molecules

Review
A Review on the Effect of Calcium Sequestering Salts on Casein
Micelles: From Model Milk Protein Systems to Processed Cheese
Gaurav Kr Deshwal 1,2,3 , Laura G. Gómez-Mascaraque 1 , Mark Fenelon 1 and Thom Huppertz 2,4, *

1 Department of Food Chemistry and Technology, Teagasc Food Research Centre, Fermoy,
P61C996 Cork, Ireland
2 Department of Agrotechnology and Food Sciences, Wageningen University, Bornse Weilanden 9,
6708 WG Wageningen, The Netherlands
3 Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal 132001, Haryana, India
4 FrieslandCampina, Stationsplein 4, 3818 LE Amersfoort, The Netherlands
* Correspondence: [Link]@[Link]

Abstract: Phosphates and citrates are calcium sequestering salts (CSS) most commonly used in
the manufacture of processed cheese, either singly or in mixtures. Caseins are the main structure
forming elements in processed cheese. Calcium sequestering salts decrease the concentration of free
calcium ions by sequestering calcium from the aqueous phase and dissociates the casein micelles
into small clusters by altering the calcium equilibrium, thereby resulting in enhanced hydration and
voluminosity of the micelles. Several researchers have studied milk protein systems such as rennet
casein, milk protein concentrate, skim milk powder, and micellar casein concentrate to elucidate
the influence of calcium sequestering salts on (para-)casein micelles. This review paper provides
an overview of the effects of calcium sequestering salts on the properties of casein micelles and
consequently the physico-chemical, textural, functional, and sensorial attributes of processed cheese.
A lack of proper understanding of the mechanisms underlying the action of calcium sequestering
salts on the processed cheese characteristics increases the risk of failed production, leading to the
waste of resources and unacceptable sensorial, appearance, and textural attributes, which adversely
affect the financial side of processors and customer expectations.
Citation: Deshwal, G.K.;
Gómez-Mascaraque, L.G.; Fenelon,
M.; Huppertz, T. A Review on the
Keywords: cheese; citrates; phosphates; casein micelles; calcium sequestering salts
Effect of Calcium Sequestering Salts
on Casein Micelles: From Model Milk
Protein Systems to Processed Cheese.
Molecules 2023, 28, 2085. https:// 1. Introduction
[Link]/10.3390/molecules28052085 Bovine casein micelles are polydisperse spherical particles having a mean radius
Academic Editors: Lin Lin and of roughly 100 nm [1]. They are primarily composed of αs1 -, αs2 -, β-, and κ-casein in
Adele Papetti the weight ratio of ~3:0.[Link]. Their integrity is held by colloidal calcium phosphate
(CCP), constituting ~7% of the micellar dry weight [2]. The attractive forces in casein
Received: 19 January 2023 may involve hydrophobic bonds, hydrogen bonds, calcium phosphate cross-links, and
Revised: 14 February 2023
electrostatic interactions, whereas repulsive interactions are basically repulsive electrostatic
Accepted: 20 February 2023
interactions, which are mainly affected by net casein charge [3]. CCP neutralizes the
Published: 23 February 2023
negatively charged phosphoseryl residues by cross-linking casein molecules and allowing
hydrophobic interaction between caseins [4]. Since αs1 -, αs2 -, and β-casein contain centers
of phosphorylation (at least three phosphoserine groups in sequence), they can readily bind
Copyright: © 2023 by the authors.
Ca2+ [5]. κ-Casein typically contains only one phosphoseryl residue and is less affected
Licensee MDPI, Basel, Switzerland. by the presence of calcium [6]. Calcium sequestering salts (CSS), such as phosphates and
This article is an open access article citrates, which are commonly used in the manufacturing of processed cheese (where they
distributed under the terms and are often referred to as emulsifying salts or melting salts), decrease the concentration of free
conditions of the Creative Commons calcium ions by sequestering calcium from the aqueous phase, and dissociate the casein
Attribution (CC BY) license (https:// micelles by altering calcium equilibrium, thereby resulting in the enhanced hydration and
[Link]/licenses/by/ volume of the micelles [7]. Upon the addition of CSS to casein micelles, CCP is removed
4.0/). and the micelles dissociate. During the manufacturing of processed cheese, the interactions

Molecules 2023, 28, 2085. [Link] [Link]

Molecules 2023, 28, 2085 2 of 17

among the casein matrices, CSS and calcium are critical factors affecting the final functional
attributes such as texture, meltability, and emulsification [8].
Processed cheese is a viscoelastic matrix consisting of cheese(s) as well as several dairy
ingredients (e.g., skim milk solids, butter, anhydrous milk fat, milk, whey powder, and
co-precipitates) and non-dairy ingredients and additives (e.g., stabilizers, acidifying and
sweetening agents, colors and flavors), to achieve the desirable functional attributes [9].
Further key ingredients in the manufacture of processed cheese are CSS, usually added
as sodium salts of monomeric or polymeric phosphates or citrates [10]. According to
the US Food and Drug Administration, 13 different CSS are permitted, either singly or
in combination, in processed cheese manufacture (Table 1) [11]. Calcium sequestration
involves the exchange of Ca2+ in the casein micelles, with monovalent cations (e.g., H+ ,
Na+ or K+ ) of the CSS [12]. The quality, textural and functional properties of processed
cheese are to a large extent determined by the composition and maturity of natural cheese,
the quantity and type of CSS added, and several other processing parameters. CSS also
plays a significant role in the formation of the required microstructure of the final product
via pH adjustment and calcium sequestration [13].

Table 1. Permitted calcium sequestering salts in processed cheese as per code of federal regulations
21CFR133.169 [9,11].

Permitted Calcium
Group Chemical Formula Molecular Weight (g/mol)
Sequestering Salts
Sodium citrate C6 H5 Na3 O7 258.07
Citrate Potassium citrate C6 H5 K3 O7 306.39
Calcium citrate C12 H10 Ca3 O14 498.40
Monosodium phosphate NaH2 PO4 119.97
Orthophosphate Disodium phosphate Na2 HPO4 141.96
Trisodium phosphate Na3 PO4 163.94
Tetrasodium pyrophosphate Na4 P2 O7 265.90
Pyrophosphate
Sodium acid pyrophosphate Na2 H2 P2 O7 221.94
Polyphosphate Sodium hexametaphosphate Na6 O18 P6 611.77
Aluminum phosphate Sodium aluminum phosphate AlNaO4 P+ 144.94
Dipotassium phosphate K2 HPO4 174.18
Potassium based CSS
Sodium potassium tartrate C4 H4 KNaO6 210.16
Tartrate Sodium tartrate C4 H4 Na2 O6 194.05

