Meat Science 58 (2001) 431–435
www.elsevier.com/locate/meatsci
Cholesterol oxidation products in irradiated raw
meat with different packaging and storage time$
K.C. Nam, M. Du, C. Jo, D.U. Ahn *
Department of Animal Science, Iowa State University, Ames, IA 50011-3150, USA
Received 6 November 2000; received in revised form 22 January 2001; accepted 22 January 2001
Abstract
The effect of irradiation and packaging conditions on the formation of cholesterol oxidation products (COPs) as well as lipid
oxidation products was determined in raw turkey leg, beef, and pork loin meat during 7 days of storage. Ground turkey leg, beef,
and pork loin muscles were prepared as patties. The patties were individually packaged either in oxygen-permeable or impermeable
bags, irradiated at 0 or 4.5 kGy using a Linear Accelerator, and stored at 4 C. The COPs such as 7a-hydroxycholesterol, 7bhydroxycholesterol, and 7-ketocholesterol were detected in fresh raw meats at 0 day at the level of 10.9 to 49.2 mg/g lipid. After 7
days of storage, other COPs such as epoxides, 20a-hyroxycholesterol, and choletanetriol were formed in mainly aerobically packaged and irradiated raw meats. Packaging effect was more crucial on the cholesterol and lipid oxidation than irradiation. In aerobically packaged and irradiated meats, turkey leg muscles had higher COPs value than beef or pork did. COPs and thiobarbituric
acid reactive substances (TBARS) values had a strongly positive correlation in turkey leg and pork. But, cholesterol oxidation in
beef proceeded in irradiated and aerobically stored samples despite of its low level of TBARS value. # 2001 Elsevier Science Ltd.
All rights reserved.
Keywords: Cholesterol oxidation products; Raw meat; Irradiation; Packaging
1. Introduction
Cholesterol oxidation products as well as fatty acid
oxidation in food system have been a concern for adult
human disease. Cholesterol oxidation products (COPs)
have been known to be more injurious to arterial cells
than pure cholesterol and are more directly connected
to the development of atherosclerosis and coronary
heart disease (Addis, 1986; Paniangvait, King, Jones, &
German, 1995). COPs deteriorated the bioavailability of
cholesterol by inhibiting cholesterol biosynthesis (Lund
& Bjorkhem, 1994) and dietary uptake of cholesterol
(Peng, Hu, & Morin, 1985). COPs also impaired a
membrane function, which results in altered membrane
permeability (Hennig & Boissonneault, 1987). On the
other hand, natural cholesterol had no atherogenic or
$
Journal Paper No. J-18976 of the Iowa Agriculture and Home
Economics Experiment Station, Ames, IA 50011. Project No. 3322,
supported by S-292 Regional Project and Iowa Egg Council.
* Corresponding author. Tel.: +1-515-294-6595; fax: +1-515-2949143.
E-mail address: duahn@iastate.edu (D.U. Ahn).
hypercholesterolemic effect and had a much lower
influence on the activities of most enzymes than COPs
(Smith, 1987).
Cholesterol is a relatively stable compound, but it can
be oxidized under harsh conditions. Oxidation of cholesterol in muscle foods can be influenced by many factors such as processing temperature, storage time,
packaging conditions, and lipid composition (Paniangvait et al., 1995). Cholesterol present in heated tallow
was degraded into 7a-hydroxycholesterol, 7b-hydroxycholesterol, 7-ketocholesterol, or a-epoxide (Park &
Addis, 1986). Smith (1987) suggested that hydroperoxides of polyunsaturated fatty acids formed during
lipid oxidation might be necessary to initiate cholesterol
oxidation, and unsaturated fat could increase the oxidation of cholesterol synergistically.
Irradiation, which has been used to improve microbial
safety of meat, can also be a crucial factor in initiating
the oxidation of cholesterol because it increases fatty
acids oxidation in the meat system. A few studies were
conducted to determine the effect of ionizing radiation
on the formation of COPs (Maerker & Jones, 1991;
Sevanian & McLeod, 1987) only using model systems.
