JPH0698787A - Production of hyaluronic acid - Google Patents

Abstract

PURPOSE:To stably produce a polysaccharide useful for a cosmetic, a medicine, etc., in a high yield by culturing a hyaluronic acid-producing microorganism belonging to Streptococcus according.to the aeration agitation method while controlling the oxygen consumption rate through the oxygen content at the gas supply opening and the gas exhaust opening of a culture tank. CONSTITUTION:A hyaluronic acid-producing microorganism (e.g. Streptococcus zooepidemicus NCTC 7023) belonging to Streptococcus is inoculated in a culture medium in a culture tank and pH is controlled to 7.0 to 7.2 with 12N NaOH at 37 deg.C under aeration agitation condition. The culture is carried out while measuring the oxygen content at the gas supply opening and the gas exhaust opening of the culture tank and automatically adjusting the addition rate of feed culture medium through a peristaltic pump so as to control the oxygen consumption rate of the microorganism within a range of 5 to 8mmol O2/L.hr, thus remarkably stably producing the objective hyaluronic acid useful for a cosmetic, a medicine, etc., in a high yield.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a stable method for producing hyaluronic acid by a microorganism.

[0002]

2. Description of the Related Art Hyaluronic acid is a type of polysaccharide having a molecular weight of several millions, and is contained in the living body, for example, in the vitreous body, skin, joint fluid, etc., and retains water, functions as a lubricant, and bacteria. It is useful for prevention of invasion of other kinds. In recent years, it is expected that the applications will be expanded to cosmetics, pharmaceuticals (arthritis remedies, eye drops, wound healing agents) and the like.

Initially, the method for producing hyaluronic acid has been intensively studied as a culture production method using a microorganism, starting from the extraction of living tissue, which is extremely expensive. As a method for culturing and producing by a microorganism, a method of agitating a hyaluronic acid-producing bacterium of the genus Streptococcus in a medium containing 3% or more of glucose by aeration and stirring (Japanese Patent Laid-Open No. 58-56692), the oxidation-reduction potential of the culture solution is -100 to- A method of aerating and agitating to 400 mV (Japanese Patent Laid-Open No. 62-51999), a method of controlling the culture solution viscosity to 100 to 800 centipoise (Japanese Patent Laid-Open No. 63-94988), and a hyaluronidase non-producing mutant strain are used. Method (JP-A-1-67196
No.) etc. have been published.

The production of hyaluronic acid is usually carried out under aeration and agitation culture conditions. Particularly, in the above-mentioned literature, the one related to aeration, that is, supply of oxygen, is a redox potential in a culture solution. The method of culturing with aeration and agitation at -100 to -400 mV (Japanese Patent Laid-Open No. 62-51999) can be mentioned. In this method, the redox potential in the culture solution was directly measured by a submerged sensor such as a platinum-calomel electrode.

[0005]

DISCLOSURE OF THE INVENTION The inventors of the present invention directly measure the oxidation-reduction potential in a culture solution by a conventional batch culture method using a platinum-calomel electrode submerged sensor as described above. When a hyaluronic acid was produced by controlling aeration and stirring so that the preferable amount would be −100 to −400 mV, in this method, a high yield and a low yield were dissimilar. Was determined to be an extremely unstable method. Further, even in accordance with the above-mentioned various known techniques, similarly to the above, stable and high-yield production cannot be achieved, and development of a new production method capable of producing hyaluronic acid stably and in high-yield has been awaited. .

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that hyaluronic acid itself is a highly viscous high-molecular substance, so that its viscosity increases as it is produced, and Specifically, the culture medium is 5,000 to 15,000.
It became extremely viscous as 00 centipoise, and because it was highly viscous, it became a non-uniform system, and feedback control by the submerged sensor was uncertain, and it was difficult to carry out stable production. We have established a manufacturing method.

