CN108410936A - A kind of buffalo's milk anti-oxidation peptide and its method for separating and preparing - Google Patents
A kind of buffalo's milk anti-oxidation peptide and its method for separating and preparing Download PDFInfo
- Publication number
- CN108410936A CN108410936A CN201810310855.2A CN201810310855A CN108410936A CN 108410936 A CN108410936 A CN 108410936A CN 201810310855 A CN201810310855 A CN 201810310855A CN 108410936 A CN108410936 A CN 108410936A
- Authority
- CN
- China
- Prior art keywords
- buffalo
- milk
- oxidation peptide
- separating
- milk anti
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 74
- 235000020246 buffalo milk Nutrition 0.000 title claims abstract description 55
- 230000003064 anti-oxidating effect Effects 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000010828 elution Methods 0.000 claims abstract description 25
- 230000002000 scavenging effect Effects 0.000 claims abstract description 13
- 238000000926 separation method Methods 0.000 claims abstract description 11
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 claims abstract description 10
- 230000007760 free radical scavenging Effects 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 claims abstract description 5
- 239000000796 flavoring agent Substances 0.000 claims abstract description 5
- 235000019634 flavors Nutrition 0.000 claims abstract description 5
- 108091005804 Peptidases Proteins 0.000 claims abstract description 4
- 239000004365 Protease Substances 0.000 claims abstract description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 4
- 238000001816 cooling Methods 0.000 claims abstract description 4
- 230000009849 deactivation Effects 0.000 claims abstract description 4
- 238000005238 degreasing Methods 0.000 claims abstract description 4
- 102000034356 gene-regulatory proteins Human genes 0.000 claims abstract description 4
- 108091006104 gene-regulatory proteins Proteins 0.000 claims abstract description 4
- 230000001954 sterilising effect Effects 0.000 claims abstract description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 4
- 239000003480 eluent Substances 0.000 claims description 19
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 238000002523 gelfiltration Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000001742 protein purification Methods 0.000 claims description 7
- 239000012505 Superdex™ Substances 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 235000013305 food Nutrition 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 230000003647 oxidation Effects 0.000 claims description 3
- 238000007254 oxidation reaction Methods 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 230000013011 mating Effects 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 7
- 230000002292 Radical scavenging effect Effects 0.000 abstract description 4
- 239000000203 mixture Substances 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 230000003078 antioxidant effect Effects 0.000 description 8
- 230000000875 corresponding effect Effects 0.000 description 6
- 238000001641 gel filtration chromatography Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- -1 hydroxyl radical free radical Chemical class 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010011756 Milk Proteins Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 101800000068 Antioxidant peptide Proteins 0.000 description 1
- 241001416153 Bos grunniens Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 102000004407 Lactalbumin Human genes 0.000 description 1
- 108090000942 Lactalbumin Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108010071421 milk fat globule Proteins 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 210000000697 sensory organ Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of buffalo's milk anti-oxidation peptide and its method for separating and preparing, belong to biotechnology.Its method for separating and preparing includes：Buffalo's milk digested through degreasing, regulatory protein matter mass fraction, sterilization, constant temperature, adjustment pH value, addition flavor protease, enzyme deactivation, obtains buffalo's milk enzymolysis product after cooling enzymolysis liquid, gel splitter is recycled to detach buffalo's milk enzymolysis product, the Detection wavelength for eluting separation process is 220nm, 280nm and 254nm, each elution fraction is collected, is freeze-dried；Wherein reducing power, DPPH free radical scavenging abilities, Hydroxyl radical-scavenging ability and the higher component of superoxide anion Scavenging activity, as buffalo's milk anti-oxidation peptide.Buffalo's milk anti-oxidation peptide preparation method simple possible of the present invention, the tracking of active peptide composition, concentration effect are good, and Product Activity is high.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of buffalo's milk anti-oxidation peptide and its method for separating and preparing.
Background technology
Lactoprotein not only contains various amino acid necessary to human body, while will produce respectively in the digestion process of gastrointestinal tract
The biologically active peptide of kind.The factors such as the generation of biologically active peptide and restriction enzyme site, protein classes and structure are significantly correlated.Water
Milk protein content is significantly higher than other species, is the important sources of high-quality protein.Studies have shown that buffalo lactalbumin and
There are significant difference, buffalo milk casein sheets for milk fat globule membrane proteins matter group and bovine ox, goat, horse, camel, yak, human milk etc.
