CN103184262A - Method for extracting and purifying pepsin-soluble collagens of black sea cucumber in East China Sea - Google Patents

Abstract

The invention provides a method for extracting and purifying pepsin-soluble collagens of black sea cucumbers in East China Sea. The method comprises the following steps: soaking a clean, massive black sea cucumber sample in East China Sea with an EDTA (Ethylene Diamine Tetraacetic Acid) solution; performing homogenized homogeneity; performing extraction in sequence by using a Tris-HCl solution, a NaOH solution and a pepsase acetic acid solution; and performing salting out and dialysis to obtain the pepsin-soluble collagens. According to the invention, the EDTA chelating agent is used to remove chemical element with high pollution concentration from sea cucumber tissues, and the removal efficiency can reach more than 90%, a crosslinking effect in chemical bonds and among the chemical bonds of a collagen albumen product can be effectively removed after pepsase is used for performing hydrolysis on collagen fibers, so that maximally extracting the solvation collagen albumen can be realized; after the purification steps of sodium chloride salting out and dialysis, and the like are carried out, and the purity of the obtained collagen albumen product by extraction can reach more than 90%, the yield can reach 56.99-61.52% (dry weight), which is 97.1-102.5% of the total collagen albumen content of the black sea cucumber in East China Sea, so that the extraction of collagen albumen content with high productivity of the black sea cucumber in East China Sea can be realized.

Description

The extracting and purifying method of a kind of East Sea crow ginseng enzyme dissolubility collagen protein

(1) technical field

The present invention relates to the extracting and purifying method of a kind of East Sea crow ginseng enzyme dissolubility collagen protein.

(2) background technology

Collagen protein is rich in amino acid such as glycine that human body needs, L-Ala, arginine, proline(Pro).Collagen protein is most important component in the extracellular matrix, it is a kind of extracellular protein, collagen protein is that 3 peptide chains twist into spiral fibrous protein, peptide chain constitutes (X: proline(Pro) by a large amount of repetition Gly-X-Y-sequences, Y: oxyproline), 15～20 amino-acid residues of end peptide (C-and N-end) do not participate in the triple helix structure, most of residues are Methionin and hydroxylysine and aldehyde radical derivative composition thereof, form intermolecular and minute in covalent crosslink, the triple helix structure relies in the peptide chain and the hydrogen bond of interchain is kept.

Collagen protein is the rich in protein of people's in-vivo content, accounts for 1/3 of whole body gross protein.Mainly be present in human body skin, bone, eyes, tooth, tendon, internal organ positions such as (comprising the heart, stomach, intestines, blood vessel), its function is to keep form and the structure of skin and histoorgan, also is the important source material material of repairing each damaged tissue.In the human body skin composition, formed by collagen protein by 70%.Extensively there is the I collagen type in nature, as Mammalss such as ox-hide, pigskin, fish, mollusk etc., extracting the collagen protein kind that obtains according to different tissues and position is divided three classes: be respectively (1) interstitial collagen I-III type, mainly be present in the tissues such as skin and tendon, it is the collagen protein between the cell, very strong anti-opening property is arranged, and wherein the II Collagen Type VI is produced by the chondrocyte.(2) IV-VII type lac gland is arranged is basement membrane collagen to basement membrane collagen, mainly is present in the middle of the internal organs.(3) the original IX of chondrin-XI type is that micro-collagen forms relevant with cartilage in the cartilage.Collagen protein is the major ingredient of tissue, and it and each organ-tissue of human body and cell have indivisible relation, so be applied to repairing and the regeneration of human organ tissue.Collagen protein mainly is various function collagen biomaterials aspect medical use, comprises collagen protein sponge, sericterium, film (surgical hemostasis; be used for cardiovascular, nerve, oral cavity, orthopaedics; skin, the woman produces operation etc.) wound dressings, artificial skin, blood vessel; heart valve, the cornea protecting materials, the injecting type collagen protein (is used for smoothing wrinkle; soft tissue is plentiful to be filled up, the treatment urinary incontinence, and urine refluxes; orthopaedics tissue regeneration filler), medicine auxiliary mechanism, purposes such as collagen matrices template.Functions such as the application moisture-keeping crease-shedding of collagen protein aspect makeup and beauty and health care is anti-oxidant, collagen protein is to keep skin consolidation and elastic major ingredient.Collagen protein also adds use in the health care of food product in addition.

