VOLATILE ORGANIC COMPOUNDS OF PARMA DRY-CURED HAM AS MARKERS OF AGEING TIME AND AGED HAM AROMA

Headnote

ABSTRACT

Extension of Parma ham ageing time resulted in several changes in volatile organic compounds in the ham headspace, enhancing signals for branched aldehydes/alcohols and ethanol/ethyl esters. The NaCl content of dry-cured hams was found to be positively related to volatile analytes with low solubility in dry muscle and molecules coming from lipid oxidation, but negatively to certain branched aldehydes originating from amino acids. In PLS regression relating volatile compounds to matured dry-cured ham aroma, branched aldehydes and several oxidation compounds were found to be influential in the sensory perception of matured ham odour. In this respect, oxidative mechanisms (lipid oxidation and oxidative degradation of amino acids) would seem to prevail over other biochemical pathways in increasing the odour of aged dry-cured ham.

- Keywords: aged dry-cured ham odour, ageing time, volatile compounds, ethyl esters, oxidation compounds, salt -

INTRODUCTION

The dry-cured ham known as 'Prosciutto di Parma' is among the most popular PDO products and its manufacture is regulated by the Consortium for its protection (Regulation EEC 2081/92).

In the early stages of traditional processing, the raw hams remain at low temperatures (0°-3°C), including the double salting with a mixture of dry and wet salt, and a resting phase for inner salt equalization and muscle dehydration. Subsequently, the hams are moved to cellars kept at room temperature for the drying and maturing phases, in a process that can take anywhere from 12 months (minimum time for brand apposition) to 30 months and beyond. Salt intake and dehydration play an important role in microbial control by reducing water activity (aw) in the outer and inner zones of dry-cured hams; in addition, temperature, relative humidity and airflow in the processing rooms are kept under control to achieve a safe and high quality product (ASEFA et al, 201 1). No addition of preservatives and ingredients other than sodium chloride is permitted.

The flavour of dry-cured ham is the result of biochemical and microbial processes which take place during the processing time, when volatile organic compounds (VOCs) are generated (TOLDRÁ et al, 1997). Most of these VOCs belong to the chemical categories of alcohols, esters, aldehydes, ketones, sulphur compounds, aromatic and aliphatic hydrocarbons. Differing VOC profiles in dry-cured hams have been ascribed to geographic origin (SANCHEZ-???? et al, 2005; SABIO et al., 1998), feeding (JURADO et al, 2007), surface microbiota (MARTIN et al, 2006), processing type and length (FLORES et al, 1997; RUIZ et al, 1999) and chemical composition (PEREZJUAN et al, 2006).

The subject of VOC dynamics during the extended ageing time of Parma ham has been tackled in the past (HINRICHSEN and PEDERSEN, 1995), but the possible effect of the ham's composition was not taken into account. Notwithstanding, due to the characteristics of this product, which features large amounts of moisture and inner aw values that continue to exceed 0.90 even after two years of processing (VIRGILI et al, 2007), the identification of headspace VOCs associated with the effective sensory perception of ageing could prove useful in detecting analytical markers of sensory quality.

Consistent with these preliminary remarks, the aim of this work is to detect markers of ageing time of Parma ham among identified VOCs, and their relationships with the sensory descriptor "matured dry-cured ham odour".

MATERIALS AND METHODS

Ham sampling

A total of 34 dry-cured hams, average deboned weight = 7.40 ± 0.5 kg, were taken in numerically homogeneous batches for each ageing time from two manufacturing plant facilities. Each facility provided 22 (8 standard maturation, 7 extended maturation, and 7 extended ageing) and 12 (3 standard maturation, 5 extended maturation, and 4 extended ageing) dry-cured hams respectively. The two plants were operating in accordance with mandatory basic regulations for Protected Designation of Origin (PDO) products, even though potential differences occurring in applied processing conditions can be a variability source for final outcomes. Both plants processed green hams obtained from the domestic heavy pigs allowed for Parma ham production (at least 9 months old at slaughtering), but no further information about the raw material (e.g. crossbreeding, feeding, slaughtering weight) was available.