There is also polymorphism, milk protein gene polymorphisms, and the amino acid sequence in albumen can be caused to change for body, and then influences albumen
With the property and function of peptide.
Having studied at present confirms that buffalo lactoprotein has certain antioxidant activity, and enzyme after food flavor enzyme directly digests
It is good to solve product sense organ, utilization can be added in food containing peptide, but do not detached further to enzymolysis product, still
The peptide composition of activity concentration is not obtained.
Invention content
High, heat that the technical problem to be solved by the invention for the present situation of prior art is to provide a kind of antioxidant activities
Stability is good and remains to keep the buffalo's milk anti-oxidation peptide of greater activity after gastroenteric environment digests.
Another technical problem to be solved by this invention is the present situation for the prior art, provides a kind of above-mentioned buffalo's milk
The preparation method of anti-oxidation peptide, this method use biological enzymolysis technology and gel filtration chromatography technical tie-up, scientific and reasonable, be easy to
Operation.
Technical method used in purpose is as follows to realize the present invention：
A kind of method for separating and preparing of buffalo's milk anti-oxidation peptide, includes the following steps：
（1）Buffalo's milk → degreasing → regulatory protein matter mass fraction → sterilization, constant temperature, adjustment pH value → addition flavor protease into
Row enzymolysis → enzyme deactivation → cooling enzymolysis liquid → obtains buffalo's milk enzymolysis product；
（2）Substance in buffalo's milk enzymolysis product is carried out by elution separation, elution point with eluent using gel filtration splitter
Detection wavelength from process is 220nm, collects each elution fraction；
（3）By step（2）Elution fraction be freeze-dried；
（4）In step（3）Each component in, screen and collect based on reducing power, DPPH free radical scavenging abilities, hydroxy radical
The higher component of synthesis oxidation resistance of Scavenging activity and superoxide anion Scavenging activity, as buffalo's milk anti-oxidation peptide.
Preferably, the step（2）Eluent be made of eluent A and eluent B, the eluent A be containing
The aqueous solution of 0.1% trifluoroacetic acid, the eluent B are the acetonitrile solution containing 0.1% trifluoroacetic acid, the eluent A and are washed
The volume ratio of de- liquid B is 7:3.
Preferably, the step（2）The Detection wavelength for eluting separation process can also be 280nm or 254nm.
Preferably, the step（2）Gel filtration splitter is 10/300 GL gels of superdex peptide point
From column, buffalo's milk enzymolysis product is passed through into 10/300 GL gel splitters of superdex peptide, buffalo's milk enzymolysis product
Sample size be 500 μ L, sample size 20mg/mL, the elution flow rate of eluent is 0.3 ~ 0.8mL/min.
Preferably, the step（2）It is to utilize the mating full-automatic collection of protein purification system to collect each elution fraction
Device carries out Fraction collection to different component sample successively according to sample elution appearance volume, continuous to collect.
Preferably, the step（3）It after freeze-drying, is redissolved using pure water, obtains buffalo's milk anti-oxidation peptide aqueous solution.
The present invention also provides a kind of buffalo's milk anti-oxidation peptide, the buffalo's milk anti-oxidation peptide is by any one described above
The method for separating and preparing is made.
Preferably, the buffalo's milk anti-oxidation peptide molecular weight is between 89.09-1245.32Da.
Preferably, the elution appearance volume of the buffalo's milk anti-oxidation peptide is 18-20mL.
The present invention further provides buffalo's milk anti-oxidation peptides in preparing health products or food with anti-oxidation efficacy
Using.
The substantive distinguishing features of the present invention and progress are：
（1）The buffalo's milk anti-oxidation peptide of the present invention has higher antioxidant activity, after pure water redissolution, peptide is prepared and contains
When amount is the buffalo's milk anti-oxidation peptide solution of 2.23mg/mL, reducing power, remove DPPH free radicals, scavenging hydroxyl with
And the clearance rate of removing ultra-oxygen anion free radical can reach 0.158,51.25%, 17.90% and 53.33% respectively.
（2）Buffalo's milk antioxidant peptide active provided by the invention is excellent, has good antioxidation, can be used as work(
Energy property ingredient, is used in antioxidant health-care product and food, and significant effect, has good market prospects.