A lot of products about collagen protein and collagen peptide or protein powder are arranged in the market, and the human consumer can't distinguish function and the effect of product, therefore is necessary from concept and application differentiation three's relation.Collagen peptide is a class by collagen protein or to contain the relatively abundanter product of collagen protein be the prepared using biological enzyme micromolecule polypeptide mixture that hydrolysis is made under certain temperature and pH condition, form with plural amino acid by two, different according to hydrolysising condition and hydrolysis degree, the amino acid of polypeptide is formed and molecular weight ranges is the principal element that determines function, and polypeptide can directly be absorbed by the body.Protein powder is formed with upper amino acid by two or two, be generally the product that contains higher protein content and pass through to add the biocatalysis zymin polypeptide mixture of hydrolysis generation under proper condition, more protein powder derives from soybean protein powder, lactalbumin powder etc. at present.Collagen protein is the material that a class has the single albumen composition of special construction and unique biological applications, needs the protein that obtains through separation and purification.Collagen protein can be used as biomedical subsidiary material, is product irreplaceable function biomaterials such as general collagen peptide or protein powder.

The natural marine product---sea cucumber and health-product market occupation rate thereof sharply rise in recent years, have a extensive future, since 2000, the output value of China sea cucumber begins to present annual 30% the speed that increases, had a surplus in 10 years up till now, the breed of sea cucumber industry and production area are based on Dalian and marine site, Shandong.Breed, processing and sales enterprise surpass 500 families, and annual value of production surpasses 20,000,000,000 yuan, have become the kind of single product output value maximum in China's fishing resources.East Sea crow ginseng (Acaudina Leucoprocta) is the important class in numerous sea cucumber kinds, coastland, the Zhejiang East Sea crow ginseng resource that is richly stored with, only Xiangshan, Zhejiang reaches 100,000 tons of scales once a band year amount of fishing, calculate according to conservative, 100,000 tons of East Sea crow ginsengs can produce 10,000,000,000 output values, therefore excavate East Sea crow ginseng resource, the relevant healthcare product of deep development will produce significance to the development of Zhejiang marine economy.Show according to Chinese biological collagen protein in 2011 market analysis and investment trend study report, Chinese collagen protein was consumed about 10000 tons in 2011, expect 2015 annual requirements and be expected to rise to 20000 tons, annual compound growth rate reaches 25%, be higher than the average amplification in the whole world, so the market potential of collagen protein is very considerable.Existing collagen protein is mainly derived from Mammalss such as ox-hide, pigskin, because the discovery of Animal diseases such as mad cow disease, foot and mouth disease, and the influence of factors such as animal source allergin and religious tradition, be badly in need of seeking alternative collagen protein source, marine prods is the maximum alternative resource of Recent study, extract purified product as the collagen protein in the tissues such as fish, mollusc, sea cucumbers, but much report and research only limits to the laboratory, can't realize large-scale process for processing, output is few, expensive, do not have the market competitiveness.

Sea cucumber is a kind of high protein, lower fat, does not contain cholesterol, is rich in sea cucumber saponin, polysaccharide isoreactivity material, is, usually is described as natural marine resource first of " marine genseng " and " eight delicacies " with functions such as nourishing and fit keeping function and medicinal healths.Collagen protein in the sea cucumber is very high, accounts for 70% of sea cucumber body total protein.The whole world has more than 1400 kind of sea cucumber approximately, and kind surplus China marine site has 100, sea cucumber kind that at present can be edible have kinds more than 20 such as Thelenota ananas (Jaeger), stichopus japonicus, black ginseng, beche-de-mer without spike, melon ginseng, beautiful foot ginseng, East Sea Wu Can.

