Small Ruminant Research 79 (2008) 210

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Small Ruminant Research
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The use of lamb rennet paste in traditional sheep
milk cheese production

M. Addis

, G. Piredda, A. Pirisi
AGRIS, Agricultural Research Agency of Sardinia, Loc. Bonassai, 07040 Olmedo, Italy
a r t i c l e i n f o
Article history:
Available online 13 August 2008
Keywords:
Lamb rennet paste
Proteolysis
Lipolysis
PDO ovine cheese
a b s t r a c t
The clotting of milk in cheese making is a key passage obtained by the use of enzymes or
rennet. Several types of rennet are commercially available, they differ both on their origin
(animal, vegetable, microbial and recombinant from genetically modied microorganism)
and their physical state (liquid, powder or paste).
Usually rennet is derived fromthe abomasa (fourth stomach or vell) of unweaned calves.
Commercial preparations of calf rennet, available as liquid or powdered form, contains
chymosin and a different number of proteases such as: pepsin A, gastricsin or pepsin B. This
rennet do not contain lipolytic enzymes, which are denatured during the activation process
of chymosin and pepsin zymogens.
In Mediterranean countries, is common the use of lamb or kid rennet paste, always
obtained from the abomasa of these small ruminants.
Lamb rennet paste, besides milk-clotting and proteolytic enzymes (chimosin and
pepsins), also contains a complex system of lipolytic enzymes. Lamb rennet paste is used
in the production of some PDOtraditional sheep milk cheeses, such as Idiazabal and Roncal
in Spain, Fiore Sardo, Pecorino Romano, Canestrato Pugliese in Italy and Feta in Greece.
The aim of this review is to summarize the current knowledge in lamb rennet paste
including methods of preparation, enzymatic composition and factors inuencing it, ana-
lytical aspects related to the enzymatic composition, proteolysis, lipolysis and sensory
characteristics of cheeses.
2008 Elsevier B.V. All rights reserved.
1. Introduction
The clotting of milk by rennet is a key passage in
cheese making that, markedly, could affect the charac-
teristics of produced cheese. Nowadays different types of
rennet are available. They differ both on their origin, ani-
mal, vegetable, microbial andrecombinant fromgenetically
modied microorganism, and their physical state, liquid,
powder or paste.

This paper is part of the special issue entitled 5th International Sym-
posiumon The Challenge to Sheep and Goats Milk Sectors Guest Edited by
Antonio Pirisi, Andr Ayerbe, Giovanni Piredda, George Psathas and Yvette
Soustre.

Corresponding author. Tel.: +39 079387229; fax: +39 079389450.