（3）The present invention is combined using biological enzymolysis technology and gel filtration chromatography technology, and whole preparation process is simple and easy to control, enzyme
It solves efficient, by gel permeation chromatography, effectively tracks and be enriched the active peptide in enzymolysis product, meanwhile, gained antioxygen
Change peptide by freeze-drying, fully removes acetonitrile and trifluoroacetic acid volatile in eluent, safety is without side-effects.
Description of the drawings
Fig. 1 is the gel permeation chromatography chromatogram of enzymolysis product.
Fig. 2 is the gel permeation chromatography chromatogram of standard substance.
Fig. 3 is the comparison of different sample component reducing powers in gel filtration separation.
Fig. 4 is the comparison of different sample component DPPH free radical scavenging abilities in gel filtration separation.
Fig. 5 is the comparison of different sample component Hydroxyl radical-scavenging abilities in gel filtration separation.
Fig. 6 is the comparison of different sample component superoxide anion Scavenging activities in gel filtration separation.
Specific implementation mode
The present invention program is described in further detail with reference to embodiment, following the description is merely to explain this hair
It is bright, its content is not defined.Experimental method used in following embodiments is conventional side unless otherwise specified
Method, material, reagent etc. used in following embodiments, is commercially available unless otherwise specified.
The method for separating and preparing of 1 buffalo's milk anti-oxidation peptide of embodiment
（1）Technology path is as follows：Buffalo's milk → degreasing → regulatory protein matter mass fraction → sterilization, constant temperature, adjustment pH value → adds
Flavor protease is added to carry out digesting → enzyme deactivation → cooling enzymolysis liquid → and obtain buffalo's milk enzymolysis product.
（2）Using the AKTA pure protein purification systems of GE Healthcare, using superdex peptide 10/
300 GL gel splitters, detach the peptide composition in enzymolysis product.Wherein, eluent A is containing 0.1% trifluoroacetic acid
Aqueous solution, B are the acetonitrile solution containing 0.1% trifluoroacetic acid, A：B=7:3（v/v）, elution flow rate 0.5mL/min, 220nm,
It is scanned simultaneously under tri- wavelength of 280nm and 254nm, sample size is 500 μ L, sample size 20mg/mL.Utilize protein purification
The full-automatic collector of system support carries out Fraction collection, even to different component sample successively according to sample elution appearance volume
Continuous to collect 120 times, freeze-drying is used for peptide content and Antioxidative Activity Determination after being redissolved using 30mL ultra-pure waters.
（3）With reference to《GBT 22492-2008 soy peptide powders》The assay method of middle peptide content, wherein acid-soluble nitrogen determination
In, TCA is formulated as 12% solution.And it measures its reducing power, the ability for removing DPPH free radicals, remove hydroxyl radical free radical
Ability and the ability for removing superoxide anion.
2 superdex peptide gel filtrations post separations of embodiment and sample collection
Gel filtration chromatography figure of the enzymolysis product at 220nm, 254nm and 280nm is shown in Fig. 1, main isolated 4 masters
Peak, wherein peak 1 are in elution volume 10mL-18mL appearances, which is named as F1, and peak 2 is in 18-20mL appearances, component name
For F2, in 20mL-22mL appearances, which is named as F3 at peak 3, and peak 4 is named as F4 in 22mL-24mL appearances, the component.
Using alanine that molecular weight is 89.09 Da and molecular weight for 1245.32Da peptide fragment TPEVDDEALEK as control, two objects
The gel filtration chromatography figure of matter is shown in Fig. 2, comparison diagram 1 and Fig. 2, it is known that the molecular weight of F2 and F3 components is in 89.09-1245.32Da
Between, 89.09 Da of molecular weight ＜ of F4 components, and molecular weight ＞ 1245.32Da and ＜ are existed simultaneously in F1 components
1245.32Da substance.
According to sample elution volume, substep receipts are carried out to different component sample using protein purification system full-automatic collector
Collection receives sample since 5mL, and it is a sample that a pipe is collected per 1mL eluents, and the elution volume when appearance that sample is No. 2-No. 24 is such as
Fig. 1, wherein the elution appearance volume of sample 2 are 5mL-6mL, the appearance volume of other samples.Each sample sets
Point collected 120 times by protein purification system, after freeze-drying, redissolved in the ultra-pure water of 30mL, carry out follow-up peptide content and
The measurement such as antioxidant activity.
