CN106434808A - Extraction method of small molecular sipunculid polypeptide - Google Patents
Extraction method of small molecular sipunculid polypeptide Download PDFInfo
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- CN106434808A CN106434808A CN201610897988.5A CN201610897988A CN106434808A CN 106434808 A CN106434808 A CN 106434808A CN 201610897988 A CN201610897988 A CN 201610897988A CN 106434808 A CN106434808 A CN 106434808A
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- Prior art keywords
- sipunculoidea
- sipunculid
- sipunculid polypeptide
- polypeptide
- small molecular
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
Abstract
The invention discloses an extraction method of small molecular sipunculid polypeptide. The method comprises the steps that a biological enzymolysis technology and a membrane technology are integrated scientifically and reasonably, biological enzymolysis is conducted on raw materials with alkaline protease, separation, concentration and purification are conducted through a ultrafiltration membrane and a nanofiltration membrane which have specific molecular weight cut-off in sequence, a concentrated solution containing the small molecular sipunculid polypeptide is obtained, spray drying is conducted on the concentrated solution finally, and a finished small molecular sipunculid polypeptide product is obtained. The extraction method is high in maneuverability, the extraction rate to the small molecular sipunculid polypeptide is 0.5%-3.9%, the prepared small molecular sipunculid polypeptide is high in purity, the biological activity of the small molecular sipunculid polypeptide can be effectively guaranteed, and the extracted small molecular sipunculid polypeptide can serve as the raw material of various high-value-added products subsequently and has wide market space and economic value.
Description
Technical field
The present invention relates to a kind of extracting method of marine product small molecular polypeptide, specifically a kind of small molecule sipunculid polypeptide
Extracting method.
Background technology
With the popularization for developing rapidly with nutrient knowledge of science and technology, and the increase of per capita income level and level of consumption
Improve, the mankind have been transitioned into trophic level from having enough to eat and wear for the requirement of food, natural, healthy, nutrition, safety have become as
The main themes of 21 century development of food industry.Collagen protein as a kind of important bio-tissue structural protein polypeptide, more
To get over the concern for causing people and interest, market potential is huge, and economic worth is higher.Compared with macro-molecular protein, collagen egg
White micromolecule active polypeptide not only has synthesis low cost, utilization rate height, toxicity and the low feature of immunogenicity, also has efficiently
Antioxidation, the pharmacological action such as antibacterial and anticancer, be widely used in medicine, aesthetic health care and food service industry.
Biological species in ocean are various, and resource is extremely enriched, and biological chain perfects substantially, and regeneration capacity is strong.Ocean
The biological protein containing the abundant health that is beneficial to man, unsaturated fatty acid, taurine, its trace element pedigree is also significantly
Better than terrestrial life, it is the fabulous source of human health food.Marine environment is very different with terrestrial environment, such as high pressure,
Low temperature, high temperature (submarine volcano mouth and near) and high salt etc..In order to be suitable for marine environment, marine protein matter no matter aminoacid
Composition or the sequence of aminoacid is all made a big difference with terrestrial life protein.Meanwhile, marine protein resource is no
By being all far longer than land protein resource in species or quantity.Miscellaneous protein amino acid sequence, with special
Protease hydrolysiss, can release the Functional Polypeptides with physiological function.Therefore, using modern separation technology, these resources are opened
Send out and utilize, will be one of current scientific research field most with prospects.
From different material extract polypeptide traditional handicraft can be divided mainly into five classes, i.e. acid system, alkaline process, enzyme process, salt method with
And Hot water extraction, its ultimate principle is all the characteristic according to polypeptide, changes the external environment of protein, polypeptide from other eggs
Separate in white matter.Acid system and alkaline process are the methods for extracting polypeptide under acid and alkaline environment respectively, and acid system exists
Apply in the extraction process of polypeptide than wide, and alkaline process report is little；Salt method be by raw material in certain density saline solution
Extract, the salt for mainly using has Sodium Chloride, potassium chloride and sodium acetate.Although three of the above method process is simple, cost
Relatively low, but due to needing in extraction process to belong to the extracting method of high energy consumption using substantial amounts of fresh water and acid-alkali salt, discharge
Acid-alkali salt can be had a negative impact to ecological environment around, do not meet green economy and sustainable development theory.And hot water leaching
Lifting manipulation needs not meeting the main flow of country and development of world economy using substantial amounts of fresh water and the energy.Biological enzyme is to utilize enzyme
The method that under certain environmental conditions polypeptide lifted out from different raw materials of gentle high efficiency of reaction, can overcome on
The deficiency of other techniques is stated, is a kind of technique of upgrading.
