CN103184262B - 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

A method for extracting and purifying enzyme-soluble collagen from black ginseng

(1) Technical field

The invention relates to a method for extracting and purifying enzymatically soluble collagen from black ginseng.

(2) Background technology

Collagen is rich in amino acids such as glycine, alanine, arginine, and proline that the human body needs. Collagen is the most important part of the extracellular matrix. It is an extracellular protein. Collagen is a fibrous protein with three peptide chains twisted into a helical shape. The peptide chain is composed of a large number of repeated Gly-X-Y-sequences (X : proline, Y: hydroxyproline), 15-20 amino acid residues of the telopeptide (C- and N-terminal) do not participate in the triple helix structure, most of the residues are lysine and hydroxylysine It is composed of its aldehyde derivatives, forming covalent crosslinks between molecules and within minutes, and the triple helix structure is maintained by hydrogen bonds within and between peptide chains.

Collagen is the most abundant protein in the human body, accounting for 1/3 of the total protein in the body. It mainly exists in human skin, bones, eyes, teeth, tendons, internal organs (including heart, stomach, intestines, blood vessels) and other parts. Its function is to maintain the shape and structure of skin, tissues and organs, and it is also an important raw material for repairing damaged tissues. . 70% of the human skin is composed of collagen. Type I collagen exists widely in nature, such as cowhide, pig skin and other mammals, fish, molluscs, etc. The types of collagen extracted from different tissues and parts are divided into three categories: (1) interstitial collagen I-III Type II, mainly present in tissues such as skin and tendons, is collagen between cells and has strong tensile properties, among which type II collagen is produced by chondrocytes. (2) Basement membrane collagen has type Ⅳ-Ⅶ colloids as basement membrane collagen, which mainly exists in organs. (3) Cartilage collagen has type Ⅸ-Ⅺ, which is related to the formation of cartilage with trace collagen in cartilage. Collagen is the main component of human tissue. It has an inseparable relationship with various organs, tissues and cells of the human body, so it is used in the repair and regeneration of human organs and tissues. Collagen is mainly a variety of functional collagen biomaterials in medical applications, including collagen sponges, silk glands, films (for surgical hemostasis, for cardiovascular, nerve, oral, orthopedic, skin, obstetrics, etc.) wound dressings, artificial Skin, blood vessels, heart valves, corneal protective materials, injectable collagen (for wrinkle removal, soft tissue plump filling, treatment of urinary incontinence, urine reflux, orthopedic tissue regeneration filler), drug-assisted mechanism, collagen matrix template, etc. . Collagen has the functions of moisturizing, anti-wrinkle and anti-oxidation in cosmetics and beauty care. Collagen is the main component to maintain skin firmness and elasticity. In addition, collagen is also used in food and health products.

Currently, there are many collagen and collagen peptides or protein powder products on the market. Consumers cannot distinguish the functions and effects of the products, so it is necessary to distinguish the relationship between the three in terms of concept and application. Collagen peptide is a kind of small molecule polypeptide mixture made of collagen or collagen-rich products as raw materials and hydrolyzed by biological enzymes under certain temperature and pH conditions. It consists of two or more amino acids. Depending on the hydrolysis conditions and degree of hydrolysis, the amino acid composition and molecular weight range of the polypeptide are the main factors determining the function, and the polypeptide can be directly absorbed by the human body. Protein powder is composed of two or more amino acids, and is generally a polypeptide mixture produced by adding biocatalytic enzyme preparations to hydrolyze under appropriate conditions for products with high protein content. At present, more protein powders come from soybean protein powder, whey protein powder etc. Collagen is a kind of substance composed of a single protein with special structure and unique biological application, which needs to be separated and purified. Collagen can be used as a biomedical auxiliary material, and it is an irreplaceable functional biomaterial for products such as general collagen peptides or protein powder.

