Small Ruminant Research 79 (2008) 2–10

Contents lists available at ScienceDirect

Small Ruminant Research

journa l homepage: www.e lsev ier .com/ locate /smal l rumres

The use of lamb rennet paste in traditional sheepmilk cheese production�

M. Addis ∗, G. Piredda, A. PirisiAGRIS, 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 pasteProteolysisLipolysisPDO 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 orrennet. Several types of rennet are commercially available, they differ both on their origin(animal, vegetable, microbial and recombinant from genetically modified microorganism)and their physical state (liquid, powder or paste).

Usually rennet is derived from the abomasa (fourth stomach or vell) of unweaned calves.Commercial preparations of calf rennet, available as liquid or powdered form, containschymosin and a different number of proteases such as: pepsin A, gastricsin or pepsin B. Thisrennet do not contain lipolytic enzymes, which are denatured during the activation processof chymosin and pepsin zymogens.

In Mediterranean countries, is common the use of lamb or kid rennet paste, alwaysobtained from the abomasa of these small ruminants.

Lamb rennet paste, besides milk-clotting and proteolytic enzymes (chimosin andpepsins), also contains a complex system of lipolytic enzymes. Lamb rennet paste is used

in the production of some PDO traditional sheep milk cheeses, such as Idiazabal and Roncalin 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 pasteincluding methods of preparation, enzymatic composition and factors influencing it, ana-lytical aspects related to the enzymatic composition, proteolysis, lipolysis and sensory

heeses

characteristics of c

1. Introduction

The clotting of milk by rennet is a key passage incheese 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 and recombinant from geneticallymodified microorganism, and their physical state, liquid,powder or paste.

� This paper is part of the special issue entitled 5th International Sym-posium on The Challenge to Sheep and Goats Milk Sectors Guest Edited byAntonio Pirisi, André Ayerbe, Giovanni Piredda, George Psathas and YvetteSoustre.

∗ Corresponding author. Tel.: +39 079387229; fax: +39 079389450.E-mail address: maraddis@tiscali.it (M. Addis).

0921-4488/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.smallrumres.2008.07.002

.© 2008 Elsevier B.V. All rights reserved.

The most used rennet derives from the abomasa (fourthstomach or vell) of unweaned calves. It is available as liquidor powder form. In Mediterranean countries, where sheepand goat breeding is largely diffused, is common the use oflamb or kid rennet paste.

The specific milk-curdling enzyme present in animalrennet is the chymosin (EC 3.4.23.4), an acid proteaseenzyme. In addition to the chymosin a different number ofgeneric 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 alsopresent. Some biochemical features of these aspartato pro-

teases are summarised in Table 2.

The most important difference between calf rennet andlamb or kid rennet paste is due to the presence of lipoly-tic enzymes in the last one. This is because the lipolyticenzymes are denatured during the activation process of

M. Addis et al. / Small Ruminant Research 79 (2008) 2–10 3

Table 1Nomenclature and sources of major proteses in rennets

Rennet type Enzyme IUB nomenclature Commercial denomination Origin

Animal (liquid) Chymosin EC 3.4.23.4 Rennin RuminantPepsin A EC 3.4.23.1 Pepsin IIGastricsin EC 3.4.23.3 Pepsin I

Animal (powder) Chymosin EC 3.4.23.4 Rennin RuminantPepsin A EC 3.4.23.1 Pepsin IIGastricsin EC 3.4.23.3 Pepsin I

Animal (paste) Chymosin EC 3.4.23.4 Rennin RuminantPepsin A EC 3.4.23.1 Pepsin IIGastricsin EC 3.4.23.3 Pepsin I

Animal Pepsin A EC 3.4.23.1 Pepsin II SwinePepsin 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 (Pfizer) Endotia parasitica

R variantvariant

cd

iTft

sRa

kpeci

2

rf

TB

GPMIppTpT

ecombinant Chymosin GeneticChymosin Genetic

hymosin and pepsin zymogens when calf rennet is pro-uced.

Lipases, whose some biochemical properties are showedn Table 3, markedly characterise cheese characteristics.hese enzymes hydrolyse milk fat triglycerides yieldingree fatty acids, which impart the characteristic piquantaste to cheese.

Some of PDO sheep milk cheeses from Southern Europe,uch as Idiazabal and Roncal in Spain, Fiore Sardo, Pecorinoomano, Canestrato Pugliese in Italy, and Feta in Greece,re produced using lamb rennet paste.

The aim of this review is to summarize the currentnowledge in lamb rennet paste including methods ofreparation, enzymatic composition and factors influ-ncing it, analytical aspects related to the enzymaticomposition, proteolysis, lipolysis and sensory character-stics of cheeses.

. Preparation of lamb rennet paste

Specifications of PDO cheeses, generally, do not includeules about the use and the preparation of the rennet. Onlyor Pecorino Romano cheese is required that stomachs used

able 2iochemical characteristics of aspartato proteinases in animal rennets

Chymosin EC 3.4.23.4

enetic forms A, B, Chosphorilation sites Noolecular weight 31,000

soelectric point (pH value) 4.5H (maximum of milk clotting activity) 5.5–6H (maximum of proteolytic activity) 4.5emperature (maximum of activity, ◦C) 40 ◦CH (value of inactivation) >6.7emperature (inactivation value, ◦C) >55 ◦C

A Chy-Max (Pfizer) Escherichia coli K12B Chymogen (Chr. Hansen) Aspergillus niger

Var. Awamori

in lamb rennet paste production come from animals raisedin the PDO zone (Scintu and Piredda, 2007).