Genus Golfingia (Sipuncula) is the common name of all vermiform oceanic invertebrates.Sipunculoidea is also known as sandworm, soil
Radix Crotalariae szemoensis, mud fourth.Sipunculoidea fossil is found in the Middle cambrian epoch.Genus Golfingia is known about 320 kinds, is divided into：2 guiding principle, 4 mesh, 6 section 17 belongs to：Leather bag
Sipunculida (Phascolosomida), including shield pipe Sipunculoidea mesh (Aspidosi-phoniformes), leather bag Sipunculoidea mesh
(Phascolosomaformes)；Sipunculus nudus guiding principle (Sipunculida), including dagger-axe sweet smell Sipunculoidea mesh (Golfingiaformes),
Sipunculus nudus (Sipunculiformes).Sipunculoidea flavour deliciousness, unique flavor, and larger in southern coastal yield, since ancient times
Just eaten by coastal resident, its taste cool in nature is trembled with fear, with YIN nourishing, the kidney invigorating, reduce internal heat, dietary function that clearing away lung-heat, eliminating the phlegm are coughed, claimed
For " animal Radix Ginseng ", the succedaneum as Cordyceps among the people.Phascolosoma (determining Sipunculoidea also known as Sipunculus nudus, sea) contains
More than 22 kinds of fatty acid, wherein content of highly unsaturated fatty acids account for total fatty acids 45.11%, arachidonic acid (AA) content
Up to 20.11%, DHA and EPA content are relatively enriched；Rich in 18 kinds of aminoacid, comprising all of essential amino acids of the mankind, its
Middle arginine content highest.
At present for the edible of Sipunculoidea, based on directly eating, and the high value added product with Sipunculoidea as raw material is less, industry
The effective method for small molecule sipunculid polypeptide extracted inside not yet is developed, and small molecule sipunculid polypeptide is extracted in industrialization,
In technology and application or blank.And for small molecule sipunculid polypeptide extraction exploitation China coast is enriched halobiontic
Develop significant.
Content of the invention
The technical problem to be solved is：A kind of extracting method of small molecule sipunculid polypeptide, the extraction side are provided
Method is workable, and its extraction ratio to small molecule sipunculid polypeptide is 0.5～3.9%, the small molecule sipunculid polypeptide for preparing
Purity height, can effectively ensure that the biological activity of small molecule sipunculid polypeptide.
The present invention solves the technical scheme that adopted of above-mentioned technical problem：A kind of extraction side of small molecule sipunculid polypeptide
Method, comprises the following steps：
(1) slightly wash：With fresh Sipunculoidea as raw material, most surface silt is washed with clear water；
(2) internal organs are removed：The Sipunculoidea for washing most surface silt is placed on smooth concrete floor, with 20 centimetres of width, diameter 60
Centimetre roundstone mill suppress Sipunculoidea repeatedly, until Sipunculoidea internal organs and silt go out to the greatest extent；
(3) clean：With tap water, the Sipunculoidea for going after internal organs is cleaned；
(4) remove the peel：With long 10 centimetres, wide 5 centimetres scraper, Sipunculoidea epidermis is scraped off；
(5) epidermis is removed：The Sipunculoidea epidermis for scraping is cleaned with tap water；
(6) digest：By the Sipunculoidea after clean epidermis and tap water or pure water with 1:3～10 weight ratio adds to enzyme
In solution tank, and alkaline protease is added in biological enzymolysis tank, the addition of alkaline protease is the Sipunculoidea weight after cleaning epidermis
The 0.5～5% of amount, is digested 3～10 hours with 40～60 DEG C of temperature, obtains enzymatic hydrolysate；
(7) separate：By the enzymatic hydrolysate Ultra filtration membrane of 5000～100000 dalton of aperture, clear liquid is removed；
(8) concentrate：Lower clear liquid to obtaining in step (7) is less than the nanofiltration membrance concentration of 80 dalton with aperture, takes supernatant
Liquid；
(9) dry：The supernatant for obtaining in step (8) is dried using spray dryer or freeze dryer, that is, is obtained
Small molecule sipunculid polypeptide finished product.
