CN104610446A - Jerk filefish skin collagen antioxidative peptide and preparation method thereof - Google Patents
Jerk filefish skin collagen antioxidative peptide and preparation method thereof Download PDFInfo
- Publication number
- CN104610446A CN104610446A CN201510052044.3A CN201510052044A CN104610446A CN 104610446 A CN104610446 A CN 104610446A CN 201510052044 A CN201510052044 A CN 201510052044A CN 104610446 A CN104610446 A CN 104610446A
- Authority
- CN
- China
- Prior art keywords
- gained
- collagen
- black scraper
- preparation
- solution
- 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.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 59
- 102000008186 Collagen Human genes 0.000 title claims abstract description 46
- 108010035532 Collagen Proteins 0.000 title claims abstract description 46
- 229920001436 collagen Polymers 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 230000003078 antioxidant effect Effects 0.000 title abstract description 25
- 241001441726 Tetraodontiformes Species 0.000 title abstract 4
- 230000036461 convulsion Effects 0.000 title abstract 4
- 241000251468 Actinopterygii Species 0.000 claims abstract description 32
- 230000007062 hydrolysis Effects 0.000 claims abstract description 16
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 150000001793 charged compounds Chemical class 0.000 claims abstract description 4
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 claims abstract description 4
- 241001676702 Thamnaconus modestus Species 0.000 claims description 54
- 230000003064 anti-oxidating effect Effects 0.000 claims description 49
- 239000000243 solution Substances 0.000 claims description 47
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 42
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 34
- 239000006228 supernatant Substances 0.000 claims description 26
- 239000011780 sodium chloride Substances 0.000 claims description 17
- 102000004190 Enzymes Human genes 0.000 claims description 16
- 108090000790 Enzymes Proteins 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 15
- 102000035101 Aspartic proteases Human genes 0.000 claims description 13
- 108091005502 Aspartic proteases Proteins 0.000 claims description 13
- 101710180012 Protease 7 Proteins 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 10
- 239000003292 glue Substances 0.000 claims description 9
- 238000010521 absorption reaction Methods 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 7
- 238000001641 gel filtration chromatography Methods 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 5
- 229920005654 Sephadex Polymers 0.000 claims description 5
- 239000012507 Sephadex™ Substances 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 238000005238 degreasing Methods 0.000 claims description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 5
- 239000002953 phosphate buffered saline Substances 0.000 claims description 5
- 238000002203 pretreatment Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- 108091005804 Peptidases Proteins 0.000 claims description 2
- 239000004365 Protease Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000013016 damping Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 238000007654 immersion Methods 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 10
- 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 abstract description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 4
- 238000012545 processing Methods 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 3
- 239000002699 waste material Substances 0.000 abstract description 3
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 230000002496 gastric effect Effects 0.000 abstract description 2
- 238000004440 column chromatography Methods 0.000 abstract 1
- 230000029087 digestion Effects 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 6
- -1 DPPH free radical Chemical class 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000007760 free radical scavenging Effects 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 101100421450 Drosophila melanogaster Shark gene Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical compound C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 230000001175 peptic effect Effects 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 101800000068 Antioxidant peptide Proteins 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 241001125843 Trichiuridae Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a jerk filefish skin collagen antioxidative peptide which is characterized in that the amino acid sequence of the antioxidative peptide is Asn-Glu-Gly-Ala-Cys-Gly, and the molecular ion peak detected by UPLC-MS is [M]+531m/z. The invention further relates to a preparation method of the jerk filefish skin collagen antioxidative peptide. According to the method provided by the invention, a double-enzyme fractional hydrolysis technique is combined with an ultrafiltration and gel column chromatography technique, the whole preparation process is simple and easy to control and high in hydrolysis efficiency, and the obtained antioxidative peptide is safe and free of side effects, has relatively high antioxidant activity, and not only has a good eliminating effect to DPPH.free radicals, but also has relatively good thermal stability. The antioxidative peptide digested by the gastrointestinal digestive tract still maintains relatively high antioxidant activity. In addition, the raw materials adopted by the antioxidative peptide prepared are processing wastes of a marine low-value fish jerk filefish, so that the environmental pollution is effectively avoided and the utilization ratio of resources is improved.