E-mail address: maraddis@tiscali.it (M. Addis).
The most used rennet derives fromthe abomasa (fourth
stomach or vell) of unweaned calves. It is available as liquid
or powder form. In Mediterranean countries, where sheep
and goat breeding is largely diffused, is common the use of
lamb or kid rennet paste.
The specic milk-curdling enzyme present in animal
rennet is the chymosin (EC 3.4.23.4), an acid protease
enzyme. In addition to the chymosin a different number of
generic proteases (Table 1) such as pepsin A (EC 3.4.23.1),
gastricsin (or pepsin B, or pepsin C) (EC 3.4.23.3) are also
present. Some biochemical features of these aspartato pro-
teases are summarised in Table 2.
The most important difference between calf rennet and
lamb or kid rennet paste is due to the presence of lipoly-
tic enzymes in the last one. This is because the lipolytic
enzymes are denatured during the activation process of
0921-4488/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.smallrumres.2008.07.002
M. Addis et al. / Small Ruminant Research 79 (2008) 210 3
Table 1
Nomenclature and sources of major proteses in rennets
Rennet type Enzyme IUB nomenclature Commercial denomination Origin
Animal (liquid) Chymosin EC 3.4.23.4 Rennin Ruminant
Pepsin A EC 3.4.23.1 Pepsin II
Gastricsin EC 3.4.23.3 Pepsin I
Animal (powder) Chymosin EC 3.4.23.4 Rennin Ruminant
Pepsin A EC 3.4.23.1 Pepsin II
Gastricsin EC 3.4.23.3 Pepsin I
Animal (paste) Chymosin EC 3.4.23.4 Rennin Ruminant
Pepsin A EC 3.4.23.1 Pepsin II
Gastricsin EC 3.4.23.3 Pepsin I
Animal Pepsin A EC 3.4.23.1 Pepsin II Swine
Pepsin A EC 3.4.23.1 Pepsin II Chicken
Microbial Proteases from M. Miehei EC 3.4.23.6 Rennilase (Novo) Hanilase
(Chr. Hansen) Fromase
(Wallerstein) Marzyme
(Miles)
Mucor Miehei
Proteases from M Pusillus Emporase (Daityland)
Meito (Meito Sangyo)
Noury (Vitex)
Mucor pusillus Var.
Lindt
Proteases from E. parasitica Sure curd Suparen (Pzer) Endotia parasitica
Recombinant Chymosin Genetic variant A Chy-Max (Pzer) Escherichia coli K12
Chymosin Genetic variant B Chymogen (Chr. Hansen) Aspergillus niger
Var. Awamori
chymosin and pepsin zymogens when calf rennet is pro-
duced.
Lipases, whosesomebiochemical properties areshowed
in Table 3, markedly characterise cheese characteristics.
These enzymes hydrolyse milk fat triglycerides yielding
free fatty acids, which impart the characteristic piquant
taste to cheese.
Some of PDOsheep milk cheeses fromSouthern Europe,
such as Idiazabal and Roncal in Spain, Fiore Sardo, Pecorino
Romano, Canestrato Pugliese in Italy, and Feta in Greece,
are produced using lamb rennet paste.
The aim of this review is to summarize the current
knowledge in lamb rennet paste including methods of
preparation, enzymatic composition and factors inu-
encing it, analytical aspects related to the enzymatic
composition, proteolysis, lipolysis and sensory character-
istics of cheeses.
2. Preparation of lamb rennet paste
Specications of PDO cheeses, generally, do not include
rules about the use and the preparation of the rennet. Only
for Pecorino Romano cheese is required that stomachs used
in lamb rennet paste production come fromanimals raised
in the PDO zone (Scintu and Piredda, 2007).
Nowadays, twotypologies: artisanal andindustrial lamb
rennet pastes are available. The artisanal one is still used to
produce some PDOcheese, such as Fiore Sardo in Italy, Ron-
cal and Idiazabal in Spain. The methods used for artisanal
lamb rennet paste preparation depends on the Countries.
In Sardinia, the manufacturing process provides that lambs
are slaughtered at 2530 days old. The abomasa are col-
lectedandthe perivisceral fat removed, thenthey are salted
and stratied in containers suitable to drain the organic
liquid. Afterward, the abomasa are dried and ripened for at
least 3months. Theabomasanallyaregroundandblended
into a paste (Pettinauet al., 1977). Inother regions of South-
ern Italy, the abomasa, removed fromlambs slaughtered at
2040days old, are lledwithmilk(50100ml) before salt-
ing, then they are dried, ripened for 2 months and ground
into a paste (Santillo et al., 2005).
The method of preparation of the lamb rennet paste is
different in Spain (Bustamante et al., 2000). Lamb stom-
achs are inspectedimmediatelyafter that animals are killed
(4 weeks old), and selected on the basis of their appear-
ance indicating that animals fedonly milk. Stomachs are air
Table 2
Biochemical characteristics of aspartato proteinases in animal rennets
Chymosin EC 3.4.23.4 Pepsin A EC 3.4.23.1 Gastricsin EC 3.4.23.3
Genetic forms A, B, C A B
Phosphorilation sites No 13 02
Molecular weight 31,000 33,000 30,000
Isoelectric point (pH value) 4.5 3.5 4.2
pH (maximum of milk clotting activity) 5.56
pH (maximum of proteolytic activity) 4.5 2 2.8
Temperature (maximum of activity,

C) 40

C 40

C 40

C
pH (value of inactivation) >6.7 >6.5 >6.5
Temperature (inactivation value,

C) >55

C >55

C >55

C
4 M. Addis et al. / Small Ruminant Research 79 (2008) 210
Table 3
Biochemical characteristics of lipolytic enzymes in animal rennets
Molecular weight pH (maximum of activity) Temperature (maximum of activity,

C) Animal species
Pregastric 49,000172,000 5.36.17.5 3040 Calf
168,000 5.56.28.6 3040 Kind
150,000 5.96.6 3040 Lamb
Gastric 7 45 Calf
driedina ventilatedroomuntil a constant weight (45days).
External fat is then removed and the stomachs are cut open
to remove any wool found inside. They are then ground,
mixed with salt and ground again to obtain a paste. Paste is
kept at 4

C in covered glass jars for up to 1 year. Artisanal

liquid lamb rennet is used in the traditional Feta cheese
production. This rennet is prepared by cutting, mixing and
extracting (with NaCl solution for 24h) dried and salted
abomasa of lambs slaughtered before weaning (Georgala
et al., 2005).
No activation procedures of chimosin are described for
artisanal lamb rennet paste. However, most of extracts
of rennet paste, particularly those prepared from the
stomachs of animals slaughtered after feeding, had pHval-
ues <4.7. This couldreplacetheactivationbyacidicationof
bovine rennet extracts. In the recent years a great increase
intheindustrial rennet pasteproductionhas beenreported.
The industrial process did not differ substantially from the
artisanal one. It provides that the abomasa, saltedandstrat-
ied in containers, are ripened, in refrigerated rooms, for 3
months at least. The abomasa are then ground and blended
to obtain a paste.
3. Enzymatic composition of lamb paste rennet
3.1. Analitical aspects
The various analytical methods used to determine the
milkclottingactivityor thelipolytic activityinlambrennets
paste, donot givefrequentlyunivocal results, becauseof the
heterogeneous enzymatic composition of this rennet.
The essential property of a rennet is milk-clotting
activity, which reect its total content of milk-clotting pro-
teinases. The Soxhlet method has been widely used to
measure the strength (total milk-clotting activity) of a ren-
net. In this method the strength is dened as the volume
of raw milk which can be clotted by one unit volume of a
rennet in 40min at 35

