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International Dairy Journal 16 (2006) 775783 www.elsevier.com/locate/idairyj

Contribution of Geotrichum candidum to the proteolysis of soft cheese

Rachel Boutrou, Liliane Kerriou, Jean-Yves Gassi
UMR 1253 Science et Technologie du Lait et de lOeuf, INRA-AgroCampus Rennes, 65 rue de Saint Brieuc, 35042 Rennes Cedex, France Received 24 July 2004; accepted 18 July 2005

Abstract To determine the action of the yeast Geotrichum candidum on the proteolysis of soft cheese, Camembert-type cheeses were manufactured with and without this surface ora. Casein degradation and the release of peptides and amino acids at the cheese surface were studied to assess overall proteolysis. The results showed extensive proteolytic activity at the surface of cheese with G. candidum, and suggested that G. candidum is able to contribute to both primary and secondary proteolysis. as1- and bA2-caseins were preferentially hydrolysed at the surface of cheese with G. candidum from the rst week of ripening. This proteolytic activity led to the production of numerous peptides that were subsequently hydrolysed, as indicated by the large increase in the concentration of free amino acids from the second week to the end of ripening. r 2005 Elsevier Ltd. All rights reserved.
Keywords: Geotrichum candidum; Proteolysis; Soft cheese; Lactococcus lactis

1. Introduction The presence of surface ora gives a specic appearance to cheese and produces a typical aroma and taste, mainly due to lipolytic and proteolytic activities. Geotrichum candidum is a yeast-like fungus, the presence of which is desirable on the surface of semihard cheeses, mould-ripened and smeared soft cheeses (Eliskases-Lechner, Kossler, & Ginzinger, 1997; Gue guen, 1984). G. candidum is used as a starter in the dairy industry and colonises nearly all surface-ripened cheeses during the early stages of ripening (Berger, Khan, guen, 1984). Molimard, Martin, & Spinnler, 1999; Gue It starts to grow on the surface of the rind of Camembert, Pont 1Eve que, Mu nster, Limburger and Livarot cheeses as well as on fresh goats and ewes milk cheeses at the beginning of the cheese ripening process, and on semi-hard cheeses such as Saint Nectaire and guen, 1984). Reblochon (Gue
Corresponding author. Tel.: +33 2 23 48 53 46; fax: +33 2 23 48 53 50. E-mail address: rachel.boutrou@rennes.inra.fr (R. Boutrou).

The growth of G. candidum during ripening on experimental Camembert cheeses is rapid for the rst week, and then the population becomes more stable at about 107 cfu per gram of cheese (Molimard, Vassal, Bouvier, & Spinnler, 1995). Similarly, G. candidum is one of the main yeast species during the manufacturing and ripening of Armada cheese, being dominant in 1and 2-week-old cheeses (Tornadijo, Fresno, Sarmiento, & Carballo, 1998). At the age of 35 days, it reaches around 20% of the total ora on the cheese surface (Wyder, 1998). On some cheeses, G. candidum is responsible for the appearance of the cheese, imparting a uniform, white, guen & Schmidt, 1992). velvety coat to the surface (Gue The strain of G. candidum that predominates on a cheese rind helps to determine the texture, cohesiveness, and thickness of the rind. Some less desirable strains create unstable rinds that disintegrate when the young cheeses are turned. Since G. candidum occurs on the surface of soft cheeses such as Camembert, and of semi-hard cheeses such as Saint Nectaire and Reblochon, a contribution to the ripening process through its biochemical activities

