Content uploaded by Aldo Di Luccia
Author content
All content in this area was uploaded by Aldo Di Luccia
Content may be subject to copyright.
Eur Food Res Technol (2012) 234:527–533
DOI 10.1007/s00217-012-1668-0
123
ORIGINAL PAPER
Proteolysis of Cacioricotta cheese made from goat milk coagulated
with capriWg (Ficus carica sylvestris) or calf rennet
M. Faccia · G. Picariello · A. Trani · P. Loizzo ·
G. Gambacorta · C. Lamacchia · A. Di Luccia
Received: 20 October 2011 / Revised: 5 January 2012 / Accepted: 6 January 2012 / Published online: 20 January 2012
© Springer-Verlag 2012
Abstract A study was undertaken on Cacioricotta, a tra-
ditional Italian goat’s cheese obtained from overheated
milk (90 °C) without use of starter. The proWle of proteoly-
sis in the artisanal type, made with vegetable coagulant
(latex released from capriWg branches) as milk clotting
agent, was compared to that of the “industrial” one, manu-
factured with calf rennet. Particular aim of the investigation
was to study the diVerences and, possibly, establish a useful
tool for distinguishing the two types of cheese. The study
was based on the quantiWcation of the water soluble, 15%
TCA soluble and amino acid nitrogen fractions, RP-HPLC
separation of low molecular weight peptides and their iden-
tiWcation by mass spectrometry (MALDI-ToF MS). The
use of Wg latex was associated to higher amounts of the
nitrogen fractions and to RP-HPLC chromatograms very
rich in peptides, in contrast to an almost complete lack of
peptides in the industrial counterpart. These results conWrm
the strong proteolyitic activity exerted by the capriWg clot-
ting enzymes in spite of the intense overheating of the milk,
which is considered to cause reduction of the rate of casein
degradation in cheese. The MS-based identiWcation of sev-
eral peptides provided a support at the molecular level for
the characterization of Cacioricotta made with this vegeta-
ble coagulant and could be useful for “tracing back”
purposes. In conclusion, the peptide pattern determined by
the use of capriWg for milk coagulation can be considered a
particular feature of the artisanal Cacioricotta, giving
conWrmation of its vocation to EU protection as typical
product.
Keywords Cacioricotta cheese · Fig coagulant ·
Proteolysis · MALDI-ToF mass spectrometry · Peptides
Introduction
Proteolysis is one of the main biochemical events occurring
in cheese ripening and has been a very popular research
subject in the last decades, since it aVects texture, intensity
of background Xavor and taste. Secondary proteolysis takes
place after that the casein matrix of cheese has been sub-
jected to primary hydrolysis by several proteases (mainly
deriving from rennet and milk) and is classically investi-
gated by chemical fractionation, chromatographic tech-
niques and, more recently, by mass spectrometry (MS) and
MS-based sequencing techniques [1, 2]. The identiWcation
of the low molecular weight peptides that are released dur-
ing ripening is a powerful tool for the molecular character-
ization of diVerent types of cheese, and a series of
information about the identiWcation of proteolytic peptides
have been reported for bovine milk cheeses, such as Grana
Padano [3], Comté [4], Provolone [5], Parmigiano Reggi-
ano [6, 7], Gouda [8] and Cheddar [9, 10]. Only few reports
have been addressed to concern ovine and goat cheeses
[11–13], which have long been considered “minor” dairy
products, due to the fragmented production, local diVusion
and poor interest by the dairy industry. However, their
marked typicality is actually more and more considered a
M. Faccia (&) · A. Trani · P. Loizzo · G. Gambacorta
Dipartimento di Biologia e Chimica Agro-Forestale ed
Ambientale, University of Bari, Via Amendola 165/A,
70126 Bari, Italy
e-mail: michele.faccia@agr.uniba.it
G. Picariello
Istituto di Scienze dell’Alimentazione—CNR,
Via Roma 52 A/C, Avellino, Italy
C. Lamacchia · A. Di Luccia
Dipartimento di Scienza degli Alimenti, Università di Foggia,
Via Napoli 25, Foggia, Italy
528 Eur Food Res Technol (2012) 234:527–533
123
chance for the producers to survive to wild market global-
ization; as consequence, there is high demand of knowledge
about their chemical, microbiological and sensorial distinc-
tive traits. In Europe, ovine and goat cheeses are mainly
manufactured in the countries bordering the Mediterranean
sea and the most part of them are still produced by tradi-
tional technologies. Among them there are cheeses
obtained by coagulation of milk with plant-derived prote-
ases. Proteases from a number of plants including Wg (Ficus
carica), cardoon (Cynara cardunculus L.), paw paw (Car-
ica papaya), pineapple (Ananas sativa) and castor oil seeds
(Ricinus communis) have been used to coagulate milk [14].
