Journal of Dairy Research (1997) 64 221230 Printed in Great Britain 221

Application of capillary electrophoresis to the study of proteolysis of

caseins
B. ISIDRA RECIO, LOURDES AMIGO, MERCEDES RAMOS
. ROSINA LOPEZ-FANDINM O
Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3,
E-28006 Madrid, Espanga
(Received 2 May 1996 and accepted for publication 23 September 1996)
Snn.v.. Capillary electrophoresis using a hydrophilically coated capillary and a
low pH buer containing urea has been used to follow the proteolytic action of
plasmin and chymosin on isolated casein fractions, whole casein and individual milk
samples selected on the basis of their genetic variants. Several of the main casein
breakdown products were identied. These included, among others,
"
-casein
(CN) A",
"
-CN A#,
"
-CN B,
"
-CN C,
#
-CN A,
"
-CN A$,
#
-CN B,
$
-CN A and

$
-CN B, as well as proteose peptones, arising from the action of plasmin on the
dierent genetic variants of -CN.
s"
-I-CN and
s"
-CN f(1-23) from
s"
-CN, and
para--CN and caseinomacropeptide from -CN produced by chymosin action were
also separated. The knowledge of their migration times provided information on the
extent and origin of casein hydrolysis in both milk and cheese, as found in samples
of proteolysed milk or fresh cheese.
Capillary electrophoresis (CE) is a fast growing technique that is nding new
applications in the analysis of milk proteins and peptides owing to its well known
advantages over electrophoretic and chromatographic methods. CE techniques
employ narrow bore capillaries (20200 m i.d.) to perform high eciency
separations of both large and small molecules. These separations are facilitated by
the use of high voltages, which may generate electro-osmotic and electrophoretic
ow of buer solutions and ionic species.
CE at basic pH with uncoated capillaries has been used for the separation and
quantication of whey proteins (Otte et al. 1994; Paterson et al. 1995; Recio et al.
1995). An uncoated capillary was also used for the analysis of caseinomacropeptide
from rennet whey (Otte et al. 1995). Van Riel & Olieman (1995) increased the
resolving power to 0n5i10'1n0i10' plate numbers\m by using a hydrophilically
coated capillary and applied it to the detection of rennet whey solids in skim milk
and acid buttermilk powder. De Jong et al. (1993) published a CE method using a
hydrophilically coated capillary that made possible the simultaneous separation of
whey proteins and caseins, including some genetic variants. The use of a coated
capillary in combination with a low pH buer containing urea and methyl-
hydroxyethyl cellulose avoided the undesirable adsorption of proteins on to the
capillary wall, usually associated with fused silica walls. Electro-osmotic ow (bulk
ow of liquid in the capillary as a consequence of the surface charge on the interior
capillary wall) was virtually zero under these conditions and the migration behaviour
of peptides and proteins depended only on their eective mobility at low pH. With
222 I. RIo . o1nv-
this method, theoretical plate numbers in the range 0n3i10'0n6i10' were obtained
(De Jong et al. 1993).
This method was recently optimized by Recio & Olieman (1996) in order to
provide a quantitative determination of the proteins separated in the electro-
pherograms, and to detect the serumprotein: casein ratio in dierent dairy products,
as well as the addition of milk powder to pasteurized milk. However, so far very few
studies have applied CE to the investigation of proteolysis in milk or cheese
(Kristiansen et al. 1994).
In an attempt to identify the CE pattern corresponding to the main degradation
products arising from the action of dierent proteolytic agents of importance in the
technology of dairy products, we have carried out a study on the action of plasmin
(EC 3;4;21;7) and chymosin (EC 3;4;23;4) on isolated casein fractions, whole
casein and individual milk samples selected on the basis of their genetic variants.
n.1vI.i- . n1no-
Samples
Isoelectric casein was prepared by precipitation from whole milk by adding 2 n-
HCl to pH 4n6, followed by centrifugation at 4500 g for 15 min. The casein precipitate
was washed three times with 1 n-sodium acetate buer, pH 4n6. The remaining fat in
the casein precipitate was removed by washing with dichloromethanesodium
acetate buer (1: 1, v\v). The nal casein precipitate was lyophilized. Casein
fractions (
s
-, - and -casein (CN)) were purchased from Sigma Chemical Co. (St
Louis, MO 63178, USA).
In order to identify the dierent genetic variants of -CN, individual milk
samples containing -CNA"A", A#A#, A#B, A"C and A"A$ variants were selected from
a pool of 631 samples.
s"
-CN f(1-23) was a gift from Dr C. Mart!n Hernandez
(Instituto del Frio). Caseinomacropeptide was prepared by purication, after
successive injections on reversed-phase HPLC, of the fraction soluble in trichlo-
roacetic acid (40 g\l) of a chymosin digest of isoelectric casein (Lo! pez-Fandin4 o et al.
1993).