In recent years, the market share of natural marine products - sea cucumbers and their health products has risen sharply, with broad prospects. Since 2000, the output value of Chinese sea cucumbers has shown an annual growth rate of 30%, and it has been more than ten years. The main production areas are Dalian and Shandong sea areas. There are more than 500 breeding, processing, and sales enterprises, with an annual output value of more than 20 billion yuan. It has become the largest single-product output value in my country's fishery resources. East China Sea black ginseng (Acaudina Leucoprocta) is one of the most important types of sea cucumbers. The coastal areas of Zhejiang are rich in East China Sea black ginseng resources. The annual catch in Xiangshan, Zhejiang alone reaches 100,000 tons. According to conservative calculations, 100,000 tons The black ginseng of the East China Sea can produce an output value of 10 billion. Therefore, mining the resources of the black ginseng in the East China Sea and in-depth development of related health care products will be of great significance to the development of Zhejiang's marine economy. According to the 2011 China Bio-Collagen Market Analysis and Investment Trend Research Report, China’s collagen consumption in 2011 was about 10,000 tons, and it is expected that the demand will increase to 20,000 tons by 2015, with a compound annual growth rate of 25%, higher than the global average. Increase, so the market potential of collagen is very considerable. Existing collagen is mainly derived from mammals such as cowhide and pigskin. Due to the discovery of animal diseases such as mad cow disease and foot-and-mouth disease, as well as the influence of factors such as animal-derived allergens and religious traditions, it is urgent to find alternative collagen sources. Marine products It is the most researched alternative resource in recent years, such as collagen extraction and purification products in fish, molluscs, sea cucumbers and other tissues, but many reports and studies are limited to laboratories, which cannot achieve large-scale production and processing, and the output is low , expensive, no market competitiveness.

Sea cucumber is a kind of high-protein, low-fat, cholesterol-free, rich in sea cucumber saponins, polysaccharides and other active substances. natural marine resources. The collagen in sea cucumber is very high, accounting for 70% of the total protein of sea cucumber. There are more than 1,400 species of sea cucumbers in the world, and there are more than 100 species in my country's sea areas. At present, there are more than 20 species of sea cucumbers available for consumption, such as plum ginseng, sea cucumber, black ginseng, light ginseng, melon ginseng, jade foot ginseng, and Donghai black ginseng. .

East China sea black ginseng (Acaudina leucoprocta): scientific name white anal sea sweet potato, is a kind of sea cucumber belonging to Echinodermata, Holothuroidea, Molpadida, Caudinidae, and Acaudina class animals. It is widely grown on the continental shelf of the East China Sea, from the Zhoushan Islands in Zhejiang Province to Hainan Island and other sea areas. In addition, it is also distributed in Iran and northwest Australia, with rich resources. Because the East China Sea black ginseng (Acaudina Leucoprocta) grows on the seabed silt for many years, the mucus secreted by its body surface is firmly combined with the seabed silt containing heavy metal deposits to form a dense and hard body surface dirt layer. With the increasing pollution of seawater Intensified, the content of harmful heavy metals such as mercury, arsenic, and lead in the dirt layer on the body surface seriously exceeded the standard. The large amount of metal elements accumulated on the body surface of Donghai black ginseng and the dense and hard tissue characteristics have seriously restricted the processing and utilization of this high-quality resource, especially in the extraction of collagen. Therefore, Donghai black ginseng has been used by local Fishermen consider it a low-value waste resource. The research and development of high value-added products of Donghai black ginseng is of great significance to the field of deep processing of low-value marine products, and at the same time provides a broad application prospect for the development and utilization of alternative resources.

The individual differences of different species of sea cucumbers are very large. The most valuable sea cucumbers along the coast of China are Liao cucumbers. Collagen Extraction Method" (CN101215315B) sea cucumber raw material is sea cucumber, and the process of collagen and collagen is described. This process is suitable for sea cucumber tissues that are easy to foam and homogenate, but not suitable for the East China Sea, which is heavily polluted by seawater Black ginseng, its tissue is hard, it is difficult to break and homogenize, the content of heavy metals and various chemical elements exceeds the standard, and the accumulation of a large number of metal elements in the tissue of East China sea ginseng seriously affects the extraction efficiency. The yield of collagen in East China sea ginseng is extracted by this patent method It is extremely low, and it is impossible to fully extract collagen, and the content of metal elements in the product exceeds the standard. Therefore, it is necessary to improve the process in the patent to be suitable for the extraction of collagen and collagen in Donghai black ginseng tissue.