Description
Technical field
The present invention relates to a kind of black scraper collagen of fish skin anti-oxidation peptide, the invention still further relates to the preparation method of this black scraper collagen of fish skin anti-oxidation peptide.
Background technology
Oxygenizement is one of major reason of mankind's catabiosis, and this viewpoint is confirmed.Research shows, cancer, aging or Other diseases etc. mostly have with excessive free radical and associate, therefore, the oxygenizement of how to resist in human body becomes one of Main way of healthcare products, makeup research and development, and seeks a kind of antioxidant that is efficient, low toxicity and become study hotspot in recent years.
Research finds, the polypeptide that proteolysis obtains has good anti-oxidant activity, and little, the easy absorption of molecular weight, safe and reliable, and therefore anti-oxidation peptide has a good application prospect at numerous areas such as food-processing, healthcare products, makeup.Application publication number is that the Chinese invention patent application " anti-oxidation peptide prepared by a kind of shark prolamine and its preparation method and application " of CN103524579A discloses a kind of anti-oxidation peptide, it utilizes, and extraction using alcohol shark protein,alcohol-soluble also utilizes enzyme engineering technology further, prepared by chromatographic technique, this anti-oxidation peptide safe without toxic side effect, has higher anti-oxidant activity.But the general thermostability of anti-oxidation peptide of the prior art is poor, and the activity of anti-oxidation peptide is vulnerable to have a strong impact on after gastroenteric environment digestion, thus reduces its antioxidant property.
Black scraper fish is also known as peeling fish, rubber fish; the marine economy fish that China is only second to hairtail; and the edible portion of black scraper fish is less; the inedible part of black scraper fish accounts for 53%; wherein fish-skin accounts for 8.5% ~ 9.4%, and black scraper fish-skin is thick and hard, and in the course of processing, fish-skin usually can be divested and abandon; so not only contaminate environment, also result in the waste of resource.In fact, containing abundant collagen protein in black scraper fish-skin, bioactive peptide is prepared if develop and utilize it, significant economic benefit and social benefit will be obtained, but, in prior art, with black scraper fish-skin for the rarely seen report of technique of collagen antioxidant peptides prepared by raw material.
Summary of the invention
Technical problem to be solved by this invention is the present situation for prior art, provides that a kind of anti-oxidant activity is high, Heat stability is good and still can keep the black scraper collagen of fish skin anti-oxidation peptide of greater activity after gastroenteric environment digestion.
Another technical problem to be solved by this invention is the present situation for prior art, a kind of preparation method of above-mentioned black scraper collagen of fish skin anti-oxidation peptide is provided, two enzyme fractional hydrolysis technology and ultrafiltration, gel filtration chromatography technical tie-up use by the method, scientific and reasonable, easy handling.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of black scraper collagen of fish skin anti-oxidation peptide, it is characterized in that the aminoacid sequence of this anti-oxidation peptide is that Asn-Glu-Gly-Ala-Cys-Gly, UPLC-MS detection provides molecular ion peak for [M]
+531m/z.
A preparation method for above-mentioned black scraper collagen of fish skin anti-oxidation peptide, is characterized in that comprising the following steps:
(1) pre-treatment of black scraper fish-skin
The black scraper fish-skin cleaned, rub is carried out skimming treatment, filters after skimming treatment and gained filter residue is cleaned;
(2) preparation of black scraper collagen of fish skin
With acetic acid by last for step (1) gained filter residue immersion, collected after centrifugation supernatant liquor, centrifugal treating after NaCl is added in this supernatant liquor, redissolved and do dialysis treatment by gained precipitation acetic acid, gained solution is black scraper Isin glue collagen, freeze-dried back;
(3) two enzyme fractional hydrolysiss of collagen protein
Step (2) gained black scraper Isin glue collagen damping fluid is configured to solution, is then hydrolyzed with Protease AAmano 2G, aspartic protease successively, collects supernatant liquor after hydrolysis for subsequent use;
(4) separation of anti-oxidation peptide
Ultrafiltration is carried out to step (3) gained supernatant liquor, collect the anti-oxidation peptide component that molecular weight is less than 5kDa, use Sephadex G-15 gel filtration chromatography again, have the component of absorption peak under being collected in 220nm, namely this component obtains described black scraper collagen of fish skin anti-oxidation peptide after lyophilize.
