Full text of DOI:10.1111/j.1365-2621.2002.tb09439.x

JFS: Food Chemistry and Toxicology
Comparison of Assays for Metmyoglobin
Reducing Ability in Beef Inside and Outside
Semimembranosus Muscle
L.M. SAMMEL, M.C. HUNT, D.H. KROPF, K.A. HACHMEISTER, AND D.E. JOHNSON
ABSTRACT: The relationships of 6 assays for metmyoglobin (Metmb) reducing ability to color stability and the
chemical differences between the inside (ISM) and outside (OSM) beef semimembranosus (SM) muscle after 5 or
1
4 d storage were investigated. The ISM had less (p < 0.05) color stability than the OSM regardless of time post
mortem, and both muscle portions were more color-stable when stored for 5 d rather than 14 d. Among the
assays, aerobic reducing ability correlated best with visual color scores (r = -0.58) and Metmb accumulation (r = -
0
.61) in the SM. The ISM had less reducing ability than the OSM, which can be attributed partially to lower oxygen
consumption rate and NAD concentrations (p < 0.05).
Keywords: color stability, semimembranosus, metmyoglobin, reducing ability
Introduction
these assays may not correctly represent muscle Metmb reduc-
NDERSTANDING MUSCLE CHEMISTRY AND THE CHANGES THAT OC- ing capacity. Measurement of Metmb reductase without the use
U
cur following slaughter is essential to maximizing meat color of artificial indicators shows a direct relationship between MRA
and color stability. In the living animal, myoglobin (Mb) must be and color stability, with the more color-stable muscles having the
in the reduced form to remain active in the transport and storage greatest reducing activity (Madhavi and Carpenter 1993; Reddy
of oxygen. Many oxidants have the capability of oxidizing Mb, and Carpenter 1991). Numerous conflicting conclusions from
thus making it physiologically inactive. However, in living sys- different MRA assays have limited the understanding of reduc-
tems, practically no metmyoglobin (Metmb) is present because ing activity in meat.
of an inherent Metmb-reducing system (Giddings 1977). Al-
The beef semimembranosus (SM) is a large, thick muscle that
though chemical properties change when muscle is converted to extends from the surface of the carcass to the femur. Because of
meat, Mb can still be reduced. However, the relationship be- its location, the outer portion (OSM) chills more rapidly than the
tween metmyoglobin reducing ability (MRA) and meat color sta- inner portion (ISM), resulting in different temperature/pH con-
bility is unclear.
ditions following slaughter. Tarrant (1977) reported pH values for
Logically, the more MRA a muscle retains, the better its color the ISM below 6 while the carcass temperature was still above
stability will be. Several different MRA assays are available, but 30 ЊC. As a result, the ISM is less color-stable than the OSM, yet it
they generate conflicting results. Early MRA research measured is unknown how slow chilling of the ISM affects MRA. Therefore,
reducing ability by oxidizing meat with potassium ferricyanide the objective of this study was to compare MRA assays and deter-
then following the subsequent pigment reduction spectrophoto- mine their relationship to chemical characteristics and color sta-
metrically. O’Keefe and Hood (1982) and Renerre and Labas bility of the ISM and OSM.
(
1987) found differing MRAs among muscles but were unable to
Materials & Methods
find a significant correlation between MRA and color stability. Po-
tassium ferricyanide facilitates electron transfer and forms a
complex with Mb when used to oxidize pigments; therefore, true
Metmb reduction capacity may not be measured (Faustman and
Cassens 1990). Ledward (1972) developed an aerobic reducing
ability (ARA) method that involved the oxidation of meat in 1%
oxygen then monitoring the subsequent pigment reduction.
This method was correlated highly to color stability; the most col-
or-stable muscles had the highest MRA. However, both methods
have been criticized because of the induction of high amounts of
Metmb on the surface of meat, which may not correspond with
the natural formation of Metmb.
Other MRA assays were developed to measure the actual en-
zyme activity. Echevarne and others (1990) and Lanari and Cas-
sens (1991) reported the highest MRA in least color-stable mus-
cles. However, they used methylene blue as an indicator, which
can be reduced by a number of nonspecific diaphorases capable
of transferring hydrogen from NADH to the redox dye. Therefore
WENTY VACUUM PACKAGED INSIDE ROUNDS (NAMP #168) WERE
T
obtained from a commercial slaughter plant and were stored
at 2 Ϯ 2 ЊC until 5 (n = 10) or 14 (n = 10) d post mortem. Following
storage, the adductor and gracilis were removed and the SM was
cut into 6 steaks, each 2.54 cm thick. Each steak was packaged on
a 4S Styrofoam tray containing a Dri-loc® meat pad (Sealed Air
Corp., Saddle Brook, N.J., U.S.A.) and overwrapped with polyvi-
2
nylchloride (23,250 cc O /m /24 h ЊC/%RH). Steaks were placed
2
in display at 0 Ϯ 2 ЊC with continuous fluorescent lighting of 1612
lux (150 foot candles; Philips, 34 Watt, Ultralume 30) in open-top
display cases (Unit Model DMF8; Tyler Refrigeration Corp., Niles,
Mich., U.S.A.). Cases were programmed to defrost at 12-h inter-
vals. The steaks from each SM were assigned randomly for analy-
sis over 6 d (d 0 through d 5). Samples were removed from both
the inside (the medial inner 1/3 closest to the femur) and outside
(
the lateral outer 1/3 closest to the surface of the carcass) por-
tions of the steak. The middle 1/3 portion was not used in the ex-
9
78 JOURNAL OF FOOD SCIENCE—Vol. 67, Nr. 3, 2002
© 2002 Institute of Food Technologists

Metmyoglobin reducing ability . . .
