CN101215315B - Method for extracting sea cucumber collagen and collagen - Google Patents

Abstract

A method for preparing sea cucumber collagen and collagen protein comprises the following steps: treating sea cucumber with an aqueous solution, a Tris-HCl solution, and a NaOH solution, and then freezing or freeze-drying the sea cucumber to obtain sea cucumber crude collagen fibers. The obtained crude collagen fibers are added with an acetic acid solution and pepsin, stirred and enzymolyzed at 0-5°C, frozen and centrifuged, and then salted out and purified, and then desalted with _Na2HPO4 , _acetic acid, and water to obtain active collagen. The obtained crude collagen fibers are added with an acetic acid solution and protease, stirred and enzymolyzed at 6-25°C, centrifuged, and then salted out and purified, and then desalted with _{Na2HPO4 ,} acetic acid, and water to obtain collagen protein.

Description

Extraction method of sea cucumber collagen and collagen

Technical field:

The present invention relates to the field of edible animal protein, and also relates to a method for obtaining collagen and collagen by using enzymes.

Background technique:

Studies have shown that collagen and collagen have a variety of physiological functions, such as protecting gastric mucosa and anti-ulcer effects, anti-allergic, lowering blood pressure, lowering cholesterol, anti-aging, promoting wound healing, enhancing bone strength and preventing osteoporosis, preventing arthritis , to promote the repair of corneal epithelial damage and promote the growth of corneal epithelial cells and other physiologically active functions. Therefore, it has a wide range of applications in the fields of medicine, chemical industry, food, etc., and more and more attention is being paid to it. More than 40% of the protein content in the body wall of sea cucumber is collagen, and the main component of its edible parts comes from collagen. Collagen contains a variety of trace elements and a variety of biologically active substances, which have anticoagulant, immune regulation, and hypolipidemic properties. , antiviral, antitumor and other pharmacological effects. In addition to the commonality of collagen, the collagen in the body wall of sea cucumbers also has certain characteristics, that is, insolubility under normal conditions, so people seldom involve its extraction.

Invention content:

The present invention uses sea cucumbers to provide a method for preparing sea cucumber collagen and collagen, which maintains the structure and physiological functions of the prepared collagen and collagen, and extracts and prepares sea cucumber body wall collagen for the processing and utilization of sea cucumbers and waste addition. The improvement of the value, especially the optimization of the existing processing method, provides a more detailed basic theoretical basis.

The technical scheme of the present invention is: divided into three steps of raw material processing, extraction and purification. In raw material treatment, sea cucumbers are treated with water, Tris-HCl solution, and sodium hydroxide solution at low temperature to remove water-soluble and salt-soluble proteins and pigments; extraction is performed with acetic acid and pepsin, and crude products are obtained through salting out; purification is The crude product was redissolved with acetic acid, and then dialyzed with disodium hydrogen phosphate solution, acetic acid and water respectively.

Technical scheme of the present invention realizes as follows:

(1).Raw material processing

Remove the viscera of fresh sea cucumbers, wash them, cut them into pieces, add 5 to 10 times of water to break the walls and homogenate with a tissue grinder, then stir at 0 to 5°C for 30 to 60 minutes, centrifuge, take the precipitate and continue to add water and stir for 30 minutes. ~120min, centrifuge to take the precipitate, add 5~10 times of 0.1mol/L Tris-HCl solution and stir for 12~24h, centrifuge to take the precipitate, stir and wash with water until neutral, then add 4~8 times of water and stir for 2~4 days, centrifuge Add 2 to 4 times more water to the precipitate and stir for 2 to 4 days, centrifuge at 10000 to 12000 rpm for 2 to 5 minutes, remove the water, and obtain the precipitate, that is, the crude collagen fibers are frozen or stored for later use;

Add the above crude collagen fibers to 10-30 times 0.05-0.20mol/L NaOH solution and stir for 2-4 days, change the solution 2-3 times in the middle, centrifuge at 13500rpm for 15-20min, wash the precipitated collagen fibers with water until neutral Post-frozen storage or freeze-dried storage for later use;

Since collagen is insoluble in water and alkaline solution, the effect of adding water, Tris-HCl solution and alkaline solution to stir in this step is to remove some water-soluble and salt-soluble proteins and pigments in sea cucumbers, and the processing capacity is 1% of the total amount of protein. 50%, while keeping collagen from being damaged.

