Bipolar Membrane Electroacidification To Produce Bovine Milk

Casein Isolate
Laurent Bazi net,
,
Franc oi s Lamarche,*
,
Deni s I ppersi el ,

and Jean Ami ot

Food Research and Devel opment Centre, Agri cul ture and Agri -Food Canada,
3600 Casavant Boul evard West, St. Hyaci nthe, Quebec, Canada J2S 8E3, and Centre de Recherche en
Sci ences et Technol ogi e du Lai t (STELA), Pavi l l on Paul -Comtoi s, Uni versi te Laval , Sai nte-Foy,
Quebec, Canada G1K 7P4
Bi pol ar membrane el ectroaci di fi cati on (BMEA) has been devel oped previ ousl y (Bazi net et al ., Report
for the Canadi an El ectri ci ty Associ ati on 9326 U 987, 1996; Bazi net et al ., J . Agric. Food Chem.
1997, 45, 2419-2425, 3788-3794) and has been used for i soel ectri c preci pi tati on of soybean protei ns.
The purpose of thi s study was to val i date the feasi bi l i ty of BMEA for the preci pi tati on of mi l k casei n
and to i nvesti gate the effect of fl ow rate. Hi gh-puri ty i sol ates contai ni ng 1.23 and 2.00% ash and
85.4 and 91.6% total protei n were obtai ned wi th fl ow rates of 0.2 and 1.2 gal /mi n. The mol ecul ar
composi ti on profi l es of the i sol ates obtai ned by HPLC showed that onl y casei ns were preci pi tated.
However, except for protei n preci pi tati on curves, the fl ow rate di d not i nfl uence the fi nal composi ti on
and puri ty of the i sol ates. These resul ts showed that BMEA i s a new al ternati ve process for the
producti on of hi gh-puri ty bovi ne mi l k casei n i sol ate.
Keywords: Electrochemical acidification; bipolar membrane; casein; milk; precipitation
I NTRODUCTI ON
Casei n i s extensi vel y used i n the manufacturi ng of
food products for i ts nutri ti onal qual i ty and functi onal
properti es. However, hi ghl y pure casei n can be obtai ned
onl y by an i nsol ubi l i zati on step, and centri fugati on can
then be used for a si mpl e separati on of the casei n from
whey (Varnam and Sutherl and, 1994).
Two mai n types of casei n usual l y produced i n the
i ndustry are rennet and aci d casei ns. I n the case of
rennet casei n, the underl yi ng mechani sm i s i denti cal
to that of the producti on of cheese curd and depends on
the uni que sensi ti vi ty of the Phe
105
-Met
106
bond of
-casei n to hydrol ysi s by aci d protei nases, the acti ve
components of rennet. For aci d casei n producti on, the
three mai n procedures used are based on i soel ectri c
preci pi tati on of casei n by chemi cal , physi cochemi cal , or
fermentati on aci di fi cati on (Segal en, 1985; Southward,
1993; Varnam and Sutherl and, 1994). Other techni ques
have been proposed for the producti on of aci d casei n:
aci di fi cati on of mi l k by i on-exchange pl us aci d (Sal mon,
1983), el ectrodi al ysi s of ski m mi l k to pH 5.0 fol l owed
by aci di fi cati on to pH 4.6 wi th aci d (Lai teri es Tri bal l at,
1979), and aci di fi cati on by water el ectrol ysi s at the
surface of monopol ar ani on- or cati on-exchange mem-
branes stacked i n an el ectrodi al ysi s cel l (Bol zer, 1985).
A procedure deri ved from el ectrodi al ysi s and usi ng
bi pol ar membranes was devel oped to preci pi tate soy-
bean protei n (Bazi net et al ., 1996, 1997a,b, 1998a).
Bi pol ar membrane el ectroaci di fi cati on (BMEA) uses a
property of bi pol ar membranes to spl i t water and the
acti on of monopol ar membranes for demi neral i zati on.
When a current i s passed across a bi pol ar membrane,
el ectri cal conducti on i s achi eved by the transport of H
+
and OH
-
i ons generated by el ectrodi ssoci ati on of water
(Mani , 1991). The protons thus generated can come i nto
contact wi th the protei ns, bri ngi ng them to thei r i so-
el ectri c poi nt and resul ti ng i n sel ecti ve separati on.