Fresh cheeses (pH 5n94), manufactured from pasteurized cows milk (72 mC, 15 s)
with the addition of suitable starter cultures (Lactococcus lactis subsp. lactis and Lc.
lactis subsp. cremoris), were purchased from a local market. Caseins from cheese were
obtained by precipitation at pH 4n6 and washed and lyophilized as above. Raw milk
samples were treated with thimerosal (0n1 g\l ; Scharlau, E-08016 Barcelona, Spain)
and incubated at 37 mC for 48 h.
Preparation of model systems
Isoelectric casein and casein fractions were dissolved to 3 mg\ml in 0n1 n-
potassium phosphate buer, pH 6n5, except for the chymosin digests where the
concentration was 25 mg\ml. Proteolysis with plasmin and calf chymosin, both from
Sigma, were carried out at enzyme: substrate ratios of 1n67i10
$ and
4n72i10
' units\mg casein respectively. Individual milk samples were digested with
plasmin at an enzyme: substrate ratio of 5i10
# units\ml.
Model systems were incubated at pH 6n5 and 37 mC for dierent periods between
0 and 2 h. Portions were withdrawn fromthe mixtures at intervals, reactions stopped
by dilution at 1: 1n5 with CE sample buer, pH 8n6p0n1 (see below), and the sample
injected without further preparation. In some cases, the pH of the reaction mixtures
was adjusted to 4n6, in order to separate products soluble and insoluble at this pH,
before the addition of CE sample buer.
Capillary electrophoresis of casein 223
Capillary electrophoresis
CE buers were prepared following the method of Recio & Olieman (1996).
Sample buer (pH 8n6p0n1) consisted of 167 mn-Tris(hydroxymethyl)-
aminomethane (reagent grade from Sigma), 42 mn-3-morpholino-propanesulphonic
acid (BioChemika MicroSelect; Fluka, CH-9470 Buchs, Switzerland), 67 mn-
ethylenedinitrilotetra-acetic acid disodium salt dihydrate (Tritiplex III; Merck, D-
64293 Darmstadt 1, Germany), 17 mn-i-dithiothreitol (Sigma), 6 n-urea(Sigma)
and methylhydroxyethyl cellulose (0n5 g\l, 30000; Serva, D-69042 Heidelberg 1,
Germany).
The electrophoresis buer was 0n32 n-citric acid20 mn-sodium citrate6 n-urea,
pH 3n0p0n1 containing 0n5 g methylhydroxyethyl cellulose\l. Before use, buers
were ltered through a 0n22 m lter (Sterile Acrodisc2 with HT Turyn membrane;
Gelman Sciences, Ann Arbor, MI 48106, USA).
CE was carried out using a Beckman P\ACE System2050 controlled by a System
Gold Software data system version 810 (Beckman Instruments Inc., San Ramon, CA
94583-0701, USA). The separations were performed using a hydrophilic-coated
fused-silica capillary column (CElect P1; Supelco, Bellefonte, PA 16823, USA) of
0n47 or 0n57 mi50 m i.d., with a slit opening of 100i800 m. Separations were as
described by Recio & Olieman (1996) with nal voltages of 20 and 25 kV respectively
for the 0n47 and 0n57 m capillaries. Detection was performed on the column at
214 nm. In the electropherograms, absorbance is represented against the time
required for a solute to migrate from the beginning of the capillary to the detector
window. When using the same capillary and running buer, the relative - for
migration times were 0n08%. However, changes in the migration times due to the
use of new capillaries or buer solutions have been observed. In addition, small
variations were noted when analysing casein fractions as compared with milk
samples, probably caused by the presence of dierent ions in the sample matrix. For
this reason, identication of milk proteins and peptides was achieved by spiking,
whenever standards were available, or by considering relative migration times.
For the purposes of identication, in addition to the CE separations all the
samples were simultaneously analysed by alkaline-PAGE (Ramos et al. 1977), SDS-
PAGE (Laemmli, 1970) and isoelectric focusing (Bovenhuis & Verstege, 1989). Thus,
a full characterization of the hydrolysates by conventional separation techniques
was achieved.
v-i1- . I---Io
Hydrolysis with plasmin
Fig. 1 shows the electropherograms of whole casein prior to hydrolysis, together
with whole casein, - and
s
-CN treated with plasmin. Certain incubation times have
been selected in order to show the most representative breakdown products arising
from the action of the enzyme. As expected, the main degradation products resulted
from the action of the enzyme on -CN (Fig. 1c; Gruerty & Fox, 1988). These
included various peaks belonging to the -CN, as well as four other peaks that
probably corresponded to hydrophilic proteose peptone components (Andrews &
Alichanidis, 1983), as they were the only degradation products from the hydrolysate
that remained soluble at pH 4n6 (marked S in Fig. 1b, c).
Owing to its high content of lysyl residues,
s#
-CN has numerous plasmin-
sensitive bonds (Le Bars & Gripon, 1989). Indeed,
s#
-CN also was rapidly cleaved
by plasmin, and disappeared from the hydrolysates of
s
-CN and whole casein after
224 I. RIo . o1nv-
0020
0015
0010
0005
0020
0015
0010
0005
0020
0015
0010
0005
0020
0015
0010
0005
0
0
10 20 30 40
Time, min
(d)
(c)
(b)
(a)
A
2
1
4

