Comparison of the volatile profiles of Arzúa-Ulloa and Tetilla cheeses manufactured from raw and pasteurized milk

LWT - Food Science and Technology 42 (2009) 1722–1728

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Comparison of the volatile profiles of Arzu´a-Ulloa and Tetilla cheeses manufactured from raw and pasteurized milk Patricia Rodrı´guez-Alonso a, Juan A. Centeno b, J. Ignacio Garabal a, * a b

˜a, Spain Dairy Science and Technology Laboratory, Agricultural Research Center of Mabegondo (CIAM), Xunta de Galicia, Apartado 10, 15080 A Corun Food Technology Laboratory, Faculty of Science, University of Vigo, Ourense, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 October 2007 Received in revised form 3 April 2009 Accepted 7 April 2009

Levels of volatile compounds in Arzu´a-Ulloa and Tetilla cheeses manufactured from raw and pasteurized milk were investigated. Analysis of volatile compounds in six raw milk (RM) starter-free cheeses (15–45 days old) and six pasteurized milk (PM) cheeses made with deliberately added starters (15–45 days old) manufactured in different dairies, was performed on an automatic dynamic headspace apparatus coupled to a GC/MS. The volatile fraction of RM cheeses displayed 46 volatile compounds (34 for PM cheeses) including fatty acids, esters, aldehydes, alcohols, ketones, hydrocarbons and sulphur compounds. Fatty acids and several esters were only detected in RM cheeses. Moreover, the highest contents of methylketones, secondary alcohols and branched-chain aldehydes and alcohols were also observed in RM cheeses. All results confirm more intense lipolysis in RM cheeses than in PM cheeses. In addition, branched-chain aldehydes and alcohols were significantly more abundant in RM than in PM cheeses, which indicates that catabolism of branched-chain amino acids was significantly higher in RM cheeses. This study has provided useful information which will allow the selection of starter and nonstarter bacteria more suitable for manufacturing Arzu´a-Ulloa and Tetilla pasteurized milk cheeses with organoleptic characteristics similar to those of traditional raw milk cheeses. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Cows’ milk cheese Arzu´a-Ulloa cheese Tetilla cheese Volatile compounds Raw milk Pasteurized milk Lipolysis

1. Introduction Cheese flavour results from the balance between large numbers of volatile and non-volatile compounds. These flavour compounds are formed during ripening, by different biochemical or chemical reactions that involve the main components of cheese: casein, fat, lactose, lactate and citrate (McSweeney & Sousa, 2000). The presence of volatile compounds and the relative concentrations of such compounds determine the quality and typical flavour of each cheese variety (McSweeney & Sousa, 2000; Smit, Smit, & Engels, 2005). More than 600 volatile compounds have been identified in cheese, but only a small fraction can be considered as ‘‘flavour-impact’’ compounds (Curioni & Bosset, 2002). Aldehydes, alcohols, carboxylic acids and esters derived from catabolism of amino acids are the most potent flavour compounds in cheese (Smit et al., 2005). It is generally agreed that pasteurization of milk promotes changes that affect the flavour of cheese. The main agents involved in flavour formation in cheese are indigenous milk enzymes, rennet

* Corresponding author. Tel.: þ34 981 647 902; fax: þ34 981 673 656. E-mail address: [email protected] (J.I. Garabal). 0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2009.04.002

and microbial enzymes from the wild microflora and/or from the commercial starters or adjunct cultures used. Some indigenous milk enzymes, such as lipoprotein lipase, are inactivated by pasteurization, and the autochthonous microflora is also partially eliminated, with a concomitant reduction in fermentation and degradation reactions (Buchin et al., 1998; Grappin & Beuvier, 1997). Differences between the volatile profile of raw and pasteurized or microfiltered milk cheeses have been reported for Manchego ˜ ez, 2002), Cheddar (Beuvier & (Ferna´ndez-Garcı´a, Serrano, & Nun Buchin, 2004; Singh, Drake, & Cadwallader, 2003), Swiss-type cheese (Demarigny, Beuvier, Buchin, Pochet, & Grappin, 1997) and Roncal (Ortigosa, Torre, & Izco, 2001). Moreover, differences in sensory aspects have also been reported. Raw milk cheeses generally have more intense aromas and contain a more diverse group of volatile compounds than cheeses made from pasteurized milk (Buchin et al., 1998; Demarigny et al., 1997). Arzu´a-Ulloa and Tetilla cheeses have Protected Designation of Origin (PDO) status and are nowadays mainly manufactured from pasteurized cows’ milk. The production area of both varieties is located in Galicia (in Northwest Spain), a region with a strong tradition in cheesemaking. One third (about 2000 million litres in 2005) of the total Spanish cows’ milk is produced in Galicia, and

