J Sci Food Agric 1997, 75, 148È154

Proteolytic and Lipolytic Changes during theRipening of a Spanish Craft Goat Cheese(Armada Variety)Jose� M Fresno,1 Mari�a E Tornadijo,1 Javier Carballo,2 Ana Bernardo1 andJosefa Gonza� lez-Prieto1*

1 Departamento de Higiene y Tecnolog•� a de los Alimentos, Universidad de Leo� n, 24071 Leo� n, Spain2 Area de Tecnolog•� a de los Alimentos, Facultad de Ciencias de Orense, Universidad de Vigo, 32004Orense, Spain

(Received 22 December 1995 ; revised version received 19 August 1996 ; accepted 14 March 1997)

Abstract : The proteolytic and lipolytic changes during the ripening process wereinvestigated in four batches of Armada goatÏs milk cheese (an artisanal varietyproduced in the North of Spain), by determining the classical nitrogen fractions,caseins and their degradation products, free amino acids, as well as the acidity ofthe fat, thiobarbituric acid (TBA) number and free fatty acids. Values obtainedfor the nitrogen fractions and for caseins and their degradation products showthat this cheese undergoes very little protein degradation. A low free amino acidscontent was observed throughout the ripening process with a predominance ofPro followed by Leu] Ile, Glu acid, Phe, His] Lys and Val. The lipid degrada-tion was very intense from the second month of ripening, only comparable tothat reported for cheeses ripened by moulds. The average free fatty acids contentincreased 20-fold during ripening, reaching Ðnal values of 44É5 g kg~1. All thefree fatty acids increased considerably during ripening, resulting in a predomi-nance of saturated and unsaturated long-chain acids, followed by medium-chainacids, principally. Short-chain fatty acid content by the end of ripening wasC10higher than that presented in other cheese varieties with a similar high degree oflipolysis.

J Sci Food Agric 75, 148È154 (1997)No. of Figures : 0. No. of Tables : 5. No. of References : 48

Key words : proteolysis, lipolysis, goat cheese, cheese ripening

INTRODUCTION

The majority of European populations of goats is foundin Mediterranean countries, where about 86% of totallivestock are situated. Spain occupies the third positionin goat milk production, after Greece and France (LeJaouen and Toussaint 1993).

A large number of homemade goatÏs cheeses are pro-duced in Spain (Ministerio de Agricultura, Pesca y Ali-mentacio� n 1990) but only a few are made on anindustrial scale. This is probably due to lack ofpublished technical information on goatÏs cheeses, in

* To whom correspondence should be addressed.Sponsor : Junta de Castilla y Leo� n.

contrast to the abundance of data on the technologicalaspects and ripening biochemistry of cowÏs milk cheeses.

Among these insufficiently studied cheeses is theArmada cheese, a hard variety traditionally manufac-tured from whole raw goatÏs milk in the North of Spain,and whose organoleptic properties (spicy and aromatictaste, greasy palate) make it highly esteemed. A betterknowledge of its biochemical and microbiological char-acteristics would favour the improvement of the manu-facturing technology, and help obtain a constant qualityproduct capable of being successfully introduced intonational and international markets.

The proteolysis and the lipolysis are among the mostimportant reactions which take place during cheeseripening. These reactions are responsible for the texture

1481997 SCI. J Sci Food Agric 0022-5142/97/$17.50. Printed in Great Britain(

Proteolysis and lipolysis in Armada cheese 149

changes and the appearance of the characteristic aromaof cheese, which are caused by the formation during theripening process of several nitrogenous compounds oflow molecular weight and free fatty acids, as well assome products derived from its degradation ; mainlyacids, alcohols, aldehydes, ketones, amines, etc.

The changes in compositional, physico-chemical andmicrobiological parameters during the Armada cheeseripening have been studied at our laboratory (Tornadijoet al 1995 ; Fresno et al 1996). The objective of this workis to obtain information on the changes throughoutripening of the di†erent nitrogen and casein fractionsand free amino acids (FAA), as well as the changes inthe lipid fraction : fat content, thiobarbituric acid (TBA)number, acidity index of the fat and free fatty acids(FFA). These results would provide essential informa-tion contributing to knowledge of the processesinvolved in the ripening of this cheese variety, which is anecessary step in order to achieve the industrial manu-facturing under controlled conditions.

