Meat Science 88 (2011) 271279

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Meat Science
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i

Oxidative stability of pork meat lipids as related to high-oleic sunower oil and
vitamin E diet supplementation and storage conditions
Vladimiro Cardenia a, Maria Teresa Rodriguez-Estrada a,, Fabio Cumella a, Luca Sardi b,
Giacinto Della Casa c, Giovanni Lercker a
a
Dipartimento di Scienze degli Alimenti, Alma Mater Studiorum-Universit di Bologna, Viale G. Fanin 40, 40127 Bologna, Italy
b
Dipartimento di Morfosiologia Veterinaria e Produzioni Animali, Alma Mater Studiorum-Universit di Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy
c
CRA Unit di ricerca per la Suinicoltura, Via Beccastecca, 345 41018 San Cesario sul Panaro (MO), Italy

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this research work was to evaluate the oxidative stability of pork meat lipids as related to dietary
Received 28 May 2010 supplementation with high-oleic sunower oil (HOSO) and/or -tocopheryl acetate (VE), as well as the
Received in revised form 16 December 2010 inuence of storage conditions. Four different diets (control; HOSO; VE; HOSO + VE), were fed to swines
Accepted 26 December 2010
until slaughtering. Meat slices were packed in vessels with transparent shrink lm and exposed to white
uorescent light for 3 days at 8 C. HOSO supplementation increased oleic acid content of pork meat. The
Keywords:
Pork meat
highest levels of peroxide value (PV) and cholesterol oxidation products (COPs) were detected in the
Lipid oxidation control group, whereas HOSO-enriched diets displayed the highest thiobarbituric reactive substance
Cholesterol oxidation (TBARs) content. After storage under light exposure, pork meat slices exhibited a decrease of PV, which
Photosensitized oxidation resulted in an increasing trend of TBARs and COPs. Feeding enrichment with both HOSO and vitamin E can
Diet be, therefore, used as an appropriate supplementation strategy to produce pork meat with a suitable
Storage oxidative stability.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
The pork meat market has, in the past few years, been subjected to
several changes inuenced by consumer needs, which have addressed
the production towards leaner and healthier meat. Special strategies
have been followed to modify the lipid fraction of pork meat, which is
known to greatly affect its overall quality. Lipid composition of pork
meat varies depending on the type muscle and muscle ber, and it is
inuenced by many factors, such as animal species, genotype, rearing
and feeding (Wood et al., 2004). Recently, swine feeding has been
formulated with a higher content of natural sources of unsaturated fatty
acids (UFA), such as n 3 series or conjugated linoleic acid (CLA), due to
human health concerns (Boselli, Pacetti, Lucci, Di Lecce, & Frega, 2008).
In fact, the low incidence of heart disease and low levels of plasma
cholesterol of the Mediterranean population have been correlated to an
increased intake of monounsaturated fat (Simopoulos, 2006) suggesting
that, in general, people should increase their relative intakes of
monounsaturated lipid. Diets rich in oleic acid have shown to reduce
levels of low density lipoproteins (LDL), without decreasing high-
density lipoproteins (HDL) in plasma and leading to a simultaneous
Corresponding author. Tel.: + 39 051 2096011; fax: + 39 051 2096017.
E-mail address: maria.rodriguez@unibo.it (M.T. Rodriguez-Estrada).
decline of oxidized LDL (Noori, Darabi, Rahimipour, Rahbani, & Abadi,
2009; Stein, Dabach, Ben-Naim, Halperin, & Stein, 2008). Since a large
proportion of the saturated fat intake in Western society arises from
animal fat, a greater unsaturation of the pork meat lipid fraction is,
therefore, expected to have a general positive impact on human health.
Several researchers have studied the effect of diets supplemented with
high UFA-containing fats on the fatty acid (FA) prole of pork meat
lipids. Diets enriched with vegetable oils (such as sunower oil, soybean
oil or corn oil) that contain an elevated UFA percentage should result in
healthier products for consumers (Mitchaothai et al., 2007); however,
the greater degree of FA unsaturation increases meat susceptibility
towards oxidation (Boselli et al., 2005). Lipid oxidation in meat products
is mainly initiated in the highly unsaturated phospholipid fraction of cell
membranes (Boselli et al., 2008), which contain other unsaturated
liposoluble molecules, such as cholesterol, that are also prone to
oxidation. Cholesterol oxidation products (COPs) have been largely
studied in several animal products (Kerry, Gilroy, & O'Brain, 2002;
Paniangvait, King, Jones, & German, 1995), since they are likely to be
involved in lipid metabolism, various chronic and degenerative diseases
and disturbance of cell functionality (Garcia-Cruset, Carpenter, Codony,
& Guardiola, 2002; Osada, 2002; Schroepfer, 2000). However, the
oxidative stability of muscle tissue also depends on the anti/prooxidant
balance (Decker & Xu, 1998), so dietary supplementation with
antioxidants, such as -tocopheryl acetate, might increase the oxidative

0309-1740/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2010.12.034
272 V. Cardenia et al. / Meat Science 88 (2011) 271279

