Meat Science 54 (2000) 385±390

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Stability of catalase and its potential role in lipid oxidation in meat

A.A. Pradhan, K.S. Rhee*, P. HernaÂndez1
Meat Science Section, Department of Animal Science, Texas A&M University, College Station, TX 77843-2471, USA

Received 25 June 1999; accepted 16 August 1999

Abstract
The activity of catalase in microbial growth-controlled and uncontrolled ground beef muscle (semimembranosus, SM) did not
change (P>0.05) during 6-day storage at 4 C. Likewise, catalase activity in ground, beef SM and longissimus dorsi (LD), pork LD,
and chicken breast (B) and thigh (T) muscles was not a€ected (P>0.05) by 2-month storage at ÿ20 C, with or without mid-month
thawing/refreezing. When sodium azide (a catalase inhibitor) was added to ground beef SM, lipid oxidation (as measured by per-
oxide values) during 4-day refrigeration was higher (P<0.05) in treated samples Ð 43 and 55% higher at day 2 and day 4,
respectively Ð than in the controls. It was concluded that catalase would be stable during meat storage/distribution and contribute
signi®cantly to the antioxidative process in raw meat products. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Meat; Lipid oxidation; Catalase

1. Introduction (Johnson, Fischer & Kays, 1992). Thus, agents, factors,

Lipid oxidation is a major nonmicrobial factor
responsible for the quality deterioration of muscle
foods. Meat contains various endogenous prooxidants
and antioxidants (Decker & Xu, 1998). Thus, the ulti-
mate lipid oxidation potential of a meat sample is
determined by the interplay of its prooxidants and
antioxidants. Ferrylmyoglobin, often referred to as
activated metmyoglobin or H2O2-activated metmyoglo-
bin, can initiate lipid oxidation (Harel & Kanner, 1985;
Xu, Asghar, Gray, Pearson, Haug & Grulke, 1990) and
is regarded as a major factor in lipid oxidation in stored
meat (Harel & Kanner, 1985; Kanner & Harel, 1985;
Rhee, 1988). Incidentally, the oxidation of oxymyoglo-
bin can yield H2O2 as well as metmyoglobin (Satoh &
Shikama, 1981; Xu et al., 1990). Metmyoglobin, when
heated, reportedly maintains the capacity to be acti-
vated by H2O2 (Harel & Kanner, 1985). H2O2 in meat,
produced either from the oxidation of oxymyoglobin or
through other routes, can also participate in the Haber±
Weiss reaction where highly reactive hydroxyl radicals
are produced from the reaction of H2O2 with superoxide
* Corresponding author. Tel.: +1-409-845-3936; fax: +1-409-845-
9454.
E-mail address: ksrhee@tamu.edu (K.S. Rhee).
1
A visiting scientist from Spain with a fellowship from the North
Atlantic Treaty Organization.
0309-1740/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0309-1740(99)00114-X
or events that can in¯uence the H2O2 level would also
a€ect the rate of lipid oxidation in meat. In this regard,
catalase (an enzyme utilizing H2O2 as substrate) could
play an important role, if stable during meat storage.
The objectives of this study were to determine the e€ects
of refrigerated and frozen storage on catalase activity in
skeletal muscles from di€erent species and to ascertain
the role of catalase in lipid oxidation in raw meat.
2. Materials and methods
2.1. Preparation of meat samples
Meats were purchased locally as needed (at di€erent
times) for each experiment. Beef semimembranosus
(SM), beef longissimus dorsi (LD), pork LD, pork bos-
ton butt, chicken breast (B), or chicken thigh (T) mus-
cles were separated from retail cuts and coarse ground
through a plate with 1.27 cm diameter holes, mixed
manually, and then reground through a plate with 0.32
cm holes. All meat preparations were done in a walk-in
cooler at 4 C.
2.2. Experiment 1
Ground beef LD was used with or without adding 30
ppm chlortetracycline hydrochloride (CTC). Twenty-gram
386 A.A. Pradhan et al. / Meat Science 54 (2000) 385±390
portions of each treatment were placed onto Petri dishes
(diameter=4 cm, height=1.5 cm) and ¯attened. Then,
each Petri dish containing meat was over-wrapped with
oxygen-permeable polyvinyl chloride (PVC) ®lm (2325
ml O2/mil/m2/24 h; thickness=0.5 mil or 12.7 mm) and
stored at 4 C for 0, 3 or 6 days. CTC solution was pre-
pared with distilled±deionized water and added at 1%
(CTC+water) of the ®nal meat mixture weight to
achieve 30 ppm CTC; meat with no CTC contained an
equivalent amount of distilled±deionized water. Stored
samples were analyzed for catalase activity as well as 2-
thiobarbituric-acid reactive substances (TBARS) values.