0309-1740/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0309-1740(01)00046-8
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K.C. Nam et al. / Meat Science 58 (2001) 431–435
The objectives of this study, therefore, were to determine the effect of irradiation and packaging on the formation of COPs and lipid oxidation in raw turkey leg,
beef, and pork during storage, and elucidate the relationship between fatty acid and cholesterol oxidation.
2. Materials and methods
2.1. Sample preparation and irradiation
Turkey leg, beef loin, and pork loin muscles were
purchased from four different local grocery stores, and
trimmed of all surface fat. Meats from each store were
used as a replication. The lean muscles were ground
separately through a 3-mm plate and patties (approximately 100 g each) were prepared. The patties were
packaged in either aerobic or vacuum bags (polyethylene vacuum bags, O2 permeability: 9.3 ml O2/m2/
24h at 0 C; Koch, Kansas City, MO) and irradiated
using a linear accelerator (Circe Thomson CSF Linac,
Saint-Aubin, France) with a dose of 0 or 4.5 kGy. The
energy and power level used were 10 MeV and 10 kW,
respectively, and the average dose rate was 92.0 kGy/
min. The maximum/minimum ratio was approximately
1.15 for 4.5 kGy. To confirm the target dose, two alanine dosimeters per cart were attached to the top and
bottom surfaces of the sample. The alanine dosimeter
was read using a 104 Electron Paramagnetic Resonance
Instrument (Bruker Instruments Inc., Billerica, MA).
The COPs were analyzed as described by Ahn, Lee, Jo,
and Sell (1999) after 0 and 7 days of storage at 4 C.
Lipid oxidation was determined by the fluorometric
analysis of TBARS according to the method of Jo and
Ahn (1998).
2.2. Lipid extraction and COPs preparation
Lipids were extracted from samples according to the
method of Folch, Lees, and Sloan-Stanley (1957). Five
grams of meat sample, butylated hydroxytoluene (BHT;
50 ml, 7.2%), and 30 ml Folch 1 solution (chloroform:
methanol=2:1) were added to a 50-ml test tube and
homogenized using a Polytron (Brinkman Instruments
Inc., Westbury, NY) at high speed for 20 s. The homogenate was filtered through a Whatman No.1 filter
paper (Whatman Inc., Clifton, NJ) into a 100-ml graduated cylinder, and the filter paper was rinsed twice with
10 ml Folch 1 solution. After adding 8 ml of 0.88%
NaCl solution to each cylinder, the cylinder was capped
with a glass stopper and the content mixed. The inside
of cylinder was washed twice with 5 ml of Folch 2
solution (chloroform: methanol: water=3:47:48). After
phase separation, the lipid layer volume was recorded,
and the upper layer (methanol and water) of the solution was completely and carefully siphoned off in order
not to contaminate the chloroform layer. The organic
layer was put in a glass scintillation vial and dried in a
block heater for 1 h at 50 C. The dried lipid was dissolved with an aliquot of hexane to make 0.1 g fat/ml
hexane and used for COPs and fatty acid analysis.
Lipid sample dissolved in hexane (0.2 g) was loaded
onto a silicic acid column prepared with a silicic acid
(100 mesh), cellite-545, and CaHPO.42H2O (10:9:1, w/w/
w) mixture in chloroform. The column was washed with
10 ml of Solvent I (hexane: ethyl acetate=9:1, v/v)
before loading a sample. Neutral lipids, cholesterol, and
phospholipids were eluted by passing 40 ml of Solvent
II (hexane: ethyl acetate=4:1, vol/vol) through the column. Then COPs were eluted with 40 ml of Solvent III
(acetone: ethyl acetate: methanol=10:10:1, vol/vol/vol)
and dried under nitrogen. The dried COPs were added
with 200 ml pyridine and 100 ml bis-trimethylsilyltrifluoroacetamide (BSTFA)+1% trimethylchlorosilane
(TMCS) and derivartized by heating in a dry bath
(80 C) for 1 h.