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Arzu´a-Ulloa and Tetilla cheeses represent about 24% (more than 4.8 million kg) of the total annual production of cows’ milk PDO cheeses manufactured in Spain. Both cheeses are obtained from (mainly) rennet curd, have a short ripening time (7–60 days), and have quite similar characteristics regarding flavour and texture. Industrial Arzu´a-Ulloa and Tetilla cheeses are made with pasteurized milk with commercial starters added, either lactococci alone or lactococci with leuconostocs. In the manufacture of artisanal raw milk cheeses, the milk is not pasteurized and starters are not deliberately added. Another difference between the cheese varieties is their shape: Tetilla cheeses have a characteristic conical shape, whereas Arzu´a-Ulloa cheeses are cylindrical. Several studies were published in the 1990s concerning the microbiological, chemical and biochemical aspects of these two varieties, including analysis of volatile free fatty acids (Centeno, Cepeda, & Rodrı´guez-Otero, 1995; Centeno, Cepeda, & Rodrı´guezOtero, 1994; Mene´ndez, Godı´nez, Centeno, & Rodrı´guez-Otero, 2001). However, no previous research regarding the volatile profile of Arzu´a-Ulloa or Tetilla cheeses has been carried out. Sensory characterization of these cheeses, mainly achieved since the 90s, has revealed that raw milk cheeses have more diverse and intense flavour notes, whereas pasteurized milk cheeses are characterized by their mild flavours (data not published). The study and definition of the volatile compounds in traditional raw milk cheeses will provide a useful tool as an ‘‘analytical fingerprint’’ for the selection of starter and non-starter lactic acid bacteria more suitable for manufacturing pasteurized milk cheeses with organoleptic characteristics very similar to those of traditional raw milk cheeses. The aim of the present work was, therefore, to determine the main volatile compounds present in both Arzu´a-Ulloa and Tetilla raw and pasteurized milk cheeses and thus to verify hypothetical differences in their volatile profiles.

Barcelona, Spain) as internal standard. Ten grams of this mixture were weighed into a glass sparger (Tekmar Dohrmann, Cincinnati, OH, USA) for extraction of volatile compounds. The samples were analysed with an automatic dynamic headspace apparatus (Tekmar Dohrmann 3100 Sample Concentrator, Cincinnati, OH, USA) connected to a gas chromatograph–mass spectrometer (Agilent 6890N Network GC System and Agilent 5953 Mass Selective Detector; Agilent Technologies, CA, USA). Cheese samples were heated to 40  C, purged with helium (40 mL min1 for 20 min), and the volatile compounds were concentrated in a Tenax Trap (Tekmar, Cincinnati, OH, USA) connected to a CO2 turbo cool device maintained at 5  C. After 20 min purging, volatile compounds were desorbed at 220  C for 1 min into the injection port, with a split ratio of 30:1. Chromatography was carried out on a DB-WAX column (60 m length  0.32 mm i.d.; 0.25-mm film thickness) (J&W Scientific, Folsom, CA, USA). Operating conditions were as follows: carrier gas (helium) at 1 mL min1; initial temperature 34  C for 10 min; 5  C min1 up to 180  C; 10  C min1 up to 225  C and maintained for 2 min. The mass detector was performed in the scan mode, from 19 to 250 amu, with the ion source at 230  C and ionization voltage at 70 eV. Cheese samples were analysed in triplicate. Data were collected by use of MSD Productivity Chemstation Software (Agilent Technologies, CA, USA), and volatile compounds were identified by comparison of spectra stored in the Nist98 library (National Institute of Standards and Technology, USA) and Wiley 275 library (Wiley & Sons, Inc., Germany). Relative abundances of compounds were expressed as percentages of their peak areas in relation to the borneol peak area.

2. Materials and methods

Dry matter, pH, protein, fat and salt (NaCl) content were determined in triplicate, following international standardized methods as reported by Mene´ndez et al. (2001).

2.1. Cheese samples A total of 30 Arzu´a-Ulloa and Tetilla cheeses (15 units of each variety), manufactured in different dairies, in Spring 2005, from raw milk without added starters, were judged by a panel of five connoisseurs of traditional RM cheeses who considered four major attributes: appearance, flavour/aroma, body and overall quality. Four Arzu´a-Ulloa and two Tetilla RM cheeses (between 15 and 45 days old) were selected for the study on the basis of their typical attributes. Pasteurized milk cheeses made with added starters (three Arzu´a-Ulloa and three Tetilla cheeses) between 15 and 45 days old and which had obtained the highest scores in the 2005 ´ a-Ulloa y Tetilla (Arzu´a-Ulloa and Tetilla Concurso de cata de Arzu cheese tasting contest – organized by their respective Cheese Regulating Councils), were purchased directly from six different manufacturers. 2.2. Volatile compounds analysis Half (500–750 g) of the raw milk cheeses remaining from the sensory test and whole (1–1.4 kg) pasteurized milk cheeses were transported to the laboratory under refrigerated conditions. Cheese samples were cut into wedges (100–150 g), then wrapped in aluminium foil, vacuum packed and stored at 20  C until analysed. Prior to analysis of the volatile compounds, cheese wedges were kept overnight at 4  C and then left for 1 h at room temperature before extraction of the volatile compounds. Five grams of the inner part of a cheese wedge were homogenized in a blender (Moulinex, Lyon, France) with 8 g anhydrous Na2SO4 (Panreac, Barcelona, Spain) and 50 mL of 13 mM L-Borneol (Panreac,

