2.2. Methods
2.2.1. Sample preparation
To process the olive samples the following steps were carried
out: (1) cleaning and leaves removal; (2) washing; (3) milling by
crusher (Rheinische Strabe 36. D. Hann. Germany, Type SK-1) to
obtain a fine paste and kept frozen until use; (4) The temperature
of the samples was adjusted to enzyme activity temperature in
warm water bath, and then the enzymes were added in the beginning
of the kneading step by using suitable doses; (5) kneading of
the resultant paste under stirring (60 min, 80 rpm); (6) centrifugation
of paste at 4500g, 20 min (BHG ROTO UNI II, Germany); (8)
heating to separation of emulsion (a thin but distinctive emulsion
layer between the oil and aqueous phases) into an oil and an aqueous
phase; and (9) mixing the serum with hexane in order to separate
the oil. The solvent was evaporated at 50 C. Reference
extractions, without employing the enzyme preparations, were
also carried out.