collected with automatic fraction collector.Merge each eluting peak eluent concentration after, by DEAE-52 from
Sub- post separation obtains antioxidant activity highest component and adopts molecular cut off carrying out desalination for the polypeptide bag filter of 300Da, collects
Obtain final product after sample lyophilizing.
Comparative example 4
(1) preparation of defat pig brain protein powder
Fresh Medulla sus domestica：Purchased from Zhenjiang market, clot and meninges (pia mater encephali and arachnoidea) are removed, with normal saline flushing,
Chopping, adds 4～6 times of boiling water, boils 5～15min, adds the 75～90% of 2～4 times of volumes ethanol after filtration, and 60
20～40min of defat at～80 DEG C, after being cooled to room temperature, 3000～6000rmp is centrifuged 10～20min, after collecting precipitation, in baking
It is dried at 50～70 DEG C to constant weight, grinding in case, preserves in drying basin, it is standby.
(2) enzymolysis prepares Medulla sus domestica polypeptide
Defat brain 1～2g of egg albumen powder is placed in 500mL conical flasks, adds the deionized water of 200～400mL, magnetic agitation
20～40min, is placed in 0.5～1.5h of insulation in 40～60 DEG C of water-baths, is completely dissolved brain egg albumen powder, is cooled to room temperature.Using
It is conventional to digest.
(3) the Medulla sus domestica polypeptide (UPCHPs) that the extra coarse sound assistance enzymolysis for obtaining are obtained, using molecular cut off (MWCO)
The ultrafilter membrane of respectively 10kDa, 5kDa carries out fractionated to UPCHPs, and control ultrafilter membrane outlet pressure is less than 5Bar.Collect
Obtained component UPCHPs-I (>10kDa), UPCHPs-II (5～10kDa)；And by each component freeze-dried back.
(4) DEAE-52 cellulose columns are isolated and purified
The polypeptide 80mg after lyophilization is weighed, 8mL pH 7.5-8.5 sodium-acetate buffers (0.02mol/L) is dissolved in, is filled
Divide 3000-5000g centrifugation 5-10min after dissolving, supernatant is taken as sample liquid is entered, it is standby.
Sample introduction drop is added in DEAE-52 chromatographic columns, successively with pH 7.5-8.5 sodium-acetate buffer (0.02mol/
L), pH 8.0Tris-HCl buffer (NaCl containing 0.0-2.0mol/L), carry out eluting, using DHL-A constant flow pumps, control
Flow velocity 0.8-1.0mL/min, is collected with automatic fraction collector.Merge each eluting peak eluent concentration after, by DEAE-52 from
Sub- post separation obtains antioxidant activity highest component and adopts molecular cut off carrying out desalination for the polypeptide bag filter of 300Da, collects
Obtain final product after sample lyophilizing.
In preparation process, the detection of following parameter is carried out：
A, degree of hydrolysis are determined
PH-stat method of the measure of degree of hydrolysis with reference to foundation such as Adler-Nissen.
In formula：DH degree of hydrolysis, NbThe concentration (mol/L) of NaOH, the volume (mL) of B NaOH, MpAminosal
Quality (g), htotPeptide bond number total in substrate protein, Medulla sus domestica albumen be 7.6mmol/g, the average dissociation degree of α.
B, peptide concentration are determined
4mL Folin-phenol (reagent A), add the sample of 0.5mL, react 10min, add 0.5mL under room temperature
Folin-phenol (reagent B), after reacting 30min, is determined its absorbance at 500nm, and is substituted with deionized water under room temperature
Sample solution does blank, and content (the BSA standard curve ranges of linearity of polypeptide in sample are calculated according to standard curve：0～
50μg/mL)。
C, ABTS+Clearance rate
ABTS+Deionized water is made into 7mM, mixes (the ABTS of 5mL with 2.45mM potassium persulfate solutions+With 88 μ L's
Potassium persulfate solution), mixture 12～16h of avoid light place at room temperature.ABTS+Solution with ethanol dilutes the (ABTS of 1mL+·
The ethanol of solution+70mL) so as to the absorbance at 734nm is 0.70 ± 0.02.100μL ABTS+Solution adds 50 μ L
The polypeptide solution of variable concentrations, after 70min under 734nm mensuration absorbance (AS).Deionized water is used as blank (AB)。VC
For positive control.So the solution same day prepare, experiment parallel testing 3 times.ABTS+Clearance rate formula is as follows：
In formula：ABBlank light absorption value；ASThe light absorption value of sample
Wherein, in enzymolysis process, per 2min, sampling, carries out the monitoring of the content and degree of hydrolysis of polypeptide.Ultra-filtration and separation
The activity of ABTS+ is removed with measure after purification step, obtained finished product carries out Tricine-SDS-PAGE electrophoresis.Finally utilize
Ground substance assistant laser ionization parsing time-of-flight mass spectrometry instrument (MALDI-TOF-TOF-MS) identifies the aminoacid sequence of polypeptide.
