Food Chemistry 128 (2011) 299–307
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Antioxidant effect of fractions from chicken breast and beef loin homogenates
in phospholipid liposome systems
Byungrok Min a, Joseph C. Cordray b, Dong Uk Ahn b,c,⇑
a
Food Science & Technology, University of Maryland Eastern Shore, Princess Anne, MD 21853, USA
Department of Animal Science, Iowa State University, Ames, IA 50011, USA
c
Department of Agricultural Biotechnology, Major in Biomodulation, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-921, South Korea
b
a r t i c l e
i n f o
Article history:
Received 4 August 2010
Received in revised form 12 January 2011
Accepted 4 March 2011
Available online 9 March 2011
Keywords:
Lipid oxidation potential
Meat fraction
Metmyoglobin
Free ionic iron
iron chelating agent
a b s t r a c t
The antioxidant effects of meat fractions from chicken breast and beef loin were compared. Five meat
fractions – homogenate (H), precipitate (P), supernatant (S), high-molecular-weight (HMW) and lowmolecular-weight (LMW) fractions – were prepared from chicken breast or beef loin. Each of the fractions
were added to a phospholipid liposome model system containing catalysts (metmyoglobin, ferrous and
ferric ion) or iron chelating agents to determine the effects of each fraction on the development of lipid
oxidation during incubation at 37 °C for 120 min. All fractions from chicken breast showed stronger antioxidant effects against iron-catalyzed lipid oxidation than those from beef loin. Iron chelating capacity of
water-soluble LMW and water-insoluble (P) fractions from both meats were responsible for their high
antioxidant capacities. High concentration of myoglobin, which served as a source of various catalysts,
was partially responsible for the high susceptibility of beef loin to lipid oxidation. Storage-stable ferric
ion reducing capacity (FRC) was detected in all fractions from both meats, and was a rate-limiting factor
for lipid oxidation in the presence of free ionic iron. Higher antioxidant capacity and lower myoglobin
content in chicken breast were primarily responsible for its higher oxidative stability than beef loin.
DTPA-unchelatable compounds, such as ferrylmyoglobin and/or hematin were the major catalysts for
lipid oxidation in beef loin, but free ionic iron and storage-stable FRC also played important roles during
prolonged storage.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Despite extensive studies for several decades, the primary catalysts for lipid oxidation in meat are still controversial. Lapidot,
Granit, and Kanner (2005) suggested that metmyoglobin is a silent
compound in the absence of hydrogen peroxide (H2O2) or lipid
hydroperoxide (LOOH). However, myoglobin appeared to be the
center compound in this controversy because myoglobin can be
converted to ferrylmyoglobin in the presence of H2O2 or LOOH
and serves as a major source of hematin and free ionic iron, which
can initiate and propagate lipid oxidation (Min & Ahn, 2005). Ferrylmyoglobin generated by the interaction of metmyoglobin with
H2O2 or LOOH can abstract a hydrogen atom from a bis-allylic carbon on a fatty acid chain and is a major initiator of lipid oxidation
(Baron & Andersen, 2002; Baron, Skibsted, & Andersen, 1997;
Hamberg, 1997). Ferrylmyoglobin can also degrade LOOH to alkoxyl or peroxyl radicals, which undergo a chain-propagation step
or are decomposed to produce secondary by-products of lipid oxi⇑ Corresponding author at: Department of Animal Science, Iowa State University,
Ames, IA 50011, USA. Tel.: +1 515 294 6595; fax: +1 515 294 9143.
E-mail address: duahn@iastate.edu (D.U. Ahn).
0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.03.018
dation (Reeder & Wilson, 1998, 2001). Free ionic iron released from
heme proteins, iron-containing proteins, or ferritin can initiate lipid oxidation in meat via the Fenton reaction in the presence of
H2O2 or LOOH and reducing agents, such as superoxide anion
(O2), ascorbic acid, NAD(P)H and thiols (Ahn & Kim, 1998; Ahn,
Wolfe, & Sim, 1993; Apte & Morrissey, 1987; Decker & Hultin,
1992; Kanner, Hazan, & Doll, 1988).
The activity of myoglobin as a major catalyst as well as a source
of free ionic iron in the processes of lipid oxidation can be affected
by the concentration of myoglobin, the presence of H2O2, LOOH,
and reducing compounds (Baron, Skibsted, & Andersen, 2002;
Gorelik & Kanner, 2001; Harel & Kanner, 1989; Lapidot et al.,
2005; Rhee, Ziprin, & Ordonez, 1987). Free ionic iron can serve as
a catalyst of lipid oxidation in the presence of reducing compounds
or O2-generating systems (Kanner, 1994; Kanner, Harel, & Hazan,
1986; Rhee, 1988; Turrens & Boveris, 1980). The status of free ionic
iron is more important than the amount of ionic iron for the development of lipid oxidation (Ahn & Kim, 1998; Ahn et al., 1993).
Water-soluble and water-insoluble components that influence
the catalytic activities of myoglobin and free ionic iron are present
in the cytosol of meat, and the balance between antioxidant and
prooxidant activities of the cytosol in muscle tissues determines
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B. Min et al. / Food Chemistry 128 (2011) 299–307
the prooxidant actions of myoglobin and free ionic iron in meat
(Min & Ahn, 2009; Min, Nam, & Ahn, 2010).
DTPA is an excellent chelating agent for both ferrous and ferric
ion. DFO chelates only ferric ion and inhibits its catalyzing activities (Kanner & Harel, 1987; Rahhal & Richter, 1989). However,
DFO serves as an electron donor, suppresses catalytic activity of
ferrylmyoglobin, and interrupts free radical chain reaction of lipid
oxidation (Kanner & Harel, 1987; Rice-Evans, Okunade, & Khan,
1989). Thus, DFO can be more efficient inhibitor of lipid oxidation
than DTPA (Gutteridge, Richmond, & Halliwell, 1979).