MATERIAL AND METHODS

Cheese samples

Four cheese batches were elaborated following the tra-ditional method using whole raw goatÏs milk, with asmall amount of whey added from previous batches,and coagulated at 30¡C by adding about 15 ml of calfrennet (strength 1/10 000) (Productos Nievi, Bilbao,Spain) per 100 litres of milk. No starters were added tothe milk. After 1 h the curd was cut and transferred tocheesecloths. These were formed into bundles and hungfor 48 h to drain the whey. The bundles were thenopened and the curd was given a rigorous kneading(“sobadoÏ). Then it was transferred to clean cheeseclothsand hung for a further 3 days, after which it waskneaded again and hand-moulded into its characteristicsquare shape. Cheeses were wrapped again in cheese-cloths and hung from the ceiling, where the ripeningprocess took place at a temperature of 10È15¡C and arelative humidity (RH) of 70È85%, depending on thetime of year. Salting was carried out by the addition ofsolid salt during kneading operations. The cheeses wereapproximately 200 mm in side and 200 mm high.

Curd samples (0 days) and cheese samples at 7, 15, 30,60 and 120 days were taken (each sample consisted ofone whole cheese) of each batch and prepared accordingto the International Dairy Federation 50B:1985 stan-dard.

Nitrogen fraction analysis

The total nitrogen content (TN) was determined by theKjeldahl method (International Dairy Federation25 :1964 standard). The procedure of Vakaleris andPrice (1959) was followed for the extraction of the total

soluble nitrogen (SN) and non-protein nitrogen (NPN),and the Johnson method (Lichstein and Oginsky 1965)was used in its determination. In the case of NPN, aprecipitation of proteins with trichloroacetic acid at12% was necessary. The method described by Ordo� n8 ez(1974) was used in the determination of ammonia nitro-gen and amino nitrogen Protein,(NH3-N) (NH2-N).casein, peptide and polypeptide nitrogen was calculatedas described by Prieto et al (1994). All analyses werecarried out in quadruplicate.

Electrophoretic analysis

The casein degradation was studied by urea-PAGEusing the Andrews procedure (1983). Electrophoresiswas performed on 0É7 mm thick plates and the bandswere stained with Naphthol Blue Black 10B (SigmaChemical Co, St Louis, MO, USA). For the identiÐca-tion and quantiÐcation of the casein fractions, the El-Shibiny and Abd El-Salam technique (1976) wasfollowed. All analyses were carried out in duplicate.

Free amino acids analysis

The extraction of amino acids was carried out from10 g of grated cheese homogenised with 40 ml 0É6 M-

in an Omni-Mixer model 17220 (Sorvall Inc,HClO4Newtown, CT, USA) at approximately 20¡C for 1 min.This preparation was centrifuged at 2500] g for 5 minand the supernatant fraction was Ðltered through aWhatman no 54 Ðlter (Whatman Biosystems, Maid-stone, UK). The pH was adjusted to 6É0 with 1 M-KOHand cooled in an ice bath for 30 min. It was then Ðlteredthrough a Whatman no 54 Ðlter and concentrated byevaporation at 40¡C under vacuum and redissolved in20 ml pH 8É5. Finally, the samples0É5 M-NaHCO3 ,were Ðltered through a 0É45 km Ðlter (Millipore,Bedford, MA, USA) and stored at [30¡C until use. ThequantiÐcation and identiÐcation of amino acids werecarried out by reversed-phase HPLC techniques on aSpectra Physics SP 8800 ternary pump (SpectraPhysics, San Jose, CA, USA), a Rheodyne 7125 injector(20 kl loop) and a Spectra Physics SP 4290 recorder-integrator according to Wiedmeier et al (1982). Aminoacid detection was carried out at 254 nm using aKontron 730 S LC (Kontron Analytic, Heston,Middlesex, UK) variable-wavelength detector.Reversed-phase column Spheri-5 ODS-224 5 km,C18 ,220 mm ] 4É6 mm and a newguard RP-8 column,7 km, 3É2 mm ] 15 mm (Brownlee Labs, Applied Bio-systems, Santa Clara, CA, USA) were used for analyticalpurposes. All analyses were carried out in duplicate.

