electron spin resonance (esr) spectroscopy study of dry-cured ham treated with electron-beam
TRANSCRIPT
Food Chemistry 133 (2012) 1530–1537
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Food Chemistry
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Electron spin resonance (ESR) spectroscopy study of dry-cured ham treatedwith electron-beam
Rosa Escudero a, Margarita Valhondo b, Juan A. Ordoñez a, Lorenzo de la Hoz a, M. Concepción Cabeza a,Raquel Velasco a, M. Isabel Cambero a,⇑a Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spainb C.A.I. de Resonancia Magnética Nuclear, Universidad Complutense de Madrid, 28040 Madrid, Spain
a r t i c l e i n f o
Article history:Received 16 May 2011Received in revised form 30 November 2011Accepted 8 February 2012Available online 15 February 2012
Keywords:Electron spin resonance (ESR)Dry-cured hamE-beam
0308-8146/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.foodchem.2012.02.045
⇑ Corresponding author. Tel.: +34 913943745; fax:E-mail address: [email protected] (M.I. Cambe
a b s t r a c t
The generation, accumulation and decay of free radicals in muscle and fat fractions from three varieties ofSpanish dry-cured ham treated (0–4 kGy) in an electron accelerator have been studied by electron spinresonance (ESR) spectroscopy. In the ESR spectra from fat fractions, a well-resolved triplet signal corre-sponding to an alkyl radical was found only in treated samples. Linear regression models (P < 0.05) wereobtained for ESR signal intensity estimation using the absorbed dose and storage time at 4 �C (from 0 to28 days). Several ESR signals were observed in the spectra from muscle fractions related to the presenceof metalloprotein complexes. However, no significant (P > 0.05) differences were found between ESRspectra from untreated (0 kGy) and treated (0–4 kGy) samples. Results suggest that the analysis of ESRspectrum in fat samples can be used to evaluate the E-beam treatment of dry-cured ham.
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1. Introduction a variety of food processing facilities (Francis, Thomas, & O‘Beirne,
Dry-cured ham is a meat product made from the whole pork leg,which has a final weight of 6–8 kg. This product has high consumeracceptance because of its particular sensory characteristics. Due toits low water activity (<0.90), dry-cured ham is a shelf-stable prod-uct and, therefore, it has a very long shelf-life. The only possiblespoilage may come from the growth of moulds and fatty acidauto-oxidation reactions when it is packed in an aerobic atmo-sphere. Likewise, dry-cured ham presents no microbial hazard if itis consumed immediately after cutting from the whole piece. Cur-rently, this product has a good track record in terms of microbialfood safety. However, today it is very usual to transform the dry-cured ham into a ready-to-eat (RTE) product. Then, it is preparedin small portions, usually slices in family-size amounts, from dry-cured pieces. During this operation, particularly if not properlydone, several pathogens may reach the product. Cutters and slicersmay be potent sources of contamination since they commonly pro-vide inaccessible sites which harbour bacteria (Garg, Curely, &Splittstoesser, 1990). Since no pathogen bacteria may growth atwater activity lower than 0.90, the organisms of most concern arethose under the ‘‘absence in 25 g’’ criterion, which is applied tothe most harmful foodborne pathogens, such as Listeria monocytog-enes, Salmonella spp., and Escherichia coli O157:H7. L. monocytogenesand Salmonella spp. are ubiquitous bacteria, which may be presentand can survive and multiply in the environment and equipment of
ll rights reserved.
+34 913943743.ro).
1999). Therefore, they can accidentally come in contact with theham slices at any time. The bovine gastrointestinal tract is believedto be the main reservoir of E. coli O157:H7 (Doyle, Zhao, Meng, &Zhao, 1997), but it is also found in mammalian meats and poultry(Samadpour et al., 1994) as well as in dry-cured sausages (wateractivity lower than 0.90); the latter were the vehicle of an outbreakwhich occurred in the states of California and Washington in 1994,and resulted in 23 victims (CDCP, 1995). The mere presence of thesebacteria in a lot is of concern, which may be the cause of condem-nation by the sanitation authorities.
Since RTE dry-cured ham production is not subjected to anylethal processing, it is not possible to consistently guarantee thatthe final product be free from the above mentioned pathogen.The application of traditional technology to sanitize sliced andpacked RTE foods, including dry-cured ham, is not viable. However,we have previously proved that the treatment of a variety of RTEfoods, including dry-cured ham (Hoz, Cambero, Cabeza, Herrero,& Ordóñez, 2008), with accelerated electrons (E-beam) is a veryuseful method for sanitizing these products (Cabeza, Cambero, dela Hoz, & Ordóñez, 2007; Cabeza, de la Hoz, Velasco, Cambero, &Ordóñez, 2009). In general, absorbed doses under 2 kGy are enoughto reduce the pathogen mentioned above to a safe level in interme-diate moisture foods, such as dry-cured ham (Hoz et al., 2008).
