Advances Study Proteolysis

International Dairy Journal 11 (2001) 327345

Advances in the study of proteolysis during cheese ripening

M.J. Sousaa, Y. Ard .ob, P.L.H. McSweeneya,*aDepartment of Food Science, Food Technology and Nutrition, University College, Cork, Ireland

bDairy Technology, Department of Dairy and Food Science, The Royal Veterinary and Agriculutral University, Rolighedsvej 30, 5, DK-1958

Frederiksberg C, Denmark

Abstract

Cheese ripening involves a complex series of biochemical, and probably some chemical events, that leads to the characteristic

taste, aroma and texture of each cheese variety. The most complex of these biochemical events, proteolysis, is caused by agents froma number of sources: residual coagulant (usually chymosin), indigenous milk enzymes, starter, adventitious non-starter microfloraand, in many varieties, enzymes from secondary flora (e.g., from Penicillium sp. in mould-ripened cheeses or Propionibacterium sp. in

Swiss cheese). Proteolysis in cheese has been the subject of active research in the last decade; there have been developments in theanalytical techniques used to monitor proteolysis and patterns of proteolysis in many cheese varieties have now been investigated.This review focuses on certain aspects of proteolysis, including proteolytic agents in cheese and specificity of some ripening enzymes,comparison of proteolysis and contribution of proteolysis to cheese flavour. r 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Cheese proteolysis and flavour; Cynara cardunculus; Cathepsin D; Cheese peptides

1. Introduction

Proteolysis in cheese during ripening plays a vital rolein the development of texture as well as flavour and hasbeen the subject of several reviews (e.g., Fox, Singh, &McSweeney, 1995b; Fox & McSweeney, 1996). Proteo-lysis contributes to textural changes of the cheesematrix, due to breakdown of the protein network,decrease in aw through water binding by liberatedcarboxyl and amino groups and increase in pH (inparticular in surface mould-ripened varieties), whichfacilitates the release of sapid compounds duringmastication. It contributes directly to flavour and tooff-flavour (e.g., bitterness) of cheese through theformation of peptides and free amino acids as well asliberation of substrates (amino acids) for secondarycatabolic changes, i.e., transamination, deamination,decarboxylation, desulphuration, catabolism of aro-matic amino acids and reactions of amino acids withother compounds.

Because proteolysis is one of the principal biochem-ical events during the ripening of cheese, it is desirable toinclude a general assay for proteolysis, e.g., pH 4.6soluble N or water soluble N as % of total N (pH 4.6

SN/TN or WSN/TN) or liberation of reactive groups, inmost ripening studies (Fig. 1). The rate and pattern ofproteolysis may be influenced by location within thecheese (e.g., surface-ripened, smear-ripened or youngbrined-salted cheeses) and a suitable sampling schemeshould consider this. Cheese variety and its character-istics (e.g. pH) should be considered when choosingmethodology. For example, since the extractability of Ncompounds varies with pH, WSN can be much higher incheeses with higher pH, so fractionation using buffers atpH 4.6 is more suitable than using water for varietiesthat are characterised by a change in pH duringripening. If the objectives of the study encompassinvestigation of the effect of one of the agents ofproteolysis in cheese, i.e., action of plasmin, differenttypes of coagulant or the effect of different starter strainsor adjunct cultures in cheese, the methodology should bechosen so as to emphasise the level of proteolysis causedby that agent. For example, comparison of the effect ofdifferent coagulants on primary proteolysis could befollowed by urea-polyacrylamide gel electrophoresis(urea-PAGE) or capillary electrophoresis (CE) of thepH 4.6-insoluble fraction (or cheese), followed byelectroblotting, sequencing and identification of theproducts of primary proteolysis. Peptide profiles of thepH 4.6-soluble fraction (or ethanol-insoluble and-soluble fractions therefrom) should be determined by

*Corresponding author.

E-mail address: [email protected] (P.L.H. McSweeney).

0958-6946/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 0 6 2 - 0

reverse phase-high performance liquid chromatography(RP-HPLC). Chemometrical analysis of the profilesobtained by urea-PAGE, CE and RP-HPLC could beperformed. As another example, analysis of the effect ofstarter or adjunct cultures should comprise RP-HPLCof the ethanol-insoluble and -soluble fractions of the pH4.6 soluble fractions of the cheese, analysis of individualamino acids, followed by multivariate analysis (e.g.,principal component analysis, PCA) of the peptideprofiles and free amino acids.

The choice of a fractionation scheme for cheese N or asuitable technique to use for assessing proteolysis incheese depends on a number of factors including (i)availability of equipment and resources, (ii) cheesevariety and (iii) objective of the study and consequentlydifferent strategies have been proposed for the fractio-nation of cheese nitrogen (Christensen, Bech, & Werner,1991; Singh, Fox, Hjrup, & Healy, 1994; Fox,McSweeney, & Singh, 1995a; McSweeney & Fox,1997; Grappin, Beuvier, Bouton, & Pochet, 1999;Ard .o, 1999a, b). Several nitrogen indices have beenproposed to study proteolysis during cheese ripening,however, proteolysis is far too complex to be describedadequately by a single index. The methodology forassessment of proteolysis has recently been reviewedextensively and will not be considered further here (Foxet al., 1995a; McSweeney & Fox, 1997; Wallace & Fox,1998; Ard .o, 1999b; B .utikofer & Ard .o, 1999; Otte, Ard .o,Weimer, & Srensen, 1999; Singh, Gripon, & Fox,1999).

Proteolysis in cheese during ripening has been anactive area for research in recent years and the literatureon the topic has increased substantially in the lastdecade. The objective of this review is to consider certain

specific aspects of proteolysis in cheese during ripening:(i) proteolytic agents in cheese and their specificity withdetailed descriptions only on new findings focused onthe plant coagulant from Cynara cardunculus and themilk protease cathepsin D; (ii) comparison of proteo-lysis within and between cheese varieties; (iii) identifica-tion of peptides in cheese and (iv) contribution ofproteolysis to cheese flavour.

2. Proteolytic agents in cheese

During ripening, proteolysis in cheese is catalysed byenzymes from (i) coagulant (e.g., chymosin, pepsin,microbial or plant acid proteinases), (ii) milk (plasminand perhaps cathepsin D and other somatic cellproteinases), (iii) enzymes from the starter, (iv) non-starter, or (v) secondary cultures (e.g., P. camemberti,P. roqueforti, Propionibacterium sp., B. linens and othercoryneforms) and (vi) exogenous proteinases or pepti-dases, or both, used to accelerate ripening (Fig. 2).

Fig. 1. Summary of some methods used to assess proteolysis in cheese during ripening. Analytical techniques are highlighted in bold.

Fig. 2. Proteolytic agents in cheese during ripening.

M.J. Sousa et al. / International Dairy Journal 11 (2001) 327345328

In many cheese varieties, the initial hydrolysis ofcaseins is caused by the coagulant and to a lesser extentby plasmin, which results in the formation of large(water-insoluble) and intermediate-sized (water-soluble)peptides which are degraded subsequently by thecoagulant and enzymes from the starter and non-startermicroflora of the cheese. The extracellular, cell envelope-associated proteinase of Lactococccus (lactocepin, PrtP)contributes to the formation of small peptides in cheeseprobably by hydrolysing the larger peptides producedfrom as1-casein by chymosin or from b-casein byplasmin, whereas the peptidases (which are intracellular)are released after the cells have lysed and are responsiblefor the degradation of short peptides and the productionof free amino acids. The final products of proteolysis arefree amino acids and their concentration in cheese at anystage of ripening is the net result of the liberation ofamino acids from casein, their degradation to catabolicproducts and perhaps some synthesis by the cheesemicroflora. This general outline of proteolysis can varysubstantially between variety (see Section 3); e.g.,coagulant is extensively or completely denatured bythe high cooking temperature used in the manufactureof Parmigiano-Reggiano and Swiss cheeses and thus thecontribution of plasmin to the initial hydrolysis ofcaseins is more pronounced than in Cheddar and Dutchvarieties.

2.1. Coagulant

Majority of cheeses produced around the world weremanufactured traditionally, and in many cases still aremanufactured, using an enzymatic coagulant extractedfrom the abomasa of milk-fed calves. This extract,known as calf rennet, consists of two proteolyticenzymes: chymosin (EC 3.4.23.4), the major component(8894% milk clotting activity, MCA) and bovinepepsin (EC 3.4.23.1; 612% MCA). The relativeproportion of these enzymes varies with individualityand age of calves, the method of rennet separation andthe conditions and pH values at which the milk clottingactivity is measured (Guinee & Wilkinson, 1992). Theprincipal role of chymosin in cheesemaking is tocoagulate milk by specifically hydrolysing the Phe105-Met106 bond of the micelle-stabilising protein, k-casein,which is many times more susceptible to chymosin thanany other bond in milk proteins and leads to thecoagulation of the milk (see Fox, Guinee, Cogan, &McSweeney, 2000).

Most of the coagulant activity added to the milk islost in the whey; only 015% of the rennet activityadded to the milk remains in the curd after manufacture,depending on factors including type of coagulant, ratioof different enzymes in blends, cooking temperature, thecheese variety and the moisture level of the final cheese(Guinee & Wilkinson, 1992). Pepsins are more sensitive

to denaturation by pH than chymosin and hence theamount of activity of these coagulants retained in thecurd is very strongly dependent on the pH of the milk atsetting and shortly thereafter (Fox &McSweeney, 1996);in fact, increasing the pH of the curds-whey mixture toaround 7 after milk coagulation using porcine pepsin isone of the methods used to produce rennet-free cheesecurd (e.g., Lane, Fox, Johnston, & McSweeney, 1997).The heat stability of rennet at the temperature usedduring cooking of curds and whey also has a large effecton the level of rennet activity remaining in the curd; inhigh cook cheeses (e.g., Emmental), chymosin isdenatured extensively and makes relatively little con-tribution to ripening (Boudjellab, Rolet-Repecaud, &Collins, 1994), while Gouda, which has a similar initialpH but which is cooked only to B371C, containsconsiderable rennet activity.

In the latter part of the last century, cheeseconsumption increased while the availability of calfrennet decreased, which led to rennet shortages andsubsequent price increases. In addition, more restrictiveethical concerns associated with production of suchanimal rennets led to a search for suitable rennetsubstitutes for cheese making. Several proteases fromanimal, microbial and plant sources were investigated aslikely substitutes and have been reviewed by Guinee andWilkinson (1992), Broome and Limsowtin (1998) andFox et al. (2000).

