Food Chemistry 122 (2010) 10271034

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Effects of stabiliser addition and in-container sterilisation on selected properties

of milk related to casein micelle stability
A. Tsioulpas, A. Koliandris, A.S. Grandison, M.J. Lewis *
School of Chemistry, Food and Pharmacy, The University of Reading, Whiteknights, P.O. Box 226, Reading RG6 6AP, UK

a r t i c l e

i n f o

Article history:
Received 9 August 2009
Received in revised form 1 February 2010
Accepted 15 March 2010

Keywords:
pH
Ionic calcium
Heat stability
Phosphate
Citrate
Casein micelle stability

a b s t r a c t
Different stabilising salts and calcium chloride were added to raw milk to evaluate changes in pH, ionic
calcium, ethanol stability, casein micelle size and zeta potential. These milk samples were then sterilised
at 121 C for 15 min and stored for 6 months to determine how these properties changed. Addition of trisodium citrate (TSC) and di-sodium hydrogen phosphate (DSHP) to milk reduced ionic calcium, increased
pH and increased ethanol stability in a concentration-dependent fashion. There was relatively little
change in casein micelle size and a slight decrease in zeta potential. Sodium hexametaphosphate (SHMP)
also reduced ionic calcium considerably, but its effect on pH was less noticeable. In contrast, sodium
dihydrogen phosphate (SDHP) reduced pH but had little effect on ionic calcium. In-container sterilisation
of these samples reduced pH, increased ethanol stability and increased casein micelle size, but had variable effects on ionic calcium; for DSHP and SDHP, ionic calcium decreased after sterilisation but, for
SHMP, it remained little changed or increased. Milk containing 3.2 mM SHMP and more than 4.5 mM
CaCl2 coagulated upon sterilisation. All other samples were stable but there were differences in browning,
which increased in intensity as milk pH increased. Heat-induced sediment was not directly related to
ionic calcium concentration, so reducing ionic calcium was not the only consideration in terms of improving heat stability. After 6 months of storage, the most acceptable product, in appearance, was that containing SDHP, as this minimised browning during sterilisation and further development of browning
during storage.
2010 Published by Elsevier Ltd.

1. Introduction
Phosphate salts or tri-sodium citrate are frequently added to
prevent coagulation during the production of evaporated milk.
Traditionally, the amount to be added is determined experimentally, by sterilising a few cans with different amounts of salts
under the conditions employed (Cronshaw, 1947). This procedure is still practised today. Most studies on heat stability have
been conducted by measuring heat coagulation time. However,
the correlation between heat coagulation time (measured by a
subjective assay) and stabilisation of milk toward commercial
sterilisation is often poor (OConnell & Fox, 2003). It appears that
pilot plant, or laboratory methods, which simulate commercial
sterilisation conditions, are better predictors of stability of milk
to commercial sterilisation, although studies in this area have
been much fewer.

* Correspondence to: M.J. Lewis, Dept. of Food and Nutritional Sciences, University of Reading, Whiteknights, PO Box 226, Reading RG6 6AP, UK. Tel.: +44 118 378
8721.
E-mail address: m.j.lewis@reading.ac.uk (M.J. Lewis).
0308-8146/$ - see front matter 2010 Published by Elsevier Ltd.
doi:10.1016/j.foodchem.2010.03.063

In terms of the effects of salts on heat stability, OConnell and

Fox (2003) concluded that, although articial alterations in the
concentrations of various salts affect the heat stability of milk, natural variations in milk salts are likely to be too small to be significant, especially in bulk milk.
Sweetsur and Muir (1980) proposed that di-sodium hydrogen
phosphate (DSHP), trisodium phosphate, tri-sodium citrate (TSC)
and sodium bicarbonate should be used if the natural pH falls on
the acid side of the heat stability maximum, while sodium dihydrogen phosphate (SDHP) or calcium chloride should be used if
the natural pH is alkaline to the maximum, with orthophosphates
being generally the most effective stabilisers.
Fox (1985) reported that addition of orthophosphates prevents
gelation of milk products, whilst chelating calcium ions. Sodium
hexametaphosphate also causes a decrease of the calcium ions,
whereas it leaves pH almost unaffected. Mittal, Hourigan, and Zadow (1990) evaluated SHMP addition to UHT recombined milk
and found that this was effective at retarding gelation. No sediment was found in UHT milk, either with or without SHMP. Montilla and Calvo (1997) observed that a mixture of NaH2PO4,
Na2HPO4 and Na3PO4 did not change the pH of raw goats milk
and increased its heat stability. Use of mixed phosphates was