A large number of governments (currently 40) permit the irradi-ation of a substantial number of food commodities. Governmentalregulation concerning irradiation of food varies considerably fromcountry to country. Most countries approve food irradiation on acase-by-case basis. Food irradiation in the United States is primarily
R. Escudero et al. / Food Chemistry 133 (2012) 1530–1537 1531
regulated by the FDA since it is considered a food additive and not afood process. However, other authorities such as the United StatesDepartment of Agriculture (USDA) also participate in the regulationof irradiation for fresh fruit, meat and poultry products (Morehouse& Komolprasert, 2004). The US set an example for the increase inpermitted food irradiation uses, as exemplified by the 1997 FDAapproval, for the irradiation of unprocessed red meat and meatproducts, for other foods such as fresh shelled eggs and for seedsfor sprouting (Morehouse & Komolprasert, 2004).
In the European Union, several specific directives (EC, 1999a,1999b) have been issued with the common aim of harmonisingthe law in relation to food irradiation of the Member States. In thiscontext, the words ‘‘irradiated’’ or ‘‘treated with ionising radiation’’must appear on the label or packaging, as well as in the documentswhich accompany treated foodstuffs or foodstuffs containing trea-ted ingredients; they must always be accurate. The frameworkDirective of the European Parliament adopted in 1999 (EC, 1999b)includes a ‘‘positive list’’ permitting irradiation only of dried aro-matic herbs, spices, and vegetable seasonings, and sets a maximumoverall average absorbed radiation dose of 10 kGy. However, anyMember State is allowed to maintain previously granted clearancesor to add a new clearance which has been granted in other MemberStates, when the EC’s Scientific Committee for Food (SCF) has givena positive vote for the specific application. Presently, seven MemberStates (Belgium, France, Italy, Netherlands, Poland, United Kingdomand Czech Republic) have adopted such provisions (EC, 2009).
From the legal and commercial points of view, it is very impor-tant to differentiate between irradiated and non-irradiated foods,not only to verify the authenticity of a given material and to detectmislabelled products, but also to build consumer confidence ofirradiated food. Various physical and chemical methods have beeninvestigated for the detection of irradiated food. The electron spinresonance (ESR) spectroscopy is the only method available to di-rectly detect paramagnetic species (i.e., free radicals and ions rad-icals) in biological systems (Shimoyama, Ukai, & Nakamura, 2007).Therefore, it is being used to evaluate free radical formation in sev-eral foods. For example, ESR can be used to detect free radicals,which are the short-lived intermediates of lipid oxidation (Ander-sen & Skibsted, 2002; Stachowicz, 1998). Moreover, ESR can beconsidered to study the effect of irradiation on different foodsand to detect irradiated foods (especially when the water contentis low). The European Union has accepted the ESR as an interna-tional standard method for the detection of irradiated food con-taining bone (EN 1786, 1996), cellulose (EN 1787, 2000) andcrystalline sugar (EN 13708, 2001).
Scientific literature (Stachowicz, 1998) shows that radicals,which result as a consequence of irradiation treatment in wet sam-ples, decay in a fraction of second with the formation of neutralmolecular products, but those induced in dry matrices (such ascould be the dry-cured ham), persist for longer periods and couldbe detected by ESR (Yarkov, Tret’yakov, & Khramov, 2000).
The present study was performed to investigate the effect ofradiation on fat and muscle fractions of dry-cured ham by ESRspectroscopy.
2. Materials and methods
2.1. Materials
Three varieties of Spanish dry-cured ham, from white pig [Teruel,a ‘‘Protected Designation of Origin (PDO)’’ from Spanish according tothe Council Regulation (EC, 2006) and Serrano, a product processedin accordance with ‘‘Traditional Speciality Guaranteed (TSG)’’ (EC,1999c)] and from black pig [Iberian, corresponding to the ProtectedDesignations of Origin ‘‘Dehesa de Extremadura’’ and belonging to
the category ‘‘bellota’’ (pigs reared in a free-range production sys-tem mainly with pasture and acorns) (MAPA, 2007)], were used,which were named Teruel, Serrano and Iberian, respectively.
The typical production process of these types of Spanish dry-cured hams includes the following steps: salting, washing-brush-ing, rest or post-salting, drying-ripening and refinement by ageing(cellar phase). However, the conditions in which each step in theprocess is managed vary according to the type of product expectedand to the corresponding Council Regulations (EC, 1999c, 2006;MAPA, 2007). However, a common practice for the three types ofdry-cured hams is the salting procedure. First, there is usually apre-salting of the fresh legs, in which the leg surfaces are rubbedmanually with curing salts. Then, the legs are covered with coarsesea salt during about 0.75–1 day per kilo of fresh material.
