An on-line method for pressurized hot water extraction and enzymatic hydrolysis of quercetin glucosides from onions

•Total quercetin content was determined in different onion varieties.•Hot water extraction was combined with hydrolysis using thermostable β-glucosidase.•Enzyme activity was highest in water containing 5% ethanol at 84 °C and pH 5.5.•The immobilized enzyme showed good stability over 72 h at the optimized conditions.•The developed method gave comparable yield of quercetin compared to conventional methods.

A novel environmentally sound continuous-flow hot water extraction and enzymatic hydrolysis method for determination of quercetin in onion raw materials was successfully constructed using a stepwise optimization approach. In the first step, enzymatic hydrolysis of quercetin-3,4′-diglucoside to quercetin was optimized using a three level central composite design considering temperature (75–95 °C), pH (3–6) and volume concentration of ethanol (5–15%). The enzyme used was a thermostable β-glucosidase variant (termed TnBgl1A_N221S/P342L) covalently immobilized on either of two acrylic support-materials (Eupergit® C 250L or monolithic cryogel). Optimal reaction conditions were irrespective of support 84 °C, 5% ethanol and pH 5.5, and at these conditions, no significant loss of enzyme activity was observed during 72 h of use. In a second step, hot water extractions from chopped yellow onions, run at the optimal temperature for hydrolysis, were optimized in a two level design with respect to pH (2.6 and 5.5), ethanol concentration (0 and 5%) and flow rate (1 and 3 mL min−1) Obtained results showed that the total quercetin extraction yield was 1.7 times higher using a flow rate of 3 mL min−1 (extraction time 90 min), compared to a flow rate of 1 mL min−1 (extraction time 240 min). Presence of 5% ethanol was favorable for the extraction yield, while a further decrease in pH was not, not even for the extraction step alone. Finally, the complete continuous flow method (84 °C, 5% ethanol, pH 5.5, 3 mL min−1) was used to extract quercetin from yellow, red and shallot onions and resulted in higher or similar yield (e.g. 8.4 ± 0.7 μmol g−1 fresh weight yellow onion) compared to a conventional batch extraction method using methanol as extraction solvent.

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