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Alcohol and Heat Stability of Milk Protein

Article  in  Journal of Dairy Science · December 1990

DOI: 10.3168/jds.S0022-0302(90)79064-9

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Alcohol and Heat Stability of Milk Protein
ABSTRACT
Recent advances have been made in
the understanding of factors that control
the destabilization of milk proteins by
alcohol and by heat. These advances are
reviewed, and argwnents are developed
supporting a physical basis for the mech-
anism of alcohol-induced precipitation.
These physical effects are contrasted with
the mainly chemical reaction-based fac-
tors controlling heat coagulation. The
benefits of these recent developments are
illusttated by case studies on cream li-
queurs and concentrated milk products.
(Key words: ethanol stability, heat stabil-
ity, milk protein)
INTRODUCTION
Stability tests based on the addition of alco-
hol or on the heating of milk. have been with us
for more than a century. Often found in city
statutes or public health regulations, they pro-
vided in the late 18008 an objective assessment
of the keeping quality of milk for retail sale (1).
These were simple, 1Dlsophisticated pass or
fail tests. If a precipitate formed when an equal
volume of 70% alcohol solution was added or
when the milk was boiled, then that batch of
milk was rejected. As late as the 19308, such
alcohol tests were accepted as indicators of
whether milk was going sour, was mixed with
colostrum, or was contaminated by milk from a
cow with a diseased udder (49).
Even as the necessity for such tests declined
with the rapidly improving bacteriological qual-
ity of milk, stability defects associated with
season, diet, and stage of lactation became
recognized. The traditional tests of alcohol and
heat stability proved to be unreliable indicators
Received September 30. 1989.
Accepted May 3, 1990.
1990 } Dairy Sci 73:36]3-3626 3613
DAVID S. HORNE and D. DONALD MUIR
Hannah Research Insll1ute
Ayr, KA6 SHL
ScoUand, UnIted Kingdom
of these defects and, in particular, of the suita-
bility of milk for processing into evaporative or
condensed products. Problems associated with
coagulation during sterilization continue to
plague the dairy industry despite intensive but
sometimes spasmodic research into milk pro-
tein stability over the years. This paper summa-
rizes some recent work carried out in the areas
of alcohol and heat stability.
ETHANOL STABILITY
Early studies of ethanol stability were based
on single-point assays at the natural pH of the
milk. When Home and Parker (24) extended
this worX, they found that artificial adjustment
of the pH provided an ethanol stability to pH
profile characteristic of an individual milk.
Such profiles (typical examples are shown in
Figure 1) were sigmoidal in shape with a mini-
mum stability at low pH and a maximum sta-
bility at high pH. In fact, increase of pH in-
creased the stability of the micelle to such an
extent that adding 100% ethanol to an equal
volume of milk often failed to induce protein
prec~~tion.HomeandParker(~)modified
the traditional alcohol test, adding 2 vol of
aqueous ethanol solution to 1 vol of milk. The
ethanol stability of the milk at the specified pH
was then reported as the concentration of the
ethanol solution inducing instantaneous precipi-
tation of the milk protein.
The typical ethanol stability to pH profile
can be characterized by four parameters: the
asymptotic values, Smin and Smax. of the two
arms of the sigmoid at low and high pH, re-
spectively; an inflection point or effective pK
value denoting the position of the profile along
the pH axis; and a gradient or slope parameter.
Continuing the analogy to an ionization pro-
cess, the experimental profiles can be fItted to
the functional fonn given by the equation be-
low, b being the slope parameter, the gradient
of its logarithmic transfonn:
 3614 HORNE AND MUIR

