Volatile compounds in dry-cured Serrano ham subjected to high pressure processing. Effect of the packaging material

Meat Science 82 (2009) 162–169

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Volatile compounds in dry-cured Serrano ham subjected to high pressure processing. Effect of the packaging material Ana Rivas-Cañedo, Estrella Fernández-García *, Manuel Nuñez Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña, km. 7, Madrid 28040, Spain

a r t i c l e

i n f o

Article history: Received 23 October 2008 Received in revised form 29 December 2008 Accepted 5 January 2009

Keywords: High pressure processing Packaging material Volatile compounds Dry-cured Serrano ham

a b s t r a c t The effect of high pressure treatment (400 MPa, 10 min at 12 °C) on the volatile profile of Spanish drycured Serrano ham, packaged with or without aluminum foil in a multilayer polymeric bag, was investigated. The analysis of the volatile fraction was carried out by dynamic headspace extraction coupled to gas chromatography–mass spectrometry. Pressure treatment only had a slight effect on the volatile fraction of Serrano ham. Most compounds affected by pressurization, such as alkanes (C9–C12), 2-undecene, 2-nonanone, 1-octen-3-one, 1-heptanol, 2-hexanol, 2-heptanol, ethyl pentanoate, benzaldehyde and styrene, presumably originated from the metabolism of moulds. A significant effect of pressurization on the migration of compounds from the plastic material was found. Linear and branched chain alkanes, alkenes as well as benzene compounds, were generally less abundant in pressurized samples than in untreated samples. A scalping effect was also observed for compounds such as butanal, pentanal, ethyl esters and pyrazines. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Dry-cured ham known as ‘Jamón Serrano’ is one of the most popular meat products in Spain. Its annual production, in continuous growth, was around 265,000 tons in 2006. The traditional manufacture of Serrano ham takes from 8 to 12 months. During the salting stage, salt and additives such as nitrate and/or nitrite, are absorbed, firstly by the external muscle Semimembranosus and next by slow diffusion through the whole ham. This is followed by a post-salting stage at a temperature below 5 °C for 1–2 months, until the water activity (aw) decreases below 0.96 to prevent growth of undesirable microorganisms. Afterwards, the temperature is generally raised from 10 to 12 °C to a maximum of 25 °C for 2–4 months during the drying stage, in which the typical flavor of this meat product develops, followed by an aging period at 12– 20 °C for 4–6 months. Proteolysis, lipolysis, as well as lipid oxidation, Maillard reactions and Strecker degradations all play a role in the development of Serrano ham flavor, especially during the ripening period (García et al., 1991; Toldrá & Flores, 1998). Biochemical reactions leading to flavor development are mainly attributed to the endogenous enzymatic systems in dry-cured ham (Toldrá, 1998). The role of microorganisms in flavor development seems limited because the microbial population (mainly Gram-positive catalase-positive cocci) inside the ham is low (Carrascosa, Marín, Avendaño, & Cornejo, * Corresponding author. Tel.: +34913476771; fax: +34913572293. E-mail address: [email protected] (E. Fernández-García). 0309-1740/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2009.01.006

1988). Consequently, the microbial enzyme activity remains at low levels (Molina and Toldrá,1992). Nowadays, Serrano ham is usually sliced and vacuum-packaged at the processing plants or at retail shops, where it might get contaminated by pathogens as well as spoilage microorganisms (Morales, Calzada, & Núñez, 2006). High pressure processing (HPP) is a non thermal treatment appropriate for eliminating post-processing contamination in food products that might be altered by heat treatments, such as readyto-eat meat products. The effectiveness of 600 MPa for 6 min at 16 °C on preventing microbial growth in Serrano ham has been proven by Garriga, Grèbol, Aymerich, Monfort, and Hugas (2004), who recorded lack of growth of Enterobacteriaceae and yeasts and delayed growth of lactic acid bacteria. The lethality of Listeria monocytogenes in Serrano ham caused by HPP has also been reported (Morales et al., 2006). One of the main advantages of HPP is supposedly no modifications in taste and flavor characteristics (Hendrickx, Ludykhuyze, Van den Broeck, & Weemaes, 1998), or changes in the nutritional value and vitamin content occurs (Cheftel & Culioli, 1997). Nevertheless, HPP may induce changes in texture or colour. Saccani, Parolari, Tanzi, and Rabbuti (2004) reported a slight discoloration of dry-cured ham after treatment at 600 MPa for 9 min, and similar results were observed by Andrés, Møller, Adamsen, and Skibsted (2004) in ham subjected to different pressures. Prior to HPP, foods must be vacuum-packaged, usually in a polymeric material. Possible interactions between food products, polymers and the environment, such as permeation, migration

