Advances in the study of proteolysis during cheese ripening

International Dairy Journal 11 (2001) 327–345

Advances in the study of proteolysis during cheese ripening M.J. Sousaa, Y. Ardo. b, P.L.H. McSweeneya,* b

a Department of Food Science, Food Technology and Nutrition, University College, Cork, Ireland Dairy 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 from a number of sources: residual coagulant (usually chymosin), indigenous milk enzymes, starter, adventitious non-starter microflora and, 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 the analytical 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 role in the development of texture as well as flavour and has been the subject of several reviews (e.g., Fox, Singh, & McSweeney, 1995b; Fox & McSweeney, 1996). Proteolysis contributes to textural changes of the cheese matrix, due to breakdown of the protein network, decrease in aw through water binding by liberated carboxyl and amino groups and increase in pH (in particular in surface mould-ripened varieties), which facilitates the release of sapid compounds during mastication. It contributes directly to flavour and to off-flavour (e.g., bitterness) of cheese through the formation of peptides and free amino acids as well as liberation of substrates (amino acids) for secondary catabolic changes, i.e., transamination, deamination, decarboxylation, desulphuration, catabolism of aromatic amino acids and reactions of amino acids with other compounds. Because proteolysis is one of the principal biochemical events during the ripening of cheese, it is desirable to include a general assay for proteolysis, e.g., pH 4.6 soluble N or water soluble N as % of total N (pH 4.6 *Corresponding author. E-mail address: [email protected] (P.L.H. McSweeney).

SN/TN or WSN/TN) or liberation of reactive groups, in most ripening studies (Fig. 1). The rate and pattern of proteolysis may be influenced by location within the cheese (e.g., surface-ripened, smear-ripened or young brined-salted cheeses) and a suitable sampling scheme should consider this. Cheese variety and its characteristics (e.g. pH) should be considered when choosing methodology. For example, since the extractability of N compounds varies with pH, WSN can be much higher in cheeses with higher pH, so fractionation using buffers at pH 4.6 is more suitable than using water for varieties that are characterised by a change in pH during ripening. If the objectives of the study encompass investigation of the effect of one of the agents of proteolysis in cheese, i.e., action of plasmin, different types of coagulant or the effect of different starter strains or adjunct cultures in cheese, the methodology should be chosen so as to emphasise the level of proteolysis caused by that agent. For example, comparison of the effect of different coagulants on primary proteolysis could be followed by urea-polyacrylamide gel electrophoresis (urea-PAGE) or capillary electrophoresis (CE) of the pH 4.6-insoluble fraction (or cheese), followed by electroblotting, sequencing and identification of the products of primary proteolysis. Peptide profiles of the pH 4.6-soluble fraction (or ethanol-insoluble and -soluble fractions therefrom) should be determined by

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

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

reverse phase-high performance liquid chromatography (RP-HPLC). Chemometrical analysis of the profiles obtained by urea-PAGE, CE and RP-HPLC could be performed. As another example, analysis of the effect of starter or adjunct cultures should comprise RP-HPLC of the ethanol-insoluble and -soluble fractions of the pH 4.6 soluble fractions of the cheese, analysis of individual amino acids, followed by multivariate analysis (e.g., principal component analysis, PCA) of the peptide profiles and free amino acids. The choice of a fractionation scheme for cheese N or a suitable technique to use for assessing proteolysis in cheese depends on a number of factors including (i) availability of equipment and resources, (ii) cheese variety and (iii) objective of the study and consequently different strategies have been proposed for the fractionation of cheese nitrogen (Christensen, Bech, & Werner, 1991; Singh, Fox, Hjrup, & Healy, 1994; Fox, McSweeney, & Singh, 1995a; McSweeney & Fox, 1997; Grappin, Beuvier, Bouton, & Pochet, 1999; . 1999a, b). Several nitrogen indices have been Ardo, proposed to study proteolysis during cheese ripening, however, proteolysis is far too complex to be described adequately by a single index. The methodology for assessment of proteolysis has recently been reviewed extensively and will not be considered further here (Fox et al., 1995a; McSweeney & Fox, 1997; Wallace & Fox, . 1999b; Butikofer . 1999; Otte, Ardo, . . 1998; Ardo, & Ardo, Weimer, & Srensen, 1999; Singh, Gripon, & Fox, 1999). Proteolysis in cheese during ripening has been an active area for research in recent years and the literature on the topic has increased substantially in the last decade. The objective of this review is to consider certain

Fig. 2. Proteolytic agents in cheese during ripening.

specific aspects of proteolysis in cheese during ripening: (i) proteolytic agents in cheese and their specificity with detailed descriptions only on new findings focused on the plant coagulant from Cynara cardunculus and the milk protease cathepsin D; (ii) comparison of proteolysis within and between cheese varieties; (iii) identification of peptides in cheese and (iv) contribution of proteolysis to cheese flavour.

2. Proteolytic agents in cheese During ripening, proteolysis in cheese is catalysed by enzymes from (i) coagulant (e.g., chymosin, pepsin, microbial or plant acid proteinases), (ii) milk (plasmin and perhaps cathepsin D and other somatic cell proteinases), (iii) enzymes from the starter, (iv) nonstarter, or (v) secondary cultures (e.g., P. camemberti, P. roqueforti, Propionibacterium sp., B. linens and other coryneforms) and (vi) exogenous proteinases or peptidases, or both, used to accelerate ripening (Fig. 2).

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In many cheese varieties, the initial hydrolysis of caseins is caused by the coagulant and to a lesser extent by plasmin, which results in the formation of large (water-insoluble) and intermediate-sized (water-soluble) peptides which are degraded subsequently by the coagulant and enzymes from the starter and non-starter microflora of the cheese. The extracellular, cell envelopeassociated proteinase of Lactococccus (lactocepin, PrtP) contributes to the formation of small peptides in cheese probably by hydrolysing the larger peptides produced from as1 -casein by chymosin or from b-casein by plasmin, whereas the peptidases (which are intracellular) are released after the cells have lysed and are responsible for the degradation of short peptides and the production of free amino acids. The final products of proteolysis are free amino acids and their concentration in cheese at any stage of ripening is the net result of the liberation of amino acids from casein, their degradation to catabolic products and perhaps some synthesis by the cheese microflora. This general outline of proteolysis can vary substantially between variety (see Section 3); e.g., coagulant is extensively or completely denatured by the high cooking temperature used in the manufacture of Parmigiano-Reggiano and Swiss cheeses and thus the contribution of plasmin to the initial hydrolysis of caseins is more pronounced than in Cheddar and Dutch varieties.

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to denaturation by pH than chymosin and hence the amount of activity of these coagulants retained in the curd is very strongly dependent on the pH of the milk at setting and shortly thereafter (Fox & McSweeney, 1996); in fact, increasing the pH of the curds-whey mixture to around 7 after milk coagulation using porcine pepsin is one of the methods used to produce rennet-free cheese curd (e.g., Lane, Fox, Johnston, & McSweeney, 1997). The heat stability of rennet at the temperature used during cooking of curds and whey also has a large effect on the level of rennet activity remaining in the curd; in high cook cheeses (e.g., Emmental), chymosin is denatured extensively and makes relatively little contribution to ripening (Boudjellab, Rolet-Repecaud, & Collins, 1994), while Gouda, which has a similar initial pH but which is cooked only to B371C, contains considerable rennet activity. In the latter part of the last century, cheese consumption increased while the availability of calf rennet decreased, which led to rennet shortages and subsequent price increases. In addition, more restrictive ethical concerns associated with production of such animal rennets led to a search for suitable rennet substitutes for cheese making. Several proteases from animal, microbial and plant sources were investigated as likely substitutes and have been reviewed by Guinee and Wilkinson (1992), Broome and Limsowtin (1998) and Fox et al. (2000).