Subsequently, ground pork boston muscle samples
without CTC were packaged as described above, stored at
4 C for 0, 2 or 4 days, and analyzed for catalase activity.
2.3. Experiment 2
Ground beef SM and LD, pork LD, and chicken B
and T muscles were formed into patties (37 g; dia-
meter=4 cm, thickness=1.5 cm) and placed on styr-
ofoam trays lined with PVC ®lm. Each tray with patties
was then over-wrapped with the oxygen-permeable PVC
®lm and stored in a ÿ20 C freezer. Catalase activity was
measured monthly for 2 months. Half the samples were
also subjected to freeze±thaw cycles at 0.5 and 1.5
months. Hence, when analyzed after 1 and 2 months,
such samples had undergone 1 and 2 freeze-thaw cycles,
respectively. In each cycle, meat was thawed at room
temperature (1.5 h at 22 C) before refreezing.
2.4. Experiment 3
Puri®ed catalase from bovine liver (Sigma Chemical
Co., St. Louis, MO) was dissolved in distilled±deionized
water and added along with CTC solution to ground
beef SM (a total of 2 ml/100 g ®nal sample) to attain
1600 or 4000 units (based on information provided by
the supplier) of catalase per gram of ®nal meat sample
and 30 ppm CTC. The control contained only CTC,
with distilled-deionized water substituted for the cata-
lase solution. Twenty-gram (for catalase assay) or 30-g
(for TBARS assay) portions of each meat mixture were
placed onto Petri dishes, covered with the oxygen-
permeable PVC ®lm, and then stored at 4 C for 0, 3 or 6
days, as in Experiment 1. Catalase activity was deter-
mined at 0, 3 and 6 days, whereas TBARS values were
determined at 1, 3 and 6 days; due to sample analysis
scheduling problems, the ®rst TBARS analysis was
done on day-1 (rather than day-0) samples.
2.5. Experiment 4
2.5.1. Part I
3-Amino-1,2,4-triazole (a catalase inhibitor) dissolved
in distilled±deionized water was added along with CTC
solution (a total of 5 ml/100 g ®nal meat mixture) to
ground beef SM to give 0.05, 0.5 or 2.5% of the inhi-
bitor and 30 ppm CTC in the meat. The control con-
tained only CTC, with distilled±deionized water
substituted for the inhibitor solution. Thirty-gram por-
tions of each treatment were placed onto Petri dishes,
covered with the oxygen-permeable PVC ®lm, and
stored at 4 C for 0, 2 or 4 days. 3-Amino-1,2,4-triazole
was found to interfere with the measurement of
TBARS; thus, lipid oxidation was determined by mea-
suring the peroxide value (PV).
2.5.2. Part II
Sodium azide was used to inhibit catalase, because
3-amino-1,2,4-triazole exhibited unexpected properties
in Part I of Experiment 4 (see 3. Results and discussion).
Sodium azide dissolved in distilled±deionized water was
added along with CTC solution (a total of 5 ml/100 g
®nal meat mixture) to ground beef SM to give 0.03%
sodium azide and 30 ppm CTC in the meat mixture. In
the control, the inhibitor solution was substituted by
plain distilled±deionized water. Ten-gram portions were
stored at 4 C, in Petri dishes covered with the PVC
®lm. Catalase activity and PV were determined at 0, 2
and 4 days.
2.6. Chemical analyses
2.6.1. TBARS assay
A distillation method was used with the addition of
propyl gallate and EDTA at the blending step (Rhee,
1978). Results were expressed as mg malonaldehyde
equivalent/kg sample.
2.6.2. PV determination
Total fat was extracted from meat samples using 2:1
chloroform±methanol, according to the procedure of
Folch, Lees and Sloane-Stanley (1957). Total fat
extracts were analyzed for PV using the International
Dairy Federation method as described by Shanta and
Decker (1994), with a slight modi®cation. Since the fat
was extracted with chloroform-methanol, extracts
(chloroform phase) were used directly in the assay, with
methanol added to attain a chloroform: methanol ratio
of 7:3, instead of evaporating the solvent and redissol-
ving the fat in 7:3 chloroform±methanol.
2.6.3. Catalase activity
The procedure of Aebi (1983) was adapted. To pre-
pare a crude enzyme extract, ground muscle (10 g) was
homogenized with 50 ml of phosphate bu€er (0.05 M,
pH=7) and the homogenate was centrifuged at 4 C for
20 min at 7000g. The supernate (0.1 ml) was reacted at
room temperature (22 C) with 2.9 ml of 30 mM H2O2
in phosphate bu€er, and the reaction (H2O2 loss) was
monitored by measuring the absorbance at 240 nm. The
 A.A. Pradhan et al. / Meat Science 54 (2000) 385±390
absorbance was measured every 5 s for 30 s. A unit of
catalase activity was de®ned as the amount of catalase
needed to decompose 1 mmol H2O2/min, as determined
from the slope of the H2O2-vs-time linear regression