Water di ssoci ati on may occur but wi th a l ower ef-
fi ci ency, usi ng monopol ar membranes stacked i n an
el ectrodi al ysi s cel l (Bazi net et al ., 1996; Korngol d, 1984;
Davi s et al ., 1997). Thi s di ssoci ati on appears onl y when
the current val ue exceeds the l i mi ti ng current val ue
(Brun, 1989; Korngol d, 1984). Bi pol ar membranes are
made to be used above l i mi ti ng current condi ti ons and
need a l ower current to reach the l i mi ti ng current val ue
than monopol ar membranes (Bazi net et al ., 1998c).
As the el ectri cal water spl i tti ng i n an el ectrodi al ysi s
cel l at the surface of monopol ar membrane i s affected,
among other factors, by the fl ow rate of the sol uti on to
be treated (Gardai s, 1990; Kl ei n et al ., 1987), thi s study
i s a part of a broader research project ai med at preci pi -
tati on of bovi ne mi l k protei n, wi thout the addi ti on of
aci ds, by decreasi ng the pH through el ectrodi al ysi s. I ts
speci fi c objecti ves were to val i date the feasi bi l i ty of
BMEA for the preci pi tati on of mi l k casei n and to
i nvesti gate the effect of fl ow rate. Both fl ow rates were
compared i n terms of el ectrodi al ysi s cel l parameters,
percentage of protei ns preci pi tated, protei n composi ti on
profi l es, and chemi cal composi ti on of i sol ates produced.
MATERI ALS AND METHODS
Material. The raw materi al used i n thi s study was com-
merci al fresh pasteuri zed and homogeni zed ski m mi l k (Que-
bon, Natrel , Longueui l , PQ, Canada).
Methods. Electroacidification Cell. The el ectroaci di fi cati on
cel l was the same as that used by Bazi net et al . (1997a,b) wi th
four Neosepta CMX cati oni c membranes and three Neosepta
BP-1 bi pol ar membranes (Tokuyama Soda Ltd., Tokyo, Japan).
* Author to whom correspondence shoul d be addressed
[e-mai l Lamarchef@em.agr.ca; tel ephone (450) 773-1105; fax
(450) 773-8461].

Agri cul ture and Agri -Food Canada.

Uni versi te Laval .

5291 J. Agric. Food Chem. 1999, 47, 52915296
10.1021/jf990524m CCC: $18.00 1999 American Chemical Society
Published on Web 11/11/1999
Thi s arrangement defi nes three cl osed l oops contai ni ng the
mi l k sol uti on, a 2 gL
-1
aqueous KCl sol uti on, and a 20 gL
-1
Na2SO4 sol uti on. Each cl osed l oop was connected to a separate
external 600 mL gl ass contai ner (School , Duran, Germany),
al l owi ng for conti nuous reci rcul ati on. The el ectroaci di fi cati on
system di d not al l ow for constant temperature control .
The anode/cathode vol tage di fference was suppl i ed by a
vari abl e 0-100 V power source, Powerstat Model 236BU-2
(The Superi or El ectri c Co., Bri stol , CO). The el ectrol ytes were
ci rcul ated usi ng three centri fugal pumps Model WMD-30RT-
220 (I waki Wal chem, Tokyo, Japan), and the fl ow rate was
control l ed usi ng Model E-03248-58 fl ow meters (Col e-Parmer
I nstrument Co., Vernon Hi l l s, I L). The anode, a di mensi onal l y
stabl e el ectrode (DSA), and the cathode, a 316 stai nl ess steel
el ectrode, were suppl i ed wi th the MP cel l .
Protocol. El ectroaci di fi cati on was performed i n batch process
usi ng a current of 2.0 A, wi th el ectrol yte vol umes of 1 L. The
el ectroaci di fi cati on was stopped after the pH reached 4.0. The
i ni ti al pH vari ed between 6.5 and 6.7. Two fl ow rates for the
mi l k sol uti on duri ng el ectroaci di fi cati on were tested (1.2 and
0.2 gal /mi n), and three repl i cates of each condi ti on were
performed i n thi s experi ment.
