International Dairy Journal 33 (2013) 135e141

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International Dairy Journal

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The effect of calcium chloride addition on the microstructure and

composition of Cheddar cheese
Lydia Ong a, b, Raymond R. Dagastine a, Sandra E. Kentish a, Sally L. Gras a, b, *
a
Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia
b
The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria 3010, Australia

a r t i c l e i n f o a b s t r a c t

Article history: The addition of calcium is widely accepted as a tool in cheese-making but the effect on the micro-
Received 1 June 2012 structure of cheese during and following manufacture is not known. In this study, cheeses made with
Received in revised form milk containing 200e600 mg L
1 of additional CaCl2 had significantly lower fat loss into the whey
19 March 2013
collected after cooking; however, the final fat composition or yield of cheese did not change. The
Accepted 21 March 2013
microstructure of the gel with 300 or 600 mg L
1 CaCl2 addition was less porous and the cooked curd
consisted of a denser protein network that may retain more fat during the early stages of manufacture. In
contrast, the cheddared curd and cheese contained more micro-pores than cheeses with lower or no
calcium addition. Such micro-pores could possibly be the channels by which fat escaped during pressing.
This study shows that calcium addition altered the microstructure and pattern of fat loss during Cheddar
manufacture.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction milk at pH 6.6 is approximately 1.2 g L
1, of which approximately

Rennet-induced coagulation is the initial step in the manufac-
ture of most hard cheeses. This coagulation can be divided into
several stages. The primary stage involves the enzymatic hydrolysis
of k-casein by rennet, which reduces the surface potential and
steric repulsion between the casein micelles (CM), allowing the CM
to aggregate and form a three-dimensional space-filling gel.
The primary stage of coagulation is known to be independent of
calcium concentration; however, the addition of calcium reduces
the pH of the milk thus indirectly increasing the rate of the enzy-
matic reaction. In contrast, the secondary (non-enzymatic) stage of
coagulation is dependent on calcium concentration. The addition of
Ca2þ reduces the surface potential of the para-casein micelles. Ca2þ
ions bind to the casein micelles via electrostatic cross linking of the
phosphate moiety of the CCP and/or direct binding to the carboxylic
acid groups of a-caseins and b-caseins (Asp, Glu), thereby neu-
tralising their charge and resulting in increased aggregation of the
renneted micelles (Dalgleish, 1983)
A clear understanding of the role of calcium within this coagu-
lation process is made difficult by the complexity of forms in which
calcium is present in milk. The concentration of total calcium in
* Corresponding author. Tel.: þ61 3 8344 6281.
E-mail address: sgras@unimelb.edu.au (S.L. Gras).
68% is present in the micelle as colloidal calcium phosphate
(CCP, w0.8 g L
1 milk) (Lucey & Fox, 1993). The CCP is closely
associated with the phosphoserine residues in the casein proteins.
Removal of CCP will cause the dissociation of CM and inhibition of
gelation. For example, renneted milk did not gel when the CCP
content of the micelles was decreased by approximately 30% at
constant Ca2þ activity (Dalgleish & Law, 1989; Udabage, McKinnon,
& Augustin, 2001; Zoon, van Vliet, & Walstra, 1988).
The remaining calcium is present in the serum, partly as free
Ca2þ ions and partly complexed with other various anions and
proteins. The total concentration of soluble calcium in milk is
approximately 0.4 g L
1, which includes 0.08 g L
1 of ionic calcium.
This calcium must also be present for coagulation to occur (Martin,
Williams, & Dunstan, 2007). The equilibrium between the soluble
and insoluble calcium is well known to be dependent on the pH,
temperature and ionic strength (Dalgleish & Law, 1989; Visser,
Minihan, Smits, Tjan, & Heertje, 1986), which vary during the
cheese-making process.
Calcium chloride is usually added to cheese-milk during
cheese-making to assist coagulation, improve the cheese-making
process and/or increase the yield with the normal range of cal-
cium addition spanning 0e0.5 g L
1 CaCl2 (Gastaldi, Pellegrini,
Lagaude, & Fuente, 1994; Okigbo, Richardson, Brown, & Ernstrom,
1985; Ustunol & Hicks, 1990; Wolfschoon-Pombo, 1997). The
addition of 0.2 g L
1 CaCl2 (1.8 mM) to milk improved the

