Meat Science 96 (2014) 1409–1416

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Determination of volatile compounds and quality parameters oftraditional Istrian dry-cured ham

Nives Marušić, Sanja Vidaček, Tibor Janči, Tomislav Petrak, Helga Medić ⁎University of Zagreb, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, Croatia

⁎ Corresponding author. Tel.: +385 1 4605126; fax: +E-mail address: hmedic@pbf.hr (H. Medić).

0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.meatsci.2013.12.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 July 2013Received in revised form 2 December 2013Accepted 9 December 2013

Keywords:AromaDry-cured hamGC–MSSPMEVolatile compoundsSensory attributes

The aim of this workwas to determine the characteristics of Istrian dry-cured ham by instrumentalmethods andsensory analysis. The aroma-active compounds of Istrian dry-cured ham from 2010 and 2012 were investigatedby using headspace-solid phasemicroextraction (SPME) and gas chromatography–mass spectrometry (GC–MS).Samples of biceps femoris were also evaluated by measuring physical and chemical characteristics. 92 volatilearoma compounds of Istrian dry-cured ham were found. Volatile compounds belonged to several chemicalgroups: aldehydes (51.4; 51.3%), terpenes (16.5; 16.4%), alcohols (15.5; 13.2%), ketones (8.6; 7.4%), alkanes(3.8; 5.7%), esters (1.3; 1.6%), aromatic hydrocarbons (0.8; 3.9%) and acids (0.6; 0.9%). Principal componentanalysis (PCA) showed that fat content, tenderness and melting texture were positively correlated. Terpeneswere strongly correlated with flavour of added spices. Sweet taste and the presence of esters were positivelycorrelated as well as negative odour, raw meat flavour and water content.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Istrian dry-cured ham is a typical dry-cured Croatian productand the tradition in its production makes it different from all otherMediterranean dry-cured hams. Croatia has recognised the importanceof its unique autochthonous products, and Istrian dry-cured ham hasreceived the designation of origin according to EU standards in 2011.The ham is produced from Landrace, Large White and Duroc pigs andtheir crossbreeds with a minimum live weight at slaughter of 160 kg.Istrian ham is processed with pelvic bones, and skin and subcutaneousfatty tissue are removed. The processing of Istrian dry-cured haminvolves four phases: salting and flavouring with garlic, pepper andlaurel, pressing, drying and ripening. The process takes between 12and 18 months (Comi, Orlic, Redzepovic, Urso, & Iacumin, 2004).

The aroma is a key attribute that impacts the overall acceptance ofdry-cured hams and is due to the presence of volatile compounds,most of them produced by lipolysis and proteolysis (Toldrá, 1998) dur-ing the maturation process (Flores, Grimm, Toldrá, & Spanier, 1997).The aroma is markedly affected by rawmaterial, processing techniques,and ageing time (Pham et al., 2008). Therefore, an understanding of thedry-cured ham aroma should include the identification and quantifica-tion of its volatiles. The flavour and aroma of dry-cured ham can alsobe determined by sensory descriptive analysis and the composition ofaroma impact compounds. Several studies have reported informationon the volatile composition of various kinds of dry-cured hams, whichare very different in their aroma, such as Corsican, Iberian, Serrano

385 1 4605072.

ghts reserved.

and Parma hams (Berdagué, Denoyer, Le Quéré, & Semon, 1991;Bolzoni, Barbieri, & Virgili, 1996; Flores et al., 1997; López et al., 1992;Pastorelli et al., 2003; Timón, Ventanas, Carrapiso, Jurado, & García,2001), but little information is available for Istrian dry-cured ham.

The objective of this research was to determine volatile compoundsin traditional Istrian dry-cured ham using solid phase microextraction(SPME) and gas chromatography–mass spectrometry (GC–MS). Anotherpurpose of this work was to determine the chemical composition andphysico-chemical aspects of Istrian dry-cured ham and their possibleconnection with the formation of volatile compounds. Comparisonof the results of sensory analysis, volatile compounds and physico-chemical parameters was also done.

2. Materials and methods

2.1. Traditional production process

Commercially produced Istrian dry-cured hamswere obtained fromLandrace, Large White and Duroc pigs and their crossbreeds with aminimum live weight at slaughter of 160 kg. It is produced with pelvicbones and without skin and the subcutaneous adipose tissue. Drysalting of hams is conducted using sea salt with an addition of groundblack pepper, garlic and laurel. Quantities of added spices vary betweenmanufacturers. Salting is conducted in cooling chambers at a tempera-ture of 0–5 °C and a relative humidity of 80–90%, for a period of21 days, including the pressing for 7 last days. Prior to drying, allhams are sprinkled with a mixture of herbs. Drying is done in dryingchambers with controlled microclimatic conditions (air circulation—10–20 cm/s; temperature—12–16 °C; humidity gradually reduced

Table 1Codes, sensory attributes and mean intensities of the sensory attributes in Istrian dry-cured ham from 2012.