periments. Only the lateral (top) 1.27 cm of each steak was ana- ciates Laboratory, Inc., Reston, Va., U.S.A.). Subsequent reduc-
lyzed for MRA. Metmyoglobin reducing ability was determined tion was determined after another 24 h in atmospheric oxygen.
using 6 assays at each evaluation time. Concentrations of NAD, The MRA was calculated as: (observed decrease in Metmb con-
NADH and Mb, and pH, were determined on d 0, and oxygen centration ÷ initial Metmb concentration) ϫ 100.
consumption rates were determined on d 1. Instrumental and vi-
sual color was measured on steaks displayed for the full 6 d.
Reduction of nitric oxide metmyoglobin: a modified proce-
dure of Watts and others (1966) involving the oxidation of muscle
with nitric oxide was used to determine MRA. Sample cubes (3 ϫ
2 ϫ 1.27 cm3, rather than 50 g of ground product) were oxidized in
Instrumental and visual color evaluations
A MiniScan XE spectrophotometer (3.18-cm-diameter aper- 50 mL of 0.3% sodium nitrite at room temperature for 30 min. The
ture and 10 degree standard observer; Hunter Associates Lab- samples then were blotted to remove excess solution, vacuum-
oratory, Inc., Reston, Va., U.S.A.) was used to determine CIE packaged, and incubated at 30 ЊC. Reflectance from 400-700 nm
L*a*b* values (CIE 1976) for Illuminant A and reflectance from was measured every 30 min for 2 h using a Hunter LabScan 2000
4
00 to 700. The spectrophotometer was calibrated using the (1.27-cm-diameter aperture. The MRA was calculated as: (ob-
white and black standard plates. Three readings on the OSM served decrease in nitric oxide Metmb concentration ÷ initial ni-
and 2 readings on the ISM (due to its smaller surface area) tric oxide Metmb concentration) ϫ 100.
were taken through the packaging film, and averages were cal-
Reduction of dichlorophenolindophenol: a 5-g sample was
culated. Hue angle [tan –1(b*/a*), detects shifts from red to homogenized in 20 mL of 0.2 mM sodium phosphate buffer (pH
brown as values decrease]; saturation index [(a* ϩ b* )1/2, 5.6) and centrifuged at 25000 ϫ g for 30 min at 4 ЊC. The superna-
greater color saturation as values increase]; a*/b* value (de- tant was decanted and filtered through a 0.45-micron syringe fil-
tects shifts from red to brown as values decrease); percent re- ter. Reduction of dichlorophenolindophenol (DCPIP) was mea-
flectance ratio, 630 nm/580 nm and reflectance difference, sured by a change in absorbance at 600 nm (Hultquist 1978).
2
2
6
30 nm–580 nm difference (both measurements indicate The DCPIP reagent consisted of 20 mL of 50 mM Tris-HCl buffer
greater redness as values increase); and percentages of oxy- (pH 8.1), 1.4 mg of 2,6-DCPIP (Sigma D-2908) and 3.7 mg of diso-
myoglobin (Oxymb) and Metmb were calculated (AMSA 1991). dium EDTA of which 1.2 mL was added to a 1.5-mL microcuvette.
Visual color was evaluated by a trained panel (n = 9) using a 5- The reaction was initiated by simultaneously adding 100 ␮L of 1
point scale (1 = very bright cherry red or very bright pinkish mM NADH (Sigma N-8129) and 200 ␮L of muscle extract to the
red; 2 = bright cherry red or bright pinkish red; 3 = dull red to cuvette. As DCPIP was reduced by the muscle extract, absor-
brown or dull pink to tan; 4 = moderately dark red to brown or bance decreased. Reducing activity was calculated from the lin-
moderately tan; 5 = dark red to brown or tan). Different de- ear phase of the assay using Beer’s law with an extinction coeffi-
scriptors were used for the ISM and OSM because 1 set of de- cient of 21,000 M–1cm–1 for DCPIP. The MRA was reported as
scriptors was not sufficient to describe the different portions. nmoles reduced/min/g of muscle.