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136 L. Ong et al. / International Dairy Journal 33 (2013) 135e141
coagulation properties of poorly coagulated milk collected in late
lactation during winter (Okigbo et al., 1985). Similarly, the addition
of 0.2 g L
1 CaCl2 improved the coagulation of winter cheese-milk
clotted with Endothia parasitica protease and increased the dry
cheese yield by 0.7% (Ustunol & Hicks, 1990). The addition of
0.25 g L
1 Ca (6.25 mM Ca) to reconstituted skim milk has also been
shown to increase the concentration of both soluble and insoluble
calcium, leading to an increased aggregation rate, preserving the
micellar structure and forming a more easily drained curd
(Gastaldi et al., 1994). High concentrations of calcium, however,
can have a negative effect. For example, the addition of a low
concentration of calcium (0.1 g L
1 CaCl2; 0.9 mM) to milk
increased the yield of Emmentaler type cheese by 0.41% but adding
more than 0.3 g L
1 CaCl2 (3 mM) increased the firmness of the
cheese (Wolfschoon-Pombo, 1997).
The mechanisms by which added calcium chloride increases
cheese yield are still unclear. The addition of CaCl2 can increase the
firmness of the gel making it less susceptible to shattering but this
addition may result in a higher degree of syneresis and hence a curd
with lower moisture content (Wolfschoon-Pombo, 1997). The
addition of calcium was suggested to increase calcium bridging
between the casein micelles, facilitating more cross linking and the
entrapment of fat in the curd during the manufacture of stirred
curd cheese with E. parasitica protease (Ustunol & Hicks, 1990).
Despite this potential to improve cheese yield, no studies have
examined the microstructural changes that may occur in curd or
cheese following CaCl2 addition.
The objective of the present study was to examine the influence
of calcium chloride addition to cheese-milk on texture and
composition of the final Cheddar cheese product, particularly on fat
retention. A second objective was to investigate the changes in the
microstructure of the gel, curd and cheese samples collected during
the manufacture of full fat Cheddar cheese as affected by calcium
chloride addition.
2. Materials and methods
2.1. Determination of ripening and cutting time
The ripening time of the starter culture in the cheese-milk was
determined in separate experiments before cheese-making. The
ripening time is the time from the addition of starter bacteria and
CaCl2 to the addition of rennet. The cheese-milk is defined as milk
prepared for Cheddar cheese manufacture, where the milk was
standardised (fat content ¼ 45.0  0.2 g kg
1 and protein
content ¼ 38.9  0.1 g kg
1) by blending whole raw milk with raw
ultrafiltered milk (UF) retentate (2 fold) before pasteurisation at
72  C for 15 s. This milk was provided by Murray Goulburn
Co-Operative Co. Ltd., Cobram, Victoria, Australia.
Different masses of CaCl2 (0, 50, 100, 200, 300, 600 or 900 mg)
(Ajax Finechem, Taren Point, NSW, Australia) were added per every
litre of cheese-milk that had been inoculated with the starter cul-
ture (0.05 g L
1) (Chr. Hansen, Bayswater, Vic, Australia) at 33  C.
The pH of the milk was measured at 5 min intervals as described
previously (Ong, Dagastine, Kentish, & Gras, 2012). The time taken
for the cheese-milk to reach pH 6.5 was recorded and used as the
ripening time in cheese-making experiments.
The viscoelastic properties of the milk during gelation were
analysed before the cheese-making experiments, as reported in a
previous study (Ong et al., 2012).