Code Sensory attributes Istrian dry-cured ham

A1 Red colour 7.2 ± 1.3A2 Marbling 5.1 ± 2.6A3 White crystals 1.1 ± 1.6A4 Positive odour 7.3 ± 1.1A5 Negative odour 2.0 ± 1.6A6 Odour typical for dry-cured ham 7.5 ± 1.1A7 Tenderness 7.0 ± 1.2A8 Melting texture 6.5 ± 1.4A9 Salty taste 6.5 ± 0.8A10 Sweet taste 2.3 ± 2.3A11 Flavour of added spices 4.0 ± 1.4A12 Raw meat flavour 0.7 ± 1.2A13 Number of positive characteristics 6.9 ± 1.1A14 Aroma duration 6.9 ± 1.2

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from90 to 70%) lasting 158 days. Ripening of hams takes place in cellarswith a stable microclimate and the possibility for complete darkness,and an air temperature which does not exceed 18 °C in summer(between 12 and 18 °C year round) and relative air humidity between65 and 75% until they become 12–18 months old (Marušić, Petrović,Vidaček, Petrak, and Medić (2011)).

2.2. Ham sampling

Samples of biceps femoris of traditional Istrian dry-cured hamwere obtained from 11 manufacturers in 2010 and from 8 manufac-turers in 2012. Samples of biceps femoris from each manufacturer wereanalysed for volatile compounds, chemical composition and physicalcharacteristics. Samples of Istrian dry-cured ham from 2012 were alsoevaluated by a sensory panel.

2.3. Chemical composition analysis

Fat, protein and ash contents were estimated according to methodsrecommended by the AOAC (1999). Moisture content and sodium chlo-ride were determined in the biceps femoris according to AOAC methods(AOAC, 1984). Two replicates of each sample were analysed and themean value was used in the data analyses. Water activity of the bicepsfemoris was determined with a precision multi-function measuringinstrument, Testo 650 (Testo Inc., New York, USA). Two replicates ofeach sample were analysed and the mean value was used in the dataanalyses.

2.3.1. Lipid oxidation by the TBARS testOxidation of lipids was assessed by the thiobarbituric acid

(TBA) assay which is based on the reaction between TBA andmalondialdehyde (MDA) and the production of a coloured pigment,the concentration of which is calculated by measuring the absorbanceat 538 nm on three replicates of each sample (Lemon, 1975). The spec-trophotometer was a Helios β (Spectronic Unicam, Cambridge, UK).A calibration curve was developed using 0, 0.01, 0.02, 0.03, 0.04and 0.05 μmol of MDA. TBARS values were expressed as mg ofmalondialdehyde equivalents/kg dry-cured ham.

2.3.2. Colour instrumental measurementColour measurements were carried out with a Minolta CM-3500d

(Osaka, Japan) spectrophotometer in the CIELAB space: lightness (L*),redness (a*) and yellowness (b*) (CIE, Commission Internationale del'Eclairage, 1976). Each sample of biceps femoris was analysed in tenreplicates, avoiding regions with excess fat to achieve representativemeasurements of the lean colour.

2.4. Analysis of volatile compounds

Analyseswere carried out by extraction of volatile compounds abovethe samples on SPME fibre and their qualification and quantification onGC/MS by the method as described by Marušić et al. (2011).

30 g of biceps femorismuscle slices from dry-cured hamwas groundwith a commercial grinder. Then dry-cured ham homogenates wereprepared by dispersing 5 g of minced muscle slices with 25 mL of dis-tilled water saturatedwith NaCl in a commercial blender. Tenmillilitresof this mixture was placed into 20 mL vials tightly capped with a PTFEseptum. Amagnetic stirrer was placed into the homogenates for stirringduring extraction.

A SPME fibre coated with 2 cm of 50/30 μm DVB/Carboxen/PDMS(Supelco, Bellefonte, PA, USA) was conditioned for 2 min at 240 °Cprior to extraction and placed above the sample mixture. Triplicate20 mL vials were placed in a water bath at 40 °C and extracted for180 min with stirring. After extraction the SPME fibre was immediatelyinjected to 6890N gas chromatograph coupled to a 5975i mass selectivedetector (Agilent Technologies, Santa Clara, CA, USA). Capillary column

DB-5ms 30 m × 0.25 mm, film thickness 0.25 μm (Agilent Technolo-gies, Santa Clara, CA, USA) was used with helium as a carrier gas at1.0 mL/min flow rate. The temperature of the injector, used in thesplitless mode, was 230 °C and desorption time was 2 min. Tempera-ture programme was at 40 °C, isothermal for 10 min, then rising to200 °C at a rate of 5 °C/min and then raising to 250 °C at a rate of20 °C/min. Final temperature was held for 5 min. The transfer linetemperaturewasmaintained at 280 °C. Themass spectrawere obtainedat 70 eV with a rate of 1 scan/s over the m/z range of 50–450.

An in-house mixture of C8–C20 n-alkanes was run under the samechromatographic conditions to calculate the retention indices (RI) ofdetected compounds. AMDIS 3.2 program version 2.62 was used foridentification of components using NIST 2005 version 2.0 spectral li-brary (NIST, Gaithersburg, MD, USA) as well as comparison of obtainedretention indices with literature values (Adams, 2001 and in-houselibrary).

2.5. Sensory analysis

Istrian dry-cured hams from eight producers from 2012 wereassessed by seven trained panellists who were selected and trainedin accordance with international standard (ISO 8586:2012). Panelmembers were situated in a private red lighted cabinet during sessions.

Samples were individually labelled and were randomly served oneat a time. All hams were evaluated in slices from the same anatomicalarea. In each sensory session, panellists evaluated 2 samples and thesensory evaluation consisted of eight sessions (each sample wasevaluated two times). Panellists were asked to indicate point of thescale corresponding to the intensity of their different feelings for eachattribute. Sensory attributes were assessed with a 10 point intensityline scale, where 0 = not detected and 9 = extremely strong. All thesamples, slices of 1.5 mm thickness, were evaluated at 20–22 °C insensory panel rooms. About 50 mL of water and 20 g of unsaltedbread were provided to assessors between successive ham samples(García-González et al., 2006).

