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Effect of ewe's-milk pasteurization on the free amino acids in Idiazábal cheese
International Dairy Journal 9 (1999) 135}141
E!ect of ewe's-milk pasteurization on the free amino acids in IdiazaH bal cheese A.I. OrdoH n ez*, F.C. IbaH n ez, P. Torre, Y. Barcina Area de Nutricio& n y Bromatologn& a, Dept de Ciencias del Medio Natural, Universidad Pu& blica de Navarra, Campus Arrosadia s/n 31006, Pamplona, Spain Received 21 January 1998; accepted 21 March 1999
Abstract This study compared the use of raw and pasteurized milk in the manufacture of IdiazaH bal ewe's-milk cheese. Two indigenous starters, A and B were tested in an attempt to "nd a starter culture potentially capable of developing the typical characteristics of IdiazaH bal cheese traditionally made from raw milk while using pasteurized milk. The nitrogen fractions and free amino acids were analysed over the ripening period. Pasteurization caused a considerable decrease in amino acid release in the cheese during ripening as re#ected by the levels of non-protein}nitrogen and total free amino acids with starter A, but not when starter B was used. Multivariate analysis con"rmed increases in asparagine, serine, and taurine and decreases in aspartic acid and glycine with pasteurization, irrespective of the starter used. The changes recorded in the levels of certain amino acids may be directly or indirectly responsible for the atypical sensory characteristics of the cheeses. Pasteurization of the milk for the manufacture of IdiazaH bal cheese is not recommended with either of the two starters tested. 1999 Elsevier Science Ltd. All rights reserved. Keywords: IdiazaH bal Ewe's-milk cheese; Starter A and B; Pasteurization
1. Introduction Pasteurization of milk to be used in cheesemaking is intended mainly to reduce microbial loads and ensure hygiene and quality, while at the same time lowering the risk that undesirable characteristics such as blowing caused by butyric fermentation or o!-#avours will subsequently appear in the cheese (GoH mez & PelaH ez, 1989). Additional advantages a!orded by pasteurization of milk for cheesemaking include enhanced control over lactic acid production during cheese manufacture, greater product consistency, standard levels of quality, and ripening at higher temperatures, thereby reducing production costs (Lau, Barbano & Rasmussen, 1991; Quintanilla & Pen a, 1991). However, milk pasteurization is known to adversely a!ect the development of many sensory characteristics of cheese, leading to alterations in texture, incomplete #avour development, and often delayed maturation, along with increased levels of the hydrophobic peptides that
* Corresponding author. Tel.: ##34-48-169106; fax: ##34-48169187. E-mail address: [email protected] (A.I. OrdoH n ez)
contribute to the formation of bitter #avours, especially in hard and semihard cheeses (Lau et al., 1991; Van den Berg & Exterkate, 1993). As a result, since concentrations of potential pathogens are known to fall below detectable levels during cheese ripening, there is a long-standing tradition of making cheeses from raw milk, particularly of ewes and goats, in most Mediterranean countries. Such cheeses have sensory characteristics that di!er substantially from those of cheeses made from pasteurized milk. Proteolysis is perhaps the most important process taking place during cheese ripening (Grappin, Rank & Olson, 1985; Rank, Grappin & Olson, 1985; Fox, 1989; Law, Fitzgerald, Uniacke-Lowe, Daily & Fox, 1993). It contributes to cheese #avour, aroma, and texture either directly through the release of peptides and free amino acids, some of which may have o!-#avours, or indirectly by the breakdown of these proteolysis products to amines, acids, thiols, thioesters, etc. (Adda, Grippon & Vassal, 1982; Stadhouders, Hup, Exterkate & Visser, 1983; Visser, Slangen, Hup & Stadhouders, 1983; Urbach, 1993). The peptide systems of the bacteria in the starter play a very important role in proteolysis, and a number of studies have been published on the e!ect of lactic starter cultures on proteolysis during cheese
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 0 3 4 - 5
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A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141 Table 1 Starter culture composition and milk treatment combinations used to produce the di!erent batches of IdiazaH bal cheese
ripening (Kamaly & Marth, 1989; Khalid & Marth, 1990; Tan & Konings, 1990; Oberg, Weimer, Moyes, Brown & Richardson, 1991; Law et al., 1992; Requena, PelaH ez & Fox, 1993; Venema, 1993). However, little has been reported comparing the e!ect of starter cultures on ripening in ewe's milk cheeses made from raw and pasteurized milk. The objective of this work was to compare two starter cultures capable of producing the typical characteristics of IdiazaH bal a traditional raw ewe's-milk cheese, for use with pasteurized milk.
2. Materials and methods
Indigenous starter cultures A and B from the Gaiker Technology Centre, Spain.
Batch
Milk treatment
Starter
(%)
Composition in starters
AR
Raw
A
10
AP
Pasteurized
A
90
BR
Raw
B
10
BP
Pasteurized
B
90
Lactococcus lactis subsp. diacetylactis GC 57 Lactobacillus casei subsp. casei GC 59 Lactococcus lactis subsp. diacetylactis GC 58 Lactobacillus casei subsp. casei GC 59
2.1. Cheese samples Four di!erent batches of cheese (AR, AP, BR, and BP), were made in a single manufacturing trial at a cheese manufacturing establishment approved by the Regulatory Board of the IdiazaH bal Cheese Appellation of Origin (ICAO). Two batches (AR and BR) were made from raw milk in accordance with the regulations of the Regulatory Board of the ICAO (Ministerio de Agricultura, Pesca y AlimentacioH n, 1993). The other two batches (AP and BP) were made following the same procedures using milk that had been pasteurized at 72}743C for 15 s. Lamb's rennet was added as 100 ml of a 12.5% (w/w) solution per 100 l of milk. Starter was added in a 1.5% (p/v) proportion. The two starter cultures employed in this study consisted of indigenous strains isolated from IdiazaH bal cheese in a previous study (PeH rez-Elortondo, Albisu & Barcina, 1993). The strains and their proportions in indigenous starters A and B are listed in Table 1. Each of the two starters contained a di!erent strain of Lactococcus lactis subsp. diacetylactis, the main bacterial species in both starters. Coagulation was achieved by holding the milk at 303C for 25 min. The coagulum was cut and heated at 363C. The curd was sliced, moulded, pressed, and the cheeses submerged in aproximately 163B brine at 103C for 24 h. After brining, the cheeses were kept in a chamber at 10}123C and 80}85% RH for one week and at 123C and 75}80% RH for an additional week. Afterwards, the cheeses were transferred to a ripening chamber, where they were stored at 63C and 80% RH; periodically, samples were collected for analysis. Four cheeses from each of the four ripening batches were sampled on each of days 1, 15, 30, 90, 120, and 180. In all, samples from a total of 96 cheeses (4;4;6) were analysed.
no. 4 (IDF, 1986). The total nitrogen (TN) content was estimated (Kjeldahl method) according to IDF standard no. 25 for cheese (IDF, 1985). ISO standard no. 334331975 (ISO, 1975) was used to determine the fat content of the cheeses. The soluble nitrogen (SN) fraction was determined by precipitating out the insoluble nitrogen fraction (caseins) in a bu!ered solution of acetic acid/acetate at pH 4.6 as per Basch, Douglas, Procino, Holsinger and Farrel (1985). The non-protein nitrogen (NPN) fraction was obtained by adding a 15% (w/w) trichloroacetic acid to precipitate out the soluble and insoluble proteins (Basch et al., 1985). The method published by Krause, Bockhardt, Neckermann, Henle and Klostermeyer (1995) was used to separate out the proteins and extract the free amino acids (FAAs) from the cheese samples. Evaluation of the extracted amino acids was made by gradient reversed-phase high-performance liquid chromatography (HPLC) using Waters (Pierce, Rockford, IL, USA) equipment that included an ULTRA WISP 715 automatic injector and a Waters M996 diode array detector. Precolumn derivatization was performed in a Waters PicoTag vacuum station (Pierce, Rockford, IL, USA) using phenyl isothiocyanate. 2.3. Sensory analysis Sensory analyses of the cheeses were carried out after 120 days of ripening by the descriptive analysis method. A 6 point scale-scoresheet was used (OrdoH n ez, IbaH n ez, PeH rez-Elortondo, Torre & Barcina, 1998). The tests were performed by a panel of expert graders, members of the tasting committe of the Apellation of Origin of IdiazaH bal cheese. Samples were cut into triangles of approximately 1 cm of thickness, avoiding the area close to the rind.