East Sea crow ginseng (Acaudina leucoprocta): the white anus of formal name used at school Haiti melon is a kind of Holothuroidea animal of Echinodermata (Echinodermata), Holothuroidea (Holothuroidea), Molpadida (Molpadida), buttocks ginseng section (Caudinidae), Acaudina (Acaudina).Grow in THE DONGHAI SEA CONTINENTAL SHELF widely, to marine sites such as Hainan Island, Iranian in addition, also there are distribution, aboundresources in the Australian northwestward from the Zhejiang Zhoushan Islands.Because East Sea crow ginseng (Acaudina Leucoprocta) grows on the bottom silt all the year round, the mucus of its body surface secretion is combined securely with the bottom silt that contains the heavy metal deposition thing, formed fine and close hard body surface schmutzband, along with the pollution of seawater increasingly sharpens the content severe overweight of its body surface schmutzband harmful heavy metal such as mercury, arsenic, lead element.A large amount of metallic elements and fine and close hard tissue signature's problem of East Sea crow ginseng body surface accumulation, seriously limited the processing and utilization of the East Sea this high-quality resource of crow ginseng, especially difficult aspect the extraction of collagen protein, so East Sea crow ginseng is considered as the waste resource of low value always by local fisherman.The East Sea crow ginseng product of research and development high added value is significant to the deep processing field of low value marine prods, and the development and use for alternative resource simultaneously provide a wide application prospect.

The sea cucumber individual difference of different genera is very big, China the famousst and precious coastal sea cucumber is the Liao Dynasty's ginseng, the market price of Thelenota ananas (Jaeger) and stichopus japonicus is higher, the patent of at present relevant holothurian collagen has two, one is " sea cucumber collagen, the extracting method of collagen protein " sea cucumber raw material in (CN101215315B) is stichopus japonicus, technology to collagen and collagen protein is described, this technology is fit to easily steep the sea cucumber tissue of sending out with homogenate, but be not suitable for by the East Sea of seawater severe contamination crow ginseng, it organizes hard, be difficult to broken homogenate, heavy metal and all kinds of chemical element content exceed standard, the accumulating of a large amount of metallic elements in the crow ginseng tissue of the East Sea has a strong impact on extraction efficiency, and it is extremely low to extract the productive rate that East Sea crow joins collagen protein with this patented method, can't realize all extracting collagen protein, therefore and metal element content exceeds standard in the product, is necessary the technology in the patent is improved, to be suitable for the extraction of collagen and collagen protein in the crow ginseng tissue of the East Sea.

Another patent be " manufacture method of a kind of East Sea crow ginseng collagen protein " (CN102251006A), this patent is the collagen protein direct hydrolysis method of East Sea crow ginseng, and the extracting method of noncollagen protein, its product is small molecules collagen polypeptide powder, and the extractive technique of collagen protein yet there are no report in the East Sea crow ginseng at present.The collagen protein that other patent that sea cucumber is relevant or document all do not relate to East Sea crow ginseng kind extracts or makes, because THE DONGHAI SEA CONTINENTAL SHELF is being contained the East Sea crow ginseng of huge output, the pollution of metallic element causes it directly to eat in the seawater, belong to the low value oceanic resources, could use after need removing chemical element, therefore be necessary it is carried out the collagen protein product that deep processing is developed as high added value.

(3) summary of the invention

At the defective that exists in the prior art, the invention provides a kind of easy and simple to handlely, cost is low, the income height, environment amenable East Sea crow ginseng collagen protein extracts the method for purifying.

The technical solution used in the present invention is:

The extracting and purifying method of a kind of East Sea crow ginseng enzyme dissolubility collagen protein, described method comprises:

(1) gets clean block East Sea crow ginseng sample with the EDTA solution soaking of 0.2M, pH8.0 5～6 days, change EDTA solution every day; The desirable fresh East Sea of crow ginseng, the block East Sea of described cleaning crow ginseng is removed silt, internal organ with 4 ℃ of water flushings, and being cut into small pieces, (silt and internal organ also can will be removed with 4 ℃ of water flushings after the freezing stripping and slicing of East Sea crow ginseng again in 5～10mm * 5～10mm);

(2) after the bulk sample washing, add ice cube, carry out the homogenate homogeneous with the stainless steel refiner after, the centrifuging and taking throw out carries out next step operation;