2.1. Coagulant Majority of cheeses produced around the world were manufactured traditionally, and in many cases still are manufactured, using an enzymatic coagulant extracted from the abomasa of milk-fed calves. This extract, known as calf rennet, consists of two proteolytic enzymes: chymosin (EC 3.4.23.4), the major component (88–94% milk clotting activity, MCA) and bovine pepsin (EC 3.4.23.1; 6–12% MCA). The relative proportion of these enzymes varies with individuality and age of calves, the method of rennet separation and the conditions and pH values at which the milk clotting activity is measured (Guinee & Wilkinson, 1992). The principal role of chymosin in cheesemaking is to coagulate milk by specifically hydrolysing the Phe105Met106 bond of the micelle-stabilising protein, k-casein, which is many times more susceptible to chymosin than any other bond in milk proteins and leads to the coagulation of the milk (see Fox, Guinee, Cogan, & McSweeney, 2000). Most of the coagulant activity added to the milk is lost in the whey; only 0–15% of the rennet activity added to the milk remains in the curd after manufacture, depending on factors including type of coagulant, ratio of different enzymes in blends, cooking temperature, the cheese variety and the moisture level of the final cheese (Guinee & Wilkinson, 1992). Pepsins are more sensitive

2.1.1. Substitutes for calf rennet The most common rennet substitutes include bovine, porcine and to a lesser extent, chicken pepsins and microbial proteases from Rhizomucor miehei, R. pusillus and Cryphonectria parasitica (see Fox & McSweeney, 1997; Fox et al., 2000). The proteolytic activities of chymosin and porcine pepsin were compared on buffalo, . cow and goat whole casein by Awad, Luthi, and Puhan (1998) and it was reported that both enzymes attacked as1 - and b-caseins in the same region as calf rennet. ! Trujillo, Guamis, Laencina, and Lopez (2000) compared some milk clotting enzymes (calf and lamb rennets, bovine chymosin and pepsin, and proteases from R. mihei and C. parasitica) on ovine casein and reported that lamb rennet and C. parasitica protease showed the lowest and the highest degree of proteolysis, respectively. These authors reported that all enzymes hydrolysed ovine casein resulting in the formation of as1 -I and b-I-caseins (the first breakdown products produced by chymosin) as initial breakdown products of as1 - and b-caseins, respectively, but C. parasitica also produced a series of degradation products with lower electrophoretic mobilities than b-casein. C. parasitica proteinase cleaves k-casein at Ser104-Phe105 rather than Phe105Met106, which is cleaved by chymosin and R. miehei proteinase (Drohse & Foltmann, 1989). Porcine pepsin tends to be more heat-sensitive, followed by

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C. parasitica, bovine pepsin, chymosin, R. pusillus protease and R. miehei protease in order of increasing heat stability (Broome & Limsowtin, 1998), although the heat stability of the microbial coagulants can be reduced after treatment with various chemical agents (Garg & Johri, 1994). The use of coagulants that are more heat stable than calf rennet should be avoided; otherwise excess proteolytic activity may remain in the curd where it may result in excessive proteolysis and bitterness unless ripening times and/or cooking temperatures are changed to compensate for the more rapid rate of proteolysis (Guinee & Wilkinson, 1992). In the last decade, genetically engineered microorganisms have been exploited increasingly for the production of commercial coagulants. The gene for chymosin has been cloned and inserted into microorganisms such as Kluyveromyces marxianus var. lactis, Aspergillus niger var. awamori or Escherichia coli which led to the development of recombinant chymosins which are now marketed commercially as Maxiren (DSM Food Specialities, Netherlands) and Chymax (Chr. Hansen, Denmark). Recombinant chymosins have been approved for commercial use in foods in many, but not all countries, and they have been used in everincreasing quantities in USA and Western Europe and now represent about 35% of the total market (Fox et al., 2000). Plant proteases have also been investigated as milk coagulants, but only a small number of aspartic proteinases from plant origin have been isolated and partially characterised (Tavaria et al., 1997; Sousa, 1998). A unique feature shared by most of these plant proteinases is an extra segment of about 100 amino acid residues which bears no sequence similarity with proteinases of mammalian or microbial origins (Faro, Ver!ıssimo, Lin, Tang, & Pires, 1995). Many aspartic and other proteinases are obtained from plants and some of them have been studied as coagulants, i.e., proteinases from Benincasa cerifera (Gupta & Eskin, 1977), Calotropis procera (Ibiama & Griffiths, 1987; Mohamed & O’Connor, 1999), Dieffenbachia maculata (Padmanabhan, Chitre, & Shastri, 1993), fruit parts of Solanum dobium (Yousif, McMahon, & Shammet, 1996), Centaurea calcitrapa (Tavaria et al., 1997) and flowers of Cynara cardunculus (Barbosa, 1983; Sousa, 1993; Sousa & Malcata, 1997a, b, 1998a, b, Sousa, 1998). Although most plant coagulant preparations were reported to have an excessively low ratio of milk clotting to proteolytic activity, which results in bitter peptides in ripened cheese, or to an excessively low clotting power that gives rise to low cheese yields. The difficulties experienced with these preparations result mainly from the unique composition of the plant extracts, which contain a complex cocktail of enzymes whose activity is difficult to control.