That is, according to the present invention, when a microorganism belonging to the genus Streptococcus and capable of producing hyaluronic acid is subjected to aeration and stirring culture, the oxygen content of the gas supply port and the gas exhaust port of the culture tank is measured to determine the oxygen content of the microorganism. 5 to consumption speed
_{8 m mol 0 2 / L ·} hr. It is a method for producing hyaluronic acid, which is characterized in that The present invention will be described in detail below.

As the microorganism used in the present invention, any strain can be used as long as it is a strain that accumulates hyaluronic acid outside the cells. In particular, serological classification by Lancefield among Streptococcus bacteria [ Bergey's Manual of Determinating Bacteriology (Bergey's Manual of)
Determinative Bacterio
l. ) 491, 1974] group C strains are preferred. Specific examples thereof include Streptococcus equi, Streptococcus excimilis, Streptococcus disgalactiae, Streptococcus zooepidemicus and the like. Particularly preferably Streptococcus zooepidemicus NCTC7023, NCTC69
63, NCTC7022 and the like. These strains are slightly positive for β-hemolysis and non-productive for hyaluronidase [J. Gen. Microbiol.
15, 485, 1956)].

In the present invention, the oxygen consumption rate is the oxygen consumption calculated from the difference between the amount of oxygen supplied and the amount not used, converted into unit time and unit liquid amount. To measure the oxygen consumption rate in the present invention, the oxygen content of the entire culture tank is obtained by measuring the oxygen content at the gas supply port and the gas exhaust port of the culture tank, and converted into unit time and unit liquid volume. The oxygen consumption rate may be calculated.

The oxygen content at the gas supply port and the gas exhaust port of the culture tank must be measured by a highly sensitive measuring method, and preferably, it can be carried out by a mass spectrometer. When measuring the oxygen content in the present invention, usually, it is preferable to measure at each of the gas supply port and the gas exhaust port,
When the oxygen supply rate is known in advance, for example, if the oxygen supply rate is controlled to be constant, the oxygen consumption rate can be calculated by measuring only the oxygen content at the gas exhaust port.

The supply gas is not particularly limited as long as it contains oxygen and does not adversely affect the culture of microorganisms.
Normally, air may be used. Although oxygen gas may be used alone, it is also preferable to appropriately adjust the oxygen concentration with air, carbon dioxide, nitrogen or the like. The oxygen concentration in the supply gas may be constant or may vary, but normally it is preferable to keep it constant for control purposes.

The oxygen consumption rate thus measured is 5 to
_{8 m mol 0 2 / L ·} hr. If it is out of the range, it can be easily controlled by adjusting the nitrogen source. For example, if the oxygen consumption rate is lower than the above range, a nitrogen source may be added to the medium.If the oxygen consumption rate is higher than this range, the addition of nitrogen source should be reduced or stopped. Good. At times, the rate of oxygen consumption can be further reduced by means such as dilution, but a method of limiting the nitrogen source in the culture composition at the start of the culture and adding a nitrogen source appropriately is usually preferable. The amount of nitrogen source added to increase the oxygen consumption rate varies depending on the strain used, culture conditions, etc., so a system for adjusting the addition amount of the nitrogen source using the oxygen consumption rate measured in real time as an index, for example, nitrogen. The method of continuously adding the source with a pump such as a peristaltic pump can be easily performed by automatically stopping or restarting the pump or increasing or decreasing the supply rate according to the oxygen consumption rate. The addition method may be performed continuously or intermittently. Further, as a method of controlling the oxygen consumption rate other than the above, it is possible to use the supply amount of water or other nutrient sources or the oxygen supply amount, but since the delicate control is often relatively difficult, It is also preferable to control in combination with addition of a source.

