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Proteolysis of Cacioricotta cheese made from goat milk coagulated with caprifig (Ficus carica sylvestris) or calf rennet

  • CIHEAM - International Center for Advanced Mediterranean Agronomic Studies of Bari-
  • Independent Researcher

Abstract and Figures

A study was undertaken on Cacioricotta, a traditional Italian goat’s cheese obtained from overheated milk (90 °C) without use of starter. The profile of proteolysis in the artisanal type, made with vegetable coagulant (latex released from caprifig branches) as milk clotting agent, was compared to that of the “industrial” one, manufactured with calf rennet. Particular aim of the investigation was to study the differences and, possibly, establish a useful tool for distinguishing the two types of cheese. The study was based on the quantification of the water soluble, 15% TCA soluble and amino acid nitrogen fractions, RP-HPLC separation of low molecular weight peptides and their identification by mass spectrometry (MALDI-ToF MS). The use of fig 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 confirm the strong proteolyitic activity exerted by the caprifig clotting 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 identification of several peptides provided a support at the molecular level for the characterization of Cacioricotta made with this vegetable coagulant and could be useful for “tracing back” purposes. In conclusion, the peptide pattern determined by the use of caprifig for milk coagulation can be considered a particular feature of the artisanal Cacioricotta, giving confirmation of its vocation to EU protection as typical product.
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Eur Food Res Technol (2012) 234:527–533
DOI 10.1007/s00217-012-1668-0
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
Keywords Cacioricotta cheese · Fig coagulant ·
Proteolysis · MALDI-ToF mass spectrometry · Peptides
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
[1113], 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
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
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 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
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
sampled out 1, 10 and 30 days after cheese making and
transported to the laboratory, where they were immediately
Chemical and microbiological analyses
Moisture, NaCl and pH were determined using Interna-
tional Dairy Federation standard methods [2426]. 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-
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].
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
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].
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)
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
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
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
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
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
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)
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
656.1 Matrix peak (also
contained in
fraction 5 and 7)
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
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 [3436]. 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-
n.i. not identiWed
m/zMolecular mass Possible
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
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.
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Supplementary resource (1)

... The results obtained in this work (Figure 2) show that the proteolytic activity of ficin extract is very high (4 times higher) than that of rennet; 139.094 μg/ml for ficin extract against 35.75 μg/ml for rennet. This excessive proteolytic activity of ficin has been reported by several authors (Oner and Akar, 1993;Fadyloglu, 2001;Nouani et al., 2009;Faccia et al., 2012). The optimal conditions of ficin activity determined in this work (Figure 3) show that the coagulant activity of the ficin strongly depends on the pH, temperature and CaCl2 salt concentration. ...
... In fact, the rennet cheese had a firmer texture than the cheeses obtained from ficin extract whose texture was creamier and unctuous. These results are consistent with those obtained by Oner and Akar, (1993) and Faccia et al. (2012), who observed the softening of cheese made with ficin extracts. This loss of hardness was due, in large part, to the proteolytic activity of the enzymatic extracts studied. ...
Full-text available
The manufacture of cheese based on goat's milk is a traditional practice using fig tree latex. The objective of this paper is to study the possibility of substituting rennet with ficin as a vegetable coagulant milk and its characterization. In addition, two trials of manufacturing fresh and soft cheese using ficin were made. The results obtained show that the coagulant activity of ficin is optimal at pH = 5, at a temperature of 75℃ and at a CaCl2 concentration of 0.03 M with 2097.90 UR., 911.79 UR., and 192.30 UR respectively. The coagulant force of this enzyme is 1/21250.911 with a proteolytic activity estimated at 139 μg of tyrosine equivalent per ml of ficin extract. The fresh cheese is white with a creamy appearance and great solubility in the mouth. The ficin soft cheese has an organoleptic quality better than that of control cheese and a softer with a more viscous texture in comparison to that of rennet. The ficin soft cheese has a penetrometry of 2.5 mm in comparison with that of a rennet of 3.6 mm. Ficin can replace rennet in cheese making and deserves to be expanded in the manufacture of different cheeses.