2.3. Gross composition analysis

2.4. Statistical analysis Volatile compounds were considered individually, or grouped in classes according to the following chemical families: fatty acids, esters, aldehydes, alcohols, ketones, hydrocarbons, sulphur compounds and terpenes. Selected volatile compounds as the most important in the flavour of cheese were used in a stepwise analysis to classify the cheeses by type of milk. Volatile compounds, including hydrocarbons and terpenes (which are present in low quantities and the amounts of which are not linked to microbial metabolism) were excluded from this analysis. Relative abundances of selected volatile compounds found in cheeses were subjected to Principal Components Analysis (PCA; Varimax rotation, Kaiser normalization), and a Hierarchical Cluster Analysis (HCA; Ward’s method, Squared Euclidean distance) was then performed to group the samples. The cluster solution was confirmed by calculation of a Wilks lambda value and a Chi-Square ratio score. Both PCA and HCA procedures were performed with the SPSS Win 15.0 programme (SPSS Inc., Chicago, IL, USA). Analysis of variance was performed by the General Lineal Model (GLM) procedure using the SAS System Win 5.1.2600 programme (SAS Institute Inc., Cary, NC, USA) considering the following sources of variation: milk type (RM/ PM), cheese type (Arzu´a-Ulloa/Tetilla), interaction (milk type  cheese type), residual variation among experimental units (replicate units), and variation among determinations within the experimental units. F-values to assess differences between the gross composition data and the relative abundances of volatile compounds according to milk type (RM vs. PM) were obtained from

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the tests of hypotheses using the Type III Mean Squares for experimental units within the combinations of milk type and cheese type as an error term. For those cases in which a volatile compound was not detected in the PM milk-type group, a t-test considering a t-critical value (one-tailed) associated with 5 d.f. (milk-type group mean averaged over six observations) was performed.

PM cheeses yielded a clear clustering, whereas no clear clustering was observed for RM cheeses (Fig. 2). Moreover, the Hierarchical Cluster Analysis correctly assigned the samples in two large clusters, with a high level of statistical significance (Wilks’ lambda ¼ 0.003; c2 ¼ 155.3; P ¼ 0.0), according to cheese milk type (raw milk compared with pasteurized milk), suggesting the homogeneity of PM cheeses compared to RM cheeses (data not shown). Alcohols were the most abundant chemical family found in both RM and PM samples, and represented more than 88% of the total volatile compounds identified (91% for RM cheeses and 76% for PM cheeses) (data related to the proportions of chemical groups are not shown). Ketones (22%) and aldehydes (1.4%) were the second most abundant group of volatiles in PM cheeses, and ketones (7%) and esters (4%) were the second most abundant in RM cheeses. Esters were notably more abundant in the volatile fraction of RM cheeses (4%) than in the same fraction of PM cheeses (0.1%), whereas fatty acids (representing 0.2% of RM cheeses) were not detected in any PM sample. The number and content of volatile compounds in RM cheeses were greater than in PM cheeses. Fifteen compounds, including fatty acids and long chain fatty acid esters, were detected exclusively in RM cheeses. The contents of 29 volatile compounds (out of a total of 46) were higher in RM than in PM cheeses, 14 of them significantly higher (P < 0.05) (butyric acid; ethyl butanoate, ethyl hexanoate, ethyl octanoate, 3-methylbutyl butanoate; 3-methylbutanal; ethanol, 1-propanol, 1-hexanol, 2-methyl 1-propanol, 3-methyl 1-butanol, 3-methyl 2-butanol and 3-methyl 3-buten-1-ol;

3. Results and discussion 3.1. Volatile fraction GC–MS analysis of the headspace of Arzu´a-Ulloa and Tetilla cheeses revealed 46 volatile compounds in the raw milk cheeses (RM) and 34 in the pasteurized milk (PM) cheeses. Chromatograms (TIC) corresponding to one representative RM cheese and one pasteurized milk (PM) cheese are shown in Fig. 1. Twenty-nine volatile compounds reported as the most important in the flavour of cheese were selected and used in a stepwise analysis (Table 1). The selected volatile compounds were extracted in six principal components (PCAs) which explained 95.8% of variance (Table 1). Fatty acids, branched-chain ketones, acetoin and secondary alcohols (isopropyl alcohol, 2-butanol) were highly positively correlated with PC1, and most of the ethyl esters were highly positively correlated with PC2. The two components together explained 66.1% of the variance (Table 1). On the other hand, when the correlation components obtained from the six major PCA components for each cheese were used as input variables to generate a cluster diagram,