~-s = lo"<PH-pK) !he identity of that active component by obseIV-

mg the effects of controlled additions of each
min
or taking logarithms component in tum. Only those experiments
producing alterations in the profile are men-
pH = pK + b 10glO [(S - Smm)!(Smax- S)] tioned below. Thus, we note here that the addi-
tion of lactose or urea at twice the naturally
The full curves in Figure 1 are least squares occurring average concentration had no effect
~ts to the experimental points using this equa- on the ethanol stability to pH profile. This
tion. contrasts with the effects of urea on heat stabil-
Cross..<fialysis experiments, in which a small ity described later.
amoWlt of milk A was dialyzed against a large The addition of Ca2+ to milk displaced the
volume of milk. B and vice versa, showed that profile to higher pH values (25), but did not
the profile was governed by diffusible compo- alter its sigmoid character nor significantly
nents ~ the serum phase (25). On dialysis, milk change the values of Smax and Smin. Removal
A acqwred the profile of milk. B and vice versa. of Ca2+ by treatment with EDTA had the re-
~ghton et al. (36) suggested an early mecha-
verse effect of shifting the entire profile to
nIsm for alcohol-induced coagulation, envision- lower pH. Addition of phosphate or citrate had
ing the alcohol denaturing whey proteins. These no effect other than to shift the stability profile
whey proteins then precipitated onto the casein in response to the chelation of ea2+ by these
micelles, which were precipitated in tum (36). additives, the level of the response being in the
The results of the crossdialysis experiments order of their Ca2+-ehelating power.
in~cated .a lack of involvement of whey pro-
Such movements of the profile, to higher pH
terns. This was confinned in experiments in when the soluble calcium level was increased
which centrifuged micelles were resuspended in and to lower pH when it was reduced by EDTA
skim milk. dialysate. The ethanol stability pro- addition, were consistent with the earlier find-
file of the resuspended system was identical to ings of Davies and White (8). Those authors
that of the original milk (25). Total removal of fOWld the ethanol stability of milk at its natural
the whey proteins had no effect on the ethanol pH to be significantly correlated with the level
stability to pH profile. of free ionic calcium in the milk. Soluble ionic
Having concluded that the position of the calcium is therefore the dominant factor in
ethanol stability to pH profile was controlled by fixing the position of the profile along the pH
diffusible serum phase components, we sought axis. Further evidence of this came from a
s~ey of !he e~ol stability of Irish creamery
milks (9) m which concurrent analysis of min-
eral constituents of ultrafiltrate permitted the
100 calculation of the soluble salt balance ratio for
-; these milks. The results of this survey showed
"- 90
> this salt balance ratio to be significantly corre-
~
BO lated with the profile pK value as shown in
Figure 2. A study of lactational trends in
........"... 70
ethanol stability carried out at the Hannah Re-
...
.Q 60 search Institute added further support to these
....II ideas on the effects of salt balance and con-
III 50 finned the role of ionic calcium (31).
:r Consideration of the actual profile suggests
0 40
.... that ionic calcium may be assigned a dominant
IIJ
30 ~le: Consider the following: the profile appears
6 6.5 7 7.5 similar to that expected from an ionization pro-
pH cess. The most obvious candidate among the
serum phase components is phosphate, which
Figure 1. Typical ethanol stability to pH profiles
demonstrating the effect of added ea'Z+ (II. 4 DIM) and has such a transition in this pH range. The
EDTA (A,S DIM) on profile position with respect to the doubly ionized form of phosphate is a much
control milt (0). more effective calcium sequestrant. Increasing
Journal of Daily Science Vol. 73, No. 12, 1990
 BORDBN SYMPOSIUM: STABIlITY OF PROTEINS IN DAIRY FOODS
0
1 the milk. This shift in mineral balance is also
....
.jJ
10
[[
• •
QJ
U
C
.8
• •
10
.....
10
lD .6
+'
.....
10
l/l
.4
6.4 6.6 6.8
Profile pK value
Figure 2. Correlation of ethaDol stability profile pI{
value with the soluble salt balaDce ratio (Ca + Mg):(inor-
game P + citrate) detenDined for a series of milks from
Irisb cows at various S1ll8Cl of Jac1ation.
the pH of the milk thus will reduce the free
calcium level and therefore increase ethanol
stability as observed. In contrast, decreasing the
pH frees ionic calcium and leads to a decrease
in ethanol stability. Further confinnation of the
role of phosphate derives from experiments in
which bulk milk was dialyzed against phos-
phate-free serum of the Jenness and Koops type
(26). The transition was lost. Conversely. a
transition in calcium caseinate systems could
only be induced by including appropriate levels
of phosphate in the mixttue (26).
The consensus view from all these diverse
studies and experiments is that the calcium ion
plays the dominant role in controlling ethanol
stability. The level of free calcium controls the
ethanol stability of the system.
Modifications mentioned earlier in this sec-
tion were deliberate manipulations of milk
composition. The major proportion of milk
consumed today is processed in some fashion
before retail sale. and some of these treatments
can cause changes that manifest themselves in
variations of the ethanol stability of the milk.
Among the processing options studied for their
effects on ethanol stability were forewarming
(28) and concentration (30). The changes in
ethanol stability brought about by forewarming
(28) can be reconciled in the above model as
consequences of reduction in soluble calcium
due to heat-induced deposition of the calcium
(as calcium phosphate) on the micellar phase of
3615
thought to be the source of the beneficial effect
of forewarming prior to concentration during
the manufacture of evaporated milk (16).
Concentration also produces marked changes
in the ethanol stability of milk. With evapora-
tive concentrates. the heat stability of the con-
centrate bears no obvious relation to that of the
original milk. This is the major problem in
predicting the heat stability of concentrated
products. In contrast, Home and Parker (30)
demonstrated that the same is not true of the
7 ethanol stability. At high pH. i.e.. the alkaline
arm of the sigmoidal pH profile, stability
decreased progressively as the milk was con-
centrated. On the acid side of the natural pH,
the ethanol stability profile was largely unaf-
fected by concentration. Both aspects of this
behavior were similar to that shown by milk of
normal concentration to which sodium chloride
had been added.
When the ethanol stability at fixed pH was
plotted against total chloride content for these
systems, linear decays in stability were ob-
served above pH 6.8. With the same milk used
throughout. the data points fell on the same line
whether the chloride content was increased by
evaporation or by direct addition. This suggests
that ionic strength of the system controls
ethanol stability of milk concentrates at pH
above the natural pH of the milk. The conse-
quences of this are twofold: 1) by adding so-
dium chloride to a milk. the ethanol stability of
its concentrate can be predicted; and 2) if that
stability is too low. it can be increased to the
desired level by reducing the chloride content
in a short dialysis step before concentration.
This new-found understanding of the stabil-
ity of milk proteins to the presence of ethanol
has been exploited most effectively in the for-
mulation of stable cream liqueurs (4).
CREAM LIQUEURS
In 1944. Arthur C. Dahlberg (6), in his
President's address to the ADSA meeting. com-
plained that no entirely new dairy product of
significant importance had been invented in the
previous half century. The major products-
cheese. butter. yogurt~ date from prehistoric
times. Even ice cream was made before modem
dairying. So almost 50 yr from Dahlberg's
address, it is a pleasure to note the emergence
Journal of Dairy Science Vol. 73. No. 12. 1990
3616 HORNE AND MUIR

