To measure PME activity, a fruit sample (10 g)
was homogenized with 25 mL of Tris-Cl 0.1 M buffer
at pH 8.0, containing 0.3 M NaCl in an Ultra
Turrax®T25 and placed in a Thermolyne Speci-Mix
agitator at 4°C for 30 min, followed by centrifugation at
9400 g for 25 min at 4°C. The enzymatic extract was
stored at -35°C until analysis and PME was determined
following the method of Rouse and Atkins (1955), with
some modifications. This method consists of the
evaluation of the activity of the enzyme through
titration, using as a substrate 25 mL of 1% pectin in
0.1N NaCl at 7.5pH, which was adjusted with 0.1N
NaOH. The pectin was placed at water bath at 30°C for
10 min and 2 mL of the extract was added. Decrement
of pH caused by the carboxylic groups, generated by the
PME during the desertification of the pectin solution
were kept constant at a 7.5 pH by titrating the solution
with 0.049N NaOH for 10 min at room temperature
(24°C). Titration was performed with an automatic
Mettler DL21 titrator. Results were expressed as a unit of
PME activity, which is defined as the amount that the
enzyme required to hydrolyze 1 μmol of carboxyl
groups, produced in 1 mL of pectin substrate per
minute.