21

A

19 17 15 13

5

3

11 9 7

4

Abundance (x106)

5

6

3 1

1

7

8

9

10

2

11

12

Br

B

8 7 6 5

3

4 3 2

4

13

1

Br

1 5

10

15

20 Time (min)

25

30

35

Fig. 1. Chromatograms corresponding to the volatile headspace of one representative RM cheese (A) and one PM cheese (B). Peak identification and retention times (min) are as follows: 1: acetaldehyde (5.16); 2: acetone (6.63); 3: ethanol (11.22); 4: diacetyl (12.68); 5: ethyl butanoate (15.67); 6: 2-methyl 1-propanol (18.79); 7: 3-methyl-2-butanol (19.86); 8: 2-heptanone (21.81); 9: 3-methyl 1-butanol (22.97); 10: ethyl hexanoate (23.69); 11: 2-heptanol (26.59); 12: 2-nonanone (28.59); 13: acetoin (25.51); Br: internal standard LBorneol (36.78).

P. Rodrı´guez-Alonso et al. / LWT - Food Science and Technology 42 (2009) 1722–1728 Table 1 Principal components values and its canonical functions (correlation coefficients) extracted from the main volatile compounds. Compounds

Principal components PC2 PC3 PC4 PC1 (36.4%)a (29.7%) (12.5%) (7.7%)

PC5 (5.2%)

Fatty acids and esters Acetic acid Butyric acid Ethyl acetate Ethyl butanoate Ethyl hexanoate Ethyl heptanoate Ethyl octanoate Ethyl decanoate 3-Methylbutyl butanoate

0.004 0.026 0.021 0.998b 0.033 0.919 0.120 0.014 0.160 0.196 0.030 0.092 0.104 0.932 0.177 0.033 0.848 0.511 0.090 0.043 0.024 0.971 0.146 0.163 0.027 0.134 0.929 0.135 0.067 0.275 0.042 0.799 0.422 0.199 0.332 0.117 0.323 0.721 0.164 0.542 0.625 0.673 0.250 0.116 0.233

Aldehydes and alcohols Acetaldehyde 3-Methylbutanal Ethanol 1-Propanol 2-Butanol 1-Hexanol 2-Methyl-propanol Isopropyl alcohol 3-Methyl-1-butanol 3-Methyl-2-butanol 3-Methyl-3-buten-1-ol

0.323 0.166 0.010 0.148 0.908 0.703 0.059 0.966 0.375 0.208 0.139

Ketones and other compounds Acetone 0.961 2-Butanone 0.974 2-Heptanone 0.063 2-Nonanone 0.079 3-Methyl-2-pentanone 0.957 3-Methyl-2-butanone 0.732 2,3-Butanedione 0.058 3-Hidroxy-2-butanone 0.962 Dimethyl disulphide 0.057

PC6 (4.3%) 0.006 0.239 0.038 0.039 0.078 0.049 0.152 0.158 0.015

0.481 0.140 0.362 0.016 0.702 0.814 0.207 0.075 0.386 0.263 0.760 0.594 0.063 0.153 0.163 0.498 0.241 0.779 0.076 0.063 0.066 0.268 0.007 0.273 0.127 0.003 0.340 0.490 0.014 0.374 0.231 0.286 0.769 0.490 0.070 0.101 0.133 0.078 0.050 0.099 0.055 0.466 0.293 0.116 0.730 0.186 0.923 0.176 0.107 0.013 0.699 0.314 0.435 0.374 0.154 0.001 0.115 0.265 0.328 0.055 0.110 0.010 0.031 0.056

0.027 0.055 0.938 0.866 0.105 0.642 0.592 0.176 0.330

0.065 0.153 0.184 0.141 0.041 0.061 0.016 0.115 0.070 0.100 0.310 0.102 0.113 0.024 0.048 0.016 0.167 0.064 0.406 0.144 0.063 0.008 0.087 0.005 0.250 0.834 0.089

Rotation converged in 10 iterations. a Percentage of variance explained by each component. b Correlation coefficients with major loadings in each component are in bolditalic.

3-methyl 2-butanone). Only five compounds (including 2,3-butanedione) were more abundant in (most of) the PM cheeses than in the RM cheeses. The relative abundances of compounds in samples of RM cheeses manufactured by different dairies were very variable, as also reported by other authors (Ferna´ndez-Garcı´a, Carbonell, & ˜ ez, 2002). Differences in chemical and especially microbiologNun ical composition of raw milk may be one of the reasons for this variability. Pasteurized milk cheeses showed less variability between samples from different dairies, probably because of the more standardized manufacture process, which involves pasteurization of milk and addition of commercial starter cultures. Differences in gross composition (dry matter, pH, protein, fat and salt contents) with intermediate and homogeneous values found for PM cheeses compared to the wider ranges observed for RM cheeses (Table 2), cannot totally explain the volatile profiles of the cheeses.