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A. Tsioulpas et al. / Food Chemistry 122 (2010) 10271034

found to be more effective than was pH adjustment by means of

NaOH. Overall, no stabiliser emerged as being the most effective.
One probable reason for this arose from seasonal variations in milk
composition (especially pH) and times of the year when milk is
naturally more unstable.
Udabage, McKinnon, and Augustin (2000) found that addition of
calcium and mixtures of SDHP and DSHP increased micellar calcium phosphate and casein, whereas addition of citrate and EDTA
had opposite effects. All these stabilisers were reported to reduce
ionic calcium activity, but no effects on heat stability or ethanol
stability were investigated. Philippe, Gaucheron, Le Graet, Michel,
and Garem (2003) investigated the addition of calcium chloride
(4.513.5 mM) and found that it caused about 80% of the calcium
to be associated with the micelle, together with an increase in inorganic phosphate and citrate in the micelle. Other changes were a
decrease in soluble casein, increase in milk protein hydrophobicity,
and decrease in zeta potential, without any change in hydrodynamic diameter. The effects of these changes on initiating heat-induced sediment formation were not investigated.
Stabilisers have also been shown to be effective at reducing sediment formation in UHT treatment, especially for goats milk
(Boumpa, Tsioulpas, Grandison, & Lewis, 2008; Zadow, Hardham,
Kocak, & Mayes, 1983), and for reducing age-onset gelation in
UHT milk (Zadow & Hardham, 1981).
This paper describes, in more detail, the effects of addition of selected phosphates and citrate to raw skim milk and subsequent
sterilisation and storage properties, such as pH, ionic calcium, average casein micelle size, zeta potential, ethanol stability and heat
stability. In this respect, heat stability refers primarily to coagulation and sediment formation, arising either during processing or
subsequent storage.
2. Materials and methods
2.1. Materials
Raw bovine milk was supplied from the Centre for Dairy Research (CEDAR), University of Reading. It was skimmed using a
centrifuge (3000g) prior to addition of stabilisers and sterilisation.
The following stabilisers were used: tri-sodium citrate (TSC), disodium hydrogen orthophosphate 2-hydrate (DSHP), sodium dihydrogen orthophosphate (SDHP), calcium chloride 2-hydrate (all
AnalaR from BDH, Poole, UK): and sodium hexametaphosphate
(SHMP) (Fisher Scientic, Loughborough).
2.2. Addition of stabilisers to raw milk
To 100 ml of skimmed milk samples, 0.05%, 0.10%, 0.15% and
0.20% (w/v) of the stabilisers were added, at 20 C. For each of
the salts, 0.2% addition corresponds to: DSHP (12.8 mM); SDHP
(16.8 mM); SHMP (3.2 mM); TSC (8.0 mM); CaCl2 (18.0 mM).
Measurements were carried out for zeta potential, average casein micelle size, pH, ionic calcium and ethanol stability.
For canning experiments, the stabilisers were added at the same
levels to 120 ml of skimmed milk, at 20 C. The milk was placed in
baby food cans (52  72 mm), sealed and sterilised at 121 C for
15 min. From each concentration and from each stabiliser, six cans
were produced. Eight more cans were produced, which contained
skimmed milk without addition of any stabiliser to act as control.
The cans were opened on day 1, day 14, day 58 and after 6 months.
On each day the samples were subjected to zeta-potential measurements, casein micelle size, ethanol stability test, pH and ionic
calcium measurements. Sediment was also measured on a dry
weight basis by centrifugation at 2800g for 30 min (Boumpa
et al., 2008). Browning was assessed visually.

2.3. Analytical methods

The concentration of ionic calcium was determined indirectly
by a Ciba Corning Ca++/pH analyser, which measures the potential
difference between a series of standard calcium solutions and that
of milk (Lin, Lewis, & Grandison, 2006). A standard calibration
curve was prepared, using ve standard solutions of ionic calcium
(0.53.0 mM), and the ionic calcium in the milk was calculated.
The measurements were done in duplicate at 20 C.
Ethanol stability was determined by mixing equal volumes
(1 ml) of milk and ethanol of various concentrations in a test tube.
The maximum concentration, where the milk sample did not exhibit occulation, was recorded as the ethanol stability of the particular milk sample. When the milk was stable at 100% of ethanol
concentration, then the milk volume was kept constant and the
ethanol volume was increased, i.e. 1 ml of milk and 1.1 ml of ethanol, giving a recorded value of 110% ethanol stability. The ethanol
stability test was carried out at 20 C.
pH was measured with a digital pH meter (Model 8520, Hanna,
Portugal). Before carrying out the pH measurements, the pH meter
was adjusted by using freshly prepared pH 7.0 and 4.0 buffer
solutions.
Zeta potential of casein micelles was measured by a Zeta Master
(Malvern Instruments, Malvern, UK). Samples of 40 ll milk were
diluted to 3 ml with distilled water. Then 2.5 ml of the diluted milk
were injected into the Zeta Master using a plastic syringe. The
measurements were performed in duplicate for all samples. Furthermore, the Zeta Master automatically performed the zeta potential measurements in triplicate for each sample.
Average casein micelle size was also measured with the Zeta Master. In a plastic cuvette, 40 ll of milk sample were diluted to 3 ml
with deionised water. The measurements were done in duplicate
for all milk samples. Furthermore, for each of the two duplicates,
the Zeta Master performed 10 sub-measurements and the output
number was a mean average of the 10 measurements. Only average
casein micelle sizes are reported. Even though micelle size distributions can be determined, these results have to be interpreted with
caution, as the samples had to be diluted. Both casein micelle size
and zeta-potential measurements were performed at 20 C.