s1

s2

s0
bB

bA
1
bA
2

s1
bA
2
bA
1

s0

s2

s
P
c
c
c
c

S
S S
S
bA
1
bA
2
bB
c
c
c
c
S
S
S
S

s1

s0

s
P
S
Fig. 1. Electropherograms of dierent protein fractions digested with plasmin, with incubation times
in parentheses: (a) whole casein (0 min), (b) whole casein (20 min), (c) -casein (CN) (10 min), (d)
s
-
CN (60 min). Separation was in a hydrophilically coated fused silica capillary (0n47 mi50 m, 0n40 m
to detection point), temperature 45 mC, injection 15 s, linear voltage gradient 020 kV in 3 min,
followed by constant voltage of 20 kV, separation buer 6 n-urea0n32 n-citric acid20 mn-sodium
citrate, pH 3n0 containing 0n5 g methylhydroxyethyl cellulose\l. , -CN;
s"
,
s"
-CN;
s!
-,
s!
-CN;

s
P, peptide derived from the action of plasmin on
s"
-CN; B, -CN B; A", -CN A"; A#, -CN A#;
, -CN; S, peptides soluble at pH 4n6 arising from the action of plasmin on
s
-CN and -CN.
Capillary electrophoresis of casein 225
0
10 20 30 40
Time, min
(b)
(a)
A
2
1
4