2.3. Gas chromatographic (GC) analysis of COPs
Analysis of COPs was performed with a Hewlett
Packard (HP) 6890 GC equipped with an on-column
capillary injector and flame ionization detector (FID,
Hewlett Packard Co., Wilmington, DE). A 30 m320
mm0.25 mm HP-5 capillary column (5% phenyl methyl
silicon, Hewlett Packard Co.) was used. A splitless inlet
was used to inject samples (0.5 ml) into the capillary
column using an autosampler (model 7683, Hewlett
Packard Co.), and a ramped oven temperature was used
(80 C for 0.25 min, increased to 230 C at 40 C/min,
increased to 270 C at 25 C/min, increased to 285 C at
1.5 C/min, and held for 8 min). Temperatures of both
the inlet and detector were 280 C. Helium was the carrier gas at constant pressure of 18.5 psi. Detector (FID)
air, H2, and make-up gas (He) flows were 300, 30, and
28 ml/min, respectively. The area of each peak (pAs)
was integrated by using Chemstation software (Hewlett
Packard Co.) and the amount of COPs was calculated
using an internal standard.
2.4. GC analysis of fatty acids composition
One milliliter of methylating reagent (boron-trifluoride methanol, Sigma Chemical Co.) was added to
100 ml of lipid extract and incubated in a 90 C water
bath for 1 h. After cooling to room temperature, 2 ml
hexane and 5 ml water were added, mixed thoroughly,
and left at room temperature overnight for phase
separation. The top hexane layer containing methylated
fatty acids was analyzed for fatty acid composition
using a GC (HP 6890, Hewlett Packard Co.). A ramped
oven temperature condition (180 C for 2.5 min,
increased to 230 C at 2.5 C/min, then held at 230 C for
K.C. Nam et al. / Meat Science 58 (2001) 431–435
7.5 min) was used. Temperatures of both the inlet and
detector were 280 C. Helium was the carrier gas at linear
flow of 1.1ml/min. Detector (FID) air, H2, and make-up
gas (He) flows were 350, 35, and 43 ml/min, respectively. Fatty acids were identified by comparison of
retention times to known standards. Relative quantities
were expressed as weight percent of total fatty acids.
2.5. Statistical analysis
The experimental design was intended to determine
the effects of irradiation, packaging condition, and storage time on lipid oxidation and cholesterol oxidation
during the 7 days of storage. Data were analyzed using
SAS software (SAS Institute, Inc., 1985) by the generalized linear model procedure; the Student–Newman–
Keuls’ multiple range test was used to compare differences among means. Mean values and standard error of
the mean (S.E.M.) were reported. Significance was
defined at P<0.05.
3. Results and discussion
3.1. Fatty acid composition
Table 1 shows the differences in fatty acids compositions of lipid from raw turkey leg, beef, and pork. Turkey leg meat had the highest proportion of unsaturated
fatty acids due to the high content of linoleic and arachidonic acid and low content of stearic acid. Beef had
the highest amounts of palmitoleic and oleic acids and
the highest content of stearic acid, but the low content
of polyunsaturated fatty acids made beef ranked to be
the most saturated among the three meat species. There
was no significant difference in total fat content among
the three meat species. In general, the amount of meat
Table 1
Fatty acid composition (%) of lipids from raw turkey, beef, and pork
meat pattiesa
Turkey Beef
Fat content (total; % of meat)
Percentage of weight
Fatty acid composition (% of total fat)
Palmitic
Palmitoleic
Margaric
Stearic
Oleic
Linoleic
Linolenic
Arachidonic
Unidentified
6.65
8.27
Pork
S.E.M.
