In/. Dair.

v Journul6 (1996) 251-272

Copyright 0 1996 Elsevier Science Limited
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ELSEVIER 0958-6946(95)00014-3

Effect of Acidification and Neutralization of Milk on Some

Physico-chemical Properties of Casein Micelles

J. A. Luceya, C. Gorrya, B. O’Kennedy”, M. Kalabb,

R. Tan-Kinita’ & P. F. Fox’
“Teagasc, National
Dairy Products Research Centre, Moorepark, Fermoy,
County Cork, Republic of Ireland
‘Centre for Food and Animal Research, Agriculture Canada, Ottawa, Ontario, Canada
“Department of Food Chemistry, University College, Cork, Republic of Ireland

(Received 15 June 1994; revised version accepted 10 March 1995)

ABSTRACT

Acidijication of milk, at low temperatures, to pH 5.0 or 4.6, followed by neutralization

to pH 6.6 (reformed milk), resulted in a reduction in the buffering maximum of’ milk
at pH -5.1; this buffering peak is caused by the solubilization of colloidal calcium
phosphate (CCP). The reduced bufjering in reformed milk suggests that little
reformation of CCP occurs on neutralization; reformed milks also had an elevated
Ca2+ activity. Acidification of milk to pH > 5,5,,followed by neutralization to pH 6.6,
hardly reduced buffering (at pH ~5.1), suggesting that either little CCP dissolved on
acidification or that reformation of CCP occurred on neutralization. Acidification of
milk to IowpH values and neutralization resulted in improved rennetingproperties and
a reduction in rennet coagulation time (RCT). Dialysis ofreformed milk resulted in a
reduction in its renneting properties which became inferior to those of’ control milk,
possibly due to its reduced CCP content or to structural changes in the micelles caused
by removal of CCP. Addition of low concentrations of CaClz to milk, at a constant pH
(6.6), improved its rennetingproperties. Electron micrographs of milk acidijied to pH
values < 5.5 prior to neutralization showed increased rlustering of casein particles,
presumably caused by the reduction in electrostatic repulsion between casein particles
during acid$cation to low pH values. The original micellar appearance was not
restored on neutralization or dialysis of rejormed milk. It is concluded that the
micellur system is not readily reversible; once disintegrated by ucid$cation, micelles
do not reform on neutralization.

INTRODUCTION

During the acidification of milk, many of the physico-chemical properties of casein

micelles undergo considerable change, especially in the pH range 5.5 to 5.0, including

257
258 J. A. Lucey et al.

a voluminosity maximum (of the sedimentable caseins), dissociation of the caseins

and reduction of the hydrodynamic diameter (Roefs et al., 1985). Acidification also
solubilizes colloidal calcium phosphate (CCP) which is an integral part of casein
micelles (Pyne & McGann, 1960; Schmidt, 1980, 1982; Walstra & Jenness, 1984;
Walstra, 1990; Holt, 1992). The extent of solubilization increases markedly below
pH 5.6 and is complete at approximately pH 5.0 (Pyne & McGann, 1960; Pierre et
al., 1983; van Hooydonk et al., 1986; Dalgleish & Law, 1989; Mariette et al., 1993).
The pH at which CCP is completely soluble presumably varies with the conditions
(e.g. temperature) of acidification. Several formulae for CCP have been suggested:
3Ca,(PO&.CaH citrate (Pyne & McGann, 1960) Cas(PO& (Schmidt, 1982)
CaHP04.2H20 (Holt et al., 1989), and an ion cluster containing two organic
phosphate groups, four inorganic phosphate ions and eight divalent cations, mainly
Ca*+ (van Dijk, 1990a, 1992). The CCP of milk can be removed to form colloidal
phosphate-free, CPF, milk by acidification to pH 4.95 followed by dialysis against
an excess of bulk milk (Pyne & McGann, 1960) or by the addition of EDTA to
chelate Ca (Morr et al., 1971). The factors responsible for maintaining the integrity
of micelles have been reviewed (Thompson & Farrell, 1973; Walstra, 1990; Creamer.
1991; Holt, 1992).
While many authors have reported on the drastic changes in casein micelles
and CCP on acidification of milk, it is not known whether these changes are
reversible if the pH is restored to its original value (-6.6). Acidified and
neutralized milk will be referred to as “reformed milk”. There are a number of
reports on the renneting properties of reformed heated milk (van Hooydonk et
al., 1987; Singh et al., 1988; Reddy & Kinsella, 1990; Lucey et al., 1993a).
However, little is known about the effects of acidification and neutralization on
the various properties of casein micelles, although the acid-base buffering and
renneting properties of reformed milk differed from those of normal milk (Lucey,
1992). Information on the reversibility of the micellar structure may provide
further insights into the factors responsible for micelle stability. The effects of
acidification and neutralization on the microstructure of casein micelles, the
buffering properties of milk and rheological properties of rennet-induced gels are
reported in this communication.