C (Soxhlet, 1877). However, due to

the variation of the raw milk characteristics, the denition
is not entirely satisfactory. The precision of this measure
considerably increased, when Berridge proposed the use of
a standardised milk powder, reconstituted in 0.01M CaCl
2
(Berridge, 1957).
Recently an IDF standard method to determine the total
milk-clotting activity in lamb or kid rennet paste has been
approved (IDF, 2006). The principle of this standard is that
the activity of the rennet is relative to the activity of an
international reference rennet powder tested on a stan-
dardised milk substrate. The result is expressed in IMCU
(International Milk Clotting Unit) per gramof rennet paste.
Beside its strength, it is also very important to know
the enzymatic composition of a given rennet. In fact the
use of a rennet with different proportions of chimosin and
pepsin, the latter having a higher proteolytic activity and
a greater pH-dependance compared to chymosin (Andren,
1998), could involve a different proteolysis both during
cheese making and ripening of cheese. The milk clotting
enzymes composition of rennet paste can be determined
adapting the IDF standard 110B (1997) conceived for a liq-
uid rennet. Piredda and Addis (1998) and Bustamante et al.
(2000) suggested a method to obtain the enzymatic extract
from rennet paste. Chymosin and pepsin contained in this
extract are chromatographically separated fromeach other
onacolumnandtheresults areexpressedas either percent-
age of chymosin activity and pepsin activity, or milligrams
of active chymosin and pepsine per litre of extract (or per
gram of paste), respectively.
To properly characterise a rennet paste, it is also neces-
sary to determine its lipolytic activity. At the time does not
exist a standard method to determine it. Various methods
(potentiometric, titrimetric, colorimetric and chromato-
graphic methods) had been used (Rampilli and Barzaghi,
1995). The pH Stat method (potentiometric method),
reported in the Food Chemical Codex (1981), was widely
used. In this method, the lipolytic activity of rennet paste is
determined against an emulsion of a synthetic substrate,
generally tributyrin, at constant pH. Many authors have
applied this method to characterise the lipolityc enzymes
of lamb rennet paste (Barzaghi and Rampilli, 1996; Piredda
and Addis, 1998; Bustamante et al., 2000; Addis et al.,
2005a). The experimental conditions, substrate and pH
(5.56.2), used in this assay, are suitable for highlighting
pregastric lipase (PGL) activity (OConnor et al., 1993; De
Caro et al., 1995).
The lipolysis evaluation of a substrate (ovine or bovine
milk fat) by means of gaschromatographic analysis, was
used to study the lipolytic enzymatic complex of ren-
net paste (Barzaghi and Rampilli, 1996; Bustamante et al.,
2000; Addis et al., 2005b). The experimental conditions of
this method, a natural mixture of triglycerides as substrate
and the pH that varied during incubation, allowed to high-
light the activity of the different lipolytic enzymes present
in the rennet paste and their specicity towards the vari-
ous fatty acids (Barzaghi and Rampilli, 1996; Addis et al.,
2005b).
3.2. Factors inuencing the enzymatic composition
Ahighly complex systemof enzymes, depending onvar-
ious factors such age and diet of the lambs at slaughtering,
has been claimed to affect both the quantity and the qual-
ity of rennet paste enzymes (Sponcet et al., 1985; Piredda
and Addis, 1998). In the young suckling or milk-fed rumi-
nants, the abomasal mucosa is dominated by the primary
M. Addis et al. / Small Ruminant Research 79 (2008) 210 5
Table 4
Total milk clotting activity and enzymatic content of lamb paste rennets
Rennet A B C D E F
Milk clotting activity (IMCU/g) 94 95 278 263 48 279
Chymosin (mg/g) 0.35 0.58 0.67 0.74 0.35 1.29
Chymosin activity (%) 100 88 77 98 82 76
Pepsin (mg/g) 0 0.13 0.29 0.02 0.12 0.59
Pepsin activity 0 12 23 2 18 24
PGL activity (LFU/g) 6.78 1.60 0.22 0.44 2.00 0.85
Adapted from Piredda and Addis (1998); Addis et al. (2005a); Pirisi et al. (2007). A=lambs fed only milk and slaughtered after suckling; B=lambs fed only
milk and slaughtered 2h after suckling; C=lambs fed only milk and slaughtered 12h after suckling; D=lambs fed milk and pasture and slaughtered after
suckling; E=lambs fed milk and pasture and slaughtered 2h after suckling; F =lambs fed milk and pasture and slaughtered 12h after suckling.
milk-clotting enzyme chymosin. In older hay or pasture-
fed animals the abomasum is dominated by pepsin, which
is also a milk-clotting enzyme, but with a lower specic
milk-clotting activity and with a higher proteolytic activ-
ity than chymosin (Andren, 1998). Furthermore, when the
unweanedruminant aresucklingmilk, thereleaseof lipases
is stimulate, particularly of pregastric enzymes (PGL) com-
ing from the oral tissues (Nelson et al., 1977; Manunta et
al., 1981). In older ruminant the lipolitic activity of the
gastric lipases, deriving from abomasal mucosa, prevails
(Richardson et al., 1971).
Only, few studies have reported on the characterisation
of the enzymatic composition of the lamb rennet paste.
Some authors (Piredda and Addis, 1998; Bustamante et
al., 2000; Addis et al., 2005a) have shown that the enzy-
matic characteristics of this rennet type are inuenced by
the lambs breeding system and also by the slaughtering
system. Piredda and Addis (1998) and Addis et al. (2005a)
reported that rennet paste, prepared from young lambs
(2530 days olds) fed exclusively milk or allowed to graze
(mixed milk-pasture diet), had 98100% of chymosin, on
condition that lambs were killed sudden after eating. Chy-
