LWT-

FoodScienceandTechnology
131(2020)109677

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LWT - Food Science and Technology

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Calcium balance during direct acidification of milk for Mozzarella cheese

production
Camilla D. Thyboa, Søren K. Lillevangb, Leif H. Skibsteda, Lilia Ahrné a,∗
a
University of Copenhagen, Department of Food Science, 1958, Frederiksberg, Denmark
b
Arla Foods Amba, Arla Innovation Center, Skejby, Denmark

A R T I CLE I N FO
A B S T R A C T

Keywords:
Mozzarella cheese
Calcium is an important nutrient but also contributes to the texture, taste and functionality of most dairy pro-
Free calcium ducts. The balance between micellar and free calcium in curd and serum phase during direct acidi fication of
Micellar calcium bovine milk was studied by changing organic acid type, pH and acidification temperature. Concentration of free
Citric acid calcium increased with decreasing pH and increasing temperature. Citric acid was more e fficient than lactic acid
Lactic acid at lowering free calcium concentration, while temperature effect was larger for acidification with lactic acid.
These findings are rationalized by the stronger binding of calcium to citrate compared to lactate with an almost
athermal binding for citrate and an endothermal binding for lactate. Curd yield decreased by decreasing pH as
solubilization of colloidal calcium phosphate causes loss of minerals into the serum phase decreasing the con-
centration of both calcium and phosphate left in the curd, while acidification temperature had little effect on
curd yield. These results are important to improve yield and tailor the properties and functionality of dairy
products, such as for example Mozzarella cheese.

1. Introduction
Mozzarella is a Pasta filata type cheese and hence, also referred to as
a fresh cheese. It has traditionally been produced from buffalo milk but
is now also produced from bovine milk. Pasta filata covers a technique
that is especially used in the manufacture of certain Italian cheeses,
including stretching the curd to obtain a smooth texture and a stringy-
like consistency of the cheese (Fasale, Patil, & Bornare, 2017; Smith,
Hindmarsh, Carr, Golding, & Reid, 2017). Mozzarella, although still
produced by hand some places in Italy, is now mainly produced on an
industrial scale.
Usually, the standardized milk for Mozzarella is pasteurized and
cooled before the acid of choice is added. The pH is in most cases ad-
justed to approXimately 5.8 before the acid coagulated milk is heated
prior to the rennet addition. Acidification and stretching of curd are
essential steps to obtain a good Mozzarella cheese (Fasale et al., 2017;
Shakeel-Ur-Rehman & Fakye, 2006). Some of the flavor characteristics
of Mozzarella are the very mild and clean flavors with a slight sourness,
depending on whether the cheese is produced from buffalo milk or
bovine milk. However, both should keep the milk-like freshness (Ah &
Tagalpallewar, 2017; Fasale et al., 2017; Seth & Bajwa, 2015).
Besides consuming Mozzarella as it is, the cheese is also used as an
ingredient in other foods. It is especially popular for its use on pizza
where good melting properties of the cheese are crucial (Zhu, Brown,
Gillies, Watkinson, & Bronlund, 2015). In addition, Mozzarella cheese is
expected to have good stretchability properties, which is significantly
affected by the type of milk and processing conditions during the
thermo-mechanical stretching step. Fasale et al. (2017) found that the
origin of milk had an influence on stretchability and buffalo milk
having higher calcium and fat content produced cheese with better
stretchability. Calcium is recognized to play an important role on
structure formation and its distribution between the serum and the curd
can be controlled during the milk acidification process. Thus, in relation
to Mozzarella production, and to achieve the desirable properties of
Mozzarella, it is important to determine the optimal amount of calcium
in the serum phase and in the curd before stretching. The effect of low
calcium in the final cheese, obtained by pre-acidification and additional
manufacturing steps, will ideally result in a cheese that meet the re-
quirements regarding stretching and melting properties.
Many of the recent studies on calcium equilibria in milk have fo-
cused primarily on the total amount of calcium in the serum. Hence,
there is a need for studies specifying the amount of free calcium in the
serum as function of type of acid, pH and temperature during the
acidification process. Thus, the objective of the present study was to
evaluate the calcium equilibria and kinetics of equilibration in milk
during direct acidification with two organic acids: lactic acid and citric

