Unveiling synergy in ultrasound-assisted enzymatic extraction: Role of treatment sequence and biomass complexity

• The impact of ultrasound on the susceptibility of different pectic substrates to enzymatic degradation was evaluated. • The effect of substrate complexity was assessed using both purified pectin and pectin-rich biomasses. • Ultrasound enhanced enzymatic degradation only in complex biomass. • The coupling mode of ultrasonic and enzymatic treatments strongly affected the enzymatic reaction. • Simultaneous ultrasonic and enzymatic treatment promoted enzymatic cofactor release, improving enzyme activity. Emerging extraction methods, such as enzyme-assisted extraction (EAE) and ultrasound-assisted extraction (UAE), are considered safer and more sustainable alternatives to conventional techniques, due to their reduced solvent usage. Nevertheless, their application remains limited due to their low efficiency and sensitivity to environmental conditions. To overcome these drawbacks, ultrasound-assisted enzymatic extraction (UAEE) has been proposed as an alternative synergistic approach to improve biomass disruption. In this study, the effect of ultrasound on the susceptibility of pectic substrates to enzymatic degradation was evaluated using both purified pectin and pectin-rich complex matrices (grapefruit peels and apple pomace). The extent of enzymatic degradation was assessed by monitoring the release of reducing sugars, while microscopic evaluation of the cell microstructure, the total phenolic release (TPC) and metal element release were quantified to support the findings. Results indicate that ultrasonic pre- and post-treatments had no significant impact on pectin hydrolysis. In contrast, when ultrasound was applied during the enzymatic reaction, the extent of hydrolysis increased, but only in the case of the complex matrices, indicating a synergistic effect. The increased release of calcium and potassium ions suggested that ultrasound induced an enzymatic cofactor release from biomass, contributing to improved enzyme activity. Similar degradation of the cell microstructure was observed in the case of ultrasonic post treatment, although without improved hydrolysis of cell-wall pectin. This implies that prior enzymatic action weakened the matrix, making it more fragile. This is the first study to investigate the impact of plant matrix structure on the synergistic effect of the ultrasound-enzyme combination.