After ham de-boning and partial removal of the rind, two adjacent ham portions, 10 mm thick, including the whole slice with intramuscular, intermuscular and subcutaneous fat (Fig. la), were cut with an electric slicer from the centre of each ham, perpendicularly to the femur bone and below the boundary trimming, for proximate composition and volatile organic compound (VOC) analyses. Both slices included the sections of muscles shown in Fig. la, intramuscular, intermuscular and exterior fat; the external fat layer was standardized to 1 cm thickness. The remaining part of the ham, from the centre to the butt, was kept for sensory analysis, while the shank was not used. Each part was separately stored in a vacuum- sealed bag; the slice for proximate composition and the portion for sensory analysis were stored at 2°C and analysed within one week from sampling, while the slice for VOC analysis was kept frozen at -20°C and analysed within three months.

Proximate composition

The whole slice was thoroughly minced and its major chemical components were determined. Moisture was determined as the weight loss of ca. 2 g per minced slice after drying at 100°-102°C for 16-18 h according to the AOAC method (2002a). Salt content was estimated at 10 g per minced slice as chlorides, which were extracted with warm water (40°C) and quantified using the Carpen ter -Volhard method, according to the AOAC (2002b). Proteins were determined as total nitrogen in ca. 2 g of minced muscle using the Kjeldal method and calculated as ? ? 6.25 following the AOAC (2002c). The estimated fat content was calculated as the difference from 100 of the summed proximate data. Moisture, salt and protein content were expressed as grams per 1 00 grams wet slice and as grams per 100 grams dry matter.

Sampling and headspace analysis

The central portion of the slice (Fig. lb), including fat and sections of biceps femoris and semimembranosus muscles, was removed from the frozen slice: this sampling position was established to minimize the variability in fat and lean distribution occurring in the whole slice. The ham portion was fragmented with a knife into particles of approximately 1 -2 mg weight. 3 g of the sample was weighed into a 20 mL glass vial tightly capped with a PTFE septum and left for 10 min at 40°C to allow temperature equilibration. Volatile components were extracted by Headspace Solid-Phase MicroExtraction technique (HS-SPME), piercing the septum covering each vial with a needle equipped with a Carboxen/PDMS/DVB (Supelco, Bellefonte, PA) coated fibre. Prior to the collection of volatiles, this fibre was preconditioned at 250°C for 40 minutes in the GC injection port and exposed to the headspace for 180 min (SÁNCHEZ-PEÑA et al, 2005) at 40°C. Then the fibre was inserted into the injector port of the GC for 1 min at 250°C using the split mode (split ratio 1:10). Temperature, time of incubation and injection were controlled by means of aTriPlus Autosampler (Thermo Electron Corporation) using Excalibur 1.4 software (Thermo Electron Corporation).

Gas chromatography-Mass spectrometry (GC-MS) analysis

The volatile compounds were separated using a SLB-5ms column (Supelco; 60 m ? 0.25 mm id ? 0.25 µ?? film thickness) installed on a Trace GC Ultra (Thermo Electron Corporation). The carrier gas was helium. The oven temperature was kept at 36°C for 15 min, programmed to rise by 10°C/min to 250°C and then held for 5 min. The GC-MS transfer line temperature was 280°C. The mass spectrometer was operating in the electron impact mode, with an electron energy of 70eV, and a scan rate of 1.4 s1 over a range of m/z from 35 to 350 in full scan mode for data collection.

The identification of volatile compounds by GC-MS was carried out using a DSQII mass-selective detector (Thermo Electron Corporation). Volatile compounds were tentatively identified by comparison with reference spectra from the NI ST 2005 version 2.0 spectral library database and with Kovats retention indices in accordance with the literature (NI ST, Gartner sburg, MD, USA).

Peak integration and relative quantification were based on the signal area, computed on an arbitrary scale, according to the Single Ion Monitoring (SIM) mode and based on a single ion selected for each compound according to its fragmentation pattern, in order to improve selectivity and remove noise due to background and coeluting peaks. Two replicates were run and averaged for each sample. An aqueous solution of ethyl propionate (0.25 mg/L) was used as external standard twice a day to correct the chromatograms according to instrumental performance.