As preferably, the detailed process of step (1) is:
It is in the Virahol of 5 ~ 15% that the black scraper fish-skin cleaned, rub is added volumetric concentration by solid-liquid weight ratio 1:10 ~ 20, cross after soak degreasing 10 ~ 20h and filter Virahol, again this filter residue being added mass concentration by solid-liquid weight ratio 1:5 ~ 10 after cleaning filter residue with distilled water is in the NaCl solution of 7 ~ 10%, filter after soaking 12 ~ 24h, for subsequent use after the cleaning of gained filter residue distilled water.
As improvement, the detailed process of step (2) is:
It is soak 12 ~ 36h in the acetic acid of 0.1 ~ 0.5mol/L that last for step (1) gained filter residue is added concentration by solid-liquid weight ratio 1:10 ~ 30, and then under the condition of 8000 ~ 10000r/min, centrifugal treating collects supernatant liquor;
In gained supernatant liquor, add NaCl to NaCl mass concentration is 6 ~ 9%, then centrifugal treating under the condition of 8000 ~ 10000r/min, after gained precipitation is redissolved with the acetic acid solution of 0.4 ~ 0.6mol/L, dialysis 24 ~ 30h, gained solution is black scraper Isin glue collagen, freeze-dried back.
Improve, the detailed process of step (3) is again:
With pH be 6.5 ~ 7.5 phosphoric acid buffer above-mentioned black scraper collagen of fish skin is made into the solution that mass concentration is 2 ~ 6%, then add Protease A Amano 2G enzymolysis 1 ~ 4h, obtain hydrolyzed solution A;
By hydrolyzed solution A dilute hydrochloric acid adjust ph to 2.2, add aspartic protease and continue hydrolysis 2 ~ 5h, obtain hydrolyzed solution B, hydrolyzed solution B is boiled and is incubated 8 ~ 15min, after cooling under the condition of 8000 ~ 10000r/min centrifugal treating to collect supernatant liquor for subsequent use.
Preferably, when having the component of absorption peak under collecting 220nm in step (4), take pH as the phosphate buffered saline buffer of 7.2 be elutriant, be separated under the flow velocity of 0.2 ~ 0.3mL/min.
In such scheme, the enzyme dosage of described Protease A Amano 2G and aspartic protease all >=3600U/g.
Compared with prior art, the invention has the advantages that:
(1) anti-oxidation peptide of the present invention has higher anti-oxidant activity, not only to DPPH free radical, there is good elimination effect, also have good thermostability, after this anti-oxidation peptide is incubated 2h respectively at 20,40,60,80,100 DEG C, anti-oxidant activity all remains on more than 90%; This anti-oxidation peptide its anti-oxidant activity conservation rate after peptic digestion digestion is 89.99%, after gastro-intestinal tract digestion, its anti-oxidant activity conservation rate is 80.53%, still maintain higher anti-oxidant activity, therefore, anti-oxidation peptide of the present invention adds in heath food, makeup as functional factor and has better application prospect;
(2) the present invention adopts two enzyme fractional hydrolysis technology and ultrafiltration, the coupling of gel filtration chromatography technology, and whole preparation process is simple and easy to control, enzymolysis efficiency is high, and gained anti-oxidation peptide has no side effect safely;
(3) raw material of the present invention is the processing waste of marine low-value fish black scraper fish, effectively prevent environmental pollution, improves resource utilization.
