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Food Control 42 (2014) 101e108

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Food Control

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Detection of as1-I casein in mozzarella Fiordilatte: A possible toolto reveal the use of stored curd in cheesemaking

M. Faccia a, A. Trani a, P. Loizzo a, R. Gagliardi c, B. La Gatta b, A. Di Luccia b,*

aDipartimento di Scienze del Suolo, della Pianta e degli Alimenti, University of Bari, Via Amendola 165/A, 70126, Bari, ItalybDipartimento di Scienze Agrarie, degli Alimenti e dell’Ambiente, University of Foggia, Via Napoli 25, Foggia, Italyc Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute, 2, 80055, Portici Napoli, Italy

a r t i c l e i n f o

Article history:Received 14 November 2013Received in revised form28 January 2014Accepted 31 January 2014Available online 11 February 2014

Keywords:FiordilatteStored curdPrimary proteolysisProteomic approachMolecular marker

* Corresponding author. Tel./fax: þ39 881589243.E-mail addresses: aldo.diluccia@unifg.it, a.scognam

http://dx.doi.org/10.1016/j.foodcont.2014.01.0440956-7135/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The assessment of the origin and quality of raw materials is pivotal for the protection and valorization oftypical dairy products. Italian high moisture mozzarella cheese, also called “Fiordilatte”, is manufacturedby stretching in hot water the curd, which can be produced inside the dairy (fresh curd) or purchased assemi-finished product from specialized companies (pre-made stored curd). The employment of storedcurd allows cheese manufacturers to obtain economic profit thanks to the reduction of the productioncosts. The use of the semi-finished product has not to be mentioned in the label according to the actualEU regulation, and this has given rise to competition between local milk farmers and curd-producingcompanies causing misinformation among the consumers, and unfair competition to the disadvantageof the traditional dairies. In the present research, a proteomic approach was used to investigate “Fior-dilatte” produced with or without the employment of stored curd. The results obtained allowed us toidentify a molecular marker which is present in very small amounts in cheese made from fresh curd andat high levels in cheese made from stored curd. The marker is as1-I casein (fragment 24e199), the mainproduct of primary proteolysis formed by the action of chymosin on as1 casein. It can be easily detectedby electrophoresis in the presence of urea (urea-PAGE) and is measured by image analysis. Even thoughthe kinetic of formation of this proteolytic product has been well known for several decades, inmozzarella it has a peculiar behavior compared to other cheeses. As a matter of fact, it originates duringthe first part of cheesemaking, and has a very slow increase during storage of the cheese, due to thedenaturation of the clotting enzyme by the hot water stretching process. The results obtained demon-strated that the quantification of as1-I fragment should be recommended to guarantee the production of“Fiordilatte” obtained from fresh milk under normal conditions, and could be the basis for the devel-opment of a method able to fully protect the traditional cheesemaking procedure.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Mozzarella cheese is the most commonly produced cheeseworldwide, due to its characteristics that allow consumption both asfresh table cheese (high moisture type, made both from buffalo andcowmilk) and as an ingredient for very popular food specialties suchas pizza (low moisture type, made from cow milk). Most of the sci-entific knowledge available on this cheese regards low moisturemozzarella, which is very different with respect to the table type. Infact, it is shaped in parallelepiped blocks weighing a few kilogramsand has amaximummoisture content of 52%; furthermore, it needs a

iglio3@tin.it (A. Di Luccia).

short ripening period to fully develop the required textural andfunctional properties, and can be preserved for more than 1 month(Gunasekaran & Ak, 2003; Joshi, Muthukumarappan, & Dave, 2004;Kindstedt, 1993; Kindstedt & Fox, 1993; Rowney, Roupas, Hickey, &Everett, 2004; Rudan, Barbano, Yun, & Kindstedt, 1999). Instead, ta-ble mozzarella cheese has a spherical shape, a weight of 10e300 gand 60e70% moisture content. In Italy it is also called “Fiordilatte”when manufactured from cow milk, it is packaged into plastic bagsor vessels with water as preserving liquid and has a very short shelf-life, ranging froma few days to twoweeks (De Candia et al., 2008; DelNobile, Gammariello, Di Giulio, & Conte, 2010; Faccia, Mastromatteo,Conte, & Del Nobile, 2012; Faccia, Trani, & Di Luccia, 2009; Minerviniet al., 2012). The functional and organoleptic properties of lowmoisture mozzarella are deeply influenced by proteolysis: softeningand changes in other functional properties during storage are