Fat characteristics

The determination of fat content was made followingthe International Dairy Federation 5B:1986 standard.

150 J M Fresno et al

The TBA number was determined as described by Tar-ladgis et al (1960). For the determination of the acidityindex of the fat the International Dairy Federation6B:1989 standard was used. All analyses were carriedout twice.

Free fatty acids analysis

The extraction and quantiÐcation of FFA was made fol-lowing the procedure of Mart•� n-Herna� ndez et al (1988).GasÈliquid chromatographic analyses were carried outin a model F-990 gas chromatograph (Perkin-Elmer Co,Norwalk, CT, USA) equipped with a stainless steelcolumn SP-2330 1É8 m ] 3É2 mm (Supelco Inc, Bella-fonte, PA, USA) packed with 10% Cyano Silicone onChromosorb W AW 100È120 mesh. Injector tem-perature was 300¡C, detector temperature 250¡C and acarrier gas Ñow-rate of 10 ml min~1. All analyses(N2)were carried out in quadruplicate.

Statistical analysis

For the comparison of the average results of the di†er-ent parameters studied in the four batches during theripening process, the analysis of variance was carriedout using the test of conÐdence intervals at 95%,according to the Statgraphics 3.0 computer programme(Statistical Graphics Corporation, Rockville, MD,USA).

RESULTS AND DISCUSSION

Changes in nitrogen fractions and casein degradation

Table 1 shows the mean values of the di†erent nitrogenfractions throughout the ripening process.

SN remained almost stable throughout ripening,whilst NPN, and contents increasedNH3-N NH2-N

signiÐcantly during the process. SN levels at the end ofripening (9É68 ^ 2É45% TN) were lower than the valuesusually described for goatÏs cheese, being only compara-ble to those of fresh goatÏs (Mart•� n-Herna� ndez et al1992), Babia-Laciana (Argumosa et al 1992) and Valde-teja (Carballo et al 1994) cheeses. NPN also showed lowÐnal values (7É34 ^ 2É03% TN), although represented76% when it was expressed as a percentage of SN,higher results than those obtained for other cheese vari-eties with moderate or intense proteolysis. andNH2-N

fractions increased more markedly, attainingNH3-Nmean values of 55É3% and 19É1% NPN respectively atthe end of ripening. These percentages are higher thanthe values reported for other goatÏs cheeses although, ifexpressed as %TN, the values obtained for both frac-tions were low.

The average values for the di†erent casein fractionsduring ripening are shown in Table 2. It can beobserved that there was little casein degradation, beingslightly more pronounced in the case of whichas-casein,declined signiÐcantly within the Ðrst 7 days, decreasingthe rate from 0É54 to 0É46. At the end of the ripen-as/bing, breakdown of and b-caseins, as a percentage ofas-content in the curd, was only 21É4% and 3%, respec-tively. The rest of the fractions did not undergo notice-able modiÐcations, with the sole exception of preas-

increasing signiÐcantly during the Ðrst week ofcaseinripening. The values obtained for and c-caseinpreas-were lower than those reported by other authors forgoatÏs cheeses such as artisanal Majorero (Fontecha etal 1990), Palmero (Go� mez et al 1991), Gredos (Medinaet al 1992) and industrial Majorero (Mart•� n-Herna� ndezet al 1992). The casein degradation is probably causedby the e†ect of the residual rennet whose action is spe-ciÐc on and, to a lesser extent, on b-caseinas-casein(Fox et al 1993). Nevertheless, its action must benotably inhibited not only by the low pH values (4É5È