The susceptibility of meat from different animal species to lipid
oxidation is different, and chicken breast is much less susceptible
to lipid oxidation than beef loin (Min & Ahn, 2009; Min, Nam,
Cordray, & Ahn, 2008). High total antioxidant capacity, high myoglobin reducing capacity, low myoglobin concentration and its
lipoxygenase-like activity, and low free ionic iron concentration
were responsible for the high oxidative stability of chicken breast
(Min & Ahn, 2009; Min, Cordray, & Ahn, 2010; Min et al., 2008).
The objective of this study was to evaluate the antioxidant and
prooxidant effects of meat fractions from chicken breast and beef
loin in a phospholipid liposome model system in the presence of
catalysts (metmyoglobin, ferrous, and ferric ions) or chelating
agents (DFO and DTPA).
2. Materials and methods
2.1. Chemicals and reagents
Metmyoglobin (from equine skeletal muscle), ferrous ammonium sulfate, ferric chloride, diethylenetriamine pentaacetic acid
(DTPA), desferrioxamine (DFO), linoleic acid, 2-thiobarbituric acid
(TBA), ferric chloride, Chelex-100 resin (50–100 dry mesh, sodium
form), and butylated hydroxytoluene (BHT) were purchased from
Sigma (St. Louis, MO). All other chemicals were of reagent grade.
Deionized distilled water (DDW) by Nanopure Infinity™ ultrapure
water system with ultraviolet (UV) light (Barnstead, Dubuque, IA)
was used for the preparation of all reagents and buffers. All DDW
and buffers were treated with Chelex-100 resin to remove any free
metal ions before use.
substances, and then used as a high molecular weight (HMW) fraction. The precipitant was re-suspended in three volumes of 50 mM
acetate buffer (pH 5.6) and centrifuged to remove remaining
water-solubles. After washing two more times with acetate buffer,
the precipitant was suspended in three volumes of 50 mM acetate
buffer (pH 5.6) and used as a precipitant (P) fraction (Fig. 1). All
fractions were stored at 4 °C until analyzed and all analyses were
finished within 3 days after preparations.
2.3. Lipid oxidation potential (LOP)
Lipid oxidation potential (LOP) of catalysts (metmyoglobin,
Fe(II), and Fe(III)), chelating agents (DFO and DTPA), fractions from
chicken breast and beef, and the mixtures of the catalysts or chelating agents with the fractions were determined in the phospholipid liposome model system. Metmyoglobin, ferrous ammonium
sulfate, ferric chloride, DTPA, and DFO solution dissolved in
50 mM acetate buffer (pH 5.6) were mixed with each fraction at
1:1 (v/v) ratio just before analyses to make their final concentrations at 1.0 mg/ml, 5 lg/ml, 5 lg/ml, 2 mM, and 2 mM, respectively. The phospholipids from egg yolk was used to prepare the
liposome model system following the method described previously
(Min & Ahn, 2009). The fatty acid composition of the phospholipids
used in this study is shown in Table 1. Briefly, an aliquot of phospholipids dissolved in chloroform were transferred to a volumetric
flask and evaporated under nitrogen gas to make a thin film on the
flask wall. Each fraction was added to the phospholipid-coated
flask and then the flask was shaken vigorously for 2 min to make
fraction-liposome solution with final concentration of 3 mg phospholipids per milliliter fraction.
The liposome solutions containing the meat fraction were
transferred to scintillation vials and incubated at 37 °C for
120 min to accelerate lipid oxidation. Lipid oxidation in the liposome solution was determined at 0, 15, 30, 60, 90, and 120 min
after incubation. After adding 10 ll of 6% BHT in ethanol to stop lipid oxidation, an aliquot (0.5 ml) of sample was mixed with 1 ml of
TBA/TCA solution (15 mM TBA/15% trichloroacetic acid (TCA; w/v))
and incubated in a boiling water bath for 15 min. After cooling, the
mixture was centrifuged at 15,000 g for 10 min. The absorbance
of the supernatant was determined at 531 nm against a reagent
2.2. Preparation of fractions from meat homogenates
Eight beef loins were obtained from a local packing plant 6d
post-slaughter. Two loins were pooled and treated as a replication.
Each loin was trimmed off any visible fat and connective tissues,
and each replication was ground separately through a 3-mm plate
twice. Twelve 8-week-old broiler chickens raised on a cornsoybean meal diets were slaughtered according to the USDA guidelines, and breast meats were separated from the carcasses 24 h
after slaughter. The breast meats from 3 birds were pooled and
used as a replication. Muscles for each replication were ground
separately through a 3-mm plate twice.
The ground meat was homogenized with three volumes of
50 mM acetate buffer (pH 5.6) using a high speed homogenizer
(Brinkman Polytron, Model PT 10/35, Westbury, NY) for 15 s at
speed setting 7. A portion of the homogenate (H) was centrifuged
at 15,000 g for 30 min at 4 °C. After centrifugation, the supernatant was filtered through a Whatman No. 1 filter paper twice and
used as a supernatant (S) fraction. A portion of S fraction was ultrafiltered by centrifugation through a Centricon Plus-20 centrifugal
filter (MW cut-off of 10,000; Millipore, Billerica, MA). The filtrate
was collected as a low-molecular-weight (LMW) fraction. The
retentate was recovered, made to the initial volume with acetate
buffer, ultrafiltered two more times through a Centricon Plus-20
centrifugal filter to remove any remaining low molecular weight
Ground chicken breast
or beef loin
(Homogenization with 3 volumes of 50mM
acetate buffer, pH 5.6)
Homogenate
H fraction
(Centrifugation at 15,000 ×g for 30 min at 4°C)
Precipitate
S fraction
Supernatant
(Ultrafiltration with Centricon,
MW cutoff 10,000)
P fraction
Retentate
Filtrate
HMW fraction
LMW fraction
Fig. 1. Flow diagram of fraction preparation from raw chicken breast and beef loin.