3. Results
3.1. Addition of phosphates, citrates and calcium chloride to raw milk
Table 1 shows the effects of addition of three different phosphate salts and TSC on some different physicochemical properties,
for two different batches of raw skimmed bovine milk. Overall,
their additions had little effect on the average casein micelle size.
Adding either SHMP or DSHP caused a decrease of ionic calcium
in a concentration-dependent fashion whereas, for SDHP addition,
it was little changed. DSHP addition increased pH, increased ethanol stability and reduced ionic calcium, and zeta potential became
slightly more negative. TSC showed effects similar to DSHP in
terms of ionic calcium, pH and ethanol stability. This is interesting
since citrate addition has been found to promote transfer of calcium and some soluble casein from the micelle, whereas phosphate addition shows the opposite effect (Udabage et al., 2000).
SHMP increased micelle size slightly, gave large reductions in ionic
calcium, but only increased pH slightly. However, addition of
3.2 mM SHMP destabilised the micelles for one of the two batches,
resulting from a large reduction in ionic calcium.
Addition of CaCl2 increased ionic calcium, had no effect on micelle size, decreased pH and ethanol stability and made zeta potential more positive. These results are in agreement with those of
Philippe et al. (2003).

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A. Tsioulpas et al. / Food Chemistry 122 (2010) 10271034

Table 1
Effect of the addition of various stabilisers on average casein micelle size, zeta potential, ionic calcium, pH and ethanol stability of raw milk before and one day after sterilisation.
Results after sterilisation are shown in parentheses.
Sample, mM

Average casein micelle size (nm)*

Raw milk

202
192
201
195
202
197
201
197
204
200
199
187
196
189
204
194
200
192
206
202
202
222
219
237
226
249
198
194
199
198
206
204
212
211

DSHP, 3.2
DSHP, 6.4
DSHP, 9.6
DSHP, 12.8
SDHP, 4.2
SDHP, 8.4
SDHP, 12.6
SDHP, 16.8
SHMP, 0.8
SHMP, 1.6
SHMP, 2.4
SHMP, 3.2
TSC, 2.0
TSC, 4.0
TSC, 6.0
TSC, 8.0

(289)
(306)
(303)
(315)
(305)
(317)
(311)
(330)
(346)
(377)
(304)
(319)
(308)
(327)
(321)
(344)
(341)
(373)
(317)
(329)
(333)
(354)
(416)
(421)
(sc)
(3835)
(313)
(319)
(316)
(326)
(338)
(351)
(402)
(412)

Zeta potential (mV)*

27.7
27.1
29.1
26.5
28.5
27.1
29.6
27.1
30.5
27.6
27.5
26.4
26.7
25.9
28.8
25.9
27.7
25.8
28.2
29.4
29.4
30.6
31.5
31.4
27.9
32.9
28.0
28.8
29.4
28.7
31.5
28.1
30.4
29.8

( 31.7)
( 30.7)
( 32.4)
( 33.2)
( 33.0)
( 32.7)
( 33.5)
( 33.4)
( 34.0)
( 33.2)
( 32.1)
( 34.4)
( 32.8)
( 33.5)
( 31.9)
( 34.1)
( 31.9)
( 33.0)
( 33.6)
( 32.5)
( 34.4)
( 32.9)
( 33.5)
( 32.7)
(sc)
( 33.1)
( 32.7)
( 33.6)
( 34.1)
( 33.0)
( 33.2)
( 33.1)
( 34.1)
( 32.7)

Ionic calcium (mM)

pH

1.87
1.88
1.64
1.59
1.45
1.36
1.27
1.27
1.11
1.06
1.85
1.74
1.87
1.86
1.89
1.90
1.90
1.91
1.49
1.28
1.18
1.07
0.73
0.82
0.49
0.46
1.57
1.37
1.36
1.31
1.18
1.16
1.00
0.99