However, a specific number on each scale represented the
Reduction of horse and bovine metmyoglobins: Reddy and
same point of discoloration or acceptability of the ISM and Carpenter (1991) reported species specificity of the reductase sys-
OSM. A score of 3.5 or greater represented the point where tem. However, bovine Metmb is not commercially available as is
panelists would discriminate against the color at retail. All horse Metmb. Therefore, assays were conducted with both horse
panelists passed the Farnsworth Munsell 100-hue test (Mac- and bovine Metmb to compare results and determine whether
beth, Newburgh, N.Y., U.S.A.) as recommended by AMSA horse Metmb can be substituted for bovine Metmb while main-
(
1991). This test requires no training, but does mandate nor- taining accuracy of reducing activity measurements. Horse Met-
mal color vision and the ability to place in correct order a series mb was obtained from Sigma Chemical Co (M-0630; St. Louis, Mo.,
of colored samples varying in hue and intensity. All panelists U.S.A.). Bovine Metmb was extracted from heart muscle by ho-
were experienced color evaluators and they were oriented to mogenizing 15 g of heart with 45 mL of buffer (10 mM Tris-HCl and
this study during sessions in which SM steaks varying in color 1 mM EDTA) and centrifuging at 10,000 ϫ g for 10 min at 4 ЊC. The
deterioration were evaluated to increase uniformity and accu- supernatant was filtered through Whatman No.1 filter paper and
racy of scoring.
concentrated to about 0.1 mM in a Millipore Centricone Plus-80
UFC5LGC02; Fisher Scientific, Atlanta, Ga., U.S.A.) by centrifu-
gating at 500 ϫ g. Two crystals of potassium ferricyanide were add-
(
Assays of metmyoglobin reducing ability
Total reducing activity: total reducing activity (TRA) was de- ed to oxidize the Mb solutions. The MRA was measured using pro-
termined as described by Lee and others (1981). Briefly, a 2-g cedures described by Madhavi and Carpenter (1993). A 5-g
sample of muscle was homogenized in buffer and mixed with po- muscle sample was homogenized in 20 mL of 0.2 mM sodium
tassium ferricyanide for 1 h. Then, ammonium sulfamate and phosphate buffer (pH 5.6) and centrifuged at 25,000 ϫ g for 30
lead acetate were added. Distilled water and trichloroacetic acid min at 4 ЊC. The supernatant was decanted and filtered through a
were added and the solution was filtered using a 0.45-micron sy- 0.45-micron syringe filter. The reaction mixture consisted of 100 ␮L
ringe filter. Absorbance of the sample and control (excluded of 5 mM EDTA, 100 ␮L of 50 mM citrate buffer, 100 ␮L of 3.0 mM
muscle homogenate) was read at 420 nm. The calculation of ac- potassium ferrocyanide and 200 ␮L of 0.75 mM horse Metmb and
tivity was modified such that it was expressed as a unitless value 200 ␮L of deionized water or 400 ␮L of 0.1 mM bovine Metmb. The
of absorbance of control - absorbance of sample filtrate.
reaction was initiated by simultaneously adding 100 ␮L of 1 mM
Aerobic reducing ability: aerobic reducing ability was deter- NADH (Sigma N-8129) and 200 ␮L of muscle extract to a 1.5-mL
3
mined as described by Ledward (1972) on a 3 ϫ 2 ϫ 1.27 cm sam- microcuvette. As Metmb was reduced by the muscle extract, the
ple. Metmyoglobin formation was induced in a 1% oxygen-99% absorbance at 580 nm increased. Reducing activity was calculated
nitrogen environment for 24 h and reflectance from 400-700 nm from the linear phase of the assay using Beer’s law with an extinc-
was measured at 24 h using a Hunter LabScan 2000 (1.27-cm-di- tion coefficient of 12,000 M–1cm–1 for Oxymb. The MRA was report-
ameter aperture and 10 degree standard observer; Hunter Asso- ed as nmoles reduced/min/g of muscle.
Vol. 67, Nr. 3, 2002—JOURNAL OF FOOD SCIENCE 979

Metmyoglobin reducing ability . . .
Oxygen consumption rate
Table 1—Mean chemical characteristics of the outside
OSM) and inside (ISM) semimembranosus
(
3
Muscle samples (3 ϫ 3 ϫ 1.3 cm ) not previously exposed to
air were removed, placed immediately in a 250-mL filtering flask Trait
with a side arm, and flushed with 100% nitrogen for 15 s. Then a
mixture of 1% oxygen and 99% nitrogen was used to flush the NADH, nmol/g
OSM
ISM
Standard Error
0.16a
0.10b
NAD, nmol/g
0.02
0.009
134
0.02
0.13
0.102a
1388
5.54b
3.64
0.124b
1013
5.59a
3.51
c
flask for 10 s to achieve an atmosphere of < l% oxygen. A rubber OCR , nmol/cube/2 h
pH
septum was placed over the side arm and sealed with vacuum
grease, and a rubber stopper was inserted in the top of the flask.
A Mocon PacCheckTM (Model 650; Mocon Inc., Minneapolis,
Myoglobin, mg/g
5
d storage 14 d storage
NADH, nmol/g
0.195a 0.031b
0.009
Minn., U.S.A.) oxygen analyzer (zirconium sensor accurate to a–b
Means within a row with a different superscript letter differ (P Ͻ 0.05).
c
3
±
0.05% at concentrations used) was used to determine the con-
OCR = oxygen consumption rate of a cube (3 ϫ 3 ϫ 1.3cm ) over 2h.
centration of oxygen in the flask. A sample of air from the flask
was removed and analyzed for oxygen by puncturing the side
arm septum with a needle attached to the automatic pump of
the analyzer. The flask was placed in an incubator (35 ЊC) and measured some chemical characteristics on both portions of the
measurements were taken over a 2-h period. Oxygen consump- SM and found the ISM was softer, and more exudative, had more
tion was reported as nmoles consumed/cube of sample/2 h.
denatured protein, and had a lower water holding capacity than
the OSM. Furthermore, succinic dehydrogenase activity, lipid oxi-
dation, Mb, heme iron, and nonheme iron concentrations, did
NAD and NADH
The NAD and NADH were extracted in acid and alkaline, re- not explain differences in color stability between the ISM and
spectively, as described by Klingenberg (1974), and their concen- OSM (Sammel and others 2002).
trations were assayed following procedures outlined by McCor-
Color stability
mick and Lemuel (1971). All reagent volumes were halved, and
the reactions were carried out in 1.5-mL microcuvettes.
