JPH062073B2 - Hyaluronic acid manufacturing method - Google Patents

Description

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, a method for producing hyaluronic acid by culturing a hyaluronic acid-producing bacterium in a culture solution containing a lytic enzyme or a lysing enzyme and a surfactant, producing and accumulating hyaluronic acid in the culture solution, and collecting the hyaluronic acid. Regarding

Hyaluronic acid exists as a constituent component in connective tissues such as joints, vitreous body, umbilical cord, cartilage, skin, and avian crest, and plays an important function in tissue flexibility, structure maintenance, and cell metabolism regulation. . In addition, the hyaluronic acid is a very large polymer substance, and its solution has a strong viscoelasticity and a water-retaining action, so that it has wide application as a cosmetic, a wound treatment agent, an eye drop, and an arthritis remedy.

Conventionally, the hyaluronic acid is industrially chicken-like,
It is obtained by extraction from the vitreous of cattle eyes, umbilical cord or cartilage of whales. 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, When the hyaluronic acid is decomposed during the extraction and purification steps to lower the molecular weight and lower the viscosity and water retention, there are some drawbacks. Therefore, an attempt to produce hyaluronic acid by a culture method is disclosed in JP-A-58-56692.

The present inventor has conducted diligent research to solve the above-mentioned problems associated with the production of hyaluronic acid, and in culturing a hyaluronic acid-producing bacterium, by using a culture solution in which serum is added to the culture solution, It was found that productivity would be greatly increased, and it was proposed as Japanese Patent Application No. 59-185150. However, this method cannot be denied that the production cost of hyaluronic acid is high because the quality of serum varies from lot to lot and is more expensive than other medium materials.

The present inventor has conducted earnest research on a production method which can significantly increase the productivity of hyaluronic acid at low cost. As a result, when culturing hyaluronic acid-producing bacteria,
By culturing a hyaluronic acid-producing bacterium using a culture solution obtained by adding a lytic enzyme or a lytic enzyme and a surfactant to the culture solution, the production cost can be reduced and the productivity of hyaluronic acid can be significantly increased. Find out what you can do,
The present invention has been completed based on this finding.

Since the lytic enzyme and the surfactant used in the present invention have little variation among the lots, it is possible to always ensure a constant quality and production amount.

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.

The present invention has the following configurations.

(1) Hyaluronic acid characterized by culturing a microorganism having a hyaluronic acid-producing ability in a culture solution to which a lytic enzyme is added, to grow and accumulate hyaluronic acid in the culture solution, and to collect this Manufacturing method.

(2) Culturing a microorganism capable of producing hyaluronic acid in a culture solution containing a lytic enzyme and a surfactant,
A method for producing hyaluronic acid, characterized in that hyaluronic acid is produced and accumulated in the culture solution, and this is collected.

Examples of hyaluronic acid-producing bacteria used in the present invention include Streptococcus pyogenes, Streptococcus equi, and Streptococcus equisimilis Streptococcus ), Streptococcus zooepidemicus, and Pasteurella multocida.

As the lytic enzyme used in the present invention, all enzymes having lytic activity can be used, but lysozyme is most preferable. The amount of the lytic enzyme added to the culture solution is not particularly limited, but is preferably 100 to 2500 units, more preferably 300 to 20000 units, and most preferably 500 per culture solution.
~ 1000 units. If the amount of the lytic enzyme added is too small, the production and accumulation of hyaluronic acid will decrease, and if the amount added is too large, the lytic action will increase and the growth of the hyaluronic acid-producing bacteria will be impaired.

Further, the lytic enzyme is added at the time when the culture solution to be added is sterilized by a pressure steam sterilization method or the like and then cooled, and aseptically added to the culture solution when the temperature reaches 45 ° C or lower. preferable.