9.38 0.929
4.01a 3.70a 2.34b 0.305
20.23b 25.18a 20.45b 0.659
1.40
1.21 1.86 0.269
9.40b 15.22a 13.17a 0.834
30.77c 39.24a 35.06b 1.267
26.16a 5.19c 16.96b 0.832
2.35b 1.34c 2.86a 0.098
4.02a 1.19b 3.26a 0.580
1.67b 7.75a 3.93b 0.746
a
Different letters within a row of the same meat are significantly
different (P<0.05). n=4.
433
cholesterol from different animal species are at the range
of 50–89 mg percent (Pikul, Leszczynski, Bechtel, &
Kummerow, 1984), thus the quantitative analysis of cholesterol of each species was not considered in this study.
3.2. Cholesterol oxidation
Previous studies showed that fresh raw meat contained
none or only trace amounts of cholesterol oxidation
products (Addis & Warner, 1991; Pie, Spahis, & Seillan,
1991). Tables 2–4, however, show that some COPs such
as 7a-hydroxycholesterol, 7b-hydroxycholesterol, and 7ketocholesterol existed in even fresh raw meat samples
before storage.
In the case of turkey leg meat (Table 2), irradiation
did not increase the oxidation of cholesterol at Day 0.
After 7 days of storage, however, aerobically packaged
turkey leg meat had more COPs content than the
vacuum-packaged one did, and irradiation also increased
the cholesterol oxidation in aerobically packaged meat.
The combined effect of irradiation and aerobic packaging increased the cholesterol oxidation synergistically.
But, irradiation had less effect on the oxidation of cholesterol compared with aerobic packaging effect. In the
aerobically packaged and irradiated raw turkey leg meat,
secondary COPs, which can be derived from primary
COPs such as a-epoxides, b-epoxides, cholestanetriol
and 20a-hydroxycholesterol, were detected. Cholestanetriol and 25-hydroxycholesterol were reported to be the
most atherogenic among oxysterols studied (Taylor,
Peng, Werthessen, Tham, & Lee, 1979). Peng et al.
(1985) reported that a remarkably acute injury to the
endothelium of rabbits resulted from 25-hydroxycholesterol and cholestanetriol.
In beef, both aerobic and vacuum packaging had no
effect on the amount of COPs at Day 0 (Table 3). Unlike
in turkey leg meat, a-epoxide was detected in beef
regardless of packaging, irradiation, and storage time.
After 7 days of storage with aerobic conditions, irradiated beef produced the highest amounts of COPs, but
the amount of total COPs was smaller than that of the
turkey leg meat. The relative increase of COPs in aerobically packaged and irradiated turkey leg meat during
the 7 days of storage was 152% compared to the initial
amount at day 0, but that in beef was only 93%. Park
and Addis (1987) reported that precooked beef product
had no detectable amounts of COPs and TBARS value
during 12 days of storage at 4 C. But, the amounts of
most COPs increased in even raw meats as storage time
increased except for 7-ketocholesterol. Secondary oxysterols, such as 20a-hydroxycholesterol and cholestanetriol, increased more than primary oxysterols after 7 days
of storage. The secondary oxysterols could be formed
from primary COPs as in turkey leg meat during storage.
Irradiation and packaging had significant effect on
the formation of COPs in pork at Day 0 (Table 4).
434
K.C. Nam et al. / Meat Science 58 (2001) 431–435
cholesterol content as in turkey leg meat and beef. Unlike
in turkey leg meat and beef, however, 20a-hydroxycholesterol was not detected in pork. Consequently, vacuumpackaged raw meats were very stable to cholesterol oxidation during storage regardless of irradiation. In some
samples, the amount of certain COPs decreased after 7
days of storage in vacuum packaging indicating that some
primary COPs could be converted to secondary COPs.
