Strain improvement of Aspergillus ficuum NRRL 3135 and optimization of conditions for phytase production for feed application

Varying amounts of fermentation medium in 500-ml flasks were used (10%, 20% and 30% v/v) to determine the effect of the volume of the medium on phytase production. Phytase production increased significantly as the fermentation volume was increased from 10% (137.67 U/ml) to 20% (180.37 U/ml). There was no significant difference between 20% and 30% (203.34 U/ml). Phytase production was found to be highest in the medium with an initial pH of 5.5 (231.1 U/ml), followed by pH 6.0 (203.04 U/ml), which was not significantly different. At neutral pH level, phytase production was significantly the lowest (62.09 U/ml). Addition of 0.2% peptone significantly increased phytase production from 136.21 U/ml to 261.05 U/ml. Results of the restricted random balance design experiments showed that rice bran and potassium chloride (KCl) had the most influence as phytase production. The new basal medium for further optimization of phytase production should have 40 grams rice bran, 0.5 grams KCl, 7.5 grams sodium nitrate, and 0.2 gram dipotassium phosphate per liter of the medium as the minimum level. Negative coefficient of influence values of glucose, ferric sulfate and magnesium sulfate means that their lower level should be the upper level or the maximum amount for the new basal medium. Time course analysis of the fermentation process revealed that the pH of the medium did not significantly affect phytase production. The pH of the medium decreased from pH 5.5 (day 0) to 3.1 during the 8-day fermentation period. A significant decrease in glucose was observed from the first day (4.82 mg/ml) to the fourth day of fermentation (0.73 mg/ml) accompanied by a corresponding increase in phytase production. Phytase was detected on the first day of fermentation (76.07 U/ml) reaching its peak on the seventh day (446.97 U/ml). The crude phytase enzyme was partially characterized. Its optimum pH is around pH 4.5 to 5.5, and it was stable at pH levels 4.5, 5.0 and 5.5. The optimum temperature of phytase activity was between 50-55 deg C. It was stable at 45deg C for three hours, but as inactivated at 60 deg C after 30 minutes of exposure. The crude phytase enzyme was stable up to one month of storage both in the freezer and in the refrigerator. A significant increase in apparent percent digestibility of phosphorus and calcium was observed in broilers fed with 135 U crude phytase enzyme per gram of feed. Phosphorus digestibility was increased from 21.45% to 42.92%, and calcium digestibility was increased from 55.99% to 70.33%, while no significant increase in crude protein digestibility was observed