2.2. Manufacture of Cheddar cheese
Six different Cheddar cheeses were prepared with the addition
of 0, 50, 100, 200, 300 or 600 mg CaCl2 L
1. The cheese-making was
replicated three times so that three batches of cheeses were pre-
pared for each treatment. The cheese was made with 4 L of the
pasteurised and standardised cheese-milk, within 2 days of milk
delivery using a 5 L stainless steel container with draining tap. The
cheese-milk was tempered to 33  C in water-bath before inocula-
tion with 0.05 g L
1 of freeze dried mixed strain direct vat set (DVS)
mesophilic starter culture and the addition of CaCl2. The ripening
time of the cheese-milk was varied in order to achieve a stand-
ardised pH 6.5 at coagulation (Section 2.1). When the pH of the
cheese-milk reached 6.5, rennet (Hannilase, 690 IMCU mL
1, Chr.
Hansen) was added to the milk at a concentration of 0.1 mL L
1. The
milk was then allowed to coagulate until it reached a storage
modulus (G0 ) value of 140 Pa (Section 2.1) before cutting with
cheese wire knives to 1 cm3. The gel was cooked by heating the
curd slowly from 33 to 38  C at a rate of 1  C rise per 12 min with
overhead stirring (Heidolph, Schwabach, Germany) at 10 revolu-
tions per minute. The cheese-making process continued as per the
method reported in a previous study (Ong, Dagastine, Kentish, &
Gras, 2011a).
2.3. Confocal laser scanning microscopy and cryo-scanning electron
microscopy of gel, curd and cheese samples
Fresh gel, curd and cheese samples collected during the cheese-
making process were prepared for observation with confocal laser
scanning microscopy (CLSM; Leica Microsystems, Heidelberg,
Germany) using the method reported in a previous study (Ong,
Dagastine, Kentish, & Gras, 2011b). The gel and curd samples
were analysed within 2 h of sample collection. The cheese samples
were analysed within 2 h of overnight pressing. Fresh samples were
also prepared for cryo-scanning electron microscopy (cryo-SEM)
analysis by slush freezing in liquid nitrogen (
210  C) followed by
freeze fracturing at
140  C under vacuum, etching at
95  C for
30 min and sputter coating with a gold and palladium alloy mixture
at a ratio of 60:40. The samples were observed using a field emis-
sion SEM (FE-SEM, Quanta; Fei Company, Hillsboro, Oregon, USA.)
at 15 kV with a backscattered detector (Ong et al., 2011b).
Image analysis of CLSM micrographs was performed using a
stereological approach, as previously described (Ong et al., 2011a).
The porosity is defined as the fraction of pore volume with respect
to the total sample volume.
2.4. Texture analysis
Texture profile analysis (TPA) was performed on cheese samples,
as previously described (Ong et al., 2012). A total of 6 readings were
obtained for each treatment; these consisted of two measurements
performed for two independent samples from each batch of cheese
and three batches of cheeses were made for each treatment.
2.5. Determination of total calcium
The total calcium content of the cheese was analysed using
Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-
OES; Varian Inc, PaloAlto, CA, USA) as previously described (Ong
et al., 2012).
2.6. Compositional analysis
Fat and protein concentration in milk, whey and cheese were
analysed as previously described (Ong et al., 2012). Total solids,
concentration of salt, moisture content and pH of the cheese were
also analysed as previously described (Ong et al., 2012). Duplicate
analyses were conducted for each cheese batch.
 L. Ong et al. / International Dairy Journal 33 (2013) 135e141 137

