CS276099B6 - Process for the fermentative production of hyaluronic acid - Google Patents

Abstract

The production method consists in the mutant strain Streptococcus zooepidemicus (syn. Streptococcus equi) DBM 1079 acting in a fermentation medium which contains, in addition to 1 to 8% w/v of fermentable carbohydrate, phosphates, magnesium and other minor ions, 0.10 to 0.30% w/v of yeast autolysate and 0.10 to 0.50% w/v of peptone, polypeptone, soy peptone or casein hydrolysate. Hyaluronic acid is used in human and veterinary medicine.

Description

The invention relates to a process for the production of hyaluronic acid by a fermentation process.

Hyaluronic acid, ie a mucosal glycosaminoglycan with a linear polymer molecule with a molecular weight of 2x10 to 1.3x10 daltons, is increasingly used in human and veterinary medicine and cosmetics. Its animal resources, such as rooster combs, are not sufficient for this need, and it has therefore begun to be produced by a fermentation process. Hitherto, species of the genus Streptococcus of serological groups A and C have been used for this purpose, i.e. Streptococcus pyogenes, Streptococcus equi and Stretococcus zooepidemicus, which is recently considered to be identical to Streptococcus equi.

The first fermentation procedure for the preparation of hyaluronic acid using hemolytic streptococcus group A is described by Holmstrom B. and Řičica J. (Appl. Microbiol. 15, 1409-1413, 1967), but the yield in calf infusion medium supplemented with 1.1% glucose was only 0. , 4 g of hyaluronic acid per liter of medium. All strains and mutants of the genus Streptococcus used in the processes described in the patent and other literature require a very rich fermentation medium containing, in addition to 3 to 8% by weight, gullucose and 0.5 to 3% by weight in order to obtain a yield of 3 to 6 g of hyaluronic acid per liter of fermentation medium. , yeast autolysate together with 2% by weight, casein hydrolysates (Nimrod A. et al., W 086/04355) or with 1 to 3% by weight, peptone (Shiseido Co. Ltd., JP 58-56692, Offenlegungsschr. DE 35 17 629, Hosoya Η., EP 0244754, Murata H., JP 62-289197, Myamori T., EP 0266578, Akasaka H., JP 61-219394, Karato et al., JP 62-51999) or even 1.5 to 2 , 5% by volume of calf serum (Morita et al. JP 61-63294, Offenlegungssch. DE 35 31 612) and in addition with the addition of certain amino acids and / or cornsteep ligorm (Murata H., JP 62-289197, Takayama K., JP 62-32893-, Karato et al., JP 62-51999, Miyazaki T., JP 62-289198). In addition, the strains used still require a relatively long fermentation time, i.e. 2 to 5 days with continuous adjustment of the pH to 7.0. If washed Streptococcus cells are used for the production of hyaluronic acid (Morita Y. et al., JP 61-15698, Miyamori T. et al. EP Appl. 0266578 A 2), these very expensive soils must be used to obtain production cells.

These shortcomings are eliminated by the process according to the invention, which uses a mutant strain of Streptococcus zooepidemicus (syn. Streptococcus equi) DBM 1079 as a production microorganism, which is stored in the collection of microorganisms of the Department of Biochemistry and Microbiology at hyaluronic acid in a fermentation medium which contains in addition to 1 to 8% w / v. of fermentable saccharide, phosphates, magnesium and other minor ions 0.10 to 0.30% w / v. yeast autolysate and 0.10 to 0.50% w / v. peptone, polypeptone, soy peptone or casein hydrolysates. The fermentable saccharide can be added in two to four portions during the fermentation process. The actual concentration of the fermentable saccharide is in the range of 0.5 to 3.5% w / v. The source of nitrogen and growth substances is added in the form of yeast autolysate. The source of phosphorus is, for example, potassium phosphate. Of the minor ions, iron, manganese, or calcium and cobalt ions are needed. Further stimulation of hyaluronic acid production can be achieved by the optional addition of 0.005 to 0.3% w / v. arginine or glutamate.

The process according to the invention proceeds as follows. The fermentation medium is inoculated with an 8 to 16 h old culture of Streptococcus zooepidemicus (syn. Streptococcus equi) DBM 1079 in the soil of the above composition. The fermentation is carried out with stirring and gentle aeration at 36 to 37 DEG C., the lactic acid and hyaluronic acid formed being continuously neutralized by the addition of potassium or sodium hydroxide solution. The fermentation time is 22 to 26 hours and its end is determined either from the cessation of the decrease in the concentration of fermentable saccharide in the medium or from the cessation of the increase in the viscosity of the medium, determined for example by the agitator load. After fermentation, the medium is immediately freed of cells, preferably by centrifugation. Heating the fermentation broth with the cells before centrifugation (eg 30 min at 85 ‘C) facilitates the centrifugation of the cells and leads to an increase in the yield of hyaluronic acid. The yield of hyaluronic acid is 4 to 5 g / l of the original medium. . »'/ F

CS 276099 B6 2

The process for the fermentation production of hyaluronic acid according to the invention is economically very advantageous. The strain used requires a substantially poorer nutrient medium for fermentation, the fermentation time lasting 22 to 26 hours with significantly higher yields of 4 to 5 g / l of nutrient medium. At the same time, the capacity of the automated fermentation plant will increase, which means savings in energy and costs for other equipment.

The invention is further elucidated on the basis of a specific example of the production of hyaluronic acid.

Example

Sterile fermentation medium containing in 1 liter 20.0 g glucose, 5.0 g peptone, 2.5 g yeast autolysate, 5.0 g K ₂ HPO ₄ , 0.1 g Na ₂ S ₂ O ₃ , 1.5 g MgSO ₄ .7H ₂ O, 0.01 g FeSO ₄ .7H ₂ O and 1.5 ml Tween 80 are inoculated with 120 ml of a 10 h culture of Streptococcus zooepidemicus DBM 1079 in soil of the same composition as the fermentation medium, which was incubated with gentle shaking at 37 c. The fermentation takes place again at 37 DEG C. with gentle stirring and aeration, the pH being maintained at 7.0 by automatic control of the metering of the 3 mol / liter NaOH solution added. After 6 hours of fermentation, 40 ml of sterile 30% w / v is added. glucose solution and the fermentation is continued. Another 40 ml of sterile 30% w / v. of glucose solution is added only after the concentration of glucose in the fermentation broth has dropped to 1.0 to 1.7% w / v. The fermentation is terminated when the glucose concentration reaches 0.3 to 0.5% w / v, which is achieved within 24 hours of total fermentation. The fermentation broth is centrifuged (30 min at 5,000 g). 4.5 g of sodium hyaluronate are isolated from the supernatant by a known method of precipitation with twice the volume of ethanol and purification of the precipitate obtained by dialysis.

Claims (2)

PATENT CLAIMS A process for the fermentation production of hyaluronic acid, characterized in that the mutant strain of Streptococcus zooepidemicus (syn. Streptococcus equi) DBM 1079 is operated in a fermentation medium which contains, in addition to 1 to 8% w / v. of fermentable saccharide, phosphates, magnesium and other minor ions 0.10 to 0.30% w / v. yeast autolysate and 0.10 to 0.50% w / v. peptone, polypeptone, soy peptone or casein hydrolyzate. 2. The method according to item 1, characterized in that the fermentable saccharide is added in 2 to 4 portions during the fermentation process.