JP4757413B2 - Sponge made of sea cucumber or starfish collagen and method for producing the same - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an invertebrate sea cucumber or starfish collagen belonging to echinoderms, more specifically, a sponge composed of high molecular weight collagen while maintaining intermolecular crosslinking in the sea cucumber or starfish body, and a method for producing the same About.
[0002]
[Prior art]
There are about 1100 species of sea cucumbers that belong to the echinoderms. Among them, for example, sea cucumbers and black sea cucumbers are used as they are for cooking dried or dried foods. Since it is not edible, it is not collected and can be said to be an almost inexhaustible resource. Similarly, starfishes, a kind of echinoderm, are not only edible, but are also troublesome for fishermen to damage shellfish.
In recent years, collagen, which is an extracellular matrix, has been used as a cosmetic raw material, a culture substrate, and a therapeutic implant, but it can usually be produced in large quantities and has a high denaturation temperature. Mammalian collagen, especially cattle and pigs, is used. Recently, however, attempts have been made to use collagen of fish (for example, Japanese Patent Application No. 05-502013, Japanese Patent Application No. 2000-148523, etc.) and birds (for example, Japanese Patent Application No. 11-202660) in addition to mammals.
It is known that sea cucumbers and starfishes, which are echinoderms, contain a large amount of collagen in their body walls, and research on the extraction of this collagen has also been conducted. However, sea cucumber or starfish collagen is highly insoluble due to the formation of many natural intermolecular crosslinks, and it takes a lot of labor and time to isolate and purify collagen. It was a problem to do.
[0003]
[Problems to be solved by the invention]
As a result of intensive studies on the use of sea cucumbers or starfish collagen, the present inventors have purified the highly insolubilized collagen contained in sea cucumbers or starfish without solubilization and use it as a sponge. The present invention has been completed and the object of the present invention is to provide a collagen sponge obtained from sea cucumbers or starfishes and a method for producing the same.
[0004]
[Means for Solving the Problems]
The gist of the present invention is an insoluble collagen sponge obtained by removing soluble components contained in sea cucumbers or starfishes. After the sea cucumbers or starfishes are mechanically pulverized, they contain a reducing agent and a salt. In a method for producing a sponge composed of sea cucumber or starfish collagen, characterized in that it is treated with a neutral solution and then the components solubilized by this treatment are removed and the insoluble components are purified, frozen and dried. is there. In purifying the insoluble component, it is preferable to treat with an alkaline solution. A chelating agent can be further added to the neutral solution.
That is, the present invention purifies without solubilizing highly insolubilized collagen contained in sea cucumbers or starfishes, and uses it as a sponge by freezing and drying, and this sponge is further solubilized. Compared to the sponge made of extracted collagen (monodispersed collagen in molecular form), or the sponge in which intermolecular crosslinking is introduced by post-treatment, it is excellent in mechanical strength, thermal stability, water retention ability, We were able to absorb the moisture quickly and obtain a good-feeling sponge.
[0005]
The present invention is described in detail below.
The sea cucumber used as a raw material is not limited as long as it is a sea cucumber belonging to the Echinodermata, sea cucumber class. Specifically, it is an edible sea cucumber, crested sea cucumber, jano sea cucumber, red sea cucumber, sea cucumber, sea bass sea cucumber, sea cucumber sea bream, sea bass sea cucumber, sea cucumber sea urchin Etc. Can do.
The starfish is not limited as long as it belongs to the starfish class. Specifically, the scallops of the scallops, the scallops, the scallops, the red starfish, the red starfish, the starfish, the starfish, the starfish, the starfish, the starfish, the starfish, the starfish, A red starfish of the family can be given.
As a part to be used, the body wall part which is the remaining part from which the internal organs such as the digestive tract are removed is used.
[0006]
The collagen sponge according to the present invention is obtained by removing soluble components contained in the bodies of sea cucumbers and starfishes, and is a sponge mainly composed of collagen, which is present in the sea cucumbers and starfishes. This sponge is characterized by leaving intermolecular crosslinks.