Fourteen traits related to sensory characteristics of dry-cured hams(Table 1) were evaluated by the quantitative-descriptive analysismeth-od. The traits were grouped into appearance (red colour, marbling, andwhite crystals), odour (positive odour, negative odour, odour typical fordry-cured ham), texture (tenderness, melting texture), taste (salty andsweet taste), flavour (flavour of added spices, raw meat flavour) andacceptability (number of positive characteristics, aroma duration).

2.6. Statistical analyses

One-way ANOVA was carried out for physical and chemical datausing the SPSS 12.0 computer programme. Statistical significance wasset at P b 0.05. Student's t-test was used to determine whether there

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were differences in volatile compounds in Istrian dry-cured ham from2010 and 2012. The results were submitted to principal componentanalysis (PCA) in order to interpret sensory attributes, volatilecompounds and physical–chemical parameters in Istrian dry-curedham. PCA analyses were performed using STATISTICA software 10.0.

3. Results and discussion

3.1. Physical and chemical analyses

Results of physical and chemical analyses of Istrian dry-cured hamfrom 2010 and 2012 aswell as Spanish (Iberian and Serrano) and Italian(Parma and San Daniele) dry-cured hams are shown in Table 2. Thewater content in the Istrian dry-cured ham was 37.9 g/100 g (2012)and 41.0 g/100 g (2010) which is with the range of values reported byother authors (37.9 g–45.0 g/100 g) (Karolyi, 2006; Krvavica et al.,2008; Marušić et al., 2011). According to the specification of origin ofIstrian dry-cured ham water content must be less than 55% and theseresults are in accordance with this specification. Istrian dry-cured hamhas lower water content than the other Mediterranean dry-curedhams because it is produced without pig's skin and subcutaneousadipose tissue. Unlike Istrian ham, hams producedwith skin and subcu-taneous fat contain higher water content like Spanish Iberian andSerrano and Italian Parma and San Daniele dry-cured hams (Table 2).

Fat content in biceps femoris of Istrian dry-cured ham ranged from13.5 to 17.0 g/100 g which is similar to the content of fat in Iberianand Parma hams while San Danielle dry-cured ham has higher fatcontent (Table 2). Fat content is believed to be one of the most crucialquality traits of cured hams (the higher the fat content, the greater theacceptability of dry-cured hams) (Jiménez-Colmenero, Ventanas, &Toldrá, 2010).

Protein content in dry-cured ham is about 30 g/100 g depending onthe extent of drying and the fat content (Toldrá, 2002). In Istrian dry-cured ham protein content is relatively high (32.4–43.1 g/100 g). Ashcontent in Istrian dry-cured ham was from 6.66 to 7.23 g/100 g arisingfrom the high salt content.

Istrian ham had a water activity from 0.80 to 0.89. Similar valueswere found for Spanish dry-cured hams while Italian had higher awvalues (Table 2).

Trend of decreasing salt content in Istrian dry-cured ham can beobserved in samples of the ham in 2012 (6.3%) in contrast to 2010(7.4%). The salt content in the Istrian dry-cured ham frommuch earlieryear determined by Marušić et al.'s (2011) paper was 9%, which ishigher compared to the results in this study. These results are inline with the trend of reductions of salt in meat products, as well as indry-cured ham, because a high proportion of NaCl is associated withcardiovascular diseases. Salt content in other types of dry-cured hamcan be seen in Table 2.

The results of colour parameters for Istrian dry-cured ham (L* a* b*values) are shown in Table 2. L* value was from 31.6 to 34.7. L* value

Table 2Physico-chemical parameters in the biceps femoris muscle in different types of dry-cured hams

Water(g/100 g)

Protein(g/100 g)

Fat(g/100 g)

Ash(g/100 g)

% NaCl mgmalondisample

Istrian 2010 41.0 ± 4.1a 32.4 ± 3.1a 13.5 ± 3.1a 7.2 ± 1.0a 7.4 ± 1.2a 0.7 ± 0Istrian 2012 37.9 ± 4.3a 43.1 ± 5.8b 17.0 ± 5.4a 6.7 ± 1.6a 6.3 ± 1.3a 0.4 ± 0Iberian 49.0 17.9–30.6 19.2 – 4.0–5.9 0.4–0.5Serrano 48.5 27.9–30.6 12.0 – 5.0–6.0 –

Parma 54.1–61.8 27.3–30.8 18.4 7.72 4.5–6.9 0.3–0.5San Daniele 54.7–60.4 27.3–30.8 23.0 – 4.5–6.9 –

⁎ 1—Experimental values-mean ± standard deviation in Istrian dry-cured ham from 2010 anCarrapiso&García, 2008; 3—Toldrá, Flores, Navarro, Aristoy, and Flores (1997); 4—Baldini et al. (Luque de Castro (2004); 7—Pugliese et al. (2006); 8—Jiménez-Colmenero et al. (2010); 9—GAntequera, Timón, and Ventanas (1998); 13—Hersleth, Lengard, Verbeke, Guerrero, and Naes (2(1999); 16—Pérez-Alvarez et al. (1999); 17—Pérez-Alvarez et al. (1998).

is associated with a thin layer on the surface of the muscle (Hunt,1980). L* values suggest that lightness in these muscles depends onthe water content (moisture) and water movement (dehydration) to-wards the surface. a* values were 7.6 and 9.7 and b* values were 6.7and 6.1. From the results shown in Table 2 it can be concluded thatIstrian ham had similar value for L* but smaller a* value than Spanishand Italian dry-cured hams. Spanish dry-cured ham had intense red col-our (higher a* values) than Istrian dry-cured ham. That is because inproduction of Spanish dry-cured ham addition of nitrates and nitritesis allowed in contrast to Istrian ham where it is not allowed.