The above-mentioned nitrogen source is not particularly limited as long as it is a nitrogen source that is assimilated by microorganisms, but an organic nitrogen source is preferable. Further, this nitrogen source is preferably water-soluble, but any nitrogen source which is eventually assimilated to be water-soluble can be used. As this nitrogen source, for example,
Examples thereof include peptone, polypeptone, yeast extract, powdered yeast, corn steep liquor, meat extract, and the like, and particularly preferable examples include peptone or yeast extract. Other nutrient sources may be appropriately added to this nitrogen source.
The nitrogen source may be added usually in the form of an aqueous solution or suspension, and its concentration is not preferable because it dilutes the culture solution when it is too thin, and when it is too thick, it should be adjusted with a pump or the like. It is not preferable because the addition of the minimum amount that can be added causes excess and cannot be adjusted practically. This concentration may be constant or varied during the supply period, but it is usually preferable to keep it constant. This concentration varies depending on the nitrogen source and conditions, but is usually about 1 to 20% for peptone and about 1 to 15% for yeast extract.

As the culture composition at the start of the culture, carbon sources, nitrogen sources, salts, vitamins and the like which are commonly used can be used. Specific examples of the carbon source include glucose, sucrose, and starch hydrolysates, and sucrose is particularly preferable in terms of productivity. The nitrogen source is as described above, and it is desirable to add an organic nitrogen source such as peptone, yeast extract, corn steep liquor and meat extract, and ammonium sulfate, ammonium chloride, ammonium nitrate and the like can be used together. In addition to these, phosphates such as sodium chloride or magnesium, potassium, iron, calcium, etc., sulfates, carbonates, etc. and trace amounts of vitamins are added as necessary. Moreover, the use of an antifoaming agent or the like can be appropriately performed.

[0015] The hyaluronic acid-producing microorganisms vary depending on the culture conditions and the like, but, for example, usually there is a growth phase of the cells by about 10 hours after culturing, and there are many patterns in which the main production period of hyaluronic acid continues thereafter. . In the present invention, the oxygen consumption rate may be controlled substantially at the production period of hyaluronic acid, and the oxygen consumption rate during the growth period of the bacterial cells is not necessarily required to be controlled. Obviously, the oxygen consumption rate is extremely low in the early stage of growth, and unless the other conditions are changed, depending on the culture composition at the start of the culture, an extremely high oxygen consumption rate increases, and once the oxygen consumption rate is 5 to 8 m. mol 0 ₂ / L · hr. However, in the present invention, it is sufficient that the oxygen consumption rate is controlled within the above range during the subsequent substantial production period of hyaluronic acid. However, if the culture composition at the start of culturing is too large, the oxygen consumption rate does not fall to the above range even at the time of substantial production of hyaluronic acid, and there are many cases where it is difficult to control. Regarding the culture composition, it is preferable to select a content that can be lowered to the above range at the time of substantial production of hyaluronic acid. The selection of the appropriate culture composition at the start of culture varies depending on the strain and culture conditions, and therefore can be easily selected by preliminary batch culture and measuring the fluctuation of the oxygen consumption rate.
In addition, it is relatively difficult to control the oxygen consumption rate when adding a new nitrogen source in anticipation of when the oxygen consumption rate is once lowered and when it falls, and it is relatively difficult to control the oxygen consumption rate beforehand. Since it often decreases sharply during the production period of acid, it is relatively difficult to control the oxygen consumption rate within the above range during the production period of hyaluronic acid, and the oxygen consumption rate falls within the above range even at the end of the growth phase. It is more preferable to limit the nitrogen source of the culture composition at the start of the culture in advance to such an extent that it is within the range, and then gradually start the addition.

The culture is carried out under aerobic conditions such as aeration and stirring, and the aeration conditions should be considered together with the stirring, but usually about 0.2 to 2.0 vvm is exemplified, and the stirring conditions include the culture medium. It may be appropriately selected depending on the viscosity and the size of the culture tank. The culture temperature is usually 25 to 42 ° C., preferably around 37 ° C., the pH is usually controlled to about 6 to 8, and hyaluronic acid is usually produced by culturing for 1 to 2 days. Usually, hyaluronic acid mainly accumulates outside the cells.