A. Rivas-Cañedo et al. / Meat Science 82 (2009) 162–169

and absorption/adsorption, have been reported (Hotchkiss, 1997; Risch & Hotchkiss, 1991). These phenomena may produce effects such as loss or imbalance of food flavor (Gremli, 1996), oxidation, package damage, microbial growth, or even safety problems (Nielsen & Jägerstad, 1994). The extent of these interactions is influenced by the properties of the polymer, the flavor molecule (molecular weight, polarity) and external conditions (Nielsen, Jägerstad, & Öste, 1992). The concentration of the sorbed material (Brody, 2002) and sorbants are also determining factors (Charara, Williams, Schmidt, & Marshall, 1992). All these facts make necessary a study of packaging material safety, as well as the changes in the overall flavor, especially when a technique such as HPP is introduced (Hugas, Garriga, & Monfort, 2002). Studies dealing with the effect of HPP and vacuum-packaging on flavor compounds are commonly performed in simulant fluids. The aim of the present work was to investigate changes in the volatile profile of Serrano ham when subjected to HPP using a multilayer plastic as packaging material, with and without intermediate wrapping in aluminum foil. 2. Materials and methods 2.1. Serrano ham, packaging materials and high pressure processing Serrano ham was purchased and sliced (approx. 1.5 mm thickness) at a local supermarket. Food-grade multilayer packaging material (MLPM) was used for vacuum-packaging (BB4L, Cryovac Sealed Air Corporation, Milano, Italy). The MLPM consists of one low-density polyethylene (LDPE) layer in contact with the food, followed by several ethylene-vinyl acetate (EVA) layers and one vinylidene chloride (VDC) layer. Treatments were conducted in triplicate. Ham slices were divided into 12 equivalent portions, six of which were directly vacuum-packaged in a MLPM bag while the other six were first wrapped in aluminum foil (AF) and then vacuum-packaged in MLPM bags. Three portions in direct contact with the MLPM and three portions shielded with AF were pressurized for 10 min at 400 MPa and 12 °C in a 3.5 L capacity discontinuous high pressure apparatus (Model ACIP 6000, ACB, Nantes, France) whereas the rest of the samples were kept untreated, as controls. After HPP, samples were stored at 4 °C for 3 days and then frozen at 35 ± 1 °C until analysis. 2.2. Volatile compound analysis Volatile compounds were analysed in an automatic dynamic headspace apparatus (Purge and Trap, HP 7695, Agilent, Palo Alto, CA) coupled to a gas chromatograph–mass spectrometer (HP-MSD HP 5973, Agilent). The fat layer surrounding the ham slices was removed to obtain more homogeneous samples. Five grams of Serrano ham were homogenized in a mechanical grinder (IKA Labortechnik, Staufen, Germany) with 5 g of anhydrous Na2SO4 (to favour a salting out effect) and 20 lL of an aqueous solution of 630 mg/L cyclohexanone (Sigma–Aldrich, Alcobendas, Spain) was added as internal standard (IS). An aliquot of the mixture (3.5 g) was subjected to dynamic headspace for 20 min at 45 °C using helium (45 mL/min) with 10 min of previous equilibration. Volatile compounds were concentrated in a Vocarb 4000 trap (Tekmar, Manson, OH) maintained at 35 °C, with 4 min dry purge, and desorbed during 2 min at 260 °C through a transfer line heated at 200 °C, directly into the injection port at 220 °C with a split ratio of 20:1 and 1.4 mL/min helium flow. Chromatography was carried out in a capillary column (60 m long; 0.25 mm i.d.; 0.5 lm film thickness; Innowax, Agilent Technologies), with 1 mL/min helium flow; with the following temperature program: 16 min at 45 °C,

163

first ramp 4 °C/min to 110 °C, 9 min at 110 °C, final ramp at 15 °C/min to 230 °C and 3 min at 230 °C. Detection was performed with electron impact ionization, with 70 eV ionization energy operating in the full-scan mode at 1.74 scans/s, with a scanning m/z range of 33–200. Source and quadrupole temperatures were 230 and 150 °C, respectively. Compound identification was carried out by injection of commercial standards, by spectra comparison using the Wiley7Nist05 Library (Wiley and Sons Inc., Germany), and/or by calculation of linear retention indexes (LRI) relative to a series of alkanes (C5–C19). The sum of abundances of up to four characteristic ions per compound was used for semi-quantitation. The areas have been referred to the IS (compound peak area multiplied by 103 and divided by IS peak area). The volatile fractions of MLPM and AF were studied elsewhere (Rivas-Cañedo, Fernández-García, & Nuñez, 2009). 2.3. Statistical analysis Statistics were performed with the SPSS Win 12.0 software (SPSS Inc., Chicago, IL). Volatile compound areas were subjected to analysis of variance (ANOVA) using packaging material and high pressure treatment as main effects. Significance was assigned at P < 0.05. A principal component analysis (PCA) with Quartimax rotation was performed with selected volatile compounds. 3. Results A total of 108 compounds were detected in the volatile fraction of Serrano ham as determined by dynamic headspace extraction. Table 1 lists the compounds ordered by chemical family, together with their chromatographic indices, the ions used for quantitation and the method used for identification. The significance of effects, high pressure processing (HPP) or packaging material (PM), together with the origin of the compounds, ham or PM, are also shown. The presence of compounds in the MLPM has been added to facilitate comprehension, although this material was analysed and results reported in a previous work (Rivas-Cañedo et al., 2009). Because of the high similarity of their fragmentation patterns, most branched chain alkanes (BCAs) found in the MLPM could not be identified with 100% certainty using the available libraries. This is the reason why BCAs have been named by their LRI in Table 1. Except for hydrocarbons, likely coming from the MPLM, the most abundant families in ham, extracted, were aldehydes and alcohols. Sixty three compounds were significantly influenced by HPP or/and by the packaging material. The results are presented considering the statistical significance of the effects. Seven compounds – some linear hydrocarbons, ethyl pentanoate and benzaldehyde – decreased significantly when HPP was applied, independently of the packaging material (Table 2), while 2-heptanone increased. Thirty compounds were influenced by the packaging material, independently of the application of HPP (Table 3). The levels of some alkanes (C9–C12) and a branched chain alcohol (LRI 1188) increased significantly when ham was directly packaged in the MLPM bag. However, the rest of compounds in Table 3, some linear and branched chain aldehydes, three ketones and one diketone, 2,3-butanedione, eight alcohols, two ethyl esters, sulphur compounds, pyrazines and furans, all decreased when directly packaged in MPLM, independently of the application of HPP. Finally, thirty four compounds were significantly influenced by both effects, HPP and PM, showing a significant HPP x PM interaction (Table 4). Three different tendencies are observed. Firstly, 15 BCAs, the most abundant being 2,2,4,6,6-pentamethylheptane, increased dramatically when ham samples were directly packaged