s1

s2

s0
bA
1
bA
2
S
004
003
002
001
0
004
003
002
001
3
4
1
2
-La
b-Lg
S
S
1
2
5
4 S

Fig. 2. Electropherograms of individual milk samples digested with plasmin for 20 min: (a) milk
containing -casein (CN) A#A#, (b) milk containing -CN A"A". -La, -lactalbumin; -Lg, -
lactoglobulin;
s#
,
s#
-CN;
s"
,
s"
-CN;
s!
,
s!
-CN; A#, -CN A#; A", -CN A"; , -CN; S, peptides
soluble at pH 4n6 arising from the action of plasmin on
s
-CN and -CN. 1,
s
P, peptide derived from
the action of plasmin on
s"
-CN; 2,
#
-CN A; 3,
"
-CN A#; 4,
$
-CN A; 5,
"
-CN A". Electrophoretic
conditions were as in Fig. 1.
15 and 20 min treatment respectively. The electrophoretic pattern of
s
-CNfollowing
plasmin treatment showed a fast migrating degradation product which was also
observed in the hydrolysate of whole casein (
s
P in Fig. 1b, d). It is likely that this
peptide was produced from
s"
-CN, which was attacked more slowly, as it was still
being released well after all the
s#
-CN had been completely hydrolysed by the
enzyme. In addition, there were some slowmoving peptides resulting fromthe action
of plasmin on
s
-CN, which were also present in the hydrolysate of whole casein. One
of those (marked S in Fig. 1d) remained soluble at pH 4n6. Plasmin acts on
s"
-CN
to yield a heterogeneous group of peptides termed -CN (Aimutis & Eigel, 1982).
Recently, Addeo et al. (1995) isolated the peptide
s"
-CN f(80-199), which derived
from the degradation of
s"
-CN by plasmin.
Plasmin hydrolysates of milks containing -CN A"A" and -CN A#A# enabled us
to identify
"
-CN A",
"
-CN A#,
#
-CN and
$
-CN (Eigel et al. 1984) as illustrated in
Fig. 2. When using a hydrophilically coated capillary, the electro-osmotic ow is
suppressed and the electrophoretic migration of proteins depends on their mass and
charge characteristics at pH 3n0 (De Jong et al. 1993). Because of its higher positive
226 I. RIo . o1nv-
003
002
001
0
003
002
001
0
002
001
0
10 20 30 40
Time, min
(b)
(a)
A
2
1
4

s1

s2
bA
1
bA
2
1
2
-La
b-Lg
S

6
7
8
3
4

s0
bB
(c)
9
2
5
4
S

s1
BC
bA
1
S
S
bA
3

s0
BC
4
10
2
1
11
+
5
bC
Fig. 3. Electropherograms of individual milk samples digested with plasmin for 20 min: (a) milk
containing -casein (CN) A#B, (b) milk containing -CN A"C, (c) milk containing -CN A"A$. -La,
-lactalbumin; -Lg, -lactoglobulin;
s"
,
s"
-CN;
s!
,
s!
-CN;
s"
BC,
s"
-CN BC;
s!
BC,
s!
-CN BC;
A#, -CN A#; B, -CN B; A", -CN A"; C, -CN C; A$, -CN A$; , -CN; S, peptides soluble at
pH 4n6 arising from the action of plasmin on
s
-CN and -CN. 1,
s
-P, peptide derived from the action
of plasmin on
s"
-CN; 2,
#
-CN A; 3,
"
-CN A#; 4,
$
-CN A; 5,
"
-CN A"; 6,
#
-CN B; 7,
"
-CN B; 8,