Nowadays, two typologies: artisanal and industrial lambrennet pastes are available. The artisanal one is still used toproduce some PDO cheese, such as Fiore Sardo in Italy, Ron-cal and Idiazabal in Spain. The methods used for artisanallamb rennet paste preparation depends on the Countries.In Sardinia, the manufacturing process provides that lambsare slaughtered at 25–30 days old. The abomasa are col-lected and the perivisceral fat removed, then they are saltedand stratified in containers suitable to drain the organicliquid. Afterward, the abomasa are dried and ripened for atleast 3 months. The abomasa finally are ground and blendedinto a paste (Pettinau et al., 1977). In other regions of South-ern Italy, the abomasa, removed from lambs slaughtered at20–40 days old, are filled with milk (50–100 ml) before salt-ing, then they are dried, ripened for 2 months and groundinto a paste (Santillo et al., 2005).

The method of preparation of the lamb rennet paste isdifferent in Spain (Bustamante et al., 2000). Lamb stom-achs are inspected immediately after that animals are killed(4 weeks old), and selected on the basis of their appear-ance indicating that animals fed only milk. Stomachs are air

Pepsin A EC 3.4.23.1 Gastricsin EC 3.4.23.3

A B1–3 0–233,000 30,0003.5 4.2

2 2.840 ◦C 40 ◦C>6.5 >6.5>55 ◦C >55 ◦C

4 M. Addis et al. / Small Ruminant Research 79 (2008) 2–10

Table 3Biochemical characteristics of lipolytic enzymes in animal rennets

Molecular weight pH (maximum of activity) Temperature (maximum of activity, ◦C) Animal species

Pregastric 49,000–172,000 5.3–6.1–7.5 30–40 Calf

168,000 5.5–6.2–8.6150,000 5.9–6.6

Gastric – 7

dried in a ventilated room until a constant weight (45 days).External fat is then removed and the stomachs are cut opento remove any wool found inside. They are then ground,mixed with salt and ground again to obtain a paste. Paste iskept at 4 ◦C in covered glass jars for up to 1 year. Artisanalliquid lamb rennet is used in the traditional Feta cheeseproduction. This rennet is prepared by cutting, mixing andextracting (with NaCl solution for 24 h) dried and saltedabomasa of lambs slaughtered before weaning (Georgalaet al., 2005).

No activation procedures of chimosin are described forartisanal lamb rennet paste. However, most of extractsof rennet paste, particularly those prepared from thestomachs of animals slaughtered after feeding, had pH val-ues < 4.7. This could replace the activation by acidification ofbovine rennet extracts. In the recent years a great increasein the industrial rennet paste production has been reported.The industrial process did not differ substantially from theartisanal one. It provides that the abomasa, salted and strat-ified in containers, are ripened, in refrigerated rooms, for 3months at least. The abomasa are then ground and blendedto obtain a paste.

3. Enzymatic composition of lamb paste rennet

3.1. Analitical aspects

The various analytical methods used to determine themilk clotting activity or the lipolytic activity in lamb rennetspaste, do not give frequently univocal results, because of theheterogeneous enzymatic composition of this rennet.

The essential property of a rennet is milk-clottingactivity, which reflect its total content of milk-clotting pro-teinases. The Soxhlet method has been widely used tomeasure the strength (total milk-clotting activity) of a ren-net. In this method the strength is defined as the volumeof raw milk which can be clotted by one unit volume of arennet in 40 min at 35 ◦C (Soxhlet, 1877). However, due tothe variation of the raw milk characteristics, the definitionis not entirely satisfactory. The precision of this measureconsiderably increased, when Berridge proposed the use ofa standardised milk powder, reconstituted in 0.01 M CaCl2(Berridge, 1957).

Recently an IDF standard method to determine the totalmilk-clotting activity in lamb or kid rennet paste has beenapproved (IDF, 2006). The principle of this standard is thatthe 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 gram of rennet paste.

Beside its strength, it is also very important to knowthe enzymatic composition of a given rennet. In fact the

30–40 Kind30–40 Lamb45 Calf

use of a rennet with different proportions of chimosin andpepsin, the latter having a higher proteolytic activity anda greater pH-dependance compared to chymosin (Andren,1998), could involve a different proteolysis both duringcheese making and ripening of cheese. The milk clottingenzymes composition of rennet paste can be determinedadapting 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 extractfrom rennet paste. Chymosin and pepsin contained in thisextract are chromatographically separated from each otheron a column and the results are expressed as either percent-age of chymosin activity and pepsin activity, or milligramsof active chymosin and pepsine per litre of extract (or pergram of paste), respectively.

To properly characterise a rennet paste, it is also neces-sary to determine its lipolytic activity. At the time does notexist 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 widelyused. In this method, the lipolytic activity of rennet paste isdetermined against an emulsion of a synthetic substrate,generally tributyrin, at constant pH. Many authors haveapplied this method to characterise the lipolityc enzymesof lamb rennet paste (Barzaghi and Rampilli, 1996; Pireddaand Addis, 1998; Bustamante et al., 2000; Addis et al.,

2005a). The experimental conditions, substrate and pH(5.5–6.2), used in this assay, are suitable for highlightingpregastric lipase (PGL) activity (O’Connor et al., 1993; DeCaro et al., 1995).

The lipolysis evaluation of a substrate (ovine or bovinemilk fat) by means of gaschromatographic analysis, wasused 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 ofthis method, a natural mixture of triglycerides as substrateand the pH that varied during incubation, allowed to high-light the activity of the different lipolytic enzymes presentin the rennet paste and their specificity towards the vari-ous fatty acids (Barzaghi and Rampilli, 1996; Addis et al.,2005b).