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.), lauryl sulfate sodium,
Triton X-100 (trade name, manufactured by Rohm and Haas Co., Ltd.), Span 80 (trade name, Kao Chemical Co., Ltd.)
Manufactured by K.K.), Span 90 (trade name, manufactured by Kao Chemical Co., Ltd.), nonion (trade name, manufactured by NOF CORPORATION), 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 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 adding the lytic enzyme or the lytic enzyme and the surfactant, a culture solution generally used for culturing hyaluronic acid-producing bacteria may be used. For example, glucose 2 0.0 to 3.0%, phosphoric acid 1
Potassium 0.3%, dipotassium phosphate 0.2%, sodium thiosulfate 0.01%, magnesium sulfate heptahydrate 0.
01%, sodium sulfite 0.002%, cobalt chloride 0.001%, manganese chloride 0.001%, antifoaming agent 0.1%.
It is possible to use a culture solution having a pH of 6.0 to 8.5 with a component containing 5%. (However,% represents weight / volume% in which weight is g and volume, and unless otherwise specified,% represents the above-mentioned weight / volume%.) The hyaluronic acid of the present invention is produced by first lysing. After sterilizing a culture solution containing no enzyme and a surfactant or a culture solution containing a surfactant but not a lytic enzyme by a pressure steam sterilization method, the temperature is lowered to 45 ° C or lower after cooling. At that time, the lytic enzyme is aseptically added to the culture solution, and then the hyaluronic acid-producing bacterium is 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 the temperature is 25 ° C to 40 ° C, preferably 30 ° C to
At a temperature of 35 ° C, pH 6.5-8.0, preferably 7.0
The hyaluronic acid-producing bacterium is cultivated for 1 to 2 days under control to 1%, and then the sugar solution is further added with 3% to further
It is cultured for 2 days to produce and accumulate hyaluronic acid. After that, the culture solution is sterilized by centrifugation or filtration, and the low molecular weight substance is removed by ultrafiltration or dialysis of the solution. Then, alcohol such as methanol and ethanol is added to the liquid from which the low molecular weight substance has been removed to precipitate a crude composition of hyaluronic acid, and the precipitated hyaluronic acid is dissolved again in water, and then cetyltrimethyme ammonium bromide is added. Addition is carried out by fractionation precipitation with the cetyltrimethylammonium bromide, followed by purification of the produced hyaluronic acid by known purification means such as ion exchange chromatography and gel perchromatography.

By using the culturing method using the culture solution containing the lytic enzyme or the lytic enzyme and the surfactant of the present invention,
It is possible to greatly improve the productivity of hyaluronic acid by 4 to 5 times per culture solution as compared with the culture method using an ordinary culture solution without adding the lytic enzyme or the lytic enzyme and a surfactant (Comparative Example). The production cost of hyaluronic acid can be reduced to 1/20 or less as compared with the culture method using serum. Also, the lytic enzymes and surfactants used have almost no variation in quality between lots, so
This is an epoch-making method for producing hyaluronic acid because it makes it possible to produce hyaluronic acid with consistently constant quality and production volume. The hyaluronic acid obtained by the present invention is a highly pure hyaluronic acid having a very low content of impurities, and can be suitably used for pharmaceutical and cosmetic applications.

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.

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

The Streptococcus zooepidemicum used in the present invention was obtained from the Animal Health Research Center of the Ministry of Agriculture, Forestry and Fisheries, and the Streptococcus equi was obtained from the Institute of Medical Science, University of Tokyo.

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
Culture solution of pH 7.0 consisting of 01% and soybean oil 0.5% 1.
5 was injected into a mini jar fermenter having an internal volume of 3.0, heat sterilized at 120 ° C. for 15 minutes, cooled to room temperature, and 0.75 mg (675 units) of egg white lysozyme was aseptically added to the culture solution. And then Streptococcus
Inoculate with 0.1 mg of pre-cultured zooepidemicus, 3 per minute
The culture was carried out for 24 hours under the conditions of 00 revolutions, an air flow rate of 0.7 vvm, and a temperature of 35 ° C. and automatically controlled to pH 7.0. Thereafter, 100 ml of a 50% aqueous solution of glucose was aseptically added to the culture solution, and the culture solution was further cultured for 26 hours under the above-mentioned culture conditions. Then, ion-exchanged water 3.2 was added to the culture solution, and the mixture was stirred. The cells were removed by centrifugation. The obtained supernatant was concentrated to 1.6 with a hollow fiber ultrafiltration machine and dialyzed against ion-exchanged water. Sodium acetate 0.5% was added to this solution, and ethyl alcohol 5 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 of ion-exchanged water, and 0.23 of a 4% aqueous cetyltrimethylammonium bromide solution was added to collect a precipitate. Then, the precipitate was dispersed in 40 ml of a 0.3 mol / concentration aqueous sodium chloride solution, centrifuged, and 120 ml of ethyl alcohol was added to the supernatant.
The formed precipitate was collected. The separated precipitate was dissolved in ion-exchanged water and then purified by ion-exchange chromatography to obtain 7.8 g of purified sodium hyaluronate white powder. The production amount was 5.2 g per culture medium. The protein content of this purified sodium hyaluronate was 0.05% by weight. The intrinsic viscosity measured by Ubbelohde viscometer is [η] = 17.3 dl / g and the molecular weight is 0.00
It was confirmed to be 5,000 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
A culture broth 1.5 having a pH 7.0 of 01% and soybean oil 0.5% was poured into a mini jar fermenter having an internal volume of 3.0, sterilized by heating at 120 ° C. for 15 minutes, and cooled to room temperature. Egg white lysozyme 0.73 mg (675 units)
Was aseptically added to the culture broth, then 0.1 preculture of Streptococcus equi was inoculated, and the cells were cultured under the culture conditions and the culture method according to Example 1. Then, the culture solution was purified by the method according to Example 1 to obtain 6.4 g of purified sodium hyaluronate white powder. Culture liquid 1
The production was 4.2 g per unit.