2.2. Chemical analysis
2.4. Statistical analysis
The pH was measured according to the method of Berdague, Grappin (1987). Total dry matter (DM) was determined according to the IDF standard
SPSS statistical package version 6.1 (SPSS, Inc., Chicago, IL, USA) was used for statistical treatment of the results. Two-way analysis of variance (a"0.05) was
A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141
employed to test for statistically signi"cant di!erences in all the variables considered over the ripening period using, as the factors, milk pasteurization and starter culture. Factorial analysis was applied to the results. Principal component analysis (PCA) was selected to extract the useful variables with the least loss of information. Kaiser's criterion (eigenvalue'1) was employed to establish the number of "nal factors from the general parameters considered. Orthogonal rotation of the factors using the &varimax' method was used to interpret the results. Stepwise discriminant analysis using Wilks' lambda selection criterion was also applied to determine the parameters explaining the variance between the raw and pasteurized-milk cheeses (Bisquerra, 1989).
3. Results and discussion For pH 4.6 soluble nitrogen, 15% TCA soluble nitrogen and total free amino acid (TFAA) variables, the percentage of the increment or decrement a!ected by the pasteurization treatment using starters A or B, was calculated by applying the following formula (Table 2): (XM !XM ) . ;100. Increment (%)" 0 XM 0 For every amino acid, the increments were calulated by substracting pasteurized milk sample content from raw milk sample content of cheeses from batches produced with starter A or B, respectively, for every day of the ripening time tested. Pasteurization did not appear to a!ect the general parameters (pH, DM) to any appreciable extent. Initially, the pH fell in all the cheese batches, attaining a minimum after 90 d of ripening in all the batches except batch BP, in which the minimum was reached earlier. The drop can be attributed to the formation of lactic acid from the residual lactose in the cheese by the lactic acid bacteria present (Fox, Lucey & Cogan, 1990). The lowest pH value, 5.13, was attained in batch AP; however, the differences among batches AR, AP, and BR were not signi"cant. In contrast, the pH values for batch BP were
137
signi"cantly higher (5.61) than the values for the other batches. After the minimum had been reached, the pH gradually increased. The increase can be ascribed to the degradation of the lactic acid following metabolization of all the residual lactose, and to the formation of alkaline compounds (Farkye & Fox, 1990). The mean percentage DM was similar for all the batches, ranging from 60.01$1.15 on day 1 to 68.38$ 0.94 on day 180. The percentage fat content in the DM did not exhibit signi"cant variations among the batches and likewise it did not vary over the ripening period; the mean fat content (as a percentage of DM) was 54.3$1.5. Apart from some small, although signi"cant, di!erences at the start of ripening, the mean total nitrogen content can be regarded as similar in all the batches, with a mean value of 5.54$0.22. Table 2 gives the values for the increments expressed as percentage in the ripening indices SN and NPN both expressed as percentage of TN and total FAAs, with milk pasteurization using starters A and B. The SN content (as a percentage of TN) increased signi"cantly (P)0.05) during ripening. The SN content di!ered between batches AR and AP and batches BR and BP, generally with higher values in the batches made from the raw milk. This might be indicative of a possible inhibitory e!ect of milk pasteurization on the SN fraction, as previously reported by Lau et al. (1991). Nevertheless, the di!erences were not signi"cant at the end of ripening. There were signi"cant di!erences in the NPN values during ripening. When starter A was used, the values for the raw-milk cheeses were, on the whole, higher than those for the pasteurized-milk cheeses. Table 2 shows that this was not the case when starter B was used, and the NPN index values increased (except at day 90) in the pasteurized-milk batch, though the increase was not signi"cant. As shown, the di!erences were negative for all the total FAA values for the batches made with starter B, whereas the sign was positive from day 15 of the ripening time when starter A was used. These results apparently indicate that starter B was more proteolytic (release of amino acids and low molecular weight peptides) when pasteurized milk was used. The published results concerning the e!ect of pasteurization on the ripening indices SN and NPN are not
Table 2 Percentage of increment [(XM !XM )/XM ;100] of the pH 4.6 soluble (SN), 15% TCA soluble (NPN), and total free amino acid (TFAA, in 0 . 0 mg 100 g\ dry matter) nitrogen fractions using starters A and B during cheese ripening (in days). Percentages were calculated from means of four replicates Starter A Days % SN/TN % NPN/TN TFAA
1 !9.6 !159.5 !36.2
Starter B 15
30
90
120
180
0.7 2.4 24.2
6.7 10.4 40.4
13.9 13.6 58.9
1.9 22.3 58.4
9.4 25.3 65.3
1 12.0 !11.8 !32.4
15
30
90
120
180
!1.3 !14.8 !68.8
4.3 !24.0 !93.7
8.5 4.0 !28.8
7.8 !2.4 !19.8
11.1 !6.2 !3.5
A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141
138
Table 3 Increments (AR content!AP content) in the free amino acids (FAAs) with milk pasteurization in the cheese batches made with added starter A during ripening (in days); Values expressed in mg 100 g\ of DM
Table 4 Increments (BR content!BP content) in the free amino acids (FAAs) with milk pasteurization in the cheese batches made with added starter B during ripening (in days); Values expressed in mg 100 g\ of DM
FAA/Days 1
15
30
FAA/Days 1
15
30
90
ASP GLU HYPRO SER ASN GLY TAU HYS GABA CIT THR ALA ARG PRO AAB TYR VAL MET CYS ILE LEU HYLYS PHE TRP ORN LYS
3.1 !9.3 !11.2 !1.5 !24.5 1.1 !0.8 0.6 38.4 !0.2 2.1 8.7 0.6 !1.5 1.8 0.5 23.1 2.8 0.6 3.2 26.3 0.0 8.0 1.5 !3.5 !5.1
7.8 5.7 0.0 !4.9 !30.5 2.4 !0.7 1.3 71.9 !3.7 3.4 16.5 0.1 !4.8 0.5 !0.7 43.2 7.8 1.0 8.1 51.3 0.3 21.0 2.2 !3.5 !4.6
ASP GLU HYPRO SER ASN GLY TAU HYS GABA CIT THR ALA ARG PRO AAB TYR VAL MET CYS ILE LEU 1H-LYS PHE TRP ORN LYS
0.7 !13.9 !11.5 !2.3 !15.5 1.1 !0.8 1.3 2.0 !1.4 !3.2 !5.1 0.6 !18.8 0.0 !2.6 !2.9 !2.5 !0.1 !4.1 !13.3 !0.2 !7.3 !0.5 !3.1 !9.6
1.2 !48.5 0.0 !4.7 !18.1 2.7 !0.7 1.7 29.0 !4.7 !7.8 !20.0 2.2 !28.8 0.0 !9.2 !26.1 !9.4 !0.1 !12.8 !36.1 !1.4 !27.0 !0.6 !12.5 !25.9
6.8 7.2 22.1 !158.1 !146.2 !157.7 0.0 9.7 0.2 !7.3 !12.8 !17.8 !24.8 !13.5 !25.9 8.4 15.3 20.6 !0.3 !0.2 !0.2 3.9 6.6 14.7 190.7 226.0 220.2 10.5 8.8 39.3 !7.0 8.4 !12.1 !32.4 !27.5 !15.9 6.2 7.7 !4.5 !67.1 !71.7 !70.6 0.0 0.0 0.0 !4.4 !4.6 !13.0 !28.1 !17.1 !7.4 !3.6 !8.2 !0.9 5.4 1.4 !0.3 !32.9 !49.9 !9.2 !84.6 !97.2 !14.7 0.0 !3.1 2.8 !32.5 !19.8 !21.9 !0.9 !1.5 !1.7 8.9 8.5 18.8 !42.6 !58.4 !26.3
0.0 !11.3 11.4 !1.4 !18.0 0.0 !0.7 !0.8 13.6 !1.4 !1.2 0.3 0.0 !12.5 0.0 !1.4 !2.9 0.9 0.0 !1.0 !3.2 0.0 !1.2 !0.2 !2.1 !6.5
90 20.9 68.0 0.0 !7.5 !17.1 7.9 !0.3 3.6 116.9 0.7 20.2 34.0 3.9 17.4 1.3 2.5 76.5 23.0 5.1 24.2 125.4 1.3 54.6 2.7 20.3 31.1
120
180
56.5 76.9 305.3 425.9 8.1 0.1 !20.9 !16.3 !43.2 !60.3 14.3 20.5 !0.2 !0.2 !1.0 14.6 177.3 263.1 !12.1 17.1 29.1 47.0 75.5 88.3 !5.7 !6.4 73.6 111.4 6.8 0.0 2.3 !1.1 113.2 155.3 39.5 69.7 22.3 4.9 31.9 85.2 173.0 208.4 1.3 0.0 69.4 92.1 !4.8 0.0 31.8 68.8 73.3 135.5
