Effects of Metmyoglobin Reducing Activity

and Thermal Stability of NADH-Dependent

Reductase and Lactate Dehydrogenase
on Premature Browning in Ground Beef
Blanchefort A. Djimsa, Anupam Abraham, Gretchen G. Mafi, Deborah L. VanOverbeke, and Ranjith Ramanathan

Abstract: Premature browning is a condition wherein ground beef exhibits a well-done appearance before reaching
the USDA recommended internal cooked meat temperature of 71.1 °C; however, the mechanism is unclear. The
objectives of this study were: (1) to determine the effects of packaging and temperature on metmyoglobin reducing
activity (MRA) of cooked ground beef patties and (2) to assess the effects of temperature and pH on thermal stability of
Food Chemistry

NADH-dependent reductase, lactate dehydrogenase (LDH), and oxymyoglobin (OxyMb) in-vitro. Beef patties (lean: fat =
85:15) were packaged in high-oxygen modified atmosphere (HiOX-MAP) or vacuum (VP) and cooked to either 65 or
71 °C. Internal meat color and MRA of both raw and cooked patties were determined. Purified NADH-dependent
reductase and LDH were used to determine the effects of pH and temperature on enzyme activity. MRA of cooked
patties was temperature and packaging dependent (P < 0.05). Vacuum packaged patties cooked to 71 °C had greater
(P < 0.05) MRA than HiOX-MAP counterparts. Thermal stability of OxyMb, NADH-dependent reductase, and LDH
were different and pH-dependent. LDH was able to generate NADH at 84 °C; whereas NADH-dependent reductase
was least stable to heat. The results suggest that patties have MRA at cooking temperatures, which can influence cooked
meat color.

Keywords: beef color, metmyoglobin reducing activity, myoglobin, NADH-dependent reductase, premature browning

Practical Application: Cooked ground beef color is not a reliable indicator of safety. The results will help the beef
processors to develop strategies to improve reducing activity of ground beef and to have a predictable internal cooked
meat color.

Introduction occurrence of either OxyMb or MetMb in the interior of patties

Cooked meat color is primarily due to the Maillard reaction and can predispose to premature browning.
myoglobin denaturation (Mottram 1998; King and Whyte 2007). The introduction of case-ready packaging has helped the meat
More specifically, the presence of either ferro- or ferrihemochro- purveyors to modify the gas compositions within the packages to
mogen will impart brown or purplish red cooked meat color, extend beef color. Greater levels of oxygen within high-oxygen
respectively (Warren and others 1996; Lytras and others 1999). (HiOX-MAP) packages allow oxygen to penetrate further into
Most consumers determine the degree of doneness by checking the interior and increase the proportion of OxyMb. Hence, the
the internal color of beef patties (USDA 1998; Ralston and others occurrence of PMB will be greater in HiOX-MAP than the tradi-
2002). This can be a food safety concern as premature brown- tional polyvinyl chloride (PVC) overwrap film or vacuum package
ing (PMB) is a condition wherein ground beef patties will have (Jayasingh and others 2002; Seyfert and others 2004).
internal brown and well-done appearance at temperatures below Metmyoglobin reducing activity (MRA) is the ability of the
the USDA recommended internal cooked meat temperature of postmortem muscle to limit the formation of MetMb by donating
71.1 °C to destroy pathogens (Hague and others 1994; Hunt and an electron to ferric heme to form DeoxyMb (Bekhit and Faust-
others 1999). A previous study estimated that 47% of ground beef man 2005). MRA can occur by both enzymatic and nonenzymatic
sold in the United States is susceptible to PMB (Killinger and pathways. An important cofactor that affect MRA is the source
others 2000). Although the mechanism of PMB is not clear, ear- of reducing equivalents such as NADH. Kim and others (2006)
lier research indicated that the myoglobin form present within reported that NADH can be regenerated by lactate dehydrogenase
the interior of patties before cooking can influence cooked meat (LDH) activity. Similarly, NADH-dependent reductase can reduce
color (Hunt and others 1999). For example, deoxymyoglobin (De- MetMb to DeoxyMb (Hagler and others 1979). Hence, meat sam-
oxyMb) is very stable to heat, whereas oxy- (OxyMb) and met- ples with greater MRA have the potential to influence myoglobin
myoglobin (MetMb) forms are less resistant to heat. Hence, the redox state. However, limited research has determined the effects
of packaging and temperature on MRA of cooked patties. Sim-
ilarly, few studies have assessed the thermal stability of purified
JFDS-2016-1156 Submitted 7/21/2016, Accepted 12/4/2016. Authors are NADH-dependent reductase and LDH. We hypothesize that dif-
with Dept. of Animal Science, Oklahoma State Univ., Stillwater, OK 74078, U.S.A. ferences in protein structure will influence the thermal stability of
Direct inquiries to author Ramanathan (E-mail: ranjith.ramanathan@okstate.edu). NADH-dependent reductase and LDH. Therefore, the objectives

C 2017 Institute of Food Technologists

 R

304 Journal of Food Science r Vol. 82, Nr. 2, 2017 doi: 10.1111/1750-3841.13606
Further reproduction without permission is prohibited
Beef reducing activity and cooked color . . .

