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Effect of added enzymes on the free amino acids and sensory characteristics in Ossau–Iraty cheese
Food Control 11 (2000) 201±207
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Eect of added enzymes on the free amino acids and sensory characteristics in Ossau±Iraty cheese J.M. Izco *, A. Irigoyen, P. Torre, Y. Barcina Department de Ciencias del Medio Natural, Area de Nutrici on y Bromatologõa, Universidad P ublica de Navarra, Campus Arrosadõa s/n, 31006 Pamplona, Spain Received 27 August 1999; received in revised form 18 October 1999; accepted 25 October 1999
Abstract A proteolytic and a lipolytic enzyme preparation were added (before clotting) to the milk used to manufacture Ossau±Iraty-type ewes'-milk cheese. The free amino acids were analysed by reversed phase-HPLC and the sulphosalicylic acid-soluble N fraction was quanti®ed by the trinitrobenzenesulphonic acid method for use as indices of proteolysis during ripening. Sensory analysis of the cheeses began after two months of ripening. The lipolytic enzyme preparation did not increase free amino acid levels, with even a slight decrease being recorded at the beginning of ripening. On the other hand, the proteolytic enzyme preparation caused an appreciable increase in proteolysis, 20% higher than in the control batch after four months of ripening. The high level of exoproteolytic activity and concomitantly higher release of free amino acids related to sweet ¯avour, such as asparagine, serine, and proline, may have masked the bitter ¯avours previously reported in the literature when proteolytic enzymes have been added. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Accelerated ripening; Enzyme; Amino acid; Sensory analysis; Ewes' cheese
1. Introduction Ossau±Iraty cheese is manufactured from raw or pasteurized ewes' milk in southwestern France and is one of the cheeses that has been awarded an Appellation of Origin in France. It is made wholly of ewesÕ milk and it is an uncooked, pressed cheese that must be aged for a minimum of 90 days (60 days in the case of a related variety, ``Petit Ossau±Iraty'' cheese). Most experiments involving accelerated ripening have been carried out on cheeses made from cows' milk, Cheddar cheese in particular. Few instances are to be found in which accelerated ripening has been employed for ewesÕ-milk cheeses (Fern andez-Garcõa & L opezFandi~ no, 1994). Various methods can be used to accelerate cheese ripening (Izco, Torre & Barcina, 1999a). All methods for accelerating cheese ripening appear to work either by increasing levels of the key enzymes involved in the ripening process or by ensuring that conditions are conducive to the activity of the native enzymes in the
*
Corresponding author. Tel.: +34-948-169141; fax: +34-948169187. E-mail address: [email protected] (J.M. Izco).
cheese (Fox, 1988). Adding the enzymes directly to the milk before coagulation yields the best distribution of the enzymes, thereby enhancing contact between the enzymes and the particles in the coagulum (FernandezGarcõa, 1986) and avoiding the risk of overripening (Fernandez-Garcõa & L opez-Fandi~ no, 1994). Lipases are used in those cheeses in which lipolysis plays a major role in the development of characteristic ¯avour, such as hard Italian cheeses, blue cheeses, Greek Feta cheese, and Egyptian Ras and Domiatti cheeses (Law & Goodenough, 1991). To a greater or lesser extent, proteolysis is a key process during ripening in all cheeses; hence research on accelerated ripening has focused mainly on the use of proteinases and peptidases (Fox & Law, 1991). Since cheese made from ewesÕ milk is less susceptible to the development of bitter ¯avours than are cheeses made from cowsÕ milk, adding proteinases to milk or to the curd is a more attractive option for ovine milk than for bovine milk (Fernandez-Garcõa & L opez-Fandi~ no, 1994). Amino acids and peptides contributing directly to cheese ¯avour and aroma are released as the caseins are broken down during cheese ripening (Visser, Hup, Exterkate & Stadhouders, 1983). Quanti®cation of the free
0956-7135/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 6 - 7 1 3 5 ( 9 9 ) 0 0 1 0 0 - 0
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amino acids (FAAs) can be quite useful for cheeses undergoing accelerated ripening for the purpose of evaluating their eect on ¯avour and aroma intensity (Kosikowski, 1988); certain FAAs are extremely important factors in ¯avour development, e.g., proline in a Swiss-type cheese (Moskowitz & Noelck, 1987). The object of this study was to establish a preliminary approximation of the eect of two enzymatic preparations on proteolysis during ripening of Ossau±Iraty-type cheese, by analysing the release of FAAs as proteolytic indices, and the sensory characteristics. Finally, the aim of this research work was to test two commercial enzymes in order to accelerate the ripening of Ossau±Iratytype cheese. 2. Materials and methods 2.1. Cheese samples Cheese samples were prepared as previously (Izco, Torre & Barcina, 1999b), in accordance with the method authorized by the Regulations of the Appellation of Origin for Ossau±Iraty cheese. The cheeses were made at a dairy approved by the Regulatory Board of the Appellation of Origin for Ossau±Iraty cheese. A proteolytic and a lipolytic commercial enzyme preparation were tested separately. The lipolytic enzyme preparation L1 (LipomodTM 187) and the proteolytic enzyme preparation PP (PromodTM 215) were added to the milk before the rennet. The enzyme preparations were supplied by Biocatalysts (Treforest Industrial Estate, Pontypridd, UK) and the doses used were 10 g/100 l. Cheese weight ranged between 2 and 3 kg. Duplicate cheese samples were collected for physicochemical analysis on days 1, 15, 30, 60, 90 and 120 of ripening. Starting on day 60, a third sample was also collected for sensory analysis. 2.2. Physicochemical analyses Dry matter (DM) was determined according to IDFFIL standard 4 (International Dairy Federation, 1985). Reversed Phase-HPLC analysis of the FAAs and sulphosalicylic acid-soluble-N (SSAN) fraction were performed and quanti®ed as previously described (Izco, Torre & Barcina, in press). 2.3. Sensory analysis All samples were evaluated by at least eight trained assessors, members of the tasting committee of the Apellation of Origin of other ewesÕ-milk cheeses (Roncal and Idiazabal cheese). A sensory analysis scoring sheet was developed speci®cally for this cheese, taking the following attributes into account: characteristic odour, sweet odour, and bitter odour (odour analysis); elasticity,