Visual color of the ISM and OSM are presented in Figure 1. On
d 0 of display, the ISM was a brighter (p < 0.05) cherry-red than
the OSM (Figure 1a). No differences were found on d 1, and the
pH
The pH was determined using a Sentron Red-Line LanceFet OSM was redder (lower visual scores) than the ISM on d 2
probe (Sentron Europe BV, Roden, The Netherlands) connected to
an Accumet portable AP61 pH meter (Fisher Scientific, Atlanta,
Ga., U.S.A.) by a Sentron 701 ISFET pH adapter. The probe was in-
serted twice in both the ISM and OSM before MRA samples were
removed, and the 2 readings were averaged for each portion.
Myoglobin concentration
A 5-g sample of pulverized muscle was homogenized in 15 mL
of deionized water at 4 ЊC. The homogenate was centrifuged at
3
0000 ϫ g for 30 min. The supernatant was filtered with a 0.45-
micron syringe filter and the absorbance was measured at 525
nm. Calculations were made using a molar extinction coefficient
of 7.6 (Bowen 1949) and a molecular weight of 16,110 Da for myo-
globin (Drabkin 1978).
Statistical analysis
Data were analyzed as a completely randomized split plot de-
sign where the SM muscle was the whole plot and storage was the
whole plot treatment. The 6 steaks from each SM were the split-
plots and muscle location (ISM versus OSM) was the split-split
plot. Proc Mixed procedures of SAS (2000) were used to deter-
mine treatment differences, and means were separated
(
P < 0.05) using least significant differences.
Results & Discussion
Characteristics of the inside and outside
semimembranosus
The ISM had less (p < 0.05) NAD, a lower oxygen consumption
rate (OCR) and a slightly higher pH than the OSM (Table 1). No
differences in Mb concentration or NADH occurred between the
c
Figure 1—Visual color scores for the semimembranosus
SM). a) Outside (OSM) and inside (ISM) SM. b) SM stored
(
for 5 d (5PM) and 14 d (14PM) post mortem
ISM and OSM; however, a significant decrease in NADH occurred a–b Values on the same display d with a different superscript
in the ISM and OSM from 5 to 14 d post mortem. Most research on letter differ (p < 0.05).
c
Score of 1 = very bright cherry red or bright pinkish red;
the SM has not differentiated between the ISM and OSM; there-
2
= bright red or bright pinkish red; 3 = dull red to brown
or dull pink to tan 4 = moderately dark red to brown or
fore, comparing these results with results of other studies is diffi-
cult. Hunt and Hedrick (1977) and Sammel and others (2002) moderately tan; 5 = dark red to brown or tan.
9
80 JOURNAL OF FOOD SCIENCE—Vol. 67, Nr. 3, 2002

Metmyoglobin reducing ability . . .
Table 2—Meang color traits for the outside (OSM) and in- Table 3—Pearson correlation coefficients (r values) for 6
a
side (ISM)semimembranos
assays for metmyoglobin (Metmb) reducing abilityb
Muscle
Portion
Display, d
2
TRA
ARA
NOMetmb
DCPIP
Horse
Trait
0
1
3
4
5
ARA
0.49
0.28
0.59
0.27
–0.18
az
ay
az
ay
az
ay
az
az
cy
by
bz
bz
by
b
40.1bz 40.2bz 41.7
az
by
ey
ez
ez
dy
39.6bz
NOMetmb
0.51
0.49
L*
OSM 42.3
41.3
49.0
28.8
27.4
22.1
23.9
82.9
cy
cy
cz
cz
cy
cy
cy
dy
dz
dz
cy
dy
cy DCPIP
0.45
NS
–0.13
ISM 51.4
OSM 32.8
ISM 35.9
OSM 24.8
ISM 28.9
48.3
25.2
21.7
19.9
21.2
69.3
48.1
22.5
18.6
18.8
20.1
56.7
50.2
18.4
15.7
15.7
18.1 19.2
29.9
48.7
17.5
15.3
16.9
c
ey Horse
NS
0.23
NS
a*
b*
ez Bovine
–0.27
NS
fz
ar values presented (p Ͻ 0.05)
TRA = total reducing activity; ARA = aerobic reducing ability;
ey NOMetmb = nitric oxide. Metmb reduction; DCPIP = dichlorophenolindophenol
reduction; Horse = horse Metmb reduction; Bovine = bovine Metmb reduction.