2.7. Measures of fat and protein recovery and cheese yield
The fat and protein collected in the whey during Cheddar
cheese-making (FL; g kg
1 and PL, g kg
1 respectively) or the fat
and protein retained within the cheese (FRet and PRet respectively;
both in g kg
1) was determined from the concentration of fat or
protein in the cheese-milk using methods described previously
(Ong et al., 2012). The total fat balance (Fbalance; g kg
1), the total
protein balance (Pbalance, g kg
1), the yield of cheese (Ya; g kg
1)
and the dry matter yield (YDM; g kg
1) were also calculated using
the methods described in Ong et al. (2012).
2.8. Statistical analysis
A statistical package from Minitab (Minitab Inc, State College,
PA, USA) was used for data analysis. A one-way analysis of variance
(ANOVA) and the Tukey’s paired comparison test were applied to
determine the differences between means. A significance level of
a ¼ 0.05 was used for all analyses.
3. Results and discussion
The addition of CaCl2 to cheese-milk induces a number of
changes during cheese-making. To obtain a constant pH at coagu-
lation, the ripening time of the cheese-milk with higher concen-
trations of CaCl2 was adjusted as shown in Table 1. A decrease in
milk pH was observed as the concentration of added CaCl2
increased and thus a shorter ripening time was required to reach
pH 6.5. In this study, CaCl2 was only added to a maximum level of
600 mg L
1. This upper limit for CaCl2 addition will allow for the
starter culture to ripen in the milk for at least 30 min before
renneting.
The stiffness of the gel at cutting as determined by the elastic
modulus was standardised to 140 Pa by varying the cutting time
(Table 1) to reduce the potential for a loss of milk solids and a
reduction in cheese yield. This stiffness was found to produce
Cheddar cheese with good texture, yield and composition for a
standard Cheddar cheese (data not shown). The microstructure of
the cheese was then assessed using two complementary micro-
scopic techniques.
3.1. Influence of CaCl2 addition on the microstructure and
properties of the gel and cheese
Sample images of the microstructure of the gels are shown in
Fig. 1. Each sample was analysed using CLSM immediately after it
reached a gel stiffness of 140 Pa. Spherical fat globules were evenly
distributed throughout the continuous protein network. Qualita-
tive differences in the microstructure of the gels were not obvious;
possibly because the gel samples were coagulated at a similar pH
and gel stiffness. Image analysis performed for the CLSM images,
however, showed that the porosity of the gels with greater levels of
calcium (300 and 600 mg L
1 of CaCl2) was significantly lower
(P < 0.05) when compared to the untreated samples (Fig. 1D),
indicating that the addition of calcium impacts on the arrangement
of the network structure.
The amount of whey expelled in the cheese vat following
cooking at 38  C was not significantly different for samples made
from milk with different CaCl2 addition (P > 0.05) (data not shown),
suggesting that any subtle differences in the protein gel network do
not have a large impact at this stage. The whey was drained when it
reached pH w6.1 and the curds milled at pH w5.4. There was no
observable trend in the cooking time or cheddaring time among
batches made with milk containing different CaCl2 addition
(Table 1). Differences in the time between starter addition to
draining or milling were mainly due to the changes in ripening time
and cutting time reported earlier. The overall cheese-making time
for cheese made with cheese-milk containing additional CaCl2 was
thus shorter.
The microstructure of the cheese formed from milk with
different concentrations of added CaCl2 is shown in Fig. 2. The fusion
of the CM during cooking, cheddaring and pressing results in the
formation of thicker protein strands. Pressing has also closed the
pores observed in the gel and the heat treatment applied during the
cheese-making process has caused the aggregation and coalescence
of fat globules. Individual fat globules are no longer visible and the
non-globular fat is evenly distributed throughout the compact
protein network. The cheese with the addition of 300 or 600 mg L
1
CaCl2 had a greater number of micron sized pores or gaps within the
cheese microstructure than cheese without any additional CaCl2.
These pores possibly indicate a reduced fusion of the cheese curd, as
a result of CaCl2 addition. The small-scale of the cheese-making
experiments could also contribute to the formation of these pores
but the absence of these pores in the cheese made without calcium
addition suggests a difference between treatments.
The addition of CaCl2 also significantly affects (P < 0.05) the
texture profile analysis (TPA) of the cheese (Fig. 3). The addition of
CaCl2 to cheese-milk at the level of 50 mg L
1 CaCl2 did not affect
the hardness of the final cheese product. Cheese made with cheese-
milk containing 100 mg L
1 CaCl2, however, was significantly
harder than cheese without the addition of CaCl2 and this trend
continued for higher calcium levels (Fig. 3). The gumminess and
chewiness of the cheeses also followed a similar trend to the
hardness (Fig. 3). No significant trend (P > 0.05) was observed in
cohesiveness and springiness of the samples with different con-
centration of added CaCl2 (data not shown). Chevanan,
Muthukumarappan, Upreti and Metzger (2006) found that the
springiness and cohesiveness of cheese with the addition of
198 mL L
1 CaCl2 was higher than the springiness or cohesiveness
in cheeses with low levels of calcium, possibly due to the greater