M. Faccia et al. / Food Control 42 (2014) 101e108102

attributed to casein hydrolysis (Creamer, 1976; Farkye, 1995; Farkye,Kiely, Allhouse, & Kindsteadt, 1991; Tunick et al., 1993). In general,primary proteolysis in this cheese is considered to progress quiteslowly, due to partial thermal inactivation of residual rennet duringthe stretching process (Mc Goldrick & Fox, 1999). However, the ratecan also be influenced by: the type and amount of rennet used incheesemaking (Farkye et al., 1991; Kindstedt, Yun, Barbano, & Larose,1995; Yun, Kiely, Kinstedt, & Barbano, 1993); the extent of itsretention in the curd (Dave, McMahon, Oberg, & Broadbent, 2003);the curd cooking temperature (Tunick et al., 1993; Yun, Kiely,Barbano, & Kinstedt, 1993); the time and temperature of stretching(Renda, Barbano, Yun, Kindstedt, & Mulvaney, 1997); and finally bypH and calcium content of cheese (Feeney, Guinee, & Fox, 2002). Asregards the role of microorganism, Yun, Barbano, Kiely, and Kinstedt(1995) reported that secondary - but not primary - proteolysis issignificantly influenced by starter populations even when it reachesvery high levels in cheese; as for the contaminantmicroflora, Baruzzi,Lagonigro, Quintieri, Morea, and Caputo (2012) and Morales,Fernandez-Garcıa, and Nunez (2003), reported that a certain rolecan be played in cheese by environmental non lactic acid-bacteriaonly if they reach very high cellular densities (more than 108). Asfar as “Fiordilatte” is concerned, no recent and sound scientific studyspecifically directed to assess proteolysis is available; however, thedifferent processing and storage conditions should give rise to adifferent pattern of casein degradation with respect to low moisturemozzarella cheese. In particular, considering the short time fromproduction to consumption, it should undergo lesser proteolysis.

Italy is the main producer of “Fiordilatte”, whose production isconcentrated in the Southern regions, but its production is rapidlyspreading in Europe. Due to the expanding market and the need ofmore efficient and competitive technological solutions, the produc-tion technology is undergoing continuous innovation regarding onthe improvement of qualityand the reduction of theproduction costs.Regarding the production costs, some dairy industries have modifiedthemanufacturing cycle by implementing theuseof “stored” curd inamixture (fresh and stored) or as a replacement of the freshly preparedone. The stored curd is a pre-made semi-finished product purchasedfrom specialized companies: different from fresh curd, it has a lowmoisture content and is long-storableby refrigerationor freezing. Thistechnology offers economic profit, since the stored curds can bepurchased at competitive costs and allow to shortening of the pro-duction cycle: “Fiordilatte” can easily then be produced only by thestretching step, and all the problems and costs related to the man-agementof liquidmilk are eliminated (or reduced). As a consequence,a sort of competition between producers of freshmilk and producersof curd has been born, it has also to be taken into consideration thatthe EU regulation does not require yet the specification in the label ofthe use of the semi-finished product.

In the view of the possible protection of “Fiordilatte”made fromfresh milk, and also considering that the EU protected type of“Fiordilatte” also exists (Traditional Specialty Guaranteed Mozza-rella cheese, EC 2527/1998), it should be necessary to have amethodcapable to discriminate between the cheesemaking methods (freshor stored curd). The aim of the present paper was to investigate thematter by a proteomic approach and to individuate a possible mo-lecular marker useful for discriminating the two types of cheese.

2. Material and methods

2.1. Curds and cheeses produced with preserved curd

Thirty-two samples of freshly produced curd and 17 samples ofstored curd were taken from 13 dairies located in the Apulia Region(Italy), and 2 wholesalers. Eight batches of “Fiordilatte” weremanufactured in a local dairy by using different amounts of stored

curd (Bad Bibra, Germany) in mixture with freshly produced curd.Twenty kg of stored curd were minced and then mixed in differentproportions (15, 20, 30, 50, 60, 80%), with fresh curd; the 6mixturesand the two curds (used as control) were manually stretched in hotwater (80 �C), obtaining 5 kg of “Fiordilatte” for each of the batches.After production, the cheeses were cooled in chilled water, pack-aged in plastic vessels and transported to the laboratorywhere theywere immediately analyzed by proteomic techniques.