The TBARS values were 0.7 and 0.4 mg MDA/kg sample whichwere in accordance with the values by Marušić et al. (2011). Resultsare comparable with the results of Iberian dry-cured ham (Table 2) inbiceps femoris at the end of the production process.

3.2. Analysis of volatile compounds

Ninety-two volatile aroma compounds of Istrian dry-cured hamwere found by gas chromatographic–mass spectrometry (GC–MS). Re-sults of the analysis are shown in Table 3. Volatile compounds belongedto several classes of chemicals: 27 terpenes, 23 aldehydes, 14 alcohols,10 ketones, 6 alkanes, 4 esters, 6 aromatic hydrocarbons and 2 acids.Chemical groups identified in Istrian dry-cured ham from 2010 and2012 were: aldehydes (51.4; 51.3%), terpenes (16.5; 16.4%), alcohols(15.5; 13.2%), ketones (8.6; 7.4%), alkanes (3.8; 5.7%), esters (1.3;1.6%), aromatic hydrocarbons (0.8; 3.9%) and acids (0.6; 0.9%). Alde-hydes, terpenes and alcohols were the major groups of compounds inthe Istrian dry-cured ham.

Proteolytic and lipolytic enzymes play an important role in theformation of volatile compounds. Most of the volatile compounds arethe result of chemical or enzymatic oxidation of unsaturated fattyacids and further interaction with proteins, peptides and free aminoacids. Also volatile compounds are formed from Strecker degradationof free amino acids (Toldrá, 1998).

Aldehydes were the most abundant group of compounds (51.4;51.3%) in Istrian dry-cured ham from 2010 and 2012, respectively.Concentration of aldehydes in this study was higher than reported byMarušić et al. (2011) (15.7% to 41.5%) for Istrian dry-cured ham. Thereason for this may be that in research by Marušić et al. (2011) saltcontent was higher (9%) and it is possible that the salt has partiallyinhibited the proteolytic and lipolytic enzymes and volatile compoundsderived from lipolysis and proteolysis (aldehydes, ketones, alkanes,alcohols etc.) were present in smaller proportions than in this study.Amount of aldehydes in San Daniele dry-cured ham was 31.5%(Gasparado, Procida, Toso, & Stefanon, 2008) which is similar value ofother types of European dry-cured ham (Berdagué et al., 1991; Garcíaet al., 1991; Ruiz, Ventanas, Cava, Andres, & Garcia, 1999). Hexanal,which is derived from oxidation of n-6 fatty acids like linoleic and ara-chidonic acids, was one of the most abundant compounds (6.4; 8.4%).Hexanal is described in the literature as the major oxidation product

.

aldehyde/kgWateractivity, aw

L⁎ a⁎ b⁎ Literature

.2a 0.80 ± 0.0a 31.6 ± 1.9a 7.6 ± 0.5a 6.7 ± 1.2a 1

.2b 0.89 ± 0.0a 34.7 ± 2.8b 9.7 ± 1.1b 6.1 ± 1.4a 10.85 38.8 18.9 7.6 2, 6, 8, 12, 14, 160.85 34.8 15.6 10.5 3, 6, 9, 13, 16, 170.94 37.9–38.0 17.7–15.9 5.9–6.1 4, 5, 7, 10, 11, 150.93 37.9–38.0 17.7–15.9 5.9–6.1 4, 5, 7, 10

d 2012 year. Different letters (a, b) indicate statistical significant difference (P b 0.05); 2—1992); 5—Laureati et al. (2014); 6—García-Rey, García-Garrido, Quiles-Zafra, Tapiador, andilles (2009); 10—D'Evoli et al. (2009); 11—Musella et al. (2009); 12—Martín, Córdoba,011); 14—Andrés, Cava, Ventanas, Thovar, and Ruiz (2004); 15—Vestergaard and Parolari

Table 3Contents of volatile compounds extracted in Istrian dry-cured ham from 2010 and 2012 (percentage of the total area).

Volatile compound RI 2010 2012 Identification P-value

Mean SD Mean SD

Aldehydes3-Methylbutanal 700 0.6 0.6 – – MS, RI 0.014Hexanal 803 6.4 3.8 8.4 3.5 MS, RI 0.181Heptanal 904 2.4 1.4 3.2 2.0 MS, RI 0.622Benzaldehyde 967 9.4 2.6 6.5 1.8 MS, RI 0.0042,4-Heptadienal 997 0.7 0.5 1.8 2.5 MS, RI 0.151Octanal 1004 6.1 2.8 3.6 2.3 MS, RI 0.663Benzeneacetaldehyde 1045 – – 7.6 10.1 MS, RI 0.009Phenylacetaldehyde 1049 7.4 13.7 1.3 0.7 MS, RI 0.2172-Octenal 1065 0.7 0.2 1.6 1.2 MS, RI 0.000Unknown aldehyde 1071 1.0 0.4 0.8 0.2 MS, RI 0.002Nonanal 1106 5.5 1.7 6.4 4.4 MS, RI 0.1152-Nonenal 1162 1.1 0.8 – – MS, RI 0.187Decanal 1205 0.5 0.4 0.4 0.3 MS, RI 0.9442.4-Nonadienal 1215 – – 0.3 0.1 MS, RI 0.0002E-Decanal 1264 2.1 1.1 2.8 1.7 MS, RI 0.1052.4-Decadienal 1294 0.5 0.4 0.6 0.5 MS, RI 0.0042-Undecanal 1365 1.2 0.7 1.5 1.3 MS, RI 0.025Tetradecanal 1612 0.3 0.1 0.2 0.1 MS, RI 0.398Pentadecanal 1714 1.2 3.0 0.2 0.1 MS, RI 0.378Hexadecanal 1818 3.5 4.0 3.6 2.0 MS, RI 0.514Unknown aldehyde 1823 0.2 0.1 – – MS, RI 0.002Heptadecanal 1922 0.1 0.1 0.1 0.1 MS, RI 0.361Octadecanal 2014 0.5 0.3 0.3 0.2 MS, RI 0.288