Various milk protein systems, including milk protein concentrate [3,14,15], skim milk
powder [16], rennet casein [12,17], and micellar casein concentrate [18,19], have been
studied to elucidate the effect of CSS on the properties of casein micelles or para-casein
micelles. On the other hand, several researchers have linked the changes in properties
of processed cheese with the influence of CSS on the properties of casein micelles. It is
likely that certain discrepancies between the research articles vis-à-vis the influence of
CSS on the properties of milk protein systems exist. This is due to inter-study differences
in CSS combinations, processing parameters (e.g., time-temperature combinations, mixer
type, and shearing rates), and matrix formulation (e.g., levels of moisture, protein, and
pH). Regardless, these research articles provide insights about the functionality of CSS
in processed cheese matrix development and its effect on rheological, functional and
physicochemical attributes. Therefore, in this review, we will focus on the different types
of CSS and their influence on the solubilization of casein fractions, casein dispersion, and
casein hydration. The changes in the viscoelastic, textural, and functional properties of
Molecules 2023, 28, x FOR PEER REVIEW 3 of 17

Molecules 2023, 28, 2085 3 of 17

cheese systems are also considered in a separate section for better insights on the func-
tionality of CSS in complex milk protein systems.
processed cheese systems are also considered in a separate section for better insights on the
[Link] of CSSofinCalcium
Different Types complexSequestering
milk protein Salts
systems.
CalciumTypes
2. Different sequestering
of Calcium salts Sequestering
have monovalent Saltscations (e.g., Na , K , and H ) and poly-
+ + +

valent anions (e.g., phosphates or citrate). The CSS most commonly used in processed
Calcium sequestering salts have monovalent cations (e.g., Na+ , K+ , and H+ ) and poly-
cheese are divided into two categories: citrates and phosphates. Commonly used citrates
valent anions (e.g., phosphates or citrate). The CSS most commonly used in processed
are trisodium citrate (TSC) and monosodium citrate (MSC), and commonly used phos-
cheese are divided into two categories: citrates and phosphates. Commonly used citrates
phates are disodium phosphate (DSP), tetrasodium pyrophosphate (TSPP), and sodium
are trisodium citrate (TSC) and monosodium citrate (MSC), and commonly used phos-
hexametaphosphate (SHMP) [20,21]. Citrate salts are obtained by replacing the hydrogen
phates are disodium phosphate (DSP), tetrasodium pyrophosphate (TSPP), and sodium
atom from the tribasic citric acid with suitable cations (e.g., Na+, K+), resulting in the for-
hexametaphosphate (SHMP) [20,21]. Citrate salts are obtained by replacing the hydrogen
mation
atom fromof mono-, di- andcitric
the tribasic trisodium citrate.
acid with Depending
suitable on the
cations dissociation
(e.g., Na+ , K+ ), steps, citricinacid
resulting the
may
formation of mono-, di- and trisodium citrate. Depending on the dissociation steps,iscitric
form citrates, hydrogen citrates, and dihydrogen citrates. Trisodium citrate the
most commonly
acid may used citrate
form citrates, CSS in
hydrogen the manufacturing
citrates, and dihydrogen of processed cheese [22].
citrates. Trisodium citrate is the
Food-grade phosphate salts are derived by the purification
most commonly used citrate CSS in the manufacturing of processed cheese [22]. of phosphoric acid
(H3POFood-grade
4). Based on the number of phosphate groups, phosphates are classified as ortho-
phosphate salts are derived by the purification of phosphoric acid (H3 PO4 ).
phosphates
Based on the (or orthophosphates)
number of phosphate (1groups,
P atom)phosphates
and polymeric phosphates
are classified (multiple phos-
as orthophosphates
phates) (>1 P atoms) [22]. The basic structure of phosphates
(or orthophosphates) (1 P atom) and polymeric phosphates (multiple phosphates) consists of each phosphorus (>1 P
atom surrounded tetrahedrally by four oxygen atoms. The
atoms) [22]. The basic structure of phosphates consists of each phosphorus molecular structure ofatom
different
sur-
types of phosphate-based
rounded tetrahedrally by calcium
four oxygensequestering
atoms. The salts are presented
molecular in Figure
structure 1. Orthophos-
of different types of
phates contain (PO
phosphate-based 4)3− anions,
calcium which may
sequestering saltshave up to three
are presented oxygen1. atoms
in Figure covalently
Orthophosphates
bonded to other3 − atoms. The oxygen atom may form linkages
contain (PO4 ) anions, which may have up to three oxygen atoms covalently bonded with phosphorus or other to
atoms (P-O-P), generating condensed phosphates (2 to ~25 P atoms).
other atoms. The oxygen atom may form linkages with phosphorus or other atoms (P-O-P), Linear condensed
phosphates have one shared
generating condensed oxygen(2atom
phosphates between
to ~25 neighboring
P atoms). PO4 groups,
Linear condensed whereas met-
phosphates have
aphosphates (also termed
one shared oxygen as cyclicneighboring
atom between phosphates)PO have three or
4 groups, more oxygen
whereas atoms shared
metaphosphates (also
by neighboring
termed as cyclic PO 4 [Link]
phosphates) Under high
three temperature
or more conditions,
oxygen atoms sharedorthophosphates
by neighboring PO or4
longer
[Link]
Underphosphates with terminal
high temperature -OH orthophosphates
conditions, groups lose water or(condensation reaction),
longer chain phosphates
thus
withbringing
terminal two-OHphosphate
groups losemolecules together to reaction),
water (condensation form polymeric phosphates
thus bringing [20]. Ma-
two phosphate
jor phosphates used in processed cheese production are orthophosphate
molecules together to form polymeric phosphates [20]. Major phosphates used in processed (e.g., disodium
phosphate (P atom are
cheese production = 1)), linear condensed
orthophosphate phosphates
(e.g., disodium such as pyrophosphates
phosphate (P atom = 1)),(e.g.,lineardiso-
con-
dium
densed pyrophosphates
phosphates such (P as
atom = 2)), and polyphosphates
pyrophosphates (e.g., disodium(Ppyrophosphates
atoms = 3–25) (e.g.,(P atom tripoly-
= 2)),
and polyphosphates
phosphate (3 P atoms)) (P[21].
atoms = 3–25) (e.g., tripolyphosphate (3 P atoms)) [21].

Molecular
[Link]
Figure structure
structure of different
of different phosphate-based
phosphate-based calcium
calcium sequestering
sequestering salts salts
used used in
in pro-
cessed cheese
processed manufacture
cheese (P and
manufacture O indicate
(P and phosphorus
O indicate and
phosphorus andoxygen
oxygenatoms,
atoms,respectively,
respectively,whereas
whereas
nnrepresents
represents the
the number
number ofof PP atoms)
atoms) (Created
(Created with
with [Link],
[Link], accessed on 13 February 2023).
Molecules 2023, 28, 2085 4 of 17