®rmness, adherence, creaminess, brittleness, deformability, and grittiness (texture analysis); characteristic taste, pungent taste, sweet taste, sour taste, salty taste, and bitter taste (taste analysis); and characteristic aftertaste, persistence, sour aftertaste, salty aftertaste, and bitter aftertaste (aftertaste analysis). Intensity of each attribute was scored according to an increasing scale from 1 (not present) to 7 (very intense). In all, a total of 21 sensory attributes were evaluated, though not all attributes contributed equally to the ®nal overall sensory score for the cheese. In order to obtain a ®nal score that would accurately re¯ect cheese quality, the sensory analysis results were weighted. The overall sensory score was calculated by multiplying the sensory analysis results by a weighting factor based on the contribution of each attribute to the sensory characteristics of this type of cheese. The maximum overall score was 100, with odour contributing 10, texture 30, taste 45 and aftertaste 15. 2.4. Statistical treatment The SPSS computer program (version 6.1, SPSS Inc., Chicago, IL, USA) was used for statistical processing. An analysis of variance with 95% con®dence intervals was run on each of the physicochemical parameters analysed to ascertain whether or not the dierences between batches on each sampling date were signi®cant. Stepwise discriminant analysis was also performed on the sensory attributes employed to ascertain which of the dierent attributes were most useful in dierentiating between batches and in classifying the cheeses according to batch. Wilk's lambda k was used as the selection criterion for the attributes. When the discriminant functions had been derived, the standardized coecients in the functions were calculated. The standardized coef®cient values were indicative of the relative importance of each of the original attributes in each of the discriminant functions. The confusion matrix was also calculated to determine the percentage of cases that were correctly classi®ed using the ®rst discriminant function only. 3. Results and discussion 3.1. Physicochemical analyses The changes in FAAs and SSAN, resulting from each of the enzymatic preparations used were calculated by substracting the enzyme-treated cheese (PP or L1) sample content from control cheese (without enzyme) sample content, for every day of the ripening time tested. As expected, Table 1 shows that the amino acid pro®le for the batch treated with added lipase (L1) was quite similar to that for the control batch over the entire ripening period and even had lower quantities of certain amino acids, especially in the early stages, with the dif-
J.M. Izco et al. / Food Control 11 (2000) 201±207
203
Table 1 Increments (control cheese content ± enzyme treated cheese content) in the FAAs (mg/100 g DM) and in the SSAN (mmol L-LEU/100 g DM) during ripening (in days); only if dierences between the mean values (p < 0.05, n 4) are signi®cant Day/Batch
1
FAAs
L1a
ASP GLU SER ASN GLY GLN TAU HIS GABA CIT THR ALA ARG PRO TYR VAL MET CYST ILE LEU HYLYS PHE TRP ORN LYS Total SSAN
15
2.1
0.7 nq 1.8 1.6 2.6 1.4 0.5 0.2
0.8 0.7 1.2 25.7
PPb 1.7 4.5
)6.4 0.9 0.5
L1 1.4
1.1 )0.5
1.1 3.1 9.1 4.6 0.6
27.4 0.4
PP )3.9 )3.2 )9.0 )8.8 0.7 )1.7 0.5 )3.0 )2.8
1.6 1.6 1.0
30
2.3
2.4 )2.3
0.2
)2.3 )1.2 )5.1
0.3
0.6
0.5
0.4 )4.4 )6.0
L1
)13.2 )0.7
)0.2
60 PP
L1
)13.7 )6.1 )22.4 )0.9 )11.6 )3.8 1.2
)5.4
)8.7 )4.3 )10.0
)64.6 )0.7
)2.8 )9.0 )4.8 )0.3 )8.6 )16.6
90 PP
L1
5.8 24.4 3.0 nqc 6.7 )1.2 11.6 nq 6.8 10.4 9.8 6.8 nq 7.9
nq
8.2
)6.2 nq )20.7
PP
L1
)42.0 )9.6 )74.1
)24.1
)4.9 nq 15.5 )11.4
120
)0.6
)19.4 )125.2 )10.6 )34.3
)18.6 )0.8 )9.7 )8.0
18.4
)11.9
19.3
)17.6 )18.6
)133.4 )0.6
202.5 0.8
)0.6
)17.8 nq )21.0 )14.2
)14.3
)31.0
)10.0 )4.1 )15.6
)17.2 )7.8 )35.0 4.0
)12.5
PP
)4.0
)1.6 )10.2 )32.6
10.2 )33.1 )49.8
)282.7
)477.6 )3.6
a
Lipolytic enzyme preparation. Proteolytic enzyme preparation. c Not quanti®ed. b
ferences decreasing at the end of ripening. The trend for the total free amino acids (TFAAs) was likewise quite similar to that in the control batch throughout ripening. The experimental results for batch L1 agreed with the ®ndings of Jolly and Kosikowski (1978). Fern andezGarcõa et al. (1994) attributed the lower proteolysis rate to the inhibitory eect of the fatty acids on the lactic acid bacteria. In contrast, Lin, Jeon, Roberts and Milliken (1987) did not record a lower proteolysis rate in Cheddar cheese made with an added animal lipase. There have been reports of appreciable increases in FAA concentrations when lipases have been added (Fernandez-Garcõa, Ramos, Polo, Ju arez & Olano, 1988). That ®nding was ascribed to contamination of the lipases with proteolytic enzymes as a result of incomplete puri®cation of the commercial lipolytic preparations; consequently, the preparations might also contain other (proteolytic) enzymes that may be activated to a greater or lesser extent depending on the conditions in the medium (pH, temperature). In fact, the lipolytic enzyme L1 increased breakdown of the casein fractions in cheeses ripened for two months (Izco et al., 1999b).
The principal ®ndings concerned batch PP. The cheeses supplemented with the protease preparation contained higher levels for many of the amino acids over the ripening period. Those ®ndings agreed with the results recorded by Zaki and Salem (1992) and FernandezGarcõa et al. (1988, 1993). In contrast, FernandezGarcõa et al. (1994) reported an increase in certain amino acids, namely, serine, glutamine, histidine, alanine, phenylalanine, isoleucine, leucine and lysine; conversely, other amino acids, namely, glutamic acid, asparagine, tyrosine, arginine, methionine and valine decreased. A possible explanation for these dierences may be that in addition to the Neutrase, a lipase was also used in that last-mentioned study, which may have generated fatty acids that inhibited proteinase activity. Likewise, Ard o and Pettersson (1988) did not observe any increase in the amounts of amino acids when Neutrase was added during the manufacture of Swedish cheese. Those ®ndings contrast with the results reported by Law and Wigmore (1982) in Cheddar cheese treated with that same enzyme, possibly due to technical differences in processing during manufacture.