∗
Corresponding author. Rolighedsvej 26, 1958, Frederiksberg, Denmark.
E-mail address: lilia@food.ku.dk (L. Ahrné ).

https://doi.org/10.1016/j.lwt.2020.109677
Received 30 January 2020; Received in revised form 1 May 2020; Accepted 27 May 2020
Availableonline13June2020
0023-6438/©2020ElsevierLtd.Allrightsreserved.
C.D. Thybo, et al.
acid at different pH values and temperatures.
2. Materials and methods
2.1. Materials
Calcium chloride dihydrate from Merck (Darmstadt, Germany) was
used for preparation of the calcium stock solution which was diluted to
different concentrations for calibration of the calcium electrode. All
dilutions were prepared using de-ionized water from a Milli-Q pur-
ification system (Milli-Q Direct, Buch & Holm, Denmark). Citric acid
and lactic acid from Sigma-Aldrich (Steinheim, Germany) were used for
acidification of 0.4% low fat milk from Arla Foods amba (Slagelse,
Denmark). Renneting of citric acid acidified milk was initiated by ad-
dition of Chymax 200 (Chr. Hansen A/S, Hørsholm, Denmark) post
acidification.
The chemicals used for digestion of ICP-OES samples were 65%
nitric acid (HNO3 ultrapure) and 32% hydrogen chloride, both from
Sigma-Aldrich (Steinheim, Germany).
2.2. Preparation of calcium chloride stock solution and standards
A stock solution of 0.1 M calcium chloride dihydrate was prepared
prior to the experiments and stored at 5 °C for use throughout the
analyses.
Based on the concentration of free calcium in milk, which is known
to be ~3 mM (Lin, Leong, Dewan, Bloomfield, & Morr, 1972), five
different standards of aqueous calcium chloride were prepared from the
stock solution for electrode calibration. The concentration of the stan-
dards was modified slightly according to preliminary tests and de-
pending on the settings of temperature and acid type used. Standard
solutions were measured under the same circumstances as the samples
and calcium activity was calculated from the electrode potential of each
sample. The ion activity was then calculated from the respective stan-
dard curve for each set of samples. Following the calculation of the
calcium activity, the calcium concentration was calculated according to
the procedure in section 2.6. Calibration was conducted 2–4 times each
measuring day.
2.3. Acidification curve
For both types of acid, a titration curve at 20 °C was made in order
to determine the required volume of acid to reach the desired pH va-
lues. Values of pH were measured using a pH meter (713 pH Meter,
Metrohm, Copenhagen, Denmark). For resemblance of the actual sam-
ples, the acidification was performed on 100 g samples of low fat milk.
During acidification, the milk was stirred using a magnetic stirrer, and
acid was added (50 μL aliquots) using a 10–100 μL automatic pipette.
Acidification was continued to a pH value below 4. The change in pH
as
a function of added lactic acid or citric acid solution was considered not
to be temperature dependent and was in agreement with buffer capa-
city expected for milk in the pH region 7 to 4 (Mcsweeney, 2007).
2.4. Preparation of milk samples for acidification and renneting
Two independent experiments were performed. For each sample,
100 g of milk was weighed into 100 mL blue cap bottles. The samples
were tempered to 5, 10, 20 or 30 °C prior to the measurement at T0 and
with no acid added. Samples at 5 and 10 °C were placed into water
baths where the temperature was controlled with ice and monitored/
adjusted continuously throughout the measurements. Samples at 20 °C
were placed in a water bath at room temperature and kept at 20 °C
using ice cubes and the samples at 30 °C were placed in pre-tempered
water bath, where the temperature was controlled throughout the
measurements. Minor fluctuations ( ± 5 °C) in temperatures might have
occurred during acid addition. After reaching the temperature, pH,