In the case of Serrano dry-cured ham (EC, 1999c), fresh legs witha minimum weight of 9.5 kg and a fat cover over 0.8 cm are em-ployed. Its production process takes an average of 12 months (finalweight around 6.1 kg). This meat product requires a final waterand salt contents of about 57% (defatted matter) and 15% (defatteddry matter), respectively. The Teruel hams (EC, 2006) were manu-factured from white pigs reared in the Teruel region (Spain). Thefresh legs (with a weight over 11 kg and a fat cover around 4–5 cm) were cured at least 800 m above sea level for a minimumof 9 months (final weight around 8–9 kg). The Iberian hams used(MAPA, 2007) were obtained from fresh legs with a minimumweight of about 9.7 kg. This type of ham requires a longer curingprocess (18–24 months), in which nearly half of its weight is lost(final weight >4.5 kg).
2.2. Sample preparation and irradiation treatment
Dry-cured ham samples from each of the above mentioned vari-eties were purchased in different marketplaces. Small portions orslices (about 0.3 cm thick and 2.5 g in weight) containing sectionsof the Biceps femoris muscle and visible subcutaneous fat were ta-ken from three different hams. The ham samples analysed in thisstudy corresponded to the left leg of the hogs and for each dry-cured ham variety three different commercial brands were used.
Several portions (12 portions from different hams and commer-cial brands, weighing about 30 g from each of the three dry-curedham varieties) were vacuum-packed to reach about 20 kPa in10 � 10 cm laminated film bags (polyamide and polyethylene, filmthickness: 90 lm) of low gas permeability (oxygen transmissionrate 35 cm3/24 h m2 bar and carbon dioxide 150 cm3/24 h m2 bar).Samples were transported (for less than 1 h) in insulated polysty-rene boxes to the processed plant (IONISOS sterilization SA, Taran-cón, Cuenca, Spain) and treated with an electron beam source,which operated at 10 MeV. The doses employed were in the rangeof 0.5–4 kGy. Some samples were also treated at 8, 12 and16 kGy. Untreated samples were used as control (0 kGy). The doseabsorbed by the samples was calculated considering the absor-bance of simultaneously treated cellulose triacetate dosimeters(ASTM, 2000). Experiments were performed at room temperature(16–18 �C) in triplicate. The temperature increase during treatmentwas less than 2 �C. After irradiation treatment, the samples werestored at 4 �C until use. The ESR measurements were carried outon days 0, 8, 18 and 28 of storage at 4 �C (consequently, three vac-uum-bags containing samples from three different commercialbrands � 3 dry-cured ham varieties � 5 absorbed doses � 4 storagetimes were used). Three irradiation treatments were carried out, atthree different times, following the procedure described above.
2.3. ESR measurements
At 0, 8, 18 and 28 days after irradiation treatment and storage at4 �C, the muscles were removed and the visible subcutaneous and
1532 R. Escudero et al. / Food Chemistry 133 (2012) 1530–1537
intermuscular fats were carefully removed from each of the cured-ham slices. The fats were frozen at �80 �C and freeze-dried at roomtemperature (18–20 �C). The freeze-dried fats from a vacuum-bagwere mixed and ground using a mortar to obtain a homogeneouspaste. Similarly, the muscle fraction of each slice was frozen,freeze-dried and ground to obtain a fine powder.
The ESR spectra were recorded at room temperature (295 K) ona Bruker EMX spectrometer. The samples were transferred to anESR capillary tube (cylindrical ER221/TUB4 Bruker quartz tubewith a 4 mm inner diameter). The fat sample mass was 60–70 mg. Typical operating parameters of the ESR spectrometer (withan HSW 10432 X-band resonator 9.5 GHz), were as follows: modu-lation frequency 100 kHz, modulation amplitude 2 G, frequency9.45 GHz, microware power, 2 mW; centerfield 3362.19 G; sweepwidth 2000 G and receiver gain, 2 � 104, time constant,327.68 ms; spectrum scan time, 41.94 s; spectrum point number1024 and scan number 32. For the muscle fractions, the ESR spec-trometer operating parameters were: time constant, 655.36 ms;spectrum scan time, 83.886 s; spectrum point number 1024 andscan number 1. The sample mass was 400 mg. The g-factors of allsamples were estimated using DPPH as standard for whichg = 2.0036 ± 0.0003 (Poole, 1996; Yordanov, 1996).
The signal intensity (ESR-SI) was calculated from the doubleintegral of the ESR spectra. The amplitude of the first derivativeof the spectrum was taken as a measurement of the relative radicalconcentration.
The g-factor was calculated using the expression: g-factor = hm/Bo lB where h is Planck constant (6.63 � 10�34 J s), m is the fre-quency, Bo is the magnetic field (Gauss) and lB corresponds to BohrMagneton (9.2740 � 10�24 J T�1).