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Changes in certain amino acids during ripening are particularly noteworthy. For instance, up to day 30 the concentration of proline in the control cheese (batch C) was higher than in the cheese batches to which enzymes were added, after which time levels converged with the values recorded in cheese batches L1 and PP. Subsequently, the proline level attained in batch PP on day 120 was 143% of the level in the control batch. Proline has been associated with sweet ¯avour in cheeses (Law & Wigmore, 1983). Those workers found that the concentration of proline was lower in Cheddar cheeses made with added Neutrase than in control cheeses; Zaki and Salem (1992) reported the same ®ndings in Edam cheese. Sood and Kosikowski (1979) recorded similar results for Cheddar cheese using a mixture of lipases plus proteinases. Asparagine was the main amino acid in the cheese samples (14.7% of the Total FAAs in the control cheeses on day 120; Izco et al., in press). The proteolytic preparation added to batch PP produced a signi®cant increase in the concentration of that amino acid (140% of the level in the control batch). Serine was aected in the same way as asparagine. The serine concentration increased in the cheeses made with added proteinase (batch PP) but did not increase in the cheeses made with added lipase (batch L1). Those two amino acids have also been related to sweet ¯avour in cheeses (Hefeli & Glaser, 1990); hence the increase in the serine and asparagine concentrations may have helped mask any bitter ¯avours that may have developed as a result of the possible formation of bitter peptides in the cheese due to the addition of the proteolytic enzymes. One method of estimating TFAA levels is by measuring the SSAN fraction using the TNBS method (Izco et al., in press). High correlations between the TFAAs and the SSAN fraction were recorded for all the cheeses over the ripening period. The correlation coecient values obtained (n 24) for the control batch, batch L1, and batch PP were 0.985, 0.990 and 0.984, respectively. In accordance with the results obtained using RPHPLC, the SSAN content in the cheeses made using added lipase (batch L1) did not dier signi®cantly from the content in the control batch on the sampling dates during ripening. Hardly any increase in the SSAN fraction was found when lipases were added during manufacture of Cheddar cheese (Lin et al., 1987) and Manchego-type cheese made from cows' milk or a blend of cows' milk and ewes' milk [30/70, v/v] (FernandezGarcõa et al., 1988, 1994). On the other hand, Fernan dez-Garcõa, Olano, Cabezudo, Martin-Alvarez and Ramos (1993) reported an increase in the SSAN fraction of cheese treated with Flavor Age (a combination of proteinase, aminopeptidase and lipase). Using that same enzymatic preparation in Cheddar cheese, Guinee, Wilkinson, Mulholland and Fox (1991) reported a slight increase in the concentration of total amino acids.
Although initial values were lower, from day 15 proteolysis was higher in batch PP than in the control batch. While dierences were not signi®cant on day 90, on day 120 the cheeses in batch PP had an SSAN concentration 20% higher than that in the control batch. Contrasting with those results, other researchers (Alkhalaf, Vassal, Desmazeaud & Gripon, 1987; Ard o & Pettersson, 1988) did not record increases in the amino acid nitrogen value (measured as SSAN or PTAN) using several dierent added proteinases. However, most workers have reported increases in those ripening index values both in cows'-milk cheeses (Law & Wigmore, 1982, 1983; Lin et al., 1987; Fernandez-Garcõa et al., 1988; Guinee et al., 1991) and in ewes'-milk cheeses ~ez (Vafopoulou, Alichanidis & Zer®nridis, 1989; N un ~ et al., 1991; Pic on, Gaya, Medina & N unez, 1994, 1995). 3.2. Sensory analysis Fig. 1(a) and (b) show that the pro®le for the control cheeses (batch C) was indicative of a cheese with a markedly sweet odour, highly creamy texture, moderate elasticity, adherence, and deformability, and low brittleness. The characteristic ¯avour of Ossau±Iraty cheese is moderately salty, with lower scores for sweet taste than for sweet odour. Bitter and pungent tastes were barely detectable, and the cheese left a characteristic, strong aftertaste after being swallowed. Batch L1 was the batch that diered the least from the control batch after 60, 90 and 120 days of ripening. On day 60, batch L1 received higher scores for pungent taste than the control batch. Perhaps the added lipase used released short-chain FFAs, as reported for pregastric esterase used to achieve the characteristic pungent overtones in certain Italian cheese varieties as Peccorino Romano, Provolone, and Parmesano (Nelson, Jensen & Pitas, 1977). However, the higher scores for pungent taste were not repeated in the cheeses after 90 and 120 days of ripening. Bitter ¯avours are one of the most common defects in cheeses made from milk or curd to which proteolytic enzymes have been added (Alkhalaf et al., 1987; Ard o& Pettersson, 1988; Fernandez-Garcõa et al., 1988; Mohamed et al., 1989) as a result of the accumulation of bitter peptides with a high proportion of hydrophobic, aromatic amino acid residues (Vafopoulou et al., 1989) and high-molecular-weight peptides as a result of casein breakdown. Even so, batch PP did not exhibit large dierences in bitter taste and aftertaste with respect to the control batch (except for bitter taste on day 120), despite the high level of casein breakdown in that batch (Izco et al., 1999b). Fig. 1(b) shows that taste panel members even awarded higher scores to the taste and aftertaste attributes for that batch than for the control ~ez et al. (1991), whole bovine batch. According to N un casein hydrolysates exhibited higher levels of bitterness
J.M. Izco et al. / Food Control 11 (2000) 201±207
Fig. 1. Sensory scores: (a) odour and texture and (b) taste and aftertaste for the Control cheese Ossau±Iraty type (C) and enzyme-treated cheeses L1 (lipolytic preparation), and PP (proteolytic preparation) on day 90 of the ripening period.
than did whole ovine casein hydrolysates, and hence there would appear to be less likelihood of the formation of bitter ¯avours in cheeses made from ewesÕ milk with added enzymes. Pic on et al. (1994, 1995) reported that the addition of encapsulated proteolytic enzymes (chymosin and BSNP) to the milk used to manufacture a ewes'-milk Manchego-type cheese did not result in the formation of bitter ¯avours. Conversely, Fern andezGarcõa et al. (1994) did report bitter ¯avour defects on adding BSNP to a blend of bovine and ovine milk (30/ 70, v/v). In addition, TFAA and SSAN values in batch PP were higher than in the control batch, possibly because of high exopeptidase activity brought about by the added enzymatic preparation, which may have led to degradation of the bitter peptides.