plot. It should be noted that the H2O2 concentration in
the assay mixture was 29 mM [vs 10 mM in the proce-
dure of Abi (1983)].
2.7. Statistical analysis
Data were analyzed using the General Linear Model
(GLM) Procedure of the SAS (1990) program to deter-
mine the signi®cance of main e€ects and interactions.
Main e€ect means were separated using the Student±
Newman±Keuls multiple range test. When there was a
signi®cant interaction between independent variables,
the mean square of the interaction term was used as the
error term to determine the signi®cance of main e€ects.
Signi®cance was established at P0.05.
3. Results and discussion
3.1. Catalase stability in refrigerated meat (experiment 1)
Endogenous catalase in aerobically refrigerated
ground beef SM was stable during 6-day storage at 4 C,
regardless of CTC treatment (Table 1). Likewise, endo-
genous catalase activity did not change notably in
ground, pork boston butt lean (comprised of multiple
muscles) stored with no CTC over 4 days. In addition,
the exogenous catalase (puri®ed catalase from bovine
liver) added in 1876 or 3582 units (as analyzed) to
ground beef SM was stable over 6-day refrigeration
(data not shown). In a previous study (Renerre, Francoise
& Gatellier, 1996) where intact beef muscles (longissimus
Table 1
Catalase activity in ground, beef SM and pork boston butt muscle
samples as a€ected by CTC treatment and/or storage time at 4 C
Meat Storage time Catalase activity (units/g meat)a
(days)
0 ppm CTC 30 ppm CTC
Beef SM 0 429 446
3 430 458
6 418 466
RSDb 37 34
Pork boston buttc 0 1144 ±
2 1092 ±
4 1048 ± Anderson & Sams, 1996)
RSD 41
a
Means in the same row or column within each meat category are
not signi®cantly di€erent (P>0.05); three samples/storage time.
b
RSD=residual standard deviation.
c
The experiment on pork was done separately (after the beef
experiment), with no CTC added to the meat.
387
lumborum, tensor fasciae latae, psoas major, and the
diaphragma) Ð not ground and not treated with any
antimicrobial agent Ð were stored aerobically at 2 C
for up to 8 days, no catalase activity changes that could
be related to the refrigerated storage were observed. Our
results, along with those of the aforementioned study,
indicate that catalase in meat is stable during refri-
gerated storage.
Beef SM was stored with or without 30 ppm CTC to
determine potential e€ects of microbial growth on the
substances analyzed. There was no interference or direct
e€ect of CTC on catalase assay or TBARS determina-
tion. During refrigerated storage, however, TBARS
values were lower in samples with no CTC (almost 50%
lower on day 3 when compared to the corresponding
samples with CTC; data not shown), con®rming our
previous observations (Rhee, Krahl, Lucia & Acu€,
1997). Previously, it was hypothesized that lower
TBARS in the microbial growth-controlled meat sam-
ples might be due to microbially mediated removal or
losses of malonaldehyde and other TBARS Ð through
direct microbial utilization of these substances and/or
through reactions between such substances and the
amine compounds produced by bacterial metabolism
(Branen, 1978) Ð as well as an elevated pH as a result
of the microbial growth (Rhee et al., 1997). Thus, CTC
was added to meat in all subsequent experiments invol-
ving refrigeration of raw samples. There have been
other studies (Greene, 1969; Jay, 1964; Rhee et al.,
1997) where CTC was used to inhibit microbial growth
in refrigerated meat samples to be assessed for non-
microbial parameters.
3.2. Frozen storage/freeze-thaw e€ects on catalase
(experiment 2)
Catalase in beef SM, beef LD, pork LD, chicken T,
and chicken B muscles proved to be stable during
3-month frozen (ÿ20 C) storage (Table 2). Lee, Mei
and Decker (1996) also found no catalase activity
change in pork muscles after 10-week storage at ÿ15 C.
Thawing and refreezing (i.e. freeze±thaw cycles) did
not a€ect catalase activity (Table 2). Samples that had
undergone a maximum of two freeze±thaw cycles during
the 2-mo storage showed no signi®cant loss of catalase
activity when compared to the frozen control. Catalase
activity was highest in pork LD and lowest in chicken B,
which was consistent with previous results of retail meat
samples that had not undergone frozen storage (Rhee,
3.3. E€ect of exogenous catalase on lipid oxidation
(experiment 3)
Puri®ed catalase (from bovine liver) added to ground
beef SM decreased TBARS values during 4 C storage
388 A.A. Pradhan et al. / Meat Science 54 (2000) 385±390

