A tentative characterization of volatile compounds from Iberian Dry-Cured Ham according to different anatomical locations. A detailed study

The elaboration of Iberian dry-cured ham is a long process of ripening (about 2-3 years). During this time, the lipids undergo a great transformation. Several studies have been carried out to see the principal changes that occur in the lipid fraction during the dry-cured processing of Iberian ham, although these studies have been done with intramuscular fat. Reactions of hydrolysis and oxidation, which produce the degradation of the lipid fraction from adipose tissue, take place during this process (López et al., 1992; Coutron-Gambotti y Gandemer, 1999). Hydrolysis mainly affects the triacylglycerols and diacylglycerols, and to a lesser affect, the monoacylglycerols and phospholipids (Narváez-Rivas et al., 2007). On the other hand, oxidation affects the fatty acids, producing oxidized compounds that have a short life; some of them are volatile and responsible for the characteristic flavor of dry-cured ham (Antequera et al., 1992). The flavor of Iberian dry-cured ham is critical to consumer acceptance and the aroma is perhaps the most important quality parameter. It has been postulated that chemical or enzymatic reactions such as lipolysis, chemical or enzymatic oxidation, proteolysis, Strecker degradation and Maillard reactions are the origin of volatile compounds (Toldrá, 1998; Toldrá et al., 2009). Due to the chemical composition of the fat in Iberian dry-cured ham, different factors like feed, breed, etc, will be able to produce changes in it (Narváez-Rivas et al., 2009). We could suppose that lipid alteration in composition of volatile compounds should be produced as a consequence of these factors. There are several scientific studies that prove this in the literature (Gandemer, 2009). Some studies have shown that volatile compounds can be affected by feed (López et al., 1992) and the conditions of drycured processing, such as its duration, salt content and temperature conditions (Ruiz et al., 1999; Andrés RESUMEN


INTRODUCTION
The elaboration of Iberian dry-cured ham is a long process of ripening (about 2-3 years).During this time, the lipids undergo a great transformation.Several studies have been carried out to see the principal changes that occur in the lipid fraction during the dry-cured processing of Iberian ham, although these studies have been done with intramuscular fat.Reactions of hydrolysis and oxidation, which produce the degradation of the lipid fraction from adipose tissue, take place during this process (López et al., 1992;Coutron-Gambotti y Gandemer, 1999).Hydrolysis mainly affects the triacylglycerols and diacylglycerols, and to a lesser affect, the monoacylglycerols and phospholipids (Narváez-Rivas et al., 2007).On the other hand, oxidation affects the fatty acids, producing oxidized compounds that have a short life; some of them are volatile and responsible for the characteristic flavor of dry-cured ham (Antequera et al., 1992).
The flavor of Iberian dry-cured ham is critical to consumer acceptance and the aroma is perhaps the most important quality parameter.It has been postulated that chemical or enzymatic reactions such as lipolysis, chemical or enzymatic oxidation, proteolysis, Strecker degradation and Maillard reactions are the origin of volatile compounds (Toldrá, 1998;Toldrá et al., 2009).

A tentative characterization of volatile compounds from Iberian Dry-Cured Ham according to different anatomical locations. A detailed study
The MS operating conditions were as follows: ion source and transfer line temperatures were 200 and 290°C, respectively.The electron energy was 70 ev a resolution of 1 and the emission current 250 μA; dwell time and inter-channel delay were 0.08 s. and 0.02 s. respectively.For GC-ion trap-MS.varian MS Workstation version 6.3 software was used for data acquisition and processing of the results.

Identification of the volatile compounds
The tentative assignment of the chromatographic peaks was done comparing the spectra with those from NIST (National Institute of Standards and Technology) and WILEY libraries and some of them were verified by standards purchased from Sigma-Aldrich and Fluka (S. Louis, MO).The peak area of the analyte was used as an analytical signal.The quantification of individual volatile compounds was carried out by evaluating the corresponding relative percentage according to the normalization area procedure, assuming an equal factor response for any species.