Table 1–Least squares means for a∗ , chroma, K/S474 ÷ K/S525, and K/S610 ÷ K/S5251 for raw surface color of ground beef patties
packaged HiOX-MAP and VP.2

a∗ chroma K/S474 ÷ K/S525 K/S610 ÷ K/S525

d 03 HiOX-MAP 31.45d 39.69d 0.871c 0.170a
VP 19.09a 24.25a 0.642b 0.372d
SE4 0.34 0.41 0.002 0.004
d2 HiOX-MAP 26.33c 33.78c 0.961d 0.192c
VP 22.24b 26.08b 0.542a 0.341c
SE 0.34 0.41 0.003 0.002
Least squares means within a column with different inline letters are significantly different (P < 0.05).
1 ∗
a , a greater a∗ value indicates more redness; chroma, indicates intensity of red color and was calculated as  (a∗2 + b∗2 ); K/S474 ÷ K/S525, a lower number means greater
amount of DeoxyMb; K/S610 ÷ K/S525, a lower number means greater amount of OxyMb.
2
HiOX-MAP, high-oxygen modified atmosphere packaging (80% oxygen and 20% carbon dioxide); VP, vacuum packaging.
3
Day 0: patties were packaged and stored in the dark for 2 h; day 2: patties were packaged and stored in the dark for 48 h.
4
SE, standard error.

of this study were (1) to determine the effects of packaging and (LID 1050 film, Cryovac sealed air, Duncan, S.C., U.S.A.)

temperature on MRA of cooked ground beef patties, and (2) to
assess the effects of temperature and pH on thermal stability of
NADH-dependent reductase, LDH, and OxyMb in vitro.
Materials and Methods
Objective 1: Effects of packaging and temperature
on cooked patties MRA
Patties preparation and packaging. Fresh coarse ground
chubs (85% lean, n = 6, 4.5 kg) were obtained from a local pur-
veyor on the day of preparation and the coarse ground beef was
fine ground using a LEM meat grinder (LEM Big Bite Grinder,
West Chester, Ohio, U.S.A.). From each chub, 9 patties (1 cm
thickness × 10.5 cm dia, 113 g) were prepared using a patty
press (Adjust-A-Burger, Boyd’s Equipment Inc., Amarillo, Tex.,
U.S.A.). Three out of the 9 patties were randomly allotted
to vacuum packaging (VP, Prime Source Vacuum Pouches,
4 mil; Koch Supplies Inc., Kansas City, Mo., U.S.A.) and 3
to high-oxygen modified atmospheric packaging (HiOX-MAP,
80% oxygen and 20% carbon dioxide). The HiOX-MAP pat-
ties were placed in Rock-Tenn DuraFreshTM rigid trays with
absorbent pads and sealed with a clear, multi-layer barrier film
Food Chemistry
using a semiautomatic Mondini tray-sealing machine (Model
CV/VG-5, G. Mondini S.P.A. Cologne, Italy) and certified gas
blends (Stillwater Steel & Welding Supply, Stillwater, Okla.,
U.S.A.). Packaged patties were stored in the dark at 0 ±
2 °C for 48 h. The remaining 3 patties were used to characterize d
0 (no packaging and storage) raw meat color, cooked color, lipid
oxidation, and MRA. Of the 3 patties assigned to each packaging
x temperature combination, 2 patties were designated to either
65 or 71 °C endpoint cooking temperatures. The third patty was
utilized to measure pH, lipid oxidation, internal raw meat color,
and raw patties MRA. The extra ground beef from each chub was
utilized to measure proximate composition.
Analysis of pH and proximate composition. The pH of
ground beef patties was determined by inserting a pH probe at
2 different locations within a patty. An Accumet combination
glass electrode connected to an Accumet 50 pH meter (Fisher
Scientific, Fairlawn, N.J., U.S.A.) was used to measure pH. The
readings were averaged for statistical analysis.
Proximate analysis was conducted using an AOAC-approved
(Official Method 2007.04) near-infrared spectrophotometer
(FOSS Food ScanTM 78800; Dedicated Analytical Solutions,

1.2 Figure 1–Effects of packaging and storage time on

d 0 - VP TBARS values of raw ground beef patties. a–b, least
b squares means with different superscripts are
Lipid oxidation (mg MDA/kg meat)