(2).Extraction

Collagen fibers were extracted by adding acetic acid of 5-15pPH2.0-3.0 and 1-15% pepsin at 0-25°C for 2-3 days, centrifuged at 13500rpm for 1h, and the supernatant was taken with a final concentration of 0.6-1mol/L NaCl Salt out and centrifuge to obtain the crude product;

Because sea cucumber collagen fibers have extensive cross-linking effects, the collagen in sea cucumbers is a highly cross-linked polymer of collagen molecules. Very little collagen dissolves without treatment of its peptide bonds. The pepsin solubilization method can break the amino acid at the end of the collagen peptide chain, so that a large amount of collagen fibers can be dissolved in the acid, which is more than 70% higher than the extraction rate of the acid solubilization method alone, so the enzymatic solubilization method can extract the most Collagen and collagen protein, the utilization rate of raw materials is maximized.

(3) Purification

The crude product is redissolved with acetic acid with pH 2.0-3.0, dialyzed with 0.02mol/L Na ₂ HPO ₄ with pH 8.0 to inactivate the enzyme, then dialyzed with acetic acid with pH 2.0-3.0, dialyzed with water and centrifuged to collect the precipitate. Store frozen or lyophilized.

In the above two process steps of (2) extraction and (3) purification: the process step of extracting active collagen is carried out at a temperature of 0-5°C, which avoids the damage of high temperature to the collagen structure and keeps the original collagen. The triple helix structure, while maintaining its activity above 80%; the temperature is 0-25 ° C is the process step of extracting collagen.

The difference between the two substances extracted in the present invention is as follows:

Collagen belongs to structural protein and exists in the form of collagen fibers in sea cucumbers. The collagen extracted at low temperature still maintains a triple helical structure, has good flexibility, elasticity and strength, good rehydration, and has biological activity. Collagen is a mixture of polypeptides, with a relative molecular mass of several thousand to tens of thousands, and a wide relative molecular mass distribution, which can be better absorbed and utilized by the human body. Collagen cannot re-form fibers and is not biologically active.

Compared with prior art, outstanding advantage of the present invention is:

1. The processing method used in the raw material processing process can remove non-collagen impurities to the greatest extent.

2. Low temperature is used to prepare active collagen, which ensures the activity and structural integrity of collagen.

3. The enzymatic solubilization method used can extract collagen and collagen protein to the maximum extent, and the utilization rate of raw materials reaches the maximum value.

4. The used raw material for preparing collagen can be fresh sea cucumber, or the remaining sea cucumber after extracting the active substances in the sea cucumber, so that the raw material has been fully utilized.

5. For the first time, sea cucumber was used to produce collagen and collagen.

Detailed ways

Example one:

1. Extraction of Active Collagen

Take fresh sea cucumbers or the remaining sea cucumbers after extracting the active substances in sea cucumbers, remove internal organs, wash the body wall, cut into pieces, add 5 times of water, use a tissue masher to break the wall, homogenate, and then stir at 4°C for 30 minutes , centrifuge, take the precipitate, continue to add water and stir for 60min, centrifuge to take the precipitate, add 10 times 0.1mol/L Tris-HCl (50mmol/L EDTA, 0.5mol/L NaCl, pH8.0) solution and stir for 12h, centrifuge to take the precipitate, stir and wash with water until Neutral, then add 6 times of water and stir for 3 days, centrifuge to collect the precipitate, add 3 times of water and stir for 3 days, centrifuge at 10000rpm for 2 minutes, remove the water, and obtain the precipitate, that is, the crude collagen fibers are stored frozen or freeze-dried for later use. Add the above crude collagen fibers to 20 times 0.1mol/L NaOH solution, stir at 4°C for 3 days, change the solution 3 times in the middle, centrifuge at 13500rpm for 20min, wash the precipitate with water until neutral, then add 10 times acetic acid of pH 2.0 and 15% The pepsin was extracted at 4°C for 2 days, centrifuged at 13500rpm for 1h, then the supernatant was salted out with a final concentration of 0.8mol/L NaCl, and the precipitate was reconstituted with pH 2.0 acetic acid after centrifugation, and 0.02mol /L pH8.0 Na ₂ HPO ₄ dialysis to inactivate the enzyme, then dialysis with pH 2.0 acetic acid, and then water dialysis to achieve the purpose of desalination, centrifuge to take the precipitate, that is, active collagen, freeze or lyophilize for storage.