6.86
6.85
6.86
6.96
6.88
7.00
6.92
7.01
6.96
7.03
6.77
6.82
6.69
6.75
6.63
6.77
6.56
6.60
6.88
6.94
6.89
6.94
6.91
6.94
6.92
6.95
6.93
6.98
6.99
7.05
7.04
7.11
7.10
7.15

(1.78)
(1.63)
(1.53)
(1.45)
(1.31)
(1.26)
(1.07)
(1.06)
(0.81)
(0.88)
(1.68)
(1.59)
(1.58)
(1.50)
(1.43)
(1.40)
(1.24)
(1.23)
(1.24)
(1.39)
(1.24)
(1.10)
(0.95)
(0.82)
(sc)
(0.47)
(1.58)
(1.45)
(1.36)
(1.29)
(1.20)
(1.13)
(1.04)
(0.98)

Ethanol stability (%)

(6.62)
(6.57)
(6.67)
(6.60)
(6.67)
(6.56)
(6.65)
(6.56)
(6.66)
(6.70)
(6.55)
(6.56)
(6.44)
(6.46)
(6.34)
(6.37)
(6.27)
(6.27)
(6.60)
(6.52)
(6.53)
(6.46)
(6.48)
(6.37)
(6.40)
(6.32)
(6.71)
(6.58)
(6.75)
(6.62)
(6.78)
(6.71)
(6.81)
(6.77)

98 (140)
98 (140)
120 (150)
120 (150)
150 (160)
140 (150)
170 (180)
150 (150)
180 (190)
160 (170)
94 (140)
100 (130)
86 (130)
120 (130)
82 (120)
130 (120)
80 (110)
140 (94)
150 (150)
100 (140)
160 (150)
120 (125)
180 (140)
120 (125)
190 (sc)
60 (55)
140 (160)
110 (150)
160 (170)
140 (150)
180 (180)
170 (160)
180 (200)
190 (170)

CaCl2, 4.5

196 (383)
188 (362)

27.4 ( 27.2)
25.9 ( 27.7)

2.67 (2.29)
2.67 (2.19)

6.73 (6.54)
6.73 (6.48)

55 (84)
62 (76)

CaCl2, 9.0

191.(sc)
189 (sc)
199 (sc)
187 (sc)
199 (sc)
188 (sc)

25.3
24.9
24.2
23.9
23.6
22.6

3.42
3.47
4.04
4.03
4.55
4.49

6.59
6.58
6.47
6.42
6.38
6.33

33
45
30
15
20
10

CaCl2, 13.5
CaCl2, 18.0

(sc)
(sc)
(sc)
(sc)
(sc)
(sc)

(sc)
(sc)
(sc)
(sc)
(sc)
(sc)

(6.36)
(6.37)
(6.24)
(6.33)
(6.13)
(6.22)

(sc)
(sc)
(sc)
(sc)
(sc)
(sc)

(sc) Sample coagulated.

Deionised water was used as the diluting medium.

SDHP addition had little effect on ionic calcium, decreased pH

slightly and produced inconsistent ethanol stability results; for
one batch, it increased whereas, in the second batch, it decreased
as SDHP concentration increased. For those compounds which decreased ionic calcium, the extent of reduction depended mainly on
calcium chelating capacity but to a lesser extent by the superimposed effect of increasing pH. Note that increasing pH of milk
has been found to decrease ionic calcium (Geerts, Bekhof, & Scherjon, 1983; Tsioulpas, Lewis, & Grandison, 2007). The changes produced for the two different batches of raw milk were
reproducible (Table 1). Apart from differences in ethanol stability
following SDHP addition and for addition of 3.2 mM SHMP. Thus
SHMP, TSC and, to a lesser extent, DSHP, are able to chelate ionic
calcium and substantially reduce ionic calcium concentration.
SDHP will also chelate calcium, but this is counteracted by an increase due to the resulting decrease in milk pH.
When data for all added compounds were collated, there was a
good relationship between ethanol stability and ionic calcium (Fig
1) and between ethanol stability and zeta potential (Fig 2). Thus,
reducing ionic calcium, increased the negative charge on the micelle, which in turn increased ethanol stability. Reducing ionic

calcium below about 1.5 mM slightly increased casein micelle size

(Fig 3).