2.1.2. Cynara cardunculus coagulant An exception from the other plant proteases is the proteases from dried flowers of C. cardunculus, which have milk-clotting activity and have been employed successfully for many centuries in the Iberian Peninsula for the manufacture of traditional cheeses, e.g., Serra da Estrela (Roseiro, 1991; Macedo, Malcata, & Oliveira, * 1993a), La Serena (Nunez, Fern!andez del Pozo, Rodriguez-Marin, Gaya, & Medina, 1991; Roa, ! Lopez, & Mendiola, 1999), Gu!ıa (Fern!andez-Salguero, Sanju!an, & Montero, 1991) and Los Pedroches (Carmona, Sanjuan, Gomez, & Fern!andez-Salguero, 1999; Fern!andez-Salguero & Sanju!an, 1999; Vioque et al., 2000). In the last years, the specificity of proteinases from 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 in cheeses manufactured from ovine milk and from ovine or caprine milk (Sousa & Malcata, 1997a, b; 1998a; Sousa, 1998). Extracts from C. cardunculus were reported to contain two proteinases, cardosin A and cardosin B (Sousa, 1993; Ver!ıssimo, Esteves, Faro, & Pires, 1995; Ver!ıssimo et al., 1996). Studies on their specificity and kinetics on the oxidised B-chain of insulin showed that cardosin A has a cleavage specificity similar to chymosin, whereas cardosin B resembles pepsin (Faro et al., 1992; Ver!ıssimo et al., 1995; Ramalho-Santos, Ver!ıssimo, Faro, & Pires, 1996). The isolation and characterisation of cDNA from flowers of C. cardunculus was reported and, more recently, cloning and characterisation of cDNA enconding cardosin A has also been reported (Cordeiro, Xue, Pietrzak, Pais, & Brodelius, 1994; Faro et al., 1999). Enzymes from extracts of dried flowers of C. cardunculus cleave the Phe105-Met106 bond of kcasein (Faro et al., 1992; Macedo, Faro, & Pires, 1993b; Sousa & Malcata, 1998b). The primary site cleaved by proteinases from C. cardunculus in bovine as1 -casein is Phe23-Phe24 (Sousa, 1993). Macedo et al. (1996) reported that proteinases from C. cardunculus were able to cleave nine bonds, viz., Phe23-Phe24, Tyr153-Tyr154, Trp164-Tyr165, Tyr165-Tyr166, Tyr166-Val167, Phe145Tyr146, Leu149-Phe150, Leu156-Asp157 and Ala163-Trp164, whereas chymosin could only cleave the Phe23-Phe24 bond of as1 -casein under the same experimental conditions. Under several ionic conditions (pH 6.5, 5.5 in the absence of NaCl and at pH 5.2 with 5% NaCl), the major cleavage sites of proteinases from C. cardunculus were Phe23-Val24 for ovine and Phe23-Val24, Trp164Tyr165 and Tyr173-Thr174, caprine as1 -casein (Sousa & Malcata, 1998b). as2 -Caseins are cleaved by proteinases from C. cardunculus at the bonds Phe88-Tyr89, and Ser9Ser10, Phe88-Tyr89 and Tyr179-Leu180 in ovine and caprine caseins, respectively (Sousa & Malcata, 1998b). Proteinases from C. carduculus cleave six bonds in

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bovine b-casein and the relative susceptibility to attack is, in decreasing order, Leu192-Tyr193, Leu191-Leu192, Leu165-Ser166, Phe190-Leu191, Ala189-Phe190 and Leu127Thr128 (Macedo et al., 1996), whereas under the same experimental conditions, Carles and Ribadeau-Dumas (1984) found that only the bonds Ala189-Phe190 and Leu192-Tyr193 were cleaved by chymosin. The major cleavage sites in ovine b-casein of proteinases from C. cardunculus at pH 6.5 or 5.5 in the absence of NaCl and at pH 5.2 with 5% NaCl were Leu127-Thr128 and Leu190-Tyr191 and in caprine b-casein were Glu100Thr101, Leu127-Thr128, Leu136-Pro137 and Leu190-Tyr191 (Sousa & Malcata, 1998b). It is worth noting the proteinases of C. cardunculus cleave all bonds in certain extremely hydrophobic regions of as1 -casein (Ala163Trp-Tyr-Tyr-Val167) and b-casein (Ala189-Phe-Leu-LeuTyr193), whereas chymosin cleaves only Trp164-Tyr165 in this region of as1 -casein and Ala189-Phe190 and Leu192Tyr193 in this region of b-casein. It was suggested that proteinases from C. cardunculus displayed a stronger preference for bonds between bulky hydrophobic residues 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 and completely inhibited by 10% NaCl, but the effect is due to modification of the substrate rather than the enzyme. Kelly, Fox, and McSweeney (1996) and . and Qvist (1999) Kristiansen, Deding, Jensen, Ardo, reported that the degradation of b-casein by the coagulant in cheese was affected by the salt content, as unsalted cheese contained less intact b-casein than the salted cheese and that the C-terminal fragment of b-casein, b-CN(f193–209), which is known to be bitter and produced by chymosin, was only formed in unsalted cheese. 2.2. Indigenous milk proteinases 2.2.1. Plasmin The dominant indigenous milk proteinase, plasmin (fibrinolysin, EC 3.4.21.7), has been the subject of much study and throughly described in recent reviews (see Bastian & Brown, 1996; Kelly & McSweeney, 2001 for reviews). Plasmin activity differs substantially between cheese varieties; Richardson and Pearce (1981) reported that Swiss and Cheddar contained 6–13 and 3–4.5 mg plasmin/g cheese, respectively and that the elevated plasmin activity in ‘‘high cook’’ cheese varieties (e.g., Swiss) has been attributed to thermal inactivation of inhibitors of plasminogen activators, resulting in the increased conversion of plasminogen, the inactive precursor of plasmin, to the active enzyme. The primary cleavage sites of plasmin on b-casein are Lys28-Lys29, Lys105-His106 and Lys107-Glu108 the cleavage of which yields b-CN(f29–209) (g1 -CN), b-CN(f106–209) (g2 -CN)

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and b-CN(f108–209) (g3 -CN) (Eigel et al., 1984). The proteose peptone (PP) components 5 (b-CN(f1–105) and b-CN(f1–107)), PP-8 fast b-CN(f1–28) and PP-8 slow (b-CN(f29–105) and b-CN(f29–107)) are the corresponding N-terminal fragments which accumulate in milk on storage. Plasmin cleaves as2 -casein in solution at eight sites. Although k-casein contains several Lys and Arg residues, it appears to be quite resistant to plasmin action. 2.2.2. Cathepsin D Milk contains an indigenous acid proteinase, first recognised by Kaminogawa and Yamauchi (1972) as cathepsin D, and later identified as procathepsin D (procathepsin is the proenzyme form of the lysosomal proteinase, cathepsin D; Larsen, Benfeldt, Rasmussen, & Petersen, 1996). The literature on cathepsin D has been reviewed recently by Hurley, Larsen, Kelly, and McSweeney (2000a) and Kelly and McSweeney (2001). Cathepsin D (EC 3.4.23.5) is an aspartic proteinase with an optimum pH of 4.0 on haemoglobin and optimum temperature of 371C (Kaminogawa & Yamauchi, 1972; Barrett, 1972). Cathepsin D produces the glycomacropeptide, k-CN(f106–169), that also is produced by chymosin by the enzymatic cleavage of the Phe105-Met106 bond (McSweeney, Fox, & Olson, 1995), and two more cleavage sites of cathepsin D on k-casein have been identified, i.e., Leu32-Ser33 and Leu79-Ser80 (Larsen et al., 1996). As the specificity of cathepsin D is similar to that of chymosin, one might expect that it possesses the ability to coagulate milk. The milk clotting potential of cathepsin D, however, has been reported to be very poor (McSweeney et al., 1995; Larsen et al., 1996). Larsen et al. (1996) reported that the enzyme was capable of coagulating milk over the pH values examined (pH 5.0– 6.5), with coagulation time decreasing as expected with decreasing pH. The level of cathepsin D present (around 0.4 mg/mL) in milk though, is far too low to be of significance with respect to milk coagulation. Cathepsin D and chymosin had similar cleavage sites on as1 -casein, i.e., Phe23-Phe24, Phe24-Val25, Leu98-Leu99 and Leu149-Phe150 (Larsen et al., 1996), cathepsin D hydrolysates of as2 -casein differ markedly from those produced by chymosin (McSweeney et al., 1995). The enzyme cleaves as2 -casein at Leu99-Tyr100, Leu123Asn124, Leu180-Lys181 and Thr182-Val183 (Larsen et al., 1996). Proteolysis of b-casein by cathepsin D is similar to that by chymosin, with b-CN(f1–192) being the primary product and b-CN(f1–163/165/167) also being formed. In total, 13 sites have been identified in b-casein that are cleaved by cathepsin D, viz., Phe52-Ala53, Leu58Val59, Pro81-Val82, Ser96-Lys97, Leu125-Thr126, Leu127Thr128, Trp143-Met144, Phe157-Pro158, Ser161-Val162, Leu165-Ser166, Leu191-Leu192, Leu192-Tyr193 and Phe205Pro206 (Larsen et al., 1996).