Accompanying drawing explanation
Fig. 1 is the UPLC-MS collection of illustrative plates of Asn-Glu-Gly-Ala-Cys-Gly in the present invention;
Fig. 2 be in the present invention Asn-Glu-Gly-Ala-Cys-Gly to the scavenging(action) figure of DPPH free radical;
Fig. 3 is the activity influence figure of temperature to Asn-Glu-Gly-Ala-Cys-Gly in the present invention;
Fig. 4 is the activity influence figure of gastro-intestinal digestion enzyme to Asn-Glu-Gly-Ala-Cys-Gly in the present invention.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment 1:
In the present embodiment, the preparation method of black scraper collagen of fish skin anti-oxidation peptide comprises the following steps:
(1) pre-treatment of black scraper fish-skin
It is in the Virahol of 5% that the black scraper fish-skin cleaned, rub is added volumetric concentration by solid-liquid weight ratio 1:20, cross after soak degreasing 10h and filter Virahol, again this filter residue being added mass concentration by solid-liquid weight ratio 1:5 after cleaning filter residue with distilled water is in the NaCl solution of 10%, filter after soaking 12h, for subsequent use after the cleaning of gained filter residue distilled water;
(2) preparation of black scraper collagen of fish skin
It is soak 12h in the acetic acid of 0.5mol/L that last for step (1) gained filter residue is added concentration by solid-liquid weight ratio 1:10, and then under the condition of 10000r/min, centrifugal treating collects supernatant liquor;
In gained supernatant liquor, add NaCl to NaCl mass concentration is 6%, then centrifugal treating under the condition of 8000r/min, after the acetic acid solution redissolution of gained precipitation 0.6mol/L, dialysis 24h, gained solution is black scraper Isin glue collagen, freeze-dried back;
(3) two enzyme fractional hydrolysiss of collagen protein
With pH be 6.5 phosphoric acid buffer above-mentioned black scraper collagen of fish skin is made into the solution that mass concentration is 6%, then add Protease A Amano 2G enzymolysis 4h, obtain hydrolyzed solution A;
By the dilute hydrochloric acid adjust ph to 2.2 of hydrolyzed solution A 2mol/L, add aspartic protease and continue hydrolysis 5h, obtain hydrolyzed solution B, hydrolyzed solution B is boiled and is incubated 8min, after cooling under the condition of 10000r/min centrifugal treating to collect supernatant liquor for subsequent use;
The enzyme dosage of Protease A Amano 2G and aspartic protease is respectively 5000U/g, 4500U/g;
(4) separation of anti-oxidation peptide
Ultrafiltration is carried out to step (3) gained supernatant liquor, collect the anti-oxidation peptide component that molecular weight is less than 5kDa, use Sephadex G-15 gel filtration chromatography again, take pH as the phosphate buffered saline buffer of 7.2 be elutriant, be separated under the flow velocity of 0.2mL/min, have the component of absorption peak under being collected in 220nm, namely this component obtains black scraper collagen of fish skin anti-oxidation peptide after lyophilize.
Embodiment 2:
In the present embodiment, the preparation method of black scraper collagen of fish skin anti-oxidation peptide comprises the following steps:
(1) pre-treatment of black scraper fish-skin
It is in the Virahol of 15% that the black scraper fish-skin cleaned, rub is added volumetric concentration by solid-liquid weight ratio 1:10, cross after soak degreasing 20h and filter Virahol, again this filter residue being added mass concentration by solid-liquid weight ratio 1:10 after cleaning filter residue with distilled water is in the NaCl solution of 10%, filter after soaking 24h, for subsequent use after the cleaning of gained filter residue distilled water;
(2) preparation of black scraper collagen of fish skin
It is soak 36h in the acetic acid of 0.1mol/L that last for step (1) gained filter residue is added mass concentration by solid-liquid weight ratio 1:30, and then under the condition of 8000r/min, centrifugal treating collects supernatant liquor;
In gained supernatant liquor, add NaCl to NaCl mass concentration is 9%, then centrifugal treating under the condition of 10000r/min, after the acetic acid solution redissolution of gained precipitation 0.4mol/L, dialysis 30h, gained solution is black scraper Isin glue collagen, freeze-dried back;
(3) two enzyme fractional hydrolysiss of collagen protein
With pH be 7.5 phosphoric acid buffer above-mentioned black scraper collagen of fish skin is made into the solution that mass concentration is 2%, then add Protease A Amano 2G enzymolysis 1h, obtain hydrolyzed solution A;
By the dilute hydrochloric acid adjust ph to 2.2 of hydrolyzed solution A 2mol/L, add aspartic protease and continue hydrolysis 2h, obtain hydrolyzed solution B, hydrolyzed solution B is boiled and is incubated 15min, after cooling under the condition of 8000r/min centrifugal treating to collect supernatant liquor for subsequent use;
The enzyme dosage of Protease A Amano 2G and aspartic protease is respectively 4500U/g, 3600U/g;
(4) separation of anti-oxidation peptide
Ultrafiltration is carried out to step (3) gained supernatant liquor, collect the anti-oxidation peptide component that molecular weight is less than 5kDa, use Sephadex G-15 gel filtration chromatography again, take pH as the phosphate buffered saline buffer of 7.2 be elutriant, be separated under the flow velocity of 0.3mL/min, have the component of absorption peak under being collected in 220nm, namely this component obtains black scraper collagen of fish skin anti-oxidation peptide after lyophilize.
