JPH0556957B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for purifying hyaluronic acid. Hyaluronic acid has the structural formula It is a type of polysaccharide represented by
It helps retain moisture, acts as a wetting agent, and prevents bacterial invasion. This substance has recently been developed as a cosmetic, a therapeutic agent for the eyes, skin, joints, etc., and a protective agent for surgery. Conventional technology It has been known for a long time that hyaluronic acid exists as a capsule component of bacteria of the genus Streptococcus [A.P. McLennan (AP
Maclennan): Journal of General
Microbiology (J.Gen.Microbicl.), 15 ,
485-491, 1956]. Alternatively, a method is known in which hyaluronic acid is produced by carrying out aerated agitation culture in a nutrient medium containing 3% or more sugar content (Japanese Patent Laid-Open No. 58-56692). In conventional technology, methods for removing high molecular compounds present as impurities in the fermentation liquid include:
Treatment with trichloroacetic acid or chloroform-isoamyl alcohol, use of proteolytic enzymes, or selective precipitation of hyaluronic acid with cetylpyridium chloride have been used. Problems to be Solved by the Invention Hyaluronic acid is increasingly being used as a raw material for cosmetics, and is also expected to be used as a pharmaceutical product. In order to use hyaluronic acid as a medicine, it is essential that it be a purified product that does not contain proteins, nucleic acids, pyrogenic substances, etc. It is also said that the larger the molecular weight and the higher the viscosity, the better the effectiveness. The conventional separation and purification method for hyaluronic acid is
This method is not industrially advantageous because the process is complicated, the chemicals used are expensive, and there are problems with the toxicity of the chemicals. Therefore, there is a need for the development of an industrially advantageous method for purifying hyaluronic acid. Means for Solving the Problems The present invention focused on the fact that hyaluronic acid and polymer compounds present as impurities are negatively charged substances, and investigated a method for purifying hyaluronic acid using an ion exchange resin. As a result, anion exchange resins generally adsorb proteins, nucleic acids, pyrogens, and hyaluronic acid well, but macroreticular anion exchange resins with a high content of divinylbenzene adsorb only polymeric hyaluronic acid. We discovered this phenomenon and completed the present invention. The present invention will be explained in detail below. The present invention provides a method for purifying hyaluronic acid characterized by using a macroreticular anion exchange resin when separating and purifying hyaluronic acid from a liquid containing hyaluronic acid. The macroreticular anion exchange resin used in the present invention is a styrene-based anion exchange resin with a high divinylbenzene content, which is also called a macroporous type or a high porous type. Diaion HPA−25,
HPA-75, IRA-900 manufactured by Amberlight,
Examples include IRA-904. The amount of anion exchange resin used is 0.7 to 1 (v/
v) degree is desirable. The present invention is preferably applied to a culture solution containing hyaluronic acid obtained by a fermentation method. Hyaluronic acid culture solution obtained by fermentation method is
Microorganisms capable of producing hyaluronic acid (e.g., Streptocatchus zooepidemics)
NCTC7023) can be obtained by culturing according to the culture method shown below. As the medium, either a synthetic medium or a natural medium can be used as long as it contains adequate amounts of carbon sources, nitrogen sources, inorganic substances, and other nutrients. As the carbon source, glucose, sucrose, waste molasses, starch hydrolyzate, etc. can be used. As a nitrogen source, it is preferable to add an organic nutrient source such as peptone, polypeptone, yeast extract, corn steep liquor, casein hydrolyzate, brain heart injection, horse serum, etc., and use in combination with ammonium sulfate, ammonium nitrate, ammonium chloride, ammonia, etc. It is also possible. Examples of inorganic salts that can be used include sodium chloride, sodium phosphate, potassium phosphate, magnesium sulfate, sodium thiosulfate, ferrous sulfate, manganese sulfate, calcium chloride, and calcium carbonate. Of course, in some cases, such as when using natural nutritional sources, it is possible to satisfy the needs only with inorganic salts contained in natural products. Further, various vitamins such as thiamine, nicotinic acid, biotin, pantothenic acid, etc. can be used as necessary. Cultivation is performed under aerobic conditions such as shaking culture and aeration culture. Culture temperature is 25~42℃, preferably 30~
38℃ is suitable. A suitable pH during culture is 5 to 9. PH adjustment is performed using ammonia water, sodium hydroxide, potassium hydroxide, calcium carbonate, etc. The culture period is usually 2 to 4 days, and hyaluronic acid is mainly accumulated outside the bacterial cells. Purification of hyaluronic acid from the hyaluronic acid culture solution obtained as described above is performed as follows. Add hyaluronic acid culture solution to water to obtain 0.5 hyaluronic acid
After diluting to about 2g/preferably 1g/adding carbon of about 1 to 10% (v/v) and stirring at 15 to 30°C for about 1 hour to mix the culture solution and water. , obtain the filtrate using Nutsuchie, etc. This carbon treatment solution is applied to a macroreticular anion exchange resin at a spatial velocity (SV) of 1 to
The treated solution is obtained by passing through the column at 2 (h ^-1 ). Adjust the pH of this treatment solution to around neutrality with hydrochloric acid, etc., and
After adding sodium chloride to the desired level,
A water-soluble organic solvent such as acetone, methanol, ethanol, n-propanol, iso-propanol, etc. is added in an amount of 1 to 4 times the amount of the treatment solution to precipitate sodium hyaluronate. The obtained precipitate was washed with 50-100% ethanol,
Purified hyaluronic acid can be obtained by vacuum drying. The purified hyaluronic acid obtained by the method of the present invention always shows a viscosity of 100 cp or more, and the molecular weight according to the limiting viscosity method [Biochim.Biohys.Acta 42 , 476, (1960)] is 200,000 cp or more. That's all. The molecular weight of hyaluronic acid present in the culture solution is not constant in all batches. Therefore, when all the hyaluronic acid in a culture solution is purified without selecting its molecular weight, its viscosity varies from about 60 cp to about 100 cp. However, according to the method of the present invention, proteins, nucleic acids, pyrogenic substances, and hyaluronic acid with a small molecular weight are adsorbed on the macroreticular anion exchange resin, so hyaluronic acid with a molecular weight of 2 million or more can be obtained. The present invention can also be used to repurify currently commercially available products. Currently commercially available hyaluronic acid is mainly extracted from chicken crests, etc., but if purified using the method of the present invention, a purified product of hyaluronic acid with a molecular weight of 1.5 million or more can be obtained, although the yield will be lower. can. To repurify a commercially available product, dissolve sodium hyaluronate in water to a concentration of about 0.5 to 2 g/g, and add it to a macroreticular anion exchange resin at a space velocity (SV) of 1 to 2.
(h ^-1 ), and purified sodium hyaltonate can be obtained in the same manner as in the purification of the hyaluronic acid culture solution. Examples of the present invention are shown below. Example 1 Streptococcus zooepidemics
NCTC 7023〔Journal of General
Microbiology (J.Gen.Microbiol.) 15 ,
485-491 (1956)] on a Brain Heart Infusion Agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37°C for 16 hours.
Seed medium (PH7.0) consisting of 1.5%, yeast extract 0.5%, corn steep liquor 1%, sodium glutamate 0.3%, dipotassium phosphate 0.2%, magnesium sulfate 0.05%, sodium thiosulfate 0.1%, and calcium carbonate 2%. It was inoculated into 300ml and cultured with shaking at 37°C for 16 hours. Add 150 ml of this seed culture to glucose 2.5%, peptone 1.5%, yeast extract 0.5%, corn steep liquor 0.5%, dipotassium phosphate 0.2
5 containing 3g of fermentation medium (PH7.2) consisting of %, magnesium sulfate 0.005%, and sodium thiosulfate 0.1%.
A hyaluronic acid culture solution was obtained by inoculating the mixture into a container and culturing it at 37°C, aeration rate of 0.3 vvm, and pH 7.0 for 26 hours. This hyaluronic acid culture solution 3
diluted to 20% with ion-exchanged water and added 200g of carbon.
was added and stirred for 1 hour at room temperature, and then filtered through Nutsuie to obtain carbon treatment liquid 20. Purified hyaluronic acid was produced from this carbon treatment liquid by the following three methods. (1) Target area A 1.2 kg of NaCl was added to carbon treatment solution 20, and 16 kg of acetone was added to obtain a precipitate of sodium hyaluronate. This precipitate was separated, washed with ethanol, and dried under vacuum to obtain 12.8 g of purified hyaluronic acid. (2) Target area B 10% cetylpyridium chloride (CPC) aqueous solution 5 was added to 20 of the carbon treatment solution to form a precipitate. After separating this precipitate and washing with water,
Dissolved in 0.3M NaCl aqueous solution 20% and acetone 30%
was added to obtain a precipitate of sodium hyaluronate. This precipitate was separated, washed with ethanol, and dried under vacuum to obtain 12.5 g of purified hyaluronic acid. (3) Test area Carbon treatment solution 20 was mixed with macroretite-like anion exchange resin Diaion HPA-
75 (manufactured by Mitsubishi Chemical Industries, Ltd.) 2.5 at a rate of 5/h to obtain a treated solution 20. The pH of this treatment solution is HC
Adjust to 7.0 and add 1.2Kg of NaCl to 16
of acetone was added to obtain a precipitate of sodium hyaluronate. This precipitate was separated, washed with ethanol, and dried in vacuum, resulting in purified hyaluronic acid.
11.5g was obtained. The protein content, nucleic acid (absorbance at 260 nm), viscosity of the purified hyaluronic acid obtained in (1) to (3) above,
The intrinsic viscosity was measured and an exothermic test was conducted. The results are shown in Table 1.