The different sample component peptide contents of embodiment 3 and antioxidant activity
2-24 samples peptide content and corresponding reducing power measurement result are shown in that Fig. 3, the higher component of sample peptide content mainly collect
In in 9-17 samples, meanwhile, in these samples reducing power also compared with other components height, wherein active highest three groups are divided into
No. 14-16, active size is followed successively by：No. 15 ＞ 16 ＞ 14, the peptide content and reducing power of No. 15 samples are highest, peptide
Content is 2.45mg/mL, and reducing power absorbance value is 0.154.Sample peptide content and corresponding DPPH free radical scavenging abilities
Measurement result is shown in Fig. 4, in the 9-17 samples that sample peptide is concentrated, No. 15, No. 16, that No. 17 samples show higher DPPH is free
Base Scavenging activity, is followed successively by：No. 15 ＞ 16 of ＞ 17, wherein No. 15 sample DPPH free radical scavenging activities reach 50.56%.Sample
Product peptide content and corresponding Hydroxyl radical-scavenging ability measurement result are shown in that Fig. 5, Scavenging activity on hydroxyl free radical are not centered at peptide and contain
Higher 9-17 samples are measured, 8-20 samples show certain Scavenging activity on hydroxyl free radical, and the higher component of activity
The 12-20 samples in addition to No. 16 components are concentrated on, activity comes the component of first three and is followed successively by：No. 15 ＞ 17 of ＞ 20,
No. 15 sample Hydroxyl radical-scavenging ability highests, reach 15.22%, and No. 20 with No. 17 Scavenging activities be respectively 11.74% and
10.43%, wherein peptide content is relatively low in No. 20 samples, from the point of view of the corresponding molecular weight ranges of appearance volume, the hydroxyl that shows
Free radical scavenging ability may be derived from other substances other than peptide.Sample peptide content and corresponding superoxide anion Scavenging activity are surveyed
Determine result and see that Fig. 6, higher three groups of ultra-oxygen anion free radical Scavenging activity are divided into No. 15, No. 16, No. 17 samples, removes
Capacity of water is followed successively by：No. 16 ＞ 17 of ＞ 15, respectively 54.88%, 46.34% and 31.71%.
To sum up, according to by Gel filtration analysis system, the peptide of different molecular weight ranges in enzymolysis product is detached, is obtained comprehensive
Antioxidant activity stronger No. 15 and No. 16 components are closed, the merging appearance elution volume of two samples is 18-20mL, is collectively formed
The F2 components that protein purification system elutes, corresponding molecular weight ranges are between 89.09-1245.32Da.Merge peptide content
No. 15, No. 16 samples of the strong elution separation of high and oxidation resistance, obtain F2 components, fully after freeze-drying, as this hair
Bright buffalo's milk anti-oxidation peptide.Pure water dissolving buffalo's milk anti-oxidation peptide is further used, it is 2.23mg/mL's that peptide content, which is prepared,
Buffalo's milk anti-oxidation peptide aqueous solution, under this peptide content, the reducing power of buffalo's milk anti-oxidation peptide aqueous solution removes DPPH freedom
Base, scavenging hydroxyl and remove ultra-oxygen anion free radical clearance rate be respectively 0.158,51.25%, 17.90% and
53.33%。
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
The change or replacement expected without creative work, should be covered by the protection scope of the present invention.Therefore, of the invention
Protection domain should be determined by the scope of protection defined in the claims.
Claims (10)
1. a kind of method for separating and preparing of buffalo's milk anti-oxidation peptide, which is characterized in that include the following steps：
（1）Buffalo's milk → degreasing → regulatory protein matter mass fraction → sterilization, constant temperature, adjustment pH value → addition flavor protease into
Row enzymolysis → enzyme deactivation → cooling enzymolysis liquid → obtains buffalo's milk enzymolysis product；
（2）Substance in buffalo's milk enzymolysis product is carried out by elution separation, elution point with eluent using gel filtration splitter
Detection wavelength from process is 220nm, collects each elution fraction；
（3）By step（2）Elution fraction be freeze-dried；
（4）In step（3）Each component in, screen and collect based on reducing power, DPPH free radical scavenging abilities, hydroxy radical
The higher component of synthesis oxidation resistance of Scavenging activity and superoxide anion Scavenging activity, as buffalo's milk anti-oxidation peptide.
2. the method for separating and preparing of buffalo's milk anti-oxidation peptide according to claim 1, which is characterized in that the step（2）'s
Eluent is made of eluent A and eluent B, and the eluent A is the aqueous solution containing 0.1% trifluoroacetic acid, the elution
Liquid B is the acetonitrile solution containing 0.1% trifluoroacetic acid, and the volume ratio of the eluent A and eluent B are 7:3.