Wherein, Tricine-SDS-PAGE electrophoresis is specially：Electrophoresis inside groove fills Cathode buffer, water jacket dress anode buffer
Liquid, is carried out under ice bath environment, constant pressure electrophoresis, prior to 30V voltages under, carry out 1～2h, after sample fully enters separation gel,
Voltage is risen to into 100V, electrophoretic voltage is risen to into 150V when sample fully enters concentration glue finally, and is proceeded about 6h's
Electrophoretic procedures, stop electrophoresis when bromophenol blue indicator reaches film bottom.
Ground substance assistant laser ionization parses time-of-flight mass spectrometry instrument, and search condition is：Retrieval type：MS/MS Ion
Search；Enzyme：Trypsin；Optional modification：Second phthalein, oxidation；Mass value：Monoisotopic peak；Molecular weight ranges：Do not limit；
Peptide quality tolerance：±0.3Da；Fragment tolerance：±0.9Da；Number is cut in maximum leakage：
Instrument type：MALDI-TOF-TOF.
As a result it is as follows：
As shown in figure 1, in front 20min, the hydrolysis reaction of conventional enzymolysis and ultrasonic wave added enzymolysis is very fast, but with
The prolongation of the time of hydrolysis, speed gradually slows down.But under same time, the protein hydrolysis degree of ultrasonic wave added enzymolysis is equal
More than conventional enzymatic isolation method, when hydrolysed between up to 90min when, compared with conventional enzymolysis, the protein hydrolysis degree of ultrasonic wave added enzymolysis is carried
It is high by 30.6%.This is because Medulla sus domestica albumen is Jing after super process sound, the peptide bond in more brain albumen is exposed, so as to be conducive to
Protease plays the hydrolysis of albumen.
As shown in Figure 2, in front 20min hydrolytic processes, with the prolongation of hydrolysis time, polypeptide obtained by two kinds of enzyme solutions
Concentration is continuously increased；But after 20 min, with time lengthening, hydrolysis gained peptide concentration is basicly stable, and this is mainly due to can be anti-
Answer the reduction of substrate quantity, enzyme self is cleared up and caused by suppressing.In from figure, in hydrolysis time 20-25min,
The concentration of ultrasound enzymolysis Medulla sus domestica polypeptide reaches maximum, is 225.6 μ g/mL.Egg in hydrolysis time 20min, the pig of conventional enzymolysis
Brain polypeptide concentration only 157.2 μ g/mL.It can be seen that, ultrasonic wave added enzymolysis can increase degree of hydrolysis, obtain more polypeptides matters.But
With the prolongation of hydrolysis time, as the increase of the degree of hydrolysis of albumen, the number of free amino acid increase, the number of peptide bond subtracts
It is few, and the quantity that centrifuged deposit is formed is consequently increased therefore follow-up with ultrasonic wave added enzymolysis and conventional enzymolysis time
The thick polypeptide (UPCHPs and PCHPs) obtained during 20min is object of study, determines its polypeptide yield and is respectively 81.5% He
66.7%.
From the figure 3, it may be seen that with the increase of UPCHPs concentration, which removes ABTS+Ability gradually strengthen.And when Medulla sus domestica is dense
Spend for 5mg/kg when, remove ABTS+Ability up to 90.2%, its EC50It is worth for 0.63mg/mL.
As shown in Figure 4, five kinds of components are obtained, respectively：UPCHPs-I(MW>10kDa), UPCHPs-II (5～10kDa),
UPCHPs-III (3～5kDa), UPCHPs- IV (1～3kDa) and UPCHPs- V (<1kDa).As a result show, UPCHPs molecules
Amount removes ABTS less than three components of 5kDa+Ability be close to, be all remarkably higher than U PCHPs-I (>10kDa) and
UPCHPs-II (5～10kDa) is with significant difference (p<0.05).