3. Effects of Calcium Sequestering Salts on the Properties of Casein Micelles

3.1. Solubilization of Individual Caseins from Casein Micelles
The protein fraction of casein micelles is composed of four different casein fractions:
αs1 -, αs2 -, β-, and κ-casein [5]. Calcium, both in colloidal and soluble form, plays a critical
role in stabilizing the casein micelle structure. de Kort et al. [14] revealed the presence
of “loosely bound calcium”, which is attached to negatively charged amino acid side
chains and phosphate groups, and “strongly bound calcium” in the CCP complexes. Ya-
mauchi et al. [23] defined approximately 40% of the colloidal calcium as “hard-to-exchange”
calcium, which is associated with the colloidal phosphate of the casein micelles in milk.
The removal of Ca2+ with CSS causes the dissociation and subsequent release of casein
protein fractions from the micelles into the serum phase [1].
The dissociation of casein micelles by CSS is described as a cooperative process, which
implies that the casein complex is either completely dissociated or largely intact; it does not
imply that all casein micelles dissociate at the same time and same CSS concentration [1,24].
The dissociation of casein micelles is a two-stage process: a rapid first stage and a slow
dissociation of the remaining casein complexes [1]. Firstly, the addition of low levels of
EDTA disrupted the easily exchangeable Ca2+ bridges and dissociated the weakly held
caseins (mainly αs - and β-casein) from the casein framework. Subsequently, the addition
of more EDTA dissociated the colloidal calcium phosphate and Ca-αs -caseinate bonds and
solubilized the framework protein (largely αs -casein [24]). With an initial decrease in Ca2+
ion activity, approximately 60% β-casein was dissociated from casein micelles without any
micellar disintegration or decrease in the size of the casein micelles. The remaining β-casein
(approximately 40%) seems to be strongly bound to αs - and κ-caseins, maintaining the
structural framework of casein micelles [25,26].
Gaucher et al. [16] reported an order of solubilization of individual caseins (αs1 > β > αs2
and κ-casein) by potassium orthophosphate at levels up to 160 mM. The preferential
solubilization of αs1 - and β-casein by phosphate-based CSS has been linked to their greater
Ca2+ binding ability due to their higher number of phosphoseryl residues in these caseins
as compared to κ-casein [16]. The increased concentration of potassium phosphate from
20 to 160 mM in skim milk showed an increasing trend of αs - and β-casein amounts in a
soluble fraction [16]. However, the ratio of individual soluble caseins was not affected by
concentration and type of CSS (disodium uridine phosphate, DSP, TSC, sodium phytate,
and SHMP) [14]. The casein fractions solubilized by CSS, as analysed in the supernatants
of micellar casein isolates, were in the same ratio as that present in milk [14,18]. Pitkowski
et al. [1] reported similar findings after the addition of EDTA and sodium polyphosphate.
The whey protein β-lactoglobulin has been reported to remain unaffected by CSS, especially
in samples without any heat treatment [16]. In heated MPC samples, CSS solubilized the
κ-casein/whey protein aggregates, represented by the formation of high molecular weight
(50–70 kDa) SDS-PAGE bands [27]. Lastly, the solubilization of casein is largely dependent
on the ratio of CSS to casein, and results in the formation of small micellar particles, which
changes with the increasing concentration of CSS.

3.2. Calcium Chelation Ability of Calcium Sequestring Salts

Calcium ions in casein micelles are part of the CCP complexes or attached to the
phosphoserine, glutamate or aspartate residues. CSS competes with the inorganic phos-
phate of CCP and phosphoserine residues for the calcium ions. The different types of CSS
have different affinities for calcium ions depending on which varying amount of CCP is
released from the micelles [28]. The effectiveness of CSS to bind Ca has been evaluated
mainly (i) by evaluating the Ca2+ ion activity using a calcium-ion selective electrode, (ii) by
non-sedimentable Ca content after (ultra)centrifugation, or (iii) by soluble Ca content after
ultrafiltration. A decreased Ca2+ ion activity and higher amount of non-sedimentable or
soluble Ca is herein indicative of the higher calcium chelation ability of CSS [18].
Molecules 2023, 28, 2085 5 of 17

The Ca binding ability of CSS exhibits the following order: long-chain phosphates > tri-
polyphosphates > pyrophosphate (triphosphates and diphosphates) > citrate > orthophos-
phate [8,10]. SHMP and sodium phytate are strong CSS and bind Ca at a ratio of 1:3 and
1:6 (phosphate:Ca), respectively. DSP also has a strong calcium binding ability, but at a
3:2 ratio. Disodium uridine phosphate forms less strong complexes with Ca and binds
Ca at a ratio of 1:1 [28]. EDTA is a stronger calcium binder than TSC [1,6], but is not
permitted for use in most products. Calcium chelates preferentially with citrate compared
to orthophosphate, because of the lower association constant of HPO4 2− (600 M−1 ) and
H2 PO4- (10 M−1 ) than citrate (Cit3− ) (105 M−1 ) for calcium [4]. Furthermore, the effective-
ness of TSC as a Ca sequestering salt was demonstrated by the reduced casein-bound Ca
and casein-bound inorganic phosphate with increasing levels of added TSC to milk protein
concentrate solution, and added citrate was not associated with casein [29]. Similarly, the
structure of phosphate molecules influenced their calcium chelation ability instead of their
organic and inorganic origin [28]. The replacement of the sodium cation of DSP and TSC
with potassium showed no significant changes in calcium ion activity [12]. The influence
of concentration of CSS, pH and temperature of the gel/solution on Ca2+ chelation is
discussed in the following sub-sections.

3.2.1. Effect of Calcium Sequestering Salt Concentration on Calcium Chelation Ability in

Casein Micelle Suspensions
The majority of the published studies on casein micelle suspensions highlight increased
calcium chelation with increasing concentration of CSS [1,15]. The calcium chelation ability
of CSS becomes constant or shows very insignificant changes after reaching a certain level
of CSS concentration [30]. For CSS such as DSP, TSC, SHMP, disodium uridine phosphate,
and sodium phytate, Ca2+ ion activity decreased with the increasing concentration of CSS
in micellar casein solutions (9%, w/v) within the concentration range of 15–60 mEq/L [18].
Since disodium uridine phosphate is a weak calcium chelator, only a slight decrease in Ca2+
ion activity was observed [14]. For phosphate-based CSS, at lower concentration, the added
phosphate does not displace micellar calcium phosphate, since the affinity of Ca2+ is higher
for micellar phosphate than for added phosphate. The addition of a higher phosphate level
induces the displacement of micellar phase calcium, leading to the demineralization and
solubilization of casein [6,16]. The level of Ca2+ chelation and solubilized casein levels
were elevated by increasing the concentration of added EDTA from 5 to 50 mM/kg of skim
milk. Moreover, the addition of 50 mM EDTA/kg skim milk disintegrated all of the casein
micelles in the milk [6].
Culler et al. [30] modelled the decrease in the turbidity of skim milk by adding
different CSS and defined C* as the threshold CSS concentration, at which rapid casein
micelles dissociation occurs. SHMP showed the greatest decrease in turbidity at the lowest
concentration of 0.33 mM, while monosodium phosphate and dipotassium phosphate had
a C* of 278.22 mM and 216 mM, respectively. In case the polyphosphate concentration
exceeds the critical level required for the complete dissociation of casein micelles, the radius
of non-micellar casein particles is reduced [1].