2. Collagen Extraction

Take fresh sea cucumbers or the remaining sea cucumbers after extracting the active substances in sea cucumbers, remove internal organs, wash the body wall, cut into pieces, add 5 times of water, use a tissue masher to break the wall, homogenate, and then stir at 4°C for 30 minutes , centrifuge, take the precipitate, continue to add water and stir for 60min, centrifuge to take the precipitate, add 10 times 0.1mol/L Tris-HCl (50mmol/L EDTA, 0.5mol/L NaCl, pH8.0) solution and stir for 12h, centrifuge to take the precipitate, stir and wash with water until Neutral, then add 6 times of water and stir for 3 days, centrifuge to collect the precipitate, add 3 times of water and stir for 3 days, centrifuge at 10000rpm for 2 minutes, remove the water, and obtain the precipitate, that is, the crude collagen fibers are stored frozen or freeze-dried for later use. Add the above crude collagen fibers to 20 times 0.1mol/L NaOH solution and stir at 4°C for 3 days, change the solution 3 times in the middle, centrifuge at 13500rpm for 20min, wash the precipitate with water until neutral, then add 10 times acetic acid with pH 2.0 and 10% NaOH Pepsin was extracted at room temperature (10°C) for 2 days, centrifuged at 13,500rpm for 1h, then the supernatant was salted out with NaCl at a final concentration of 0.8mol/L, and the precipitate was reconstituted with acetic acid at pH 2.0 after centrifugation. 0.02mol/L Na ₂ HPO ₄ at pH 8.0 to inactivate the enzyme, then dialyze with acetic acid at pH 2.0, and then dialyze with water to achieve the purpose of desalting, centrifuge to collect the precipitate, freeze or lyophilize for storage.

Example two:

1. Extraction of Active Collagen

Take fresh sea cucumbers or the remaining sea cucumbers after extracting the active substances in sea cucumbers, remove internal organs, wash the body wall, cut into pieces, add 8 times of water, use a tissue masher to break the wall, homogenate, and then stir at 5°C for 40 minutes , centrifuge, take the precipitate and continue to add water and stir for 90min, centrifuge to take the precipitate, add 8 times 0.1mol/L Tris-HCl (50mmol/L EDTA, 0.5mol/L NaCl, pH8.0) solution and stir for 18h, centrifuge to take the precipitate, stir and wash with water until Neutral, then add 8 times of water and stir for 2 days, centrifuge to collect the precipitate, add 4 times of water and stir for 2 days, centrifuge at 11000rpm for 3 minutes, remove the water, and obtain the precipitate, that is, the crude collagen fibers are stored frozen or freeze-dried for later use. Add the above crude collagen fibers to 30 times of 0.05mol/L NaOH solution and stir at 4°C for 3 days, change the solution twice in the middle, centrifuge at 13500rpm for 20min, wash the precipitate with water until neutral, add 5 times of acetic acid of pH 3.0 and 12 % pepsin was extracted at 5°C for 2 days, centrifuged at 13500rpm for 1h, and then the supernatant was salted out with a final concentration of 0.7mol/L NaCl, and the precipitate was reconstituted with acetic acid of pH 3.0 after centrifugation and washed with 0.02 mol/L Na ₂ HPO ₄ at pH 8.0 to inactivate the enzyme, then dialyzed with acetic acid at pH 3.0, and then dialyzed with water to achieve the purpose of desalting, centrifuge to collect the precipitate, freeze or lyophilize for storage.