Example 3 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
Culture solution of pH 7.0 consisting of 01% and soybean oil 0.5% 1.
5 Tween 80 (trade name) 0.7 as a surfactant
After adding g, the culture solution was poured into a mini jar fermenter having an internal volume of 3.0, sterilized by heating at 120 ° C. for 15 minutes, and cooled to room temperature, and then 0.4 mg of egg white lysozyme was added.
(360 units) was aseptically added, and then a preculture solution of Streptococcus zooepidemicus 0.1 was inoculated and cultured under the culture conditions and the culture method according to Example 1. Then, the culture solution was purified by the method according to Example 1 to obtain 8.0 g of purified sodium hyaluronate powder. The production amount was 5.3 g per culture solution. The protein content of this purified sodium hyaluronate was 0.04% by weight. Further, it was confirmed that the intrinsic viscosity by an Ubbelohde viscometer was [η] = 15.0 dl / g and the molecular weight was 837,000 daltons.

Example 4 Sugar 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
Culture solution 1.5 having a pH of 7.0, to which 01%, soybean oil 0.5% and 1.5 g of Tween 80 as a surfactant were added
Was injected into a mini jar fermenter having an internal volume of 3.0, heat sterilized at 120 ° C. for 15 minutes, cooled to room temperature, and 0.73 mg (675 units) of egg white lysozyme was aseptically added to the culture solution, Then, 0.1 of the preculture liquid of Streptococcus equi was inoculated and cultured under the culture conditions and the culture method according to Example 1. Then, the culture solution was purified by the method according to Example 1 to obtain 6.9 g of white powder of purified sodium hyaluronate. The production amount was 4.6 g per culture solution.

Comparative Example 1 2.0% butter sugar, 0.5% yeast extract, 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 a culture solution 1.5 having a pH of 01% and soybean oil 0.5% into a mini jar fermenter having an internal volume of 3.0, sterilized by heating at 120 ° C for 15 minutes and cooled to room temperature, Culture conditions in accordance with Example 1, inoculated with a preculture liquid 0.1 of Streptococcus zooepidemicus,
It was cultured by the culture method. Then, the culture solution was purified by the method according to Example 1 to obtain 1.5 g of white powder of purified sodium hyaluronate. 1.0g per culture
It was the production amount of. The protein content of this purified sodium hyaluronate was 0.03% by weight. It was also confirmed that the intrinsic viscosity measured by an Ubbelohde viscometer was [η] = 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
A culture broth 1.5 having a pH 7.0 of 01% and soybean oil 0.5% was poured into a mini jar fermenter having an internal volume of 3.0, sterilized by heating at 120 ° C. for 15 minutes, and cooled to room temperature. The preculture liquid 0.1 of Streptococcus equi was aseptically inoculated and cultured according to Example 1. Thereafter, the culture solution was purified according to Example 1,
1.4 g of white powder of purified sodium hyaluronate was obtained. The production amount was 0.93 g per culture medium. The protein content of this purified sodium hyaluronate is 0.0
It was 3% 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.

Claims (4)

[Claims] 1. A method for culturing a microorganism having a hyaluronic acid-producing ability in a culture solution to which a lytic enzyme is added, to produce and accumulate hyaluronic acid in the culture solution, which is collected. Method for producing hyaluronic acid. 2. A method of culturing a microorganism having a hyaluronic acid-producing ability in a culture solution to which a lytic enzyme and a surfactant have been added, to produce and accumulate hyaluronic acid in the culture solution, and to collect the microorganism. A method for producing hyaluronic acid, comprising: 3. The method for producing hyaluronic acid according to claim 1, wherein lysozyme is used as a lytic enzyme. 4. The method for producing hyaluronic acid according to claim 2, wherein lysozyme is used as a lytic enzyme.