RESULTS AND DISCUSSION
Figure 1 shows a representative chromatogram of the total volatile compounds obtained under the conditions described above.A total of 109 volatile compounds have been identified by GC-MS.The assignation of each peak with its corresponding compound was carried out using NIST and WILEY libraries, and in some cases using standards.
Due to the high number of identified volatile compound, they have been classified into seven et al., 2007).In addition to these factors, the genotype and sex of the animals have an effect on these volatile compounds (Ramírez and Cava, 2007).
There are many volatile compounds in Iberian drycured ham that have been characterized by different authors (aldehyde, ketone, alcohol, alkane, esters, etc).Several volatile compounds, like hexanal and heptanal, are always present.However, there are others, such as limonene, that has been associated with feed (Luna et al., 2006).Also, many compounds show values that are significantly different, depending on the animals´ feed (López et al., 1992).
The aims of this work were to carry out a characterization of the volatile fraction of 23 Iberian dry-cured hams by GC-ion-trap-MS.Two different locations, subcutaneous fat and muscle, from the slices taken parallel to the femur from each ham have been analyzed.

Ham samples
Twenty-three samples of dry-cured hams from castrated males, fourteen months old, pure Iberian and processed in an industry for 24 months, were analyzed.The animals received a fattening diet based exclusively on acorn (Quercus ilex, Q. suber and Q. faginea) and pasture for 90 days prior to slaughter.They were kindly provided by the Designation of Origin "Los Pedroches".All the animals included in the study were reared extensively.
The slices were taken parallel to the femur and at different depths in each ham.Each slice contained adipose tissue, semimembranosus and semitendinosus muscles.The samples were stored in vacuum plastic bags at -18°C until they were needed for the analytical studies.
In each slice, the semimembranosus and semitendinosus muscles were selected, minced and mixed, in order to increase the interface between the sample and the striping gas during the concentration step.The same treatment was applied to the adipose tissue from these slices.

Volatile compounds analysis
The volatile compounds were isolated from three grams of sample prepared as indicated above by the dynamic headspace technique and adsorbed on a Tenax trap, using a Purge and Trap Concentrator apparatus Tekmar velocity XPT (Thousand Oaks, A TENTATIvE CHARACTERIzATION OF vOLATILE COMPOUNDS FROM IBERIAN DRY-CURED HAM ACCORDING TO… In this research work, ethyl-benzene, styrene, (1-methyl-propyl)-benzene, 1-propenyl-benzene and 1-methyl-4-(1-methyl-ethenyl)-benzene, have also been detected.However, they are not included as they could be contaminants from the plastic packing (Reineccius, 2006).

Alcohols
Alcohols do not usually contribute to aroma, but if they have high concentrations (ppm) or are unsaturated alcohols, they can usually be smelled.In Table 2, all alcohols detected in the volatile fraction of the samples are shown.They were detected in both locations, with the exception of 2-etoxi-ethanol, which was not detected in the adipose tissue.
In this group, the 4-methyl-5-decanol was the major alcohol in the subcutaneous fat and muscle, the percentage found in adipose tissue being higher than that found in the muscle.In addition, 4-methyl-5-decanol was the major compound among the volatiles identified in this work.This result is did not agree with a previous report where the major compounds found were hexanal, octanal or hexanoic acid (López et al., 1992;Sabio et al.,1998;Ramírez et al., 2007;García-González et al., 2008).Finally, significant differences for the alcohols due to anatomical location have not been found.

Aldehydes
In Table 3, the detected aldehydes in the volatile fraction from Iberian dry-cured ham are shown.groups according to their functional group (Tables 1-7).The different groups of volatile compounds identified are shown in each table together with the peak number, mean values and Standard Deviation (S.D.).
In order to explore whether there were significant differences between both anatomical locations, main effects analysis of variance (ANOvA) was performed according to the general lineal model procedure.