$
-CN B; 9,
"
-CN C; 10,
"
-CN A$; 11,
#
-CN A$. Electrophoretic conditions were as in Fig. 1.
charge,
#
-CN had the lowest migration time, followed by
"
-CN A" (Fig. 2b) and
"
-
CN A# (Fig. 2a). (In this case the substitution of His'( in -CN A" for Pro'( in -CN
A# made it possible to separate them at acidic pH.)
$
-CN had the longest migration
time.
The electrophoretic patterns resulting from the action of plasmin on milks
containing -CN A#B, -CN A"C and -CN A"A$ variants are shown in Fig. 3. The
substitution of Ser"## in -CN A for Arg"## in -CN B, in addition to the presence of
His'(, as in -CN A" (Eigel et al. 1984), rendered
#
-CN B,
$
-CN B and
"
-CN B more
electropositive than their variant A counterparts (Fig. 3a). Similarly, the
Capillary electrophoresis of casein 227
003
002
001
0
10 20 30 40
Time, min
(b)
(a)

s1
bA
1
bA
2
p-

s0
(c)

s1
-I

s1
-X
CMP

s2

s
f(1-23)
p-
CMP
005
004
003
002
001
0
012
010
008
006
004
002
0
004

s1
-X

s1
-I

s0

s1

s2

s1
f(1-23)
A
2
1
4
Fig. 4. Electropherograms corresponding to dierent protein fractions digested with chymosin, with
incubation times in parentheses: (a) whole casein (10 min), (b) -casein (CN) (10 min), (c)
s
-CN
(120 min).
s#
,
s#
-CN;
s"
,
s"
-CN;
s!
,
s!
-CN; A", -CN A"; A#, -CN A#; p-, para--CN; CMP,
caseinomacropeptide;
s"
-I,
s"
-I-CN;
s"
-X, peptide derived from
s
-CN;
s"
f(1-23),
s"
-CN f(1-23).
Electrophoretic conditions were as in Fig. 1.
substitutions of SerP$& and Glu$( in -CN A" for Ser$& and Lys$( in -CN C, together
with the presence of His'( (Eigel et al. 1984), contributed to the high positive charge
of
"
-CN C and thus to its low migration time (Fig. 3b).
"
-CN A$ was tentatively
identied in view of the amino acid substitutions Pro'( and Gln"!' of -CN A$, which
make it less electropositive than
"
-CN A" and
"
-CN A# (Fig. 3c). However,
#
-CN
A$, which should appear as a peak with lower migration time than
#
-CNA" by virtue
of the substitution of His"!' for Gln"!', was not found in the plasmin digest of milk
containing -CN A"A$, maybe owing to coelution with
"
-CN A". The presence of
s"
-
CN BC and
s!
-CN BC in the electropherogram illustrated in Fig. 3(c) should be
noted. Trieu-Cuot & Gripon (1982) separated -CN A" and -CN A# by isoelectric
228 I. RIo . o1nv-
(a)
008
006
004
002
0
10 20 30
Time, min
S
S S
S
4
3
5
1
2

s1

s0

s2
-La
-Lg

bA
1
bA
2
A
2
1
4
(b)
007
005
004
002
0
10 20 30
Time, min
S
S
S
4
2

s1

s0

s2
-La
p-
bA
1
bA
2
A
2
1
4
006
003
001
001
-Lg
5
7
6

s1
-I
bB
40
Fig. 5. Electropherograms corresponding to (a) milk incubated for 48 h at 37 mC in the presence of 0n1 g
thimerosal\l (electrophoretic conditions were as in Fig. 1), (b) casein from a fresh cheese clotted
enzymically (electrophoretic conditions were as in Fig. 1 except that the total length of the capillary
was 0n57 m, and the nal voltage 25 kV). -La, -lactalbumin; -Lg, -lactoglobulin;
s#
,
s#
-casein
(CN) ;
s"
,
s"
-CN;
s!
,
s!
-CN;
s"
-I,
s"
-I-CN; B, -CN B; A", -CN A"; A#, -CN A#; , -CN;
p-, para--CN; S, peptides soluble at pH 4n6 arising fromthe action of plasmin on
s
-CN and -CN. 1,

s
P, peptide derived from the action of plasmin on
s"
-CN; 2,
#
-CN A; 3,
"
-CN A#; 4,
$
-CN A; 5,