3.2. Factors influencing the enzymatic composition

A highly complex system of enzymes, depending on var-

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; Pireddaand 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) 2–10 5

Table 4Total 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 279Chymosin (mg/g) 0.35 0.58 0.67 0.74 0.35 1.29Chymosin activity (%) 100 88 77 98 82 76Pepsin (mg/g) 0 0.13 0.29 0.02 0.12 0.59Pepsin activity 0 12 23 2 18 24P 0.22 0.44 2.00 0.85

A 2007). A = lambs fed only milk and slaughtered after suckling; B = lambs fed onlym htered 12 h after suckling; D = lambs fed milk and pasture and slaughtered afters g; F = lambs fed milk and pasture and slaughtered 12 h after suckling.

mfimiuiiag(

oSamtsr((cmaa(lmr(

pspA

TF

F

S

F

AD

GL activity (LFU/g) 6.78 1.60

dapted from Piredda and Addis (1998); Addis et al. (2005a); Pirisi et al. (ilk and slaughtered 2 h after suckling; C = lambs fed only milk and slaug

uckling; E = lambs fed milk and pasture and slaughtered 2 h after sucklin

ilk-clotting enzyme chymosin. In older hay or pasture-ed animals the abomasum is dominated by pepsin, whichs also a milk-clotting enzyme, but with a lower specific

ilk-clotting activity and with a higher proteolytic activ-ty than chymosin (Andren, 1998). Furthermore, when thenweaned ruminant are suckling milk, the release of lipases

s stimulate, particularly of pregastric enzymes (PGL) com-ng from the oral tissues (Nelson et al., 1977; Manunta etl., 1981). In older ruminant the lipolitic activity of theastric lipases, deriving from abomasal mucosa, prevailsRichardson et al., 1971).

Only, few studies have reported on the characterisationf the enzymatic composition of the lamb rennet paste.ome authors (Piredda and Addis, 1998; Bustamante etl., 2000; Addis et al., 2005a) have shown that the enzy-atic characteristics of this rennet type are influenced by

he lambs’ breeding system and also by the slaughteringystem. Piredda and Addis (1998) and Addis et al. (2005a)eported that rennet paste, prepared from young lambs25–30 days olds) fed exclusively milk or allowed to grazemixed milk-pasture diet), had 98–100% of chymosin, onondition that lambs were killed sudden after eating. Chy-osin decreased to 88%, if lambs were slaughtered 2 h

fter suckling, or dropped to 76% of the total coagulatingctivity if the lambs had fasted 12 h before slaughteredTable 4). Bustamante et al. (2000), in contrast found that, inamb rennet paste, prepared from lambs 4 weeks old, chy-

osin represented 75–80% of the total coagulating activity,egardless of the state of the stomach when lamb was killedempty or full of milk).

Many authors agree to affirm that, lamb rennet pasterepared from suckling animals, slaughtered sudden afteruckling, had a significantly higher lipase activity than thatrepared from animals with empty stomachs (Piredda andddis, 1998; Bustamante et al., 2000; Addis et al., 2005a).

able 5ree fatty acids (FFAs) (mean ± S.D.) in sheep milk cream substrate incubated wit

eeding Rennets

A C

laughtering conditions 0 h 12 h

atty acidsC4:0 12.78 ± 0.11a 5.43SCFFA/TFFA (%) 87.03 ± 0.31a 53.80MCFFA/TFFA (%) 9.85 ± 0.14c 27.08LCFFA/TFFA (%) 3.12 ± 0.45c 19.12

dapted from Addis et al. (2005b). A = lambs fed only milk and slaughtered afte= lambs fed milk and pasture and slaughtered after suckling; F = lambs fed milk

Fig. 1. Lipolytic activity at differents pH values of the artisanal rennet A(lambs fed only milk and slaughtered after suckling) and D (lambs fed milkand pasture and slaughtered after suckling) or industrial rennet (G).

Considering that, the lipase activity showed high values atpH 5–6 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. Pireddaand Addis (1998) reported that grazing can partially inhibitthe production of PGL in suckling lambs and can enhancethe activity of other lipases. Addis et al. (2005b) determinedthe specificity of the rennet pastes’ lipolytic enzymatic sys-tem against a sheep milk cream substrate. They observedthat the lipase system of rennet paste, obtained from suck-ling lambs only and slaughtered after suckling, showed a

high specificity towards the short chain fatty acids (SCFAs),in particular butyric and caproic acids, concluding thatthis specificity correspond closely to that of PGL (Table 5).These results are in agreement with those reported byBustamante et al. (2000). The enzymatic activity of PGL

h 80 LU mL−1 of extract of rennet A, C, D and F and determined after 24 h

D F

0 h 12 h

± 0.78b 6.74 ± 0.45b 5.00 ± 0.20b± 1.64c 67.50 ± 0.53b 51.40 ± 0.09c± 0.88a 20.51 ± 0.69b 29.08 ± 1.25a± 0.76a 11.99 ± 1.22b 19.52 ± 1.34a

r suckling; C = lambs fed only milk and slaughtered 12 h after suckling;and pasture and slaughtered 12 h after suckling.

minant R

6 M. Addis et al. / Small Ru

is high on triglycerides containing SCFAs (O’Connor et al.,1993; De Caro et al., 1995), especially those containing

butyric acid, which is predominantly bound on the sn-3position of the triglycerides in sheep milk fat (Pitas andJensen, 1970; Ha and Lindsay, 1993). Addis et al. (2005b)

also found higher levels of medium and long chain fattyacids (MCFAs and LCFAs) in sheep milk cream substratehydrolysed by a paste rennet obtained both from lambslaughtered after being fasted and from grazing lambs.The authors conclude that fasting or changes in the lambsdiet (grazing vs. milk-suckling) may depress PGL activ-ity and enhance the activity of other lipases (gastric ormicrobials). As know microbial lipases hydrolysed fromtriglycerides preferably medium and long chain fatty acidssuch 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 orpowdered calf rennet.