2. Collagen Extraction

Take fresh sea cucumbers or the remaining sea cucumbers after extracting the active substances in sea cucumbers, remove internal organs, wash the body wall, cut into pieces, add 8 times of water, use a tissue masher to break the wall, homogenate, and then stir at 5°C for 40 minutes , centrifuge, take the precipitate and continue to add water and stir for 90min, centrifuge to take the precipitate, add 8 times 0.1mol/L Tris-HCl (50mmol/L EDTA, 0.5mol/LNaCl, pH8.0) solution and stir for 18h, centrifuge to take the precipitate, stir and wash with water until medium Then add 8 times of water and stir for 2 days, centrifuge to collect the precipitate, add 4 times of water and stir for 2 days, centrifuge at 11000rpm for 3 minutes, remove the water, and obtain the precipitate, that is, the crude collagen fibers are stored frozen or freeze-dried for later use. Add the above crude collagen fibers to 30 times of 0.05mol/L NaOH solution and stir at 4°C for 3 days, change the solution twice in the middle, centrifuge at 13500rpm for 20min, wash the precipitate with water until neutral, add 5 times of acetic acid of pH 3.0 and 8 % Pepsin was extracted at room temperature (15°C) for 2 days, centrifuged at 13,500rpm for 1h, then the supernatant was salted out with NaCl at a final concentration of 0.7mol/L, and the precipitate was reconstituted with pH 3.0 acetic acid after centrifugation , dialyzed with 0.02mol/L Na ₂ HPO ₄ pH 8.0 to inactivate the enzyme, then dialyzed with acetic acid with pH 3.0, and then dialyzed with water to achieve the purpose of desalting, centrifuged to collect the precipitate, frozen or freeze-dried for storage.

Example three:

1. Extraction of Active Collagen

Take fresh sea cucumbers or the remaining sea cucumbers after extracting the active substances in sea cucumbers, remove the internal organs, wash the body walls, cut them into pieces, add 10 times of water, use a tissue masher to break the walls, homogenate, and then stir at 0°C for 60 minutes , centrifuge, take the precipitate and continue to add water and stir for 120min, centrifuge to take the precipitate, add 5 times of 0.1mol/L Tris-HCl (50mmol/L EDTA, 0.5mol/L NaCl, pH8.0) solution and stir for 24h, centrifuge to take the precipitate, stir and wash with water until Neutral, then add 4 times of water and stir for 4 days, centrifuge to collect the precipitate, add 2 times of water and stir for 4 days, centrifuge at 12000rpm for 5 minutes, and obtain the precipitate, that is, the thick collagen fibers are stored frozen or freeze-dried for later use. Add the above crude collagen fibers to 10 times 0.2mol/L NaOH solution and stir at 0°C for 4 days, change the solution twice in the middle, centrifuge at 13500rpm for 20min, wash the precipitate with water until neutral, then add 15 times acetic acid with pH 2.0 and 10% The pepsin was extracted at 0°C for 3 days, centrifuged at 13500rpm for 1h, then the supernatant was salted out with a final concentration of 0.9mol/L NaCl, and the precipitate was reconstituted with pH 2.0 acetic acid after centrifugation, and 0.02mol /L Na ₂ HPO ₄ at pH 8.0 to inactivate the enzyme, then dialyze with acetic acid at pH 2.0, and then dialyze with water to achieve the purpose of desalting, centrifuge to collect the precipitate, freeze or lyophilize for storage.