3.2. Effects of heat treatment

The effects of in-container sterilisation are shown for two different batches of milk in Table 1 (values in parentheses). The main
trends were that heat treatment increased average casein micelle
size, decreased zeta potential (more negative), reduced ionic calcium, and in most cases, increased ethanol stability. Ethanol stability has rarely been reported in heat-treated milk products and it
will be inuenced by the pH, ionic calcium and amount of calcium
phosphate which precipitates on the surface of the micelle. However, changes in pH and ionic calcium were different for each of
the stabilisers. Also, some samples showed poor heat stability
and coagulated after sterilisation, namely those with more than
4.5 mM calcium chloride and one of the samples containing
3.2 mM SHMP. Coagulation was the main manifestation of poor
heat stability observed for in-container sterilisation. Some sediment was observed after 6 months of storage (Table 3), but this

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A. Tsioulpas et al. / Food Chemistry 122 (2010) 10271034

200
raw milk 1
raw milk 2

180
160

ethanol stability (%)

140
120
100
80
60
40
20
0
0

0.5

1.5

2.5

3.5

4.5

ionic calcium (mM)

Fig. 1. The relationship between ethanol stability and ionic calcium for two batches of raw milk, following stabiliser additions.

200
180

ethanol stability (%)

160
140
120

raw milk 1
raw milk 2

100
80
60
40
20
0
-35

-30

-25

-20

-15

zeta potential (mV)

Fig. 2. The relationship between ethanol stability and zeta potential for two batches of raw milk, following stabiliser additions.

700

casein micelle size (nm)

600

500

raw milk 1
raw milk 2
heated milk 1
heated milk 2

400

300

200

100

0
0

0.5

1.5

2.5

3.5

4.5

ionic calcium (mM)

Fig. 3. The relationship between casein micelle size and ionic calcium for raw milk and heated milk for stabiliser additions.

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A. Tsioulpas et al. / Food Chemistry 122 (2010) 10271034

Table 2
Effect of the addition of various stabilisers on average casein micelle size, zeta potential, ionic calcium, pH and ethanol stability of sterilised milk, one day and 6 months after
sterilisation (results in parentheses are after 6 months).
Sample, mM

Average casein micelle size* (nm)

Zeta potential* (mV)

Ionic calcium (mM)

pH

Ethanol stability** (%)

Raw milk
Control
DSHP, 3.2
DSHP, 6.4
DSHP, 9.6
DSHP, 12.8
SDHP, 4.2
SDHP, 8.4
SDHP, 12.6
SDHP, 16.8
SHMP, 0.8
SHMP, 1.6
SHMP, 2.4
SHMP, 3.2
TSC, 2.0
TSC, 4.0
TSC, 6.0
TSC, 8.0
CaCl, 4.5
CaCl, 9.0
CaCl, 13.5
CaCl, 18.0

194.7
306 (288.3)
315.0 (302)
317.5 (319)
330.3 (326)
377.4 (379)
318.6 (287)
326.9 (287)
344.3 (294)
373.9 (330)
328.2 (319)
354.2 (344)
420.7 (468)
3835.0
319.2 (308)
325.5 (333)
351.2 (366)
412.3 (422)
362.3 (369)
SC (SC)
SC (SC)
SC (SC)

29.45
30.65 ( 26.3)
33.15 ( 28.2)
32.65 ( 28.7)
33.45 ( 28.3)
33.2
34.35 ( 27.8)
33.5 ( 27.7)
34.15 ( 27.8)
33 ( 27.1)
32.45 ( 27.9)
32.9 ( 27.4)
32.65 ( 26.3)
33.1 (SC)
33.55 ( 26.9)
33 ( 27.9)
33.15 ( 26.8)
32.7 ( 26.0)
27.7
SC (SC)
SC (SC)
SC (SC)

1.91
1.63 (1.64)
1.45 (1.41)
1.26 (1.22)
1.06 (1.00)
0.88
1.59 (1.53)
1.50 (1.35)
1.40 (1.17)
1.23 (1.03)
1.39 (1.41)
1.10 (1.2)
0.82 (1.0)
0.47 (0.84)
1.45 (1.43)
1.29 (1.24)
1.13 (1.08)
0.98 (0.97)
2.19 (2.45)
SC (SC)
SC (SC)
SC (SC)

6.87
6.57 (6.40)
6.6 (6.39)
6.56 (6.40)
6.56 (6.39)
6.7
6.56 (6.29)
6.46 (6.20)
6.37 (6.11)
6.27 (6.04)
6.52 (6.40)
6.46 (6.37)
6.37 (6.26)
6.32
6.58 (6.41)
6.62 (6.45)
6.71 (6.50)
6.77 (6.53)
6.48 (6.27)
6.37
6.33
6.22

96
140
150
150
150
165
130
130
120
94
140
125
125
55
150
150
160
170
76
Sample clotted
Sample clotted
Sample clotted

(sc) Sample coagulated.