0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.07.007

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can be expected. Indeed, it produces several enzymes for the breakdown of protein and fat, resulting in the production of important aroma compounds (Jollivet, Chataud, Vayssier, Bensoussan, & Belin, 1994). For example, G. candidum plays a key role in ripening because it contributes to the typical avours of Camembert, Pont lEve que and Livarot cheeses (Hemme, Bouillanne, Metro, & Desmazeaud, 1982). However, the proteolytic system of G. candidum remains quite unknown. In addition, its role in cheese proteolysis remains unclear, in part because G. candidum is not the only yeast growing on cheese. Thus, proteolysis of Camembert cheese by G. candidum is difcult to determine but is clearly lower than that of Penicillium camembertii, a mould growing at the cheese surface after G. candidum (Gripon, 1993). In fact, it is commonly accepted that for Camembert cheese, proteolytic activity is mainly from microbial origin, especially from the mould P. camembertii, whereas the deamination activities of some bacteria and yeasts such as G. candidum lead to the formation of ammonia, aldehydes and organic acids that have a role in cheese avour (Lenoir, Lamberet, & Schmidt, 1983). However, Geotrichum isolated from surfaces of cheeses grew in a casein (azocasein, as-, b-casein) liquid medium, clearly showing proteolytic activity (Chen & Ledford, 1972). Knowledge of the proteolytic enzymes of G. candidum is dispersed, fragmented and controversial (for a review guen, 2005). First of all, the see Boutrou & Gue proteolytic enzymes produced vary, depending on the G. candidum strains. With respect to extracellular proteolytic activity, two sub-groups have been distin guen & Lenoir, 1975): one group (the guished (Gue majority of the strains studied) is characterised by rather weak activity, while the second group is characterised by strong extracellular activity. However, extracellular proteolytic activity is low compared with intracellular activity. Indeed, the extent of variation for the extraand intracellular proteolytic activities has been shown to guen & be about 115 in 83 strains of G. candidum (Gue Lenoir, 1976). These authors characterised in vitro the extra- and intracellular proteolytic systems of G. candidum strains that exhibited different proteolytic guen & Lenoir, 1976). It is of note that levels (Gue guen (1985) found no extracellular Hannan and Gue activity when they studied endopeptidase activity during growth of G. candidum, and found only low membrane activity. Similarly, Vincente-Soler and Gacto (1991) found that proteinase activity was not detectable in the culture medium when growing G. candidum, but demonstrated the presence of an intracellular serinetype protease active on casein, as did Litthauer, Louw, and Du Toit (1996) in G. candidum. Studies of G. candidum peptidases are scarce. Auber` re (1997) investigated the aminoger, Lenoir, and Berge and carboxypeptidase systems of G. candidum. The

mycelial and extracellular aminopeptidase activity corresponded to two different enzymatic systems. The carboxypeptidase system, which is mainly mycelial bound or endocellular, seems to be due to only one enzyme or to two closely related components. Most of the proteolytic activities of G. candidum have been studied in vitro. To better understand the role of G. candidum in the proteolysis of soft cheese, we have chosen to study in situ the ability of G. candidum to contribute to primary and secondary proteolysis. Thus, the aim of the present study was to dene the proteolytic activity of G. candidum at the surface of a white, soft cheese, i.e., where it grows during ripening. This knowledge is all the more important in that G. candidum is one of the rst microorganisms to colonise and grow at the surface of soft cheese. Model soft cheese was manufactured using Lactococcus lactis as lactic starter, with or without the addition of G. candidum Geo17 as the surface ora. The extent and nature of proteolysis were determined at the surface of the cheeses throughout 27 days of ripening.

2. Materials and methods 2.1. Cheese-making microorganisms and soft cheese manufacture The lactic ferment used was L. lactis subsp. lactis -Saint-Romain, strain MM101 (Rhodia Food, Dange France) grown overnight at 20 1C in heat-treated (100 1C for 15 min) reconstituted low-heat skim milk powder (10 g 100 mL1). The strain G. candidum Geo17 (Rhodia -Saint-Romain, France) was selected beFood, Dange cause of its particular use in the dairy industry. Freeze-dried spores were maintained at 20 1C until inoculation. Soft cheeses were made in duplicate as described by Boutrou et al. (2002). Raw skim milk was micro-ltered te at 35 1C through a 1.4 mm Sterilox membrane (Socie ramiques Techniques, F-65460 Bazet, France). des Ce This process allows milk ltration with bacterial retention rates of the contaminant ora in the discarded et al., 1991). The microretentate above 99.9% (Trouve ltered milk was inoculated at 34 1C with a 1.5% (v/v) inoculum of L. lactis prepared as described above. G. candidum was inoculated in milk at renneting at 1.6 108 viable spores (25% to +50%) per 1000 L of milk. A control without surface ora, and thus containing only L. lactis as maturing agent, was also manufactured. After demoulding, curds were salted for 22 min at 12 1C in saturated brine acidied at pH 4.6 using lactic acid. They were thereafter ripened at 12 1C and 97% relative humidity for 10 days, in a ripening room previously disinfected using a fumispore tte Soge bul, Poligny, France) to prevent base (Socie