3.2.2. Effect of pH on Calcium Chelation Ability

The addition of CSS, acid or base to casein micelle containing dairy systems generates
a pH change, which influences the Ca2+ sequestering ability of CSS. The addition of
150 mEq/L of sodium dihydrogen phosphate to milk protein concentrate dispersions (5%,
w/w) decreased the pH from 6.9 to 6.2, whereas TSC caused a pH increase, and SHMP
showed no changes in pH for 15–150 mEq/L [27]. The pH change created by added
potassium dihydrogen phosphate to skim milk (pH 6.65 at 4.5 mM and 5.85 at 157 mM)
induced the shift of acid-based equilibrium of the phosphate from HPO4 2− towards the
H2 PO4 − form. Due to the higher affinity for calcium of HPO4 2− (600 M−1 ) compared to
H2 PO4 − (10 M−1 ), the former is considered to be an effective Ca2+ binder [16]. DSP and
TSC concentrations of 20 and 40 mM/L showed a decreasing influence on Ca2+ ion activity,
Molecules 2023, 28, 2085 6 of 17

with a pH change from 6.7 to 8.0. However, at very high concentrations (60–100 mM/L),
non-significant influences of pH are reported [12].
In evaluating the effect of pH (5.0–8.8) on calcium chelation by ten different CSS,
Culler et al. [30] reported the highest calcium chelation at pH 5.8 and 6.8. Similarly, with
increasing pH from 6.7 to 7.3, the calcium ion activity of DSP, TSC and SHMP was found to
decrease, but the decrease became smaller with increasing pH [18]. This is credited to more
significant calcium phosphate complex formation and enhanced electrostatic repulsion
between caseins at higher pH [31,32].
As pH values shift closer towards the isoelectric point of casein, a decrease in calcium
chelation ability of CSS is also observed. Culler et al. [30] reported the lowest casein
dispersion by different CSS at pH 5.0. Likewise, sodium phytate showed no significant
binding of calcium below pH 5.0, but between pH 5.0 to 8.0, calcium binding occurs in the
ratio of 6:1 (calcium:phytate) [33]. At very low pH values, especially near the isoelectric
pH of casein (pH 4.6), the CCP and probably calcium-pyrophosphate complexes from
TSPP might dissolve [3]. The casein micelle structure becomes more compact near the
isoelectric pH. This tighter micelle structure is more dependent on aggregation from the
protein charge than calcium phosphate bridges, reducing its susceptibility to dissociation
caused by the calcium chelation of CSS [30].

3.2.3. Effect of Heat Treatment on Calcium Chelation Ability

Heat treatment does not change the composition of aqueous solution of orthophos-
phate or orthophosphate solution in the presence of Ca [34]. At temperatures below 100 ◦ C,
hydrolysis of polyphosphate is almost negligible in water at pH 7 and pH 5.6; however,
their composition is affected by the presence of Ca and temperatures above 120 ◦ C [35,36].
The presence of Ca increased the hydrolysis of short-chain and long-chain polyphosphates
(>4 phosphorus atoms) into trimetaphosphates and orthophosphates. This is linked to the
increase in the positive charge of phosphorus atoms in long-chain phosphates by the pres-
ence of Ca, which may promote hydrolytic degradation [36]. Hydrolysis occurs through
the nucleophilic reaction of water on the terminal phosphate unit leading to the breaking
of the P-O-P bond and the formation of orthophosphates as end products [37].
The heating (>85 ◦ C) of milk protein systems after adding CSS induces the rapid
hydrolysis of linearly condensed phosphates to tripolyphosphates and pyrophosphates,
and then more slowly to orthophosphates [9]. By heating micellar casein isolate solutions
to 126 ◦ C, SHMP hydrolyzes into sodium trimetaphosphate and sodium orthophosphate in
acidic conditions, which induces a decrease in pH, the release of Ca ions, and increased
calcium-ion activity. As a result, SHMP forms cross-links between caseins that are released
during heating [18]. The hydrolysis of phosphate-based CSS increases the ratio of short-
chain to long chain phosphates, thereby affecting the calcium chelation [9]. The heat
treatment (128 ◦ C for 5 min) of milk prior to the addition of sodium citrate and EDTA
diminished the ease of solubilization of micellar calcium phosphate [6]. Generally, heating
leads to the hydrolysis of phosphate-based CSS into short chain components, causing lower
Ca chelation possibilities.
Overall, phosphates and citrates have very different effects on Ca chelation in sim-
plified model milk protein systems, with each CSS demonstrating strong dependence on
concentration, pH and temperature, as the role of CSS in a concentrated casein-based
matrix may be more complex than previously believed. The influence of different calcium
sequestering salts and their increasing concentration on the properties of casein micelles is
presented in Table 2.
Molecules 2023, 28, 2085 7 of 17

Table 2. Effect of different calcium sequestering salts on the properties of casein micelles.

Properties Effect of Calcium Sequestering Salts (CSS) Increasing CSS Level Reference
• Order of solubilization- αs1 > β > αs2
Solubilization of individual and κ-casein
↑ [14,16]
casein fractions • Ratio of individual soluble caseins is not affected
by type of CSS
Long-chain phosphates > tri-polyphosphates
Calcium chelation ability > pyrophosphate (triphosphates and di-phosphates) ↑ [8,10]
> citrate > orthophosphate
Disodium uridine phosphate < disodium phosphate
Casein dispersion < trisodium citrate < sodium phytate < sodium ↑ [14,18]
hexametaphosphate
Tripolyphoshates > pyrophosphates > polyphosphates
Emulsion droplet size > citrates ≈ orthophosphates ≈ sodium - [9,38,39]
aluminium phosphates
• Phosphates: Increased casein hydration with
Casein hydration increasing chain length of sodium phosphates ↑ [38,39]
• Citrates: Lower than ortho- and pyro-phosphates

• Phosphates: Insoluble Ca phosphate complexes

Complex between CSS and Ca - [1,12]
• Citrates: Soluble Ca citrate complexes
The general effects of different calcium sequestering salts are summarized from the published literature. However,
the precise effects of changing them may depend on several factors, including their interactions.

3.3. Casein Dispersion

The degree of casein dispersion within a processed cheese system is linked to the
ability of CSS to bind calcium complexes and disrupt the calcium phosphate crosslinks in
the para-casein matrix [3,7]. The flexible hydrophilic parts of caseins can be immobilized
by calcium phosphate, which imparts a more rigid structure to casein micelles [40]. Casein
dispersion has been evaluated by measuring the optical density/turbidity of model protein
system (or processed cheese). Lower optical density or decreased turbidity represents more
extensive casein dispersion or removal of CCP from the micelles [7,14]. The dissociation
rate and extent is largely determined by the ratio of CSS to casein [1]. Panouillé et al. [2]
and Pitkowski et al. [1] observed that calcium chelators induce the dissociation of intact
casein micelles into smaller casein particles containing 10–15 casein proteins. A mixture of
CSS (sodium polyphosphate and sodium citrate) dissociated casein micelles into smaller
particles, with a diameter of 12 nm [2].
Based on the findings of different studies, phosphates with longer chain length and a
higher proportion of polyphosphates in the CSS mixture showed higher casein dispersion [7,10].
de Kort et al. [14] and de Kort et al. [18] reported that casein micelles were dissociated
in the order of disodium uridine phosphate < DSP < TSC < sodium phytate < SHMP.
Casein micelles were not dispersed by the addition of DSP to a milk protein concentrate
solution [15]. At a concentration of ≥45 mEq/L of SHMP, most of the casein micelles
were dissociated, induced by pH decrease, the increased net negative charge of casein
micelles, and the depletion of CCP from the casein micelles [18]. Similarly, the addition of
150 mEq/L of TSC to transglutaminase-treated skim milk removed all of the CCP from the
casein micelles, while the micelle remained intact [5,41].
Overall, the casein dispersion ability of different CSS follows a trend similar to the
Ca2+ sequestering ability of the CSS, but the ability of some CSS to cross-link caseins
simultaneously creates differences in casein dispersion ability. SHMP and sodium phytate
have six homogeneously distributed and twelve (clustered in pairs) negative charges
around the molecule, respectively. This led to a stronger calcium sequestering ability of
sodium phytate than of SHMP. The immediate strong chelation of Ca by sodium phytate
may leave no charge or free calcium ions available for the cross-linking of caseins. Thus,
Molecules 2023, 28, 2085 8 of 17