... Coagulant agents from plant extracts are rarely used, due to the low organoleptic characteristics of the resulting cheese such as bitter taste and cheese texture which are less attractive to consumers due to the high value of its proteolytic activity. For this reason, it is necessary to further search for other plant extracts that are suitable for use as coagulant agents in cheese production without affecting its organoleptic characteristics [4]. ...
... No coliform counts were determined in the HS1 and HS2 cheeses on days 60 and 90, while a considerable decrease occurred in the RS cheeses due to the starter culture activity. Similar coliform counts were reported for Cacioricotta cheese (Faccia et al., 2012) and raw milk goat cheese ( € Oner & Sarıda g, 2019). The counts of yeasts and moulds ranged between 1.075 and 4.646 log cfu g À1 in the cheeses (Table 3). ...
... Physical structure and molecular weight of protein also might affect antioxidant activity reflected by decreases in DPPH RSA. Indeed, most studies on antioxidant properties group peptide fractions into three categories as < 2 kDa, 3-10 kDa, and >10 kDa (Banihashemi, Nikoo, Ghasempour, & Ehsani, 2020;Faccia et al., 2012;Mushtaq et al., 2016;Timón et al., 2014;Yasar & Guzeler, 2011;Yates et al., 2010). Decreased antioxidant potential is consistent with Timón, Andrés, Otte, and Petrón (2018) who concluded that peptide fractions between 3 and 10 kDa displayed higher DPPH RSA compared with <2 kDa, and >10 kDa fractions. ...
This study evaluated the incorporation of Lactobacillus or an Enterococcus probiotic bacteria into Wagashi cheese with a focus on their viability during production and storage, and their significance on key quality features evaluated using standard methods and a reference frame specific for Wagashi. Probiotic bacteria were added following heat treatment. Addition of probiotic strains did not affect protein content in contrary to fat, dry matter, pH and titratable activity, which were influenced by strain and storage time. Final bacteria count exceeded 7 log cfu/g suggested for probiotic cheeses. Yeast and mould counts decreased in all cheese, except for RwLc. Probiotic cheese samples, except RwLc, were similar considering their mesophilic lactic acid bacteria (LAB) counts conversely to the differences denoted for their thermophilic LAB counts. All cheeses exhibited antioxidant activity. Total phenolic content was highest in the control (Rc). Butyric, ascorbic, formic, citric, propionic, malic and lactic acid were found in the cheeses. All cheeses were scored higher than 5, and RwLp, RwLr had a significantly higher acceptability. Key cheese features were not negatively affected during storage, highlighting the suitability of Wagashi cheese as a dietary source of probiotic bacteria.
... The first source for their formation was β-CN, and for the most part originated from AA regions 70 to 120 and 200 to 220. Higher presence of β-CN-derived peptides in hard cheese with respect to those deriving from other caseins was reported by Singh et al. (1995) in Cheddar and by Faccia et al. (2012) in ripened Cacioricotta, whereas Ferranti et al. (1997) reported α S1 -CN as the main source of low MW peptides in 21-mo-old Grana Padano. It must be underlined that the peptide profile of mature hard cheeses depends on a huge number of variables, including the ripening time, gross composition and weight of the cheese, type of rennet used, and salt concentration, and making a comparison among different types is very difficult. ...