The utilization of these coagulants in cheese making has
remained quite limited due to disproportionate proteolytic
action relative to their milk-clotting activity [15]. Excessive
proteolysis can lead to a decrease in cheese yield (due to
excessive non-speciWc proteolysis in the cheese vat and loss
of peptides in the whey) and defects in the Xavour (e.g., bit-
terness) and texture (e.g., softness) of ripened cheese [16].
Nevertheless, there is a number of cheeses for which pro-
duction is historically based on the use of plant-derived
milk clotting enzymes, both for traditional dairy practices
(mainly in Europe) and ethical concerns (India and Israel).
Cheeses clotted with plant extract are Serra and Serpa from
Portugal; Los Pedroches, La Serena, Torta del Casar,
Los Ibores, Murcia al Vino and Flor de Guia from Spain
[17, 18]; CacioWore and Cacioricotta from Italy [19, 20];
Gaziantep [21] an Teleme cheese [22] from Turkey. Extracts
of Cynara cardunculus are mainly used in Spain and Portu-
gal, whereas Wg latex is used in Italy and Turkey. ScientiWc
information about Wg latex cheeses is very limited: it is
known that the proteolytic agent is Wcin, which is a cysteine
protease that acts on bonds involving uncharged and/or aro-
matic amino acids. According to Akar and Fadiloglou [22]
Wcin (EC 3.4.22.3) contains two groups of proteolytic
enzymes: one has high milk clotting but low proteolytic
activity, the other expresses higher aspeciWc proteolytic
eVect. CapriWg latex is used in Italy for producing the above
mentioned Cacioricotta. This cheese, made from over-
heated goat’s milk, is shaped like a Xat cylinder and
weights about 0.5 kg; it has a soft texture if used as fresh
cheese, but it becomes hard when subjected to ripening.
During the last years, besides the traditional technology, an
“industrial” manufacturing protocol has been developed, in
which the only variation is the use calf rennet instead of the
capriWg coagulant. The new protocol derives from the exi-
gency of standardizing the product and diYculties in insert-
ing the use of Wg latex in the HACCP protocols (high risk
of microbiological contaminations). The products obtained
by the two technologies are sold without any speciWcation
and are not diVerentiated under a commercial point of view,
and there is no scientiWc information about their features.
However, the use of diVerent milk coagulants should lead
to diVerent characteristics; this consideration also derives
from the fact that, due to the particular drying process [23],
ripening of this cheese mainly occurs during the Wrst weeks
of storage, when the role of residual rennet (i.e. the part
remaining into the curd) is crucial.
The aim of the present research was to investigate prote-
olysis of Cacioricotta via quantiWcation and chromato-
graphic characterization of the soluble nitrogen fractions. In
addition, the MALDI-TOF MS-based identiWcation of the
water soluble peptides was carried out in an attempt of
establishing the role of the coagulant and distinguishing at a
molecular level the traditional from the industrial cheese. In
our knowledge, it represents the Wrst proteolysis study per-
formed on a molecular basis about cheeses made with
plant-derived milk clotting enzymes.
Materials and methods
All of the chemicals used to prepare buVers or reagents
were of analytical grade (Merck, Darmstadt, Germany).
HPLC grade water was obtained with a Milli-Q water puri-
Wcation system (Millipore Corp., Bedford, MA, USA). The
solvents used for chromatographic analyses (HPLC grade)
were Wltered on a 0.45 m cut-oV membrane (Filtron Tech-
nology Corp., Northborough, MA) and then degassed with
helium.
Cheese samples
Four batches of goat’s Cacioricotta cheese (two trials £2
replicates) were produced in a dairy farm. The two trials
diVered as to the type of clotting enzyme employed for
coagulating milk: capriWg coagulant (CC), and calf rennet
(CR, 1:12,500, Clerici-Sacco group, Cadorago, CO, Italy).