Embodiment 3:
In the present embodiment, the preparation method of black scraper collagen of fish skin anti-oxidation peptide comprises the following steps:
(1) pre-treatment of black scraper fish-skin
It is in the Virahol of 10% that the black scraper fish-skin cleaned, rub is added volumetric concentration by solid-liquid weight ratio 1:15, cross after soak degreasing 16h and filter Virahol, again this filter residue being added mass concentration by solid-liquid weight ratio 1:7 after cleaning filter residue with distilled water is in the NaCl solution of 8%, filter after soaking 18h, for subsequent use after the cleaning of gained filter residue distilled water;
(2) preparation of black scraper collagen of fish skin
It is soak 25h in the acetic acid of 0.3mol/L that last for step (1) gained filter residue is added concentration by solid-liquid weight ratio 1:20, and then under the condition of 9000r/min, centrifugal treating collects supernatant liquor;
In gained supernatant liquor, add NaCl to NaCl mass concentration is 8%, then centrifugal treating under the condition of 9000r/min, after the acetic acid solution redissolution of gained precipitation 0.5mol/L, dialysis 28h, gained solution is black scraper Isin glue collagen, freeze-dried back;
(3) two enzyme fractional hydrolysiss of collagen protein
With pH be 7 phosphoric acid buffer above-mentioned black scraper collagen of fish skin is made into the solution that mass concentration is 4%, then add Protease A Amano 2G enzymolysis 3h, obtain hydrolyzed solution A;
By the dilute hydrochloric acid adjust ph to 2.2 of hydrolyzed solution A 2mol/L, add aspartic protease and continue hydrolysis 4h, obtain hydrolyzed solution B, hydrolyzed solution B is boiled and is incubated 12min, after cooling under the condition of 8500r/min centrifugal treating to collect supernatant liquor for subsequent use;
The enzyme dosage of Protease A Amano 2G and aspartic protease is respectively 3600U/g, 4000U/g;
(4) separation of anti-oxidation peptide
Ultrafiltration is carried out to step (3) gained supernatant liquor, collect the anti-oxidation peptide component that molecular weight is less than 5kDa, use Sephadex G-15 gel filtration chromatography again, take pH as the phosphate buffered saline buffer of 7.2 be elutriant, be separated under the flow velocity of 0.25mL/min, have the component of absorption peak under being collected in 220nm, namely this component obtains black scraper collagen of fish skin anti-oxidation peptide after lyophilize.
As shown in Figure 1, the anti-oxidation peptide molecular ion peak that prepared by the embodiment of the present invention is [M]
+531m/z, 5 fragment ion peaks are respectively 474m/z ([M-57]
+), 345m/z ([M-57-129]
+), 288m/z ([M-57-129-57]
+), 217m/z ([M-57-129-57-71]
+), 114m/z ([M-57-129-57-71-103]
+), fragment ion peak 114m/z ([M-57-129-57-71-103]
+) being speculated as Asn residue, other fragment ion peak is respectively and loses that Gly, Glu, Gly, Ala, Cys residue formed, and the material that namely prepared by the present invention is six peptides, and its aminoacid sequence is Asn-Glu-Gly-Ala-Cys-Gly.