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Cathepsin D was considered to be a relatively heat labile enzyme and Kaminogawa and Yamauchi (1972) reported its complete inactivatation at 701C  10 min. Recent studies on heat stability reported that B8% cathepsin D activity in skim milk survived pasteurisation (721C  15 s) (Larsen et al., 2000; Hayes et al., 2001) suggesting that this enzyme may play a minor proteolytic role in dairy products prepared from pasteurised milk. In rennet free-cheeses, formation of as1 -CN(f24– 199) has been attributed to the activity of cathepsin D (Visser & de Groot-Mostert, 1977). Wium, Kristiansen, and Qvist (1998) reported cathepsin D-like activity in Feta cheese made without rennet addition, from milk that was pasteurised (721C  15 s), ultrafiltered (501C), pasteurised again, stored at 41C overnight and pasteurised again and homogenised, confirming that some cathepsin D activity can survive pasteurisation. The presence of procathepsin D in the ripened UF-Feta was confirmed using immunological methods (Larsen et al., 2000). Hurley, Larsen, Kelly, and McSweeney (2000b) reported the presence of cathepsin D or procathepsin D in Quarg cheese samples produced from raw, pasteurised or raw ultrafiltered skim (except those to which pepstain had been added) and peptides thought to be produced as result of cathepsin D were observed in cheese made from raw and pasteurised milk. Cathepsin D activity is obvious in cheese varieties where no rennet was added, but it is hard to quantify the contribution of cathepsin D to the ripening of cheese varieties such as Cheddar, wherein the activity would be masked by far greater levels of chymosin (Hurley, 1999; Hayes et al., 2001). In Swiss cheese, chymosin is believed to be substantially inactivated by the high cooking regime (temperatures 53–551C for up to 1 h) and the absence of rennet-like activity with little or no degradation of as1 casein to as1 -I-casein might be expected; however, the presence of as1 -I-casein has been reported in Swiss cheese (Beuvier et al., 1997; McGoldrick & Fox, 1999) which may be, at least partly, a result of cathepsin D activity. 2.2.3. Other milk proteases In addition to cathepsin D, other proteolytic enzymes are present in lysosomes of somatic cells and may contribute to proteolysis in cheese. One of the principal enzymes found in polymorphonuclear granulocytes (PMN cells or neutrophils) is the serine proteinase, elastase (Verdi & Barbano, 1991). Elastase has a broad specificity on b- and as1 -caseins, cleaving 19 and 25 sites, respectively (Considine, Healy, Kelly, & McSweeney, 1999, 2000). Elastase hydrolyses b-casein and some of the cleavage sites are identical to or near those cleaved by plasmin, chymosin or cell envelope-associated proteinases of several strains of Lactococcus. Most of the cleavage sites were found to be located near the N- 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, Lys97Val98, Ala101-Met102, Glu108-Met109, Phe119-Thr120, Glu131-Asn132, Leu163-Ser164, Ala189-Phe190, Phe190Leu191 and Pro204-Phe205 (Considine et al., 1999). Therefore, it is possible that indigenous elastase in milk may be of significance to the proteolysis of milk proteins. Recently, we have identified immunoreactive procathepsin B in milk (Magboul, Larsen, McSweeney, & Kelly, 2001). Cathepsin B is capable of extensively degrading as1 - and b-caseins in vitro and its specificity is known (Considine, 2000). Indigenous proteolytic enzymes and their role in cheese ripening is discussed in more detail in Kelly and McSweeney (2001). 2.3. Starter proteinases and peptidases from Lactococcus and Lactobacillus The starter cultures commonly used in cheese manufacture include mesophilic Lactococcus and Leuconostoc species, thermophilic Lactobacillus species and Streptococcus thermophilus. The principal role of the starter culture is in the production of lactic acid, causing a decrease in pH. Although lactic acid bacteria (LAB) are weakly proteolytic, they possess a very comprehensive proteinase/peptidase system (Fig. 3) capable of hydrolysing oligopeptides to small peptides and amino acids and this subject has been studied extensively and reviewed 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 this review. LAB possess a cell envelope-associated proteinase (CEP, lactocepin, PrtP), intracellular oligoendopeptidases (PepO) and (PepF), at least three general aminopeptidases (PepN, PepC, PepG), glutamyl aminopeptidase (PepA), pyrolidone carboxylyl peptidase (PCP), leucyl aminopeptidase (PepL), X-prolyldipepti-

Fig. 3. Schematic representation of the proteolytic system of lactococcus.

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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 a peptide and amino acid transport systems (Fig. 3). This proteolytic system is necessary to enable the LAB to grow to high numbers in milk (109–1010 cfu/mL), which contains only low levels of small peptides and free amino acids. The physiological role of individual peptidases has been investigated using single and multiple peptidase deletion mutants constructed in L. helveticus and L. lactis and a general decrease in growth rates has been reported when the mutants were evaluated in milk (Christensen et al., 1999). McGarry et al. (1994) studied the role of PepN in cheese ripening by manufacturing Cheddar cheese using strains of Lactococcus engineered to overproduce PepN; these authors reported no significant changes in terms of body, texture, or flavour characteristics between cheeses made with a PepN superproducer and control strains. Likewise, Christensen, Johnson, and Steele (1995) manufactured Cheddar cheese using strains engineered to overproduce PepN. Although higher levels of free amino acids were found in this cheese relative to the control made with a wild-type starter, no significant sensory differences were found. However, Meyer and Spahni (1998), who studied the influence of PepX on Gruy"ere cheese ripening by making model cheeses using PepX+ and PepX strains of L. delbrueckii subsp. lactis, reported that PepX influenced proteolysis and sensory characteristics of this variety. 2.4. Non-starter lactic acid bacteria proteolytic systems During the maturation of Cheddar and many other cheeses, the starter lactococcal population declines and the initially small population of adventitious non-starter lactic acid bacteria (NSLAB) ultimately becomes the dominant bacterial population in the maturing cheese (Peterson & Marshall, 1990; Martley & Crow, 1993; Fox, McSweeney, & Lynch, 1998). NSLAB, although present initially at low numbers (o50 cfu/g in Cheddar made from pasteurised milk), grow rapidly to reach B107 cfu/g within 4 weeks and this number remains relatively constant thereafter (Folkertsma, Fox, & McSweeney, 1996). Thus, depending on the rate of death of the starter, NSLAB can dominate the viable microflora of Cheddar throughout most of the ripening period. The proteolytic activity of NSLAB appears to supplement that of the starter, producing peptides with generally similar molecular weights, and free amino acids (Lane & Fox, 1996; Lynch, McSweeney, Fox, Cogan, & Drinan, 1997; Williams & Banks, 1997; Williams, Xavier, & Banks, 1998; Muehlenkamp-Ulate & Warthesen, 1999). Peptidolytic strains of NSLAB may therefore be considered for use as adjuncts in