Duri ng each treatment, 1.5 mL sampl es of the mi l k sol uti on
were taken, i n the outl et l i ne outsi de of the BMEA cel l and
just before the mi l k reservoi r, at every 0.2 pH uni t decrease
from i ni ti al pH (6.6) to pH 4.0. The ti me requi red to reach
pH 4.0, the anode/cathode vol tage di fference, the conducti vi ty,
and the temperature were recorded as the treatment pro-
gressed. The concentrati on of sol ubl e protei n was determi ned
on freshl y aci di fi ed 1.5 mL sampl es. At the end of each run,
500 mL of pH 4.0 mi l k was taken. These sampl es were
centri fuged for 10 mi n at 4 C, at 500g (centri fuge Model J2-
21, rotor type JA-10, Beckman I nstruments I nc., Pal o Al to,
CA). The preci pi tate was washed twi ce wi th di sti l l ed water
before bei ng l yophi l i zed for 48 h at room temperature (Model
Freezone 4.5, Labconco, Kansas Ci ty, MO). The l yophi l i zed
i sol ates were stored at 4 C before total protei n determi nati on,
protei n composi ti on profi l es, and ash content measurements
were performed.
Analysis Methods. (1) Ash Content. I n accordance wi th
Method 930-30 (AOAC I nternati onal , 1995), 1.5 g of l yoph-
i l i zed sampl e was added to the cool ed cruci bl es, and the mass
was recorded. The sampl es were then ashed at 550 C for 16
h and wei ghed agai n when they reached room temperature.
(2) Soluble Protein and Total Protein Determination. The
protei n concentrati on determi nati on was done usi ng an FP-
428 LECO apparatus (LECO Corp., Sai nt Joseph, MI ). The
i nstrument was cal i brated each ti me wi th ethyl enedi ami ne-
tetraaceti c aci d (EDTA) as a ni trogen standard. A prel i mi nary
compari son of the LECO and Kjel dahl methods has demon-
strated a very good correl ati on between both methods (R
2
)
0.986 and 0.996 for the hi gh and l ow ranges of protei n,
respecti vel y).
For sol ubl e protei n determi nati on the LECO condi ti ons were
the fol l owi ng:
Condi ti ons for total protei n determi nati on were the same as
for sol ubl e protei n, except for l oop sel ect (hi gh range), fl ow
constant (hi gh, 10 s; hi gh, 30 s; hi gh, end) and mass of EDTA
(150 mg).
(3) Molecular Profiles. The chromatographi c anal ysi s of
protei n composi ti on profi l e of the l yophi l i zed protei n i sol ate
and ski m mi l k was performed by reverse-phase HPLC accord-
i ng to the method of Jaubert and Marti n (1992). Separati on
of Rs1-, Rs2-, -, and -casei ns was carri ed out on a 15-cm Vydac
C4 col umn (Model 214 TP 5415, Vydac, Hesperi a, CA) coupl ed
wi th a Vydac Protei n C4 guard col umn (Model 214 FSK 54,
Vydac). El uti on at 1 mL/mi n was achi eved usi ng a l i near
gradi ent from 37 to 57%sol vent B [0.096%(v/v) tri fl uoroaceti c
aci d i n 80% (v/v) acetoni tri l e] and from 63 to 43% sol vent A
[0.1% (v/v) tri fl uoroaceti c aci d i n water] for a total run ti me of
38 mi n at room temperature. The detecti on wavel ength was
214 nm, and a 50 L sampl e vol ume was i njected.
Before anal ysi s, freeze-dri ed sampl es of i sol ates (55 mg) and
l yophi l i zed ski m mi l k (LSM) (135 mg) were added to 4 mL of
buffer sol uti on (100 mM Tri s-HCl , 8 M urea, 1.3% tri sodi um
ci trate, pH 7.0). The mi xture was reduced i n 10 mM 1,4-
di thi othrei tol (DTT) and mai ntai ned for 1 h at 37 C. Before
i njecti on, the homogenate sampl e was di l uted 10 ti mes wi th
sol vent A and fi l tered on a 0.45 m Mi l l i pore fi l ter.
Statistical Analyses. The durati on of the el ectroaci di fi cati on,
the vol tage, the conducti vi ty, and the percent sol ubl e protei n
as a functi on of pH were subjected to a spl i t-pl ot anal ysi s of
vari ance usi ng SAS software (SAS, 1989). Regressi on equa-
ti ons and curve fi tti ng were cal cul ated for the vol tage, dura-
ti on, and percent sol ubl e protei ns as a functi on of pH usi ng
Si gmaPl ot (versi on 3.0 for Wi ndows, Jandel Sci enti fi c, Corte
Madera, CA). The ash content and the percent total protei n
data were anal yzed by an anal ysi s of vari ance and exami ned
by Duncan tests to determi ne the si gni fi cance of di fferences
between the di fferent sampl es.