Another patent is "A Method for Manufacturing Collagen from Black Ginseng of the East China Sea" (CN102251006A), which is a direct hydrolysis method of collagen from black ginseng of the East China Sea, not a method of extracting collagen. The product is a small-molecule collagen peptide powder. At present, there is no report on the extraction technology of collagen in black ginseng of the East China Sea. None of the other patents or documents related to sea cucumbers involves the extraction or production of collagen of the East China Sea black cucumber variety. Because the East China Sea continental shelf contains a huge amount of East China Sea black cucumber, the pollution of metal elements in the seawater makes it impossible to eat directly, and it is a low-value marine resource. , needs to be removed from the chemical elements before it can be used, so it is necessary to develop it into a high value-added collagen product through deep processing.

(3) Contents of the invention

Aiming at the defects existing in the prior art, the present invention provides a method for extracting and purifying collagen from black ginseng of the East China Sea, which is easy to operate, low in cost, high in profit, and environmentally friendly.

The technical scheme adopted in the present invention is:

A method for extracting and purifying enzymatically soluble collagen from black ginseng, the method comprising:

(1) Soak the clean blocky black ginseng sample in 0.2M, pH 8.0 EDTA solution for 5-6 days, and replace the EDTA solution every day; the clean blocky black ginseng can be washed with fresh Donghai black ginseng with water at 4°C , remove the silt and viscera, cut into small pieces (5-10mm×5-10mm), or freeze and cut the Donghai black cucumber into pieces, and then rinse with 4°C water to remove the silt and viscera;

(2) After the blocky sample is washed with water, add ice cubes, use a stainless steel homogenizer to homogenize the slurry, and then centrifuge to collect the sediment for the next step;

(3) Step (2) Precipitation Add 10 to 20 times the volume (ratio of solution volume in milliliters to precipitation mass in grams, the same below) of 0.1M, pH 8.0 Tris-HCl solution, stir for 2 to 4 days, and fully Dissolve the tissue protein, and take the precipitate after centrifugation for the next step; Tris-HCl solution can also be added with 0.5M NaCl, 4-50mM EDTA and 0.2M β-mercaptoethanol, or without β-mercaptoethanol, or NaCl and EDTA, There was no significant difference in the extraction results. This method can effectively remove polluting chemical elements, and at the same time fully dissolve and expand collagen fibers without affecting the structure and properties of collagen;

(4) Step (3) Add 10-20 times the volume of 0.1M NaOH solution 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, centrifuge to take the precipitate for the next step, or wash the precipitate with water until neutral and freeze-dry directly to obtain crude collagen fibers for the next step of enzymatic hydrolysis; this step can remove non-collagenous substances and The activity of endogenous biological enzymes in sea cucumbers can effectively remove non-collagenous substances such as saponins, polysaccharides, and miscellaneous proteins, increase the yield of collagen, and at the same time inhibit the degradation of biologically active substances in sea cucumbers;

(5) The precipitate (or crude collagen fiber) obtained in step (4) is added to a sufficient amount (more than 20 times the volume) of 0.5M acetic acid solution containing 300w-800w U/L pepsin (derived from pig gastric mucosa) to extract for 2-4 days, Centrifuge to obtain supernatant 1, add enough 0.5M acetic acid solution containing 300w~800w U/L pepsin to the precipitate to extract for 24 hours, and centrifuge to obtain supernatant 2; crude collagen fibers are enzymolyzed with pepsin from pig gastric mucosa The extraction of collagen fibers can effectively weaken and remove the interaction of chemical bonds between collagen fibers, so that the insoluble collagen fibers can be fully dissolved in the acidic solution, and the insoluble collagen fibers can be converted into soluble collagen.