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cheesemaking both to manipulate the overall flavour profile of the cheese and to accelerate the rate of flavour formation (Fox et al., 1998; Fox & Tobin, 1999; Madkor, Tong, & El Soda, 2000). The criteria for selection of adjuncts are often not defined and frequently isolates from a good-quality cheese have been selected for evaluation. However, there is a need to identify the proteolytic and lipolytic enzyme systems of the NSLAB that could potentially contribute to the overall maturation process. Recently, particular interest has been shown in the characterisation of individual proteolytic enzymes produced by strains of non-starter Lactobacillus spp. isolated from cheese (reviewed by Kunji et al., 1996; Christensen et al., 1999). 2.5. Proteolytic agents of cultures for specific cheese varietiesFP. roqueforti, P. camemberti, B. linens, Propionibacterium and yeasts In many cheese varieties, secondary cultures are added intentionally and/or encouraged to grow by controlling environmental conditions. These cultures have a diverse range of functions, depending on the organisms, but the main difference between them and the starter cultures is that they are not added to acidify the cheese, i.e., to produce lactic acid. These secondary cultures can grow on the surface in the case of smearripened (Tilsit, Gruy"ere, Appenzeller, Limburger, etc.) and mould-ripened (e.g., Camembert, Brie) cheeses or produce CO2 (eye formation), propionate, and acetate in the case of Swiss varieties (e.g., Emmental and Comt!e). The principal secondary microorganisms contributing to cheese ripening are P. roqueforti (Blue mould cheese), P. camemberti (surface mould cheese such as Camembert and Brie), B. linens, Arthrobacter and other coryneform bacteria and several species of yeasts (Geotrichum candidum, Kluyveromyces marxianus and Debaryomyces hansenii) in surface smear-ripened cheeses, Propionibacterium freudenreichii subsp. shermanii (Swiss-type cheese). Nowadays, the milk for mould-ripened varieties is inoculated with a pure culture of P. roqueforti, in the case of Blue cheeses, or P. camemberti, in the case of Camembert and Brie, at the same time as starters. For surface- or smear-ripened cheeses, like Tilsit, Munster and Limburger are dipped, sprayed or brushed with aqueous suspensions of G. candidum and B. linens as soon as the cheeses are removed from the brine and are then ripened at 10–151C at high relative humidity. However, the Gram-positive bacterial flora, which grows on the cheese surface is very complex and contains many adventitious strains. The surface microflora of smear-ripened cheese has two important functions: (i) production of enzymes (lipases, proteinases and peptidases) and (ii) deacidification of first the cheese surface and then the cheese body.

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During the first few days of ripening of smear-ripened cheese, yeast and moulds grow on the cheese surface and deacidify it by oxidizing the lactate to H2O and CO2 (Reps, 1993; Eliskases-Lechner & Ginzinger, 1995). Yeasts grow particularly well because of their tolerance to 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 and temperature (12–241C) in the ripening rooms (Welthagen & 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 be important bacteria growing on the surface of smearripened cheeses; for this reason D. hansenii and B. linens are commonly used as ripening starters and enzymes from the latter have been studied. B. linens secrete an extracellular proteinase and an aminopeptidase, possess a number of intracellular peptidases, which may be released on cell lysis (Rattray & Fox, 1997, 1999) and the specificity of the extracellular proteinases from B. 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 of its aromatic and proteolytic properties and its bright orange pigments, but considering the low proportion of brevibacteria on many semi-hard smear-ripened cheeses, its direct contribution to proteolysis might be rather limited (Bockelmann et al., 1997a,b; Bockelmann, Hoppe-Seyler, Lick, & Heller, 1998). P. roqueforti and P. camemberti secrete aspartyl and metalloproteinases, 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 but strongly peptidolytic and they are particularly active on proline-containing peptides during the ripening of Swiss-type cheeses, which may contribute to the characteristic flavour of these cheeses. PepN and PepI were detected in Propionibacterium and PepX and three endopeptidases have been isolated and characterised from at least one strain of P. freudenreichii subsp. shermanii which may be active in cheese during ripening (El-Soda, Chen, Riesterer, & Olson, 1991; Ezzat, ElSoda, & El-Shafei, 1994; Tobiassen, Stepaniak, & Srhaug, 1996; Fern!andez-Espl!a & Fox, 1997; Stepaniak, Gobbetti, Pripp, & Srhaug, 1998a; Stepaniak, Tobiassen, Chukwu, Pripp, & Srhaug, 1998b).

3. Comparison of proteolysis within and between cheese varieties Proteolysis has been considered by some researchers as a basis for the classification of cheese. Several indices

of proteolysis could be useful for classification, however an obvious difficulty is the fact that cheese is a dynamic system and therefore the results obtained depend to a large extent on the age of the cheese when analysed ! & Fern!andez-Salguero, 1979; (Marcos, Esteban, Leon, Smith & Nakai, 1990; Fox, 1993; McGoldrick & Fox, . 1999a; Pripp, Stepaniak, & Srhaug, 1999; Ardo, 2000c). Several studies have shown differences in proteolysis between cheese varieties. Marcos et al. (1979) compared proteolysis in several cheese varieties by analysing gel electrophoregrams and reported that, in general, as1 -casein was degraded more extensively than b-casein. These authors reported that in cheeses in which b-casein was degraded less extensively (e.g., Parmesan, Emmental, Gruyere and Tilsit), the concentrations of g1 - and g2 -caseins were high, while in cheeses were almost all b-casein had been degraded (i.e., Roquefort), less g1 -casein and more g2 - and in particular, g3 -casein were present indicating more extensive plasmin action. The ratio of b- to g-caseins has been suggested as a basis for cheese classification (Fox, 1993). Smith and Nakai (1990) classified Cheddar, Edam, Gouda, Swiss, and Parmesan cheeses by multivariate analysis of their HPLC profiles. Fox (1993) reported distinct intervarietal differences between Cheddar, Emmental, Gouda, Parmesan, Brie, and Appenzeller based on their RP-HPLC profiles. McGoldrick and Fox (1999) studied proteolysis in different varieties of cheese (Cheddar, British Territorial, Dutch, Swiss and Italian varieties) by ureaPAGE and by RP-HPLC and reported that RP-HPLC of the 70% ethanol-soluble or insoluble fractions of the cheese was more effective than urea-PAGE when classifying cheese according to variety. These authors reported that urea-PAGE of the water-insoluble fraction of cheese was unable to distinguish Emmental from Parmesan and both of these from Cheddar and Dutch types, but urea-PAGE of the 70% ethanol-soluble fraction showed large differences between cheese varieties but there were also differences within the same variety. Dellano, Polo, and Ramos (1995) separated artisanal cheeses (Afuega’Pitu, Beyos, Vidiago, Cabrales and Peral) based on their peptide profile throughout ripening. Zarmpoutis, McSweeney, and Fox (1997) compared proteolysis in blue-veined cheese varieties (Stilton, Gorgonzola, Danablu, and the Irish farmhouse varieties Cashel and Chetwynd) and reported that all cheeses showed high extent of proteolysis, but Gorgonzola showed higher levels of amino acids than the other varieties. Grappin et al. (1999) compared proteolysis in Swiss cheeses and reported that Emmental showed the highest 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 of as1 -CN(f24–199) were found in Emmental (indicating lower plasmin and higher chymosin activity, respec-