Sensory analysis

Samples were evaluated by quantitative descriptive analysis (MEILGAARD et al., 1999). The panel, consisting of 8 members, were trained (ISO 8586-1: 1993; ISO 8586-2: 1994) in three preliminary sessions in the use of the attribute "odour of aged dry-cured ham" defined as the complex and characteristic dry-cured ham odour related to the presence of aged fat and aged muscle (GUARDIA et al., 2010). This attribute was scored on a non-structured 0-9 intensity scale (0= not detected, 9= maximum perception of the attribute) and anchored to the given scale values by providing the panel with dry-cured hams corresponding to perceptions of the attributes covering most of the intensity scale. Dry-cured ham slices 1.0 mm thick with 1 cm of covering fat, obtained using an electric slicer a few minutes before testing, were presented at 15°C, according to a randomized order. Analysis of the 34 hams took no. 7 sessions (no. 5 samples per session). At the beginning of the session, each dry-cured ham was sliced and presented to the panellists in an open tray. At the end of the testing sessions, the sensory rating for each ham sample was averaged over the panel (to be kept, scores had to fall within the panel mean ± 3 standard deviations).

Statistical analysis

SPSS 14 for Windows was used for statistical analysis, running the procedures ANOVA (one-way analysis of variance) and General Linear Model (GLM); in GLM, the processing length was the sole main effect, while salt content was included as covariate. The Least Square Means (LSM) of volatile compounds of each group of processing length were estimated and the Bonferroni test was performed to statistically separate them [P< 0.05). Partial-Least-Square (PLS) analysis (Unscrambler ver. 9.7, CAMO Software AS, Norway) was applied to relate sensory scores of "aged ham odour" (dependent variable) with the set of HS volatiles (independent variables) and Martens' uncertainty test was run to detect significant independent variables; two PLS components were run to achieve variance reduction of the dependent variables (calibration model) by means of the Full Cross Validation Method.

RESULTS AND DISCUSSION

Identified VOCs and proximate composition

Identified VOCs belonged to chemical categories of aldehydes, alcohols, ketones, esters, acids, sulphur compounds, aliphatic and aromatic hydrocarbons are reported in Table 1 . Most of the more than 60 identified volatile compounds have been previously reported in studies dealing with dry-cured ham odour (with the exception of 3-heptanone and 2,3-dimethylphenol), and their origin and olfactory notes have been thoroughly discussed (THERON et ed., 2010; GARCÍAGONZÁLEZ et ed., 2008; MARCO et ed., 2006). Amino acid catabolism, auto- and ß -oxidation of lipids, esterase activity and carbohydrate catabolism are the biochemical pathways leading to the generation of most of the VOCs reported in Table 1.

The amount of volatile molecules in the headspace is related to their concentration in the substrate as well as interaction with food components (DE ROOS, 2000). Applying the same experimental conditions to all tested samples, the portion of the analyte absorbed by the fibre is related to its original concentration in the sample and to the analyte distribution coefficient sample/headspace as reported by WANG et cd. (2005).

In general, lipids and proteins reduce the volatility of aroma compounds, while salt, through the salting-out phenomenon (FLORES et al, 2007), can influence the release of analytes into the headspace depending on the nature of the VOCs (PEREZ- JUAN et ed., 2007). As a consequence, the composition of dry-cured hams is a potential source of variability in the release of volatile compounds into the headspace, because of the sample/headspace distribution coefficient and the analytes absorbed by the fibre; all to be taken into account when differences due to ageing times are investigated.