The anti-oxidation peptide Asn-Glu-Gly-Ala-Cys-Gly of above-mentioned preparation is carried out to the removing experiment of DPPH free radical, judge its anti-oxidant activity with this.Concrete, the anti-oxidation peptide obtained is mixed with the solution of different concns, gets 1mL and 0.1mmoL DPPH solution and mix according to the ratio of 1:1, after room temperature lucifuge reaction 20min, centrifugal treating 10min under 4000r/min condition; Under 517nm, survey light absorption value Ai, contrast and background simultaneously, contrast Aj is sample and alcohol mixed solution, and background A0 is sample and deionized water mixing solutions.
Clearance rate calculation formula: clearance rate (%)=[1-(Ai-Aj)/A0] × 100%; EC
50the DPPH free radical scavenging activity that value is determinand reaches concentration when 50%, according to measured each concentration samples to DPPH free radical scavenging activity, draw the typical curve of clearance rate and sample concentration, draw fit equation, go out the concentration of sample when clearance rate is 50% again according to Equation for Calculating, be EC
50value.
As shown in Figure 2, gained anti-oxidation peptide of the present invention is to the EC of DPPH free radical
50value is 1.8mg/mL, has good DPPH free radical scavenging effect, shows higher anti-oxidant activity.
The anti-oxidation peptide prepared the present invention at different temperatures anti-oxidant activity is tested, anti-oxidation peptide is incubated 2h respectively at 20 DEG C, 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C, as shown in Figure 3, result shows, the activity of anti-oxidation peptide all remains on more than 90%, shows good thermostability.
Invention emulates external gastro-intestinal tract enzyme and situation is affected on anti-oxidation peptide activity resistent, as shown in Figure 4, result shows, anti-oxidation peptide prepared by the present invention its anti-oxidant activity conservation rate after peptic digestion digestion is 89.99%, after gastro-intestinal tract digestion, anti-oxidant conservation rate is 80.53%, all maintains higher anti-oxidant activity.
Enzyme Protease A Amano 2G manufacturer in the various embodiments described above is Japanese Amano company, and aspartic protease manufacturer is Beijing Solarbio company.
Claims (7)
1. a black scraper collagen of fish skin anti-oxidation peptide, is characterized in that the aminoacid sequence of this anti-oxidation peptide is that Asn-Glu-Gly-Ala-Cys-Gly, UPLC-MS detection provides molecular ion peak for [M]
+531m/z.
2. a preparation method for black scraper collagen of fish skin anti-oxidation peptide described in claim 1, is characterized in that comprising the following steps:
(1) pre-treatment of black scraper fish-skin
The black scraper fish-skin cleaned, rub is carried out skimming treatment, filters after skimming treatment and gained filter residue is cleaned;
(2) preparation of black scraper collagen of fish skin
With acetic acid by last for step (1) gained filter residue immersion, collected after centrifugation supernatant liquor, centrifugal treating after NaCl is added in this supernatant liquor, redissolved and do dialysis treatment by gained precipitation acetic acid, gained solution is black scraper Isin glue collagen, freeze-dried back;
(3) two enzyme fractional hydrolysiss of collagen protein
Step (2) gained black scraper Isin glue collagen damping fluid is configured to solution, is then hydrolyzed with Protease AAmano 2G, aspartic protease successively, collects supernatant liquor after hydrolysis for subsequent use;
(4) separation of anti-oxidation peptide
Ultrafiltration is carried out to step (3) gained supernatant liquor, collect the anti-oxidation peptide component that molecular weight is less than 5kDa, use Sephadex G-15 gel filtration chromatography again, have the component of absorption peak under being collected in 220nm, namely this component obtains described black scraper collagen of fish skin anti-oxidation peptide after lyophilize.
