Volatile and Amino Acid Profiling of Dry Cured Hams from Different Swine Breeds and Processing Methods

Molecules 2013, 18, 3927-3947; doi:10.3390/molecules18043927 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Volatile and Amino Acid Profiling of Dry Cured Hams from Different Swine Breeds and Processing Methods Diego L. García-González *, Ramón Aparicio and Ramón Aparicio-Ruiz Instituto de la Grasa (CSIC), Padre García Tejero, 4, Sevilla E-41012, Spain; E-Mails: [email protected] (R.A.); [email protected] (R.A.-R.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +34-954-611-550; Fax: +34-954-616-790. Received: 27 February 2013; in revised form: 28 March 2013 / Accepted: 28 March 2013 / Published: 3 April 2013 Abstract: The flavor of dry cured ham explains the high appreciation of this product and it determines consumer acceptance. Volatile compounds provide valuable information about the odor and sensory quality of dry cured hams. Since amino acids are the origin of some volatile compounds of dry cured ham, the volatile and amino acid compositions of forty-one dry cured hams from Spain and France were determined to establish associations between them. The samples included different pig breeds (non Iberian vs. Iberian), which were additionally affected by different maturation times and feeding types (acorn vs. fodder). Results showed that 20 volatile compounds were able to distinguish Iberian and non Iberian hams, and 16 of those had relevant sensory impact according to their odor activity values. 3-Methylbutanol, 2-heptanol and hexanal were among the most concentrated volatile compounds. In the case of non-volatile compounds, the concentrations of amino acids were generally higher in Iberian hams, and all the amino acids were able to distinguish Iberian from non Iberian hams with the exception of tryptophan and asparagine. A strong correlation of some amino acids with volatile compounds was found in the particular case of alcohols and aldehydes when only Iberian hams were considered. The high correlation values found in some cases proved that proteolysis plays an important role in aroma generation. Keywords: dry-cured ham; volatile compounds; amino acids; aroma; HS-GCMS; HPLC OPEN ACCESS Molecules 2013, 18 3928 1. Introduction The unique flavor of dry cured ham is the result of a long manufacturing process that produces changes in its aroma and taste. The dry cured ham aroma is markedly affected by the raw material and several parameters during the production process. Raw hams undergo several stages, such as salting with dry salt, washing, post-salting for salt equalization and ripening-drying. In the course of this long process, over 24 months in some cases, the temperature and humidity are controlled to reduce the risk of bacterial spoilage [1]. However, the final products obtained by this process are heterogeneous and there are many kinds of hams whose overall quality depends on diverse factors such as pig breed, age and feeding. The heterogeneity also occurs within a single ham sample since locations in the ham (i.e., muscles and fat) are characterized by diverse concentrations of flavor compounds. The aroma of dry-cured hams is due to the presence of many volatile compounds, most of them produced by chemical and enzymatic mechanisms during the post-mortem process [2]. Lipolysis and proteolysis are the main biochemical reactions involved in the generation of these compounds, producing a wide range of volatiles and precursors [3]. Therefore, a better understanding of dry-cured ham aroma should include the identification and quantification of the volatile compounds present in the samples. Several studies have reported information on the volatile composition of Iberian [4], Parma [5], French [6] and Spanish Serrano hams [2]. Some of the published papers have pointed out the variability of the quantified volatiles, which can be due to the fact that the hams are not homogeneous products and the muscles and subcutaneous fat of the samples may differ in their composition. Furthermore, it has been established that chemical changes occurring in different muscles during ripening influence the ham flavor [7]. Nevertheless, the contribution of the main volatiles of each muscle to the ham