3.2. Fatty acids and esters The method of analysis used in the present study allowed detection only of fatty acids with no more than four carbon atoms. Both acetic and butyric acids were found exclusively in RM cheeses (Table 3). Butyric acid is derived mainly from lipolysis of milk fat by the action of lipases in milk (lipoprotein lipase), although it can also originate from clostridial fermentation. Acetic acid originates from

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metabolism of lactose, citrate and/or amino acids (Marilley & Casey, 2004; McSweeney et al., 2000). Enterococci and micrococci have been isolated from Arzu´a-Ulloa and Tetilla raw milk cheeses (Centeno et al., 1994; Mene´ndez et al., 2001) and were shown to exhibit exogenous lipase activity (Centeno et al., 1995). Moreover, other microbial groups, such as yeasts, are also present in high levels in these cheese varieties (Centeno et al., 1994; Mene´ndez et al., 2001), and may also contribute to the production of free fatty acids. Biosynthesis of esters is achieved by esterification of alcohols and free fatty acids, either by different microorganisms (e.g. yeasts, lactic acid bacteria, Micrococcaceae spp. and coryneforms) or by chemical reactions (Buchin et al., 1998; Ferna´ndez-Garcı´a, Carbonell et al., 2002). The greatest amounts of fatty acid esters (ethyl acetate, ethyl butanoate, ethyl hexanoate and ethyl octanoate) were found in all or most of the RM cheeses analysed (Table 3). Among the esters identified, some medium-chain fatty acid esters with even numbers of C atoms (C2n) (ethyl hexanoate and ethyl octanoate) were exclusively found in RM cheeses. This suggests that lipolysis was more extensive in RM cheeses probably because of (i) higher activity of lipoprotein lipase (LPL) and/or (ii) higher activity of lipases in the indigenous microflora present in RM cheeses. It is well known that LPL is completely inactivated by pasteurization (Grappin & Beuvier, 1997). Nevertheless, some authors (Choisy, Desmazeaud, Gripon, Lamberet, & Lenoir, 1997; McSweeney, Fox, Lucey, Jordan, & Cogan, 1993) have reported that in comparison with microbial enzymes, LPL contributes relatively little to cheese lipolysis. Other varieties of raw milk cheeses have also been found to contain greater amounts of esters than pasteurized or microfiltered milk cheeses (Beuvier & Buchin, 2004; Ferna´ndez-Garcı´a, Serrano et al., 2002; Shakeel-Ur-Rehman et al., 2000). 3.3. Aldehydes and alcohols Acetaldehyde and 3-methylbutanal represented about 80% of all aldehydes detected. In PM cheeses, acetaldehyde represented nearly 80% of the aldehydes. Acetaldehyde is formed from lactate metabolism by lactic acid bacteria (McSweeney et al., 2000), and is also derived from reversible pathways in which acetaldehyde is produced by the action of threonine aldolase on threonine (Ardo¨, 2006). Branched and linear chain aldehydes are probably derived from microbial degradation of amino acids, either by transamination followed by decarboxylation or by Strecker degradation (Curioni & Bosset, 2002). Linear aldehydes are also formed from metabolism of unsaturated fatty acids. Aldehydes are transitionary compounds that may be easily reduced to the corresponding alcohols in the low redox environment of cheese (Marilley & Casey, 2004; McSweeney et al., 2000), and, therefore, low amounts of aldehydes are expected in ripened cheeses (Ama´rita, De La Plaza, Ferna´ndez de Palencia, Requena, & Pela´ez, 2006). Most of the RM cheeses analysed contained significantly higher (P < 0.05) amounts of 3-methylbutanal, which is derived from catabolism of leucine and easily reduced to 3-methylbutanol, than the PM cheeses (Table 3). This compound was not detected in most of the PM samples. The relative abundance of 3-methylbutanol in RM cheeses, was 20–60 times higher than that of 3-methylbutanal. This appears to be consistent with the conversion of aldehyde to alcohols, which mainly takes place during cheese ripening (Ama´rita et al., 2006). Centeno, Tomillo, ˜ ez (2002) reported that inoculaFerna´ndez-Garcı´a, Gaya, and Nu´n tion of wild lactococci producing branched-chain volatile compounds into raw ewes’ milk promoted the formation of branched-chain volatile compounds such as 2-methylpropanol or 3-methylbutanol in cheese. Moreover, Morales, Ferna´ndez-Garcı´a, ˜ ez (2003) reported that production of large amounts Gaya, and Nun

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Rescaled Distance Cluster Combine 0 5 10 15 20 25 +---------+---------+---------+---------+---------+