RESULTS AND DI SCUSSI ON
After el ectroaci di fi cati on of the ski m mi l k sol uti on,
the fi nal product was sti l l mi l ky. However, after the
sol uti on was l eft to settl e, the preci pi tate was composed
of smal l and soft cl umps of casei n, and the supernatant
was cl ear. I n the BMEA cel l , a sl i ght foul i ng appeared
due to the formati on of a casei n network i n the spacer
turbul ence promoter formed by a doubl e l ayer of wi re
mesh. The casei n fi l l ed the 2 2 mm square of the
turbul ence promoter to form a soft whi te curd. However,
the foul i ng produced by reci rcul ati on of these smal l
parti cl es di d not al ter the yi el d of casei n recovered
because the curd was formed onl y at the end of the
process, when al l of the casei n was preci pi tated. An on-
l i ne centri fuge shoul d al l ow the recovery of the casei n
parti cl es and decreased foul i ng.
Electroacidification Parameters: Duration, An-
ode/Cathode Voltage Difference, and Conductiv-
ity. Resul ts of the anal ysi s of vari ance i ndi cated that
the fl ow rate of the mi l k sol uti on had no si gni fi cant
effect on the durati on of el ectroaci di fi cati on (P > 0.10),
on the anode/cathode vol tage di fference (P > 0.92), and
on the conducti vi ty (P > 0.74) duri ng the el ectroaci di -
fi cati on process. However, pH (P < 0.001) and the dual
i nteracti on of pH and fl ow rate (P <0.0009) had a hi ghl y
si gni fi cant effect on the anode cathode vol tage di ffer-
ence. The fi rst-order l i near regressi on cal cul ated for the
durati on and the thi rd-order regressi on curves cal cu-
l ated for the anode/cathode vol tage di fference as a
functi on of pH produced coeffi ci ents of determi nati on
i n the range of 0.950-0.999.
Duration of Electroacidification. To decrease the pH
of ski m mi l k from 6.6 to 4.0, the durati ons were the
same wi th 17.8 and 18.1 mi n at 1.2 and 0.2 gal /mi n,
respecti vel y (Fi gure 1). The fl ow rate does not i nfl uence
the durati on of el ectroaci di fi cati on. However, at the
begi nni ng of the process, the ti mes to decrease the pH
sampl e si ze 75 mg
anal ysi s constants
oxi dati on furnace temp 900 C
oxi dati on standby temp 650 C
purge cycl es 3
mi ni mum ti meout 30
comparator l evel 1.00
l oop sel ect l ow range
fl ow constants hi gh, 30 s
hi gh, 30 s
hi gh, end
gases O
2, 99.99%; He, 99.99%
cal i brati on standard 75 mg of EDTA (no. 502-092,
9.56 ( 0.03% N, LECO
5292 J. Agric. Food Chem., Vol. 47, No. 12, 1999 Bazinet et al.
from 6.6 to 6.4 were di fferent: 2.1 and 3.4 mi n for 1.2
and 0.2 gal /mi n, respecti vel y. Thi s del ay i n aci di fi cati on
between 1.2 and 0.2 gal /mi n was found from pH 6.4 to
5.0 and di sappeared at pH 4.8. Consi deri ng that the
system was at sol uti on equi l i brati on at pH 6.6, the
aci di fi cati on process was found to be l i near i n ti me,
whi ch was confi rmed by the regressi on coeffi ci ents
cal cul ated i n Fi gure 1. From pH 6.4 to 4.0, del ay i n
aci di fi cati on was progressi vel y di mi ni shed to di sappear
at the end of the process. Thi s di fference i n ti me shoul d
be due to a faster ci rcul ati on of the H
+
produced at the
bi pol ar membrane at hi gh fl ow rate, resul ti ng i n a better
mi xi ng of H
+
conti nuousl y produced wi th the mi l k
protei n sol uti on i n the bul k contai ner. Moreover, the
di sappearance of del ay i n aci di fi cati on between 1.2 and
0.2 gal /mi n coul d be expl ai ned by a strong preci pi tati on
of mi l k protei n between pH 5.0 and 4.8. These resul ts
are confi rmed by the fact that the producti on of H
+
and,
consequentl y, the ti me of aci di fi cati on were previ ousl y
shown to mai nl y depend on protei n concentrati on and
current densi ty (Bazi net et al ., 1997a,b, 1998b; Mani ,
1991). When protei n concentrati on and current densi ty
were mai ntai ned constant, the ti me of aci di fi cati on was
the same whatever the other condi ti ons, except i n the
case of compl ete foul i ng of the el ectrodi al ysi s cel l
spacers.