2.4. Statistical analysis
The fraction of fat or muscle from a vacuum-bag containingabout 30 g weight of dry-cured ham was the experimental unit.To check the normal distribution (90% confidence) of samples,the Shapiro–Wilks test was applied. When samples fitted thenormal distribution, the one-way ANOVA analysis was per-formed. When samples did not fit the normal distribution, theKruskal–Wallis test was used to determine the null hypothesis,that the medians of the variable within each of the sample levelswere the same. Duncan’s test for multiple mean comparisonswas applied to ascertain differences among means. Simpleregression analyses (using a Durbin–Watson statistics test, at a95% confidence level) were performed to determine the relation-ships between absorbed dose and signal intensity in the ESRspectra.
The statistical analysis was carried out using a Statgraphics Plusversion 5.0. Data were presented as the means and the standarddeviations (SD) of each radiation treatment.
3. Results and discussion
3.1. ESR spectrometry of dry-cured ham fat
An example of ESR spectra from the fat fractions (both visiblesubcutaneous and intermuscular fat) of untreated and E-beam irra-diated (0.5–4 kGy) dry-cured ham samples after treatment (0 days)are shown in Fig. 1. It is noteworthy that no ESR signals were ob-tained when untreated (0 kGy) fat samples were analysed (Fig. 1and Table 1). However, the treated fat samples (in the range of0.5 and 4 kGy) showed one signal at g-factor of 2.003. These resultsindicated that the ESR detected paramagnetic species were notpresent in untreated fat fractions; however, the E-beam treatmentinduced their formation.
As shown in Fig. 1(a–c), similar ESR spectra were obtained forthe fat fractions of the different varieties of hams studied. Never-theless, significant differences (P < 0.05) were detected betweenthe intensity of the signal at g-factor of 2.003 that corresponds tothe three varieties of dry-cured ham after the E-beam treatment(0 days) with the same doses (Fig. 1 and Table 1). These resultsare probably related to the differences detected in the fatty acidprofiles of each of the varieties of the Spanish dry-cured hams(Fernández et al., 2007).
Research has been conducted to study the free radical mecha-nisms of chemical reactions induced by the radiolysis of lipids(Sevilla, Morehouse, & Swarts, 1981; Sevilla, Swarts, & Sevilla,1983). Taub (1984) reported that the types of radicals and radicalreactions described for palmitates can be considered as being rep-resentative for other saturated and unsaturated fatty acid estersand triglycerides. The formation of alkyl, carbonyl, allyl, acyl andacyloxi radicals from fatty acids has been proven or presumed inirradiated food (Sevilla et al., 1983; Taub, 1984). The result ob-tained in the present study suggests that the radical at a g-factorof 2.003 recorded in the ESR spectra of dry-cured ham fat wasthe most stable in the environmental conditions mentioned previ-ously and was possibly the result of several radiolytic reactions.
The g-factor is an intrinsic characteristic of the paramagneticcentre and its local coordination. The characteristics of the g-factorprovide enough information to distinguish between the carbon,nitrogen, and sulphur centred radical (Schaich, 1980). At a g-factorof 2.003 (Fig. 1) is observed a quintet spectrum including a tripletwhose components were coupled by 22 G split by a doublet of53 G. The total spectral width is 97 G at 25 �C. The slight asymme-try of this signal, -five-line spectrum- (reflecting the interaction ofthe unpaired electron with the single hydrogen as well as with thetwo hydrogens on the adjacent carbon) is typical for abstractionradicals (Sevilla et al., 1981). The spectra obtained were similarto those reported by Sevilla et al. (1983) for the radicals formedfollowing c-irradiation of several lipid compounds and may be
identified as the alkyl radical: R—O— C
Ojj
— C�
H—CH2—R1. The major
radiolytic reactions in fats thus involve preferential cleavages inthe vicinity of the carbonyl group, which lead to the formation ofspecific, relatively abundant, free radicals. If a double bond is pres-ent in the fatty acid molecule, the electron deficiency would belocalised in both the carboxyl group and in the centre of unsatura-tion. The various free radicals produced by irradiation may thenengage in a number of reactions leading to the formation of stableradiolytic compounds (Sevilla et al., 1983).
In all the samples after E-beam treatment (0 days), the radicalformed was proportional to the dose absorbed (Table 1 and Fig. 1)and a high uniformity and reproducibility of the ESR measurementswere obtained. A significant (P < 0.05) increase of the ESR signalintensity was observed when the absorbed dose was increased(Table 1). A high significant linear regression (P < 0.001) was foundbetween the dose absorbed (x) and the intensity of the ESR signal (y)in Teruel (y = 5.51x + 0.064; R2 = 0.92), Serrano (y = 8.14x + 0.13;R2 = 0.96) and Iberian (y = 6.14x + 0.13; R2 = 0.92) dry-cured hams.