Table 1
Cheddar cheese-making process parameters and adjustments.a

CaCl2 Ripening pH at Cutting pH at Cooking pH at Time between Cheddaring pH at Time between

(mg L
1) time (min) renneting time (min) cutting time (min) draining starter addition time (min) milling starter addition
to drain (min) to milling (min)
0 60 6.53  0.02A 45 6.46  0.03A 115  10AB 6.14  0.05A 220  10A 85  5A 5.35  0.07A 305  5.0A
50 60 6.51  0.02A 40 6.47  0.01A 100  7.0B 6.06  0.05A 201  5.0B 87  3A 5.25  0.09A 288  3.0B
100 55 6.53  0.03A 34 6.48  0.02A 112  8.0AB 6.12  0.04A 201  8.0B 83  8A 5.36  0.07A 284  10BC
200 50 6.54  0.03A 28 6.49  0.02A 118  8.0A 6.14  0.08A 196  8.0B 82  8A 5.37  0.06A 278  5.0C
300 45 6.50  0.04A 26 6.45  0.04A 100  10AB 6.12  0.05A 171  10C 75  9A 5.36  0.06A 246  5.0D
600 35 6.48  0.05A 15 6.42  0.05A 108  10AB 6.14  0.05A 158  10D 82  3A 5.34  0.03A 240  10D
a
Results are expressed as the mean  the standard deviation of mean (n ¼ 3); means in a single column with different superscript letters are significantly different
(P < 0.05). Ripening time is time from the addition of starter bacteria and CaCl2 to the addition of rennet; cutting time is the time when the gel is firm enough to start cutting
the gel (as the G0 reach 140 Pa); cooking and cheddaring time were adjusted based on the pH of the curds during Cheddar cheese manufacture.
138 L. Ong et al. / International Dairy Journal 33 (2013) 135e141

Fig. 1. The microstructure of rennet-induced gels prepared using cheese-milk with the addition of (A) 0, (B) 300, or (C) 600 mg CaCl2 per litre of milk. Panel D gives the porosity of
the rennet-induced gels. Results are the mean  the standard error of mean (n ¼ 6). The Nile Red stained fat appears red and the Fast Green FCF stained protein appears green in
these images. The scale bars are 10 mm in length. Means with different superscript letters for the same series are significantly different (P < 0.05). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

number of cross-linkages induced by Ca addition, which increase
the casein intermolecular associations in the cheese network. This
trend was not observed here suggesting that the role of Ca may be
more complex.
3.2. Influence of CaCl2 addition on the composition of cheese
There was no observable trend in the amount of protein lost in
the whey (Table 2) or in the protein content of the final cheese
(Table 3). These results are consistent with Wolfschoon-Pombo
(1997) who investigated the effect of adding calcium chloride
(100 mg L
1) on the yield of Emmentaler cheese over a period of
one year and found that the non-fat milk solids composition of
the cheeses were not significantly affected. There was an
increasing trend in the concentration of total calcium in the
final cheeses when more CaCl2 was added to the cheese-milk
(Table 3).
The final cheese yield and yield in dry matter for all cheeses was
also unaffected by calcium (Table 2). This is contrary to the findings
of Wolfschoon-Pombo (1997), who reported an increased yield of
Emmentaler type cheese by 0.41% when 100 mg L
1 CaCl2 was
added to the cheese-milk. Similarly, Ustunol and Hicks (1990)
observed an increased yield when 200 mg L
1 calcium was added
to a cheese made with E. parasitica.

Fig. 2. The microstructure of Cheddar cheese prepared using cheese-milk with the addition of (A) 0, (B) 300 or (C) 600 mg CaCl2 per litre of milk. The Nile Red stained fat appears
red and the Fast Green FCF stained protein appears green in these images. The scale bars are 10 mm in length. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
 L. Ong et al. / International Dairy Journal 33 (2013) 135e141 139

added. Table 4 confirms, however, that the fat loss into the whey
a continued after the first draining stage, with significantly greater
60 ab
ab (P < 0.05) amounts of fat loss occurring when CaCl2 is added during
Texture parameters (N)

