JPH0630604B2 - Hyaluronic acid manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a method for producing hyaluronic acid (hereinafter abbreviated as HA) by a fermentation method, and is close to naturally occurring HA desired for cosmetics or pharmaceuticals. It relates to a method for producing high molecular weight HA.

[Conventional technology and its problems]

Conventionally, HA has been obtained by extraction from chicken mackerel, vitreous or umbilical cord of bovine eyes. However, separation and purification from living organisms requires complicated steps due to the presence of mucopolysaccharides such as contaminating proteins and chondroitin sulfate, and in addition, it was originally high in molecular weight during the separation and purification steps by hyaluronidase in living bodies. There was a drawback that HA was decomposed and the molecular weight was lowered, and the viscosity and heat retention were lowered. So H
Various methods have been devised to obtain a higher molecular weight HA by suppressing the decomposition of A (for example, the trade name "Healon" manufactured by Pharmacia Co. has a molecular weight of 230 calculated by an intrinsic viscosity method).
However, it was difficult to say that it was sufficient, and it was also a factor of cost increase. As a solution to these drawbacks, a microorganism of the genus Streptococcus having HA-producing ability is aerobically cultivated in a liquid medium containing nutrient sources such as glucose, yeast extract and polypeptone while adjusting the pH to around 7, and HA is removed from the culture solution. Isolation methods are disclosed in JP-A-58-56692, JP-A-60-133894, JP-A-61-63294 and the like. These methods have the advantages that they are easier to isolate and purify and are cheaper than the extraction methods. However, compared with the extraction method HA, HA obtained by the fermentation method generally has a lower molecular weight (generally 1.5 million or less) and is said to be inferior in the viscosity and moisturizing effect, which are the characteristics of HA. No method has yet been found for producing hyaluronic acid of close high molecular weight. Therefore, its use in medicinal applications such as ophthalmology and arthritis remedies requiring a high molecular weight close to natural has been limited.

[Means for solving problems]

As a result of studies to solve these problems, the present inventors have found that a high molecular weight HA can be obtained by a fermentation method by controlling the pH of a culture solution, and completed the present invention. Specifically, as a result of detailed examination of the relationship between the pH of the culture solution and the molecular weight of the accumulated hyaluronic acid, it was remarkably high when the pH of the culture solution was maintained on the alkaline side rather than near neutrality of pH 7 which is the conventional technique. It was found that high molecular weight hyaluronic acid was accumulated.

That is, the present invention, when aerobically culturing a microorganism of the genus Streptococcus having HA-producing ability,
A method for producing hyaluronic acid by a fermentation method, which comprises producing and accumulating high-molecular-weight hyaluronic acid in a culture solution by controlling pH in the range of 7.5 to 9.0.

The present invention will be described in detail below.

Examples of the HA-producing bacterium used in the present invention include Streptococcus equi (Streptococcus equisimoces) and Streptococcus esculia dysgalactia (Streptococcus equisimilicus) Streptococcus esculia dysgalactia. However, Streptococcus equi is preferable. In addition, mutant strains derived from these strains can also be used.

The medium used in the present invention may be one used for culturing ordinary microorganisms, and includes carbon sources such as glucose, fructose, galactose, and sucrose, primary potassium phosphate, secondary potassium phosphate, magnesium sulfate, sodium sulfite, and naosulfate. Inorganic salts such as soda and ammonium phosphate, polypeptone, casamino acid, yeast extract, corn steep liquor, organic nutrient sources such as soybean hydrolyzate, and various amino acids, vitamins and the like are preferably used if necessary. These medium components can be used either as a batch or as a divided addition.

The culture of the present invention may be performed by a known method such as aeration and stirring culture, and the culture temperature is preferably 30 to 35 ° C.

Since the pH of the culture solution decreases with the growth of the bacterium, pH adjusters such as caustic soda, caustic potash, and ammonia are added to pH 7.5.
~ 9.0, preferably pH 8.0 ~ 8.7 can achieve the object of the present invention. If the pH is less than 7.5, the molecular weight of the produced HA will be 2,000,000 or less, and if the pH exceeds 9.0, the growth of HA-producing bacteria will be remarkably reduced, resulting in an extremely low HA yield, which is not preferable.