In the recent years there has been a great increasein production of rennet paste in specialised industrialplants. These plants collect the abomasa from larger slaugh-terhouses, where the lambs wait a long time on emptystomachs before being slaughtered. Moreover lambs,nowadays, are weaned earlier to anticipate delivery of milkto dairies. These changes induce an increase of the pepsincontent in the lamb’s stomach and a parallel decrease ofchymosin and pregastric lipases (Piredda and Addis, 1998;Addis et al., 2005a; Pirisi et al., 2007). Thus, often the indus-trial lamb paste rennet preparations contain more pepsinand less pregastric lipases than those artisanal ones. Asthe amount of rennet is usually regulated on the basis ofa fixed milk clotting time, the use of an industrial pasterennet could upset the optimal ratio of lipolytic to clottingactivity. For this reason, the use in cheese making of indus-trial lamb rennet paste could influence the characteristics ofPDO cheeses in unpredictable and undesirable way (Addiset al., 2005a; Pirisi et al., 2007).

Recently, in Sardinia, some industrial manufacturerdiversified their production selecting abomasa full of milk.This rennet is often destined to produce the PDO Fiore Sardocheese.

4. Traditional ewe milk cheeses produced withlamb rennet paste

The lamb rennet paste is used in certain Southern Euro-pean countries to produce sheep milk cheeses, such asIdiazabal and Roncal in Spain, Pecorino Romano, FioreSardo and Canestrato Pugliese in Italy, and Feta in Greece.

4.1. PDO Idiazabal cheese

Idiazabal cheese is a typical product of the BasqueCountry region of Northen Spain and is manufacturedfrom raw ovine milk according to the specifications of its

Denomination of Origin Regulatory Board (Ministerio deAgricoltura, Pesca y Alimentation, 1993). Traditionally, Idi-azabal cheese has been manufactured with artisanal lambrennet, although commercial calf rennets are nowadayswidely used by artisanal and industrial manufacturers.

esearch 79 (2008) 2–10

Nevertheless, cheeses made with artisanal lamb rennetshow specific sensory characteristics distinguishable fromthe cheeses made with commercial calf rennet. The regu-latory board does not specify the type of rennet to use ascoagulant for Idiazabal cheese making, although it is par-ticularly interested in promoting the use of artisanal lambrennet 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 firstSpanish cheese to be granted an Appellation of Origin in1981. It is an uncooked, pressed-curd cheese, with a mini-mum ripening time of 4 months. The regulations of the PDOpermit the addition of artisanal lamb or commercial calfrennet. Traditionally, Roncal cheese has been made usingartisanal 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 thatmust be made exclusively from whole sheep milk and lambrennet paste as coagulant. By the law it can be producedin Sardinia, Latium and Tuscany (Province of Grosseto).This cheese have a particular flavour, which has beenattributed to the use of lamb rennet paste. The specifi-cations for the Pecorino Romano production require thatthe stomachs used to produce the rennet have to comefrom animals raised in the PDO zone (Decreto Ministeriale,1995).

4.4. PDO Fiore Sardo cheese

Fiore Sardo PDO is a traditional Italian uncooked sheepmilk cheese, produced exclusively in Sardinia from rawmilk. The requirements of the cheese making protocol ofFiore Sardo cheese permit the use of traditional lamb orkid rennet paste. The sensory characteristics, and in partic-ular the piquant taste developed during ripening, are dueto the large lipolysis induced by the enzymes contained inthe rennet. Traditionally, and to a great extent up till now,the rennet used in the Fiore Sardo cheese making is directlyproduced at the farm by shepherds themselves, slaughter-ing lactating lambs a short time after suckling and removingthe abomasa full of milk. (Addis et al., 2005b; Scintu andPiredda, 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 flocks. Since1985 it has had the Appelation of Origin status, which

has been acknowledged by the European Community (C.D.1996). It is manufactured from winter to spring and its pro-duction protocol (DPR 1986) provides the use of animalrennet for milk coagulation, but do not specify the type(Santoro and Faccia, 1998).

minant R

4

(mtmmmmri2

5r

idTttps

yhpiafs(

ccmbibitr

VMpPIcw

rnhicnet

M. Addis et al. / Small Ru

.6. PDO Feta cheese

Feta cheese is a Greek PDO white pickled cheeseripened in tins filled with brine) made from ewe or a

ixture of ewe and goat milk. Nowadays it is manufac-ured, most of all, in modern dairies, from pasteurised

ilk with the use of mesophilic starter cultures and com-ercial calf or bovine rennet. Feta cheese is also stillanufactured in small dairies, traditionally from ther-ised milk, using yoghurt as starter culture, and artisanal

ennet from lambs’ and kids’ abomasa and it is ripenedn wooden barrels without brine addition (Georgala et al.,005).

. Proteolysis of cheeses produced using lambennet paste

Proteolysis is widely considered as the main biochem-cal and the most complex transformation taking placeuring cheese ripening (Fox, 1989; Farkye and Fox, 1990).he main proteolytic agents in cheese ripening process arehe natural proteases of milk (plasmin and cathepsin D),he rennet or clotting enzymes retained in the curd, theroteases and the peptidases produced by starter and non-tarter bacteria.

In the early stage of ripening, caseins are hydrol-sed, mainly by enzymes coming from rennet, yieldingigh-molecular-weight peptides. This process is know asrimary proteolysis (Grappin et al., 1985) and results in

mportant alterations in the texture of the cheeses (Creamernd Olson, 1982). Successively these polypeptides areurther degraded by the proteinase-peptidase system oftarter and non-starter bacteria to peptides and amino acidsFox, 1989).

The use of a rennet with different proportions ofhimosin and pepsin brings about different proteolytichanges in the cheese. As well-know, the proportion of chi-osin decreases as the animal age increases, accompanied

y a concomitant increase in the pepsin content. Pepsins a highly active proteolytic, non-specific enzyme that aseen held responsible for the production of bitter flavour

n cheese (Guinee and Wilkinson, 1992). A review of litera-ure has disclosed little works concerning the effect of lambennet paste on proteolysis process in ovine cheeses.

Santoro and Faccia (1998), Irigoyen et al. (2000, 2002),icente et al. (2001a,b), Bustamante et al. (2003) andoatsou et al. (2004) studied the effect of lamb rennet

aste or bovine rennet on the proteolysis in Canestratougliese, Roncal, Idiazabal and Feta cheeses, respectively.n general, there are no indications about the total milklotting activity added to the vats during milk coagulation,hich does not facilitate comparison among the results.