mosin decreased to 88%, if lambs were slaughtered 2h
after suckling, or dropped to 76% of the total coagulating
activity if the lambs had fasted 12h before slaughtered
(Table 4). Bustamante et al. (2000), incontrast foundthat, in
lamb rennet paste, prepared from lambs 4 weeks old, chy-
mosin represented 7580% of the total coagulating activity,
regardless of the state of the stomachwhenlamb was killed
(empty or full of milk).
Many authors agree to afrm that, lamb rennet paste
prepared from suckling animals, slaughtered sudden after
suckling, had a signicantly higher lipase activity than that
prepared from animals with empty stomachs (Piredda and
Addis, 1998; Bustamante et al., 2000; Addis et al., 2005a).
Fig. 1. Lipolytic activity at differents pH values of the artisanal rennet A
(lambs fedonly milk andslaughteredafter suckling) andD(lambs fedmilk
and pasture and slaughtered after suckling) or industrial rennet (G).
Considering that, the lipase activity showed high values at
pH 56 and sharply decreasing to pH 7, as showed in Fig. 1.
This activity has been ascribed to the presence of pregas-
tric lipase (Bustamante et al., 2000; Addis et al., 2005a).
The feeding system can affect quantitatively and qualita-
tively the lipolytic activity of lamb rennet paste. Piredda
and Addis (1998) reported that grazing can partially inhibit
the production of PGL in suckling lambs and can enhance
the activityof other lipases. Addis et al. (2005b) determined
the specicity of the rennet pastes lipolytic enzymatic sys-
tem against a sheep milk cream substrate. They observed
that the lipase system of rennet paste, obtained from suck-
ling lambs only and slaughtered after suckling, showed a
high specicity towards the short chain fatty acids (SCFAs),
in particular butyric and caproic acids, concluding that
this specicity correspond closely to that of PGL (Table 5).
These results are in agreement with those reported by
Bustamante et al. (2000). The enzymatic activity of PGL
Table 5
Free fatty acids (FFAs) (meanS.D.) in sheep milk cream substrate incubated with 80LUmL
1
of extract of rennet A, C, D and F and determined after 24h
Feeding Rennets
A C D F
Slaughtering conditions 0h 12h 0h 12h
Fatty acids
C4:0 12.780.11a 5.430.78b 6.740.45b 5.000.20b
SCFFA/TFFA (%) 87.030.31a 53.801.64c 67.500.53b 51.400.09c
MCFFA/TFFA (%) 9.850.14c 27.080.88a 20.510.69b 29.081.25a
LCFFA/TFFA (%) 3.120.45c 19.120.76a 11.991.22b 19.521.34a
Adapted from Addis et al. (2005b). A=lambs fed only milk and slaughtered after suckling; C=lambs fed only milk and slaughtered 12h after suckling;
D=lambs fed milk and pasture and slaughtered after suckling; F =lambs fed milk and pasture and slaughtered 12h after suckling.
6 M. Addis et al. / Small Ruminant Research 79 (2008) 210
is high on triglycerides containing SCFAs (OConnor et al.,
1993; De Caro et al., 1995), especially those containing
butyric acid, which is predominantly bound on the sn-3
position of the triglycerides in sheep milk fat (Pitas and
Jensen, 1970; Ha and Lindsay, 1993). Addis et al. (2005b)
also found higher levels of medium and long chain fatty
acids (MCFAs and LCFAs) in sheep milk cream substrate
hydrolysed by a paste rennet obtained both from lamb
slaughtered after being fasted and from grazing lambs.
The authors conclude that fasting or changes in the lambs
diet (grazing vs. milk-suckling) may depress PGL activ-
ity and enhance the activity of other lipases (gastric or
microbials). As know microbial lipases hydrolysed from
triglycerides preferably medium and long chain fatty acids
such as palmitic and oleic acids (Barzaghi and Rampilli,
1996).
These results emphasize the complexity of the enzy-
matic system of lamb rennet paste compared to liquid or
powdered calf rennet.
In the recent years there has been a great increase
in production of rennet paste in specialised industrial
plants. Theseplants collect theabomasafromlarger slaugh-
terhouses, where the lambs wait a long time on empty
stomachs before being slaughtered. Moreover lambs,
nowadays, are weaned earlier to anticipate delivery of milk
to dairies. These changes induce an increase of the pepsin
content in the lambs stomach and a parallel decrease of
chymosin and pregastric lipases (Piredda and Addis, 1998;
Addis et al., 2005a; Pirisi et al., 2007). Thus, oftenthe indus-
trial lamb paste rennet preparations contain more pepsin
and less pregastric lipases than those artisanal ones. As
the amount of rennet is usually regulated on the basis of
a xed milk clotting time, the use of an industrial paste
rennet could upset the optimal ratio of lipolytic to clotting
activity. For this reason, the use in cheese making of indus-
trial lambrennet pastecouldinuencethecharacteristics of
PDO cheeses in unpredictable and undesirable way (Addis
et al., 2005a; Pirisi et al., 2007).
Recently, in Sardinia, some industrial manufacturer
diversied their production selecting abomasa full of milk.
This rennet is oftendestinedtoproducethePDOFioreSardo
cheese.
4. Traditional ewe milk cheeses produced with
lamb rennet paste
The lamb rennet paste is used in certain Southern Euro-
pean countries to produce sheep milk cheeses, such as
Idiazabal and Roncal in Spain, Pecorino Romano, Fiore
Sardo and Canestrato Pugliese in Italy, and Feta in Greece.
4.1. PDO Idiazabal cheese
Idiazabal cheese is a typical product of the Basque
Country region of Northen Spain and is manufactured
from raw ovine milk according to the specications of its
Denomination of Origin Regulatory Board (Ministerio de
Agricoltura, Pesca y Alimentation, 1993). Traditionally, Idi-
azabal cheese has been manufactured with artisanal lamb
