The whey protein microbeads formed by cold set gelation were
manufactured as described by Egan et al. (2013a). Whey protein
was rehydrated in deionised water (containing 0.02% sodium
azide) under magnetic stirring to 10% w/w on a protein basis and
left to stand overnight at 4 C to allow hydration of the proteins.
The solution was denatured by heating to 80 C and holding for
30 min in a shaking water bath (Fisher Scientific, model DMS360)
and subsequently cooled to 25 C in ice water. The heat denatured
whey protein solution (15 ml, pH 6.8) was dispensed at 20 C
through a 10 ll pipette tip using a variable speed transfer pump
at a rate of 0.6 ml per minute (Gilson minipuls 2, Villiers-le-Bel,
France) into 100 ml of 100 mMcalcium chloride cross-linking solution
to produce microbeads 1–2 mmin diameter. These beads were
stored in the calcium chloride for 24 h at 4 C followed by multiple
rinsing steps in deionised water.