sodium phytate does not cross-link caseins, whereas SHMP does, thereby contributing
to higher casein dispersion by SHMP [14,28]. Similarly, tetrasodium pyrophosphate with
four homogenously distributed charges around its molecule cross-links casein more easily
than SHMP [3]. Ultimately, the commencement and degree of casein dispersion is largely
dependent on the type and concentration of CSS, which could be helpful in controlling
the viscosity, turbidity and heat stability of dairy systems. Furthermore, the insights on
the effect of the mixtures of CSS on casein dispersion could provide new opportunities for
modifying the desirable properties of casein-based matrices.

3.4. Formation of Complexes between Calcium Sequestering Salts and Calcium

Phosphate-based CSS may associate with dispersed caseins, while caseins dispersed
by TSC may not necessarily aggregate and form gels [3]. Interestingly, TSC chelates Ca
from indigenous CCP and forms soluble Ca citrate complexes, while DSP chelates Ca
and forms an insoluble Ca phosphate complex, which may be trapped within the protein
matrix [12]. EDTA also forms soluble complexes with Ca, which are less soluble than citrate-
Ca complexes [1]. The tendency of TSC to form soluble complexes decreases the amount of
casein-bound Ca and P. Instead, in the case of phosphate-based CSS, the formation of Ca
phosphate complexes increases the casein bound Ca and P [15].
At a lower concentration (0.1%) of SHMP and TSPP, insoluble Ca-phosphate com-
plexes were observed [29]. At a higher concentration of SHMP (0.5–0.7%), soluble CaHMP
complexes were formed. This is attributed to the excessive charge repulsion resulting from
the multiple negative charges introduced by SHMP molecules [15]. The addition of tetra-
sodium pyrophosphate dispersed the caseins and formed casein-calcium pyrophosphate
complexes [29]. Shirashoji et al. [42] reported the formation of insoluble casein-calcium
pyrophosphate complexes at higher concentrations of TSPP (>1%), with a portion of TSPP
remaining in the soluble phase. The binding of calcium pyrophosphate complexes with
dispersed casein could reduce the charge repulsion, thus facilitating the hydrophobic
interactions between hydrophobic segments of caseins and promoting aggregation [3].
Condensed phosphates such as pyrophosphate, tripolyphosphate and hexametaphos-
phate form more stable casein-Ca-phosphate complexes than with orthophosphate. The
addition of tetrasodium pyrophosphate (7.6 mM) to milk protein concentrate solution
(51 g/L) dispersed the casein and formed casein-Ca pyrophosphate complexes [29]. The
dispersion of casein is due to the loss of calcium phosphate cross-links, which may expose
charged phosphoserine groups, thus increasing electrostatic repulsion between caseins [43].
The formation of stable casein-Ca-phosphate complexes could be attributed to complexes
of condensed phosphates, with dispersed casein leading to a reduction in the electrostatic
repulsion between casein molecules [3]. The stability constant (the equilibrium constant for
complex formation, which measures the strength of interaction between reagents involved
in complex formation), indicates the relative efficiency of chelators’ complexation with Ca2+
and is also higher for pyrophosphate (5.0) than citrate (3.5) [44]. Furthermore, in processed
cheese systems, phosphates associated with casein micelles might act as cross-linking
agents within or between casein micelles, thus affecting their functional attributes such as
low meltability and higher firmness.

3.5. Casein Hydration

Rennet casein is insoluble in water owing to the presence of calcium mediated cross-
bridges [17]. In order to add more water to the processed cheese, casein locked in the
micellar structure has to be released by the usage of CSS [45]. Ca chelation by CSS swells
and partially hydrates the insoluble casein and converts it to water soluble caseinate. This is
achieved by the exchange of Ca2+ in the casein network with monovalent cations (e.g., Na+ ,
H+ , K+ ) of CSS, also leading to the increased negative charge on caseins [12,14]. Owing to
calcium chelation, the concentration of free calcium ions decreased in the continuous phase,
thereby increasing the negative charge and electrostatic repulsion of the casein micelles,
finally leading to more hydrated and swollen casein micelles [14]. The extensive hydration
Molecules 2023, 28, 2085 9 of 17

of casein molecules permits their interaction with the oil phase, promoting the stabilization
and emulsification of fat globules [45].
Cavalier-Salou et al. [38] suggested an increase in para-casein hydration with the
increasing chain length of sodium phosphates added to cheese analogues. TSC gives lower
casein hydration than ortho- and pyro-phosphates [39]. Huppertz et al. [41] also showed
the swelling of casein micelles induced by trisodium citrate (0–50 mM) in cross-linked
casein micelles suspension caused by the dissociation of micellar calcium phosphate. The
addition of sodium citrate (238 mM) and sodium phosphate (173 mM) was linked to
better rehydration rates and the higher moisture content of the native phosphocaseinate
suspension. CSS solubilized the casein micelles, and water bound to micellar casein was
more difficult to remove than water bound to soluble casein [46].
Sodium cations are more effective binders than potassium to carboxylate anions of
amino acid residues in casein. The change of sodium cation with potassium in CSS leads to
less cation binding and consequently less hydrogen ion displacement, eventually causing
a higher pH. This higher pH leads to enhanced electrostatic repulsion between casein
molecules, which may facilitate better protein hydration. It is also suggested that smaller
hydrated potassium ion sizes facilitate higher calcium chelation and the easier hydration
of the casein matrix [47]. A small variation in moisture content causes large changes
in textural, rheological and functional attributes of processed cheese, especially at low
moisture levels. Thus, the degree of casein hydration could have a significant role in
achieving the maximum level of moisture content and the final desirable attributes of
processed cheese.

