International Dairy Journal 73 (2017) 116e127

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International Dairy Journal

journal homepage: www.elsevier.com/locate/idairyj

Contribution of fluorescence spectroscopy and independent

components analysis to the evaluation of NaCl and KCl effects on
molecular-structure and fat melting temperatures of Cantal-type
cheese
M. Loudiyi a, b, R. Karoui c, D.N. Rutledge d, R. Lavigne b, M.-C. Montel b,
A. Aït-Kaddour a, b, *
a
VetAgro Sup, 89 Avenue de L'Europe, 63370, Lempdes, France
b
Universit
e Clermont Auvergne, INRA, UMRF, F-15000, Aurillac, France
c
Universit
e D'Artois, EA 7394, Institut Charles VIOLLETTE, Lens, F-62300, France
d
UMR Ing enierie Proced
es Aliments, AgroParisTech, INRA, Universit
e Paris-Saclay, 91300, Massy, France

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

Article history: One of the main objectives of the present study was to investigate by different analytical techniques
Received 14 June 2016 (physicochemical analysis, dynamic rheology, and synchronous fluorescence spectroscopy) the impact of
Received in revised form the NaCl reduction and its substitution by KCl on the molecular structure and fat melting of Cantal-type
8 May 2017
cheese. Molecular structure changes were investigated on five cheese sample formulations from 20 to
Accepted 9 May 2017
60
C with five offsets using SF spectroscopy coupled with independent components analysis. Results
Available online 12 June 2017
showed that significant differences were observed for protein, Cl, Ca, Na and K contents of cheeses.
Complex viscosity decreased as the temperature increased for the different cheeses. SF spectroscopy
provided relevant information related to protein and fat structures with varying salt concentrations and
type during melting, allowing investigation of molecular structure changes of the cheeses. In addition,
similar fat melting temperatures for each cheese were obtained regardless the technique used (dynamic
rheology and fluorescence).
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Cheese is growing in commercial importance because of its use
as an ingredient in cooked dishes (e.g., topping on pizzas, sliced in
sandwiches). Processed cheeses are the most used, but a growing
proportion of natural cheeses have the same trend. Although many
cheeses exhibit sensorial and technological properties that lend
themselves to direct use as an ingredient (e.g., Emmental, Comte ),
specific manufacturing has emerged recently for the same appli-
cation. It is likely that in the future, cheese manufacturing will
evolve to meet specific ingredient uses.
This increase in commercial importance is facing an emerging
health recommendation: reducing sodium intake in the human
diet. Indeed, processed products, such as baked goods (24% of salt
* Corresponding author. Tel.: þ33 473981378.
E-mail address: abderrahmane.aitkaddour@vetagro-sup.fr (A. Aït-Kaddour).
intake), soups (16.5% of salt intake), meats (12.5% of salt intake) and
cheeses (7e11% of salt intake) are among the richest dietary sources
of salt (Henderson et al., 2003). The minimum adult requirement
for NaCl is about 0.5 g per day, but the average total daily intake in
developed countries is up to 25 times greater (Dillon, 1987; IFT,
1980). Consuming a high quantity of sodium contributes to the
development of hypertension, a predecessor for health disorders
(e.g., cardiovascular problems) (Appel et al., 2012; Cotugna &
Wolpert, 2011; Doyle & Glass, 2010). Due to its adverse health ef-
fects, a great demand exists to reduce NaCl level in cheese products
(Rulikowska et al., 2013).
For reducing NaCl two main strategies exist. The first is to simply
reduce NaCl content. The second is to use salt substitutes con-
taining little or no sodium (e.g., KCl, MgCl2), but giving a taste
similar to NaCl (Fitzgerald & Buckley, 1985; Johnson, Kapoor,
McMahon, McCoy, & Narasimmon, 2009; McMahon et al., 2014).
Choosing either of the above strategies will have an impact on
cheese manufacturing and qualities due to the effect of NaCl on