"
-CN A"; 6,
#
-CN C; 7,
"
-CN B.
Capillary electrophoresis of casein 229
focusing, as well as
"
-CN A",
"
-CN A#,
#
-CN and
$
-CN. However, to
our knowledge the separation of the degradation products of -CN A$, -CN B and
-CN C has not so far been achieved by conventional electrophoresis.
Hydrolysis by chymosin
The electrophoretic pattern of whole casein hydrolysed with chymosin is
illustrated in Fig. 4(a). The main degradation products included para--CN and
caseinomacropeptide (identied by spiking with the puried peptide), resulting from
the action of the enzyme on the Phe"!&Met"!' bond of -CN, which is the primary
cleavage site of chymosin action and leads to the enzymic coagulation of milk (Fig.
4b; Dalgleish, 1987).

s"
-CN was also hydrolysed rapidly by chymosin at the Phe#$Phe#% bond, giving
rise to
s"
-I-CN(
s"
-CNf(24-199)) and
s"
-CNf(1-23) (Fig. 4c). This is one of the most
important events that takes place during the early stages of ripening of most cheese
varieties and is responsible for the softening of cheese texture (Creamer & Olson,
1982).
s"
-I-CN is further hydrolysed by chymosin during cheese ripening, giving rise
to degradation products of high electrophoretic mobility in urea-PAGE at alkaline
pH(Grappin et al. 1985; McSweeney et al. 1993). The electropherograms of
s
-CNand
whole casein treated with chymosin (Fig. 4a, c) showed peaks of longer migration
time than
s"
-I-CN, which were produced at a rate similar to the hydrolysis of
s"
-
I-CN.
Proteolysis in milk and cheese
Identication of the degradation products arising from dierent enzymes makes
this method suitable for studying proteolysis in milk and cheese, as illustrated in Fig.
5. The main breakdown products arising from plasmin action were observed in the
electropherogram of a sample of raw milk that had been kept at 37 mC for 48 h in the
presence of bacterial inhibitors to promote protein degradation by native proteinases
(Fig. 5a). The presence of para--CN, which might have been formed by the action
of residual psychrotroph enzymes, cannot be excluded as it co-migrates with -
lactoglobulin.
Fig. 5(b) shows the casein fraction of a fresh cheese sample made frompasteurized
milk. Heat-denatured whey proteins can be observed in the electropherogram (Recio
& Olieman, 1996), with the two genetic variants of -lactoglobulin appearing as a
double shoulder on the para--CN peak. In addition to para--CN, the main
degradation products were -CN,
s"
-I-CN and proteose peptone components.
The present results showed that capillary electrophoresis could be an ecient tool
for following the hydrolysis of caseins caused by various proteinases in milk and
cheese. This method provided a very high resolution of proteins and peptides
diering in just one amino acid substitution and, unlike conventional electro-
phoresis, there is no limitation in the size of the components to be separated. Several
of the main casein breakdown products have been identied.
The authors thank Ms C. Talavera for skilful technical assistance. This work has
been supported by project CAM COR0035\94.
vv-
Ao, F., G.vvo, G., I1ovI., N., PiiovIo, L., R-nII, P. & CnI.-, L. 1995 Gel electrophoresis
and immunoblotting for the detection of casein proteolysis in cheese. Journal of Dairy Research 62 297309
230 I. RIo . o1nv-
AIn1I-, W. R. & EIoi, W. N. 1982 Identication of -casein as plasmin-derived fragments of bovine
s"
-