Overall authors observe an increase in the formationate of the water-soluble nitrogen (WSN) and non-proteinitrogen (NPN) fractions with the use of lamb rennet. Thisas been related both to the high retention of chymosin

n the curd when lamb rennet was used and to a greateromplexity of lamb rennet than bovine rennet. Lamb ren-et exhibite a greater proteolytic activity against �s-casein,specially �s1-casein than calf rennet. In consequence bothhe formation and the subsequent degradation of �s1-I

esearch 79 (2008) 2–10 7

casein took place more quickly in cheese made using lambrennet (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 inproteolysis are also qualitative, being the ratios betweenhydrophilic and hydrophobic compounds closely related tothe chymosin/pepsin ratio (Santoro and Faccia, 1998).

Although lamb rennet paste or their acqueous extractare not very well characterised, three proteases havebeen reported for this rennet, which can explicate thegreater proteolytic activity compared to that of calf rennet(Foltman, 1993).

Bustamante et al. (2003) studied the proteolytic processin Idiazabal cheeses made using a traditional lamb rennetpaste (chymosin/pepsine ratio, 80:20) and a commercialcalf rennet (chymosin/pepsine ratio, 90:10) in high andlow level. In disagreement with the previous cited works,they found that rennet type did not significantly affect thepercentages of any nitrogen fraction. This, if comparableamounts of total milk clotting activity are used in cheesemaking and chymosin to pepsin ratio is similar in rennetpreparations. They also observed that hydrolysis of �s1-casein was not significantly affected by the rennet type andthat �s1-I casein fraction was greater in cheese made withbovine 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 commercialcalf rennet on the characteristics of Feta cheese. Both ren-nets had a similar chymosin and pepsin ratio (70:30) andwere used in same quantities with reference to the totalmilk clotting activity. Although no significant differenceswere detected, the accumulation of WSN in cheese madewith artisanal rennet was faster, the same was also for thechanges in residual �s1-casein.

The shown results are frequently contradictory becausechymosin to pepsin ratio of the rennets and the differenttotal milk clotting activity added to the cheese milk werenot sometimes equals.

Studies comparing the effect of the use of lamb ren-net paste with different milk clotting activity and differentenzymatic composition on the proteolysis during cheeseripening, are scarce, probably due to the highly localised useof rennet paste (Irigoyen et al., 2001; Addis et al., 2005a,b;Pirisi et al., 2007).

Irigoyen et al. (2001) studied the effect of two lambpaste rennets, having different milk-clotting activity anddifferent levels of chymosin and pepsine enzymes (78:22and 54:46, respectively), on the proteolysis in Roncalcheeses. The authors found that cheeses made with therennet with the highest milk clotting activity and highestchymosin 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 rennetpaste preparations in Fiore Sardo cheese manufacture. Ren-

nets coming from lambs differently fed (milk only or milkand grass mixture) and slaughtered at full or empty stom-ach, differed in total milk clotting activity and in chymosinpepsin ratio (100:0, 98:2, 77:23, 76:24, respectively). Dur-ing cheese making, rennets were used with a standardised

minant R

8 M. Addis et al. / Small Ru

milk clotting activity. Authors did not find any difference inWSN and NPN fractions. These results were in agreementwith those found by Bustamante et al. (2003). The sameauthors (Addis et al., 2005a) studied the effect of two tradi-tional lamb rennets paste, prepared from lambs differentlyfed (milk only or milk and grass mixture) (88:12 and 82:18chymosin/pepsin ratio, respectively) compared with acommercial lamb rennet paste (64:36 chymosin/pepsinratio), on PDO Pecorino Romano cheese. According tothe previous work, they did not find differences in theproteolysis indices due to the used rennet type.

Pirisi et al. (2007) studied the influence of the use of twolamb rennets paste type on PDO Fiore Sardo cheese char-acteristics. Rennets were a traditional one, produced fromthe abomasa of lambs fed only milk and slaughtered aftersuckling (100:0 chymosyn/pepsin ratio) when another onewas pruduced at industrial level coming from stomachs oflambs weaned and slaughtered on a empty stomach (60:40chymosyn/pepsin ratio). Since the total coagulating activ-ity added to the milk in the manufacture of the cheeses wasthe same, the higher WSN development in cheese, causedby the industrial lamb rennet paste preparation, suggestan increased proteolytic activity due to the co-presenceof pepsine, which increases the general proteolytic activ-ity on casein. The cheeses made with industrial rennetunderwent a more rapid �s1-casein degradation, whichconfirms the higher efficiency exhibited by pepsin con-taining industrial rennet preparation in producing WSN.This also means that the amount of residual �s1-caseinwas higher in cheeses made with traditional lamb rennetpaste. These results are in disagreement with those foundby Irigoyen et al. (2001). No significant differences depend-ing on the rennet type were found for �-casein degradation.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 specific to the PDO Fiore Sardo cheese. InGrana Padano cheese, where calf liquid rennet is used ascoagulant, the only SerP residue subjected to dephospho-rylation is located at the N-terminus of phosphopeptides.In the PDO Fiore Sardo cheese, manufactured using rennetpaste, no apparent difference in susceptibility to dephos-phorylation was found amongst differently located SerPpeptide residues. This obviously depends on the nature andspecificity of phosphatase(s) prevalently active in the twocheese varieties. The different levels of the alkaline phos-phatase activity present in liquid or paste rennet could beaffect, in a different way, the mechanism of casein peptidesdephosphorylation (Pirisi et al., 2007).