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tively) than in Comt!e and Beaufort. These authors suggested that secondary proteolysis was lower in Emmental than in Comt!e and Beaufort and that Comt!e had a higher level of medium sized peptides in comparison with Beaufort cheese. Six varieties of cheeses, Beaufort, Parmigiano-Reggiano, Appenzeller, Fontina, Comt!e and Mahon, were compared in terms of indices of proteolysis (nitrogen content of different fractions and urea-PAGE) by cluster analysis allowing qualitative and quantitative comparison between cheeses (Lopez-Fandino, Martin-Alvarez, Pueyo, & Ramos, 1994). Belgian cheeses (Passendale, Wijnendale, Nazareth and Oud Brugge) were differentiated based on SDS-PAGE of the pH 4.6-insoluble fractions of cheese (Dewettinck, Dierckx, Eichwalder, & Huyghebaert, 1997), allowing not only qualitative, but also quantitative classification, resulting in a clear separation of Nazareth and Oud Brugge and to a lesser extent of Passendale and Wijnendale. Indices of proteolysis are also useful to discriminate within a particular variety between cheeses of different quality or made with different starters. O’Shea, Uniacke-Lowe, and Fox (1996) compared Cheddar cheeses varying in age and quality in terms of their peptide profiles by HPLC, total free amino acid contents and additional information from compositional analysis and urea-PAGE. Using this approach, these authors discriminated between mild, mature and extra-mature Cheddar cheeses. Grappin et al. (1999) reported on the comparison of 20 Comt!e cheeses made in five cheese plants, with either wild starters made locally or with the same commercial starter, and ripened under the same conditions, and were able to discriminate them according to their physico-chemical variables, proteolysis, microbiological counts and sensory characteristics, showing the importance of milk characteristics and cheesemaking conditions to the final characteristics of cheese. In recent years, a promising approach in the area of cheese ripening research is the application of multivariate analysis (e.g., PCA and PLS) to proteolytic patterns to model quantitative relationships (No.el et al., 1998; Molina, Martin-Alv!arez, & Ramos, 1999a; Pripp, Stepaniak, & Srhaug, 2000c). As well as discriminating between cheeses, chemometrical analysis of proteolytic profiles can be used generally as a powerful method to better understand proteolysis in cheese and how this process is influenced by factors including type of starter, physico-chemical variables, microbial counts, cheesemaking parameters, age, quality and sensory characteristics. Using multivariate statistical analysis of heights or areas of peaks in the CE and RP-HPLC profiles, the quantitative contribution of rennet and bacterial proteolytic enzymes to proteolysis in model sodium caseinate systems under cheese-like conditions was demonstrated,

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as well as the effect of single strains of Lactococcus on proteolysis 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, McSweeney, & Fox, 1999; Pripp, McSweeney, Srhaug, & Fox, 2000a; Pripp, Shakeel-Ur-Rehman, McSweeney, Srhaug, & Fox, 2000b).

4. Identification of peptides and patterns of proteolysis in cheese The extent and type of proteolysis in a number of the principal cheese varieties has been characterised. However, complete characterisation of proteolysis in cheese requires isolation and identification of individual peptides. Using various extraction techniques and methods to isolate individual peptides (i.e., urea-PAGE, HPLC and CE) (Fig. 1), many of the water-insoluble and water-soluble peptides have been isolated from cheese and identified, using amino acid sequencing and mass spectrometry. 4.1. Cheddar Proteolysis in Cheddar cheese is well characterised as reviewed by Fox, Singh, and McSweeney (1994), McSweeney, Pochet, Fox, and Healy (1994), Singh et al. (1994) Singh, Fox, and Healy (1995, 1997), Fern!andez, Singh, and Fox (1998) and Mooney, Fox, Healy, and Leaver (1998) and peptides isolated are summarised in Fig. 4 and 5. The major water-insoluble peptides are produced either from as1 -casein by chymosin or from b-casein by plasmin and some are degraded further by the lactococcal CEP (Fig. 4). as1 -Casein in Cheddar cheese is rapidly and completely hydrolysed by chymosin at the Phe23-Phe24 bond. The larger peptide, as1 -CN(f24–199) produced by the cleavage of Phe23Phe24, is further hydrolysed by chymosin at the bond Leu101-Lys102 and, to a lesser extent, at Phe32-Gly33 and Leu109-Glu110, and perhaps by plasmin at Lys103-Tyr104 and Lys105-Val106. The large C-terminal peptides, as1 -CN(f24–199), as1 -CN(f33–199), as1 -CN(f102–199) and as1 -CN(f110–199) and as1 -CN(f99–199), as1 CN(f104–199) and as1 -CN(f106–199) have been identified in the water-insoluble fraction of Cheddar cheese (McSweeney et al., 1994; Mooney et al., 1998). The complementary N-terminal peptides (e.g., as1 -CN(f24– 98), as1 -CN(f24–101) and as1 -CN(f24–109)) could not be identified in the water-insoluble fraction, but since these are highly phosphorylated, they may be in the watersoluble extract. Surprisingly, the bond Trp164-Tyr165, which is hydrolysed rapidly by chymosin in as1 -casein in solution (McSweeney, Olson, Fox, Healy, & Hjrup, 1993b), does not appear to be hydrolysed in cheese; at

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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).

least a peptide with Tyr165 as its N-terminal has not yet been identified neither in water-soluble fraction nor in model cheese systems (Singh et al., 1994, 1995, 1997; Exterkate, Alting, & Slangen, 1995; Exterkate, Lagerwerf, Haverkamp, & Schalkwijk, 1997; Fern!andez et al., 1998). Perhaps, the bond Trp164-Tyr165 is difficult for chymosin to access in cheese, perhaps due to intermolecular interactions. The peptide as1 -CN(f1–23) is hydrolysed rapidly by the lactococcal CEP at the bonds Gln9-Gly10, Gln13Glu14, Glu14-Val15 and Leu16-Asn17, and probably at other sites, depending on the specificity of the enzyme (Exterkate & Alting, 1993; Exterkate et al., 1995). The peptides as1 -CN(f1–9), as1 -CN(f1–13) and as1 -CN(f1– 14) accumulate and dominate the RP-HPLC chromatograms of UF permeable or 70% ethanol-soluble fraction of the WSF of Cheddar, whereas degradation products of the complementary C-terminal peptides have been identified in the UF permeate, and some of them have been partially hydrolysed by an aminopeptidase, releasing amino acids (Singh et al., 1994, 1995, 1997; Fern!andez et al., 1998). In Cheddar and many other cheeses, b-casein is much more resistant to hydrolysis than as1 -casein; only B50% of b-casein in Cheddar cheese is hydrolysed. b-Casein is hydrolysed mainly by plasmin at Lys28-Lys29, Lys105-Gln106 and Lys107-

Glu108, producing the fragments b-CN(f29–209), b-CN(f106–209) and b-CN(f108–209) (g1 -, g2 -, and g3 -, respectively). The g-caseins are present in the waterinsoluble fraction of Cheddar (McSweeney et al., 1994; McGoldrick & Fox, 1999), whereas degradation products of proteose peptone 8 fast (b-CN(f1–28)), 8 slow (b-CN(f29–105/107)) and 5 (b-CN(f1–105/107)), are present in the UF retentate or 70% ethanol-insoluble fraction of the WSE (Fox & Wallace, 1997). Most of the peptides that have been identified in the UF retentate (or 70% ethanol-insoluble fraction of the WSE are produced from b-casein by the action of lactococcal CEP, probably on proteose peptones rather than on intact b-casein, since none of the peptides identified contained an intact plasmin cleavage site (Singh et al., 1997; Mooney et al., 1998; Fox & Wallace, 1997). The concentration of as2 -casein appears to decrease during ripening but no large peptides derived from as2 casein have yet been reported (Mooney et al., 1998), and only a few small peptides derived from as2 -casein have been identified in the WSE (Singh et al., 1995, 1997, 1999). During the last few years, considerable progress has been made on fractionating and characterizing the water-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

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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).