stability of pork meat lipids (Boselli et al., 2008; Eder, Mller, Kluge,
Hirche, & Brandsch, 2005; Guo et al., 2006). Previous studies reported
that feeding high levels of vitamin E (VE) (300700 mg/kg) resulted in
reduced lipid oxidation as measured by thiobarbituric acid reactive
substances (TBARs) (Corino, Oriani, Pantaleo, Pastorelli, & Salvatori,
1999; Jensen et al., 1997).
Besides dietary supplementation, storage conditions (such as
irradiation and packaging) play a determinant role on the onset of
lipid and cholesterol oxidation in meat (Boselli, Rodriguez-Estrada,
Fedrizzi, & Caboni, 2009; Boselli, Rodriguez-Estrada, Ferioli, Caboni, &
Lercker, 2010). In particular, under light exposure, excited singlet
oxygen (1O2) is formed and directly reacts with the double bond of
unsaturated lipids by a nonradical mechanism and without any
induction period (Lercker & Rodriguez-Estrada, 2002). Cholesterol
photosensitized oxidation has been evaluated in different muscle
tissues as related to packaging and storage conditions (Boselli et al.,
2005; Boselli et al., 2009; Boselli et al., 2010). In general, white
uorescent light induced a higher COP formation, which could be
partially masked by the oxygen concentration (Boselli et al., 2009) or
reduced when a suitable protective lm has been employed (Boselli
et al., 2010). To the authors' knowledge, no study has been performed
on the photosensitized oxidation of pork meat, as related to the pig's
dietary supplementation.
The aim of the current work was to evaluate the effect of both dietary
supplementation (with high-oleic sunower oil and/or -tocopheryl
acetate) and light exposure on the oxidative stability of pork meat slices
during storage in a commercial retail bench refrigerator.
2. Materials and methods
2.1. Reagents and solvents
Ammonium thiocyanate (NH4SCN, 97.5%), barium chloride
dehydrate (BaCl22H2O, 99%), ethylenediamine-tetraacetic acid
(EDTA) disodium salt (100% 1%), iron(II) sulfate heptahydrate
(FeSO47H2O, 99.0%), trichloroacetic acid (99%), diethyl ether,
pyridine, hexamethyldisilazane, trimethylchlorosilane and double
distilled water, were supplied by Carlo Erba Reagenti (Rodano,
Italy). Chloroform, n-hexane, methanol and ethanol were purchased
from Merck (Darmstadt, Germany). Anhydrous sodium sulfate and
potassium hydroxide were purchased from BDH (Poole, England) and
Prolabo (Fontenay, France), respectively. The standard mixture of
fatty acid methyl esters (GLC 463) was supplied by Nu-Chek (Elysian,
MN, USA). Tridecanoic acid methyl ester, cholest-5-en-3,19-diol
(19-hydroxycholesterol) (purity: 99%) and cholest-5-en-3,7-diol
(7-hydroxycholesterol, 7-HC) (purity: 99%) were purchased from
Steraloids (Newport, RI, USA). (24S)-ethylcholest-5,22-dien-3-ol
(stigmasterol) (purity: 95%), cholest-5-en-3,7-diol (7-hydroxy-
cholesterol, 7-HC) (purity: 90%), 5,6-epoxy-cholestan-3-ol
(-epoxycholesterol, -CE) (purity: 87%), 5,6-epoxy-cholestan-
3-ol (-epoxycholesterol, -CE) (purity: 80%), cholestan-3,5a,6-
triol (cholestanetriol) (purity: 99%), cholest-5-en-3-ol-7-one
(7-ketocholesterol, 7-KC) (purity: 99%) and cholest-5-en-3-ol
(cholesterol) (purity: 99%), were purchased from Sigma (St. Louis,
MO, USA). No. 1 lters (70 mm diameter) were used (Whatmann,

Table 1
Percent, chemical and fatty acid composition of three different feed formulations (g/kg) used, according to the swine weight. Vitamin E supplementation is not reported in this table.

Ingredients Live weight

7090 kg 90120 kg 120160 kg

Control HOSO Control HOSO Control HOSO

Maize meal (g/kg) 550.0 530.0 550.0 530.0 550.0 530.0

Barley meal (g/kg) 173.5 180.8 189.2 195.1 204.4 210.7
Wheat bran (g/kg) 100.0 60.0 100.0 60.0 100.0 60.0
Soybean meal (g/kg) 145.0 165.0 130.0 150.0 115.0 135.0
HOSO (g/kg) 30.0 30.0 30.0
HCl lysine (g/kg) 2.0 2.4 1.8 2.1 1.6 1.8
DL-Methionine (g/kg) 0.5 0.5 0.5 0.2
Calcium carbonate (g/kg) 12.0 11.0 12.0 11.5 12.0 11.5
Dicalcium phosphate (g/kg) 8.0 11.0 8.0 11.5 8.0 11.5
Sodium chloride (g/kg) 4.0 4.0 4.0 4.0 4.0 4.0
Premixa (g/kg) 5.0 5.3 5.0 5.3 5.0 5.3

Chemical analysis
Dry matter (%) 89.2 89.2 89.4 89.6 88.2 88.5
Crude protein (%) 14.2 14.4 13.6 13.9 13.1 13.3
Ether extract (%) 2.69 5.25 2.69 5.24 2.96 5.67
Crude ber (%) 4.26 4.03 4.24 4.01 4.22 3.99
Ash (%) 5.08 5.02 4.82 5.10 4.99 5.10
Lysine (%) 0.80 0.86 0.75 0.80 0.70 0.75
Net energy (MJ/kg) 2288 2454 2292 2457 2296 2461
Lys/NE 3.50 3.51 3.28 3.27 3.05 3.04

Fatty acids
C14:0 (%) 0.002 0.003 0.002 0.003 0.007 0.007
C16:0 (%) 0.378 0.471 0.337 0.405 0.584 0.782
C16:1 (%) 0.003 0.006 0.003 0.005 0.007 0.010
C18:0 (%) 0.063 0.150 0.055 0.125 0.076 0.139
C18:1 (%) 0.651 3.082 0.612 2.779 0.631 2.821
C18:2 n 6 (%) 1.103 1.361 1.011 1.135 1.544 1.704
C18:3 n 3 (%) 0.052 0.070 0.041 0.049 0.093 0.142
C20:0 (%) 0.009 0.019 0.009 0.016 0.008 0.013
C20:4 (%) 0.007 0.021 0.009 0.014 0.008 0.012
a
Vitamins and minerals provided the following per kilogram of premix: vitamin A, 3,000,000 IU; vitamin D3, 400,000 IU; vitamin E acetate (-tocopherol 91%), 10,000 mg; vitamin K3
(menadione), 500 mg; vitamin PP (niacinamide), 5000 mg; vitamin B1 (thiamin mononitrate), 400 mg; vitamin B2, 1000 mg; D-pantothenic acid, 3000 mg; vitamin B6 (piridossin chloridrate),
800 mg; vitamin B12, 6 mg; folic acid, 200 mg; vitamin H (D-biotin), 30 mg; choline chloride, 80,000 mg; iron (ferrous sulfate monohydrate), 30,000 mg; copper (copper sulfate pentahydrate),
3000 mg; manganese (manganese oxide), 5000 mg; cobalt (basic cobalt carbonate monohydrate), 80 mg; zinc (zinc oxide), 20,000 mg; iodine (calcium iodide anhydrous), 300 mg; and
selenium (sodium selenite), 20 mg.
 V. Cardenia et al. / Meat Science 88 (2011) 271279
Maidstone, England). Silica solid-phase extraction (SPE) cartridges
(Strata NH2-55 mm, 500 mg/3 ml) from Phenomenex (Torrence, CA,
USA) were utilized for sterol oxide purication.
The phosphate buffer used for the TBARs determination, was
prepared adding 65.8 ml of 0.5 M NaH2PO4 and 111 ml of 0.5 M
Na2HPO4H2O (water solutions) in a 500 ml volumetric ask. pH was
controlled, taken to neutrality (either with 1N HCl or 1N NaOH
solutions), and made up to volume with water. To delay oxidation and
prevent the pro-oxidative effect of metals, proper amounts of EDTA
and ascorbic acid were added to the phosphate buffer to reach a nal
concentration of 0.1% (w/v) of both of them.
The silylation mixture was prepared with dried pyridine,
hexamethyldisilazane and trimethylchlorosilane at a ratio of 5:2:1
by volume.
2.2. Sampling, packaging and photoxidation experiment
A total of 32 Duroc Large White pigs (16 castrated males and 16
intact females) of initial average body weight of 70 kg, were
homogeneously (on the basis of litter, age and body weight) allotted
to four experimental groups:
1) control group (C) (feed containing maize/soybean without oil
addition);
2) O group (control diet supplemented with 3% of high-oleic
sunower oil (HOSO) (85% oleic acid and 6.9% linoleic acid));
3) E group (control diet supplemented with 250 mg/kg -tocopheryl
acetate);
4) OE group (control diet supplemented with 3% HOSO + 250 mg/kg
-tocopheryl acetate).
To meet the pigs' requirements, three different feed formulations
were used according to the swine weight (7090 kg, 90120 kg and
120160 kg). Table 1 reports the percent composition and chemical
Fig. 1. Sampling design for experiment.
273
analysis of such diets. The diets (CE vs OOE) had different amount of
lysine and net energy (NE), whilst lysine to NE ratio was the same
across the four groups. In addition, pigs belonging to the oil groups
(O and OE) were given a lower amount of feed (91%) than the non-
oil groups (C and E). Feed was provided as liquid (meal to water ratio
1:3) and pigs were fed at the rate of 9% of their metabolic live weight
up to a maximum of 3.3 kg per head per day. Pigs were slaughtered at
about 160 kg live weight, after a 12-h fast. At 24-h post mortem, the
loin (longissimus dorsi muscle) of the right side was cut and
subsamples were obtained; the meat slices were 1 cm thick and had
a weight that ranged between 100 and 150 g. Two meat slices were
placed in a polyethylene tray without blotter, which was wrapped with
a transparent shrink lm (74 m thickness) with an oxygen transmis-
sion rate at 20 C and 65% relative humidity of 1.5 ml/m2 dbar (ASTM,
1988). The packed slices were subjected to the following storage
conditions:
1) sixteen vessels were immediately frozen ( 20 C), representing
T0 samples;
2) sixteen vessels were stored in the dark at 8 C for 3 days (T3D);
3) sixteen vessels were stored at 8 C under a daylight lamp for 3 days
(T3L).
A bench refrigerator was used to keep samples at 8 C, as in a retail
shop. The daylight lamps had a color temperature and power of
3800 K and 36 W (Osram, Milan, Italy), respectively, and were located
1.5 m above the samples. After photosensitized oxidation (T3D and
T3L), packed meat slices were stored at 18 C until analysis.
The experimental plan and sample legend are displayed in Fig. 1.
2.3. Lipid extraction
The lipids were extracted according to a modied version of the
method described by Folch, Lees, and Sloane-Stanley (1957). The
274 V. Cardenia et al. / Meat Science 88 (2011) 271279