2
LWT-
FoodScienceandTechnology1
calcium activity and conductivity were measured on the untreated milk
samples prior to acid addition at time zero.
2.5. Direct acidification
1.0 M citric acid was added to milk samples at T0, in the amounts
required to obtain pH values 6.4, 6.1, 5.8, 5.5, 5.2 or 4.6 at the tem-
peratures 5, 10, 20 or 30 °C. For lactic acid, samples were acidified
with
1.0 M lactic acid, and acidification at 5 °C and 30 °C was tested. Acid
was added according to the titration curve (see section 2.3). Im-
mediately after acid addition, samples were stirred vigorously on a
magnetic stirrer for 10 s to obtain an even distribution of acid
throughout the milk.
2.6. Electrochemical measurements of calcium ion acitivity to determine
free calcium
The free calcium was defined as the concentration of calcium that
is not in the micelles and not bound to any other constituents in milk,
and can therefore be measured using a calcium electrode that has
been calibrated using the five different standards of aqueous calcium
chloride previously described. To determine calcium concentration,
measurements for pH, calcium ion activity and conductivity were per-
formed up to 3 h after 10, 20, 30, 60, 90, 120, 150 and 180 min. The
calcium ion activity was determined using an ISE25Ca calcium selective
electrode and a REF 251 reference electrode (Radiometer, Copenhagen,
Denmark). Calibration of the electrode was performed using calcium
chloride standard solutions with concentrations of 1.00 X 10−4,
1.00 × 10−3, 4.00 × 10−3, 8.00 × 10−3 and 1.00 × 10−2 M. All
standard solutions were prepared from the 0.1 M stock solution. The
activity of the calcium ion (aCa2+) was calculated according to the
Nernst equation. The relationship between the calcium ion activity and
the concentration is given as:
aCa2+ = cCa2+ ⋅ γ2+ (1)
with cCa2+ being the concentration of calcium in the solution and γ2+
being the activity coefficient, calculated from Davies' equation (Davies,
1962):
I
log γ2+ = −A z2 ⎛ − 0.3 ⋅I ⎞
⎜ ⎟
I
DH (2)
A is the Debye-Hü ⎝ 1ckel
+ ⎠
constant which is temperature dependent, z
DH
is the ion charge (Manov, Bates, Roger, Hamer, & Acree, 1943;
Vavrusova, Liang, & Skibsted, 2014), and I is the ionic strength:
1
I= ⋅ ∑ ci⋅zi2
2 (3)
The ionic strength is calculated considering the concentration, ci,
of all ions present in the solution which was prepared using de-
ionized water.
All calcium concentration calculations for the samples were based
on the linear relationship between ionic strength and conductivity in
mS, which was introduced by Griffin and Jurinak for 25 °C (Griffin &
Jurinak, 1973):
I ≅ 0.013 ⋅ λ (4)
2.7. pH and conductivity measurements
Measurements of pH were performed with a 713 pH Meter and a
602 combination glass electrode (Metrohm, Denmark), calibrated each
day towards the international pH standards pH 4.000, 7.000 and 9.240.
Conductivity measurements for each sample were performed by using a
4-pole-conductivity cell model CDC866T (Radiometer, Copenhagen,
Denmark).
2.8. Rennet coagulation
Rennet was added to all samples after the 180 min of acid coagu-
lation and after being placed in a heated water bath (35 °C) for ap-
proXimately 5 min. Rennet was added to the sample after diluting 10
times in 60 μL aliquots into 100 g samples. Samples were stirred vig-
orously on a magnetic stirrer after rennet addition, in order to distribute
the rennet into the acidified milk. Samples were left for renneting in a
35 °C water bath until they were ready for cutting. Whether the curd
was ready for cutting or not was tested by performing a “clean break
test” where a single small cut was made to assure a clean break and
visible separation of whey.
2.9. Whey drainage and pressing of curd
After cutting and resting the coagulum for 10 min, the cut coagulum
