Dairy Chemistry And Biochemistry

MILK PROTEINS 155

4.3.10

Highly purified whey protein preparations, referred to as whey protein

isolates (containing 90-95% protein), are prepared industrially from whey

by ion exchange chromatography. Denatured (insoluble) whey proteins,

referred to as lactalbumin, may be prepared by heating whey to 95°C for

10-20 min at about pH 6.0; the coagulated whey proteins are recovered by

centrifugation. The whey proteins may also be precipitated using FeCl, or

polyphosphates (section 4.15.6).

Other methods for the preparation of whey proteins

4.4 Heterogeneity and fractionation of casein

Initially, casein was considered to be a homogeneous protein. Heterogeneity
was first demonstrated in the 1920s by Linderstrsm-Lang and co-workers,
using fractionation with ethanol-HC1, and confirmed in 1936 by Pedersen,
using analytical ultracentrifugation, and in 1939 by Mellander, using free
boundary electrophoresis. Three components were demonstrated and
named a-, ,!?- and y-casein in order of decreasing electrophoretic mobility
and represented 75, 22 and 3%, respectively, of whole casein. These caseins
were successfully fractionated in 1952 by Hipp and collaborators based on
differential solubilities in urea at c. pH 4.6 or in ethanol/water mixtures; the
former is widely used although the possibility of forming artefacts through
interaction of casein with cyanate produced from urea is of concern.
In 1956, Waugh and von Hippel showed that the a-casein fraction of
Hipp et al. contained two proteins, one of which was precipitated by low
concentrations of Ca2+ and was called a,-casein (s = sensitive) while the
other, which was insensitive to Ca2+, was called k--casein. a,-Casein was
later shown to contain two proteins which are now called uSl- and
a,,-caseins. Thus, bovine casein contains four distinct gene products, desig-
nated ctsl-, rs2-, /I- and K-caseins which represent approximately 37, 10,^35
and 12% of whole casein, respectively.
Various chemical methods were developed to fractionate the caseins but
none gives homogeneous preparations. Fractionation is now usually
achieved by ion-exchange chromatography on, for example, DEAE-cellu-
lose, using urea-containing buffers; quite large (e.g. 10 g) amounts of
caseinate can be fractionated by this method, with excellent results (Figure
4Sa, b). Good results are also obtained by ion-exchange chromatography
using urea-free buffers at 2-4°C. High performance ion-exchange
chromatography (e.g. Pharmacia FPLCTM on Mono Q or Mono S) gives
excellent results for small amounts of sample (Figure 4.5c, d). Reversed-
phase HPLC or hydrophobic interaction chromatography may also be used
but are less effective than ion-exchange chromatography.