extraction procedure was the same reported in a previous work
(Boselli, Velazco, Caboni, & Lercker, 2001). The frozen samples were
minced and 15 g were homogenized with 200 ml of a chloroform:
methanol solution (1:1, v/v) in a glass bottle with screw-cap. The
bottle was kept in an oven thermostated at 60 C for 20 min before
adding 100 ml chloroform. After 3 min of homogenization, the
content of the bottle was ltered through lter paper to eliminate
the solid residue, which consisted mostly of proteins. The ltrate was
mixed thoroughly with 100 ml of a 1 M KCl solution and left overnight
at 4 C in order to obtain phase separation. The lower phase
containing the lipids was collected and dried with a rotary evaporator.
The fat content was determined gravimetrically. Two lipid extractions
were performed for each sample.
temperatures were both set at 250 C. Helium was used as carrier
gas at a constant ow of 0.7 ml/min. The split ratio was 1:70.
Tridecanoic acid methyl ester was used as internal standard for FA
quantication, and peak identication was carried out by comparing
the peak retention times with those of the GLC 463 FAME standard
mixture. The GC response factor of each fatty acid was calculated by
using the GLC 463 FAME standard mixture and the internal standard
(C13:0). The limit of detection (LOD) of FAMEs was 0.004 mg,
whereas the limit of quantication (LOQ) was 0.01 mg. LOD and
LOQ were calculated as signal-to-noise ratios equal to 3:1 and 10:1,
respectively.
2.7. Gas-chromatographic determination of total sterols and total COPs

2.4. Spectrophotometric determination of peroxide value A 200 mg lipid subfraction of the Folch extract was added with
known amounts of the internal standard solution (0.140 mg of
PV was determined in 50 mg of lipid extract, as suggested by Shantha betulinol and 0.0125 mg of 19-hydroxycholesterol for the determi-
and Decker (1994). This method is based on the ability of peroxides to nation of total sterols and COPs, respectively). Subsequently, the
oxidize ferrous ions to ferric ions. Ammonium thiocyanate reacts with sample was dried under nitrogen and treated with 10 ml of 1N KOH
ferric ions, resulting in a colored complex that can be measured solution in methanol to perform saponication at room temperature
spectrophotometrically. PV was evaluated at 500 nm with a double- for 18 h (Sander, Addis, Park, & Smith, 1989). For the extraction of the
beam UVvisible spectrophotometer (Jasco model V-550, Jasco Inter- unsaponiable matter, 10 ml of water and 10 ml of diethyl ether were
national, Tokyo, Japan), and it was calculated from the absorbance. For added to the samples, which were shaken, and the diethyl ether
the quantitative determination of PV, a Fe(III) standard calibration fraction was separated; the extraction with 10 ml of diethyl ether was
curve was used with a concentration range of 0.15 g/ml repeated twice. The three portions of diethyl ether were pooled,
(y = 0.0282x 0.0003; r2 = 0.999). PV was expressed as milliequi- treated with 5 ml of a 0.5 N KOH solution, and extracted. The resulting
valents of O2 per kilogram of fat. Three replicates were run per ethereal extract was washed twice with 5 ml of water. The ether
sample. solution was nally evaporated in a rotary evaporator, after
elimination of excess water by addition of anhydrous sodium sulfate.
2.5. Spectrophotometric determination of thiobarbituric reactive One-tenth of the unsaponiable matter was used for the determina-
substances (TBARs) tion of total sterols, whereas the remaining part was utilized for COP
analysis.
TBARs value was determined in 2 g of sample (ground meat)
according to a modied method of Witte, Krause, and Bailet (1970). Table 2
This method is based on the reaction between the thiobarbituric acid Fatty acid composition of raw pork meat (as % of total fatty acids), as related to diets and
with aldehydes that derive from secondary oxidation of lipids present in storage conditions.
the sample, resulting in a colored complex that can be measured Factor C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:4
spectrophotometrically. TBARs were evaluated at 530 nm with a n6 n3
double-beam UVvisible spectrophotometer (Jasco model V-550, Jasco Diet
International, Tokyo, Japan), and they were calculated from the C 1.4ab 26.1a 2.8 13.7a 41.7b 7.3 0.3 0.2 0.6bc
absorbance. For the quantitative determination of TBARs, a 1,1,3,3- O 1.5ab 25.4a 2.5 12.6ab 43.8a 7.5 0.3 0.2 0.5c
tetramethoxypropane standard calibration curve was used with a E 1.5a 25.8a 2.9 13.9a 40.7b 8.3 0.3 0.2 0.7a
OE 1.4b 24.1b 2.5 11.8b 44.7a 8.1 0.3 0.2 0.7ab
concentration range of 0.045-0.113 g/ml (y = 0.0087x 0.0051;
Stat. signif. ** *** *** *** ***
r2 = 0.999). TBARs value was expressed as milligrams of malonylalde-
hyde (MDA) per kilogram of sample. Three replicates were run per Storage condition
sample. T0 1.4 25.2 2.5 13.3 42.4 8.4a 0.3 0.2 0.6
T3D 1.4 25.1 2.7 12.7 43.3 7.7ab 0.3 0.2 0.6
T3L 1.5 25.7 2.8 13 42.5 7.4b 0.3 0.2 0.6
2.6. Gas-chromatographic determination of total fatty acids Stat. signif. *