TABLE 1. Effect of calcium ion "inactivation" on the 24

••
shelf-life of cream liqueur at 4S·C. a.
u •
Shelf-life Apparent U 20
• •
Treatment (d) pH 0
0 • •
ru
• • •
• •... ...... ...
Control 8 6.72
Anhydrous milk fat 63 6.61 •
>-
16
• •
• •• •
Washed cream 78 6.82 ...
Trisodium citrate 1 +'
Citric acid estCl' of GMS 2
74
71
6.80
6.95
....
OJ
0 12
.............
........
lAdded at 10 mmol/L of product. u
OJ
It . . 00000 0
201ycerol mODOstcarate. added at .5%/L of product; .... 0000 0000
added caseinate reduced from. 3 to 1%. > 8
0 5 10 15 20
Storage Time (d @ 45°C)
of just such a new product. Even though mix- FlgUI'e 3. Effect of ionic strength (added NaCl) on the
tures of alcohol with milk or cream have their stability of high proof cream liqueurs, alcohol content
origins in antiquity, the modem equivalents- 192% wt/wt. Control (0); + 10 mM NaCl (A); + 20 mM
cream liqueurs-bave been successfully deve- (.); + 50 mM<->; + 100 mM (e).
loped only in the last decade.
The earliest of these products gained im-
mediate consumer acceptance but suffered from each of these options cleansing the system of
defects that led to a limited shelf-life. First, a residual calcium (3).
plug of fat in the neck of bottles was commonly More recent work has focused attention on
found. This problem was the result of ineffi- the production of cream liqueurs with alcohol
cient homogenization; large fat globules readily contents up to 24% by volume. At high alcohol
cream on storage. The cure, once the problem
levels, not only is the presence of calcium
was recognized, was careful control of homog-
deleterious, but all ions have a detrimental ef-
enization conditions during manufacture.
fect on emulsion stability and product quality.
The liqueur is an emulsion of milk fat in an
aqueous alcohol solution. The second defect With viscosity increase used as an indicator of
manifested itself as a slow coagulation, ulti- destabilization, the deleterious effect of added
mately separating into a curd and a clear serum. sodium chloride becomes apparent during
The emulsion droplets were stabilized by a prolonged storage at 45"C (Figure 3). Measure-
coating of casein. Casein micelles could be ments cease at high salt concentrations due to
regarded as protein droplets similarly stabilized the occurrence of complete coagulation accom-
by a protein coat, again casein. We now know panied by serum separation. Once more, the
that the chief agent in controlling the ethanol emulsion behavior of liqueurs paralleled effects
stability of casein micelles is ionic calcium. seen in milks in which ionic effects predomi-
The cream, derived from milk, contains signifi- nated in stable regions in which high concentra-
cant levels of residual soluble milk compo- tions of ethanol were required to induce precip-
nents, including calcium. These low concen- itation. The solution to this problem is that
trations-about one-sixth of those found in already mentioned in dealing with soluble cal-
milk-are in sufficient quantity to affect the cium, either to remove the ionic constituents of
long-term stability of the product liqueur. The the cream by washing with water or to use
solution to this defect, calcium-associated insta- anhydrous milk fat (3).
bility, was to incorporate a calcium sequestrant
such as citrate into the formulation (4). Ac- CORRELATION BETWEEN ALCOHOL
celerated stability tests, based on storage of AND HEAT STABILITIES
product liqueur at 45"C, demonstrated the effi-
cacy of modest additions of citrate (5 to 10 The general shape of the ethanol stability to
mM) in extending shelf-life (fable 1). Similar pH profile is characteristically sigmoidal. The
increases were obtained by using washed cream work of Rose (53, 54) is frequently quoted
or anhydrous milk fat as the fat component, when discussing the effects of pH adjustment