The proximate composition data of ham slices, expressed on a wet and dry matter basis and grouped according to assayed ageing times are reported in Table 2. Dry-cured hams with scheduled processing times showed no difference in estimated fat and protein content; the expected decrease in moisture due to progressive dehydration occurring during protracted ageing, was not significant (P > 0.05). These findings can be ascribed to variability in ham slices in terms of lean and fat distribution masking moisture changes occurring in single muscles during ageing (VIRGILI et al., 2007). The most aged hams were the least salty (on a dry matter basis), as a result of the common practice of using less salt for hams manufactured for extended ageing, in order to prevent excessive salt concentration in the dried muscles. Even if dry-cured ham groups differed only in salt content, the sensory score given to the descriptor "aged ham aroma" was higher in longer-aged hams than in lesser aged ones (6.38 us 5.76, ? < 0.05). Consequently, the volatile release into the headspace may be affected by salt differences between dry-cured ham groups, masking the actual concentration in the substrate. Furthermore, during ham processing, salt, well-known as a proteolysis inhibitor, may be decreasing the production of VOCs generated by amino acid catabolism (PEREZ- JUAN et ed., 2006), and accelerating lipid peroxidation by acting as a pro-oxidant agent (KANNER et ed., 1991).

Comparison of VOCs detected in Parma hams with different ageing times

Differences in volatile compounds due to extended ageing times were evaluated by means of GLM analysis: the processing length was the sole main effect of the model, while salt was included as covariate to estimate salt coefficients for HS- VOCs (Table 3): a significant covariate estimate coefficient (P < 0.05), denotes a relationship between ham salt content and the corresponding volatile compound. Further sources of uncontrolled bias in dry-cured hams with different ageing times may be due to the process- ing conditions applied in the plants of origin, on top of raw material properties.

Most branched aldehydes originating from amino acid catabolism proved to be positive- ly influenced by maturing time: 3-methyl-bu- tanal and benzeneacetaldehyde rose sharply in the most aged samples, according to previous findings (JURADO et ed., 2007). Branched vola- tile compounds represent the final step of the proteolytic events that occur during dry-cured ham maturation and produce large amounts of free amino acids (FAA). Both chemical oxidative Strecker degradation and microbial amino acid catabolism (Erlich pathway) have been postu- lated as accounting for the formation pathway of branched volatile compounds in dry-cured ham (MARTIN et al., 2006; ANDRADE et al, 2009). During the maturation of Parma ham up to 24 months, a progressive increase in free amino acids (FAA) has been detected (VIRGILI et al., 2007), matching the increase in amino acid-related volatile compounds found in the present study. Among branched VOCs, salt estimate coefficient was significant for 2-methyl-propanal and 3-methyl-butanal: the negative sign may be accounted for by the inhibition exerted by sodium chloride on proteolytic activities (TOLDRÀ et al., 1997; JURADO et ed., 2007), proteolysis, and free amino acid release in dry-cured ham (VIRGILI et al., 1999). In this respect, the above-mentioned branched aldehydes, having low odour thresholds and odour notes relevant for drycured meat (THERON et al., 2010; CARRAPISO et al., 2010), which would be expected to increase in the headspace of dry-cured hams with low salt and high free amino acid content. 2-methyl1-butanol and characterized by malty olfactory notes coming from the microbial reduction of 2-methyl-butanal, remarkably increased in the headspace of the longest aged dry-cured hams. Toluene, ascribed to the catabolism of phenylalanine (BERDAGUÉ et al., 1991), or deriving from the cyclization of unsaturated carboxylic chains produced by lipid degradation (MIN et al., 1977), increased in the headspace as a result of both ageing and salt (Table 3). The positive sign of the salt covariate can be explained by the salting-out phenomenon enhancing the release of analytes which are poorly soluble in dried muscle such as toluene; in the same way, ethyl benzene and 2,3-dimethyl-phenol, generated by amino acid catabolism, could be increased in the headspace of more salty dry-cured hams, masking the expected variations due to ageing time.

Most volatile compounds generated by fatty acid oxidation either decreased during the ageing of dry-cured ham (1-pentanol and hexanal) (BOLZONI et al., 1996), or remained fairly constant, e.g. octanoic acid.

Among the VOCs originating from ß-oxidation, l-penten-3-ol decreased during ageing; the positive sign of the salt covariate for 2-pentanone may be increasing this molecule in the headspace of the saltiest samples, mainly belonging to the least aged group (Table 1). Oxidation compounds characterized the first ageing deadline (14-15 months), with the exception of nonane, which displayed a significant increase during the extended ageing of assayed hams. Several VOCs generated by oxidation pathways had positive significant relationships with salt content (1-hexanol, ?-hexalactone, tetradecane, 2-pentanone, 2-heptanone).