cdy
b
ey
Oxymyo- OSM 94.4
27.6
j
globin
%
c
Not significant
ay
78.0b 51.2
cz
34.9dz 14.2
46.5
ez
bz
ez
az
ISM 106.7
Metmyo- OSM 13.9
13.6
64.0
fy
ez
dz
cz
27.4
37.0
56.1
j
globin
%
ISM
9.6fz
36.3
36.2
36.5
37.2
41.2
5.3
4.0
21.4
24.1
ey
b
56.4dy 68.7
cy
74.6
24.1
24.2
40.7
49.6
2.5
1.7
11.3
9.1
by
e
79.6
ay
h
az
ay
cz
fy
cy
d
24.2e
24.7e
Saturation OSM 41.4
index ISM 46.3
Hue angle OSM 37.4
32.2
30.4
38.5
44.5
4.2
2.6
16.7
16.1
29.1
27.7
39.7
47.8
3.4
postmortem treatment of the muscle is modified.
b
cz
bcz
dy
cy
cz
c
d
e
i
cz
ey
by
bz
bz
by
bz
cy
dy
dz
dy
dz
bz
by
ey
ez
ey
ez
az
44.3
51.8
2.4
ay Metmyoglobin reducing assays
ISM
OSM
ISM
OSM 27.8
ISM 40.5
39.0
7.5
6.9
ay
az
az
ay
ey
6
5
30nm/
80nm
Identifying an MRA assay that correlates highly to color stabil-
ity was a prime objective of this study. Results from 6 assays are
ez
2.1
1.6
6
5
30nm-
80nm
14.1
11.7
9.1f
presented in Figure 2 to 7. The TRA assay is not specific for MRA
or any other muscle-reducing chemistry, but it does indicate the
muscle’s overall reductive state. Few differences existed between
the ISM and OSM at the same time post mortem for TRA (Figure
2). However, this assay did identify significant decreases in re-
ducing capacity between SM displayed after 5 and 14 d of stor-
age. Muscles stored for 5 d exhibited a higher TRA on d 0 of dis-
play, but it decreased significantly until d 4, whereas muscles
stored for 14 d had a low initial TRA which decreased only slightly
with display. These results, as expected, indicated that reducing
ability of the SM was greater with shorter postmortem storage.
This corresponds with visual scores, indicating lower color stabili-
ty of muscles stored for 14 rather than 5 d, but not with visual dif-
ferences between the OSM and ISM.
c
e
7.5
a–f
g
Means within a row with a different superscript letter differ (P < 0.05).
Standard errors: L* = 1.24; a* = 0.72; b* = 0.58; oxymyoglobin = 3.85;
metmyoglobin = 2.26; saturation index = 0.79; hue angle = 0.69; 630 nm/580
nm = 0.18; 630 nm-580 nm = 1.03.
h
i
j
2
2 –1/2
Saturation index = (a + b ) .
tan–1
Hue angle = (b/a)
.
Estimates for percentage oxymyoglobin and metmyoglobin are accurate to
only 6 to 7%, therefore their sum may be over 100% on a given day.
y–z
a
Means within a trait Pearson correlation coefficients (r values) for 6
b
assays for metmyoglobin (Metmb) reducing ability
through 5, indicating a slower rate of discoloration. A score of 3.5
or greater represented panelist rejection based upon visual color.
By d 2 of display, the ISM was determined visually unacceptable
by panelists (color score = 3.8), whereas the OSM still had a score
of 3.6 (borderline acceptable) on d 3 and was not severely discol-
ored until d 4. Panelists also detected differences in discoloration
due to storage post mortem. As expected, muscles stored for only
The ARA spectrophotometrically measures the muscle’s abili-
ty to reduce Metmb; therefore, it is not surprising that this assay
differentiated the ISM and OSM, which visually discolor at differ-
ent rates. The OSM stored for 5 d had significantly higher reduc-
ing ability for 3 display d than the OSM stored for 14 d or the ISM
5
d had less discoloration (lower scores) on d 0 through 3 of dis-
play than muscles stored for 14 d (Figure 1b). On d 2 of display,
SM stored for 5 d scored below 3.5, whereas SM stored for 14 d
scored above 3.5 (p < 0.05).
On d 0 of display, the ISM had more Oxymb and less Metmb
than the OSM (Table 2). Apparently, the lower OCR of the ISM al-
lowed more oxygen penetration into the muscle, resulting in a
greater bloom. Furthermore, MacDougall (1977) showed that
slow chilling of the ISM denatured proteins, causing them to
scatter more light and thus a paler appearance. However, after d
0
of display, the color of the ISM deteriorated rapidly. The ISM
had more Metmb and less Oxymb than the OSM on d 2 through d
of display. By d 2, the ISM had 56% Metmb and appeared
5
brown, whereas the OSM had only 37% Metmb and still retained
red color. In addition, hue angles and percent reflectance 630
nm/ 580 nm ratios for the ISM and OSM were different (p < 0.05)
over display, denoting the faster discoloration of the ISM.
Other researchers determined that different chilling rates sig-
nificantly impacted the color traits of the ISM and OSM (Nichols
and Cross 1980; Taylor and others 1980-81). Sammel and others
(
2002) found that the ISM was exposed 2 to 4 times longer to a pH
6
.0 and a meat temperature of 25 ЊC than the OSM. Although the Figure 2—Total reducing activity of the outside (OSM) and
inside (ISM) semimembranosus after postmortem storage
OSM retained acceptable color longer than the ISM, consumers
discriminate against the whole SM once the ISM is discolored. a–d
of 5 d (5PM) and 14 d (14PM).