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water-insoluble peptides, the water-soluble peptide profiles are unique to the variety and presumably reflect the specificity of the starter and non-starter proteinases and peptidases (from Lactococcus and Lactobacillus; Fox & McSweeney, 1996). Most of the peptides from the WSF of Cheddar are derived from the N-terminal half of b-casein (especially from residues 53 to 91), with a smaller number from the N-terminal half of as1 -casein (Singh et al., 1997) (see Fig. 5c). The N-terminal of most of these peptides corresponds to a cleavage site for chymosin (as1 -casein), plasmin (b-casein), or lactococcal CEP. Alli, Okoniewska, Gibbs, and Konishi (1998) reported the identification in Cheddar cheese of 13 peptides from as1 -casein, 7 from b-casein and 5 from k-casein, by electrospray ionisation mass spectrometry. Thus, the large water-insoluble peptides in Cheddar are from the C-terminal segments of as1 -casein produced mainly by chymosin or from b-casein (g-caseins) produced by plasmin. Para-k-casein (k-CN(f1–105)) is not hydrolysed during ripening. The small watersoluble peptides appear to be peptides produced by the action of the lactococcal CEP, or perhaps endopeptidases, from products of chymosin or plasmin action. NSLAB supplement the peptidolytic activity of the starter, especially in the production of amino acids. 4.2. Blue cheese Very extensive proteolysis occurs in blue-mould cheeses; both as1 - and b-caseins are hydrolysed completely, and most of the principal water-insoluble peptides have different mobilities from those in Cheddar and some have been identified partially (Gripon, 1993). Initial proteolysis is due mainly to chymosin, and as1 -CN(f24–199) is the major large peptide produced; but following sporulation of P. roqueforti at about 15 days, its extracellular proteinases become dominant and peptides with very low mobility are produced as a result of their proteolytic activity (Gripon, 1993). However, only little work has been done on the small (pH 4.6soluble) peptides of blue cheese, i.e., PTA-soluble peptides from Gamonedo Blue cheese (Gonz!alez de Llano, Polo, & Ramos, 1991) and the production and identification of PTA-soluble peptides in blue cheese by HPLC (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 amino acids represent B25% of total N), probably due mainly to its long ripening time and the action of thermophilic Lactobacillus proteinases and plasmin since chymosin is largely inactivated during cooking (Battistotti & Corradini, 1993; Bertozzi & Panari, 1993). In Parmigiano-

Reggiano, urea-PAGE showed limited breakdown of as1 -casein but a peptide with mobility similar to as1 -CN(f24–199) could have been produced by a low level of residual chymosin or by cathepsin D (Fox & McSweeney, 1996). Electrophoregrams showed that b-casein is hydrolysed rapidly during the first months of ripening (Bertozzi & Panari, 1993); consequently, the g2 - and g3 -caseins continue to increase, and g1 -casein tends to decrease, confirming the important role of plasmin in maturation (Fox & McSweeney, 1996). Addeo et al. (1992) reported the isolation and identification of low molecular mass peptides formed during the ripening of Parmagiano-Reggiano cheese. Using fast atom bombardment-mass spectrometry (FAB-MS), it was found that the majority of the oligopeptides are produced from regions 1–20 and 6–28 of b-casein (i.e., b-CN(f1–20), b-CN(f7–28), b-CN(f2–6), b-CN(f8–28), b-CN(f9–28), b-CN(f2–20), b-CN(f3–20), b-CN(f4–20), b-CN(f5–20), b-CN(f5–14), b-CN(f15–28)), 5 phosphopeptides were produced from the region 64–84 of as1 -casein (as1 -CN(f63–74), as1 -CN(f64–74), as1 -CN (f70–78), as1 -CN(f71–78), as1 -CN(f64–84)), 3 phosphopeptides from the region 1–21/24 of as2 -casein (as2 -CN (f6–18), as2 -CN(f7–18), as2 -CN(f8–18) and 1 peptide from the C-terminal part of as2 -casein (as2 -CN(f172–178)) (Addeo et al., 1992, 1994, 1995). Ferranti et al. (1997) isolated a total of 45 phosphopeptides from GranaPadano; 24 originated from b-casein, 16 from as1 -casein and 5 from as2 -casein. The casein phosphopeptides were reported to consist of a mixture of components derived from three parent peptides, b-CN(f7–28)4P, as1 -CN(f61– 79)4P and as2 -CN(f7–21)4P.

4.4. Serra da Estrela Serra da Estrela has primary proteolysis of about 35% of total N (TN) soluble in water, but secondary proteolysis of about 6% of TN soluble in 12% trichloroacetic acid; 1.2% of TN is soluble in 5% phosphotungstic acid. Lower extent of primary proteolysis was found when milk was coagulated with proteinases from C. cardunculus than with animal rennet (Sousa & Malcata, 1997a). The primary cleavage sites were reported to be at Leu190-Tyr191 in ovine b-casein and Phe23-Val24 in ovine as1 -casein, producing the peptides b-CN(f1–190) and as1 -CN(f24–191), respectively; however the bond Phe23-Val24 in as1 -casein was cleaved earlier in cheese manufactured with plant rennet than with animal rennet, thus producing the peptides as1 -CN(f24–191) and as1 -CN(f24–165), respectively, perhaps with some implication for cheese texture. Significant effects of the type of milk on bovine, ovine and caprine cheeses with respect to primary and secondary proteolysis (Sousa & Malcata, 1997b) and on their peptide profiles and specificity were found in

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these cheeses manufactured with plant proteinases (Sousa & Malcata, 1997a, 1998a; Sousa, 1998). 4.5. Feta In Feta cheese, most of the peptides from the WSF was shown to originate from as1 -casein (as1 -CN(f1–14), as1 -CN(f4–14), as1 -CN(f24–30), as1 -CN(f24–32), as1 -CN (f40–49), as1 -CN(f91–98), as1 -CN(f102–109)), 2 peptides originated from the C-terminal of b-casein (b-CN (f164–180), b-CN(f191–205)) and 1 peptide from k-casein (k-CN(f96–105)). Most of the peptides could be explained on the basis of known specificity of chymosin and the lactococcal CEP (Michaelidou, Alchinidis, Urlaub, Polychroniadou, & Zerfiridis, 1998).