Total 51.4 Total 51.3

Terpenesp-Xylene 871 0.7 1.0 0.1 0.2 MS, RI 0.273α-Thujene 934 0.1 0.1 – – MS, RI 0.000α-Pinene 943 2.4 1.7 – – MS, RI 0.001Sabinene 979 1.0 0.5 – – MS, RI 0.000β-Pinene 983 0.7 0.2 1.0 0.4 MS, RI 0.034β-Myrcene 995 0.3 0.3 3.9 1.6 MS, RI 0.000α-Terpinene 1016 – – 0.5 0.3 MS, RI 0.000o-Cymene 1025 0.3 0.2 – – MS, RI 0.000p-Cymene 1028 0.9 1.0 1.0 2.4 MS, RI 0.045D-Limonene 1033 1.6 0.9 1.4 0.6 MS, RI 0.934Terpinolene 1089 0.3 0.4 1.2 0.7 MS, RI 0.048Linalool 1100 1.8 1.1 1.7 1.1 MS, RI 0.000Terpinen-4-ol 1181 2.1 1.5 1.4 0.9 MS, RI 0.191p-Cymen-8-ol 1186 – – 0.4 0.1 MS, RI 0.000β-Fenchyl alcohol 1192 – – 0.3 0.2 MS, RI 0.004Piperidine 1194 0.5 0.4 0.8 0.4 MS, RI 0.045Piperonal 1334 0.4 0.2 0.2 0.1 MS, RI 0.002α-Copaene 1376 0.3 0.1 0.1 0.1 MS, RI 0.031β-Elemene 1392 0.1 0.1 – – MS, RI 0.026Italicene 1394 0.4 0.4 0.6 0.0 MS, RI 0.082β-Caryophyllene 1421 1.0 0.7 0.3 0.2 MS, RI 0.078α-Caryophyllene 1458 0.2 0.1 0.7 1.8 MS, RI 0.011β-Selinene 1490 0.3 0.2 0.1 0.0 MS, RI 0.062α-Selinene 1497 0.3 0.4 – – MS, RI 0.087β-Bisabolene 1510 0.2 0.2 0.2 0.2 MS, RI 0.099D-Cadiene 1523 0.5 0.3 0.6 1.0 MS, RI 0.853Unknown sesq alcohol 1648 0.2 0.2 0.1 0.1 MS, RI 0.398

Total 16.5 Total 16.4

Alcohols1-Pentanol 774 0.6 0.3 0.6 0.4 MS, RI 0.8462-Pentanol 852 0.7 0.9 0.1 0.2 MS, RI 0.1221-Hexanol 875 0.2 0.3 0.7 1.3 MS, RI 0.7852-Heptanol 883 0.6 0.6 – – MS, RI 0.042Unknown alcohol 957 0.3 0.3 – – MS, RI 0.0001-Octen-3-ol 986 6.9 2.7 6.1 2.0 MS, RI 0.3272-Ethyl-1-hexanol 1034 – – 1.5 0.9 MS, RI 0.000Benzyl alcohol 1037 1.3 0.4 0.9 0.5 MS, RI 0.002Octanol 1075 1.8 1.2 1.6 0.6 MS, RI 0.858Phenylethyl alcohol 1112 2.3 1.4 1.0 1.0 MS, RI 0.023Undecan-1-ol 1164 0.2 0.2 0.3 0.1 MS, RI 0.328Dodecanol 1254 0.2 0.1 0.3 0.3 MS, RI 0.709Tetradecanol 1483 0.1 0.1 0.0 0.0 MS, RI 0.001Octadecanol 1996 0.3 0.1 0.2 0.1 MS, RI 0.922

Total 15.5 Total 13.2

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Table 3 (continued)

Volatile compound RI 2010 2012 Identification P-value

Mean SD Mean SD

Ketones2-Heptanone 892 0.6 0.4 0.7 0.4 MS, RI 0.5111-Butoxy-2-propanone 950 0.4 0.3 0.1 0.2 MS, RI 0.0001-Octen-3-one 983 – – 1.1 1.4 MS, RI 0.0003-Octanone 989 0.8 0.5 0.1 0.3 MS, RI 0.0012.3-Octanedione 991 0.8 0.6 1.0 0.5 MS, RI 0.1382-Nonenone 1062 5.0 2.4 0.9 1.0 MS, RI 0.000Acetophenone 1066 – – 1.4 2.4 MS, RI 0.0002-Nonanone 1092 0.8 0.4 1.2 1.3 MS, RI 0.333g-Nonalactone 1362 0.3 0.3 0.9 0.9 MS, RI 0.2005,9-Undecadien-2-one 1447 – – 0.2 0.0 MS, RI 0.000

