Synergistic effects of ethanol pretreatment and sequential enzymatic treatments on structure and functionality of porous cassava starch.

Granular cassava starch possesses a naturally smooth surface, making it inherently resistant to pore formation within the granular form. This study aimed to develop porous cassava starch through a combination of hot ethanol pretreatment (Et) and enzymatic hydrolysis using maltogenic α-amylase (MA), amyloglucosidase (AMG), or their combination (MA + AMG). Ethanol pretreatment effectively collapsed the granular surface, enhancing enzymatic accessibility, as confirmed by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) analyses. The sequential Et➔MA + AMG process resulted in the most pronounced structural changes, yielding the lowest recovery and highest sugar release. Ethanol pretreatment followed by enzymatic hydrolysis resulted in a high specific surface area, total pore volume, increased relative crystallinity, reduced amylose content and pasting parameters, particularly in the Et➔MA and Et➔MA + AMG treatments. Differential scanning calorimetry revealed increased gelatinization temperatures and narrower transition ranges, reflecting improved granular stability. Furthermore, Et➔MA + AMG sample demonstrated superior encapsulation efficiency (72.71%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition (82.38%) for gallic acid, indicating enhanced functionality and antioxidant protection. These findings highlight the synergistic impact of ethanol pretreatment and enzymatic hydrolysis, especially the combination of MA and AMG, in enhancing starch porosity and functionality. The resulting porous cassava starch demonstrates strong potential as an effective encapsulant in food, pharmaceutical, and nutraceutical applications.