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contamination of surface cheese. We have previously shown that the combination of micro-ltering the milk and ripening room disinfection is effective in ensuring that no other microorganisms grow signicantly on the cheese surface (R. Boutrou, unpublished). Cheeses were turned after 2 and 6 days to ensure the growth of the ora on the whole surface of the cheese. Cheeses were cooled at 6 1C for 2 h before being packed in a cellophane-based lm and ripened further at 6 1C up to 27 days. 2.2. Sample preparation for proteolysis At each time point in the ripening period (0, 5, 8, 12, 15, 18, 21 and 27 days), two cheeses were taken for analysis. To focus on the role of G. candidum, only the surface of the cheese was sampled. The cheese surface samples were extracted with sodium citrate solution as described by Gripon, ` re (1975). A cheese Desmazeaud, Le Bars, and Berge surface sample (10 g) was suspended in 40 mL of 0.5 M sodium citrate solution, pH 7.0, at 40 1C and homogenised twice with an Ultra-Turrax disperser (Bioblock, F-67403 Illkirch, France) for 30 s each time. The volume of the solution was adjusted to 200 mL with distilled water. Insoluble and soluble fractions were prepared from 20 mL of cheese surface extract that had been acidied to pH 4.6 by dropwise addition of 1 M HCl under constant agitation. The acidied extract was centrifuged at 10 900 g for 20 min at 4 1C. The supernatant comprised the soluble fraction whereas the pellet, which was dissolved in 8.75 M urea (20 mL nal volume), comprised the insoluble fraction. 2.3. Nitrogen content The soluble nitrogen (SN) content in the soluble fraction was measured in duplicate by the Kjeldahl method. 2.4. Urea-polyacrylamide gel electrophoresis Samples were analysed by urea-polyacrylamide gel electrophoresis (urea-PAGE) using 18% (w/v) acrylamide gels comprising 4.33 M urea and 0.0375 M Tris-HCl buffer, pH 8.8, according to the method of Andrews (1983). A Mini Protean II gel system (BioRad, F-94203 Ivry-sur Seine, France) was used. One hundred microlitres of cheese surface extract were diluted to 1/10 in distilled water, and 40 mL of the diluted extract were denatured with 20 mL 0.094 M Tris-HCl buffer, pH 6.8, containing, 3.3 M urea, 5% (v/v) b-mercaptoethanol, 10% (v/v) glycerol and 0.05% (w/v) bromophenol blue for 1 h at 37 1C. Twenty microlitres of sample were loaded into each lane. Electrophoresis was continued for 2.5 h at 20 1C and 200 V constant voltage, using 0.5 M