(3) step (2) precipitation adds 10～20 times of volumes (liquor capacity milliliter number and the ratio that precipitates the quality grams, the Tris-HCl solution of down together) 0.1M, pH8.0, stirred 2～4 days, and fully dissolved tissue protein, centrifugal back taking precipitate carries out next step operation; Tris-HCl solution also can add 0.5M NaCl, and 4～50mM EDTA and 0.2M beta-mercaptoethanol also can not contain beta-mercaptoethanol, or NaCl and EDTA, extracts the result and does not have significant difference.This method can effectively be removed the chemical element of pollution, makes collegen filament fully dissolve expansion simultaneously, but does not influence structure and the character of collagen protein;

(4) the 0.1M NaOH solution stirring of 10～20 times of volumes of step (3) precipitation adding is 2～4 days, change NaOH solution every day, centrifugal, get the 0.1MNaOH that precipitation adds 10～20 times of volumes again and stir 24h, centrifugal, get the precipitation wash with water to neutrality, the centrifuging and taking throw out carries out next step operation, perhaps this throw out is washed to the directly freeze-drying of neutral back, obtains thick collegen filament and carry out next step enzymolysis operation; This step can be removed non-Collagen material and be removed sea cucumber endogenous biological enzyme activity, can effectively remove non-Collagen materials such as saponin, polysaccharide, foreign protein, improves the productive rate of collagen protein, plays the Degradation that suppresses biologically active substance in the sea cucumber body simultaneously;

(5) step (4) gained precipitation (or thick collegen filament) adds the 0.5M acetic acid solution extraction 2～4 days that capacity (20 times more than the volume) contains 300w～800w U/L stomach en-(pig stomach mucous membrane source), the centrifugal supernatant liquor 1 that obtains, throw out adds capacity again and contains the pepsic 0.5M acetic acid solution extraction of 300w～800w U/L 24h, the centrifugal supernatant liquor 2 that obtains; The stomach en-enzymolysis and extraction collegen filament that thick collegen filament are originated with the pig stomach mucous membrane, can effectively weaken and remove the mutual cross action of the intermolecular chemical bond of collegen filament, the insoluble collagen fiber fully is dissolved in the acidic solution, insoluble collegen filament is changed into the collagen protein of solubility.

(6) merge supernatant liquor 1 and supernatant liquor 2, adding 4M NaCl to NaCl final concentration is 0.8M, saltouts, leave standstill 1～2h after, centrifugal, taking precipitate;

(7) step (6) gained throw out adds the 0.5M acetic acid solution dissolving of 9～10 times of volumes, dialyses with the dialysis membrane of molecular weight cut-off 7000Da, uses the Na of 0.02M, pH8.0 earlier ₂HPO ₄Solution dialysis 48h changes 0.1M acetic acid solution dialysis 48h, changes deionized water dialysis 48h again, and the product freeze-drying is described enzyme dissolubility collagen protein;

Saltout and the dialysis membrane dialysis with NaCl, effective collagen purification protein product, through protein electrophoresis and gel chromatography chromatographic determination, do not contain foreign protein in the collagen protein product, through determination and analysis such as amino acid, metal element content, productive rates, the extraction purification technique of this method can obtain high yield and highly purified collagen protein product, is can scale operation and abundant alternative Mammals source sea-food high-quality collagen protein resource.

All carry out under 4 ℃ above-mentioned steps (1)～(7).

Preferably, also be added with in step (3) the Tris-HCl solution: 0.5M NaCl, 4～50mMEDTA and 0.2M beta-mercaptoethanol.

Beneficial effect of the present invention is mainly reflected in: adopt the EDTA sequestrant to remove the chemical element of high pollution concentration in the sea cucumber tissue, removal efficiency reaches more than 90%, collagen protein product behind the pepsin hydrolysis collegen filament has effectively been removed in the chemical bond, cross-linked effect between key, can extract the dissolving collagen protein to greatest extent, saltout through sodium-chlor, purification steps such as dialysis, the protein electrophoresis analysis of collagen protein product and gel chromatography chromatography chromatographic determination, the collagen protein product purity that extraction obtains reaches more than 90%, productive rate is 56.99%～61.52%(dry weight), account for 97.1%～102.5% of total collagen content in the crow ginseng albumen of the East Sea, realized that high yield extracts the technology of collagen content in the crow ginseng of the East Sea.