205
After weighting the sensory analysis results, the overall scores for each of the cheeses were calculated. Those scores provide a general idea of the ®nal rating for each cheese. Table 2 shows that all the cheeses had quite similar scores, suggesting that on the whole the enzymatic preparations did not adversely aect the organoleptic characteristics of this cheese. The discriminant analysis of the sensory analysis results was designed to select only those attributes that minimized the Wilk's k value, namely, the ones most useful in discriminating between batches. Firmness, creaminess, salty taste, and characteristic odour were the most eective attributes in discriminating between batches. Table 3 gives the correlation values for each sensory attribute in each of the discriminant functions calculated and furnishes an idea of the relative importance of each of the attributes considered in the sensory evaluations in the discriminant functions. Function 1 was more closely correlated with the sensory attributes for odour and taste, characteristic aftertaste, bitterness, ®rmness, brittleness, and adherence. Fig. 2 shows that the function was capable of dierentiating between all the cheese batches. Taste-related attributes, such as pungent taste, had negative correlation coecient values, causing batch L1 to be located in the negative region of the ®gure, since as already mentioned above, that batch had a distinctly more pungent taste than the other batches on day 60. Sweet taste, with a positive correlation coecient value (Table 3), was extremely important in function 1. The proteolytic enzyme preparation added to batch PP caused the release of the amino acids asparagine, proline, and serine, related to sweet taste, which was responsible for the location of batch PP on the plot. However, the function also included such texture attributes as ®rmness and, to a somewhat lesser extent, brittleness. The greater intensity of proteolysis caused by the addition of proteinases to accelerate cheese ripening has sometimes resulted in a more brittle texture (El Soda & Saada, 1986; Fedrick, Aston, Nottingham & Dulley, 1986; Alkhalaf et al., 1987) and reduced elasticity (Zaki & Salem, 1992; Pic on et al., 1994). On other occasions, in contrast, it did not give rise to any brittleness defects, and indeed elasticity has sometimes even been higher in the experimental batch than in the control batch (Pic on et al., 1995). Function 2 clearly distinguished the control batch from the batches with added enzymes but did not discriminate between batches L1 and PP. This function included the remaining aftertaste and texture attributes, especially creaminess. The present paper contributes new data on the effect of adding enzymatic preparations to the milk used in the manufacture of French Ossau±Iraty cheese to help ®ll the gap in available knowledge on the application of accelerated ripening methods in ewes'-milk cheeses.
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Table 2 Overall sensory scores for the dierent cheeses at days 60, 90 and 120 of ripening Ripening time
Cheese batch C
L1
PP
p
Day 60 Day 90 Day 120
83 80 88
79 77 89
78 87 84
nsa ns ns
a
Not signi®cant dierences between the mean values (p < 0.05, n 8). Table 3 Pooled within-group correlations between discriminating attributes and canonical discriminant functions (attributes ordered by withinfunction correlation value) Attribute
Function 1
Function 2
Firmness Salty taste Sweet taste Pungent taste Characteristic odour Sour taste Brittleness Sweet taste Characteristic aftertaste Bitter aftertaste Sour odour Bitter taste Characteristic taste Adherence Creaminess Persistence Grittiness Elasticity Salty aftertaste Deformability Sour aftertaste
0.61979a )0.46882a 0.40848a )0.39541a 0.39441a )0.31492a 0.26810a 0.21444a 0.20313a )0.18767a )0.17488a )0.16435a 0.14482a )0.05534a )0.46632 )0.01520 0.12080 0.11311 )0.16019 )0.12470 0.01180
0.30029 )0.35241 0.05787 0.09241 0.02526 )0.16700 0.08244 0.11994 )0.02055 )0.06968 )0.01471 0.01541 )0.06020 0.04343 0.78470a )0.32628a )0.30012a )0.29332a )0.27999a 0.21887a )0.14309a
a
Denotes largest absolute correlation between each attribute and any discriminant function.
The results of the physicochemical analyses performed showed that, on the whole, lipolytic enzyme preparation L1 did not cause an increase in the release of amino acids. Indeed, a decrease in certain amino acids was even observed at the beginning of ripening, possibly due to an increase in the release of FAAs that then acted to inhibit the proteolytic activity of the lactic acid bacteria. However, the dierences with respect to the control batch decreased with ripening, and on day 120 there were practically no signi®cant dierences in the levels of any of the amino acids. Similarly, there were no appreciable alterations in the sensory characteristics of the cheeses, though certain abnormal results were observed on certain sampling dates, such as the pungent taste on day 60 and the decrease in sweet odour on day 90. The main ®ndings were in relation to batch PP. Addition of the enzymatic preparation to that batch resulted in an increase in the proteolytic indices analysed, e.g., a 20% increase in the SSAN value with respect to the control batch on day 120. The higher level of proteolysis had no adverse impact on the organoleptic characteristics of the Ossau±Iraty type cheese. One of the most commonly encountered defects caused by the addition of proteinases is the appearance of bitter ¯avours through the formation of bitter peptides. That defect may have been oset by the high level of exoproteolytic activity of the enzymatic preparation used, resulting in the breakdown of such peptides. The higher levels of such free amino acids as asparagine, serine, and proline, which are related to sweet ¯avour in cheeses, may also have masked bitter ¯avours in the cheese samples.
Acknowledgements This research work is part of the project ``PL921298'' titled ``Design and production of an enzymatic and microbial mixture to improve the processing of ewesÕ cheese (Spain, France, Italy and Portugal), its safety and quality and to get a novel functional food as a response to European demand for new products low in cholesterol and protein enriched'' of the AAIR Program. The author is grateful to the Government of Navarra for the ®nancial support provided for this study.
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Fig. 2. Plot of the canonical discriminant functions obtained using the sensory analysis results. Classing Control cheese Ossau±Iraty type (C) and enzyme-treated cheeses L1 (lipolytic preparation), and PP (proteolytic preparation).