10urnal of Dairy Science VoL 73. No. 12, 1990

 BORDEN SYMPOSWM: STABILITY OF PR01EINS IN DAIRY FOODS
on heat stability. Rose classified two fonns of
the heat stability curve, the Type A with the
minimum and the monotonically increasing
Type B (53, 54). Neither curve bears any
marked resemblance to the ethanol stability
profile. However, in the paper that introduced
the subjective heat stability test, Miller and
Sommer (40) also examined the effects of
acidifying milk on its heat stability.
The parameter measured in their study (40)
was coagulation temperature, the temperature to
which the pH-adjusted sample of milk had to
be heated to induce instantaneous coagulation,
or, in practice, within 2 min. It therefore differs
from the modem test in which the time required
to induce clot formation at a prechosen fixed
temperature is quoted as a measure of heat
stability. This distinction is important.
Darling (7) described the increase in coagu-
lation temperature with increasing pH, seen by
Miller and Sommer (40), as approximately line-
ar. But when individual heat stability to pH
profiles (Figure 4) are considered, a remarkable
similarity to the expected ethanol stability
counterpart can be discerned. The profiles are
apparently sigmoidal, tending to a minimum
stability at low pH and a maximum stability at
high pH. Indeed, the transition can be fitted by
an equation of the fonn used to fit ethanol
stability profiles, and the full curve is the best
fit of that function to the data points shown.
The pK value for the control curve is somewhat
lower for this curve than normally encountered
for ethanol stability profiles, but we know that
forewarming, heating at 9O"C, increases ethanol
stability by shifting the profile to lower pH
values (28). The act of heating could produce a
similar effect in a heat stability situation. We
also see fairly marked shifts in ethanol stability
profiles through lactation and we know nothing
of the history of the milk used by Miller and
Sommer in this respect.
What we do know from their published data
is the effect of added calcium or phosphate on
the position of the heat stability profile. Again,
exemplary data have been extracted from their
paper and plotted in Figure 4. The response to
the addition or removal of calcium is similar to
that shown by the ethanol stability profile
(Figure I), namely, a decrease in stability and a
shift of the profile to higher pH when calcium
level is increased. Conversely, an increase in
stability and a shift in profile to lower pH occur
3617
oU 170
OJ
150
L
::J
-I.l
10 130
L
OJ
0-
E 110
OJ
I--
90
Cl
10
8 70
5.7 5.9 6.1 6.3 6.5 6.7
pH
Figure 4. Reworked heat stability data of Miller and
Sommer (40) demonstrating the fit of the sigmoidal profile
equation, previously used in the treatment of ethanol stabil-
ity data, to their coaguIation (Coag.) temperature versus pH
data. Curves and data points are drawn for control millc (0),
millc pius added calcium (.), and millc plus added phos-
pbate <-).
when the effective calcium content of the milk
is reduced by the addition of a calcium seques-
trant. In this instance, the data of Miller and
Sommer show phosphate to be very effective in
shifting the profile. Addition of citrate (5 mM)
was equally effective. Nevertheless, in some of
their other examples, phosphate addition had no
detectable effect on the calculated profile pK
value. They give at least four examples with
calcium addition in their paper. All of them
show the shift to higher pH demonstrated in the
example shown in Figure 4.
Marked similarities are detected between
ethanol stability and this particuIar method of
measuring heat stability. They demonstrate a
similar response to pH adjustment. They re-
spond at least qualitatively in the same manner
to increase or decrease in available calcium. Of
course, similarities exist in the protocols fol-
lowed in both tests. Both tests monitor the
results of an almost instantaneous reaction. We
added ethanol of sufficiently high concentration
to bring about the immediate appearance of
clots in the milk. No lower concentrations of
ethanol interested us. In fact, the 2% increment
in ethanol concentration between successive
test solutions is sufficiently wide and the differ-
ence in rate of coagulation sufficiently great
that generally no clots become visible in the
next lower solution even on prolonged stand-
Journal of Dairy Science Vol. 73, No. 12, 1990
3618 HORNE AND MUIR
ing. In the heat stability test of Miller and
Sommer (40), they too, were only interested in
a rapid reaction and were seeking to determine
that temperature at which heat coagulation oc-
curred within 2 min of the sample being in-
serted into the hot oil bath.
The similarities in protocols defining the test
situations and the similarities in response to pH
and calcium adjustment suggest that in both
tests a similar, if not identical, reaction pathway
might be followed. The following simple argu-
ment gives some idea of how this behavior may
be satisfied.
Colloids-and casein micelles are colloidal
particles-are stable because a sufficiently large
repulsive energy barrier exists between particles
to prevent coagulation. The Arrhenius equation
relates the rate constant (k) for a reaction, or a
reaction step, to the system temperature through
the equation, shown below in its logarithmic
form,
In k = In A - ElRT
where:
A = collisional frequency factor,
R = molar gas constant, and
T = temperature ("K).
In this sample argument, the activation energy,
E, is equated with the energy barrier height.
Current thinking concerning casein micelles in-
cludes a steric stabilization component in the
repulsive energy contributing to this barrier-the
"hairy micelle" concept of Holt (18) and WaI-
stra (65). But, for destabilization induced by
addition of ethanol, the light scattering studies
of Home (20, 21, 23) have shown the collapse
of the hairs and the consequent loss of the
sterlc component with subcritical amounts of
ethanol. Even with this loss, an electrostatic
component still remains a possibility. As evi-
dence of its existence, we may cite the follow-
ing: the observation that the effects produced
by substituting a range of alcohol is merely one
of dielectric constant (27); the effects of chemi-
cal modification that can be reconciled as a
change in protein charge (29); the influence of
calcium and pH on the kinetics of aggregation
(Home, unpublished); and the direct measure-
ment of electrophoretic mobility over the crit-
ical range of ethanol concentration (Home, un-
published). This evidence points to the presence
Journal of Dairy Science Vol. 73, No. 12, 1990
of a repulsive electrostatic component that must
be overcome before aggregation can proceed.
Simplistically, this energy is estimated as
proportional to the square of the electrostatic
surface potential, '1', and to the dielectric con-
stant, E. The log of the rate constant is then
given by the equation:
where:
No = Avogadro's number.
Thus, as the ethanol concentration is increased,
the dielectric constant and the term on the
right-hand side of the equation is decreased
until rapid coagulation occurs. Equally, the
right-hand tean can be reduced simply by in-
creasing the temperature. However, this would
assume that the surface potential was the same
in each situation at the instant coagulation oc-
curs, not necessarily before the alcohol was
added or the heating commenced, and this we
have yet to prove. But the speculation is in-
teresting nevertheless.
More interesting are the differences between
the Miller and Sommer heat stability profiles
and those we generally see in current studies.
This is discussed in the following section.
HEAT STABILITY
In this short review of heat stability, we
cannot do justice to all that has been published
on the subject over the last half century. For
more detailed infonnation on aspects not cov-
ered here, refer to the excellent reviews of Fox
and Morrissey (14), Fox (12), and Muir (42).
This section concentrates on the following:
first, an explanation of why the sigmoidal pH
profile observed by Miller and Sommer (40), so
reminiscent of the ethanol stability to pH pro-
file, changes when the method of defining heat
stability is altered; and second, a reassessment
of the situation regarding the origins of the
minimum in the Type A heat stability curve.
pH Dependence of Heat Stability
Miller and Sommer (40) defined heat stabil-
ity as the temperature required to instantane-
 BORDEN SYMPOSIUM: STABnlTY OF PROTPlNS IN DAIRY FOODS
ously coagulate milk protein (effectively within
2 min). Today, the accepted definition of heat
stability is the time taken for coagulation to
occur under standard conditions, usually 14O"C
for milks of nonna! concentration or 120"C for
concentmted milks. As first observed by Rose
(53, 54), this difference of time versus tempera-
ture leads to a manifestly different heat stability
to pH curve (Figure 5). Rose found that coagu-
lation time to pH curves fell into two classes.
The Type A milk, with the pronounced mini-
mum, and the Type B, with the progressive
increase in coagulation time as pH is increased,
both differing from the sigmoidal behavior
found by Miller and Sommer (40) on acidifying
milk.
When milk is heated. several competitive
and often interdependent reactions occur. Fox
(11) reviewed the heat-induced changes in milk
preceding coagulation and classified them into
five groups: acid development, precipitation of
calcium phosphate, Maillard reactioos, casein
modification, and interaction of sulfhydryl
groups, the latter including whey protein
denaturation.
3619
Many of these reactions proceed concurrent-
ly. Probably not all reactions lead directly to
coagulation, but each has its own temperature
dependence, and the products of one reaction
may influence the course of another. Thus, as
an example, the Maillard reaction is pH depen-
dent. Other reactions acidifying the milk and
lowering the system pH will thus slow the
Maillard reaction. The more rapid reactions,
such as whey protein denaturation and calcium
phosphate precipitation, are complete within 5
min at 14O"C. Changes in the latter reaction
with pH are the most likely candidates for
increasing heat stability in the Miller and Som-
mer scenario (40) along the lines Home (22)
previously explored for ethanol stability.
Coagulation times are generally longer than 5
min, however, and during the prolonged heat-
ing prior to precipitation, all of the reactions
detailed progress to a greater or lesser extent.
Each reaction, of course, is influenced by
changes in milk composition, although in many
instances detailed studies are lacking. We still
await an all-encompassing mechanism for the
heat-induced destabilization of milk protein.

