CN103805663A - Method for separating, purifying and extracting bioactive peptide from marine product - Google Patents
Method for separating, purifying and extracting bioactive peptide from marine product Download PDFInfo
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- CN103805663A CN103805663A CN201310618049.9A CN201310618049A CN103805663A CN 103805663 A CN103805663 A CN 103805663A CN 201310618049 A CN201310618049 A CN 201310618049A CN 103805663 A CN103805663 A CN 103805663A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 23
- 230000000975 bioactive effect Effects 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 14
- 241000237502 Ostreidae Species 0.000 claims abstract description 48
- 235000020636 oyster Nutrition 0.000 claims abstract description 47
- 238000004108 freeze drying Methods 0.000 claims abstract description 15
- 239000000287 crude extract Substances 0.000 claims abstract description 9
- 239000006228 supernatant Substances 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 7
- 239000012153 distilled water Substances 0.000 claims abstract description 6
- 239000002002 slurry Substances 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims abstract description 4
- 108090000790 Enzymes Proteins 0.000 claims abstract description 4
- 239000000284 extract Substances 0.000 claims description 11
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 11
- 238000000926 separation method Methods 0.000 claims description 9
- 229920001184 polypeptide Polymers 0.000 claims description 8
- 238000010612 desalination reaction Methods 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 7
- 239000002893 slag Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 238000001641 gel filtration chromatography Methods 0.000 claims description 4
- 238000013016 damping Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000003814 drug Substances 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 239000012506 Sephacryl® Substances 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 abstract description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 abstract description 3
- 238000004440 column chromatography Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 239000000047 product Substances 0.000 abstract description 2
- 238000000108 ultra-filtration Methods 0.000 abstract description 2
- 206010028980 Neoplasm Diseases 0.000 abstract 1
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- 201000011510 cancer Diseases 0.000 abstract 1
- 230000009849 deactivation Effects 0.000 abstract 1
- 238000011033 desalting Methods 0.000 abstract 1
- 238000000605 extraction Methods 0.000 abstract 1
- 230000003311 flocculating effect Effects 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 238000005406 washing Methods 0.000 abstract 1
- 238000011160 research Methods 0.000 description 5
- 229920005654 Sephadex Polymers 0.000 description 4
- 239000012507 Sephadex™ Substances 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000013543 active substance Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002386 leaching Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 229920002271 DEAE-Sepharose Polymers 0.000 description 2
- 229920002527 Glycogen Polymers 0.000 description 2
- 102000015636 Oligopeptides Human genes 0.000 description 2
- 108010038807 Oligopeptides Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 229940088623 biologically active substance Drugs 0.000 description 2
- 229940096919 glycogen Drugs 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
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- 230000001681 protective effect Effects 0.000 description 2
- 241000237519 Bivalvia Species 0.000 description 1
- 241000548230 Crassostrea angulata Species 0.000 description 1
- 241000991587 Enterovirus C Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000538 anti-polioviral effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
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- 210000000496 pancreas Anatomy 0.000 description 1
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- 241000712461 unidentified influenza virus Species 0.000 description 1
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- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for separating and purifying oyster bioactive peptide from a marine product oyster on the basis of enzymatic hydrolysis of oyster protein and ultra filtration column chromatography. the method comprises the following steps: removing the shells of oysters, then washing and homogenating the oysters, and then adding distilled water to obtain oyster slurry, extracting at a low temperature; flocculating and centrifuging for residue removal; taking supernatant and freeze-drying the supernatant; then separating by using a Sephacryl s-100 HR column, or carrying out enzymolysis and enzyme deactivation on the oyster slurry, centrifuging for residue removal, and freeze-drying to obtain a crude extract; carrying out gel column stratographic analysis on the crude extract; collecting all the peaks and then separating and purifying by using ion-exchange chromatography; and collecting bioactive peaks, freeze-drying, and then desalting by using a gel column to obtain oyster natural purified bioactive peptide. The oyster bioactive peptide obtained according to the method has high purity and cancer resistant activity, is a naturally-extracted substance, and can be used as a medicine for development and application as the bioactivity of the oyster peptide is protected during extraction.
Description
Technical field:
The invention belongs to marine natural product processing technique field, relate to a seed oyster and produce the method for bioactive peptide from marine prods, utilize enzymatic hydrolysis Oyster Protein and ultrafiltration column chromatography technology separation and purification oyster active peptides.