http://dx.doi.org/10.1016/j.idairyj.2017.05.004
0958-6946/© 2017 Elsevier Ltd. All rights reserved.
 M. Loudiyi et al. / International Dairy Journal 73 (2017) 116e127
controlling microbial growth, removal of whey, development of
specific flavour, texture, structure and functional properties of
cheeses through the control of proteolysis, lipolysis, and glycolysis
(Johnson et al., 2009; Murtaza et al., 2012; Pastorino, Hansen, &
McMahon, 2003; Rulikowska et al., 2013), thus fulfilling determi-
nant roles towards consumer satisfaction.
Texture is widely recognised as one of the most important at-
tributes in determining identity and quality of cheese (Creamer &
Olson, 1982; Euston, Piska, Wium, & Qvist, 2002; Jack, Piggolt, &
Paterson, 1993). It is known that textural characteristics of cheese
are affected by their structural properties, composition, fat distri-
bution, making process and proteolysis (Lobato-Calleros, Vernon-
Carter, & Hornelas-Uribe, 1998). A reduction in NaCl without sub-
stitution with other salts adversely affects cheese quality. Fitzgerald
and Buckley (1985) reported that the texture profile of Cheddar
cheese made with a NaCl and KCl mixture was similar to that of the
control (only NaCl). Ayyash and Shah (2011) and Katsiari, Voutsinas,
Alichanidis, and Roussis (1997, 1998) reported similar results for
halloumi, feta and kefalograviera cheeses. Meltability is one of the
most important physical properties of cheeses used for their heat-
induced functional properties. On cooking, cheese may be required
to melt, flow, stretch, and oil-off to varying degrees depending on
its application. Effect of salt on cheese melting seems to vary ac-
cording to cheese type (e.g., mozzarella and Munster) (Pastorino
et al., 2003), age (e.g., few days compared with several weeks
old), content of salt (e.g., 0.5% compared with 2.0%), and with
cheese composition (e.g., calcium 0.4% compared with 0.7%) and fat
contents (Pastorino et al., 2003; Paulson, McMahon, & Oberg, 1998).
Different methodologies have been used to evaluate the texture
and meltability of cheeses. Dynamic low amplitude oscillatory
rheology is probably one of the most widely used techniques. It is
generally applied for determining rheological properties of cheese
(e.g., G0 , G00 , tan (d)) and used to determine the effect of different (i)
salt contents (i.e., concentration and substitution of NaCl) and (ii)
heating temperatures on cheese texture characteristics (Karoui,
Laguet, & Dufour, 2003a; Lobato-Calleros, Vel azquez-Varela,
Sanchez-García, & Vernon-Carter, 2003; Mounsey & O'Riordan,
1999; Subramanian & Gunasekaran, 1997).
A different technique, fluorescence spectroscopy, offers several
advantages for the characterisation of molecular interactions and
reactions, molecule environments and has been used to charac-
terise molecular structure and texture properties of cheeses (Karoui
& Dufour, 2006; Tang, Chen, Chen, Xie, & Li, 2008). Indeed, the
emission of tryptophan residues in protein and vitamin A can be
used as an indicators for protein conformational changes
(Lakowicz, 1983), physical state of triglycerides and proteinelipid
interactions in dairy products (Dufour, Lopez, Riaublanc, &
Mouhous Riou, 1998; Dufour, Devaux, Fortier, & Herbert, 2001).
This method was used in classical front-face fluorescence (FFF) and
synchronous fluorescence (SF) excitation modes to investigate
changes of milk component structures and interactions during
heating and acidification (Boubellouta & Dufour, 2008), for struc-
tural changes during cheese melting (Boubellouta & Dufour, 2012;
Karoui et al., 2003a) and for predicting some chemical components
of cheeses (Abbas, Karoui, & Aït-Kaddour, 2012). SF spectroscopy
presents an interesting advantage compared with FFF spectroscopy.
A SF spectrum retains information related to several fluorophores,
compared with a FFF spectroscopy emission spectrum that is
mainly specific of a sole fluorophore (Aït-Kaddour et al., 2016;
Boubellouta & Dufour, 2008; Divya & Mishra, 2007).
As far as we know FFF and SF spectroscopy have not been used
for studying the effect of salts on molecular structure of cheeses.
Therefore, the main objective of the present study is to investigate
by different analytical techniques namely physicochemical analysis,
rheology, and SF spectroscopy the impact of NaCl reduction and its
117
substitution by KCl on the molecular structure and meltability of an
uncooked pressed model cheese (Cantal-type cheese).
2. Materials and methods
2.1. Cantal-type cheeses manufacture
Raw milk collected from an agricultural school farm (ENILV,
Aurillac, France) was used to prepare model cheeses with cylin-
drical shape (size, 12 cm  5.5 cm; weight, 861 ± 27 g) based on an
uncooked pressed cheese process (Cantal-type cheese) in an
experimental dairy plant (INRA, UR545 Fromage res, Aurillac,
France). The entire cheese-making process was carried out using
sterile tools and equipment. Raw milk (fat, 40.50 ± 0.00 g L1;
protein, 32.70 ± 0.00 g L1) was standardised at a fat/protein ratio
of 1.10, pasteurised (72
C, 30 s), cooled (32
C for 12 min), then
inoculated with commercial lactic starters (0.05 g L1, Flora Danica,
Chr. Hansen, Arpajon, France), yeasts and mould culture (Penbac
0.002 mL L1 and Monilev 0.002 mL L1, LIP, Aurillac, France) and
CaCl2 was added at 0.30%. Calf rennet (0.1% chymosin, LCP food
ingredients, Le Launay, France) was added at the same temperature
(i.e., 32
C) to vats at 0.35 mL L1, 25 min after microbial
inoculations.
Coagulation proceeded for 45 min; at pH 6.70 ± 0.01 the curd
was cut for 8 min to produce pellets of 5e6 mm in diameter,
blended for 12 min and gently stirred for 4 min to separate the
whey from the curd. Curd was placed in a pressing tray, pressed for
105 min using a sequence of 4.90 KPa (30 min), 9.81 KPa (30 min)
and 14.71 KPa (45 min). Between each pressuring sequence the
curd was cut into 15 cm sided cubes and turned manually 3 times
(i.e., total of 9 times) to promote whey extraction from the cheese.
After pressing, the curd cubes were left to drain for 24 h at 22
C
and were automatically ground at pH 5.17 ± 0.02 into grains of
20 mm in diameter with a curd grinder.
Curd was divided into 5 equivalent parts and salted at the
following rates: 2% NaCl (control, coded with the letter C in the
text), 0.5% NaCl (coded A in the text), and 1% (coded B in the text),
1.5% NaCl/0.5% KCl (coded D in the text) and 1% NaCl/1% KCl (coded
E in the text). Salts were mixed for 5 min into the curd, 4 times by
hand interspersed by 15 min breaks. Curd was equally distributed
into cylindrical moulds (13 cm internal diameter  15 cm high,
Doryl, France). The curd was drained by pressure using a pressure
sequence of 80, 180, 280, and 350 KPa each for 15 min (RH, 90%;
15
C). Then, the curd was kept under 450 KPa pressure for 23 h.
After pressing, each cheese was ripened for 5 days (RH, 96%;
9
C); then the cheeses were vacuum packed and transported 2 h at
4
C to the laboratory for analysis (VetAgro Sup, Lempdes, France).
The samples were rapidly frozen to 18 ± 2
C (Liebherr, Ober-
hausen, Germany) until analysis for physicochemical, dynamic
oscillatory experiment and fluorescence spectroscopy because it
was not technically possible to perform all the experiments at the
same time on all cheeses. Before analysis, cheese samples were
thawed during 16 h at 4
C and then equilibrated at room tem-
perature for 1 h. The experiment was repeated twice allowing 2
cheeses per formulation to be obtained, giving a total of 10 cheeses.
2.2. Synchronous fluorescence spectroscopy