And the manufacturing method of this collagen sponge is described concretely. First, the part to be used as a raw material is mechanically pulverized and cut into minced or small pieces. For example, the body wall of sea cucumbers or starfishes from which the internal organs have been removed is made into minced or small pieces using a homogenizer, a cutter or the like. If the degree of mincing and fragmentation is not sufficient, it cannot be isolated and purified by the following neutral solution treatment. Moreover, when this process is carried out too much, the strength of the resulting sponge will decrease. Specifically, the desired state of mince and fragmentation is preferably such that it becomes ground as a result of grinding without adding water.
Next, a neutral solution containing a reducing agent and a salt is added to this mince, fragment, specifically mercaptoethanol or thioglycolic acid as a reducing agent, NaCl as a salt to adjust the pH to 6-9, Leave at 0-40 ° C for 1 day or more. This treatment time varies depending on the temperature, mince, and fragmentation state, but it is necessary to carry out treatment for one day or more.
Further, inorganic substances such as Ca can be removed by adding a chelating agent to the neutral solution. As chelating agents, EDTA (ethylenediaminetetraacetic acid), NTA (nitrilotriaceteic acid), HIDA (hydroyethyliminodiacetic acid), HEDTA (hydroxyethylene ethylenediaminetriacetic acid), DTRP (diethylene triamine pentaacetic acid), TTHA (triethylene tetraminehexaacetic acid), GLDA (dicarboxymethyleglutamic acid tetrasodium salt) , DHEG (dihydroxyethyl glycine), PDTA (1.3-propane diaminetetraacetic acid), DPTA-OH (1,3-Diamino-2-hydroxypropane tetraacetic acid), HEDP (hydroxyethylidene diphosphonic acid), NTMP (nitrilotris (methylene phosphonic acid), PBTC ( phosphonobutane tricaroxylic acid) etc. Especially in the case of starfishes, it is desirable to add a chelating agent to the neutral solution because Ca is contained in the body wall at a higher concentration than in the case of sea cucumbers.
By treatment with this neutral solution, proteins and polysaccharides other than collagen can be extracted into the solution in a free state, and at the same time, the color of sea cucumbers or starfish body walls can be largely removed by this treatment. . Insoluble collagen purified by centrifugation or decantation can be obtained from this treatment solution at a concentration of about 0.5% to 2%, but the concentration is higher by isolation by centrifugation and more reliably. be able to.
[0007]
Further, after this treatment, an acidic solution such as acetic acid is added to adjust the pH to 2 to 5, whereby purified insolubilized collagen having a higher concentration can be obtained. This is because insoluble collagen aggregates due to this treatment.
In this way, only highly insolubilized collagen can be precipitated without precipitation of proteins and polysaccharides other than collagen released by treatment with a reducing agent and salt or chelating agent under neutral conditions. Attempts were made to measure the molecular weight of the resulting precipitate by SDS-PAGE, but there was no component that could also enter the upper gel with a gel concentration of 3%. From this, the highly insolubilized collagen obtained as this precipitate was It is thought that many collagen molecules (molecular weight: about 300,000) are bound.
In this SDS-PAGE, collagen is electrophoresed after heat denaturation, and the α chain with a molecular weight of 100,000, which is a component of collagen molecules, can be observed. However, the collagen according to the present invention is highly insolubilized and the components are also Since it has a large molecular weight, it did not enter the gel even after denaturation. In addition, proteins other than collagen were not observed by SDS-PAGE, and it was confirmed that insoluble collagen was sufficiently purified.
Further, it was found that a soft sponge can be obtained by treating the precipitate obtained by the above treatment at a higher pH. That is, after putting the previous precipitate in a 0.1 NaOH solution under refrigerated conditions for 3 days and then collecting the precipitate again by centrifugation or decantation, the precipitate is thoroughly washed with water to become a soft sponge raw material. I found it.