Tris-HCl, pH 8.3, containing 0.192 M glycine as the electrode buffer. The bands were revealed with R250 Coomassie blue staining. 2.5. Reverse-phase high-performance liquid chromatography Caseins present in the insoluble fraction of the cheese surface extract were analysed by reverse-phase HPLC on a Spectra Physics SP 8800 system (Spectra Physics, , CA, USA) with a Vydac C4 column San Jose (150 4.6 mm ID, Touzart et Matignon, Vitry sur Seine, France). The insoluble fraction was diluted to 1/ 10 in 8.75 M urea and reduced with b-mercaptoethanol (16 mM nal concentration) for 1 h at 37 1C. Triuoroacetic acid (TFA) was added (1% (v/v) nal concentration) to the samples before ltration through a 0.45 mm lter. Samples (100 mL) were injected onto the column maintained at 40 1C and equilibrated with 63% solvent A (0.106% (v/v) TFA in MilliQ water). A linear gradient was run in 37.5 min from 37% to 57% buffer B (0.1% (v/v) TFA in 80% (v/v) acetonitrile in MilliQ water) at a ow rate of 1 mL min1. The absorbance of the eluent was measured at 214 nm. Samples were run in duplicate. The identity of the HPLC peaks corresponding to each casein was determined as described by , Herrouin, and Le onil (2001). The peak Gagnaire, Molle area corresponding to each casein was determined. Peptides were analysed by reverse-phase HPLC using a C18 Vydac column (250 4.6 mm ID). The soluble fraction was diluted 1:1 with 0.3% TFA solution before ltration through a 0.45 mm lter. The chromatographic system and solvents A and B were the same as those used for casein measurement. The elution was done at a ow rate of 1 mL min1 at 40 1C, with a linear gradient from 5% to 68% solvent B in 57 min. Samples were run in duplicate. 2.6. Free amino acid analysis Free amino acids (FAAs) produced throughout proteolysis were determined from the soluble fraction after precipitation of whey proteins and casein fragments with 10% sulphosalicylic acid for 1 h at 4 1C followed by centrifugation (3000 g, 10 min). The supernatant was ltered through a 0.45 mm lter and injected onto a Pharmacia LKB-Alpha Plus series 2 amino acid analyser (Pharmacia Biotech., Saclay, France). FAA analyses were done in duplicate. The concentration of each of the 20 FAAs analysed was summed; individual FAAs were also considered. 2.7. pH The pH of the cheese surface was measured in triplicate using a penetrating electrode (Ingold) linked

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Peak area (arbitrary units)

to a Hi 9025 pH-meter, and placed between two slices of the cheese surface.

1.E+05 0.E+00 -1.E+05 -2.E+05 -3.E+05 -4.E+05 -5.E+05 -6.E+05 0 5 10 15 20 Ripening time (days) 25 30

3. Results During the rst days of ripening, G. candidum grew at the cheese surface, and a little white down was visible from day 6 (D6). 3.1. Primary proteolysis An overall view of cheese proteolysis was given by urea-PAGE proles of the cheese surface extracts (Fig. 1), thus comprising both the soluble and the insoluble fractions. Very little hydrolysis of caseins occurred over the whole ripening period in the control cheese, which did not have surface ora. In contrast, the intensity of as1- and b-casein bands decreased markedly from D5 to D15 in cheese manufactured with G. candidum, and remained constant thereafter. In general, the intensity of all the casein bands decreased in cheese made with G. candidum, while numerous products of proteolysis appeared. This suggests a high and diversied proteolytic activity of G. candidum after the rst week of ripening. Hydrolysis of caseins at the cheese surface was measured using HPLC. The peak areas of caseins in the proles obtained from the control cheese were subtracted from the corresponding peak areas of the prole from the surface-ripened cheese (Fig. 2). It was seen that the presence of G. candidum results in signicant hydrolysis of caseins from D8 to D15. Thereafter, the peak areas representative of each casein remained almost unchanged. At the end of the ripening period, 69% and 59% of the as1- and bA2-caseins were hydrolysed, respectively. In contrast, bA1- and para-kcaseins remained virtually undegraded in the surfaceripened cheese throughout the ripening period compared with the control cheese, and as2-casein was only weakly degraded.

Fig. 2. Caseins present in the pH 4.6-insoluble fraction of sodium citrate-extracted cheese surface. Samples throughout ripening were separated using C4 RP-HPLC and the area of each corresponding peak was determined. The difference in peak area between control and G. candidum cheese is presented for as1 (m), as2 (), bA1 ( ), bA2 (K) and para-k (E) caseins. Standard deviations for each sampling are shown.

35 pH 4.6 SN (g kg-1 cheese) 30 25 20 15 10 0 5 10 15 20 Ripening time (days) 25 30

Fig. 3. Nitrogen content of the pH 4.6-soluble fraction derived from the surface of the control (&) and surface-ripened cheese (K) during ripening. Standard deviations for each sampling are shown.

