JPS6163294A - Production of hyaluronic acid through fermentation process - Google Patents

Abstract

PURPOSE:A microorganism producing hyaluronic acid is cultured, as the culture medium is controlled within a specific pH range, and, a saccharide is further added, when the pH exceeds a certain value to continue the cultivation, whereby mass production of hyaluronic acid becomes possible. CONSTITUTION:In the cultivation of a microorganism producing hyaluronic acid such as Streptococcus pyogeness, the cultivation is carried out so that the pH of the medium may not lower than a specific value ranging from 6.5-7.5, preferably 7.0. When the pH exceeds 7.5, a saccharide is added to continue the cultivation to effect accumulation of hyaluronic acid in the medium. Since the pH of the culture mixture becomes lower as hyarulonic acid is formed, it is controlled by adding aqueous alkali. When the growth of the microorganism stops, the pH rises and at this time, 2-7%, preferably 5% of a saccharide is added.

Description

[Detailed description of the invention] The present invention relates to a method for producing hyaluronic acid by fermentation.

Hyaluronic acid exists as a component in connective tissues such as joints, vitreous body, vitreous tissue, cartilage, skin, and the crest of birds, and has important functions such as maintaining the flexibility and structure of these connective tissues, and regulating cell metabolism. is fulfilled. In addition, hyaluronic acid is a very large polymeric substance, and its solution has strong viscoelasticity, so it has a wide range of uses as cosmetics, wound treatment agents, ophthalmic agents, and arthropathy treatment agents.

Conventionally, the hyaluronic acid has been obtained by extraction methods from chicken combs, cow eye vitreous or fake tissue, synovial fluid, and the like.

However, it is very difficult to separate and purify hyaluronic acid obtained from these living organisms because it forms a complex with proteins and mucopolysaccharides such as chondroitin. Since hyaluronic acid often contains hyaluronidase, the hyaluronic acid is decomposed during the extraction and purification process, resulting in a decrease in molecular weight, resulting in a disadvantage of low viscosity and water retention.

In order to improve these shortcomings, a method of culturing microorganisms that have the ability to produce hyaluronic acid (hereinafter referred to as hyaluronic acid-producing bacteria), and directly extracting and purifying the hyaluronic acid from the culture solution has been disclosed. It is disclosed in Publication No. 58-56692.

Since this method obtains hyaluronic acid by culturing hyaluronic acid-producing bacteria that do not contain hyaluronidase, it has the advantage of being simpler and cheaper than the above-mentioned extraction method from living organisms. However, this fermentation method for producing hyaluronic acid requires a long time for culturing, that is, 4 to 6 days, and the production amount of hyaluronic acid per culture solution 1) is 0.
．． 3-1. The drawback is that it is only ON.

The present inventors have conducted extensive research in an effort to improve this low productivity of hyaluronic acid. As a result, when culturing hyaluronic acid-producing bacteria, the pH of the culture solution is automatically and continuously controlled so as not to fall below a specified pH in the range of 6.5 or more and less than 7.5. We have discovered that the productivity of hyaluronic acid can be greatly improved by culturing acid-producing bacteria and adding saccharides to the culture solution when the pH reaches 7.5 or higher, The invention has been completed.

As is clear from the above description, an object of the present invention is to provide a method for producing hyaluronic acid by a fermentation method, which can greatly improve the productivity of hyaluronic acid.

The present invention has the following configuration.

When culturing microorganisms that have the ability to produce hyaluronic acid in a culture solution, the pH of the culture solution is automatically continuously controlled so that it does not fall below a specified pH of 6.5 or more and less than 7.5. A method for producing hyaluronic acid by a fermentation method, characterized in that hyaluronic acid is produced and accumulated in the culture solution by further adding saccharides and culturing when the pH reaches 7.5 or more.

The hyaluronic acid-producing bacteria used in the present invention include 8 Streptococcus pyogenes (Streptococcus pyogenes).
cuspyogenes), Streptococcus
Streptococcus equi
, Streptococcus Q equisimilis (Strept.
ococcus equisimil is), Streptococcus dysgalactiae (Streptococcus
coccusdysgalactiae), Streptococcus zooepidemicus (5treptococcus
ccus zooepidemicus ), Pasteurella multocida (Pasteurella multocida)
cida), etc.

The culture solution used in the present invention may be a culture solution used for the cultivation of ordinary microorganisms, and serum may be added as a component of the culture solution. A specific example of the culture solution is, for example, 2.0% glucose, 0:3% monopotassium phosphate.
, dipotassium phosphate 0.2%, sodium thiosulfate 0.01%, magnesium sulfate heptahydrate o, o 1
%, sodium sulfite (0.002 t) (however, the above amount should be 1.5 t/vol.) and 1.5 t/t of revitalized serum can be used.