2.1.1. Substitutes for calf rennetThe most common rennet substitutes include bovine,

porcine and to a lesser extent, chicken pepsins andmicrobial proteases from Rhizomucor miehei, R. pusillusand Cryphonectria parasitica (see Fox & McSweeney,1997; Fox et al., 2000). The proteolytic activities ofchymosin and porcine pepsin were compared on buffalo,cow and goat whole casein by Awad, L .uthi, and Puhan(1998) and it was reported that both enzymes attackedas1- and b-caseins in the same region as calf rennet.Trujillo, Guamis, Laencina, and L !opez (2000) comparedsome milk clotting enzymes (calf and lamb rennets,bovine chymosin and pepsin, and proteases fromR. mihei and C. parasitica) on ovine casein and reportedthat lamb rennet and C. parasitica protease showed thelowest and the highest degree of proteolysis, respec-tively. These authors reported that all enzymes hydro-lysed ovine casein resulting in the formation of as1-I andb-I-caseins (the first breakdown products produced bychymosin) as initial breakdown products of as1- andb-caseins, respectively, but C. parasitica also produced aseries of degradation products with lower electrophore-tic mobilities than b-casein. C. parasitica proteinasecleaves k-casein at Ser104-Phe105 rather than Phe105-Met106, which is cleaved by chymosin and R. mieheiproteinase (Drohse & Foltmann, 1989). Porcine pepsintends to be more heat-sensitive, followed by

M.J. Sousa et al. / International Dairy Journal 11 (2001) 327345 329

C. parasitica, bovine pepsin, chymosin, R. pusillusprotease and R. miehei protease in order of increasingheat stability (Broome & Limsowtin, 1998), althoughthe heat stability of the microbial coagulants can bereduced after treatment with various chemical agents(Garg & Johri, 1994). The use of coagulants thatare more heat stable than calf rennet should beavoided; otherwise excess proteolytic activity mayremain in the curd where it may result in excessiveproteolysis and bitterness unless ripening times and/orcooking temperatures are changed to compensate forthe more rapid rate of proteolysis (Guinee & Wilkinson,1992).

In the last decade, genetically engineered microorgan-isms have been exploited increasingly for the productionof commercial coagulants. The gene for chymosin hasbeen cloned and inserted into microorganisms such asKluyveromyces marxianus var. lactis, Aspergillus nigervar. awamori or Escherichia coli which led to thedevelopment of recombinant chymosins which are nowmarketed commercially as Maxiren (DSM FoodSpecialities, Netherlands) and Chymax (Chr. Hansen,Denmark). Recombinant chymosins have beenapproved for commercial use in foods in many, butnot all countries, and they have been used in ever-increasing quantities in USA and Western Europeand now represent about 35% of the total market(Fox et al., 2000).

Plant proteases have also been investigated as milkcoagulants, but only a small number of asparticproteinases from plant origin have been isolated andpartially characterised (Tavaria et al., 1997; Sousa,1998). A unique feature shared by most of these plantproteinases is an extra segment of about 100 amino acidresidues which bears no sequence similarity withproteinases of mammalian or microbial origins (Faro,Ver!ssimo, Lin, Tang, & Pires, 1995). Many aspartic andother proteinases are obtained from plants and some ofthem have been studied as coagulants, i.e., proteinasesfrom Benincasa cerifera (Gupta & Eskin, 1977),Calotropis procera (Ibiama & Griffiths, 1987; Mohamed& OConnor, 1999), Dieffenbachia maculata (Padma-nabhan, Chitre, & Shastri, 1993), fruit parts of Solanumdobium (Yousif, McMahon, & Shammet, 1996), Cen-taurea calcitrapa (Tavaria et al., 1997) and flowers ofCynara cardunculus (Barbosa, 1983; Sousa, 1993; Sousa& Malcata, 1997a, b, 1998a, b, Sousa, 1998). Althoughmost plant coagulant preparations were reported tohave an excessively low ratio of milk clotting toproteolytic activity, which results in bitter peptides inripened cheese, or to an excessively low clotting powerthat gives rise to low cheese yields. The difficultiesexperienced with these preparations result mainly fromthe unique composition of the plant extracts, whichcontain a complex cocktail of enzymes whose activity isdifficult to control.

2.1.2. Cynara cardunculus coagulantAn exception from the other plant proteases is the

proteases from dried flowers of C. cardunculus, whichhave milk-clotting activity and have been employedsuccessfully for many centuries in the Iberian Peninsulafor the manufacture of traditional cheeses, e.g., Serra daEstrela (Roseiro, 1991; Macedo, Malcata, & Oliveira,1993a), La Serena (Nu *nez, Fern!andez del Pozo,Rodriguez-Marin, Gaya, & Medina, 1991; Roa,L !opez, & Mendiola, 1999), Gu!a (Fern!andez-Salguero,Sanju!an, & Montero, 1991) and Los Pedroches (Car-mona, Sanjuan, Gomez, & Fern!andez-Salguero, 1999;Fern!andez-Salguero & Sanju!an, 1999; Vioque et al.,2000). In the last years, the specificity of proteinasesfrom C. cardunculus were studied in solutions of bovine(Faro, Moir, & Pires, 1992; Sousa, 1993; Macedo, Faro,& Pires, 1996) ovine and caprine caseins (Sousa &Malcata, 1998b), as well as primary proteolysis incheeses manufactured from ovine milk and from ovineor caprine milk (Sousa & Malcata, 1997a, b; 1998a;Sousa, 1998). Extracts from C. cardunculus werereported to contain two proteinases, cardosin A andcardosin B (Sousa, 1993; Ver!ssimo, Esteves, Faro, &Pires, 1995; Ver!ssimo et al., 1996). Studies on theirspecificity and kinetics on the oxidised B-chain of insulinshowed that cardosin A has a cleavage specificity similarto chymosin, whereas cardosin B resembles pepsin (Faroet al., 1992; Ver!ssimo et al., 1995; Ramalho-Santos,Ver!ssimo, Faro, & Pires, 1996). The isolation andcharacterisation of cDNA from flowers of C. carduncu-lus was reported and, more recently, cloning andcharacterisation of cDNA enconding cardosin A hasalso been reported (Cordeiro, Xue, Pietrzak, Pais, &Brodelius, 1994; Faro et al., 1999).

Enzymes from extracts of dried flowers ofC. cardunculus cleave the Phe105-Met106 bond of k-casein (Faro et al., 1992; Macedo, Faro, & Pires, 1993b;Sousa & Malcata, 1998b). The primary site cleaved byproteinases from C. cardunculus in bovine as1-casein isPhe23-Phe24 (Sousa, 1993). Macedo et al. (1996)reported that proteinases from C. cardunculus were ableto cleave nine bonds, viz., Phe23-Phe24, Tyr153-Tyr154,Trp164-Tyr165, Tyr165-Tyr166, Tyr166-Val167, Phe145-Tyr146, Leu149-Phe150, Leu156-Asp157 and Ala163-Trp164,whereas chymosin could only cleave the Phe23-Phe24bond of as1-casein under the same experimental condi-tions. Under several ionic conditions (pH 6.5, 5.5 in theabsence of NaCl and at pH 5.2 with 5% NaCl), themajor cleavage sites of proteinases from C. cardunculuswere Phe23-Val24 for ovine and Phe23-Val24, Trp164-Tyr165 and Tyr173-Thr174, caprine as1-casein (Sousa &Malcata, 1998b). as2-Caseins are cleaved by proteinasesfrom C. cardunculus at the bonds Phe88-Tyr89, and Ser9-Ser10, Phe88-Tyr89 and Tyr179-Leu180 in ovine andcaprine caseins, respectively (Sousa & Malcata, 1998b).Proteinases from C. carduculus cleave six bonds in


bovine b-casein and the relative susceptibility to attackis, in decreasing order, Leu192-Tyr193, Leu191-Leu192,Leu165-Ser166, Phe190-Leu191, Ala189-Phe190 and Leu127-Thr128 (Macedo et al., 1996), whereas under the sameexperimental conditions, Carles and Ribadeau-Dumas(1984) found that only the bonds Ala189-Phe190 andLeu192-Tyr193 were cleaved by chymosin. The majorcleavage sites in ovine b-casein of proteinases fromC. cardunculus at pH 6.5 or 5.5 in the absence of NaCland at pH 5.2 with 5% NaCl were Leu127-Thr128 andLeu190-Tyr191 and in caprine b-casein were Glu100-Thr101, Leu127-Thr128, Leu136-Pro137 and Leu190-Tyr191(Sousa & Malcata, 1998b). It is worth noting theproteinases of C. cardunculus cleave all bonds in certainextremely hydrophobic regions of as1-casein (Ala163-Trp-Tyr-Tyr-Val167) and b-casein (Ala189-Phe-Leu-Leu-Tyr193), whereas chymosin cleaves only Trp164-Tyr165 inthis region of as1-casein and Ala189-Phe190 and Leu192-Tyr193 in this region of b-casein. It was suggested thatproteinases from C. cardunculus displayed a strongerpreference for bonds between bulky hydrophobicresidues than does chymosin (Macedo et al., 1996).Hydrolysis of bovine b-casein by chymosin (Fox &Walley, 1971) and by proteinases from C. cardunculus(Sousa, 1993) is strongly inhibited by 5% NaCl andcompletely inhibited by 10% NaCl, but the effect isdue to modification of the substrate rather than theenzyme. Kelly, Fox, and McSweeney (1996) andKristiansen, Deding, Jensen, Ard .o, and Qvist (1999)reported that the degradation of b-casein by thecoagulant in cheese was affected by the salt content, asunsalted cheese contained less intact b-casein than thesalted cheese and that the C-terminal fragment ofb-casein, b-CN(f193209), which is known to be bitterand produced by chymosin, was only formed in unsaltedcheese.

2.2. Indigenous milk proteinases

2.2.1. PlasminThe dominant indigenous milk proteinase, plasmin

(fibrinolysin, EC 3.4.21.7), has been the subject of muchstudy and throughly described in recent reviews (seeBastian & Brown, 1996; Kelly & McSweeney, 2001 forreviews). Plasmin activity differs substantially betweencheese varieties; Richardson and Pearce (1981) reportedthat Swiss and Cheddar contained 613 and 34.5 mgplasmin/g cheese, respectively and that the elevatedplasmin activity in high cook cheese varieties (e.g.,Swiss) has been attributed to thermal inactivation ofinhibitors of plasminogen activators, resulting in theincreased conversion of plasminogen, the inactiveprecursor of plasmin, to the active enzyme. The primarycleavage sites of plasmin on b-casein are Lys28-Lys29,Lys105-His106 and Lys107-Glu108 the cleavage of whichyields b-CN(f29209) (g1-CN), b-CN(f106209) (g2-CN)

and b-CN(f108209) (g3-CN) (Eigel et al., 1984). Theproteose peptone (PP) components 5 (b-CN(f1105) andb-CN(f1107)), PP-8 fast b-CN(f128) and PP-8 slow(b-CN(f29105) and b-CN(f29107)) are the corre-sponding N-terminal fragments which accumulate inmilk on storage. Plasmin cleaves as2-casein in solution ateight sites. Although k-casein contains several Lys andArg residues, it appears to be quite resistant to plasminaction.

2.2.2. Cathepsin DMilk contains an indigenous acid proteinase, first

recognised by Kaminogawa and Yamauchi (1972) ascathepsin D, and later identified as procathepsin D(procathepsin is the proenzyme form of the lysosomalproteinase, cathepsin D; Larsen, Benfeldt, Rasmussen,& Petersen, 1996). The literature on cathepsin D hasbeen reviewed recently by Hurley, Larsen, Kelly, andMcSweeney (2000a) and Kelly and McSweeney (2001).Cathepsin D (EC 3.4.23.5) is an aspartic proteinase withan optimum pH of 4.0 on haemoglobin and optimumtemperature of 371C (Kaminogawa & Yamauchi, 1972;Barrett, 1972).