b
c c
40
a a a a
b after salting in order to minimise the amount of fat lost in the whey.
b
20 b ab a Australia, 8.5% of the total fat is lost in the whey, of which about
c c
0 Cogan, & McSweeney, 2000). In our study, 6% of the total fat was
0 100 200 300 400
Addition of calcium chloride (mg
Fig. 3. The effect of CaCl2 addition on the texture of Cheddar cheese: hardness (),
gumminess (-) and chewiness (:). Results are the mean  the standard error of
mean (n ¼ 6). Means with different superscript letters for the same series are signif-
icantly different (P < 0.05).
More importantly, the addition of CaCl2 was observed to
significantly decrease the amount of fat lost in the whey during
cheese-making (Fig. 4). Cheeses made with milk containing an
additional 200e600 mg L
1 CaCl2 had significantly lower (P < 0.05)
fat lost into the whey. Ustunol and Hicks (1990) also found that the
addition of 500 mg L
1 CaCl2 to cheese-milk coagulated with highly
proteolytic E. parasitica protease increased the fat content by about
0.5%. They suggested that the addition of Ca increased calcium
bridging between the caseins and lead to more cross linking thus
allowing for better fat entrapment in the curd.
Contrary to the work of Ustunol and Hicks (1990), the increase
in fat entrapped in the curd on our experiments did not result in a
final cheese with a higher fat content. There were no significant
differences (P > 0.05) in the amount of fat retained in the final
cheese (Table 2), the fat in dry matter or in the final fat composition
(Table 3). The composition of fat, fat in dry matter, and moisture
content of the cheeses were also not significantly affected (P > 0.05)
by the addition of CaCl2 (Table 3). All measured parameters were
within ranges usually observed for Cheddar cheese. This indicates
that the fat trapped in the curd was lost after whey draining;
possibly during cheddaring or pressing. To confirm if this was the
case, the cheese-making process was repeated for the cases with
50 mg L
1 and 300 mg L
1 added CaCl2 and the amount of fat lost in
the whey after the initial draining (after cooking), after cheddaring
and pressing were measured (Table 4).
The amount of protein lost in the whey at any of the processing
stages was not significantly (P > 0.05) affected by the different
concentration of added CaCl2. Consistent with the results in Fig. 4,
less fat is lost in the whey collected after cooking when CaCl2 is
the pressing of curd. The same total amount of fat is ultimately lost
at any calcium level but at higher calcium levels it is lost later in the
cheese-making process.
These results highlight the need to control the pressing stage
a In some industrial cheese-making operations, such as those in
76%, 17%, 5% and 2% is lost in the whey collected from the cheese
vat, cheddaring tower, salting belt and block former (Fox, Guinee,
500 600 lost in the whey, of which 9% and 28% of the total fat loss was from
L-1) the whey collected during pressing of cheese when 50 mg L
1 and
300 mg L
1 CaCl2 was added, respectively. The higher fat loss re-
ported for samples made from milk containing higher concentra-
tions of CaCl2 could be due to some difference in the microstructure
of the curds before pressing (after cheddaring). Minimising the fat
loss during pressing would thus help to preserve the higher amount
of fat retained earlier in the process as a result of calcium addition.
When the mass of fat and protein in the samples withdrawn
from the milk, cooked curd and cheddared curd for analyses is
estimated (126 g kg
1 and 104 g kg
1 for cheese with 50 mg L
1
CaCl2 addition; 127 g kg
1 and 96 g kg
1 for cheese with
300 mg L
1 CaCl2 addition), the total fat or protein balance is close
to 1000 g kg
1.
3.3. Microstructure of intermediate samples collected during
cheese-making
Any differences in microstructure of the gels with 50 and
300 mg L
1 added CaCl2 were not apparent in CLSM or cryo-SEM
images (Figs. 5 and 6). However, quantitative analysis of the gel
porosity in CLSM images indicated that the gels were less porous
in the presence of added calcium (0.39  0.02 and 0.31  0.03 for
gels made with 50 and 300 mg L
1 additional CaCl2, respectively).
The micrographs of the curd after cooking for samples made with
300 mg L
1 CaCl2 addition also show a much denser microstruc-
ture, possibly due to the increased calcium bridging between
casein proteins (Fig. 6B and F), consistent with the less porous gel
network observed for these samples. This denser microstructure
possibly allows for better fat entrapment within the curd,
resulting in the lower fat lost in the whey collected after cooking
(Table 4).
CSLM images taken after cheddaring appear to show the for-
mation of micro-pores in the curd made with the higher calcium
cheese-milk (Fig. 5G). The increased calcium concentration in the
curd may have increased the fusion of para-casein particles possibly
resulting in a contraction of the protein matrix and pore formation.