4. Effect of Calcium Sequestering Salt on the Properties of Processed Cheese

The manufacturing of processed cheese involves the selection of different natural
cheeses, the cleaning and size reduction of natural cheeses into small curd particles, the
mixing with water and sequestering salts, the shearing of the blend under the influence
of heat, and hot packaging and cooling. The conversion of milk into natural cheese
involves the destabilization of the milk protein network, mostly calcium phosphate, into a
concentrated para-casein network occluding fat [32]. The protein in natural cheese curd
occurs as para-casein micelles fused in a network, which is rendered insoluble by inter-
protein linkages mediated by calcium, colloidal calcium phosphate, and hydrophobic
interactions between uncharged amino acid residues. On the conversion of natural cheese
into processed cheese, the calcium phosphate para-casein network is deconstructed with
partial protein solubilization, enabling it to bind water and emulsify free fat released
during the heating and shearing stage [21]. This is confirmed by the increased level of
water-soluble protein from 5–20% in natural cheese to 60–80% in processed cheese [48].
The “loose” oil-in-water (o/w) emulsion of natural cheese consisting of the concen-
trated gelled calcium phosphate para-casein network changes into a “finer” o/w emulsion
in a concentrated casein(ate) dispersion in processed cheese. The hydrated casein/para-
casein immobilizes free serum and emulsifies free fat into emulsified fat globules and
creates a stabilized processed cheese. The fat globules in natural cheese are naturally emul-
sified by the native fat globule membrane consisting of protein and phospholipids, whereas
in processed cheese, fat is emulsified with a reformed layer of re-hydrated para-caseinate.
The fat globules in processed cheese are considered as pseudo-protein particles, which may
interact with other emulsified fat globules [9]. Depending on the formulation, processing
conditions and type of sequestering salts, the final processed cheese product may vary
from firm and sliceable to soft and spreadable. However, differences in the functionality of
CSS affects the properties of processed cheese and offers the ability to cheese manufactur-
ers to customize the properties of the final product. CSS facilitates the processed cheese
manufacturing process by sequestering calcium, enabling the swelling and hydrating of
casein, casein peptisation, the emulsifying of free fat, the demineralising of casein, and pH
adjustment [21]. A myriad of research studies have compared the effect of different types,
combinations and levels of CSS on different physiochemical and functional properties of
Molecules 2023, 28, 2085 10 of 17

processed cheese. This section describes the influence of CSS on various physiochemical,
functional, rheological, and sensory properties of processed cheese.

4.1. Emulsion Droplet Size

Caseins are the major emulsifiers in the processed cheese matrix, and a lower degree
of emulsification is revealed by the larger diameter of fat globules [49]. The ability of
CSS to promote emulsification generally coincides with the trend of calcium sequestra-
tion. The emulsification potential of CSS follows the following order: tripolyphoshates
> pyrophosphates > polyphosphates > citrates ≈ orthophosphates ≈ sodium aluminium
phosphates [9,38,39]. A higher degree of calcium sequestration causes more intensive casein
dispersion developing the emulsification and hydration properties of these proteins. These
proteins stabilize the fat globules by acting as a membrane leading to fat emulsification and
higher casein cross-links [10].
During the manufacturing of processed cheese, free fat is separated during the initial
heating stage, which is re-emulsified by CSS under the influence of heating [50]. Emulsified
fat globules have a lower tendency to coalesce on reheating and are generally thermostable,
contributing to the lower meltability of processed cheese [51]. Processed cheese samples
prepared with polyphosphates and pyrophosphates had a higher number of fat globules
compared to those with citrates and orthophosphates [50]. Low levels of emulsification
results in softer processed cheese, and well emulsified processed cheese shows higher
hardness and reduced meltability [52].

4.2. Textural Properties

In the course of processed cheese manufacturing, the addition of CSS in combination
with heating and high-speed shearing disperses the insoluble casein matrix. During the
cooling stage, these dispersed casein strands reassociate, besides CSS-Ca complexation,
which significantly affect the textural properties of processed cheese [43]. A higher extent
of casein dispersion in the processed cheese matrix increases the emulsifying and hydrating
potential of caseins, which is responsible for the stabilisation of fat and water available in
processed cheese. Concurrently, better protein hydration and fat emulsification generates a
higher intensity of casein cross-links and a harder processed cheese [3,8].

4.2.1. Effect of Calcium Sequestering Salt Type on Textural Attributes of Processed Cheese
Trisodium citrate provided a higher value of processed cheese hardness as compared
to disodium phosphate and orthophosphates, but a lower value than polyphosphates and
pyrophosphates [51,53]. These differences were mainly linked to the bigger fat globule size
observed in DSP-based cheeses, which have low surface area and less interactions with
protein [51]. During processing (melting stage), polyphosphates undergo rapid hydrolysis
into triphosphates and diphosphates, causing a substantial increase in the hardening of
processed cheese [52]. This is attributed to the higher ability of products of hydrolysis
(especially triphosphates) to aggregate casein and emulsify fat, thus forming a 3-D network
and leading to a more rigid and elastic processed cheese [9,29,52]. About 50% of the added
phosphates are hydrolysed during the melting procedure, and the remainder is hydrolysed
after 7 to 10 weeks of storage [54]. The products of hydrolysis possess a greater ability
to induce aggregation by the formation of the caseinate-Ca phosphate complex and form
a more rigid and elastic structure. The ability to form a rigid structure and support a
three-dimensional network follows the following order: orthophosphate < polyphosphate
< diphosphate < triphosphate [9,29]. Furthermore, diphosphates and triphosphates cause
better fat emulsification, resulting in the higher firmness of processed cheese [50]. It has
also been suggested that citrates dissociate on cooling post-manufacture, subsequently
acting as a calcium ion source. This also means that some of the insoluble Ca-citrate present
in processed cheese can dissociate/dissolve on cooling. These calcium ions cross-link the
CSS anions attached to casein and increase the hardness of processed cheese [13,51,55].
Molecules 2023, 28, 2085 11 of 17

Within the phosphate CSS category, the hardness of processed cheese generally in-
creases with the increasing number of phosphorus atoms present in CSS [56]. Orthophos-
phates are low molecular weight substances with the ability to permeate among cross-linked
caseins and strongly bind water. In addition, orthophosphates have very low calcium ion
exchange ability, resulting in the lowest hardness of processed cheese [7,52]. Diphosphates
act as cross-linking agents by forming complexes with calcium ions (caseinate-Ca phos-
phate complexes) and reduce charge repulsion, thus inducing the gel formation of casein
proteins and higher hardness values [55,56]. The excessive incorporation of diphosphates in
processed cheese binds too much calcium, making it unavailable for diphosphate-calcium
cross-linking interactions, and resulting in the lower hardness of processed cheese [56].
Long chain polyphosphates can not only bind calcium strongly, but also disperse casein
effectively. Polyphosphates bind to casein fractions and provide them with strong multi-
ple negative charges [10]. These extensively charged casein fractions disallow sufficient
gel formation, thus generating difficulties in casein re-association through hydrophobic
segments [3,8]. In phosphate based emulsifying salts, ion exchange ability (Na+ for Ca2+ )
increases with the increasing number of phosphorus atoms linearly bound in a phosphate
molecule. Long-chain polyphosphates have the highest ion-exchange ability, resulting in
the greatest degree of hardness of processed cheese. When polyphosphates are used in
combination with orthophosphate and/or diphosphate up to 50–60%, greater hardness is
observed. In the case of a higher proportion of polyphosphates (60% or more) in CSS mix,
the hardness of processed cheese is decreased [52,56]. This could be explained by the ability
of polyphosphates to give caseins a multiple negative charge, which reduces the specific
effects of orthophosphates and diphosphates, and the formation of a three-dimensional
network of the melt dominates. As a result, the ability of long-chain polyphosphates to
strongly disperse casein chains prevail, leading to increased casein hydration and better
fat emulsification. Thus, orthophosphates and/or diphosphates in a lower amount cannot
show their specific properties, and yields processed cheese with a lower hardness [57].
The use of SHMP imparted higher hardness to processed cheese in comparison to
TSC or orthophosphates. This is attributable to the enhanced casein dispersion (hydration,
peptization, or swelling) and the Ca chelation ability of SHMP. SHMP disperses the casein
molecules, which results in greater cross-linking during the cooling stage of processed
cheese and a firmer cheese [8]. Another study reported highest hardness of SHMP in-
corporated processed cheese followed by tetrasodium pyrophosphate, trisodium citrate
and disodium phosphate cheese, respectively [53]. However, processed cheese prepared
with tetrasodium pyrophosphate (TSPP) had higher hardness in comparison to DSP and
SHMP, which was linked with the higher fat particle size reduction ability of TSPP. Smaller
fat globules offer a higher number of interaction points with protein, thus making the
network firmer [13].