MATERIALS AND METHODS

Fresh, pasteurized (72°C for 15 s), homogenized (6.9 MPa, single stage) whole
milk from the Moorepark Friesian Herd was used, unless otherwise stated. Raw
skim milk was used for measurements of Ca*+ activity. To prevent bacterial
growth 100 mg kgg’ thiomersal (C2H5HgSC6H4COONa, BDH Chemicals Ltd,
Poole, UK) were added to all milk samples used for rheological analysis.

Acidification and neutralization

Milk was cooled rapidly to 2°C before acidification to pH values in the range 6.5-
4.6 with 0.5 M HCl and neutralized immediately to pH 6.6 with 0.5 M NaOH at
2°C. Milk samples were then warmed to 20°C readjusted to pH 6.6 with 0.5 M
NaOH and held at 31°C for 1 h before further analysis. An Autotitrator (Mettler
DL 21, Greifenese, Switzerland) was used for pH adjustments.
 Effect of acidification and neutralization of milk 259

Dialysis of reformed milk

Milk was acidified to pH 4.6 with 0.5 M HCl and reneutralized to pH 6.6 with 0.5
M NaOH at 2°C. This reformed milk was warmed to 20°C readjusted to pH 6.6
with 0.5 M NaOH and then dialysed against 20 volumes of original milk for 2
days at 4°C.

Titrations

Acid-base titrations were performed as described by Lucey et al. (1993b).

Rheological measurements

Rheological properties of rennet-induced milk gels were determined using a

Bohlin VOR Rheometer (Bohlin Rheologi, Lund, Sweden), as described by
Bohlin et al. (1984) and van Vliet et al. (1991). Rennet-induced milk gels are
viscoelastic (van Dijk, 1982; Zoon, 1988); the storage modulus (G’) was
determined on renneted milks subjected to sinusoidal oscillation. G’ is the elastic
component of the gel and is a measure of the energy stored and released per
deformation cycle (i.e. “stiffness“).
The amplitude of oscillation was kept sufficiently low (y <0.030) to ensure
linear behaviour (van Vliet et al., 1991); the frequency of oscillation (0) was 6.28
rad s-‘. Temperature of renneting was 31 f O.l”C. Oscillation commenced 4 min
after the addition of rennet. To prevent evaporation, gels were covered with
vegetable oil. Each experiment was carried out on one milk sample, three
replicates were performed for each treatment and results are reported as means
and standard deviation (error bars).

Determination of calcium activity

Standard solutions containing 2, 4, 6, 8 or 10 mmol L-’ CaC12 and 1M KC1 were

used to calibrate the calcium ion electrode (Radiometer F2112Ca) connected to a
Radiometer pH meter (PHM82) fitted with a calomel reference electrode
(Radiometer K401). The Ca2+ activity coefficient was taken as 0.4 (van Kreveld
& van Minnen, 1955; Geerts et al., 1983). A constant stirring rate and
temperature were used to avoid changes in electrode sensitivity. Regular
calibration was performed during analysis of samples.
Raw skim milks samples (100 mL) were tempered at 2°C resulting in an
increase in the pH of milk from 6.7 to 6.8. Milk samples were acidified to pH
values in the range 6.8 to 4.4 by the slow addition of 0.5 M HCl and an
equilibrium time of at least 30 min was allowed at the final pH value. Milk
samples were then neutralized to pH 6.8 at 2°C and warmed to 30°C for -1 h and
held at 23°C for ~1 h before analysis. Results are reported for two milk samples
from seasonal milk analysed at different times of the year.
After determination of the calcium ion activity, these milk samples were
dialysed at 24°C for 3 days against two lo-volume changes of the original milk
containing 0.05% NaNs. All calcium ion activity measurements were determined
at 23°C.
260 J. A. Lucey et al.