As shown in Figure 5, the protein peak peak type Jing after DEAE-52 separation is symmetrical, and separating effect is preferable.Collect peak value pipe, Jing
Dialysis, after lyophilization, is redissolved with the corresponding buffer of 2mL, determines which and remove ABTS+Vigor.One component is in 0.5mg/mL
When remove ABTS+For 72.4%, in each component Scavenging ability it is most strong when 0.5mg/mL to ABTS+For
93.3%, name Scavenging ability in each component most strong as F3, and F3 is main antioxidation in UPCHPs-III
Component, therefore select F3 to carry out next step identification and analysis.
As shown in fig. 6, No. 1 band is Medulla sus domestica enzymolysis polypeptide Jing Tricine-SDS-PAGE electrophoretic analysiss, in 5856-
Bands of a spectrum are appeared below with 3313Da between 3313Da, and the position bands of a spectrum between 5856～3313Da are wider, color is deeper；
Be distributed slightly narrow in 3313Da position below bands of a spectrum, color is shallower, thus can draw UPCHPs-F3 that enzymolysis obtains mainly by
<Polypeptide composition between 5kDa.Therefore, further the UPCHPs-F3 that ultrafiltration component molecular amount is less than 5kDa is carried out secondary
The separation of DEAE-52 fibre resins.No. 2 bands are Medulla sus domestica enzymolysis polypeptide Jing after DEAE-52 fibre resin secondary separations
Tricine-SDS-PAGE electrophoretic analysiss.<Obvious bands of a spectrum are occurred in that at 3kDa, its distribution is slightly narrow, and color is deeper, and polypeptide contains
Amount large percentage.Secondly exist<Obvious bands of a spectrum are occurred in that at 5kDa, two target stripes are respectively designated as F31 and F32.
As shown in Figure 7, UPCHPs-F31 and UPCHPs-F32 components remove ABTS+EC50Value is respectively 0.185 He
The EC of 0.241mg/mL, UPCHPs-F3150Value is the 29% of unpurified UPCHPs, so UPCHPs-F31 is for next step point
Analysis.
By known to Fig. 8 and Fig. 9, by the LIFT tandem mass spectrum collection of illustrative plates of Ultra MALDI-TOF/TOF-MS, Bio is used
Tools4.0 software analysis m/z：451.67 and 1105.59Da peptide fragment, aminoacid sequence are respectively Ile-Tyr-Arg (IY R)
With Glu-Ala-Ser-Ile-Glu-Trp-Ser-Arg-Lys (EASIEWSRK), exactly correspond to SAW760 161-163 and
81-98 peptides site.
Although embodiment of the present invention is disclosed as above, which is not restricted to listed by description and embodiment
With, it can be applied to various suitable the field of the invention completely, for those skilled in the art, can be easily
Other modification is realized, therefore under the general concept limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the legend with description.
Claims (6)
1. a kind of preparation method of Medulla sus domestica protein antioxidant peptide, it is characterised in that comprise the following steps：
Step one, pretreatment：
Take fresh Medulla sus domestica to rinse, chopping, through defat, drying and pulverize, defat brain egg albumen powder is obtained standby；
Step 2, ultrasonic Treatment pig brain protein powder：
It is 1 that defat brain egg albumen powder obtained in step one is taken according to volume ratio:200 ratio adds deionized water, magnetic agitation 20-
40min, is placed in 40-60 DEG C of water-bath and is incubated 0.5-1.5h, is completely dissolved brain egg albumen powder；
The process of adverse current pulse ultrasonic wave：The brain egg albumen powder that will be cooled to room temperature is placed in process in adverse current pulse ultrasonic wave processor,
Wherein, supersonic frequency is 20kHz, and power is 60-100W, ultrasonic 4-6min, period ultrasonic time 2s, intermittent time 2s；
Step 3, the thick Medulla sus domestica polypeptide of enzymolysis and extraction：Medulla sus domestica protein solution after ultrasound adds alkaline protease to be digested, wherein
Enzyme bottom ratio is 1000-3000U/g；PH value is 8.0-9.0, and hydrolysis temperature is 30-50 DEG C, and enzymolysis time is 80-100min；Enzymolysis
Immediately enzymolysis solution is placed in boiling water bath afterwards and stands 5-15min；Room temperature is cooled to, 10- is centrifuged in 8000-12000rpm
20min, takes supernatant, and Medulla sus domestica polypeptide crude extract is obtained；
Step 4, separates：
The Medulla sus domestica polypeptide crude extract that ultrasonic wave added enzymolysis is obtained, adopts molecular cut off and is classified for the ultrafilter membrane of 3-5kDa
Separate, after collecting products therefrom lyophilization, Medulla sus domestica polypeptide is obtained, wherein, control ultrafilter membrane outlet pressure is less than 5Bar；
Step 5, purification：
Medulla sus domestica polypeptide prepared by step 4 is carried out using DEAE-52 fibre resins chromatography post secondarily purified；Prepared Medulla sus domestica albumen
Anti-oxidation peptide.