When culturing is performed in this manner, the viscosity of the culture solution gradually increases as HA is produced. When the carbon source used is consumed in the culture solution, the culture is stopped, and after sterilization by centrifugation,
High-purity, high-molecular-weight HA can be obtained by a simple known purification method such as precipitation with an organic solvent such as alcohol and desalting by ultrafiltration.

〔Example〕

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1 From the same medium to a culture solution 1 of pH 8.5 consisting of glucose 2%, potassium potassium phosphate 0.2%, magnesium sulfate heptahydrate 0.05%, sodium naosulfate 0.1%, polypeptone 1.0% and yeast extract 0.5%. Streptococcus Exhaust (AT
CC 9527) inoculated with 10m of pre-cultured solution, aeration volume 1.
5 vvm, stirring 200 rpm, temperature of 33 ° C with caustic soda
The culture was carried out for 15 hours while controlling the pH to 8.5.

The culture solution was adjusted to pH 4 with hydrochloric acid, diluted 2-fold with distilled water, and centrifuged to remove bacteria. Ethyl alcohol is added to the obtained sterilized solution to precipitate HA soda. After separating this by filtration, it is dissolved in water, cetylpyridinium chloride is added,
The formed precipitate is collected by filtration, redissolved in water, and precipitated again with ethyl alcohol. The obtained HA soda was dried under reduced pressure at room temperature to obtain 1.8 g of white HA soda per culture 1.

This HA soda was used as a 0.2 M sodium chloride solution, and the average molecular weight was calculated by the limiting viscosity method. The molecular weight is measured by the Japanese Pharmacopoeia General Test Method 28: Viscosity Measurement Method, and the reduced viscosity for each concentration is calculated by the following formula: A graph is drawn with the reduced viscosity on the vertical axis and the corresponding concentration (g / 100m) on the horizontal axis. The extension of the line connecting each point and the intersection of the vertical axis are the intrinsic viscosity [η], and the Wang's formula [R .Clel, J.
L. Wang, Biopolymer, 9 , 799 (1970)] Molecular weight M = ([η] /0.000228) ^1.225 .

As a result, the average molecular weight was 3.3 million.

Comparative Example Table 1 shows the same results as in Example 1 except that the pH during culture was set to pH 7.0 and pH 9.2.

Example 2 Culture was started in the same manner as in Example 1 except that the pH during the culture was adjusted to pH 8.0 and pH 9.0, and the residual glucose concentration in the culture solution was 0.5.
Culture was continued for 40 hours while adding glucose to a concentration of ˜1%, and these culture solutions were treated in the same manner as in Example 1 to obtain HA soda. The results are shown in Table 2.

[Effects of the Invention] As described above, according to the present invention, it is possible to obtain a high molecular weight HA which has not been obtained by the fermentation method.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Haruhisa Saegusa 3-5-1 Asahimachi, Machida-shi, Tokyo Denka Kagaku Kogyo Co., Ltd. Central Research Laboratory Examiner Hiroshi Taniguchi

Claims (3)

[Claims] 1. When aerobically culturing a microorganism of the genus Streptococcus capable of producing hyaluronic acid, the pH of the culture broth is controlled to be in the range of 7.5 to 9.0, whereby high molecular weight hyaluronic acid is contained in the culture broth. A method for producing hyaluronic acid by a fermentation method, which comprises producing and accumulating. 2. The production of hyaluronic acid according to claim 1, wherein the high molecular weight hyaluronic acid produced and accumulated in the culture medium is hyaluronic acid having an average molecular weight of 2,000,000 to 4,000,000 as determined by the limiting viscosity method. Law 3. A microorganism belonging to the genus Streptococcus having hyaluronic acid-producing ability is Streptococcus equi (Strept.
ococcus equi) The method for producing hyaluronic acid according to claim 1.