2.2. Two dimensional gel electrophoresis (2DGE) of curd

For 2DGE, an immobilized pH gradient (IPG) was used as the firstdimension and an 8%e18% SDS-PAGE orthogonal pore gradient asthe second dimension (O’Farrell, 1975). Sixty micrograms of curdtotal protein were dissolved in a rehydration buffer (8 M urea, 1 Mthiourea, 50 mM DTT, 2% CHAPS, 2%IPG buffer pH 3e10) and loadedonto a 13 cm IPG Dry Strip pH 3e10 (Amersham Bioscience, Uppsala,Sweden). The first dimension was provided using the Multiphor IIelectrophoresis unit (Amersham Bioscience) in 4 steps: 500 V/h,1000 V/h, a gradient to 8000 V over 110min, and 8000 V for 3 h. Afterthe IPG electrophoresis, the strips were equilibrated for 25 min in asolution containing 1.5M TriseHCl, pH 6.8, 6M urea, 30% glycerol (p/v), 2% SDS, 64 mM dithiothreitol, 135 mM iodoacetamide, and a traceof bromophenol blue. The equilibrated IPG Dry Stryps were loadedon top of the 8e18% SDS-PAGE gradient gel (14 � 16 cm), and elec-trophoresis was carried out overnight at a constant voltage of 100 Vand at 15 �C. The 2-dimensionalmapswere stainedwith Brillant BlueCoomassie G250, destainedwith double distilledwater, and analyzedusing the Image Master Platinum software (Amersham Bioscience).

2.3. Mass spectrometry identification

Protein spots from 2 DGE were manually excised and an in situdigestionwas applied. They were destained, reduced, and alkylatedby carboxymethylation and then digested in situ by trypsin (SigmaAldrich, St. Louis, MO, USA) overnight at 37 �C, according to theprocedure of Shevchenko, Wilm, Vorm, and Mann (1996). Thepeptides resulting from the digestion step were extracted 3 timeswith 40 mL of AcN, water, and formic acid solution (50:45:5, vol/vol/vol) and then concentrated by vacuum centrifugation for MSanalysis. Finally, the peptide mixtures were dissolved in aqueous50% (vol/vol) AcN containing 1% (vol/vol) formic acid. Duplicatealiquots of peptide mixture solution were mixed (1:1) with matrixsolution (a-cyano-4-hydroxy-cinnamic acid in 50% v/v acetonitrilecontaining 0.5% v/v trifluoroacetic acid). This mixture (0.3 ml) wasapplied in duplicate (two different spots or bands) on a sample slidetray and allowed to dry in air. Mass spectra were acquired inreflection mode, using an Ettan MALDI-ToF Pro mass spectrometer(Amersham Biosciences). Calibration of the time-to-mass scale wasperformed using two external standard peptides (ile7AngIII, MþH897.531, monoisotopic, and hACTH 18e39, MþH 2465.191, mono-isotopic). MALDI-ToF analysis was carried out in triplicate and alsousing the internal standard peptide hACTH 18e39.

2.4. Polyacrylamide gel electrophoresis in the presence of urea(urea-PAGE) of curd and cheese

The samples were dissolved in 9 M urea and loaded onto adiscontinuous gel polyacrylamide in the presence of urea(16 � 18 cm). Electrophoresis was conducted as reported byAndrews (1983) and the gels were stained with Silver Bleu(Candiano et al., 2004) and destained in water. The image of thedestained gel was scanned and subjected to densitometry analysisusing software Quantity One (Biorad, Hercules, CA, USA). The op-tical densities obtained, were expressed in relative quantity (RQ)

M. Faccia et al. / Food Control 42 (2014) 101e108 103

compared to the total area of the identified bands in the electro-phoretic pattern of each sample.

2.5. Kinetic study on as1-I casein in fresh curd and cheese producedat laboratory level

Pasteurized whole bovine milk was purchased from a local shopto prepare fresh curd at the laboratory (five replicates were carriedout by using 2 L of five different milk brands). Processing involveddirect acidification of the milk to pH 5.75 with citric acid and coag-ulation by addition of single (0.18 mL/L) or triple (0.54 mL/L) dose ofrennet (1:10,000 strenght, chymosin/pepsin ratio 75:25, Sacco srl,Cadorago, Italy). Each of the curds obtained were split into two partsthat were kept at different conditions: in whey at 37 �C for 6 h andout of whey at 10 �C for 50 h (Fig. 1). The as1-I-CN formation wasmonitored at 1, 2, 4, 6 h of storage inwhey and at 20, 26, 44 and 50 hof storage out of whey; after collection, the samples were immedi-ately dissolved in urea 9 M and stored at 4 �C until the series wascompleted for analysis. From the curd stored in whey, samples of“Fiordilatte” were made by stretching under controlled conditions(1:1water/curd ratio, temperature of the paste 60 �C) aliquots of curdtaken at 1 and 6 h. The cheeses obtained were stored in pot water at4 �C and analyzed at 1, 2 and 5 days by urea-PAGE as above indicated.