Background technology:
Oyster (Oyster) is a kind of marine shellfish.Oyster belongs to Mollusca in classification, lamellibranchiata, and Anisomyaria, oyster superfamily, Ostreidae, it is a kind of bivalve sea-food of food and medicament dual-purpose.Coastal in China north and south all have a cultivation, particularly Guangdong Province developed comparatively fast in recent years, oyster meat is milky white, delicate, nutritious, except containing the nutritive ingredients such as rich in protein, VITAMIN and carbohydrate, also contains ten multiple amino acids, the mineral matter nutritional composition etc. of needed by human.Now research also proves, contains abundant glycogen in oyster, is that human inner cell carries out the metabolism energy, can improve heart and blood circulation function, and the function that can promote liver, has hepatoprotective effect.Glycogen can be directly for body absorbs, thereby alleviate pancreas burden, very favourable to the control of diabetes.Concha Ostreae extract has anti-microbial effect, and poliovirus and influenza virus are had to restraining effect, and its water soluble component still can improve animal body immunizing power etc.In oyster, contain abundant biologically active substance, there is significant medicinal health care function.
The research of extracting active substance from oyster early has report, and the oyster protective foods with remarkable medical care effect has had mass selling.If Shenzhen Hai Wang group is in the development research of oyster related products, obtain significant economic benefit and social benefit, in order further to advance the industrialized development of this project, making great efforts to research and develop efficient oyster protective foods and medicine.Patent documentation " a kind of preparation method of oyster active peptides ", is take Pacific oyster as raw material, adopts enzymatic hydrolysis nano-filter desalination concentration technology to prepare the method for oyster active peptides, belongs to technical field of food biotechnology, does not relate to field of medicaments.In recent years, the developed countries such as Europe, Japan, the U.S. are increasing to the research of biologically active substance in oyster, but really from oyster, find the material with notable biological activity, and not appearing in the newspapers of developing as medicine.
Summary of the invention:
The object of the present invention is to provide a kind of preparation method with bioactive polypeptide protein class material who derives from marine prods oyster.The oyster active polypeptide purity obtaining by preparation method of the present invention is higher; and this bioactive peptide is the material of natural extract; do not pass through any chemically modified, in leaching process, its biological activity has been carried out to effective protection, can be used as drug ingredient and developed.
In order to realize foregoing invention object, the present invention is achieved through the following technical solutions:
Oyster is peeled off, cleaning, homogenate, adds distilled water to obtain oyster slurries extract at low temperature below 10 ℃; Reflocculation, centrifugal removing slag, get supernatant liquor, by after supernatant liquor freeze-drying, with dextrane gel (Sephacryl) the s-100HR post separation (NH that pH value is 7.4
4hCO
3buffer solution elution), collect respectively and got bioactive peak behind each peak and carry out again ion-exchange chromatography, collect activated each peak, after freeze-drying, using gel column desalination, obtain oyster natural biological polypeptide sterling, is the active substance without the natural extract of any chemically modified; Or by oyster slurries enzymolysis, the enzyme that goes out, centrifugally remove slag, freeze-drying obtains crude extract, crude extract carries out gel filtration chromatography with dextrane gel Sephadex G25, collect respectively and got bioactive peak behind each peak and carry out again ion-exchange chromatography and carry out separation and purification, collect activated each peak, after freeze-drying, use gel column desalination, obtain oyster natural biological polypeptide sterling, be the active substance of natural extract, belong to the oligopeptides of molecular weight.
The present invention is because the biological activity of it being extracted to composition at leaching process has been carried out effective protection; make gained natural active matter show that through anticancer experiment in vitro the natural biological polypeptide that the present invention extracts has anti-tumor activity, likely become a kind of novel antitumor drug.
Embodiment:
Below in conjunction with embodiment, the present invention will be further described.
Embodiment 1:
Oyster is peeled off, get Oyster, after cleaning with 4 ℃ of water, homogenate, add distilled water low temperature (4 ℃) to extract after 2 hours according to the part by weight of 1:1, flocculation, centrifugal removing slag, get supernatant liquor, by after supernatant liquor freeze-drying, with Sephacryl s-100HR post separation (0.03mol/lNH
4hCO
3buffer solution elution), collect respectively behind each peak, get bioactive peak and used again negatively charged ion sepharose DEAE-Sepharose – FF ion-exchange chromatography (PH8.5Tris-HCl damping fluid, 0～0.3mol/lNaCl gradient elution), collect activated each peak, after freeze-drying, with the desalination of dextrane gel Sephadex G15 gel column, obtain oyster natural biological polypeptide sterling.