Anode/ CathodeVoltageDifference. At both fl ow rates,
the evol uti on of anode/cathode vol tage di fference was
the same: a drop fol l owed by an i ncrease (Fi gure 2).
Duri ng the aci di fi cati on of ski m mi l k, at 1.2 and 0.2 gal /
mi n, respecti vel y, the vol tage dropped from 73.7 to 49.7
V and from 71.0 to 48.7 V and then i ncreased from 49.7
to 65.0 V and from 48.7 to 73 V. Thi s phenomenon was
previ ousl y observed by Bazi net et al . (1997a,b) on
soybean protei n. The decrease i n vol tage woul d be the
resul t of the hi gher conducti vi ty of H
+
generated at the
bi pol ar membrane i n the repl acement of i ons mi grati ng
across the cati oni c membrane, to mai ntai n the el ectri cal
neutral i ty of the sol uti on (Bazi net et al ., 1998a): The
mol ar conducti vi ty of H
+
i s much hi gher than that of
al l the other i ons (Brett and Ol i vei ra-Brett, 1994). Thi s
repl acement i n part of the mi l k cati ons by H
+
i n the
protei n sol uti on and the mi grati on of these cati ons from
the protei n compartment to the KCl and Na
2
SO
4
compartments across the cati oni c membrane, coupl ed
i n the KCl compartment wi th generati on of OH
-
at the
ani oni c exchange l ayer of the bi pol ar membrane, i n-
duced a decrease i n the overal l resi stance of the system
and, consequentl y, a decrease i n anode/cathode vol tage
di fference (Bazi net et al ., 1998a). Thi s phenomenon
progressed duri ng the enti re process, but when the
protei n began to preci pi tate, the overal l resi stance of
the system i ncreased because of a sl i ght foul i ng i n the
spacers of the cel l .
Conductivity. Resul ts of the stati sti cal anal ysi s i ndi -
cate that the fl ow rate di d not affect the conducti vi ty
(Fi gure 3). The conducti vi ty changed i n the same way
duri ng el ectroaci di fi cati on whatever the fl ow rate, as
shown i n Fi gure 3. Thi s demonstrates that conducti vi ty
at a l evel of 2.6-2.4 mS/cm does not appear to be a
l i mi ti ng factor i n the el ectroaci di fi cati on of mi l k protei n.
Figure1. Effect of the fl ow rates, 1.2 and 0.2 gal /mi n, on the
ti me requi red to decrease the pH by bi pol ar membrane
el ectroaci di fi cati on of a ski m mi l k sol uti on run at 2.0 A
constant current.
Figure2. Effect of the fl ow rates, 1.2 and 0.2 gal /mi n, on the
anode/cathode vol tage di fference duri ng bi pol ar membrane
el ectroaci di fi cati on of a ski m mi l k sol uti on run at 2.0 A
constant current.
Figure3. Effect of the fl ow rates, 1.2 and 0.2 gal /mi n, on the
conducti vi ty of ski m mi l k sol uti on duri ng bi pol ar membrane
el ectroaci di fi cati on run at 2.0 A constant current.
Bipolar Membrane Electroacidification for Production of Casein Isolate J. Agric. Food Chem., Vol. 47, No. 12, 1999 5293
The apparent l ower decrease i n conducti vi ty, 0.1-
0.2 mS/cm duri ng el ectroaci di fi cati on, was si mi l ar to the
resul ts obtai ned by Bazi net et al . (1997b) on soybean
protei n.
SolubleProtein. The anal ysi s of vari ance of the data
shows that the fl ow rate (P > 0.50) had no si gni fi cant
effect, whereas pH (P <0.0001) and the dual i nteracti on
of pH and fl ow rate (P < 0.0009) had hi ghl y si gni fi cant
effects on the amount of sol ubl e protei ns. The equati ons
of the curves representi ng percentage of sol ubl e protei ns
as a functi on of pH were cal cul ated and model ed; R
2
ranged between 0.993 and 0.997.