BrieXy, for both trials raw caprine milk (pH 6.65) was
heated to 90 °C, then cooled at 45 °C and: (i) coagulated by
immersing Wve small branches (25 cm length) of capriWg
(Ficus carica sylvestris) in 500 L milk for 3 min (artisanal
type); (ii) coagulated at the same temperature by calf rennet
(0.5 mL/L of milk, industrial type). No starter was used. It
was not possible to compare the amount of milk clotting
agents, nevertheless, coagulation was completed almost in
the same time (25 and 27 min, for Wg latex and calf rennet,
respectively). The coagulated mass was very Wnely cut
(about 0.2 cm), the grains were allowed to sediment and
permit draining, then the curd was placed into cylindrical
moulds, pressed and left for 24 h at room temperature. The
cheeses were then salted by rubbing salt on both surfaces,
and 24 h later were placed at 11 °C in a ventilated room
(65% RH) for 7 days to get fast decrease of moisture.
Finally, they were washed with diluted brine, packaged
under vacuum and stored at 4 °C. Samples of cheese were
Eur Food Res Technol (2012) 234:527–533 529
123
sampled out 1, 10 and 30 days after cheese making and
transported to the laboratory, where they were immediately
analysed.
Chemical and microbiological analyses
Moisture, NaCl and pH were determined using Interna-
tional Dairy Federation standard methods [24–26]. Fat was
quantiWed by the Soxhlet method, total nitrogen (TN),
water-soluble nitrogen (WSN, obtained throughout aqueous
extraction as indicated by Kuchroo and Fox [27], and
non-casein nitrogen (NCN, obtained after separation in
15% trichloracetic acid according to Basch et al. [28]),
were quantiWed by the Kjeldhal method. The ripening index
(RI) was calculated and expressed as grams WSN per 100 g
of TN. Free aminoacids (FAAs) were extracted and deter-
mined by the EZ: faast method (Phenomenex Inc., Torrance,
CA, USA). All analyses were carried out in triplicate. As to
microbiological analyses, the total viable counts were
determined on plate count agar (Oxoid Ltd., Basingstoke,
UK) at 30 °C for 72 h, total coliforms on violet red bile
agar (Oxoid) at 30 °C for 24 h, mesophilic and thermo-
philic lactobacilli on MRS agar (Oxoid) at 30 °C and 45 °C
for 48 h under anaerobiosis, respectively, lactococci on
M17 agar (Oxoid) at 30 °C for 48 h, yeasts and molds on
Worth agar (Oxoid) at 30 °C for 72 h. All determinations
were made in duplicate and expressed as log colony-form-
ing units per gram of cheese.
RP-HPLC analysis
Fifty milligrams of WSN were redissolved in 1 mL deion-
ized water and ultraWltered on 5,000 Da cut-oV membranes
(Amicon, Millipore Corp., Bedford, MA, USA). The perme-
ate was loaded onto a Simmetry C18 reversed phase column,
5m, 250 mm £4.6 i. d. (Waters, Milford, MA, USA),
installed on a Waters HPLC composed of 600E pumps and a
996 Diode Array detector. The chromatographic separation
was conducted at 25 °C, at a Xow rate of 1 mL min¡1
with the following binary gradient: 0–45 min, 0–35% B;
45–60 min, 35–70% B; 60–70 min, 70–0% B, monitoring
the eZuent at = 220 and 280 nm. Eluent A was 0.1% tri-
Xuoroacetic acid in water and eluent B was 0.1% triXuoro-
acetic acid (TFA) in acetonitrile. HPLC analyses were
carried out in triplicate analysis at least to check for repeat-
ability. The peptide fractions were manually collected and
utilized for MS analysis. The fractions were dried out using
a centrifugal concentrator and frozen at ¡20 °C until use.
Mass spectrometry
OV-line matrix assisted laser desorption ionization-time of
Xight mass spectrometry (MALDI-ToF MS) analyses were
performed on a Voyager DE-Pro spectrometer (PerSeptive
BioSystems, Framingham, MA, USA) equipped with an N2
laser (= 337 nm). For the analysis of peptides separated
by HPLC, -cyano-4-hydroxycinnamic acid, prepared by
dissolving 10 mg mL¡1 in 50% (v/v) acetonitrile containing
0.1% (v/v) TFA, was used as the matrix. The instrument
operated with an accelerating voltage of 20 kV and typi-
cally 400 laser shots were averaged for each spectrum.