A multiparameter study was performed to evaluate the effect of fondaco, a traditional ripening cellar without any artificial temperature and relative humidity control, on the chemical, microbiological, and sensory characteristics of Protected Geographical Indication Canestrato di Moliterno cheese. Ripening in such a nonconventional environment was associated with lower counts of lactococci, lactobacilli, and total viable bacteria, and higher presence of enterococci, in comparison with ripening in a controlled maturation room. Moreover, fondaco cheese underwent accelerated maturation, as demonstrated by faster casein degradation, greater accumulation of free AA, and higher formation of volatile organic compounds. Secondary proteolysis, as assessed by liquid chromatography-mass spectrometry of free AA and low molecular weight peptides, did not show any qualitative difference among cheeses, but fondaco samples evidenced an advanced level of peptidolysis. On the other hand, significant qualitative differences were observed in the free fatty acid profiles and in the sensory characteristics. Principal component analysis showed a clear separation of the fondaco and control cheeses, indicating that ripening in the natural room conferred unique sensory features to the product.
Plant-derived proteases to coagulate milk in cheese making have gained considerable attention. This is mainly due to greater demand to diversify the cheese making and limitations associated with animal-derived coagulants, e.g., rennet (chymosin and pepsin). Improved understanding of plant proteolytic enzymes’ structure, function, and technological properties have made it possible to use coagulants from diverse plant sources in the cheese industry. Proteases in Ficus carica L. (common fig) have been thoroughly characterized and evaluated for their milk-clotting ability. Ficins (sometimes called ficain) are the key proteolytic enzymes in F. carica used in milk coagulation due to their well-characterized structures, functions, and properties. This section critically examines the current understanding of the activity of ficins from F. carica in milk coagulation and its performances under varying physicochemical contexts. Extraction and characterization of ficin and other proteolytic constituents in F. carica also deepen our understanding. Apart from the coagulation and subsequent gel formation ability of F. carica in cheese manufacturing, numerous other health-promoting effects due to its phytochemicals are also highlighted in this chapter.
Thermal inactivation of ficin, bromelain, and papain was studied in solution and upon their immobilization on matrices of medium- (200 kDa) and high-molecular-weight (350 kDa) chitosans. Native ficin was inactivated at 70°C after a 10-min exposure; bromelain in solution retained up to 40% of its activity after a 60-min incubation at 60 and 70°C; and the enzymatic activity of free papain remained constant at 70°C over the total exposure period (60 min). Ficin, bromelain, and papain in solution were fully inactivated at 80 or 90°C after 10-min incubation. Immobilization on chitosan matrices of ether type increased the thermal stability of ficin, which retained at least 20% of its enzymatic activity at 70°C. After immobilization, ficin was completely inactivated at 80 and 90°C after a 10-min incubation, while papain and bromelain retained more than 10% of their initial catalytic activity in these conditions.
The heat-induced interactions between whey proteins and casein micelles were investigated by defining the final product of the reaction when milk was heated at temperatures up to 90°C. By looking at the changes of the interactions in skim milk and in resuspended casein micelles, to which different amounts of whey protein had been added, information on the mechanisms that determine the heat-induced protein–protein interactions in milk was derived. The ratio of α-lactalbumin and β-lactoglobulin to κ-casein and the ratio of α-lactalbumin to β-lactoglobulin found in the micellar pellet were used as indices of these heat-induced reactions occurring in milk. The results suggested that at these low temperature (70–90°C) with batch heating conditions, whey proteins form soluble complexes which act as intermediates in the heat-induced association of α-lactalbumin and β-lactoglobulin with the micelles. The presence of β-lactoglobulin was necessary for any association of whey protein with casein micelles to occur; furthermore, the amount of β-lactoglobulin found in the micellar pellet after heating seemed to be limited by a discrete number of binding sites available on the micelles.
Danbo 45+ was manufactured from milk subjected to heat treatment at 72, 80 or 90°C for 15, 30 and 60s. The effect of heat treatment on the activity of plasmin in the resulting cheeses and the subsequent proteolysis of caseins during ripening were examined. This revealed that the plasmin activity decreased as the temperature and the holding time increased. In addition, the cheeses showed decreased digestion of β- and αs2-caseins during ripening, which was explained by variations in plasmin activity. The digestion of para-κ casein was slightly affected by heat treatment, which may be explained by steric hindrance due to the formation of thermally induced complexes with whey proteins. The digestion of αs1-casein, however, was unaffected by the heat treatment and probably results primarily from the action of residual rennet. It is suggested that the altered proteolytic digestion of casein during ripening of cheese manufactured from heated milk results from reduced plasmin activity in the cheese as a consequence of thermal inactivation of the plasminogen activation system and thermally induced interactions between the components of the plasminogen activation system and β-lactoglobulin.