Butanoic acid, generated by carbohydrate fermentation, showed a rather slight decrease during the extended ageing of dry-cured hams, as a possible consequence of the decrease in carbohydrate availability, while ethanol was characterised by a sharp increase during ageing. Ethanol has been recognized as most likely originating from pyruvate (SPAZIANI et al., 2009) produced by the microbial metabolism of free organic acids and amino acids (LIU, 2003). The accumulation of amino acid sources during ageing is consistent with the progressive increase in ethanol during ham ageing.

Ethyl ester formation was related to the presence of ethanol and was reported as being the result of microbial enzymatic mechanisms such as esterification and alcoholysis (TALON et al., 1998; LIU, 2003). The enzymes needed for ethyl esters biosynthesis are present in Micrococcaceae (MONTEL et al., 1996) and in many species of yeast and moulds (JELÉN and WASOWICZ, 1998), i.e., microbial populations detected at high levels in Parma hams (LORI et al., 2005; SIMONCINI et al., 2007). The present investigation found a rise in ethyl esters during the extended ageing of Parma dry-cured hams, unaffected by salt variations. SABIO et al. (1998), in comparing the volatile compounds of six types of European drycured hams, reported that Parma hams were more characterised by ethyl esters than other types of ham. According to the data shown in Table 3, ethyl esters can be regarded as a class of volatile compounds highly representative of fully-aged Parma dry-cured hams: the processing method (PAROLARI, 1996), the aw values still around 0.90 after two years of processing (VIRGILI et al., 2007) and the absence of preservatives other than salt, may account for the increased microbial esterase activities in comparison with other dry-cured ham types (HINRICHSEN and PEDERSEN, 1995).

Relationship between

"matured ham smell" and volatile compounds

Several volatile markers of the investigated time range were evidenced, but it may be that molecules highly representative of increasing ageing time had a different influence on the sensory perception of aged ham aroma. Since the sensory quality of dry-cured ham is influenced by descriptors related to the intensity of drycured ham flavour and aroma (RESANO et al., 2010), the relationship between volatile molecules corresponding to scheduled ageing times and the sensory perception of the matured drycured ham smell was investigated.

The algorithm used was the PLS regression (MARTENS and MARTENS, 1986); this technique constrains the components obtained by linear combination of the x-variables (volatile compounds listed in Table 1) to be orthogonal, thereby overcoming the problem of related variables; the use of the full cross validation method prevents non-significant relations even if the model is run on more variables than objects. The regression model achieved by means of two PCs, gave a reduction in % Y-variance corresponding to 42.7 (PCI) and 67.6 (PCI + PC2). Table 4 presents the results of PLS analysis, expressed by the loadings of the dependent and independent variables: the coefficients of the first and second factor were reported (only coefficients of PCI or PC2 > 0. 1 are listed). In general, the size of the regression coefficients provides an approximate assessment of the importance: if the regression coefficient is greater than 0.2 in absolute value, the effect of the variable is most probably important, while if it is smaller than 0. 1 then the effect is negligible. Significant independent variables (Martens' uncertainty test) are reported in italics in Table 4.

The loadings of "matured ham smell" are positive on PCI and PC2: the first factor highlighted the opposition between volatiles with negative loadings such as linear alcohols (1-pentanol, 1-hexanol), methyl ketones (2-propanone, 2-butanone and 2-pentanone), hydrocarbons like heptane, octane, ethylbenzene, 2-pentyl furane, and volatiles with positive scores like branched aldehydes, ethanol/ethyl esters, diacetyl and acetoin and long-chain alkanes nonane and tetradecane. Ethyl esters, though having positive loadings along PC 1 are in opposition along PC2 (accounting for nearly 25% of the total explained variance), indicating a less important role than branched aldehydes in enhancing "matured drycured ham smell". Furthermore, four branched aldehydes but no ester passed the test for a significant effect on the Y variable (Table 4). On PC2, oxidation compounds such as linear aldehydes, lactones and tetradecane, and some branched aldehydes, positively counteracted ethyl esters and organic acids.