rennet, although commercial calf rennets are nowadays
widely used by artisanal and industrial manufacturers.
Nevertheless, cheeses made with artisanal lamb rennet
show specic sensory characteristics distinguishable from
the cheeses made with commercial calf rennet. The regu-
latory board does not specify the type of rennet to use as
coagulant for Idiazabal cheese making, although it is par-
ticularly interested in promoting the use of artisanal lamb
rennet in order to achieve a more distinct and typical prod-
uct (Vicente et al., 2001a).
4.2. PDO Roncal cheese
Roncal cheese, made from raw ewe milk, was the rst
Spanish cheese to be granted an Appellation of Origin in
1981. It is an uncooked, pressed-curd cheese, with a mini-
mumripening time of 4months. The regulations of the PDO
permit the addition of artisanal lamb or commercial calf
rennet. Traditionally, Roncal cheese has been made using
artisanal lamb rennet even if nowadays most cheese mak-
ers prefer to use commercial calf one (Irigoyen et al., 2001;
Irigoyen et al., 2002).
4.3. PDO Pecorino Romano cheese
Pecorino Romano cheese is an Italian PDO cheese that
must be made exclusively fromwhole sheep milk and lamb
rennet paste as coagulant. By the law it can be produced
in Sardinia, Latium and Tuscany (Province of Grosseto).
This cheese have a particular avour, which has been
attributed to the use of lamb rennet paste. The speci-
cations for the Pecorino Romano production require that
the stomachs used to produce the rennet have to come
fromanimals raised in the PDO zone (Decreto Ministeriale,
1995).
4.4. PDO Fiore Sardo cheese
Fiore Sardo PDO is a traditional Italian uncooked sheep
milk cheese, produced exclusively in Sardinia from raw
milk. The requirements of the cheese making protocol of
Fiore Sardo cheese permit the use of traditional lamb or
kid rennet paste. The sensory characteristics, and in partic-
ular the piquant taste developed during ripening, are due
to the large lipolysis induced by the enzymes contained in
the rennet. Traditionally, and to a great extent up till now,
the rennet used in the Fiore Sardo cheese making is directly
produced at the farm by shepherds themselves, slaughter-
inglactatinglambs ashort timeafter sucklingandremoving
the abomasa full of milk. (Addis et al., 2005b; Scintu and
Piredda, 2007; Pirisi et al., 2007).
4.5. PDO Canestrato Pugliese cheese
Canestrato Pugliese cheese is a traditional cheese man-
ufactured in Puglia, from sheep milk of local ocks. Since
1985 it has had the Appelation of Origin status, which
has been acknowledged by the European Community (C.D.
1996). It is manufactured fromwinter to spring and its pro-
duction protocol (DPR 1986) provides the use of animal
rennet for milk coagulation, but do not specify the type
(Santoro and Faccia, 1998).
M. Addis et al. / Small Ruminant Research 79 (2008) 210 7
4.6. PDO Feta cheese
Feta cheese is a Greek PDO white pickled cheese
(ripened in tins lled with brine) made from ewe or a
mixture of ewe and goat milk. Nowadays it is manufac-
tured, most of all, in modern dairies, from pasteurised
milk with the use of mesophilic starter cultures and com-
mercial calf or bovine rennet. Feta cheese is also still
manufactured in small dairies, traditionally from ther-
mised milk, using yoghurt as starter culture, and artisanal
rennet from lambs and kids abomasa and it is ripened
in wooden barrels without brine addition (Georgala et al.,
2005).
5. Proteolysis of cheeses produced using lamb
rennet paste
Proteolysis is widely considered as the main biochem-
ical and the most complex transformation taking place
during cheese ripening (Fox, 1989; Farkye and Fox, 1990).
The main proteolytic agents in cheese ripening process are
the natural proteases of milk (plasmin and cathepsin D),
the rennet or clotting enzymes retained in the curd, the
proteases and the peptidases produced by starter and non-
starter bacteria.
In the early stage of ripening, caseins are hydrol-
ysed, mainly by enzymes coming from rennet, yielding
high-molecular-weight peptides. This process is know as
primary proteolysis (Grappin et al., 1985) and results in
important alterations inthetextureof thecheeses (Creamer
and Olson, 1982). Successively these polypeptides are
further degraded by the proteinase-peptidase system of
starter andnon-starter bacteriatopeptides andaminoacids
(Fox, 1989).
The use of a rennet with different proportions of
chimosin and pepsin brings about different proteolytic
changes in the cheese. As well-know, the proportion of chi-
mosin decreases as the animal age increases, accompanied
by a concomitant increase in the pepsin content. Pepsin
is a highly active proteolytic, non-specic enzyme that as
been held responsible for the production of bitter avour
in cheese (Guinee and Wilkinson, 1992). A reviewof litera-
ture has disclosedlittle works concerning the effect of lamb
rennet paste on proteolysis process in ovine cheeses.
Santoro and Faccia (1998), Irigoyen et al. (2000, 2002),
Vicente et al. (2001a,b), Bustamante et al. (2003) and
Moatsou et al. (2004) studied the effect of lamb rennet
paste or bovine rennet on the proteolysis in Canestrato
Pugliese, Roncal, Idiazabal and Feta cheeses, respectively.
In general, there are no indications about the total milk
clotting activity added to the vats during milk coagulation,
which does not facilitate comparison among the results.
Overall authors observe an increase in the formation
rate of the water-soluble nitrogen (WSN) and non-protein
nitrogen (NPN) fractions with the use of lamb rennet. This
has been related both to the high retention of chymosin