5. Contribution of proteolysis to the development of taste and flavour compounds Compounds which contribute to cheese flavour are added or are produced during manufacture (e.g., lactic acid and NaCl) but are mainly formed as consequence of the many biochemical changes which occur during ripening; cheese taste is an important organoleptic attribute and the correct balance of sapid compounds is vital to cheese quality (McSweeney, 1997). Proteolysis contributes to the taste of cheese by the production of peptides and free amino acids and the sapid flavour compounds generally partition into the soluble fraction on extraction of cheese with water. Large peptides do not contribute directly to cheese flavour, but are important for the development of the correct texture; however, large peptides can be hydrolysed by proteinases to shorter peptides that may be sapid. Engels and Visser (1994) analysed water-soluble fractions from Cheddar, Edam, Gouda, Gruyere, Maasdam, Parmesan and Proosdij cheeses and suggested that low-molecular (o500 Da) compounds (small peptides, amino acids, free fatty acids or their breakdown products) were responsible for the basic taste of cheese. The exact role of these medium- and small-sized peptides in cheese flavour is not clear, although it is likely that they contribute to the background flavour of Cheddar, at least to a brothy or savoury flavour (see review by McSweeney, 1997). * (1999b) Molina, Ramos, Alonso, and Lopez Fandino further fractionated the water soluble fraction of cheeses made from cow’s, ewe’s and goat’s milk and assessed the contribution of small peptides, free amino acids and volatile components to cheese flavour. Differences were reported in intensity and predominance of individual tastes in the various fractions of cheeses made from milk of the three species; it was suggested that bovine milk cheeses were mainly salty and sour, ovine milk cheeses had predominant umami taste and caprine milk cheese

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was umami, astringent and bitter and the highest cheese flavour intensity was found in the fractions with the highest concentration of amino acids and volatile compounds (Molina et al., 1999b). g-Glutamyl peptides have been implicated in cheese flavour, although, no g-glutamyl bonds occur in the caseins, but g-Glu-Phe, g-Glu-Leu and g-Glu-Tyr (9, 20 and 70 mg/kg, respectively) were isolated from Comt"e cheese (RoudotAlgaron, Kerhoas, Le Bars, Eibhorn, & Gripon, 1994). The composition of the amino acid fraction and the relative proportions of individual amino acids are thought to be important for the development of the characteristic flavour (e.g., Broome, Krause, & Hickey, 1990; Engels & Visser, 1994; Molina et al., 1999b). However, the relative proportion of individual amino acids appears to be similar in many varieties and increasing the concentration of free amino acids in cheese does not necessarily accelerate ripening nor flavour intensity (McGarry et al., 1994; Christensen, et al., 1995). Fox and Wallace (1997) suggested that cheese flavour and the concentration of free amino acids could not be correlated, since different cheeses (e.g., Cheddar, Gouda and Edam) have very different flavours, although the concentration and relative proportions of free amino acids were generally similar. Bitterness in cheese is most often due to hydrophobic peptides and is generally regarded as a defect, although bitter notes may contribute to the desirable flavour of mature cheese. The literature concerning bitterness in dairy products has been reviewed by Lemieux and Simard (1991, 1992) and McSweeney, Nursten, and Urbach (1997) and this topic is only briefly summarised here from these reviews. Bitter peptides are formed mainly by the action of coagulant and starter proteinases and bitterness occurs in cheese when such peptides accumulate to an excessive concentration, either as a result of over production or of inadequate degradation by microbial enzymes. Certain sequences in the caseins are particularly hydrophobic and, when excised by proteinases, can lead to bitterness. Bitter peptides from as1 -casein are predominantly from the region of residues 14–34, 91–101 and 143–151, while bitter peptides from b-casein are mostly from the region of residues 46–90, and particularly from the hydrophobic C-terminus. Chymosin (or rennet substitutes) is important in the production of bitter peptides, since residual coagulant is the principal proteinase in many cheese varieties and its primary action on b-casein releases extremely hydrophobic peptides. Thus factors that affect the retention and activity of coagulant in the curd (e.g., pH or salt) may influence the development of bitterness. Bitter peptides may also be produced directly by starter proteinases and then accumulate in cheese due to the absence of peptidases from starter. ‘‘Bitter’’ starters may be unable to hydrolyse bitter peptides to non-bitter peptides that are too small to be perceived as bitter. Salt

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may also decrease bitterness by inhibiting lactococcal CEP and thereby promote the aggregation of large nonbitter, hydrophobic regions of the caseins (e.g., the C-terminal region of b-casein) and perhaps peptides, which would otherwise be degraded to bitter peptides. The addition of exogeneous proteinases, e.g., to accelerate ripening, often causes bitterness, while peptidases have been used to reduce the intensity of bitterness. Low-fat cheeses are susceptible to the development of bitterness, perhaps because, in full-fat cheese, hydrophobic bitter peptides are less likely to be perceived as being bitter due to partitioning into the fat phase. In addition to peptides, a number of other compounds can contribute to bitterness in cheese, including amino acids, amines, amides, substituted amides, longchain ketones and some monoglycerides (Adda, Gripon, & Vassal, 1982). Several amino acids are bitter, mainly those with non-polar or hydrophobic side chains, such as Ile, although Lys (potentially charged) and Tyr (polar but normally uncharged) are also considered to be bitter (McSweeney, 1997). Pro and Lys are reported to be bitter/sweet and Arg to be bitter (although this residue has a low hydrophobicity) whereas, Ala, Gly, Ser, and Thr are sweet; Glu, His and Asp are sour and Asp and Glu have the lowest taste thereshold. Catabolism of free amino acids plays an important role in flavour development in most varieties and general pathways for the catabolism 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 free amino acids can result in a number of compounds, including ammonia, amines, aldehydes, phenols, indole and alcohols, all of which may contribute to cheese flavour.

6. Future perspectives In the future, work may be expected to develop further methodology for studying proteolysis. Although there have been notable advances in their application and in data interpretation (chemometrics), the common analytical techniques for proteolysis (e.g., urea-PAGE, RP-HPLC and quantification of soluble N and free amino acids) have remained relatively unchanged over the last number of years. Technical development of instruments for capillary electrophoresis (CE) has advanced and is currently very promising. The trend of the application of analytical techniques developed for protein chemistry to cheese analysis will continue. A notable trend in recent years, which we feel will continue in the future, has been the study of proteolysis in different cheese varieties. This includes many cheese varieties produced in smaller quantities or in restricted geographical areas that now have been characterised

with respect to proteolysis, including the isolation and identification of some significant peptides. The identification of peptides from cheese using mass spectrometry and amino acid sequencing has only begun. Future work on the identification of peptides has the potential to increase our understanding of the ongoing processes in cheese and, as a consequence, also how to control them. The kinetics of peptide production and degradation is not well understood and thus it is likely that mathematical modelling techniques will be applied to study proteolysis in cheese during ripening. The genetics of lactic acid bacteria has been an area of very active research in the last decade. One product of this research has been the characterisation of the genes encoding the proteolytic system of cheese starter bacteria. This has led to a greater understanding of the contribution of individual enzymes to the growth of lactic acid bacteria in milk and their contribution to proteolysis in cheese during ripening. On-going research in this area will effect a much clearer picture of the role of these enzymes to cheese ripening. Finally, as discussed above, proteolysis contributes to cheese flavour mainly by producing free amino acids, which act as precursor compounds for further catabolic reactions. This will encourage further research into the link between proteolysis, i.e., the liberation of peptides and free amino acids from the caseins, and amino acid catabolism.

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