External mass calibration was performed through a separate
acquisition of a mixture of low molecular mass peptides
(Sigma-Aldrich, St. Louis, MO, USA). The mass spectra
were acquired in the positive reXector ion mode using the
Delay Extraction (DE) technology, thereby achieving an
accuracy in the measurement of the peptide mass higher
than 75 ppm. Raw data were elaborated using the software
program Data Explorer 4.0 supplied by Applied Biosys-
tems.
Casein sequences and database searches
Signals in the mass spectra were assigned by comparison
with the expected peptide masses using the the GPmaw 5.0
software (Lighthouse data, Odense, Denmark). The
sequences of the goat caseins were extracted from the
Swiss-Prot data bank; goat (Capra hircus) S1-casein and
variants are coded as P18626, S2-casein and variants are
P33049, -casein is P33048, and -casein and variants are
P02670. The most frequent S1-casein B2 and A variants
were considered in the compute of the expected peptide
masses [29].
Results
Cheese characteristics and proteolysis
The gross composition and the microbiological pattern
found were typical of Cacioricotta, and no signiWcant diVer-
ences were found between the industrial and artisanal type
(Tables 1 and 2). The high pH values at 1 day demonstrate
the typical poor acidiWcation that characterizes this cheese;
it derives from the low viable bacterial counts in milk
caused by overheating and from the absence of starters. The
fast moisture decrease observed after 10 days of ripening is
consequent to the forced drying process; afterwards, the
moisture content remains almost stable since the cheeses
are wrapped under vacuum and stored at 4 °C. DiVerently
from gross composition, the nitrogen fractions resulting
from proteolysis strongly diVered (Table 3): proteolysis
was in general higher in cheeses prepared from milk coagu-
lated by capriWg proteases. The WSN content was signiW-
cantly higher throughout the entire ripening period,
whereas NCN and FAAs were higher from day 10 onward.
530 Eur Food Res Technol (2012) 234:527–533
123
The ripening index at 30 days was about 17 g WSN per
100 g of TN in industrial cheese, whereas it was about two
times higher (about 40 g WSN per 100 g of TN) in that
obtained with capriWg coagulant. The RI value in CC
cheese is comparable with those commonly found in long
ripened cheeses such as Parmigiano Reggiano and Fossa
[30]. The proportions of FAAs in NCN in matured Caciori-
cotta were about 50% and only 33% in CR and CC cheeses,
respectively. Full conWrmation of the poor proteolysis in
the calf rennet cheese was oVered by the chromatographic
study of soluble nitrogen: the comparison of the proWles of
the two samples (Fig. 1) clearly shows the presence of
remarkable diVerences. The chromatogram of CR cheese is
almost Xat, whereas that of CC is quite complex, with most
part of peptide species concentrated in the centre of the
chromatogram, revealing intermediate characteristics of
hydrophobicity. The soluble nitrogen fraction extracted
from CC cheese was investigated by MALDI-ToF MS.
Even though the ultraWltration step allowed the simpliWca-
tion of the mixture, the proWle appears to be rather complex
(Fig. 1). The fractions for which molecular weight was
determined are labelled (1–7) in Fig. 2. MS analysis
allowed to identify 46 peptides, among which 9 ranged
from tetrapeptide to octapeptide with molecular masses
from m/z662.4 to 998.3 g/mol for the (M + H)+ ion
(Table 4). Due to the occurrence of interfering matrix ions,
the MW range below 500 Da was not explored by MALDI-
ToF MS. Furthermore, the MS analysis of complex peptide
mixtures can be aVected by ion suppression phenomena.
Therefore, too short sequences or peptides with a low
intrinsic ionization eYciency most likely escaped identiW-
cation. The largest number of identiWed peptides came from
the degradation of -casein, followed by s1- and para
-casein. The occurrence of peptides from para k-casein
in cheese has not been frequently reported in the literature
[2, 31].
