The calcium concentration was determined using a flame
atomic absorption spectrophotometer (Model A Analyst 400,
Perkin Elmer, Santa Clara, California, USA), using a lumina hollow
cathode lamp (Perkin Elmer) based on the adapted methodology
of AOAC – Association of Official Analytical Chemists. (1995).
Approximately 6 g of fresh samples (without treatment) and 2 g
of treated samples, i.e., freeze dried and previously ground, were
weighed in a 50 ml glazed, porcelain crucible placed in a muffle
furnace and heated up to 550 C until complete ignition. Then,
the porcelain crucibles were cooled in desiccators, where 20 ml
of chloride acid (0.1 M) was added to the capsules with fresh samples
and 30 ml was added to the treated samples. The ash was dissolved,
and then, an aliquot of this solution was quantitatively
taken and diluted 8 times (fresh samples) or 100 times (treated
samples) with 0.1 M chloride acid. Standard calcium solutions
between 2 and 20 ppm were used to determine a calibration curve
of absorbance versus ppm of calcium.