JPH0593000A - Production of high-purity fish scale collagen soluble in acid - Google Patents

Abstract

PURPOSE:To provide a process for producing a high-purity acid-soluble collagen from fish scale in a state free from gelatin which is a depolymerized and denaturated collagen. CONSTITUTION:The objective process for producing an acid-soluble fish-scale collagen comprises the steps of deashing the fish scale; extracting an acid-soluble collagen from the deashed fish scale with an acidic aqueous solution; and recovering the extracted acid-soluble collagen, wherein the above steps are carried out at <=15 deg.C.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing acid-soluble collagen which is useful as various biomedical materials for medical use, cosmetic materials, etc., from fish scales which are organic industrial wastes.

[0002]

2. Description of the Related Art Fish such as sardines have a large amount of scales peeled off when they are landed or processed and are mixed in drainage water. In order to prevent environmental pollution due to wastewater, it is necessary to collect fish scales in wastewater by filtration. However, fish scales have no utility value as they are, and most of them are currently discarded.

Fish scales usually contain about 50% or more ash and about 40% or less protein. Most of the ash is composed of calcium phosphate (hydroxyapatite), which is about 80% of protein.
Is known to be collagen. Conventionally known methods for extracting collagen from fish scales include a method of directly extracting fish scales with hot water and a method of directly extracting acetic acid with acetic acid [Journal of Fisheries Society of Japan 54 (11), 1987-199].
2 (1988)].

[0004]

In the method of directly extracting collagen from fish scales with hot water, low-molecular-weight collagen gelatin is obtained, and in the method of directly extracting collagen with acetic acid, gelatin is contained in collagen. Therefore, there is a problem that the purity of collagen is low. An object of the present invention is to provide a method for producing high-purity acid-soluble collagen from fish scales, which does not contain gelatin, which is a denatured low molecular weight product of collagen.

[0005]

The present invention comprises the steps of decalcifying fish scales, extracting acid-soluble collagen from the decalcified fish scales with an acidic aqueous solution, and recovering the extracted acid-soluble collagen. The present invention also provides a method for producing acid-soluble fish scale collagen, which comprises performing the above steps at 15 ° C or lower.

The present invention focuses on the fact that collagen extracted from fish scales behaves sensitively to temperature and is denatured into low-molecular-weight gelatin, and the main step, preferably all steps, of producing acid-soluble fish scale collagen. Is carried out under a temperature condition of 15 ° C or lower, more preferably 4 to 5 ° C.

The fish scale used as a raw material maintains freshness,
Refrigerated storage, preferably frozen storage is preferred.
Since the collected fish scale is contaminated with considerable impurities such as dorsal fin and caudal fin, it is preferable to wash them with water to remove them in advance. Further, in order to remove the surplus protein adhering to the surface of the fish scale, it is preferable to wash with an 8 to 12% by weight, preferably 9 to 11% by weight sodium chloride aqueous solution for 10 to 48 hours, preferably for 24 to 48 hours. Since the waste liquid after cleaning becomes a considerable suspension, it is preferable to repeat it until the waste liquid does not become cloudy. Preferably 3
It is recommended to change the washing solution 5 times to wash. It is preferable to appropriately change the washing time and the number of washings by observing the washing state of the fish scale. The washing step is also preferably performed at 15 ° C. or lower.

A buffer solution prepared with a chelating agent is used in the decalcification step of the washed fish scales. Examples of the chelating agent include ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium, and ethylenediaminetetraacetic acid tetrasodium (hereinafter collectively referred to as EDTA). The EDTA buffer solution preferably has a pH in the range of 6.0 to 8.0. More preferably, the pH is 7.0 to 7.5. When the pH is lower or higher than the above range, collagen denaturation is likely to occur, which is not preferable. The EDTA concentration in the buffer solution is 0.1.
3 to 0.6 mol / liter, more preferably 0.4 to 0.
It is 5 mol / liter.

Add washed fish scales to EDTA buffer,
Decalcify. The ratio of fish scale to EDTA buffer is adjusted to be 1:15 to 1:25 by weight / volume ratio, more preferably 1:18 to 1:23. Decalcification is carried out for 24 to 48 hours, more preferably 36 to 48 hours. If decalcification is insufficient, the amount of acid-soluble fish scale collagen extracted will also be insufficient. Therefore, it is preferable to repeat the decalcification step 3 to 5 times. About 90 wt% is deashed in the first deashing step, and about 95 wt% or more is deashed in the third deashing step.
The waste liquid after decalcification is very viscous. For this reason, it is preferable to use centrifugation, filtration, or the like to separate the sample and the waste liquid. A preferred operation is centrifugation followed by filtration. This is because the time required for filtration can be shortened and the loss of the sample can be reduced.

The crude fish scale collagen obtained by decalcification is preferably washed with a buffer solution to adjust the pH before entering the collagen extraction step. As the buffer solution, for example, a mol / 15 phosphate buffer solution (pH about 8.0) prepared from monosodium hydrogen phosphate and disodium hydrogen phosphate is used. Then, the acid-soluble fish scale collagen is extracted by shaking with an acidic aqueous solution. The acidic aqueous solution used is preferably adjusted to pH 1.0 to 5.0, preferably pH 2.0 to 4.0. The acidic aqueous solution may be either an organic acid or a mineral acid,
For example, acetic acid, hydrochloric acid, etc. are exemplified. When the pH is lower than the above range, it is gelatinized, and when it exceeds the above range, the yield is lowered, which is not preferable. The ratio of coarse fish scale collagen to acidic aqueous solution is 1:15 to 1: 2 by weight / volume ratio.
A range of 5, preferably 1:18 to 1:23 is preferably employed.