Effect of CaC12 on the rheological properties of milk

Calcium chloride (1 M) was added to normal milk at concentrations ranging

from 0.5 to 5 mmol L-‘. The pH of the Ca-supplemented milks was adjusted to
pH 6.6 at 20°C and held at 31°C for 1 h before analysis.

Transmission electron microscopy (TEM)

Milk samples were adjusted to pH 6.6, held at 31°C for 1 h, mixed with 12.5%
glutaraldehyde to give a final concentration of 1.25% and held for a further 30
min. The pH of the glutaraldehyde was adjusted to pH 6.6 to minimise structural
alterations during fixation. Fixed milk was mixed with molten agar (4.8%) and
cut into cubes of about 1 mm3. Between 20 and 30 cubes were taken for each milk
sample. These cubes were placed in plastic vials which were tilled with 1%
glutaraldehyde and closed to avoid retention of air bubbles. The samples were
mailed to the laboratory of the Centre for Food and Animal Research, Ottawa,
Canada, where they were embedded in Spurr’s low-viscosity resin (J. B. EM
Service, Inc., Pointe Claire-Dorval, Quebec, Canada), sectioned (-90 nm thick
sections), stained with uranyl acetate and lead citrate solutions, and examined in
a Philips EM300 transmission electron microscope (Kalab et al., 1988). Each
individual sample was embedded in duplicate, i.e. in two separate resin blocks.
Both blocks were sectioned and approximately 20 sections were obtained.
Between 8810 sections from each sample were examined. Multiple fields were
viewed and typical fields photographed.

RESULTS

Buffering curves of reformed milk

The buffering curves for reformed milks on titration with acid (0.5 M HCl) are
shown in Fig. 1. The buffering properties of all milk samples at pH values ~4.5
were virtually identical. Acidification of milk to pH values in the range 6.6 to 4.6,
followed by neutralization to pH 6.6, resulted in a reduction in the buffering peak

k
= 0.010
a
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

PH PH

Fig. 1. Buffering curves of milk titrated from the initial pH (6.6)‘to 3.0 with 0.5 N HCI;
control milk (O), milk acidified to pH 6.0 (O), 5.5 (A), 5.0 (X), or 4.6 (*) and neutralized
to pH 6.6; and milk acidified to pH 4.6, neutralized to pH 6.6 with 0.5 N NaOH and
dialysed against control milk (0).
 Effect of acidification and neutralization of milk 261

at pH -5.1, indicating that the buffering properties of reformed (especially

samples acidified to pH values < 5.5) milk differed from those of the original
milk. Dialysis of milk that had been acidified to pH 4.6 against an excess of the
original milk resulted in a slight increase in buffering at pH -5.1 (Fig. 1). The
buffering maxima at pH -5.1, for a large number of different milk samples which
were acidified to pH values in the range 6.5 to 4.6 and then neutralized to pH 6.6,
decreased with the extent of acidification (r = 0.918) (Fig. 2).
Odagiri & Nickerson (1965) also found that acidification of milk to pH values
65.5 altered the turbidity and CCP content of milk which were not restored by
neutralization to pH 6.5.
The buffering curves for acidified reformed milk on titration with base (0.5 M
NaOH) were similar to those for normal milk (Fig. 3). However, the buffering
peak at pH 6.3 was slightly reduced for milks acidified to pH values < 5.0 prior to
neutralization; dialysis of this milk against control milk had little effect on its
buffering properties. The buffering maximum at pH -6.3 during the back
titration of many different reformed milk samples decreased slightly with the
extent of acidification (Y= 0.614) (Fig. 4).

Rheological properties of renneted reformed milk

Further evidence that the structure and properties of casein micelles were not
restored after acidification and neutralization was provided by the rheological
properties of renneted reformed milk. Acidification of milk and reneutralization
resulted in an increase in G’ (Fig. 5) of rennet-induced milk gels. There was little
difference between the rheological properties (1 h after addition of rennet) of
milks acidified to pH values ~6.0, although the rennet coagulation time (RCT)
decreased with the extent of acidification.
Dialysis of reformed (acidified to pH 4.6) milk against an excess of bulk milk
resulted in a reduction in G’ (especially 1 h after addition of rennet) and an
increased RCT. This suggests that the improved renneting properties of reformed
milk may have been due to an elevated Ca*+ activity. Dialysed reformed milk had
a longer RCT and lower G’ value than control milk, possibly due to a reduction in
the CCP content or to dilution of milk during reforming and dialysis.