(4) description of drawings

Fig. 1 is the SDS-PAGE spectrogram of embodiment 3 collagen proteins, PSC: East Sea crow ginseng enzyme dissolubility collagen protein, M: molecular weight marker thing, CSC: cow leather collagen standard reference material.

(5) embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1:

1. the black ginseng in the fresh East Sea is (commercial, originating from Xiangshan, Zhejiang one band) sample washes with 4 ℃ of tap water, after removing silt, internal organ, 5mm*5mm is cut into small pieces, get 200g bulk sample 1000mL0.2M EDTA(pH8.0) soaking and stirring 5 days, change solution every day, remove pollution element and residual impurities such as calcium, iron, manganese, magnesium, lead.

2. after the washing, add ice cube 100g(solution temperature be down to below 4 ℃), with stainless steel refiner (IKA T25Homogenizer) homogenate homogeneous, 18000rpm, 20min, centrifugal back taking precipitate A.

3. throw out A adds the 0.1M Tris-HCl(pH8.0 of 20 times of volumes (solution 20mL/1g precipitation)), stir 48h, fully dissolve tissue protein, centrifugal back taking precipitate B.

4. sediment B adds the 0.1M NaOH stirring 48h of 20 times of volumes, changes solution every day, removes non-Collagen material and removes the endogenous biological enzyme activity, the centrifugal 30min of 12000G.Abandoning supernatant, throw out C stirs 24h with the 0.1M NaOH of 20 times of volumes again, and the throw out after centrifugal washes to neutrality the centrifuging and taking sediment D with water.

Sediment D add 20 times of volumes contain the 0.5g/L stomach en-(the Sigma stomach en-, 0.5M acetic acid solution 600-1200units/mg) extracted 3 days, the centrifugal 30min of 12000G, get supernatant liquor 1, throw out extracts 24h again, and the centrifugal 30min of 12000G gets supernatant liquor 2.

6. merge twice supernatant solution 1 and 2, add 4M NaCl to solution NaCl final concentration 0.8M, saltout, leave standstill 1h after, the centrifugal 20min of 12000G, taking precipitate E.

7. throw out E dissolves with the 0.5M acetic acid solution of 9 times of volumes (9mL solution/1g precipitation), dialysis membrane (molecular weight cut-off: 7000Da) dialysis, the Na of 0.02M, pH8.0 of elder generation ₂HPO ₄Solution dialysis 48h changes 0.1M acetic acid solution dialysis 48h, changes deionized water dialysis 48h again, and the product freeze-drying is enzyme dissolubility collagen protein.

Embodiment 2:

1. fresh East Sea crow ginseng sample directly is cut into particle (size is 5mm*5mm) after freezing, clean stirring three times with frozen water, after removing impurity such as silt, internal organ, get 200g bulk sample 2000mL0.2M EDTA(pH8.0) soaking and stirring 5 days, change solution every day, remove pollution elements such as calcium, iron, manganese, magnesium, lead.

2. after the washing, add ice cube 200g(solution temperature be down to below 4 ℃), with stainless steel refiner (IKA T25Homogenizer), 18000rpm homogenate 20min, centrifugal back taking precipitate A.

3. throw out A adds 0.1M, the pH8.0Tris-HCl+0.5M NaCl+50mMEDTA+0.2M beta-mercaptoethanol of 20 times of volumes, stirs and extracts 72h, centrifugal back taking precipitate B.

4. the 0.1M NaOH of 20 times of volumes of sediment B adding stirs 72h and changes solution every day, removes non-Collagen material and removes the endogenous biological enzyme activity, the centrifugal 30min of 12000G.Abandoning supernatant, throw out C is washed to neutrality, and (can add small amount of acetic acid and adjust pH), the product freeze-drying gets thick collegen filament.

5. the thick collegen filament of getting 5g dissolve with the 5L0.5M acetic acid solution, add 0.1g stomach en-(3200-4500units/mg) and stir extraction 72h, the centrifugal 30min of 12000G, sediment D is extracted 24h, the centrifugal 30min of 12000G with containing the pepsic 0.5M acetic acid solution of 0.1g/L again.