Cheeses were sliced into thin samples (2.5 cm length  0.9 cm
width  0.4 cm thickness) and placed in a quartz cell. Fluorescence
spectra were recorded in a synchronous excitation mode by using a
FluoroMax-4 spectrofluorimeter (JobineYvon, Longjumeau,
France), presenting a 3000:1 signal to noise ratio, equipped with a
solid sample holder accepting a quartz cell (30  1  1 mm) and
presenting a 60
incidence angle for the excitation radiation to
118 M. Loudiyi et al. / International Dairy Journal 73 (2017) 116e127
minimise reflected light, scattered radiation and depolarisation
phenomena. During acquisition, the excitation wavelength (lex)
and emission wavelength (lem) were scanned simultaneously
(synchronously), maintaining a constant interval, named offset (Dl)
between lex and lem. In this study, six Dl (20, 40, 60, 80, 100, and
120 nm) were used. All the excitation fluorescence spectra were
recorded between 250 and 550 nm at different temperatures on the
same sample during heating from 20 to 60
C with a temperature
step gap of 5
C. Spectral data collection and manipulation were
performed using FluorEssence (2010) software, version 3.5, from
Horiba scientific (JobineYvon, Longjumeau, France). For each
cheese, 2 kinetic temperatures were performed using different
samples (i.e., 4 temperature kinetics per cheese formulation).
2.3. Independent components analysis
Independent components analysis (ICA) is one of the most
powerful techniques for blind source separation (BSS). It aims to
extract the pure underlying signals from a set of mixed signals
(mixed signals are considered as weighted sums of pure source
signals (Wang, Ding, & Hou, 2008); the weights being proportional
to the contribution of the corresponding pure signals to each
mixture) by estimating a linear transformation that maximises the
statistical independence among the extracted source signals
(Bouveresse, Benabid, & Rutledge, 2007). In the case of ICA, max-
imising independence means maximising the non-Gaussianity of
the signals. According to the central limit theorem, the observed
signals, which are considered to be combinations of independent
pure signals, tend to be normally distributed (more Gaussian).
Therefore, the objective of ICA is to search for the least Gaussian
possible sources, i.e., the most independent.
Given that observed signals are organised into a data matrix X
(s  v) where s are the spectra corresponding to the rows of X, and v
the variables corresponding to the columns (wavelengths in the
case where signals are spectra), the general model of ICA can be
described as:
X ¼AS
where A is the matrix of proportions of pure signals (contributions
of pure signals to the observed ones), or the so called mixing ma-
trix; and S the matrix of pure source signals (the independent
components ICs). In other terms, this means that observed signals
(spectra ¼ rows of X) are linear mixtures of pure source signals
(rows of S) weighted by corresponding values of A.
ICA aims at determining both A (proportions) and S (ICs),
knowing only X. It attempts to achieve this objective by estimating
a demixing matrix called W ¼ A1, so that pure source signals (ICs)
may be recovered from the matrix of observed signals (X) according
to the following equation:
S¼W X
The mixing matrix A can then be calculated as:
 1
A ¼ XST SST
To achieve this estimation, several algorithms are available like
FastICA and InfoMax. In this paper, the joint approximate diago-
nalisation of eigen matrices (JADE) algorithm was used (Rutledge &
Bouveresse, 2013).
The choice of the number of independent components is a very
crucial step to consider in an ICA data treatment. In our case, we
used ICA-by-blocks to achieve a pertinent choice. This method
consists of splitting the data matrix into B blocks of samples of
approximately equal number of rows in a way that each block is
representative of the whole data matrix. For each of these pre-
defined blocks, Amax ICA models are computed (Amax corresponds to
the maximum number of ICs to be computed and which needs to
exceed the expected optimal number of ICs). ICs corresponding to
true source signals should be found in all blocks. Such true ICs
should be highly correlated while noisy ICs will have low correla-
tions with ICs extracted from other blocks (Bouveresse, Moya-
Gonzalez, Ammari, & Rutledge, 2012).
2.4. Independent components analysis on synchronous fluorescence
spectra
To reduce differences in global intensities of the fluorescence
signals and to reduce noise, all spectra were preprocessed by
Standard Normal Variates and mean centring before ICA analysis.
All those transformations were performed with Matlab software
R2013b (The MathWorks, Natick, Massachusetts, USA) and the PLS-
toolbox 7.0.3 released in January 2013 (Eigenvector Research,
Manson, Washington, USA). ICA was performed on the full 3D
matrix containing 540 samples  301 excitation wavelengths  6
offsets. Sample dimension includes the 20 sample measurements
(i.e., 10 cheeses with 2 sampling per cheese)  9 temperatures
measurements and 3 replicates per cheese sample. The JADE al-
gorithm needs data to be in the form of a matrix (2D-array).
Therefore, the 540  301  6 cube was unfolded into a 540  1806
data matrix. After ICA, the matrix S (matrix of pure source signals
extracted from the synchronous spectra with the optimal number
of ICs) was refolded to obtain spectra-chemically interpretable ICs.
All computations were performed using MATLAB R2013b (The
MathWork).
2.5. Dynamic oscillatory experiments
Cheeses were sliced in the centre part into thin disks
(2 mm ± 0.05 thick, 20 mm diameter) with a cheese slicer equipped
with a millimetre guide and a die cutter of 20 mm diameter (TA
Instruments, Leatherhead, UK). Before sampling, sliced samples
(1)
were placed into a plastic box (60  15 mm), surrounded by
aluminium paper in its interior to prevent dehydration and stored a
maximum of 1 h at 5
C before analysis. Dynamic oscillatory ex-
periments were performed with a rheometer (Kinexus proþ,
rSpace for Kinexus 1.61 Software, Malvern Instruments, Malvern,
Worcs., UK) equipped with a plate geometry of 20 mm diameter
and a serrated probe to prevent any slippage during the experi-
ments. Before analysis, the sample size was controlled with an
electronic digital caliper to ensure again that the sample dimension
were in the expected range. Oscillation experiments were per-
formed in the linear viscoelastic region (measurement not shown),
determined before performing the actual experiment, by applying a
shear strain ranging from 0.001 to 100% at a constant frequency of
(2) 1 Hz. The temperature of the sample was increased from 20 to 60
C
(rate, 3
C min1) by a Peltier heating element equipped with an
Active Hood Peltier Plate cartridge. This device prevented thermal
loss from the sample environment and hence minimised sample
(3) thermal gradients. Measurement was performed at a shearing
strain of 0.05% and a constant frequency of 1 Hz. The parameters
measured were elastic modulus (G0 ), loss modulus (G0 ), loss angle
(d) and complex viscosity (h*) versus temperature. The log (h*)
versus temperature (
C) curve was the only parameter considered
further to determine the melting point of fat in the cheese. The
curve shows two distinct linear regions and the intersection point
was identified as the melting point of fat in the cheese based on
previous work (Boubellouta & Dufour, 2012; Karoui et al., 2003a).
For each cheese formulation, six experiments were performed.
 M. Loudiyi et al. / International Dairy Journal 73 (2017) 116e127 119

