Khongkomolsakul, W.; Buathong, P.; Yang, E.; Dadmohammadi, Y.; Zhou, Y.; Li, P.; Yang, L.; Frazier, P.; Abbaspourrad, A.

Phytase (phyA) breaks down phytate, which can help with nutrient absorption in a plant-based seed diet or high-phytate food. Unfortunately, it is prone to denaturation at food preparation temperatures and is easily inactivated by pepsin during gastric digestion. To protect phyA for use in high-temperature processes (100 ) and gastric digestion, chitosan (CS) was used to complex phyA. Bayesian optimization, a machine learning technique, was used to demonstrate how to expedite the optimization process. Thermal stability of the optimized complex increased from 20% (Control: phyA in the native state) up to 74% at 4:1 CS to phyA (CS-phyA) complex and 52% at the 1:1 CS-phyA complex as measured by phytase activity assay. Chitosan complexation also improved the retention of enzyme activity after thermal and gastric digestion by 13-fold, retaining residual activity at 40% for the 4:1 CS to phyA and 22% for the 1:1 CS-phyA complexes compared to the enzyme itself, which only retained 3% residual activity. Molecular docking and circular dichroism were used to investigate the underlying interaction mechanism between CS and phyA and the secondary structure of the enzyme after heat treatment. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) confirmed the complexation of phyA with CS and revealed complex morphology. With improved enzyme stability, there is great potential for efficiently expanding phytase applications in a high plant-based seed food matrix.