Cathepsin D produces the glycomacropeptide,k-CN(f106169), that also is produced by chymosin bythe enzymatic cleavage of the Phe105-Met106 bond(McSweeney, Fox, & Olson, 1995), and two morecleavage sites of cathepsin D on k-casein have beenidentified, i.e., Leu32-Ser33 and Leu79-Ser80 (Larsen et al.,1996). As the specificity of cathepsin D is similar to thatof chymosin, one might expect that it possesses theability to coagulate milk. The milk clotting potential ofcathepsin D, however, has been reported to be very poor(McSweeney et al., 1995; Larsen et al., 1996). Larsenet al. (1996) reported that the enzyme was capable ofcoagulating milk over the pH values examined (pH 5.06.5), with coagulation time decreasing as expected withdecreasing pH. The level of cathepsin D present (around0.4 mg/mL) in milk though, is far too low to be ofsignificance with respect to milk coagulation.

Cathepsin D and chymosin had similar cleavage siteson as1-casein, i.e., Phe23-Phe24, Phe24-Val25, Leu98-Leu99and Leu149-Phe150 (Larsen et al., 1996), cathepsin Dhydrolysates of as2-casein differ markedly from thoseproduced by chymosin (McSweeney et al., 1995). Theenzyme cleaves as2-casein at Leu99-Tyr100, Leu123-Asn124, Leu180-Lys181 and Thr182-Val183 (Larsen et al.,1996). Proteolysis of b-casein by cathepsin D is similarto that by chymosin, with b-CN(f1192) being theprimary product and b-CN(f1163/165/167) also beingformed. In total, 13 sites have been identified in b-caseinthat are cleaved by cathepsin D, viz., Phe52-Ala53, Leu58-Val59, Pro81-Val82, Ser96-Lys97, Leu125-Thr126, Leu127-Thr128, Trp143-Met144, Phe157-Pro158, Ser161-Val162,Leu165-Ser166, Leu191-Leu192, Leu192-Tyr193 and Phe205-Pro206 (Larsen et al., 1996).


Cathepsin D was considered to be a relatively heatlabile enzyme and Kaminogawa and Yamauchi (1972)reported its complete inactivatation at 701C 10min.Recent studies on heat stability reported that B8%cathepsin D activity in skim milk survived pasteurisa-tion (721C 15 s) (Larsen et al., 2000; Hayes et al., 2001)suggesting that this enzyme may play a minor proteo-lytic role in dairy products prepared from pasteurisedmilk. In rennet free-cheeses, formation of as1-CN(f24199) has been attributed to the activity of cathepsin D(Visser & de Groot-Mostert, 1977). Wium, Kristiansen,and Qvist (1998) reported cathepsin D-like activity inFeta cheese made without rennet addition, from milkthat was pasteurised (721C 15 s), ultrafiltered (501C),pasteurised again, stored at 41C overnight and pas-teurised again and homogenised, confirming that somecathepsin D activity can survive pasteurisation. Thepresence of procathepsin D in the ripened UF-Feta wasconfirmed using immunological methods (Larsen et al.,2000). Hurley, Larsen, Kelly, and McSweeney (2000b)reported the presence of cathepsin D or procathepsin Din Quarg cheese samples produced from raw, pas-teurised or raw ultrafiltered skim (except those to whichpepstain had been added) and peptides thought to beproduced as result of cathepsin D were observed incheese made from raw and pasteurised milk. CathepsinD activity is obvious in cheese varieties where no rennetwas added, but it is hard to quantify the contribution ofcathepsin D to the ripening of cheese varieties such asCheddar, wherein the activity would be masked by fargreater levels of chymosin (Hurley, 1999; Hayes et al.,2001). In Swiss cheese, chymosin is believed to besubstantially inactivated by the high cooking regime(temperatures 53551C for up to 1 h) and the absence ofrennet-like activity with little or no degradation of as1-casein to as1-I-casein might be expected; however, thepresence of as1-I-casein has been reported in Swisscheese (Beuvier et al., 1997; McGoldrick & Fox, 1999)which may be, at least partly, a result of cathepsin Dactivity.

2.2.3. Other milk proteasesIn addition to cathepsin D, other proteolytic enzymes

are present in lysosomes of somatic cells and maycontribute to proteolysis in cheese. One of the principalenzymes found in polymorphonuclear granulocytes(PMN cells or neutrophils) is the serine proteinase,elastase (Verdi & Barbano, 1991). Elastase has a broadspecificity on b- and as1-caseins, cleaving 19 and 25 sites,respectively (Considine, Healy, Kelly, & McSweeney,1999, 2000). Elastase hydrolyses b-casein and some ofthe cleavage sites are identical to or near those cleavedby plasmin, chymosin or cell envelope-associatedproteinases of several strains of Lactococcus. Most ofthe cleavage sites were found to be located near theN- or C-termini of the molecule, viz., Ile26-Asn27, Gln40-

Thr41, Ile49-His50, Phe52-Ala53, Gln56-Ser57, Leu58-Val59,Asn68-Ser69, Val82-Val83, Val95-Ser96, Sesr96-Lys97, Lys97-Val98, Ala101-Met102, Glu108-Met109, Phe119-Thr120,Glu131-Asn132, Leu163-Ser164, Ala189-Phe190, Phe190-Leu191 and Pro204-Phe205 (Considine et al., 1999).Therefore, it is possible that indigenous elastase in milkmay be of significance to the proteolysis of milkproteins. Recently, we have identified immunoreactiveprocathepsin B in milk (Magboul, Larsen, McSweeney,& Kelly, 2001). Cathepsin B is capable of extensivelydegrading as1- and b-caseins in vitro and its specificity isknown (Considine, 2000). Indigenous proteolytic en-zymes and their role in cheese ripening is discussed inmore detail in Kelly and McSweeney (2001).

2.3. Starter proteinases and peptidases from Lactococcusand Lactobacillus

The starter cultures commonly used in cheesemanufacture include mesophilic Lactococcus and Leu-conostoc species, thermophilic Lactobacillus species andStreptococcus thermophilus. The principal role of thestarter culture is in the production of lactic acid, causinga decrease in pH. Although lactic acid bacteria (LAB)are weakly proteolytic, they possess a very comprehen-sive proteinase/peptidase system (Fig. 3) capable ofhydrolysing oligopeptides to small peptides and aminoacids and this subject has been studied extensively andreviewed recently (e.g., Fox & McSweeney, 1996; Kunji,Mierau, Hagting, Poolman, & Konings, 1996; Law &Haandrikman, 1997; Christensen, Dudley, Pederson, &Steele, 1999) and will only be described briefly in thisreview.

LAB possess a cell envelope-associated proteinase(CEP, lactocepin, PrtP), intracellular oligoendopepti-dases (PepO) and (PepF), at least three generalaminopeptidases (PepN, PepC, PepG), glutamyl amino-peptidase (PepA), pyrolidone carboxylyl peptidase(PCP), leucyl aminopeptidase (PepL), X-prolyldipepti-

Fig. 3. Schematic representation of the proteolytic system of lacto-

coccus.


dyl aminopeptidase (PepX), proline iminopeptidase(PepI), aminopeptidase P (PepP), prolinase (PepR),prolidase (PepQ), general dipeptidases (PepV, PepD,PepDA) and general tripeptidase (PepT) as well as apeptide and amino acid transport systems (Fig. 3). Thisproteolytic system is necessary to enable the LAB togrow to high numbers in milk (1091010 cfu/mL), whichcontains only low levels of small peptides and free aminoacids. The physiological role of individual peptidaseshas been investigated using single and multiple peptidasedeletion mutants constructed in L. helveticus and L.lactis and a general decrease in growth rates has beenreported when the mutants were evaluated in milk(Christensen et al., 1999). McGarry et al. (1994) studiedthe role of PepN in cheese ripening by manufacturingCheddar cheese using strains of Lactococcus engineeredto overproduce PepN; these authors reported nosignificant changes in terms of body, texture, or flavourcharacteristics between cheeses made with a PepNsuperproducer and control strains. Likewise, Christen-sen, Johnson, and Steele (1995) manufactured Cheddarcheese using strains engineered to overproduce PepN.Although higher levels of free amino acids were found inthis cheese relative to the control made with a wild-typestarter, no significant sensory differences were found.However, Meyer and Spahni (1998), who studied theinfluence of PepX on Gruy"ere cheese ripening bymaking model cheeses using PepX+ and PepX strainsof L. delbrueckii subsp. lactis, reported that PepXinfluenced proteolysis and sensory characteristics of thisvariety.

2.4. Non-starter lactic acid bacteria proteolytic systems

During the maturation of Cheddar and many othercheeses, the starter lactococcal population declines andthe initially small population of adventitious non-starterlactic acid bacteria (NSLAB) ultimately becomes thedominant bacterial population in the maturing cheese(Peterson & Marshall, 1990; Martley & Crow, 1993; Fox,McSweeney, & Lynch, 1998). NSLAB, although presentinitially at low numbers (o50 cfu/g in Cheddar madefrom pasteurised milk), grow rapidly to reach B107 cfu/gwithin 4 weeks and this number remains relativelyconstant thereafter (Folkertsma, Fox, & McSweeney,1996). Thus, depending on the rate of death of the starter,NSLAB can dominate the viable microflora of Cheddarthroughout most of the ripening period.

The proteolytic activity of NSLAB appears tosupplement that of the starter, producing peptides withgenerally similar molecular weights, and free aminoacids (Lane & Fox, 1996; Lynch, McSweeney, Fox,Cogan, & Drinan, 1997; Williams & Banks, 1997;Williams, Xavier, & Banks, 1998; Muehlenkamp-Ulate& Warthesen, 1999). Peptidolytic strains of NSLABmay therefore be considered for use as adjuncts in

cheesemaking both to manipulate the overall flavourprofile of the cheese and to accelerate the rate of flavourformation (Fox et al., 1998; Fox & Tobin, 1999;Madkor, Tong, & El Soda, 2000). The criteria forselection of adjuncts are often not defined andfrequently isolates from a good-quality cheese havebeen selected for evaluation. However, there is a need toidentify the proteolytic and lipolytic enzyme systems ofthe NSLAB that could potentially contribute to theoverall maturation process. Recently, particular interesthas been shown in the characterisation of individualproteolytic enzymes produced by strains of non-starterLactobacillus spp. isolated from cheese (reviewed byKunji et al., 1996; Christensen et al., 1999).

2.5. Proteolytic agents of cultures for specific cheesevarietiesFP. roqueforti, P. camemberti, B. linens,Propionibacterium and yeasts

In many cheese varieties, secondary cultures areadded intentionally and/or encouraged to grow bycontrolling environmental conditions. These cultureshave a diverse range of functions, depending on theorganisms, but the main difference between them andthe starter cultures is that they are not added to acidifythe cheese, i.e., to produce lactic acid. These secondarycultures can grow on the surface in the case of smear-ripened (Tilsit, Gruy"ere, Appenzeller, Limburger, etc.)and mould-ripened (e.g., Camembert, Brie) cheeses orproduce CO2 (eye formation), propionate, and acetate inthe case of Swiss varieties (e.g., Emmental and Comt!e).