PM cheeses TP8 TP9 TP10 A5C A1C A2C

RM cheeses

A22 T4 T2 A7 A5´ AR8

Wilks’lambda= 0.003 =155.3; P=0.0 Fig. 2. Hierarchical cluster analysis (Ward method) generated with the six major PCA’s correlation components from cheese samples: pasteurized milk (PM) (TP8, TP9, TP10, A1C, A2C A5C) and raw milk (RM) (A50 , A7, A8, A22, T2, T4) cheeses. The level of statistical significance of discrimination between RM and PM cheeses is indicated by a dashed line.

of branched-chain aldehydes and alcohols during manufacture and ripening of fresh cheese is common when wild strains of Lactococcus lactis are present. The higher contents of 3-methylbutanal detected in RM cheeses in the present study may, therefore, be explained by a greater presence of wild lactic acid bacteria strains that produce branched-chain volatile compounds (i.e. strains that display aminotransferase activity) (Ama´rita et al., 2006). Alcohols were the most abundant chemical group in both RM and PM cheeses (Table 3). Ethanol was the most abundant of all volatiles detected. This compound is mainly derived from lactose degradation by heterofermentative lactic acid bacteria (leuconostocs and heterofermentative lactobacilli) (Marilley & Casey, 2004; McSweeney et al., 2000). The fact that acetic acid was found exclusively in the RM cheeses analysed (Table 3) and that these cheeses contained the greatest amounts of ethanol (P < 0.05) may suggest a greater presence of heterofermentative lactic acid bacteria in RM cheeses than in PM cheeses. Numbers of heterofermentative LAB (leuconostocs, obligatory heterofermentative lactobacilli, facultative heterofermentative lactobacilli) in RM cheeses and characterization of several isolates among those microbial groups have been determined in previous works (Garabal, Rodrı´guez-Alonso, & Centeno, 2008; Mene´ndez et al., 2001). Moreover, predominant LAB in PM cheeses were the lactococci from the starters (data not reported). Although ethanol does not contribute directly to the flavour of cheese, it is involved in the formation of ethyl esters, which are important in cheese aroma (McSweeney et al., 2000). The greater presence of ethyl esters in

RM cheeses (Table 3) may be associated with the greater amounts of ethanol also detected in the RM cheeses. Other authors (Buchin et al., 1998; Ortigosa et al., 2001; Shakeel-Ur-Rehman et al., 2000) have also reported that raw milk cheeses contain greater amounts of alcohols than pasteurized milk cheeses. Moreover, 7 out of 15 alcohols detected, including branched-chain (2-methylpropanol, 3methylbutanol, 3-methyl 2-butanol, 3-methyl 3-buten-1-ol) and primary alcohols (ethanol, 1-propanol, 1-hexanol) were present in significantly higher amounts (P < 0.05) in most of the RM cheeses than in the PM cheeses. Most of the RM cheeses analysed contained significantly higher amounts (P < 0.05) of 2-methylpropanol and 3-methylbutanol, produced by the reduction of 2-methylpropanal and 3-methylbutanal, respectively, than the PM cheeses (Table 3). Because 2alkanols result from the reduction of previously formed compounds, this is also consistent with the fact that the raw milk microflora induces faster cheese ripening (Beuvier & Buchin, 2004). Morales et al. (2003) reported significantly greater amounts of methyl aldehydes and methyl alcohols in fresh, raw ewes’ milk cheeses manufactured with wild lactococci than in control raw milk cheeses. This also suggests that the catabolism of branched-chain amino acids is more accentuated in RM samples. 3.4. Ketones and other compounds The contents of ketones in Arzu´a-Ulloa and Tetilla cheeses are summarized in Table 3. The ketones, 2-nonanone, 3-methyl-2-

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Table 2 Gross composition data (means of triplicate experiments) of the cheeses made from raw milk (RM cheeses) and pasteurized milk (PM cheeses). Arzu´a-Ulloa

pH Salt (NaCl %, w/w) Dry matter (%, w/w) Fat/dry matter (%, w/w) Protein/dry matter (%, w/w) a b

Tetilla

Statistic results RM vs. PMa

Total (mean  S.D.)

RM (n ¼ 4)

PM (n ¼ 3)

RM (n ¼ 2)

PM (n ¼ 3)

RM (n ¼ 6)

PM (n ¼ 6)

F

Significance level

5.2 1.3 53.1 49.2 38.2

5.5 1.0 51.1 53.3 35.3

5.0 0.9 50.5 49.7 39.2

5.2 1.1 52.8 53.9 34.8

5.1  0.15 1.2  0.29 52.21  4.33 49.4  5.23 38.5  5.46

5.4  0.51 1.1  0.07 51.9  1.22 53.6  0.74 35.1  0.64

18.73 0.07 0.01 2.54 1.69

P < 0.01 NSb NS NS NS

Statistic results for the analysis of variance by ANOVA’s F-test between RM and PM cheeses. NS: non significant (P  0.05).