Values on the same display d with different superscript
Therefore, the SM has a display life of only 1 to 2 d unless the letters differ (p < 0.05).
Vol. 67, Nr. 3, 2002—JOURNAL OF FOOD SCIENCE 981

Metmyoglobin reducing ability . . .
a
Table 4—Overall Pearson correlation coefficients (r values) half of its reducing ability during storage. The OSM stored for 14
of assays for metmyoglobin (Metmb) reducing abilityb and
d 3 display correlations for aerobic reducing ability (ARA)
the differences were significant only at d 1 and 3 of display. No
and reduction of nitric oxide metmyoglobin (NOMetmb) and
color traits
d had greater ARA than the ISM at either storage time, although
differences (p>0.05) in ARA due to postmortem storage were de-
tected in the ISM. The results for ARA correlated with color traits
showing that the OSM was more color-stable than the ISM re-
gardless of storage time.
Reduction of nitric oxide Metmb is another method for mea-
suring MRA that Watts and others (1966) used as an alternative to
oxidizing meat with potassium ferricyanide. They observed a
slower and more linear reduction when nitric oxide was used as
the oxidizing agent. However, they used this method only on
ground beef and did not make comparisons among muscles. Our
results for the reduction of nitric oxide Metmb are comparable to
those for ARA. The MRA’s of OSM stored for 5 and 14 d were
equivalent until d 2 of display, and then the OSM stored for 14 d
TRA
DCPIP
Horse
Bovine
Visual
L*
a*
b*
Oxymyoglobin, %
Metmyoglobin, %
a*/b* value
Hue angle
Saturation index
–0.58
NS
–0.53
–0.18
0.52
0.39
0.52
–0.51
0.47
–0.46
0.49
0.53
–0.13
0.20
0.14
0.31
0.11
–0.09
–0.13
0.13
0.21
0.18
0.17
0.48
NS
–0.45
–0.29
–0.45
0.48
–0.46
0.46
–0.41
–0.40
–0.44
c
0.57
0.55
0.55
–0.54
0.36
–0.36
0.58
0.58
0.51
6
6
30 nm/580 nm
30 nm–580 nm
0.42
Overall
d 3 display
ARA
–0.58
–0.28
0.56
0.35
0.56
NOMetmb
–0.42
–0.47
0.40
NS
0.42
–0.49
0.59
–0.57
0.31
ARA NOMetmb had significantly less activity (Figure 4). No MRA differences
Visual
L*
a*
b*
–0.46
0.51
0.43
0.41
0.45
–0.61
0.71
–0.68
NS
–0.68
–0.49
0.63
NS
0.66
–0.70
0.70
0.70
0.48
0.70
0.41
(
p>0.05) were found between ISM stored for 5 and 14 d, and ac-
tivities were lower (p < 0.05) than those of the OSM during dis-
play, except on d 5.
Reduction of DCPIP by Metmb reductase is a quick and easy
assay to measure enzyme activity without having to isolate Mb.
On day 0 and 1 of display, the OSM stored for 5 d had higher
Oxymyoglobin, %
Metmyoglobin, % –0.61
a*/b* value
Hue angle
Saturation index
0.57
–0.55
0.50
0.64
0.42
(
p < 0.05) reductase activity than the ISM stored for 5 d; activities
were equivalent on d 2 and 3, and then the OSM activity was sig-
nificantly higher on d 4 and 5 of display (Figure 5). The OSM and
ISM stored for 14 d exhibited no differences in reductase activity,
which was lower than activity of muscle stored for 5 d.
6
6
30 nm/580 nm
30 nm–580 nm
0.48
0.24
0.54
NS
a
b
r values presented (p Ͻ 0.05).
TRA = total reducing activity; DCPIP = dichlorophenolindophenol reduction;
Horse = horse Metmb reduction; Bovine = bovine Metmb reduction.
c
Reductase activity measured with horse and bovine Metmb
showed inconsistent results, which may have been due to meth-
odology (Figure 6 and 7). The reductase reaction is influenced by
the amount of Metmb and enzyme added to the reaction cuvette
Not significant.
with either storage time (Figure 3). The ARA of the OSM stored for (Hagler and others 1979). As display time increased, Metmb con-
d remained high for 1 d of display before decreasing to a level centrations in the muscles and muscle homogenates increased,
5
similar to that of the ISM at d 4 of display. Before display, the thus, possibly changing the rate of reduction in the reaction cu-
OSM stored for 5 d had more reducing ability than the OSM vette. Metmyoglobin concentrations in ISM and OSM homoge-
stored 14 d. Therefore, the OSM stored 5 d had a larger decline in nates also varied, making it difficult to determine whether differ-
ARA once displayed whereas the OSM stored 14 d had a smaller ences in MRA were due to differences in reducing capacity of the
decline in ARA during display because it had already exhausted muscles or just differences in Metmb concentrations. Other re-
searchers (Reddy and Carpenter 1991; Madhavi and Carpenter
1
993) oxidized the muscle homogenate with potassium ferricya-
Figure 3—Aerobic reducing ability of the outside (OSM) and Figure 4—Reduction of nitric oxide metmyoglobin on the
inside (ISM) semimembranosus after postmortem storage outside (OSM) and inside (ISM) semimembranosus after
of 5 d (5PM) and 14 d (14PM).
postmortem storage of 5 d (5PM) and 14 d (14PM).
a–d
a–d
Values on the same display d with different superscript
Values on the same display d with different superscript
letters differ (p < 0.05).
letters differ (p < 0.05).
