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An integrated process was developed to hydrolyze the phytates in light steep water (LSW) and to simultaneously isolate inorganic phosphate (Pi) and myo-inositol products. The proposed integrated process is helpful in resolving the environmental and nutritional concerns in the use of corn gluten feed (CGF) in the animal diets. This process comprised of partial and total hydrolysis of LSW and intermediate anion exchange separation technique. The phytates in LSW were initially degraded to negatively charged myo-inositol phosphates (InsP2 - InsP5). The optimized experimental parameters for the partial hydrolysis of LSW were determined to be 2 h hydrolysis with 1 FTU A. niger/g substrate at 35°C. The negatively charged species of the partially hydrolyzed substrate were separated on a strong base anion exchange resin. The negatively charged species, retained by the resin, were eluted with 1M NaCl solution and were subjected to complete hydrolysis with E. coli, A. niger-derived phytases and their respective combinations. The maximum amount of myo-inositol released from the anion exchange column was detected after 48 h reactions catalyzed by 100 FTU E. coli, 150 FTU E. coli, and 150 FTU the combination of A. niger and E. coli. The time course of Pi released showed a similar trend to that of myo-inositol and the released Pi reached a maximum amount after 48 h incubation at the enzyme loadings for which the maximum concentration of myo-inositol were reached.
An isocratic HPLC method was developed for routine analysis of myo-inositol and Pi. For myo-inositol, the limit of detection (LOD) was 0.01 mg/ml, and the limit of quantification (LOQ) was 0.04 mg/ml. The linear range of this method for myo-inositol was 0-20 mg/ml. The HPLC method is also a fast method for Pi quantification in the hydrolysate. The linear range of this method for Pi was 0-10 mg/ml. The LOD and LOQ were 0.05 and 0.17 mg/ml, respectively.
A size-exclusion chromatography packed was developed for isolating and purifying myo-inositol and Pi, from the mixture with E.coli phytase and Cl-.
Adviser: Hossein Nouredddini