Aerobically packaged and irradiated samples had the
highest amounts of 7a- plus 7b-hydroxycholesterol and
total COPs, and vacuum-packaged and irradiated pork
had the lowest amount of total COPs. The predominant
COPs in raw pork were 7a- or 7b-hydroxycholesterol, 7ketocholesterol, and a-epoxide. Therefore, a-epoxide
were found in mainly beef or pork rather than in turkey
leg meat. Pie et al. (1991) reported that total COPs
increased after storage and C-7 oxidized products were
predominant with 7-ketocholesterol. Both aerobic packaging and irradiation were crucial in the formation of
COPs in pork. After 7 days of storage with aerobic
packaging, irradiated pork samples had the highest value
of 7a- plus 7b-hydroxycholesterol, b-epoxide, and total
3.3. Fatty acid oxidation
Irradiation and packaging influenced the TBARS
values of turkey and pork, but not beef. After 7 days of
storage, aerobically packaged turkey and pork developed
Table 2
Content of cholesterol oxidation products (COPs) in raw turkey leg meat with different irradiation, packaging, and storage timea
COPs
7a- & 7b-Hydroxycholesterol
b-Epoxide
a-Epoxide
20a-Hydroxycholesterol
Cholestanetriol
7-Ketocholesterol
Total
Day 0 (mg COPs/g lipid)
Day 7 (mg COPs/g lipid)
V-C
V-IR
A-C
A-IR
S.E.M.
V-C
V-IR
A-C
A-IR
S.E.M.
31.2
0
0.6
0
0.6
1.2
33.5
34.3
0
0
0
0
2.6
36.8
36.4
0
1.0
0.4
0.4
1.8
39.9
43.2
0
3.9
0.4
0
6.0
53.4
4.61
0
1.67
0.27
0.35
1.41
6.82
36.0c
0b
0c
0b
0
2.7c
38.7c
30.0c
0b
0c
0b
0
1.5c
31.6c
51.9b
0b
6.4b
0b
0
19.0b
77.3b
86.7a
7.2a
11.5a
1.4a
1.0
27.1a
134.7a
3.01
1.35
0.77
0.24
0.29
2.02
3.13
a
Different letters within a row of the same meat are significantly different (P<0.05). n=4. A, aerobic packaging; V, vacuum packaging; C, nonirradiated; IR, irradiated at 4.5 kGy dose; chol., cholesterol; S.E.M., standard error of the mean.
Table 3
Content of COPs in raw beef with different irradiation, packaging, and storage timea
COPs
7a- & 7b-Hydroxycholesterol
b-Epoxide
a-Epoxide
20a-Hydroxycholesterol
Cholestanetriol
7-Ketocholesterol
Total
Day 0 (mg COPs/g lipid)
Day 7 (mg COPs/g lipid)
V-C
V-IR
A-C
A-IR
S.E.M.
V-C
V-IR
A-C
A-IR
S.E.M.
7.8
0.8
5.6
0.6
0.6
7.7
23.0
5.6
1.1
3.6
0.8
0
5.3
16.3
19.0
0
7.0
0
0
13.4
39.4
15.1
0
8.3
0
0
12.8
36.1
3.54
0.67
2.20
0.50
0.31
2.70
7.96
9.2
3.7b
9.0c
3.6
0.3b
6.8
32.7b
8.3
3.1b
9.6c
5.8
0b
7.7
33.3b
11.8
3.7b
16.1b
5.3
0.3b
8.4
45.6b
17.3
7.5a
25.1a
5.6
2.1a
12.5
69.9a
2.37
0.75
1.89
0.85
0.34
1.84
4.66
a
Different letters within a row of the same meat are significantly different (P<0.05). n=4. A, aerobic packaging; V, vacuum packaging; C, nonirradiated; IR, irradiated at 4.5 kGy dose; chol, cholesterol; S.E.M., standard error of the mean.
Table 4
Content of COPs in raw pork with different irradiation, packaging, and storage timea
COPs
7a- or 7b-Hydroxycholesterol
b-Epoxide
a-Epoxide
20a-Hydroxycholesterol
Cholestanetriol
7-Ketocholesterol
Total
a
Day 0 (mg COPs/g lipid)
Day 7 (mg COPs/g lipid)
V-C
V-IR
A-C
A-IR
S.E.M.