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Jolly, R. C., & Kosikowski, F. V. (1978). Eects of added microbial and animal lipases on protein hydrolysis in blue cheese made with pasteurized milk. Journal of Dairy Science, 61, 536±541. Kosikowski, F. V. (1988). Enzyme behavior and utilization in dairy technology. Journal of Dairy Science, 71, 557±573. Law, B. A., & Wigmore, A. (1982). Accelerated cheese ripening with food grade proteinases. Journal of Dairy Research, 49, 137±146. Law, B. A., & Wigmore, A. (1983). Accelerated ripening of Cheddar cheese with a commercial proteinase and intracellular enzymes from starter streptococci. Journal of Dairy Science, 50 (5), 519±525. Law, B.A., & Goodenough, P.W. (1991). In G. A. Tucker & L. F. J. Woods, Emzymes in food processing (pp. 99±127). Blackie and Son Ltd., London. Lin, J. C. C., Jeon, I. J., Roberts, H. A., & Milliken, G. A. (1987). Eects of commercial food grade enzymes on proteolysis and textural changes in granular Cheddar cheese. Journal of Food Science, 52 (3), 620±625. Moskowitz, G. J., & Noelck, S. S. (1987). Enzyme-modi®ed cheese technology. Journal of Dairy Science, 70 (8), 1761±1769. Nelson, J. H., Jensen, R. G., & Pitas, R. E. (1977). Pregastric esterase and other oral lipases ± a review. Journal of Dairy Science, 60 (3), 327±362. ~ez, M., Guillen, A. M., Rodrõguez-Marõn, M. A., Marcilla, A. M., N un Gaya, P., & Medina, M. (1991). Accelerated ripening of eweÕs milk manchego cheese the eect of neutral proteinases. Journal of Dairy Science, 74 (12), 4108±4118. ~ez, M. (1994). The eect of Pic on, A., Gaya, P., Medina, M., & N un liposome encapsulation of chymosin derived by fermentation on Manchego cheese ripening. Journal of Dairy Science, 77 (1), 16±23. ~ez, M. (1995). The eect of Pic on, A., Gaya, P., Medina, M., & N un liposome-encapsulated Bacillus subtilis neutral proteinase on Manchego cheese ripening. Journal of Dairy Science, 78 (6), 1238±1247. Sood, V. K., & Kosikowski, F. V. (1979). Accelerated Cheddar cheese ripening by added microbial enzymes. Journal of Dairy Science, 62, 1865±1872. Vafopoulou, A., Alichanidis, E., & Zer®ridis, G. (1989). Accelerated ripening of Feta cheese with heat-shocked cultures or microbial proteinases. Journal of Dairy Research, 56, 285±296. Visser, S., Hup, G., Exterkate, F. A., & Stadhouders, J. (1983). Bitter ¯avour in cheese. II. Model studies on the formation and degradation of bitter peptides by proteolytic enzymes from calf rennet, starter cells and starter cell fractions. Netherlands Milk Dairy Journal, 37, 169±180. Zaki, N., & Salem, S. A. (1992). Eect of proteolytic enzymes on accelerated ripening of Edam cheese. Indian Journal of Dairy Science, 45 (6), 303±312.
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Eect of added enzymes on the free amino acids and sensory characteristics in Ossau±Iraty cheese J.M. Izco *, A. Irigoyen, P. Torre, Y. Barcina Department de Ciencias del Medio Natural, Area de Nutrici on y Bromatologõa, Universidad P ublica de Navarra, Campus Arrosadõa s/n, 31006 Pamplona, Spain Received 27 August 1999; received in revised form 18 October 1999; accepted 25 October 1999
Abstract A proteolytic and a lipolytic enzyme preparation were added (before clotting) to the milk used to manufacture Ossau±Iraty-type ewes'-milk cheese. The free amino acids were analysed by reversed phase-HPLC and the sulphosalicylic acid-soluble N fraction was quanti®ed by the trinitrobenzenesulphonic acid method for use as indices of proteolysis during ripening. Sensory analysis of the cheeses began after two months of ripening. The lipolytic enzyme preparation did not increase free amino acid levels, with even a slight decrease being recorded at the beginning of ripening. On the other hand, the proteolytic enzyme preparation caused an appreciable increase in proteolysis, 20% higher than in the control batch after four months of ripening. The high level of exoproteolytic activity and concomitantly higher release of free amino acids related to sweet ¯avour, such as asparagine, serine, and proline, may have masked the bitter ¯avours previously reported in the literature when proteolytic enzymes have been added. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Accelerated ripening; Enzyme; Amino acid; Sensory analysis; Ewes' cheese
1. Introduction Ossau±Iraty cheese is manufactured from raw or pasteurized ewes' milk in southwestern France and is one of the cheeses that has been awarded an Appellation of Origin in France. It is made wholly of ewesÕ milk and it is an uncooked, pressed cheese that must be aged for a minimum of 90 days (60 days in the case of a related variety, ``Petit Ossau±Iraty'' cheese). Most experiments involving accelerated ripening have been carried out on cheeses made from cows' milk, Cheddar cheese in particular. Few instances are to be found in which accelerated ripening has been employed for ewesÕ-milk cheeses (Fern andez-Garcõa & L opezFandi~ no, 1994). Various methods can be used to accelerate cheese ripening (Izco, Torre & Barcina, 1999a). All methods for accelerating cheese ripening appear to work either by increasing levels of the key enzymes involved in the ripening process or by ensuring that conditions are conducive to the activity of the native enzymes in the
*
Corresponding author. Tel.: +34-948-169141; fax: +34-948169187. E-mail address: [email protected] (J.M. Izco).
cheese (Fox, 1988). Adding the enzymes directly to the milk before coagulation yields the best distribution of the enzymes, thereby enhancing contact between the enzymes and the particles in the coagulum (FernandezGarcõa, 1986) and avoiding the risk of overripening (Fernandez-Garcõa & L opez-Fandi~ no, 1994). Lipases are used in those cheeses in which lipolysis plays a major role in the development of characteristic ¯avour, such as hard Italian cheeses, blue cheeses, Greek Feta cheese, and Egyptian Ras and Domiatti cheeses (Law & Goodenough, 1991). To a greater or lesser extent, proteolysis is a key process during ripening in all cheeses; hence research on accelerated ripening has focused mainly on the use of proteinases and peptidases (Fox & Law, 1991). Since cheese made from ewesÕ milk is less susceptible to the development of bitter ¯avours than are cheeses made from cowsÕ milk, adding proteinases to milk or to the curd is a more attractive option for ovine milk than for bovine milk (Fernandez-Garcõa & L opez-Fandi~ no, 1994). Amino acids and peptides contributing directly to cheese ¯avour and aroma are released as the caseins are broken down during cheese ripening (Visser, Hup, Exterkate & Stadhouders, 1983). Quanti®cation of the free
0956-7135/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 6 - 7 1 3 5 ( 9 9 ) 0 0 1 0 0 - 0