About 20 mg of lipid extract were methylated with 200 l of Diet storage condition
diazomethane (Fieser & Fieser, 1967); 1.01 mg of tridecanoic acid C T0 1.4 26.0 2.8 13.6 41.7bcd 7.5 0.3 0.1 0.7abc
O T0 1.4 25.0 2.4 12.6 44.0ab 8.1 0.3 0.2 0.5bc
methyl ester was added (as internal standard), and the mixture was
E T0 1.5 26.1 2.6 14.9 39.2d 9.3 0.3 0.2 0.6bc
transmethylated with 40 l of 2N KOH in methanol (European OE T0 1.4 23.8 2.3 12 44.7a 8.7 0.3 0.2 0.6bc
Commission. Allegate X. B. regulation L128/14, 2002), vortexed for C T3D 1.4 25.9 2.7 13.7 41.7bc 7.4 0.3 0.2 0.5c
1 min, left standing for 5 min, and centrifuged at 1620 g for 5 min. O T3D 1.5 25.6 2.5 12.8 43.4ab 7.5 0.3 0.2 0.5c
Supernatant was transferred to a vial before being injected into a gas E T3D 1.5 25.0 3.1 12.6 43.0ab 7.5 0.3 0.2 0.8a
OET3D 1.4 24.0 2.5 11.8 44.9a 8.2 0.3 0.2 0.7abc
chromatograph coupled to a ame ionization detector (GC-FID).
C T3L 1.4 26.3 2.8 13.7 41.7bcd 7.1 0.3 0.2 0.6bc
The GC-FID instrument was a GC8000 series (Fisons Instruments, O T3L 1.5 25.5 2.7 12.5 43.9ab 7 0.3 0.2 0.6bc
Milan, Italy) interfaced with a computerized system for data E T3L 1.6 26.2 2.9 14.3 40.0 cd 8 0.3 0.2 0.7abc
acquisition (Chromcard Data System, ver. 2.3.1, Fisons Instruments). OE T3L 1.4 24.6 2.8 11.6 44.6a 7.5 0.3 0.2 0.7ab
Stat. signif. ** ***
A J&W HP88 fused-silica column (100 m 0.25 mm 0.2 m lm
thickness) (Agilent Technologies, Santa Clara, CA, USA) coated with Each value is the average of three replicates for each diet storage condition.
88% cyanopropyl aryl siloxane was used. Oven temperature was Abbreviations: C, control diet; O, diet supplemented with HOSO; E, diet supplemented
with VE; OE, diet supplemented with HOSO+ VE; T0, fresh raw meat; T3D, samples stored
programmed from 100 C to 180 C at a rate of 3 C/min, kept at 180 C at dark for 3 days; T3L, samples exposed at light for 3 days; Stat. signif., statistical
for 10 min, and then taken to 240 C at a rate of 3 C/min; the nal signicance (ns, not signicant; *, signicant at P 0.05; **, signicant at P 0.01; ***,
temperature was kept for 30 min. The injector and detector signicant at P 0.001); a, b, c, d, statistically different means (Tukey's test; P 0.05).
 V. Cardenia et al. / Meat Science 88 (2011) 271279 275

Table 3 standards, a solution of derivatized sterol standards, the silylated

Fatty acid classes (as % of total fatty acids) and their ratios present in raw pork meat, as sterol and puried COP fractions from pork meat samples, was
related to diets and storage conditions.
performed in the split mode, at a 1:10 split ratio. The lament
Factor SFA MUFA UFA/SFA PUFA/MUFA n 6/n 3 PUFA emission current was 70 eV. A mass range from m/z 40 to 650 was
Diet scanned at a rate of 1500 amu/s.
C 41.8a 49.4bc 1.4b 0.2 29.5a 8.8 Sterols and COPs were quantied with the internal standard
O 40.7a 50.5b 1.5b 0.2 28.9ab 8.8 method, using betulin and 19-hydroxycholesterol, respectively. Their
E 41.8a 48.2c 1.4b 0.2 29.5a 9.9
response factors were evaluated with respect to the corresponding
OE 38.0b 52.2a 1.6a 0.2 27.1b 9.8
Stat. signif. *** *** *** * internal standards. GC-FID LOD and LOQ of sterols were 1.96 and
6.53 g, respectively, whereas those of COPs were 0.25 and 0.84 g,
Storage condition respectively. LOD and LOQ were calculated as signal-to-noise ratios
T0 40.5 49.5 1.5 0.2 28.6 10.0a equal to 3:1 and 10:1, respectively.
T3D 40.3 50.5 1.5 0.2 28.4 9.1ab
T3L 40.8 50.2 1.5 0.2 29.3 9.0a
Stat. signif. *
2.8. Statistical analysis
Diet storage condition
C T0 41.6 49.3cd 1.4 0.2 29.9 9.1
The data are reported as mean values of three independent replicates
O T0 39.6 50.9abc 1.5 0.2 27.9 9.5
E T0 43.1 46.1e 1.3 0.2 30.0 10.8 (n =3) of each analytical determination (Tables 2-5). Factorial ANOVA
OE T0 37.9 51.7abc 1.6 0.2 26.5 10.4 was performed for data from the crossed treatments, in order to study the
C T3D 41.7 49.4bcd 1.4 0.2 28.5 8.8 inuence of the different diets and storage conditions, as well as their
O T3D 42.3 49.2cd 1.4 0.2 29.3 8.5 interaction, on several composition and oxidative parameters (FA
E T3D 39.6 51.0abc 1.5 0.2 28.9 9.3
composition, cholesterol, campesterol, -sitosterol, total sterols, PV,
OE T3D 37.7 52.4ab 1.7 0.2 26.5 9.9
C T3L 42.1 49.4cd 1.4 0.2 30.2 8.5 TBARs, 7-HC, 7-HC, -CE, -CE, 7-KC, total COPs, oxidized cholesterol).
O T3L 40.1 51.5abc 1.5 0.2 29.6 8.4 Tukey's honest signicance test was carried out at a 95% condence level
E T3L 42.7 47.6de 1.3 0.2 28.3 9.7 (p 0.05), in order to separate means of statistically-different parameters
OE T3L 38.3 52.5a 1.6 0.2 28.3 9.2
and interactions. Pearson correlation coefcients (=0.05) were used to
Stat. signif. ***
examine possible relationships between the lipid composition and
Each value is the average of three replicates for each diet storage condition. oxidative parameters of pork meat slices over the whole data set
Abbreviations: C, control diet; O, diet supplemented with HOSO; E, diet
(n =36). Statistical analysis of the data was performed by SPSS 16.0.1
supplemented with VE; OE, diet supplemented with HOSO + VE; T0, fresh raw meat;
T3D, samples stored at dark for 3 days; T3L, samples exposed at light for 3 days; Stat. (2007, IBM-SPSS Inc., Chicago, Illinois, USA).
signif., statistical signicance (ns, not signicant; *, signicant at P 0.05; **, signicant
at P 0.01; ***, signicant at P 0.001); a, b, c, d, statistically different means (Tukey's
test; P 0.05).