Table 2
Catalase activity according to storage time at ÿ20 C for beef LD and SM, pork LD, and chicken B and T samples with or without freeze±thaw
cycles (FT)

Storage time (days) Catalase activity (units/g sample)b

Beef Pork Chicken

LD LD(FT) SM SM(FT) LD B B(FT) T T(FT)

0 527 527 410 410 751 148 148 555 555

30 543 557 414 459 689 123 107 520 522
60 493 464 406 429 680 138 112 573 531
RSD 88 69 57 51 60 40 32 73 63
Overall meansa 520b 519b 410c 433c 705a 132d 115d 545b 535b
a
Means within the same row bearing the same letter are not signi®cantly di€erent (P>0.05).
b
Means within the same column (among storage days) are not signi®cantly di€erent (P>0.05); two samples/storage time.

(Table 3). However, in spite of the high concentrations
of the added catalase (as high as >9 times the activity
of endogenous catalase), the TBARS value decreases,
though statistically signi®cant (P<0.05), were not
marked. The overall di€erence (averaged over storage
days) in TBARS content between the controls and
samples containing the added catalase was only about
8%. Lee et al. (1996), who added puri®ed catalase Ð at
concentrations similar to endogenous catalase
concentrations Ð to cooked meat (ground turkey thigh
muscles), found that the decline in lipid oxidation (as
measured by TBARS values) during 4 C storage was
not remarkable. Reasons for the ine€ectiveness of the
added catalase in inhibiting lipid oxidation are not
clear. It is possible that, in raw meat, the endogenous
catalase (proven stable during refrigerated storage;
Table 1), along with the endogenous glutathione perox-
idase, may have been able to control H2O2 concentra-
tions in the samples. Another possibility is that the
added catalase may not have e€ectively reached the site
of the substrate (H2O2) in muscle, allowing H2O2 to
interact with metmyoglobin to produce ferrylmyoglobin,
which can initiate lipid oxidation. H2O2 was reported to
react rapidly with metmyoglobin to form ferrylmyoglobin
radicals (Kanner & Harel, 1985). Besides ferrylmyoglobin
radicals, highly reactive hydroxyl radicals could have
been formed from the H2O2 in meat, through Haber±
Weiss/Fenton reaction (Johnson et al., 1992).
3.4. E€ects of inhibition of endogenous catalase
(experiment 4)
The addition of 3-amino-1,2,4-triazole at 2.5% (w/w)
of sample weight completely inhibited catalase in beef
SM (Table 4). However, at that concentration, the inhi-
bitor severely interfered with the TBARS assay
(TBARS value of 0.08 vs 1.18 for the control). There-
fore, PV was used as a measure of lipid oxidation. The
inhibition experiment using 3-amino-1,2,4-triazole gave
unexpected results (Table 5). One may expect that PV
would be higher for samples containing the inhibitor
than for the control, because the antioxidative function
of catalase would no longer be carried out with the
inhibitor addition. On the contrary, there was no sig-
ni®cant PV increase in the treated samples after 2 days
at 4 C, while PV for the control increased; on day 4,
treated and untreated samples had similar values. More
research would be required to clarify the unexpected
behavior of this inhibitor and its interaction with meat
constituents.

