Thiamine Hydrochloride → L-Leucine

Temperature

121 °C

Brevibacterium flavum AJ 3686 (FERM-BP 755) was inoculated on agar plate medium containing 1% yeast extract, 1% peptone, 0.5% sodium chloride and 0.5% glucose followed by culturing at 31° C. for 24 hours. In 30 Sakaguchi flasks, each having a 500 ml volume, were charged 25 ml each of medium (pH 7.0) composed of 3% glucose, 0.1% potassium phosphate, 0.04% magnesium sulfate, 0.001% iron sulfate, 0.001% manganese sulfate, 0.3% urea, 10 μg/l biotin, 200 μg/l vitamin B1 and 0.085% (calculated as total nitrogen) of soybean protein hydrolyzate. After sterilization by heating at 121° C. for 15 minutes, the temperature was lowered to room temperature and one platinum loopful of the cells of Brevibacterium flavum AJ 3686 previously grown on the agar plate medium was inoculated on the sterilized medium. By shake culturing at 31° C. for 24 hours, a seed culture broth was prepared. In a fermentation tank of 20 liter volume, was charged 10 liters of medium (pH 7.0) composed of 15% glucose, 0.1% potassium phosphate, 0.04% magnesium sulfate, 0.001% iron sulfate, 1% ammonium sulfate, 50 μg/l biotin, 300 μg/l vitamin B1 and 0.01% (calculated as isoleucine) of soybean protein hydrolyzate. After sterilization by heating at 121° C. for 20 minutes, the temperature was lowered to room temperature and the seed culture broth previously cultured in the flask was added to the medium followed by culturing under conditions: culture temperature at 31° C., controlled pH of 7, aeration amount of 1/2 vvm, a stirring number of 600 rpm and inner pressure of 0.2 kg/cm2. Immediately before the glucose in the culture broth was consumed, a solution (sterilized) containing 45 g/l of glucose was replenished to the culture tank to control the glucose concentration in the culture broth to always keep it between 0.1 to 1.5%. At and from the time period when the amount of leucine accumulated exceeded its solubility and the crystals of leucine began to crystallize in the culture broth, a part of the culture broth was aseptically taken out of the system, passed through a small-sized liquid cyclone to isolate the crystals. Then the culture broth was returned to the culture tank. Culture for 60 hours by this method gave 0.24 kg (moisture content of 16.6%) of the slurry of leucine crystals and 14 liters of the culture broth containing 2.6% leucine. The obtained culture broth was crystallized by concentration. The crystals were isolated and combined with the slurry of crystals obtained during the culture. By repeating dissolution and crystallization three times, 0.42 kg of leucine crystals was obtained in a high purity.

1. additioncontaining 1% yeast
2. extractionextract
3. additionIn 30 Sakaguchi flasks, each having a 500 ml volume, were charged 25 ml each of medium (pH 7.0)
4. customwas lowered to room temperature
5. waitBy shake culturing at 31° C. for 24 hours
6. customa seed culture broth was prepared
7. additionIn a fermentation tank of 20 liter volume, was charged 10 liters of medium (pH 7.0)
8. temperatureAfter sterilization by heating at 121° C. for 20 minutes
9. customwas lowered to room temperature
10. additionwas added to the medium
11. customat 31° C.
12. customImmediately before the glucose in the culture broth was consumed
13. additiona solution (sterilized) containing 45 g/l of glucose
14. concentrationthe glucose concentration in the culture broth
15. customto crystallize in the culture broth
16. customto isolate the crystals

ORD record

ord-8f91d83e76bb42769739973e67b38a05

Source dataset

uspto-grants-1996_08

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US05547858