casein. Journal of Dairy Science 65 175181
Avv-, A. T. & AiIn.II-, E. 1983 Proteolysis of caseins and the proteose-peptone fraction of bovine
milk. Journal of Dairy Research 50 275290
BovnI-, H. & Vv-1o, A. J. M. 1989 Improved method for phenotyping milk protein variants by
isoelectric focusing using PhastSystem. Netherlands Milk and Dairy Journal 43 447451
Cv.nv, L. K. & Oi-o, N. F. 1982 Rheological evaluation of maturing Cheddar cheese. Journal of Food
Science 47 631636, 646
D.ioiI-n, D. G. 1987 The enzymatic coagulation of milk. In Cheese: Chemistry, physics and microbiology, vol.
1, pp 6396 (Ed. P. F. Fox). London: Elsevier Applied Science Publishers
D Joo, N., VI--v, S. & OiIn., C. 1993 Determination of milk proteins by capillary electrophoresis.
Journal of Chromatography A 652 207213
EIoi, W. N., B1iv, J. E., Ev-1von, C. A., F.vvii, H. M., H.vv.ii.v, V. R., J--, R. &
WnI1., R. ML. 1984 Nomenclature of proteins of cows milk: fth revision. Journal of Dairy Science 67
15991631
Gv.III, R., R.i, T. C. & Oi-o, N. F. 1985 Primary proteolysis of cheese proteins during ripening. A
review. Journal of Dairy Science 68 531540
Gvv1., M. B. & Fox, P. F. 1988 Review article. Milk alkaline proteinase. Journal of Dairy Research 55
609630
KvI-1I.-, K. R., O11, J., Z.iov., M. & QvI-1, K. B. 1994 Capillary electrophoresis used to monitor the
enzymatic hydrolysis of caseins and the fractionation of hydrolysis products. Milchwissenschaft 49 683687
L.nniI, U. K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature 227 680685
L B.v-, D. & GvIIo, J.-C. 1989 Specicity of plasmin towards bovine
s#
-casein. Journal of Dairy Research
56 817821
Lo! Iz-F.I4 o, R., Ao, M. I. & R.no-, M. 1993 Comparative study by HPLC of caseinomacropeptides
from cows, ewes and goats milk. Journal of Dairy Research 60 117121
MSv., P. L. H., Oi-o, N. F., Fox, P. F., H.i., A; . & HuavI, P. 1993 Proteolytic specicity of
chymosin on bovine
s"
-casein. Journal of Dairy Research 60 401412
O11, J. A. H. J., KvI-1I.-, K. R., Z.iov., M. & QvI-1, K. B. 1994 Separation of individual whey
proteins and measurements of -lactalbumin and -lactoglobulin by capillary zone electrophoresis.
Netherlands Milk and Dairy Journal 48 8197
O11, J., MI1o..v, L. & QvI-1, K. B. 1995 Analysis of caseinomacropeptide(s) by free solution capillary
electrophoresis. Milchwissenschaft 50 7579
P.1v-o, G., HIii, J. P. & O11v, D. E. 1995 Separation of -lactoglobulin A, B and C variants of bovine
whey using capillary zone electrophoresis. Journal of Chromatography A 700 105110
R.no-, M., M.v1I!z-C.-1vo, I. & J.vz, M. 1977 Detection of cows milk in Manchego cheese. Journal of
Dairy Science 60 870877
RIo, I., MoiI., E., R.no-, M. & Fv1o-, M. 1995 Quantitative analysis of major whey proteins by
capillary electrophoresis using uncoated capillaries. Electrophoresis 16 654658
RIo, I. & OiIn., C. 1996 Determination of denatured serum proteins in the casein fraction of heat-
treated milk by capillary zone electrophoresis. Electrophoresis 17 12281233
TvI-Co1, P. & GvIIo, J.-C. 1982 A study of proteolysis during Camembert cheese ripening using
isoelectric focusing and two-dimensional electrophoresis. Journal of Dairy Research 49 501510
V. RIi, J. & OiIn., C. 1995 Determination of caseinomacropeptide with capillary zone electrophoresis
and its application to the detection and estimation of rennet whey solids in milk and buttermilk powder.
Electrophoresis 16 529533