2.6. Mineral dialyzability
A modification of the widespread in vitro Miller, Schricker,
Rasmussen, and Van Campen (1981) method, according to Drago,
Binaghi, and Valencia (2005) was followed. The samples were
ground previously and then prepared to 10 g solid/100 g dispersion
using deionizedwater. Aliquots (25 g) of homogenized sampleswere
adjusted to pH 2.0 with 4 mol/L HCl and after addition of 0.8 mL
pepsin digestionmixture (160 g/L pepsin (Sigma P-7000) solution in
0.1mol/LHCl),were incubated at 37 C for 2 h in a shakingwater bath.
At the end of pepsin digestion, dialysis bags containing 20 mL
0.15 mol/L PIPES (piperazine-N,N0-bis [2-ethane-sulfonic acid] disodium
salt) buffer (Sigma P-3768) were placed in each flask and were
incubated for 50min in a shakingwater bath at 37 C. Pancreatin-bile
mixture (6.25 mL of 25 g/L bile (Sigma B-8631), 4 g/L pancreatin
(Sigma P-1750) solution in 0.1mol/LNaHCO3)was then added to each
flask and the incubation continued for another 2 h. Then, bag contents
were weighed and analyzed for its mineral content by flame atomic
absorption spectroscopy. Mineral dialyzability was calculated from
theamount of each dialyzedmineral expressed as a percentage of the
total amount present in each sample.
Dialyzable Mineral ðDM%Þ ¼ ½D=ðW AÞ 100
Where: D is the total amount of dialyzed mineral (mg); W is the
weight of sample (g) and A is the concentration of each mineral in
the sample (mg/g).