Table 4
Effects of diets and storage conditions on the average cholesterol, campesterol, -
The determination of total sterols (sum of free and esteried) was sitosterol, and total sterol contents (as mg/kg of lipids) of raw pork meat.
achieved by GC-FID after silylation (Sweeley, Beutley, Mokita, & Wells, Factor Cholesterol Campesterol -sitosterol Total sterols
1963). The GC instrument was an HRGC 5300 model (Carlo Erba,
Diet
Milan, Italy), which was equipped with a splitsplitless injector, a C 8051.4 118.7a 63.7 8265.2
ame ionization detector and interfaced with a computerized system O 7740.0 91.2b 33.6 7881.4
for data acquisition (Turbochrom Navigator software Ver.6.1.1.0.0: E 7580.7 105.2ab 45.6 7747.4
K20, Perkin Elmer, Norwalk, CT, USA). A CP-SIL 5CB Low Bleeds/MS OE 7497.5 94.2b 40.0 7657.6
Stat. signif. ns * ns ns
(30 m 0.32 mm i.d. 0.25 m lm thickness) (Varian Chrompack,
Middelburg, The Netherlands) was used. The oven temperature was Storage condition
programmed from 265 C to 280 C at 0.5 C/min and then from T0 6642.6 86.6b 36.6 6788.5
280 C to 325 C at 4 C/min; the injector and detector temperatures T3D 8180.6 110.4a 51.4 8367.4
were both set at 325 C. Helium was used as carrier gas at a ow of T3L 8329.1 109.9a 50.1 8507.8
Stat. signif. ns * ns ns
2.9 ml/min; the split ratio was 1:15.
Regarding the determination of COPs, the remaining 9/10 of the Diet storage condition
unsaponiable matter were puried by NH2-SPE (Rose-Sallin, Hugget, C T0 8516.4 123.0 57.6 8732.2
Bosset, Tabacchi, & Fay, 1995); COPs were eluted with acetone. The O T0 7196.7 83.9 25.2 7329.0
E T0 5578.9 74.4 30.9 5695.3
puried fraction was then silylated (Sweeley et al., 1963), dried under
OE T0 5278.3 65.1 32.7 5406.5
nitrogen stream and dissolved in 50 l of n-hexane. One microliter of C T3D 7922.6 119.7 74.5 8151.2
the silylated sterol oxides was injected into GC-FID under the same O T3D 7826.3 91.8 37.8 7977.9
conditions as reported for the determination of total sterols. E T3D 8120.0 115.9 45.0 8300.7
The identication of sterols and COPs was conrmed by comparing OE T3D 8853.4 114.3 48.4 9040.0
C T3L 7715.3 113.5 59.1 7912.2
their retention times and mass spectra with those of sterols and COP
O T3L 8197.0 97.8 39.0 8346.3
standards. GC-mass spectrometry (MS) identication of sterols and E T3L 9043.2 125.2 60.8 9246.3
COPs was performed with a GC Hewlett-Packard 6890 coupled to a OE T3L 8360.8 103.2 39.0 8526.2
5973 mass selective detector (Agilent Technologies, Palo Alto, CA, Stat. signif. ns ns ns ns
USA). The system was tted with a capillary SPB-5 column Each value is the average of three replicates for each diet storage condition.
(30 m 0.25 mm i.d. 0.25 m lm thickness) (Supelco, Bellefonte, Abbreviations: C, control diet; O, diet supplemented with HOSO; E, diet
PA, USA), and helium was used as the carrier gas (1 ml/min). The oven supplemented with VE; OE, diet supplemented with HOSO + VE; T0, fresh raw meat;
T3D, samples stored at dark for 3 days; T3L, samples exposed at light for 3 days; Stat.
temperature was programmed from 250 to 310 C at 0.8 C/min. The signif., statistical signicance (ns, not signicant; *, signicant at P 0.05; **, signicant
injector and transfer line temperatures were set at 310 and 280 C, at P 0.01; ***, signicant at P 0.001); a, b, c, d, statistically different means (Tukey's
respectively. Manual injection of 1 l of a solution of derivatized COP test; P 0.05).
276 V. Cardenia et al. / Meat Science 88 (2011) 271279

Table 5
Effects of diets and storage conditions on the average of peroxide value (PV, meq O2/kg fat), TBARs (mg MDA/kg meat), single and total COP contents (mg/kg fat), and cholesterol
oxidation ratio (OR, %) of raw pork meat.

Factor PV TBARs 7-HC 7-HC -CE -CE 7-KC Total COPs Cholesterol OR

Diet
C 4.4 1.1 2.0a 2.9a 3.8a 3.0a 4.0a 15.7a 0.2a
O 2.8 1.3 1.2b 1.8b 2.4b 2.3b 2.5b 10.2b 0.1b
E 1.9 0.8 1.3ab 2.1ab 2.3b 2.4ab 2.2b 10.4b 0.1b
OE 2.3 0.8 1.6ab 2.3ab 2.6b 2.3b 2.4b 11.3b 0.2ab
Stat. signif. ns ns * * ** * *** *** **

Storage condition
T0 1.9 0.3b 1.7 2.1 2.9 2.4ab 2.9 12.0 0.2a
T3D 3.3 0.8b 1.3 2.2 2.5 2.3b 2.8 11.0 0.1b
T3L 1.9 2.0a 1.7 2.6 2.9 2.8a 2.7 12.7 0.2b
Stat. signif. ns *** ns ns ns * ns ns **