was poured into a funnel shaped separation filter that was made from a
finely meshed nylon net and placed over a beaker for whey drainage.
The whey was drained for 15 min by gravity combined with light
manual pressing of the curd. Manual pressing (4 × 10 s) was applied
for each sample, in order to decrease the amount of whey in the curd.
After separation, the curd yield was found by weighing the curd. After
weighing, the curd and whey were analyzed. Calcium electrode po-
tential, pH and conductivity were measured for the whey, while it was
only possible to measure calcium electrode potential and pH of the
curd.
2.10. ICP-OES of curd
For preparation of samples for ICP-OES, 1.00 g of sample (curd)
was weighed into HVT50 vessels. For digestion, 8 mL analytical grade
HNO3 (65%) was added to the samples followed by 2 mL analytical
grade HCl (32%). Post acid addition, the vessels were closed and
loaded into a Multiwave GO Rotor 12HVT50 digestion system for
digestion of the samples by microwaves (Anton Paar, Austria).
Digested samples were analyzed in a 5100 ICP-OES auto sampling
system from Agilent Technologies (Santa Clara, California, USA)
according to standard curves at wavelengths 422 nm for Ca and 213
nm for P. Standards were prepared from standard solutions of
calcium and phosphorus from Sigma-Aldrich (Steinheim, Germany).
3. Results and discussion
3.1. Calcium equilibria
The calcium equilibrium behavior during acidification with citric or
lactic acids was investigated by periodic measurements of pH, calcium
ion activity and conductivity of the acidified milk. Calcium binds to
hydroXycarboXylates like lactate and citrate in a 1:1 ratio despite
structural differences and numbers of carbo Xylate groups in fast reac-
tions at the pH of relevance for dairy products (Vavrusova et al., 2014).
At a pH of 4 and higher most of the hydro XycarboXylates are present as
their anions as will be evident from the pKa value of lactic acid of 3.85
and the first dissociation of citric acid to form the mono negative anion
with pKa 3.13 (Belitz, Grosh, & Schieberle, 2009). The time dependence
of free calcium in the milk serum during acidification at 5, 10, 20 and
30 °C for citric and lactic acid are shown in Figs. 1 and 2, respectively.
An initial increase in concentration of free calcium within 10 min of
acidification followed by minor fluctuations was noticed for all milk
samples, especially when pH < 5.8. The effects of pH were significant,
while effects of temperature were less pronounced. Only small differ-
ences in free calcium with a slight increase with acidification were
observed at high pH (6.4 and 6.1). Further acidification increased the
amount of free calcium in the serum.
For milks acidified with citric acid as shown in Fig. 1, fluctuations
were seen at low temperatures, while the equilibrium occurred faster at
the higher temperatures. Solubilization of colloidal calcium phosphate
(CCP) is inversely dependent on temperature (Gaucheron, 2005), and
the solubility is higher at lower temperatures. The higher solubility
could possibly cause more free ions to form complexes with caseins or
chelating agents causing slower adjustment of the equilibrium. How-
ever, at lower temperatures electrode stabilization is slower.
There were slight differences in the initial concentration of free
calcium ions in milk from different batches. The variations of free
calcium in milk has been reported in a number of studies, and Lin et al.
(1972) found it to range between 2.5 and 3.4 mM.
After pH adjustment, it was observed that pH 5.8–4.6 creates an
initial peak showing an overshoot in the free calcium concentration,
before a decrease depending on pH and temperature. When milk is
directly acidified with a concentrated acid instead of acidification with
a bacterial culture, the milk system is rapidly perturbed (Jeantet,