V-C
V-IR
A-C
A-IR
S.E.M.
10.6b
3.0
8.4ab
0
0
8.8ab
30.8b
9.1b
1.9
3.0b
0
0
3.2b
17.2c
10.3b
5.0
7.0ab
0
0
8.4ab
30.7b
15.7a
3.5
13.4a
0
0
10.5a
48.4a
0.83
0.93
1.92
–
–
1.70
2.55
8.8c
1.8b
7.6
0
0.7
9.1ab
27.9c
10.6c
0c
5.8
0
1.7
6.6b
24.6c
29.2b
0c
7.6
0
1.1
15.0a
55.6b
48.8a
5.7a
8.3
0
2.2
11.6ab
76.5a
4.03
0.33
1.75
–
1.00
1.96
5.97
Different letters within a row of the same meat are significantly different (P<0.05). n=4. A, aerobic packaging; V, vacuum packaging; C, nonirradiated; IR, irradiated at 4.5 kGy dose; chol, cholesterol; S.E.M., standard error of the mean.
435
K.C. Nam et al. / Meat Science 58 (2001) 431–435
Table 5
Effect of irradiation, packaging, and storage time on lipid oxidation (TBARS) of raw turkey leg, beef, and porka
Treatment
V-C
V-IR
A-C
A-IR
S.E.M.
Turkey meat (TBARS [ppm])
Beef (TBARS [ppm])
Pork (TBARS [ppm])
Day 0
Day 7
S.E.M.
Day 0
Day 7
S.E.M.
Day 0
Day 7
S.E.M.
17.7
18.1
17.3y
18.0y
1.14
18.3c
17.7c
24.7bx
52.9ax
1.42
1.31
1.36
0.54
1.66
10.6
11.8
10.4
11.0
1.74
7.6
9.9
10.6
15.2
2.11
1.39
1.63
1.95
2.56
4.0b
4.3b
4.0b
5.5ay
0.26
4.1c
4.6c
17.3b
31.6ax
2.93
0.26
0.14
2.10
3.57
a
Different letters within a column are significantly different (P<0.05). n=4. Different letters within a row of the same meat are significantly different (P<0.05). A, aerobic-packaging; V, vacuum packaging; C, non-irradiated; IR, irradiated at 4.5 kGy dose; S.E.M., standard error of the mean.
higher TBARS than vacuum-packaged ones, and aerobically packaged and irradiated turkey leg and pork
produced higher TBARS than the non-irradiated
(Table 5). De Vore (1988) reported that significant levels
of TBARS and 7-ketocholesterol were observed in raw
ground beef patties after four days of storage, but irradiation, packaging, and storage had no effect on the
TBARS value of beef in the current study (Table 3). On
the other hand, turkey leg meat, which had a higher
proportion of unsaturated fatty acids than beef
(Table 1), produced higher amounts of TBARS and
COPs after 7 days of storage regardless of packaging
and irradiation. The strongly positive correlation
between cholesterol and fatty acid oxidation was found
in turkey leg and pork, not in beef. Smith (1987) postulated that cholesterol oxidation in food and biological
systems was initiated by the peroxyl or alkoxyl radicals
of oxidized neighboring polyunsaturated fatty acids
(phospholipids) in the membrane.
4. Conclusions
A considerable amount of cholesterol oxidation products was found in fresh raw meats. The amount and
composition of cholesterol oxidation products in meat
varied significantly depending on animal species and
storage time. Although the packaging conditions of
meat during storage were critical for the oxidation of
cholesterol in raw meat, irradiation synergistically
increased it. Vacuum packaging of raw meats was
enough to protect cholesterol and fatty acids from oxidation regardless of irradiation dose. Cholesterol was
oxidized in beef without fatty acid oxidation, but fatty
acid oxidation accelerated the oxidation of cholesterol
in turkey and pork.
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