0 5

8 12

15 18

21 27

12 15

18 21

27

s2 s1 s1-I

As a consequence of primary proteolysis, large and medium-sized fragments of caseins were released. The amount of pH 4.6-soluble nitrogen was similar in control and surface-ripened cheeses from the time of demoulding to D12 (Fig. 3). Thereafter, the amount of soluble nitrogen at the surface of cheese ripened with G. candidum increased rapidly up to D18, and then more slowly until the end of the ripening. In contrast, the amount of soluble nitrogen at the surface of the control cheese was almost unchanged from D12 to the end of ripening. At the end of ripening, the amount of soluble nitrogen at the surface of the G. candidum cheese was 38% higher than in the control cheese. 3.2. Secondary proteolysis

Fig. 1. Eighteen percent acrylamide urea-PAGE proles of cheese surface extract samples taken throughout ripening from the control cheese (left) and cheese with G. candidum (right). Numbers at the top of the proles indicate the time of ripening (in days). Bands corresponding to caseins are shown on the left.

The release of peptides, i.e., small casein fragments, was followed using C18 RP-HPLC proles. A few peaks appeared throughout ripening in the control cheese and the peak areas increased slightly up to D27 (Fig. 4). In

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0.4

0.4

D27 Absorbance (arbitrary units at 214 nm) 0.3 D18

D27

0.3

D18

0.2 D12

0.2

D12

0.1 D0

0.1

D0

0 0 10 20 30 40 Elution time (min) 50 60

0 0 10 20 30 40 Elution time (min) 50 60

Fig. 4. Reverse-phase-HPLC proles of the pH 4.6-soluble fraction of sodium citrate-extracted cheese surface samples taken throughout ripening from the control cheese (left) and surface-ripened cheese (right). Samples were prepared with 0.3% (v/v) TFA and ltered before injection. Analysis was done on a Vydac C18 column using elution conditions as described in the text.

8.E+05 Total FAA (mmol kg-1 cheese) Total peak area (arbitrary units) 6.E+05 4.E+05 2.E+05 0.E+00 0 5 10 15 20 Ripening time (days) 25 30

35 30 25 20 15 10 5 0 0 5 10 15 20 Ripening time (days) 25 30

Fig. 5. Total peak area calculated from reverse-phase-HPLC chromatograms of the pH 4.6-soluble fraction of sodium citrate-extracted cheese surface samples of control (&) and surface-ripened cheese (K) during ripening. Standard deviations for each sampling are shown.

Fig. 6. Total free amino acids in the pH 4.6-soluble fraction of sodium citrate-extracted cheese surface samples taken throughout ripening from the control cheese (&) and surface-ripened cheese (K). Standard deviations for each sampling are shown.

the surface-ripened cheese, the proles became increasingly complex from demoulding to D18 with numerous peaks appearing. Thus, the peak areas increased gradually. The area of some peaks continued to increase thereafter while that of others decreased. The sum of all peak areas was quantied to give a view of the overall change throughout the ripening (Fig. 5). The total amount of peptides present in the soluble fraction from the G. candidum cheese increased rapidly from D8 to D15, and was 80% higher than in the control cheese at D12. However, by D27, the total peak area had decreased and the total amount of peptides was only

15% higher at the surface of G. candidum cheese than at the surface of the control cheese. The concentration of FAAs was also determined in all cheese surface samples during ripening. After a slight increase during the rst week of ripening, the concentration of total FAAs remained constant until the end of the ripening in the control cheese (Fig. 6). The situation was the same at the surface of the cheese with G. candidum during the rst week of ripening. Thereafter, the concentration of FAAs at the surface of cheese

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ripened with G. candidum increased rapidly up to D18 and then remained almost constant through to D27. Thus, at the end of the ripening, there were three times more FAAs at the surface of the G. candidum cheese than at the surface of the control cheese. The concentrations of glutamic acid, glutamine, proline, alanine, lysine, histidine and arginine were signicantly higher from D12 to the end of ripening at the surface of cheese manufactured with G. candidum than was the case with the control cheese (data not shown). The concentration of each FAA at D27 in both cheese samples is presented in Fig. 7. 3.3. Change of pH at the surface of the cheese pH remained about 4.8 at the surface of the control cheese whereas it increased gradually at the surface of cheese manufactured with G. candidum up to the end of the ripening (Fig. 8). Thus, the surface pH was higher in cheese manufactured with G. candidum where it reached 7.0 at the end of the ripening than in cheese without this microorganism. The sudden raising of pH from D5 coincided with the growth of G. candidum.
FAA concentration (mmol kg-1 cheese) 6 5 4 3 2 1 Met Tyr Leu Phe Lys His Ile Asp Thr Asn Arg Glu Gln Pro Gly Ser Ala Val Cys 0