Croguennec, Schuck, & Brulé , 2016). As discussed by Carr (2015, pp. 13–
15), the protons when added as a weak acid into solutions affects
the equilibrium between micellar and free calcium depending on type
of acid and its acid dissociation constant as well as the resulting pH of
the solution (Simon & Beevers, 1951). The binding of protons on the
surface of the casein micelle neutralizes the negative charge, thus
weakening the structure and inducing dissociation of CCP and other
constituents of the casein micelle (Broyard & Gaucheron, 2015). When
CCP is dissociated, it enters the serum phase as inorganic phosphate and
calcium ions. Consequently, the presence of free calcium in the serum
phase is expected to increase at the expense of calcium bound to citrate,
phosphate and chloride salts or other milk components, like proteins or
peptides. Thus, the rapid changes in milk properties in general from the
direct addition of citric acid or lactic acid were assigned to the rapid
increase in the concentration of free calcium.
For milk acidified with lactic acid, at 5 °C only slight effects of
changes in pH were seen. For pH 5.2, a slight overshoot at 10 min
after acidification was observed. As in the case of citric acid samples,
the overshooting might be attributed to a rapid addition of protons
causing the most readily available calcium to dissociate into the
serum phase. The subsequent decrease in concentration was probably
caused by migration of acid into the casein micelles, causing some
calcium to be reorganized. At 30 °C there was a clear effect of pH on
the free calcium concentration. The highest concentration of free
calcium ions was ob- tained at pH 4.6 while the lowest was obtained
for pH 6.4. A significant effect of temperature was observed as milk
acidified at 30 °C reached much higher concentrations of free calcium.
An effect of acidification on free calcium concentration was also
observed. For both tempera- tures, stabilization was reached within
30 min. Both temperature and pH influenced the solubilization of CCP
and after cooling to 5 °C, the solubilization of CCP increased
(Gaucheron, 2005). In addition, the
dissociation of especially β-casein is assumed to have taken place as the
hydrophobic interactions are weakened during decreasing temperature.
At high temperatures, the solubilization of CCP was reversed.
The phenomenon of increasing concentration of free calcium with
increasing temperature was unexpected, as the relationship between
solubilization of CCP and temperature was normally found to be in-
verse. At lower temperatures, more calcium enters the serum phase due
to increased solubilization of colloidal calcium phosphate. Although, it
was found by Koutina, Knudsen, Andersen, and Skibsted (2014), that
most of the calcium liberated from the colloidal phase remains as free
calcium, it still has to be considered that some calcium is bound to
different milk components. The binding of calcium to citrate was found
by Singh, Yeboah, Pambid, and Debayle (1991) to be exothermic and
close to thermoneutral. Calcium binding to citrate is slightly exothermic
with ΔH = - 5.1 kJ/mol, which is close to thermoneutral. The binding is
strong with Ka = 3.6 × 104 at 25 °C and only decreasing slightly with
temperature. However, the binding to hydrogen citrate although
weaker with Ka = 357 will contribute at lower pH because of the
shifting acid/base equilibria. The binding to hydrogen citrate is not
thermoneutral, but strongly exothermic with ΔH = - 22 kJ/mol.
In
Fig. 1. Concentration of free calcium during acidification with citric acid as function of time. Acidification at 5 °C, 10 °C, 20 °C and 30 °C. Acidi fication was
performed at pH 6.4 ( ), 6.1 ( ), 5.8 ( ), 5.5 ( ), 5.2 ( ) and 4.6 ( ). Error bars are based on standard deviation calculated from duplicate
experiments.