Discussion
The peculiar traits of proteolysis observed in the two types
of Cacioricotta could be explained on the basis of a series
of considerations. First of all, we need to consider the
impact of the strong heat treatment on the endogenous pro-
tease system of milk. Since heating of the milk was carried
out at 90 °C directly in the vats (about 1 h needed for
Table 2 Viable counts of bacterial groups for 30 days matured
Cacioricotta cheese
CC capriWg coagulant, CR calf rennet
Coagulant Log CFU g¡1 (SD)
Total viable CC 6.05 (0.22)
CR 6.02 (0.09)
Total coliforms CC 3.87 (0.12)
CR 4.04 (0.31)
Mesophilic lactobacilli CC 6.01 (0.17)
CR 6.11 (0.05)
Termophilic lactobacilli CC 5.44 (0.04)
CR 5.78 (0.25)
Lactococci CC 6.33 (0.33)
CR 6.17 (0.08)
Yeasts and molds CC 4.12 (0.08)
CR 4.40 (0.22)
Table 3 Mean values (§SD) for the nitrogen fractions in Cacioricotta
cheese during ripening
CC capriWg coagulant, CR calf rennet, WSN water soluble nitrogen,
NCN non casein nitrogen, FAAs free amino acids, (g kg¡1)
a,b,c,d Means within same column not sharing common superscripts are
diVerent (P< 0.05)
WSN NCN WSN-NCN FAAs
CC
1 day 11.2 (§0.4)b3.1 (§1.6)a,b 8.1 (§1.2)d1.0 (§0.3)a
10 days 18.6 (§0.7)c11.5 (§0.4)c7.1 (§0.3)d3.7 (§0.5)b
30 days 21.6 (§1.0)d19.9 (§1.6)d1.7 (§0.6)b6.5 (§0.3)d
CR
1 day 7.1 (§1.1)a1.3 (§0.3)a5.8 (§0.8)c0.5 (§0.3)a
10 days 10.7 (§0.5)b4.8 (§0.9)b5.9 (§0.4)c1.4 (§0.7)a
30 days 11.3 (§1.4)b10.8 (§0.9)c0.5 (§0.3)a5.3 (§0.3)c
Table 1 Mean values (§SD)
for the gross composition in
Cacioricotta cheese during
ripening (g kg¡1)
Time pH Moisture Protein Fat NaCl
CC
1 day 6.01 (§0.04)a423.1 (§5.1)a253.3 (§4.2)a240.9 (§8.5)a32.8 (§1.9)a
10 days 5.44 (§0.05)b347.2 (§4.4)b275.6 (§2.1)b269.9(§4.2)b38.5 (§0.5)b
30 days 5.25 (§0.04)c349.8 (§6.5)b276.7 (§1.8)b272.5 (§5.9)b36.7 (§1.1)b
CR
1 day 6.09 (§0.05)a434.5 (§7.6)a248.9 (§3.0)a251.1 (§7.1)a34.7 (§0.6)a
10 days 5.37 (§0.03)b352.4 (§7.0)b272.3 (§5.4)b275.8 (§5.1)b40.2 (§3.2)c
30 days 5.21 (§0.02)c357.3 (§10.1)b270.6 (§6.1)b287.8 (§9.3)b,c 37.8 (§0.9)b
CC capriWg coagulant, CR calf
rennet
a,b,c Means within same column
not sharing common super-
scripts are diVerent (P<0.05)
Eur Food Res Technol (2012) 234:527–533 531
123
Fig. 1 HPLC chromatograms of 30 days ripened Cacioricotta made
with capriWg coagulant (a) or calf rennet (b)
Fig. 2 Peptides collected and utilized for MS analysis (insight of
chromatogram A)
A
Table 4 MALDI-ToF MS analysis of the RP-HPLC peaks of the water
soluble fractions of Cacioricotta cheese made with capriWg coagulant
m/zMolecular mass Possible
casein
fragment
HPLC
fraction
656.1 Matrix peak (also
contained in
fraction 5 and 7)
6
662.4 662.4 s1-(19–23) 3
780.2 780.4 k-(97–102) 1
791.4 791.4 s1-(18–23) 2
827.4 827.5 k-(95–101) 3
851.2 851.4 k-(96–102) 1
905.4 905.5 s1-(17–23) 3
964.4 964.5 k-(96–103) 2
981.3 981.6 -(74–82) 1
998.3 998.6 s1-(1–8) 1
1080.3 1080.7 -(74–83) 1
1101.4 n.i. 2
1117.6 1117.6 s1-(15–23) 3
1126.4 1126.7 s1-(1–9) 1
1169.4 1168.6 -(126–135) 1
1174.6 1174.6 s1-(17–26) 3
1183.4 1183.6 s1-(1–10) 1.2
1252.6 1252.7 k-(76–86) 7
1296.4 1296.7 s1-(1–11) 1
1364.0 1363.8 -(195–207) 5
1414.7 1414.7 s1-(12–23) 3
1450.4 1450.7 -(120–132) 1
1490.9 1490.9 -(193–206) 4
1505.8 1505.8 -(192–205) 3
1507.3 n.i. 1
1527.7 1527.8 s1-(11–23) 3
1555.8 1555.8 -(191–204) 3.7
1584.8 1584.8 s1-(10–23) 3
1589.9 1589.9 -(193–207) 7
1590.0 1589.9 -(193–207) 5
1609.7 1609.8 s1-(1–14) 2
1618.9 1618.9 -(192–206) 4
1718.1 1718.0 -(192-207) 5.7
1781.9 1781.9 s1-(12–27) 3
1783.0 1783.0 -(191–206) 4
1881.2 1882.1 -(191–207) 5.7
1896.1 1896.1 -(190–206) 4
1919.8 1920.2 s1-(1–17) 2
1994.3 1995.2 -(190–207) 5.7
2104.9 2104.9 k-(1–17) pyro-Glu 4
2107.4 2108.4 -(189–207) 5.7
2123.3 2124.9 k-(1–17) 4
2163.9 2164.1 s1-(106–124) 2
532 Eur Food Res Technol (2012) 234:527–533
123
heating, about 40 min for cooling at 45 °C), inactivation of
both plasmin and cathepsin should have occurred [32, 33].