In the culture of the present invention, any of culture with aeration and stirring of the culture solution, culture with shaking, or culture with gentle stirring without aeration may be used. When the culture solution is aerated and stirred, it is preferable to add 0.5% (weight/volume) of an antifoaming agent to the culture solution. The culture temperature is 30-40°C, preferably 35-30°C.
Keep warm at 8℃.

Since the pH of the culture solution decreases due to the organic acid produced as the hyaluronic acid producing bacteria grow, add an alkaline aqueous solution such as sodium hydroxide or aqueous ammonia to adjust the pH of the culture solution to 6.5 or higher.7 Automatically and continuously control the specified PH1, preferably PH7.0, in a range of less than .5 using a PH controller.

When cultured in this manner for 2 to 3 days, the growth of the hyaluronic acid-producing bacteria almost stops, and the pH of the culture solution rises to 7.5 or higher. At this point, add 2 more sugars to the culture solution.
~7%, preferably 5% (weight/volume %) is added.

The saccharide to be used is one or a mixture of two or more of saccharides such as glucose, fructose, galactose, lactose, sucrose, and blackstrap molasses.

When the saccharide is added, the viscosity of the culture solution gradually increases as hyaluronic acid is produced.

After the addition of the saccharide, when the increase in the viscosity of the culture solution has stopped, the culture solution is diluted by adding ion-exchanged water in an amount equal to or less than 10 times the volume of the culture solution, and then centrifuged or filtered. The bacterial cells of the hyaluronic acid bacteria are removed by filtration. Purified hyaluronan liquor can be obtained by purifying the hindlimb filtrate by ultrafiltration, precipitation fractionation using ethyl alcohol, fractionation using an interfacial agent, ion exchange chromatography, gel filtration chromatography, etc. can.

According to the hyaluronic acid lining of the present invention described above,
The amount of hyaluronic acid C produced by the conventional culture method is 1)
Compared to C, which is 0.3 to 1.09 per cent, culture solution 1)"
It was found that a production amount of 4.5 to 61 times more than 5 times could be obtained, confirming the great effect of the present invention.

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

, Example 1, Comparative Example 1 Glucose 2.0%, Newborn Bovine Blood F4 1.5% by volume, Potassium phosphorus ciIX 0.3%5 Dipotassium phosphate 0.2
From H, sodium thiosulfate o, o1)%, magnesium sulfate heptahydrate 0.01%, sodium sulfite 0.002H and soybean oil 1% (however, H other than φ in serum represents weight/volume H). Culture solution 1,51 (PH7,O
) to preculture solution of Streptococcus equi roomj
inoculated, 300 revolutions per minute, ventilation ft0.7 vvm
, and cultured at a temperature of 35°C.

A 6N aqueous sodium hydroxide solution was added under automatic and continuous control using a P)I controller so that the pH of the culture solution did not fall below 6.94.

After 40 hours, add glucose 50 wt/vol f water solution som.
1 was added aseptically to the culture solution and cultured for 10 hours. The viscosity of the culture solution at this time was 2 cp for 10 hours.
cpK increased. Ion-exchanged water for hindlimb culture medium 3.2
j was added and the bacterial cells were removed by centrifugation. Dilute hydrochloric acid was added to the resulting supernatant to adjust the pH to 4.0, the pH was concentrated to 0.61 using a hollow fiber ultrafilter, and the mixture was further dialyzed against ion-exchanged water. Next, known methods such as precipitation fractionation using ethyl alcohol, treatment with cetylpyridinyl ammonium chloride, and chromatography using ion exchange cellulofine, etc.
It was purified by the method described above to obtain a white powder of sodium hyaluronate with a concentration of 8.5I.

The production amount was 5.6 I per liter of culture solution.

The protein content of the hyaluronic acid thus obtained was 0.051
The molecular weight was determined by gel filtration chromatography using Sepharose 6B to ensure that the pH was 2x10. Even when the temperature reached 5 or higher, the culture was continued for another 10 hours without adding glucose.

The culture solution was treated and purified in the same manner as in Example 1, and 0.
9 g of purified sodium hyaluronate white powder was obtained.

The production amount of culture solution 1) was 90.6 II.

that's all

Claims (2)

[Claims] (1) When culturing microorganisms that have the ability to produce hyaluronic acid in a culture solution, the pH of the culture solution should be 6.5 or more and 7.
．． By culturing while automatically continuously controlling the pH so that it does not fall below a specified pH of less than 5, and when the pH reaches 7.5 or higher, further sugars are added and cultured. A method for producing hyaluronic acid using a fermentation method, which is characterized by producing and accumulating hyaluronic acid. (2) A method for producing hyaluronic acid by the fermentation method according to claim (1), using one or more selected from glucose, fructose, galactose, lactose, sucrose, or blackstrap molasses as the saccharide.