The fl ow rate had no effect on the amount of sol ubl e
protei n, whi ch decreased from 100% to 21.5-24.4%
(Fi gure 4). However, the rates of decrease of the sol ubl e
protei n from pH 6.6 to 4.0 were di fferent between the
two fl ow rates. Thus, at the begi nni ng of the procedure,
from pH 6.6 to 5.6, sol ubl e protei n amounts were the
same for both fl ow rates, rangi ng between 93 and 100%.
When the pH dropped from 5.6 to 5.2, at 0.2 gal /mi n,
sol ubl e protei n decreased to 82.9% at pH 5.4 and to
70.9%at pH 5.2, whereas at 1.2 gal /mi n, sol ubl e protei n
was constant at 93-100%. At pH 5.0, sol ubl e protei n
was comparabl e for both fl ow rates at 31.3 and 33.2%.
As the el ectroaci di fi cati on conti nued from pH 4.8 to 4.0,
sol ubl e protei n was constant at 24%whatever the fl ow
rate.
Decreasi ng the fl ow rate accel erated the preci pi tati on
of protei n by i ncreasi ng the resi dence ti me of the protei n
i n the el ectrodi al ysi s cel l . Consequentl y, at 0.2 gal /mi n,
as the generati on of H
+
was constant and as a l ower
vol ume of protei n sol uti on was i n contact wi th more H
+
,
the pH decreased more rapi dl y and more protei n
preci pi tati on occurred. Al so, as protei n was l onger i n
contact wi th the el ectrogenerated H
+
, a pH gradi ent
occurred between the i nl et of the el ectrodi al ysi s cel l ,
correspondi ng to the bul k contai ner sol uti on, and the
outl et of the cel l . At hi gh fl ow rate, however, the shorter
resi dence ti me was not suffi ci ent to create a measurabl e
pH gradi ent between the i nl et and the outl et of the cel l .
Then, as the mi xi ng between the outl et sol uti on and the
bul k sol uti on was hi gh, suppl ementary passes through
the cel l were requi red to obtai n a pH cl ose to the
i soel ectri c poi nt and to al l ow protei n preci pi tati on. The
di fferent evol uti on of sol ubl e protei n i s confi rmed by the
cal cul ati ons of the i nfl ecti on poi nts and the wi dth of
transi ti on of the sol ubi l i ty curves: pH 5.08 and 0.04 pH
uni t and pH 5.16 and 0.11 pH uni t at 1.2 and 0.2 gal /
mi n, respecti vel y. The 24% sol ubl e protei n remai ni ng
woul d correspond to the whey protei ns, whi ch are
sol ubl e i n the pH range from 4.8 to 4.0. Thi s i s confi rmed
by percentages of whey protei n i n mi l k composi ti on ci ted
i n the l i terature rangi ng from 14 to 24% (Cheftel et al .,
1985; Brunner, 1981; Swai sgood, 1982; Lori ent, 1991).
Chemical Composition. The chemi cal composi ti on,
i n terms of ash content and total protei n, of i sol ates
obtai ned at pH 4.0 after el ectroaci di fi cati on was com-
pared to that of an LSM.
The ash content of i sol ates was l ower i n compari son
wi th LSM: 1.23 ( 0.01 and 2.00 ( 0.60% for 0.2 and
1.2 gal /mi n el ectrochemi cal i sol ates i n compari son wi th
8.05 ( 0.01% for LSM. The ash content of LSM i s i n
accordance wi th data found i n the l i terature, 8.05 versus
7.9% (Hargrove and Al ford, 1974; Bassette and Acosta,
1988; Renner et al ., 1996), whereas the ash content of
the el ectroaci di fi ed i sol ate was l ower than data ci ted i n
the l i terature, 2.0-3.8 and 8.0-10.5% for commerci al
casei n and copreci pi tated casei n, respecti vel y (Hargrove
and Al ford, 1974; Bassette and Acosta, 1988; Al ai s,
1984; Wal stra and Jenness, 1984). Thi s di fference i n ash
content i ndi cates a demi neral i zati on phenomenon acti ng
duri ng el ectroaci di fi cati on. I ndeed, to mai ntai n the mi l k
sol uti on el ectri cal l y neutral , one cati oni c charge must
cross the cati oni c membrane for each H
+
produced at
the bi pol ar membrane (Bazi net et al ., 1997a,b, 1999).