2. the preparation method of Medulla sus domestica protein antioxidant peptide as claimed in claim 1, it is characterised in that described in the pretreatment
Defat is specifically included：Fresh Medulla sus domestica after by chopping boils 5-15min in adding the boiling water of its volume 4-6 times, after filtration again plus
Enter defat 20-40min at the ethanol of the 75-90% of 2-4 times of volume, 60-80 DEG C, be cooled to room temperature, after 3000-
6000rmp is centrifuged 10-20min, collects precipitation；The drying is referred specifically to, 50-70 DEG C in the baking oven at be dried to constant weight.
3. the preparation method of Medulla sus domestica protein antioxidant peptide as claimed in claim 1, it is characterised in that the ultrasound of the step 2
PH value in ripple processing procedure with the 0.1-0.5mol/L NaOH solutions regulation Medulla sus domestica protein solution is constant for 8.0-9.0.
4. the preparation method of Medulla sus domestica protein antioxidant peptide as claimed in claim 1, it is characterised in that the purification of the step 5
Specially：The Medulla sus domestica polypeptide 50-100mg of step 4 preparation is weighed, the 0.02mol/L acetic acid of 5-10mL pH 7.5-8.5 is dissolved in
Sodium buffer, is centrifuged 5-10min in 3000-5000rpm, takes supernatant as entering sample liquid；
Sample introduction drop is added in DEAE-52 fibre resins chromatography post, is delayed with the 0.02mol/L sodium acetates of pH 7.5-8.5 successively
Liquid, 8.0 Tris-HCl buffer of pH are rushed, eluting is carried out, using DHL-A constant flow pumps, coutroi velocity 0.8-1.0mL/min, is used
Automatic fraction collector is collected, and collection liquid adopts molecular cut off carries out desalination for the polypeptide bag filter of 300Da；Freeze after desalination
It is dried prepared Medulla sus domestica protein antioxidant peptide.
5. the preparation method of Medulla sus domestica protein antioxidant peptide as claimed in claim 4, it is characterised in that 8.0 Tris- of the pH
HCl buffer contains the NaCl of 0.0-2.0mol/L.