In the case of separating and purifying hyaluronic acid from the culture solution, for example, first, after the culture is completed, the culture solution is kept at 60 ° C.
Heat treatment for 30 minutes to decompose β-hemolytic substance, then dilute with water to reduce its viscosity to 100 centipoise or less, add a filter aid and remove cells by centrifugation or filtration,
It is purified from the supernatant using a conventionally known method for separating and purifying polysaccharides, that is, ultrafiltration, dialysis, an ion exchange resin, an organic solvent precipitation method, etc., and hyaluron is used by means such as freeze-drying, vacuum filtration and spray drying. You can get acid.

[0018]

EXAMPLES The present invention will be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

[0019]

[Example 1] Glucose (manufactured by Nippon Sanyo Kogyo Co., Ltd.) 1.0
%, Peptone (Far East Pharmaceutical Co., Ltd.) 1.5%, (manufactured by Far East Pharmaceutical Co., Ltd.) yeast extract 0.5% (manufactured by Shiraishi, Inc.) CaC0 ₃
Streptococcus zooepidemicus NCTC7 lyophilized and preserved in a medium sterilized by autoclaving 1 L of a medium containing 0.2% in a 2 L Erlenmeyer flask.
023 was inoculated, and static culture was performed at 37 ° C. for 25 hours to obtain a seed culture.

The main culture was a 30 L jar fermenter with 0.4% peptone, 0.3% yeast extract and BC-
51% (Nippon Yushi Co., defoaming agent) 0.05% was sterilized, and 6% of sucrose (manufactured by Mitsui Sugar Co., Ltd.) sterilized by another autoclave was added to 18 L to make the seed culture 0.04. % Inoculation, aeration of 1vv at 37 ° C
m, stirring was increased from 200 rpm as the viscosity increased and sufficiently stirred, and the pH was adjusted to 7.0 to 7.
It was controlled to 2 and cultured for 24 hours.

Peptone 8.4%, yeast extract 4.9
The mixed solution containing 100% was used as the feed medium. The oxygen content of each of the supply port and the exhaust port was measured with a mass spectrometer (WSMR type, manufactured by Westlon Co., Ltd.), and the oxygen consumption rate calculated from the measured values was 5 to 8 mmol mol 0 ₂ / L.
-Hr. From the 10th hour after the start of the main culture, the feed medium was continuously fed to the jar fermenter while the addition rate was automatically adjusted by a peristaltic pump so as to fall within the range. About 24 hours after the start of the main culture, about 2 L of the feed medium was added to complete the culture.

A portion (2.75 L) of the culture solution was added at 60 ° C,
After heat treatment for 30 minutes, 4 L of water was added to dilute, the pH was adjusted to 4 with hydrochloric acid, 50 g of filter aid perlite was added, and the mixture was filtered through Nutze. The filtered cake was washed with water, and the obtained filtrate and washings were combined to obtain 7.1L. Sodium chloride (44 g) was dissolved in this solution, and then acetone (8 g) was added.
L was added, and hyaluronic acid was separated by precipitation. This precipitate was collected by filtration, dissolved in 5 L of 0.5 molar sodium chloride solution, 10 L of ethanol was added, the precipitate formed was collected by filtration, and the precipitate was thoroughly washed with 75% ethanol water and then with ethanol, and then dried in vacuum. To obtain 19.8 g of hyaluronic acid. The production amount of 7.2 g per liter of the culture broth was about 1.7 million (viscosity method).

[0023]

Comparative Example 1 The feed medium of Example 1 was mixed with peptone 2.
Instead of a feed medium consisting of a mixed solution of peptone-yeast extract containing 4% of yeast extract and 1.4% of yeast extract, the oxygen consumption rate was 1 to 4 mmol mol 0 ₂ / L · hr. The culture was carried out by controlling so as to be within the range. 24 after the start of main culture
About 2 L of feed medium was added over time to complete the culture.
As a result, the yield of hyaluronic acid was extremely low as compared with Example 1.