2.6. Cheese composition
Moisture content was determined by oven drying 3 g of product
at 105
C (Memmert, 854 Schwabach, Germany) in compliance
with the ISO 5534 standard (ISO, 2004a). Fat content was evaluated
by van Gulik method according to the ISO 1735 (ISO, 2004b). The pH
was measured by using a pH-meter CG 840 (Schott, Mainz, Ger-
many) equipped with a glass electrode. Total nitrogen (TN) and
water soluble nitrogen (WSN) were determined using a Kjeldahl
Unit (Büchi, Flawil, Switzerland) according to Kjeldahl method in
compliance with the NF ISO 8968-1 standard (ISO, 2014). Nitrogen
content was then converted into protein using the conversion
factor of 6.38. Those measurements were used to calculate the
proteolytic index (WSN/TN).
Contents of Ca, P, Mg, K and Na in cheese samples were
measured from ashes dissolved in 1 mL of nitric acid followed by
atomic measure using a spectrophotometer AA 240 FS (Varian, Les
Ulys, France) in compliance with the NF ISO 8070 standard (ISO,
2007a). The first step of the analysis was incineration of 1 g of
cheeses at 550
C for 6 h (Fours Nagat, Bretagne, France). Ash

(A)
4.5
20nm
4
40nm
60nm

3.5
× 10-5)

3
(a.u.(a.u)

2.5
Fluorescence
Fluorescence

1.5

0.5

0
250 300 350 400 450 500 550
Wavelength (nm)
Wavelength (nm)
(B)
4.5
80nm
4
100nm
120nm

3.5
× 10-5)

3
(a.u.(a.u)

2.5
Fluorescence
Fluorescence

1.5

0.5

0
250 300 350 400 450 500 550
Wavelength (nm)
Wavelength (nm)

Fig. 1. Average synchronous fluorescence spectra recorded with different offsets or Dl (20 nm, line with squares, 40 nm, grey line; 60 nm, heavy black line) (A) and Dl (80 nm,
dashed line; 100 nm, grey line; 120 nm, heavy black line) (B) at 20
C with an excitation range of 250e550 nm for the control cheese (2% NaCl).
120
obtained was dissolved in 1 mL of nitric acid (25%; VWR Interna-
tional, Fontenay-sous-Bois, France) and then made up to 250 mL
with demineralised water. After dilution, the solution was injected
in a Fast Sequential Atomic Absorption Spectrometer (Varian,
AA240FS, Palo Alto, CA, USA) according to ISO (2007a) to assay Ca,
K, Mg, and Na contents. The total P was assessed with the col-
ourimetric nitro-vanado-molybdate method from the same ash
solution on a spectrophotometer (Secomam, Domont, France) at
430 nm wavelength. The Cl content in cheeses was measured by
potentiometric titration (Titroline Easy Potentiometer, SI Analytics,
Weilheim, Germany) with 0.1 N silver nitrate (VWR International,
Fontenay-sous-Bois, France) according to the NF ISO 5943 standard
(ISO, 2007b). All the physicochemical measurements were per-
formed in triplicate.
2.7. Statistical analysis
First, a one-way analysis of variance (ANOVA) was applied to the
experimental data using the PROC ANOVA function with the Dun-
nett's test, to investigate the significant differences between the
physicochemical parameters and fat melting temperatures of the
2% NaCl cheese (control, C) compared with the other salted cheeses
(A, B, D and E). Second, a student test was applied using the PROC
TTEST function to compare the fat melting temperature results
obtained with fluorescence and rheological methods. All functions
were carried out using the SAS statistical software package (Version
9.3, SAS institute Inc., Cary, NC, USA). All differences were consid-
ered as statistically significant at p  0.05.
3. Results
3.1. Effect of offsets on synchronous fluorescence spectra
Fluorescence spectra recorded at the different Dl (20, 40, 60, 80,
100 and 120 nm) at 20
C on the C cheese are presented in Fig. 1A
4.5
4
3.5
× 10-5)
3
(a.u.(a.u)
2.5
Fluorescence
Fluorescence
1.5
0.5
0
250 300
Fig. 2. Effect of heating from 20 to 60
C (20
C, heavy black line; 30
C, thin black line; 40
C, dashed black line; 50
C, heavy grey line; 60
C dashed grey line) on synchronous
fluorescence spectra recorded at Dl ¼ 80 nm for the control cheese (C: 2% NaCl).
M. Loudiyi et al. / International Dairy Journal 73 (2017) 116e127
and B. Similar spectra were obtained for cheeses containing the
other salts (A, B, D and E). The spectra recorded with Dl ¼ 20 nm
presented one prominent peak at 380 nm (emission, 400 nm) and a
smaller one between 449 and 525 nm (emission between 469 and
545 nm) with a maximum at 491 nm (emission, 511 nm). Those
maximum bands shifted to lower wavelengths, 376 and 472 nm,
respectively, for spectra scanned with Dl ¼ 40 nm.
The SF spectra acquired with Dl ¼ 60 nm presented four bands
centred at 308, 323, 365 and 467 nm (emission: 368, 383, 425 and
527 nm, respectively). The offset of 80 nm depicted the highest
number of fluorescence bands and gave spectra with band maxima
at 304, 321, 364 and 449 nm (emission: 384, 401, 444 and 529 nm,
respectively). When SF spectra were recorded with 100 and 120 nm
offsets the bands located at 304, 364 and 449 nm tended to
disappear, while a new shoulder and distinct bands appeared at 297
(397 nm emission for the 100 nm offset), 308 (428 nm emission for
the 120 nm offset) and 352 nm (452 and 472 nm emission for 100
and 120 nm offsets, respectively).
3.2. Effect of soft heating on synchronous fluorescence spectra
The effect of temperature increase was studied at the 80 nm
offset that depicted the highest number of fluorescence bands in
the SF spectra whatever the cheese (Abbas et al., 2012; Boubellouta,
Karoui, Lebecque, & Dufour, 2010). As an example, mean SF spectra
recorded on the control cheese during heating from 20 to 60
C are
presented in Fig. 2. Sharp and intense bands maximum at 304, 321,
364 and 449 nm (emission: 384, 401, 444 and 529 nm, respectively)
reacted to temperature increase as previously reported (Abbas
et al., 2012; Boubellouta et al., 2010). The 321 and 449 nm band
intensities decreased drastically during heating. In addition, a blue
shift of 4 nm (i.e., to 360 nm) was observed for the 364 nm band
compared with the other bands. Moreover, a different rate of
decrease can be identified for the 321 nm band compared with the
other bands. The band at 304 nm presented a modification in
20°C
30°C
40°C
50°C
60°C
350 400 450 500 550
Wavelength (nm)
Wavelength (nm)
 M. Loudiyi et al. / International Dairy Journal 73 (2017) 116e127 121