4. Discussion Most information on the proteolytic enzymes of G. candidum has been previously obtained in vitro on pure substrates. However, cheese is a complex and biphasic system in which the enzymatic activities depend on the environment and the available substrates. Therefore, we studied in situ the proteolytic activity of G. candidum. To determine the contribution of G. candidum on the proteolysis at the surface of soft cheese, Camembert-type cheese with G. candidum as sole surface ora was manufactured. Thus, an assessment of the proteolytic activity of G. candidum was not complicated by the presence of other yeasts and moulds. To obtain an overall view of the proteolytic activities in situ, different techniques were applied to assess primary and secondary proteolysis. The primary proteolytic activity observed in control cheese was from chymosin, the coagulant enzyme used in our study. If one considers that optimal breakdown of as1-casein by rennet occurs at pH 5.0 in simulated soft cheese (Noomen, 1978), it can be inferred that pH conditions in control cheese (Fig. 8) permit the action of chymosin on that casein till the end of the ripening time. This action was visualised through the increased intensity of the as1-I band up to D15 (Fig. 1). In contrast, the lactococcal proteinase activity in control cheese might be low. Indeed, the proteinase puried from L. lactis NCDO 763 is active at pH 6.06.5 on casein (Monnet, Le Bars, & Gripon, 1987), and the optimum pH for the proteinase measured for casein hydrolysis is 6.4 (Laan & Konings, 1989). The action of plasmin, the milk endoproteinase, at the surface of either the control or the G. candidum cheese was probably negligible. The pH at demoulding was 4.8 in our experimental cheeses, and at pH values o4.6 plasmin has been reported (Grufferty & Fox, 1988) to be released from the caseins, indicating that it may be completely removed in whey during cheese manufacture. In addition, the optimal pH for the activity of the residual plasmin is about 7.5 (Bastian & Brown, 1996; McSweeney, Olson, Fox, Healy, & Hojrup, 1993). This is conrmed by the nding that the concentration of gcaseins that results from the proteolysis of b-casein by plasmin did not increase during ripening of either cheese (Fig. 1). Chymosin was also active at the surface of G. candidum cheese up to D8. Thereafter, its action might be minimised as far as the pH increased. The decreased intensity of casein bands on electrophoretic proles from D8 to D15 indicated that G. candidum is capable of primary proteolysis at the surface of soft cheese (Figs. 1 and 2). This result is in agreement with the activity of G. candidum proteinases that have optimal pH from 5.5 to 6.2 (Chen & Ledford, 1972; guen & Lenoir, 1976). Gue

Fig. 7. Concentration of free amino acids in the pH 4.6-soluble fraction of sodium citrate-extracted cheese surface samples taken throughout ripening in control (white bars) and surface-ripened cheese (black bars) after 27 days of ripening.

7.0 6.6 6.2 pH 5.8 5.4 5.0 4.6 0 5 10 15 20 Ripening time (days) 25 30

Fig. 8. pH of cheese surface samples taken throughout ripening from the control cheese (&) and surface-ripened cheese (K). Standard deviations for each sampling are shown.