0.11
4.5
I
5.0
I
5.5
I
6.0
I
6.5
I
7.0

PH

Fig. 2. Effect of pH of acidification (prior to reformation) on the value of the buffering

maximum at pH -5.1 during the acid titration of reformed milk; (r = 0.918).
 262 J. A. Lucey et al.

c
P
"a 0.04

%
- 0.03
.E
P
.e 0.02
0"
'e 0.01
G
m1 0.00

PH PH

Fig. 3. Buffering curves of milk titrated from pH 2.0 to 10.0 with 0.5 N NaOH; control
milk (a), milk acidified to pH 6.0 (O), 5.5 (A), 5.0 (X), or 4.6 (*) and neutralized to pH
6.6; and milk acidified to pH 4.6, neutralized to pH 6.6 with 0.5 N NaOH and dialysed
against control milk (0).

O.iI I I I I I
4.5 5.0 5.5 6.0 6.5 7.0
PH
Fig. 4. Effect of pH of acidification (prior to reformation) on the value of the buffering
maximum at pH -6.3 during the back titration of acidified reformed milk; (r = 0.614)

Time after rennet addition (s)

Fig. 5. Storage modulus (G’) during the rennet coagulation of milk; control milk (a), milk
acidified to pH 6.0 (b), 5.5 (c), 5.0 (d), or 4.6 (e) and neutralized to pH 6.6; and milk
acidified to pH 4.6, neutralized to pH 6.6 and dialysed against control milk (t). The
coagulation temperature was 31°C and (I) was 6.28 rad s-l. Mean and standard deviation
from three replicates of a single milk sample.
 Effect of acidification and neutralization of milk 263

Ca-activity of reformed milks

The results shown in Fig. 6, for two separate experiments, show that the Ca2+
activity increased on acidification in the pH range 6.8 to 4.6 followed by
neutralization to pH 6.8 at 2°C. Dialysis of the reformed milks against a large
excess of bulk milk for 2 days at 2°C restored the Ca*+ activity of these milk
samples to that of the bulk milk (data not shown). These results suggest that the
improved renneting properties of reformed milks were due to increased Ca*+
activity.

Rheological properties of Ca-supplemented milks

To support the hypothesis that the improved rheological properties of reformed

milk could be due to elevated Ca2+ activity, an additional experiment was carried
out. Various levels of CaCl* were added to normal milk and the Ca-supplemented
samples adjusted to pH 6.6. Addition of CaC12 to milk (at pH 6.6) resulted in a
reduction in RCT and an increase in G’ (Fig. 7). This was expected as the
beneficial effect of Ca*+ on the renneting properties of milk is well known (see
Lucey & Fox, 1992).

Electron microscopy

Electron micrographs of milks that had been acidified and neutralized (Fig. 8)
showed increased clustering of casein particles. Solubilization of CCP and the
concomitant increase in Ca2+ activity may be partially responsible for the
increased aggregation in reformed milks; however, reformed milks that were
dialysed to remove excess Ca2+ also had a clustered microstructure. It is likely

2.15 -

h 2.25 -
.S
.,
2
+
*$ 1.75 -
a
;
1.25 -

0.75 I I I I I J
4.0 4.5 5.0 5.5 6.0 6.5 7.0
pH of acidification prior to neutralization

Fig. 6. Ca2+ activity in two separate sets of milk samples acidified to pH values in the
range 6.8 to 4.4 at 2”C, neutralized to pH 6.8 at 2°C and then warmed to 30°C for 1 h.
264 J. A. Lucey et al.

Time after rennet addition (s)

Fig. 7. Effect of Ca on the storage modulus (G’) during the rennet coagulation at pH 6.6
and 31°C of milk supplemented with 0 (a), 1 (b), 3 (c) or 5 (d) mmol Ca L-‘. The o was
6.28 rad se’. Mean and standard deviation from three replicates of a single milk sample.

that clustering was due to the reduction in electrostatic repulsion between casein
particles during acidification to low pH values. Homogenization resulted in the
attachment of casein particles to fat globules. Agar gel, which was used to
immobilize casein micelles, is noticeable in the micrographs in the form of tibres.