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Table 1 Volatile compounds identified in Serrano ham subjected to high pressure processing (HPP), and in the plastic packaging material (PM). Volatile compound

Alkanes Pentane Hexane Heptane Octane Nonane Decane Undecane Dodecane Branched-chain alkane 2,2,4,6,6-Pentamethylheptane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Branched-chain alkane Alkenes 1,3-Pentadiene 1-Octene 1-Undecene 2-Undecene Aldehydes Ethanal Propanal 2-Propenal Butanal Pentanal Hexanal Heptanal Octanal Nonanal 2-Methylpropanal 2-Methyl-2-propenal 2-Methylbutanal 3-Methylbutanal 2-Methyl-2-butenal 2,4-Decadienal Ketones 2-Propanone 2-Butanone 2-Pentanone 2-Heptanone 2-Octanone 2-Nonanone 2,3-Butanedione 3-Methyl-2-pentanone 2,3-Pentanedione 1-Octen-3-one 2-Cyclohexen-1-one Alcohols Ethanol 1-Butanol 1-Pentanol 1-Hexanol 1-Heptanol 1-Octanol 2-Propanol 2-Butanol 2-Pentanol 2-Hexanol 2-Heptanol 2-Methyl-1-propanol 1-Methoxy-2-propanol

LRIa

QIb

IDc

Origind

500 600 700 800 900 1000 1100 1200 660 947 963 967 995 997 1012 1017 1039 1042 1049 1067 1079 1084 1093 1095

42, 57, 43, 43, 57, 43, 71, 57, 57, 57, 57, 56, 57, 57, 99, 57, 56, 71, 56, 57, 57, 71, 85, 57,

41, 57, 72 41, 56, 42 57, 71, 41 57,85,71 41, 85, 56 57, 71, 85 43, 57, 85 43, 71, 85 56, 41, 43 56, 71, 85 56, 71, 85 57, 71, 41 56, 41, 71 56, 41, 71 56, 41 43, 71, 41 57, 43, 41 43, 57, 41 57, 43, 71 41, 71, 97 71, 85, 43 57, 43, 70 57, 84 43, 71, 98

ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS ST, MS MS ST, MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS

H, H, H, H, H, H, H, H, H, H, H, H, H H, H, H, H H, H H, H, H, H, H,

642 837 1147 1164

67, 55, 70, 69,

68, 70, 83, 70,

53, 39 41, 83 41, 97 154, 83

708 802 851 885 990 1103 1208 1306 1416 818 892 919 923 1119 1843

44, 58, 56, 43, 58, 56, 70, 57, 57, 41, 39, 57, 44, 84, 81,

43, 57 55, 41, 44, 57, 44, 56, 98, 43, 70, 41, 58, 55, 67,

42, 41

821 909 987 1206 1302 1408 990 1033 1082 1272 1475 940 1172 1264 1360 1472 1570 929 1041 1149 1237 1326 1121 1160

PM PM PM PM PM PM PM PM PM PM PM PM

Significance of effectse HPP

PM

HPP x PM

ns ns ns ns

ns ns ns ns

*

**

ns ns ns ns ns ns

*

*

***

***

***

*

***

ns

ns

ns ns

**

***

**

***

***

***

*

***

*

*

***

*

*

***

*

***

***

**

***

***

**

**

***

**

***

***

**

***

***

**

PM PM PM PM PM

***

***

**

***

***

***

**

***

**

***

***

**

*

***

*

MS ST, MS MS MS

H H, PM H, PM H, PM

ns

ns

ns

***

***

***

***

***

***

***

***

***

MS MS MS MS MS ST, MS MS MS MS ST, MS MS ST, MS ST, MS MS MS

H, PM H H H H, PM H, PM H H H H H H H H H

ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns

*

*

ns ns

ns ns ns ns ns

ns ns ns ns ns ns ns ns ns

***

*

*

ns ns ns ns

58, 43, 42, 39 43, 72, 57 43, 41, 86, 57 43, 58, 71, 85 43, 58, 71, 59 58, 43, 71, 59 43, 86, 42, 87 43, 57, 72, 100 100, 43, 42 43, 57, 99, 72 68, 96, 39, 40

MS ST, MS MS MS MS ST, MS MS MS MS MS MS

H H H H H H H H H H H

ns ns ns

45, 56, 42, 56, 70, 56, 45, 45, 45, 45, 45, 42, 45,

MS MS MS MS MS MS MS MS MS MS MS ST, MS MS

H H H H H H H H H H H H H

ns ns ns ns

53 72, 41, 72 43, 55, 70, 72 41, 58, 41, 53, 55,

39 57 55 84 82 43 39 43 83 95

46, 43, 41 41, 43, 42 55, 41, 70 55, 69, 41 56, 55, 69 55, 69, 70 43, 41, 59 59 73 43, 69, 57 55, 43, 41 43,41, 74 47, 43, 75