6. Lipolysis of cheeses produced using lamb rennetpaste

Lipolysis is one of the major biochemical changes thatoccur during cheese ripening. The released of free fattyacids (FFAs) during lipolysis is considered to influence the

flavour of cheeses (Woo et al., 1984). This influence can bedirect, as in some Italian cheese varieties where short chainfatty acids themselves contribute directly to the taste andflavour (Woo and Lindsay, 1984; Addis et al., 2005a,b; Pirisiet al., 2007), or indirect considering FFAs as precursors for

esearch 79 (2008) 2–10

compounds such as methyl ketones, alkanes, lactones andesters (Urbach, 1991). Changes in FFAs composition dur-ing ripening have been suggested as index of ripening incow milk cheese, even if it is reported as less useful thanproteolytic and glycolytic indicators (Farkye and Fox, 1990).

The lipolytic agents presents in cheese are lipolyticenzymes coming naturally from milk (milk lipases), fromrennet (pregastric and gastric lipases) and from microfloraactivity (Collins et al., 2003).

The contribution of the rennet depends on the rennettype. Commercial calf and bovine rennets, the most com-monly used, are normally lipolytic activity free. On theother hand, traditional paste rennets, made from abomasaof lambs unweaned and slaughtered at full stomach, havehigh lipolytic activity due to their content of PGL (Pireddaand Addis, 1998; Bustamante et al., 2000). PGL prefer-

entially hydrolyse short chain fatty acids esterified in thesn-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 flavourfound in Italian cheeses like Pecorino Romano, Fiore Sardoand Provolone, have been attributed to their high butyricacid content and to the use of this rennet type (Barzaghi etal., 1997; Addis et al., 2005a,b).

Only few studies 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).PDO specifications of Idiazabal and Roncal cheeses allow

the use of bovine or lamb rennet as coagulant. Severalauthors (Larrayoz et al., 1999; Virto et al., 2003; Hernandezet al., 2005) reported that the concentration of total freefatty acids (TFFAs) in Idiazabal cheese manufactured withlamb paste rennet was significantly higher than in cheesesmanufactured with bovine rennet. They also observed thatlipolysis in the former cheeses increased with the total unitsof lipolytic activity added to the milk. The FFAs compositionof these cheeses was also dependent on the rennet type,with short-chain fatty acids (SCFAs) accounting for morethan 65% in cheese made with lamb paste rennet but only45% in cheese made with bovine rennet. Virto et al. (2003)suggest that, the increase in total lipolysis accompained byan increase in the level of SCFAs, as a percentage of TFFAs,greater than 50 or 55 �mol 100 �mol−1, can be used as areliable index to infer the presence of pregastric lipase inthe paste rennets. Among SCFAs, butyric acid was the mostabundant. Idiazabal cheese made with lamb paste rennetreceived significantly higher scores for odour and flavourintensity (Virto et al., 2003; Hernandez et al., 2005).

Addis et al. (2005b) used four different lamb paste ren-nets having different enzymatic characteristics to assesthe influence of paste rennet on lipolytic process and onquality of Fiore Sardo cheese. Results showed significantlydifferences in the levels of TFFAs, particularly of SCFAs,among rennets. In agreement with Virto et al. (2003) andHernandez et al. (2005) the authors concluded that, this

lipolytic pattern in cheeses was related to the high PGL con-tent in rennet coming from lambs fed only milk, being PGLsecretion stimulated by milk suckling. These authors alsofound that changes in the lamb diet, from milk to herbage,and the absence of food in the stomachs at slaughtering,

minant R

rphppBnaaatpStcsdlccmaacw

7l

stya(

amoti

fian

iRtrwha

sar

o

M. Addis et al. / Small Ru

educed the amount of PGL in the abomasa, and then in theaste rennet. The use of this rennet type gave cheeses withigher in MCFA and LCFA content. In the recent years theroduction of lamb paste rennet in specialised industriallants is increased (Addis et al., 2005a; Pirisi et al., 2007).oth lipolytic activity and chymosin content of this ren-et type, often was lower than traditional rennet. Addis etl. (2005a) and Pirisi et al. (2007), found both qualitativend quantitative differences for FFA in Pecorino Romanond in Fiore Sardo cheeses made with artisanal or indus-rial lamb paste rennet. Cheese made using traditional lambaste rennet had higher content of TFFAs due to the highCFAs level and, above all, to the butyric acid content. Onhe contrary, percentage of MCFA and LCFA were higher inheese made using industrial rennet. Georgala et al. (2005)tudied the evolution of lipolysis in traditional Feta cheeseuring the ripening, made with artisanal liquid rennet from

ambs’ and kids’ abomasa or a mixed artisanal rennet withalf rennet. The TFFA content in cheeses differed signifi-antly. The levels of individual FA were the highest in cheeseade with traditional lamb rennet. These results were in

greement with those of Moatsou et al. (2004) who foundhigher C4:0-C10:0 FFA content in ripened industrial Feta

heese made with artisanal rennet than in cheeses madeith calf rennet.

. Sensory characteristics of cheeses produced usingamb rennet paste

Flavour and texture of cheese are the outcome of aeries of chemical, biochemical, and microbiological eventshat occur during ripening. The FFAs released during lipol-sis contribute, together with the volatile compoundsnd the proteolysis products, directly to cheese flavourMcSweeney and Sousa, 2000).

The SCFAs play an important role in the sensory char-cteristics of the cheeses made with lamb paste rennet,ainly due to their high levels and low perception thresh-

lds (Larrayoz et al., 1999). Hernandez et al. (2005) foundhat the percentage of SCFAs is linearly correlated with thentensity score for the attribute “pungent flavour”.

Several authors agreed on affirm that cheeses manu-actured with lamb paste rennet had significantly higherntensity scores in “strong” attributes such as overall odournd flavour intensity, sharp odour, pungent flavour, ren-et odour and flavour 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 desirablen the case of certain cheeses such as Idiazabal, Pecorinoomano, Fiore Sardo, Feta cheeses, because they are part ofheirs characteristic flavour and odour. The same authorseported that the piquant taste observed in cheese madeith artisanal lamb rennet paste was associated with theigh SCFAs content and specifically with the high butyriccid content.