The principal secondary microorganisms contributingto cheese ripening are P. roqueforti (Blue mould cheese),P. camemberti (surface mould cheese such as Camem-bert and Brie), B. linens, Arthrobacter and othercoryneform bacteria and several species of yeasts(Geotrichum candidum, Kluyveromyces marxianus andDebaryomyces hansenii) in surface smear-ripenedcheeses, Propionibacterium freudenreichii subsp. sherma-nii (Swiss-type cheese).

Nowadays, the milk for mould-ripened varieties isinoculated with a pure culture of P. roqueforti, in thecase of Blue cheeses, or P. camemberti, in the case ofCamembert and Brie, at the same time as starters. Forsurface- or smear-ripened cheeses, like Tilsit, Munsterand Limburger are dipped, sprayed or brushed withaqueous suspensions of G. candidum and B. linens assoon as the cheeses are removed from the brine and arethen ripened at 10151C at high relative humidity.However, the Gram-positive bacterial flora, whichgrows on the cheese surface is very complex andcontains many adventitious strains.

The surface microflora of smear-ripened cheese hastwo important functions: (i) production of enzymes(lipases, proteinases and peptidases) and (ii) deacidifica-tion of first the cheese surface and then the cheese body.


During the first few days of ripening of smear-ripenedcheese, yeast and moulds grow on the cheese surface anddeacidify it by oxidizing the lactate to H2O and CO2(Reps, 1993; Eliskases-Lechner & Ginzinger, 1995).Yeasts grow particularly well because of their toleranceto low pH and high NaCl concentrations of the curd,their ability to use lactate as a fermentable substrate,and because there is a high relative humidity andtemperature (12241C) in the ripening rooms (Weltha-gen & Viljoen, 1998; Wyder & Puhan, 1999a; Fox et al.,2000) and because some of them are very proteolytic(Wyder & Puhan, 1999b).

For many years, B. linens have been known to beimportant bacteria growing on the surface of smear-ripened cheeses; for this reason D. hansenii and B. linensare commonly used as ripening starters and enzymesfrom the latter have been studied. B. linens secrete anextracellular proteinase and an aminopeptidase, possessa number of intracellular peptidases, which may bereleased on cell lysis (Rattray & Fox, 1997, 1999) andthe specificity of the extracellular proteinases fromB. linens on as1- and b-caseins has been determined(see Fox et al., 1995b; Rattray, Fox, & Healy, 1996,1997). B. linens is essential for smear cheese because ofits aromatic and proteolytic properties and its brightorange pigments, but considering the low proportion ofbrevibacteria on many semi-hard smear-ripened cheeses,its direct contribution to proteolysis might be ratherlimited (Bockelmann et al., 1997a,b; Bockelmann,Hoppe-Seyler, Lick, & Heller, 1998).

P. roqueforti and P. camemberti secrete aspartyl andmetalloproteinases, which have been well characterised,including their specificity on as1- and b-caseins (Gripon,1993). Intracellular acid proteinases and exopeptidases(amino and carboxy) are also produced (Gripon, 1993).

Propionibacterium spp. are weakly proteolytic butstrongly peptidolytic and they are particularly active onproline-containing peptides during the ripening ofSwiss-type cheeses, which may contribute to thecharacteristic flavour of these cheeses. PepN and PepIwere detected in Propionibacterium and PepX and threeendopeptidases have been isolated and characterisedfrom at least one strain of P. freudenreichii subsp.shermanii which may be active in cheese during ripening(El-Soda, Chen, Riesterer, & Olson, 1991; Ezzat, El-Soda, & El-Shafei, 1994; Tobiassen, Stepaniak, &Srhaug, 1996; Fern!andez-Espl!a & Fox, 1997; Stepa-niak, Gobbetti, Pripp, & Srhaug, 1998a; Stepaniak,Tobiassen, Chukwu, Pripp, & Srhaug, 1998b).

3. Comparison of proteolysis within and between cheesevarieties

Proteolysis has been considered by some researchersas a basis for the classification of cheese. Several indices

of proteolysis could be useful for classification, howeveran obvious difficulty is the fact that cheese is a dynamicsystem and therefore the results obtained depend to alarge extent on the age of the cheese when analysed(Marcos, Esteban, Le !on, & Fern!andez-Salguero, 1979;Smith & Nakai, 1990; Fox, 1993; McGoldrick & Fox,1999; Ard .o, 1999a; Pripp, Stepaniak, & Srhaug,2000c).

Several studies have shown differences in proteolysisbetween cheese varieties. Marcos et al. (1979) comparedproteolysis in several cheese varieties by analysing gelelectrophoregrams and reported that, in general,as1-casein was degraded more extensively than b-casein.These authors reported that in cheeses in which b-caseinwas degraded less extensively (e.g., Parmesan, Emmen-tal, Gruyere and Tilsit), the concentrations of g1- andg2-caseins were high, while in cheeses were almost allb-casein had been degraded (i.e., Roquefort), lessg1-casein and more g2- and in particular, g3-casein werepresent indicating more extensive plasmin action. Theratio of b- to g-caseins has been suggested as a basis forcheese classification (Fox, 1993). Smith and Nakai(1990) classified Cheddar, Edam, Gouda, Swiss, andParmesan cheeses by multivariate analysis of theirHPLC profiles. Fox (1993) reported distinct intervarietaldifferences between Cheddar, Emmental, Gouda, Par-mesan, Brie, and Appenzeller based on their RP-HPLCprofiles. McGoldrick and Fox (1999) studied proteolysisin different varieties of cheese (Cheddar, BritishTerritorial, Dutch, Swiss and Italian varieties) by urea-PAGE and by RP-HPLC and reported that RP-HPLCof the 70% ethanol-soluble or insoluble fractions of thecheese was more effective than urea-PAGE whenclassifying cheese according to variety. These authorsreported that urea-PAGE of the water-insoluble fractionof cheese was unable to distinguish Emmental fromParmesan and both of these from Cheddar and Dutchtypes, but urea-PAGE of the 70% ethanol-solublefraction showed large differences between cheese vari-eties but there were also differences within the samevariety. Dellano, Polo, and Ramos (1995) separatedartisanal cheeses (AfuegaPitu, Beyos, Vidiago, Cabralesand Peral) based on their peptide profile throughoutripening. Zarmpoutis, McSweeney, and Fox (1997)compared proteolysis in blue-veined cheese varieties(Stilton, Gorgonzola, Danablu, and the Irish farmhousevarieties Cashel and Chetwynd) and reported that allcheeses showed high extent of proteolysis, but Gorgon-zola showed higher levels of amino acids than the othervarieties. Grappin et al. (1999) compared proteolysis inSwiss cheeses and reported that Emmental showed thehighest average proportion of degradation of caseins(33%) followed by Comt!e (21.6%) and Beaufort (19%),although a lower level of g-caseins and a higher level ofas1-CN(f24199) were found in Emmental (indicatinglower plasmin and higher chymosin activity, respec-


tively) than in Comt!e and Beaufort. These authorssuggested that secondary proteolysis was lower inEmmental than in Comt!e and Beaufort and thatComt!e had a higher level of medium sized peptides incomparison with Beaufort cheese. Six varieties ofcheeses, Beaufort, Parmigiano-Reggiano, Appenzeller,Fontina, Comt!e and Mahon, were compared in terms ofindices of proteolysis (nitrogen content of differentfractions and urea-PAGE) by cluster analysis allowingqualitative and quantitative comparison betweencheeses (Lopez-Fandino, Martin-Alvarez, Pueyo, &Ramos, 1994). Belgian cheeses (Passendale, Wijnendale,Nazareth and Oud Brugge) were differentiated based onSDS-PAGE of the pH 4.6-insoluble fractions of cheese(Dewettinck, Dierckx, Eichwalder, & Huyghebaert,1997), allowing not only qualitative, but also quantita-tive classification, resulting in a clear separation ofNazareth and Oud Brugge and to a lesser extent ofPassendale and Wijnendale.

Indices of proteolysis are also useful to discriminatewithin a particular variety between cheeses of differentquality or made with different starters. OShea, Uni-acke-Lowe, and Fox (1996) compared Cheddar cheesesvarying in age and quality in terms of their peptideprofiles by HPLC, total free amino acid contents andadditional information from compositional analysis andurea-PAGE. Using this approach, these authors dis-criminated between mild, mature and extra-matureCheddar cheeses. Grappin et al. (1999) reported on thecomparison of 20 Comt!e cheeses made in five cheeseplants, with either wild starters made locally or with thesame commercial starter, and ripened under the sameconditions, and were able to discriminate them accord-ing to their physico-chemical variables, proteolysis,microbiological counts and sensory characteristics,showing the importance of milk characteristics andcheesemaking conditions to the final characteristics ofcheese.

In recent years, a promising approach in thearea of cheese ripening research is the application ofmultivariate analysis (e.g., PCA and PLS) to proteo-lytic patterns to model quantitative relationships(No.el et al., 1998; Molina, Martin-Alv!arez, & Ramos,1999a; Pripp, Stepaniak, & Srhaug, 2000c). Aswell as discriminating between cheeses, chemometricalanalysis of proteolytic profiles can be used generallyas a powerful method to better understand proteolysis incheese and how this process is influenced by factorsincluding type of starter, physico-chemical variables,microbial counts, cheesemaking parameters, age,quality and sensory characteristics. Using multivariatestatistical analysis of heights or areas of peaks inthe CE and RP-HPLC profiles, the quantitativecontribution of rennet and bacterial proteolyticenzymes to proteolysis in model sodium caseinatesystems under cheese-like conditions was demonstrated,

as well as the effect of single strains of Lactococcus onproteolysis in miniature Cheddar-type model cheeses(Pripp et al., 1998; Pripp, Shakeel-Ur-Rehman,McSweeney, & Fox, 1999a; Pripp, Stepaniak, &Srhaug, 1999b; Shakeel-Ur-Rehman, Pripp, McSwee-ney, & Fox, 1999; Pripp, McSweeney, Srhaug, & Fox,2000a; Pripp, Shakeel-Ur-Rehman, McSweeney,Srhaug, & Fox, 2000b).

4. Identification of peptides and patterns of proteolysisin cheese

The extent and type of proteolysis in a number of theprincipal cheese varieties has been characterised. How-ever, complete characterisation of proteolysis in cheeserequires isolation and identification of individual pep-tides. Using various extraction techniques and methodsto isolate individual peptides (i.e., urea-PAGE, HPLCand CE) (Fig. 1), many of the water-insoluble andwater-soluble peptides have been isolated from cheeseand identified, using amino acid sequencing and massspectrometry.