(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, Change the 0.1M acetic acid solution for dialysis for 48 hours, then change the deionized water for 48 hours, and freeze-dry the product, which is the enzyme-soluble collagen;

Using NaCl salting out and dialysis membrane dialysis, the collagen product can be effectively purified. After protein electrophoresis and gel chromatography, the collagen product does not contain impurity proteins. According to the determination and analysis of amino acid, metal element content and yield, the product The extraction and purification technology of the method can obtain high-yield and high-purity collagen products, and is a high-quality collagen resource that can be produced in a large scale and can replace marine products derived from mammals.

The above steps (1) to (7) were all carried out at 4°C.

Preferably, in step (3) the Tris-HCl solution is further added: 0.5M NaCl, 4-50mM EDTA and 0.2M β-mercaptoethanol.

The beneficial effect of the present invention is mainly reflected in: the use of EDTA chelating agent to remove the chemical elements with high pollution concentration in the sea cucumber tissue, the removal efficiency reaches more than 90%, and the collagen product after the collagen fiber is hydrolyzed by pepsin effectively removes the chemical bonds in and between the bonds The cross-linking effect can maximize the extraction and dissolution of collagen. After purification steps such as sodium chloride salting out and dialysis, protein electrophoresis analysis and gel chromatography of the collagen product, the purity of the extracted collagen product reaches More than 90%, the yield is 56.99% to 61.52% (dry weight), accounting for 97.1% to 102.5% of the total collagen content in Donghai black ginseng protein, realizing the high yield extraction technology for collagen content in Donghai black ginseng.

(4) Description of drawings

Fig. 1 is the SDS-PAGE spectrogram of the collagen in Example 3, PSC: enzymatically soluble collagen from black ginseng, M: molecular weight marker, CSC: bovine skin collagen standard control substance.

(5) Specific implementation methods

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

Example 1:

1. Fresh Donghai black ginseng (commercially purchased, produced in Xiangshan, Zhejiang) samples were washed with tap water at 4°C to remove sediment and internal organs, cut into small pieces of 5mm*5mm, and 200g of block samples were washed with 1000mL0.2M EDTA (pH8 .0) Soak and stir for 5 days, replace the solution every day to remove calcium, iron, manganese, magnesium, lead and other pollution elements and residual impurities.

2. After washing with water, add 100g of ice cubes (to reduce the temperature of the solution to below 4°C), homogenize with a stainless steel homogenizer (IKA T25Homogenizer), 18000rpm, 20min, and take the precipitate A after centrifugation.

3. Add 0.1M Tris-HCl (pH 8.0) 20 times the volume (solution 20mL/1g precipitate) to precipitate A, stir for 48 hours to fully dissolve tissue protein, and take precipitate B after centrifugation.

4. Add 20 times the volume of 0.1M NaOH to the precipitate B and stir for 48 hours, replace the solution every day to remove non-collagenous substances and the activity of endogenous biological enzymes, and centrifuge at 12000G for 30 minutes. The supernatant was discarded, and the precipitate C was stirred again with 20 times the volume of 0.1M NaOH for 24 hours. After centrifugation, the precipitate was washed with water until neutral, and the precipitate D was collected by centrifugation.

5. Add 20 times the volume of precipitate D to 0.5M acetic acid solution containing 0.5g/L pepsin (Sigma pepsin, 600-1200units/mg) to extract for 3 days, centrifuge at 12000G for 30min, take supernatant 1, and precipitate again Extract for 24 hours, centrifuge at 12000G for 30 minutes, and take supernatant 2.

6. Combine the two supernatant solutions 1 and 2, add 4M NaCl to the final NaCl concentration of the solution to 0.8M, salt out, let stand for 1h, centrifuge at 12000G for 20min, and take the precipitate E.

7. Dissolve the precipitate E with 9 times the volume (9mL solution/1g precipitate) of 0.5M acetic acid solution, dialyze with a dialysis membrane (molecular weight cut-off: 7000Da), first dialyze with 0.02M, pH8.0 Na ₂ HPO ₄ solution for 48h, Change 0.1M acetic acid solution for dialysis for 48 hours, then change deionized water for 48 hours, and freeze-dry the product, which is enzyme-soluble collagen.

