JPH0753117B2 - Hyaluronic acid manufacturing method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing hyaluronic acid by a microorganism capable of producing hyaluronic acid (hereinafter referred to as hyaluronic acid-producing bacterium). More specifically, it relates to a method for producing hyaluronic acid by culturing a hyaluronic acid-producing bacterium in a culture solution containing a surfactant, producing and accumulating hyaluronic acid in the culture solution, and collecting the hyaluronic acid.

Hyaluronic acid exists as a constituent component in connective tissues such as joints, vitreous body, umbilical cord, cartilage, skin and avian crest, and has an important function for tissue flexibility, structure maintenance, cell metabolism regulation and the like. Is fulfilling. Further, the hyaluronic acid is a very large polymer substance, and its solution has strong viscoelasticity and water retention action, so
It has a wide range of uses as a wound treatment agent, eye drops, and arthritis treatment agent.

[0003]

2. Description of the Related Art Conventionally, the hyaluronic acid has been industrially obtained by an extraction method from chicken bone, vitreous body of cattle, cartilage of umbilical cord or whale. However, hyaluronic acid obtained from these living organisms by an extraction method forms a complex with a mucopolysaccharide such as protein or chondroitin, and therefore requires a complicated step for separation and purification, and since hyaluronidase is often mixed, There is a drawback that the hyaluronic acid is decomposed in the extraction and purification steps to lower the molecular weight and lower the viscosity and water retention. For this reason, an attempt to produce hyaluronic acid by a culturing method has been disclosed in JP-A-58-5
It is disclosed in Japanese Patent No. 6692.

The present inventor has conducted diligent research to solve the above-mentioned problems associated with the production of hyaluronic acid, and when culturing a hyaluronic acid-producing bacterium, by using a culture solution containing serum added to the culture solution, It was found that the productivity of hyaluronic acid was drastically increased, and Japanese Patent Application No. 59-1851
Proposed as No. 50. However, this method cannot be denied that the production cost of hyaluronic acid is high because serum varies in lot-to-lot quality and is more expensive than other medium materials.

[0005]

DISCLOSURE OF THE INVENTION The inventor of the present invention has conducted earnest research on a production method capable of significantly increasing the productivity of hyaluronic acid at low cost. As a result, when culturing a hyaluronic acid-producing bacterium, by culturing the hyaluronic acid-producing bacterium using a culture solution obtained by adding a surfactant to the culture solution, the production cost is reduced, and the productivity of hyaluronic acid is reduced. I found that it can be greatly increased,
The present invention has been completed based on this finding. Since the surfactant used in the present invention has almost no lot-to-lot variation,
It is possible to always maintain a certain quality and production volume. As is clear from the above description, an object of the present invention is to provide a method for stably and significantly increasing the productivity of hyaluronic acid, and at a low cost, for producing hyaluronic acid.

[0006]

The present invention has the following configuration. Production of hyaluronic acid characterized by culturing a microorganism having a hyaluronic acid-producing ability in a culture solution containing a surfactant to produce and accumulate hyaluronic acid in the culture solution, and collecting this Law.

The hyaluronic acid-producing bacterium used in the present invention is Streptococcus py.
ogenes), Streptococcus equi
equi), Streptococcus equisimilis
occus equisimilis), Streptococcus dysgalactiae, Streptococcus zooepidemic
us), Pasteurella multocida
And so on.

As the surfactant used in the present invention, cetyltrimethylammonium bromide, cetylpyridinium chloride, Tween 80 (trade name, manufactured by Kao Chemical Co., Ltd.), Tween 90 (trade name, manufactured by Kao Chemical Co., Ltd.) , Sodium lauryl sulfate, Triton X-100 (trade name, manufactured by Rohm and Haas Co., Ltd.), Span 80 (trade name, manufactured by Kao Kagaku Co., Ltd.), Span 90 (trade name, Kao Kagaku Co., Ltd.)
Manufactured), nonion (trade name), diethylhexyl sulfosuccinate, and the like. The amount of the surfactant added to the culture solution is preferably 0.5 to 10 g, more preferably 0.5 to 3 g, and most preferably 1.0 to 2.0 g per liter of the culture solution. The surfactant is added to the culture solution before sterilizing the culture solution with pressurized steam.