9
82 JOURNAL OF FOOD SCIENCE—Vol. 67, Nr. 3, 2002

Metmyoglobin reducing ability . . .
nide before running the reaction, but that oxidation was omitted are altering the inherent reducing system before or during mea-
in this study. However, Madhavi and Carpenter (1993) were un- surement. Aerobic reducing activity is a relatively simple assay
able to show a decrease in MRA in the LD and PM over 7 d of dis- that can differentiate between muscles of different color stabili-
play. Addition of NADH to the reaction cuvette could be respon- ty and changes with time post mortem, while correlating well
sible for the inconclusive results. It may be the limiting factor in with visual color scores. However, the assay does not specifically
the reduction reaction in vivo, because it is lost quickly post mor- measure the reducing enzyme, and it takes 48 h to get results.
tem (DeVore and Solberg 1975). However, the reaction will not
run in vitro without the addition of NADH (Reddy and Carpenter say that can be conducted in about 2 h. Although the assay’s over-
991). Therefore, the reduction of horse or bovine Metmb in vitro all correlations with color traits were lower than those for ARA, it
Reduction of nitric oxide Metmb is another relatively easy as-
1
may differentiate reducing capacity among muscles; but chang- had a high correlation with many of the color traits on d 3 of dis-
es in MRA over display were not evident because the limiting fac- play. Therefore, the reduction of nitric oxide Metmb may be the
tor was present in excess. From d 0 of display, before Metmb con- best assay for distinguishing MRA differences between muscles at
centrations in the ISM and OSM differed greatly, the activities for different stages of discoloration. Reduction of DCPIP had a slight-
the reduction of horse Metmb were: OSM stored for 5 d > OSM ly lower correlation to visual color panels than ARA, yet the assay
stored for 14 d > ISM stored for 14 d > ISM stored for 5 d. When bo- measures Metmb reductase activity without isolating Metmb and
vine Metmb was reduced, the OSM with both storage times re- takes about 45 min. Correlations between MRA assays and color
duced more (p < 0.05) Metmb than the ISM with both storage traits were not as high as expected, possibly because many other
times. Therefore, our results indicated the OSM had more MRA factors including Mb autoxidation and OCR influence discolora-
than the ISM, although we were unable to detect how MRA tion. However, MRA assays that do not involve potassium ferricya-
changed over display time.
nide or methylene blue show a direct relationship with color stabil-
ity, indicating the importance of MRA in fresh meat (Madhavi and
Carpenter 1993; Reddy and Carpenter 1991).
Relationships among MRA, muscle chemistry, and
color stability
Differences in NAD and OCR between the ISM and OSM may
Pearson correlation coefficients for MRA assays are given in be responsible for the differences in MRA and color stability. Oxy-
Table 3. Total reducing activity and the reduction of DCPIP were gen consumption rate and ARA were correlated significantly on d
the most highly correlated (r = .59), and ARA and reduction of ni- 0 (r = 0.39) and d 3 (r = 0.50, data not shown) of display. Although
tric oxide Metmb had a slightly lower correlation (r = .51). Reduc- the correlations were not high, they do indicate a definite rela-
tion of horse and bovine Metmb did not correlate well to the oth- tionship which has been observed by other researchers (Watts
er 4 assays.
and others1966; Atkinson and Follett 1973; Ledward 1985; Ariha-
Overall Pearson correlation coefficients for MRA assays and ra and others 1996). The significant differences in NAD concen-
color traits are given in Table 4 as well as correlation coefficients tration within the SM may be partially responsible for the differ-
for ARA and reduction of nitric oxide Metmb with color traits on d ences in MRA, because Metmb reductase requires NADH as
3
. Among the MRA assays, TRA and ARA correlated best with vi- reducing equivalents (Arihara and other 1990). Although mus-
sual color scores. The ARA also correlated best with %Metmb ac- cles with greater OCR have been characterized as having lower
cumulation and the 630 nm/580 nm ratio. Furthermore, ARA had color stability (Lanari and Cassens 1991; O’Keefe and Hood 1982;
a high correlation with a*/b* value and hue angle on d 3 of dis- Renerre and Labas 1987), the citric acid cycle utilizes NAD and
play when the OSM was slightly discolored and the ISM was se- generates NADH, which can be used in oxidative phosphoryla-
verely discolored. Although ARA has been criticized because of tion or the reduction of Metmb. Arihara and others (1996) inhib-
the induction of high amounts of Metmb on the surface of meat, ited Metmb reduction with the addition of glycolytic pathway in-
it is measured on intact muscle and does not require the addition
of any chemicals to measure activity. Thus, it is not likely that we
Figure 5—Reduction of dichlorophenolindophenol on the Figure 6—Reduction of horse metmyoglobin on the outside
outside (OSM) and inside (ISM) semimembranosus after (OSM) and inside (ISM) semimembranosus after postmor-
postmortem storage of 5 d (5PM) and 14 d (14PM).
tem storage of 5 d (5PM) and 14 d (14PM).
a–d
a–d
Values on the same display d with different superscript
Values on the same display d with different superscript
letters differ (p < 0.05).
letters differ (p < 0.05).