2.6. Determination of as1-I-casein in industrial cheese

Samples of “Fiordilatte” were produced at a local dairy from thesame batch of fresh milk (24 h from milking) by two different

Fig. 1. Protocol of production of curd

technologies: direct acidification with citric acid and acidificationwith natural whey starter culture as described by Faccia,Gambacorta, Quinto, and Di Luccia (2010). After production thecheeses were cooled, packaged in plastic vessels filled with chilledpot water, and transported at the laboratory where they werestored at 10 �C for 15 days. Sampling was made at 0, 7, 10 and 15days of storage. Besides these samples produced under controlledconditions, 81 samples of freshly produced “Fiordilatte” wererandomly collected from the storage warehouse of 27 dairies afterverification of the lot number/production date and storage tem-perature (4 � 2 �C). Out of 81 samples, 48 were declared by theproducers as manufactured from fresh curd and 33 with addedpreserved curd. All samples were subjected to one-dimensionalelectrophoresis analysis, as previously reported.

2.7. Statistical analysis

All data showed were means obtained from at least threeanalytical determinations. Descriptive statistics, regression analysisand t Student test were performed by SPSS v.19 (IBM, NewYork,USA).

3. Results and discussion

3.1. Proteomic investigation

In Fig. 2 the 2DGEmaps of fresh and preserved curds are shown;for identification, all the gel spots were cut and digested with

and Fiordilatte at the laboratory.

Fig. 2. Two dimensional electrophoresis analysis of two curd samples: freshly produced (A) and stored (B). Identification of the bands is reported in Table 1.

M. Faccia et al. / Food Control 42 (2014) 101e108104

tripsin. By the peptide mass fingerprints (PMF) obtained byMALDI-TOF mass spectrometry and by the position of the spots in terms ofmolecular weight (MW) and isoelectric point (pI) identificationswere provided. As shown in table 1, PMFs of spot 1 and spot 3 wererecognized as as1-casein (CN) and as1-I-CN, respectively. The actual,spectrum of whole as1-CN (inset of Fig. 3) shows two higher signalat 1267 and 1760 m/z than as1-I-CN, whose spectrum shows theabsence of the signal at 1760 m/z corresponding to the peptide 8e22, and of the signal at 1384 corresponding to the peptide 23e34.By comparing the maps, the main difference regards spot 3, whichis characterized by MW of about 20 kDa and pI of 4.10: it is veryintense in the preserved curd sample, whereas it is slightlydetectable in the fresh one.

Fragment of as1-I-CN is themain proteolytic product obtained byrennet digestion of this casein (Mc Sweeney, Olson, Fox, Healy, &Højrup, 1993; Mulvihill & Fox, 1979). In cheesemaking, the pro-duction of this large peptide is closely associatedwith chymosin: itsformation takes place already at the earliest stages of production,but proceeds during the whole ripening period at a rate that de-pends on temperature, water activity, amount of rennet resituatedin the paste, pH, NaCl concentration, etc. The results of the 2DGEanalysis are fully explainable by the different ages of the curdsanalyzed, and were expected: the higher the age of the curd, thehigher the presence of products of proteolysis. Several samples offresh and preserved curd were then analyzed by alkaline urea-PAGE, the most widely used electrophoretic technique to investi-gate primary proteolysis in cheese and able to reveal several largecasein fragments. As shown in Fig. 4 the fragment was confirmed tobe always detectable at much higher intensities in the stored curdsamples than in the fresh ones; the intensity among the sampleswas different, probably depending on the age and manufacturing/storage conditions. Through the electrophoretic mobility and PMFobtained by MALDI-TOF mass spectrometry, the identity of theband was confirmed, and the casein fragment was called “ALMI�”,according to the Italian Patent n. 0001397624, for application in this

Table 1Data identification of protein spots by peptide mass fingerprinting.

Spot N. Casein N Peptidesa Phosphate MW [KDa] pI

1 as1-CN 8/25 8e9 23.5-23.9 4.42e4.372 as2-CN 10/41 1e12 24.3e25.3 4.86e8.343 as1-I Cn f(24e199) 6/19 8e9 20.6e20.7 4.13e4.074 b-CN 7/20 5 23.9 5.135 g1-Cn 6/19 0e1 20.4e20.5 5.47e5.76 para-k-CN 3/9 0 12.29 9.337 g2-Cn 5/16 0 11.82 6.44

a Number of identified peptides/number of expected peptides.

work. In order to ascertain in cheese the relationships between theALMI fragment and the amounts of stored curd used, we alsoanalyzed samples of “Fiordilatte” made by mixing increasingamounts (from 0 to 80%) of stored curd with freshly prepared curd.The results shown in Fig. 5 indicated that the intensity of the bandwas almost linearly related to the amount of stored curd used, andby the image analysis it was ascertained that the value of the opticaldensity was about 4 RQ in the fresh curd control cheese (Fig. 6).

