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O A RIGIN
19992
Journal of Applied Scien
nces Research, 7(12): 1992-20000, 2011
ISSN 18199-544X
This is a referreed journal and alll articles are profeessionally screenedd and reviewed
ORIGIN
NAL ARTIICLES
Evaluatiion of the Structural
S
C
Changes
off Cantal Ch
heese Throu
ughout Ripeening by
Synchroonous Fluorrescence Sp
pectroscopyy and Rheollogy Methoods
1
1
2
Shaima H.
H Othman, 2Khaled A. Abbas,
A
A. Lebecque, 1R. Bayoumi, 3G
G.A. Ibrahim
m and
M.A. Deegheidi
1
Dairy Scieence and Techn
nology Departtment, Faculty of Agriculture,, Fayoum Univversity, Egypt.
U.R. CAL
LITYSS, VetAgrro Sup, Campuus agronomiquue de Clermonnt-Ferrand, 899 avenue de l''Europe -BP 3563370 Lem
mpdes, France.
3
Dairy Scieence Departmeent, National Research
R
Centrre, Dokki, Cairro, Egypt.
2
ABSTRAC
CT
The co
ompositional, physical (coloour and texturee) and structurral changes off 12 samples of
o Cantal cheeese
representattive different ripening periood (30, 120 annd 200 days) were evaluateed by chemicaal, rheology annd
synchronouus fluorescencce spectroscoppy (SFS) methods. Synchronnous fluorescennce spectra were
w
recorded on
o
cheese sam
mples from 25
50 to 500 nm with offset ∆
∆λ= 80 nm folllowed by a cllassification of samples usinng
principal component annalysis (PCA)) and factoriaal discriminannt analysis (F
FDA). All thee compositionnal
characterisstics of Cantall cheese increeased significaantly (P<0.05) over ripeningg, except for the decrease in
calcium an
nd moisture conntents. Proteoly
ysis was the m
most important biochemical
b
chhanges of Canttal cheese durinng
ripening as revealed from the increase in the wateer soluble/totall nitrogen ration (WSN/TN %). The wateersoluble nittrogen to total nitrogen ratioo increased siggnificantly during the ripeninng period. Thee changes in thhe
rheologicaal characteristiccs and colour values
v
reflecteed the biochem
mical changes in
i Cantal cheeese. The G’, G’’,
tan δ and *
 values of chheese increased significantlyy as the ripeninng processes, but
b exhibited an
a opposite trennd
over 120 days
d
as comparred to 200 dayss. Ripening ledd to a decrease of L* and b* vvalues and a sliight increase inn a* value. The
T change in the
t fluorescencce intensity at 229 , 322 and 355
3 nm reflectss the physicochhemical changges
of cheese matrix
m
and, in particular, stru
uctural changees in the proteiin network thrroughout ripeniing period. Thhe
spectral paattern associateed with the firstt two PCs show
ws the importaance of the band with a maxim
mum at 295, 3222
and 355 nm
m which are the most suitablee for separatingg the spectra. PCA
P
and FDA show that SF spectra
s
of Canttal
cheese are clearly separated and the corrrect classificaation of 100% was
w observed. These results suggest
s
that SF
FS
in combinnation with multivariate
m
daata analysis ccould be conssidered as a fingerprint, alllowing a goood
characterizzation and classification of chheese based onn their structuraal changes throoughout ripenin
ng period.
Key wordss: Cantal cheeese, Texture, Rheology, Strructure, Colouur, synchronouus fluorescencce spectroscoppy,
chemometriics.
Introductiion
D
of
Cantall cheese is a haard-uncooked, pressed cheesee variety grantted the status oof a Protected Denomination
Origin (PD
DO) by Europpean Commisssion and produuced in the Auvergne
A
regioon in France, with an annuual
productionn of 19 000 T (CNIEL,
(
2009). Its making process
p
is veryy similar to Chheddar cheese. It is made froom
either raw or pasteurized
d cow’s milk annd commerciallized as “youngg” (ripened forr at least one month),
m
“betweeen
the two” (rripened for 2 to
t 6 months) or
o “old” (ripenned for over 6 months).
m
Canttal cheese is ch
haracterized ass a
cylinder-shhaped (round wheels)
w
cheese with a dry cruust; its weight ranges
r
betweenn 35 to 40 kg, 40
4 cm height, 36
3
to 42 cm diameter.
d
The dry
d matter conntent and the F
Fat/dry matter ratio
r
must be, respectively, at
a least 57% annd
45% resp.
Qualitty attributes off food productts are closely related to struucture. Cheese structure can be described as
protein unnits (mostly caaseins) held toogether by phhysical forces with fat, and moisture (contains mineralls,
vitamins and
a organic acids) dispersed throughout thhis structure (D
David and Auty, 2008). Mu
uch of the major
changes inn cheese struccture, which ultimately
u
affects final qualiity, occur durring ripening process.
p
Cheeese
ripening is complex prrocess of phy
ysical, chemical and microbbiological chaanges affectinng the princippal
componentts (i.e., proteinn, fat, carbohyddrate…etc) of ccheese matrix that
t affect the structure and texture
t
of cheeese
Correspond
ding Author: Shhaima, H. Othman, Dairy Sciencce and Technoloogy Depatment, Faculty
F
of Agricculture, Fayoum
U
University,
Egyptt.
E
E-mail:
[email protected]
19993
J. Appl. Sci. Res., 7(12): 199
92-2000, 2011
(Fox et al.,, 1990). Texture is the primaary quality attriibute of cheesees: it is a reflecction of cheesee structure at thhe
microscopiic and molecullar levels (Dufo
four et al., 20011).
In cheeese factories, evaluation off ripening stagge is carried by
y the cheese maker
m
on the basis of limiteed
measuremeents (pH and weight)
w
as welll as on the baasis of visual and
a tactile exaamination. In addition,
a
severral
analytical techniques
t
havve been develooped to follow cheese ripenin
ng at the laborratory level. Alll these methods
are relativeely expensive, time-consuming, require higghly skilled opperators and are not easily addapted to on-linne
monitoringg (Karoui and De Baerdemaaeker, 2007). F
For this reason
n, there is a need
n
to develoop new methods
which are rapid,
r
non-desttructive, relativvely low-cost aand monitoringg the cheese rippening process.
Rheoloogical propertties obtained in the linear vviscoelastic reggion are usefuul tools for thee food industrry.
Elastic andd viscous contrributions to the internal struccture of the ch
heese can be obbtained perform
ming oscillatory
measuremeents (Konstancce and Holsingger, 1992). Succh studies provvide an insight into the fundam
mental nature of
the physicaal basis of foodd texture (Gunaasekaran and A
Ak, 2000).
Synchhronous fluoresscence is a typee of spectroscoopy which deteects so-called ffluorophores, molecules
m
withh a
structure that
t
allows em
mission of ligght when relaxxing to the ground
g
state fr
from an exciteed singlet statte.
Synchronoous fluorescencce spectrum reecorded on a cheese
c
samplee following exxcitation betweeen 250-500 nnm
(offset 80 nm)
n gave inforrmation on sevveral intrinsic fluorophores
f
fo
ounded in cheeese and may bee considered ass a
characterisstic fingerprintt which allowss the sample too be identifiedd (Boubelloutaa and Dufour, 2010). The beest
known fluuorescent moleecules in dairry products innclude: tryptopphan residues of proteins, vitamin A annd
riboflavin, which all havee been reported
d to be affected during structtural changes in cheese (Dufo
our et al., 20011).
ften used as a reference grouup for protein structure channges, binding of
Tryptophann fluorescencee spectra is oft
ligands an
nd protein-prottein interactionns (Herbert et al., 2000). Moreover,
M
usingg vitamin A excitation,
e
as an
a
intrinsic fluuorescent prob
be, can also proovide informattion on the phy
ysical state of triglycerides and
a protein–lippid
interactionns (Dufour et al.,
a 2000). Ribo
oflavin can be used for the evaluation
e
of oxidative
o
changges in processeed
cheese durring storage (W
Wold et al., 20002).
The ob
bjective of thiss research werre to evaluate cchanges in com
mpositional (pH
H value, moistture, protein, faat,
WSN/TN%
%, salt, Ca annd ash contennts) and physiical (color andd texture) chaaracteristics off Cantal cheeese
throughoutt ripening proccess. And to evvaluate the pottential of synchhronous fluoreescence spectrooscopy to folloow
the ripeninng phenomena of Cantal cheeese. In order to
t discriminatee between thesse cheeses in term
t
of ripeninng
period, thee principal com
mponent analy
ysis (PCA) annd factorial discriminant anaalysis (FDA) were applied to
synchronou
us fluorescencee data.
Materials and Methods
Cheese Sam
mples:
Twelv
ve samples of Cantal
C
cheese varying in ripeening period (3
30, 120 and 2000 days) were supplied by tw
wo
different chheese-manufaccturing plants location in thee Auvergne reg
gion in Francee. Samples (weeighting 2-3 kgg.)
were cut off
o in the midd
dle of the cheesse height at 2 cm
c from the riind. About 9000 g was gratedd and thoroughhly
homogenizzed for physicoo-chemical, rheeological and ssynchronous flu
uorescence anaalysis.
Physicocheemical Analysiis:
pH vaalues were meaasured by a pH
H meter (Schottt, CG840, Parris, France) aft
fter grating 10 g of cheese annd
dispersing it in 50 ml. off ionized waterr. The moisturee content was determined
d
by desiccation at 105°C for 24 h,
2
The tottal
and fat coontent was meaasured by Gerrber method aaccording to French standardds (AFNOR, 2004).
nitrogen was
w determinedd by Kjeldahl method
m
(FIL-IIDF standard 20B;
2
(IDF, 19993). Cheese ex
xtract for wateersoluble niitrogen (WSN
N) was prepareed according to (Bouton et
e al., 1994). Briefly, 3 g of cheese was
w
homogenizzed with 50 ml
m of distilled water for 5 m
min with a laaboratory blendder (Stomacheer MIX 1, AE
ES
Laboratoire, Combourg, France) and thhe resulting hoomogenate wass maintained foor 1 h in a watter bath at 40°C
C.
The insoluuble material was
w centrifugedd at 1200 for 330 min. at 4°C
C. The supernattant was filtereed through glaass
wool, and nitrogen conttent was deterrmined on a fi
filtrate aliquot by (Kjeldahl method IDF, 1993). The saalt
content off cheese was determined
d
according to Frennch standard (A
AFNOR: NF IISO 5843) usin
ng an automattic
titrator (TiitroLine easy, Model III, Scchott, France) which is based on Volhardd titrimetric teest according to
(Marchall method IDF, 2003).
2
The ashh content was ddetermined aftter incinerationn of a sample (5
( g) in a mufffle
d by using an aatomic absorptiion spectroscoppy
furnace at 550°C for 6 h.. The total calccium of cheese was measured
m
± standaard
as describeed by (IDF, 20003). All analyyses were donee in triplicate and the resultss reported as mean
deviation.
1994
J. Appl. Sci. Res., 7(12): 1992-2000, 2011
Colour Measurments:
Cheese colour was measured using a colorimeter CR-400 (Konica Minolta, Tokyo, Japan). The L*, a*, and
b* colour measurements were determined according to the CIELAB colour space (CIE ,1976) with reference to
D65 (natural daylight, the colour warmth of 6500K) and observation angle 10°. The following parameters were
determined; L* (lightness or whiteness; L*=0 for black and L*=100 for white colour), a*(red-green
components, - a*=greenness and + a*= redness) and b* (yellow-blue components, - b*= blueness and
+b*=yellowness). The colorimeter was calibrated with a white standard plate 3.5 cm thick layer (X = 0.3155, Y
=0.3319, Z=94.0) before the measurements. Colour measurements were made 5 times, 1 on the middle and 4 on
different parts of cheese surface after removing a 0.5 cm layer of upper surface.
Rheological Measurments:
For rheological characterization, cheeses were sliced into thin disks (2 mm.thick and 20 mm. diameter) with
a cheese slicer. The dynamic oscillatory analyses were performed with a rheometer (CP 20, TA Instrument,
Guyancourt, France) with a plate geometry of 20 mm. diameter. Temperature sweep tests were used to
determine the viscoelastic characteristics of the cheeses in the linear viscoelastic region by applying force (0.5
N) at a constant frequency of 1Hz as a function of temperature according to (Karoui et al., 2003) Parameters
describes the viscoelastic characteristics of the cheeses included the elastic component G’ (storage modulus), the
viscous component G" (loss modulus), the phase angle (Tan δ), and the complex viscosity (η*). Three
cylindrical specimens were tested for each cheese sample.
Synchronous Fluorescence Spectroscopy:
Synchronous fluorescence spectra were recorded using a FlyotoMax-2 spectrofluorimeter (Spex-Jobin
Yvon, Longjumeau, France) mounted with a front-surface accessory. The incidence angle of the excitation
radiation was set at 56° to ensure that reflected light, scattered radiation and depolarization phenomena were
minimized. Spectra of cheese slices (2 cm long,1 cm wide, 0.2 cm think) mounted between two quartz slides
were recorded at 20°C with emission and excitation slits set at 4 nm SF spectra were recorded in the 250-500
nm excitation wavelength range using offsets of 80 nm (Boubellouta and Dufour, 2010) between excitation and
emission monochromators. For each cheese sample, three spectra were recorded on 3 different slices.
Statistical Analysis:
One-Way ANOVA was carried out for the chemical and rheological data in order to assess significant
differences among the samples throughout ripening and results reported as mean ± standard deviation. The
Fisher least square difference (LSD) test was performed for each significant factor at a level significance of 5%.
All calculations were carried out with XLSTAT software version 2007 (Addinsoft, France).
Principal components analysis (PCA) and Factorial discriminate analysis (FDA) were the two chemometric
tools used in the multivariate evaluation of fluorescence data; both techniques based on a linear decomposition
of data.
PCA (Wold et al., 1987) provides an approximation of a data matrix, X into a few vectors, in terms of the
product of two sets of vectors, T (scores) and P (loadings). These vectors capture the essential patterns of X, and
are called latent variables or principal components (PC). PCA of the fluorescence data was applied in order to
obtain the best possible overview of the spectral structure and distribution of samples. Score plots visualize the
relationship between cheese samples for each PC, while loadings plots were used for interpretation of the
corresponding spectral variation (Bertrand et al., 1987).
FDA technique (Safar et al., 1994) aim to predict the membership of an individual to a qualitative group
defined as a preliminary. FDA assesses new synthetic variables called ‘‘discriminant factors”, which are linear
combinations of the selected PCs, and allows a better separation of the centres of gravity of the considered
groups. FDA was applied on the first 5 PCs performed on spectral data set to evaluate the potential of SFS to
discriminate cheeses according to structural changes throughout ripening. A group was created for each ripening
period (i.e. 30, 120 and 200 days). Synchronous fluorescence spectra were not subjected to any kind of
preprocessing before analysis. PCA and FDA were performed by using MATLAB version 6.5 software (The
Mathworks Inc., Natica, MA, USA).
Results and Discussion
Compositional Changes of Cantal Cheese Throughout Ripening Periods:
Table (1) indicated that as ripening progressed, fat, protein, salt, WSN/TN % and ash contents of Cantal
cheese continuously increased, as a result of the significant decrease in the moisture content, whereas the
calcium and fat in dry matter contents decreased. This can be related to cheese ripening, released amino acids
1995
J. Appl. Sci. Res., 7(12): 1992-2000, 2011
raise pH value to a somewhat higher level (Waagner, 1993). The WSN/TN % of cheeses increased during the
ripening period, indicating progressive proteolysis. It has also been reported that there is an appreciable
reduction in the amount of calcium content in cheese during the ripening period because of the solubilization of
colloidal Ca phosphate (CCP). The reduction in the amount of calcium associated with casein molecules (i.e.,
CCP) and hydrolysis of casein would be expected to alter cheese texture (Lucey et al., 2003; 2005).
Table 1: Mean (±SD) of chemical characteristics and texture of Cantal cheese throughout the ripening periods.
Cantal cheese
Young
Mild
Old
Parameters
(30 days)
(120 days)
(200 days)
pH
5.23(±0.01)c
5.41(±0.02)b
5.79(±0.01)a
Moisture (%)
42.92 (±0.06)c
38.61(±0.05)b
34.90(±0.05)a
Protein (%)
24.43(±0.08)c
25.11(±0.19)b
28.40(±0.46)a
Fat (%)
29.92(±0.14)c
31.83(±0.14)b
32.50(±0.00)a
Fat in dry matter (%)
52.41(±0.28)a
51.85(±0.21)b
49.92(±0.04)c
WSN/TN (%)
11.27(±2.30)c
25.62(±0.64)b
39.95(±0.51)a
b
b
Salt (%)
1.34(±0.03)
1.43(±0.08)
1.63(±0.01)a
Ash (%)
4.20(±0.03)b
4.33(±0.02)b
4.62(±0.03)a
Total Ca (%)
0.765(±1.41)a
0.743(±2.30)ab
0.717(±2.22)b
Texture attributes
G' (KPa)
12.69(±2.09)b
58.64(±4.33)a
51.15 (±1.56)a
G'' (KPa)
4.36(±0.74)b
19.21(±1.15)a
18.02 (±0.54)a
Tan δ (G’’/G’)
0.34(±0.00)ab
0.33(±0.00)b
0.35 (±0.02)a
η* (KPa.s)
2.14(±0.35)b
9.83(±0.71)a
8.14 (±0.26)a
One-Way ANOVA was applied to data and values in the same row with different superscript letter are significantly different (P<0.05, LSD
test)
Physical Characteristics:
The Changes of Colour Values in Cantal Cheese Throughout Ripening Periods:
No significant differences were observed in the colour values (L* and a* values) of Cantal cheese samples
ripened for at 30, 120, and 200 days, although a slightly lower values for L* and a* values were found in aged
samples (200 days) (Figure 1). The values of L* and b* indicate that the young cheeses had a light yellow
colour which aquired more darker colour as the ripening progress. Regarding the a* parameter the cheeses had,
in general, negative values. Negative numbers for the a* value indicate that cheeses are more green than red.
The values of the b* confirms that the predominant colour of the cheeses was yellow.
The cheese whiteness is influenced by several factors including light scattering of fat and protein particles
(Rudan et al., 1998) and whey pockets (Paulson et al., 1998). As ripening progressed, whey in serum pockets
diffused, from cheese body out, as seen in moisture loss. The surface area occupied by light-scattering centers
was therefore decreased. Thus, changes in Cantal colour throughout the ripening was probably and mainly
attributed to the loss of moisture content which in turn increase the dry matter content and in parallel to changes
in the decreased light scattering, and hence, lower L* and b* values.
Our results are in agreement with other who described a decrease in both lightness (L*) and yellowness (b*)
and a slight increase in redness (a*) during cheese ripening (Rohm and Jaros, 1996; Pillonel et al., 2002).
Fig. 1: Changes in colour values (L*, a* and b*) of Cantal cheese ripened for 30, 120 and 200 days.
Rheological changes in Cantal cheese throughout the ripening periods.
Changes in the rheological characteristics i.e. storage modulus (G’), loss modulus (G’’), the phase angle
(tan δ) and viscosity complex (η*) of Cantal cheese during the ripening are presented in Table (1) and fig. (2).
1996
J. Appl. Sci. Res., 7(12): 1992-2000, 2011
In general, at any ripening stage the G’(index of the firmness), was always higher than G’’ which indicates
the predominating solid character of cheeses (Ustunol et al., 1995). Moreover, the value of tan δ for Cantal
cheese was less than 1.0 indicating that the elasticity nature (G’) of the samples was higher than their viscous
nature (G’’), an indication that the cheese exhibited solid-like behaviour (Tunick et al.,1993).
One-way ANOVA showed that there were significant differences (P < 0.05) in all textural properties
between the cheeses ripened for 30 and 120 days, but the further increase of the ripening period to 200 days
slightly decreased in the G’ and G” as compared to 120-days-old cheeses. The increase in the viscosity complex
(*) observed throughout ripening can be attributed to degradation of protein (Visser, 1991; Olson et al., 1996).
Fig. 2: Changes in rheological characteristics (G’, G’’, Tan  and *) of Cantal cheese ripened for 30, 120 and
200 days.
These differences in rheological parameters could be explained by a magnitude of two opposite effects
(weakening of cheese matrix due to proteolysis and firming effect due to moisture loss) throughout ripening
period which would be predominant depending on the extent of proteolysis, pH and water content.
Differences between the cheese ripened for 30 and 120 days was probably attributed to, in the early stages
of ripening time, the degree of curd fusion and contact area between casein particles was low which we believe
to be responsible for the increase in the rheological parameters. The long-ripened cheese (120 and 200 days) the
rheological parameters were decreased, but this decrease did not statistically important, due to extended
proteolysis (Khosroshahi et al., 2006), a gradual breakage of the network calcium bonds (Ehsani et al.,1999) and
the loss of water available of solvation of the protein chains and the consequent formation of a more compact
cheese matrix (firmness cheese). Similar results were in agreement with those obtained for soft cheese (Karoui
and Dufour, 2003) and Cheddar cheese (Wick et al.,2004).
Synchronous fluorescence spectroscopy :
The ripening of Cantal cheese was studied in terms of various structural changes at the molecular levelprotein structure and interactions associated with protein and protein-lipids interactions by following the
changes in the intrinsic fluorophores (tryptophan, vitamin A and riboflavin) exist in cheese by SFS.
4
18
x 10
30 days
120 days
200 days
Fluorescence intensity (a.u.)
16
14
12
10
8
6
4
2
0
250
300
350
400
Wavelength (nm)
450
500
Fig. 3: Changes in synchronous fluorescence spectra of young (30 days), mild (120 days) and old (200 days)
Cantal cheese collected in the 250-500 nm excitation wavelength range using offsets of Δλ 80 nm.
The synchronous scans performed on Cantal cheese throughout the ripening period showed the presence of
three major fluorophores, namely; 295, 322 and 355 nm, as shown in Figure (3). The synchronous fluorescence
spectra showed slightly different shapes between the investigated cheeses and the fluorescence intensity
decreased in accordance with the degree of ripening period.
1997
J. Appl. Sci. Res., 7(12): 1992-2000, 2011
From figure (3), for all cheeses, the highest synchronous fluorescence peak was obtained with excitation at
295 nm (emission at 375 nm), followed by that at 322 nm (emission at 402 nm), and that at 355 nm (emission at
435 nm). Apart from these three major peaks, smaller peaks were observed at around 449-490 nm The band
observed at 295 nm could be attributed to tryptophan residues of proteins (Karoui et al., 2004), while the band
appeared at 322 nm (emission 402 nm) was probably related to vitamin A (Karoui, 2004) and the band appeared
at 355 nm (emission at 435 nm) was probably related to riboflavin (Karoui et al., 2007c). Finally, the bands
around 449-490 nm could be assigned to some coenzymes (e.g. NADH, FADH) (Kulmyrzaev et al., 2005),
riboflavin found in milk (Boubellouta & Dufour, 2008) and Maillard-reaction products (Kristensen et al., 2001;
Karoui et al., 2007).
The observed differences for tryptophan (band at 295 nm) and vitamin A fluorescence spectra (band at 322
nm) are consistent with changes of cheese matrix structure and lipid structure throughout the ripening period,
respectively (Dufour and Riaublanc, 1997; Dufour et al., 2000). Concerning the changes in the band at 355 nm
excitation fluorescence spectra, this could be attributed to the lipid oxidation of cheeses throughout ripening
which could contribute to the change observed on the riboflavin spectra.
The ability of synchronous spectra data to discriminate Cantal cheeses ripened for different periods was
analyses by principal component analysis (PCA) and factorial discriminat analysis (FDA), respectively.
Firstly, PCA was applied to the set (24 objects and 251 variables) of synchronous fluorescence spectra recorded
on Cantal cheese throughout the ripening period. The first two principal components accounted for 94 % of the
total variance with a large predominance of the principal component 1 (explains 76.36%). Figure (4 a) shows the
score plot of PC1 (explains 76.36% of total variance) versus PC2 (explains 17.92% of total variance) of PCA
plot applied on the synchronous fluorescence spectra of young (30 days), between the two (120 days) and old
(200 days) Cantal cheeses.
Three groups of cheese were observed; the first group (30 days) can be seen in the upper right quadrant
which have high PC1 values; the second group (120 days) can be seen in the lower right quadrant of the low PC1
values and the third group (200 days) can be seen in the upper left quadrant according to PC1. It appeared that
the first and second groups exhibited positive values according to PC1, the third group (200 days) showed
negative values according to PC1 and positive values according to PC2 (Figure 4 a). These differences reflected
changes in the structure of cheese matrix, the physical state of triglycerides and protein-lipid interactions
throughout cheese ripening. It was concluded that one (or more) continuous phenomenon, taking place during
the ripening, was detected when the fluorescence of the intrinsic was considered.
In order to point out which wavelengths were involved in the discrimination of the cheese samples, the
factor loadings associated with the PC1 and PC2 were analyzed (Figure 4 b). The factor loadings for PC1 and PC2
shows the importance of the bands with maxima at 295 (assigned to tryptophan) at 322 (assigned to vitamin A)
and 355 nm (assigned to riboflavin), and they describes changes in these bands throughout ripening.
Factor loadings 1 (Figure 4 b) characterized the samples on the right of the map (Figure 6 a) which it were
characterized by a relatively higher fluorescence intensity than those on the left side. It indicated that during the
ripening process, the main components of cheese (casein and fat) are subject to physical and chemical changes,
which effect on the fluorescence intensity of tryptophan, vitamin A and riboflavin, resulting in changes in the
structure of casein micelles. These structural changes can induce a more hydrophilic environment of the
tryptophan of caseins in accordance with the red shift of the maximum for the older cheeses and change in the
shape of vitamin A spectra which was found to correlate with lipid oxidation of cheese. Moreover, ripening
involves mainly an increase in pH value, a change in protein-protein and the physical state of triglycerides and
protein-lipid interactions. The pH of 30, 120 and 200 days-old cheeses were 5.23, 5.41 and 5.79, respectively
(Table 1).
Factor loadings 2 (Figure 4 b) indicated that the shape of fluorescence spectra was larger for cheeses
located on the positive side (30 and 120 days) than for those on the negative side (200 days). It appeared that
changes in fluorescence spectra observed could be due to different protein-protein/fat interactions and different
network structures resulting from the ripening process. Our results confirm previous findings (Herbert, 1999;
Dufour et al., 2001; Mazerolles et al., 2001; Karoui et al., 2007; Karoui et al.,2007) reporting that three intrinsic
fluorophores presented in the cheese could be considered as fingerprints allowing a good identification of
changes in the cheeses as a function of their ripening time.
Secondly, in order to find out the differences between cheeses at the molecular level-protein structure and
interactions throughout the ripening, the FDA was applied on the first 5 PCs of the PCA performed on the
synchronous fluorescence spectra of Cantal cheese throughout ripening. The similarity map of the FDA allowed
a good discrimination of the investigated cheeses. The map defined by the discriminant factors 1 and 2
represented 100 % of the total variance with discriminant factor 1 accounting for 82.10 % (Figure 5).
Considering discriminant factor 1, Cantal cheeses ripened for 120 days and 200 days exhibited negative scores,
whereas Cantal cheeses ripened for 30 days had positive score values. The discriminant factor 2 which took into
1998
J. Appl. Sci. Res., 7(12): 1992-2000, 2011
account 17.90 % of the total variance differentiated between 120-days-old and 200-days-old Cantal cheeses. A
correct classification of 100 % was obtained.
Fig. 4: (a) Principal component analysis similarity map (score plot) determined by principal components 1 (PC1)
and principal component 2 (PC2) and (b) factor loadings corresponding to PC1 and PC2 performed on
the synchronous fluorescence spectra of the Cantal cheese ripened for 30, 120 and 200 days. The
lines in (b) indicate: PC1 (solid) and PC2 (dotted).
0.3
0.2
200 days
F2 (17.90%)
0.1
0
-0.1
30 days
120 days
-0.2
-0.2
-0.1
0
0.1
F1 (82.10%)
0.2
0.3
Fig. 5: Discriminant analysis similarity map determined by discriminant factors 1 (F1) and 2 (F2) for the
factorial discriminant analysis (FDA) performed on the first 5 principal components (PCs) of the
principal component analysis (PCA) applied to the synchronous fluorescence spectra of Cantal cheeses
ripened for 30, 120 and 200 days.
Conclusion:
The compositional characteristics (pH value, fat, protein, salt, WSN/TN% and ash contents) increased
significantly during the ripening period but calcium and the moisture contents decreased to some extent.
Ripening significantly influenced colour, resulting in a decrease of L* and b*, but it was observed a slight
increase in a* value over ripening. Rheological characteristics increased with the ripening period, showing that
ripening contributed to changes in the structure of cheese matrix, where the differences in G’ and G” were
observed. The results of FDA performed on PCs, showed a good discrimination of the cheeses from their
spectral data. Synchronous fluorescence spectroscopy presents a suitable alternative for monitoring changes in
the chemical characteristics of Cantal cheese throughout the ripening period compared with the routine analysis.
SFS could be considered as a fingerprint, allowing a good identification of cheese based on their structural
changes throughout ripening period.
1999
J. Appl. Sci. Res., 7(12): 1992-2000, 2011
Acknowledgments
We would like to thank the VetAgro Sup, Campus Agronomique, Clermont-Ferrand, France for funding
this study and providing samples and Delphine Guerinon is thanked for her technical assistance.
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