DISCUSSION

Lucey et al. (1993b) reported that the buffering curves during the acidification
and back titration of milk were not similar; when milk was acidified, maximum
buffering occurred at pH -5.1 and when acidified milk was back titrated with
base, maximum buffering occurred at pH -6.3. The removal of CCP from milk
resulted in the disappearance of the buffering maxima at pH -5.1 (which
occurred during the acidification of milk) and at pH -6.3 (during the back
titration of acidified milk). Milk contains many other buffering substances,
including protein residues and salts, which contribute to buffering over a wide pH
range. These buffering groups buffer strongly at their respective pK, values, e.g.
in the presence of excess calcium, phosphoric acid has its pK,z at 5.8 and pKa3 at
6.6 (Walstra & Jenness, 1984). The buffering peaks in milk at pH 5.1 and 6.3
cannot be due to protein residues or soluble salts as these substances always
buffer at a particular pK, and the buffering curve would be unaffected by the
titration sequence. In this paper we are interested mainly in changes that occur in
the buffering peaks at pH 5.1 and 6.3 in reformed milks and how changes in the
form of calcium phosphate may be responsible.
In normal milk, the buffering peak at pH -5.1 is due to solubilization of CCP
which results in the formation of phosphate ions which can combine with H+,
resulting in buffering (Lucey et al., 1993b). Solubilization of CCP involves the
protonation of inorganic P (van Dijk, 1990b) and a reduction in the proton
relaxation rate of milk (Mariette et al., 1993). When acidified (pH 2) normal milk is
titrated with base (0.5 M NaOH), buffering is low at pH 5.1 but a buffering peak
 265
Effect ofacidification and neutralization ofmilk

(4

(b)
Fig. 8. Transmission electron micrographs of milk samples; control milk (a), milk acidified
to pH 6.0 (b), 5.5 (c), 5.0 (d), or 4.6 (e) and neutralized to pH 6.6; and milk acidified to pH
4.6, neutralized to pH 6.6 and dialysed against control milk (f). Scale bar is 1 pm
266 J. A. Lucey et al.

L * *
0
I* 1 *
.

(4
Fig. 8. -Contd.
Effect of acidification and neutralization of milk 267

(e)

V-l
Fig. 8. -Contd.
268 J. A. Lucey et al.

occurs at pH 6.3 due to the formation of insoluble calcium phosphate and the release
of H+ which can combine with OH-, resulting in buffering (Lucey et al., 1993b).
The pH at which insoluble calcium phosphate is formed when the pH of
solutions containing calcium and phosphate is increased depends mainly on the
temperature and concentration of calcium and phosphate. During the titration of
phosphoric acid with Ca(OH)2, when the concentration of calcium increases to
such a point as to cause the formation of a precipitate, further increments of
Ca(OH)2, instead of raising the pH, merely precipitate calcium phosphate. This
flattening of the titration curve occurs just below pH 6 (Holt et al., 1925). Boulet
& Marier (1961) reported that in order to induce precipitation of calcium
phosphate at 21°C in solutions containing between 2 and 24 mmol L-’ of calcium
and phosphate (ionic strength O.OS), the pH of these solutions had to be adjusted
to between 6 and 8 with NaOH. Schmidt & Both (1987) found that at 25°C
precipitation of calcium phosphate did not commence until pH 6.1 (for
concentrations of phosphate and Catotal of < 10 mmol L-‘) while at 50°C
precipitation of calcium phosphate occurred in the pH range 5 to 7 (for
concentrations of phosphate and Ca totai of between 8 and 12 mmol L-l).
The absence of the buffering peak at pH -5.1 in reformed (acidified to pH
values < 5.5) milk can be explained as follows: reducing the pH of milk leads to
the solubilization of CCP; when the pH is increased subsequently, dicalcium
phosphate dihydrate (DCPD) or octacalcium phosphate (OCP) may precipitate
(Madsen & Thorvardarson, 1984; Schmidt & Both, 1987). Little formation of
CCP appears to occur on neutralization to pH 6.6 as the buffering peak at pH
-5.1 was absent during the acid titration of reformed milks (Fig. 1).
The buffering peak at pH 6.3 during the back titration of acidified milks is
attributed to the precipitation of calcium phosphate which is likely to result in the
production of H+ due to the deprotonation of H2P04- or HPOd2- (van Dijk,
1990~). During the production of artificial casein micelles, protons are also
liberated (Schmidt et al., 1977), presumably due to formation of CCP. The
buffering curves of normal and reformed milks during the back titration of
acidified milks were similar (Fig. 3), which suggests that the precipitation of
calcium phosphate (presumably DCPD or OCP) is affected little by the previous
pH cycling treatment of the milk.
Acidification of milk solubilizes CCP and concomitantly increases Ca2+
activity; however, if CCP is not reformed during the neutralization step, reformed
milks are likely to have an elevated Ca2+ activity, which was also reported by
Singh et al. (1988), although other calcium phosphates may precipitate.
The large difference in the initial Ca*+ activity of the control milks reflects the
variation in the calcium equilibrium of seasonal milk (see White & Davies, 1958).
An elevated Ca*+ activity is the most likely factor responsible for the improved
rennet coagulation properties of reformed milk, as dialysis eliminated this effect.
It is unlikely that an elevated ionic strength, due to increased concentrations of
NaCl, are responsible as addition of NaCl results in an increased RCT but little
other effects on the rheological properties of rennet-induced milk gels, except at
very high concentrations and after long ageing periods (Zoon, 1988).
The reduction in RCT and increased G’ of Ca-supplemented milks was also
reported by Zoon (1988). This demonstrates, as is well known, that Ca2+ play an
important role in reducing repulsion between negatively charged caseins at pH
-6.7 (e.g. Dalgleish, 1984) and consequently increases the aggregation rate
 Effect of acidification and neutralization of milk 269