6. merge supernatant solution twice, add 4M NaCl to solution NaCl final concentration 0.8M, saltout, leave standstill 1h after, the centrifugal 20min of 12000G, taking precipitate E.

7. throw out E dissolves with the 0.5M acetic acid solution of 9 times of volumes, dialysis membrane (molecular weight cut-off: 7000Da) dialysis, the Na of 0.02M, pH8.0 of elder generation ₂PO ₄Solution dialysis 48h changes 0.1M acetic acid solution dialysis 48h, changes deionized water dialysis 48h again, and the product freeze-drying is enzyme dissolubility collagen protein.

Embodiment 3:

1. the black ginseng in the fresh East Sea is organized water ratio 83.61 ± 0.2%, ash content 3.9 ± 0.05%, protein 13.71 ± 0.1%, fat 0.9 ± 0.05%.The contents of heavy metal elements severe overweight, see the following form:

By the table in as seen, the content of As, Pb, the Cr 0.5mg/Kg of standard is head and shoulders above limited the quantity of.

2. the black ginseng in fresh East Sea epidermis is seriously polluted, be yellow, part is by serious rust red color, it organizes the hard homogenate that is difficult for, with organizing of-20 ℃ of freezing preservations is freezing be cut into small-particle after, behind deionized water wash, be immersed among the 0.2M EDTA of pH8.0, change solution every day, soak after 5 days, the decreasing ratio of metallic elements such as Ca, Fe, Mn, Mg, Zn, Sn is more than 90%, tissue is softening the expansion obviously, is easy to homogenate, adds frozen water, 18000rpm homogenate 20min, the centrifugal back of 12000G taking precipitate A.

3. throw out A uses the 0.1M Tris-HCl(pH8.0 of 20 times of volumes) dissolving stirring 72h, this step can make tissue particles thing expansion cracking, and collegen filament are fully dissolved.

4. centrifugal back taking precipitate B adds the 0.1M NaOH solution stirring 72h of 20 times of volumes, removes non-Collagen material, and suppresses the endogenous biological enzyme of East Sea crow ginseng, and is middle centrifugal and change solution, can effectively remove metallic element impurity.

5. centrifugal back taking precipitate C, be washed to neutrality, directly freeze-drying obtains thick collegen filament, with the thick collegen filament dry product of 0.5M acetic acid dissolving 1g of 1L volume, the stomach en-that adds 0.1g/L,, stirred enzymolysis 3 days, the centrifugal supernatant liquor that goes, sediment D homogenate adding again contains the pepsic 0.5M acetic acid solution extraction of 0.1g/L 24h, merges extracted twice liquid.

6. saltout: add 4M NaCl to strength of solution 0.8M, precipitated product is the transparence collagen protein, leaves standstill 2h, centrifuging and taking throw out E.

7. dialyse: throw out E adds the 0.5M acetic acid dissolving of 9 times of volumes, dialysis membrane (molecular weight cut-off: 7000Da) dialysis, the Na of 0.02M, pH8.0 of elder generation ₂PO ₄Solution dialysis 48h, again with 0.1M acetic acid dialysis 48h, with the deionized water 48h that dialyses, the product freeze-drying obtains enzyme dissolubility collagen protein again.

8. collagen content is 70% of total protein content in the East Sea crow ginseng, the extraction yield of collagen protein is 9.34%～10.08%(weight in wet base), being converted into dry weight is 56.99%～61.52%(butt), the collagen protein that is higher than in the huge Red sea ginseng (Parastichopus californicus) extracts yield 20.8%(epidermis) and the 24.3%(tissue).To be converted to collagen protein initial content (account for total protein 70%) be 97.1～102.5% to the extraction yield of enzyme dissolubility collagen protein in the crow ginseng of the East Sea, therefore the inventive method can realize extracting fully the collagen protein in the crow ginseng of the East Sea, uses this method to extract the collagen protein heavy metal content that obtains and has all reached national limit standard.

