MPC80 was mixed into Millipore water, agitated by an overhead
mixer to a final concentration of 5 g protein 100 g1. Mixing ceased
when all protein powder was visibly hydrated and the MPC80
dispersion was then stored overnight at 4 C in a sealed vessel to
allow for complete hydration.
MPC80 dispersion was weighed into 4-L Erlenmeyer shake
flasks, equilibrated to room temperature, and adjusted to the optimum
hydrolysis pH using hydrochloric acid and sodium hydroxide.
For trypsin and chymotrypsin hydrolysis, the pH of the MPC80
dispersion was adjusted to 8.0. MPC80 dispersions for papain hydrolysis
were adjusted to pH 6.8 and those for pepsin hydrolysis
were adjusted to pH 2.0. After pH adjustment, the MPC80 dispersion
temperaturewas equilibrated to 37 C for hydrolysis by trypsin
or pepsin, 50 C for chymotrypsin, and 60 C for papain.
Enzyme solutions were prepared separately in Millipore water
for chymotrypsin, trypsin, and papain, and in 0.01 mol L1 hydrochloric
acid for pepsin at concentrations of 100 g L1. Enzyme solution
was added to provide 1 g enzyme for every 100 g protein
being hydrolyzed. The sealed 4-L flask was agitated in a shaker
incubator at controlled temperature and times (Table 1). Two hydrolysis
times were selected for each enzyme based on preliminary
hydrolysis work (data not shown). At the end of hydrolysis, the
reactions were stopped by placing the flasks in a 95 C water bath
for 10 min. A non-hydrolyzed MPC80 control solution prepared at
5 g protein 100 g1 was also heated at 95 C for 10 min (MPCFD).
MPCFD and the MPC80 hydrolysates were freeze dried and ground