Vol. 67, Nr. 3, 2002—JOURNAL OF FOOD SCIENCE 983

Metmyoglobin reducing ability . . .
protein (OM cytochrome b) in rat muscles as a metmyoglobin reducing system
component. Jpn J Zootech Sci 61(9):837-842
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(
E-1.3.1) 1978, 1971/(TC-1-3). Commission Internationale de Eclairage, Par-
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Figure 7—Reduction of bovine metmyoglobin on the out-
side (OSM) and inside (ISM) semimembranosus after post-
mortem storage of 5 d (5PM) and 14 d (14PM).
a–d
Values on the same display d with different superscript
letters differ (p < 0.05).
Hunt MC, Hedrick HB. 1977. Profile of fiber types and related properties of five
bovine muscles. J Food Sci 42(2):513-517.
Klingenberg M. 1974. Nicotinamide-adenine dinucleotides (NAD, NADP, NADH,
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bility. J Food Sci 56(6):1476-1479.
hibitors to cell suspensions. Thus, without some mitochondrial
activity, NADH will not be generated. Furthermore, NADH was
correlated to DCPIP reduction (d 0: r = 0.43; d 3: r = 0.67) and
TRA (d 0: r = 0.85; d 3: r = 0.47, data not shown). However, high
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Ledward DA. 1972. Metmyoglobin reduction and formation in beef during aer-
obic storage at 1 ЊC. J Food Sci 37(4):634-635.
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in beef muscles. Meat Sci 15(2):149-171.
and Hood 1982). Perhaps the significantly lower OCR of the ISM
Lee M, Cassens RG, Fennema OR. 1981. Effect of metal ions on residual nitrite.
indicates less generation of NADH as display time increases,
leading to less MRA, while the OSM still has the capability of gen-
erating NADH. Therefore, a very high or very low OCR may have
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5
9-69.
a negative impact on color stability, whereas an intermediate Madhavi DL, Carpenter CE. 1993. Aging and processing affect color, metmyoglo-
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OCR may positively affect color stability.
9
42.
McCormick DB, Lemuel DW. 1971. Nicotinic acid: Analogs and coenzymes. In:
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Conclusion
EROBIC REDUCING ABILITY CORRELATED BEST WITH COLOR Nichols JE. Cross HR. 1980. Effects of electrical stimulation and early postmor-
tem muscle excision on pH decline, sarcomere length and color in beef mus-
cles. J Food Prot 43(7):514-519.
O’Keefe M, Hood DE. 1982. Biochemical factors influencing metmyglobin for-
mation on beef from muscles of differing colour stability. Meat Sci 7(3):209-
A
stability over display and appeared to be the best current
method for measuring reducing ability. Reduction of nitric oxide
Metmb correlated well with d 3 color traits when discoloration dif-
ferences between the ISM and OSM were greatest. Direct mea-
surement of the reductase enzyme did not correlate to color sta-
bility possibly because addition of NADH in vitro does not
2
28.
Reddy IM, Carpenter CE. 1991. Determination of metmyoglobin reductase ac-
tivity in bovine skeletal muscles. J Food Sci 56(5):1161-1164.
Renerre M, Labas R. 1987. Biochemical factors influencing metmyoglobin for-
mation in beef muscles. Meat Sci 19(2):151-165.
represent the actual Metmb reducing environment in muscle. Sammel LM, Hunt MC, Kropf DH, Hachmeister KA, Kastner CL. 2002. Influence
of chemical characteristics of beef inside and outside semimembranosus on
color traits. J Food Sci (Forthcoming).
The ISM had lower OCR and less NAD, MRA and color stability
than the OSM, which may have been due to slow chilling rates. SAS. 2000. SAS user’s guide: Basic statistical analysis. SAS Institute, Inc., Cary,
NC.
Both the ISM and OSM were more color-stable after storage of 5 d
rather than 14 d. Early postmortem treatment of the ISM should
be modified to enhance its chilling and slow its decline in pH
Tarrant VP. 1977. The effect of hot-boning on glycolysis in beef muscle. J Sci Food
Agric 28(10):927-930.
Taylor AA, Shaw BG, MacDougall DB. 1980-81. Hot deboning beef with and with-
out electrical stimulation. Meat Sci 5(2):109-123.
Watts BM, Kendrick J, Zipser MW, Hutchins B, Saleh B. 1966. Enzymatic reducing
pathways in meat. J Food Sci 31(1):855-861.
(
Sammel and others 2002) to improve its color stability and shelf
life.
MS20010169 Submitted 12/4/00, Accepted 7/23/01, Received 7/31/01
References
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AMSA. 1991. Guidelines for meat color evaluation. Recip Meat Conf Proc 44:1- Industry, 232Weber Hall, Manhattan; KS 66506-0201. Please address corre-
1
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84 JOURNAL OF FOOD SCIENCE—Vol. 67, Nr. 3, 2002