Preferably, described fresh Sipunculoidea is Sipunculus nudus.
Preferably, the inlet temperature that described spray dryer dries is 125～148 DEG C, described freeze dryer dries
Temperature be subzero 60 DEG C.
Compared with prior art, it is an advantage of the current invention that：A kind of extraction of small molecule sipunculid polypeptide that the present invention is provided
Method, biological enzymolysis technology and membrane technology is carried out scientific and reasonable integration, carries out enzyme by alkaline protease to raw material
Solution, then successively through ultrafilter membrane and the nanofiltration membrane separation concentration purification of specific molecular cut off, obtain many containing small molecule Sipunculoidea
The concentrated solution of peptide, is finally spray-dried to the concentrated solution, that is, obtain small molecule sipunculid polypeptide finished product.Extracting method of the present invention
Workable, its extraction ratio to small molecule sipunculid polypeptide is 0.5～3.9%, the small molecule sipunculid polypeptide for preparing
Purity height, can effectively ensure that the biological activity of small molecule sipunculid polypeptide, subsequently can have as the raw material of various high value added products
There are the wide market space and economic worth.
Description of the drawings
Fig. 1 is the chromatogram of the small molecule sipunculid polypeptide of embodiment 1.
Specific embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
The extracting method of the small molecule sipunculid polypeptide of embodiment 1, comprises the following steps：
(1) slightly wash：With fresh Sipunculus nudus as raw material, most surface silt is washed with clear water；
(2) internal organs are removed：The Sipunculoidea for washing most surface silt is placed on smooth concrete floor, with 20 centimetres of width, diameter 60
Centimetre roundstone mill suppress Sipunculoidea repeatedly, until Sipunculoidea internal organs and silt go out to the greatest extent；
(3) clean：With tap water, the Sipunculoidea for going after internal organs is cleaned；
(4) remove the peel：With long 10 centimetres, wide 5 centimetres scraper, Sipunculoidea epidermis is scraped off；
(5) epidermis is removed：The Sipunculoidea epidermis for scraping is cleaned with tap water；
(6) digest：By the Sipunculoidea after clean epidermis and tap water or pure water with 1:4 weight ratio adds to biological enzymolysis tank
In, and alkaline protease is added in biological enzymolysis tank, the addition of alkaline protease is the Sipunculoidea weight after cleaning epidermis
0.5%, digested 8 hours with 60 DEG C of temperature, obtain enzymatic hydrolysate；
(7) separate：By the enzymatic hydrolysate Ultra filtration membrane of 5000～100000 dalton of aperture, clear liquid is removed；
(8) concentrate：Lower clear liquid to obtaining in step (7) is less than the nanofiltration membrance concentration of 80 dalton with aperture, takes supernatant
Liquid；
(9) dry：The supernatant for obtaining in step (8) is dried using spray dryer, that is, obtains embodiment 1
Small molecule sipunculid polypeptide finished product.After testing, the extraction ratio of the small molecule sipunculid polypeptide of embodiment 1 is 3.9%.