Example 2:

1. Fresh black ginseng samples from the East China Sea were frozen and cut into granules (size: 5mm*5mm), washed with ice water and stirred three times to remove impurities such as sediment and viscera, then took 200g block samples and washed them with 2000mL0.2M EDTA (pH8. 0) Soak and stir for 5 days, replace the solution every day to remove calcium, iron, manganese, magnesium, lead and other polluting elements.

2. After washing with water, add 200g of ice cubes (to lower the temperature of the solution to below 4°C), use a stainless steel homogenizer (IKA T25Homogenizer) to homogenize at 18000rpm for 20min, and take the precipitate A after centrifugation.

3. Add 20 times the volume of 0.1M, pH8.0 Tris-HCl+0.5M NaCl+50mM EDTA+0.2M β-mercaptoethanol to the precipitate A, stir and extract for 72 hours, and take the precipitate B after centrifugation.

4. Add 20 times the volume of 0.1M NaOH to the precipitate B and stir for 72 hours. Change the solution every day to remove non-collagenous substances and the activity of endogenous biological enzymes, and centrifuge at 12000G for 30 minutes. The supernatant was discarded, and the precipitate C was washed with water until neutral (a small amount of acetic acid can be added to adjust the pH), and the product was lyophilized to obtain coarse collagen fibers.

5. Take 5g of crude collagen fiber and dissolve it with 5L0.5M acetic acid solution, add 0.1g pepsin (3200-4500units/mg) and stir for extraction for 72h, centrifuge at 12000G for 30min, and then use 0.5 M acetic acid solution was extracted for 24 hours, and centrifuged at 12000G for 30 minutes.

6. Combine the two supernatant solutions, add 4M NaCl to the final NaCl concentration of 0.8M, salt out, let stand for 1h, centrifuge at 12000G for 20min, and take the precipitate E.

7. Dissolve the precipitate E with 9 times the volume of 0.5M acetic acid solution, dialyze with a dialysis membrane (molecular weight cut-off: 7000Da), first dialyze with 0.02M, pH8.0 Na ₂ PO ₄ solution for 48 hours, then change to 0.1M acetic acid solution for 48 hours , and then dialyzed with deionized water for 48 hours, and the product was freeze-dried, which was enzymatically soluble collagen.

Embodiment 3:

1. The tissue water content of fresh black ginseng is 83.61±0.2%, the ash content is 3.9±0.05%, the protein is 13.71±0.1%, and the fat is 0.9±0.05%. The content of heavy metal elements seriously exceeds the standard, as shown in the table below:

It can be seen from the table that the contents of As, Pb and Cr far exceed the standard limit of 0.5mg/Kg.

2. The epidermis of fresh Donghai black ginseng is seriously polluted, yellow in color, and partly has a serious rust red color. In 0.2M EDTA with pH 8.0, the solution is replaced every day. After soaking for 5 days, the removal rate of Ca, Fe, Mn, Mg, Zn, Sn and other metal elements is above 90%, and the tissue is obviously softened and swollen, and it is easy to homogenize. Add ice water, homogenize at 18000rpm for 20min, centrifuge at 12000G and take precipitate A.

3. Precipitate A was dissolved with 20 times the volume of 0.1M Tris-HCl (pH8.0) and stirred for 72 hours. This step can swell and crack the tissue particles and fully dissolve the collagen fibers.

4. Take the precipitate B after centrifugation, add 20 times the volume of 0.1M NaOH solution and stir for 72 hours to remove non-collagenous substances and inhibit the endogenous biological enzymes of Donghai black ginseng. Centrifuge and replace the solution in the middle, which can effectively remove metal element impurities .

5. After centrifugation, take the precipitate C, wash it with water until it is neutral, and freeze-dry it directly to obtain crude collagen fibers. Dissolve 1 g of dry crude collagen fibers with 0.5 M acetic acid in 1 L volume, add 0.1 g/L pepsin, and stir the enzyme After 3 days of solution, centrifuge to remove the supernatant, and the precipitate D can be homogenized again and added to 0.5M acetic acid solution containing 0.1g/L pepsin for extraction for 24 hours, and the two extracts are combined.