aroma is scarcely unknown [8]. The study of the aroma, and the volatile compounds of dry cured hams, implies the examination of the factors that leads to characteristic sensory notes. Pig feeding and breeding are among the most remarkable factors affecting dry cured ham aroma [9]. Large farm pigs are usually fed with fodder (e.g., mixture of maize with other cereals), while higher quality is achieved if they are fed with acorns instead. Pig breed is also an outstanding characteristic that determine price and quality. Usually the pigs that are fed with acorns are also from the particular Iberian breed. Therefore, the feeding and breed types are two factors that are usually studied together (Iberian+acorn feeding vs. non Iberian+fodder feeding). This work displays the results of a thorough study of the volatiles produced by each one of the four well-known ham locations (biceps femoris, semimembranosus and semitendinosus muscles, and subcutaneous fat) in hams corresponding to different feeding and breed types. Amino acids, on the other hand, are not only responsible for texture, and partially for taste, of hams, but also they are at the origin of some volatile compounds determined in the muscles [10,11]. For that reason, amino acids, together with creatine and creatinine, have been determined in dry cured hams to find their relationship with the occurrence of some volatile compounds. Although some previous studies have addressed the simultaneous analyses of volatiles and amino acids of dry cured hams, they are based on few samples and/or centered in a particular breed/feeding type [12,13]. The objectives of this work were: (i) the evaluation of the amount of each volatile produced at each ham location, (ii) the understanding of the potential contribution of the volatiles released in these locations to dry cured-ham aroma, and Molecules 2013, 18 3929 (iii) the relationship between the concentration of amino acids and volatiles quantified in the 4 ham locations. The work was carried out with Spanish (Iberian and non Iberian breeds) and French (non Iberian breeds) hams. The characterization of the samples was carried out with the assistance of statistical procedures taking into account only a classification criterion: Iberian vs. non Iberian hams. Information from odor threshold and GC-sniffing/olfactometry (henceforth, GC-O) was taken into consideration for the chemical interpretation of dry cured ham aroma. 2. Results and Discussion The high heterogeneity of ham samples is undoubtedly the main hurdle towards a representative aroma analysis. In order to obtain a representative sample from the ham pieces, 350 g of the part located along and behind the femur was collected from each one of the above described French and Spanish hams. The samples were collected from four well differentiated locations: biceps femoris (BF), semimembranosus (SM) and semitendinosus (ST) muscles, and subcutaneous fat (SF). 2.1. Amino Acids and Related Compounds Dry-cured hams contain a large number of free amino acids and derivatives resulting from extensive proteolysis, which is characteristic of all types of hams, though the extent of amino acid release depends on the processing time [14,15]. The responsible enzymes are aminopeptidases that act on the N-terminal of peptides and proteins [14]. Table 1 shows that lysine, followed by others such as valine, isoleucine, leucine and phenylalanine, are the amino acids determined in higher amounts. The values of amino acids are, in general, higher in Iberian hams, which explains that all the amino acids can be used to distinguish Iberian from non Iberian hams with the exception of tryptophan and asparagine. Thus, results show leucine, isoleucine and lysine are higher in Iberian hams whose drying step is longer, which agrees with Toldrá et al. [15]. Toldrá [3] described that glutamic acid, aspartic acid, histidine, arginine, valine, methionine, isoleucine, leucine, tryptophan and lysine were strongly correlated with the length of the drying process [3]. Our results agree with those results, except for tryptophan, when hams were classified into four groups: 7 months, 8–11 months, 12–17 months and more the 18 months (Iberian hams). Those amino acids were positively correlated (p < 0.001) to the drying length with adjusted-R2 regression coefficients higher than 0.70. Coefficient values between 0.65 and 0.70 were also determined for tyrosine, aspargine, taurine, serine, and glycine. 