consistent. Lau et al. (1991) reported that pasteurization did not a!ect the soluble fraction in Cheddar cheese, while the percentage NPN in cheeses made from pasteurized milk decreased during ripening. On the other hand, Gaya, Medina, RodrmH guez-MarmH n and NuH n ez (1990) recorded higher levels of degradation of the a- and b-caseins in cheeses made from pasteurized milk and higher SN contents at pH 4.6 in cheeses made from raw milk. Tables 3 and 4 show the changes for all the amino acids analysed. No variations were recorded in the values for such amino acids as phosphoethanolamine, b-alanine, b-aminoisobutyric acid, 1-M-histidine, 3-M-histidine, and 2-hydroxylysine, probably because the levels of these amino acids in the cheeses were very low and occasionally were below the detection threshold, hence those amino acids have not been included in the Tables. Pasteurization of the milk seemed to exert a distinct e!ect on the levels of certain amino acids in the cheese over the ripening period, irrespective of the starter used. This was the case for serine, asparagine, and taurine, whose levels increased. Higher levels of asparagine and serine as a consequence of pasteurization have been observed by other workers in Cheddar cheese (Kosikowski, 1951; Bullock & Irvine, 1956). Aspartic acid, glycine, and histidine decreased as a result of pasteurization, as did
0.0 !9.6 12.6 0.3 !10.8 0.0 !0.7 !0.7 8.3 0.4 !1.1 !2.6 0.0 !7.5 0.0 !1.7 !3.8 !1.4 0.0 !2.0 !5.9 0.0 !3.1 !0.2 !2.8 !8.6
120
180
c-aminobutyric acid, an amino acid formed by decarboxylation of glutamic acid. Other studies have also described negative e!ects of pasteurization on the levels of biogenic amines formed by the action of decarboxylase on the respective amino acids in IdiazaH bal cheese (OrdoH n ez, IbaH n ez, Torre & Barcina, 1997) and in Gouda cheese (Joosten, 1987). The contents of ornithine, a nonprotein amino acid, were slightly higher in the pasteurized batches at the start of ripening, but by the end of ripening the levels were higher in the batches made from the raw milk. The levels of the amino acids that made the greatest contribution to the total FAAs, such as glutamic acid, valine, methionine, leucine, phenylalanine, and lysine, followed di!ering trends depending on the starter employed. For starter A, levels of the above-mentioned amino acids were higher in the cheeses made from the raw milk, whereas for starter B, levels were higher in the batches made from the pasteurized milk. Higher levels of proteolysis in cheeses made from pasteurized milk compared to cheeses made from raw milk have been described by OrdoH n ez and Burgos (1977), and this may be related to the speci"c peptidase and aminopeptidase activity of the strains in the starter cultures used. On the other hand, the higher levels of FAAs in batch BP compared to batch BR in this study also needs to be related
A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141
to the higher pH values recorded in the former batch throughout the ripening period. Following veri"cation of the adequacy of the data matrix, PCA was applied to the data set and reduced the 38 variables considered to three main factors that explained 74.4% of the variance. The "rst factor (explaining 58.5% of the variance) appeared to represent variability as a result of ripening time, particularly from day 90 on (Fig. 1). The previously mentioned amino acids with the highest levels during ripening appears to make a signi"cant contribution to that factor. Factor 2 (explaining 7.2% of the variance) mainly comprised the amino acids that were positively or negatively a!ected by pasteurization of the milk. Nevertheless, the distribution of the samples in the space de"ned by these two factors (Fig. 1) suggested that there was an additional contributor to factor 2, possibly the e!ect of the starter used. Subsequent stepwise discriminant analysis correctly classi"ed the samples and indicated that leucine, asparagine, threonine, aspartic acid, taurine, citrulline, arginine, ornithine, and glycine were the amino acids most a!ected by pasteurization of the milk. The e!ect of the starter used on the levels of certain amino acids and on the ripening indices can be associated with the results of the sensory analysis of the cheeses carried out after 120 days of ripening by a panel of seven trained judges using the 6 point scale scoresheet
139
Fig. 1. Distribution of cheese samples in a PCA plot of "rst and second principal components.
issued by the ICAO for purposes of quality control (OrdoH n ez et al., 1998). The AR cheeses were awarded the highest scores, the cheeses in batch BP the lowest (Table 5). On the whole, the cheeses made from the pasteurized milk were characterized by a sweet aroma
Table 5 Means of the sensory scores, for the four 120 days ripened cheeses, obtained on a 6-point scale (0) not present, (1) very weak, (2) weak, (3) medium, (4) intense, (5) very intense Descriptor
AR
AP
BR
BP
Odour
Pungent Acid Sweet Characteristic
1.0 2.1 0.0 3.0
0.8 1.3 2.9 2.4
1.5 1.4 0.5 2.3
0.8 1.0 1.3 1.8
Texture
Elasticity Creaminess Firmness Grainy
3.1 3.8 2.4 0.9
2.1 2.9 3.4 1.6
3.0 3.4 2.8 1.6
3.8 2.6 1.9 1.8
Taste
Pungent Sweet Acid Bitter Salty Characteristic
2.1 0.0 2.0 0 2.9 3
1.3 2.6 1.1 0.5 2.5 2.1
2.8 0.3 2.0 1.1 2.8 2.5
1.6 2.5 1.1 1.6 2.8 1.1
Aftertaste
Pungent Acid Bitter Characteristic
3.0 1.9 0.0 3.9
1.4 1.1 0.5 2.4
2.6 1.9 0.9 2.6
1.6 0.8 1.1 1.1
49 Well maturated
38.5 Tasteless
33.5 Tasteless
12.5 Odourless O!-#avour Plastic
Total score Observations
140
A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141
and #avour. The characteristic IdiazaH bal cheese #avour was not as strong in the pasteurized-milk cheeses, which were also somewhat less creamy, less pungent and less acid than the raw-milk cheeses. Certain observations noted on the sensory scoresheets indicated that batch AP lacked #avour, and some of the judges recorded o!-#avours in batch BP. This may be related to the high amino acid contents. Various researchers have described how pasteurization slows and alters the development of the sensory characteristics of cheese (Gaya et al., 1990; Lau et al., 1991; McSweeney, Fox, Lucey, Jordan & Cogan, 1993). Some of the characteristics perceived in these cheeses may have been related to the increased levels of certain amino acids. Proline has been associated with a sweet #avour in Edam cheese (Zaki & Salem, 1992) and asparagine and serine have also been associated with sweet #avours (Haefeli & Glaser, 1990); pasteurization raised the levels of those amino acids. Arginine and, particularly, peptide-containing aromatic amino acids have been associated with bitter #avours in cheese (Lau et al., 1990). GoH mez, Garde, Gaya, Medina and NuH n ez (1997) reported that heat treatments applied to the milk had no e!ect on the levels of the hydrophobic and hydrophilic peptides in the water-soluble fraction. None of the cheeses in this study presented appreciable bitter #avour overtones.
4. Conclusions Pasteurization substantially decreased the level of free amino acids in the cheeses made using starter A as re#ected by the NPN index and total FAA values during ripening. The levels of those same fractions were not a!ected by milk pasteurization when starter B was used, though this may have been related to resulting pH values. Pasteurization raised the levels of such amino acids as asparagine, serine, and taurine and lowered the levels of such other amino acids as aspartic acid and glycine, regardless of the starter used. The changes observed in the levels of certain amino acids could be directly or indirectly responsible for the sensory characteristics of the various cheeses. The characteristic IdiazaH bal cheese #avour was more highly developed in the batches made from the raw milk, especially when starter A was used. Pasteurization of milk to be used in making IdiazaH bal cheese is not recommended with either of the starters considered in this study, because, even though a high level of proteolysis was achieved using starter B, sensory characteristics were indicative of developments atypical of IdiazaH bal cheese.
Acknowledgements The authors wish to thank the Department of Education of the Government of the Autonomous Community
of Navarre and the Interministerial Commission for Science and Technology (ALI93-0895-CO2-01) for the funding provided and the Regulatory Board of the IdiazaH bal Cheese Appellation of Origin for providing cheese samples.