during the rennet coagulation of milk (Green & Marshall, 1977; Dalgleish, 1983).
It is unknown why the renneting properties of dialysed reformed milk were
inferior to those of the control milk, although reducing the CCP content of milk,
while maintaining the Ca2+ activity, results in the inhibition of rennet
coagulation (McGann & Pyne, 1960; Pyne & McCann, 1962; Shalabi & Fox,
1982; Zoon, 1988). This is probably due mainly to structural changes in the
micelle caused by the removal of CCP. The role of calcium in the rennet
coagulation of milk has been reviewed (Lucey & Fox, 1992).
It may well be that the original micelle structure is unique (Pyne, 1962) since
the original properties of milk are not restored completely by the re-introduction
of colloidal phosphate into CPF milk (McGann & Pyne, 1960), or by the
exhaustive dialysis of urea-treated milk (McGann & Fox, 1974) or dispersions of
casein submicelles (Ono et al., 1983) against the bulk milk. However, artificial
casein micelles, formed by the addition of Ca, Mg, phosphate and citrate to
casein at pH 6.7 and 37°C may produce micelles with properties similar to those
of native micelles (Schmidt et al., 1977; Schmidt, 1979).
Presumably, the clustering of acidified casein particles caused by acidification
and neutralization in the cold is due to the reduction in electrostatic repulsion which
facilitates aggregation by van der Waals attractions and hydrophobic interactions
(Roefs, 1986). During the acidification of milk, casein particles aggregate as the pH
decreases and gel at approximately pH 5 (at 30°C) which does not depend on the
solubilization of CCP or Ca2+ mteractions as sodium caseinate solutions also gel at
a similar pH (Roefs, 1986). Dissociation of /?-casein from the micelle occurs when
milk is cooled to low temperature (Rose, 1968; Downey & Murphy, 1970; Davies &
Law, 1983) e.g. during the cold acidification and neutralization procedure.
However, considerable reassociation occurs when milk is subsequently warmed
(Creamer et al., 1977; Ali et al., 1980; Davies & Law, 1983). The original casein
micellar structure is absent in reformed milks presumably because all the caseins
and CCP do not reassociate when acidified milks were neutralized.

CONCLUSIONS

The buffering properties of milk can be used to monitor changes in the colloidal
composition of milk. CCP is not reformed when acidified milk is neutralized,
other calcium phosphates are formed which have different acid-base buffering
properties compared to normal milk. Acidification of milk, followed by
neutralization, resulted in improved renneting properties, probably due to an
elevated Ca2+ activity. It was demonstrated that addition of CaC12, at constant
pH, resulted in improved renneting properties of milk. The micellar system does
not appear to be readily reversible; once disintegrated by acidification, micelles
are not reformed by neutralization.

ACKNOWLEDGEMENTS

The authors would like to thank Miss Gisele Laroque for technical assistance with
electron microscopy. The Electron Microscope Unit, Research Branch, Agriculture
Canada in Ottawa provided facilities. Contribution 2217 from CFAR in Ottawa.
270 J.A. Luceyet al.

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