Diet storage condition

C T0 2.0 0.4 2.0 2.7 3.4ab 2.8 3.7abc 14.6ab 0.2ab
O T0 2.8 0.3 1.4 1.4 2.7ab 2.3 2.5abc 10.1ab 0.1ab
E T0 1.1 0.2 1.4 2.2 2.7ab 2.4 2.2bc 10.8ab 0.2ab
OE T0 1.5 0.2 1.9 2.3 2.9ab 2.3 3.1abc 12.5ab 0.3a
C T3D 5.4 0.7 2.0 3.2 3.9a 3.0 4.4a 16.3a 0.2ab
O T3D 3.5 1.0 1.3 2.0 2.3ab 2.0 2.4abc 9.9ab 0.1b
E T3D 2.7 0.7 1.1 1.6 1.6b 2.2 2.0bc 8.5b 0.1b
OE T3D 1.8 0.6 0.9 1.9 2.1ab 2.2 2.3abc 9.3ab 0.1b
C T3L 6.0 2.0 2.1 2.9 4.1a 3.2 3.8ab 16.1a 0.2ab
O T3L 2.0 2.7 1.3 2.0 2.3ab 2.6 2.7abc 10.8ab 0.1ab
E T3L 1.9 1.6 1.3 2.7 2.5ab 2.9 2.4abc 11.8ab 0.1b
OE T3L 3.7 1.5 2.1 2.7 2.9ab 2.6 1.7c 12.1ab 0.1ab
Stat. signif. ns ns ns ns * ns *** ** ***

Each value is the average of three replicates for each diet storage condition. Abbreviations: C, control diet; O, diet supplemented with HOSO; E, diet supplemented with VE; OE, diet
supplemented with HOSO + VE; T0, fresh raw meat; T3D, samples stored at dark for 3 days; T3L, samples exposed at light for 3 days; Stat. signif., statistical signicance (ns, not
signicant; *, signicant at P 0.05; **, signicant at P 0.01; ***, signicant at P 0.001); a, b, c, d, statistically different means (Tukey's test; P 0.05).

3. Results and discussion
3.1. Total fatty acid composition
Lipid content of pork muscle ranged from 3.8 to 10.3% (data not
shown), due to different animals' sex and fat trimming of meat slices.
Table 2 reports the effects of diet and storage conditions on the fatty
acid composition (expressed as percent) of the meat. In general, the
most abundant FA was oleic acid (~3945% of total FA), followed by
palmitic (~ 2426%), stearic (~ 1215%), palmitoleic (~ 3%), and
myristic acids (~12%). Among long-chain PUFA, arachidonic acid
was the most abundant, whereas docosahexaenoic (DHA) and
eicosapentaenoic (EPA) acids were detected at trace levels (b0.01 mg).
FA composition data here obtained agreed with those reported by Eder
et al. (2005), as well as with those found by Miller, Shackelford, Hayden,
and Reagan (1990) in meat from swine fed high-oleic lipids or oils.
The factorial analysis of the single FA content shows that when
HOSO was added to swine feeding (diets O and OE), the oleic acid
level in meat was signicantly higher with respect to samples
obtained with the other two diets (control and E); however, there
was a signicant interaction of samples obtained with OE diet under
all storage conditions, so oleic acid levels were affected by both diet
and storage conditions. Diets had also signicant effects on the
amount of myristic, palmitic, stearic and arachidonic acids, the rst
three FA being lower in samples obtained with OE diet, whereas
arachidonic acid was more abundant in meat from E and OE diets;
however, the latter FA also displayed a signicant interaction
(E T3D). Regarding the storage condition factor, no signicant
differences were observed except for the linoleic acid content,
which signicantly decreased when meat was subjected to photo-
sensitized oxidation; this might be related to the fact that unsaturated
FA are more prone to oxidation.
Saturated (SFA), monounsaturated (MUFA), and polyunsaturated
(PUFA) fatty acids accounted for 3843, 4653, and 811% of total FA,
respectively (Table 3), which were in agreement with data reported
by Mitchaothai et al. (2007). However, some researchers observed
different distribution of FA classes in pork meat obtained with and
without -tocopheryl acetate feed supplementation (Mason et al.,
2005); in fact, SFA and MUFA contents were lower (3537% and 16
23%, respectively) than those found in the present study, which might
be due to different feeding (composition, amount and time of
supplementation) and swine characteristics (breed, sex, age). On
the other hand, UFA/SFA ratio ranged from 1.3 to 1.7, whereas PUFA/
MUFA ratio was similar in all pork meat slices (0.2); the results of both
FA ratios are similar to those reported by Guo et al. (2006). The n 6/
n 3 FA ratio varied from 27 to 30, which reects the current Western
diet trend, being much higher than the corresponding nutritional
recommendations (1 b n 6/n 3 b 4) (Simopoulos, 1999).
The factorial analysis of the FA classes shows that OE diet resulted in a
signicantly lower SFA and a higher MUFA. However, it must be noted
that several signicant crossed treatment interactions were found for
MUFA, where OE T3L and ET0 exhibited signicantly higher and
lower levels with respect to the other treatments; this, therefore,
conrms that MUFA levels were affected by both diet and storage
conditions. On the other hand, UFA/SFA ratio was signicantly higher also
in OE diet as compared with the other diets, which is probably related to
the higher oleic acid level. No signicant effects of diets on PUFA and
PUFA/MUFA ratio, were observed. Diets C and E led to a signicantly
higher n 6/n 3 FA ratio with respect to OE diet. On the other hand,
storage conditions had no signicant effects on FA classes and their ratios.
In general, pork meat FA composition was affected by feedings,
since HOSO supplementation resulted in a higher oleic acid content.
However, no signicant differences in FA composition were found
when VE was added to feeding, which is in agreement with previous
studies (Guo et al., 2006).
3.2. Sterol content
Total sterol content ranged from 5407 to 9246 mg/kg lipids, which
corresponded to 387 and 742 mg sterols/100 g meat (Table 4). Total
 V. Cardenia et al. / Meat Science 88 (2011) 271279
cholesterol content of the meat slices ranged from 5278 to 9043 mg/
kg lipids (~378726 mg/100 g meat), which was in agreement with
literature data (Bragagnolo & Rodriguez-Amaya, 2002); it must be
noted that a large variation in the cholesterol content was registered,
due to the heterogeneous structure of the muscle body in relationship
to animal's sex, as well as to the different fat trimming of meat slices.
Some researchers reported an hypocholesteronemic inuence of n 3
fatty acid on muscle cholesterol concentration (Barowicz, Brzoska, &
Pietras, 2000), whereas other studies stated that MUFA- or PUFA-
enriched diets had not such effect (Barowicz, Brzoska, Pietras, &
Gasior, 1997; Zanardi, Novelli, Ghiretti, & Chizzolini, 2000).
Total cholesterol was about 97% of total sterols, followed by
campesterol (60%70% of phytosterols), -sitosterol (26%), 5-
avenasterol (11%) and stigmasterol (traces). It must be noted that
the relative presence of the single phytosterols in the raw meat does
not correspond to the sterol composition of the dietary oil sources
(HOSO), where -sitosterol is about 24 times higher than campes-
terol (Stazione Sperimentale per le Industrie degli Oli e dei Grassi,
2002). It might be possible that phytosterols have been selectively
absorbed in different amounts according to their molecular structure,
form (free or esteried) and physico-chemical status (Rozner & Garti,
2006) and/or have been partially metabolized/degraded by swine,
converting them into other compounds.
No signicant effects (p N 0.05) of diets, storage conditions and
crossed treatment interactions on the single and total sterols were
found, except for campesterol. The latter was signicantly higher in
meat slices obtained with control diet, as well as in those stored
3 days.
3.3. Lipid oxidation
The effect of photosensitized oxidation on lipids from pork meats
obtained with different diets was evaluated by PV and TBARs
(Table 5). PV ranged from 1.1 to 6.0 meq O2/kg lipid, but no signicant
effects (p N 0.05) of diets, storage conditions and crossed treatment
interactions on PV were found. However, it is possible to observe
some general oxidative trends. When VE and HOSO + VE were added
to swine feeding, PV was lower as compared with those of samples
obtained with control diet. In addition, storage at dark for 3 days
(T3D) led to a PV increase, regardless of the feeding, which could be
ascribed to autoxidation and enzymic oxidation mechanisms (Min,
Nam, Cordray, & Ahn, 2008). Similarly to the untreated samples,
photoxidized meat displayed the lowest average PV, which is related
to hydroperoxide breakdown induced by light.
Regarding secondary oxidation products, TBARs ranged from 0.2 to
2.7 mg of MDA/kg of meat. The factorial analysis shows no signicant
differences due to dietary treatments. However, TBARs were lower in
diets E and OE, regardless of the storage condition. On the other hand,
light exposure led to a signicant increase in TBARs, with respect to
untreated meat and samples stored for 3 days at dark; such TBARs
levels were higher than the generally established values for thresh-
olds of rancidity odor in fresh meats (Fernndez, Prez-lvarez, &
Fernndez-Lpez, 1997). The higher TBARs formation rate in light-
exposed meat is due to hydroperoxide breakdown, which is converted
into secondary oxidation products, such as aldehydes. The factorial
analysis showed no signicant differences (p N 0.05) due to crossed
treatment interaction, though.
PV and TBARs data obtained in the present work were higher than
those found in pork chops obtained by feed supplementation with
vitamin E and HOSO (PV = 0.20.3 meq O 2 /kg lipid and
TBARs ~ 0.1 mg MDA/kg meat) (Zanardi et al., 1998) and those
detected in longissimus dorsi muscle from Landrace and Duroc pigs
subjected to restricted feeding and antioxidant supplementation
(TBARs = 0.10.2 mg MDA/kg meat) (Mason et al., 2005). These
differences on oxidative stability could be attributed to several factors,
such as swine characteristics (breed, sex, age), feeding (composition,
277
amount and time of supplementation), slaughtering conditions,
postmortem history, sample preparation and storage conditions.
Although the overall oxidative stability of pork meat, in the
present study, was not signicantly affected by the dietary treat-
ments, VE and HOSO + VE supplementation of swine feeding led to a
lower oxidative degree of meat lipids. Similar results were reported by
Eder et al. (2005) and Guo et al. (2006), who added diverse types of
lipid and levels of vitamin E to swine feeding and investigated the
effect of three levels of dietary -tocopherol acetate and different
feeding duration on meat quality and lipid oxidation, respectively. In
fact, supplementation of high levels of VE (300700 mg/kg) resulted
in reduced lipid oxidation as measured by TBARs (Corino et al., 1999;
Jensen et al., 1997).
3.4. Cholesterol oxidation
Table 5 shows the effects of diet and storage conditions on the
single and total COP contents of pork meat samples, as well as on their
cholesterol oxidation rates. Total COP content of the pork slices
ranged from 8.5 to 16.3 mg/kg of lipids (~0.51.1 mg/kg meat), which
agrees with data found by Thurner, Razzazi-Fazeli, Wagner, Elmadfa,
and Luf (2007) on fresh pork meat samples. Other researchers were
not able to detect COPs in raw pork meat (Hur, Park, & Joo, 2007),
whereas Monahan et al. (1992) reported that COPs were present in
some pork meat samples only after 8 days of refrigerated storage.