Embodiment 2:
By oyster peel off, cleaning, homogenate, according to 1, 1 part by weight adds distilled water, and add trypsinase according to substrate 2% weight ratio, 45 ℃ of enzymolysis 4 hours, enzyme goes out, centrifugal removing slag, freeze-drying obtains enzymolysis crude extract, crude extract carries out gel filtration chromatography with dextrane gel Sephadex G25, collect each peak, carry out separation and purification (PH8.5Tris-HCl damping fluid with negatively charged ion sepharose DEAE-Sepharose – FF ion-exchange chromatography again, 0～0.3mol/lNaCl gradient elution), collect activated each peak, after freeze-drying, with the desalination of Sephadex G15 gel column, obtain oyster natural biological polypeptide sterling.This bioactive peptide is the material of natural extract, belongs to the oligopeptides of molecular weight, and leaching process has carried out effective protection to its biological activity.
Claims (3)
1. from marine prods, the method for bioactive peptide is extracted in separation and purification, it is characterized in that preparing according to following steps: oyster is peeled off, cleaning, homogenate, adds distilled water to obtain oyster slurries extract at low temperature below 10 ℃; Reflocculation, centrifugal removing slag, get supernatant liquor, by after supernatant liquor freeze-drying, separates NH through post
4hCO
3buffer solution elution or by oyster slurries enzymolysis, the enzyme that goes out, centrifugally remove slag, freeze-drying obtains crude extract, crude extract carries out gel filtration chromatography; Collect respectively and got bioactive peak behind each peak and carry out again ion-exchange chromatography, collect activated each peak, after freeze-drying, use gel column desalination, obtain oyster natural biological polypeptide sterling.
According to claim 1 a kind of from marine prods separation and purification extract the method for bioactive peptide, it is characterized in that oyster to peel off, get Oyster, with after 4 ℃ of water cleanings, homogenate, adds distilled water at 4 ℃, to extract 2 hours according to the part by weight of 1:1.
According to claim 1 a kind of from marine prods separation and purification extract the method for bioactive peptide, it is characterized in that oyster homogenate supernatant liquor separates through post, the NH that pH value is 7.4
4hCO
3buffer solution elution or enzymolysis crude extract gel filtration chromatography are collected behind each peak, have got bioactive peak and have used ion-exchange chromatography again, use PH8.5Tris-HCl damping fluid, 0～0.3mol/lNaCl gradient elution, collect activated each peak, after freeze-drying, gel column desalination.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310618049.9A CN103805663A (en) | 2013-11-27 | 2013-11-27 | Method for separating, purifying and extracting bioactive peptide from marine product |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310618049.9A CN103805663A (en) | 2013-11-27 | 2013-11-27 | Method for separating, purifying and extracting bioactive peptide from marine product |
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| Publication Number | Publication Date |
|---|---|
| CN103805663A true CN103805663A (en) | 2014-05-21 |
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|---|---|---|---|
| CN201310618049.9A Pending CN103805663A (en) | 2013-11-27 | 2013-11-27 | Method for separating, purifying and extracting bioactive peptide from marine product |
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| Country | Link |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755268A (en) * | 2017-03-22 | 2017-05-31 | 中国科学院海洋研究所 | The method that in-vitro screening has the oyster active peptides for improving memory |
| WO2021082312A1 (en) * | 2019-10-29 | 2021-05-06 | 中国食品发酵工业研究院有限公司 | Oyster peptide having sexual function improving effect and preparation method therefor |
| CN113337565A (en) * | 2021-06-22 | 2021-09-03 | 中国科学院南海海洋研究所 | Marine biological active peptide with obvious skin sunburn protection effect and preparation method and application thereof |
-
2013
- 2013-11-27 CN CN201310618049.9A patent/CN103805663A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755268A (en) * | 2017-03-22 | 2017-05-31 | 中国科学院海洋研究所 | The method that in-vitro screening has the oyster active peptides for improving memory |
| CN106755268B (en) * | 2017-03-22 | 2020-07-17 | 中国科学院海洋研究所 | Method for in vitro screening oyster active peptide with function of improving memory |
| WO2021082312A1 (en) * | 2019-10-29 | 2021-05-06 | 中国食品发酵工业研究院有限公司 | Oyster peptide having sexual function improving effect and preparation method therefor |
| CN113337565A (en) * | 2021-06-22 | 2021-09-03 | 中国科学院南海海洋研究所 | Marine biological active peptide with obvious skin sunburn protection effect and preparation method and application thereof |
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| C06 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140521 |