Type A Type B
35 3l!!

30 30

C
.rf 25 2!5
E

20 20
Gl
E
.rf
t- 15 1!5

01
III 10 10
0
U
5

0 0
6.3 6.6 6.7 6.9 7.1 7.3 15.3 B.e e.7 e.1i 7.1 7.3

pH pH
Figure 5. Typical Type A llDd Type B coagulation time to pH beat stability curves.

Journal of Dairy Science Vol. 73, No. 12, 1990

 3620 HORNE AND MUIR
TABLE 2. Methods of interchanging form of heat stability profile.
Conversion
Type A is couverted to Type B when
Assay temperature is decreased from 150 to 120·C
lC-Casein is added
Colloidal calcium phosphate is removed
Phosphate is replaced by aDO~ anion
Type A milk is dialyzed against excess Type B milk
Type B is converted to Type A when
Assay temperature is rai8cd from 130 to 150·C
The milk is forewanned at SO·C for 30 min
j:l-Lactoglobulin is added
Divalent cations (ea2+, ~+, or both) arc added
Type B milk is dialyzed against excess Type A milk
Type A Coagulation TIme
to pH Curves
Rather than review each individual reaction
and attempt to assess its contribution to the
overall integrated coagulation process, discus-
sion will concentrate on the occurrence of the
minimum in the Type A curve. A few more
facts are first considered
The frequency of occurrence of the two
types of profile varies widely. In Scotland. all
bulk milk i:l of Type A. and, for individual
cows, Type B curves are ObselVed in 1 to 3%
of all cases (17). Feagan et al. (10) and Fox and
Morrissey (14) report a similar or lower propor-
tion for milks in Australia and Ireland. respec-
tively, whereas Miyabe [quoted in Fox and
Morrissey (14)], found that in Japan approxi-
mately 70% of all bovine milk. was Type B in
character.
The measurement of coagulation time by
visual observation is a subjective test, the accu-
racy of which depends on the operator's ability
to detect the first signs of coagulation. White
and Davies (67) proposed the total nitrogen
depletion curve as an objective measure, the
amount of protein remaining in the supematant
on gentle centrifugation decreasing sharply at
the coagulation time.
Later, Parker and Dalgleish (50) showed that
such curves could be modeled by treating the
aggregating micelles as trifunctional units and
applying the theory of branching processes
originally derived to describe polymer conden-
sation and gelation. Such single-step depletion
curves were given by Type B milks or by Type
A milks outside (above or below) the coagula-
tion time minimum. Within the minimum, how-
Iournal of Daily Science Vol. 73, No. 12, 1990
Reference
(63)
(64)
(55)
(41)
(14, 41)
(63)
(63)
(64)
(41)
(14, 41)
ever, a two-stage, N depletion curve was ob-
served (63). The branching process model was
modified to reproduce this behavior: in this
region two distinct types of active particles
were assumed to exist, each of which could
react only with its own type with few or no
crossreaction products (51). The first drop in
the depletion curve is the observed visual
coagulation time. The second is visually un-
detectable. A theoretical model on which to
base heat coagulation studies is thus available.
A further point requiring explanation is the
observation that the coagulum first formed in
the minimum is soluble in 6 M urea, whereas
that formed outside the minimum in a Type A
milk or in a Type B milk is insoluble in this
dissociating medium (51). Again, this points to
the occurrence of two reactions in the region of
the cr minimum.
Milk showing Type A characteristics can be
converted into milk. showing a Type B profile,
or vice versa. Table 2 is a list of some ways
this can be done. Some of these are controver-
sial in that not every researcher has found them
to be operative in every milk on every occa-
sion. Among this group is the effect seen on
raising assay temperature. Sweetsur and White
(63) found that most milks were Type B at
130·C but assumed Type A characteristics with
increasing temperature, whereas Fox and Hearn
(13) were unable to confirm that assay tempera-
ture had any major influence on the shape of
the heat stability curve. Conversion from Type
B to Type A on raising temperature is easily
explainable as a consequence of the occurrence
of two reactions with different activation ener-
gies. The first reaction in the minimum has to
 BORDEN SYMPOSIUM: STABILITY OF PROTEINS IN DAIRY FOODS
have the higher activation energy, and it is, in
fact, observed to have an activation energy 30
to 40 kllmol higher than that for the coagula-
tion of micelles at the pH of maximum stability
(44). However, reaction mtes are also concen-
tration dependent, and failure to observe the
temperature effect on every occasion is proba-
bly associated with compositional differences
affecting the availability of reactants for the
two pathways. Genemlly, researchers agree that
the presence of the minimum in a Type A curve
is associated with the formation of a complex
between 13-lactoglobulin and lC-easein. Many
Type B milks are low in lactoglobulin and can
be converted to Type A by the addition of this
whey protein (10, 64). The lC-easein partner,
however, must be micellar. Addition of free lC-
casein to the milk. allows the complex forma-
tion to take place in the serum phase, and a
Type B curve results (64). Similarly, forcing
the dissociation of lC-casein from the micelle
into the serum phase by the addition of NaCI
produces a Type B profile (64).
Although 13-lactoglobulin is the principal
whey protein, other whey proteins such as a-
lactalbumin and bovine serum albumin can
destabilize milk. subjected to heating. The effect
of a-lactalbumin is similar to that of P-lac-
toglobulin, whereas addition of bovine serum
albumin results in a more general destabiliza-
tion (13).
If we postulate an equilibrium between se-
rom and micellar lC-easein mediated by calcium
ion activity, we can mtionalize a few more of
the observations in Table 2. For example, if a
Type B milk. were low in calcium, then higher
levels of calcium favoring the binding of lC-
casein to the micelle would induce a conversion
to Type A. The behavior of dialyzed milks is
also explicable on the basis of calcium move-
ment, if the Type A milk. has the higher free
calcium level, which transfers to the Type B,
and induces conversion. As to the effect of
colloidal calcium phosphate depletion, this
could be considered as a buffer regulating the
amount of free calcium ion. Removal of this
sink or of the phosphate anion itself reduces the
amount of bound calcium, increases micellar
charge and stability, and results in a Type B
profile. Much of this last paragmph is highly
speculative. Many of the observations, which
are by no means as clear cut as in the ethanol
stability situation, could equally be explained
3621
by postulating an influence of calcium ion on
the actual formation reaction for the protein
complex.
The evidence seems incontrovertible that the
formation of the l3-lactoglobulin and lC-casein
complex is the switch that triggers entry into
the minimum in the Type A cure. Before 1985,
researchers commonly accepted that this sur-
face coating of l3-lactoglobulin/lC-casein com-
plex sensitized the micelle to coagulation, at-
tributed by Morrissey (41) to heat-induced
precipitation of calcium phosphate. The in-
volvement of calcium phosphate in this fashion
seems unlikely in view of the decreasing stabil-
ity of calcium phosphate over the entire pH
range at which milk. is genemlly increasing in
stability.
In 1985, Singh and Fox (57) published the
first of a series of papers that confirmed the
earlier work of Creamer and Matheson (5), who
found that the lC-casein and j3-lactoglobulin
complex dissociates from the micelle above pH
6.9. Concurring with the earlier view of Kudo
(35), Singh and Fox suggested that the denuded
micelle is calcium-sensitive and that the origin
of the Type A minimum thus lies in the actual
dissociation of the complex from the micelle.
At pH greater than 7, heat stability increases
once more, presumably because of an increased
micellar negative charge, and also, we would
suggest, decreased availability of ionic calcium.
A comparison of the pH dependence of the
Singh and Fox data (56) for complex release
with any of their Type A heat stability curves
shows the following features. The plot for N-
acetyl neuraminic acid, an indicator of lC-casein,
parallels that for total nonsedimentable nitro-
gen, both horizontal to the pH axis until pH 6.8
and rising thereafter at higher pH. The Type A
coagulation time profiles of Singh and Fox (56,
57) are maximal by pH 6.7, well into the
minimum by pH 6.8, and minimal at pH 6.9 to
7.0 before rising again. Therefore, the micelles
exhibit marked levels of sensitization and insta-
bility before any signs of dissociation are dis-
cernible. This lack of alignment in pH depen-
dence raises doubts as to the validity of the
mechanism proposed by Singh and Fox (57).
More convincing, if largely circumstantial, evi-
dence favors the original idea of the micellar-
bound complex sensitizing the milk protein to
coagulation. This involves accommodating into
the mechanistic framework the observations re-
10urnal of Dairy Science Vol. 73, No. 12, 1990
3622 HORNE AND MUlR