4.1. Cheddar

Proteolysis in Cheddar cheese is well characterised asreviewed by Fox, Singh, and McSweeney (1994),McSweeney, Pochet, Fox, and Healy (1994), Singhet al. (1994) Singh, Fox, and Healy (1995, 1997),Fern!andez, Singh, and Fox (1998) and Mooney, Fox,Healy, and Leaver (1998) and peptides isolated aresummarised in Fig. 4 and 5. The major water-insolublepeptides are produced either from as1-casein by chymo-sin or from b-casein by plasmin and some are degradedfurther by the lactococcal CEP (Fig. 4). as1-Casein inCheddar cheese is rapidly and completely hydrolysed bychymosin at the Phe23-Phe24 bond. The larger peptide,as1-CN(f24199) produced by the cleavage of Phe23-Phe24, is further hydrolysed by chymosin at the bondLeu101-Lys102 and, to a lesser extent, at Phe32-Gly33 andLeu109-Glu110, and perhaps by plasmin at Lys103-Tyr104and Lys105-Val106. The large C-terminal peptides,as1-CN(f24199), as1-CN(f33199), as1-CN(f102199)and as1-CN(f110199) and as1-CN(f99199), as1-CN(f104199) and as1-CN(f106199) have been identi-fied in the water-insoluble fraction of Cheddar cheese(McSweeney et al., 1994; Mooney et al., 1998). Thecomplementary N-terminal peptides (e.g., as1-CN(f2498), as1-CN(f24101) and as1-CN(f24109)) could not beidentified in the water-insoluble fraction, but since theseare highly phosphorylated, they may be in the water-soluble extract. Surprisingly, the bond Trp164-Tyr165,which is hydrolysed rapidly by chymosin in as1-casein insolution (McSweeney, Olson, Fox, Healy, & Hjrup,1993b), does not appear to be hydrolysed in cheese; at


least a peptide with Tyr165 as its N-terminal has not yetbeen identified neither in water-soluble fraction nor inmodel cheese systems (Singh et al., 1994, 1995, 1997;Exterkate, Alting, & Slangen, 1995; Exterkate, Lager-werf, Haverkamp, & Schalkwijk, 1997; Fern!andez et al.,1998). Perhaps, the bond Trp164-Tyr165 is difficult forchymosin to access in cheese, perhaps due to inter-molecular interactions.

The peptide as1-CN(f123) is hydrolysed rapidly bythe lactococcal CEP at the bonds Gln9-Gly10, Gln13-Glu14, Glu14-Val15 and Leu16-Asn17, and probably atother sites, depending on the specificity of the enzyme(Exterkate & Alting, 1993; Exterkate et al., 1995). Thepeptides as1-CN(f19), as1-CN(f113) and as1-CN(f114) accumulate and dominate the RP-HPLC chromato-grams of UF permeable or 70% ethanol-soluble fractionof the WSF of Cheddar, whereas degradation productsof the complementary C-terminal peptides have beenidentified in the UF permeate, and some of them havebeen partially hydrolysed by an aminopeptidase, releas-ing amino acids (Singh et al., 1994, 1995, 1997;Fern!andez et al., 1998). In Cheddar and many othercheeses, b-casein is much more resistant to hydrolysisthan as1-casein; only B50% of b-casein in Cheddarcheese is hydrolysed. b-Casein is hydrolysed mainly byplasmin at Lys28-Lys29, Lys105-Gln106 and Lys107-

Glu108, producing the fragments b-CN(f29209),b-CN(f106209) and b-CN(f108209) (g1-, g2-, and g3-,respectively). The g-caseins are present in the water-insoluble fraction of Cheddar (McSweeney et al., 1994;McGoldrick & Fox, 1999), whereas degradation pro-ducts of proteose peptone 8 fast (b-CN(f128)), 8 slow(b-CN(f29105/107)) and 5 (b-CN(f1105/107)), arepresent in the UF retentate or 70% ethanol-insolublefraction of the WSE (Fox & Wallace, 1997). Most of thepeptides that have been identified in the UF retentate (or70% ethanol-insoluble fraction of the WSE are pro-duced from b-casein by the action of lactococcal CEP,probably on proteose peptones rather than on intactb-casein, since none of the peptides identified containedan intact plasmin cleavage site (Singh et al., 1997;Mooney et al., 1998; Fox & Wallace, 1997).

The concentration of as2-casein appears to decreaseduring ripening but no large peptides derived from as2-casein have yet been reported (Mooney et al., 1998), andonly a few small peptides derived from as2-casein havebeen identified in the WSE (Singh et al., 1995, 1997, 1999).

During the last few years, considerable progress hasbeen made on fractionating and characterizing thewater-soluble peptides in Cheddar cheese (Singh et al.,1994, 1995, 1997; Breen, Fox, & McSweeney, 1995;Fern!andez et al., 1998) (Fig. 5) and in contrast to the

Fig. 4. Principal water-insoluble peptides derived from as1-casein (A) and b-casein (B) isolated from Cheddar cheese (McSweeney et al., 1994;Mooney et al., 1998).


Fig. 5. Water-soluble peptides derived from as1-casein (A), as2-casein (B), and b-casein (C) isolated from Cheddar cheese. The principal chymosin,plasmin and lactococcal cell-envelope proteinase cleavage sites are indicated (Singh et al., 1994, 1995, 1997; Breen et al., 1995; Fern!andez et al., 1998).


water-insoluble peptides, the water-soluble peptideprofiles are unique to the variety and presumably reflectthe specificity of the starter and non-starter proteinasesand peptidases (from Lactococcus and Lactobacillus;Fox &McSweeney, 1996). Most of the peptides from theWSF of Cheddar are derived from the N-terminal halfof b-casein (especially from residues 53 to 91), with asmaller number from the N-terminal half of as1-casein(Singh et al., 1997) (see Fig. 5c). The N-terminal of mostof these peptides corresponds to a cleavage site forchymosin (as1-casein), plasmin (b-casein), or lactococcalCEP. Alli, Okoniewska, Gibbs, and Konishi (1998)reported the identification in Cheddar cheese of 13peptides from as1-casein, 7 from b-casein and 5 fromk-casein, by electrospray ionisation mass spectrometry.Thus, the large water-insoluble peptides in Cheddar arefrom the C-terminal segments of as1-casein producedmainly by chymosin or from b-casein (g-caseins)produced by plasmin. Para-k-casein (k-CN(f1105))is not hydrolysed during ripening. The small water-soluble peptides appear to be peptides produced bythe action of the lactococcal CEP, or perhaps endo-peptidases, from products of chymosin or plasminaction. NSLAB supplement the peptidolytic activityof the starter, especially in the production of aminoacids.

4.2. Blue cheese

Very extensive proteolysis occurs in blue-mouldcheeses; both as1- and b-caseins are hydrolysed com-pletely, and most of the principal water-insolublepeptides have different mobilities from those in Cheddarand some have been identified partially (Gripon, 1993).Initial proteolysis is due mainly to chymosin, andas1-CN(f24199) is the major large peptide produced;but following sporulation of P. roqueforti at about 15days, its extracellular proteinases become dominant andpeptides with very low mobility are produced as a resultof their proteolytic activity (Gripon, 1993). However,only little work has been done on the small (pH 4.6-soluble) peptides of blue cheese, i.e., PTA-solublepeptides from Gamonedo Blue cheese (Gonz!alez deLlano, Polo, & Ramos, 1991) and the production andidentification of PTA-soluble peptides in blue cheese byHPLC (Dellano et al. (1991)).

4.3. Parmigiano-Reggiano and Grana Padano

Parmigiano-Reggiano undergoes extensive proteolysis(>35% of the total N is soluble in water and free aminoacids represent B25% of total N), probably due mainlyto its long ripening time and the action of thermophilicLactobacillus proteinases and plasmin since chymosin islargely inactivated during cooking (Battistotti & Corra-dini, 1993; Bertozzi & Panari, 1993). In Parmigiano-

Reggiano, urea-PAGE showed limited breakdown ofas1-casein but a peptide with mobility similar toas1-CN(f24199) could have been produced by a lowlevel of residual chymosin or by cathepsin D (Fox &McSweeney, 1996). Electrophoregrams showed thatb-casein is hydrolysed rapidly during the first monthsof ripening (Bertozzi & Panari, 1993); consequently, theg2- and g3-caseins continue to increase, and g1-caseintends to decrease, confirming the important role ofplasmin in maturation (Fox & McSweeney, 1996).Addeo et al. (1992) reported the isolation and identifica-tion of low molecular mass peptides formed during theripening of Parmagiano-Reggiano cheese. Using fastatom bombardment-mass spectrometry (FAB-MS), itwas found that the majority of the oligopeptides areproduced from regions 120 and 628 of b-casein (i.e.,b-CN(f120), b-CN(f728), b-CN(f26), b-CN(f828),b-CN(f928), b-CN(f220), b-CN(f320), b-CN(f420),b-CN(f520), b-CN(f514), b-CN(f1528)), 5 phospho-peptides were produced from the region 6484 ofas1-casein (as1-CN(f6374), as1-CN(f6474), as1-CN(f7078), as1-CN(f7178), as1-CN(f6484)), 3 phospho-peptides from the region 121/24 of as2-casein (as2-CN(f618), as2-CN(f718), as2-CN(f818) and 1 peptidefrom the C-terminal part of as2-casein (as2-CN(f172178))(Addeo et al., 1992, 1994, 1995). Ferranti et al. (1997)isolated a total of 45 phosphopeptides from Grana-Padano; 24 originated from b-casein, 16 from as1-caseinand 5 from as2-casein. The casein phosphopeptides werereported to consist of a mixture of components derivedfrom three parent peptides, b-CN(f728)4P, as1-CN(f6179)4P and as2-CN(f721)4P.

4.4. Serra da Estrela

Serra da Estrela has primary proteolysis of about35% of total N (TN) soluble in water, but secondaryproteolysis of about 6% of TN soluble in 12%trichloroacetic acid; 1.2% of TN is soluble in 5%phosphotungstic acid. Lower extent of primary proteo-lysis was found when milk was coagulated withproteinases from C. cardunculus than with animal rennet(Sousa & Malcata, 1997a). The primary cleavage siteswere reported to be at Leu190-Tyr191 in ovine b-caseinand Phe23-Val24 in ovine as1-casein, producing thepeptides b-CN(f1190) and as1-CN(f24191), respec-tively; however the bond Phe23-Val24 in as1-casein wascleaved earlier in cheese manufactured with plant rennetthan with animal rennet, thus producing the peptidesas1-CN(f24191) and as1-CN(f24165), respectively,perhaps with some implication for cheese texture.Significant effects of the type of milk on bovine, ovineand caprine cheeses with respect to primary andsecondary proteolysis (Sousa & Malcata, 1997b) andon their peptide profiles and specificity were found in


these cheeses manufactured with plant proteinases(Sousa & Malcata, 1997a, 1998a; Sousa, 1998).

4.5. Feta

In Feta cheese, most of the peptides from the WSFwas shown to originate from as1-casein (as1-CN(f114),as1-CN(f414), as1-CN(f2430), as1-CN(f2432), as1-CN(f4049), as1-CN(f9198), as1-CN(f102109)), 2 peptidesoriginated from the C-terminal of b-casein (b-CN(f164180), b-CN(f191205)) and 1 peptide fromk-casein (k-CN(f96105)). Most of the peptides couldbe explained on the basis of known specificity ofchymosin and the lactococcal CEP (Michaelidou,Alchinidis, Urlaub, Polychroniadou, & Zerfiridis, 1998).