Alternate methods for quantitation of caseins and whey proteins in milk products were investigated. The Harland-Ashworth and Leighton procedures, which are used for routine determinations of soluble whey proteins in milk, could not be adapted satisfactorily to quantitation of whey protein in blends of nonfat dry milk solids and whey protein concentrates because of problems of precipitation techniques. Gel electrophoresis in sodium dodecyl sulfate does not require fractionation prior to analysis and works well for nonfat dry milk solids, whey protein concentrates, and blends of these products, as well as total milk protein concentrates. Use of thiourea and hydrogen peroxide as gel catalysts improves band resolution and allows for easy handling and better quantitation. This method, which is an adaptation of the Laemmli procedure, may be of use for detecting adulteration of nonfat dry milk solids or even fluid milk with whey protein concentrates and may find other applications; 10 major milk proteins can be visualized and quantitated on one gel electrophoretogram.
A worldwide shortage of calf rennet for cheese production has existed for several decades. Bovine pepsin and to a certain extent porcine pepsin and plant coagulants have been used as rennet substitutes, but these have not been commercially successful owing to their extensive proteolytic nature and other inherent drawbacks. Investigations to develop other alternatives have resulted in the introduction of various microbial coagulants which have found markets in several countries despite certain shortcomings when compared with traditional calf rennet. However, in some countries, microbial coagulants have not been accepted for regular cheese manufacture because they are believed to result in a reduced yield and a lower quality product; this is particularly applicable to cheddar cheese manufacture. Immobilization of proteases for milk coagulation has received renewed thrust; this may convert renneting of milk to a continuous operation. However, this technique has yet to be applied on an industrial scale; such a process is not likely to be successful since immobilized enzyme technology is dependent on the ability of a small, rapidly diffusing substrate to move quickly around the immobilized enzyme. In contrast, casein micelles are large and diffusion is slow, so the proteolysis rate is very slow. Microbial rennets offer an attractive target for genetic engineers since it may be possible to alter their structure/function characteristics to match those of calf rennet; application of site‐directed mutagenesis could be particularly rewarding. The term “calf rennet,” in the cheese industry, generally refers to an enzyme extract obtained from the fourth stomach (abo‐masum) of 10‐ to 30‐day‐old calves and used to coagulate milk for cheese production. The purified milk‐clotting enzyme present in crude rennet preparations is known as “rennin” or “chymosin.” The designation “chymosin” is now recommended in the international enzyme nomenclature (renin is associated with hypertension and is derived from the kidney). Calf rennet is referred to as “animal rennet” frequently, the term “chymase” appears in the older literature as a synonym for rennet. In more general usage, however, any milk‐clotting enzyme preparation yielding a relatively stable curd is designated as rennet. Christian Hansen is credited with the first industrial production of rennet in 1874 (1). This enzyme was called chymosin (EC; it belongs to the group of aspartic proteases and is the standard against which all other types of milk‐clotting enzymes are compared. Since the origin of cheese production, the manufacturing process has always needed soluble enzymes to clot the milk; it has been adapted to the properties of calf rennet, an essential enzyme for this purpose. Rennet coagulates milk rapidly at its natural pH with little further degradation of the milk proteins. The zymogen, prorennin, is converted to chymosin at pH below 5.0, but optimally at pH 2.0 (2). Chymosin, a strong protease, has been crystallized (3) and detailed crystallographic studies of the enzyme have been reported by Bunn et al. (4). Besides a slightly high proteolytic activity, another disadvantage of this enzyme is that it is extracted from the abomasum of the unweaned calf. As the calf ages, chymosin is replaced by pepsin, although in cattle, the secretion of chymosin never comes to a complete stop. Although pepsin can clot milk, it has a tendency to result in higher fat losses because the curd formed has a more open, looser structure than that formed with chymosin; the cheese produced also has a softer body than desired. Pepsin and a commercial product under the trade name Metroclot (5, 6) have, however, been used for the production of a variety of cheeses in the past. The kinetic properties and amino acid composition of chymosin and pepsin have been extensively studied. There is more or less a consensus of opinion in favor of chymosin as the enzyme for cheese making; presently three companies are producing calf chymosin through recombinant DNA technology. The cloned chymosin preparations produced by different microorganisms have been tested in various countries for cheese manufacture. No major differences could be detected among cheeses made with cloned chymosin and those made with the natural enzyme. This review describes the past, present, and future status of rennet substitutes utilized as milk coagulants and applications of modern biological tools to strain improvement and process development.