References Adda, J., Grippon, J. C., & Vassal, J. (1982). The chemistry of #avour and texture generation in cheese. Food Chemistry, 9, 115}129. Basch, J. J., Douglas Jr, F. W., Procino, L. G., Holsinger, V. H., & Farrel Jr, H. M. (1985). Quantitation of caseins and whey proteins of processed milks and whey protein concentrates, application of gel electrophoresis, and comparison with Harland}Ashwort procedure. Journal of Dairy Science, 68, 23}31. Berdague, J. L., & Grappin, R. (1987). A$nage et qualiteH du Gruye`re de ComteH . II. In#uence de l'a$nage sur l'eH volution des caracteH ristiques physico-chimiques des fromages. Lait, 67, 237}241. Bisquerra, R. (1989). IntroduccioH n conceptual al anaH lisis multivariable. Un enforque informaH tico con los paquetes SPSS-X, BMDP, LISREL y SPAD. Editorial PPU.S.A. Barcelona, Spain. Bullock, D. H., & Irvine, O. R. (1956). A chromatographic study of Cheddar cheese ripening. Journal of Dairy Science, 39, 1229}1234. Farkye, N. Y., & Fox, P. F. (1990). Objective indices of cheese ripening. Trends in Food Science and Technology, 1, 37}40. Fox, P. F. (1989). Proteolysis during cheese manufacture and ripening. Journal of Dairy Science, 72, 1379}1408. Fox, P. F., Lucey, J. A., & Cogan, T. M. (1990). Glycolysis and related reactions during cheese manufacture and ripening. Food Science and Nutrition, 29, 237}253. Gaya, P., Medina, M., RodrmH guez-MarmH n, M. A., & NuH n ez, M. (1990). Accelerated ripening of ewe's milk Manchego cheese: the e!ect of elevated ripening temperatures. Journal of Dairy Science, 73, 26}32. GoH mez, R., & PelaH ez, C. (1989). Lactic starters in the cheese industry. Alimentacio& n, Equipos y Tecnologn& a, 1, 193}202. GoH mez, M. J., Garde, S., Gaya, P., Medina, M., & NuH n ez, M. (1997). Relationship between level of hydrophobic peptides and bitterness in cheese made from pasteurized and raw milk. Journal of Dairy Research, 64, 289}297. Grappin, R., Rank, T. C., & Olson, N. F. (1985). Primary proteolysis of cheese proteins during ripening. A review. Journal of Dairy Science, 68, 531}540. Haefeli, R. J., & Glaser, D. (1990). Taste responses and thresholds obtained with primary amino acids in humans. Lebensmittel Wissenschaft & Technologie, 23, 523}527. IDF (1985). Cheese and processed cheese products. Determination of total protein. Kjeldahl method. International Dairy Federation Brussels, Belgium (FIL-IDF standard no. 25). IDF (1986). Cheese and processed cheese products. Determination of dry matter. International Dairy Federation. Brussels, Belgium (FIL-IDF standard no. 4). ISO (1975). Cheese: determination of fat matter. Van Gulik method. International Organization for Standarization. Geneva, Switzerland. (Standard 33433). Joosten, H. M. L. J. (1987). Conditions allowing the formation of biogenic amines in cheese. 3. Factors in#uencing the amounts formed. Netherland Milk and Dairy Journal, 41, 329}357. Kamaly, K. M., & Marth, E. H. (1989). Enzyme activities of lactic streptococci and their role in maturation of cheese: a review. Journal of Dairy Science, 72, 1945}1966. Khalid, N. M., & Marth, E. H. (1990). Lactobacilli: their enzymes and role in ripening and spoilage of cheese: a review. Journal of Dairy Science, 73, 2669}2684.
A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141 Kosikowski, F. V. (1951). The liberation of free amino acids in raw and pasteurized milk Cheddar cheese during ripening. Journal of Dairy Science, 34, 235}241. Krause, I., Bockhardt, A., Neckermann, H., Henle, T., & Klostermeyer, H. (1995). Simultaneous determination of amino acids and biogenic amines by RP-HPLC of the dansyl derivative. Journal of Chromatography A, 715, 67}79. Lau, K. L., Barbano, M., & Rasmussen, R. R. (1991). In#uence of pasteurization of milk on protein breakdown in Cheddar cheese during aging. Journal of Dairy Science, 74, 727}740. Law, J., Fitzgerald, G. F., Daily, C., Fox, P. F., & Farkye, N. Y. (1992). Proteolysis and #avor development in Cheddar cheese made with the single starter strains Lactococcus lactis ssp lactis UC317 or Lactococcus lactis ssp cremoris HP. Journal of Dairy Science, 75, 1173}1185. Law, J., Fizgerald, G. F., Uniacke-Lowe, T., Daily, C., & Fox, P. F. (1993). The contribution of lactococcal starter proteinases to proteolysis in Cheddar cheese. Journal of Dairy Science, 76, 2455}2467. McSweeney, P. H. L., Fox, P. F., Lucey, J. A., Jordan, K. N., & Cogan, T. M. (1993). Contribution of the indigenous micro#ora to the maturation of Cheddar cheese. International Dairy Journal, 3, 613}634. Ministerio de Agricultura, Pesca y AlimentacioH n. (1993). Regulatory Board of the IdiazaH bal Cheese Appellation of Origin. Boletin Oxcial del Estado, 289, 34 591}34 596. Oberg, C. J., Weimer, B. C., Moyes, L. V., Brown, R. J., & Richardson, G. H. (1991). Proteolytic characterization of Lactobacillus delbrueckii subsp. bulgaricus strains by the o-phtaldialdehyde test and amino acid analysis. Journal of Dairy Science, 74, 398}403. OrdoH n ez, A. I., IbaH n ez, F. C., Torre, P., & Barcina, Y. (1997). Formation of biogenic amines in IdiazaH bal ewe's milk cheese: e!ect of ripening, pasteurization and starter. Journal of Food Protection, 60, 1}6. OrdoH n ez, A. I., IbaH n ez, F. C., PeH rez-Elortondo, F. J., Torre, P., & Barcina, Y. (1998). Application of multivariate analysis to sensory characterization of ewes' milk cheese. Journal of Sensory Studies, 13, 45}55.
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OrdoH n ez, J. A., & Burgos, J. (1977). Ulloa cheese. Free amino acids formed during ripening. Lait, 57, 416}420. PeH rez-Elortondo, F. J., Albisu, M., & Barcina, Y. (1993). Changes in the micro#ora of IdiazaH bal cheese with addition of commercial lactic starters. Australian Journal of Dairy Technology, 48, 10}14. Quintanilla, M. A., & Pen a, A. E. (1991). Cheesemaking yield. Alimentaria, 3, 39}42. Rank, T. C., Grappin, R., & Olson, N. (1985). Secondary proteolysis of cheese during ripening: a review. Journal of Dairy Science, 68, 801}805. Requena, T., PelaH ez, C., & Fox, P. F. (1993). Peptidase and proteinase activity of Lactococcus lactis, Lactobacillus casei and Lactobacillus plantarum. Zeitschrift fu( r Lebensmittel Untersuchung und Forschung, 196, 351}355. SPSS. Base System User's guide. VersioH n 6.1, Edition (1994). SPSS Inc. Chicago. IL. Stadhouders, J., Hup, G., Exterkate, F. A., & Visser, S. (1983). Bitter #avour in cheese. I. Mechanism of the formation of bitter #avour defects in cheese. Netherland Milk and Dairy Journal, 37, 157}169. Tan, P., & Konings, W. N. (1990). Puri"cation and characterization of an aminopeptidase from Lactococcus lactis subsp cremoris Wg2. Applied Environmental Microbiology, 56, 526}532. Urbach, G. (1993). Relations between cheese #avour and chemical composition. International Dairy Journal, 3, 389}422. Van den Berg, G., & Exterkate, F. A. (1993). Technological parameters involved in cheese ripening. International Dairy Journal, 3, 485}507. Venema, G. (1993). Molecular biology and genetic modi"cation of Lactococci. Journal of Dairy Science, 76, 2133}2144. Visser, S., Slangen, K. J., Hup, G., & Stadhouders, J. (1983). Bitter #avour in cheese. 3. Comparative gel-chromatographic analysis of hydrophobic peptide fractions from twelve Gouda-type cheeses and identi"cation of bitter peptides isolated from a cheese made with Streptococcus cremoris strain HP 1, 2. Netherlands Milk and Dairy Journal, 37, 181}187. Zaki, N., & Salem, S. A. (1992). E!ect of proteolytic enzymes on accelerated ripening of Edam cheese. Indian Journal of Dairy Science, 45, 303}312.