Fig. 2. Concentration of free calcium during acidification with lactic acid as function of time. Acidification at 5 °C and 30 °C. Acidification was performed at pH 6.4
( ), 6.1 ( ), 5.8 ( ), 5.5 ( ), 5.2 ( ) and 4.6 ( ). Error bars are based on standard deviation calculated from duplicate experiments. Note
that the maximum concentration of free calcium is not the same for plot A and B.
Fig. 3. Concentration of free calcium ions at times 10 ( ), 90 ( ) and 180 ( ) minutes for milk acidified with citric acid. Acidification at 5 °C, 10 °C, 20 °C and 30 °C.
Error bars are based on standard deviations calculated from duplicate experiments.

contrast, calcium binding to lactate is weak with K a = 49, but strongly

solubilization of CCP at pH ~5 (Dalgleish & Law, 1988; Lucey, 2017).
endothermic and accordingly increasing with temperature (Vavrusova
Acidification time did not have a large influence as there was no
et al., 2014). The endothermic binding to lactate is unusual compared
marked difference between the times for acidification at each pH.
most other calcium complexes including citrate (Mekmene &
Thus, it can be concluded that most of the calcium was released within 10
Gaucheron, 2011). The temperature effect for milk acidified with citric
min of acidification.
acid is accordingly as expected due to the weaker binding at higher
For lactic acid acidification there was a clear effect of temperature,
temperatures. For lactate, calcium binding dominates by other ligands
as the milk acidified at 30 °C reached a significantly higher calcium
and the temperature effect is different from what you would expect concentration than those at 5 °C. This was found for the low pH values
from lactate alone.
in particular. At pH 4.6, the calcium concentration was 5–6 times
For a better understanding of the effect of pH on the amount of free
higher for acidified milk at 30 °C. The free calcium concentration at
calcium, the concentration of free calcium was compared after 10, 90
30 °C did not level out around pH 5.2, as it was observed for the other
and 180 min for citric acid (Fig. 3) as well as for lactic acid (Fig. 4). The
temperatures, and which could be expected due to total solubilization
pattern for all temperatures showed an initial increase in free calcium
of CCP. This increase in concentration of free calcium beyond the re-
from pH 6.4 and down to 5.5–5.2. Following the increase, for most
ported total solubilization point of CCP could moreover be attributed to
cases the curve started leveling out or decreasing as they reached the calcium still remaining in the micelle. While the calcium in CCP and the
point of all the CCP being solubilized at pH ~5 (Dalgleish & Law, 1988;
total amount of inorganic phosphate is thought to be fully solubilized
Lucey, 2017). The effect of time at each pH was only minor. With that
around pH 5.2, the remaining calcium is totally solubilized at pH 3.5
in mind, it is feasible to assume that most of the calcium is released
(Le Graet & Brulé , 1993). The remaining calcium is associated with the
from the micelle within the first 10 min. The results are also compar-
organic phosphate in the phosphoserine residues of the casein mole-
able to a study by Law and Leaver (1998) who found serum con-
cules (Gaucheron, 2005).
centrations of calcium and phosphate to increase rapidly after pH ad-
The difference between citric and lactic acid acidification can again
justment with HCl.
be assigned to the difference in binding of calcium to lactate and citrate
For milk acidified with citric acid, the pattern for all temperatures
and the difference temperature effect on chelation.
was similar, showing an initial increase in free calcium from pH 6.4 and
down to 5.5–5.2. Following the increase, the curve started flattening
out or decreasing as it reached the point of apparent complete
Fig. 4. Concentration of free calcium at times 10 ( ), 90 ( ) and 180 ( ) minutes for milks acidified with lactic acid. Acidification at 5 °C and 30 °C. Error bars are
based on standard deviations calculated from duplicate experiments.

3.2. Curd yield and total calcium in curd

As lactic acid showed not to be as effective as citric acid for the

purpose of lowering the free calcium concentration, the effects of
curd yield were assessed only for citric acid acidification. Fig. 5 shows
the effects of pH and temperature on the curd yield. The total calcium
concentration for the respective curd sample can be obtained from
Fig. 6. At pH 4.6 and 5.2, results at 5 °C are not presented as there
were no separation of curd and whey.
The results indicate that the curd yield is decreasing with decreasing
pH. In addition, it appears that there is correlation between acidifica-
tion temperature and curd yield. Previously Metzger, Barbano, and
Kindstedt (2000) reported that yield decreased in pre-acidified milk.
Solubilization of casein micelles from acidification leads to
dissociation
of colloidal calcium phosphate but this solubilization can also cause
dissociation of β-casein into the serum (Dziuba & Muziñ ska, 1998). This
is especially seen at low temperatures. However, the increased solubi-
lization at lower pH could influence the dissociation of β-casein into the
serum phase, even at higher temperatures. Liu, Weeks, Dunstan, and
Martin (2013) found less dissociation of β-casein with temperatures
increasing between 10 and 40 °C. As the dissociation of β-casein at low

Fig. 6. Variations in total calcium in the curd as a reaction of acidification from

pH 6.4 to 4.6 with citric acid. Total calcium is compared between the acid-
ification temperatures 5 °C ( ), 10 °C ( ) 20 °C ) and
( 30 °C ( ).