garding the influence of milk urea levels on cyanate-sulfhydryl reaction offers a second
heat stability. pathway to coagulation, one that prevents the
Urea plays a marked role in increasing the lC-easein and P-Iactoglobulin reaction, the route
heat stability of milk when a single-stage to premature coagulation in the Type A mini-
coagulation occurs, as in a Type B milk or in a mum.
Type A milk. at pH values outside the mini- To summarize, urea and P-lactoglobulin pro-
mum, but is less effective when two-stage vide two separate and mutually exclusive reac-
coagulation dominates, as within the minimum tion partners for the two sulfhydryl groups of
of a Type A profile (43). lC-casein. Outside the minimum, the cyanate-
Because the natural pH of milk usually falls sulfhydryl reaction must be favored, because
outside the minimum and generally corresponds heat stability here correlates so well with urea
to that of the maximum in a Type A profile for level. Inside the minimum, the P-lactoglobulin-
most of the year in Scotland, Holt et aI. (19) lC-casein reaction is dominant and the balance
were able to demonstrate a very highly signifi- can be shifted only by greatly increasing the
cant correlation between the natural coagulation urea level. Something must happen near pH 6.8
time of farm and creamery bulk milk with the to enhance the P-lactoglobulin and lC-casein
urea content of milk. Subsequent studies in reaction. The most obvious candidate is the
Ireland (34) confinned these observations; 89% known and extensively studied dissociation and
of the seasonal variance in natural coagulation conformation change in the p-lactoglobulin.
time was accounted for by variations in urea Recent results (33) indicate that although the
level. Clear evidence shows that the urea level thiol group is on the solvent accessible surface
in milk is controlled by feeding practice (32). of the P-lactoglobulin monomer, it is buried in
In two separate serles of experiments, the direct the dimer, the solution state below pH 6.8. In
link between diet, blood urea level, milk urea our view, this increase in accessibility and reac-
concentration, and heat coagulation time was tivity following dissociation, with the conse-
clearly established (2). Urea level, the single quent increase in complex formation, induces
most important factor in controlling heat stabil- instability above pH 6.8 and thereby creates the
ity at the natural pH of milk, is subject to minimum in the Type A heat stability profile.
dietary manipulation and control. However, the This is the source of the instability mther than
coagulation time within the minimum of a Type the dissociation of the complex from the
A heat stability profile can only be increased by micelle.
the addition of very large amounts of urea (44). As to why the coated micelle should be
Fox et al. (15) have suggested that urea acts unstable, much has been said in recent years
simply by the buffering action of its reaction concerning the presence of a sterlc stabilization
products produced on heating. O'Reilly and component in the repulsive intermicellar poten-
Kelly (48), using 14c-labeled urea, found that a tial. Electrostatic repulsion due to micellar
significant proportion of the radioactive label charge has never been considered sufficient in
was incorporated into the protein after heating, itself (52). The external coat of lC-casein has
the proportion decreasing if the milk was pre- been assigned the role of providing this
treated with formaldehyde or N-ethyl malei- component-the so-called "hairy" micelle mod-
mide. Manson and Annan (38), discussing the el, proposed independently by Holt (18) and
chemistry of heated milk, focused attention on Walstm (65). The loss of this sterlc stabiliza-
the potential reaction between sulfhydryl tion component has been demonstrated as a
groups and cyanate, a rearrangement product of preliminary step in both rennet coagulation,
urea produced on heating. Such a reaction during which the lC-easein hairs are shaved by
would be wholly consistent with the observed the enzyme (66), and in ethanol-induced precip-
heat stability phenomena. First, it accounts for itation, during which the hairs are collapsed or
the chemical incorpomtion of radioactive label combed (20, 21, 23). The subsequent coagula-
after heating. Second, the enhancement of reac- tion reactions are still influenced by the miner-
tivity at high temperatures is accommodated, al, particularly calcium, content of the milk
because native P-Iactoglobulin has one free because of the remaining electrostatic potential.
sulfhydryl group and only after heating are the In the case of heat stability, the possibility
other sulfhydryl groups exposed. Third, the arises that the intemction of lC-casein with 13-