Variable loadings and sample scores were normalized to the interval ± 1 and projected onto the PC1-PC2 plane (bi-plot), to display variables and sample interrelationships; each quarter of the bi-plot corresponding to different combinations of PCI and PC2 signs was named from Ql to Q4. Ham samples were labelled by processing the time group number (1 or 2 or 3) and the symbol surrounding the number identifies the sensory score as low (< 5.5), medium-low (5.5 - 6.0), medium-high (6.0 - 6.5) and high (> 6.5) (Fig. 2).

As a consequence of the overall decrease in VOCs, the samples projected onto Q3 (Fig. 2) and belonging to ageing groups 1 or 2, warranted low scores for "aged ham odour". On the contrary, most samples placed in Q4, though having low ageing times on average, achieved increased scores thanks to volatiles yielded by oxidation mechanisms. Samples located in Ql are characterised by high scores for "aged ham odour": these mostly correspond to ageing groups 2 and 3, even if two samples from group 1 also earned a high rating. Carbonyl compounds, including branched and linear aldehydes, lactones, ketones, resulted the dominant chemical class involved with the sensory perception of "matured dry-cured ham smell" in Parma hams. GARCÍAGONZÁLES et cd. (2008), reported for the "acorn odour" descriptor, a relevant contribution of ben - zaldehyde, 2-heptanone and 3-methylbutanal, in accordance with our results for matured ham smell, as shown in Fig. 2. Even if the abovementioned attributes describe different sensory perceptions (acorn odour rates the acorn flavour perception mainly due to pig feeding), both of these are positively related to dry-cured ham quality and acceptability (RESANO et al, 2010).

The volatiles loadings projected onto the Q2 area of the bi-plot include ethyl esters, organic acids, and molecules related to carbohydrate fermentation such as ethanol, acetoin, diacetyl and butanoic acid. Though all coming from ageing groups 2 and 3, samples that fell into Q2 decreased their ratings if compared to Q 1 cases.

Even if the model reported in Table 4 does not explain the 32.4% in aged ham odour variability, it does seem that, although both branched aldehydes and ethyl esters are positive markers of ageing time extension, the former contributes to aged ham odour perception more than the latter. A recent GC-O study on odour-active compounds of Bayonne ham found that fruity notes due to ethyl esters were masked by the overall flavour of dry-cured ham (THERON et al., 2010). Moreover, volatiles generated by lipid oxidation, although not related to extended ageing time, can improve aged ham odour perception. Salt, being positively associated with some oxidation compounds and molecules like toluene, ethylbenzene, tetradecane, and 2,3-dimethyl-phenol (salting-out effect), may enhance aged ham aroma when aged odour -impact molecules such as branched aldehydes, that need extended ageing time to increase, are still at low levels.

CONCLUSIONS

The results of the present investigation suggest that, among the volatile molecules identified, the markers of Parma dry-cured ham extended ageing time do not overlap the markers of "matured ham smell" sensory perception. The main information provided by the present study is that the volatile compounds increased by the ageing time of Parma ham are not equally aged-odour active, or involved in processes that positively contribute to matured ham smell perception. In the case of some dry-cured hams with shorter processing times, the achievement of a high "aged odour" score can more be ascribed to VOCs generated through oxidative processes than to volatiles molecules increasing with ageing time. As a consequence, even if the extension of ageing time does have a positive influence on aged ham odour perception, further ways should be investigated for their effectiveness in yielding aged odour-active volatile profile. Environmental factors in maturing rooms, their effects on ham composition and on microbial populations growing in the outer and inner layers of Parma hams, could be key parameters in throwing light on mechanisms suitable for selective VOC generation.

ACKNOWLEDGEMENTS

The support of the "Consorzio del Prosciutto di Parma" through the participation of the co-author T. Toscani and the cooperation of the operators at the dry-cured ham production plants is acknowledged.

Sidebar

Paper received September 20, 2011 Accepted February 17, 2012

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