5. Contribution of proteolysis to the development of tasteand flavour compounds

Compounds which contribute to cheese flavour areadded or are produced during manufacture (e.g., lacticacid and NaCl) but are mainly formed as consequenceof the many biochemical changes which occur duringripening; cheese taste is an important organolepticattribute and the correct balance of sapid compoundsis vital to cheese quality (McSweeney, 1997). Proteolysiscontributes to the taste of cheese by the production ofpeptides and free amino acids and the sapid flavourcompounds generally partition into the soluble fractionon extraction of cheese with water. Large peptides donot contribute directly to cheese flavour, but areimportant for the development of the correct texture;however, large peptides can be hydrolysed by protei-nases to shorter peptides that may be sapid. Engels andVisser (1994) analysed water-soluble fractions fromCheddar, Edam, Gouda, Gruyere, Maasdam, Parmesanand Proosdij cheeses and suggested that low-molecular(o500Da) compounds (small peptides, amino acids,free fatty acids or their breakdown products) wereresponsible for the basic taste of cheese. The exact roleof these medium- and small-sized peptides in cheeseflavour is not clear, although it is likely that theycontribute to the background flavour of Cheddar, atleast to a brothy or savoury flavour (see review byMcSweeney, 1997).

Molina, Ramos, Alonso, and Lopez Fandi *no (1999b)further fractionated the water soluble fraction of cheesesmade from cows, ewes and goats milk and assessed thecontribution of small peptides, free amino acids andvolatile components to cheese flavour. Differences werereported in intensity and predominance of individualtastes in the various fractions of cheeses made from milkof the three species; it was suggested that bovine milkcheeses were mainly salty and sour, ovine milk cheeseshad predominant umami taste and caprine milk cheese

was umami, astringent and bitter and the highest cheeseflavour intensity was found in the fractions with thehighest concentration of amino acids and volatilecompounds (Molina et al., 1999b). g-Glutamyl peptideshave been implicated in cheese flavour, although, nog-glutamyl bonds occur in the caseins, but g-Glu-Phe,g-Glu-Leu and g-Glu-Tyr (9, 20 and 70mg/kg, respec-tively) were isolated from Comt"e cheese (Roudot-Algaron, Kerhoas, Le Bars, Eibhorn, & Gripon, 1994).

The composition of the amino acid fraction and therelative proportions of individual amino acids arethought to be important for the development of thecharacteristic flavour (e.g., Broome, Krause, & Hickey,1990; Engels & Visser, 1994; Molina et al., 1999b).However, the relative proportion of individual aminoacids appears to be similar in many varieties andincreasing the concentration of free amino acids incheese does not necessarily accelerate ripening norflavour intensity (McGarry et al., 1994; Christensen,et al., 1995). Fox and Wallace (1997) suggested thatcheese flavour and the concentration of free amino acidscould not be correlated, since different cheeses (e.g.,Cheddar, Gouda and Edam) have very differentflavours, although the concentration and relativeproportions of free amino acids were generally similar.

Bitterness in cheese is most often due to hydrophobicpeptides and is generally regarded as a defect, althoughbitter notes may contribute to the desirable flavour ofmature cheese. The literature concerning bitterness indairy products has been reviewed by Lemieux andSimard (1991, 1992) and McSweeney, Nursten, andUrbach (1997) and this topic is only briefly summarisedhere from these reviews. Bitter peptides are formedmainly by the action of coagulant and starter protei-nases and bitterness occurs in cheese when such peptidesaccumulate to an excessive concentration, either as aresult of over production or of inadequate degradationby microbial enzymes. Certain sequences in the caseinsare particularly hydrophobic and, when excised byproteinases, can lead to bitterness. Bitter peptides fromas1-casein are predominantly from the region of residues1434, 91101 and 143151, while bitter peptides fromb-casein are mostly from the region of residues 4690,and particularly from the hydrophobic C-terminus.Chymosin (or rennet substitutes) is important in theproduction of bitter peptides, since residual coagulant isthe principal proteinase in many cheese varieties and itsprimary action on b-casein releases extremely hydro-phobic peptides. Thus factors that affect the retentionand activity of coagulant in the curd (e.g., pH or salt)may influence the development of bitterness. Bitterpeptides may also be produced directly by starterproteinases and then accumulate in cheese due to theabsence of peptidases from starter. Bitter starters maybe unable to hydrolyse bitter peptides to non-bitterpeptides that are too small to be perceived as bitter. Salt


may also decrease bitterness by inhibiting lactococcalCEP and thereby promote the aggregation of large non-bitter, hydrophobic regions of the caseins (e.g., theC-terminal region of b-casein) and perhaps peptides,which would otherwise be degraded to bitter peptides.The addition of exogeneous proteinases, e.g., to accel-erate ripening, often causes bitterness, while peptidaseshave been used to reduce the intensity of bitterness.Low-fat cheeses are susceptible to the development ofbitterness, perhaps because, in full-fat cheese, hydro-phobic bitter peptides are less likely to be perceived asbeing bitter due to partitioning into the fat phase.

In addition to peptides, a number of other com-pounds can contribute to bitterness in cheese, includingamino acids, amines, amides, substituted amides, long-chain ketones and some monoglycerides (Adda, Gripon,& Vassal, 1982). Several amino acids are bitter, mainlythose with non-polar or hydrophobic side chains, suchas Ile, although Lys (potentially charged) and Tyr (polarbut normally uncharged) are also considered to be bitter(McSweeney, 1997). Pro and Lys are reported to bebitter/sweet and Arg to be bitter (although this residuehas a low hydrophobicity) whereas, Ala, Gly, Ser, andThr are sweet; Glu, His and Asp are sour and Asp andGlu have the lowest taste thereshold. Catabolism of freeamino acids plays an important role in flavour devel-opment in most varieties and general pathways for thecatabolism of free amino acids have been reviewed(Aston & Douglas, 1983; Fox et al., 1995b; Fox &Wallace, 1997; Christensen et al., 1999; McSweeney &Sousa, 2000; Yvon & Rijnen, 2001). Catabolism of freeamino acids can result in a number of compounds,including ammonia, amines, aldehydes, phenols, indoleand alcohols, all of which may contribute to cheeseflavour.

6. Future perspectives

In the future, work may be expected to developfurther methodology for studying proteolysis. Althoughthere have been notable advances in their applicationand in data interpretation (chemometrics), the commonanalytical techniques for proteolysis (e.g., urea-PAGE,RP-HPLC and quantification of soluble N and freeamino acids) have remained relatively unchanged overthe last number of years. Technical development ofinstruments for capillary electrophoresis (CE) hasadvanced and is currently very promising. The trendof the application of analytical techniques developed forprotein chemistry to cheese analysis will continue.

A notable trend in recent years, which we feel willcontinue in the future, has been the study of proteolysisin different cheese varieties. This includes many cheesevarieties produced in smaller quantities or in restrictedgeographical areas that now have been characterised

with respect to proteolysis, including the isolation andidentification of some significant peptides. The identifi-cation of peptides from cheese using mass spectrometryand amino acid sequencing has only begun. Future workon the identification of peptides has the potential toincrease our understanding of the ongoing processes incheese and, as a consequence, also how to control them.The kinetics of peptide production and degradation isnot well understood and thus it is likely that mathema-tical modelling techniques will be applied to studyproteolysis in cheese during ripening.

The genetics of lactic acid bacteria has been an area ofvery active research in the last decade. One product ofthis research has been the characterisation of the genesencoding the proteolytic system of cheese starterbacteria. This has led to a greater understanding of thecontribution of individual enzymes to the growth oflactic acid bacteria in milk and their contribution toproteolysis in cheese during ripening. On-going researchin this area will effect a much clearer picture of the roleof these enzymes to cheese ripening. Finally, asdiscussed above, proteolysis contributes to cheeseflavour mainly by producing free amino acids, whichact as precursor compounds for further catabolicreactions. This will encourage further research into thelink between proteolysis, i.e., the liberation of peptidesand free amino acids from the caseins, and amino acidcatabolism.

References

Adda, J., Gripon, J.-C., & Vassal, L. (1982). The chemistry of flavour

and texture generation in cheese. Food Chemistry, 9, 115129.

Addeo, F., Chianese, L., Sacchi, R., Spagna-Musso, S., Ferranti, P., &

Malorni, A. (1994). Characterization of the oligopeptides of

Parmigiano-Reggiano cheese soluble in 120 g trichloroacetic

acid/l. Journal of Dairy Research, 61, 365374.

Addeo, F., Chianese, L., Salzano, A., Sacchi, R., Cappuccio, U.,

Ferranti, P., & Malorni, A. (1992). Characterization of the 12%

trichloroacetic acid-soluble oligopeptides of Parmigiano-Reggiano

cheese. Journal of Dairy Research, 59, 401411.

Addeo, F., Garro, G., Intorcina, N., Pellegrino, L., Resmini, P., &

Chianese, L. (1995). Gel electrophoresis and immunoblotting for

the detection of casein proteolysis in cheese. Journal of Dairy

Research, 62, 297309.

Alli, I., Okoniewska, M., Gibbs, B. F., & Konishi, Y. (1998).

Identification of peptides in Cheddar cheese by electrospray

ionization mass spectrometry. International Dairy Journal, 8,

643649.

Ard .o, Y. (1999a). General introduction. Bulletin 337, International

Dairy Federation, Brussels, pp. 3.

Ard .o, Y. (1999b). Evaluating proteolysis by analysing the N content of

cheese fractions. Bulletin 337, International Dairy Federation,

Brussels, pp. 49.

Aston, J. W., & Douglas, K. (1983). The production of volatile sulphur

compounds in Cheddar cheeses during accelerated ripening.

Australian Journal of Dairy Technology, 38, 6670.

Awad, S., L .uthi, Q.-Q., & Puhan, Z. (1998). Proteolytic activities of

chymosin and porcine pepsin on buffalo, cow, and goat whole and


b-casein fractions. Journal of Agriculture and Food Chemistry,46, 49975007.

Barbosa, M. (1983). Cardo (Cynara cardunculus L) as vegetable

rennet. Boletim do Departamento de Tecnologia e Ind !ustria

Alimentar, 58(45), 111.

Barrett, A. J. (1972). Lysosomal enzymes. In J. T. Dingle (Ed.),

Lysosomes, a Laboratory Handbook (pp. 46135). Amsterdam:

North-Holland Publishing Company.

Bastian, E. D., & Brown, R. J. (1996). Plasmin in milk and dairy

products: an update. International Dairy Journal, 6, 435457.

Battistotti, B., & Corradini, C. (1993). Italian cheese. In P. F. Fox,

Cheese: Chemistry, Physics and Microbiology, Vol. 2 (pp. 221243).

London: Chapman & Hall.

Bertozzi, L., & Panari, G. (1993). Studies on proteolysis in

Parmigiano-Reggiano cheese. International Dairy Journal, 3, 46.

Beuvier, E., Berthaud, K., Cegarra, S., Dasen, A., Pochet, S., Buchin,

S., & Duboz, G. (1997). Ripening and quality of Swiss-type cheese

made from raw, pasteurized or microfiltered milk. International

Dairy Journal, 7, 311323.

Bockelmann, W., Hoppe-Seyler, T., Krush, U., Hoffmann, W., &

Heller, K. J. (1997a). The microflora of Tilsit cheese. Part 2.