3. the method for separating and preparing of buffalo's milk anti-oxidation peptide according to claim 1, which is characterized in that the step（2）It washes
The Detection wavelength of de- separation process can also be 280nm or 254nm.
4. the method for separating and preparing of buffalo's milk anti-oxidation peptide according to claim 1, which is characterized in that the step（2）
Gel filtration splitter is 10/300 GL gel splitters of superdex peptide, and buffalo's milk enzymolysis product is passed through
The sample size of 10/300 GL gel splitters of superdex peptide, buffalo's milk enzymolysis product is 500 μ L, and sample size is
The elution flow rate of 20mg/mL, eluent are 0.3 ~ 0.8mL/min.
5. the method for separating and preparing of buffalo's milk anti-oxidation peptide according to claim 1, which is characterized in that the step（2）
It is using the mating full-automatic collector of protein purification system, according to sample elution appearance volume, successively to collect each elution fraction
Fraction collection is carried out to different component sample.
6. the method for separating and preparing of buffalo's milk anti-oxidation peptide according to claim 1, which is characterized in that the step（3）
It after freeze-drying, is redissolved using pure water, obtains buffalo's milk anti-oxidation peptide aqueous solution.
7. a kind of buffalo's milk anti-oxidation peptide, which is characterized in that the buffalo's milk anti-oxidation peptide is by claim 1-6 any one
The method for separating and preparing is made.
8. buffalo's milk anti-oxidation peptide according to claim 7, which is characterized in that the buffalo's milk anti-oxidation peptide molecular weight exists
Between 89.09-1245.32Da.
9. buffalo's milk anti-oxidation peptide according to claim 7, which is characterized in that the buffalo's milk anti-oxidation peptide elutes
Peak volume is 18-20mL.
10. the buffalo's milk anti-oxidation peptide described in claim 7 is being prepared with the application in anti-oxidation efficacy health products or food.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810310855.2A CN108410936A (en) | 2018-04-09 | 2018-04-09 | A kind of buffalo's milk anti-oxidation peptide and its method for separating and preparing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810310855.2A CN108410936A (en) | 2018-04-09 | 2018-04-09 | A kind of buffalo's milk anti-oxidation peptide and its method for separating and preparing |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108410936A true CN108410936A (en) | 2018-08-17 |
Family
ID=63134771
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810310855.2A Pending CN108410936A (en) | 2018-04-09 | 2018-04-09 | A kind of buffalo's milk anti-oxidation peptide and its method for separating and preparing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108410936A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283234A (en) * | 2019-08-01 | 2019-09-27 | 四川旅游学院 | It is a kind of derived from the anti-oxidation peptide and its preparation of Yak Blood and application |
| CN113461778A (en) * | 2021-05-21 | 2021-10-01 | 广西大学 | Buffalo milk characteristic peptide and buffalo milk identification method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101768209A (en) * | 2009-01-05 | 2010-07-07 | 北京林业大学 | Hypotensive peptide with high in-vivo activity and preparation and purification method thereof |
| CN106520878A (en) * | 2016-11-25 | 2017-03-22 | 邵阳学院 | Method for preparing active peptide from waste fermented grains |
| CN107027886A (en) * | 2017-05-10 | 2017-08-11 | 广西壮族自治区水牛研究所 | A kind of preparation method of the coagulating type flavor fermentation buffalo's milk rich in whey anti-oxidation peptide |
| CN107183181A (en) * | 2017-05-10 | 2017-09-22 | 广西壮族自治区水牛研究所 | A kind of preparation method of buffalo lactalbumin peptide lactic acid bacteria beverage |
-
2018
- 2018-04-09 CN CN201810310855.2A patent/CN108410936A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101768209A (en) * | 2009-01-05 | 2010-07-07 | 北京林业大学 | Hypotensive peptide with high in-vivo activity and preparation and purification method thereof |
| CN106520878A (en) * | 2016-11-25 | 2017-03-22 | 邵阳学院 | Method for preparing active peptide from waste fermented grains |