in the curd when lamb rennet was used and to a greater
complexity of lamb rennet than bovine rennet. Lamb ren-
net exhibite a greater proteolytic activity against
s
-casein,
especially
s1
-casein than calf rennet. In consequence both
the formation and the subsequent degradation of
s1
-I
casein took place more quickly in cheese made using lamb
rennet (Irigoyen et al., 2002).
The rennet type did not affect the extent of degrada-
tion of -casein (Fox and Law, 1991; Fontecha et al., 1994;
Trujillo et al., 2000; Papoff et al., 2004).
Results on WSN fraction show that the differences in
proteolysis are also qualitative, being the ratios between
hydrophilic and hydrophobic compounds closely related to
the chymosin/pepsin ratio (Santoro and Faccia, 1998).
Although lamb rennet paste or their acqueous extract
are not very well characterised, three proteases have
been reported for this rennet, which can explicate the
greater proteolytic activity compared to that of calf rennet
(Foltman, 1993).
Bustamante et al. (2003) studied the proteolytic process
in Idiazabal cheeses made using a traditional lamb rennet
paste (chymosin/pepsine ratio, 80:20) and a commercial
calf rennet (chymosin/pepsine ratio, 90:10) in high and
low level. In disagreement with the previous cited works,
they found that rennet type did not signicantly affect the
percentages of any nitrogen fraction. This, if comparable
amounts of total milk clotting activity are used in cheese
making and chymosin to pepsin ratio is similar in rennet
preparations. They also observed that hydrolysis of
s1
-
casein was not signicantly affected by the rennet type and
that
s1
-I casein fraction was greater in cheese made with
bovine rennet than in cheeses made with lamb one.
Moatsou et al. (2004) compared the effect of an arti-
sanal liquid rennet from kids and lambs and a commercial
calf rennet on the characteristics of Feta cheese. Both ren-
nets had a similar chymosin and pepsin ratio (70:30) and
were used in same quantities with reference to the total
milk clotting activity. Although no signicant differences
were detected, the accumulation of WSN in cheese made
with artisanal rennet was faster, the same was also for the
changes in residual
s1
-casein.
The shown results are frequently contradictory because
chymosin to pepsin ratio of the rennets and the different
total milk clotting activity added to the cheese milk were
not sometimes equals.
Studies comparing the effect of the use of lamb ren-
net paste with different milk clotting activity and different
enzymatic composition on the proteolysis during cheese
ripening, arescarce, probablyduetothehighlylocaliseduse
of rennet paste (Irigoyen et al., 2001; Addis et al., 2005a,b;
Pirisi et al., 2007).
Irigoyen et al. (2001) studied the effect of two lamb
paste rennets, having different milk-clotting activity and
different levels of chymosin and pepsine enzymes (78:22
and 54:46, respectively), on the proteolysis in Roncal
cheeses. The authors found that cheeses made with the
rennet with the highest milk clotting activity and highest
chymosin content showed the highest WSN and NPN con-
tents. These results agree with those Santoro and Faccia
(1998). Addis et al. (2005b) used four different lamb rennet
paste preparations inFiore Sardo cheese manufacture. Ren-
nets coming from lambs differently fed (milk only or milk
and grass mixture) and slaughtered at full or empty stom-
ach, differed in total milk clotting activity and in chymosin
pepsin ratio (100:0, 98:2, 77:23, 76:24, respectively). Dur-
ing cheese making, rennets were used with a standardised
8 M. Addis et al. / Small Ruminant Research 79 (2008) 210
milk clotting activity. Authors did not nd any difference in
WSN and NPN fractions. These results were in agreement
with those found by Bustamante et al. (2003). The same
authors (Addis et al., 2005a) studied the effect of two tradi-
tional lamb rennets paste, prepared fromlambs differently
fed (milk only or milk and grass mixture) (88:12 and 82:18
chymosin/pepsin ratio, respectively) compared with a
commercial lamb rennet paste (64:36 chymosin/pepsin
ratio), on PDO Pecorino Romano cheese. According to
the previous work, they did not nd differences in the
proteolysis indices due to the used rennet type.
Pirisi et al. (2007) studied the inuence of the use of two
lamb rennets paste type on PDO Fiore Sardo cheese char-
acteristics. Rennets were a traditional one, produced from
the abomasa of lambs fed only milk and slaughtered after
suckling (100:0 chymosyn/pepsin ratio) when another one
was pruduced at industrial level coming from stomachs of
lambs weaned and slaughtered on a empty stomach (60:40
chymosyn/pepsin ratio). Since the total coagulating activ-
ity added to the milk in the manufacture of the cheeses was
the same, the higher WSN development in cheese, caused
by the industrial lamb rennet paste preparation, suggest
an increased proteolytic activity due to the co-presence
of pepsine, which increases the general proteolytic activ-
ity on casein. The cheeses made with industrial rennet
underwent a more rapid
s1
-casein degradation, which
conrms the higher efciency exhibited by pepsin con-
taining industrial rennet preparation in producing WSN.
This also means that the amount of residual
s1
-casein
was higher in cheeses made with traditional lamb rennet
paste. These results are in disagreement with those found
by Irigoyen et al. (2001). No signicant differences depend-
ingonthe rennet type were foundfor -caseindegradation.
This is consistent with the results of Papoff et al., 2004 indi-
cating a lower level of -casein degradation products than

