JPH01115902A - Production of hyaluronic acid - Google Patents

Abstract

PURPOSE:To obtain in high yield high-viscosity and high-purity hyaluronic acid useful as a base material for drugs and cosmetics, by chopping and making the raw material into a paste while keeping in a freezed state and sterilizing the product at high temperatures for a short time followed by quenching and then making a protease treatment. CONSTITUTION:A raw material containing large quantities of hyaluronic acid such as cockscombs, skins or synovial fluid is rapidly freezed followed by chopping with, e.g., a meat chopper and then making the product into a paste using, e.g., a homogenizer, ultrafine grinder. This paste is then sterilized normally at 70-140 deg.C for pref. 1sec-10min followed by quenching and then performing treatment with a protease (e.g., papain, trypsin, alkalase), thus obtaining the objective hyaluronic acid. This hyaluronic acid can be prevented from quality deterioration due to hyaluronidase or prolonged heating, thus enabling improvement of its quality and yield along with significant reduction in its treatment time.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing high-molecular hyaluronic acid with high purity and high yield.

(Prior art and its problems) Hyaluronic acid is a type of complex polysaccharide consisting of amino sugar and uronic acid. It is widely distributed in animal tissues, especially mesenchymal tissues, and is abundant in the vitreous body, aqueous fluid, umbilical cord, joint fluid, pleural fluid, skin, chicken comb (so-called comb), etc. is a binding substance that forms a gel as a free acid and salt and fills the spaces between cells and fibers.

Hyaluronic acid, which has properties such as viscosity, water retention, and lubricity, is in high demand as a base material for medicines and cosmetics, and high viscosity and high purity are required.

Conventionally, the basic method for obtaining hyaluronic acid is to extract the raw material with water, precipitate it with cetylpyridinium chloride from the aqueous solution, dissolve it in a lium chloride solution, and then reprecipitate with ethanol. It is being

In order to produce large quantities of high-yield, high-quality hyaluronic acid, it is necessary to pre-treat raw materials, and many methods have been proposed. For example, connective tissue is heated to approximately 70 to 130°C in its original form, then treated with proteolytic enzymes, and then treated using conventional methods (Japanese Patent Publication No. 60-904).
No. 2 Publication No. 1), a method in which a cock's comb is left in its shape or cut into pieces, heated at 35 to 65°C, treated with a leukolytic enzyme, and then treated using a conventional method (Japanese Patent Publication No. 8083/1983); A method in which hyaluronic acid-containing raw materials that have been heat-treated in advance are made into a paste and then treated with protease (Japanese Patent Laid-Open No. 60-2
4194) etc.

All of these methods are simple and efficient methods for mass production, but hyaluronidase mixed into the raw materials and during the manufacturing process reduces the molecular weight of hyaluronic acid, and they require long-term heating. There were problems such as deterioration of quality.

An object of the present invention is to provide a method for producing hyaluronic acid with good yield and preventing such quality deterioration caused by hyaluronidase.

(Means for solving the problems) The method for producing hyaluronic acid of the present invention includes rapidly freezing raw materials,
It is characterized in that it is shredded and made into a paste in a frozen state, then sterilized at high temperature for a short time, rapidly cooled, treated with protease, and then processed by a conventional method.

As a raw material, connective tissue containing a large amount of hyaluronic acid is used. Such raw materials include chicken comb, skin, nuchal ligament,
Examples include synovial fluid, zona, blood vessel walls, cartilage, vitreous body, etc., and cockscomb is particularly preferred since it is easily available in large quantities.

Raw materials are quickly frozen immediately after collection. This is to prevent the quality of raw materials from deteriorating due to hyaluronidase over time after they are collected.

Do not thaw frozen raw materials (shred them as they are,
Make a paste. For shredding, a conventional method can be used; for example, a meat chopper or the like is sufficient. To make a paste, use a homogenizer, an ultrafine crusher (such as a supermass colloider), and add water or ice flakes as necessary to maintain fluidity. In order to suppress the rise in temperature of the paste, it is preferable to operate at a temperature of about 10°C or less, particularly preferably 2°C or less. The efficiency of extracting hyaluronic acid can be improved by cutting it into pieces and making it into a paste.

Next, the paste is sterilized at high temperature for a short time, preferably at a temperature of 70 to 140° C. for a short time of 1 second to 10 minutes. After sterilization at high temperatures for a short period of time, it is rapidly cooled. In this step, hyaluronidase is completely inactivated, and quality deterioration during subsequent operations can be prevented. Moreover, since the process is carried out in a short time, there is no deterioration in quality due to long heating times as in conventional methods.