As consequence, the formation of WSN, and the diVerences
observed in the two types of cheese, can likely be ascribed
to diVerent contribution of the milk coagulants. However,
WSN contains both products of primary and secondary pro-
teolysis, and, in order to make a hypothesis about the con-
tribution of the coagulants, we have calculated the
diVerence between WSN and NCN. It essentially expresses
the concentration of the high molecular weight peptides,
giving indirect information about primary proteolysis, and
clearly conWrms an higher level in CC cheese through the
entire ripening period. The level of primary proteolysis in
both cheeses dramatically decreases from 10 to 30 days. It
is probable that the activities of the residual coagulants are
fully expressed in the Wrst phase of ripening, when the
cheese still has a high moisture level, then tends to slow
down. As regards secondary proteolysis, from the data of
Table 2 it appears that the main agent responsible for the
formation of small peptides and amino acids, namely
microXora, was found at too low levels (and at similar via-
ble counts in the two types of cheese) to exert signiWcant
inXuence. This was not unexpected, since the heat treat-
ment was very severe and no starter had been added. Once
more, the diVerences observed should be assigned to the
remarkable non-speciWc proteolytic activity of Wcin on
caseins, with release of signiWcant amounts of free amino
acids, compared to the scarce aspeciWc proteolytic power of
calf rennet, which are well documented [34–36]. Besides
the diVerent proteolytic activity, it should also be taken into
account the thermal stability of the clotting enzymes and
their retention into the curd. The extreme time/temperature
conditions used for milk coagulation (25–27 min, 45 °C)
determines a thermal proWle of cheesemaking that is com-
parable with that used in middle-cooked curd cheeses:
under these conditions chymosin is likely to be partially
inactivated [37], whereas Wcin is heat resistant [38]. As to
the retention of the enzymes, it is known that for chymosin
it is closely related to the pH of milk at draining. In the case
of Cacioricotta, pH was too high (more than 6.0) to allow
good retention of chymosin into the curd [39]. A compari-
son with Wcin is not possible due to lacking of studies about
the mechanism and the rate of recovery of this coagulant in
cheese; nevertheless, the isoelectric point is known to be
within the range 9–10 [40] and, therefore, Wcin should be
poorly recovered into the curd, as well. Finally, it has to
be taken into account the composition of the cheese protein
fraction: the presence of remarkable amounts of whey pro-
teins in the paracaseinate network is a particular feature of
Cacioricotta [23, 41, 42], and is due to the overheating pro-
cess, which causes denaturation and association to casein
micelles in milk [43]. This characteristic is likely to have
inXuenced proteolysis in CR cheese, since the casein–whey
protein interactions have detrimental eVects on the activity
of residual rennet [44, 45].
The MS identiWcation of peptides arising from all the
casein families clearly conWrms that Wcin is able to hydro-
lyze all caseins. Furthermore, the prevalence of peptides
arising from hydrolysis at level of hydrophobic residues
(Table 4) suggests that Wcin exhibits some cleavage speci-
Wcity for hydrophobic amino acids. In detail, the proteolytic
peptides of -CN mainly belong to the hydrophobic N-ter-
minus portion (sequences 1–19, 23–45 and 76–86) and less
to the terminal part (96–103) of para-k-CN. As to s1-C,
apart peptide 106–124 that has been found both in the phos-
phorylated and dephosphorylated form, most of the pep-
tides found arise from the N-terminal hydrophobic region;
the peptides derive from the B2 hard allele, probably
because of both the elevated allelic frequency in caprine
Xocks of Southern Italy and the high level of expression.