Table 3
TBARS values (mg malonaldehyde equivalents/kg meat)a for beef SM samples with 30 ppm CTC as a€ected by added catalase and storage at 4 C

Storage time (days) Added catalase (units) Overall meanb

0 1600 4000

1 3.39 2.91 3.41 3.23c

3 6.06 5.83 5.79 5.90b
6 7.96 6.96 7.10 7.34a
Overall meanb 5.80a 5.23b 5.43b
a
Means within the same column or row bearing the same letter are not signi®cant di€erent (P>0.05). Two samples/storage time/catalase
treatment.
b
RSD=0.23 for the overall model. The interaction between added catalase and storage time was not signi®cant (P>0.05).
 A.A. Pradhan et al. / Meat Science 54 (2000) 385±390 389

Table 4 Sodium azide inhibits catalase by reacting with the

E€ect of 3-amino-1,2,4-triazole (AT) on the activity of catalase in beef heme prosthetic group of the enzyme (Smith, 1964).
SM with 30 ppm CTC
This means that it could also inhibit the prooxidant
AT conc. (%, w/w) Catalase activity (units/g meat)a % Inhibition activity of heme pigments as it binds to their heme
groups; the binding would decrease the ability of heme
0.0 226.51a 0
0.05 150.08b 34
pigments to complex with ligands like peroxides and
0.5 46.93c 79 oxygen. Nevertheless, lipid oxidation (PV) increased
2.5 No discernable activity 100 with the addition of sodium azide, despite its ability to
RSD 26.71 inhibit the prooxidant activity of heme pigments. With-
out such ability of sodium azide, the PV increase in the
a
Means (n=3 for each mean) within the same column with di€er- treated samples (Fig. 1) would have been even greater.
ent letters are signi®cantly di€erent (P<0.05).
In other words, the observed increase in lipid oxidation
due to the sodium azide addition would likely be an
Table 5
underestimation of the ability of catalase to protect the
E€ect of 3-amino-1,2,4-triazole (2.5%) on peroxide value of refri- meat from lipid oxidation.
gerated ground beef SM stored with 30 ppm CTCa

Storage time (days) Peroxide value (meq peroxides/kg fat)b RSD

4. Conclusions
Control Treated

0 3.02a,B 2.02a,B 0.51

Catalase in meat was stable during refrigerated and
2 5.33a,A 2.27b,B 0.40 frozen storage, and seemed to play an important role in
4 5.95a,A 5.74a,A 0.78 modulating lipid oxidation in uncooked meat. Lipid
RSD 0.58 0.59 oxidation in refrigerated, ground beef muscle increased
a
Means in the same row bearing the same letters are not sig-
markedly when its catalase was inhibited. If the endo-
ni®cantly di€erent (P>0.05). Means in the same column bearing the genous catalase were not an important part of the anti-
same letter are not signi®cantly di€erent (P>0.05). oxidative process in the meat, the inhibition of the
b
Two samples/storage/treatment. enzyme would not have produced such a di€erence.

Fig. 1. E€ect of sodium azide on peroxide values of ground beef SM
stored at 4 C. a,bMeans within the same day having di€erent letters are
signi®cantly di€erent (P<0.05). Two samples/storge time/treatment.
RSD: 0.18, 0.32 and 0.28 at days 0, 2 and 4, respectively.
When sodium azide (0.03% in the meat) was used to
inhibit catalase, PV was signi®cantly higher in the trea-
ted beef SM samples than in the controls (Fig. 1); the
treated samples had 43 and 55% higher values on days 2
and 4, respectively. There was no discernible catalase
activity in the samples treated with sodium azide.
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