9. amino acidanalyser is measured 18 seed amino acids such as aspartic acid, glycine, L-Ala, L-glutamic acid, proline(Pro), oxyproline, Methionin, hydroxylysine in fresh sea cucumber tissue, the gained enzyme dissolubility collagen protein, and amino acid composition and the content with cow leather collagen, stichopus japonicus (Stichopus japanious) and huge Red sea ginseng (Parastichopus californicus) compares respectively.The content of proline(Pro) directly influences the content of collagen protein, and oxyproline is the important imido acid of keeping collagen structure, and the glycine in the sea-food and L-glutamic acid content are higher.Glycine accounts for 1/3rd of total amino acid in the crow ginseng of the fresh East Sea, the peptide chain of measurement result and collagen protein is matched by the character that the Gly-X-Y-sequence of a large amount of repetitions constitutes, 133 (Saito of 141.0 ± 2.9 (in 1000 amino-acid residues) a little higher than stichopus japonicus of proline(Pro) and hydroxyproline content (Stichopus japanious) wherein, M., Kunisaki, N., Urano, N. ， ﹠amp; Kimura, S. (2002) .Collagen as the major edible component of sea cucumber (Stichopus japonicus) .Journal of Food Science, 67 (4), 1319-1322.), proline(Pro) in the enzyme dissolubility collagen protein that extraction obtains and hydroxyproline content 160.6 ± 2.5 (in 1000 amino-acid residues) are higher than 153 of huge Red sea ginseng (Parastichopus californicus), and (Stichopus japanious) is similar with stichopus japonicus, but are lower than 215 and 220 (Park of pigskin of ox-hide, S.Y., Lim, H.K., Lee, S., Hwang, H.C., Cho, S.K. ， ﹠amp; Cho, M. (2012) .Pepsin-solubilised collagen (PSC) from Red Sea cucumber (Stichopus japonicus) regulates cell cycle and the fibronectin synthesis in HaCaT cell migration.Food Chemistry, 132 (1), 487-492.).The oxyproline of collagen extraction protein product accounts for 38.4% of proline(Pro) total amount in the crow ginseng of the East Sea, and oxyproline accounts for main effect (Bachinger, H.P., Morris, N.P. ， ﹠amp in keeping the triple helix structure of collagen protein; Davis, J.M. (1993) .Thermal-Stability And Folding Of the Collagen Triple Helix And the Effects Of Mutations In Osteogenesis Imperfecta on the Triple Helix Of Type-I Collagen.American Journal Of Medical Genetics, 45 (2), 152-162.), therefore the total amount comparing analysis result from oxyproline and proline(Pro) can infer, the stability of the collagen protein of East Sea crow ginseng may than mammiferous have slightly inferior, but similar with similar sea cucumber kind.Amino acid whose concrete measurement result is as shown in the table:

Median ± relative standard deviation of three sample determination results.

*（Ahmad,M.,Benjakul,S.,&Nalinanon,S.(2010).Compositional?and?physicochemical?characteristics?of?acid?solubilized?collagen?extracted?from?the?skin?of?unicorn?leatherjacket(Aluterus?monoceros).Food?Hydrocolloids,24(6-7),588-594.）；

**（Liu,Z.Y.,Oliveira,A.C.M.,&Su,Y.C.(2010).Purification?and?Characterization?of?Pepsin-Solubilized?Collagen?from?Skin?and?Connective?Tissue?of?Giant?Red?Sea?Cucumber(Parastichopus?californicus).Journal?of?Agricultural?and?Food?Chemistry,58(2),1270-1274.）；

***（Cui,F.X.,Xue,C.H.,Li,Z.J.,Zhang,Y.Q.,Dong,P.,Fu,X.Y.,&Gao,X.(2007).Characterization?and?subunit?composition?of?collagen?from?the?body?wall?of?sea?cucumber?Stichopus?japonicus.Food?Chemistry,100(3),1120-1125.）