Nam, Du, Jo, and Ahn (2001) detected higher oxysterol levels (17.2
48.4 g COPs/g fat) in irradiated and not irradiated fresh pork meat
packed under different conditions, as compared with those shown in
the present study.
The factorial analysis shows that samples obtained with control
diet had a signicantly higher total COP level as compared with the
other dietary supplementations. However, several signicant crossed
treatment interactions were found, where C T3D and C T3L
exhibited a signicantly higher level with respect to the other
treatments, and E T3D displayed a signicantly lower total COP
content; in these cases, therefore, COP levels were affected by both
diet and storage conditions. The higher antioxidant efcacy of vitamin
E during storage at dark might be related to its different scavenging
activity concerning radicals and singlet oxygen (Kim & Min, 2008); in
fact, vitamin E has been found to display a lower scavenging afnity
for the latter, being thus a more suitable antioxidant for autoxidized
and/or thermoxidized samples rather than for photoxidized ones. No
signicant effect of the storage conditions on the total COP content
was found, even though light-exposed meat slices exhibited a slightly
higher level of oxysterols. Despite that COP are secondary oxidation
products, they were not signicantly affected by storage conditions as
TBARs did, which might be due to COP decomposition and/or
interaction with amino compounds or other components (Olkkonen
& Hynynen, 2009; Rodriguez-Estrada, Penazzi, Caboni, Bertacco, &
Lercker, 1997), leading to the formation of derivatives that are not
detectable under the analytical conditions used for COP determina-
tion. This particular behavior has been observed in various muscle
foods that have been subjected to different oxidation conditions
(Boselli et al., 2010; Rodriguez-Estrada et al., 1997).
COPs determined in all samples were 7-HC (0.92.1 mg/kg
lipids), 7-HC (1.43.2 mg/kg lipids), -CE (1.64.1 mg/kg lipids),
-CE (2.03.2 mg/kg lipids), and 7-KC (1.74.4 mg/kg lipids). The
most abundant COPs were 7-KC (20.327.7% of total COPs) and -CE
(18.625.5%) followed by -CE (17.725.1%), 7-HC (18.122.6%)
and 7-HC (8.517.5%); no triol was detected. This further conrms
that 7-KC can be employed as a suitable marker of the extent of
cholesterol oxidation in raw muscle foods (Boselli et al., 2005; Boselli
et al., 2009; Boselli et al., 2010; Lercker & Rodriguez-Estrada, 2000),
since it usually represents about 3050% of total COPs. The 7-hydroxy
derivatives reached the most stable form from the thermodynamics
standpoint (Lercker & Rodriguez-Estrada, 2002), since 7-HC is
278 V. Cardenia et al. / Meat Science 88 (2011) 271279
always higher than 7-HC regardless of the diet and storage
conditions. The COP level found in the present study was lower
than those reported in literature for ground pork meat (Nam et al.,
2001). Regarding the effect of feeding treatment on the single
oxysterol levels, a signicantly higher content of each COP was
found in samples obtained with the control diet as compared with
those from the other dietary treatments. No signicant effect of
storage conditions on the single COP were observed, except for -CE
whose content was signicantly higher in meat slices exposed to light
than in those kept at dark. Factorial analysis of crossed treatment
interaction showed that -CE was signicantly higher in C T3L and
C T3D with respect to E T3D, being thus affected by both diet and
storage conditions. A similar trend was displayed by 7-KC, which was
signicantly higher in C T3D with respect to samples obtained with
VE (E T0, E T3D) and HOSO + VE (OE T3L) supplementation
under different storage conditions. Antioxidant efcacy of vitamin E
toward an actual rise of the main single COP during storage at dark,
was also conrmed.
The cholesterol oxidation rate (calculated as % COPs/cholesterol;
OR) varied from 0.1 to 0.3%, which is in agreement with those
reported by Lercker and Rodriguez-Estrada (2000). Signicant effects
of both diet and storage conditions were observed on cholesterol
oxidation rate, being higher in control diet and T0, respectively.
Regarding crossed treatment interaction, only OE T0 was found to be
signicantly higher than most samples stored at dark for 3 days
(except for C T3D) and E T3L; the latter means that there are some
interactions between feeding and storage conditions.
The different degrees of oxidation induced by light exposure can
be attributed in part to the lamp radiation energy (expressed as K),
which is inversely proportional to the wavelength (Francis &
Clydesdale, 1975); in fact, the temperature and wavelength of the
daylight are 3800 K and 480 nm respectively. Meat pigments
(hemoglobin and myoglobin) also play an important role, since they
act as photosensitizers and tend to absorb more in the blue and green
parts of the light spectrum (Boselli et al., 2005). Such absorption
characteristics will depend on the level of total iron, its oxidation
status, the occurrence of covalent and ionic complexes with molecular
oxygen and water, respectively (Francis, 1985). Therefore, the higher
oxidation degree of meat exposed to the daylight lamp is a
consequence of the absorption characteristics of the meat pigments,
as well as the amount of blue light and energy of radiation of this type
of irradiation source (Boselli et al., 2005).
Although the average total COP contents of fresh and photoxidized
pork meat slices were relatively low (8.516.3 mg/kg lipids), they
might represent a risk for human health according to the threshold of
toxicological concern (TTC) for unclassied compounds, which
corresponds to 0.15 g per person per day (Kroes et al., 2004).
However, the extent of the diverse biological effects of COPs have
been reported to vary according to their molecular structure, the
different concentrations of the single oxysterols or their mixtures, and
the in vitro or in vivo systems where COPs have been tested and
measured (Schroepfer, 2000). Therefore, further research is required
to better ascertain the toxicity levels of the single COPs, as well as the
possible interactions and synergic effects among them.
The formation of relevant amounts of epoxy derivates might be
partly due to the interaction of sterols with hydrogen peroxide, which
is released by microbial enzymes naturally present in muscle tissues
(Hur et al., 2007).
3.5. Correlations between composition and oxidation parameters of raw
pork meat obtained with different dietary treatments and subjected to
several storage conditions
A correlation study (Pearson test, = 0.05) was performed on the
results obtained for lipid composition, oxidative parameters and the
twelve crossed treatments deriving from the four types of feedings
and three storage conditions. For better data comprehension, only
signicant correlations are discussed here.
Palmitoleic acid and arachidonic acid contents were directly
correlated to -CE (r = 0.379, p = 0.023) and TBARs (r = 0.411,
p = 0.013), respectively, which conrms the important role of FA
unsaturation degree on the generation of secondary oxidation
products. A positive, linear correlation was found between PV and
TBARs (r = 0.480, p = 0.001), which supports the well-known strict
interdependence between hydroperoxides and their demolition/
evolution compounds. Regarding COPs, 7-HC was directly correlated
to 7-HC (r = 0.651, p b 0.001), -CE (r = 0.728, p b 0.001), and 7-KC
(r = 0.538, p b 0.001), whereas -CE was also positively correlated to
7-HC (r = 0.523, p b 0.001), -CE (r = 0.609, p b 0.001) and 7-KC
(r = 0.710, p b 0.001). It must be noted that these direct correlations
between COPs were not conditioned by the oxidation pathways they
derive from. In fact, the 7-oxysterols (7-HC, 7-HC and 7-KC)
originate from 7-hydroperoxide demolition, whereas cholesterol
epoxides are formed from the bimolecular interaction between a
cholesterol molecule and a hydroperoxyl radical (Lercker and
Rodriguez-Estrada, 2002).
4. Conclusions
This study evaluated the effects of four different diets on the extent
of lipid and cholesterol oxidation in fresh raw pork meat (T0), as well
as in raw meat slices stored at dark (T3D) and under light (T3L) for
3 days at 8 C. Meat FA composition was affected by feeding, since
HOSO supplementation resulted in a higher oleic acid content of pork
meat, whereas no signicant differences in FA composition were found
when VE was added to feeding. The highest PV and total COPs were
detected in the group fed with the control diet, whereas HOSO-
enriched diets displayed the highest TBARs content; VE and HOSO+VE
supplementation led to a lower oxidative degree of meat lipids, which
conrms the antioxidant effect of vitamin E. After storage under light
exposure, pork meat slices exhibited a decrease of PV, which resulted
in an increasing trend of TBARs and COPs. Although cholesterol
oxidation rate did not exceed 0.3%, further research is required about
toxicity levels of the single COPs, as well as the possible interactions
and synergic effects among them, to better ascertain that COPs levels
found in fresh and photoxidized meats (49107 g/100 g of meat) do
not represent a risk for human health. In the overall picture, however,
feeding enrichment with both HOSO and vitamin E proved to be an
appropriate supplementation strategy to produce pork meat with a
suitable oxidative stability, which could be further enhanced by using
alternative protective packaging and lighting conditions during
commercial retail storage.
Acknowledgments
The authors would like to thank Prof. Dr. Lorenzo Barbanti for the
assistance on data statistical analysis. This work was supported by the
Italian Ministry of University and Scientic Research (PRIN 2004 prot.
2004077052).
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