Deionised water was used as the diluting medium.
**
After 6 months of storage, all samples had ethanol stabilities above 100% except for 0.2% SDHP (96%) and 0.05% CaCl2 (94%).
*

was not considered to be an excessive amount, compared to that

observed in some UHT treatments (Boumpa et al., 2008).
After sterilisation, SDHP produced a big reduction in ionic calcium and pH, but the ethanol stability increased and exceeded
100%. Samples containing DSHP showed a slight reduction in ionic
calcium and pH. SHMP gave the greatest reduction in ionic calcium
and the sample with 3.2 mM addition coagulated. Also, it produced
the greatest reduction in pH following heat treatment and the largest increase in casein micelle size. TSC produced very little reduction in ionic calcium but caused zeta potential to become more
negative and this may be its chief means of micelle stabilisation.
Calcium chloride addition caused a considerable reduction in
heat stability; samples with 9.0 mM and higher concentrations of
calcium chloride coagulated, forming a thick rm gel. Another
noticeable change for all samples was the increase in micelle size
brought about by the sterilisation process (see Fig 3).
Ethanol stability increased as ionic calcium was reduced, up to a
certain point. For instance, milk with 3.2 mM SHMP produced the
greatest reduction in ionic calcium but an ethanol stability of only
60%. Zeta potential decreased as a result of heat treatment (comparison of raw against control) and further decreased with the
addition of the stabilisers.
Heat treatment caused considerable browning; differences
were observed immediately after heat treatment, but these became more noticeable after 6 months of storage.
3.3. Changes after 6 months of storage
Table 2 shows selected properties, 1 day and 6 months after sterilisation. Micelle size for DSHP and TSC addition was little changed over 6 months. For SDHP, micelle size was reduced. For
SHMP there was little change at low levels of addition but large increases at higher levels. Zeta potential became less negative for all
samples and pH was signicantly reduced for all samples. This
could have contributed to reduction in zeta potential. Ethanol stability was still over 100% in all samples, except for 16.8 mM SDHP
and 4.5 mM CaCl2.
Ionic calcium concentrations, for DSHP and TSC, were largely
unchanged throughout storage, but were reduced for SDHP and in-

Table 3
Sediment formation (%) after 6 months of storage (the control contained 1.3% db).
Concentration (%)

DSHP
% db

SDHP
% db

SHMP
% db

TSC
% db

CaCl2
% db

0.05
0.1
0.15
0.2

1.2
1.4
1.7
1.5

0.5
0.9
0.6
0.7

1.9
1.6
2.4
6.4

0.8
1.0
1.5
1.4

1.2
Sc
Sc
Sc

0.2% Addition corresponds to: DSHP (12.8 mM); SDHP (16.8 mM); SHMP (3.2 mM);
TSC (8.0 mM); CaCl2 (18.0 mM).
db dry weight basis.

creased for SHMP. Results for SDHP were interesting because they
produced the least browning and the least amount of sediment
(Table 3). Table 2 also shows that SDHP gave the smallest micelle
size of the stabilisers evaluated. In fact, SDHP was the only stabiliser to give a decreasing micelle size on storage, showing some
reversibility toward the increase in size induced by heat treatment.
Ionic calcium concentration also decreased during storage for
DSHP. However, SDHP was found not to be effective for reducing
sediment in UHT goats milk (Boumpa et al., 2008).
For DSHP addition, the micelles appeared not to change size
during storage: zeta potential became less negative, pH decreased
slightly, but ionic calcium decreased. After 6 months, the milks
were very brown, showing that considerable Maillard browning
had occurred.
TSC produced quite large micelles, considerable sediment, high
ethanol stability and little change in ionic calcium during storage.
Micelle size hardly changed during storage and samples were very
brown. TSC and DSHP showed similar characteristics in terms of
observed pH and ionic calcium changes during storage. Note that
changes in pH and ionic calcium are probably caused more by
Maillard reactions, rather than solubilisation of calcium phosphate
during storage.
SHMP caused an increase in micelle size during storage, which
was greater as concentration increased: zeta potential became less
negative and the ionic calcium increased slightly. A large amount
of sediment was found in the sample containing 3.2 mM SHMP.
This is interesting and suggests that reducing ionic calcium too

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A. Tsioulpas et al. / Food Chemistry 122 (2010) 10271034