The extracting method of the small molecule sipunculid polypeptide of embodiment 2, comprises the following steps：
(1) slightly wash：With fresh Sipunculus nudus as raw material, most surface silt is washed with clear water；
(2) internal organs are removed：The Sipunculoidea for washing most surface silt is placed on smooth concrete floor, with 20 centimetres of width, diameter 60
Centimetre roundstone mill suppress Sipunculoidea repeatedly, until Sipunculoidea internal organs and silt go out to the greatest extent；
(3) clean：With tap water, the Sipunculoidea for going after internal organs is cleaned；
(4) remove the peel：With long 10 centimetres, wide 5 centimetres scraper, Sipunculoidea epidermis is scraped off；
(5) epidermis is removed：The Sipunculoidea epidermis for scraping is cleaned with tap water；
(6) digest：By the Sipunculoidea after clean epidermis and tap water or pure water with 1:9 weight ratio adds to biological enzymolysis tank
In, and alkaline protease is added in biological enzymolysis tank, the addition of alkaline protease is the Sipunculoidea weight after cleaning epidermis
0.2%, digested 4 hours with 50 DEG C of temperature, obtain enzymatic hydrolysate；
(7) separate：By the enzymatic hydrolysate Ultra filtration membrane of 5000～100000 dalton of aperture, clear liquid is removed；
(8) concentrate：Lower clear liquid to obtaining in step (7) is less than the nanofiltration membrance concentration of 80 dalton with aperture, takes supernatant
Liquid；
(9) dry：The supernatant for obtaining in step (8) is dried using freeze dryer, that is, obtains little point of embodiment 2
Sub- sipunculid polypeptide finished product.After testing, the extraction ratio of the small molecule sipunculid polypeptide of embodiment 1 is 3.8%.
The extracting method of the small molecule sipunculid polypeptide of embodiment 3, comprises the following steps：
(1) slightly wash：With fresh Sipunculus nudus as raw material, most surface silt is washed with clear water；
(2) internal organs are removed：The Sipunculoidea for washing most surface silt is placed on smooth concrete floor, with 20 centimetres of width, diameter 60
Centimetre roundstone mill suppress Sipunculoidea repeatedly, until Sipunculoidea internal organs and silt go out to the greatest extent；
(3) clean：With tap water, the Sipunculoidea for going after internal organs is cleaned；
(4) remove the peel：With long 10 centimetres, wide 5 centimetres scraper, Sipunculoidea epidermis is scraped off；
(5) epidermis is removed：The Sipunculoidea epidermis for scraping is cleaned with tap water；
(6) digest：By the Sipunculoidea after clean epidermis and tap water or pure water with 1:6 weight ratio adds to biological enzymolysis tank
In, and alkaline protease is added in biological enzymolysis tank, the addition of alkaline protease is the Sipunculoidea weight after cleaning epidermis
1.5%, digested 10 hours with 48 DEG C of temperature, obtain enzymatic hydrolysate；
(7) separate：By the enzymatic hydrolysate Ultra filtration membrane of 5000～100000 dalton of aperture, clear liquid is removed；
(8) concentrate：Lower clear liquid to obtaining in step (7) is less than the nanofiltration membrance concentration of 80 dalton with aperture, takes supernatant
Liquid；
(9) dry：The supernatant for obtaining in step (8) is dried using spray dryer, that is, obtains embodiment 3
Small molecule sipunculid polypeptide finished product.After testing, the extraction ratio of the small molecule sipunculid polypeptide of embodiment 1 is 3.4%.
In embodiments above, dry using spray dryer or freeze dryer, the inlet temperature that spray dryer dries
For 125～148 DEG C, the temperature that freeze dryer dries is subzero 60 DEG C.
For the small molecule sipunculid polypeptide finished product of embodiment 1, by high performance liquid chromatograph and according to GB/T 22729-
2008《Ocean fish oligopeptide powder》Detection peptide molecular weight distribution, the chromatogram for obtaining is shown in Fig. 1, and molecular weight detection the results are shown in Table 1.
The molecular weight detection result of the small molecule sipunculid polypeptide finished product of 1 embodiment 1 of table
As seen from Table 1, in the small molecule sipunculid polypeptide finished product of embodiment 1, molecular weight is less than the polypeptide of 2000 dalton
Weight ratio is 99.35%.