Coarse fish scale collagen was added to an acidic aqueous solution,
The acid-soluble fish scale collagen is extracted by immersion for 24 to 48 hours, preferably 36 to 48 hours. The extraction is preferably carried out 1 to 5 times, preferably 3 to 5 times by exchanging the acidic aqueous solution. About 80% by weight of acid-soluble fish scale collagen can be extracted even by the first extraction operation.

Separation of the acid-soluble fish scale collagen dissolved in the acidic aqueous solution and the residue may be carried out by an ordinary physical separation means, but a centrifugal separation method is preferably adopted. The method for fibrating / precipitating and recovering collagen from the acid-soluble fish scale collagen solution from which the residue has been separated may be carried out in the same manner as the method used for pig skin or cow skin collagen. That is, sodium chloride is added to the above solution to increase the salt concentration and to form fibrosis. Alternatively, sodium hydroxide may be added to adjust the pH to around neutrality and fibrosis.

The fibrillated collagen is recovered by, for example, a centrifugation method, then dissolved again in an appropriate amount of an acidic aqueous solution, and dialyzed with a neutral solution such as a mol / 15 phosphate buffer solution (pH 7.5) or ion-exchanged water. Then, the acid-soluble fish scale collagen is recovered. After dialysis, freeze-drying gives high-purity acid-soluble fish scale collagen.

Hereinafter, the present invention will be described in more detail based on examples. In the examples,% means% by weight unless otherwise specified.

Example 100 g of fresh sardine fish scales were washed with water at about 5 ° C.,
After removing impurities, the mixture was placed in 2 liters of a 10% sodium chloride aqueous solution and shaken under a temperature condition of 4 to 5 ° C. for 24 hours,
The supernatant was separated and removed by centrifugation and filtration. This operation was repeated 3 times by exchanging the liquid and then washed with water to obtain washed fish scales.

The washed fish scale was treated with 0.5 mol of tetrasodium ethylenediaminetetraacetate and 0.05 mol of tris-hydrochloric acid (p.
H7.5) and 0.5 mol of EDTA buffer 2 prepared from
The mixture was placed in a liter and shaken under a temperature condition of 4 to 5 ° C. for 48 hours. After that, the supernatant was separated and removed by centrifugation and filtration. Repeat this operation by replacing the EDTA buffer
After repeated times, the product was washed with water to obtain 30.5 g of crude fish scale collagen.

600 ml of a mol / 15 phosphate buffer solution (pH 8.0) was added to the crude fish scale collagen, and
After washing with shaking at a temperature of 5 ° C to separate and remove the supernatant, 600 ml of a 0.5 mol acetic acid aqueous solution was added, and the mixture was shake-extracted at a temperature of 4-5 ° C for 48 hours to extract acid-soluble fish scales. A solution of collagen was obtained. 1 of this solution
Centrifugation was performed at 8,000 rpm for 30 minutes to separate the acid-soluble fish scale collagen solution and the residue. The residue was washed with water, and then shaken and extracted again with a 0.5 mol acetic acid solution. This was repeated 3 times to obtain about 1.5 liters of acid-soluble fish scale collagen solution.

The salt concentration was adjusted by adding sodium chloride to the acid-soluble fish scale collagen solution so that the final concentration was 5%. The fibrous precipitate produced by this was added to 4-5
The mixture was centrifuged at 18,000 rpm for 30 minutes under the temperature condition of ° C. The obtained precipitate was again dissolved in a 0.5 mol acetic acid aqueous solution, and the solution was dialyzed with a mol / 15 phosphate buffer (pH 7.5) for 72 hours under a temperature condition of 4 to 5 ° C. After freeze-drying, 2 g of acid-soluble fish scale collagen fibrosis was obtained.

This acid-soluble fish scale collagen fibrous material is
It was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGB) and amino acid analysis. In SDS-PAGB, α
The band pattern peculiar to collagen such as ₁ chain, α ₂ chain and β chain was shown, and gelatin was not mixed. The amino acid analysis also showed an amino acid composition peculiar to collagen, and it was confirmed that the acid-soluble fish scale collagen has high purity. Table 1 shows the amino acid composition of pig skin collagen and acid-soluble fish scale collagen.

Table 1 Amino acid residues Pig skin Acid-soluble fish scale Collagen Collagen Hydroxyproline 125 63 Aspartic acid 45 52 Threonine 17 24 Serine 36 40 Glutamic acid 70 73 Proline 112 108 Glycine 328 324 Alanine 99 125 1/2 cystine 0 0 Valine 22 19 Methionine 4 10 Isoleucine 12 3 2 8 Phenyltyrosine 25 8 13 22 Hydroxylysine 5 8 Lysine 30 32 Histidine 7 19 Arginine 47 52 Note: A-chain amino acid composition. The values are shown per 1000 amino acid residues.

[0021]

EFFECTS OF THE INVENTION According to the present invention, there is provided a method for producing highly pure acid-soluble fish scale collagen as compared with the conventional method for extracting acid-soluble fish scale collagen from fish scale.

Claims (2)

[Claims] 1. A step of decalcifying fish scales, a step of extracting acid-soluble collagen from the decalcified fish scales with an acidic aqueous solution, and a step of recovering the extracted acid-soluble collagen. A method for producing an acid-soluble fish scale collagen, which comprises performing 2. The method for producing an acid-soluble fish scale collagen according to claim 1, wherein decalcification is carried out with ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium or ethylenediaminetetraacetic acid tetrasodium.