The percentage of total protei n i s 2.3-2.5 ti mes
hi gher for el ectroaci di fi ed i sol ate i n compari son wi th
LSM. Thi s resul t confi rms the effi ci ency of BMEA for
the preci pi tati on and the separati on of mi l k casei n.
Total protei n i n LSM i s i n accordance wi th the l i tera-
ture, 36.9 versus 36.2% for regul ar nonfat dri ed mi l k
(Hargrove and Al ford, 1974; Bassette and Acosta, 1988;
Renner et al ., 1996), whereas the percent total protei n
of el ectroaci di fi ed i sol ates (91.6 ( 4.6 and 85.4 ( 4.1%
for 0.2 and 1.2 gal /mi n i sol ates, respecti vel y) i s si mi l ar
to or sl i ghtl y hi gher than data found i n the l i terature,
83.0-88.5 and 83.0-85.0% for commerci al casei n and
copreci pi tated casei n, respecti vel y (Hargrove and Al ford,
1974; Bassette and Acosta, 1988; Al ai s, 1984). The
di fference i n ash content coul d expl ai n the di fference
i n percent total protei n observed for the i sol ates ob-
tai ned by BMEA versus commerci al i sol ates.
Molecular ProfileAnalysis. The compari son of the
mol ecul ar profi l es obtai ned by HPLC showed that
BMEA al l ows the separati on of hi gh-puri ty bovi ne mi l k
casei n (Fi gure 5). Whey protei n peaks, mai nl y R-l actal -
bumi n and -l actogl obul i n, wi th retenti on ti mes rangi ng
from about 35 to 39 mi n, do not appear or appear i n a
very l ow quanti ty on the i sol ate profi l es (Fi gure 5).
BMEA al l ows a good separati on of casei n from raw mi l k.
Moreover, the fl ow rate does not i nfl uence the puri ty
and the mol ecul ar profi l e of the fi nal i sol ate obtai ned
by BMEA.
CONCLUSI ON
Resul ts obtai ned i n thi s study show that BMEA i s a
new al ternati ve process for the producti on of hi gh-puri ty
casei n bovi ne mi l k i sol ate. Moreover, except for protei n
preci pi tati on curves, fl ow rate does not i nfl uence the
fi nal composi ti on and puri ty of the i sol ate.
Figure4. Effect of the fl ow rates, 1.2 and 0.2 gal /mi n, on the
percentage of sol ubl e protei ns i n the ski m mi l k sol uti on duri ng
bi pol ar membrane el ectroaci di fi cati on run at 2.0 A constant
current.
5294 J. Agric. Food Chem., Vol. 47, No. 12, 1999 Bazinet et al.
The hi gh puri ty of the i sol ate i s expl ai ned by the
demi neral i zati on process coupl ed wi th the acti on of
bi pol ar membranes. Bi pol ar membrane decreases the
pH, wi thout the addi ti on of aci d, and cati ons mi grate
accross the cati on-exchange membrane (CEM) to de-
crease the ash content of the fi nal product i n order to
mai ntai n the el ectroneutral i ty of the mi l k sol uti on.
However, a deposi t, probabl y cal ci um hydroxi de,
appears on the CEM si de i n contact wi th the base. The
cal ci um i on mi grati ng from the mi l k sol uti on coul d
preci pi tate wi th OH
-
produced on the ani oni c si de of
the bi pol ar membrane to form a foul i ng compl ex on the
CEM. Hence, further research i s needed to study the
wi de possi bi l i ti es of BMEA and to understand the
membrane phenomena associ ated wi th the mi grati on
of cati ons.
ACKNOWLEDGMENT
We thank Mr. Chri stopher Barr for revi ewi ng the
manuscri pt.
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Recei ved for revi ew May 18, 1999. Revi sed manuscri pt recei ved
September 13, 1999. Accepted September 17, 1999. Fi nanci al
and techni cal support of thi s research furni shed by the
Laboratoi re des Technol ogi es El ectrochi mi ques et des El ec-
trotechnol ogi es, Hydro-Quebec, Shawi ni gan (PQ), Canada, and
Novalait I nc., Quebec (PQ), Canada, is gratefully acknowledged.
JF990524M
5296 J. Agric. Food Chem., Vol. 47, No. 12, 1999 Bazinet et al.