Finally, the majority of -casein peptides identiWed origi-
nate from the hydrophobic C-terminal end of the molecule,
in the 114–141 and 187–207 regions. Peptide attributable to
degradation of whey proteins were not observed, conWrm-
ing the resistance of these proteins to the cheese proteases
[46]. The results obtained suggest an increased exposition
of hydrophobic domains at the surface of the micelles, and
this could be explained by the fact that, under intense heat-
ing, caseins tend to expose domains that are normally hid-
den. In these conditions Wcin, which selectively cleaves
sites containing hydrophobic uncharged and aromatic
amino acids, should be particularly favoured [47]. Never-
theless, it has to be evidenced that hydrophobic randomly-
cleaved casein peptides are generally much more detectable
by MALDI-ToF MS than hydrophilic ones. Thus, the pre-
dominance of hydrophobic fragments have to be ascribed at
least in part to an enhanced ionization of these species, and
the presence of peptides arising from hydrophilic casein
domains could be underestimated.
Table 4 continued
List of peptides detected and corresponding possible fragment identi-
ties
n.i. not identiWed
m/zMolecular mass Possible
casein
fragment
HPLC
fraction
2243.9 2244.1 s1-(106–124) P 2
2387.4 2387.0 k-(1–19) 4
2784.7 2785.4 k-(25–45) ? 4
2984.7 2984.5 k-(23–45) ? 4
3030.8 3031.6 -(115–141) ? 4
3192.9 3194.6 -(114–141) ? 4
3679.3 3679.9 s1-(1–32) 6
Eur Food Res Technol (2012) 234:527–533 533
123
Conclusions
The results obtained in this study give a contribution to the
understanding of the biochemical events occurring during
ripening of cheese made with Wg latex, and demonstrate
that this type of milk coagulant deeply inXuences the prote-
olytic proWle, favouring an enhanced degree of secondary
proteolysis if compared to calf rennet. The HPLC proWle of
soluble peptides allows to easily distinguish artisanal Caci-
oricotta from the industrial one, providing a reliable basis
for developing analytical strategies for authentication pur-
poses. The MALDI-ToF MS characterization of several
proteolytic peptides speciWcally marked the process of pro-
duction, and supports at a molecular level the expected
traits of typicity traditional of Cacioricotta, coagulated by
enzymes present in Wg latex, which is widely considered in
the territory as a suitable cheese for a PDO protection label.
Further work is now needed to ascertain the relationships
between proteolysis and the organoleptic features of the
two types of Cacioricotta cheese.
References
1. McSweeney PLH, Fox PF (1997) Lait 77:41–76
2. Sousa MJ, Ardö Y, McSweeney PLH (2001) Int Dairy J 11:327–
345
3. Ferranti P, Itolli E, Barone F, Malorni A, Garro G, Laezza P, Chi-
anese L, Migliaccio F, Stingo V, Addeo F (1997) Lait 77:683–697
4. Roudot-Algaron F, Bars DL, Kerhoas L, Einhorn J, Gripon JC
(1994) J Food Sci 59:544–547
5. Santoro M, Faccia M (1991) It J Food Sci 3:307–312
6. Addeo F, Chianese L, Salzano A, Sacchi R, Cappuccio U, Ferranti
P, Malorni A (1992) J Dairy Res 59:401–411
7. Addeo F, Chianese L, Sacchi R, Spagna Musso S, Ferranti P,
Malorni A (1994) J Dairy Res 61:365–374
8. Igoshi K, Jiromaru N, Kobayashi H, Arima S (1997) Anim Sci
Technol 68:385–388
9. Sing TJ, Fox PF, Healy A (1995) J Dairy Res 62:629–640
10. Sing TJ, Fox PF, Healy A (199) J Dairy Sci 64:433–443
