Optimized Formation of Benzyl Isothiocyanate by Endogenous Enzyme and its Extraction from <i>Carica Papaya</i> Seed

Abstract

Purpose : To use endogenous myrosinase in Carica papaya seed to convert benzyl glucosinolate (BG) to benzyl isothiocyanate (BITC) and then extract it for further studies. Methods : Process variables including seed powder particle size, sample-to-solvent ratio, pH of buffer solution, enzymolysis temperature, enzymolysis time were investigated using single-factor experiments and response surface methodology coupled with Box-Behnken design for further optimization of conversion conditions. The formed benzyl isothiocyanate was extracted by steam distillation method and purified by thin-layer chromatography (TLC). Relevant process variables were also studied. Finally, the purified benzyl isothiocyanate was analyzed by gas chromatography-mass spectrometer (GC-MS) and compared to BITC standard. Results : Optimum enzymolysis conditions were seed powder particle size, 90 - 120 µm; sample-tosolvent ratio, 1:20; pH of buffer solution, 4.8; enzymolysis temperature, 40 0C; and enzymolysis time, 27 min. Enzymolysis liquid was steam-distilled for 2 h, extracted twice using dichloromethane (DCM) (using 3 times the volume of distillate each time) as extractant. Finally, 1:20 was adopted as the optimal dichloromethane-petroleum ether (DCM-PE) elution proportion to wash out the unnecessary compounds. Conclusion : The result showed that benzyl isothiocyanate was formed under the optimized conditions and was no doubt the basic component of the extraction. Keywords : Carica papaya, Benzyl isothiocyanate, Endogenous myrosinase, Enzymolysis, Extraction