E!ect of ewe's-milk pasteurization on the free amino acids in IdiazaH bal cheese A.I. OrdoH n ez*, F.C. IbaH n ez, P. Torre, Y. Barcina Area de Nutricio& n y Bromatologn& a, Dept de Ciencias del Medio Natural, Universidad Pu& blica de Navarra, Campus Arrosadia s/n 31006, Pamplona, Spain Received 21 January 1998; accepted 21 March 1999
Abstract This study compared the use of raw and pasteurized milk in the manufacture of IdiazaH bal ewe's-milk cheese. Two indigenous starters, A and B were tested in an attempt to "nd a starter culture potentially capable of developing the typical characteristics of IdiazaH bal cheese traditionally made from raw milk while using pasteurized milk. The nitrogen fractions and free amino acids were analysed over the ripening period. Pasteurization caused a considerable decrease in amino acid release in the cheese during ripening as re#ected by the levels of non-protein}nitrogen and total free amino acids with starter A, but not when starter B was used. Multivariate analysis con"rmed increases in asparagine, serine, and taurine and decreases in aspartic acid and glycine with pasteurization, irrespective of the starter used. The changes recorded in the levels of certain amino acids may be directly or indirectly responsible for the atypical sensory characteristics of the cheeses. Pasteurization of the milk for the manufacture of IdiazaH bal cheese is not recommended with either of the two starters tested. 1999 Elsevier Science Ltd. All rights reserved. Keywords: IdiazaH bal Ewe's-milk cheese; Starter A and B; Pasteurization
1. Introduction Pasteurization of milk to be used in cheesemaking is intended mainly to reduce microbial loads and ensure hygiene and quality, while at the same time lowering the risk that undesirable characteristics such as blowing caused by butyric fermentation or o!-#avours will subsequently appear in the cheese (GoH mez & PelaH ez, 1989). Additional advantages a!orded by pasteurization of milk for cheesemaking include enhanced control over lactic acid production during cheese manufacture, greater product consistency, standard levels of quality, and ripening at higher temperatures, thereby reducing production costs (Lau, Barbano & Rasmussen, 1991; Quintanilla & Pen a, 1991). However, milk pasteurization is known to adversely a!ect the development of many sensory characteristics of cheese, leading to alterations in texture, incomplete #avour development, and often delayed maturation, along with increased levels of the hydrophobic peptides that
* Corresponding author. Tel.: ##34-48-169106; fax: ##34-48169187. E-mail address: [email protected] (A.I. OrdoH n ez)
contribute to the formation of bitter #avours, especially in hard and semihard cheeses (Lau et al., 1991; Van den Berg & Exterkate, 1993). As a result, since concentrations of potential pathogens are known to fall below detectable levels during cheese ripening, there is a long-standing tradition of making cheeses from raw milk, particularly of ewes and goats, in most Mediterranean countries. Such cheeses have sensory characteristics that di!er substantially from those of cheeses made from pasteurized milk. Proteolysis is perhaps the most important process taking place during cheese ripening (Grappin, Rank & Olson, 1985; Rank, Grappin & Olson, 1985; Fox, 1989; Law, Fitzgerald, Uniacke-Lowe, Daily & Fox, 1993). It contributes to cheese #avour, aroma, and texture either directly through the release of peptides and free amino acids, some of which may have o!-#avours, or indirectly by the breakdown of these proteolysis products to amines, acids, thiols, thioesters, etc. (Adda, Grippon & Vassal, 1982; Stadhouders, Hup, Exterkate & Visser, 1983; Visser, Slangen, Hup & Stadhouders, 1983; Urbach, 1993). The peptide systems of the bacteria in the starter play a very important role in proteolysis, and a number of studies have been published on the e!ect of lactic starter cultures on proteolysis during cheese
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 0 3 4 - 5
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A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141 Table 1 Starter culture composition and milk treatment combinations used to produce the di!erent batches of IdiazaH bal cheese
ripening (Kamaly & Marth, 1989; Khalid & Marth, 1990; Tan & Konings, 1990; Oberg, Weimer, Moyes, Brown & Richardson, 1991; Law et al., 1992; Requena, PelaH ez & Fox, 1993; Venema, 1993). However, little has been reported comparing the e!ect of starter cultures on ripening in ewe's milk cheeses made from raw and pasteurized milk. The objective of this work was to compare two starter cultures capable of producing the typical characteristics of IdiazaH bal a traditional raw ewe's-milk cheese, for use with pasteurized milk.
2. Materials and methods
Indigenous starter cultures A and B from the Gaiker Technology Centre, Spain.
Batch
Milk treatment
Starter
(%)
Composition in starters
AR
Raw
A
10
AP
Pasteurized
A
90
BR
Raw
B
10
BP
Pasteurized
B
90
Lactococcus lactis subsp. diacetylactis GC 57 Lactobacillus casei subsp. casei GC 59 Lactococcus lactis subsp. diacetylactis GC 58 Lactobacillus casei subsp. casei GC 59
2.1. Cheese samples Four di!erent batches of cheese (AR, AP, BR, and BP), were made in a single manufacturing trial at a cheese manufacturing establishment approved by the Regulatory Board of the IdiazaH bal Cheese Appellation of Origin (ICAO). Two batches (AR and BR) were made from raw milk in accordance with the regulations of the Regulatory Board of the ICAO (Ministerio de Agricultura, Pesca y AlimentacioH n, 1993). The other two batches (AP and BP) were made following the same procedures using milk that had been pasteurized at 72}743C for 15 s. Lamb's rennet was added as 100 ml of a 12.5% (w/w) solution per 100 l of milk. Starter was added in a 1.5% (p/v) proportion. The two starter cultures employed in this study consisted of indigenous strains isolated from IdiazaH bal cheese in a previous study (PeH rez-Elortondo, Albisu & Barcina, 1993). The strains and their proportions in indigenous starters A and B are listed in Table 1. Each of the two starters contained a di!erent strain of Lactococcus lactis subsp. diacetylactis, the main bacterial species in both starters. Coagulation was achieved by holding the milk at 303C for 25 min. The coagulum was cut and heated at 363C. The curd was sliced, moulded, pressed, and the cheeses submerged in aproximately 163B brine at 103C for 24 h. After brining, the cheeses were kept in a chamber at 10}123C and 80}85% RH for one week and at 123C and 75}80% RH for an additional week. Afterwards, the cheeses were transferred to a ripening chamber, where they were stored at 63C and 80% RH; periodically, samples were collected for analysis. Four cheeses from each of the four ripening batches were sampled on each of days 1, 15, 30, 90, 120, and 180. In all, samples from a total of 96 cheeses (4;4;6) were analysed.
no. 4 (IDF, 1986). The total nitrogen (TN) content was estimated (Kjeldahl method) according to IDF standard no. 25 for cheese (IDF, 1985). ISO standard no. 334331975 (ISO, 1975) was used to determine the fat content of the cheeses. The soluble nitrogen (SN) fraction was determined by precipitating out the insoluble nitrogen fraction (caseins) in a bu!ered solution of acetic acid/acetate at pH 4.6 as per Basch, Douglas, Procino, Holsinger and Farrel (1985). The non-protein nitrogen (NPN) fraction was obtained by adding a 15% (w/w) trichloroacetic acid to precipitate out the soluble and insoluble proteins (Basch et al., 1985). The method published by Krause, Bockhardt, Neckermann, Henle and Klostermeyer (1995) was used to separate out the proteins and extract the free amino acids (FAAs) from the cheese samples. Evaluation of the extracted amino acids was made by gradient reversed-phase high-performance liquid chromatography (HPLC) using Waters (Pierce, Rockford, IL, USA) equipment that included an ULTRA WISP 715 automatic injector and a Waters M996 diode array detector. Precolumn derivatization was performed in a Waters PicoTag vacuum station (Pierce, Rockford, IL, USA) using phenyl isothiocyanate. 2.3. Sensory analysis Sensory analyses of the cheeses were carried out after 120 days of ripening by the descriptive analysis method. A 6 point scale-scoresheet was used (OrdoH n ez, IbaH n ez, PeH rez-Elortondo, Torre & Barcina, 1998). The tests were performed by a panel of expert graders, members of the tasting committe of the Apellation of Origin of IdiazaH bal cheese. Samples were cut into triangles of approximately 1 cm of thickness, avoiding the area close to the rind.