PM PM PM PM

** *

ns *

ns ns

*

*

ns

ns

ns ns ns ns ns

***

***

***

ns ns ns

**

ns

ns ns ns

**

**

***

ns

ns

ns

ns

ns ns ns ns

***

ns

***

* ** **

*

***

*

ns ns ns ns

ns

ns ns ns ns

*

ns **

**

*

*

**

***

**

ns ns

*

ns ns

ns

165

A. Rivas-Cañedo et al. / Meat Science 82 (2009) 162–169 Table 1 (continued) Volatile compound

LRIa

QIb

IDc

Origind

1-Penten-3-ol Branched chained alcohol 3-Methyl-1-butanol 2-Butoxyethanol 1-Octen-3-ol 2-Ethyl-1-hexanol Esters Ethyl acetate Ethyl butanoate Ethyl 2-methylbutanoate Ethyl pentanoate Ethyl hexanoate Ethyl octanoate 2-Ethylhexyl 2-propenoate Ethyl decanoate Benzene compounds Toluene Ethylbenzene p-Xylene m-Xylene o-Xylene p-Ethylphenol Ethyltoluene Styrene Trimethylbenzene 1,3-Bis(1,1-dimethylethyl)benzene Benzaldehyde Phenylacetaldehyde Acetophenone Miscellaneous Methanethiol Carbon disulfide Dimethyl disulfide Dimethyl trisulfide Tetrahydrofuran Acetonitrile 2-Butylfuran 2-Pentylfuran Limonene Dimethylpyrazine Trimethylpyrazine 2,2-Dichloroethanol Naphthalene 2-Methylnaphthalene

1184 1188 1227 1425 1466 1499

57, 55, 55, 57, 57, 57,

41, 41, 70, 87, 72, 43,

MS MS ST, MS MS ST, MS MS

H H H H H H

899 1057 1073 1157 1250 1457 1460 1651

43, 88, 57, 57, 88, 88, 55, 88,

45, 61, 70 73, 89, 61 102, 85, 41 85, 88, 60 99, 43, 60 101, 127, 57 70, 57, 41 101, 157, 73

MS ST, MS MS ST, MS ST, MS ST, MS MS ST, MS

H H H H H H H H

ns ns ns

1061 1147 1157 1163 1208 1240 1246 1279 1303 1452 1562 1679 1690

91, 92, 65, 93 91, 106, 65, 51 91, 106 91, 106, 105, 77 91, 106, 105, 77 107, 122, 77 105, 120, 41 104, 103, 78, 77 105, 120, 77, 91 175, 190, 176, 147 77, 105, 106, 51 91, 65, 92, 120 105, 120, 77

MS MS ST, MS ST, MS ST, MS MS MS ST, MS MS MS ST, MS ST, MS ST, MS

H H H, H, H, H H H H H, H H H

ns ns ns ns ns ns ns

682 735 1095 1412 867 1022 1152 1248 1216 1348 1434 1629 1802 1933

47, 48, 45, 46 76, 78, 77, 64 94, 79, 46 126, 45, 47, 79 42, 72, 41, 71 40, 41, 39 81, 124, 82 81, 82, 138, 53 68, 93, 67, 121 108, 42 122, 42, 81, 39 49, 79, 114, 83 128, 127, 129, 102 142, 141, 115, 143

MS MS MS MS MS MS MS MS MS MS MS MS MS MS

H H H H H H H H, PM H, PM H H H H H

43, 43, 42, 41 55 55,

88 69 43

70

Significance of effectse HPP

PM

ns

ns

ns

**

***

**

ns ns ns ns

*

ns ns ns ns

PM PM PM

**

ns PM

HPP x PM

ns

ns

*

*

*

ns ns

***

ns ns ns ns ns ***

ns *

ns ns ns ns ns ns ns ns

ns ns ns ns ns

ns ns ns ns ns

***

***

ns

ns

**

**

ns

ns

**

***

**

*

*

ns

ns

ns ns

**

**

*

ns

ns

ns *

***

*

ns ns ns ns

**

ns ns ns ns

*

ns ns

*

**

**

ns ns ns

**

ns ns ns

*

***

*

**

***

***

ns ns

ns

ns ns

ns *

*

a

LRI: linear retention indexes, calculated in relation to the retention time of n alkane (C5–C19) series. QI, ions used for quantification library. c ID, peak identification: ST, comparison of spectra and retention time with commercial standards; MS, tentatively identified by spectra comparison using the Wiley Library. d Origin of compounds: H, dry cured ham; PM, multilayer packaging material. e ns, Non significant. * P < 0.05. ** P < 0.01. *** P < 0.001. b

Table 2 Mean abundancesa (±SD) of the volatile compounds significantly influenced by high pressure processing (HPP) in Serrano ham not showing a significant interaction with the packaging material (PM). Compound

Control (n = 6)

HPP (n = 6)

Nonane Decane Undecaneb Dodecane 2-Undeceneb 2-Heptanone Ethyl pentanoate Benzaldehyde

2.24 ± 0.77 4.52 ± 1.55 18.69 ± 0.80 0.59 ± 0.53 0.82 ± 0.07 14.84 ± 2.05 1.37 ± 0.29 12.11 ± 1.46

1.35 ± 0.79 2.90 ± 1.15 11.42 ± 1.92 0.26 ± 0.31 0.28 ± 0.06 18.01 ± 2.82 0.78 ± 0.41 9.76 ± 2.08

a Calculated as the sum of abundances of up to four characteristic ions and referred to the IS (compound area x 1000/IS area). b n = 3, Only detected in samples packed directly in plastic material.

in MLPM, but the increase was less pronounced when HPP had been applied. Instead of presenting individual results for the 14 minor BCAs, the total sum is given, since they all show the same trend. Similar results were observed for 1-undecene and for the benzene compound 1,3-bis(1,1-dimethylethyl)benzene. The latter compound together with BCAs were identified in the volatile fraction of MLPM (Table 1), as reported elsewhere (Rivas-Cañedo et al., 2009). A second tendency was shown by ethanal and 2-methyl-2-propenal. HPP caused an increase of these compounds if ham slices had been previously wrapped in AF, and a decrease when directly packaged in MLPM. The third tendency was that of 1-octen-3-one, 2-nonanone, 1-heptanol, 2-hexanol, 2-heptanol, styrene, acetophenone, 2-butylfurane, carbon disulfide and trimethylpyrazine. The abundances of these compounds were significantly higher in the non-treated samples which had been previously wrapped in AF than in any of the other samples.