Total 8.6 Total 7.4

AlkanesHeptane 720 1.2 0.6 4.1 0.7 MS, RI 0.000Dodecane 1200 0.6 0.2 0.4 0.2 MS, RI 0.114Tridecane 1300 0.3 0.2 0.2 0.1 MS, RI 0.207Tetradecane 1400 0.6 0.3 0.3 0.2 MS, RI 0.013Pentadecane 1500 0.8 0.4 0.6 0.3 MS, RI 0.207Hexadecane 1600 0.3 0.2 0.1 0.1 MS, RI 0.025

Total 3.8 Total 5.7

EstersOctanoic acid. ethyl ester 1196 – – 0.6 0.4 MS, RI 0.000Nonanyl acetate 1314 0.3 0.2 0.5 0.6 MS, RI 0.407Hexyl-hexanoate 1347 1.0 0.6 0.1 0.0 MS, RI 0.002Pentanedioic acid. dimethyl ester 1575 – – 0.3 0.2 MS, RI 0.000

Total 1.3 Total 1.6

Aromatic hydrocarbonsMethoxy-phenyl oxime 916 – – 1.2 1.0 MS, RI 0.007Trimethyl-pirazine 999 – – 1.5 2.9 MS, RI 0.0062,4-Dimethoxytoluene 1237 0.6 0.5 0.6 0.3 MS, RI 0.0045-Penthyl-2(3H) furanone 1337 – – 0.2 0.2 MS, RI 0.000Eugenol 1351 0.2 0.1 0.2 0.1 MS, RI 0.623Cyclohexane 1389 0.1 0.1 0.1 0.1 MS, RI 0.146

Total 0.8 Total 3.9

Acidsn-Decanoic acid 1178 0.6 0.3 0.7 0.2 MS, RI 0.003Butanedioic acid 1476 – – 0.2 0.1 MS, RI 0.000

Total 0.6 Total 0.9

Compounds stated in bold are significant compounds within class.

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in other dry-cured meat products (Ramirez & Cava, 2007). The mostpotent odourants found in Iberian ham were hexanal (green), (Z)-3-hexenal (acornlike), 3-methylbutanal (malty, nutty, toasted), 1-octen-3-one (mushroom), 1-octen-3-ol (mushroom, rustlike), hydrogen sulfide(boiled or rotten eggs, sewagelike), methanethiol (rotten eggs or meat,sewagelike) and 2-methyl-3-furanthiol (nutty, dry-cured hamlike,toasted) (Carrapiso & Garcia, 2004). 3-Methylbutanal, 1-octen-3-oneand 1-octen-3-ol were also found in Istrian dry-cured ham.

Nonanal contributes to flavour with sweet and fruity aroma (Nunes,Coimbra, Saraiva, & Rocha, 2008). In Istrian dry-cured ham nonanalwas present in an amount of 5.5%; 6.4%. Aldehydes have a low odourthreshold value and present in small amounts contribute significantlyto theflavour of dry-curedham.Amongother aldehydes present in higheramounts in Istrian dry-cured hamwere: phenylacetaldehyde (7.4%; 1.3%)and octanal (6.1%; 3.6%). Benzaldehyde found in Istrian dry-cured hamoriginates from laurel (Maars & Visscher, 1989), which originates fromspices added in the salting phase of the production process.

In Istrian dry-cured ham straight-chain aldehydes were the mostabundant chemical class. Specifically, n-aldehydes occur mostly in theproduction stage between 7 and 12 monthswhile their share decreasesafter 12 months and increases the proportion of methyl branchedaldehydes (Gasparado et al., 2008). Branched aldehydes are formed byoxidative deamination via Strecker-degradation (Sabio, Vidal-Aragon,Bernalte, & Gata, 1998). Branched aldehydes present in the Istriandry-cured ham were 3-methylbutanal (0.6%) and 2, 4-heptadienal

(0.7%; 1.8%). Branched aldehydes such as 2-methylbutanal and 3-methylbutanal are the largest contributor to the flavour of Italiandry-cured hams (Careri et al., 1993).

Aldehydes like hexanal, heptanal; 2,4-heptadienal, phenylacetal-dehyde, nonanal, 2-nonenal, decanal, 2E-decanal, tetradecanal,pentadecanal, hexadecanal, heptadecanal and octadecanal are typicalfor Istrian dry-cured ham (P-value N 0.05).

Alcohols represented 13–15% of the total area of the identified com-pounds. The most abundant alcohols were: 1-octene-3-ol (6.9%; 6.1%),phenylethyl alcohol (2.3%; 1.0%), octanol (1.8%; 1.6%) and benzylalcohol (1.3%; 1.0%). Alcohols have a higher threshold value than alde-hydes and their impact on the flavour is small, exceptions are alcoholssuch as 1-octene-3-ol and 1-penten-3-ol, which have a low thresholdvalue. 1-Octene-3-ol is present in Istrian dry-cured ham in high propor-tions (6.9%; 6.1%).

Unsaturated alcohols (1-octene-3-ol, 1-penten-3-ol) and pentanolare the most abundant compounds in Corsican dry-cured hams whilethe branched alcohols (2-methylpropanol, 2- and 3-methylbutanol)and ethanol in French Bayonne and Spanish Serrano dry-cured hamsItalian San Daniele dry-cured ham is characterized with ethanol,isobutanol, 1-propanol and 1-penten-3-ol (Gasparado et al., 2008).Generally, a higher content of alcohols occurs if there is a higher degreeof lipid oxidation, which can be confirmedwith high content of hexanal.