Journal of Dairy Science Vol. 73. No. 12. 1990

 BORDEN SYMPOSIUM: STABILITY OF PROTEINS IN DAIRY FOODS
lactoglobulin could make the 1(~ins hairs
rigid, a crosslinldng reaction that would neu-
tralize their steric stabilizing activity and render
the micelles prone to a calcium-moderated pre-
cipitation.
Although this theory has the advantage of
providing a unifying theme for the mechanism
of micellar destabilization, however this is in-
duced, it is somewhat speculative. Conclusive
evidence as to what is actually taking place in
heat coagulation over this narrow range of pH
will require in-depth study of the physical state
of the micelle, together with full determination
of the nitrogen depletion curves and quantita-
tive measurement of partitioning of all protein
components between serum and micellar
phases. The rewards are great. Not only will
micellar heat stability be explained, but the
knowledge gained will also assist in overcom-
ing problems associated with the incorporation
of whey proteins into cheese and in the manu-
facture of yogurt, in which heat treatment is
applied without full understanding of its
benefits.
CASE STUDY: CONCENTRATED MILKS
Research into the heat stability of milk was
undertaken with the aim of predicting the abil-
ity of evaporated concentrates to withstand in-
can sterilization. The initial failure to fulfill this
goal led to the dairy industry's adoption of ad
hoc empirical procedures to overcome prob-
lems in the manufacture of these products.
Forewarming, homogenization, and addition of
permitted additives, such as citrate or poly-
phosphates, all became part of the annory of
fire-fighting techniques to improve stability. So
successful were these empirical solutions that
comparatively little research has been carried
out on evaporated milks in recent times, a
situation exacerbated by a declining market for
most products. A recent exception to this
decline has been a growing demand for a full-
cream product for export from the United King-
dom for sale in warmer climates. The necessity
for countering a more severe temperature regi-
men and the extension of the shelf-life require-
ment from 1 to 2 yr has provided the impetus
for recent studies on concentrated milk
products.
As skim milk is concentrated, progressive
changes occur in the nature of the heat stability
3623
to pH profile (45). The minimum of the curve
deepens with increasing concentration until, at
concentrations greater than about 17% total
solids, the milk becomes highly unstable when
heated at pH values greater than 7.0. With
further concentration, the maximum coagula-
tion time, which occurs at about pH 6.6, contin-
ues to decrease. In addition, the stabilizing
action wrought by increasing the urea level has
progressively less effect. Muir and Sweetsur
(45) also found that even at the maximum of
the heat stability profile, coagulation takes
place in a two-stage process, i.e., in the same
manner as in the minimum of a Type A prome
from an unconcentrated milk.
The major influence of whey proteins on the
pH dependence of unconcentrated milk has
been detailed in the preceding section. Muir
and Sweetsur (45) demonstrated that concen-
trates prepared with a high level of 13-lac-
toglobulin were significantly less stable than
those with lower amounts of the whey protein
and that this destabilization took place over the
entire pH range. They also showed that addition
of 1(~ein increased the heat stability of the
concentrates. Additional confinnation regarding
the influence of whey proteins comes from the
work of Newstead et al. (46), who found that
selective reduction of whey protein content by
ultrafJJ.tration increased heat stability, whereas
increasing whey protein content was very
detrimental to heat stability.
The influence of the mineral content of milk
on the heat stability of concentrated milk has
long been recognized (58, 59), although the
actual mechanism is still unclear. Recent
studies of Hardy et al. (16) disclosed new evi-
dence to suggest that mineral equilibria in
evaporated milk probably act on heat stability
through differences in the concentration of
soluble calcium. During processing of milk,
they found a progressive transfer of both cal-
cium and phosphate from the soluble to the
colloidal phase. The effect of adding phosphate
prior to forewarming led to further progressive
reductions in soluble calcium and indicated a
possible cause of the stabilizing action of this
additive. Such results are consistent with many
reports that addition of calcium destabilizes
concentrated milk (45, 61) and with previous
observations that, over an entire season, the
heat stability of concentrated skim milk is sig-
nificantly correlated with soluble calcium in
Journal of Dairy Science Vol. 73, .No. 12, 1990
3624 HORNE AND MUIR.
raw milk (61). Nieuwenhuijse et al. (47), ex-
tending the study of calcium and phosphate
partition in concentrated milk to include the
influence of pH variation, demonstrated a
monotonic decrease in calcium ion activity,
ultrafilterable calcium, and inorganic phosphate
with pH. This led them to conclude that the
typical heat stability profile with its maximum
cannot be related to the variation of any of
these parameters with pH, although Ca2+ activ-
ity appears to be an essential factor for the heat
stability of concentrated milk at pH values be-
low the maximum.
In summary, an extensive body of evidence
can be interpreted to show that as milk is
concentrated, premature coagulation-of the
kind found in the Type A heat stability profile
for unconcentrated milk-becomes predominant.
Soluble calcium then becomes one determinant
of heat stability and. with other factors con-
stant, may become dominant.
Milk can be both selectively concentrated
and concomitantly reduced in soluble calcium
by ultrafiltration. A significant improvement in
heat stability is obtained and products concen-
trated by a factor of 5 can be canned and
sterilized using traditional methods (60). The
protein:carbohydrate ratio can be restored to
that of the original milk by the addition of
reducing sugars without significant loss of sta-
bility (62). This methodology opens a route to a
completely new generation of sterile milk
products with stability ensured by control of
calcium ion activity.
A further significant advance has been made
in the production of homogenized products.
Without adequate homogenization, creaming of
milk fat occurs on storage. Homogenization
usually results in a marked decrease of initial
heat stability. Studies of Hardy et aI. (17) ex-
tended the original work of Maxcy and Som-
mer (39) and Leviton and Pallansch (37) to
demonstrate that addition of low concentrations
of lecithin (.2% wt/wt) can compensate for this
decrease in stability. Hardy et al. (17) also
demonstrated that the use of lecithin completely
or partially eliminated the need for phosphate
inclusion to ensure stability during sterilization.
Lecithin incorporation also permitted the appli-
cation of higher than usual homogenization
pressures with a consequent improvement in
emulsion fineness and inhibition of creaming.
Further studies in pursuit of the mechanism of
Joumal of Dairy SciCIICC Vol 73, No. 12. 1990
its operation are still underway. Preliminary
results using model milks prepared from skim
milk powder and anhydrous milk fat indicate
that 1) the phospholipids bind to the fat parti-
cles but do not displace protein already on the
fat surface, and 2) from experiments using iso-
lated soya lecithin fractions, acidic phospholi-
pids are responsible for the increase in heat
stability (McCrae-Homsma and Muir, unpub-
lished).
CONCLUSIONS
This paper has attempted to summarize the
present state of our knowledge of ethanol sta-
bility and some related aspects of the heat
stability of milk protein. Considerable advances
have been made in our understanding of the
factors controlling ethanol stability, particularly
of the central role played by ionic calcium. The
mechanism postulated for ethanol-induced
coagulation is dominated by physical interac-
tions. In this respect, ethanol and heat stability
tests differ fundamentally because the
prolonged heating of the heat stability test al-
lows a multitude of concurrent chemical reac-
tions to occur, some undoubtedly advantageous,
as with urea, but others, as with whey proteins,
deleterious. Such reactions proceed at tempera-
ture-dependent rates, and, if curtailed, pennit a
behavioral similarity to ethanol stability to be
achieved.
We have also tried to provide evidence of
the validity of the postulate that the calcium
sensitivity of the P-lactoglobulin and K-casein
complex is central to heat-induced coagulation
and frequently becomes dominant in concen-
trated systems. This suggests that the ultimate
coagulation pathways of the two routes to
destabilization may be closer than we presently
realize. Further research is needed before a
complete unified theory of milk protein stabil-
ity can be fully developed.
In the meantime, the newfound understand-
ing of the ethanol-induced destabilization of
milk protein has pennitted the formulation of a
novel, high value product, the cream liqueur.
This emulsion of cream, alcohol, and sugar is a
prime example of product design technology.
Equally, the advancing knowledge of heat sta-
bility is pointing the way toward greater control
of stability in milk concentrates, providing
scope for new or improved products.
 BORDEN SYMPOSIUM: STABILITY OF PR.oTBINS IN DAIRY FOODS
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