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Table 3 Mean abundances (±SD) of the volatile compounds in Serrano ham significantly influenced by the packaging material not showing a significant interaction with the high pressure processing. Volatile compound Hydrocarbons Nonane Decane Undecane Dodecane Aldehydes and ketones Ethanal Butanal Pentanal 2-Methylbutanal 2-Methyl-2-butenal 2-Butanone 2-Heptanone 2,3-Butanedione 3-Methyl-2-pentanone Alcohols and esters 1-Butanol 1-Pentanol 1-Hexanol 2-Propanol 2-Pentanol 2-Methyl-1-propanol 2-Ethyl-1-hexanol 1-Octen-3-ol Branched chain alcohol LRI 1188 Ethyl octanoate Ethyl decanoate Miscellaneous Dimethyl disulfide Dimethyl trisulfide Dimethylpyrazine Benzaldehyde 2-Pentylfuran 2-Methylnaphthalene a

AF + MLPMa (n = 6)

MLPM (n = 6)

1.19 ± 0.60 2.81 ± 1.11 0.00 ± 0.00 0.07 ± 0.18

2.38 ± 0.69 4.62 ± 1.45 15.06 ± 0.42 0.77 ± 0.33

257.7 ± 37.65 2.24 ± 0.33 53.59 ± 8.79 68.65 ± 9.12 8.56 ± 1.42 21.81 ± 1.76 17.97 ± 2.10 105.75 ± 6.44 1.65 ± 0.55

215.4 ± 39.74 1.34 ± 0.42 41.94 ± 3.58 54.71 ± 7.14 7.05 ± 0.74 16.25 ± 1.93 14.88 ± 2.83 94.82 ± 6.45 0.17 ± 0.42

10.08 ± 0.51 43.75 ± 4.33 25.32 ± 3.44 234.8 ± 40.38 24.79 ± 2.97 12.68 ± 2.12 5.10 ± 0.81 10.09 ± 1.59 0.00 ± 0.00 4.44 ± 0.41 2.79 ± 0.25

9.32 ± 0.59 37.29 ± 1.53 19.09 ± 1.47 194.1 ± 18.55 19.23 ± 1.46 9.70 ± 1.28 3.14 ± 0.53 7.91 ± 0.82 1.86 ± 0.24 3.41 ± 0.23 2.32 ± 0.34

6.18 ± 1.15 1.75 ± 0.61 4.41 ± 0.77 12.06 ± 1.73 5.91 ± 1.08 1.93 ± 0.62

4.27 ± 0.74 0.77 ± 0.82 3.37 ± 0.62 9.52 ± 1.90 4.26 ± 0.66 1.21 ± 0.44

Table 4 Mean abundances (±SD) of the volatile compounds in Serrano ham significantly influenced by high pressure processing (HPP), showing a significant interaction with the packaging material (PM). Compound Hydrocarbons 2,2,4,6,6-Pentamethylheptane Sum of branched chain alkanesc 1-Octene 1-Undecene Aldehydes and ketones Ethanal 2-Methyl-2-propenal 1-Octen-3-one

AF, aluminum foil; MLPM, multilayer packaging material.

2-Nonanone Alcohols 1-Heptanol 2-Hexanol 2-Heptanol 2-Butoxyethanol 2,2-Dichloroethanol Benzene compounds Styrene p-Ethylphenol 1,3-Bis(1,1dimethylethyl)benzene Acetophenone

3.1. Principal component analysis (PCA) Fig. 1 represents the principal component analysis of some significant volatile compounds found in dry-ham. The loading plots of the factor scores extracted by the PCA are represented in Fig. 2. The levels of some compounds presumably migrating from the MLPM, mainly linear and branched chain alkanes and undecene together with the benzene compound 1,3-bis(1,1-dimethylethyl)benzene build function 1, explaining 54.9% of the variance. Some compounds showing significant differences between control and HP treated samples (Tables 2 and 4), namely 2-hexanol, 2-heptanol, 1-heptanol, 1-octen-3-one, 2-nonanone, pentanoic acid ethyl ester and styrene, make up function 2, explaining 35.2% of the variance. PC1 divides the samples according to the packaging material and PC2 can be related to HP treatment. 4. Discussion As shown in Fig. 2, the individual ham samples are distributed into four well-defined groups by the principal component analysis. PC1 could be named ‘packaging material’. Samples directly packaged in MLPM are pushed to the right side of the plane, mainly due to their higher contents of branched chain hydrocarbons and benzene compounds. PC1 also separates the samples directly packaged in MPLM into two distinct groups, according to the HP treatment, since the treated samples directly packaged in MLPM underwent lower levels of migration from plastic components. PC2 could be designated as ‘HP treatment’, the treated samples occupying the lower part of the plane (Fig. 2). This function sepa-

Miscellaneous Carbon disulfide 2-Butylfuran Trimethylpyrazine

PM

Controla (n = 3)

HPPa (n = 3)

AF + MLPMb MLPM AF + MLPM

13.80 ± 4.27 3625 ± 489 1.43 ± 2.47

10.60 ± 1.08 2270 ± 223.24 0.63 ± 0.25

MLPM AF + MLPM MLPM AF + MLPM MLPM

742.5 ± 100.18 7.50 ± 1.37 2.57 ± 1.33 1.41 ± 1.26 15.45 ± 0.83

408.9 ± 48.7 17.97 ± 0.64 1.53 ± 0.39 2.04 ± 0.17 9.52 ± 0.42

AF + MLPM MLPM AF + MLPM MLPM AF + MLPM MLPM AF + MLPM MLPM

235.9 ± 45.43 241.0 ± 30.21 28.99 ± 1.28 28.92 ± 1.60 1.30 ± 0.22 0.42 ± 0.08 1.41 ± 0.21 0.59 ± 0.03

279.6 ± 7.04 189.8 ± 32.69 33.66 ± 1.82 24.13 ± 2.14 0.42 ± 0.08 0.56 ± 0.24 0.68 ± 0.05 0.68 ± 0.05

AF + MLPM MLPM AF + MLPM MLPM AF + MLPM MLPM AF + MLPM MLPM AF + MLPM MLPM

2.47 ± 0.28 1.36 ± 0.11 3.41 ± 0.75 1.91 ± 0.02 4.47 ± 0.71 2.23 ± 0.40 3.26 ± 0.57 1.92 ± 0.02 0.88 ± 0.04 0.37 ± 0.04

1.83 ± 0.20 1.44 ± 0.17 1.68 ± 0.44 1.74 ± 0.31 2.31 ± 0.02 1.91 ± 0.42 2.54 ± 0.28 2.22 ± 0.33 0.45 ± 0.08 0.38 ± 0.12

AF + MLPM MLPM AF + MLPM MLPM AF + MLPM

1.12 ± 0.25 0.40 ± 0.02 1.36 ± 0.07 0.48 ± 0.12 0.15 ± 0.15

0.32 ± 0.10 0.39 ± 0.14 1.09 ± 0.17 1.03 ± 0.17 0.00 ± 0.00

MLPM AF + MLPM MLPM

32.88 ± 3.82 1.14 ± 0.16 0.76 ± 0.03

20.31 ± 3.52 0.70 ± 0.07 0.69 ± 0.18

AF + MLPM MLPM AF + MLPM MLPM AF + MLPM MLPM

262.55 ± 11.55 44.46 ± 5.59 0.64 ± 0.20 0.10 ± 0.02 7.14 ± 0.39 5.16 ± 0.20

104.77 ± 94.84 39.67 ± 16.01 0.22 ± 0.06 0.22 ± 0.02 5.94 ± 0.37 5.13 ± 0.70

a Mean abundances are separately given for Serrano ham samples packaged with or without AF. b AF, aluminum foil; MLPM, multilayer packaging material. c Sum of LRI 963, 995, 997, 1012, 1017, 1039, 1042, 1049, 1067, 1079, 1084, 1093 and 1095.

rates the control from the HP treated samples wrapped in AF, due to the higher contents in control samples of compounds such as ketones and alcohols, which we have related to mould metabolism (see discussion below). However, the HP-treated and nontreated samples directly packaged in MLPM are not well separated by PC2. This is because the compounds building PC2 seem to have increased during the cold storage only in the control (not treated) samples wrapped in AF, but not in those directly packaged in MLPM. 4.1. Effect of HPP on the volatile fraction The effect of HPP on the volatile fraction of Serrano ham was limited, with only a few compounds being significantly affected, in comparison with the results obtained for fresh meats (RivasCañedo et al., 2009). Physicochemical conditions in Serrano ham

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Fig. 1. Loading plot of Serrano ham volatile compounds in the rotated space determined by the principal components. BCA, branched chain alkanes; bis-DMEB, 1,3-bis(1,1-dimethylethyl)benzene; PMH, 2,2,4,6,6-pentamethylheptane.

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ketones, are commonly found in food with mould growth (Collins, McSweeney, & Wilkinson, 2003). Aliphatic alkanes can be related to lipid oxidation processes (Frankel, 1984), but also to mould metabolism. Martín, Córdoba, Aranda, Córdoba, and Asensio (2006) studied the volatile profile of cured ham inoculated with Penicillium chrysogenum and Debaryomyces hansenii and reported a significant relationship between mould inoculation and the levels of hydrocarbons with C > 7, besides other compounds, such as 3octen-2-one. Sunessen, Trihaas, and Stahnke (2004) also reported increasing amounts of alkanes when Penicillium nalgiovense was used as starter in dry sausage manufacture. In the case of styrene, some moulds can produce this compound from unsaturated fatty acids under starvation conditions, (Jollivet, Chateaud, Vayssier, Bensoussan, & Belin, 1994). The lower levels of those compounds observed for HPP samples could be related to the inhibition of mould growth or mould metabolism by pressurization, mostly in the samples packaged in AF, as shown by the PCA. Since the storage period was too short to allow significant mould growth, we can only ascribe the differences found to the inhibition of mould metabolism. It has been shown that moulds are inactivated by pressures of 200–300 MPa, and their spores by pressures of 400 MPa (Aymerich, Picouet, & Monfort, 2008). Rubio, Martínez, García-Cachán, Rovira, and Jaime (2007) reported a delay in mould growth, along with other microorganisms, in dry-cured beef meat after a 500 MPa for 5 min treatment. However, to our knowledge, no information is available on the inhibition of mould metabolism by high pressure in meat products. Benzaldehyde, which may originate from phenylalanine catabolism (McSweeney & Sousa, 2000), was also reduced in HPP treated samples. Hugas et al. (2002) reported that a 600 MPa for 6 min treatment did not modify the non-protein nitrogen fraction in both cooked and dry-cured ham and suggested that pressurization does not induce proteolysis. In our opinion, benzaldehyde could also come from mould metabolism. Some authors have described a slight reduction in antioxidant enzyme activities (superoxide dismutase, catalase and glutathione peroxidase) in dry-cured ham after treatments of 400–600 MPa (Serra et al., 2007). This effect, which might favour lipid oxidation, was not observed in the present study. 4.2. Effect of packaging material

Fig. 2. Loading plot of the factor scores of the Serrano ham samples in the plane defined by principal components. d – Control samples directly packaged in MLPM. s – Control samples previously wrapped in AF. j – HPP samples directly packaged in MLPM. h – HPP samples previously wrapped in AF.

(high salt content, low water activity) do not favour bacterial growth, and the production of metabolic volatile compounds by microorganisms is somehow restricted. Only on the ham surface is the growth of yeasts and molds, especially from the genera Penicillium and Aspergillus, relevant (Huerta, Sanchis, Hernández, & Hernández, 1987). Volatile compounds such as unsaturated ketones, 2-alkanols and styrene have generally been associated with the metabolism of moulds. Probably, 1-octen-3-one originates from linoleic and linolenic catabolism by moulds (Mollimard & Spinnler, 1996) or from methyl linoleate autooxidation (Ullrich & Grosch, 1987). 2-Alkanols, coming from the reduction of methyl-

Migration seemed to be the main phenomenon taking place in Serrano ham directly packaged in MLPM, confirming previous results reported for fresh meats (Rivas-Cañedo et al. 2009). Nevertheless, much higher levels of compounds coming from the plastic material, especially BCAs and benzene compounds, were observed in the present study on ham as compared with fresh meats. This enhanced migration is possibly due to the higher fat content together with the lower water content of ham (Castle, Sharman, & Gilbert, 1988; Tice, 1993), and most likely also to the fact that sliced ham had a higher surface/volume ratio than fresh meat. According to Gremli (1996), the degree of sorption is directly related to the surface area in contact with the food. Thus, interchange phenomena between food and plastic are expected to be enhanced when food is packaged in thin layers rather than in pieces or bulks. It is worth highlighting that, when pressure was applied, migration of compounds from the plastic material was significantly less intense than in non-treated samples, as reflected by PCA (Fig. 2). The observed impaired migration caused by HPP may reside in a change of the structural properties of the MLPM. Supporting these results, López-Rubio et al. (2005) reported a slight improvement in crystalline morphology which resulted in better barrier properties of ethylene-vinyl alcohol copolymers (EVOH) treated with HPP.

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Kübel, Ludwig, Marx, and Tauscher (1996) observed a lower degree of acetophenone and p-cymene migration from model aroma solutions through the package, presumably due to a glass transition phase forming in the polymer caused by HPP. However, Le-Bail, Hamadami, and Bahuaud (2006) observed no effect on the water barrier properties of BB4L plastic, used in the present study, when subjected to HPP. Schauwecker, Balasubramaniam, Sadler, Pascall, and Adhikari (2002) stated that there were no HPP-induced molecular changes in the treated plastics, although they observed better barrier properties to 1,3-propanediol of treated EVOH. Previously on fresh meats no significant effect of HPP on compound migration from MLPM was observed (Rivas-Cañedo et al., 2009). As the migration levels observed in that study were much lower, possible interactions between packaging material and HPP were probably undetectable. Besides migration, other phenomena are worth noticing, such as absorption/adsorption of some compounds from food to the MLPM (scalping) or permeation. Lower levels of compounds, such as aldehydes, 2,3-butanedione, 3-methyl-2-pentanone, some ethyl esters, some alcohols, sulphur compounds and pyrazines, were found in Serrano ham samples directly packaged in MLPM (Table 3), which could have been adsorbed into the packaging material or lost by permeation. Cilla, Martínez, Beltrán, and Roncalés (2006) observed a decrease in the aroma and flavor of dry-cured ham after longterm vacuum-packaging, which could be due to scalping. Regarding the factors affecting scalping of compounds, some studies on model systems have reported a positive correlation between absorption and molecular weight (MW) up to 10 carbon atoms (Shimoda, Ikegami, & Osajima, 1988), and a negative correlation between absorption and polarity (Charara et al., 1992; Sajilata, Savitha, Singhal, & Kanetkar, 2007). However, our data show the opposite trend, since the relative decrease of the affected compounds, which would point to scalping or permeation phenomena, declines with MW and rises with polarity within a given chemical family. For example, the decrease of butanal was 33% while the decrease of pentanal was 20% (Table 3), in spite of the higher MW and the lower polarity of the latter. Similarly, the observed decrease of ethyl octanoate was 22% against 17% of the less polar and higher MW ethyl decanoate, and the decrease of dimethylpyrazine was 26% against 22% (mean values) of the less polar and higher MW trimethylpyrazine (Table 3). A possible explanation for these conflicting results could be that the cited studies on scalping were carried out on model systems or diluted aqueous solutions, while fat and protein are the main ham constituents. Van Willige, Linssen, and Voragen (2000a) reported that ß-lactoglobulin and casein bind to volatile compounds like decanal and limonene and decrease the absorption of these compounds from model solutions into linear low-density polyethylene (LLDPE) packages. The same authors (Van Willige, Linssen, & Voragen, 2000b) observed that the absorption of volatile compounds from model solutions into LLDPE diminished with increasing oil concentrations, and that this effect was more pronounced for the more apolar molecules, which would agree with our results. These authors reported that fat-containing foods undergo lower volatile loss by absorption into the plastic material since numerous compounds in the product are highly lipophylic. In our study pentanal, ethyl decanoate and trimethylpyrazine would have been adsorbed to a lesser extent by the MLPM because, at least partly, of their more apolar and lipophylic nature which favours their retention by the fat phase of ham. Regarding the effect of HPP on scalping, Caner, Hernandez, Pascall, Balasubramaniam, and Harte (2004) reported no effect or even a lower degree of limonene scalping from food simulating liquids into LLDPE when treated with HPP. Schauwecker et al. (2002) also reported lower levels of 1,2-propanediol permeation from the pressure-transmitting fluid into a food simulating liquid through EVOH pouches treated with HPP. These authors speculated that