3.2. Kinetic study of as1-I casein on laboratory-manufacturedsamples

For a possible utilization of the ALMI fragment as a sort of“molecular marker” of quality, the kinetic of its formation in curdand cheese has to be well understood. Since it is a very complexargument, adequate deepening was carried out both by studyingthe literature and by a specifically addressed experimentation. Theliterature reports that as1-I-CN increases very slowly in low-moisture mozzarella, and even though it can be detected at sig-nificant amounts after several weeks of ripening, it is only slightlypresent in fresh cheese (Costabel, Pauletti, & Hynes, 2007; Sheehan,O’sullivan, & Guinee, 2004). As for “Fiordilatte”, as already said,information about proteolysis is scarce, but it seems that the as1-Ifragment is even less abundant than in the low moisture type, andis barely detectable in few days-aged cheeses (Baruzzi et al., 2012;Di Matteo, Chiovitti, & Addeo, 1982). Following these consider-ations, we postulated the hypothesis that the presence of thefragment could be suitable to detect the use of stored curd ifanalysis is performed in freshly produced cheeses. If it is found at ahigh level of intensity already in the first days after production, theuse of semi-finished products should be revealed. For the sake ofprecision, little controversial information does exist: Feeney et al.(2002) found the presence of this casein fragment already at day1 after production under certain conditions. In our opinion, thisfinding does not conflict with our hypothesis since it should derivefrom the different conditions of cheesemaking with respect to highmoisture mozzarella (Guinee, Feeney, Auty, & Fox, 2002). In Table 2the main technological differences between the two cheeses aresummarized: as can be seen, the processing cycle of the lowmoisture type, involves higher processing temperature, longerholding times before the stretching of the curd and slower coolingof the finished product. The specifically addressed experimentationaimed to verify if such technological differences can cause adifferent kinetic of formation of the “ALMI” fragment in “Fiordi-latte”, and if a sort of “tolerance level” for its presence in the cheeseobtained from fresh curd could be fixed. To do so, the kinetic was

Fig. 3. MALDI-ToF profiles of tryptic digest relative to spots 1 and 3, identified as as1-I-CN and as1-CN. The MALDI spectrum of as1-CN is displayed into the inset of as1-I-CN spectrum.The table reports the comparison among theoretical mass of peptides from tryptic cut of as1-CN sequence and those observed from in situ digestion of the spots.

M. Faccia et al. / Food Control 42 (2014) 101e108 105

investigated both in curd and cheese, which were produced in thelaboratory and stored under normal and “forced” conditions (singleor triple dose of rennet, short or long keeping of the curd at opti-mum conditions for proteolysis). Figs. 7 and 8 show the kinetic offormation of the fragment (expressed as RQ of the electrophoreticband) in fresh curd maintained under whey at þ 37 �C for 6 h andat þ 10 �C for 50 h, respectively. The RQ values in the curd main-tained in whey were negligible but, as expected, they increased

when a triple dose of rennet was used, reaching level 7 after 6 h:this value exceeded level 4 previously found for fresh curd “Fior-dilatte” (Fig. 6). This could appear aweak point to propose the ALMIfragment as molecular marker of quality, but such a rennet dose isnot used in “Fiordilatte” cheesemaking, since it is not compatiblewith the attainment of a soft cheese. The kinetic study at þ10 �Cconfirmed the slow formation of the ALMI marker, whose RQ valueremained under level 4 after 50 h storage. The urea-PAGE pattern of

Fig. 4. Urea-PAGE patterns of freshly produced (F1, F2, F3) and stored (P) curds. P1, P2and P3 were taken at the arrival at the wholesaler, P4, P5, P6 and P7 were taken fromthe storage warehouse and had various ages. Identification of the bands is the same asindicated in Table 1.

Fig. 5. Urea-PAGE analysis of Fiordilatte made using different percentages of fresh andstored curds. Lanes 1e7: cheeses made with 0, 15, 20, 30, 50, 60, 80% of stored curd,respectively; lane 8: stored curd used.

Fig. 6. Regression curve obtained by the results of image analysis of Fig. 5.

Table 2Main technological differences between the production processes (direct acidifica-tion) of Fiordilattea and low moisture mozzarella.b

Fiordilatte Low moisturemozzarella

Temperature of milk on adding acid (�C) 6 29Rennet addition temperature (�C) 35 36Temperature at scald (�C) No scalding 42Surface temperature at end of cooling (�C) 10 24Core temperature at end of cooling (�C) 10 <45Rennet addition-to-drain time (min) 30 82Drain-to-stretching time (min) 15 69Cooling time (min) 20 30Total processing time (min) 65 181

a Average cheesemaking conditions.b Taken from Guinee et al. (2002).

Fig. 7. Kinetic of formation of the ALMI fragment in curds produced with single ortriple dose of rennet and stored in whey at þ 37 �C for 6 h. The amounts are expressedas relative quantity (RQ) of the electrophoretic bands (means of 5 repetitions).