s1
-casein. Pirisi et al. (2007) also observed that some pep-
tides seem to be specic to the PDO Fiore Sardo cheese. In
Grana Padano cheese, where calf liquid rennet is used as
coagulant, the only SerP residue subjected to dephospho-
rylation is located at the N-terminus of phosphopeptides.
In the PDO Fiore Sardo cheese, manufactured using rennet
paste, no apparent difference in susceptibility to dephos-
phorylation was found amongst differently located SerP
peptide residues. This obviously depends on the nature and
specicity of phosphatase(s) prevalently active in the two
cheese varieties. The different levels of the alkaline phos-
phatase activity present in liquid or paste rennet could be
affect, in a different way, the mechanismof casein peptides
dephosphorylation (Pirisi et al., 2007).
6. Lipolysis of cheeses produced using lamb rennet
paste
Lipolysis is one of the major biochemical changes that
occur during cheese ripening. The released of free fatty
acids (FFAs) during lipolysis is considered to inuence the
avour of cheeses (Woo et al., 1984). This inuence can be
direct, as in some Italian cheese varieties where short chain
fatty acids themselves contribute directly to the taste and
avour (Woo and Lindsay, 1984; Addis et al., 2005a,b; Pirisi
et al., 2007), or indirect considering FFAs as precursors for
compounds such as methyl ketones, alkanes, lactones and
esters (Urbach, 1991). Changes in FFAs composition dur-
ing ripening have been suggested as index of ripening in
cow milk cheese, even if it is reported as less useful than
proteolytic andglycolytic indicators (Farkye andFox, 1990).
The lipolytic agents presents in cheese are lipolytic
enzymes coming naturally from milk (milk lipases), from
rennet (pregastric and gastric lipases) and from microora
activity (Collins et al., 2003).
The contribution of the rennet depends on the rennet
type. Commercial calf and bovine rennets, the most com-
monly used, are normally lipolytic activity free. On the
other hand, traditional paste rennets, made from abomasa
of lambs unweaned and slaughtered at full stomach, have
high lipolytic activity due to their content of PGL (Piredda
and Addis, 1998; Bustamante et al., 2000). PGL prefer-
entially hydrolyse short chain fatty acids esteried in the
sn-3 position, releasing high level of butyric acid (C4:0)
(Pitas and Jensen, 1970; Nelson et al., 1977; Ha and Lind-
say, 1993). The characteristic piquant or pungent avour
found in Italian cheeses like Pecorino Romano, Fiore Sardo
and Provolone, have been attributed to their high butyric
acid content and to the use of this rennet type (Barzaghi et
al., 1997; Addis et al., 2005a,b).
Only fewstudies are available on the effect of lamb ren-
net paste on lipolysis in ovine cheese (Larrayoz et al., 1999;
Virto et al., 2003; Addis et al., 2005a,b; Hernandez et al.,
2005; Georgala et al., 2005; Pirisi et al., 2007).
PDOspecications of Idiazabal andRoncal cheeses allow
the use of bovine or lamb rennet as coagulant. Several
authors (Larrayoz et al., 1999; Virto et al., 2003; Hernandez
et al., 2005) reported that the concentration of total free
fatty acids (TFFAs) in Idiazabal cheese manufactured with
lamb paste rennet was signicantly higher than in cheeses
manufactured with bovine rennet. They also observed that
lipolysis intheformer cheeses increasedwiththetotal units
of lipolytic activity addedtothe milk. The FFAs composition
of these cheeses was also dependent on the rennet type,
with short-chain fatty acids (SCFAs) accounting for more
than 65% in cheese made with lamb paste rennet but only
45% in cheese made with bovine rennet. Virto et al. (2003)
suggest that, the increase in total lipolysis accompained by
an increase in the level of SCFAs, as a percentage of TFFAs,
greater than 50 or 55mol 100mol
1
, can be used as a
reliable index to infer the presence of pregastric lipase in
the paste rennets. Among SCFAs, butyric acid was the most
abundant. Idiazabal cheese made with lamb paste rennet
received signicantly higher scores for odour and avour
intensity (Virto et al., 2003; Hernandez et al., 2005).
Addis et al. (2005b) used four different lamb paste ren-
nets having different enzymatic characteristics to asses
the inuence of paste rennet on lipolytic process and on
quality of Fiore Sardo cheese. Results showed signicantly
differences in the levels of TFFAs, particularly of SCFAs,
among rennets. In agreement with Virto et al. (2003) and
Hernandez et al. (2005) the authors concluded that, this