2. Collagen Extraction

Take fresh sea cucumbers or the remaining sea cucumbers after extracting the active substances in sea cucumbers, remove the internal organs, wash the body walls, cut them into pieces, add 10 times of water, use a tissue masher to break the walls, homogenate, and then stir at 0°C for 60 minutes , centrifuge, take the precipitate and continue to add water and stir for 120min, centrifuge to take the precipitate, add 5 times of 0.1mol/L Tris-HCl (50mmol/L EDTA, 0.5mol/L NaCl, pH8.0) solution and stir for 24h, centrifuge to take the precipitate, stir and wash with water until Neutral, then add 4 times of water and stir for 4 days, centrifuge to collect the precipitate, add 2 times of water and stir for 4 days, centrifuge at 12000rpm for 5 minutes, and obtain the precipitate, that is, the thick collagen fibers are stored frozen or freeze-dried for later use. Add the above crude collagen fibers to 10 times 0.2mol/L NaOH solution and stir at 0°C for 4 days, change the solution twice in the middle, centrifuge at 13500rpm for 20min, wash the precipitate with water until neutral, then add 8 times acetic acid with pH 2.0 and 10% Pepsin was extracted at room temperature (20°C) for 2 days, centrifuged at 13,500rpm for 1h, then the supernatant was salted out with NaCl at a final concentration of 0.9mol/L, and the precipitate was reconstituted with acetic acid at pH 2.0 after centrifugation. Dialyze with 0.02mol/L Na ₂ HPO ₄ pH 8.0 to inactivate the enzyme, then dialyze with pH 2.0 acetic acid, and then dialyze with water to achieve the purpose of desalting, centrifuge to collect the precipitate, freeze or lyophilize for storage.

Claims (5)

1. the extracting method of sea cucumber collagen, collagen protein is characterized in that technological process is: (1) Raw material handling Remove the viscera of fresh sea cucumbers, wash them, cut them into pieces, add 5 to 10 times of water to break the wall and homogenate with a tissue grinder, then stir at 0 to 25°C for 30 to 60 minutes, centrifuge, take the precipitate and continue to add water and stir for 30 minutes. ~120min, centrifuge to take the precipitate, add 5~10 times of 0.1mol/L Tris-HCl solution and stir for 12~24h, centrifuge to take the precipitate, stir and wash with water until neutral, then add 4~8 times of water and stir for 2~4 days, centrifuge Add 2 to 4 times more water to the precipitate, stir for 2 to 4 days, centrifuge at 10,000 to 12,000 rpm for 2 to 5 minutes, remove the water, and obtain the precipitate, that is, the crude collagen fibers are frozen or stored for later use; Add the above crude collagen fibers to 10-30 times 0.05-0.20 mol/L NaOH solution, stir at 0-5°C for 2-4 days, change the solution 2-3 times in the middle, centrifuge at 13500rpm for 15-20min, and precipitate the obtained collagen fibers. After washing with water until neutral, freeze or freeze-dry for storage until use; (2) Extraction Add 5-15 times acetic acid with pH 2.0-3.0 and 1-15% pepsin to extract collagen fibers at 0-25°C for 2-3 days, centrifuge at 13500rpm for 1h, and take the supernatant with a final concentration of 0.6-1mol/L NaCl salting out, centrifugation to obtain the crude product; (3) Purification Reconstitute the crude product with acetic acid with pH 2.0-3.0, dialyze with 0.02mol/L Na ₂ HPO ₄ with pH 8.0 to inactivate the enzyme, then dialyze with acetic acid with pH 2.0-3.0, then dialyze with water and centrifuge to collect the precipitate, freeze or freeze dry storage. 2. the extracting method of sea cucumber collagen and collagen protein as claimed in claim 1, is characterized in that the extracting method of active collagen, (2) extraction and (3) purifying steps are carried out at 0～5 ℃ in the process. 3. the extracting method of sea cucumber collagen as claimed in claim 1, collagen protein, it is characterized in that the extraction method of collagen protein, (2) extraction and (3) purifying steps are to carry out at 6～25 ℃ in the technological process. 4. as claimed in claim 1, 2 or 3 described sea cucumber collagen, the extracting method of collagen protein, it is characterized in that (1) the Tris-HCl solution described in raw material processing is: EDTA, NaCl, and its pH value is 8.0. 5. the method for extracting sea cucumber collagen and collagen as claimed in claim 1, characterized in that the raw material in the raw material processing is the remaining sea cucumber after extracting the active substance in the sea cucumber.