[0008]
A sponge can be obtained by washing the precipitate obtained by the above treatment with water and then lyophilizing it. In addition, the density of the resulting sponge can be adjusted by diluting the precipitate with water as necessary during lyophilization. The obtained sponge is a sponge having high mechanical strength because highly insolubilized collagen as a raw material is derived from natural intermolecular cross-linking in sea cucumbers or starfish. In general, other collagens have lower heat denaturation temperatures than mammalian collagen, and this is expected from the low content of hydrixyproline, which is an amino acid unique to collagen by amino acid analysis. The sponge had a high denaturation temperature due to highly formed natural intermolecular crosslinking, and the denaturation temperature of the resulting sponge was 52 ° C.
This sponge can be used by taking advantage of the features of the sponge, such as cosmetic use such as a pack sponge and facial mask, research use such as a cell culture carrier, and medical use such as a wound covering agent.
[0009]
【Example】
The following examples are given to illustrate the present invention, but the present invention is not limited thereby.
Example 1
After opening the body wall of the mushroom, removing the built-in part and cutting it to about 5 mm square, a cut piece (50 g) containing the body wall as a main component was added to a neutral solution (NaCl 29.22 g), 2-mercaptoethanol (15.63 g). ), Tris (hydroxymethyl) aminomethane (12.12 g) was dissolved in water to make the total volume 1 L), and the mixture was stirred for 3 days in a refrigerator with a stirrer. The muddy emulsion obtained by this treatment was centrifuged (10,000 rotations, 30 minutes) to recover 1000 g of insoluble collagen as a precipitate. The collagen concentration in the precipitate was about 0.5%. This precipitate was transferred to a tray and freeze-dried to obtain 5 g of a sponge composed of sea cucumber collagen.
The denaturation temperature of the obtained sponge was measured. When the denaturation temperature was measured by DSC after sufficiently absorbing the sponge, a beak was observed at 52 ° C. Further, when water was dropped on the sponge, water droplets on the sponge were immediately absorbed by the sponge. From the above, it was found that this sponge has high thermal stability and high water absorption.
Example 2
EDTA · 2Na (18.61 g) was added to the neutral solution of Example 1, and after the same treatment, a sponge composed of sea cucumber collagen was obtained by lyophilization.
Example 3
500 g of insoluble collagen having a collagen concentration of about 0.5% obtained by the treatment of Example 1 was stirred for 3 days at 4 ° C. with 5 L of 0.1 M NaOH solution. Insoluble collagen was recovered as a precipitate from this alkaline solution by centrifugation. The precipitate was thoroughly washed with water, and then the washed precipitate was transferred to a tray and freeze-dried to obtain a sponge composed of sea cucumber collagen. The sponge obtained here was whiter and more flexible than the sponge obtained in Example 1 or 2.
Example 4
By using the same starfish as a raw material (50 g as a body wall), 4.5 g of a sponge made of starfish collagen was obtained in the same manner as in Example 2.
[0010]
【The invention's effect】
As described above, the present invention maintains the intermolecular cross-linkage in the sea cucumber or starfish body by a simple operation of removing soluble components from the sea cucumber or starfish collagen belonging to the echinoderms that have been less useful. High molecular weight collagen sponge can be obtained, and the resulting collagen sponge is derived from natural intermolecular cross-linking in sea cucumbers or starfish, so it has high mechanical strength, high water absorption, and high heat denaturation temperature. Have

Claims (4)

An insoluble collagen sponge obtained by removing soluble components contained in sea cucumbers or starfishes. After sea cucumbers or starfishes are mechanically pulverized, they are treated with a neutral solution containing a reducing agent and a salt, and then the components solubilized by this treatment are removed. A method for producing a sponge comprising sea cucumber or starfish collagen, characterized by purification, freezing and drying. The method for producing a sponge comprising sea cucumber or starfish collagen according to claim 2, wherein a chelating agent is further added to the neutral solution. 4. A method for producing a sponge comprising sea cucumber or starfish collagen, wherein the obtained insoluble component is washed with an alkaline solution having a pH of 13 in the production method according to claim 2.