Enzyme-Assisted Supercritical Fluid Extraction of Flavonoids from Apple Pomace (Malus×domestica)

Herein an enzyme-assisted supercritical fluid extraction (EA-SFE) was developed using the enzyme mix snailase to obtain flavonols and dihydrochalcones, subgroups of flavonoids, from globally abundant waste product apple pomace. Snailase, a commercially available mix of 20-30 enzymes, was successfully used to remove the sugar moieties from quercetin glycosides, kaempferol glycosides, phloridzin and 3-hydroxyphloridzin. The resulting flavonoid aglycones quercetin, kaempferol, phloretin and 3-hydroxyphloretin were extracted using supercritical carbon dioxide (scCO₂) and minimum amounts of polar cosolvents. A sequential process of enzymatic hydrolysis and supercritical fluid extraction was developed, and the influence of the amount of snailase, pre-treatment of apple pomace, the time for enzymatic hydrolysis, the amount and type of cosolvent and the time for extraction, was studied. This revealed that even small amounts of snailase (0.25 %) provide a successful cleavage of sugar moieties up to 96 % after 2 h of enzymatic hydrolysis followed by supercritical fluid extraction with small amounts of methanol as cosolvent, leading up to 90 % of the total extraction yields after 1 h extraction time. Ultimately, a simultaneous process of EA-SFE successfully demonstrates the potential of snailase in scalable scCO₂ extraction processes for dry and wet apple pomace with satisfactory enzyme activity, even under pressurized conditions.