A: 0.5% NaCl
2.5 B: 1% NaCl
C: 2% NaCl
2 D: 0.5% KCl/1.5% NaCl
E: 1% NaCl/1% KCl

1.5
Fluorescence (a.u.)
(a.u)

1
Fluorescence

0.5

-0.5

-1

-1.5
250 300 350 400 450 500 550
Wavelength (nm)
Wavelength (nm)

Fig. 3. Effect of NaCl concentration and its substitution by KCl on standard normal variate synchronous fluorescence spectra recorded on cheeses A (0.5% NaCl; thin black line), B (1%
NaCl; black squares), C (2% NaCl; heavy black line), D (1.5% NaCl/0.5% KCl; grey squares) and E (1% NaCl/1% KCl; heavy grey line) at 20
C and Dl ¼ 80 nm.

fluorescence intensity: a decrease between 20 and 30
C, and then
an increase from 30 to 50
C and finally a decrease from 50 to 60
C
was observed. A blue shift during heating was also noted for this
band (i.e., 304 to 300 nm).
3.3. Effect of salts concentration on synchronous fluorescence
spectra
Fig. 3 presents the effects of NaCl concentration and its substi-
tution by KCl on SF spectra recorded with Dl ¼ 80 nm at 20
C.

Fig. 4. Random_ICA with B ¼ 2 blocks and k ¼ 30 repetitions: boxplot of the lowest correlations as a function of the number of independent components (ICs) ranging from 1 to 20
ICs.
122 M. Loudiyi et al. / International Dairy Journal 73 (2017) 116e127

Fig. 5. Source signals (i.e., “loadings”; a, b, c, d, e), variation of emission intensity associated to each loading (i.e., a', b', c', d', e') and proportions of signal source (i.e., “scores”; a'', b'',
c'', d'', e'') obtained after ICA on synchronous fluorescence spectra of the five cheese formulations, A (0.5% NaCl: ), B (1% NaCl: ), C (2% NaCl; ), D (1.5% NaCl/0.5% KCl: ) and E (1%
NaCl/1% KCl: ), during soft heating from 20 to 60
C.
 M. Loudiyi et al. / International Dairy Journal 73 (2017) 116e127 123

Table 1
Evaluation of the melting temperature of fats in model cheeses using synchronous
fluorescence spectra and rheological data.a
Salt treatment Fat melting temperature (
C)
SFS (IC2) Rheology (log (h*))
b cheese: 0.5% to E cheese: 1%) of cheese. The second signal source
A 33.32 ± 0.53 34.19 ± 0.67b
B 34.49 ± 0.35b 35.10 ± 1.81b
C 36.04 ± 0.74a 35.87 ± 0.32a
D 34.81 ± 0.58b 34.49 ± 0.51a
± 0.57a ± 0.46b
E 36.20 34.64
a
Salt treatments are: A, 0.5% NaCl; B, 1% NaCl; C, (control), 2% NaCl; D, 1.5% NaCl/
0.5% KCl; E, 1% NaCl/1% KCl. Means in each column with the same superscript letter
with the control cheese did not differ significantly (P > 0.05).
Compared with the control cheese (i.e., 2% NaCl), the substitution of
NaCl by KCl induced changes in SF spectra. Chesses formulated with
0.5% NaCl (A) and 0.5% KCl/1.5% NaCl (D) presented the highest
intensity at 304, 321, and 364 nm and the lowest at 449 nm.
Cheeses B and E presented quite similar spectra compared with the
control at 364 and 449 nm bands. Nonetheless, cheeses containing
the highest KCl content (E) presented the lowest intensity at
304 nm, while the B cheese exhibited almost similar intensity
compared with the control. A red shift of the 304 nm band was also
observed when NaCl was partly replaced by KCl. When considering
this band, an increase of 3 (i.e., 307 nm) and 2 nm (i.e., 306 nm)
were noted respectively for the D and E cheeses.
3.4. Independent components analysis
To select the number of ICs, the Random_ICA procedure was
applied with B ¼ 2 blocks, F max ¼ 20 and k ¼ 30 repetitions. The
correlation boxplot (Fig. 4) shows that after extracting 5 ICs, the
correlation values started to decrease from IC ¼ 6 to IC ¼ 10.
However, the addition of an 11th IC increased the correlation again
to nearly 0.9. Therefore, 11 ICs were chosen as being the optimal
number of ICs. The reason why the correlation decreased from IC 6
to IC 9 models is that the order of extraction is different in the two
blocks (IC of block 1 can correspond to an IC that is not yet
extracted from block 2, and vice-versa). ICA with 11 ICs, as deter-
mined by Random_ICA, was applied to the data matrix to analyse
the ICA outcomes. Fig. 5 presents results obtained after ICA was
applied on SF spectra of the different cheeses (A, B, C, D, and E). It
depicted the relevant source signals (i.e., “loadings”) (Fig. 5aee),
the variation of emission intensity associated with each loading
depended on the offset value (Fig. 5a'ee') and the proportions of
signal source (i.e., “scores”) (Fig. 5a''ee'') for each cheese sample
according to temperature increase. The 1st loading (Fig. 5a) pre-
sented a large band centred at 468 nm. The optimum emission
offset (Fig. 5a') for this fluorophore was identified at 120 nm
(emission maximum, 588 nm). The variation of the score values for
IC1 shows that IC1 enabled discrimination of the A cheese from the
others (Fig. 5a''). The scores on IC1 tended to increase when
increasing both NaCl (A cheese, 0.5% to C cheese, 2%) and KCl (D
IC2 presented bands with maximum excitation and emission at
321 nm (Fig. 5b) and 401 nm (Fig. 5b'), respectively. Fig. 5b'' of IC2
scores depicted that IC2 tended to discriminate the scores based on
temperature. An increase in temperature from 20 to 60
C induced
a steady state decrease of the scores with an inflection point
whatever the cheese formulation. Moreover, the decreasing rate of
the scores curve during heating was noted larger before the in-
flection point compared with the terminal region of the curve,
whatever the cheese. The 3rd signal source presented a thin band
with maximum excitation and emission at 301 (Fig. 5c) and
381 nm (Fig. 5c'), respectively. Cheeses presented scores almost
shaped like “a bell shape curve” on IC3 except for the E cheese
(Fig. 5c''). Based on the signal profile, the IC3 tended to separate
cheese samples by their maximum scores. The 4th signal source
presented a band centred at 295 nm (Fig. 5d). The optimum
emission offset of this fluorophore was identified at 100 nm
(emission maximum, 395 nm) (Fig. 5d'). As with scores on IC3, the
scores on IC4 presented almost “a bell shape curve” for all cheeses
except for the E cheese. The IC4 tended to discriminate samples
based on their maximum scores value.
The 5th signal source presented an excitation band centred at
380 nm (Fig. 5e) with an optimum emission at 400 nm (Fig. 5e').
Scores on IC5 increased from the control cheese (2% NaCl, C) to the
A cheese (0.5% NaCl) (Fig. 5e''). Compared with the C cheese, the
substitution of a part of the NaCl content by KCl did not induce a
huge modification of the score values (cheeses D and E). The IC11
was not analysed due to its noisy shape (results not shown).
3.5. Melting temperatures of fat
Table 1 presents the fat melting temperatures of the different
cheeses derived from IC2 scores obtained after ICA applied on
synchronous fluorescence data and the dynamic oscillatory
experiments.
In each IC2 scores kinetic, two distinct linear regions were
observed, and the intersection point was identified as the melting
temperature of fat in the cheese. No significant difference was
observed between the results obtained with the two methods (i.e.,
fluorescence and dynamic oscillatory experiment) at a level of 5%.
Considering the rheology data, significant difference (P < 0.05)
was noted between the control and the other cheeses except for

Table 2
Mean and standard deviation of the chemical properties of model cheeses with different salt treatments.a

Parameter Salt treatment

A B C D E

Moisture (%, w/w) 40.7 ± 0.3b 39.7 ± 0.4b 38.4 ± 0.3a 38.7 ± 0.2a 38.9 ± 0.1b
Protein (%, w/w) 43.1 ± 0.5b 42.3 ± 0.5a 42.4 ± 0.4a 42.3 ± 0.7a 42.6 ± 0.4a
Fat (%, w/w) 49.8 ± 0.6a 49.8 ± 0.3a 49.5 ± 0.2a 49.3 ± 0.3a 49.8 ± 0.3a
WSN/TN (%, w/w) 4.7 ± 0.4a 5.0 ± 0.7a 4.5 ± 0.2a 4.9 ± 0.4a 4.7 ± 0.5a
pH 5.30 ± 0.01a 5.33 ± 0.08a 5.35 ± 0.01a 5.40 ± 0.02a 5.40 ± 0.03a
Cl (mg 100 g1) 546.0 ± 8.9b 983.2 ± 19.9b 1703.8 ± 10.6a 1597.9 ± 11.9b 1399.2 ± 9.6b
Ca (mg 100 g1) 1063.8 ± 19.7a 1048.0 ± 16.6a 1049.4 ± 22.5a 1109.3 ± 18.7b 1074.6 ± 9.0b
Na (mg 100 g1) 332.8 ± 9.4b 544.2 ± 15.8b 932.7 ± 9.9a 766.6 ± 9.5b 529.6 ± 12.4b
K (mg 100 g1) 153.4 ± 10.3a 148.7 ± 7.1a 149.9 ± 7.5a 383.5 ± 5.3b 614.2 ± 15.3b
Mg (mg 100 g1) 57.3 ± 2.1a 60.5 ± 7.6a 56.5 ± 1.8a 57.3 ± 0.9a 58.3 ± 1.1a
P (mg 100 g1) 897.4 ± 7.9a 887.9 ± 12.0a 894.9 ± 12.9a 883.0 ± 10.1a 891.0 ± 16.7a
a
Salt treatments are: A, 0.5% NaCl; B, 1% NaCl; C, (control), 2% NaCl; D, 1.5% NaCl/0.5% KCl; E, 1% NaCl/1% KCl. Results are expressed based on dry matter (except moisture) as
mean values ± SE of 6 trials; means in each row with the same superscript letter as the control cheese did not differ significantly (P > 0.05).
124 M. Loudiyi et al. / International Dairy Journal 73 (2017) 116e127
Table 3
Excitation and emission maximum of five independent components identified after
ICA on SFS.
Component l max (nm)
Excitation Emission
1 (IC1) 468 588
2 (IC2) 321 401
3 (IC3) 301 381
4 (IC4) 284 384
5 (IC5) 380 400
the D cheese (1.5% NaCl/0.5% KCl). Almost the same results were
obtained from the fluorescence data, no significant difference was
observed between the control cheese (C) and the other cheeses
except for the cheese (E) formulated with 1% NaCl/1% KCl
(P < 0.05).
3.6. Physicochemical analysis
Results of the physicochemical analyses are depicted in Table 2.
Compared with the control cheese (2% NaCl, C), analyses of variance
show that salt treatments had no significant effect on fat, proteo-
lytic index (WSN/TN), pH, Mg and P contents. Concerning the Cl and
Na contents, significant difference was observed between the
control cheese and the other cheeses (A, B, D and E) (P < 0.05). For
the Ca and K contents a significant effect was noted only between
control cheese and the cheeses with added KCl (D, and E cheeses).
Na and Cl contents decreased (P < 0.05) in model cheese with
increases in the reduction or substitution of NaCl with KCl. Indeed,
Na and Cl contents in model cheeses ranked in the order
C > D > B > E > A, which is in accordance with the amount of Na and
Cl content in cheeses. Potassium contents were significantly higher
(P < 0.05) in cheeses salted with KCl (D and E) compared with the
control (C), expectedly. Compared with the control (C), cheese
prepared with lower NaCl (A, B and E) were consistently higher in
moisture (P < 0.05). Regarding protein content, the only significant
difference (P < 0.05) was observed between low salted cheese (A)
and the control (C).
4. Discussion
In addition to aw, pH and lactic acid content, NaCl is one of the
factors that guarantee the quality of cheese. A reduction of NaCl
level in cheese during manufacturing can lead to an alteration of
flavour, texture and functional properties. This work investigated
the potential use of fluorescence spectroscopy combined with
multivariate data analysis to identify, at a molecular level, the
modification induced by NaCl and its substitution by KCl on cheeses
produced at the pilot scale. Few studies have been published on the
use of fluorescence spectroscopy to monitor the effect of heating on
cheese samples (Boubellouta & Dufour, 2012; Karoui et al., 2003a).
These studies reported that classical FFF and SF spectroscopies can
be used to investigate the effect of heating at the molecular
structure of hard French cheeses (Cantal, Comte, and raclette). They
also demonstrated that both fat and cheese melting temperatures
can be identified by fluorescence spectroscopy since no significant
difference was observed between the data obtained with SF spec-
troscopy and rheology measurements. Compared with similar
published studies on the same subject, this study brings new in-
sights regarding the effect of NaCl and KCl concentrations on the
molecular structure of cheese. Most the published studies used
fundamental and empirical rheology methods to characterise the
effect of NaCl and its substitution with KCl.
4.1. Effect of soft heating on synchronous fluorescence spectra
Our study reported the same fluorescence bands as those
identified by Boubellouta and Dufour (2012). The bands around 304
and 321 nm were attributed to tryptophan residues of proteins and
vitamin A, respectively, while those observed at 364 and 449 nm
were assigned to riboflavin, NADH, FADH, and/or Maillard reaction
compounds. The increase of temperature from 20 to 60
C drasti-
cally changes the intensity and position of the fluorescence bands.
 and raclette cheeses, depending
As previously reported on Comte
on the temperature values, fluorescence bands tend to decrease
and increase (Boubellouta & Dufour, 2012). It was previously
demonstrated that heat treatment can affect the composition of the
milk fat globule. For example, Dufour et al. (1998) demonstrated
that the shape of the vitamin A excitation spectra in reconstituted
milk fat globules is temperature dependent and therefore depends
on the liquid or crystalline state of triglycerides. It was also reported
that the amount of protein associated with fat globules membrane
increased and the phospholipids and triacylglycerides decreased
during heating (Corredig & Dalgleish, 1996; Houlihan, Goddard,
Kitchen, & Masters, 1992). Finally, it was noted a high correlation
(R2 ¼ 0.997) between the fluorescence data and lipid level in the
liquid state determined by differential scanning calorimetry
(Dufour et al., 1998). So, the decrease of the band assigned to
vitamin A (321 nm) during heating could be attributed to modifi-
cation of the structure of the milk fat globules.
The fluorescence emission of riboflavin is highly sensitive to its
local environment, and can be used as an indicator group for the
oxidation of dairy products. It was also identified as a marker of
protein conformation and interaction changes in cheese matrices
during ripening since it interacts with proteins (Karoui & Dufour,
2006). Thus, the decrease of the band centred at 364 nm during
heating can underline a modification of the protein matrix struc-
ture (Boubellouta & Dufour, 2008). The increase in intensity and
wavelength shift of the tryptophan band (304 nm) during heating
can be related to more exposure of tryptophan residues to the
aqueous phase of cheese (Karoui et al., 2003a).
4.2. Effect of salts concentration on synchronous fluorescence
spectra
The NaCl addition in cheese modifies the intensity, width and/or
position of the different fluorescence bands as identified by SF
spectroscopy. Modifications of tryptophan (304 nm) and riboflavin
(364 nm) fluorescence bands are related to an increase in casein
hydration as well as the volume of the matrix and solubilisation of
casein through calcium displacement by sodium ions (Guinee,
2004). Indeed, addition of salts impacts proteinewater in-
teractions, through their abilities to hydrate, and alter water
structure. It has been reported by Cervantes, Lund, and Olson
(1983) and Ramkumar, Creamer, Johnston, and Bennett (1997)
that NaCl alters the water-binding properties of casein within the
cheese matrix and, thus, influences the physical properties of the
cheese. These observations overlap with physicochemical results
exposed in the present study and findings of Kosikowski (1983) and
Schroeder, Bodyfelt, Wyatt, and McDaniel (1988). These authors
reported that reduction of NaCl results in higher moisture, texture
defects and lack of flavour in Cheddar cheese due to a combination
of increased moisture and enzymatic activity. Nonetheless, this
observation is inconsistent with the findings of Guo and Kindstedt
(1995) on mozzarella cheeses due to differences in processing
technology. Indeed, Guo and Kindstedt (1995) demonstrated that
unsalted blocks of part-skim mozzarella cheese had higher levels of
expressible serum than did brined part-skim mozzarella cheese,
suggesting that salting of the cheese increases the water-holding
 M. Loudiyi et al. / International Dairy Journal 73 (2017) 116e127
capacity of the cheese matrix by increasing the hydration of the
casein molecules that make up the cheese matrix.
Modifications in the width of the tryptophan (304 nm) and
vitamin A (321 nm) fluorescence bands were also observed from
one cheese to another. These modifications were attributed to
difference in the network structure due to differences in pro-
teineprotein, proteinefat, and/or proteinewater interactions
(Herbert et al., 2000). Proteins in cheese not only interact among
themselves, but also with water and fat, the nature and extent of
these interactions depends on the ionic environment of cheese and
processing conditions. It has been reported that: (i) proteinefat
interactions induced changes in both vitamin A and tryptophan
fluorescence spectra (Karoui, Schoonheydt, Decuypere, Nicolaï, &
De Baerdemaeker, 2007) and (ii) interaction of NaCl and pH pa-
rameters have a significant effect (P < 0.05) on the size of fat
droplets in a model lipoproteic matrix mimicking hard-type cheese
(Floury et al., 2009) and imitation cheeses (El-Bakry, Beninati,
Duggan, O'Riordan, & O'Sullivan, 2011).
Different studies conducted on partial replacement of NaCl with
KCl reported no modification on cheese composition, proteolysis,
and texture. For example, Fitzgerald and Buckley (1985) reported
that substitution of NaCl with KCl (up to 50%) did not influence
proteolysis rates in Cheddar cheese, consistent with our study.
Reddy and Marth (1993) also found no significant differences in
proteolysis in cheeses made with NaCl or KCl used alone or in
mixtures. Regarding kefalograviera cheese, partial substitution of
NaCl by KCl did not affect the proteolysis during 6 months of ageing
(Katsiari, Voutsinas, Alichanidis, & Roussis, 2001), but disagrees
with Rulikowska et al. (2013) who observed a decrease in bacterial
peptidase activity in lower-salt cheeses, resulting in notably higher
bitterness.
In our study the partial substitution of NaCl by KCl induced a
modification of fluorescence spectra that could be due to changes
occurring at the molecular level. Those modifications can be related
to possible modification of water availability to caseins. Indeed, a
lower moisture content was observed in the cheese containing the
highest level of K (E cheese). Another explanation could arise from
the highest values of pH (D and E cheeses). Dufour et al. (2001)
reported that structure of casein is strongly modified by pH in-
crease during a fluorescence study of soft cheese. The modifications
occurring in the shape of the fluorescence spectra can also be
related to changes in triglycerides, proteinelipid and/or lipidelipid
interactions. Indeed, it has been reported that the shape of the
vitamin A spectra depends on the physical state (liquid or solid) of
triglycerides and proteinelipid and lipidelipid interactions (Dufour
et al., 2001; Karoui, Mazerolles, & Dufour, 2003b). Moreover, pre-
vious investigations demonstrated that NaCl and KCl salts can
drastically modify the viscosity of a caseinate solution corre-
sponding to their order in the Hofmeister series of approximately
decreasing hydration tendency (Carr, Munro, & Campanella, 2002),