TABLE 1Changes in the nitrogen fractions (g kg~1 total nitrogen) and in total nitrogen (g kg~1 total solids) of Armada cheese during

ripening (average values of four batches)a

Days of ripening SEDMb

0 7 15 30 60 120

TN 51É1a 51É0a 52É5a 53É8a 52É8a 54É9a 0É6SN 75É0a 90É4a 79É6a 71É2a 76É4a 96É8a 4É0NPN 25É2a 50É0b 48É9b 47É3b 50É1b 73É4c 6É2NH3-N 0É4a 1É9a 2É6a 5É8a 5É6a 14É0b 1É9NH2-N 9É4a 14É7a 19É7a 18É3a 22É3a 40É6b 4É3Protein N 974É8a 950É0b 951É1b 952É7b 949É1b 926É6c 6É2Casein N 925É0a 909É6a 920É4a 928É8a 923É6a 903É2a 4É0Polypeptide N 49É8a 40É4ab 30É7ab 23É9b 27É0ab 23É4b 4É3Peptide N 15É4a 33É4b 26É6bc 23É2abc 22É2ac 18É8ac 2É5

a Means in the same row and parameter group during ripening without a common following letter are signiÐcantly di†erent(P\ 0É05).b Standard error of the di†erence for treatment means.

Proteolysis and lipolysis in Armada cheese 151

TABLE 2Changes in casein fractionsa during ripening of Armada cheese (average values of four batches)b

Days of ripening SEDMc

0 7 15 30 60 120

c-CN 1É72a 3É35a 2É94a 2É51a 3É24a 5É30a 0É22b-CN 62É36a 61É44a 61É62a 61É83a 61É63a 61É02a 0É16as-CN 33É79a 28É49b 28É47b 29É58b 29É53b 26É54b 0É82Pre-as-CN 2É08a 6É73b 7É06b 6É08b 5É60b 7É13b 0É79as/b 0É54a 0É46b 0É46b 0É48b 0É48b 0É44b 0É01

a Expressed as % of total absorbance.b Means in the same row and parameter group during ripening without a common following letter are signiÐcantly di†erent(P\ 0É05).c Standard error of the di†erence for treatment means.

5É0) but also by the high salt/moisture concentrations(4È10%) present during ripening in this cheese (Fresnoet al 1996). Mulvihill and Fox (1978) and Noomen(1978) have demonstrated that the action of rennet on

and b-casein is restrained at pHs below 5É8 and 6É4,a-srespectively. On the other hand, Fox and Walley (1971)and Thomas and Pearce (1981) have shown that salt/moisture concentrations above 4% limit its action.

The contribution of the microbial proteinases to thedegradation of Armada cheese caseins seems to be verysmall, provided that its action is fundamentally circum-scribed to the degradation of b-casein (McSweeney et al1993) which, in our case, remains almost intact.However, the role of its peptidases in the formation oflow-molecular-weight peptides and free amino acids(FAA) (Law et al 1993), essential components of aminonitrogen, is more important. We have found a high

rate, around 3, during ripening, whichNH2-N/NH3-Ncould be related to the residual activity presented bythese enzymes at the low pH values of this cheese(Visser 1993).

Changes in free amino acids

The evolution of FAA during the ripening process ofArmada cheese is shown in Table 3. In the curd, thepredominant amino acid was proline, a phenomenonalso observed in Taleggio (Resmini et al 1969) and Te� le� -me� (Polychroniadou and Vlachos 1979) cheeses, fol-lowed by glutamic acid, valine, leucine] isoleucine andhistidine ] lysine, which in all made up 60% of the totalFAA content. At the end of ripening, proline continuedas the quantitatively most important amino acid,together with leucine] isoleucine, glutamic acid, valine,phenylalanine and histidine] lysine. Such proÐle con-curred basically with those reported in Taleggio(Resmini et al 1969), Te� le� me� (Polychroniadou andVlachos 1979), Caciocavallo (Santoro et al 1983), semi-hard type Edam (Baltadjieva et al 1985), Maho� n (Polo

et al 1985) and Stilton (Madkor et al 1987) cheeses, eventhough all of them had much lower proline percentagesthan Armada cheese, in which this amino acid made up30% of the total free amino acids.

This amino acid proÐle is typical of cheeses in whichthe predominant Ñora consists of L actococcus and L ac-tobacillus (Puchades et al 1989), which have speciÐcpeptidases for hydrophobic peptides rich in proline,such as prolidases, aminopeptidases P, prolyldipepti-dases, etc (Booth et al 1990a,b ; Visser 1993).