Development of a surface smear starter culture. Nahrung-Food, 41,

213218.

Bockelman, W., Hoppe-Seyler, T., Lick, S., & Heller, K. J. (1998).

Analysis of casein degradation in Tilsit cheeses. Kieler Milch-

wirtschafttliche. Forschungsberichte, 50, 105113.

Bockelmann, W., Krush, U., Engel, G., Klijn, N., Smit, G., & Heller,

K. J. (1997b). The microflora of Tilsit cheese. Part 1. Variability of

the smear flora. Nahrung-Food, 41, 208212.

Boudjellab, N., Rolet-Repecaud, O., & Collins, J. C. (1994). Detection

of residual chymosin in cheese by an enzyme-linked immunosor-

bent assay. Journal of Dairy Research, 61, 101109.

Breen, E. D., Fox, P. F., & McSweeney, P. L. H. (1995). Fractionation

of peptides in a 10 kDa ultrafiltration retentate of a water-soluble

extract of Cheddar cheese. Italian Journal of Food Science, 7,

211220.

Broome, M. C., Krause, D. A., & Hickey, M. W. (1990). The use of

non-starter lactobacilli in Cheddar cheese manufacture. Australian

Journal of Dairy Technology, 45, 6773.

Broome, M. C., & Limsowtin, G. K. Y. (1998). Milk coagulants.

Australian Journal of Dairy Technology, 53, 188190.

B .utikofer, U., & Ard .o Y. (1999). Quantitative determination of free

amino acids in cheese. Bulletin 337, International Dairy Federation,

Brussels, pp. 2432.

Carles, C., & Ribadeau-Dumas, B. (1984). Kinetics of action of

chymosin (rennin) on some peptide bonds of bovine b-casein.Biochemistry, 23, 68396843.

Carmona, M. A., Sanjuan, E., Gomez, R., & Fern!andez-Salguero, J.

(1999). Effect of starter cultures on the physico-chemical and

biochemical features in ewe cheese made with extracts from flowers

of Cynara cardunculus L. Journal of the Science of Food and

Agriculture, 79, 737744.

Christensen, J. E., Dudley, E. G., Pederson, J. A., & Steele, J. L.

(1999). Peptidases and amino acid catabolism in lactic acid

bacteria. Antoine van Leeuwenhoek, 76, 217246.

Christensen, J. E., Johnson, M. E., & Steele, J. L. (1995). Production

of Cheddar cheese using Lactococcus lactis ssp. cremoris SK11

derivative with enhanced aminopeptidase activity. International


Christensen, T. M. I. E., Bech, A. M., & Werner, H. (1991). Methods

for crude fractionation (extraction and precipitation) of nitrogen

components in cheese. Bulletin 261, International Dairy Federation,

Brussels, pp. 49.

Considine, T. (2000). Role of Somatic Cell Proteinases in Dairy Product

Quality. Ph.D. Thesis, National University of Ireland, Cork,

Ireland.

Considine, T., Healy, A., Kelly, A. L., & McSweeney, P. L. H. (1999).

Proteolytic specificity of elastase on bovine b-casein. FoodChemistry, 66, 463470.

Considine, T., Healy, A., Kelly, A. L., & McSweeney, P. L. H. (2000).

Proteolytic specificity of elastase on bovine as1-casein. FoodChemistry, 69, 1926.

Cordeiro, M. C., Xue, Z. T., Pietrzak, M., Pais, M. S., & Brodelius, P.

E. (1994). Isolation and characterization of cDNA from flowers of

Cynara cardunculus encoding cyprosin (an aspartic proteinase) and

its use to study the organ-specific expression of cyprosin. Plant

Molecular Biology, 24, 733741.

Dellano, D. G., Polo, M. C., & Ramos, M. (1991). Production,

isolation and identification of low-molecular mass peptides from

blue cheese by high performance liquid chromatography. Journal of

Dairy Research, 58, 363372.

Dellano, D. G., Polo, M. C., & Ramos, M. (1995). Study of proteolysis

in artisanal cheeses-high performance liquid chromatography of

peptides. Journal of Dairy Science, 78, 10181024.

Dewettinck, K., Dierckx, S., Eichwalder, P., & Huyghebaert, A.

(1997). Comparison of SDS-PAGE profiles of four Belgian cheeses

by multivariate statistics. Lait, 77, 7789.

Drohse, H. B., & Foltmann, B. (1989). Specificity of milk-clotting

enzymes towards bovine k-casein. Biochimica et Biophysica Acta,995, 221224.

Eigel, W. N., Butler, J. E., Ernstrom, C. A., Farrell Jr., H. M.,

Harwalkar, V. R., Jenness, R., & Whitney, R. McL. (1984).

Nomenclature of proteins of cows milk: fifth revision. Journal of

Dairy Science, 67, 15991631.

Eliskases-Lechner, F., & Ginzinger, W. (1995). The yeast flora of

surface ripened cheeses. Michwissenschaft, 50, 458462.

El-Soda, M., Chen, C., Riesterer, B., & Olson, N. (1991). Acceleration

of low-fat cheese ripening using lyophilized extracts or freeze

shocked cells of some cheese related microorganisms. Milchwis-

senschaft, 46, 358360.

Engels, W. J. M., & Visser, S. (1994). Isolation and comparative

characterization of components that contribute to the flavour of

different cheese types. Netherlands Milk and Dairy Journal, 48,

127140.

Exterkate, F. A., & Alting, A. C. (1993). The conversion of the

as1-casein-(1-23)-fragment by the free and bound form of the cell-envelope proteinase of Lactococcus lactis subsp. cremoris under

conditions prevailing in cheese. Systematic and Applied Microbiol-

ogy, 16, 18.

Exterkate, F. A., Alting, A. C., & Slangen, C. J. (1995). Conversion of

the as1-casein-(24-199)-fragment and b-casein under cheese condi-tions by chymosin and starter peptidases. Systematic and Applied

Microbiology, 18, 712.

Exterkate, F. A., Lagerwerf, F. M., Haverkamp, J., & Schalkwijk, S.

(1997). The selectivity of chymosin action on as1- and b-caseinsin solution is modulated in cheese. International Dairy Journal, 7,

4754.

Ezzat, N., El-Soda, M., & El-Shafei, H. (1994). Partial purification and

characterization of peptide hydrolase system in several

cheese-related bacteria. Egyptian Journal of Dairy Science, 22,

217231.

Faro, C. J., Moir, A. J., & Pires, E. V. (1992). Specificity of a milk-

clotting enzyme extracted from the thistle Cynara cardunculus L:

Action on oxidized insulin and k-casein. Biotechnology Letters, 14,841846.

Faro, C. J., Ramalho-Santos, M., Vieira, M., Mendes, A., Simoes, I.,

Andrade, R., Verissimo, P., Lin, X. L., Tang, J., & Pires, E. (1999).

Cloning and characterization of cDNA encoding cardosin A, all

RGD-containing plant aspartic proteinase. Journal of Biological

Chemistry, 274, 2872428729.

Faro, C. J., Ver!ssimo, P., Lin, Y., Tang, J., & Pires, E. V. (1995). In K.

Takahashi (Ed.), Aspartic proteinases: Structure, Function, Biology


and Biomedical Implications (pp. 373377). New York, USA:

Plenum Press.

Fern!andez, M., Singh, T. K., & Fox, P. F. (1998). Isolation and

identification of peptides from the diafiltration permeate of the

water-soluble fraction of Cheddar cheese. Journal of Agricultural

and Food Chemistry, 46, 45124517.

Fern!andez-Espl!a, M. D., & Fox, P. F. (1997). Purification and

characterization of a X-prolyl dipeptidyl aminopeptidase from

Propionibacterium shermanii NCDO 853. International Dairy

Journal, 7, 2329.

Fern!andez-Salguero, J., & Sanju!an, E. (1999). Influence of vegetable

and animal rennet on proteolysis during ripening in ewes milk

cheese. Food Chemistry, 64, 177183.

Fern!andez-Salguero, J., Sanju!an, E., & Montero, E. (1991). A

preliminary study of the chemical composition of Gu!a cheese.

Journal of Food Composition and Analysis, 4, 262269.

Ferranti, P., Barone, F., Chianese, L., Addeo, F., Scaloni, A.,

Pellegrino, L., & Resmini, P. (1997). Phosphopeptides from Grana

Padano cheese: Nature, origin, and changes. Journal of Dairy

Research, 64, 601615.

Folkertsma, B., Fox, P. F., & McSweeney, P. L. H. (1996).

Acceleration of Cheddar cheese ripening at elevated temperatures.

International Dairy Journal, 6, 11171134.

Fox, P. F. (1993). Cheese: An overview. In P. F. Fox, Cheese:

Chemistry, Physics and Microbiology, Vol. 1 (pp. 136). London:

Chapman & Hall.

Fox, P. F., Guinee, T. P., Cogan, T. M., & McSweeney, P. L. H.

(2000). Fundamentals of Cheese Science. Gaithersburg Maryland:

Aspen Publishers, Inc.

Fox, P. F., & McSweeney, P. L. H. (1996). Proteolysis in cheese during

ripening. Food Reviews International, 12, 457509.

Fox, P. F., & McSweeney, P. L. H. (1997). Rennets: Their role in milk

coagulation and cheese ripening. In B. A. Law (Ed.), Microbiology

and Biochemistry of Cheese and Fermented Milk (2nd ed.)

(pp. 1249). London: Chapman and Hall, Blackie Academic &

Professional.

Fox, P. F., McSweeney, P. L. H., & Lynch, C. M. (1998). Significance

of non-starter lactic acid bacteria in Cheddar cheese. Australian

Journal of Dairy Technology, 53, 8389.

Fox, P. F., McSweeney, P. L. H., & Singh, T. K. (1995a). Methods for

assessing proteolysis in cheese during ripening. In E. L. Malin, &

M. H. Tunick (Eds.), Chemistry of Structure/Function Relationships

in Cheese (pp. 161194). London: Plenum Press.

Fox, P. F., Singh, T. K., & McSweeney, P. L. H. (1994). Proteolysis in

cheese during ripening. In A. T. Andrews, & J. Varley (Eds.),

Biochemistry of Milk Products (pp. 131). Cambridge: Royal

Society of Chemistry.

Fox, P. F., Singh, T. K., & McSweeney, P. L. H. (1995b). Biogenesis of

flavour compounds in cheese. In E. L. Malin, & M. H. Tunick

(Eds.), Chemistry of Structure/Function Relationships in Cheese

(pp. 5998). London: Plenum Press.

Fox, P. F., & Tobin, J. (1999). Acceleration and modification of

proteolysis. Proceedings of the 36th Annual Marshall Cheese

Symposium, Gateway to Success, Santa Clara, USA, September

910, pp. 1447.

Fox, P. F., & Wallace, J. M. (1997). Formation of flavour compounds.

Advances in Applied Microbiology, 45, 1785.

Fox, P. F., & Walley, B. F. (1971). Influence of sodium chloride on the

proteolysis of casein by rennet and pepsin. Journal of Dairy

Research, 38, 165170.