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amino acids (FAAs) can be quite useful for cheeses undergoing accelerated ripening for the purpose of evaluating their eect on ¯avour and aroma intensity (Kosikowski, 1988); certain FAAs are extremely important factors in ¯avour development, e.g., proline in a Swiss-type cheese (Moskowitz & Noelck, 1987). The object of this study was to establish a preliminary approximation of the eect of two enzymatic preparations on proteolysis during ripening of Ossau±Iraty-type cheese, by analysing the release of FAAs as proteolytic indices, and the sensory characteristics. Finally, the aim of this research work was to test two commercial enzymes in order to accelerate the ripening of Ossau±Iratytype cheese. 2. Materials and methods 2.1. Cheese samples Cheese samples were prepared as previously (Izco, Torre & Barcina, 1999b), in accordance with the method authorized by the Regulations of the Appellation of Origin for Ossau±Iraty cheese. The cheeses were made at a dairy approved by the Regulatory Board of the Appellation of Origin for Ossau±Iraty cheese. A proteolytic and a lipolytic commercial enzyme preparation were tested separately. The lipolytic enzyme preparation L1 (LipomodTM 187) and the proteolytic enzyme preparation PP (PromodTM 215) were added to the milk before the rennet. The enzyme preparations were supplied by Biocatalysts (Treforest Industrial Estate, Pontypridd, UK) and the doses used were 10 g/100 l. Cheese weight ranged between 2 and 3 kg. Duplicate cheese samples were collected for physicochemical analysis on days 1, 15, 30, 60, 90 and 120 of ripening. Starting on day 60, a third sample was also collected for sensory analysis. 2.2. Physicochemical analyses Dry matter (DM) was determined according to IDFFIL standard 4 (International Dairy Federation, 1985). Reversed Phase-HPLC analysis of the FAAs and sulphosalicylic acid-soluble-N (SSAN) fraction were performed and quanti®ed as previously described (Izco, Torre & Barcina, in press). 2.3. Sensory analysis All samples were evaluated by at least eight trained assessors, members of the tasting committee of the Apellation of Origin of other ewesÕ-milk cheeses (Roncal and Idiazabal cheese). A sensory analysis scoring sheet was developed speci®cally for this cheese, taking the following attributes into account: characteristic odour, sweet odour, and bitter odour (odour analysis); elasticity,
®rmness, adherence, creaminess, brittleness, deformability, and grittiness (texture analysis); characteristic taste, pungent taste, sweet taste, sour taste, salty taste, and bitter taste (taste analysis); and characteristic aftertaste, persistence, sour aftertaste, salty aftertaste, and bitter aftertaste (aftertaste analysis). Intensity of each attribute was scored according to an increasing scale from 1 (not present) to 7 (very intense). In all, a total of 21 sensory attributes were evaluated, though not all attributes contributed equally to the ®nal overall sensory score for the cheese. In order to obtain a ®nal score that would accurately re¯ect cheese quality, the sensory analysis results were weighted. The overall sensory score was calculated by multiplying the sensory analysis results by a weighting factor based on the contribution of each attribute to the sensory characteristics of this type of cheese. The maximum overall score was 100, with odour contributing 10, texture 30, taste 45 and aftertaste 15. 2.4. Statistical treatment The SPSS computer program (version 6.1, SPSS Inc., Chicago, IL, USA) was used for statistical processing. An analysis of variance with 95% con®dence intervals was run on each of the physicochemical parameters analysed to ascertain whether or not the dierences between batches on each sampling date were signi®cant. Stepwise discriminant analysis was also performed on the sensory attributes employed to ascertain which of the dierent attributes were most useful in dierentiating between batches and in classifying the cheeses according to batch. Wilk's lambda k was used as the selection criterion for the attributes. When the discriminant functions had been derived, the standardized coecients in the functions were calculated. The standardized coef®cient values were indicative of the relative importance of each of the original attributes in each of the discriminant functions. The confusion matrix was also calculated to determine the percentage of cases that were correctly classi®ed using the ®rst discriminant function only. 3. Results and discussion 3.1. Physicochemical analyses The changes in FAAs and SSAN, resulting from each of the enzymatic preparations used were calculated by substracting the enzyme-treated cheese (PP or L1) sample content from control cheese (without enzyme) sample content, for every day of the ripening time tested. As expected, Table 1 shows that the amino acid pro®le for the batch treated with added lipase (L1) was quite similar to that for the control batch over the entire ripening period and even had lower quantities of certain amino acids, especially in the early stages, with the dif-
J.M. Izco et al. / Food Control 11 (2000) 201±207
203
Table 1 Increments (control cheese content ± enzyme treated cheese content) in the FAAs (mg/100 g DM) and in the SSAN (mmol L-LEU/100 g DM) during ripening (in days); only if dierences between the mean values (p < 0.05, n 4) are signi®cant Day/Batch
1
FAAs
L1a
ASP GLU SER ASN GLY GLN TAU HIS GABA CIT THR ALA ARG PRO TYR VAL MET CYST ILE LEU HYLYS PHE TRP ORN LYS Total SSAN
15
2.1
0.7 nq 1.8 1.6 2.6 1.4 0.5 0.2
0.8 0.7 1.2 25.7
PPb 1.7 4.5
)6.4 0.9 0.5
L1 1.4
1.1 )0.5
1.1 3.1 9.1 4.6 0.6
27.4 0.4
PP )3.9 )3.2 )9.0 )8.8 0.7 )1.7 0.5 )3.0 )2.8
1.6 1.6 1.0
30
2.3
2.4 )2.3
0.2
)2.3 )1.2 )5.1
0.3
0.6
0.5
0.4 )4.4 )6.0
L1
)13.2 )0.7
)0.2
60 PP
L1
)13.7 )6.1 )22.4 )0.9 )11.6 )3.8 1.2
)5.4
)8.7 )4.3 )10.0
)64.6 )0.7
)2.8 )9.0 )4.8 )0.3 )8.6 )16.6
90 PP
L1
5.8 24.4 3.0 nqc 6.7 )1.2 11.6 nq 6.8 10.4 9.8 6.8 nq 7.9
nq
8.2
)6.2 nq )20.7
PP
L1
)42.0 )9.6 )74.1
)24.1
)4.9 nq 15.5 )11.4
120
)0.6
)19.4 )125.2 )10.6 )34.3
)18.6 )0.8 )9.7 )8.0
18.4
)11.9
19.3
)17.6 )18.6
)133.4 )0.6
202.5 0.8
)0.6
)17.8 nq )21.0 )14.2
)14.3
)31.0
)10.0 )4.1 )15.6
)17.2 )7.8 )35.0 4.0
)12.5
PP
)4.0
)1.6 )10.2 )32.6
10.2 )33.1 )49.8
)282.7
)477.6 )3.6
a
Lipolytic enzyme preparation. Proteolytic enzyme preparation. c Not quanti®ed. b
ferences decreasing at the end of ripening. The trend for the total free amino acids (TFAAs) was likewise quite similar to that in the control batch throughout ripening. The experimental results for batch L1 agreed with the ®ndings of Jolly and Kosikowski (1978). Fern andezGarcõa et al. (1994) attributed the lower proteolysis rate to the inhibitory eect of the fatty acids on the lactic acid bacteria. In contrast, Lin, Jeon, Roberts and Milliken (1987) did not record a lower proteolysis rate in Cheddar cheese made with an added animal lipase. There have been reports of appreciable increases in FAA concentrations when lipases have been added (Fernandez-Garcõa, Ramos, Polo, Ju arez & Olano, 1988). That ®nding was ascribed to contamination of the lipases with proteolytic enzymes as a result of incomplete puri®cation of the commercial lipolytic preparations; consequently, the preparations might also contain other (proteolytic) enzymes that may be activated to a greater or lesser extent depending on the conditions in the medium (pH, temperature). In fact, the lipolytic enzyme L1 increased breakdown of the casein fractions in cheeses ripened for two months (Izco et al., 1999b).