Fig. 2 shows the evolution of the alkyl radical during the storageof the samples at 4 �C. In the three varieties of dry-cured ham itwas significatively found (P < 0.05) that the higher the increase ofthe storage time after E-beam treatment the lower the ESR signalintensity (Fig. 2). Nevertheless, for the two varieties of dry-curedhams from white pigs (Teruel and Serrano), significant differences(P < 0.05) were always detected between untreated and E-beamtreated fat samples (Fig. 2) during the whole storage period (aboutone month). Following E-beam treatment, these types of dry-curedhams also showed significant determination coefficients (R2 < 0.80,P < 0.05) for the simple linear regression between the intensity of
0 days 18 days 30 days a) b) c)
3200 3300 3400 3500 3600
-2.0x106
-1.5x106
-1.0x106
-5.0x105
0.0
5.0x105
1.0x106
1.5x106
2.0x106
ES
R s
igna
l Int
ensi
ty (
a.u)
1.5 kGy
0.5 kGy
0 kGy
4 kGy
Magnetic Field (Gauss)
2 kGy
3200 3300 3400 3500 3600
-1.0x106
-5.0x105
0.0
5.0x105
1.0x106
1.5x106
ES
R s
igna
l Int
ensi
ty (
a.u)
1.5 kGy
0.5 kGy
0 kGy
4 kGy
Magnetic Field (Gauss)
2 kGy
3200 3300 3400 3500 3600
0kGy
0.5 kGy
4 kGy
2 kGy
Maagnetic Field (Gauss)
1.5 kGy
3200 3300 3400 3500 3600
0.5 kGy
0 kGy
4 kGy
1.5 kGy
2 kGy
Magnetic Field (Gauss)
3200 3300 3400 3500 3600
4 kGy
2 kGy
0 kGy
Magnetic Field (Gaus)
1.5 kGy
3200 3300 3400 3500 3600
-1.0x106
-5.0x105
0.0
5.0x105
1.0x106
1.5x106
0 kGy
4 kGy
1.5 kGyES
R s
igna
l Int
ensi
ty (
a.u.
)
Magnetic Field (Gauss)
2 kGy
3200 3300 3400 3500 3600
1.5 kGy
2 kGy
0 kGy
Magnetic Field (Gauss)
4 kGy
3200 3300 3400 3500 3600
-1.0x106
-5.0x105
0.0
5.0x105
1.0x106
1.5x106
4 kGy
1.5 kGy
0 kGy
ES
R s
igna
l Int
ensi
gy (
a.u.
)
Magnetig Fiel d (Gauss)
2 kGy
3200 3300 3400 3500 3600
4 kGy
1.5 kGy
0 kGy
Magnetic Field (Gauss)
2 kGy
3200 3300 3400 3500 3600
1.5 kGy
2 kGy 0 kGy
Campo Magnético (Gauss)
4 kGy
0 days 18 days 30 days 0 days
18 days
28 days
(a) (b) (c)
Each spectrum is displaced vertically for clarity
Fig. 1. Electron spin resonance (ESR) spectra of the fat fraction (visible subcutaneous and intramuscular fat) from selected varieties of Spanish dry-cured ham ((a) Teruel, (b)Serrano, (c) Iberian) during storage (0, 18 and 28 days) and after an E-beam treatment in the range 0–4 kGy (a.u.: arbitrary units).
R. Escudero et al. / Food Chemistry 133 (2012) 1530–1537 1533
ESR signals and the dose absorbed (kGy) during the four weeks ofstorage at 4 �C. However, in the treated Iberian dry-cured ham, thistrend was only observed during the first 8 days after E-beamtreatment.
These results are in line with the findings of several authors(Stachowicz, 1998) which state that free radicals in the dry foodcomponents (such as dry-cured ham fat fractions), persist for long-er periods of time. Several authors (Stachowicz, 1998; Taub, 1984)
Table 1Apparent yields of radical with an ESR signal at g-factor of 2.003 in fat of dry-curedham after E-beam treatment at different doses.
Absorbed dose(kGy)
ESR signal intensity (peak to peak)a
Selected varieties of Spanish dry-cured ham
Teruel Serrano Iberian
0 n.d. n.d. n.d.0.5 2.19 ± 0.7d; b 1.51 ± 0.4d; b 4.65 ± 1.3d; a1 5.46 ± 1.3c; a 7.68 ± 2.1c; a 6.03 ± 1.1c, d; a1.5 7.2 ± 1.1c; b 14.0 ± 2.6b; a 8.3 ± 1.6b, c; b2 13.2 ± 2.9b; b, v 17.7 ± 2.3b; a, b 9.3 ± 2.1b; v3 17.3 ± 3.2a, b; b 25.0 ± 2.8a; a 18.9 ± 3.7a; a, b4 21.0 ± 3.7a; b 30.8 ± 2.9a; a 25.5 ± 2.9a; a, b
n.d.: Not detected.Different letters in the same column indicate significant differences (P < 0.005) a, b,c.Different letters in the same row indicate significant differences (P < 0.005) a, b, c.
a (Arbitrary units � 105).
1 2 3 40
1020
30
0
10
20
30
40
01 2 3 4
010
2030
10
20
30
40
01 2 3 4
010
2030
10
20
30
40
(a)
(b)
(c)
ESR
sig
nal i
nten
sity
(ar
bitr
ary
unit
s x
105
)
Absorbed dose (kGy)Storage ti
me at 4
ºC (d
ay)
Fig. 3. Response surface plot showing the relationship between the E-beamtreatment (absorbed dose) and storage time at 4 �C for the presence of radicalwith an ESR signal at g-value of 2.003 in the fat of Teruel (a), Serrano (b) and Iberian(c) dry-cured hams.
1534 R. Escudero et al. / Food Chemistry 133 (2012) 1530–1537
have reported that the stability of radicals induced by radiationtreatment is very different and varies depending on their locationand structure. Some of them decay within several minutes whileothers decay after several days or even weeks. A very low concen-tration of stabilised paramagnetic species has been detected in ri-gid matrices, but their presence is high enough to be detected byESR, such is the case of irradiated bones (Stachowicz, 1998; Yarkov,Tret’yakov, & Khramov, 2000). In irradiated dried fruits, mush-rooms and other foods of vegetable origin, the ESR signals havebeen described as being unstable and decaying slowly within afew days of storage (Stachowicz, 1998).
In the case of the dry-cured ham fat fraction, the stability of theESR signal at g-factor of 2.003 was probably favored by the limitedoxygen due to the vacuum-package during the irradiation treat-ment and subsequent storage. Following 18 days of storage, onlythe E-beam treated Iberian dry-cured ham samples (Figs. 1 and 2)showed similar spectra to untreated samples (radicals were not de-tected after 28 days of storage at 4 �C). Moreover, this dry-curedham type showed a lower ESR signal intensity than the samplesfrom the white pigs (Serrano and Teruel) treated at the same irradi-ation doses and storage times (Fig. 2). These results could be asso-ciated to the scavenging of the radical because of the high contentof a- and b-tocopherol present in Iberian pigs fed on acorns andpasture (Rey, López-Bote, Daza, & Lauridsen, 2010; Soto et al.,2008). The antioxidant effect of tocopherol is due to its depositionin cellular and subcellular membranes rich in phospholipids(Buckley, 1989) and highly susceptible to oxidative stress. Severalauthors (Rey et al., 2010; Soto et al., 2008) have studied thesusceptibility of meat and meat products to oxidative processes,
02
02 4
0
5
10
15
20
a
b,c
ce
e
ee c
c b
b
c
b
b,c
ee c
c
Absorbed dose (kGy)
Teruel ham
Serr
ESR
sig
nal i
nten
sity
(arb
itrar
y un
its x
105 )
1.5
1.5
Fig. 2. Changes in the radical with an ESR signal at g-value 2.003 in the fat fraction ofcolumn with different letters (a, b, c, d, e) indicates significant differences (P < 0.005).
which depends on the fatty acid composition of lipids, the levelof antioxidants (tocopherol) and its balance in the muscle. Increas-ing a-tocopherol tissue levels have been shown to be an effective
02 4
4e
b
bb
b b
b
ee e
ee
c cb,c
d,e dc
8
18
28
Stor
age tim
e
at4
ºC(d
ay)
ano ham
Iberian ham
1.5
dry-cured ham during storage at 4 �C after E-beam treatment at different doses. A
R. Escudero et al. / Food Chemistry 133 (2012) 1530–1537 1535
method for reducing oxidation in fresh, cooked and stored meat(Buckley, 1989; Soto et al., 2008).
For a better understanding of the effect of the irradiation treat-ment and the storage time on the presence of radicals in the dry-cured ham fat fraction, the estimated response surface for intensity(peak to peak) of the ESR signal at g-factor of 2.003, using the ab-sorbed dose (X1) and the storage time at 4 �C (X2), is shown inFig. 3. The regression equations obtained for the three varietiesof Spanish dry-cured hams were:
(a) Teruel ham, ESR signal intensity ¼ 0:49þ 4:43X1 � 0:12X2þ0:23X2
1 þ 0:004X21 � 0:12X1X2½P < 0:0001; R2 : 0:90; R2 ðadjusted
for d:f :Þ : 0:89�.(b) Serrano ham, ESR signal intensity¼1:89þ7:83X1� 0:60X2
2þ0:21X2
1 þ 0:02X22�0:20X1X2 ½P<0:0001;R2 :0:88;R2ðadjusted
for d:f :Þ : 0:86�.(c) Iberian ham, ESR signal intensity ¼ 1:82þ 6:70X1� 0:71X2
2þ0:33X2
1 þ0:02X22�0:31X1X2 ½P<0:0001;R2 :0:82;R2ðadjusted
for d:f :Þ : 0:81�.