Fat characteristics

Table 4 shows mean results for fat content, TBAnumber and acidity index of the fat throughout ripen-ing. TBA number remained constant throughout theripening process. The Ðnal values (0É423 mg malonal-dehyde kg~1 of cheese) turned out to be slightly higherthan those reported for Babia-Laciana cheese(Argumosa et al 1992), Valdeteja (Carballo et al 1994)and Leo� n cowÏs milk cheese (Prieto et al 1994).However, taking into account the analyses made in ourlaboratory for goatÏs milk and rancid butter givingvalues of 0É085 and 3É2 mg malonaldehyde kg~1,respectively, it could be concluded that in Armadacheese, the degree of autooxidative rancidity of fat wasweak.

The acidity index of the fat increased markedlythroughout ripening. The average Ðnal values(26É31 ^ 15É13 mg KOH g~1 fat matter) revealed theexistence of very intense lipolysis. These values weremuch higher than those described elsewhere for otherripened goatÏs milk cheese varieties, as Babia-Laciana(Argumosa et al 1992), Majorero (Mart•� n-Herna� ndez etal 1992), Valdeteja (Carballo et al 1994) cheeses, beingonly comparable to those observed in blue cheeses suchas Blue Danish (Vanbelle et al 1978) and Cabrales(Alonso et al 1987).

152 J M Fresno et al

TABLE 3Changes in free amino acids (mg kg~1 total solids) in Armada cheese during ripening (average values of four batches)a

Days of ripening SEDMb

0 7 15 30 60 120

Taurine 64É0a 58É4a 33É6b 27É6bc 19É5bc 13É7c 8É4Anserine 10É1a 24É1a 18É7a 13É3a 10É2a 8É7a 2É4Glutamine 13É1a 43É1a 52É1a 48É9a 29É1a 22É7a 8É3Serine 64É6a 64É3a 85É8a 56É3a 51É1a 62É0a 4É8Aspartic acid 25É7a 58É6ab 93É8bc 115É0c 80É0bc 69É9b 11É0Glutamic acid 126É0a 341É1ab 352É5ab 489É9b 374É3ab 307É1ab 48É4Glycine 31É6a 52É3a 55É0a 44É1a 39É2a 42É1a 3É5Threonine 16É1a 32É7a 107É8a 83É9a 85É0a 121É7a 17É5Alanine 32É3a 171É8b 174É4b 148É9b 113É2b 110É3b 21É7Proline 139É3a 179É8a 174É7a 284É3a 438É4a 1094É6b 148É7Methionine Èc 27É9ab 59É9bc 77É5bc 81É0c 110É6c 16É2Cystine 33É3a 72É1a 62É3a 93É0a 80É8a 29É7a 10É4Valine 78É9a 125É7ab 148É1ab 186É5ab 216É8ab 274É9b 28É3Phenylalanine 59É2a 177É4ab 247É1bc 297É7bc 265É7bc 340É8c 40É9Tryptophan 21É5a 50É1a 53É4a 62É1a 46É0a 43É6a 5É6Leucine] isoleucine 111É2a 335É1ab 606É6bc 719É3c 603É7bc 666É4c 95É9Histidine ] lysine 80É2a 221É7ab 334É0b 374É3b 299É7ab 223É0ab 42É8Tyrosine 31É4ab 39É4ab 43É0ab 50É3b 29É5ab 16É6a 4É8Total free amino acids 936É9a 2075É5ab 2703É1bc 3172É8bc 2863É0bc 3558É4c 381É4

a Means in the same row and amino acid group during ripening without a common following letter are signiÐcantly di†erent(P\ 0É05).b Standard error of the di†erence for treatment means.c È, Not detected.

Changes of free fatty acids

Table 5 shows the mean proÐle for the main FFA in thecurd and cheeses along ripening.

The changes in FFA content during ripening agreedwith that followed by the acidity index of the fat, anincrease of 20-fold being observed from the curd to the120 days cheese, in which a mean value of 44É5 ^ 22É3 gof FFA kg~1 of cheese was found. This Ðnal value washigher than current data for di†erent goatÏs cheeses,such as Majorero (Fontecha et al 1990), goatÏs freshcheese and washed curd semi-hard cheese (Mart•� n-Her-na� ndez et al 1992), Cendrat del Montsec (Carretero et al

1992), La Vera (Fuente et al 1993), and only comparableto values presented in mould ripened cheeses such asRoquefort (Woo et al 1984), Cabrales (Alonso et al1987) and Gamonedo (Gonza� lez de Llano et al 1992).