butanone and 3-methyl-2-pentanone were not detected in any of the PM samples. Ketones were relatively more important in PM cheeses (22% of total volatile compounds compared with 4% for raw milk cheeses) because of the relative abundance of 2,3-butanedione (diacetyl) in the volatile profile of the PM samples (19% compared with 2% for RM cheeses). Methylketones (acetone, 2-heptanone and 2-nonanone) containing odd numbers of C atoms are produced from b-oxidation of fatty acids and reduced to the corresponding secondary alcohols (McSweeney et al., 2000). The highest amounts of ketones, butyric acid and fatty acid esters were mainly detected in the RM cheeses, with significant (P < 0.05) differences found for a number of these compounds (Table 3). The fact that lipolysis and catabolism of free fatty acids were more accentuated in RM cheeses is probably explained by the high lipase and esterase activities of the autochthonous microflora.

2,3-Butanedione (diacetyl) and its reduction products 3-hydroxy2-butanone (acetoin), 2-butanone, 2,3-butanediol and 2-butanol are mainly formed through citrate metabolism by lactic acid bacteria (McSweeney et al., 2000). In the present study, diacetyl was the only compound for which significantly (P < 0.01) higher contents were determined in the PM cheeses. Nevertheless, there was high variability in the contents of diacetyl and its reduction products in RM samples (Table 3), and, therefore, it cannot be concluded whether these compounds are linked to a certain type of cheese (RM or PM cheese). Mene´ndez et al. (2001) have reported that diacetyl is not usually present in high concentrations in Tetilla raw milk cheeses (2–3 weeks old) (mean content 31 mg diacetyl-acetoin kg1). Moreover, other studies have reported higher contents of diacetyl and/or acetoin in pasteurized and/or microfiltered milk cheeses made with commercial starters than in raw milk cheeses (Ferna´ndez-Garcı´a, Serrano et al., 2002; Ortigosa et al., 2001; Shakeel-Ur-Rehman et al., 2000).

Table 3 Relative abundances (means of triplicate experiments) of main volatile compounds found in Arzu´a-Ulloa and Tetilla cheeses made from raw milk (RM cheeses) and pasteurized milk (PM cheeses). Compounds

Arzu´a-Ulloa

Tetilla

Statistic results RM vs. PMa

Total (mean  S.D.)

RM (n ¼ 4)

PM (n ¼ 3)

RM (n ¼ 2)

PM (n ¼ 3)

Fatty acids and esters Acetic acid Butyric acid Ethyl acetate Ethyl butanoate Ethyl hexanoate Ethyl heptanoate Ethyl octanoate Ethyl decanoate 3-Methylbutyl butanoate

56.4 38.8 861.0 668.1 337.9 1.5 56.3 4.8 7.7

NDb ND 7.8 8.9 ND ND ND ND ND

12.0 39.5 214.2 676.3 575.0 2.9 52.5 ND 20.4

ND ND 0.3 ND 0.1 ND ND ND ND

Aldehydes and alcohols Acetaldehyde 3-Methybutanal Ethanol 1-Propanol 2-Butanol 1-Hexanol 2-Methyl 1-propanol Isopropyl alcohol 3-Methyl 1-butanol 3-Methyl 2-butanol 3-Methyl 3-buten-1-ol

68.6 52.6 41966.4 274.8 234.5 8.6 786.3 618.5 1740.7 144.7 7.7

156.4 2.3 10052.8 23.1 3.3 ND 2.0 19.6 12.7 ND ND

312.1 255.5 39488.1 195.2 73.1 4.0 841.7 23.8 1581.5 8.7 11.2

57.2 ND 4424.5 3.6 23.2 0.3 ND 131.3 12.4 0.6 1.3

149.8  148.66 120.2  110.43 41140.3  24998.30 248.3  113.04 180.7  240.40 7.1  4.10 804.7  453.29 420.3  673.36 1687.7  1007.76 99.4  91.43 8.9  4.44

109.7  55.19 1.2  1.94 7404.2  3294.59 13.9  10.26 12.7  18.54 0.1  0.42 1.1  2.41 72.2  116.46 12.6  10.36 0.3  0.45 0.6  1.39

2.77 37.32 7.12 20.90 2.03 15.34 13.27 1.10 11.04 6.95 23.52

NS P < 0.001 P < 0.05 P < 0.01 NS P < 0.01 P < 0.01 NS P < 0.05 P < 0.05 P < 0.01

308.4 55.3 9.1 ND ND ND 3057.5 151.8 2.0

417.7 3.6 13.7 4.9 14.1 ND 1566.3 186.1 ND

87.7 10.7 2.5 ND ND ND 485.4 60.5 13.0

617.3  763.99 62.0  151.36 127.3  132.39 65.9  87.51 110.5  112.54 29.8  51.72 780.7  695.26 280.5  428.61 10.9  14.71