6. Salting out: Add 4M NaCl to the solution concentration of 0.8M, the precipitated product is transparent collagen, let it stand for 2 hours, and centrifuge to get the precipitate E.

7. Dialysis: Add 9 times the volume of 0.5M acetic acid to dissolve the precipitate E, dialyze with a dialysis membrane (molecular weight cut-off: 7000Da), first dialyze with 0.02M, pH8.0 Na ₂ PO ₄ solution for 48 hours, and then dialyze with 0.1M acetic acid 48 hours, and then dialyzed with deionized water for 48 hours, and the product was freeze-dried to obtain enzyme-soluble collagen.

8. The collagen content in black ginseng is 70% of the total protein content, and the extraction yield of collagen is 9.34%-10.08% (wet weight), which is 56.99%-61.52% (dry basis) when converted into dry weight. The yield of collagen extracted from giant red sea cucumber (Parastichopus californicus) is 20.8% (epidermal) and 24.3% (tissue). The extraction rate of enzymatically soluble collagen in the black ginseng of the East China Sea is converted into the initial content of collagen (70% of the total protein) and is 97.1 to 102.5%. Therefore, the method of the present invention can realize the complete extraction of the collagen in the black ginseng of the East China Sea. Using this method The heavy metal content of the extracted collagen has reached the national limit standard.

9. Amino acid analyzer to measure aspartic acid, glycine, alanine, glutamic acid, proline, hydroxyproline, lysine, hydroxylysine, etc. in fresh sea cucumber tissue and obtained enzyme-soluble collagen 18 The amino acids were compared with the amino acid composition and content of bovine collagen, sea cucumber (Stichopus japanious) and giant red sea cucumber (Parastichopus californicus). The content of proline directly affects the content of collagen, and hydroxyproline is an important imidic acid to maintain the structure of collagen, and the content of glycine and glutamic acid in seafood is relatively high. Glycine accounts for one-third of the total amino acids in fresh Donghai black ginseng, and the measurement results are consistent with the nature of the collagen peptide chain consisting of a large number of repeated Gly-X-Y-sequences. The content of proline and hydroxyproline is 141.0 ±2.9 (based on 1000 amino acid residues) slightly higher than 133 in Stichopus japanious (Saito, M., Kunisaki, N., Urano, N., & Kimura, S. (2002). Collagen as the major edible component of seacucumber (Stichopus japonicus). Journal of Food Science, 67(4), 1319-1322.), the content of proline and hydroxyproline in the extracted enzymatic collagen is 160.6±2.5 ( Based on 1000 amino acid residues), it is higher than the 153 of the giant red sea cucumber (Parastichopus californicus), similar to that of sea cucumber (Stichopus japanious), but lower than the 215 of cowhide and 220 of pig skin (Park, S.Y., Lim, H.K. ,Lee,S.,Hwang,H.C.,Cho,S.K.,&Cho,M.(2012).Pepsin-solubilized collagen(PSC)fromRed Sea cucumber(Stichopus japonicus)regulates cell cycle and thefibronectin synthesis in HaCaT cell migration.Food Chemistry 132(1), 487-492.). The hydroxyproline of the collagen extract protein product in Donghai black ginseng accounts for 38.4% of the total proline, and hydroxyproline plays a major role in maintaining the triple helix structure of collagen (Bachinger, H.P., Morris, N.P., & 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 OfType-I Collagen.American Journal Of Medical Genetics, 45(2), 152-16), therefore From the comparative analysis results of the total amount of hydroxyproline and proline, it can be inferred that the stability of the collagen of the black cucumber of the East China Sea may be slightly inferior to that of mammals, but it is similar to that of the same species of sea cucumber. The specific determination results of amino acids are shown in the table below:

The median ± relative standard deviation of the results of the three samples.