The response surfaces obtained for the samples from white pigshowed a similar radical evolution, thus suggesting that the effectof the absorbed dose was higher than that of the storage time at4 �C. Although, in general, the presence of the alkyl radical de-creased with the increase of storage time after E-beam radiationtreatment, it was only the treated samples from the Iberian pig thatshowed ESR spectra without a signal (according to the regressionequation obtained for treatment at 4 kGy when the storage timeexceeded 22 days). Therefore, the analysis of the ESR spectra offat samples can be used to evaluate the irradiation treatment ofdry-cured ham.
In order to determine the stability of the alkyl radical at high ab-sorbed doses, some dry-cured ham samples were treated at 8 kGyand even at 12 and 16 kGy, above the 10 kGy dose limit recom-mended by international organizations (FAO, IAEA, WHO) and Co-dex Alimentarius (WHO, 1981). The ESR spectra from these
-3,0x106
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ES
R s
igna
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ensi
gy (
a.u.
)
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ES
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igna
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gy (
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)
3200-3,0x106
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0,0
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3,0x106
2 kGy
3 kGy
0.5 kGy
0
ES
R s
igna
l Int
ensi
gy (
a.u.
)
Magnetic3200 3300 3400
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0.0
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3.0x106
0 kGy
0.5 kGy
4 kGy 2 kGy
ES
R s
igna
l Int
ensi
ty (
a.u.
)
Magnetic Field (G)
1.5 kGy
3200 3300
Magnetic Field
(a) (b)
Fig. 4. Electron spin resonance (ESR) spectra of muscle from selected varieties of Spandifferent doses (a.u.: arbitrary units).
samples showed a similar signal shape (data not shown) to theone obtained for samples treated with lower doses. This suggestsan analogous formation and evolution along storage time of theradicals, regardless of the dose absorbed by the fatty material.
3.2. ESR spectrometry of muscle dry-cured ham
The ESR spectra of samples of muscle are shown in Fig. 4. Thespectra from the untreated (0 kGy) and E-beam irradiated (0.5–4 kGy) muscle fractions of the three varieties of the dry-curedhams showed a similar signal shape. No new ESR signals associatedto E-beam treatment in the range 0.5–4 kGy were detected. There-fore, only native or endogenous ESR signals were observed. A largedispersion of data (and high standard deviations) was obtained forthe ESR signal intensity regardless of the dose applied (0–4 kGy)and of the storage time at 4 �C (Table 2), and no significant differ-ences (P > 0.05) were detected between samples of the same typeof dry-cured ham. The ESR spectra from the muscle fraction dif-fered depending on the selected varieties of Spanish dry-curedham considered. The ESR spectra corresponding to the dry-curedhams from white pigs showed two signals at g-factors 1.99 andfrom 2.06 to 2.01 (with a central g-factor of 2.035). The ESR spectraof the muscle fraction from Iberian dry-cured hams only showedthe signal at g-factor of 1.99. These ESR signals from the musclefractions can be attributed to some radicals which are consideredto be stable, (such as several free radicals from myoglobin), espe-cially in food with limited free water (such as dried products likedry-cured ham) in which the dispersion of ions and free radicalsare lower (Taub, 1984). In accordance with previous observations(Møller, Adamsen, & Skibsted, 2003), the ESR signal at g-factor of1.99 has been associated with the presence of different chemicalconformations of nitrosylmyoglobin [MbFe(II)NO]. Nevertheless,the ESR signal at g-factor from 2.06 to 2.01 could be related tothe presence of the dinitrosyl iron–sulphur complex (Fe–NO–S)(Cammack, Shergill, Inalsingh, Ananda, & Martin, 1998) or othermetalloprotein complexes (Brazzolotto et al., 2006).
-3,0x106
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ES
R s
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ty (
a.u.
)
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ES
R s
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l Int
ensi
ty (
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)
3300 3400
1.5 kGy
kGy
Field (Gauss)3200 3300 3400
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0,0
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2,0x106
2,5x106
3,0x106
0.5 kGy
4 kGy 2 kGy
1.5 kGy 0 kGy
ES
R s
igna
l Int
ensi
ty (
a.u.
)
Magnetic Field (Gauss)
3400
4 kGy
2 kGy
0 kGy
0.5 kGy
(Gauss)
1.5 kGy
(c)
Each spectrum is displaced
vertically for clarity
ish dry-cured ham ((a) Teruel, (b) Serrano, (c) Iberian) after E-beam treatment at
Table 2Apparent yields of radicals detected by ESR in muscle of dry-cured ham after E-beamtreatment at different doses.