2.2. Volatile Compounds Table 2 shows the volatile compounds identified in the samples clustered according to their breeding (non Iberian and Iberian), while Figure 1 displays the chromatograms of three dry-cured hams. The formation of these volatiles is caused by the intense degradation processes that happen in lipids and proteins during the processing of dry-cured hams. Most of the volatiles identified (hydrocarbons, aldehydes, alcohols, ketones, esters, carboxylic acids, etc.) derive from lipid oxidation that is probably the main source of ham volatiles as in almost all fat products [16]. Other volatiles, such as 3-methylbutanol and benzaldehyde, derive from reactions between amino acids and reducing Molecules 2013, 18 3930 sugars referred to as the Maillard reaction [16]. An intense proteolysis, and hence a larger release of amino acids in hams, promotes the Strecker degradation, and is the implication of reactive carbonyls in the Strecker degradation of free amino acids, which would explain the concentrations of the branched aldehydes (e.g., 2-methylbutanal derived from isoleucine) quantified in dry-cured hams. Table 1. Amino acids and related compounds (mg/100 g) determined in non Iberian and Iberian hams. Column with p-value lower than 0.05 indicates amino acids distinguish between hams. Values are expressed as the mean and the standard error of the mean. Code Compound Non Iberian a Iberian a p A1 Tryptophan 23 ± 1 21 ± 1 0.30 A2 Phenyalanine 252 ± 55 286 ± 67 <0.01 A3 Tyrosine 103 ± 3 150 ± 11 <0.01 A4 Tyramine 12 ± 2 2 ± 1 0.02 A5 Isoleucine 300 ± 8 391 ± 10 <0.01 A6 Leucine 267 ± 7 353 ± 11 <0.01 A7 Methionine 157 ± 1 209 ± 1 <0.01 A8 Valine 316 ± 1 402 ± 1 <0.01 A9 Creatine 1637 ± 19 118 ± 23 <0.01 A10 Proline 146 ± 4 197 ± 6 <0.01 A11 Creatinine 29 ± 1 36 ± 1 <0.01 A12 Glutamic acid 543 ± 162 739 ± 25 <0.01 A13 Arginine 195 ± 6 301 ± 9 <0.01 A14 Asparagine 185 ± 50 195 ± 15 0.50 A15 Taurine 57 ± 1 86 ± 8 <0.01 A16 Histidine 165 ± 5 226 ± 6 <0.01 A17 Serine 181 ± 7 255 ± 5 <0.01 A18 Glycine 92 ± 3 119 ± 2 <0.01 A19 Lysine 686 ± 22 966 ± 29 <0.01 Note: a mean ± standard deviation. Table 2. Codes and relative retention times (Rt) of the volatile compounds quantified in the hams, mean concentration and standard deviation of volatiles determined in non Iberian and Iberian hams, and p values of each volatile compound classifying the hams by their breeds (non Iberian vs. Iberian). Odor threshold values (OT) in mg/kg and sensory descriptions obtained by GC-olfactometry (GC-O) are displayed as well. Code Rt Volatile compound Non Iberian a Iberian a p OT GC-O V1 0.16 Hexane 0.36 ± 0.03 0.29 ± 0.03 0.20 1.50 Spicy V2 0.17 Heptane 0.22 ± 0.03 0.23 ± 0.03 0.85 0.67 Sweety, alkane V3 0.20 Octane 2.23 ± 0.42 3.00 ± 0.41 0.35 0.94 Sweety, alkane V4 0.21 2-Propanone 1.72 ± 0.13 2.21 ± 0.32 0.10 500 Fruity, apple, cooked meat V5 0.27 2-Butanone 0.34 ± 0.04 0.20 ± 0.02 0.03 40 Ethereal V6 0.29 3-Methylbutanal 0.13 ± 0.02 0.38 ± 0.06 <0.01 0.08 Acorn, fruity, cheesy, salty V7 0.31 2-Propanol 0.07 ± 0.01 0.07 ± 0.01 0.95 26 Alcoholic, dry, buttery-taste V8 0.32 Ethanol 1.31 ± 0.20 1.52 ± 0.26 0.61 30 Alcohol, sweet Molecules 2013, 18 3931 Table 2. Cont. Code Rt Volatile compound Non Iberian a Iberian a p OT GC-O V9 0.34 2-Ethylfuran 0.09 ± 0.04 0.07 ± 0.01 0.74 - Sweet V10 0.38 2-Pentanone + 3-Pentanone 0.79 ± 0.08 0.48 ± 0.10 0.05 70 b Sweet, fruity, green V11 0.39 2,3-Butanedione 0.36 ± 0.07 0.35 ± 0.10 0.95 - Vanilla/caramel-like, buttery V12 0.46 α-Pinene 0.09 ± 0.01 0.08 ± 0.02 0.57 0.02 Sharp, pine V13 0.51 Methyl benzene 0.10 ± 0.01 0.12 ± 0.01 0.03 0.33 Plastic, glue, strong V14 0.53 2-Methyl-3-buten-2-ol 0.06 ± 0.01 0.07 ± 0.01 0.74 0.48 Earthy V15 0.60 Dimethyl disulfide 0.02 ± 0.00 c 0.02 ± 0.00 c 0.39 0.01 Cauliflowers, vegetable V16 0.61 Butyl acetate 0.01 ± 0.00 c 0.01 ± 0.00 c 0.80 0.30 Fruity, banana, apple V17 0.64 Hexanal 1.18 ± 0.20 3.76 ± 0.64 <0.01 0.08 Green, grassy, fatty V18 0.69 2-Methyl propanol 0.11 ± 0.01 0.32 ± 0.04 <0.01 1.00 Wine, penetrating V19 0.75 2-Butanol 0.03 ± 0.01 0.02 ± 0.00 0.16 0.50 Winey V20 0.78 Ethyl benzene 0.03 ± 0.00 c 0.02 ± 0.00 c 0.81 - Dry, glue, unpleasant V21 0.90 Butanol 0.04 ± 0.02 0.15 ± 0.01 0.01 0.04 Fruity, medicinal V22 1.05 2-Heptanone 1.56 ± 0.17 1.24 ± 0.28 0.36 0.30 Spicy, acorn, blue cheese V23 1.06 Heptanal 1.03 ± 0.33 1.47 ± 0.21 0.49 0.50 Fatty, greasy, ham-like V24 1.09 Limonene 0.59 ± 0.10 2.68 ± 0.56 <0.01 0.25 Citric, fresh V25 1.21 3-Methylbutanol 1.33 ± 0.11 5.27 ± 1.15 <0.01 0.10 Woody, acorn, pleasant green V26 1.31 2-Pentylfuran 0.49 ± 0.08 0.94 ± 0.22 0.01 0.10 Green fruity, butter V27 1.43 Octan-3-one 1.25 ± 0.07 0.63 ± 0.08 <0.01 0.01 Spicy, mushroom, dirty V28 1.46 Pentanol 1.25 ± 0.15 1.26 ± 0.06 0.99 0.47 Pungent, strong, balsamic V29 1.59 (E,E)-2,4-Decadienal 0.46 ± 0.06 0.03 ± 0.01 <0.01 2.50 Fatty, rancid V30 1.61 2-Octanone 2.20 ± 0.37 0.72 ± 0.12 0.03 0.51 Fruity, floral, green, fresh V31 1.63 Octanal 4.28 ± 0.88 7.38 ± 1.10 0.04 0.32 Meat-like, green, fresh V32 1.84 (E)-2-Heptenal 1.33 ± 0.33 0.71 ± 0.19 0.31 0.05 Green, fatty, fruity, almonds V33 1.89 2-Heptanol 0.55 ± 0.06 0.70 ± 0.13 0.27 0.01 Oily, sweety V34 2.09 Hexanol 1.74 ± 0.18 4.10 ± 0.59 <0.01 0.40 Fruity, green V35 2.30 2-Nonanone 1.13 ± 0.22 1.69 ± 0.52 0.26 0.10 Floral, fruity, blue cheese V36 2.33 Nonanal 2.50 ± 0.44 4.33 ± 0.67 0.04 0.15 Rancid, fatty, tallowy V37 2.55 (E)-2-Octenal 0.84 ± 0.16 2.47 ± 0.58 <0.01 0.00 d Leaves, pungent, fatty, fruity V38 2.76 1-Octen-3-ol 2.72 ± 0.20 1.66 ± 0.21 0.01 0.00 d Mushroom-like, earthy, dust V39 3.02 Decanal 0.26 ± 0.02 0.28 ± 0.03 0.74 0.65 Citrus, waxy V40 3.11 Benzaldehyde 0.97 ± 0.09 1.78 ± 0.21 <0.01 0.06 Bitter almonds, penetrating V41 3.22 (E)-2-Nonenal 2.03 ± 0.37 3.88 ± 0.92 0.03 0.15 Fatty, waxy V42 3.47 Octanol 0.42 ± 0.05 0.98 ± 0.12 <0.01 0.03 Fatty, sharp V43 3.94 Butanoic acid 0.57 ± 0.05 0.61 ± 0.09 0.69 0.65 Cheesy, rancid V44 4.13 Nonanol 0.19 ± 0.01 0.26 ± 0.04 0.08 0.28 Fatty green V45 4.14 Isobutyric acid 6.49 ± 0.46 4.99 ± 0.67 0.12 8.10 Iron, fishy V46 4.35 Hexanoic acid 4.76 ± 0.58 4.90 ± 0.88 0.12 0.70 Fatty, cheese, sweaty Note: a mean ± standard deviation; b odor threshold of 3-pentanone; c standard deviations dimethyl disulfide (0.004 and 0.003), butyl acetate (0.001 and 0.001), 2-butanol (0.002), and ethyl benzene (0.003 and 0.003); d E-2-Octenal (0.004), and 1-octen-3-ol (0.001). Molecules 2013, 18 3932 Figure 1. Chromatograms of volatiles of the subcutaneous fat of two non Iberian hams from different geographical origins: (a) Iberian ham from PDO “Jamón de Huelva”; (b) Non Iberian ham from PDO “Jamón de Teruel”; and (c) Non Iberian ham from Parlan, Auvergne (France). Note: codes are described in Table 1. Chromatographic method is described in Experimental section. 10 20 30 40 50 60 70 80 Minutes 5 10 15 20 mVolts 5 10 15 20 mVolts 5 10 15 20 mVolts 1 2 3 4 5 6 7 8 9 12 3 4 5 67 10 11 12 13 14 15 16 17 18 22 23 24 25 26 2728 31 32 34 35 36 37 38 39 40 42 43 44 45 46 1 2 3 45 6 78 9 10 11 12 13 1415 1718 1920 21 22 23 24 25 26 27 28 30 31 32 33 34 35 36 37 38 3940 42 43 44 45 46 10 13 1415 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3031 32 33 34 3536 37 38 39 40 42 43 44 45 9 8 11 12 20 21 30 33 41 41 46 19 41 29 a) b) c) In terms of total concentrations of the most abundant series (alcohols, aldehydes, hydrocarbons and ketones) quantified in Iberian hams, the highest concentrations corresponded to alcohols followed by ketones, aldehydes and hydrocarbons [17], while ketones showed the highest concentrations in non Iberian hams, followed by alcohols, aldehydes and hydrocarbons [18]. The concentration of these compounds depends on the location where they are quantified. Thus, Table 3 shows the concentration of the volatile compounds quantified in the four ham locations displayed in chemical series. Table 3. Volatile compounds (mg/kg) quantified in four locations of dry cured ham samples (SF, subcutaneous fat, BF, biceps femoris, SM, semimembranosus, ST semitendinosus muscles) and distributed in chemical series. Presented data are mean values from all samples (non Iberian and Iberian), while figures within brackets are minimum and maximum concentrations. Chemical series Volatile compounds BF a SF a SM a ST a Hydrocarbons Hexane 0.40 (0.03–1.77) 0.27 (0.04–0.66) 0.37 (0.03–0.97) 0.38 (0.04–1.25) Heptane 0.16 (0.03–1.13) 0.26 (0.05–0.70) 0.29 (0.03–2.99) 0.15 (0.02–0.58) Octane 1.66 (0.10–7.91) 3.22 (0.52–10.81) 2.12 (0.39–5.30) 1.37 (0.40–3.81) Methyl benzene 0.14 (0.04–0.37) 0.15 (0.02–0.44) 0.19 (0.07–0.42) 0.13 (0.06–0.24) Ethyl benzene 0.12 (tr-0.35) 0.20 (tr-0.73) 0.17 (0.01–0.74) 0.14 (0.01–0.71) Limonene 0.40 (0.01–4.71) 0.51 (tr-3.16) 1.42 (tr-14.60) 0.56 (tr-5.14) α-Pinene 0.19 (tr-1.61) 0.10 (tr-0.82) 0.28 (tr-1.38) 0.22 (0.02–1.56) Molecules 2013, 18 3933 Table 3. Cont. Chemical series Volatile compounds BF a SF a SM a ST a Alcohols 2-Propanol 0.32 (0.01–2.64) 0.11 (tr-0.52) 0.33 (tr-3.25) 0.29 (tr-1.62) Ethanol 0.70 (0.01–5.86) 0.40 (0.01–3.52) 0.83 (0.01–8.90) 0.79 (0.01–5.46) 2-Methyl-3-buten-2-ol 0.04 (tr-0.24) 0.11 (tr-0.42) 0.04 (tr-0.19) 0.05 (tr-0.40) 2-Methyl propanol 0.13 (tr-0.63) 0.08 (tr-0.26) 0.15 (0.01–0.75) 0.12 (tr-0.70) 2-Butanol 0.02 (tr-0.27) 0.03 (tr-0.13) 0.03 (tr-0.49) 0.03 (tr-0.40) Butanol 0.28 (tr-8.50) 0.05 (tr-0.16) 0.09 (tr-0.37) 0.09 (tr-0.79) 3-Methyl butanol 3.10 (0.06–21.31) 0.65 (0.04–6.13) 1.76 (0.02–1 0.63) 2.04 (0.02–17.99) Pentanol 1.19 (0.07–7.66) 1.48 (0.28–4.81) 1.26 (0.09–7.43) 1.17 (0.11–8.57) 2-Heptanol 0.66 (tr-5.06) 0.71(0.04–3.12) 0.72 (0.09–3.76) 0.66 (0.05–3.27) Hexanol 1.56 (0.14–11.31) 2.84 (0.03–10.13) 2.10 (0.45–6.32) 2.55 (0.34–9.77) 1-Octen-3-ol 2.48 (tr-7.73) 2.27 (0.07–8.40) 0.54 (0.11–1.65) 0.65 (0.09–2.54) Octanol 0.56 (tr-3.02) 0.79 (0.13–2.90) 0.52 (tr-2.13) 0.53 (tr-2.25) Nonanol 0.23 (0.02–1.02) 0.38 (0.08–0.92) 0.19 (0.03–0.49) 0.19 (0.02–1.09) Aldehydes 3-Methylbutanal 0.20 (0.02–1.23) 0.13 (0.01–046) 0.40 (tr-2.23) 0.21 (0.01–1.19) Hexanal 0.80 (0.05–9.41) 3.29 (0.03–15.58) 1.20 (0.08–11.40) 0.74 (0.06–7.17) Heptanal 0.82 (tr-6.11) 2.51 (0.01–8.76) 0.87 (tr-4.01) 0.77 (tr-4.89) (E,E)-2,4-Decadienal 0.36 (tr-2.45) 0.25 (tr-1.56) 0.26 (tr-2.97) 0.43 (tr-4.80) Octanal 1.70 (tr-8.60) 1 0.45 (0.04–37.40) 5.10 (0.04–28.46) 4.11 (tr-30.95) (E)-2-Heptenal 1.89 (0.01–50.59) 1.68 (0.01–15.22) 2.69 (0.04–10.67) 1.97 (0.01–35.65) Nonanal 4.57 (tr-67.17) 5.05 (0.03–18.97) 4.91 (tr-23.51) 4.05 (tr-31.84) (E)-2-Octenal 0.43 (0.02–3.01) 2.23(0.03–18.61) 0.59 (0.02–7.65) 0.33 (0.02–3.24) Decanal 0.17 (tr-0.82) 0.18 (tr-0.77) 0.20 (0.05–0.57) 0.16 (tr-2.15) Benzaldehyde 1.27 (0.21–5.74) 0.86 (0.12–2.29) 1.54 (0.01–5.42) 1.10 (0.2–3.84) (E)-2-Nonenal 1.14 (0.01–9.22) 4.64 (0.14–25.14) 1.18 (0.14–8.18) 0.87 (0.08–7.94) Ketones 2-Octanone 1.89 (tr-13.36) 0.93 (tr-4.17) 0.67 (tr-7.27) 1.88 (tr-16.27) 2-Propanone 2.88 (0.09–9.45) 1.53 (0.12–6.67) 2.73 (0.301 0.29) 2.78 (0.03–12.47) 2-Butanone 0.28 (0.02–1.16) 0.13 (0.05–0.30) 0.41 (0.01–2.34) 0.27 (0.08–0.82) 2-Pentanone b 0.70 (tr-2.46) 0.57 (tr-3.54) 0.70 (tr-3.67) 0.80 (tr-3.82) 2,3-Butanedione 0.47 (tr-7.51) 0.57 (tr-3.83) 0.41 (tr-6.84) 0.47 (tr-4.71) 2-Heptanone 185 (tr-7.77) 1.53 (tr-10.14) 2.01 (0.20–9.30) 2.45 (tr-14.48) Octen-3-one 0.77 (0.05–2.25) 0.89 (0.08–2.44) 1.07 (0.02–3.19) 0.79 (tr-2.01) 2-Nonanone 1.46 (0.08–1 0.73) 2.47 (tr-11.16) 2.17 (0.15–18.46) 2.31 (0.20–13.75) Acids Butanoic acid 0.47 (0.03–1.48) 0.64 (0.04–2.67) 0.49 (0.06–1.78) 0.56 (0.06–1.72) Isobutyric acid 6.10 (0.48–20.72) 4.45 (0.32–11.55) 4.41 (0.50–17.58) 5.37 (0.56–12.85) Hexanoic acid 2.70 (0.24–18.07) 7.60 (0.10–28.98) 4.01 (0.25–24.37) 4.39 (0.27–44.19) Furans 2-Pentylfuran 0.34 (tr-2.28) 1.15 (0.03–5.09) 0.63 (tr-3.09) 0.23 (tr-1.43) 2-Ethylfuran 0.17 (tr-5.58) 0.11 (0.01–0.29) 0.09 (tr-0.31) 0.06 (tr-0.28) Esters Butyl acetate 0.03 (tr-0.28) 0.01 (tr-0.04) 0.02 (tr-0.07) 0.02 (tr-0.07) Sulfur compounds Dimethyl disulfide 0.03 (tr-0.22) 0.02 (tr-0.13) 0.05 (tr-1.10) 0.03 (tr-0.14) Note: a mean (minimum-maximum) values; b 3-pentanone; tr, traces. Molecules 2013, 18 3934 2.2.1. Hydrocarbons The concentration of the volatile compounds distributed in the different chemical series pointed out that the amount of hydrocarbons was higher in SM and SF and lower in BF and ST. Among the hydrocarbons, limonene and octane were the most abundant in the four locations. Hydrocarbons were at higher concentrations in SF than in the muscles with the exception of limonene and α-pinene. According to diverse authors [19,20], the presence of limonene in the hams has been associated with the pig feeding and it contributes with “lemon” sensory notes to ham flavor. Taking into account the odor thresholds, octane and limonene are, among the quantified hydrocarbons, the major contributors to ham odors although they do not contribute in same manner to all the hams. Thus, while the octane concentrations are higher than its odor threshold (OT) in samples from all breeds—Table 2 shows high p-value when comparing non Iberian vs. Iberian hams, the concentration of limonene has been quantified in amounts higher than its OT in all the Iberian hams, whichever the analyzed muscle, the highest values corresponding to SM (Table 3). Limonene, in concentrations higher than its OT, were also determined in all the muscles of Gascon and Basque hams, which agrees with Sabio et al. [19], and in two samples from PDO Teruel. This compound always contributed to lemon odor in hams with a maturation time higher than 12 months (Iberian hams maturation time was ≥ 18 months). Limonene was not perceived, on the contrary, with hams with short processing times, such as some American hams [21]. The concentration of α-pinene is higher than its OT in most of the samples, which means this compound contributes to ham aroma with the pine sensory note regardless the pig breed. The high concentration of hydrocarbons in Iberian hams was also observed for the total sum of the concentrations of hydrocarbons. 2.2.2. Alcohols The identified alcohols, linear and branched, were the most abundant volatiles because they are among the main lipid oxidation products. The methyl branched alcohols can also be derived from the Strecker degradation of amino acids. It is known that branched alcohols originate from microbial degradation of the respective branched aldehydes [22]. Thus, the formation and release of branched alcohols is affected by the salting conditions due to the antimicrobial activity of NaCl. Thus, a higher production of branched alcohols is observed when NaCl is partially replaced by other formulations [23]. The highest concentration of total alcohols was found in BF muscle (11.27 mg/kg) while the lowest concentration corresponded to SM (8.56 mg/kg). 3-Methyl-1-butanol was by far the most abundant alcohol in the three muscles. The high concentration of 3-methyl-1-butanol (i.e., 3.10 mg/kg in BF) can be due to the activity of the microorganisms present in the ham. Microorganisms can act on 3-methylbutanal formed by Strecker degradation of amino acids during proteolysis to give rise to 3-methylbutanol [24]. The alcohol with the highest concentration in SF was hexanol. In SF, where lipids mean 89.7% [25], most of alcohols are produced by lipid oxidation and the proteolysis mechanism does not occur at great extent. Because of the high OT of some alcohols (2-propanol, ethanol, 2-methyl-3-buten-2-ol, 2-mehyl propanol, 2-butanol, and nonanol) in comparison with their concentrations (Table 2), their impact on aroma has been considered minor [26]. Table 2 also shows that, in general, alcohols contribute to ham Molecules 2013, 18 3935 flavor with herbaceous, woody and fatty notes [27]. 3-Methyl-1-butanol has a significant sensory impact and it is a marker of Iberian hams because its high concentration in these hams compared to other breeds. The contribution of this compound to aroma depends on its concentration as it varies from green [8] to dried fruits, and particularly to acorns in the case Iberian hams (Table 2). Another odor compound whose concentration is higher in Iberian hams is hexanol, which contributes to fruity-green odor perception. Its concentration in the SF is mostly responsible of its odor perception. Among the alcohols which contribute to aroma, pentanol and 2-heptanol, for instance, would not be able to distinguish between non Iberian and Iberian hams (p < 0.05). The low odor threshold of 1-octen-3-ol indicates that it contributes with a strong mushroom aroma (Table 2) to almost all the hams. The concentration of this compound significantly rises as the amount of curing salt increases [28], which may explain the differences between samples. The study of the relationship between alcohols and amino acids showed that there was much more relationship between them in non Iberian than in Iberian hams. The values of the adjusted-R2 when regressing both data sets were lower in the former, with the exception of 2-butanol from ST with histidine (0.96). On the contrary, high correlation values between the concentrations of amino acids and volatiles were observed in Iberian hams because the increase of free amino acids in long-processed hams [11]. Thus, we found that 2-methyl propanol was the only alcohol that was not related with amino acids while butanol was highly correlated with tyrosine (0.89) and taurine (0.93). Table 4 shows the correlation values between alcohols and amino acids. Ethanol showed a fine correlation exclusively with creatine for all the analyzed locations while 3-methyl-1-butanol was highly correlated with tyramine (0.92). Creatine was also well correlated with 2-heptanol (0.92) and 1-octen-3-ol (0.93). Table 4. Correlation between alcohols and amino acids determined in Iberian hams with an adjusted-R2 regression coefficient higher than 0.75 with p < 0.05. Notes: Codes of volatile compounds are described in Table 2; BF, biceps femoris muscle; ST, semitendinosus muscle; SM, semimembranosus muscle; SF, subcutaneous fat. Amino acids Volatile compounds V7 V8 V14 V19 V21 V25 V28 V34 V33 V38 V42 V44 Tryptophan SF:0.75 BF:0.79 BF:0.80 Tyrosine BF:0.85 ST:0.80 SM:0.89 ST:0.76 ST:0.81 Tyramine ST:0.92 SF:0.79 Creatine BF:0.85 SF:0.79 BF:0.82 SF:0.89 BF:0.92 BF:0.83 ST:0.79 SM:0.93 BF:0.75 BF:0.82 ST:0.87 Asparagine BF:0.77 Taurine ST:0.82 ST:0.87 BF:0.93 BF:0.81 ST:0.80 ST:0.75 Glycine BF:0.85 Molecules 2013, 18 3942 standards were used for determining their odor thresholds and their odor sensory descriptors in a matrix of fully deodorized olive oil. The standard solutions were also used for determining the response factors using the same deodorized olive oil. 3.3. Response Factors Concentrations in the range of 0.1–5.0 mg/kg and 0.5–20 mg/kg were analyzed under the analytical conditions described above. These two ranges allowed analyzing the recovery of volatile compounds at two different concentration levels. The absolute response factors of the standard compounds were calculated as the slopes of the linear regressions obtained from the ratio of total peak area as a function of concentration (averaged value from the two studied ranges). Relative response factors were obtained as the ratio of the absolute response factor of each compound to that of the internal standard (4-methyl-2-pentanol). 3.4. Odor Threshold of Volatile Compounds Fully deodorized edible oil was the matrix for the assessment of the odor threshold values; the absence of volatile compounds in the matrix was checked by the SPME-GC procedure described above. The sensory assessment was carried out in a test room arranged for evaluating sensory characteristics. Five assessors with a large experience of odor recognition in fat food products carried out the evaluation. Three samples were presented to the assessors following the triangle test whose results were statistically analyzed. Each sample (15 mL) was kept in standardized glasses at 29 °C ± 2 °C for 15 min and then tested. The samples were diluted until none of the assessors was able to classify the samples by odor intensity. The odor activity values (OAVs) of the volatile compounds, defined as the ratio of the concentration to the odor threshold [33], were calculated to determine their sensory significance. Thus, only those volatiles with OAV ≥ 1.0 contribute to the sensory perceptions [33]. 3.5. GC-Olfactometry (GC-O) GC-O was applied to assess the aroma notes corresponding to ham volatile compounds. It was performed with a GC (Varian 3900) connected to an olfactory port (OP275; GL Sciences Inc., Tokyo, Japan). Purified helium (purity > 99.99%) was used as the carrier gas at a constant flow rate of 3.8 mL/min. The inlet pressure was 170 kPa, and the inlet system was in split/splitless mode. The oven temperature program was that already described in a previous section. The effluent of the GC column was split 1–10 to the detector and the sniffing port, respectively. The olfactory detection was performed during the chromatographic separation by three assessors with a large experience in odor recognition and sniffing of dry-cured hams. Damp air was continuously passed