3. preparation method according to claim 2, is characterized in that the detailed process of step (1) is:
It is in the Virahol of 5 ~ 15% that the black scraper fish-skin cleaned, rub is added volumetric concentration by solid-liquid weight ratio 1:10 ~ 20, cross after soak degreasing 10 ~ 20h and filter Virahol, again this filter residue being added mass concentration by solid-liquid weight ratio 1:5 ~ 10 after cleaning filter residue with distilled water is in the NaCl solution of 7 ~ 10%, filter after soaking 12 ~ 24h, for subsequent use after the cleaning of gained filter residue distilled water.
4. preparation method according to claim 2, is characterized in that the detailed process of step (2) is:
It is soak 12 ~ 36h in the acetic acid of 0.1 ~ 0.5mol/L that last for step (1) gained filter residue is added concentration by solid-liquid weight ratio 1:10 ~ 30, and then under the condition of 8000 ~ 10000r/min, centrifugal treating collects supernatant liquor;
In gained supernatant liquor, add NaCl to NaCl mass concentration is 6 ~ 9%, then centrifugal treating under the condition of 8000 ~ 10000r/min, after gained precipitation is redissolved with the acetic acid solution of 0.4 ~ 0.6mol/L, dialysis 24 ~ 30h, gained solution is black scraper Isin glue collagen, freeze-dried back.
5. preparation method according to claim 2, is characterized in that the detailed process of step (3) is:
With pH be 6.5 ~ 7.5 phosphoric acid buffer above-mentioned black scraper collagen of fish skin is made into the solution that mass concentration is 2 ~ 6%, then add Protease A Amano 2G enzymolysis 1 ~ 4h, obtain hydrolyzed solution A;
By hydrolyzed solution A dilute hydrochloric acid adjust ph to 2.2, add aspartic protease and continue hydrolysis 2 ~ 5h, obtain hydrolyzed solution B, hydrolyzed solution B is boiled and is incubated 8 ~ 15min, after cooling under the condition of 8000 ~ 10000r/min centrifugal treating to collect supernatant liquor for subsequent use.
6. preparation method according to claim 2, is characterized in that: when having the component of absorption peak under collecting 220nm in step (4), take pH as the phosphate buffered saline buffer of 7.2 is elutriant, is separated under the flow velocity of 0.2 ~ 0.3mL/min.
7. preparation method according to claim 5, is characterized in that: the enzyme dosage of described Protease A Amano 2G and aspartic protease all >=3600U/g.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510052044.3A CN104610446B (en) | 2015-02-02 | 2015-02-02 | A kind of black scraper collagen anti-oxidation peptide and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510052044.3A CN104610446B (en) | 2015-02-02 | 2015-02-02 | A kind of black scraper collagen anti-oxidation peptide and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104610446A true CN104610446A (en) | 2015-05-13 |
| CN104610446B CN104610446B (en) | 2018-02-23 |
Family
ID=53145121
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510052044.3A Active CN104610446B (en) | 2015-02-02 | 2015-02-02 | A kind of black scraper collagen anti-oxidation peptide and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104610446B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350556A (en) * | 2016-08-26 | 2017-01-25 | 青岛琛蓝海洋生物工程有限公司 | Method for preparing high-purity medical fish skin collagen |
| CN109207541A (en) * | 2018-09-20 | 2019-01-15 | 福建省水产研究所 | River Puffer collagen peptide extraction process and application |
| CN110016076A (en) * | 2019-04-11 | 2019-07-16 | 浙江万里学院 | A kind of black scraper fish skin collagen peptide and preparation method thereof with ACE inhibitory activity |
| CN114106151A (en) * | 2021-12-10 | 2022-03-01 | 中基惠诚科技有限公司 | Preparation method of antioxidant active collagen peptide |
| CN114539387A (en) * | 2021-12-31 | 2022-05-27 | 浙江万里学院 | Marine-source antifreeze peptide preparation and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103524597A (en) * | 2012-07-03 | 2014-01-22 | 浙江海洋学院 | Antioxidant peptide prepared from shark prolamin and preparation method and application thereof |
-
2015