| CN107027886A (en) * | 2017-05-10 | 2017-08-11 | 广西壮族自治区水牛研究所 | A kind of preparation method of the coagulating type flavor fermentation buffalo's milk rich in whey anti-oxidation peptide |
| CN107183181A (en) * | 2017-05-10 | 2017-09-22 | 广西壮族自治区水牛研究所 | A kind of preparation method of buffalo lactalbumin peptide lactic acid bacteria beverage |
Non-Patent Citations (5)
| Title |
|---|
| MAHMOUD ABDEL-HAMID等: "Angiotensin I-converting enzyme inhibitory activity and antioxidant capacity of bioactive peptides derived from enzymatic hydrolysis of buffalo milk proteins", 《INTERNATIONAL DAIRY JOURNAL》 * |
| 唐艳 等: "水牛乳干酪副产物乳清抗氧化肽基料的制备技术", 《食品工业》 * |
| 姜锡瑞 等: "《生物发酵产业技术》", 31 May 2016, 中国轻工业出版社 * |
| 李玲 等: "直接酶解技术制备水牛乳抗氧化肽基料", 《中国乳品工业》 * |
| 钟兴业 等: "固定化中性蛋白酶制备水牛乳抗氧化活性肽的研究", 《食品工业科技》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283234A (en) * | 2019-08-01 | 2019-09-27 | 四川旅游学院 | It is a kind of derived from the anti-oxidation peptide and its preparation of Yak Blood and application |
| CN110283234B (en) * | 2019-08-01 | 2022-11-29 | 四川旅游学院 | Antioxidant peptide derived from yak blood and preparation and application thereof |
| CN113461778A (en) * | 2021-05-21 | 2021-10-01 | 广西大学 | Buffalo milk characteristic peptide and buffalo milk identification method |
| CN113461778B (en) * | 2021-05-21 | 2022-09-27 | 广西大学 | Buffalo milk characteristic peptide and buffalo milk identification method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105586379B (en) | A kind of preparation method with the collagen active peptide for inhibiting cancer cell multiplication effect | |
| Brew et al. | The characterization of the whey proteins of guinea-pig milk: The isolation and properties of α-lactalbumin | |
| CN104450839B (en) | The preparation method of the rice bran protein peptide with ACE inhibitory activity | |
| US20080319163A1 (en) | Method for Isolating and Purifying Immuno-Modulating Polypeptide from Cow Placenta | |
| JPS6030291B2 (en) | HGI glycoprotein that promotes differentiation and proliferation of human granulocytes, method for producing HGI glycoprotein, and therapeutic agent for leukopenia containing HGI glycoprotein | |
| CN110964087B (en) | Fugu active peptide and preparation method thereof | |
| JPS6011424A (en) | Fibroblast growth factor and characteristics display | |
| CN104479001A (en) | Preparation and application of kappa-casein derived bioactive peptides | |
| CN102088862A (en) | Angiogenin-enriched milk fractions | |
| CN111100186B (en) | Puffer fish polypeptide with ACE inhibitory activity and preparation method thereof | |
| CN104710524A (en) | Bovine alpha s2-casein source bioactive peptides preparation and application thereof | |
| CN111004309B (en) | ACE inhibitory peptide prepared from Takifugu flavidus fish skin and preparation method thereof | |
| CN111072756A (en) | Tetrodotoxin ACE inhibitory peptide and preparation method thereof | |
| CN108410936A (en) | A kind of buffalo's milk anti-oxidation peptide and its method for separating and preparing | |
| CN104693272B (en) | Yak milk lactalbumin has the tripeptides of antioxidant activity and application thereof and compositions | |
| CN107574214A (en) | A kind of preparation method of the whey protein peptide of anti-aging | |
| CN106520877A (en) | Method for preparing pig cerebral protein antioxidative peptide | |
| CN101747409B (en) | Cobra-venom factor and cobra-venom neurotoxin combined separation and purification method | |
| Dimino et al. | Purification of bovine hemoglobin via fast performance liquid chromatography | |
| CN109748951B (en) | Angelica sinensis antioxidant polypeptide and preparation method and application thereof | |
| CN103695518A (en) | Product capable for reducing serum cholesterol level by using whey protein peptide | |
| CN102372765A (en) | Ruditapes philippinarum enzymatic polypeptide and preparation method thereof | |
| CN107245094A (en) | A kind of anti-oxidation peptide and its method for separating and preparing and purposes | |
| CN115109817B (en) | Almond peptide with antioxidant and immune activities and preparation method and application thereof | |
| CN105085651A (en) | Casein phosphopeptide monomers and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |