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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. 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