Abbreviations: H, homogenate fraction; P, precipitate fraction; S, supernatant
fraction; HMW, high molecular weight fraction from supernatant fraction; LMW,
low molecular weight fraction from supernatant fraction; MW, molecular weight.
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B. Min et al. / Food Chemistry 128 (2011) 299–307
lications. The data were analyzed using the JMP software (SAS
Institute Inc., Cary, NC) and reported as means and standard deviation. Differences among means were assessed by Tukey’s method
(P < 0.05).
Table 1
Fatty acid composition of phospholipids extracted from
egg yolk by ethanol.
Fatty acid
Content (%)
Myristic acid
Palmitic acid
Palmitoleic acid
Margaric acid
Margaroleic acid
Stearic acid
Oleic acid
trans-Vaccenic acid
Linoleic acid
c-Linolenic acid
Gondoic acid
Arachidonic acid
DTA
DPA
DHA
0.19 ± 0.03
28.70 ± 0.22
1.28 ± 0.18
0.28 ± 0.01
0.12 ± 0.03
16.25 ± 0.14
27.01 ± 0.20
1.59 ± 0.15
15.38 ± 0.14
0.17 ± 0.02
0.21 ± 0.03
6.68 ± 0.09
0.41 ± 0.08
0.14 ± 0.02
1.59 ± 0.04
3. Results and discussion
Means were expressed with standard deviation. n = 4.
Abbreviations: DTA, all cis-7, 10, 13, 16-docosatetraenoic acid; DPA, all-cis-7, 10, 13, 16, 19-docosapentaenoic acid, DHA, all cis-4,7,10,13,16,19-docosahexaenoic
acid.
blank. The level of lipid oxidation in the liposome solution was expressed as 2-thiobarbituric acid reactive substances (TBARS) value
(mmol malondialdehyde (MDA)/kg phospholipid) calculated using
the molar extinction coefficient of 1.56 105 M1 cm1. The TBARS
value after 120 min incubation was used as LOP. The lipid oxidation potential (LOP) was defined as the capacity of each catalyst
(metmyoglobin, Fe(II), Fe(III)), fractions from raw chicken breast
or beef loin, or combinations of each catalyst and fraction, to increase the TBARS values in phospholipid liposome system after
the 120 min-incubation period. The TBARS values (mmol MDA/kg
meat) of chicken breast and beef loin used in this study was 2.64
and 3.61, respectively, which were not different from each other
(P > 0.05).
2.4. Statistical analysis
A factorial design (5 fractions 2 meats 6 treatments) was
used in this study. All the analyses were performed with four rep25.0
Fig. 2 shows the LOP of each catalyst in the liposome system
without any meat fractions. The LOP of each catalyst indicates its
own catalytic capacity for lipid oxidation in the liposome system
and the patterns of increases in TBARS values during the incubation vary depending on the mode of action of each catalyst. Fe(III)
showed extremely low LOP (0.68 mmol MDA/kg phospholipid),
which was not different from that of the phospholipid control
(P > 0.05). This indicates that Fe(III) is not a catalyst of lipid oxidation in the absence of reducing compounds. The LOP of metmyoglobin (22.55 mmol MDA/kg phospholipid) was significantly
higher than that of Fe(II) (14.77 mmol MDA/kg phospholipid)
(P < 0.05). The pattern of TBARS increase by metmyoglobin in the
liposome system was different from that of Fe(II). Metmyoglobin
increased the TBARS values linearly during the incubation probably
due to the linear production of ferrylmyoglobin and/or hematin
from metmyoglobin throughout the incubation (Min & Ahn,
2005). Fe(II) increased the TBARS value rapidly at the beginning,
but the rate of TBARS increase during incubation was much slower
than that of metmyoglobin. Fe(II) showed a very strong catalytic
activity but its activity decreased as Fe(II) is converted to Fe(III)
during the incubation. Different prooxidant activities between
Fe(III) and Fe(II) are consistent with the previous result (Ahn &
Kim, 1998), which suggested that the status of free ionic iron is
more important than the amount. The presence of reducing agents
is critical for the conversion of Fe(III) to Fe(II) for the continuous
catalysis of lipid oxidation (Decker & Hultin, 1992; Min & Ahn,
2005).
Figs. 3–7 show the TBARS and LOP of liposomes containing each
fraction from chicken breast or beef loin added with catalysts and
iron chelating agents during incubation. The LOP of each fraction is
closely associated with the interactions between pro- and antioxidant factors in each fraction. Therefore, the comparison of
changes in the LOPs of fractions from each meat by the addition
of catalysts and iron chelating agents can provide useful
PL
TBARS value (mmol MDA / kg phospholipid)
Fe(II)
Fe(III)
20.0
Mb
15.0
10.0
5.0
0.0
0
20
40
60
80
100
120
Reaction time (min)
Fig. 2. Lipid oxidation potential of myoglobin (Mb, 1 mg/ml liposome solution), and free ionic irons (Fe(II) and Fe(III), 5 lg/ml liposome solution, respectively), in the
phospholipid liposome model system during incubation at 37 °C for 120 min (TBARS values, mmol malondialdehyde (MDA)/kg phospholipid). The phospholipid liposome
model system with 50 mM acetate buffer (pH 5.6) was used as a control (PL). Means with standard deviations are indicated (n = 4).
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B. Min et al. / Food Chemistry 128 (2011) 299–307
(B) Beef loin
(A) Chicken breast
25.0
25.0
PL
PL
Ct rl
Ct rl
Fe(II)
20.0
TBARS value (mmol MDA / kg phospholipid)
TBARS value (mmol MDA / kg phospholipid)
Fe(II)
Fe(III)
Mb
DT PA
DFO
15.0
10.0
5.0
0.0
20.0
Fe(III)
Mb
DT PA
DFO
15.0
10.0
5.0
0.0
0
20
40
60
80
100
120
0
20
Reaction time (min)
40
60
80
100
120
Reaction time (min)
Fig. 3. Lipid oxidation potential of homogenate (H) fractions from chicken breast (A) and beef loin (B) treated with myoglobin (Mb, 1 mg/ml liposome solution), free ionic
irons (Fe(II) and Fe(III), 5 lg/ml liposome solution, respectively), or chelating agents (desferrioxamine (DFO, 2 mM; final conc.) and diethylenetriamine pentaacetic acid
(DTPA, 2 mM; final conc.)) in phospholipid model system during incubation at 37 °C for 120 min (TBARS values, mmol malondialdehyde (MDA)/kg phospholipid). The
phospholipid liposome model system with each fraction was used as a control (Ctrl) and with 50 mM acetate buffer (pH 5.6) was as a blank control (PL). Means with standard
deviations are indicated (n = 4).
(A) Chicken breast
(B) Beef loin
25.0
25.0
PL
PL
Ct rl
Ct rl
20.0
Fe(II)
TBARS value (mmol MDA / kg phospholipid)
TBARS value (mmol MDA / kg phospholipid)
Fe(II)
Fe(III)
Mb
DT PA
DFO
15.0
10.0
5.0
0.0
20.0
Fe(III)
Mb
DT PA
DFO
15.0
10.0
5.0
0.0
0
20
40
60
80
Reaction time (min)
100
120
0
20
40
60
80
100
120
Reaction time (min)
Fig. 4. Lipid oxidation potential of precipitate (P) fractions from chicken breast (A) and beef loin (B) treated with myoglobin (Mb, 1 mg/ml liposome solution), free ionic irons
(Fe(II) and Fe(III), 5 lg/ml liposome solution, respectively), or chelating agents (desferrioxamine (DFO, 2 mM; final conc.) and diethylenetriamine pentaacetic acid (DTPA,
2 mM; final conc.)) in phospholipid model system during incubation at 37 °C for 120 min (TBARS values, mmol malondialdehyde (MDA)/kg phospholipid). The phospholipid
liposome model system with each fraction was used as a control (Ctrl) and with 50 mM acetate buffer (pH 5.6) was as a blank control (PL). Means with standard deviations are
indicated (n = 4).
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B. Min et al. / Food Chemistry 128 (2011) 299–307
(A) Chicken breast
(B) Beef loin
25.0
25.0
PL
PL
Ct rl
Ct rl
Fe(II)
20.0
20.0
Fe(III)
TBARS value (mmol MDA / kg phospholipid)
TBARS value (mmol MDA / kg phospholipid)
Fe(II)
Mb
DT PA
DFO
15.0
10.0
5.0
Fe(III)
Mb
DT PA
DFO
15.0
10.0
5.0
0.0
0.0
0
20
40
60
80
100
120
0
20
Reaction time (min)
40
60
80
100
120
Reaction time (min)
Fig. 5. Lipid oxidation potential of supernatant (S) fractions from chicken breast (A) and beef loin (B) treated with myoglobin (Mb, 1 mg/ml liposome solution), free ionic irons
(Fe(II) and Fe(III), 5 lg/ml liposome solution, respectively), or chelating agents (desferrioxamine (DFO, 2 mM; final conc.) and diethylenetriamine pentaacetic acid (DTPA,
2 mM; final conc.)) in phospholipid model system during incubation at 37 °C for 120 min (TBARS values, mmol malondialdehyde (MDA)/kg phospholipid). The phospholipid
liposome model system with each fraction was used as a control (Ctrl) and with 50 mM acetate buffer (pH 5.6) was as a blank control (PL). Means with standard deviations are
indicated (n = 4).
(A) Chicken breast
(B) Beef loin
25.0
25.0
PL
PL
Ct rl
Ct rl
Fe(II)
20.0
TBARS value (mmol MDA / kg phospholipid)
TBARS value (mmol MDA / kg phospholipid)
Fe(II)
Fe(III)
Mb
DT PA
DFO
15.0
10.0
5.0
0.0
20.0
Fe(III)
Mb
DT PA
DFO
15.0
10.0
5.0
0.0
0
20
40
60
80
Reaction time (min)
100
120
0
20
40
60
80
100
120
Reaction time (min)
Fig. 6. Lipid oxidation potential of high molecular weight (HMW) fractions from chicken breast (A) and beef loin (B) treated with myoglobin (Mb, 1 mg/ml liposome solution),
free ionic irons (Fe(II) and Fe(III), 5 lg/ml liposome solution, respectively), or chelating agents (desferrioxamine (DFO, 2 mM; final conc.) and diethylenetriamine pentaacetic
acid (DTPA, 2 mM; final conc.)) in phospholipid model system during incubation at 37 °C for 120 min (TBARS values, mmol malondialdehyde (MDA)/kg phospholipid). The
phospholipid liposome model system with each fraction was used as a control (Ctrl) and with 50 mM acetate buffer (pH 5.6) was as a blank control (PL). Means with standard
deviations are indicated (n = 4).
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B. Min et al. / Food Chemistry 128 (2011) 299–307
information for identifying factors affecting different oxidative stability between meats and, ultimately, better understanding the
mechanisms of lipid oxidation in meat.
Table 2
Antioxidant or prooxidant potential of each fraction from raw chicken breast and beef
loin for metmyoglobin (metMb), Fe(II), and Fe(III) in the phospholipid liposome
model system.
Fraction
3.1. The LOP of homogenate (H) fraction
The LOP of homogenate (H) fraction from chicken breast
(2.40 mmol MDA/kg phospholipid) was significantly lower than
that from beef loin (4.29 mmol MDA/kg phospholipid) (P < 0.05;
Fig. 3) probably due to higher storage-stable total antioxidant
capacity (TAC) in chicken breast. In our previous study (Min &
Ahn, 2009), TAC was significantly higher in H fraction from chicken
breast than that from beef loin. The TAC in chicken breast did not
change during the 10-day storage but that in beef loin decreased
significantly (Min & Ahn, 2009). Addition of Fe(III) to the H fraction
significantly increased the LOPs (P < 0.05), but its increase with
beef fraction (3.79 mmol MDA/kg phospholipid) was more than 3
times higher than that from chicken breast (1.12; P < 0.05). As
shown in Fig. 2, Fe(III) itself was not a catalyst in liposome model
system, and should be reduced to Fe(II) to catalyze lipid oxidation.
Our previous study (Min & Ahn, 2009) indicated that ferric ion
reducing capacity (FRC) of H fraction from chicken breast was
around 2 times higher than that from beef loin, but the FRC of H
fraction from chicken breast decreased rapidly at the initial stage
of the storage to the same level of that from beef loin. Therefore,
storage-unstable FRC of H fraction from chicken breast appeared
to be responsible for a rapid increase in the initial TBARS value.
Storage-stable FRCs accounted for continuous increases of TBARS
values of Fe(III)-added H fractions from both meats during incubation. The differences in LOP of H fractions from both meat with Fe
(III) was likely caused by the differences in their storage-stable TAC
(Min & Ahn, 2009). In addition, lipid oxidation increased drastically
(A) Chicken breast
metMb
%
H
P
S
HMW
LMW
Prooxidant
potentialb
Fe(II)
Fe(III)
Chicken
breast
Beef
loin
Chicken
breast
Beef
loin
Chicken
breast
Beef
loin
79.68
35.43
75.53
47.83
58.11
83.78
62.53
82.60
90.42
49.54
81.19
72.95
77.05
68.83
83.65
54.83
66.88
50.71
43.67
70.77
164.98
122.10
116.72
88.48
10.25
560.40
138.00
472.04
429.39
221.17
Abbreviations: H, homogenate fraction; P, precipitate fraction; S, supernatant fraction; HMW, high molecular weight fraction from supernatant fraction; LMW, low
molecular weight fraction from supernatant fraction.
a
The percentage inhibiting rate of each fraction from raw chicken breast and beef
loin for metmyoglobin- or Fe(II)-mediated lipid oxidation in phospholipid liposome
model system. See Eq. (1).
b
The percentage increasing rate of each fraction from raw chicken breast and
beef loin for lipid oxidation in Fe(III)-contained phospholipid liposome model
system. See Eq. (2).
from 90 min to 120 min in Fe(III)-added H fraction from beef loin
probably due to a significant increase in the concentration of free
ionic iron released from myoglobin. A significant increase of free
ionic irons was detected in H fraction from beef loin after 5-day
storage, but not that from chicken breast (Min & Ahn, 2009). Therefore, the presence of storage-stable FRC and lower TAC can make
beef loin more susceptible to lipid oxidation than the chicken
breast in the presence of free ionic iron.
(B) Beef loin
25.0
25.0
PL
PL
Ct rl
Ct rl
20.0
Fe(II)
TBARS value (mmol MDA / kg phospholipid)
Fe(II)
TBARS value (mmol MDA / kg phospholipid)
Antioxidant potentiala
Fe(III)
Mb
DT PA
DFO
15.0
10.0
5.0
0.0
20.0
Fe(III)
Mb
DT PA
DFO
15.0
10.0
5.0
0.0
0
20
40
60
80
Reaction time (min)
100
120
0
20
40
60
80
100
120
React ion t ime (min)
Fig. 7. Lipid oxidation potential of low molecular weight (LMW) fractions from chicken breast (A) and beef loin (B) treated with myoglobin (Mb, 1 mg/ml liposome solution),
free ionic irons (Fe(II) and Fe(III), 5 lg/ml liposome solution, respectively), or chelating agents (desferrioxamine (DFO, 2 mM; final conc.) and diethylenetriamine pentaacetic
acid (DTPA, 2 mM; final conc.)) in phospholipid model system during incubation at 37 °C for 120 min (TBARS values, mmol malondialdehyde (MDA)/kg phospholipid). The
phospholipid liposome model system with each fraction was used as a control (Ctrl) and with 50 mM acetate buffer (pH 5.6) was as a blank control (PL). Means with standard
deviations are indicated (n = 4).
B. Min et al. / Food Chemistry 128 (2011) 299–307
Addition of Fe(II) also increased the LOPs of H fractions from
both meats significantly, but its increase from beef loin (6.67 mmol
MDA/kg phospholipid) was over 2 times higher than that from
chicken breast (2.78) (P < 0.05; Fig. 3) probably due to higher storage-stable TAC in the H fraction from chicken breast (Min & Ahn,
2009). The antioxidant potential of H fraction from chicken breast
(81.19%) against Fe(II)-mediated lipid oxidation was 1.48 time
higher than that from beef loin (54.83%) (P < 0.05; Table 2). These
results suggested that the lower TAC in beef loin is a critical factor
for its higher susceptibility to lipid oxidation than chicken breast.
Addition of metMb to the liposome system containing H fraction increased LOPs of H fractions from both meats, but the increase of LOP with H fraction from chicken breast (4.58 mmol
MDA/kg phospholipid) was significantly higher than that from beef
loin (3.66) (P < 0.05; Fig. 3). The amount of metmyoglobin is a critical factor for its prooxidant activity in the presence of LOOH or
fatty acid: metmyoglobin acts as a strong prooxidant at a low concentration in lipid system (Baron & Andersen, 2002; Lapidot et al.,
2005). Our previous study (Min et al., 2010) also showed that the
level of lipid oxidation significantly decreased as the metmyoglobin concentration increased from 0.25 to 2 mg/ml in phospholipid
liposome model system. The myoglobin concentrations of liposome solutions in the metMb-added H fraction from beef loin
and chicken breast were 1.85 and 1.13 mg/ml liposome,
respectively.
The addition of chelating agents (DTPA or DFO) completely suppressed the LOPs of H fraction from chicken breast (Fig. 3), indicating that free ionic iron-catalyzed lipid oxidation may be the main
mechanism in chicken breast. Both DTPA and DFO significantly decreased the LOP of H fraction from beef loin, but DFO was more
efficient suppressor than DTPA (P < 0.05). Addition of DTPA lowered approximately 25% of LOP of H fraction from beef loin, indicating
that
DTPA-unchelatable
compounds,
such
as
ferrylmyoglobin and/or hematin, could be major catalysts of lipid
oxidation in raw beef loin.
3.2. The LOP of precipitate (P) fraction
The LOP of precipitate (P) fraction from chicken breast
(1.00 mmol MDA/kg phospholipid) was not different from that
from beef loin (0.13 mmol MDA/kg phospholipid, P > 0.05; Fig. 4).
Also, the LOPs of Fe(II)- and Fe(III)-added P fractions from chicken
breast (4.98 and 1.81 mmol MDA/kg phospholipid, respectively),
were not different from those from beef loin (5.03 and 1.07 mmol
MDA/kg phospholipid, respectively). However, the LOP of metMbadded P fractions from chicken breast (15.54 mmol MDA/kg
phospholipid) was significantly higher than that from beef loin
(8.58 mmol MDA/kg phospholipid, P < 0.05). The LOP of P fraction
from chicken breast and beef loin were not increased by adding
Fe(III) (P > 0.05) even though storage-stable FRCs were detected
in the P fractions from both meats. This could be related to the iron
chelating capacity of water-insoluble proteins, such as hemosiderin. The addition of Fe(II) increased the LOP of P fraction from both
meats (4.00 and 4.90 mmol MDA/kg phospholipid). The addition of
metMb significantly increased the LOP of P fraction from both
chicken breast and beef loin, but the LOP of P fraction from chicken
breast was greater than that from beef loin (14.56 vs 8.45 mmol
MDA/kg phospholipid). This indicated that the primary mode of
antioxidant action of P fraction from chicken breast is due to its
iron chelating capacity.
3.3. The LOP of supernatant (S) fraction
The LOP of supernatant (S) fraction from chicken breast
(2.97 mmol MDA/kg phospholipid) was ½ of the beef loin
(6.65 mmol MDA/kg phospholipid, P < 0.05; Fig. 5) because of high-
305
er TAC and lower myoglobin content in the S fraction from chicken
breast than beef loin (Min & Ahn, 2009). The addition of Fe(III) did
not increase the LOP of S fraction from chicken breast, but significantly increased that from beef loin. The pattern of TBARS increase
in model system with Fe(III)-added S fraction from both meats was
similar to that with Fe(III)-added H fraction (Fig. 3).
The addition of Fe(II) increased significantly the LOP of S fraction from both meats (Fig. 5), but the increase of LOP with S fraction from beef loin (7.28 mmol MDA/kg phospholipid) was
approximately 2 times higher than that from chicken breast
(3.39 mmol MDA/kg phospholipid, P < 0.05). This suggested that
the S fraction from chicken breast had higher antioxidant capacity
against Fe(II)-catalyzed lipid oxidation than that from beef loin.
However, the addition of metMb increased the LOP of S fraction
from chicken breast (5.52 mmol MDA/kg phospholipid) more than
that from beef loin (3.93 mmol MDA/kg phospholipid, P < 0.05).
This may be partially attributed to the myoglobin concentration effect to lipid oxidation. Our previous study (Min et al., 2010) found
that TBARS values of metMb-added raw chicken breast patty did
not increase and around 73% of metMb in the patty were reduced
during 10 days of storage. However, both P and S fractions from
chicken breast could not prevent metMb-mediated lipid oxidation
(Figs. 4 and 5). This suggested that the interaction between reducing components in both P and S fractions is essential to prevent
metMb-mediated lipid oxidation in chicken breast.
Addition of iron chelating agent, such as DTPA or DFO completely suppressed the LOP of S fraction from chicken breast
(Fig. 5), indicating that the presence of free ionic iron is the primary
cause of the LOP of S fraction from chicken breast. Both DTPA and
DFO significantly decreased the LOP of S fraction from beef loin,
but DFO was a more efficient suppressor than DTPA. Addition of
DTPA lowered the LOP of the S fraction from beef loin by about
50%, suggesting that DTPA-chelatable (free ionic iron) and unchelatable compounds (ferrylmyoglobin and/or hematin) were equally
responsible for the LOP of S fraction from beef loin. The contribution of free ionic iron to the LOP of S fraction from beef loin
(50%) was much higher than that of H fraction from beef loin
(25%). This difference is likely due to the difference in the availability of free ionic iron, resulting from the presence of iron chelating
capacity of water-insoluble compounds such as hemosiderin in P
fraction from beef loin. The nonheme iron content in the P fraction
from beef loin significantly increased during 10-day storage as the
myoglobin content decreased (Min & Ahn, 2009).
3.4. The LOP of high molecular weight (HMW) fraction
The LOP of HMW fraction from chicken breast (0.36 mmol
MDA/kg phospholipid) was much lower than that of HMW fraction
from beef loin (12.57 mmol MDA/kg phospholipid, P < 0.05; Fig. 6).
The LOP of HMW fraction from beef loin was significantly higher
than that of H and S fractions (4.29 and 6.65 mmol MDA/kg
phospholipid, respectively, P < 0.05), suggesting that the P and
LMW fraction inhibited the prooxidant potential of myoglobin during incubation. The addition of Fe(III) did not increase the LOP of
HMW fraction from chicken breast, but significantly increased that
from beef loin. The addition of Fe(II) significantly increased the LOP
of HMW fraction from chicken breast, but it was significantly lower
than that from beef loin. However, addition of myoglobin significantly increased the LOP of HMW fraction from chicken breast.
The addition of DFO and DTPA decreased the LOP of HMW fraction from beef loin by 75% (Fig. 6), which was greater than that of H
and S fractions from beef loin (25% and 50%, Figs. 3 and 5, respectively). This indicated that DTPA-chelatable compounds (i.e. free
ionic irons) in the presence of storage-stable FRC are the primary
catalysts in the HWP fraction from beef loin, rather than DTPAunchelatable compounds (i.e. ferrylmyoglobin and/or hematin).
306
B. Min et al. / Food Chemistry 128 (2011) 299–307
This also suggested that most of the free ionic irons released from
myoglobin were chelated by the P and LMW fraction from beef
loin. The LOPs of DTPA-added H, S, and HWM fractions from beef
loin were similar (3.21, 3.29, and 3.19 mmol MDA/kg phospholipid,
respectively). Thus, the catalytic activity of DPTA-unchelatable catalysts from myoglobin was stable and was not inhibited by the
antioxidant potentials in P and LMW fractions. The organic compounds containing phosphate groups, such as ADP and ATP, may
be the primary iron chelating agents in LMW fraction (Erickson,
Hultin, & Borhan, 1990), and the hemosiderin in P fraction (Decker
& Hultin, 1992). However, the ability of low molecular weight chelating agents to inhibit the development of lipid oxidation varied
significantly depending upon the concentrations of chelating
agents and free ionic iron (Graf, Mahoney, Bryant, & Eaton,
1984). Continuous increase of free ionic iron content would exceed
the iron chelating capacity of LMW fraction from beef loin after
prolonged storage and turn pre-existing iron chelating agents into
prooxidants. This synergistic effect explains a rapid increase of
TBARS values in model system with metMb- and Fe(III)-added H
fraction from beef loin between 90 and 120 min of incubation
(Fig. 3). Therefore, we suggest that DTPA-unchelatable compounds,
such as ferrylmyoglobin and/or hematin, derived from myoglobin
rather than released free ionic irons may be the major catalysts
of lipid oxidation in raw beef loin under normal storage conditions.
However, exogenous oxidative stresses such as prolonged storage,
NaCl addition, temperature abuse, cooking, and various other processes, which facilitate the release of free ionic iron from myoglobin in beef loin beyond its chelating capacity, can also result in an
exponential increase of lipid oxidation in beef loin (Min & Ahn,
2005).
3.5. The LOP of low molecular weight (LMW) fraction
The LOP of LMW fraction from chicken breast was significantly
higher than that from beef loin (2.34 vs 0.98 mmol MDA/kg
phospholipid, P < 0.05) (Fig. 7). Lipid oxidation by the LMW fractions from both meat increased rapidly from 0 to 15 min, and then
did not change during the rest of incubation because of rapid
depletion of low molecular weight reducing compounds, such as
ascorbic acid, NAD(P)H, glutathione, thiols, etc. (Min & Ahn,
2005) in LMW fractions. These results are consistent with those
of Kanner, Salan, Harel, and Shegalovich (1991) who indicated that
LMW fraction from turkey meat showed a prooxidant effect in the
model system containing ascorbic acid and ferric ion, whereas
HMW fraction suppressed lipid oxidation in the same system.
Our previous study (Min & Ahn, 2009) showed that the FRC of
LMW fraction from chicken breast was around 2 times higher than
that from beef loin at Day 0 but rapidly decreased during storage.
Therefore, a difference between the FRCs of LMW fractions from
both meats were responsible for the difference in the LOP of
LMW fractions between the two meats.
Addition of Fe(III) did not increase the LOP of LMW fraction
from chicken breast (Fig. 7A). Lipid oxidation of model system with
H and S fractions from chicken breast increased gradually after a
rapid increase at an early stage of incubation, and the addition of
Fe(III) increased the LOP of H and S fractions (Figs. 3A and 5A).
The storage-unstable FRC of LMW fraction should be responsible
for the rapid increase at the early stage of incubation, and the storage-unstable FRC of P and HMW fractions for the gradual increase
of TBARS. Reducing compounds can act as prooxidants or antioxidants, depending on the concentration of iron: prooxidants at high
concentrations and antioxidants at low concentrations of free ionic
iron (Decker & Hultin, 1992). Chicken breast had low free ionic iron
sources (i.e. heme pigments) and most of the free ionic irons released from iron-containing sources were chelated in P and S fractions (Min & Ahn, 2009). Therefore, the stable and unstable
reducing systems in chicken breast appeared to serve as antioxidants rather than prooxidants.
The addition of Fe(III) significantly increased the LOP of LMW
fraction from beef loin (P < 0.05) because lipid oxidation increased
gradually throughout the incubation (Fig. 7B). This indicates the
presence of storage-stable FRC in the LMW fraction from beef loin.
The storage-stable FRC in the LMW fraction from beef loin was
around 2 times greater than that from chicken breast after 10day storage and a significant amount of free ionic irons was detected in the LMW fraction from beef loin after 10-day storage at
4 °C (Min & Ahn, 2009). Therefore, the storage-stable FRC in the
LMW fraction from beef loin can contribute to the development
of lipid oxidation after prolonged storage of beef loin.
The addition of Fe(II) increased the LOP of LMW fraction from
beef loin significantly, and the value was significantly higher than
that from chicken breast (P < 0.05; Fig. 7). LMW fraction from
chicken breast showed the highest antioxidant potential (83.65%)
for Fe(II)-catalyzed lipid oxidation among fractions (Table 2). The
high concentration of carnosine and anserine in chicken breast
(Chan & Decker, 1994) may be partially responsible for the high
TAC of its LMW fraction. The addition of both DTPA and DFO completely suppressed the LOP of LMW fractions from both meats.
3.6. Anti- and prooxidant potential of the fractions
Anti- and prooxidant potential of fractions from chicken breast
and beef loin. The LOPs of fractions from chicken breast and beef
loin were increased by addition of catalysts. However, their increases varied depending upon the catalysts, composition of proand antioxidant factors in the fraction, the mode of reactions between catalysts, and the pro- and anti-oxidant factors in each fraction. The LOP of each fraction increased by metmyoglobin and
Fe(II), but their increases were lower than that of metmyoglobin
or Fe(II) alone (Fig. 2). This indicated that each fraction had antioxidant potentials to reduce the prooxidant capacity of metmyoglobin or Fe(II). However, the LOPs of each fraction increased by
Fe(III) even though Fe(III) does not show the prooxidant capacity
in liposome system (Fig. 2). This indicated that each fraction had
storage-stable FRC, which can convert Fe(III) to Fe(II) for continuous increase of lipid oxidation during incubation. Table 2 summarizes the prooxidant or antioxidant potential of each fraction in the
liposome system, which was calculated as follow:
ðLOP CF LOP FÞ
Antioxidant potentialð%Þ ¼ 1 100
ðLOP C LOP BÞ
Prooxidant potentialð%Þ ¼
ðLOP CF LOP FÞ
100
ðLOP C LOP BÞ
ð1Þ
ð2Þ
where, LOP_CF is the LOP of catalyst-added fraction, LOP_F is the
LOP of fraction without catalyst, LOP_C is the LOP of catalyst, and
LOP_B is the LOP of phospholipid liposome blank.
The antioxidant potentials of fractions from chicken breast
against the catalytic activity of Fe(II) were higher than those of
counterpart fractions from beef loin. The prooxidant potentials of
fractions from beef loin in the presence of Fe(III) were much higher
than those of its counterpart fractions from chicken breast probably because of lower TAC and antioxidant potentials in beef loin.
However, the antioxidant potentials of the H, S, and HMW fractions
from beef loin against the catalytic activity of metMb were greater
than their counterparts from chicken breast. Studies (Baron &
Andersen, 2002; Lapidot et al., 2005; Min et al., 2010) indicated
that the prooxidant activity of metMb is dependent upon its relative concentration to fatty acid or LOOH: the lower its concentration, the higher its prooxidant activity. Our previous study (Min
et al., 2010) demonstrated that an increase of metmyoglobin con-
B. Min et al. / Food Chemistry 128 (2011) 299–307
centration from 0.25 to 2 mg/ml in the liposome solution decreased the level of lipid oxidation. The inclusion of metMb into
the H, S, and HMW fractions from beef loin doubled the concentration of metMb in the reaction solution, resulting in the decreased
prooxidant activity of metMb. This is the reason why their antioxidant potential were higher despite their lower TAC compared with
their counterparts from the chicken breast.
4. Conclusions
Higher antioxidant potential and TAC and lower myoglobin concentration were the major contributors for higher oxidative stability of chicken breast than beef loin. The antioxidant potential of
chicken breast was attributed to iron chelating capacity and the
synergistic interactions between antioxidant agents in the LMW
and P fractions. Most of the fractions contained storage-stable
FRC, which could continuously increase lipid oxidation in the presence of free ionic iron. Although the P and LMW fractions from beef
loin showed significant iron chelating capacities, the amount of
free ionic iron released from myoglobin during storage exceeded
their iron chelating capacity. Major catalysts for lipid oxidation
in beef loin were DTPA-unchelatable compound, ferrylmyoglobin
and/or hematin in ‘‘normal’’ storage conditions because most of
the free ionic irons released from myoglobin in beef loin were chelated by LMW and P fractions. However, free ionic iron reduced by
storage-stable FRC could be a major catalyst under prolonged storage conditions where free ionic iron content exceeds the iron chelating capacity of beef loin.
Acknowledgements
The work has been supported by the National Integrated Food
Safety Initiative/USDA (USDA Grant 2002-5110-01957), Washington DC, and WCU (World Class University) program (R31-10056)
through the National Research Foundation of Korea funded by
the Ministry of Education, Science and Technology.
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