*（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 ConnectiveTissue of Giant Red Sea Cucumber(Parastichopus californicus).Journal of Agricultural istry and Food Chemistry, 5( 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 electrophoresis spectrum analysis, the extracted product enzymatically soluble collagen product has only one main band on 7.5% SDS-PAGE, the molecular weight is 138kDa, compared with bovine skin collagen standard reference substance, it is α1 chain, and there are two other bands. The two lighter bands are γ chain and β chain respectively, and the extracted product is type 1 collagen, which is similar to the collagen products of sea cucumber and giant red sea cucumber (Parastichopus californicus), without α ₂ chain, which is (α ₁ ) ₃ Triple helical structure, different species of sea cucumbers may contain similar collagen structures, due to different habitats, sea cucumber tissue characteristics and amino acid composition and structure will be different (Gomez-Guillen, MC, Turnay, J., Fernandez- Diaz, MD, Ulmo, N., Lizarbe, MA, & 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. Viscosity measurement results of enzymatically soluble collagen extracts from black cucumber in the East China Sea show that the denaturation temperature Td is 25.4°C, which is higher than that of the giant red sea cucumber (Parastichopus californicus) epidermis (Parastichopus californicus) at 18.5°C and tissue at 17.9°C, and lower than that of cowhide collagen at 37°C. ℃; The thick collagen fibers in the black ginseng of the East China Sea are easily soluble in acidic solution and contain pepsin solution. After enzymatic modification with pepsin, the collagen can be completely released and extracted. The optimal dissolution condition of enzymatically soluble collagen is pH2.66 . The crude collagen fibers can hardly be directly dissolved in the acetic acid solution. This phenomenon indicates that there are strong cross-linking reactions in the molecular chains and between the chains of the crude collagen fibers in Donghai black ginseng, which is consistent with the roughness of the body surface and the firmness of the tissue structure. Therefore, the extraction rate of acid-soluble collagen is extremely low, which is significantly different from other fish thick collagen fibers (easy to extract acid-soluble collagen).

Claims (2)

1. an extracting and purifying method for black sea cucumbers from East China Sea pepsin-solubilized collagen, described method comprises:

(1) get the EDTA solution soaking 5 ~ 6 days of clean block black sea cucumbers from East China Sea 0.2M, pH8.0, change EDTA solution every day;

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

(3) step (2) precipitation adds the Tris-HCl solution of 0.1M, pH8.0 of 10 ~ 20 times of volumes, stirs 2 ~ 4 days, fully dissolves tissue protein, and centrifugal rear taking precipitate carries out next step operation;

(4) step (3) precipitation add 10 ~ 20 times of volumes 0.1M NaOH stir 2 ~ 4 days, every day changes NaOH solution, centrifugal, get the 0.1M NaOH that precipitation adds 10 ~ 20 times of volumes again and stir 24h, centrifugal, get precipitation and wash with water to neutrality, centrifuging and taking throw out carries out next step operation; (5) step (4) gained precipitation adds enough containing the pepsic 0.5M acetic acid solution extraction of 300w ~ 800w U/L 2 ~ 4 days, centrifugally obtain supernatant liquor 1, throw out again adds and enoughly extracts 24h containing the pepsic 0.5M acetic acid solution of 300w ~ 800w U/L, centrifugally obtains supernatant liquor 2;

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

(7) step (6) gained throw out adds the 0.5M acetic acid solution dissolving of 9 ~ 10 times of volumes, dialyses, first use the Na of 0.02M, pH8.0 with the dialysis membrane of molecular weight cut-off 7000Da ₂hPO ₄solution dialysis 48h, changes 0.1M acetic acid solution dialysis 48h, then changes deionized water dialysis 48h, and product freeze-drying, is described pepsin-solubilized collagen; Above-mentioned steps (1) ~ (7) are all carried out at 4 DEG C.

2. the method for claim 1, is characterized in that also being added with in step (3) Tris-HCl solution: 0.5M NaCl, 4 ~ 50mM EDTA and 0.2M beta-mercaptoethanol.