The principal ®ndings concerned batch PP. The cheeses supplemented with the protease preparation contained higher levels for many of the amino acids over the ripening period. Those ®ndings agreed with the results recorded by Zaki and Salem (1992) and FernandezGarcõa et al. (1988, 1993). In contrast, FernandezGarcõa et al. (1994) reported an increase in certain amino acids, namely, serine, glutamine, histidine, alanine, phenylalanine, isoleucine, leucine and lysine; conversely, other amino acids, namely, glutamic acid, asparagine, tyrosine, arginine, methionine and valine decreased. A possible explanation for these dierences may be that in addition to the Neutrase, a lipase was also used in that last-mentioned study, which may have generated fatty acids that inhibited proteinase activity. Likewise, Ard o and Pettersson (1988) did not observe any increase in the amounts of amino acids when Neutrase was added during the manufacture of Swedish cheese. Those ®ndings contrast with the results reported by Law and Wigmore (1982) in Cheddar cheese treated with that same enzyme, possibly due to technical differences in processing during manufacture.
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Changes in certain amino acids during ripening are particularly noteworthy. For instance, up to day 30 the concentration of proline in the control cheese (batch C) was higher than in the cheese batches to which enzymes were added, after which time levels converged with the values recorded in cheese batches L1 and PP. Subsequently, the proline level attained in batch PP on day 120 was 143% of the level in the control batch. Proline has been associated with sweet ¯avour in cheeses (Law & Wigmore, 1983). Those workers found that the concentration of proline was lower in Cheddar cheeses made with added Neutrase than in control cheeses; Zaki and Salem (1992) reported the same ®ndings in Edam cheese. Sood and Kosikowski (1979) recorded similar results for Cheddar cheese using a mixture of lipases plus proteinases. Asparagine was the main amino acid in the cheese samples (14.7% of the Total FAAs in the control cheeses on day 120; Izco et al., in press). The proteolytic preparation added to batch PP produced a signi®cant increase in the concentration of that amino acid (140% of the level in the control batch). Serine was aected in the same way as asparagine. The serine concentration increased in the cheeses made with added proteinase (batch PP) but did not increase in the cheeses made with added lipase (batch L1). Those two amino acids have also been related to sweet ¯avour in cheeses (Hefeli & Glaser, 1990); hence the increase in the serine and asparagine concentrations may have helped mask any bitter ¯avours that may have developed as a result of the possible formation of bitter peptides in the cheese due to the addition of the proteolytic enzymes. One method of estimating TFAA levels is by measuring the SSAN fraction using the TNBS method (Izco et al., in press). High correlations between the TFAAs and the SSAN fraction were recorded for all the cheeses over the ripening period. The correlation coecient values obtained (n 24) for the control batch, batch L1, and batch PP were 0.985, 0.990 and 0.984, respectively. In accordance with the results obtained using RPHPLC, the SSAN content in the cheeses made using added lipase (batch L1) did not dier signi®cantly from the content in the control batch on the sampling dates during ripening. Hardly any increase in the SSAN fraction was found when lipases were added during manufacture of Cheddar cheese (Lin et al., 1987) and Manchego-type cheese made from cows' milk or a blend of cows' milk and ewes' milk [30/70, v/v] (FernandezGarcõa et al., 1988, 1994). On the other hand, Fernan dez-Garcõa, Olano, Cabezudo, Martin-Alvarez and Ramos (1993) reported an increase in the SSAN fraction of cheese treated with Flavor Age (a combination of proteinase, aminopeptidase and lipase). Using that same enzymatic preparation in Cheddar cheese, Guinee, Wilkinson, Mulholland and Fox (1991) reported a slight increase in the concentration of total amino acids.
Although initial values were lower, from day 15 proteolysis was higher in batch PP than in the control batch. While dierences were not signi®cant on day 90, on day 120 the cheeses in batch PP had an SSAN concentration 20% higher than that in the control batch. Contrasting with those results, other researchers (Alkhalaf, Vassal, Desmazeaud & Gripon, 1987; Ard o & Pettersson, 1988) did not record increases in the amino acid nitrogen value (measured as SSAN or PTAN) using several dierent added proteinases. However, most workers have reported increases in those ripening index values both in cows'-milk cheeses (Law & Wigmore, 1982, 1983; Lin et al., 1987; Fernandez-Garcõa et al., 1988; Guinee et al., 1991) and in ewes'-milk cheeses ~ez (Vafopoulou, Alichanidis & Zer®nridis, 1989; N un ~ et al., 1991; Pic on, Gaya, Medina & N unez, 1994, 1995). 3.2. Sensory analysis Fig. 1(a) and (b) show that the pro®le for the control cheeses (batch C) was indicative of a cheese with a markedly sweet odour, highly creamy texture, moderate elasticity, adherence, and deformability, and low brittleness. The characteristic ¯avour of Ossau±Iraty cheese is moderately salty, with lower scores for sweet taste than for sweet odour. Bitter and pungent tastes were barely detectable, and the cheese left a characteristic, strong aftertaste after being swallowed. Batch L1 was the batch that diered the least from the control batch after 60, 90 and 120 days of ripening. On day 60, batch L1 received higher scores for pungent taste than the control batch. Perhaps the added lipase used released short-chain FFAs, as reported for pregastric esterase used to achieve the characteristic pungent overtones in certain Italian cheese varieties as Peccorino Romano, Provolone, and Parmesano (Nelson, Jensen & Pitas, 1977). However, the higher scores for pungent taste were not repeated in the cheeses after 90 and 120 days of ripening. Bitter ¯avours are one of the most common defects in cheeses made from milk or curd to which proteolytic enzymes have been added (Alkhalaf et al., 1987; Ard o& Pettersson, 1988; Fernandez-Garcõa et al., 1988; Mohamed et al., 1989) as a result of the accumulation of bitter peptides with a high proportion of hydrophobic, aromatic amino acid residues (Vafopoulou et al., 1989) and high-molecular-weight peptides as a result of casein breakdown. Even so, batch PP did not exhibit large dierences in bitter taste and aftertaste with respect to the control batch (except for bitter taste on day 120), despite the high level of casein breakdown in that batch (Izco et al., 1999b). Fig. 1(b) shows that taste panel members even awarded higher scores to the taste and aftertaste attributes for that batch than for the control ~ez et al. (1991), whole bovine batch. According to N un casein hydrolysates exhibited higher levels of bitterness
J.M. Izco et al. / Food Control 11 (2000) 201±207
Fig. 1. Sensory scores: (a) odour and texture and (b) taste and aftertaste for the Control cheese Ossau±Iraty type (C) and enzyme-treated cheeses L1 (lipolytic preparation), and PP (proteolytic preparation) on day 90 of the ripening period.
than did whole ovine casein hydrolysates, and hence there would appear to be less likelihood of the formation of bitter ¯avours in cheeses made from ewesÕ milk with added enzymes. Pic on et al. (1994, 1995) reported that the addition of encapsulated proteolytic enzymes (chymosin and BSNP) to the milk used to manufacture a ewes'-milk Manchego-type cheese did not result in the formation of bitter ¯avours. Conversely, Fern andezGarcõa et al. (1994) did report bitter ¯avour defects on adding BSNP to a blend of bovine and ovine milk (30/ 70, v/v). In addition, TFAA and SSAN values in batch PP were higher than in the control batch, possibly because of high exopeptidase activity brought about by the added enzymatic preparation, which may have led to degradation of the bitter peptides.