In the present invention, as a component of the culture solution before the addition of the surfactant, a culture solution usually used for culturing hyaluronic acid-producing bacteria may be used, for example glucose 2.0 to 3.0%, 1 potassium phosphate 0.3
%, Dipotassium phosphate 0.2%, sodium thiosulfate 0.01%, magnesium sulfate heptahydrate 0.01%, sodium bisulfate 0.002%, cobalt chloride 0.001
%, Manganese chloride 0.001%, and antifoaming agent 0.5%, and a culture solution having a pH of 6.0 to 8.5 can be used. (However,% means weight / volume% where weight is g and volume is liter, and% means the above-mentioned weight / volume% unless otherwise specified.)

The hyaluronic acid of the present invention is produced by sterilizing a culture solution containing no surfactant or a culture solution containing a surfactant by a pressure steam sterilization method or the like, and then cooling the temperature of the culture solution. When the temperature becomes 45 ° C. or lower, hyaluronic acid-producing bacteria are aseptically inoculated into the culture solution. Then, the culture broth inoculated with the hyaluronic acid-producing bacterium is aerated with stirring or allowed to stand, and at a temperature of 25 ° C to 40 ° C, preferably 30 ° C to 35 ° C, a pH of 6.5 to 8.0, preferably 7.0. After controlling and culturing the hyaluronic acid-producing bacterium for 1 to 2 days, 3% of a sugar component is further added to the culture solution and further culturing for 1 to 2 days to generate and accumulate hyaluronic acid. Thereafter, the culture solution is sterilized by centrifugation or filtration, and then the low molecular weight substance is removed by ultrafiltration or dialysis of the furnace solution. Then, alcohol such as methanol or ethanol is added to the furnace liquid from which the low molecular weight substances have been removed to precipitate a crude product of hyaluronic acid, and the precipitated hyaluronic acid is dissolved again in water, and then cetyltrimethylammonium bromide is added. Fractional precipitation with the cetyltrimethylammonium bromide, followed by ion exchange chromatography,
The hyaluronic acid thus produced is purified by a known purification means such as gel filtration chromatography.

By using the culturing method using the culture solution containing the surfactant of the present invention, hyaluronic acid can be obtained as compared with the culturing method using a normal culture solution containing no surfactant (comparative example). 3 ~ per liter of acid-producing medium
The production cost of hyaluronic acid can be greatly improved to 4 times, and the production cost of hyaluronic acid is 1/20 compared with the culture method using serum.
It has become possible to lower it below. In addition, since the surfactant used has little variation in quality between lots, it is possible to always produce hyaluronic acid with a constant quality and production amount, which is an epoch-making method for producing hyaluronic acid. The hyaluronic acid obtained by the present invention has a very low content of impurities, is a high-purity hyaluronic acid,
It can be suitably used for cosmetics. As described above, it was confirmed that the method for producing hyaluronic acid according to the present invention can stably produce highly pure hyaluronic acid with extremely high productivity.

[0012]

EXAMPLES The present invention will be specifically described below with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1 Glucose 2.0%, yeast extract 0.5%, peptone 1.
5%, 1 potassium phosphate 0.3%, 2 potassium phosphate 0.2%, sodium thiosulfate 0.011%, magnesium sulfate heptahydrate 0.01%, sodium sulfite 0.005
2%, cobalt chloride 0.001%, manganese chloride 0.0
Inject 1.5 liter of pH 7.0 culture medium containing 01%, soybean oil 0.5% and Tween 80 (trade name) 1.5 g as a surfactant into a mini jar fermenter having an internal volume of 3.0 liter. After heat sterilization at 120 ° C. for 15 minutes and cooling to room temperature, a preculture liquid of Streptococcus equi is aseptically inoculated, 300 rpm, aeration volume 0.7 vvm, pH 7. The culture was carried out for 24 hours by automatically controlling it to 0. Then 50 of glucose
100 ml of 100% aqueous solution is added to the culture solution aseptically,
After further culturing for 26 hours under the above-mentioned culturing conditions, 3.2 liters of ion-exchanged water was added to the culture broth, and the mixture was stirred and then centrifuged to remove cells. The obtained supernatant was concentrated to 1.6 liters using a hollow fiber ultrafilter and further dialyzed against ion-exchanged water. Sodium acetate 0.5% was added to this solution, and ethyl alcohol (5 liters) was further added to precipitate a hyaluronic acid-containing polysaccharide, which was then separated by centrifugation. The separated polysaccharide containing hyaluronic acid was dissolved in 0.5 liter of ion-exchanged water, and 0.23 liter of a 4% aqueous cetyltrimethylammonium bromide solution was added to collect a precipitate. Then, the precipitate was treated with an aqueous solution of sodium chloride having a concentration of 0.3 mol / liter 4
After dispersing in 0 ml and centrifuging, 120 ml of ethyl alcohol was added to the supernatant to separate the formed precipitate.
The separated precipitate was dissolved in ion-exchanged water and purified by ion-exchange chromatography to obtain 6.1 g of white powder of sodium hyaluronate. The production amount was 4.1 g per liter of the culture solution. The protein content of this purified hyaluronic acid was 0.03% by weight. Also, the intrinsic viscosity measured by Ubbelohde viscometer is 12.0 dl / g.
Was confirmed to have a molecular weight of 628,000 daltons.