- 2015-02-02 CN CN201510052044.3A patent/CN104610446B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103524597A (en) * | 2012-07-03 | 2014-01-22 | 浙江海洋学院 | Antioxidant peptide prepared from shark prolamin and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 张昊等: "天然抗氧化肽的研究进展", 《食品科学》 * |
| 曹少谦等: "马面鱼皮胶原蛋白的提取及其特性研究", 《中国食品学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350556A (en) * | 2016-08-26 | 2017-01-25 | 青岛琛蓝海洋生物工程有限公司 | Method for preparing high-purity medical fish skin collagen |
| CN109207541A (en) * | 2018-09-20 | 2019-01-15 | 福建省水产研究所 | River Puffer collagen peptide extraction process and application |
| CN110016076A (en) * | 2019-04-11 | 2019-07-16 | 浙江万里学院 | A kind of black scraper fish skin collagen peptide and preparation method thereof with ACE inhibitory activity |
| CN114106151A (en) * | 2021-12-10 | 2022-03-01 | 中基惠诚科技有限公司 | Preparation method of antioxidant active collagen peptide |
| CN114539387A (en) * | 2021-12-31 | 2022-05-27 | 浙江万里学院 | Marine-source antifreeze peptide preparation and preparation method and application thereof |
| CN114539387B (en) * | 2021-12-31 | 2023-06-27 | 浙江万里学院 | Marine source antifreeze peptide preparation and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104610446B (en) | 2018-02-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104610446A (en) | Jerk filefish skin collagen antioxidative peptide and preparation method thereof | |
| CN104710525B (en) | Tuna bone collagen source zinc chelated collagen peptide and preparation method and application thereof | |
| WO2021142880A1 (en) | Method for producing clam active peptide | |
| CN104774896B (en) | Preparation method of hairtail fishbone iron-chelated collagen peptide | |
| CN103992387B (en) | A kind of mussel boiling liquid bioactive peptide and its preparation method and application | |
| CN104342040B (en) | A kind of high glue melts the preparation method of temperature fish scale gelatin | |
| CN103103244B (en) | Walnut blood pressure-lowering active peptide, its preparation method and application | |
| CN103204906A (en) | Mussel meat protein antioxidative peptide and preparation method thereof | |
| CN103088097A (en) | Preparation method of matreel active peptide | |
| CN104250286B (en) | Navodon septentrionalis fish-skin antioxidant collagen peptide and preparation method and use thereof | |
| AU2021105562A4 (en) | Preparation Method of Tilapia Protein Peptide with Antioxidant and Antifatigue Activities | |
| CN107164447A (en) | A kind of method that utilization cod processing accessory substance prepares anti-oxidation peptide | |
| CN103333940A (en) | Method for preparing dipeptidyl peptidase IV (DPP-IV) inhibitory peptide through using hairtail | |
| CN102477004A (en) | Method for extracting natural taurine from oyster by ultrafiltration technology | |
| CN105695545A (en) | Method for preparing sea cucumber fucoidan and sea cucumber glycoprotein | |
| CN109504732A (en) | A kind of preparation method of oyster active peptides | |
| CN109943612B (en) | Method for producing high-activity minced fillet and product antifreeze agent by enzymolysis of silver carp scales | |
| CN114214384A (en) | Marine organism-derived collagen peptide, and extraction method and application thereof | |
| CN104152518A (en) | Preparation method of hepatopathy complementary-food cod skin collagen peptide | |
| CN103992386A (en) | Pseudosciaena crocea fish scale oxidation-resistant collagen peptide, and preparation method and application thereof | |
| CN102936611B (en) | Collagen extracted from black butterflyfish skins with scales and application of collagen | |
| CN107177650B (en) | Preparation method of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid | |
| CN105567772B (en) | A kind of high antioxidant protein peptides and the preparation method and application thereof | |
| CN104892750A (en) | Preparation method of acid-soluble fish scale collagen | |
| CN105648004B (en) | Preparation method of dasyatis akajei cartilage antioxidative collagen peptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |