Meat Science 85 (2010) 402–409

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Meat Science
journal homepage: www.elsevier.com/locate/meatsci

Protein oxidation in emulsified cooked burger patties with added fruit

extracts: Influence on colour and texture deterioration during chill storage
Rui Ganhão a, David Morcuende b, Mario Estévez b,*
a
Food Science Department, School of Maritime Technology, Polytechnic Institute of Leiria, Peniche, Portugal
b
Animal Production and Food Science Department, Faculty of Veterinary Science, University of Extremadura, Cáceres, Spain

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

Article history: The influence of protein oxidation, as measured by the dinitrophenylhydrazine (DNPH) method, on colour
Received 23 September 2009 and texture changes during chill storage (2 °C, 12 days) of cooked burger patties was studied. Extracts
Received in revised form 3 February 2010 from arbutus-berries (Arbutus unedo L., AU), common hawthorns (Crataegus monogyna L., CM), dog roses
Accepted 8 February 2010
(Rosa canina L., RC) and elm-leaf blackberries (Rubus ulmifolius Schott., RU) were prepared, added to bur-
ger patties (3% of total weight) and evaluated as inhibitors of protein oxidation and colour and texture
changes. Negative (no added extract, C) and positive control (added quercetin; 230 mg/kg, Q) groups
Keywords:
were also considered. The significant increase of protein carbonyls during chill storage of control burger
Antioxidant
Fruit phenolics
patties reflect the intense oxidative degradation of the muscle proteins. Concomitantly, an intense loss of
Functional product redness and increase of hardness was found to take place in burger patties throughout refrigerated stor-
Protein oxidation age. Most fruit extracts as well as Q significantly reduced the formation of protein carbonyls and inhibited
Meat redness colour and texture deterioration during chill storage. Likely mechanisms through which protein oxidation
Meat hardness could play a major role on colour and texture changes during chill storage of burger patties are discussed.
Chill storage Amongst the extracts, RC was most suitable for use as a functional ingredient in processed meats since it
enhanced oxidative stability, colour and texture properties of burger patties with no apparent drawbacks.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction inactivation of muscle proteases (Rowe, Maddock, Lonergan, &

Oxidation of lipids and proteins are major threats to meat qual-
ity. The onset of oxidative reactions in muscle foods during han-
dling, processing and storage of leads to undesirable sensory
changes and serious health concerns (Gray & Pearson, 1994; Ladi-
kos & Lougovois, 1990; Xiong, 2000). Whereas the influence of lipid
oxidation on quality traits such as meat colour and odour is well
recognized, the impact of protein oxidation on meat quality re-
quires further attention. In the presence of reactive oxygen species
(ROS), protein oxidation is manifested by free radical chain reac-
tions similar to hose of lipid oxidation, including initiation, propa-
gation and termination stages (Gardner, 1979). Amongst other
effects, the oxidative degradation of muscle proteins involves mod-
ification of the amino acid side chains, with the formation of car-
bonyl compounds being the most marked change in oxidizing
proteins (Stadtman & Levine, 2003; Xiong, 2000). It is generally ac-
cepted that the oxidation of myofibrillar proteins affects their tech-
nological properties (reviewed by Xiong, 2000) but the
consequences of such reactions on particular quality traits is not
well understood. Some initial approaches reported the effect of
early post-mortem protein oxidation on beef tenderisation through
* Corresponding author. Tel.: +34 927257122; fax: +34 927257110.
E-mail address: mariovet@unex.es (M. Estévez).
Huff-Lonergan, 2004). Estévez, Ventanas, and Cava (2005) sug-
gested a plausible reason for the effect of protein oxidation on col-
our and texture of frankfurters during refrigerated storage. Lund,
Lametsch, Hviid, Jensen, and Skibsted (2007) reported that fresh
pork tenderness is affected by oxidizing proteins during chill stor-
age of meat and Ventanas, Ventanas, Tovar, García, and Estévez
(2007) described the likely impact of protein oxidation on colour,
texture and flavour of dry-cured products. Some other meat prod-
ucts, such as burger patties, are very susceptible to oxidation as
mincing, cooking and the addition of salt, promotes the formation
of ROS and hence, the occurrence and intensity of the oxidative
reactions (Ladikos & Lougovois, 1990). Nevertheless, the occur-
rence of protein oxidation during processing and storage of these
meat products has not been studied and the impact of these reac-
tions on particular quality traits remains unknown.
The commitment to avoiding the unpleasant effects of oxidative
reactions in muscle foods and in emphasizing the physiological
functions of meat explains the growing interest amongst scientists
in the development of functional meat products and the plant
kingdom has been recognised as a versatile source of materials
with proven functional properties. When added to a particular
meat product, plant materials provide dietary fibre and a large
variety of minerals, vitamins and phenolic compounds (Jiménez-
Colmenero, Carballo, & Cofrades, 2001). Plant phenolics are known

0309-1740/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2010.02.008
 R. Ganhão et al. / Meat Science 85 (2010) 402–409
to act as effective antioxidants in meat systems through radical
scavenging and metal-chelating activities (Rice-evans, Miller, Bol-
well, Bramley, & Pridham, 1995). In addition, plant phenolics might
enhance the nutritional value of muscle foods as they display anti-
oxidant activity in vivo as well as anti-inflammatory and anti-car-
cinogenic properties (Bravo, 1998). A large variety of wild fruits
and berries are found in the Mediterranean forest such as arbu-
tus-berries (Arbutus unedo L., AU), common hawthorns (Crataegus
monogyna L., CM), dog roses (Rosa canina L., RC) and elm-leaf black-
berries (Rubus ulmifolius Schott., RU). These fruits have been found
to have large amounts of phenolic compounds and their antioxi-
dant potential has been demonstrated in vitro (Ganhão, Estévez,
& Morcuende, 2008). However, the influence of the addition of ex-
tracts of these fruits in meat products, in terms of antioxidant ef-
fects and impact on meat quality traits is unknown. The
protective role played by fruit extracts and phenolic compounds
towards food proteins has been seen in model systems (Estévez,
Kylli, Puolanne, Kivikari, & Heinonen, 2008a; Salminen, Jaakkola,
& Heinonen, 2008); the effect on real meat products deserves fur-
ther attention. The present study aimed to evaluate the effect of
phenolic-rich extracts of selected Mediterranean wild fruits on tex-
ture and colour changes during refrigerated storage of cooked bur-
ger patties. The likely impact of protein oxidation on these changes
is also addressed.
2. Material and methods
2.1. Chemicals
All chemicals and reagents used were purchased from Panreac
(Panreac Química, S.A., Barcelona, Spain), Merck (Merk, Darmstadt,
Germany) and Sigma Chemicals (Sigma–Aldrich, Steinheim,
Germany).
2.2. Fruits
Samples of strawberry tree (A. unedo L., AU), common hawthorn
(C. monogyna L., CM), dog rose (R. canina L., RC) and elm-leaf black-
berry (R. ulmifolius Schott., RU) cultivars were collected at full ripe-
ness in the Cáceres region, Spain (altitude = 450 m) during the
summer and autumn of 2007. After hand-harvest, the samples
were immediately transferred to the laboratory, cleaned and sorted
to eliminate damaged and shrivelled fruits and then frozen at
80 °C.
2.3. Preparation of fruit extracts for burger patties
Fruits (30 g), including peel and pulp, were cut into pieces while
the seeds were carefully removed. The fruit was ground, dispensed
in a falcon tube and homogenized with 10 volumes (w/v) of abso-
lute ethanol. The homogenates were centrifuged at 2600g for
10 min at 6 °C. The supernatants were collected and the residue
was re-extracted once more following the procedure previously
described. The two supernatants were combined, evaporated using
a rotaevaporator and redissolved using 250 g of distilled water.
Water solutions from each fruit were prepared and stored under
refrigeration until used for the manufacture of porcine burgers
(less than 24 h). No insoluble fragments or residues were observed
in the water solutions.
2.4. Manufacture of burger patties
Six types of porcine burger patties were prepared with the addi-
tion of different fruit extracts (AU, CM, RC, RU) including negative
(no added extract, C) and positive controls (added quercetin;
403
230 mg/kg, Q). In the basic formulation, the ingredients per kg of
patty were as follows: 725 g meat (porcine longissimus dorsi mus-
cle), 250 g distilled water, and 25 g sodium chloride. In the formu-
lation of the treated patties, the 250 g of distilled water were
replaced by 250 g of a water solution containing the corresponding
fruit extracts or the quercetin. All ingredients were minced in cut-
ter until a homogeneous raw batter was obtained. Sixteen burger
patties per batch were prepared in two independent processes
(eight patties per batch each time). Burger patties were formed
using a conventional burger-maker (100 g/patty), to give average
dimensions of 10 cm diameter and 1 cm thickness. Preliminary
cooking trials were performed to establish the cooking conditions
required to achieve a meat core temperature of 73 °C. Patties were
placed on trays and cooked at 170 °C for 18 min in a forced-air
oven. The cooking loss was calculated as: Cooking loss =
[(Wb Wa)/Wb]  100 where Wb and Wa are the weights of the
burger patties before and after cooking, respectively. The cooked
burger patties were dispensed in polypropylene trays, wrapped
with PVC film and subsequently stored for 12 days at + 2 °C under
white fluorescent light (620 lux), simulating retail display condi-
tions. At sampling (days 1, 4, 8 and 12), four burger patties per
batch were taken out of the refrigerator and analysed for carbonyl
gain and colour and texture. Storage loss was calculated as the
weight loss during refrigerated storage of cooked burger patties
as follows: storage loss = [(W1 W12)/W1]  100 where W1 and
W12 are the weight of the cooked burger patties at days 1 and
12, respectively.
2.5. Proximate composition of burger patties
Moisture and total protein contents were determined using offi-
cial methods (AOAC, 2000a,b). The method of Folch, Lees, and Stan-
ley (1957) was used for determining the fat content of the patties.
2.6. Determination of total carbonyls by the DNPH-method
Protein oxidation, as measured by the total carbonyl content,
was evaluated by derivatisation with DNPH as described by Oliver
et al. (1987) with slight modifications. Burger patties (1 g) were
minced and then homogenized 1:10 (w/v) in 20 mM sodium phos-
phate buffer containing 6 M NaCl (pH 6.5) using an ultraturrax
homogenizer for 30 s. Two equal aliquots of 0.2 mL were taken
from the homogenates and dispensed in 2 mL eppendorf tubes.
Proteins were precipitated by cold 10% TCA (1 mL) and subsequent
centrifugation for 5 min at 4200g. One pellet was treated with 1 mL
2 M HCl (protein concentration measurement) and the other with
an equal volume of 0.2% (w/v) DNPH in 2 M HCl (carbonyl concen-
tration measurement). Both samples were incubated for 1 h at
room temperature. Afterwards, samples were precipitated by 10%
TCA (1 mL) and washed three times with 1 mL ethanol:ethyl ace-
tate (1:1, v/v) to remove excess DNPH. The pellets were then dis-
solved in 1.5 mL of 20 mM sodium phosphate buffer containing
6 M guanidine HCl (pH 6.5), stirred and centrifuged for 2 min at
4200g to remove insoluble fragments. Protein concentration was
calculated from the absorption at 280 nm using BSA as standard.
The amount of carbonyls was expressed as nmol of carbonyl per
mg of protein using an absorption coefficient of 21.0 nM 1 cm 1
at 370 nm for protein hydrazones. The percent inhibition of fruit
extracts against carbonyl gain was calculated as follows:
[100  (C12 T12)/C12)] where T12 and C12 are the carbonyl con-
tents of the treated and control samples at day 12.
2.7. Colour measurements
Surface colour measurements of cooked burger patties were
performed using a Minolta Chromameter CR-300 (Minolta Camera
404 R. Ganhão et al. / Meat Science 85 (2010) 402–409

Corp., Meter Division, Ramsey, NJ) which consisted of a measuring Table 1

head (CR-300), with an 8 mm diameter measuring area and a data Chemical composition and cooking and storage losses of cooked burger patties with
added fruit extracts and quercetin.
processor (DP-301). Before each session the chromameter was cal-
ibrated on the CIE colour space system using a white tile. The L* va- C RC CM RU AU Q SEMA
lue indicates lightness (L* = 0 darkness, L* = 100 lightness); a* value Moisture B
70.84 72.46 70.63 70.67 70.48 70.93 0.19
indicates redness (+60 = red, 60 = green) and b* value indicates FatB 2.19 1.86 2.19 1.97 1.96 2.53 0.06
yellowness (+60 = yellow, 60 = blue). Colour measurements were ProteinB 23.03 23.38 23.70 23.01 23.50 22.53 0.02
Cooking lossC 22.06 23.59 22.71 21.73 22.73 22.24 0.30
made on the surface of each patty in triplicate at three randomly Storage lossC 3.23 2.85 3.09 2.95 2.37 4.37 0.18
selected locations. Colour measurements were made at room tem-
perature (22 °C) with illuminant D65 and a 0° angle observer at C: control; RC: Rosa canina; CM: Crataegus monogyna; RU: Rubus ulmifolius; AU:
Arbutus unedo; Q: quercetin.
days 1, 4, 8, and 12 of storage. Saturation index and Hue angle
Values with a different letter (a–c) within a row are significantly different (P < 0.05).
(h°) values were obtained by the following equations: saturation A
Standard error of the mean (n = 72; except for storage loss, n = 24).
index = (a*2 + b*2)0.5; h° = arctg b*/a*. A numerical total colour dif- B
Expressed as g/100 g burger patty.
C
ference (DE) between burgers at day 1 and day 12 of storage was Expressed as percentage.
calculated by: DE1–12 = [(L12 L1)2 + (a12 a1)2 + (b12 b1)2)]1/2.
The percent inhibition of fruit extracts against loss of redness
was calculated as: 100 [100  (T1 T12)/(C1 C12)] where T ties (Table 2). This increase was considerably more intense in the
and C are the redness values of the treated and control samples, control samples (Ddays1–12 = 5.8 nmol carbonyls/mg protein) than
respectively, and subscripts represents freshly cooked (1) and in the treated counterparts (Ddays1–12 = 1.1–1.7 nmol carbonyls/
cooked and chilled samples (12). mg protein). The addition of fruit extracts significantly inhibited
the formation of protein carbonyls in the burger patties. At all sam-
pling days, control patties had significantly higher amounts of pro-
2.8. Texture measurements
tein carbonyls than the treated ones. No differences in
effectiveness between fruit extracts was observed as all treated
Texture profile analysis (TPA) was performed at room tempera-
burgers had similar amounts of protein carbonyls by the end of
ture with a Texture Analyser TA-XT2i (Stable Micro Systems, Sur-
storage. In fact, all fruit extracts and quercetin showed similar per-
rey, UK). Three cylindrical samples (2.5 cm diameter) were taken
cent inhibitions against the formation of protein carbonyls (Fig. 1).
from random locations in each patty and subjected to a two-cycle
The addition of RU extracts had a significant effect on the colour
compression test. The samples were compressed to 40% of their
displayed by cooked burger patties (Table 3). Burger patties with
original height with a cylindrical probe of 5 cm diameter and a
added RU were darker (lower L*-value) and had a redder colour
cross-head speed of 5 mm/s. Texture profile parameters were
(higher a*-value) than the patties from the other batches. In addi-
determined following descriptions by Bourne (1978) and the SMS
tion, RU patties displayed a less intense colour (lower saturation
manual (Stable Micro Systems, Surrey, UK). All analyses were per-
index) which was significantly closer to the red axis (lower hue an-
formed in triplicate. The percent inhibition of fruit extracts against
gle value) than the other patties. In general, RC, CM and AU extracts
hardness increase was calculated as: 100 [100  (T12 T1)/
had no effect on colour parameters whereas the addition of Q led to
(C12 C1)] where T and C are the hardness values for the treated
decreased L* values and increased b* and saturation index values.
and control samples, respectively, and subscripts represents
Subsequent refrigerated storage significantly influenced colour
freshly cooked (1) and cooked and chilled samples (12).
parameters with the redness and hue angle values from control
and RU patties being the most affected. Redness significantly de-
2.9. Statistical analysis creased in control (from 4.16 to 0.54) and in RU patties (from
9.36 to 8.07) but this parameter was unaffected in the remaining
Four burger patties per batch and per storage day were pro- patties. In addition, hue angle values significantly increased in con-
duced and used as experimental units. All analyses were per- trol and RU patties while no change was observed in samples from
formed in triplicate in each burger patty (4 burger patties  3 the other batches. Consistently, the total colour difference mea-
analysis; n = 12 per batch and storage day). Analyses of variance sured between day 1 and day 12 of refrigerated storage revealed
(ANOVA) and Tukey tests by SPSS for Windows (v. 15.0) were car- significant differences between samples. Control patties suffered
ried out to study the effect of the addition of fruit extracts and the most intense colour modification followed by RU samples
quercetin on the measured parameters. Differences were consid- whereas samples with added RC, AU, Q and CM displayed signifi-
ered significant at p 6 0.05. Relationships amongst protein oxida-
tion measurement and instrumental colour and texture
parameters were calculated using Pearson’s correlation
Table 2
coefficients.
Protein hydrazonesA gain during refrigeration of cooked burger patties with added
fruit extracts and quercetin.
3. Results C RC CM RU AU Q SEMB
a,z b,y b,y c,y b,y c,y
Day 1 3.68 2.58 2.56 2.14 2.60 2.17 0.13
The proximate composition of cooked burger patties with added 4 5.70a,y 3.33b,xy 3.52b,xy 2.48b,xy 3.27b,xy 3.05b,xy 0.18
fruit extracts and Q is shown in Table 1. Burger patties were ana- 8 6.30a,y 4.03b,x 3.51bc,xy 2.58c,xy 3.92b,x 3.18bc,xy 0.23
lysed for moisture (70.5–72.5 g/100 g), fat (1.9–2.5 g/100 g) and 12 9.52a,x 4.21b,x 3.97b,x 3.23b,x 4.29b,x 3.73b,x 0.36
SEMC 0.51 0.17 0.17 0.16 0.17 0.12 –
protein (22.5–23.7 g/100 g) contents. The addition of fruit extracts
did not affect the chemical composition as no significant differ- C: control; RC: Rosa canina; CM: Crataegus monogyna; RU: Rubus ulmifolius; AU:
ences were found amongst batches. The cooking and storage losses Arbutus unedo; Q: quercetin.
Values with a different letter (a–c) within a row of the same storage day are sig-
ranged from 21.7 to 23.6 g/100 g and from 2.4 to 4.4 g/100 g,
nificantly different (P < 0.05). Values with a different letter (x–z) within a column of
respectively. The loss of weight after cooking and chill storage of the same batch are significantly different (P < 0.05).
burger patties was not affected by added fruit extracts. A
Expressed as nmol hydrazones/mg protein.
B
Chill storage had a significant effect on protein oxidation as the Standard error of the mean within the same storage day (n = 72).
C
amount of carbonyl compounds increased significantly in all pat- Standard error of the mean within the same batch (n = 48).
 R. Ganhão et al. / Meat Science 85 (2010) 402–409 405

Fig. 1. Percentage inhibition of fruit extracts against the formation of protein hydrazones, loss of redness and increase of hardness during refrigerated storage of cooked
burgers. a,b,c Different letters between groups denote significant differences (p < 0.05); ns = non significant.

Table 3
Instrumental colour measured during refrigerated storage of cooked burger patties with added fruit extracts.

C RC CM RU AU Q SEMA
L*
Day 1 68.11a 67.45ab 66.76ab 59.25c,x 66.91ab,xy 66.23b 0.38
4 69.19a 68.67a 67.63ab 60.30c,x 67.73ab,x 66.82b 0.39
8 67.69a 68.07a 66.48a 60.04b,x 68.04a,x 66.34a 0.48
12 67.91a 67.47a 66.17a 57.34b,y 65.24a,y 66.52a 0.80
SEMB 0.26 0.33 0.28 0.24 0.27 0.26 –
a*
Day 1 4.16bc,x 4.46b 4.31bc 9.36a,x 4.25bc 3.95c 0.23
4 2.23c,y 4.37b 4.07b 8.74a,y 4.15b 3.95b 0.25
8 1.13c,z 4.38b 4.04b 8.42a,yz 3.90b 3.77b 0.31
12 0.54c,z 4.32b 3.88b 8.07a,z 4.07b 3.98b 0.46
SEM 0.24 0.10 0.09 0.10 0.09 0.07 –
b*
Day 1 13.07b 13.59ab 14.48a 8.28c 13.23b 14.22a 0.26
4 13.21a 13.38a 13.68a 9.34b 13.04a 14.15a 0.24
8 13.18a 13.03a 13.59a 9.54b 13.38a 13.74a 0.26
12 13.43a 13.24a 14.15a 9.62b 13.32a 14.40a 0.39
SEM 0.11 0.25 0.19 0.21 0.17 0.15 –
Saturation index
Day 1 13.73c 14.31abc 15.11a 12.54d 13.90bc 14.76ab 0.13
4 13.41ab 14.07ab 14.28ab 12.85b 13.69ab 14.70a 0.16
8 13.23 13.75 14.18 12.75 13.94 14.25 0.17
12 13.44 13.93 14.68 12.57 13.93 14.94 0.27
SEM 0.11 0.27 0.20 0.12 0.18 0.15 –
Hue angle
Day 1 72.44ab,z 71.80b 73.43ab 41.32c,y 72.21ab 74.46a 1.39
4 80.40a,y 72.04b 73.53b 46.52c,xy 72.46b 74.46b 1.33
8 85.08a,x 71.50c 73.47bc 48.42d,xy 73.81bc 74.70b 1.61
12 87.74a,x 71.99b 74.67b 49.97c,x 73.05b 74.58b 2.35
SEM 1.01 0.19 0.27 0.90 0.27 0.24 –
DE *
4.20a 2.16c 1.02d 2.95b 1.73c 2.14c 0.13

C: Control; RC: Rosa canina; CM: Crataegus monogyna; RU: Rubus ulmifolius; AU: Arbutus unedo; Q: quercetin.
Values with a different letter (a–c) within a row of the same storage day are significantly different (P < 0.05). Values with a different letter (x–z) within a column of the same
batch are significantly different (P < 0.05).
A
Standard error of the mean within the same storage day (n = 72).
B
Standard error of the mean within the same batch (n = 48).

cantly smaller changes. Once again, all fruit extracts and Q were
similarly efficient at inhibiting the loss of redness during refriger-
ated storage of cooked burger patties. Most colour parameters sig-
nificantly correlated with protein oxidation measurements (Table
4).
In general, the addition of fruit extracts significantly increased
the hardness of cooked burger patties and other related parameters
such as chewiness (Table 5). It is particularly remarkable the in-
tense texture modifications suffered by the control and CM burger
patties during the 12 days of storage. Hardness and chewiness sig-
406 R. Ganhão et al. / Meat Science 85 (2010) 402–409

Table 4 bitions against hardness increase during chill storage. RC was the
Pearson’s correlation coefficients (R2)a between protein oxidation (Pox) as measured most effective fruit extract at inhibiting the increase of hardness,
by protein hydrazones and the instrumentally measured colour and texture
parameters.
followed by RU, AU, Q and finally, CM. Various other texture
parameters, namely, springiness, cohesiveness and resilience
Pox p-value showed significant correlations with protein oxidation
L* 0.33 <0.001 measurements.
a* 0.60 <0.001
b* 0.22 0.048
Saturation index 0.21 0.284
Hue angle 0.50 <0.001
4. Discussion
Hardness 0.01 0.988
Adhesiveness 0.18 0.230 4.1. Protein oxidation
Springiness 0.48 <0.001
Cohesiveness 0.44 <0.001
The protein carbonyl content was used as a measure of the ex-
Chewiness 0.12 0.375
Resilience 0.31 <0.001 tent of oxidative reactions affecting muscle proteins during refrig-
erated storage of burger patties. The onset of lipid oxidation in
a
n = 48 Cooked burger patties for correlation coefficients calculated from mea-
cooked and refrigerated meats and the impact of these reactions
surements on day 1 and day 12 of refrigerated storage.
on meat quality (i.e. warmed-over flavour) have been extensively
studied (reviewed by Ladikos & Lougovois (1990) and Gray & Pear-
son (1994)) whereas research on the onset and extent of protein
nificantly increased in the aforementioned groups whereas these
oxidation in processed meat products is limited. The increase of
parameters were unaffected in burger patties with added Q and
protein carbonyls show that muscle proteins in cooked patties
RC, AU and RU extracts. At day 12, burger patties with added CM
are susceptible to oxidative reactions leading to carbonyl gain. Car-
displayed significantly higher values for hardness and chewiness.
bonyl compounds are formed as a result of the oxidative degrada-
Fruit extracts and Q displayed significantly different percent inhi-
tion of side chains of lysine, proline, arginine and histidine residues

Table 5
Texture parametersA measured during refrigerated storage of cooked burger patties with added fruit extracts and quercetin.

C RC CM RU AU Q SEMB
Hardness
Day 1 23.28c,y 31.45ab 29.00ab,z 32.65a 28.48ab 24.36bc 0.81
4 28.89ab,xy 34.58a 32.83ab,yz 35.81a 30.99ab 25.62b 0.88
8 29.39ab,xy 33.99a 36.17a,xy 36.08a 32.55ab 25.64b 0.89
12 34.08bc,x 33.45bc 42.14a,x 36.29b 32.39bc 27.11c 0.91
SEMC 1.04 0.98 1.16 0.99 0.92 0.89 –
Adhesiveness
Day 1 -0.02 -0.61 -0.35 -0.26 -0.43 -0.17 0.07
4 0.10 -0.08 -0.21 0.04 -0.24 0.15 0.07
8 0.21 -0.27 0.09 -0.14 -0.33 0.14 0.07
12 0.19 -0.09 0.02 -0.22 -0.18 0.12 0.08
SEM 0.06 0.09 0.09 0.08 0.10 0.10 –
Springiness
Day 1 0.96y 0.94y 0.95 0.94y 0.96 0.96 <0.01
4 0.96y 0.95xy 0.95 0.95xy 0.97 0.97 <0.01
8 0.97y 0.96xy 0.95 0.95xy 0.95 0.95 <0.01
12 1.02a,x 0.98ab,x 0.98ab 0.97ab,x 0.95b 0.99ab <0.01
SEM <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 –
Cohesiveness
Day 1 0.66a 0.64b 0.65ab 0.65ab 0.65ab 0.66a <0.01
4 0.66 0.65 0.66 0.65 0.66 0.65 <0.01
8 0.67 0.65 0.66 0.65 0.65 0.66 <0.01
12 0.67 0.65 0.65 0.66 0.66 0.66 <0.01
SEM <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 –
Chewiness
Day 1 14.58b,y 18.94ab 18.82ab,y 20.27a 17.74ab 15.37b 0.50
4 18.16ab,xy 21.17ab 20.61ab,y 21.96a 22.07a 16.23b 0.59
8 19.87x 21.15 22.48y 22.32 20.16 17.49 0.52
12 22.30ab,x 22.76ab 27.65a,x 23.00ab 20.43b 17.83b 0.67
SEM 0.77 0.67 0.81 0.56 0.71 0.59 –
Resilience
Day 1 0.46a 0.42b,y 0.43ab 0.43ab 0.44ab 0.46a <0.01
4 0.45a 0.41b,y 0.43ab 0.42b 0.45a 0.45a <0.01
8 0.46a 0.41c,y 0.42bc 0.42bc 0.43abc 0.45ab <0.01
12 0.48 0.45x 0.44 0.45 0.46 0.48 <0.01
SEM <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 –

C: control; RC: Rosa canina; CM: Crataegus monogyna; RU: Rubus ulmifolius; AU: Arbutus unedo; Q: quercetin.
Values with a different letter (a–c) within a row of the same storage day are significantly different (P < 0.05). Values with a different letter (x–z) within a column of the same
batch are significantly different (P < 0.05).
A
Units for texture parameters in Section 2.
B
Standard error of the mean within the same storage day (n = 72).
C
Standard error of the mean within the same batch (n = 48).
 R. Ganhão et al. / Meat Science 85 (2010) 402–409
(Stadtman & Levine, 2003). The formation of specific protein car-
bonyls, a-amino adipic semialdehyde (AAS) and c-glutamic semi-
aldehyde (GGS), during in vitro oxidation of myofibrillar proteins
has been confirmed (Estévez, Ollilainen, & Heinonen, 2009). These
semialdehydes could have contributed to the carbonyl gain found
as they account for around 70% of total protein carbonyls in oxi-
dized proteins (Requena, Chao, Levine, & Stadtman, 2001).
The large amount of protein carbonyls in the present study de-
notes more intense oxidative reactions than described in previous
studies on raw pork and beef subjected to chill storage (Lund,
Lametsch, et al. (2007); Lund, Hviid, & Skibsted, 2007; Martinaud
et al., 1997). The initiation of muscle proteins oxidation requires
further clarification although it is generally accepted that heme
and non-heme iron as well as oxidizing lipids play major roles
(Estévez, Kylli, Puolanne, Kivikari, & Heinonen, 2008b; Estévez
et al., 2008a; Kroger-Ohlsen, Ostdal, & Andersen, 2003; Salminen
et al., 2008). In the present experiments, mincing and the high
temperatures applied during cooking could have enhanced protein
oxidation in the patties. Disruption of the tissues leads to the re-
lease of pro-oxidants naturally present in the muscle and enhance
the incorporation of oxygen in the system. Heating degrades myo-
globin causing the release of iron which is believed to increase its
pro-oxidant potential in cooked meats (Kristensen & Purslow,
2001; Schrickler & Miller, 1983). In fact, it is probable that non-
heme iron is a main initiator of protein oxidation in cooked meat
systems as the formation of the major carbonyls, AAS and GGS, re-
quires the presence of metals (Requena et al., 2001) and iron has
been shown to be an effective promoter of carbonyl formation in
myofibrillar proteins (Estévez et al., 2009).
The results confirm the ability of the selected fruit extracts to
act as efficient inhibitors of muscle protein oxidation. The protect-
ing effect of added fruit extracts against protein oxidation can be
attributed to the phenolic compounds naturally present in RC
(11.8 mg gallic acid equivalents (GAE)/g fruit), CM (20.7 mg GAE/
g fruit), RU (5.0 mg GAE/g fruit) and AU (4.3 mg GAE/g fruit) (Gan-
hão et al., 2008). These fruits were found to contain high amounts
of phenolic compounds and display intense antioxidant activities
in vitro against DPPH and ABTS radicals (Ganhão et al., 2008). Spe-
cifically, RC and CM contains high amounts of proanthocyanidins,
RU is particularly rich in ellagic acid, ellagetannins and anthocya-
nins and AU has high contents of catechins and benzoic acids
(Bahorun, Luximon-Ramma, Crozier, & Aruoma, 2004; Dall’Acqua,
Cervellati, Loi, & Innocenti, 2008; Hellström, Törrönen, & Mattila,
2009). Previous studies have reported antioxidant activities of phe-
nolic compounds and diverse plant materials against protein car-
bonyl formation in various meat products such as beef patties
(Lund, Hviid, et al., 2007), frankfurters (Estévez et al., 2005) and li-
ver pâtés (Estévez, Ventanas, & Cava, 2006). Plant phenolics also
display antioxidant potential against other protein oxidations such
as tryptophan depletion in myofibrillar proteins (Estévez et al.,
2008a; Estévez et al., 2008b) and loss of thiol groups in turkey
meat (Mercier, Gatellier, Viau, Remignon, & Renerre, 1998). As re-
dox-active compounds, plant phenolics also display pro-oxidant
actions towards myofibrillar and other food proteins (Viljanen,
Kivikari, & Heinonen, 2004; Estévez et al., 2008a). This effect, how-
ever, was not observed in the present study. In fact, fruit extracts
displayed higher percent inhibitions against carbonyl gain than se-
lected plant phenolics added at similar GAE concentrations (Esté-
vez et al., 2008a).
The precise mechanisms involved in the antioxidant actions of
plant phenolics on myofibrillar proteins are not well understood.
Phenolics from the tested fruits are known to act as efficient radical
scavengers and could have blocked the pro-oxidant action of ROS
on proteins. Additionally, some phenolics such as cyanidin-3-glu-
coside, which is present in RC and RU, display metal-chelating
activities (Rice-Evans et al., 1995). This might be particularly rele-
407
vant in cooked meats as phenolics could have hindered the pro-
oxidant action of non-heme iron by chelation. Both antioxidant
mechanisms would explain the results obtained since certain
ROS such as hydroxyl radicals and iron are directly involved in
the formation of protein carbonyls from myofibrillar proteins
(Estévez et al., 2009). By inhibiting protein carbonyl formation,
fruit extracts enhance the nutritional value of porcine burgers. Be-
sides the loss of essential amino acids, the formation of carbonyl
compounds from oxidizing protein might have additional reper-
cussions on particular quality traits, as discussed below.
4.2. Colour deterioration
The red-brownish colour of cooked meats is mainly determined
by the presence of denatured-globin hemochromes formed as a re-
sult of high temperatures (Fox, 1994). During cooking, the heme
protein is denatured and the iron is oxidized into its ferric form
and the hematin pigment remains intact (Lawrie, 1998). Addition-
ally, the formation of coloured Maillard products on heating, the
physico-chemical state of proteins and other meat components
and the addition of colorants are also influential (Fox, 1994; Law-
rie, 1998). Amongst the fruit extracts, RU was the only one to sig-
nificantly influence the colour of freshly cooked burger patties (day
1). Anthocyanins, which are major pigment constituents of RU, lit-
erally dyed burger patties with a distinct purplish colour. The im-
pact of RU pigments on burger patties led to an intense increase of
redness and a decrease of the other colour parameters compared to
burger patties from the other batches. In contrast to RU, the other
fruits contain small quantities of anthocyanins which explain their
lack of influence on the colour of cooked patties. An intense red-
brownish colour is expected and generally preferred by cooked
meat consumers (Aaslyng et al., 2007; Berry, 1998). In the absence
of sensory evaluation of the samples, the impact of the peculiar
colour displayed by patties with added RU extracts on consumer’s
acceptance remains unknown.
Control burger patties underwent intense discolouration during
chill storage. According to Francis and Clydesdale (1975), the col-
our modifications instrumentally measured can be considered as
noticeable visual changes when the total colour difference (DE1–
12) values are higher than 2. Thus, colour changes in the control
patties during chill storage would be very noticeable to consumers
whereas the addition of fruit extracts and Q preserved the colour of
freshly made patties. The loss of redness and increase of hue angle
values have been described in raw and cooked meats subjected to
refrigerated and frozen storage (Estévez, Morcuende, & Cava, 2003;
Georgantelis, Blekas, Katikou, Ambrosiadis, & Fletouris, 2007;
Hassaballa, Mohamed, Ibrahim, & Abdelmageed, 2009; Ramírez,
Morcuende, Estévez, & Cava, 2004). The discolouration of raw bur-
ger patties is generally attributed to the oxidation of ferrous heme–
iron (Fe2+) into its ferric form (Fe3+) induced by lipid oxidation
products (Yin & Faustman, 1993). Under these circumstances, oxy-
myoglobin is transformed into metmyoglobin and the colour shifts
from a pleasant bright red to an undesirable brownish colour. The
specific reason for the discolouration of cooked meats is not clear
(Fox, 1994). Ramírez et al. (2004) adapted the classical theory for
raw meat discoloration to cooked meats by claiming that the col-
our deterioration in cooked pork during refrigerated storage was
caused by lipid oxidation products. Unfortunately, these authors
provide no valid arguments or further details about the mecha-
nism. Fernández-Ginés, Fernández-López, Sayas-Barberá, Sendra,
and Pérez-Alvarez (2003) suggested that colour deterioration dur-
ing refrigerated storage of cured and cooked meats is explained by
the oxidative degradation of certain nitrosopigments. Estévez and
Cava (2004) and Estévez et al. (2005) reported significant correla-
tions between protein oxidation and discolouration of cooked liver
pâtés and frankfurters subjected to chill storage. According to these
408 R. Ganhão et al. / Meat Science 85 (2010) 402–409
authors, the oxidative degradation of the denatured-globin and the
oxidative cleavage of the hematin pigment would lead to the re-
lease of iron from the heme molecule causing the eventual discol-
ouration of the cooked meat. It is reasonable that the colour
changes reported in the present study are caused by oxidative
reactions since the addition of substances with proven antioxidant
activity inhibited the discoloration of cooked, chilled patties.
Although lipid-derived products could have induced pigment oxi-
dation and meat discolouration, the impact of protein oxidation
might be considerably higher as the present patties had 10-fold
more protein than lipid (20% protein vs. 2% lipid). Protein radi-
cals and other protein oxidation products are known to induce li-
pid oxidation (Viljanen, Kivikari, & Heinonen, 2004) and hence,
heme pigments in cooked meats could also be affected by their
pro-oxidant action. The significant negative correlation between
redness and protein oxidation measurements ( 0.60; p < 0.001)
support this hypothesis. The discoloration suffered by patties with
added RU might not be due to oxidative changes in muscle pig-
ments and proteins as this fruit extract was highly effective against
protein oxidation. The oxidative degradation of anthocyanins,
which are mainly responsible for the colour of RU patties, has been
well documented (Rein & Heinonen, 2004) and would explain the
slight discolouration recorded for these samples during chill
storage.
Besides the effect of oxidation on meat and fruit pigments,
physico-chemical changes affecting other meat components could
have enhanced the colour changes. In fact, protein carbonyls, as-
sessed in this study, as a measure of protein oxidation, are involved
in the formation of protein cross links through further oxidative
reactions (Requena et al., 2001; Xiong, 2000). The formation of
cross links and protein aggregates as a result of protein oxidation
during chill storage of burger patties could have affected light
reflection and hence, could have contributed to the discolouration.
Hassaballa et al. (2009) have recently ascribed colour changes in
cooked tuna muscle to the aggregation of myofibrillar proteins
during frozen storage.
4.3. Texture deterioration
The increase of hardness and other related texture parameters
such as chewiness in chilled control burgers is highly undesirable
as this could have a great impact on consumer acceptability. Hard-
ness increases during refrigerated storage of burger patties and
other cooked meat products has been reported (Estévez et al.,
2005; Fernández-Ginés et al., 2003; Fernández-López, Sayas-Bar-
berá, Sendra, & Pérez-Alvarez, 2004; Hassaballa et al., 2009). In
agreement with the effect of added fruit extracts on colour
changes, the addition of most fruit extracts and Q inhibited the tex-
ture deterioration seen in the control samples. Although loss of
moisture during storage could explain the increase of hardness in
burger, this is not applicable in the present study since all samples
underwent similar weight losses during storage. In addition, the
proximate composition of control and treated patties was similar
at day 1 and at day 12 (data not shown). On the other hand, oxida-
tive damage to proteins has an impact on protein solubility, lead-
ing to the aggregation and complex formation due to cross link
formation (Karel, Schaich, & Roy, 1975). It is plausible that protein
oxidation led to an increase of hardness in burger patties through
the formation of protein carbonyls, the loss of protein functionality
and the formation of cross links between proteins. Phenolics from
fruit extracts would have reduced hardness increases in patties
through the inhibition of protein oxidation during refrigerated
storage. The lack of significance for the correlation between protein
oxidation measurements and instrumental hardness is likely due
to the contradictory effect on CM. This fruit extract was efficient
against protein oxidation and particularly effective at inhibiting
colour changes but patties with added CM suffered marked texture
changes. Unlike the other fruits, Crataegus spp. contains large
amounts of pectins with highly efficient gelling capacities (Kuliev
& Poletaeva, 1982). The texture changes in patties with added
CM are probably induced by physico-chemical modifications of
CM polysaccharides during chill storage. The retrogradation of
these gums during storage probably led to the increase of hardness
in the food matrixes. Regarding the influence of fruit extracts on
texture changes, RC had the most intense effect against hardness
increase compared to the other fruit extracts.
5. Conclusions
The wild Mediterranean fruits tested displayed intense antioxi-
dant activity against protein oxidation and could play an important
role as functional ingredients in burger patties by improving their
oxidative stability and quality. The addition of R. ulmifolius would
affect the colour displayed by burger patties whereas the addition
of C. monogyna would supply some negative texture properties to
these products. Some other fruits such as R. canina improve the oxi-
dative stability, colour and texture of cooked burger patties with
no apparent drawbacks. Protein oxidation might play a major role
in the nutritional and sensory quality of meat products. Further
investigations should confirm some of the likely hypothesis and
mechanisms reported. The development of more sensitive and spe-
cific methods for assessing protein oxidation are required to shed
light on the chemical nature of these complex reactions.
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