【table】

[Table] Example 2 1 g of sodium hyaluronate manufactured by Kewpie Corporation was dissolved in 1 part of water, and the solution was passed through 200 ml of Diaion HPA-75 (OH) manufactured by Mitsubishi Chemical Corporation at a flow rate of 400 ml/h to obtain treatment liquid 1.2. Ta. The amount of sodium hyaluronate contained in the treatment solution is 312 mg, approximately 70%
was adsorbed and removed by the resin. The pH of the treatment solution was adjusted to 7.0 with hydrochloric acid, 70 g of NaCl was added, and then 1 acetone was added to obtain a precipitate of sodium hyaluronate. This precipitate was separated and dried under vacuum to obtain 305 mg of purified sodium hyaluronate. The viscosity, intrinsic viscosity, and molecular weight of the sample before and after purification were measured using the same method as in Example 1, and the results are as shown in Table 2, demonstrating the effectiveness of the method of the present invention for hyaluronic acid derived from natural products. Proven.

[Table] Effects of the Invention According to the present invention, purified hyaluronic acid having a molecular weight of 2 million or more and containing no proteins, nucleic acids, or pyrogens can be obtained.

Claims (1)

[Claims] 1. A method for purifying hyaluronic acid, which comprises using a macroreticular anion exchange resin when separating and purifying hyaluronic acid from a liquid containing hyaluronic acid.