Absorbeddose (kGy)
ESR signal intensity (peak to peak)a
Selected varieties of Spanish dry-cured ham
Teruel Serrano Iberian
g-Factor
2.035 1.99 2.035 1.99 2.035 1.99
0 14.9 ± 9.7 9.34 ± 7.12 23.8 ± 11.0 20.4 ± 14.1 n.d. 8.01 ± 3.40.5 28.9 ± 10.1 7.42 ± 4.6 39.4 ± 11.3 5.2 ± 3.7 n.d. 18.6 ± 9.71 19.8 ± 8.5 5.9 ± 3.4 34.5 ± 10.2 8.6 ± 4.4 n.d. 10.7 ± 6.51.5 5.91 ± 3.05 14.9 ± 7.1 12.5 ± 7.8 17.8 ± 8.5 n.d. 12.9 ± 7.02 5.49 ± 2.54 14.8 ± 6.6 10.4 ± 6.41 17.1 ± 9.3 n.d. 10.1 ± 6.63 15.5 ± 9.3 5.81 ± 3.7 27.1 ± 9.7 9.8 ± 5.7 n.d. 7.73 ± 4.74 7.35 ± 4.46 17.6 ± 6.6 16.2 ± 6.64 25.3 ± 9.9 n.d. 16.2 ± 7.5
n.d.: Not detected.a (Arbitrary units � 105).
1536 R. Escudero et al. / Food Chemistry 133 (2012) 1530–1537
Free radicals derived from myoglobin have been implicated invarious processes (such as thermolysis, photolysis, enzymaticcatalysis and radiolysis) and in meat systems containing peroxidiz-ing lipids (Nakhost & Karel, 1983; Taub, 1984). Several studies sug-gest that free radicals generated by irradiation can react withmyoglobin or residual haemoglobin which results in changes inthe colour of irradiated samples (Jo, Jin, & Ahn, 2000). Changes inredness induced by irradiation have been the object of studiesin both aerobic and vacuum-packaged raw and cooked meat(Brewer, 2004). However, irradiation has no effect on the colourof meat treated with nitrite or a nitrite-free curing system (pre-formed cooked cured-meat pigment, sodium ascorbate and sodiumtripolyphosphate with or without sodium acid pyrophosphate)(Shahidi, Pegg, & Shamsuzzaman, 1991). This finding could be re-lated with the results obtained in the present work because nonew ESR signals and no significant differences for the ESR signalintensity associated to irradiation treatment were detected. Never-theless, the main problem (Stachowicz, 1998) when using the ESRto study the irradiation effects and the detection of irradiated foodis the appearance of the ESR signals in untreated foodstuffs (nativeor endogenous ESR signal). Since this is the situation of the ESRspectra of the muscle fractions of the dry-cured hams, it is possiblethat the E-beam treatment induced weak ESR signals and, there-fore the native signals and those produced by radiation were com-parable. On the other hand, the influence of irradiation on thenative signals in foods is not well known. It is common for irradi-ated food to have poorly resolved ESR spectra, and to show theenvelope of several EPR signals in one.
Because untreated samples showed no native or endogenousESR signals, the fat fractions of dry-cured ham are considered tobe a better substrate with which to study the formation of theparamagnetic species induced by irradiation. Consequently, thisfraction can be used to evaluate the irradiation effect during pro-cessing and the subsequent storage. In addition, the relative stabil-ity of the ESR signals detected in the fat fractions could allow theuse of ESR spectroscopy as a method with which to detect E-beamtreated dry-cured ham. Extrapolation of the results may be possi-ble to other cured meat products because the types of radicalswould be similar although the predominance of some radicals overothers would depend on the specific endogenous factors of theproduct (i.e., its structure and composition) and the storage condi-tions. Further work is needed in this regard.
4. Conclusions
The ESR spectra from untreated (0 kGy) and E-beam irradiated(0–4 kGy) muscle portions of dry-cured ham were similar. How-
ever, in the ESR spectra from fat fractions a well-resolved tripletsignal corresponding to an alkyl radical was only found in irradi-ated samples. Moreover, a positive correlation was detected be-tween the radiation doses and this ESR signal intensity, althoughit decreased with longer storage time. The successive measure-ments performed at different time intervals allowed the construc-tion of kinetic decay curves for the alkyl radical to be used as anindicator of irradiation in dry-cured ham and to indicate the detec-tion period for this radical. Linear regression models for estimatingthe ESR signal intensity were obtained using the absorbed dose(kGy) and the storage time at 4 �C (days) for the three varietiesof Spanish dry-cured hams.
These results suggest that the analysis of the electron spin res-onance (ESR) spectrum of fat samples can be used to evaluate theirradiation treatment of dry-cured ham.
Acknowledgements
This work was funded by the Projects AGL2007-65235-CO2-02,CARNISENUSA (CSD2007-00016) included in CONSOLIDER-INGE-NIO 2010 and AGL2010-19158.
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