M. Faccia et al. / Food Control 42 (2014) 101e108106

the cheeses confirmed negligible detection of the ALMI fragmentboth after stretching (Fig. 9) and until the 5 days storage at þ10 �C(Fig. 10). As can be seen, the rate of formation during cheese stor-age, dramatically dropped with respect to curd, due to chymosindenaturation, and the intensity of the band remains under an RQvalue of 4 up to 5 days. On the whole, considering that thesemanufacturing conditions exceed those normally adopted intraditional cheesemaking, the RQ level 4 (found under our elec-trophoretic conditions) could be fixed as the tolerance limit for“Fiordilatte” obtained from freshly prepared curd.

3.3. Determination of as1-I-casein in industrial cheese

The evolution of the ALMI fragment was then investigated inindustrial “Fiordilatte” stored at commercial temperature

Fig. 8. Kinetic of formation of the ALMI fragment in curd stored out of whey at 10 �Cfor 50 h. The amounts are expressed as relative quantity (RQ) of the electrophoreticbands (means of 5 repetitions).

Fig. 9. Urea-PAGE pattern of samples of Fior di latte obtained from curd stored in wheyat þ37 �C for 6 h. Lanes 1e4 and 5e8 are two series of cheeses obtained from curd at1,2, 4,6 h; lane 9 is a fresh curd; lane 10 is a frozen curd (12 months of storage).Identification of the bands is the same as indicated in Table 1.

Fig. 10. Kinetic of formation of the ALMI fragment during storage at þ10 �C of Fior-dilatte obtained by stretching of the curd after 1 and 6 h holding in whey.

Fig. 11. Urea-PAGE of samples of industrial Fior di latte manufactured by direct acid-ification with citric acid or acidification with natural whey starter culture, and storedat þ10 �C. Lanes 1, 3, 5, 7: direct acidification samples at 0, 5, 7, 15 days; lanes 2,4,6,8:starter culture samples at 0, 5, 7, 15 days. Identification of the bands is the same asindicated in Table 1.

Fig. 12. Boxplot of the results obtained by analysis of 81 samples of industrial Fior dilatte, freshly produced.

M. Faccia et al. / Food Control 42 (2014) 101e108 107

conditions (þ10 �C). The urea-PAGE pattern shown in Fig. 11 allowsa comparison between two different cheesemaking technologies(direct acidification and fermentation with natural whey starterculture). The results confirm that the fragment is only slightlydetectable until 5 days of storage and remains under the level of 4RQ independently from the type of acidification used. After 5 daysthe band becomes more intense, exceeding the value 4, althoughdifferences between the two production technologies are onlydetectable at day 15. We concluded that the production technologyhas no significant effect on primary hydrolysis of as1 casein, and ourmethod can be applied to both types of “Fiordilatte” if analyzedwithin a few days from manufacture. The suitability of the methodwas finally tested on 81 freshly produced “Fiordilatte” samplestaken from different dairies, as reported in the materials andmethods section. On the whole, 95% of the samples from fresh curdfell within the RQ range 4.18e6.19, and the mean RQ value of ALMIfragment for fresh curd cheese was 5.3 (higher than that found inthe laboratory experimentation) and that for those containing

preserved curd was 16.6. The Boxplot reported in Fig. 12 demon-strates that the cheeses declared to be made with or without theemployment of preserved curd were well discriminated: the dif-ference between the two groups is significant by Student t test, at

M. Faccia et al. / Food Control 42 (2014) 101e108108

p < 0.001. Nevertheless, in a small part of Boxplot the two groupsappear to overlap, giving rise to a sort of a “grey zone” in which thediscrimination does not seem to be certain. However, we found thatShapiroeWilk test of normality and skewness showed an abnormaldistribution of data in fresh products. More precisely the RQ ofALMI fragment had a positive skewness and, consequently, themedian was lower than the mean. The sampling method couldaccount for this statistical behavior and, in particular, the absence ofinformation about milk used at the dairies. Eventually some slightproteolysis inmilk during long storage before cheesemaking shouldoverlap to that caused by rennet: more investigation on this subjectis actually in progress.