On the other hands intensity scores for milder attributes,

uch as milk odour, butter odour and flavour, nutty, toastednd sweet flavour were lower in cheeses made with lambennet paste (Virto et al., 2003).

As concern the proteolysis products, the caseinligopeptides could have an important role in the cheese

esearch 79 (2008) 2–10 9

flavour intensity. The prevalence of the hydrophobic pep-tides over hydrophilic ones could generate bitternessduring 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), showedthat hydrophilic peptides fraction prevails in cheeses madeusing lamb paste rennets with a high chymosin content.

8. Conclusions

In conclusion, the enzymatic composition of the rennetlamb paste markedly influence the biochemical pathwaysof proteins and fat degradation during cheese ripening.Since the enzymatic (lipolytic and proteolytic) composi-tion of rennet paste depends on the diet and slaughteringconditions of lambs, it may be necessary to standard-ise these conditions when establishing norms for rennetpaste production, especially for the production of PDOcheeses.

References

Addis, M., Piredda, G., Pes, M., Di Salvo, R., Scintu, M.F., Pirisi, A., 2005a.Effect of the use of three different lamb paste rennets on lipolysis ofthe PDO Pecorino Romano cheese. Int. Dairy J. 15, 563–569.

Addis, M., Pirisi, A., Di Salvo, R., Podda, F., Piredda, G., 2005b. The influenceof the enzymatic composition of lamb rennet paste on some propertiesof experimentally produced PDO Fiore Sardo cheese. Int. Dairy J. 15,1271–1278.

Andren, A., 1998. Milk-clotting activity of various rennets and coagulants:background and information regarding IDF standards. Bull. IDF 332,9–14.

Barzaghi, S., Rampilli, M., 1996. Evaluation of the enzymic characteristicsof commercial rennet pastes. Sci. Tec. Latt. Cas. 47, 171–181.

Barzaghi, S., Davoli, E., Rampilli, M., Contarini, G., 1997. Lipolysis inProvolone cheese: Effect of rennet-paste. Sci. Tec. Latt. Cas. 48,146–156.

Berridge, N.J., 1957. Some observation on the determination of the activityrennet. Analyst 77, 57–62.

Bustamante, M.A., Chavarri, F., Santisteban, A., Ceballos, G., Hernandez,I., Miguelez, M.J., Aramburu, I., Barron, L.J.R., Virto, M., De Renobales,M., 2000. Coagulating and lipolytic activities of artisanal lamb rennetpaste. J. Dairy Res. 67, 393–402.

Bustamante, M.A., Virto, M., Aramburu, M., Barron, L.J.R., Perez-Elortondo,F.J., Albisu, M., de Renobales, M., 2003. Lamb rennet paste in ovinecheese (Idiazabal) manufacture. Proteolysis and relationship betweenanalytical and sensory parameters. Int. Dairy J. 13, 547–557.

Collins, Y.F., McSweeney, P.L.H., Wilkinson, M.G., 2003. Lipolysis and freefatty acids catabolism in cheese: a review of current knowledge. Int.Dairy J. 13, 841–866.

Creamer, L.K., Olson, N.F., 1982. J. Food Sci. 47, 631.De Caro, J., Ferrato, F., Verger, R., De Caro, A., 1995. Purification and molec-

ular characterisation of lamb pregastric lipase. Biochim. Biophys. Acta1252, 321–329.

Farkye, N.Y., Fox, P.F., 1990. Objective indices of cheese ripening. TrendsFood Sci. Technol. 1, 37–42.

Foltman, B., 1993. General and molecular aspect of rennets. In: Fox, P.F.(Ed.), Cheese: Chemistry, Physics and Microbiology, vol. 1, 2nd ed.Chapman & Hall, London, pp. 37–68.

Fontecha, C., Pelaez, C., Juarez, M., 1994. Biochemical characteristicsof a semi-hard ewe’s-milk cheese. Lebbensm. Unters. Forsch. 198,24–28.

Food Chemical Codex, 1981. Lipase/Esterase Forestomac Activity, 13th ed.NRC, NAS, Washington, DC, pp. 493–494.

Fox, P.F., 1989. J. Dairy Sci. 72, 1379.Fox, P.F., Law, J., 1991. Enzymology of cheese ripening. Food Biotechnol. 5,

239–262.

Georgala, A., Moschopoulou, E., Aktypis, A., Massouras, T., Zoidou, E.,

Kandarakis, I., Anifantakis, E., 2005. Evolution of lipolysis during theripening of traditional Feta cheese. Food Chem. 93, 73–80.

Grappin, R., Rank, T.C., Olson, N.F., 1985. J. Dairy Sci. 68, 531.Guinee, T.P., Wilkinson, M.G., 1992. Rennet coagulation and coagulants in

cheese manufacture. J. Soc. Dairy Technol. 45, 94–104.

minant R

10 M. Addis et al. / Small Ru

Ha, J.K., Lindsay, R.C., 1993. Release of the volatile branched-chain andother fatty acids from ruminant milk fats by various lipases. J. DairySci. 76, 677–690.

Hernandez, I., de Renobales, M., Virto, M., Perez-Elortondo, F.J., Barron,L.J.R., Flanagan, C., Albisu, M., 2005. Assessment of industrial lipases forflavour development in commercial Idiazabal (ewe’s raw milk) cheese.Enzyme Microbial Technol. 36, 870–879.

IDF, 1997. Calf rennet & adult bovine rennet—determination of chymosin &bovine pepsin content (chromatographic method), International DairyFederation, Standard 110B, Brussels.

IDF, 2006. Milk and milk products – ovine and caprine rennets – determi-nation of total milk-clotting activity, International Dairy Federation,Standard 199/ISO 23058, Brussels.

Irigoyen, A., Izco, J.M., Ibanez, F.C., Torre, P., 2000. Evaluation of the effectof rennet type on casein proteolysis in an ovine milk cheese by meanscapillary electrophoresis. J. Chromatogr. A 881, 59–67.

