METHOD OF PREPARING AN IMMUNITY STIMULATING AGENT USED AT DIFFERENT DISEASES Russian patent published in 1998 - IPC

FIELD: microbiology, biotechnology. SUBSTANCE: invention relates to preparing an immunostimulating agent from cultural fluid of strain-producer Vibrio cholerae. Method involves culturing a producer on nutrient medium containing carbon and nitrogen sources and mineral salts, additional feeding with glucose, aeration and stirring in the process of culturing. At the end of log-phase of producer growth vibrios are rendered safe followed by isolation of toxin from cultural fluid supernatant. In the process of producer culturing temperature is maintained at the level 36-37 C for the first 2-3 h and at 34.25-35.75 C - for the last hours and at pH - 7.3-7.9 and 7.8-8.2, respectively. Glucose feeding is 5-7 ml/min for the first 4-5 h, 7.5-12.5 ml/min at the next 2 h and 15-20 ml/min - for the last 1-2 h (as measured for 1 l nutrient medium). Additional feeding is carried out with ammonia (3 ml/min at the first 2-4 h). In the process of culturing aeration is carried out at the rate 400 ml/min for the first 3 h, at 500 ml/min for the next 3-4 h and at 500-600 ml/min for the last 1-2 h. Formalin (final concentration is 0.6 vol.%) is added to cultural fluid to convert toxin to anatoxin. Cultural fluid supernatant is removed and kept at 10-12 C for 25-34 days at pH 6.7-7.3 at constant formalin concentration 0.2 vol.%, not less. After keeping anatoxin is precipitated from supernatant with ammonium sulfate at saturation 38-80 vol.%. Obtained precipitate is separated, resuspended and purified by dialysis. Method ensures to obtain ecologically harmless, nontoxic and highly active immunostimulating agent. EFFECT: increased yield of the end product, simplified method of preparing. 1 tbl