Although within the Ðrst 30 days the FFA evolutionwas quantitatively similar to that of other goatÏscheeses, during the last stage of ripening, a markedincrease was observed concomitantly to a great devel-opment of moulds, mainly Penicillium roqueforti (Fresnoet al 1994), that even at the low pH values and highsalt/moisture concentrations in Armada cheese, shows acertain lipolytic activity (King and Clegg 1979). Thehigh short-chain FFA content throughout(C4ÈC8)

TABLE 4Changes in fat content (g kg~1 total solids), TBA number (mg of malonaldehyde kg~1 of cheese) and acidity index of the fat (mg

KOH g~1 of fat) in Armada cheese during ripening (average values of four batches)a

Days of ripening SEDMb

0 7 15 30 60 120

Fat 619É8a 592É1a 620É0a 606É5a 601É9a 595É7a 5É0TBA number 0É196a 0É163a 0É192a 0É122a 0É336a 0É423a 0É048Acidity index of the fat 1É20a 4É19a 8É00a 7É97a 17É64ab 26É31b 3É82

a Means in the same row and parameter group during ripening without a common following letter are signiÐcantly di†erent(P\ 0É05).b Standard error of the di†erence for treatment means.

Proteolysis and lipolysis in Armada cheese 153

TABLE 5Changes in free fatty acids content (mg kg~1 of cheese) in Armada cheese during ripening (average values of four batches)a

FFA Days of ripening SEDMb

0 7 15 30 60 120

C4 138a 382ab 631ab 824ab 1087bc 1838c 245C6 141a 325a 546a 734a 1189ab 2460b 346C8 112a 312a 549a 669a 1122ab 2156b 219C10 271a 776a 1346a 1690a 3145ab 5927b 847C12 134a 275a 450a 599a 1100a 2158b 306C14 175a 443a 803a 983a 1967a 4070b 588C16 456a 1166a 2007a 2310a 4596a 9413b 1346C18 213a 549a 1041a 1124a 2318ab 4819b 693C18 > 1 ] C18 > 2 387a 1683ab 4021ab 4063ab 6780bc 11 565c 1633

Total FFA 2027a 5899a 11 397a 12 995a 23 303ab 44 529b 6290

a Means in the same row and free fatty acid group during ripening without a common following letter are signiÐcantly di†erent(P\ 0É05).b Standard error of the di†erence for treatment means.

ripening as well as the predominance of capric acidover myristic acid could be also due to the speciÐcactivity of some lipolytic agents, principally fungicallipases (Ha and Lindsay 1993), on and positionssn1 sn3of the triglycerides, a place where short-chain fatty acids(Olivecrona 1979) and caproic acid (Kuksis et al 1973)are mainly situated.

Finally, in contrast to what happens in many goatÏscheeses, oleic acid content was always higher than pal-mitic acid content, a fact which can be explained, atleast in the Ðrst two months, by the presence in Armadacheese of Geotrichum candidum as the major organism(Fresno et al 1994), provided that this mould has certainspeciÐcity in the liberation of fatty acids with a doublelink in the position 9 (Tahoun et al 1982). Besides, thesituation of oleic acid in the position (and in tosn3 sn1a lesser extent), in contrast to the position andsn1 sn2of palmitic acid, could facilitate a higher liberationduring the ripening process (Kuksis et al 1973).

ACKNOWLEDGEMENTS

The authors gratefully acknowledge Drs ManuelaJua� rez, M Carmen Mart•� n-Herna� ndez and MiguelAngel de la Fuente (Instituto del Fr•� o, CSIC, Madrid,Spain) for his help in the determinations of FFA. Thisstudy was funded by the Junta de Castilla y Leo� n (TheRegional Government). Jose� Mar•� a Fresno wasawarded a grant from the Consejeri� a de Agricultura yGanaderi� a of the Junta de Castilla y Leo� n.

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