11. Sousa MJ, Malcata FX (1998) J Agric Food Chem 46:4034–4041
12. Sommerer N, Salles C, Promé D, Promé JC, Le Quéré JL (2001) J
Agric Food Chem 49:402–408
13. Pirisi A, Pinna G, Addis M, Piredda G, Mauriello R, De Pascale S,
Caira S, Mamone G, Ferranti P, Addeo F, Chianese L (2007) Int
Dairy J 17:143–156
14. Garg SK, Johri BN (1994) Food Rev Int 10:313–355
15. Walstra P, Geurts TJ, Noomen A, Jellema A, van Boekel MAJS
(1999) Dairy technology: principles of milk properties and pro-
cesses. Marcel Dekker Inc., New York
16. Fox PF, McSweeney PLH (1998) Dairy chemistry and biochemis-
try. Blackie Academic & Professional, London
17. Fernandez-Salguero J, Sanjuaan E (1999) Food Chem 64:177–183
18. Bivar Roseiro L, Barbosa M, Ames JM, Wilbey RA (2003) Int J
Dairy Technol 56:76–85
19. Delforno G (1987) Cacio Fiore cheese (Mondo del Latte) 39:774–
776
20. Italian National Research Council (1996) In: Tecnos SRL (ed)
I prodotti caseari del Mezzogiorno, vol II. Milan
21. Öner MD, Akar B (1993) Lebensm Wissensch Technol 26:318–
321
22. Akar B, Fadiloglu S (1999) J Food Qual 22:671–680
23. Faccia M, Gambacorta G, Caponio F, La Gatta B, Di Luccia A
(2007) Int J Food Sci Technol 42:427–433
24. International Dairy Federation (1986) IDF standard no 4. FIL-IDF,
Brussels
25. International Dairy Federation (1988) IDF standard no 88A.
FIL-IDF, Brussels
26. International Dairy Federation (1989) Standard no 115A. FIL-IDF,
Brussels
27. Kuchroo CN, Fox PF (1982) Milchwissenschaft 37:331–335
28. Basch JJ, Douglas FW Jr, Procino LG, Holsinger VH, Farrel HM
Jr (1985) J Dairy Sci 68:23–31
29. Sacchi P, Chessa S, Budelli E, Bolla P, Ceriotti G, Soglia D, Rase-
ro R, Cauvin E, Caroli A (2005) J Dairy Sci 88:1561–1568
30. Gobbetti M, Folkertsma B, Fox PF, Corsetti A, Smacchi E, De
Angelis M, Rossi J, Kilcawley K, Cortini M (1999) Int Dairy J
9:763–773
31. Piraino P, Upadhyay VK, Rossano R, Riccio P, Parente E, Kelly
A, McSweeney PLH (2007) Food Chem 101:964–972
32. McSweeney PLH (2004) Int J Dairy Technol 57:127–144
33. Benfeldt C, Sorensen J, Ellegard KH, Petersen TE (1997) Int Dairy
J 7:723–731
34. Low YH, Agboola S, Zhao J, Lim MY (2006) Int Dairy J 16:335–
343
35. Alirezaei M, Gheisari MAHR, Tavana M (2011) Eur J Food Res
Rev 1:43–51
36. Benfeldt C, Sørensen J (2001) Int Dairy J 11:567–574
37. Upadhyay VK, McSweeney PLH, Magboul AAA, Fox PF (2004)
In: Fox PF, McSweeney PLH, Cogan TM, Guinee TP (eds) Cheese
chemistry, physics and microbiology, VIII edn, vol 1. Elsevier
Ltd, Oxford
38. Katsaros GI, Katapodis P, Taoukis PS (2009) J Food Engin
91:42–48
39. Holmes DG, Duersch JW, Ernstrom CA (1977) J Dairy Sci
60:862–869
40. Khan IA, Abourashed EA (2010) In: Leung’s encyclopedia of
common natural ingredients: used in food, drugs and cosmetics, III
edn. Wiley, Hoboken
41. Caponio F, Pasqualone A, Gomes T (2001) Eur Food Res Technol
213:178–182
42. Blaiotta G, Moschetti G, Simeoli E, AndolW R, Villani F, Coppola
S (2001) J Dairy Res 68:139–144
43. Corredig M, Dalgleish DG (1999) Int Dairy J 9:233–236
44. Lau KY, Barbano DM, Rasmussen RR (1991) J Dairy Sci
74:727–740
45. Christensen TMIE, Kristiansen KR, Werner H (1991) Milchwis-
senschaft 46:279–283
46. Manzo C, Pizzano R, Addeo F (2008) J Agric Food Chem
56:7929–7933
47. Trujillo AJ, BuVa M, Casals I, Fernandez P, Guamis B (2002) Inn
Food Sci Emerg Technol 3:309–319