Content of linear and branched alcohol increases with the lengthof ageing (Bolzoni et al., 1996; Martín, Córdoba, Benito, Aranda, &

A1

A2 A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13A14

aldehydes

alcoholsketones

alkanes

terpenes

esteres

acids

aromatic hydrcarbones

NaCl

water

fat

proteinashL*

a*b*

aw

TBARS

-1 -1 0 1 1

Factor 1 : 30.28%

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

Fac

tor

2 : 2

0.84

% A1

A2 A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13A14

aldehydes

alcoholsketones

alkanes

terpenes

esteres

acids

aromatic hydrcarbones

NaCl

water

fat

proteinashL*

a*b*

aw

TBARS

Fig. 1. Plot of principal component analysis of the sensory attributes, volatile compoundsand physical–chemical parameters of Istrian dry-cured ham from 2012. Note: Codes inTable 1.

1414 N. Marušić et al. / Meat Science 96 (2014) 1409–1416

Asensio, 2003) and a high proportion of linear alcohols can be identifiedwith the length of ageing. In Istrian dry-cured ham branched-chainalcohols that can derive from microbial activity were not found inlarge quantities, most likely due to the antimicrobial effect of NaCl.

Based on results of this study and results from literature it couldbe concluded that alcohols like 2-methylpropanol, butanol, and3-methyl-1-butanol characterize Iberian dry-cured ham (García-González, Aparicio, & Aparicio-Ruiz, 2013) while 2-pentanol, undecan-1-ol, dodecanol and octadecanol characterize Istrian dry-cured ham.1-Hexanol, aswell as 1-octen-3-ol and octanol are found both in Iberianand Istrian dry-cured hams. The low odour threshold of 1-octen-3-olindicates that it contributes with a strong mushroom aroma to almostall the hams as well as Istrian.

The most abundant ketone in Istrian dry-cured hamwas 2-nonenone(5.0%; 0.9%), followed by: 3-octanone (0.8%; 0.1%), 2,3-octanedione(0.8%; 1.0%), 2-nonanone (0.8%; 1.2%) and 2-heptanone (0.6%; 0.7%).2-Propanone was the most abundant ketone in French and Spanishdry-cured hams. Content of 2-propanone was 35.7 to 42.9% of totalketones in Spanish and from 47.9 to 59.0% in the French dry-curedham (Sánchez-Peña, Luna, García-González, & Aparicio, 2005). Theamount of 2-propanone is however dependent on the raw materialand the process of production (Flores et al., 2006). 3-Octanone, ketonein Istrian dry-cured ham arrives from rosemary added in the saltingphase of the production process (Maars & Visscher, 1989). Methylketones such as 2-propanone, 2-butanone, 2-heptanone, 2-octanoneand 2-nonanone are responsible for the aroma of blue cheeses andhave an intense odour. Methyl ketones are formed by chemicalreactions in the presence of a large number of microorganisms, buthigh concentration of ketones is a symptom of bad quality of dry-cured ham (Pastorelli et al., 2003). In dry-cured ham number of micro-organisms in relatively low, so it is assumed that these compounds canbe formed by other chemical reactions (Sabio et al., 1998). Two ketones(2-heptanone and 2-nonanone) present also in the Istrian dry-curedham contribute to blue cheese aroma (Creuly, Laroche, & Gros, 1992).Iberian dry-cured ham is characterized with 2-butanone, octen-3-oneand 2-octanone (García-González et al., 2013) while Istrian with 2,3-octadienone and γ-nonalactone. Esters are formed by esterification ofcarboxylic acids and alcohols (Sabio et al., 1998). Esters are describedin dry-cured hams at the end of the maturation process and it seemsthat NaCl concentration affects the ester production through the activa-tion of esterases (Armenteros, Toldrá, Aristoy, Ventanas, & Estevez,2012). Esters were found in low amounts (1.32%; 1.56%). The lowportion of esters is probably related to the antimicrobial activity of sodi-um chloride to the long curing period (Gasparado et al., 2008). Thesecompounds have fruity notes, mainly those formed from short-chainacids. Esters formed from long-chain acids have fat odour. Ester,nonanyl acetate found in Istrian dry-cured ham can be one compoundon which is possible distinguishing Istrian from Iberian dry-cured ham.

Terpenes are generally associated with the addition of spices, inparticular pepper (Hinrichsen & Pedersen, 1995) and some of themhave been found in meat as a consequence of their presence in animalfeedstuffs (Ansorena, Gimeno, Astiasaran, & Bello, 2001). Terpenes inIstrian dry-cured ham are the second group of compounds by the con-tent of total volatile compounds (16.46%; 16.37%). Terpenes with highconcentration were: α-pinene (2.4%), β-pinene (0.7%; 1.0%), sabiene(1.0%), d-limonene (1.6%; 1.4%), linalool (1.8%; 0.3%), β-caryophyllene(1.0%; 0.3%) and p-cymene (0.9%; 1.0%). Other terpenes were found inlower concentrations. Some terpenes were found in Bayonne andCorsican hams, due to the black pepper treatment on the surfaceof the ham during processing, because these compounds constitute90% of pepper essential oil (Sabio et al., 1998). Terpenes such as α-terpinene, terpinolene, limonene, α- and β-pinene, α-thujene,sabinene, α- and β-selinene, β-caryophyllene, α-copaene and linaloolare derived from the added pepper, laurel and rosemary. Terpenic alco-hol such as p-cymene-8-ol andβ-bisabolene is derived frompepper androsemary, while β-frenchyl alcohol is derived from added rosemary.