4. Conclusions

The results obtained indicate that the use of stored curd in “Fior-dilatte” cheesemaking can be revealed by quantification of fragment-24-199 of as1-CN. Its formation in this cheese takes place in twophases, at different rates: it is rapid in the curd (first phase, untilstretching), since chymosin is fully active, whereas it is slow in cheese(second phase, from stretching until consumption), since chymosin ismore or less denatured. If we consider that highmoisture mozzarellais stored at low temperature and has a short shelf-life, the intensity ofthepeptide in thefinished productmainly dependson thefirst phase,and time and temperature conditions at which the curd is storedbecome critical points. Under normal conditions, the formation of thecasein fragment is negligible, since the curd is promptly stretched; inthe case of stored curd, the formation becomes as more intense asstorage time is prolonged. A level of about 4 RQ band intensity couldbe the “tolerance level” that must not be exceeded in “Fiordilatte”obtained from fresh curd. However, being densitometry a semi-quantitative method, the tolerance level should be always verifiedby using a standard sample. Even if the evaluation of the ALMI frag-ment seems already useful in obtaining an indication about the use ofsemi-finished products in cheesemaking, further investigation isneeded to evaluate the feasibility of the method and, in particular, tobetter establish the RQ tolerance level for cheese from fresh curd. Atthe moment, the method is useful to certify the attainment of “Fior-dilatte” under controlled technological conditions of production andstorage, as is often required by the official protocol ofproductionofEUtypical products (DOP, IGP, GTS).

References

Andrews, A. T. (1983). Proteinases in normal bovine milk and their action on ca-seins. Journal of Dairy Research, 50, 45e55.

Baruzzi, F., Lagonigro, R., Quintieri, L., Morea, M., & Caputo, L. (2012). Occurrence ofnon-lactic acid bacteria populations involved in protein hydrolysis of cold-stored high moisture Mozzarella cheese. Food Microbiology, 30, 37e44.

Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, B.,et al. (2004). Blue silver: a very sensitive colloidal Coomassie G-250 staining forproteome analysis. Electrophoresis, 25, 1327e1333.

Commission Regulation (EEC) No 2527/98 of 25 November 1998 supplementing theAnnex to Regulation (EC) No 2301/97 on the entry of certain names in the‘Register of certificates of specific character’ provided for in Council Regulation(EEC) No 2082/92 on certificates of specific character for agricultural productsand foodstuffs. Official Journal of the European Communities, L 317, 41.

Costabel, L., Pauletti, M. S., & Hynes, E. (2007). Proteolysis in mozzarella cheesesmanufactured by different industrial processes. Journal of Dairy Science, 90,2103e2112.

Dave, R. I., McMahon, D. J., Oberg, C. J., & Broadbent, J. R. (2003). Influence ofcoagulant level on proteolysis and functionality of mozzarella cheeses madeusing direct acidification. Journal of Dairy Science, 1, 114e126.

De Candia, S., De Angelis, M., Dunlea, E., Minervini, F., McSweeney, P. L. H., Faccia, M.,et al. (2008). Molecular identification and typing of natural whey starter culturesand microbiological and compositional properties of related traditional Mozza-rella cheeses. International Journal of Food Microbiology, 119, 182e191.

Del Nobile, A. M., Gammariello, D., Di Giulio, S., & Conte, A. (2010). Active coating toprolong the shelf life of Fior di latte cheese. Journal of Dairy Research, 77, 50e55.

Di Matteo, M., Chiovitti, G., & Addeo, F. (1982). Changes in Mozzarellacheese composition at different conditions of storage. Scienza e Tecnica LattieroCasearia, 33, 197e213.

Faccia, M., Gambacorta, G., Quinto, M., & Di Luccia, A. (2010). Lead and cadmium insome milk and milk-based mozzarella cheeses from Apulia, Italy. Italian Journalof Food Science, 22, 150e155.

Faccia, M., Mastromatteo, M., Conte, A., & Del Nobile, M. A. (2012). Influence of thedifferent sodium chloride concentrations on microbiological and physico-chemical characteristic of mozzarella cheese. Journal of Dairy Research, 79,390e396.

Faccia, M., Trani, A., & Di Luccia, A. (2009). Relationships between milk quality andacidification in the production of table Mozzarella without starters. Journal ofDairy Science, 92, 4211e4217.

Farkye, N. Y., Kiely, L. J., Allhouse, R. D., & Kindsteadt, P. S. (1991). Proteolysis inmozzarella cheese during refrigerated storage. Journal of Dairy Science, 174,1433e1438.

Farkye, N. Y. (1995). Contribution of milk-clotting enzymes and plasmin to cheeseripening. In E. L. Malin, & M. H. Tunick (Eds.), Chemistry of structureefunctionrelationships in cheese (pp. 195e208). New York: Plenum Press.

Feeney, E. P., Guinee, T. P., & Fox, P. F. (2002). Effect of pH and calcium concentrationon proteolysis in Mozzarella cheese. Journal of Dairy Science, 85, 1646e1654.

Guinee, T. P., Feeney, E. P., Auty, M. A. E., & Fox, P. F. (2002). Effect of pH and calciumconcentration on some textural and functional properties of Mozzarella cheese.Journal of Dairy Science, 85, 1655e1669.