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bA2- and as1-caseins were much more readily hydrolysed than the other caseins in the presence of G. candidum at the surface of the cheese. The question arises as to whether this is due to facile accessibility of these two caseins in the cheese by proteases or to the actual specicity of G. candidum proteinase(s). Protease specicity is of importance in cheese ripening. Dluzewski and Bruderer (1963) observed signicant changes in the casein of cheese ripened for 24 h at 22 1C with G. candidum. These authors found that the proteolytic activity of G. candidum does not seem to be conned to the a-fraction of casein, as a study of casein separated from cheese has suggested the hydrolysis of b-casein by this microorganism. An in vitro study showed that G. candidum isolated from the surface of Limburger cheese and grown in a casein medium degraded as- and b-caseins within two days, with preferred hydrolysis of b-casein (Chen & Ledford, 1972). Like other microorganisms, G. candidum uses organic nitrogen such as casein and peptones, i.e., a tryptic hydrolysate of caseins. Adour, Couriol, Amrane, and Prigent (2002) and Aldarf, Amrane, and Prigent (2002) studied growth of G. candidum on liquid and solid media, respectively, where the sole sources of carbon and nitrogen were peptones. The consumption of peptones always started simultaneously with growth. However, in our cheese surface with G. candidum, peptides were not available at the beginning of G. candidum growth. Thus, it rst had to hydrolyse caseins to assure its supply of peptides. In cheese with a complex surface ora, G. candidum presumably does not have to hydrolyse caseins into peptides as these can be provided by the proteolytic activity of other yeasts. This could explain why it is commonly accepted that the contribution of G. candidum to proteolysis in cheese is mainly peptidolytic, rather than proteolytic, in nature. In particular, G. candidum is known to produce aminopeptidases and carboxypeptidases and to reduce cheese bitterness by the breakdown of bitter peptides (Lenoir, Lamberet, Schmidt, & Tourneur, 1985; Moli` re, & Vassal, 1983; mard et al., 1994; Mourgues, Berge Wyder, 1998). The lactococcal cell-bound envelope proteinase (lactocepin) might also play a role in proteolysis at the surface of G. candidum cheese, as an intermediate activity which converts primary, chymosin-generated , & Le onil, 2001; Exterkate, peptides (Boutrou, Molle 1995; Exterkate, Slangen, & Siezen, 2001). The pH conditions prevalent in G. candidum cheese are parallel to those that seem to confer stability upon the lactocepin of L. lactis (Exterkate, 2000; Laan & Konings, 1989). In addition, the proteinase release is almost completely inhibited in presence of free Ca2+ in the medium (Laan & Konings, 1989), as it was the case at surface of Camembert-type cheese (Le Grae t & , 1988). The pH increase during ripening may Brule

affect the activity of lactocepin both directly and through changes in the concentration of free calcium. This in turn may impact on the potential contribution of lactocepin to surface proteolysis. As a consequence of primary proteolysis, large and medium-sized casein fragments and peptides were produced after 12 days of ripening at the surface of G. candidum cheese (Figs. 1 and 4). The rate of production decreased from D18, suggesting a lower production of these products of hydrolysis and/or the degradation of a part of them. After two weeks of ripening, the amount of peptides decreased (Fig. 5) while the level of FAAs increased at the surface of G. candidum cheese (Fig. 6). Secondary proteolysis involves the production of small peptides and FAAs, which G. candidum continued to produce until the end of the ripening. This suggests that most of the peptides were degraded to amino acids through the activity of G. candidum peptidases, whose optimal pH is from 6.5 to 9.5 (Auberger et al., 1997). It is of note that, given their pH optima (Monnet, Chapot-Chartier, & Gripon, 1993), lactococcal peptidases might also be increasingly active during ripening as the surface pH increased. Given that the released FAAs are potential avour precursors, this result is consistent with the knowledge that G. candidum contributes to an aroma similar to that of traditional Normand Camembert (Ribadeau-Dumas, 1984) through the degradation of certain amino acids (Jollivet et al., 1994). A large amount of FAAs was present at the surface of cheese with G. candidum. The large accumulation of proline, glutamine and glutamic acid at the surface of cheese manufactured with G. candidum suggests that this microorganism is unable to metabolise all of the amino acid pool (Fig. 7). However, studies of the cultivation of G. candidum in synthetic liquid media containing a single amino acid, either glutamic acid or proline, showed these FAAs to be convenient nitrogen and carbon sources for G. candidum (glutamine was not tested) (Plihon, Le Doujet, Amrane, & Prigent, 1998). We hypothesise that the combination of the relatively high levels of glutamic acid and proline in caseins and the proteolytic activity of G. candidum results in signicant amounts of the free forms of these amino acids at the surface of our Camembert-type cheese, such amounts being in excess of the requirements of G. candidum. Similarly, there was a signicant amount of glutamic acid and aspartic acid in Limburger-type cheeses (Greenberg & Ledford, 1979). The concentrations of leucine and alanine in our surface-ripened cheese, which were similar and higher than in control cheese, respectively, indicate the action of the extracellular system studied by Auberger et al. (1997).

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