much might result in a decrease in heat stability. This has also been
suggested by Boumpa et al. (2008), when using stabilisers to improve heat stability of UHT goats milk.
4. Discussion
Stabilisers used were added at the same concentrations, which
resulted in different molar additions and therefore made direct
comparisons less straightforward. SHMP required the smallest molar addition to be effective in reducing ionic calcium, although it
had no noticeable effect on pH. However, the molecule does contain six atoms of P. At about 3.2 mM addition, DSHP only reduced
ionic calcium by 13%, whereas SHMP reduced it by 73%. In comparison, SDHP did not reduce ionic calcium and also resulted in a
reduction in pH. TSC and DSHP showed almost similar effects,
overall, when compared in molar terms, with TSC being slightly
more effective at reducing ionic calcium.
The increases in casein micelle size, found after heating in this
study, were higher than those found by Anema and Li (2003),
who considered that whey proteins played a major role in this increase: due either to the association of whey proteins with the micelle surface or to the partial aggregation of whey protein-coated
casein micelles. An alternative explanation is that the smaller micelles were coagulating and hence increasing the average micelle
size. Ono, Yoshida, Tanaami, and Ohkosi (1999) produced micelles
of various sizes and found that the large micelles remained the
same and the size of medium and small micelles increased. In
our study, micelle size appears to be slightly inuenced by ionic
calcium, more so in heat-treated milks, where reducing ionic calcium resulted in an increase in micelle size. However, coagulation
occurred when ionic calcium was reduced too much, for example
when adding 3.2 mM SHMP, and also when it was increased too
much, as when calcium chloride was added. This larger micelle size
might also explain why more sediment was produced in milks with
lower ionic calcium levels. According to OConnell and Fox (2000),
heat stability is inversely proportional to the micelle size. Milk
with small micelles was found to be more heat-stable and did
not exhibit a minimum in the HCT-pH prole, supporting the theory that the minimum in the HCT-pH prole is due to premature
coagulation of big micelles. Furthermore the small micelles contained more j-casein and this may explain why they are more
heat-stable since they become less sensitive to Ca2+ induced precipitation. The fact that small micelles exhibit weaker van der

Waals attractive forces may also be important (Fox & McSweeny,

1998). However, Fox and McSweeny (1998) used different methods
to evaluate heat stability.
Zeta potential became more negative on addition of DSHP, SHMP
and TSC, probably due to a reduction in ionic calcium (Fig 4) and, for
DSHP and TSC, probably due to an increase in pH; this was accompanied by an increase in ethanol stability. This decrease in zeta potential may render casein micelles less sensitive to heat and calcium
ions. Darling and Dickson (1979) showed that heating milk at
135 C for 50 min left zeta potential almost unaffected. Dalgleish
(1984) showed that the zeta potential of native casein micelles
and Ca2+ concentration were inversely related and that, at higher
temperatures, the absolute value of the zeta potential was higher
for samples having the same Ca2+ concentration, Anema and
Klostermeyer (1996) found that reconstituted skim milk (10% TS),
when heated at 140 C at pH 6.5, showed an increase in the zeta potential at rst, which decreased with prolonged heating, whereas
heating at 80 or 100 C at the same pH, left zeta potential almost
the same. Heating the same sample at 140 C at pH 7.1 caused an
initial decrease in the zeta potential which then remained stable.
Citrate addition has been shown to increase soluble calcium,
whereas phosphate addition decreases it (Ozcan-Yilsay, Lee, Horne,
& Lucey, 2007; Tessier & Rose, 1958). Udabage et al., 2000 looked at
the effects of phosphates, citrate, EDTA and calcium addition on
the movement of casein and minerals into and out of micelles.
Addition of calcium and mixtures of SDHP and DSHP increased
micellar calcium phosphate and casein, whereas addition of citrate
and EDTA had opposite effects. All of these stabilisers were reported to reduce ionic calcium activity, but no effects on heat stability, ethanol stability or casein micelle size were investigated.
Calcium addition to milk is important in terms of calcium supplementation. Addition of calcium chloride to milk reduced pH, increased ionic calcium and decreased ethanol stability, which in
turn reduced its heat stability. Calcium carbonate addition has
been found to be useful in improving the heat stability of calcium-supplemented milk powder (Vyas & Tong, 2004), the explanation being that it could exert a buffering capacity. Measuring
ionic calcium routinely may help provide a better understanding
of the process. Stabilisers appear to be used in calcium-fortied
products, especially those subject to UHT treatment (De la Fuente,
Belloque, & Juarez, 2004).
Throughout storage, there were further changes in most of the
properties but no further samples coagulated. After 6 months, all

raw milk 1
raw milk 2
heated milk 1
heated milk 2

-5

zeta potential (mV)

-10

-15

-20

-25

-30

-35

-40
0

ionic calcium (mM)

Fig. 4. The relationship between zeta potential and ionic calcium for raw milk and heated milk for stabiliser additions.