Terpenes found in the Istrian dry-cured ham like β-myrcene, β-elemene and piperidine are derived from the added pepper and laurel(Maars & Visscher, 1989).

Phenols are not only the most classical smoke components butcan also originate from the added spices like eugenol (4-allyl-2-metoxyphenol) found in this research that originates from the addedspices (pepper, bay leaves and rosemary) (Maars & Visscher, 1989).

Volatile compounds provide valuable information about the odourand sensory quality of dry-cured hams. The aroma perception in meatproducts depends on the concentration and odour threshold of volatilecompounds and on their interactions with other food components thatwill affect its gas phase concentration (Guichard, 2002). Compoundswhich can be regarded as contributors to the aroma are those whichare present in food at concentrations higher than the associated odourthresholds. Particular attention is also given to those compounds thatprovide the characteristic aroma of food and are known as the keyodourants (Belitz, Grosch, & Schieberle, 2009).

Based on the results of this study it can be concluded that dominantcompounds in Istrian dry-cured ham are: aldehydes hexanal, heptanal;2,4-heptadienal, phenylacetaldehyde, nonanal, 2-nonenal, decanal, 2E-decanal, tetradecanal, pentadecanal, hexadecanal, heptadecanal andoctadecanal; alcohols: 2-pentanol, undecan-1-ol, dodecanol andoctadecanol; ketones 2,3-octadienone and g-nonalactone; esternonanyl acetate and terpenes: p-xylene, d-limonene, terminen-4-ol,italicene, β-caryophyllene, β-selinene, α-selinene, β-bisabolene andd-cadiene.

3.3. Relationship between chemical and sensory parameters and volatilecompounds

Table 1 shows the sensory attributes evaluated by the assessors andtheir mean intensities evaluating Istrian dry-cured ham from 2012.

In order to analyse the whole sensory assessment and to determinethe relationship between chemical and sensory parameters and volatilecompounds, the multivariate statistical procedure of principal compo-nent analysis (PCA) was performed using data obtained from thechemical and sensory characterisation of muscle biceps femoris fromdry-cured hams. Results are shown in Fig. 1.

The two principal components account for 30.28% and 20.84% of thevariance, respectively, (51.12% in total). Fat content, tenderness and

1415N. Marušić et al. / Meat Science 96 (2014) 1409–1416

melting texture were positively correlated, which means that alarger proportion of fat affects the tenderness and melting texture ofdry-cured ham. Terpenes were strongly correlated with flavour ofadded spices. Terpenes constitute a significant proportion of fractionsof vegetable oils. Spices like pepper, bay leaves and rosemary areadded in salting phase of the production of Istrian dry-cured ham andterpenes found in the aroma analysis of Istrian dry-cured ham originatefrom these spices. Sweet taste and the presence of esterswere positivelycorrelated. Esters are formed by esterification of carboxylic acids and al-cohols and have fruity notes especially those that arise from short chainacids (higher concentration of esters, more pronounced sweetness ofthe products) (Sabio et al., 1998) and were identified in this study.Red colour of dry-cured hamwaspositively correlatedwith a* (redness)value. Salt content (and ash) was positively correlated with the saltytaste. Negative odour was positively correlated with the raw meatflavour. Negative odour, raw meat flavour and water content werepositively correlated. In dry-cured hams with higher water content(younger ham, shorter ripening process) raw meat flavour was morepronounced. Sweet taste and salty taste were negatively correlated.

4. Conclusions

92 volatile aroma compounds of Istrian dry-cured ham werefound by gas chromatographic–mass spectrometry (GC–MS). Volatilecompounds belonged to several chemical groups: aldehydes (51.4;51.3%), terpenes (16.5; 16.4%), alcohols (15.5; 13.2%), ketones (8.6;7.4%), alkanes (3.8; 5.7%), esters (1.3; 1.6%), aromatic hydrocarbons(0.8; 3.9%) and acids (0.6; 0.9%). Aldehydes, terpenes and alcoholswere the major groups of compounds in the Istrian dry-cured ham.These compounds originate from lipolysis and proteolysis and play acrucial role in the formation of aroma of dry-cured ham. In addition tovolatile compounds derived from lipolysis and proteolysis terpenesthat originate from spices added in the salting phase of the productionprocess were also found in large quantities in Istrian dry-cured ham.

Principal component analysis (PCA) showed that fat content, tender-ness andmelting texturewere positively correlated,whichmeans that alarger proportion of fat affects the tenderness and melting texture ofdry-cured ham. Terpenes were strongly correlated with flavour ofadded spices. Spices like pepper, bay leaves and rosemary are addedin salting phase of the production of Istrian dry-cured ham and terpenesfound in the aroma analysis of Istrian dry-cured ham originate fromthese spices. Sweet taste and the presence of esters were positivelycorrelated. Negative odour was positively correlated with the rawmeat flavour. Negative odour, raw meat flavour and water contentwere positively correlated. In dry-cured hams with higher watercontent (younger ham, shorter ripening process) raw meat flavourwas more pronounced.

Acknowledgement

The authors would like to thank the “Producers association of theIstrian dry cured ham” and the International fair of dry cured hams(www.isap.hr) for the samples of Istrian dry-cured ham.

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