2.2. Chemical analysis
2.4. Statistical analysis
The pH was measured according to the method of Berdague, Grappin (1987). Total dry matter (DM) was determined according to the IDF standard
SPSS statistical package version 6.1 (SPSS, Inc., Chicago, IL, USA) was used for statistical treatment of the results. Two-way analysis of variance (a"0.05) was
A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141
employed to test for statistically signi"cant di!erences in all the variables considered over the ripening period using, as the factors, milk pasteurization and starter culture. Factorial analysis was applied to the results. Principal component analysis (PCA) was selected to extract the useful variables with the least loss of information. Kaiser's criterion (eigenvalue'1) was employed to establish the number of "nal factors from the general parameters considered. Orthogonal rotation of the factors using the &varimax' method was used to interpret the results. Stepwise discriminant analysis using Wilks' lambda selection criterion was also applied to determine the parameters explaining the variance between the raw and pasteurized-milk cheeses (Bisquerra, 1989).
3. Results and discussion For pH 4.6 soluble nitrogen, 15% TCA soluble nitrogen and total free amino acid (TFAA) variables, the percentage of the increment or decrement a!ected by the pasteurization treatment using starters A or B, was calculated by applying the following formula (Table 2): (XM !XM ) . ;100. Increment (%)" 0 XM 0 For every amino acid, the increments were calulated by substracting pasteurized milk sample content from raw milk sample content of cheeses from batches produced with starter A or B, respectively, for every day of the ripening time tested. Pasteurization did not appear to a!ect the general parameters (pH, DM) to any appreciable extent. Initially, the pH fell in all the cheese batches, attaining a minimum after 90 d of ripening in all the batches except batch BP, in which the minimum was reached earlier. The drop can be attributed to the formation of lactic acid from the residual lactose in the cheese by the lactic acid bacteria present (Fox, Lucey & Cogan, 1990). The lowest pH value, 5.13, was attained in batch AP; however, the differences among batches AR, AP, and BR were not signi"cant. In contrast, the pH values for batch BP were
137
signi"cantly higher (5.61) than the values for the other batches. After the minimum had been reached, the pH gradually increased. The increase can be ascribed to the degradation of the lactic acid following metabolization of all the residual lactose, and to the formation of alkaline compounds (Farkye & Fox, 1990). The mean percentage DM was similar for all the batches, ranging from 60.01$1.15 on day 1 to 68.38$ 0.94 on day 180. The percentage fat content in the DM did not exhibit signi"cant variations among the batches and likewise it did not vary over the ripening period; the mean fat content (as a percentage of DM) was 54.3$1.5. Apart from some small, although signi"cant, di!erences at the start of ripening, the mean total nitrogen content can be regarded as similar in all the batches, with a mean value of 5.54$0.22. Table 2 gives the values for the increments expressed as percentage in the ripening indices SN and NPN both expressed as percentage of TN and total FAAs, with milk pasteurization using starters A and B. The SN content (as a percentage of TN) increased signi"cantly (P)0.05) during ripening. The SN content di!ered between batches AR and AP and batches BR and BP, generally with higher values in the batches made from the raw milk. This might be indicative of a possible inhibitory e!ect of milk pasteurization on the SN fraction, as previously reported by Lau et al. (1991). Nevertheless, the di!erences were not signi"cant at the end of ripening. There were signi"cant di!erences in the NPN values during ripening. When starter A was used, the values for the raw-milk cheeses were, on the whole, higher than those for the pasteurized-milk cheeses. Table 2 shows that this was not the case when starter B was used, and the NPN index values increased (except at day 90) in the pasteurized-milk batch, though the increase was not signi"cant. As shown, the di!erences were negative for all the total FAA values for the batches made with starter B, whereas the sign was positive from day 15 of the ripening time when starter A was used. These results apparently indicate that starter B was more proteolytic (release of amino acids and low molecular weight peptides) when pasteurized milk was used. The published results concerning the e!ect of pasteurization on the ripening indices SN and NPN are not
Table 2 Percentage of increment [(XM !XM )/XM ;100] of the pH 4.6 soluble (SN), 15% TCA soluble (NPN), and total free amino acid (TFAA, in 0 . 0 mg 100 g\ dry matter) nitrogen fractions using starters A and B during cheese ripening (in days). Percentages were calculated from means of four replicates Starter A Days % SN/TN % NPN/TN TFAA
1 !9.6 !159.5 !36.2
Starter B 15
30
90
120
180
0.7 2.4 24.2
6.7 10.4 40.4
13.9 13.6 58.9
1.9 22.3 58.4
9.4 25.3 65.3
1 12.0 !11.8 !32.4
15
30
90
120
180
!1.3 !14.8 !68.8
4.3 !24.0 !93.7
8.5 4.0 !28.8
7.8 !2.4 !19.8
11.1 !6.2 !3.5
A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141
138
Table 3 Increments (AR content!AP content) in the free amino acids (FAAs) with milk pasteurization in the cheese batches made with added starter A during ripening (in days); Values expressed in mg 100 g\ of DM
Table 4 Increments (BR content!BP content) in the free amino acids (FAAs) with milk pasteurization in the cheese batches made with added starter B during ripening (in days); Values expressed in mg 100 g\ of DM
FAA/Days 1
15
30
FAA/Days 1
15
30
90
ASP GLU HYPRO SER ASN GLY TAU HYS GABA CIT THR ALA ARG PRO AAB TYR VAL MET CYS ILE LEU HYLYS PHE TRP ORN LYS
3.1 !9.3 !11.2 !1.5 !24.5 1.1 !0.8 0.6 38.4 !0.2 2.1 8.7 0.6 !1.5 1.8 0.5 23.1 2.8 0.6 3.2 26.3 0.0 8.0 1.5 !3.5 !5.1
7.8 5.7 0.0 !4.9 !30.5 2.4 !0.7 1.3 71.9 !3.7 3.4 16.5 0.1 !4.8 0.5 !0.7 43.2 7.8 1.0 8.1 51.3 0.3 21.0 2.2 !3.5 !4.6
ASP GLU HYPRO SER ASN GLY TAU HYS GABA CIT THR ALA ARG PRO AAB TYR VAL MET CYS ILE LEU 1H-LYS PHE TRP ORN LYS
0.7 !13.9 !11.5 !2.3 !15.5 1.1 !0.8 1.3 2.0 !1.4 !3.2 !5.1 0.6 !18.8 0.0 !2.6 !2.9 !2.5 !0.1 !4.1 !13.3 !0.2 !7.3 !0.5 !3.1 !9.6
1.2 !48.5 0.0 !4.7 !18.1 2.7 !0.7 1.7 29.0 !4.7 !7.8 !20.0 2.2 !28.8 0.0 !9.2 !26.1 !9.4 !0.1 !12.8 !36.1 !1.4 !27.0 !0.6 !12.5 !25.9
6.8 7.2 22.1 !158.1 !146.2 !157.7 0.0 9.7 0.2 !7.3 !12.8 !17.8 !24.8 !13.5 !25.9 8.4 15.3 20.6 !0.3 !0.2 !0.2 3.9 6.6 14.7 190.7 226.0 220.2 10.5 8.8 39.3 !7.0 8.4 !12.1 !32.4 !27.5 !15.9 6.2 7.7 !4.5 !67.1 !71.7 !70.6 0.0 0.0 0.0 !4.4 !4.6 !13.0 !28.1 !17.1 !7.4 !3.6 !8.2 !0.9 5.4 1.4 !0.3 !32.9 !49.9 !9.2 !84.6 !97.2 !14.7 0.0 !3.1 2.8 !32.5 !19.8 !21.9 !0.9 !1.5 !1.7 8.9 8.5 18.8 !42.6 !58.4 !26.3
0.0 !11.3 11.4 !1.4 !18.0 0.0 !0.7 !0.8 13.6 !1.4 !1.2 0.3 0.0 !12.5 0.0 !1.4 !2.9 0.9 0.0 !1.0 !3.2 0.0 !1.2 !0.2 !2.1 !6.5
90 20.9 68.0 0.0 !7.5 !17.1 7.9 !0.3 3.6 116.9 0.7 20.2 34.0 3.9 17.4 1.3 2.5 76.5 23.0 5.1 24.2 125.4 1.3 54.6 2.7 20.3 31.1
120
180
56.5 76.9 305.3 425.9 8.1 0.1 !20.9 !16.3 !43.2 !60.3 14.3 20.5 !0.2 !0.2 !1.0 14.6 177.3 263.1 !12.1 17.1 29.1 47.0 75.5 88.3 !5.7 !6.4 73.6 111.4 6.8 0.0 2.3 !1.1 113.2 155.3 39.5 69.7 22.3 4.9 31.9 85.2 173.0 208.4 1.3 0.0 69.4 92.1 !4.8 0.0 31.8 68.8 73.3 135.5
