JPS6143999A - Preparation of acid dispersion liquid containing collagen fiber - Google Patents

Abstract

PURPOSE:To obtain the titled acid dispersion liquid having high activity and suitable as a clinical examination reagent, especially a reagent for the determination of the platelet coagulation activity and platelet adhesion activity, by treating the Achilles tendon of an animal with a protein hydrolase, followed by other specific treatments. CONSTITUTION:The Achilles tendon of an animal is treated with a protein hydrolase free from collagenase. The treated product is washed with a aqueous solution of an inorganic salt, dried, and pulverized. The obtained fine powder of collagen fiber is swollen by immersing in an acid, and the swollen fiber is further pulverized to obtain the objective acid dispersion of collagen fiber.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing an acid dispersion containing collagen fibers suitable for use as a clinical test reagent, such as a reagent for measuring platelet aggregation ability or platelet adhesion ability.

BACKGROUND OF THE INVENTION Conventionally, collagen fiber-containing acid dispersions have been commercially available and used as clinical test reagents, particularly as reagents for platelet adhesion measurement and platelet adhesion measurement.

Problems to be Solved by the Invention However, conventionally commercially available collagen-containing M acid dispersions have poor activity as reagents, and also have the drawback that their activity deteriorates significantly when they are used after being stored for a long period of time. Was.

Friendly Means to Solve the Problems The present invention has been made in view of the above-mentioned problems. (1) Collagen is produced by treating animal Achilles key with a proteolytic enzyme other than collagenase. (2) washing the above-mentioned processed material with an aqueous inorganic salt solution and removing sugars contained in small amounts in the Achilles key; (3) drying the above-mentioned washed material; a step of pulverizing to obtain a fine collagen fiber powder; (4j1) a step of immersing the collagen fine fiber powder in an acid and subjecting it to swelling; (5) a step of further pulverizing the swollen collagen fiber powder, respectively. This is what we have prepared.

That is, in order to obtain a highly active collagen fiber-containing acid dispersion, the present inventors focused on Achilles' hematoma, which has a greater fiber cohesive force and greater fiber orientation than animal dermis, bone, etc. This is how the present invention was accomplished.

Collagen fibers from fresh animal Achilles membranes have more directional fibers than collagen fibers from other parts of the dermis, such as the mid-bones of the dermis, and even after being finely powdered and dispersed in acid, they return to the neutral range. The fiber cohesive force at 9 o'clock is extremely large. Therefore, since they have large aggregation and adhesive activities even in small amounts for measuring platelet aggregation ability and platelet adhesion ability, they are convenient for use as clinical test drugs. However, because the fiber regeneration power of the animal Achilles' collagen fibers is larger than that of collagen fibers in other parts of the body, even if the collagen fibers are purified and crushed into fine collagen fiber powder of 100 μm or less, It is difficult to obtain a uniform acid dispersion g8 by simply immersing the dispersion in an acid, and if such a dispersion is returned to a neutral region, fibers aggregate more than necessary.

Visually, large fiber bundles can become hot and inconvenient. In the process of making the adhesion measurement reagent,
In particular, a dispersion that maintains fine collagen fibers in a neutral region is required.

Therefore, in the present invention, a fine collagen fiber powder is immersed in $1 to swell it and then pulverized to obtain a uniform acid dispersion.

Collagen is a biomaterial and, like other proteins, is unstable to heat and easily corrodes, so each of the above steps is preferably carried out under semi-sterile conditions.

For example, when the purified collagen powder that has gone through the steps (1) to (3) above is swollen with an acid aqueous solution, ethanol sterilization,
Alternatively, ethylene oxide side gas sterilization may be used, and the acid aqueous solution used in the above-mentioned swelling process is also preferably sterilized by filtration or boiling.

In the method of the present invention, it is preferable to use a weakly basic vertical sodium hydride aqueous solution or a sodium carbonate aqueous solution as the inorganic salt aqueous solution.

Generally, animal Achilles collagen content is high, but other proteins and other components of Achilles pyogenes can be solubilized and removed in Steps 1 and 21 of the present invention.

The pH of the acid aqueous solution is preferably 1 to 5, and the pH is 2 to 4.
It is more preferable that

Example The present invention will be specifically explained below with reference to Examples.

Take out the fresh beef Achilles M while pre-rinsing it with cold water.
Cut into approximately 5cm squares using a utility knife.

The chopped beef Achilles m was soaked in cold water and crushed using a micro cutter (manufactured by Stefan GmbH, West Germany), and the crushed product was thoroughly washed with cold water. 1 piece of washed crushed material
Protein hydrolase other than collagenase, such as pancreatin, was added in an amount of about inn% based on the dry Achilles recommended standard to hydrolyze proteins other than collagen contained in the cow's Achilles oboro, solubilize them in water and remove them. A 1% aqueous solution of sodium bicarbonate was added to the protein hydrolyzed and crushed product to elute and remove the sugars contained in small amounts in the Achilles nut. After sufficiently repeatedly washing insoluble matter with cold water, the immersion solvent was replaced with one portion of ethanol.

The pulverized material subjected to the ethanol immersion treatment is immersed in a mixed solvent of ethanol:ether = 1:1, and then immersed in ethanol.Thus, the fat content in the collagen fibers is removed. Removed. After this solvent replacement treatment, the insoluble matter was immersed in a solvent system mainly consisting of ethanol, which has a lower boiling point than water.