4.2.2. Effect of Calcium Sequestering Salt Concentration on Textural Attributes of

Processed Cheese
Generally, increasing the concentration of CSS has been linked with the increasing
hardness of processed cheese. An increase in the level of TSC, TSPP, STPP, SHMP and
DSP from 1 to 3% showed an increase in hardness values [13]. Shirashoji et al. [51] also
reported the increased hardness of processed cheese with the increasing concentration of
TSC from 0.25% to 0.75%. The increased concentration of CSS improves fat emulsification
and casein dispersion. Obviously, at a lower CSS concentration, fat globules are poorly
emulsified [55]. With increased CSS concentration coupled with shearing treatment during
the cooking stage of processed cheese manufacturing, fat globules are emulsified and
covered as casein. These casein-covered fat globules behave like large pseudo-protein
particles and are actively incorporated into the casein network of processed cheese. This
allows the formation of a more reinforced network of protein stabilized fat globules and
the higher hardness of processed cheese [9].
Molecules 2023, 28, 2085 12 of 17

Overall, several researchers present conflicting findings for textural attributes of

phosphate- and citrate-based processed cheese. These discrepancies are related to the
differences in blend formulation, processing conditions, pH, and type and concentration
of CSS. The effect of different CSS on physico-chemical, textural, functional and sensorial
attributes of processed cheese are summarized in Table 3.

Table 3. Effect of different calcium sequestering salts on physico-chemical, functional and sensorial
properties of processed cheese.

Properties Effect of Calcium Sequestering Salts Reference

Hardness Strong effect of concentration and type of CSS- lot of contradictory reports [51–53]
Less rigid structure (lower G0 ) of orthophosphate < polyphosphate < diphosphate
Viscoelastic [51,52]
< triphosphate.
• Higher meltability of TSC-based processed cheese than DSP, tetrasodium
pyrophosphate, and sodium aluminium phosphate
Melting ability [9,58]
• Sodium aluminium phosphate ≈ trisodium citrate > disodium phosphate > sodium
tripolyphosphates ≈ tetrasodium pyrophosphate > long chain polyphosphates

• Increasing pyrophosphate content: shinier and whiter processed cheese

Color [59,60]
• Whiter processed cheese with SHMP than TSC and tetrasodium pyrophosphate

• Sodium polyphosphate (5.8) < disodium phosphate (6.6) < sodium tripolyphosphate
pH (6.7) < tetrasodium pyrophosphate (6.8) < trisodium phosphate (6.9) [52,56]
• Decrease in pH of processed cheese with increase in length of phosphate chain of CSS

• Phosphates: Soapy, chemical or salty flavour

Sensory • Pyrophosphates: Bitterness at 2% (w/w) level [61–63]
• Potassium based CSS: salty, metallic or bitter taste

4.3. Viscoelastic Properties

The viscoelastic properties of processed cheese have been evaluated and described
by mainly performing two different oscillatory rheological tests, i.e., temperature sweep
and frequency sweep. During temperature sweep, the storage or elastic modulus (G0 )
and loss or viscous modulus (G00 ) are measured at a fixed frequency and a specified rate
of temperature increase. A sample may show liquid-like behavior (G00 > G0 ), solid-like
behavior (G00 < G0 ), and point of heat-induced state (solid ↔ liquid) transition (G00 = G0 ).
The ratio of G00 to G0 is termed as the phase angle (δ), and a phase angle equal to 45◦
(tan δ = 1) represents the gel-sol transition point, while δ greater than 45◦ indicates a
liquid-like (or melt) behavior (Lee & Anema, 2009). The breakage of intermolecular casein
interactions by CSS creates the potential for new interactions between caseins via hydrogen,
hydrophobic, and electrostatic bonds [51]. Lucey et al. [43] reported that lower values of
the loss tangent for processed cheese indicates less bond mobility.
The calcium sequestration leads to the swelling of casein micelles, followed by their
enhanced hydration and voluminosity, which in turn increases the viscous modulus [12].
Processed cheese samples prepared with DSP and TSC exhibited more liquid like behavior,
and TSPP and pentasodium tripolyphosphate based processed cheese showed solid-like
behavior [53]. The formation of a more rigid structure (higher G0 ) of processed cheese by
different CSS showed the following order: orthophosphate < polyphosphate < diphosphate
< triphosphate. Processed cheese with a higher number of phosphate groups permits
interactions within or between casein molecules, especially via calcium bridges resulting in
a more rigid structure [52]. The increasing concentration of DSP from 0.75 to 3.40 g/100 g
processed cheese reduced the maximum tan δ and increased G0 (Guinee & O’Kennedy,
2012). Shirashoji et al. [51] and Shirashoji et al. [8] publicized similar trends for trisodium
citrate and sodium hexametaphosphate at added levels of 0.25–2.75 g/100 g, respectively.
The textural and rheological properties of processed cheese are largely dependent on similar
Molecules 2023, 28, 2085 13 of 17

factors and show a similar kind of variation. It would be interesting to study the correlation
among different measurable variables defining these properties.