through the head of the port during operation; the flow rate was 30 mL/min. Elution of each aroma compound through the sniffing port was recorded by writing the beginning and end of the entire sensation of any odorant as well as its odor properties. The final aromagram (sensory description vs. Rt) is the result of merging the information from the individual analyses of the assessors. Table 2 shows the perception of the assessors (GC-O) at retention times. Molecules 2013, 18 3943 3.6. Amino Acids and Related Compounds The analysis of amino acids and related compounds was carried out according to the method described by Ruiz et al., 1999 [10]. Samples with an internal standard (norleucine 10 mg/mL) were mixed with 5% sulfosalicylic acid and homogenized with an Omnimixer. Homogenized samples were stored at 4 °C for 15 h and they were centrifuged at 15,300 g for 10 min and filtered through Whatman No. 54 paper. The pH of the filtrates was adjusted to 6 with 4 N NaOH, and then 50 μL were added to 200 μL solution of ethanol-water-triethylamine-phenyl tioisocyanate 7:1:2:1, and after 10 min the mixture was evaporated at cold temperature for 20 min, and reconstituted with a 0.5 M sodium phosphate buffer (500 μL), pH 7.4 and 5% acetonitrile. An aliquot (20 μL) was injected onto the HPLC system (LaChrom Elite, Tokyo, Japan), equipped with a UV diode array detector. The column was a Supelcosil LC-18 containing octadecyldimethylsilyl (25 × 4.6 mm; 5 μm particle size; Supelco), maintained at 35 °C. The gradient elution, at a flow rate of 1.0 mL/min, was achieved by using the following mobile phases: 0.03 M sodium acetate and 0.05% triethylamine, pH 6.80 (solvent A), and 90:10 acetonitrile-water (solvent B). The solvent gradient was programmed as follows: initial 96.8% (A)-3.2% (B) for 0.5 min; from 96.8 (A)-3.2% (B) to 95.5 (A)-4.5% (B) in 5 min; 90% (A)-10% (B) in 9.5 min; 81% (A)-19% (B) in 7 min; 73% (A)-27% (B) in 10 min; 1% (A)-99% (B) in 5 min. The chromatographic signals were obtained at 254 nm. The identification and response factors were based on the analysis of solutions (1 mg/mL) of standard amino acids obtained from Sigma Chemical: L-Ala, L-Arg HCl, L-Asn, L-Asp, L-Cys, L-Glu, L-Gln, Gly, L-His HCl, Pro (4-OH), L-Ile, L-Leu, L-Lys HCl, L-Met, L-Phe, L-Pro, L-Ser, L-Thr, L-Trp, L-Tyr and L-Val. Creatine and creatinine were analyzed by using the method described by Mora et al. [51]. 3.7. Statistical Analyses Univariate and multivariate algorithms have been used by means of Statistica 8.0 (Statsoft, Tulsa, OK, USA). An ANOVA allowed selecting the volatile compounds and amino acids that better characterize the samples by breed. Correlation was used to determine the relationship between sensory attributes and chemical compounds while the first screening of the relationship between those two set of variables was carried out by principal component analysis (PCA). 4. Conclusions The results comparing the volatile composition of Iberian and non Iberian hams showed that 20 volatile compounds had significant differences (p < 0.05) in their concentration depending on the combined effect of breed, feeding and curing process. Sixteen out of these 20 compounds have a sensory impact on dry cured ham aroma given that their concentrations were higher than their odor threshold. The amino acids composition of Iberian and non Iberian hams were also different for all the amino acids excepting tryptophan and asparagine. In this case, the concentration values were higher in Iberian hams in all cases probably due to their longer curing process compared to the other samples. Carnitine analyses showed, on the contrary, lower concentration values for Iberian hams due to the degradation of this compound during the processing [51]. The correlation between volatile compounds and amino acids provided satisfactory results (adjusted-R2 > 0.70) when only Iberian hams were Molecules 2013, 18 3944 including in the data set, the highest correlation coefficients corresponding to alcohols and aldehydes. The branched alcohols and aldehydes, typically produced from amino acid degradation [29], gives a chemical support to the relationship established between amino acids and volatiles. 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