After removing most of the soaking solvent by suction filtration, the remaining insoluble ground material was air-dried. Since the air-drying conditions are not necessarily constant, the moisture content of the insoluble pulverized material can be determined by measuring the nitrogen content using the Kehrmehr method and determining the moisture content by determining the difference between the nitrogen content before and after air-drying. The ratio was set to 5% or less. In case f, vacuum drying was performed after air drying.

Purified collagen with a water content of 5 qb or less was still in the form of fiber bundles. Therefore, this purified collagen pulverized product was re-pulverized using a Wiley pulverizer model W-100S (manufactured by Ikeda Rika).
The mixture was fractionated through a 2-mesh sieve to obtain collagen fiber powder having a length of 1 ws or less. The mixture was further fractionated through a fine sieve to collect shredded collagen fibers having a length of about 100 μm or less. Naturally, this shredding and fractionation could be repeated, and shredded collagen fibers with a length of 100 μm or less could be obtained. The shredded collagen fiber powder thus obtained was stored frozen in a container such as polyethylene to prevent it from absorbing moisture.

Purified and shredded cow Achilles recommended fiber powder is sterilized with ethanol or ethylene oxide gas as necessary, and then sterilized by filtration or boiling at a temperature of 10C or less. pH~3)
The mixture was soaked in an amount such that the collagen content was about 0.4 to 0.5% to make it swell. In addition, the type of animal used is necessary for the shredded collagen fibers to contain 1/10% water and to swell sufficiently to produce a fine and uniform acid dispersion as described below. Depending on factors such as age, pension, gender, living environment, etc., it takes approximately 1 to 10 days.

The swollen 1'c collagen fiber base acid dispersion (
Collagen content: 0.4% N50id, capacity 10011
Put it in a Ll homogenizer container, and use a homogenizer (manufactured by Nippon Seiki: Universal HEDI type) with 800 Or, p
, m for 20 minutes to obtain a homogeneous acid dispersion containing even finer collagen fibers. The resulting acid dispersion had an egg white-like viscosity. At this time, the homogenizer was operated while the contents of the homogenizer were externally cooled (preferably to 4 C or less) with iced water or the like to avoid the risk of thermal denaturation of the collagen due to the generation of heat accompanying mechanical dispersion. The aggregation and adhesive strength of collagen fibers are influenced by the quaternary structure of collagen molecules, and the collapse and disturbance of the quaternary structure due to denaturation directly affects the aggregation and adhesive strength of collagen fibers.

The acid dispersion obtained in this way has an egg white shape and is viscous, so after the dispersion is completed, the dispersion and the bud dispersion are heated again in a homogenizer at a rotation speed of 5000 to 8000 r, p, m for an operating time of 2. It was homogenized under conditions of ~3 minutes to obtain a uniform acid dispersion with a collagen content suitable for storage and use.

When we measured the resulting uniform acid dispersion and measured its change over time, we found that it is stable for 1 to 2 years when packed in a bottle and stored in a refrigerator at a temperature of 2 to 8C. Obtained. At this time, when the dispersion liquid freezes, fiber aggregation occurs and the activity decreases, so it is necessary to store it under refrigeration.

When the platelet aggregation ability and platelet adhesion ability of the collagen fiber acid dispersion thus produced were measured, it was found to have about 10 times more activity than the conventional collagen fiber acid dispersion I.

Preservatives such as methylparaben and ethylparaben may also be added to the resulting uniform acid dispersion.

In addition, when a storage test was conducted on the purified collagen dry powder before swelling with 0.01N lactic acid aqueous solution,
Simply sealing the dry powder in a polyethylene bag and storing it in the freezer did not lead to a decrease in activity.

As described in detail of the invention, in the present invention, an acid dispersion liquid 8 is prepared using Achilles' collagen, which has a stronger fiber directionality and an extremely large fiber cohesive force compared to collagen fibers in other parts. Therefore, it is possible to obtain extremely highly active reagents for use in measuring platelet aggregation ability, platelet adhesion ability, etc.

Moreover, since the collagen fiber powder that has been finely ground is immersed in acid to swell and peel, it is ground again to obtain a uniform dispersion, so that the acid dispersion can be returned to a neutral range especially for measurement. Sometimes larger fibers aggregate than necessary, causing inconvenience! I've never had one.

Claims (1)

[Claims] 1. (1) A step of treating the Achilles tendon of an animal with a protein hydrolase that does not contain collagenase, (2).
a step of washing the above-mentioned treated material with an aqueous inorganic salt solution; (3) a step of pulverizing the above-mentioned washed material in a dry state to obtain collagen fine fiber powder; (4) a step of washing the above-mentioned collagen fine fiber powder with an acid. A method for producing an acid dispersion containing collagen fibers, comprising the following steps: (5) pulverizing the swollen collagen fiber powder. 2. The method according to claim 1, wherein each step is carried out under semi-sterile conditions. 3. The method according to claim 1 or 2, characterized in that sodium hydrogen carbonate or a sodium carbonate aqueous solution is used as the inorganic salt aqueous solution. 4. The method according to any one of claims 1 to 3, characterized in that an acetic acid aqueous solution or a lactic acid aqueous solution is used as the acid dispersion medium.