Table 2
Protein lost in whey, fat and protein retained in cheese and the final Cheddar cheese yield of samples prepared using cheese-milk with added CaCl2.a

CaCl2 Protein lost in whey Fat and protein retained in cheese Total fat and protein balance Cheddar cheese yield
(mg L
1)
1
1
1
1
1
PL (g kg ) FRet (g kg ) PRet (g kg ) Fbalance (g kg ) Pbalance (g kg ) Ya (g kg
1) YDM (g kg
1)
A A A A A A
0 230  6.8 849  22 724  16 896  25 954  18 109  1 70.5  0.3A
50 222  3.5B 830  7.1A 724  14A 880  9.8A 946  18A 110  0.3A 70.0  0.3A
100 228  3.9A 830  17A 720  24A 877  16A 948  26A 111  2A 71.0  1A
200 225  1.4B 828  18A 713  14A 868  20A 937  13A 110  0.4A 77.3  9A
300 215  1.7A 817  17A 711  22A 852  19A 925  22A 109  0.3A 70.3  0.8A
600 231  2.8A 819  18A 743  10A 848  21A 974  10A 110  0.3A 71.7  0.2A
a
Results are expressed as the mean  standard error of mean (n ¼ 6); means in a single column with different superscript letters are significantly different (P < 0.05).
Abbreviations are: PL, protein lost in whey; FRet, fat retained in cheese; PRet, protein retained in cheese; Fbalance, the sum of fat loss and fat retained in cheese (fat
loss þ FRet); Pbalance, the sum of protein loss and protein retained in cheese (PL þ PRet); Ya, total cheese yield per kg of cheese-milk; YDM, yield in dry matter per kg of
cheese-milk. PL, FRet, PRet, Fbalance and Pbalance were calculated on the basis of protein or fat levels in the cheese-milk.
140 L. Ong et al. / International Dairy Journal 33 (2013) 135e141

Table 3
Composition of Cheddar cheese prepared using cheese-milk with added CaCl2.a

CaCl2 (mg L
1) Fat (g kg
1) Protein (g kg
1) Salt (g kg
1) pH Moisture (g kg
1) S/M (g kg
1) FDM (g kg
1) Calcium (g kg
1)
A A B AB A B A
0 349  7 258  7 14.7  0.6 5.38  0.05 354  9 41.5  4.1 541  15 5.76  0.74C
50 343  3A 252  4A 15.2  0.6AB 5.41  0.05A 362  0.9A 41.9  2.8AB 537  3.5A 6.80  0.21BC
100 339  9A 253  2A 14.5  0.3B 5.32  0.01B 357  8A 40.7  2.0B 526  2.5A 6.48  0.40BC
200 341  7A 257  0.9A 14.8  0.4B 5.36  0.01AB 356  2A 42.0  5.3AB 528  1.2A 6.74  0.49ABC
300 339  8A 254  5A 15.2  0.4B 5.38  0.03AB 352  6A 43.6  1.9AB 524  9.4A 7.04  0.36AB
600 337  8A 260  5A 16.8  0.8A 5.41  0.04A 346  2A 48.6  4.6A 516  12A 7.66  0.37A
a
The results are expressed as the mean  the standard error of the mean (n ¼ 6); means in a single column with different superscript letters are significantly different
(P < 0.05). Abbreviations are: S/M, salt in moisture; FDM, fat in dry matter.

60 moisture content of the cheese reported by Pastorino was not

Fat loss in whey at drainage (g kg-1)

observed in our study (Table 3). Interestingly, the micro-pores

a a
a within the cheddared curd were not readily observed using cryo-
50 SEM (Fig. 6G). This is probably due to the different observation
b
scales produced by CLSM and cryo-SEM.

40 bc

c
30

20
0 100 200 300 400 500 600

Addition of calcium chloride (mg L-1)

Fig. 4. The effect of CaCl2 addition on the fat loss in the whey collected at whey
drainage after cooking. The fat loss was calculated on the basis of fat levels in the
cheese-milk. Results are the mean  the standard error of mean (n ¼ 6). Means with
different superscript letters for the same series are significantly different (P < 0.05).