Example 2 Glucose 2.0%, yeast extract 0.5%, peptone 1.
5%, 1 potassium phosphate 0.3%, 2 potassium phosphate 0.2%, sodium thiosulfate 0.011%, magnesium sulfate heptahydrate 0.01%, sodium sulfite 0.005
2%, cobalt chloride 0.001%, manganese chloride 0.0
To a mini jar fermenter with an internal volume of 3.0 liters, 1.5 liters of a pH 7.0 culture solution containing 01%, soybean oil 0.5% and Tween 80 (trade name) 1.5% as a surfactant. Inject, heat sterilize at 120 ° C. for 15 minutes, inoculate with 0.1 liter of preculture solution of Streptococcus zooepidemicus, 300 revolutions per minute, aeration rate of 0.
The cells were cultured at 7 vvm and a temperature of 33 ° C. for 2 days while automatically controlling the pH to 7.0. Then, the culture solution was purified according to Example 1 to obtain 6.3 g of purified sodium hyaluronate. The amount was 4.2 g per liter of culture solution.

Comparative Example 1 Glucose 2.0%, yeast extract 0.5%, peptone 1.
5%, 1 potassium phosphate 0.3%, 2 potassium phosphate 0.2%, sodium thiosulfate 0.011%, magnesium sulfate heptahydrate 1.01%, sodium sulfite 0.005
2%, cobalt chloride 0.001%, manganese chloride 0.0
1.5 liter of a culture solution of 01% and soybean oil 0.5% and having a pH of 7.0 was poured into a mini jar fermenter having an internal volume of 3.0 liter, sterilized by heating at 120 ° C. for 15 minutes, and cooled to room temperature. Then, 0.1 liter of a pre-cultured solution of Streptococcus equi was aseptically inoculated and cultured according to Example 1. Then, the culture solution was purified according to Example 1 to obtain 1.4 g of white powder of purified sodium hyaluronate. 0.9 per liter of culture solution
The production amount was 3 g. The protein content of this purified sodium hyaluronate was 0.03% by weight. Further, it was confirmed by an Ubbelohde viscometer that the intrinsic viscosity was 12.0 dl / g and the molecular weight was 628,000 daltons.

Comparative Example 2 Glucose 2.0%, yeast extract 0.5%, peptone 1.
5%, 1 potassium phosphate 0.3%, 2 potassium phosphate 0.2%, sodium thiosulfate 0.011%, magnesium sulfate heptahydrate 0.01%, sodium sulfite 0.005
2%, cobalt chloride 0.001%, manganese chloride 0.0
After injecting 1.5 liters of a culture solution containing 01% and soybean oil 0.5% and having a pH of 7.0 into a mini jar fermenter having an internal volume of 3.0 liters, heat sterilize at 120 ° C for 15 minutes and cool to room temperature. After that, Streptococcus
Aseptic inoculation was performed with 0.1 liter of the pre-cultured solution of Zooepidemicus, and the cells were cultured under the culture conditions and the culture method according to Example 2. Then, the culture solution was purified by the method according to Example 2 to obtain 1.5 g of purified sodium hyaluronate white powder. The production amount was 1.0 g per liter of the culture solution.

[0017]

INDUSTRIAL APPLICABILITY The method for producing hyaluronic acid according to the present invention is a method capable of stably producing highly pure hyaluronic acid with extremely high productivity.

Claims (1)

[Claims] 1. A culture solution containing a surfactant,
A method for producing hyaluronic acid, which comprises culturing a microorganism capable of producing hyaluronic acid, causing hyaluronic acid to be produced and accumulated in the culture solution, and collecting this.