4.4. Melting Properties

Meltability is the physical characteristic of cheese highlighting the melting properties
of fat globules and the reorganisation of the protein structure (the major weakening of the
bond between proteins) [52]. Tatsumi et al. [64] determined a decrease in meltability of
processed cheese with an increase in insoluble casein, determined by the centrifugation
of model cheese dispersions containing sodium caseinate, butter fat, and water. However,
the studied model’s processed cheese systems did not contain sequestering salts, but the
decrease in meltability was linked to the formation of insoluble casein at 80 ◦ C due to
the heat-induced aggregation of casein. When the number of casein-casein interactions
decreases due to the extensive proteolysis of natural cheese or the chelation of Ca, the
meltability of the processed cheese is increased. The reduced number of colloidal cal-
cium phosphate cross-links and increased electrostatic repulsions by exposure of negative
charges of phosphoserine residues increased the meltability of processed cheese added
with TSC [43]. The addition of disodium oxalate to the CSS mix for rennet casein-based
processed cheese considerably improved the meltability than that prepared with disodium
phosphate and tetrasodium pyrophosphate individually. Oxalate not only acted as a cal-
cium sequestering agent but also reduced the emulsification, which was indicated by the
size of the large fat globules [58].
The TSC-incorporated model rennet casein-based processed cheese showed higher
meltability than that prepared with DSP, tetrasodium pyrophosphate, and sodium alu-
minium phosphate, owing to the greater casein dispersion by TSC. Phosphate-based CSS
demonstrates extra casein-CSS interactions, reducing the melt [58]. The melting proper-
ties of processed cheese prepared with different CSS shows the following trend: sodium
aluminium phosphate ≈ trisodium citrate > disodium phosphate > sodium tripolyphos-
phates ≈ tetrasodium pyrophosphate > long chain polyphosphates [9]. With the increasing
concentration of disodium phosphate from 0.5 to 4.0 g/100 g processed cheese, a decreased
trend of meltability was reported [48]. Similarly, the highest concentration of TSC at the
level of 2.75% exhibited the lowest meltability in processed cheese [51]. The reduction in
meltability was due to increased fat emulsification and greater immobilisation and struc-
turing of the aqueous phase [48]. A higher degree of fat emulsification produces a higher
number of casein-covered emulsified fat particles, positively contributing to the structural
integrity of the processed cheese matrix [13,48]. Overall, the hardness and meltability of
processed cheese seem to be inversely related. General trends emerge showing the desirable
melting properties of orthophosphates, citrates and sodium aluminium phosphates. In
contrast, condensed phosphates have poor melting properties.

4.5. pH
The pH of processed cheese commonly varies between 5.5 and 6.0, depending on com-
positional and processing factors. High pH processed cheese products are moist and elastic,
while a low pH leads to dry, short (more brittle) and crumbly processed cheese with a high
susceptibility to fat separation (correlated with less fat emulsification) [61,65]. At a pH of
5.2, granular processed cheese with less emulsification and large protein aggregates was
obtained [65]. CSS plays a significant role in the final pH adjustment and stabilization of pro-
cessed cheese owing to its buffering capacity [21,56]. The pH of 1% aqueous solution of a few
commonly used CSS such as trisodium citrate, disodium phosphate, tetrasodium pyrophos-
phate, and sodium hexametaphosphate was found to be 6.2–6.3, 8.9–9.1, 10.2–10.4, and 6.0–7.5,
respectively [21]. The pH of processed cheese (without adjustment) increased in the order
of sodium polyphosphate (5.8) < disodium phosphate (6.6) < sodium tripolyphosphate
(6.7) < tetrasodium pyrophosphate (6.8) < trisodium phosphate (6.9) [52].
It has been established that sodium salts of phosphates increase the pH of processed
cheese in all but a few exceptions, such as disodium pyrophosphate at the rate of 3 g/100 g
Molecules 2023, 28, 2085 14 of 17

processed cheese, which yielded a pH of 4.7 in the final product [52]. A few other examples
of acidic CSS are monosodium citrate, monosodium phosphate, and sodium hexametaphos-
phate, which resulted in processed cheese with a pH of 5.2 or lower, having mealy, dry
and crumbly textures [61]. The pH of the processed cheese samples decreased with the in-
creasing amount of polyphosphates ((NaPO3 )n , where n was 15–20)) in the ternary mixture
of CSS [56]. Nagyová et al. [10] reported a decrease in pH of processed cheese with the
increase in length of the phosphate chain of CSS. This was credited to the higher availability
and release of hydrogen cations in long phosphates as compared to shorter phosphate
CSS. Owing to the acidic pH of monosodium citrate (pH of 1% aqueous solution—3.75)
and disodium citrate, these are suggested to achieve the desired pH of processed cheese,
when high-pH cheese, skim milk solids, or mature natural cheese are used in the processed
cheese blend [9].

4.6. Color
The color of processed cheese is largely dependent on the type, composition and
properties of natural cheese, processing parameters, colorants (majorly annatto and pa-
prika), and other ingredients. The color of processed cheese usually varies from yellow to
orange; in some circumstances where mould cheeses are used it could have a blue, green
or greyish tinge [66]. Fat content and the size of the fat globules dispersed in the cheese
matrix also influence the color of the processed cheese. Smaller fat globules disperse more
light, leading to a whiter color of processed cheese [59]. The lightness values (L*) decrease
upon the addition of CSS due to Ca binding and the dissociation of casein micelles. The
addition of sodium phosphate to milk protein concentrate dispersions showed no change
in L* values, indicating no dissociation of casein micelles. Since SHMP also binds colloidal
calcium and dissociates casein micelles, SHMP showed a higher decrease in L* values
than TSC within the similar concentration range of 15–150 mEq/L [27]. With increasing
pyrophosphate content, shinier and whiter processed cheese samples were obtained due to
their higher soluble protein content [60]. Processed cheese prepared with SHMP was whiter
than TSC and tetrasodium pyrophosphate, which was linked with the smaller fat globule
size of SHMP and the absence of interactions between citrate and casein in TSC-based
processed cheese [59].

4.7. Sensory Properties

There are very few studies pertaining to the influence of CSS on the sensory evaluation
of processed cheese. The replacement of sodium-based CSS by potassium-based CSS up to
50% showed no adverse influence on the sensory qualities of processed cheese [62]. The
replacement with potassium-based CSS above 50% led to a certain salty flavor and metallic
or bitter taste [63]. A soapy, chemical or salty flavor has been associated with phosphates.
At a level of 2% (w/w), pyrophosphates may impart bitterness in processed cheese [61].
Sodium citrates generally impart a “clean” flavor to processed cheese, whereas potassium
citrate may cause bitterness [9]. The use of sodium potassium tartrate caused grittiness due
to the formation of calcium tartrate [61].

5. Conclusions
The influence of a large number of variables (processing as well as compositional) on
the rheological, functional and textural attributes of processed cheese manifests that it is a
challenge to have a single method and set of processing conditions. To better control pro-
cessed cheese quality, there needs to be a better understanding of ingredient functionality
that influences the possible chemical interactions responsible for its quality. The effect of
CSS on pH, calcium chelation, the degree of casein dissociation, and fat emulsification is
interlinked and greatly explains the variations in processed cheese quality attributes.
Molecules 2023, 28, 2085 15 of 17

Author Contributions: Conceptualization, G.K.D., L.G.G.-M., M.F. and T.H.; literature review and
writing original draft—G.K.D.; manuscript review, corrections and editing—T.H. and L.G.G.-M.;
supervision, L.G.G.-M., M.F. and T.H.; project administration, L.G.G.-M., M.F. and T.H.; funding
acquisition, L.G.G.-M. and T.H. All authors have read and agreed to the published version of
the manuscript.
Funding: Gaurav Kr Deshwal is a recipient of a Walsh Scholarship from Teagasc, the Irish Agricultural
and Food Development Authority (Ref 2020213).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare that they have no conflict of interest.
Sample Availability: Not applicable.

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