These micro-pores could possibly become the channels from which

the fat escapes from the curds to the whey during pressing. These
micro-pores appear to remain in the cheese, as observed in Figs. 2
and 5H. Fewer pores were observed in the microstructure of the
cheddared curd and cheese with 50 mg L
1 added CaCl2 (Fig. 5C and
D) or where calcium was not added (data not shown).
The formation of the micro-pockets in the cheese made with
calcium supplemented milk was also observed by Pastorino,
Hansen, and McMahon (2003), although the decrease in the

Table 4
Fat and protein lost in whey collected during various stages of cheese-making.a

Stageb Fat lost (g kg
1) Protein lost (g kg
1)

1
1
50 mg L 300 mg L 50 mg L
1 300 mg L
1
CaCl2 CaCl2 CaCl2 CaCl2
Whey 1 53.0  2.0A 39.0  3.4B 197  9.3A 206  5.4A
Whey 2 2.4  0.5A 3.1  0.6A 15.1  1.2A 15.6  0.90A
Whey 3 5.80  0.20A 16.5  5.10B 6.4  0.50A 6.0  1.8A
FRet or PRet 828  15.9A 840  3.60A 663  4.1A 654  3.2A
Fbalance or 886  14.0A 893  6.5A 872  15A 879  6.6A
Pbalance
a
The results are expressed as the mean  standard error of mean (n ¼ 6). Means
in a row for fat or protein with different superscript upper case letters are signifi-
cantly different (P < 0.05). Fig. 5. Confocal laser scanning microscopy images of samples (A, E: gel; B, F: cooked
b
Abbreviations are: Whey 1, whey collected during draining; Whey 2, whey curd; C, G: cheddared curd and D, H: cheese) collected during Cheddar cheese
collected during Cheddaring; Whey 3, whey collected during pressing; FL, fat lost in manufacturing. AeD and EeH are samples made with cheese-milk with the addition
whey; PL, protein lost in whey; FRet, fat retained in cheese; PRet, protein retained in of 50 or 300 mg L
1 CaCl2 L
1, respectively. The Nile Red stained fat appears red and
cheese; Fbalance, the sum of fat loss and fat retained in cheese (Fat Loss þ FRet); the Fast Green FCF stained protein appears green in these images. The scale bars are
Pbalance, the sum of protein loss and protein retained in cheese (PL þ PRet). PL, FRet, 10 mm in length. Arrows indicate micro-pores. (For interpretation of the references
PRet, Fbalance and Pbalance were calculated on the basis of protein or fat levels in to colour in this figure legend, the reader is referred to the web version of this
the cheese-milk. article.)
 L. Ong et al. / International Dairy Journal 33 (2013) 135e141
Fig. 6. Cryo-SEM images of samples (A, E: gel; B, F: cooked curd; C, G: cheddared curd
and D, H: cheese) collected during Cheddar cheese manufacturing. AeD and EeH are
samples made with cheese-milk with the addition of 50 or 300 mg L
1 CaCl2 L
1,
respectively. The scale bars are 5 mm in length.
4. Conclusion
The addition of CaCl2 to cheese-milk increased the hardness of
the final cheese. Cheeses made with milk containing an additional
200e600 mg L
1 CaCl2 had less fat loss into the whey collected
after cooking but this CaCl2 did not affect the final fat composition,
cheese yield or yield in dry matter. This indicates that the fat
retained during cooking may be ultimately lost in the latter stages
of the cheese-making process, especially during the pressing of
curds. The denser microstructure within the cooked curd made
with high calcium cheese-milk appears to retain fat. The contrac-
tion of the protein matrix and formation of micro-pores observed in
the curd collected after cheddaring may also provide channels that
allow fat to escape from the curds to the whey during pressing.
141
Acknowledgements
This work was funded by Dairy Innovation Australia Limited
and the Australia Research Council (LP0883300). The authors
would like to thank the Bio21 Molecular Science & Biotech-
nology Institute and the Particulate Fluids Processing Centre,
which is a Special Research Centre of the Australian Research
Council at The University of Melbourne, for access to equipment.
We would also like to thank Murray Goulburn, Warrnambool
Cheese and Butter, Lion and Burra Foods for their involvement.
We appreciate the technical input from Mr Roger Curtain (Bio21
Molecular Science and Biotechnology Institute, The University of
Melbourne) and his help operating the scanning electron mi-
croscope in cryo mode.
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