6. the preparation method of the Medulla sus domestica protein antioxidant peptide as described in any one of claim 1-5, it is characterised in that including following
Step：
Step one, pretreatment：
Take fresh Medulla sus domestica to rinse, chopping, through defat, drying and pulverize, defat brain egg albumen powder is obtained standby；The defat
Specifically include：Fresh Medulla sus domestica after by chopping boils 5-15min in adding the boiling water of its volume 4-6 times, and 2-4 is added after filtration
The ethanol of the 75-90% of times volume, at 60-80 DEG C, defat 20-40min, is cooled to room temperature, be centrifuged after 3000-6000rmp
10-20min, collects precipitation；The drying is referred specifically to, 50-70 DEG C in the baking oven at be dried to constant weight；
Step 2, ultrasonic Treatment pig brain protein powder：
It is 1 that defat brain egg albumen powder obtained in step one is taken according to volume ratio:200 ratio adds deionized water, magnetic agitation 20-
40min, is placed in 40-60 DEG C of water-bath and is incubated 0.5-1.5h, is completely dissolved brain egg albumen powder；Use 0.1-0.5mol/LNaOH solution
The pH value for adjusting the Medulla sus domestica protein solution is constant for 8.0-9.0；
The process of adverse current pulse ultrasonic wave：The brain egg albumen powder that will be cooled to room temperature is placed in process in adverse current pulse ultrasonic wave processor,
Wherein, supersonic frequency is 20kHz, and power is 60-100W, ultrasonic 4-6min, period ultrasonic time 2s, intermittent time 2s；
Step 3, the thick Medulla sus domestica polypeptide of enzymolysis and extraction：Medulla sus domestica protein solution after ultrasound adds alkaline protease to be digested, wherein
Enzyme bottom ratio is 1000-3000U/g；PH value is 8.0-9.0, and hydrolysis temperature is 30-50 DEG C, and enzymolysis time is 80-100min；Enzymolysis
Immediately enzymolysis solution is placed in boiling water bath afterwards and stands 5-15min；Room temperature is cooled to, 10- is centrifuged in 8000-12000rpm
20min, takes supernatant, and Medulla sus domestica polypeptide crude extract is obtained；
Step 4, separates：
The Medulla sus domestica polypeptide crude extract that ultrasonic wave added enzymolysis is obtained, adopts molecular cut off and is classified for the ultrafilter membrane of 3-5kDa
Separate, after collecting products therefrom lyophilization, Medulla sus domestica polypeptide is obtained, wherein, control ultrafilter membrane outlet pressure is less than 5Bar；
Step 5, purification：
Medulla sus domestica polypeptide prepared by step 4 is carried out using DEAE-52 fibre resins chromatography post secondarily purified；Prepared Medulla sus domestica albumen
Anti-oxidation peptide；The purification is specially：The Medulla sus domestica polypeptide 50-100mg of step 4 preparation is weighed, 5-10mL pH7.5-8.5 are dissolved in
0.02mol/L sodium-acetate buffers, 5-10min is centrifuged in 3000-5000rpm, supernatant is taken as entering sample liquid；
Sample introduction drop is added in DEAE-52 fibre resins chromatography post, is delayed with the 0.02mol/L sodium acetates of pH 7.5-8.5 successively
Liquid, 8.0 Tris-HCl buffer of pH are rushed, eluting is carried out, using DHL-A constant flow pumps, coutroi velocity 0.8-1.0mL/min, it
Afterwards by the liquid collected be added drop-wise to again DEAE-52 fibre resins chromatography post in carry out it is secondarily purified after collection liquid, collect
Liquid adopts molecular cut off carries out desalination for the polypeptide bag filter of 300Da；Lyophilization after desalination is obtained Medulla sus domestica protein antioxidant
Peptide；8.0 Tris-HCl buffer of the pH contains the NaCl of 0.0-2.0mol/L.
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| CN108576366A (en) * | 2018-05-09 | 2018-09-28 | 江苏省农业科学院 | The preparation method of high foaming fowl orgotein powder |
| CN111363771A (en) * | 2020-03-18 | 2020-07-03 | 湖北瑞邦生物科技有限公司 | Production process of pig brain extract with strong oxidation resistance and pig brain extract |
| US10941432B1 (en) | 2019-08-15 | 2021-03-09 | Santiago De Urbina Gaviria | Method for the preparation of low molecular weight porcine lympho-reticular polypeptides |
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Cited By (6)
| 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 |
| CN107099573B (en) * | 2017-06-13 | 2020-12-15 | 江苏省农业科学院 | Method for preparing antioxidant peptide by optimizing degreasing mode and combining ultrasonic-assisted enzymolysis of duck liver |
| CN108576366A (en) * | 2018-05-09 | 2018-09-28 | 江苏省农业科学院 | The preparation method of high foaming fowl orgotein powder |
| US10941432B1 (en) | 2019-08-15 | 2021-03-09 | Santiago De Urbina Gaviria | Method for the preparation of low molecular weight porcine lympho-reticular polypeptides |
| CN111363771A (en) * | 2020-03-18 | 2020-07-03 | 湖北瑞邦生物科技有限公司 | Production process of pig brain extract with strong oxidation resistance and pig brain extract |
| CN111363771B (en) * | 2020-03-18 | 2023-03-14 | 湖北瑞邦生物科技有限公司 | Production process of pig brain extract with strong oxidation resistance and pig brain extract |
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