Hydrocarbons
This is the most numerous group present in the volatile fraction of slices from Iberian dry-cured ham.Table 1 show all the hydrocarbons identified in these samples.It can be observed that all of them are in the two different sample locations, adipose tissue and muscle, except octyl-cyclohexane, which appears only in the muscle.
Although the presence of 6-nonenal has not been All of them were found in the adipose tissue and muscle.

Esters and Ethers
Different esters and ethers identified in the volatile fraction of the samples are shown in table 5, where the presence of all compounds can be observed in the adipose tissue and muscle, except eucalyptol which was not detected in the adipose tissue.

Nitrogen compounds
In Table 6 the different nitrogen compounds identified in this type of sample are shown.1H-pyrrole was not identified in the adipose tissue.The rest of the nitrogen compounds were detected in both locations.shows that only 2-ethenyl-pyridine has significant differences (p<0.001) between locations.

Others compounds
Other compounds identified in the volatile fraction of the samples are shown in Table 7.They were detected in the adipose tissue and muscle, as can be observed.All these compounds, carbon disulfide, butanoic acid and dimethyl disulfide have been described as volatile compounds of the Iberian ham by other authors (López et al., 1992;Timón et al., 1998;Ruíz et al., 1998;Sabio et al., 1998;Ruíz et al., 1999;Andrés et al., 2002;Sánchez-Peña et al., 2005;Ramírez et al., 2007;García-González et al., 2008;Andrade et al., 2009) In this fraction, carbon disulfide had the major mean value although significant differences between anatomical locations was only found for dimethyl disulfide (p<0.01).
Figure 1.GC-ion-trap-MS chromatogram in full scan mode of the total volatile compound fraction from Iberian dry-cured ham: A, from 0.0 to 17.5 minutes; B, from 17.5 to 35.0 minutes; C, from 35.0 to 52.5 minutes and D, from 52.5 to 70.0 minutes.

Table 1 Means expressed as % and standard deviation (S.D.) of Volatile hydrocarbons detected by GC-MS technique of Iberian dry-cured ham from different anatomical locations
A TENTATIvE CHARACTERIzATION OF vOLATILE COMPOUNDS FROM IBERIAN DRY-CURED HAM ACCORDING TO…

Table 2 Means expressed as % and standard deviation (S.D.) of Volatile alcohols detected by GC-MS technique of Iberian dry-cured ham from different anatomical locations
*Compound detected for the first time in the volatile fraction of Iberian dry-cured ham.

Table 3 Means expressed as % and standard deviation (S.D.) of Volatile aldehydes detected by GC-MS technique of Iberian dry-cured ham from different anatomical locations
Compound detected for the first time in the volatile fraction of Iberian dry-cured ham. a p<0,05; b p<0,01; c p<0,00001. *

Table 4 Means expressed as % and standard deviation (S.D.) of Volatile ketones detected by GC-MS technique of Iberian dry-cured ham from different anatomical locations
*Compound detected for the first time in the volatile fraction of Iberian dry-cured ham. a p<0,05; b p<0,01.A TENTATIvE CHARACTERIzATION OF vOLATILE COMPOUNDS FROM IBERIAN DRY-CURED HAM ACCORDING TO…

Table 5 Means expressed as % and standard deviation (S.D.) of Volatile esters and ethers detected by GC-MS technique of Iberian dry-cured ham from different anatomical locations
*Compound detected for the first time in the volatile fraction of Iberian dry-cured ham. a p<0,05; b p<0,01; c p<0,001; d p<0,00001

Table 6 Means expressed as % and standard deviation (S.D.) of Volatile nitrogen compounds detected by GC-MS technique of Iberian dry-cured ham from different anatomical location
*Compound detected for the first time in the volatile fraction of Iberian dry-cured ham. a p<0,001.

Table 7 Means expressed as % and standard deviation (S.D.) of other volatile compounds detected by GC-MS technique of Iberian dry-cured ham from different anatomical locations
MóNICA NARváEz-RIvAS, EMERENCIANA GALLARDO, JOSé JULIAN RíOS AND MANUEL LEóN-CAMACHO