1033

A. Tsioulpas et al. / Food Chemistry 122 (2010) 10271034

4.5

sediment (dry weight) %

4
3.5
3
2.5
2
1.5
1
0.5
0

0.5

1.5

2.5

ionic calcium (mM)

Fig. 5. The relationship between sediment (dry weight) and ionic calcium in sterilised milk after 6 months of storage .

samples showed some sediment, which is another manifestation of

micelle instability. The amount of sediment increased as ionic calcium in the milk prior to sterilisation decreased and correlated
quite well with casein micelle size (R2 = 0.76) (Figs. 5 and 6), which
is contrary to that observed when stabilisers were used to control
sediment formation in UHT goats milk (Boumpa et al., 2008). The
stabiliser which best controlled sediment was DHSP; this also gave
the least browning. However, this stabiliser was the least effective
at controlling sediment in UHT goats milk (Boumpa et al., 2008).
Thus heat stability mechanisms may be different during UHT and
in-container sterilisation. One important difference arises from
the higher temperature used in UHT, leading to the speculation
that heat stability is inuenced by the pH and ionic calcium concentration achieved at the sterilisation temperature, which in turn
is also inuenced by the buffering capacity of the milk. Salaun,
Mietton, and Gaucheron (2005) have reviewed the factors affecting
buffering capacity, but have not suggested any effect of buffering
capacity on milk stability. This is currently being further
investigated.
Heat stability of in-container sterilised milk, at its normal concentration, has been studied much less than has heat coagulation
time (HCT). Rose (1963) attributed this to the procedure being
cumbersome. However, correlations between HCT and other measurements of heat stability or compositional factors have not
been established. Also, HCT is determined at 140 C in unconcen-

trated milk, whereas sterilisation takes place at 115 to 120 C. The

milk used in this study showed good heat stability toward in-container sterilisation; only samples containing 3.2 mM SHMP and
more than 4.5 mM CaCl2 coagulated on heat treatment. Coagulation during sterilisation is more likely to occur in concentrated
milks.
Milk sterilisation resulted in a reduction in pH. During sterilisation, milk pH would decrease, probably reaching below 6.0 at the
sterilisation temperatures used here (Walstra & Jenness, 1984).
On cooling, pH will increase and, for HTST pasteurisation and
UHT treatment, it is virtually fully reversed. However, for in-container sterilisation, this is not the case. This is most probably
caused by degradation of lactose and by hydrogen ions liberated
during the Maillard reaction pathways. Also, calcium phosphate
will associate with the casein micelle, which in turn will also lower
the pH. Note that the fall in pH appeared to increase as the level of
stabiliser increased.
Heat treatment of milk has also been reported to reduce ionic
calcium, rst observed by Geerts et al. (1983). This partially recovers during subsequent storage. However, following in-container
sterilisation, ionic calcium was found to increase for some samples
and decrease for others. One explanation is that the lower pH
found in sterilised milk will in turn increase ionic calcium, which
may account for the increase in ionic calcium found in some
samples.

4.5

sediment formation (%)

4
3.5
3
2.5
2
1.5
1
0.5
0

100

200

300

400

500

600

700

casein micelle size (nm)

Fig. 6. The relationship between sediment formation and casein micelle size in sterilised milk after 6 months of storage.

1034

A. Tsioulpas et al. / Food Chemistry 122 (2010) 10271034

Stabiliser addition to milk, and effects on heat treatment, have

been shown to be complex. Adding stabilisers, such as phosphates
and citrates, for reducing problems caused by poor heat stability,
was recognised in the 1920s. Either DSHP or TSC addition was used
to prevent coagulation during the production of evaporated milk,
and the amount to be used was determined experimentally, by
sterilising a few tins with different amounts of salts under the conditions employed (Cronshaw, 1947). This procedure is still practised today. Routinely measuring ionic calcium and pH prior to
sterilisation might help to better understand the processes that
are taking place. Stabilisers which are effective are those which
bring about a moderate reduction in ionic calcium, without
increasing the pH unduly, as pH increase leads to an increase in
both the extent of browning during sterilisation and the further
development of browning during storage. For sterilised milks, combinations of DSHP and SDHP might be effective. One suggestion,
worthy of evaluation, is to use a combination that would not
change the natural pH of the milk, which can show considerable
variations.
For all compounds evaluated, there was a good correlation between ionic calcium and ethanol stability (Fig 1). Most of the stabilisers increase ethanol stability to values above 100%.
5. Conclusions
The effects of addition of different phosphates and citrates to
raw milk were evaluated, before and after in-container sterilisation
and during storage for 6 months. Properties such as pH, ionic calcium, average casein micelle size, zeta potential, ethanol stability
and heat stability were measured. In many respects, DSHP and
TSC showed similar effects, increasing pH and reducing ionic calcium. A negative effect of these two stabilisers was the excessive
browning during storage, caused by the higher pH.
SHMP was more variable in its effects, whereas SDHP decreased
pH and had little effect on ionic calcium. However, this caused
much less browning.
Good quality raw milk is unlikely to coagulate under normal
sterilisation conditions, provided ionic calcium is neither too high
nor too low. However, milk may be susceptible to some sediment
formation during storage, which is inuenced by ionic calcium
concentration. Thus, monitoring pH, ionic calcium and heat stability as part of a quality assurance programme could provide a better
understanding of their role and thereby improve product quality.
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