205
After weighting the sensory analysis results, the overall scores for each of the cheeses were calculated. Those scores provide a general idea of the ®nal rating for each cheese. Table 2 shows that all the cheeses had quite similar scores, suggesting that on the whole the enzymatic preparations did not adversely aect the organoleptic characteristics of this cheese. The discriminant analysis of the sensory analysis results was designed to select only those attributes that minimized the Wilk's k value, namely, the ones most useful in discriminating between batches. Firmness, creaminess, salty taste, and characteristic odour were the most eective attributes in discriminating between batches. Table 3 gives the correlation values for each sensory attribute in each of the discriminant functions calculated and furnishes an idea of the relative importance of each of the attributes considered in the sensory evaluations in the discriminant functions. Function 1 was more closely correlated with the sensory attributes for odour and taste, characteristic aftertaste, bitterness, ®rmness, brittleness, and adherence. Fig. 2 shows that the function was capable of dierentiating between all the cheese batches. Taste-related attributes, such as pungent taste, had negative correlation coecient values, causing batch L1 to be located in the negative region of the ®gure, since as already mentioned above, that batch had a distinctly more pungent taste than the other batches on day 60. Sweet taste, with a positive correlation coecient value (Table 3), was extremely important in function 1. The proteolytic enzyme preparation added to batch PP caused the release of the amino acids asparagine, proline, and serine, related to sweet taste, which was responsible for the location of batch PP on the plot. However, the function also included such texture attributes as ®rmness and, to a somewhat lesser extent, brittleness. The greater intensity of proteolysis caused by the addition of proteinases to accelerate cheese ripening has sometimes resulted in a more brittle texture (El Soda & Saada, 1986; Fedrick, Aston, Nottingham & Dulley, 1986; Alkhalaf et al., 1987) and reduced elasticity (Zaki & Salem, 1992; Pic on et al., 1994). On other occasions, in contrast, it did not give rise to any brittleness defects, and indeed elasticity has sometimes even been higher in the experimental batch than in the control batch (Pic on et al., 1995). Function 2 clearly distinguished the control batch from the batches with added enzymes but did not discriminate between batches L1 and PP. This function included the remaining aftertaste and texture attributes, especially creaminess. The present paper contributes new data on the effect of adding enzymatic preparations to the milk used in the manufacture of French Ossau±Iraty cheese to help ®ll the gap in available knowledge on the application of accelerated ripening methods in ewes'-milk cheeses.
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J.M. Izco et al. / Food Control 11 (2000) 201±207
Table 2 Overall sensory scores for the dierent cheeses at days 60, 90 and 120 of ripening Ripening time
Cheese batch C
L1
PP
p
Day 60 Day 90 Day 120
83 80 88
79 77 89
78 87 84
nsa ns ns
a
Not signi®cant dierences between the mean values (p < 0.05, n 8). Table 3 Pooled within-group correlations between discriminating attributes and canonical discriminant functions (attributes ordered by withinfunction correlation value) Attribute
Function 1
Function 2
Firmness Salty taste Sweet taste Pungent taste Characteristic odour Sour taste Brittleness Sweet taste Characteristic aftertaste Bitter aftertaste Sour odour Bitter taste Characteristic taste Adherence Creaminess Persistence Grittiness Elasticity Salty aftertaste Deformability Sour aftertaste
0.61979a )0.46882a 0.40848a )0.39541a 0.39441a )0.31492a 0.26810a 0.21444a 0.20313a )0.18767a )0.17488a )0.16435a 0.14482a )0.05534a )0.46632 )0.01520 0.12080 0.11311 )0.16019 )0.12470 0.01180
0.30029 )0.35241 0.05787 0.09241 0.02526 )0.16700 0.08244 0.11994 )0.02055 )0.06968 )0.01471 0.01541 )0.06020 0.04343 0.78470a )0.32628a )0.30012a )0.29332a )0.27999a 0.21887a )0.14309a
a
Denotes largest absolute correlation between each attribute and any discriminant function.
The results of the physicochemical analyses performed showed that, on the whole, lipolytic enzyme preparation L1 did not cause an increase in the release of amino acids. Indeed, a decrease in certain amino acids was even observed at the beginning of ripening, possibly due to an increase in the release of FAAs that then acted to inhibit the proteolytic activity of the lactic acid bacteria. However, the dierences with respect to the control batch decreased with ripening, and on day 120 there were practically no signi®cant dierences in the levels of any of the amino acids. Similarly, there were no appreciable alterations in the sensory characteristics of the cheeses, though certain abnormal results were observed on certain sampling dates, such as the pungent taste on day 60 and the decrease in sweet odour on day 90. The main ®ndings were in relation to batch PP. Addition of the enzymatic preparation to that batch resulted in an increase in the proteolytic indices analysed, e.g., a 20% increase in the SSAN value with respect to the control batch on day 120. The higher level of proteolysis had no adverse impact on the organoleptic characteristics of the Ossau±Iraty type cheese. One of the most commonly encountered defects caused by the addition of proteinases is the appearance of bitter ¯avours through the formation of bitter peptides. That defect may have been oset by the high level of exoproteolytic activity of the enzymatic preparation used, resulting in the breakdown of such peptides. The higher levels of such free amino acids as asparagine, serine, and proline, which are related to sweet ¯avour in cheeses, may also have masked bitter ¯avours in the cheese samples.
Acknowledgements This research work is part of the project ``PL921298'' titled ``Design and production of an enzymatic and microbial mixture to improve the processing of ewesÕ cheese (Spain, France, Italy and Portugal), its safety and quality and to get a novel functional food as a response to European demand for new products low in cholesterol and protein enriched'' of the AAIR Program. The author is grateful to the Government of Navarra for the ®nancial support provided for this study.
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Fig. 2. Plot of the canonical discriminant functions obtained using the sensory analysis results. Classing Control cheese Ossau±Iraty type (C) and enzyme-treated cheeses L1 (lipolytic preparation), and PP (proteolytic preparation).
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