high pressures might act to compress the void spaces and thus decrease permeate penetration through the microchannels of the polymers. However, in our study HPP seemed to favour the scalping or permeation of a few compounds (ethanal and 2-methyl-2propenal), which decreased with HPP only in the samples directly packaged in MLPM (Table 4). 5. Conclusions The main conclusion is that the application of high pressure to sliced Serrano ham, to increase product shelf life, does not markedly modify its volatile fraction. Only a few compounds decreased with treatment, very likely as the result of an impairment of mould metabolism. Compound migration from plastic packaging material into the ham seemed to be reduced by the application of high pressure. Nevertheless, the high levels of migration found make it necessary to carefully select the type and characteristics of the packaging material for high pressure applications. Acknowledgments This work was supported by projects CPE03-012-C3-1 (INIA), S0505/AGR/0314 (Comunidad de Madrid) and CSD 2007-00016 (Consolider). The authors thank INIA for granting Ana Rivas-Cañedo, and C. Juez, B. Rodríguez and M. de Paz for their valuable technical assistance. References Andrés, A. I., Møller, J. S. K., Adamsen, C. E., & Skibsted, L. H. (2004). High pressure treatment of dry-cured Iberian ham. Effect on radical formation, lipid oxidation and colour. European Food Research and Technology, 219, 205–210. Aymerich, T., Picouet, P. A., & Monfort, J. M. (2008). Decontamination technologies for meat products. Meat Science, 78, 114–129. Brody, A. L. (2002). Flavor scalping: Quality loss due to packaging. Food Technology, 56, 124–125. Caner, C., Hernandez, R. J., Pascall, M., Balasubramaniam, V. M., & Harte, B. R. (2004). The effect of high-pressure food processing on the sorption behaviour of selected packaging materials. Packaging Technology and Science, 17, 139–153. Carrascosa, A. V., Marín, M. E., Avendaño, M. C., & Cornejo, I. (1988). Jamón Serrano. Cambios microbiológicos y fisico-químicos durante el curado rápido. Alimentaria, 194, 9–12. Castle, L., Sharman, M., & Gilbert, J. (1988). Analysis of the epoxidized soya bean oil additive in plastic bags by gas chromatography. Journal of Chromatography A, 437, 274–280. Charara, Z. N., Williams, J. W., Schmidt, R. H., & Marshall, M. R. (1992). Orange flavor absorption into various polymeric packaging materials. Journal of Food Science, 57, 963–969. Cheftel, J. C., & Culioli, J. (1997). Effects of high pressure on meat: A review. Meat Science, 46, 211–236. Cilla, I., Martínez, L., Beltrán, J. A., & Roncalés, P. (2006). Effect of low-temperature preservation on the quality of vaccum-packaged dry-cured ham: Refrigerated boneless ham and frozen cuts. Meat Science, 73, 12–21. Collins, Y. F., McSweeney, L. H., & Wilkinson, M. G. (2003). Lipolysis and free fatty acid catabolism in cheese: A review of current knowledge. International Dairy Journal, 13, 841–866. Frankel, E. N. (1984). Recent advances in the chemistry of rancidity of fats. In A. J. Baikey (Ed.), Recent advances in the chemistry of meat (pp. 87–118). Burlington House, London: The Royal Society of Chemistry. García, C., Berdagué, J. L., Antequera, T., López-Bote, C., Córdoba, J. J., & Ventanas, J. (1991). Volatile components of dry-cured Iberian ham. Food Chemistry, 41, 23–32. Garriga, M., Grèbol, N., Aymerich, M. T., Monfort, J. M., & Hugas, M. (2004). Microbial inactivation after high-pressure processing at 600 MPa in commercial meat products over its shelf life. Innovative Food Science and Emerging Technologies, 5, 451–457. Gremli, H. (1996). Flavor changes in plastic containers: A literature review. Perfumer and Flavorist, 21, 1–8. Hendrickx, M., Ludykhuyze, L., Van den Broeck, I., & Weemaes, C. (1998). Effects of high pressure on enzymes related to food quality. Trends in Food Science and Technology, 9, 197–203. Hotchkiss, J. H. (1997). Food-packaging interactions influencing quality and safety. Food Additives and Contaminants, 14, 601–607. Huerta, T., Sanchis, V., Hernández, J., & Hernández, E. (1987). Mycoflora of dry-salted Spanish ham. Microbiologie, Aliments, Nutrition, 5, 247–248. Hugas, M., Garriga, M., & Monfort, J. M. (2002). New mild technologies in meat processing: High pressure as a novel technology. Meat Science, 62, 359–371.

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