Claims (3)
1. a kind of extracting method of small molecule sipunculid polypeptide, it is characterised in that comprise the following steps：
（1）Slightly wash：With fresh Sipunculoidea as raw material, most surface silt is washed with clear water；
（2）Remove internal organs：The Sipunculoidea for washing most surface silt is placed on smooth concrete floor, with 20 centimetres of width, 60 centimetres of diameter
Roundstone mill suppress Sipunculoidea repeatedly, until Sipunculoidea internal organs and silt go out to the greatest extent；
（3）Cleaning：With tap water, the Sipunculoidea for going after internal organs is cleaned；
（4）Peeling：With long 10 centimetres, wide 5 centimetres scraper, Sipunculoidea epidermis is scraped off；
（5）Epidermis is removed：The Sipunculoidea epidermis for scraping is cleaned with tap water；
（6）Enzymolysis：By the Sipunculoidea after clean epidermis and tap water or pure water with 1:3～10 weight ratio adds to biological enzymolysis tank
In, and alkaline protease is added in biological enzymolysis tank, the addition of alkaline protease is the Sipunculoidea weight after cleaning epidermis
0.5～5 %, is digested 3～10 hours with 40～60 DEG C of temperature, obtains enzymatic hydrolysate；
（7）Separate：By the enzymatic hydrolysate Ultra filtration membrane of 5000～100000 dalton of aperture, clear liquid is removed；
（8）Concentrate：To step（7）In the lower clear liquid that obtains with aperture less than the nanofiltration membrance concentration of 80 dalton, take supernatant；
（9）Dry：To step（8）In the supernatant that obtains be dried using spray dryer or freeze dryer, that is, obtain little point
Sub- sipunculid polypeptide finished product.
2. the extracting method of a kind of small molecule sipunculid polypeptide according to claim 1, it is characterised in that described is fresh
Sipunculoidea is Sipunculus nudus.
3. a kind of extracting method of small molecule sipunculid polypeptide according to claim 1 and 2, it is characterised in that described spraying
The inlet temperature that drying machine dries is 125～148 DEG C, and the temperature that described freeze dryer dries is subzero 60 DEG C.
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| CN201610897988.5A CN106434808A (en) | 2016-10-14 | 2016-10-14 | Extraction method of small molecular sipunculid polypeptide |
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| CN201610897988.5A CN106434808A (en) | 2016-10-14 | 2016-10-14 | Extraction method of small molecular sipunculid polypeptide |
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| CN201610897988.5A Withdrawn CN106434808A (en) | 2016-10-14 | 2016-10-14 | Extraction method of small molecular sipunculid polypeptide |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108887634A (en) * | 2018-06-28 | 2018-11-27 | 钦州学院 | Sandworm nutritive soup blend packet and preparation method thereof |
| CN109938349A (en) * | 2019-03-29 | 2019-06-28 | 宁波御坊堂生物科技有限公司 | A kind of oligomeric peptide functional food and preparation method thereof |
| CN114027427A (en) * | 2021-11-26 | 2022-02-11 | 中国热带农业科学院农产品加工研究所 | Sipunculid collagen antioxidant peptide food and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101210261A (en) * | 2006-12-29 | 2008-07-02 | 宁波大学 | Method for preparing sipunculid polypeptide and application thereof |
| CN102336806A (en) * | 2011-09-23 | 2012-02-01 | 华东理工大学 | Angiotensin converting enzyme (ACE) inhibitor |
| CN104473185A (en) * | 2014-12-27 | 2015-04-01 | 广西壮族自治区海洋研究所 | Effervescent tablet containing vitamin C and sipunculusnudus enzymatic protein |
-
2016
- 2016-10-14 CN CN201610897988.5A patent/CN106434808A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101210261A (en) * | 2006-12-29 | 2008-07-02 | 宁波大学 | Method for preparing sipunculid polypeptide and application thereof |
| CN102336806A (en) * | 2011-09-23 | 2012-02-01 | 华东理工大学 | Angiotensin converting enzyme (ACE) inhibitor |
| CN104473185A (en) * | 2014-12-27 | 2015-04-01 | 广西壮族自治区海洋研究所 | Effervescent tablet containing vitamin C and sipunculusnudus enzymatic protein |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108887634A (en) * | 2018-06-28 | 2018-11-27 | 钦州学院 | Sandworm nutritive soup blend packet and preparation method thereof |
| CN109938349A (en) * | 2019-03-29 | 2019-06-28 | 宁波御坊堂生物科技有限公司 | A kind of oligomeric peptide functional food and preparation method thereof |
| CN114027427A (en) * | 2021-11-26 | 2022-02-11 | 中国热带农业科学院农产品加工研究所 | Sipunculid collagen antioxidant peptide food and preparation method thereof |
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