explaining the modification of the fluorescence spectra.
4.3. Independent components analysis of synchronous fluorescence
spectra
The aforementioned patterns in the fluorescence landscapes
were investigated further by the use of ICA with the objective to
extract information from the data sets, i.e. estimate the maximum
excitation and emission profiles of fluorophores directly from the
fluorescence landscapes. The excitation/emission maximum for the
five compounds permits identification of different fluorophores
(Table 3). The assignment of the first signal profiles is questionable
since it could be attributed to the riboflavin, Coenzyme and/or
fluorescent Maillard reaction products (Karoui & De Baerdemaeker,
2007; Kristensen, Hansen, Arndal, Trinderup, & Skibsted, 2001).
125
Comparing the second signal source profile (321 nm/401 nm) with
the fluorescence profiles previously reported on cheese samples
(Dufour et al., 1998; Herbert et al., 2000; Karoui & Dufour, 2006),
vitamin A is an obvious suggestion for this component. This is also
underlined by the fact that similar results have been reported
during cheese heating (Boubellouta & Dufour, 2008; Karoui &
Dufour, 2003).
The score plot of the vitamin A variation during cheese heating
shows two distinct linear regions; the gradient of the kinetic scores
was larger initially compared with the terminal region whatever
the cheese formulation. This finding indicates a transition zone, for
which the cheese softened and started to flow under constant force
(Karoui & Dufour, 2003). This transition zone represented the
softening of the cheese, regardless of the formulation. After
regression transformations as depicted by Karoui and Dufour
(2003), the inflection points were found to be affected by NaCl
concentration and KCl substitution level (P < 0.05). To date and to
the best of our knowledge, no study has investigated the effect of
NaCl and its substitution by KCl on fat melting temperature of
cheeses. Nonetheless, different studies reported that NaCl content
induces a modification of cheese melting (El-Bakry et al., 2011). Our
observation certainly indicated that salts impact fat droplets size
and protein hydration (Floury et al., 2009; Guo & Kindstedt, 1995)
The fourth signal source profile corresponds quite well with the
characteristics of tryptophan, whereas the excitation maximum of
the third component seems a little too high for tryptophan since
the latter emitted generally in the 330e350 nm range as reported
by Karoui and Dufour (2006). A similar observation was discussed
by Christensen, Povlsen, and Sørensen (2003) during a fluorescence
study of processed cheese during storage. Having the rather low
resolution of 20 nm in the synchronous excitation mode in mind
and knowing that the fluorescence properties of protein-bound
amino-acids are known to be affected by the structure of protein
(Lakowicz, 1999), Christensen et al. (2003) suggested that the sec-
ond spectral profile (321 nm/401 nm) can also be due to tryptophan
fluorescence, but simply shifted due to the inclusion of different
protein structures. Another explanation could arise from the
transfer of energy between vitamin and tryptophan. The last signal
profile (i.e., the fifth) corresponds quite well with the characteris-
tics of riboflavin (380 nm/400 nm). Like other fluorophores, the
fluorescence emission of riboflavin is highly sensitive to its local
environment, and it can be used as an indicator for protein
conformation and interaction changes in cheese matrices during
ripening since it interacts with proteins (Karoui & Dufour, 2006).
Those observations suggested that the signal profiles 1, 3, 4 and 5
are derived from protein structure modification during heating,
while the signal profile 2 can be assigned to change in the viscosity
of fat (Table 3).
Finally, to clearly demonstrate that salts (NaCl and KCl) have an
effect on the molecular structure of control cheeses during heat
treatment a support vector machine discriminant analysis (SVMDA)
with 5 fold cross-validation splits was conducted on the 5 extracted
ICs (IC1, IC2, IC3, IC4 and IC5). The SVMDA gave good results with
correct classification, specificity, and sensitivity higher than 96%,
and an error lower than 1.9% after cross-validation for each cheese.
The similar melting temperature of fats derived from rheology and
synchronous fluorescence after ICA analysis of the IC2 confirms the
relationship between the molecular structure of cheese, as inves-
tigated by SF spectroscopy, and the macroscopic structure assessed
using dynamic testing rheology.
5. Conclusion
This study showed the potential of SF spectroscopy coupled with
independent components analysis (ICA) to delineate the structure
126 M. Loudiyi et al. / International Dairy Journal 73 (2017) 116e127
changes of model cheeses with different salt contents (KCl and
NaCl) during heating. Indeed, results obtained demonstrated that
NaCl concentration and its substitution by KCl modifies the mo-
lecular structure (intensity, width and/or position of the fluores-
cence bands). In addition, it was possible to identify similar melting
temperatures of cheese fats from both spectral and rheology data,
thereby demonstrating a good relationship between the structure
of the cheese at the molecular level and the texture. Our results and
hypothesis still remain to be validated with further analysis (e.g.,
image microscopy) to prove the identification of the fluorophores
and the effects of salts on casein and fat globules, but this investi-
gation certainly underlines the potential of fluorescence spectros-
copy in combination with chemometrics, as a fast, non-destructive
innovative method, that can be applied to monitoring thermal and
melting behaviour of cheeses.
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