temperature and pH causes a decreased protein recovery, it will like-

wise result in a decreased yield. The loss of minerals, i.e. calcium and
phosphate, at low pH, will also cause the yield to decrease in agreement
with the hydrophobic binding of β-casein to other caseins. Thus, when
hydrophobic interactions are weakened during cooling, dissociation is
observed for β-casein (Dziuba & Muziñ ska, 1998; Zhang et al., 2018).
As illustrated by Figs. 6 and 7, the concentrations of calcium and
phosphorus as determined by ICP-OES both decreased in the curd with
a decrease in pH. The decrease in total calcium in the curd is the result
of increasing serum calcium with acidification and simultaneous solu-
bilization of CCP. According to Kindstedt (2007) and Guinee, Feeney,
Auty, and FoX (2002), a decrease in calcium content is desirable due to
increased protein hydration, which improves the functional properties.
It will be relevant to perform further studies focusing on this. For both
calcium and phosphorus, the decrease in concentration was most pro-
found at higher pH while there were only minor changes in both be-
tween pH 5.2 and 4.6. This is likely caused by the solubilization of CCP,
Fig. 5. Curd yield for milks acidified with citric acid at 5, 10, 20 and 30 °C and
which according to Lucey (2017) is complete around pH 5.0, depending
pH 6.4, 6.1, 5.8, 5.5, 5.2 and 4.6) before renneting. Error bars are based on
on the temperature.
standard deviations for duplicate experiments.
Fig. 7. Variations in total phosphorus in the curd as a reaction of acidification from 6.4 to 4.6 with citric acid. Total phosphorus is compared between the acid-
ification temperatures 5 °C ( ), 10 °C ( ) 20 °C ( ) and 30 °C ( ).

The phosphorus remaining in the curd was found lower than cal- concentration of free calcium.
cium at all pH values. It is generally accepted that CCP is a complex of
calcium and phosphate and according to FoX (1993), the Ca/P ratio of
CCP is approXimately 1.5. In comparison, the Ca/P ratio for 10 °C at
pH
6.4 in this study was found to be appro Ximately 1.7, explaining why less
phosphate is left in the curd after whey drainage.

4. Conclusions

In this study, the influence of temperature (5–30 °C), pH (6.4–4.6)

and type of acid (citric acid and lactic acid) on the calcium equilibrium
and kinetics of equilibration between the serum and colloidal phase
during direct acidification of milk for Mozzarella cheese were studied.
Most of the calcium was dissociated into the serum phase within the
first 10 min after acidification. Between pH 5.2 and pH 4.6, the free
calcium concentration was mostly stable caused by the solubilization of
colloidal calcium phosphate. However, calcium continued to increase in
lactic acid acidified milk at 30 °C. The amount of free calcium was
influenced by both pH and temperature. In general, and for both acids,
a decrease in pH and increase of temperature leads to an increasing
 For acidification with citric acid, pH had a strong effect on the
amount of free calcium, while temperature had less effect. For lactic
acid acidification, a considerable effect of pH and temperature was
observed as explained by less extensive binding of calcium to lactate
compared to citrate. Compared to lactate, calcium at low temperatures
had a stronger binding to other molecules like proteins or peptides in
contrast to citrate.
The curd yield for citric acid acidification generally decreased
with pH and increased with the temperature. A possible explanation
for lower yield at lower temperature and pH is the dissociation of β-
casein
into the serum, hence causing loss in protein recovery. A comparison of
the total concentration of calcium in the curd with the concentration of
free calcium in the acidified milk after 180 min showed that milk
acidified at 5 °C had a lower concentration of free calcium compared to
the milk acidified at 30 °C for the same concentration of total calcium in
the curd. A possible explanation is the reaction between calcium and
citrate being exothermic with calcium binding to dissociated β-
casein
from the casein micelles at low temperature and pH.
Acknowledgements
The present study is part of the Platform for Novel Gentle Processing
supported by the Danish Dairy Research Foundation (DDRF), University
of Copenhagen and Arla Foods.
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