Enzymes used for protease treatment include, for example, plant-derived enzymes such as papain, ficin, and bromelain, animal-derived enzymes such as trypsin and chymotrypsin, prolysin,
Examples include microbial enzymes such as actinase, alcalase, and neutrase. Although it is possible to use only one type of these enzymes alone, if multiple types of enzymes with different properties are mixed and used together, the treatment will be more effective than when treated with one type of enzyme or multiple times with multiple types of enzymes. This method is particularly advantageous because it can shorten processing time and improve purity and yield.

After the protease treatment, a conventional method is used.

After purification, for example by centrifugation or filtration, a quaternary ammonium salt (eg cetylpyridinium chloride) is added to the supernatant or filtrate. The obtained precipitate is redissolved in a saline solution or the like, and after adding ethanol, the precipitate is collected and dried.

(Effects of the Invention) According to the present invention, the raw material is shredded and made into a paste in a frozen state, sterilized at high temperature for a short time, and treated with protease. It is possible to prevent quality deterioration, improve quality and yield, and significantly shorten processing time.

(Examples) Example 1 A chicken comb collected immediately after slaughter was quickly frozen in liquid nitrogen using a spray freezing device. 1 kg of the frozen chicken comb was shredded using a meat chopper. Add 2k flaky ice to this
g was added thereto, and the mixture was made into a paste using Super Mascolloider (manufactured by Masuko Sangyo Al). The resulting paste was flash sterilized at 130° C. for 3 seconds and immediately rapidly cooled. Water was added at 21°C to adjust the pH to 7.0, and 300ml of actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.) was added as a protease, followed by incubation at 50°C for 3 hours. The resulting solution was then filtered to obtain 3.21 ml of filtrate. To this filtrate was added 600 i of 5 (w/v)% cetylpyridinium chloride solution. The formed precipitate was collected by filtration and redissolved in 0.5M sodium chloride solution.

Ethanol was added to this solution at a concentration of 60 (v/v)%, and the resulting precipitate was collected by filtration and dried.

Example 2 Using a paste obtained by processing in the same manner as in Example 1, 1
It was flash sterilized at 00°C for 300 seconds and immediately rapidly cooled.

Add 2β of water, adjust the pH to 7.0, and use 15 mg of actinase E and 0.5 L of Neutrase (manufactured by Novo Industries Japan) as protease.
．． 5 ml was added and incubated at 55°C for 2 hours. The resulting solution was then filtered and treated as in Example 1.

Comparative Example 1 Example 1 except that the resulting paste was sterilized at 60°C for 30 minutes instead of being instantaneously sterilized at 130°C for 3 seconds.
Hyaluronic acid was obtained in the same manner as above.

In this comparative example, hyaluronidase was insufficiently deactivated.

Comparative Example 2 Hyaluronic acid was obtained in the same manner as in Example 1, except that slowly thawed frozen chicken combs were used as the raw material.

Comparative Example 3 11 parts of water was added to 1 kg of frozen chicken comb, and the mixture was heated at 95°C for 45 minutes. After removing the solids and mincing them, add 41 liters of water.
After the protease treatment, hyaluronic acid was obtained in the same manner as in Example 1, except that the pH was adjusted to 7.0.

Table 1 shows the yield, purity, and molecular weight of hyaluronic acid obtained in Examples and Comparative Examples.

Table 1 Note 1) The amount of uronic acid measured by the carbazole sulfuric acid method is
．． Calculated by multiplying by 06.

2) Calculated from the intrinsic viscosity measured using an Ubbelohde viscometer.

Example 3 Quick-frozen pig skin (1 kg) was used as a raw material, heat treatment was performed at 100°C for 30 seconds, and protease treatment was performed using water for 32 seconds.
After addition Alcalase 0.6L, Neutrase 0.5L
(All manufactured by Novo Industries Japan) 1 liter each
Hyaluronic acid derived from pig skin was obtained by carrying out a series of operations according to Example 1, except for adding ml and incubating at 50° C. for 7 hours. The yield is 3.
1 g, the purity measured in the same manner as in the above example was 89.6%, and the molecular weight was 170,000.

Claims (1)

[Claims] A method for producing hyaluronic acid, which is characterized in that raw materials are rapidly frozen, shredded and made into a paste in a frozen state, then sterilized at high temperature for a short period of time, rapidly cooled, treated with protease, and then treated by a conventional method.