[0024]

Comparative Example 2 The feed medium of Example 1 was mixed with peptone 1
The same procedure as in Example 1 was carried out except that the feed medium was replaced by a feed medium consisting of a mixed solution of peptone-yeast extract containing 4.4% and 8.4% yeast extract. About 24 hours after the start of the main culture, about 2 L of the feed medium was added to complete the culture. As a result, the yield of hyaluronic acid was lower than that in Example 1.

[0025]

Example 2 As a result of repeating the culture 5 times in the same manner as in Example 1, the yield per 1 L of the culture solution was 7.
The amounts were 2 g, 7.5 g, 7.0 g, 7.1 g, and 7.3 g, and the average values were 7.2 g / L and the coefficient of variation was 2.7, showing extremely stable productivity.

[0026]

Comparative Example 3 Of the main culture medium of Example 1, only peptone and yeast extract were added at 1.2% and 0.7%, respectively.
In the middle of culturing, no new nutrient source is fed,
The culture was performed without controlling the oxygen consumption rate at all. The oxygen consumption rate reaches about 19 at about 10 hours, and then decreases sharply 1
Values of ~ 4 were shown. The yield per 1 L of the culture solution after culturing 5 times was 5.6 g, 5.3 g, 7.5 g, and
The amounts were 4.5 g, 4.8 g and 5.7 g, the average value was 6.1 g / L, and the variation coefficient was 21.2.

[0027]

Example 3 The feed medium of Example 1 was mixed with peptone 12
% Aqueous solution, and the oxygen consumption rate was 5 to 8 mmol mol 0 ₂ / L · hr. From the 10th hour after the start of the main culture, the rate of addition of the feed medium was automatically adjusted by a peristaltic pump so as to be within the range. About 24 hours after the start of the main culture, about 2 L of the feed medium was added to complete the culture. The yield per liter of culture broth was 7.4 g / L on average, and the coefficient of variation of 5 times was small, showing very stable productivity.

[0028]

[Example 4] The feed medium of Example 1 was replaced with a 7% aqueous solution of yeast extract, and the oxygen consumption rate was 5 to 8 mmol mol 0 ₂ / L · hr. From the 10th hour after the start of the main culture, the rate of addition of the feed medium was automatically adjusted by a peristaltic pump so as to be within the range. About 24 hours after starting the main culture, feed medium
L was added to complete the culture. The average yield of the culture broth was 7.2 g / L, and the coefficient of variation of 5 times was small, and the productivity was very stable.

[0029]

Example 5 The feed medium of Example 1 was used as meat extract 15
% Aqueous solution, and the oxygen consumption rate was 5 to 8 mmol mol 0 ₂ / L · hr. From the 10th hour after the start of the main culture, the rate of addition of the feed medium was automatically adjusted by a peristaltic pump so as to be within the range. About 24 hours after the start of the main culture, about 2 L of the feed medium was added to complete the culture. The yield was high and the productivity was very stable.

[0030]

Example 6 The feed medium of Example 1 was mixed with powdered yeast 10
% Aqueous suspension, and the oxygen consumption rate was 5 to 8 mmol mol 0 ₂ / L · hr. From the 10th hour after the start of the main culture, the rate of addition of the feed medium was automatically adjusted by a peristaltic pump so as to be within the range. Approximately 2 L of feed medium 24 hours after the start of main culture
And the culture was completed. The yield was high and the productivity was very stable.

[0031]

INDUSTRIAL APPLICABILITY According to the present invention, hyaluronic acid can be produced and supplied in an extremely stable and high yield.

Claims (1)

[Claims] 1. When a microorganism belonging to the genus Streptococcus and having the ability to produce hyaluronic acid is subjected to aeration stirring culture, the oxygen content at the gas supply port and the gas exhaust port of the culture tank is measured to determine the oxygen consumption rate of the microorganism. ~ 8 mmol
0 ₂ / L · hr. A method for producing hyaluronic acid, characterized in that