consistent. Lau et al. (1991) reported that pasteurization did not a!ect the soluble fraction in Cheddar cheese, while the percentage NPN in cheeses made from pasteurized milk decreased during ripening. On the other hand, Gaya, Medina, RodrmH guez-MarmH n and NuH n ez (1990) recorded higher levels of degradation of the a- and b-caseins in cheeses made from pasteurized milk and higher SN contents at pH 4.6 in cheeses made from raw milk. Tables 3 and 4 show the changes for all the amino acids analysed. No variations were recorded in the values for such amino acids as phosphoethanolamine, b-alanine, b-aminoisobutyric acid, 1-M-histidine, 3-M-histidine, and 2-hydroxylysine, probably because the levels of these amino acids in the cheeses were very low and occasionally were below the detection threshold, hence those amino acids have not been included in the Tables. Pasteurization of the milk seemed to exert a distinct e!ect on the levels of certain amino acids in the cheese over the ripening period, irrespective of the starter used. This was the case for serine, asparagine, and taurine, whose levels increased. Higher levels of asparagine and serine as a consequence of pasteurization have been observed by other workers in Cheddar cheese (Kosikowski, 1951; Bullock & Irvine, 1956). Aspartic acid, glycine, and histidine decreased as a result of pasteurization, as did
0.0 !9.6 12.6 0.3 !10.8 0.0 !0.7 !0.7 8.3 0.4 !1.1 !2.6 0.0 !7.5 0.0 !1.7 !3.8 !1.4 0.0 !2.0 !5.9 0.0 !3.1 !0.2 !2.8 !8.6
120
180
c-aminobutyric acid, an amino acid formed by decarboxylation of glutamic acid. Other studies have also described negative e!ects of pasteurization on the levels of biogenic amines formed by the action of decarboxylase on the respective amino acids in IdiazaH bal cheese (OrdoH n ez, IbaH n ez, Torre & Barcina, 1997) and in Gouda cheese (Joosten, 1987). The contents of ornithine, a nonprotein amino acid, were slightly higher in the pasteurized batches at the start of ripening, but by the end of ripening the levels were higher in the batches made from the raw milk. The levels of the amino acids that made the greatest contribution to the total FAAs, such as glutamic acid, valine, methionine, leucine, phenylalanine, and lysine, followed di!ering trends depending on the starter employed. For starter A, levels of the above-mentioned amino acids were higher in the cheeses made from the raw milk, whereas for starter B, levels were higher in the batches made from the pasteurized milk. Higher levels of proteolysis in cheeses made from pasteurized milk compared to cheeses made from raw milk have been described by OrdoH n ez and Burgos (1977), and this may be related to the speci"c peptidase and aminopeptidase activity of the strains in the starter cultures used. On the other hand, the higher levels of FAAs in batch BP compared to batch BR in this study also needs to be related
A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141
to the higher pH values recorded in the former batch throughout the ripening period. Following veri"cation of the adequacy of the data matrix, PCA was applied to the data set and reduced the 38 variables considered to three main factors that explained 74.4% of the variance. The "rst factor (explaining 58.5% of the variance) appeared to represent variability as a result of ripening time, particularly from day 90 on (Fig. 1). The previously mentioned amino acids with the highest levels during ripening appears to make a signi"cant contribution to that factor. Factor 2 (explaining 7.2% of the variance) mainly comprised the amino acids that were positively or negatively a!ected by pasteurization of the milk. Nevertheless, the distribution of the samples in the space de"ned by these two factors (Fig. 1) suggested that there was an additional contributor to factor 2, possibly the e!ect of the starter used. Subsequent stepwise discriminant analysis correctly classi"ed the samples and indicated that leucine, asparagine, threonine, aspartic acid, taurine, citrulline, arginine, ornithine, and glycine were the amino acids most a!ected by pasteurization of the milk. The e!ect of the starter used on the levels of certain amino acids and on the ripening indices can be associated with the results of the sensory analysis of the cheeses carried out after 120 days of ripening by a panel of seven trained judges using the 6 point scale scoresheet
139
Fig. 1. Distribution of cheese samples in a PCA plot of "rst and second principal components.
issued by the ICAO for purposes of quality control (OrdoH n ez et al., 1998). The AR cheeses were awarded the highest scores, the cheeses in batch BP the lowest (Table 5). On the whole, the cheeses made from the pasteurized milk were characterized by a sweet aroma
Table 5 Means of the sensory scores, for the four 120 days ripened cheeses, obtained on a 6-point scale (0) not present, (1) very weak, (2) weak, (3) medium, (4) intense, (5) very intense Descriptor
AR
AP
BR
BP
Odour
Pungent Acid Sweet Characteristic
1.0 2.1 0.0 3.0
0.8 1.3 2.9 2.4
1.5 1.4 0.5 2.3
0.8 1.0 1.3 1.8
Texture
Elasticity Creaminess Firmness Grainy
3.1 3.8 2.4 0.9
2.1 2.9 3.4 1.6
3.0 3.4 2.8 1.6
3.8 2.6 1.9 1.8
Taste
Pungent Sweet Acid Bitter Salty Characteristic
2.1 0.0 2.0 0 2.9 3
1.3 2.6 1.1 0.5 2.5 2.1
2.8 0.3 2.0 1.1 2.8 2.5
1.6 2.5 1.1 1.6 2.8 1.1
Aftertaste
Pungent Acid Bitter Characteristic
3.0 1.9 0.0 3.9
1.4 1.1 0.5 2.4
2.6 1.9 0.9 2.6
1.6 0.8 1.1 1.1
49 Well maturated
38.5 Tasteless
33.5 Tasteless
12.5 Odourless O!-#avour Plastic
Total score Observations
140
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and #avour. The characteristic IdiazaH bal cheese #avour was not as strong in the pasteurized-milk cheeses, which were also somewhat less creamy, less pungent and less acid than the raw-milk cheeses. Certain observations noted on the sensory scoresheets indicated that batch AP lacked #avour, and some of the judges recorded o!-#avours in batch BP. This may be related to the high amino acid contents. Various researchers have described how pasteurization slows and alters the development of the sensory characteristics of cheese (Gaya et al., 1990; Lau et al., 1991; McSweeney, Fox, Lucey, Jordan & Cogan, 1993). Some of the characteristics perceived in these cheeses may have been related to the increased levels of certain amino acids. Proline has been associated with a sweet #avour in Edam cheese (Zaki & Salem, 1992) and asparagine and serine have also been associated with sweet #avours (Haefeli & Glaser, 1990); pasteurization raised the levels of those amino acids. Arginine and, particularly, peptide-containing aromatic amino acids have been associated with bitter #avours in cheese (Lau et al., 1990). GoH mez, Garde, Gaya, Medina and NuH n ez (1997) reported that heat treatments applied to the milk had no e!ect on the levels of the hydrophobic and hydrophilic peptides in the water-soluble fraction. None of the cheeses in this study presented appreciable bitter #avour overtones.
4. Conclusions Pasteurization substantially decreased the level of free amino acids in the cheeses made using starter A as re#ected by the NPN index and total FAA values during ripening. The levels of those same fractions were not a!ected by milk pasteurization when starter B was used, though this may have been related to resulting pH values. Pasteurization raised the levels of such amino acids as asparagine, serine, and taurine and lowered the levels of such other amino acids as aspartic acid and glycine, regardless of the starter used. The changes observed in the levels of certain amino acids could be directly or indirectly responsible for the sensory characteristics of the various cheeses. The characteristic IdiazaH bal cheese #avour was more highly developed in the batches made from the raw milk, especially when starter A was used. Pasteurization of milk to be used in making IdiazaH bal cheese is not recommended with either of the starters considered in this study, because, even though a high level of proteolysis was achieved using starter B, sensory characteristics were indicative of developments atypical of IdiazaH bal cheese.
Acknowledgements The authors wish to thank the Department of Education of the Government of the Autonomous Community
of Navarre and the Interministerial Commission for Science and Technology (ALI93-0895-CO2-01) for the funding provided and the Regulatory Board of the IdiazaH bal Cheese Appellation of Origin for providing cheese samples.