Gunasekaran, S., & Ak, M. M. (2003). Mozzarella cheese. In Cheese reology andtexture (pp. 247e251). Boca Raton: CRC press.

Kindstedt, P. S. (1993). Effect of manufacturing factors, composition, and proteolysison the functional characteristics of mozzarella cheese. Critical Reviews in FoodScience and Nutrition, 33, 167e187.

Kindstedt, P. S., & Fox, P. F. (1993). Mozzarella and pizza cheese. In P. F. Fox (Ed.),Major cheese groups: Vol. 2. Cheese: Chemistry, physics and microbiology (pp.337e362). London: Chapman & Hall.

Kindstedt, P. S., Yun, J. J., Barbano, D. M., & Larose, K. L. (1995). Mozzarella cheese:impact of coagulant concentration on chemical composition, proteolysis andfunctional properties. Journal of Dairy Science, 78, 2591e2597.

Creamer, L. K. (1976). Casein proteolysis in mozzarella-type cheese. New ZealandJournal of Dairy Science and Technology, 11, 130e131.

Joshi, N. S., Muthukumarappan, K., & Dave, R. I. (2004). Effect of calcium onmicrostructure and meltability of part skim mozzarella cheese. Journal of DairyScience, 8, 1975e1985.

Mc Goldrick, M., & Fox, P. F. (1999). Intervarietal comparison of proteolysis incommercial cheese. Z Lebensm Unters Forsch A, 208, 90e99.

Mc Sweeney, P. L., Olson, N. F., Fox, P. F., Healy, A., & Højrup, P. (1993). Proteolyticspecificity of chymosin on bovine alpha s1-casein. Journal of Dairy Research, 60,401e412.

Minervini, F., Siragusa, S., Faccia, M., Dal Bello, F., Gobbetti, M., & De Angelis, M.(2012). Manufacture of Fior di Latte cheese by incorporation of probiotic lac-tobacilli. Journal of Dairy Science, 95, 508e520.

Morales, P., Fernandez-Garcıa, E., & Nunez, M. (2003). Caseinolysis in cheese byEnterobacteriaceae strains of dairy origin. Letters in Applied Microbiology, 37,410e414.

Mulvihill, D. M., & Fox, P. F. (1979). Proteolytic specificity of chymosin on bovineas1-casein. Journal of Dairy Research, 46, 641e651.

O’Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins.Journal of Biological Chemistry, 25, 4007e4021.

Renda, A., Barbano, D. M., Yun, J. J., Kindstedt, P. S., & Mulvaney, S. J. (1997). Influ-ence of screw speeds of the mixer at low temperature on characteristics ofmozzarella cheese. Journal of Dairy Science, 80, 1901e1907.

Rowney, M. K., Roupas, P., Hickey, M. W., & Everett, D. W. (2004). Salt-inducedstructural changes in 1-day old mozzarella cheese and the impact upon free oilformation. International Dairy Journal, 14, 809e816.

Rudan, M. A., Barbano, D. M., Yun, J. J., & Kindstedt, P. S. (1999). Effect of fatreduction on chemical composition, proteolysis, functionality, and yield ofmozzarella cheese. Journal of Dairy Science, 82, 661e672.

Sheehan, J. J., O’sullivan, K., & Guinee, T. P. (2004). Effect of coagulant type andstorage temperature on the functionality of reduced-fat mozzarella cheese. Lait,84, 551e566.

Shevchenko, A., Wilm, M., Vorm, O., & Mann, M. (1996). Mass spectrometricsequencing of proteins silver-stained polyacrylamide gels. Analytical Chemistry,68, 850e858.

Tunick, M. H., Malin, E. L., Smith, P. W., Shieh, J. J., Sullivan, B. C., Mackey, K. L., et al.(1993). Proteolysis and rheology of low fat and full fat mozzarella cheesesprepared from homogenized milk. Journal of Dairy Science, 76, 3621e3628.

Yun, J. J., Kiely, L. J., Kinstedt, P. S., & Barbano, D. M. (1993). Mozzarella cheese:impact of coagulant type on composition and proteolysis. Journal of Dairy Sci-ence, 76, 3648e3656.

Yun, J. J., Kiely, L. J., Barbano, D. M., & Kinstedt, P. S. (1993). Mozzarella cheese:impact of cooking temperature on chemical composition, proteolysis andfunctional properties. Journal of Dairy Science, 76, 3664e3673.

Yun, J. J., Barbano, D. M., Kiely, L. J., & Kinstedt, P. S. (1995). Mozzarella cheese:Impact of Rod:Coccus ratio on composition, proteolysis, and functional prop-erties. Journal of Dairy Science, 78, 751e760.