lipolytic patternincheeses was relatedto the highPGL con-
tent in rennet coming from lambs fed only milk, being PGL
secretion stimulated by milk suckling. These authors also
found that changes in the lamb diet, from milk to herbage,
and the absence of food in the stomachs at slaughtering,
M. Addis et al. / Small Ruminant Research 79 (2008) 210 9
reduced the amount of PGL in the abomasa, and then in the
paste rennet. The use of this rennet type gave cheeses with
higher in MCFA and LCFA content. In the recent years the
production of lamb paste rennet in specialised industrial
plants is increased (Addis et al., 2005a; Pirisi et al., 2007).
Both lipolytic activity and chymosin content of this ren-
net type, often was lower than traditional rennet. Addis et
al. (2005a) and Pirisi et al. (2007), found both qualitative
and quantitative differences for FFA in Pecorino Romano
and in Fiore Sardo cheeses made with artisanal or indus-
trial lambpaste rennet. Cheese made usingtraditional lamb
paste rennet had higher content of TFFAs due to the high
SCFAs level and, above all, to the butyric acid content. On
the contrary, percentage of MCFA and LCFA were higher in
cheese made using industrial rennet. Georgala et al. (2005)
studied the evolution of lipolysis in traditional Feta cheese
during the ripening, made withartisanal liquidrennet from
lambs and kids abomasa or a mixed artisanal rennet with
calf rennet. The TFFA content in cheeses differed signi-
cantly. Thelevels of individual FAwerethehighest incheese
made with traditional lamb rennet. These results were in
agreement with those of Moatsou et al. (2004) who found
a higher C4:0-C10:0 FFA content in ripened industrial Feta
cheese made with artisanal rennet than in cheeses made
with calf rennet.
7. Sensory characteristics of cheeses produced using
lamb rennet paste
Flavour and texture of cheese are the outcome of a
series of chemical, biochemical, and microbiological events
that occur during ripening. The FFAs released during lipol-
ysis contribute, together with the volatile compounds
and the proteolysis products, directly to cheese avour
(McSweeney and Sousa, 2000).
The SCFAs play an important role in the sensory char-
acteristics of the cheeses made with lamb paste rennet,
mainly due to their high levels and low perception thresh-
olds (Larrayoz et al., 1999). Hernandez et al. (2005) found
that the percentage of SCFAs is linearly correlated with the
intensity score for the attribute pungent avour.
Several authors agreed on afrm that cheeses manu-
factured with lamb paste rennet had signicantly higher
intensity scores in strong attributes such as overall odour
and avour intensity, sharp odour, pungent avour, ren-
net odour and avour and piquant taste (Bustamante et al.,
2003; Virto et al., 2003; Hernandez et al., 2005; Addis et al.,
2005a; Georgala et al., 2005). These attributes are desirable
in the case of certain cheeses such as Idiazabal, Pecorino
Romano, Fiore Sardo, Feta cheeses, because they are part of
theirs characteristic avour and odour. The same authors
reported that the piquant taste observed in cheese made
with artisanal lamb rennet paste was associated with the
high SCFAs content and specically with the high butyric
acid content.
Ontheother hands intensityscores for milder attributes,
such as milk odour, butter odour and avour, nutty, toasted
and sweet avour were lower in cheeses made with lamb
rennet paste (Virto et al., 2003).
As concern the proteolysis products, the casein
oligopeptides could have an important role in the cheese
avour intensity. The prevalence of the hydrophobic pep-
tides over hydrophilic ones could generate bitterness
during cheese ripening. High levels of hydrophobic pep-
tides have been detected in bitter cheeses. Pirisi et al.
(2007), in agreement with Santoro et al. (1998), showed
that hydrophilic peptides fraction prevails in cheeses made
using lamb paste rennets with a high chymosin content.
8. Conclusions
In conclusion, the enzymatic composition of the rennet
lamb paste markedly inuence the biochemical pathways
of proteins and fat degradation during cheese ripening.
Since the enzymatic (lipolytic and proteolytic) composi-
tion of rennet paste depends on the diet and slaughtering
conditions of lambs, it may be necessary to standard-
ise these conditions when establishing norms for rennet
paste production, especially for the production of PDO
cheeses.
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