Irigoyen, A., Izco, J.M., Ibanez, F.C., Torre, P., 2001. Influence of rennet milk-clotting activity on the proteolytic and sensory characteristics of anovine cheese. Food Chem. 72, 137–144.

Irigoyen, A., Izco, J.M., Ibanez, F.C., Torre, P., 2002. Influence of calf or lambrennet on the physicochemical, proteolytic, and sensory characteris-tics of an ewe’s-milk cheese. Int. Dairy J. 12, 27–34.

Larrayoz, P., Martinez, M.T., Barron, L.J.R., Torre, P., Barcina, Y., 1999. Theevolution of free fatty acids during the ripening of Idiazabal cheese:influence of rennet type. Eur. Food Res. Technol. 210, 9–12.

Manunta, G., Naitana, S., Spada, B., Calaresu, G., 1981. The pregastric lipasein lamb. Atti IV convegno SIPAOC, 191.

McSweeney, P.H.L., Sousa, M.J., 2000. Biochemical pathways for the pro-duction of flavour compounds in cheeses during ripening. A review.Le Lait 80, 293–324.

Moatsou, G., Moschopoulou, E., Georgala, A., Zoidou, E., Kandarakis, I.,Kaminarides, S., Anifantakis, E., 2004. Effect of artisanal liquid rennetfrom kids and lambs abomasa on the characteristics of Feta cheese.Food Chem. 88, 517–525.

Nelson, J.H., Jensen, R.G., Pitas, R.E., 1977. Pregastric esterase and other orallipase—a review. J. Dairy Sci. 60, 327–362.

O’Connor, C.J., Manuel, R.D., Turner, K.W., 1993. Calf and lamb linguallipases as catalyst for the hydrolysis of tributyrin, triolein and 4-nitrophenylacetate. J. Dairy Sci. 76, 3674–3682.

Papoff, C.M., Mauriello, R., Pirisi, A., Piredda, G., Addis, M., Chianese, L.,

2004. Proteolytic activity of animal rennet on ovine casein. Milchwis-senschaft 59, 414–417.

Pettinau, M., Nuvoli, G., Podda, F., Ledda, A., 1977. Influenza del metododi preparazione del caglio di agnello sulla conservazione delle sueproprietà coagulanti, proteolitiche e lipolitiche. Sci. Tec. Latt. Cas. 28,101–114.

esearch 79 (2008) 2–10

Piredda, G., Addis, M., 1998. Effect of diet on enzyme activity in lamb rennetpaste. Sci. Tec. Latt. Cas. 49, 129–138.

Pirisi, A., Pinna, G., Addis, M., Piredda, G., Mauriello, R., De Pascale, S., Caira,S., Mamone, G., Ferranti, P., Addeo, F., Chianese, L., 2007. Relationshipbetween enzymatic composition of lamb rennet paste and proteolytic,lipolytic pattern and texture of PDO Fiore Sardo ovine cheese. Int. DairyJ. 17, 143–156.

Pitas, R.E., Jensen, J.H., 1970. Action of pregastric esterase on synthetictriglycerides containing butyric acid. J. Dairy Sci. 53, 1083.

Rampilli, M., Barzaghi, S., 1995. Rennet paste: analytical aspects. Il Latte20, 517–521.

Richardson, G.H., Nelson, J.H., Farnham, M.G., 1971. Gastric lipase char-acterisation and utilisation in cheese manufacture. J. Dairy Sci. 54,643–647.

Santillo, A., D’Angelo, F., Caroprese, R., Marino, R., Albenzio, M., 2005. Influ-ence of diet and of lamb slaughtering age on the coagulating propertiesof rennet paste. Ital. J. Anim. Sci. 2 (Suppl.), 336–338.

Santoro, M., Faccia, M., 1998. Influence of mould size and rennet on prote-olysis and composition of Canestrato Pugliese cheese. Ital. J. Food Sci.10, 217–228.

Scintu, M.F., Piredda, G., 2007. Typicity and biodiversity of goat and sheepmilk products. Small Rumin. Res. 68, 221–231.

Soxhlet, F., 1877. Die Darstellung haltbarer Labflüssigkeiten. Milch-Ztg. 6,497–501.

Sponcet, M., Sifflet, P., Michalet, A., 1985. Current development and under-standing of rennet use in the dairy industry. Dairy Ind. Int. 50,27–30.

Trujillo, A., Guamis, B., Laencina, J., Lopez, M.B., 2000. Proteolytic activ-ity of some milk clotting enzymes on ovine casein. Food Chem. 71,449–457.

Urbach, G., 1991. Butter flavour in food systems. Food Res. 51, 50–54.Vicente, M.S., Ibanez, F.C., Barcina, Y., Barron, L.J.R., 2001a. Changes in the

free amino acid content during ripening of Idiazabal cheese: influenceof starter and rennet type. Food Chem. 72, 309–317.

Vicente, M.S., Ibanez, F.C., Barcina, Y., Barron, L.J.R., 2001b. Casein break-down during ripening of Idiazabal cheese: influence of starter andrennet type. J. Sci. Food Agric. 81, 210–215.

Virto, M., Chavarri, F., Bustamante, M.A., Barron, L.J.R., Aramburu, M.,Vicente, M.S., Perez-Elortondo, F.J., Albisu, M., de Renobales, M., 2003.Lamb rennet paste in ovine cheese manufacture. Lipolyisis and flavour.

Int. Dairy J. 13, 391–399.

Woo, A.H., Kollodge, S., Lindsay, R.C., 1984. Quantification of major freefatty acids in several cheese varieties. J. Dairy Sci. 67, 874–878.

Woo, A.H., Lindsay, R.C., 1984. Concentration of major free fatty acidsand flavour development in Italian cheese varieties. J. Dairy Sci. 67,960–968.