References Adda, J., Grippon, J. C., & Vassal, J. (1982). The chemistry of #avour and texture generation in cheese. Food Chemistry, 9, 115}129. Basch, J. J., Douglas Jr, F. W., Procino, L. G., Holsinger, V. H., & Farrel Jr, H. M. (1985). Quantitation of caseins and whey proteins of processed milks and whey protein concentrates, application of gel electrophoresis, and comparison with Harland}Ashwort procedure. Journal of Dairy Science, 68, 23}31. Berdague, J. L., & Grappin, R. (1987). A$nage et qualiteH du Gruye`re de ComteH . II. In#uence de l'a$nage sur l'eH volution des caracteH ristiques physico-chimiques des fromages. Lait, 67, 237}241. Bisquerra, R. (1989). IntroduccioH n conceptual al anaH lisis multivariable. Un enforque informaH tico con los paquetes SPSS-X, BMDP, LISREL y SPAD. Editorial PPU.S.A. Barcelona, Spain. Bullock, D. H., & Irvine, O. R. (1956). A chromatographic study of Cheddar cheese ripening. Journal of Dairy Science, 39, 1229}1234. Farkye, N. Y., & Fox, P. F. (1990). Objective indices of cheese ripening. Trends in Food Science and Technology, 1, 37}40. Fox, P. F. (1989). Proteolysis during cheese manufacture and ripening. Journal of Dairy Science, 72, 1379}1408. Fox, P. F., Lucey, J. A., & Cogan, T. M. (1990). Glycolysis and related reactions during cheese manufacture and ripening. Food Science and Nutrition, 29, 237}253. Gaya, P., Medina, M., RodrmH guez-MarmH n, M. A., & NuH n ez, M. (1990). Accelerated ripening of ewe's milk Manchego cheese: the e!ect of elevated ripening temperatures. Journal of Dairy Science, 73, 26}32. GoH mez, R., & PelaH ez, C. (1989). Lactic starters in the cheese industry. Alimentacio& n, Equipos y Tecnologn& a, 1, 193}202. GoH mez, M. J., Garde, S., Gaya, P., Medina, M., & NuH n ez, M. (1997). Relationship between level of hydrophobic peptides and bitterness in cheese made from pasteurized and raw milk. Journal of Dairy Research, 64, 289}297. Grappin, R., Rank, T. C., & Olson, N. F. (1985). Primary proteolysis of cheese proteins during ripening. A review. Journal of Dairy Science, 68, 531}540. Haefeli, R. J., & Glaser, D. (1990). Taste responses and thresholds obtained with primary amino acids in humans. Lebensmittel Wissenschaft & Technologie, 23, 523}527. IDF (1985). Cheese and processed cheese products. Determination of total protein. Kjeldahl method. International Dairy Federation Brussels, Belgium (FIL-IDF standard no. 25). IDF (1986). Cheese and processed cheese products. Determination of dry matter. International Dairy Federation. Brussels, Belgium (FIL-IDF standard no. 4). ISO (1975). Cheese: determination of fat matter. Van Gulik method. International Organization for Standarization. Geneva, Switzerland. (Standard 33433). Joosten, H. M. L. J. (1987). Conditions allowing the formation of biogenic amines in cheese. 3. Factors in#uencing the amounts formed. Netherland Milk and Dairy Journal, 41, 329}357. Kamaly, K. M., & Marth, E. H. (1989). Enzyme activities of lactic streptococci and their role in maturation of cheese: a review. Journal of Dairy Science, 72, 1945}1966. Khalid, N. M., & Marth, E. H. (1990). Lactobacilli: their enzymes and role in ripening and spoilage of cheese: a review. Journal of Dairy Science, 73, 2669}2684.
A.I. Ordo& nJ ez et al. / International Dairy Journal 9 (1999) 135}141 Kosikowski, F. V. (1951). The liberation of free amino acids in raw and pasteurized milk Cheddar cheese during ripening. Journal of Dairy Science, 34, 235}241. Krause, I., Bockhardt, A., Neckermann, H., Henle, T., & Klostermeyer, H. (1995). Simultaneous determination of amino acids and biogenic amines by RP-HPLC of the dansyl derivative. Journal of Chromatography A, 715, 67}79. Lau, K. L., Barbano, M., & Rasmussen, R. R. (1991). In#uence of pasteurization of milk on protein breakdown in Cheddar cheese during aging. Journal of Dairy Science, 74, 727}740. Law, J., Fitzgerald, G. F., Daily, C., Fox, P. F., & Farkye, N. Y. (1992). Proteolysis and #avor development in Cheddar cheese made with the single starter strains Lactococcus lactis ssp lactis UC317 or Lactococcus lactis ssp cremoris HP. Journal of Dairy Science, 75, 1173}1185. Law, J., Fizgerald, G. F., Uniacke-Lowe, T., Daily, C., & Fox, P. F. (1993). The contribution of lactococcal starter proteinases to proteolysis in Cheddar cheese. Journal of Dairy Science, 76, 2455}2467. McSweeney, P. H. L., Fox, P. F., Lucey, J. A., Jordan, K. N., & Cogan, T. M. (1993). Contribution of the indigenous micro#ora to the maturation of Cheddar cheese. International Dairy Journal, 3, 613}634. Ministerio de Agricultura, Pesca y AlimentacioH n. (1993). Regulatory Board of the IdiazaH bal Cheese Appellation of Origin. Boletin Oxcial del Estado, 289, 34 591}34 596. Oberg, C. J., Weimer, B. C., Moyes, L. V., Brown, R. J., & Richardson, G. H. (1991). Proteolytic characterization of Lactobacillus delbrueckii subsp. bulgaricus strains by the o-phtaldialdehyde test and amino acid analysis. Journal of Dairy Science, 74, 398}403. OrdoH n ez, A. I., IbaH n ez, F. C., Torre, P., & Barcina, Y. (1997). Formation of biogenic amines in IdiazaH bal ewe's milk cheese: e!ect of ripening, pasteurization and starter. Journal of Food Protection, 60, 1}6. OrdoH n ez, A. I., IbaH n ez, F. C., PeH rez-Elortondo, F. J., Torre, P., & Barcina, Y. (1998). Application of multivariate analysis to sensory characterization of ewes' milk cheese. Journal of Sensory Studies, 13, 45}55.
141
OrdoH n ez, J. A., & Burgos, J. (1977). Ulloa cheese. Free amino acids formed during ripening. Lait, 57, 416}420. PeH rez-Elortondo, F. J., Albisu, M., & Barcina, Y. (1993). Changes in the micro#ora of IdiazaH bal cheese with addition of commercial lactic starters. Australian Journal of Dairy Technology, 48, 10}14. Quintanilla, M. A., & Pen a, A. E. (1991). Cheesemaking yield. Alimentaria, 3, 39}42. Rank, T. C., Grappin, R., & Olson, N. (1985). Secondary proteolysis of cheese during ripening: a review. Journal of Dairy Science, 68, 801}805. Requena, T., PelaH ez, C., & Fox, P. F. (1993). Peptidase and proteinase activity of Lactococcus lactis, Lactobacillus casei and Lactobacillus plantarum. Zeitschrift fu( r Lebensmittel Untersuchung und Forschung, 196, 351}355. SPSS. Base System User's guide. VersioH n 6.1, Edition (1994). SPSS Inc. Chicago. IL. Stadhouders, J., Hup, G., Exterkate, F. A., & Visser, S. (1983). Bitter #avour in cheese. I. Mechanism of the formation of bitter #avour defects in cheese. Netherland Milk and Dairy Journal, 37, 157}169. Tan, P., & Konings, W. N. (1990). Puri"cation and characterization of an aminopeptidase from Lactococcus lactis subsp cremoris Wg2. Applied Environmental Microbiology, 56, 526}532. Urbach, G. (1993). Relations between cheese #avour and chemical composition. International Dairy Journal, 3, 389}422. Van den Berg, G., & Exterkate, F. A. (1993). Technological parameters involved in cheese ripening. International Dairy Journal, 3, 485}507. Venema, G. (1993). Molecular biology and genetic modi"cation of Lactococci. Journal of Dairy Science, 76, 2133}2144. Visser, S., Slangen, K. J., Hup, G., & Stadhouders, J. (1983). Bitter #avour in cheese. 3. Comparative gel-chromatographic analysis of hydrophobic peptide fractions from twelve Gouda-type cheeses and identi"cation of bitter peptides isolated from a cheese made with Streptococcus cremoris strain HP 1, 2. Netherlands Milk and Dairy Journal, 37, 181}187. Zaki, N., & Salem, S. A. (1992). E!ect of proteolytic enzymes on accelerated ripening of Edam cheese. Indian Journal of Dairy Science, 45, 303}312.