To investigate the amount of encapsulated calcium in the final
particles, the release of calcium in the aqueous phase was measured
for each washing step. Fig. 4 summarizes the results, in
which letters A to E refer to sequential washing steps. Note the differences
in scale between the different graphs. The results are
expressed as mg calcium per g of protein. Control sample still contained
approximately 0.6 mg calcium/g of protein. For particles
containing calcium concentrations up to 30.0 mM, a large part of
the calcium was retained during washing and redispersion. The
proteins of the samples prepared with 7.4 mM, 15 mM and
30 mM calcium retained approximately 100%, 92% and 62% of the
initial calcium, respectively. Higher concentrations of calcium in
the initial protein solution resulted in significant calcium losses.
The particles prepared with 100 mM calcium decreased to 30% of
its initial calcium content bound per protein, and the 400 mM sample
decreased to 11% of its initial calcium content bound per protein.
In both samples, most calcium was immediately lost in the
first washing step, and the calcium content decreased slightly in
subsequent washing steps. No significant further losses of calcium
were found upon storage for a week (data not shown).