10. collagen protein electrophoretogram map analysis, extract product enzyme dissolubility collagen protein product and have only a main band at 7.5%SDS-PAGE, the molecular weight size is 138kDa, compare with the cow leather collagen standard reference material, be α 1 chain, have two lighter bands to be respectively γ chain and β chain in addition, extracting product is a collagen type, the collagen protein product of joining (Parastichopus californicus) to stichopus japonicus with huge Red sea is similar, does not contain α ₂Chain is (α ₁) ₃The triple helix structure, kinds different in the Holothuroidea may contain similar collagen structure, because the difference of habitat, sea cucumber tissue characteristics and the meeting of amino acid The Nomenclature Composition and Structure of Complexes variant (Gomez-Guillen, M.C., Turnay, J., Fernandez-Diaz, M.D., Ulmo, N., Lizarbe, M.A. ， ﹠amp; Montero, P. (2002) .Structural and physical properties of gelatin extracted from different marine species:a comparative study.Food Hydrocolloids, 16 (1), 25-34.), as shown in Figure 1.

11. the viscosimetric analysis result of the enzyme dissolubility collagen protein extraction product in the crow ginseng of the East Sea shows that its denaturation temperature Td is 25.4 ℃ and is higher than 18.5 ℃ of huge Red sea ginseng (Parastichopus californicus) epidermis and organizes 17.9 ℃, is lower than 37 ℃ of cow leather collagen; Thick collegen filament in the crow ginseng of the East Sea easily are dissolved in acidic solution and contain pepsin solution, and after pepsic enzyme was cut modification, collagen protein can be discharged fully to be extracted, and enzyme dissolubility collagen protein optimal dissolution condition is pH2.66.Thick collegen filament almost can not directly be dissolved in acetic acid solution, this phenomenon shows the crosslinking reaction that thick collegen filament molecular chain is interior and the interchain existence is stronger in the crow ginseng of the East Sea, match with the hard phenomenon of the coarse and weave construction of its body surface, therefore the extraction yield of solubility in acid collagen protein is extremely low, has marked difference with other the thick collegen filament character of fish (being easy to extract the solubility in acid collagen protein).

Claims (2)

1. a method for extracting and purifying the enzymatic collagen of black ginseng from the East China Sea, said method comprising: (1) Soak the clean bulky black ginseng in 0.2M, pH8.0 EDTA solution for 5-6 days, and replace the EDTA solution every day; (2) After washing with water, add ice cubes, use a stainless steel homogenizer to homogenize the slurry, and then centrifuge to collect the precipitate for the next step; (3) Step (2) Precipitation Add 10-20 times the volume of 0.1M, pH 8.0 Tris-HCl solution, stir for 2-4 days, fully dissolve the tissue protein, and take the precipitate after centrifugation for the next step; (4) Step (3) Add 10-20 times the volume of 0.1M NaOH to the precipitate and stir for 2-4 days, replace the NaOH solution every day, centrifuge, take the precipitate, add 10-20 times the volume of 0.1M NaOH and stir for 24 hours, centrifuge, take Wash the precipitate with water until neutral, and centrifuge to take the precipitate for the next step; (5) The precipitate obtained in step (4) is extracted by adding a sufficient amount of 0.5M acetic acid solution containing 300w ~ 800w U/L pepsin for 2 to 4 days, and centrifuged to obtain For supernatant 1, add enough 0.5M acetic acid solution containing 300w ~ 800w U/L pepsin to extract the precipitate again for 24 hours, and centrifuge to obtain supernatant 2; (6) Combine supernatant 1 and supernatant 2, add 4M NaCl until the final concentration of NaCl is 0.8M, salt out, let stand for 1 to 2 hours, centrifuge and take the precipitate; (7) The precipitate obtained in step (6) was dissolved by adding 9 to 10 times the volume of 0.5M acetic acid solution, and dialyzed with a dialysis membrane with a molecular weight cut-off of 7000Da, first dialyzed with a 0.02M, pH8.0 Na ₂ HPO ₄ solution for 48 hours, Dialyze with 0.1M acetic acid solution for 48 hours, then with deionized water for 48 hours, and freeze-dry the product, which is the enzyme-soluble collagen; the above steps (1) to (7) are all carried out at 4°C. 2. The method according to claim 1, characterized in that step (3) Tris-HCl solution is also added with: 0.5M NaCl, 4-50mM EDTA and 0.2M β-mercaptoethanol.

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