Garg, S. K., & Johri, B. N. (1994). Rennet: Current trends and future

research. Food Reviews International, 10, 313355.

Gonz!alez de Llano, D., Polo, C., & Ramos, M. (1991). Production,

isolation and identification of low molecular mass peptides from

Blue cheese by high performance liquid chromatography. Journal

of Dairy Research, 58, 363372.

Grappin, R., Beuvier, E., Bouton, Y., & Pochet, S. (1999). Advances in the

biochemistry and microbiology of Swiss-type cheeses. Lait, 79, 322.

Gripon, J. C. (1993). Mould-ripened cheeses. In P. F. Fox, Cheese:

Chemistry, physics and microbiology, Vol. 2 (pp. 111136). London:

Chapman & Hall.

Guinee, T. M., & Wilkinson, M. G. (1992). Rennet coagulation and

coagulants in cheese manufacture. Journal of the Society of Dairy

Technology, 45, 94104.

Gupta, S. K., & Eskin, N. A. M. (1977). Potential use of vegetable

rennet in the production of cheese. Food Technology, 31, 6264.

Hayes, M. G., Hurley, M. J., Magboul, A. A. A., Larsen, L. B.,

Heegaard, C. W., Oliveira, J. C., McSweeney, P. L. H., & Kelly, A.

L. (2001). Thermal inactivation kinetics of bovine cathepsin D.

Journal of Dairy Research, 68, 267276.

Hurley, M. J. (1999). Studies on Aspartic Proteinases in Cheese. M.Sc.

Thesis, National University of Ireland, Cork.

Hurley, M. J., Larsen, L. B., Kelly, A. L., & McSweeney, P. L. H.

(2000a). The milk acid proteinase cathepsin D: A review.


Hurley, M. J., Larsen, L. B., Kelly, A. L., & McSweeney, P. L. H.

(2000b). Cathepsin D activity in Quarg. International Dairy

Journal, 10, 453458.

Ibiama, E., & Griffiths, M. W. (1987). Studies on a milk-coagulating

enzyme, calotropain, obtained from sodom apple (Calotropis

procera). Journal of the Science of Food and Agriculture, 1, 157162.

Kaminogawa, S., & Yamauchi, K. (1972). Acid protease of bovine

milk. Agricultural and Biological Chemistry, 36, 23512356.

Kelly, M., Fox, P. F., & McSweeney, P. L. H. (1996). Influence of salt-

in-moisture on proteolysis in Cheddar-type cheese. Milchwis-

senschaft, 51, 498501.

Kelly, A. L., & McSweeney, P. L. H. (2001). Indigenous proteinases.

In: P. F. Fox, & P. L. H. McSweeney (Eds.), Advanced Dairy

Chemistry-1. Proteins. (3rd ed.), Gaithersburg, MD: Aspen

Publishers (in press).

Kristiansen, K. R., Deding, A. S., Jensen, D. F., Ard .o, Y., & Qvist, K.

B. (1999). Influence of salt content on ripening of semi-hard round-

eyed cheese of Danbo-type. Michwissenschaft, 54, 1923.

Kunji, E. R. S., Mierau, I., Hagting, A., Poolman, B., & Konings, W.

N. (1996). The proteolytic system of lactic acid bacteria. Antonie

van Leeuwenhoek, 70, 187221.

Lane, C. N., & Fox, P. F. (1996). Contribution of starter and added

lactobacilli to proteolysis in Cheddar cheese during ripening.


Lane, C. N., Fox, P. F., Johnston, D. E., & McSweeney, P. L. H.

(1997). Contribution of coagulant to proteolysis and textural

changes in Cheddar cheese during ripening. International Dairy

Journal, 7, 453464.

Larsen, L. B., Benfeldt, C., Rasmussen, L. K., & Petersen, T. E. (1996).

Bovine milk procathepsin D and cathepsin D: Coagulation and

milk protein degradation. Journal of Dairy Research, 63, 119130.

Larsen, L. B., Wium, H., Benfeldt, C., Heegaard, C. W., Ard .o, Y.,

Qvist, K. B., & Petersen, T. E. (2000). Bovine milk procathepsin D:

Presence and activity in heated milk and extracts of rennet free UF-

feta. International Dairy Journal, 10, 6774.

Law, J., & Haandrikman, A. (1997). Proteolytic enzymes of lactic acid

bacteria. International Dairy Journal, 7, 111.

Lemieux, L., & Simard, R. E. (1991). Bitter flavour in dairy products.

I. A review of the factors likely to influence its development, mainly

in cheese manufacture. Lait, 71, 599636.

Lemieux, L., & Simard, R. E. (1992). Bitter flavour in dairy products.

II. A review of bitter peptides from the caseins: Their formation,

isolation and identification, structure masking and inhibition. Lait,

72, 335382.

Lopez-Fandino, R., Martin-Alvarez, P. J., Pueyo, E., & Ramos, M.

(1994). Proteolysis assessment of several cheese varieties using

different methods. Milchwissenschaft, 49, 315318.


Lynch, C. M., McSweeney, P. L. H., Fox, P. F., Cogan, T. M., &

Drinan, F. D. (1997). Contribution of starter lactococci and non-

starter lactobacilli to proteolysis in Cheddar cheese with controled

microflora. Lait, 77, 441459.

Macedo, A., Malcata, F. X., & Oliveira, J. C. (1993a). The technology,

chemistry, and microbiology of Serra cheese: A review. Journal of

Dairy Science, 76, 17251739.

Macedo, I. Q., Faro, C. J., & Pires, E. V. (1993b). Specificity and

kinetics of the milk-clotting enzyme from Cardoon (Cynara

cardunculus L.) toward bovine k-casein. Journal of Agriculturaland Food Chemistry, 41, 15371540.

Macedo, I. Q., Faro, C. J., & Pires, E. V. (1996). Caseinolytic

specificity of cardosin, an aspartic protease from the cardoon

Cynara cardunculus L: Action on bovine as- and b-casein andcomparison with chymosin. Journal of Agricultural and Food

Chemistry, 44, 4247.

Madkor, S. A., Tong, P. S., & El Soda, M. (2000). Ripening of

Cheddar cheese with attenuated adjunct cultures of lactobacilli.

Journal of Dairy Science, 83, 16841691.

Magboul, A. A. A., Larsen, L. B., McSweeney, P. L. H., & Kelly, A. L.

(2001). Cysteine protease activities in bovine milk. International

Dairy Journal, (in press).

Marcos, A., Esteban, M. A., Le !on, F., & Fern!andez-

Salguero, J. (1979). Electrophoretic patterns of European cheese:

Comparison and quantification. Journal of Dairy Science, 62,

892900.

Martley, F. G., & Crow, V. L. (1993). Interactions between non-starter

microorganisms during cheese manufacture and ripening. Interna-

tional Dairy Journal, 3, 461483.

McGarry, A., Law, J., Coffey, A., Daly, C., Fox, P. F., & Fitzgerald,

G. F. (1994). Effect of genetically modifying the lactococcal

proteolytic system on ripening and flavour development in

Cheddar cheese. Applied and Environmental Microbiology, 60,

42264233.

McGoldrick, M., & Fox, P. F. (1999). Intervarietal comparison of

proteolysis in commercial cheese. Zeitschrift f .ur Lebensmittel

Untersuchung und-Forchung, 208, 9099.

McSweeney, P. L. H. (1997). The flavour of milk and dairy products:

III. cheese taste. International Journal of Dairy Technology, 50,

123128.

McSweeney, P. L. H., & Fox, P. F. (1997). Chemical methods for the

characterization of proteolysis in cheese during ripening. Lait, 77,

4176.

McSweeney, P. L. H., Fox, P. F., & Olson, N. F. (1995). Proteolysis of

bovine caseins by cathepsin D: Preliminary observations and

comparison with chymosin. International Dairy Journal, 5,

321336.

McSweeney, P. L. H., Nursten, H. E., & Urbach, G. (1997). Flavours

and off-flavours in milk and dairy products. In: P. F. Fox (Ed.),

Advanced Dairy ChemistryFLactose, Water, Salts and Vitamins,Vol. 3, (2nd ed.), (pp. 403468). London: Chapman & Hall.

McSweeney, P. L. H., Olson, N. F., Fox, P. F., Healy, A., & Hjrup,P. (1993b). Proteolytic specificity of chymosin on bovine as1-casein.Journal of Dairy Research, 60, 401412.

McSweeney, P. L. H., Pochet, S., Fox, P. F., & Healy, A. (1994).

Partial identification of peptides from the water-insoluble fraction

of Cheddar cheese. Journal of Dairy Research, 61, 587590.

McSweeney, P. L. H., & Sousa, M. J. (2000). Biochemical pathways

for the production of flavour compounds in cheese during ripening:

A review. Lait, 80, 293324.

Meyer, J., & Spahni, A. (1998). Influence of X-prolyl-dipeptidyl-

aminopeptidase of Lactobacillus debrueckii subsp. lactis on

proteolysis and taste of Swiss Gruy"ere cheese. Milchwissenschaft,

53, 449453.

Michaelidou, A., Alichanidis, E., Urlaub, H., Polychroniadou, A., &

Zerfiridis, G. K. (1998). Isolation and identification of some major

water-soluble peptides in Feta cheese. Journal of Dairy Science, 81,

31093116.

Mohamed, M. A., & OConnor, C. B. (1999). Calotropis procera with

emphasis on its use as a milk coagulating agent. A review. Egyptian

Journal of Dairy Science, 27, 112.

Molina, E., Martin-Alv!arez, P. J., & Ramos, M. (1999a). Analysis of

cows, ewes and goats milk mixtures by capillary electrophoresis:

Quantification by multivariate regression analysis. International


Molina, E., Ramos, M., Alonso, L., & Lopez-Fandino, R. (1999b).

Contribution of low molecular weight water soluble compounds to

the taste of cheeses made of cows, ewes and goats milk.


Mooney, J. S., Fox, P. F., Healy, A., & Leaver, J. (1998). Identification

of the principal water-insoluble peptides in Cheddar cheese.


Muehlenkamp-Ulate, M. R., & Warthesen, J. J. (1999). Evaluation of

several nonstarter lactobacilli for their influence on Cheddar cheese

slurry. Journal of Dairy Science, 82, 13701378.

No.el, Y., Ard .o, Y., Pochet, S., Hunter, A., Lavanchy, P., Luginnbuhl,

W., Le Bars, D., Polychroniadou, A., & Pellegrino, L. (1998).

Characterisation of protected denomination of origin cheeses:

Relationships between sensory texture and instrumental data. Lait,

78, 569588.

Nu *nez, M., Fern!andez del Pozo, B., Rodriguez-Marin, M. A.,

Gaya, P., & Medina, M. (1991). Effect of vegetable and animal

rennet on chemical, microbiological, rheological and sensory

characteristics of La Serena Cheese. Journal of Dairy Research,

58, 511519.

OShea, B. A., Uniacke-Lowe, T., & Fox, P. F. (1996). Objective

assessment of Cheddar cheese quality. International Dairy Journal,

6, 11351147.

Otte, J., Ard .o, Y., Weimer, B., & Srensen, J. (1999). Capillaryelectrophoresis used to measur

Documents

Advances Study Proteolysis