Fig tree latex (ficin) was stepwise purified by ion exchange chromatography on carboxymethyl (CM)-cellulose and gel filtration chromatography on Sephadex G-100, and then utilized in the production of teleme. Following ion exchange chromatography, the milk clotting to proteolytic activity ratio (MCA/PA) increased from 1.97 to 3.1 and following gel filtration, to 7.4. The purified fraction gave better chemical and sensory properties than teleme made by fig tree latex. The protein content of teleme made by fig tree latex and the purified fraction were 3.90 and 6.50%, respectively. Syneresis in teleme decreased from 95% to 85% upon purification of the proteolytic enzymes. Exclusion of proteolytic activity appears to be essential to improve the quality of teleme.
Regular and ultrafiltered (UF; 1×, 2× and 4× concentrated) skim milk samples were treated with a range of enzymes including calf rennet, ficin and papain. The clotting properties, curd casein profiles and free amino acid (FAA) contents were determined. In general, UF milk samples coagulated faster and formed firmer curds irrespective of protein concentration. Furthermore, both ficin and papain had a more significant effect on proteolysis in curd formed from regular and 1× UF milk than on 2× or 4× UF milk. Cardoon extract and calf rennet had very similar clotting properties, although the former caused both the capillary electrophoresis profile of caseins and FAA measurements to show slightly more extensive hydrolysis in the curd. The results suggest that the UF process may cause structural changes to proteins or other milk constituents with a resultant change in clotting properties and proteolysis of the casein molecules.
A linear diffusion test capable of measuring milk clotting enzyme activity at concentrations of 1 x 10 -4 to 1 x 10 -l rennin units per ml is described. The distribution of rennet activity between curd and whey in freshly coagulated milk was measured at 72% in the whey and 31% in the curd at pH 6.6. At pH 5.2, the whey contained 17% and the curd 86%. Total recovery of activity was 102 -+ 5%. Distribution of milk clotting enzymes from Mucor pusillus var. Lindt and Mucor miebei between curd and whey in freshly coagulated milk was independent of pH with approximately 83% in the whey and 17% in the curd. Porcine pepsin was unstable, precluding accurate assessment of enzyme distribution. During the manu- facture of Cheddar cheese approximately 35% of the rennet activity was destroyed up to the time the whey was drained, and 6% remained in the cheese following pressing. Neither of the microbial en- zymes lost activity during cheese making. Most of the activity remained in the whey while only 2 to 3% was detected in the cheese after pressing. I NT RODUCTI ON
The isolation and identification of low molecular mass peptides formed during the ripening of Parmigiano-Reggiano cheese is described. A strategy was used based on the fractionation of nitrogenous material using chemical methods followed by HPLC to isolate peptides and fast atom bombardment-mass spectrometry to identify them. It was found that the majority of cheese oligopeptides arose from the proteolysis of β-casein. Several phosphopeptides and oligopeptides known in vivo to be biologically active have also been identified during the ripening of cheese.(Received July 18 1991)(Accepted February 26 1992)