204.6  133.73 34.3  23.83 6.0  4.34 ND ND ND 1847.1  1390.48 108.8  116.09 7.2  9.47

1.62 0.07 3.87

NS NS NS NS P < 0.05 NS P < 0.01 NS NS

Ketones and other compounds Acetone 717.1 2-Butanone 91.1 2-Heptanone 184.1 2-Nonanone 96.5 3-Methyl 2-butanone 158.8 3-Methyl 2-pentanone 44.6 2,3-Butanedione 387.9 3-Hydroxy-2-butanone 327.6 Dimethyl disulphide 16.4 a b c

RM (n ¼ 6) 41.6  77.55 39.0  34.83 645.4  972.52 670.8  460.83 417.0  339.11 1.9  2.58 55.0  35.62 3.2  5.76 11.9  12.28

Statistic results for the analysis of variance by ANOVA’s F-test or for t-test, between RM and PM cheeses. ND: Compounds not detected. NS: non significant (P  0.05).

PM (n ¼ 6) ND ND 4.3  4.23 4.7  10.60 0.0  0.13 ND ND ND ND

F

t

Significance level

1.31 2.74

NSc P < 0.05 NS P < 0.05 P < 0.05 NS P < 0.01 NS P < 0.05

1.54 9.05 8.22 1.80 3.78 1.36 2.37

1.84 2.40 1.41 11.47 0.75 0.03

1728

P. Rodrı´guez-Alonso et al. / LWT - Food Science and Technology 42 (2009) 1722–1728

Hydrocarbons are possible secondary products of lipid autoxidation (Barbieri et al., 1994), and do not make a major contribution to cheese aroma, although these components may serve as precursors for the formation of other aromatic compounds (Arora, Cormier, & Lee, 1995). The low contents (<10 units of Relative abundance) of aromatic hydrocarbons and terpenes detected in both Arzu´a-Ulloa and Tetilla RM and PM cheeses (data not shown) suggest that these groups of compounds are not affected by the presence of the native raw milk microflora. Dimethyl disulphide, the only sulphur compound detected, was present at very low quantities in both RM and PM cheeses (Table 3). Contrary to our findings, the results of other studies showed higher contents of sulphur compounds in ripened raw milk cheeses than in pasteurized milk cheeses (Beuvier & Buchin, 2004), which indicates that inactivation of native enzymes by heat treatment may be more important in preventing the formation of sulphur compounds than the elimination of the native microflora (Beuvier & Buchin, 2004). 4. Conclusions Arzu´a-Ulloa and Tetilla cheeses manufactured from raw and pasteurized milk showed differences in their volatile profiles (on the basis of their volatile components). Moreover, RM cheeses showed more diverse volatile compounds than PM cheeses. Likewise, RM cheeses contained greater amounts of most volatile components. The fact that esters and free fatty acids were exclusively detected in Arzu´a-Ulloa and Tetilla RM samples suggests that lipolysis was more common in RM cheeses. This had been previously suggested (Mene´ndez et al., 2001), but not confirmed. Moreover, the higher contents of methyl ketones and secondary alcohols in RM cheeses also indicate more intense lipolysis in this type of cheese. In addition, branched-chain aldehydes and alcohols were significantly more abundant in RM than PM cheeses, which indicates that catabolism of branched-chain amino acids was significantly higher in RM cheeses. Statistical analysis of data allowed us to discriminate between RM and PM samples on the basis of their volatile profiles. Although only 12 cheeses were analysed because of the current difficulty in finding RM starter-free cheeses with typical sensory qualities, the results provide us with some useful information. The analysis of volatile compounds on these traditional RM cheeses can help to build an ‘‘analytical fingerprint’’ which could be useful in the selection of (previously isolated) LAB and secondary microflora for the manufacture of pasteurized milk cheeses with more typical flavours. The RM samples showed a more diverse flavour profile than PM cheeses, which may be mainly attributed to the large diversity of the indigenous microflora present in these type of cheeses (as they were manufactured in different dairies within the production area). Furthermore, the volatile profile of PM cheeses was clearly defined. It appears that industrial or semi-industrial manufacture of cheese ensures a more homogeneous product, rather different from RM cheeses, in relation to the presence of specific volatile compounds. Acknowledgements Our grateful thanks to Raquel Lage and Cristina Fena´ndez for technical assistance and Julio Lo´pez for skilful assistance with statistical methods. The authors also thank Dr. E. Ferna´ndezGarcı´a (Department of Food Technology, INIA) and Dr. J. MorenoGonza´lez (Department of Coordination and Technological Development, CIAM) for expert and helpful advice about the statistical methods used in this study. This study was financially supported by the Instituto Nacional de Investigacio´n y Tecnologı´a Agraria y Alimentaria, Spain (INIA) (project RM02-004) and

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