1.0
d 2 - VP different (P < 0.05). d 0, patties were stored for 2 h in
the dark. d 2, patties packaged in high oxygen
d 2 - HiOX-MAP modified atmosphere (HiOX-MAP) or in vacuum (VP)
were stored in the dark for 48 h.
0.8

0.6

a
0.4 a

0.2

0.0
Days of storage and packaging

Vol. 82, Nr. 2, 2017 r Journal of Food Science 305

 Beef reducing activity and cooked color . . .
DK-3400 Hillerod, Denmark). The compositional values were
reported on a percent (%) basis.
Raw surface and internal color measurement. Raw sur-
face color was recorded on day 0 (2 h) and day 2 (48 h). Following
surface color readings, patties were butterflied, and the internal
color was recorded immediately. A HunterLab MiniScan spec-
trophotometer (2.54-cm diameter aperture, illuminant A, and 10°
standard observer; HunterLab Associates, Reston, Va., U.S.A.) was
used to record color. For both VP and HiOX-MAP patties, values
of the Commission Internationale de l’Eclairage (CIE) L∗ , a∗ , and
b∗ , and reflectance from 400 to 700 nm were recorded at 2 random
locations within a patty. Values of a∗ and b∗ were used to calculate
chroma (C) = ( [a∗2 + b∗2 ]). Reflectance from 400 to 700 nm
was used to determine K/S values at 474 and 610 nm (AMSA
2012) to measure DeoxyMb and OxyMb, respectively.
Metmyoglobin reducing activity. MRA was determined on
Food Chemistry
day 0 (2 h) and day 2 (48 h) according to a methodology described
by Sammel and others (2002). Briefly, fresh cut interior surface
was immersed in a 0.3% sodium nitrite solution (Sigma Aldrich,
St. Louis, Mo., U.S.A.) for 20 min to oxidize OxyMb/DeoxyMb
to MetMb. After incubation, patties were removed, blotted
dry, vacuum packaged (Prime Source Vacuum Pouches, 4 mil;
Koch Supplies Inc.), and immediately scanned for pre-incubation
MetMb reflectance values from 400 to 700 nm with a HunterLab
Miniscan XE Plus spectrophotometer. Metmyoglobin reduction
was accomplished by incubating the samples at 30 °C for 2 h.
Samples removed from the incubator were rescanned for postincu-
bation reflectance spectra. MRA was estimated as (% reflectance at
630 nm – % reflectance at 580 nm; AMSA 2012).
Lipid oxidation. Thiobarbituric acid (TBA) reactive sub-
stances (TBARS) values were measured according to the procedure
30
c
25
65°C 71°C
20
a* values
15
a a
10
0
HiOX-MAP VP
Packaging
306 Journal of Food Science r Vol. 82, Nr. 2, 2017
of Witte and others (1970) as an indicator of lipid oxidation. On
day 0 and day 2, 5 g of ground beef that contained surface and
interior was mixed with 25 mL trichloroacetic acid (TCA) solu-
tion (11% [w/v]; Sigma Aldrich), blended with a Waring table
top blender (Dynamics Corp. of America, New Hartford, Conn.,
U.S.A.) for 30 s. The homogenate was filtrated through a What-
man (No. 1) filter paper (Fisher Scientific, Bohemia, N.Y., U.S.A.).
The filtrate (1 mL) was mixed with 20 mM TBA and incubated
in a boiling (100 °C) water bath (Isotemp; Fisher Scientific) for
10 min. The absorbance at 532 nm was measured using a Shimadzu
UV-2600 UV-Vis spectrophotometer (Shimadzu Inc., Columbia,
M.D., U.S.A.). The blank consisted of a mixture of TCA and TBA
solutions (1:1, v/v).
Cooking. Patties were cooked to an internal endpoint temper-
ature of either 65 or 71°C using a George Foreman clam-shell grill
(Salton Inc., Columbia, Mo., U.S.A.). The internal temperature
of each patty was measured using a handheld probe thermome-
ter (AccuTuff 340, Atkins, Gainesville, Fla., U.S.A.) inserted into
the geometric center. The temperature at the upper and lower
grill surfaces was maintained at 170 to 180 °C. After reaching
the desired temperature, patties were placed into vacuum pouches
(Prime Source Vacuum Pouches, 4 mil; Koch Supplies Inc.) and
held in ice to avoid postcooking temperature rise.
After 5 min in ice, patties were bisected parallel to the grilled
surfaces and color of the freshly cut interior was measured using a
HunterLab Miniscan XE Plus spectrophotometer. Internal a∗ and
b∗ values were used to calculate chroma.
Modified MRA for cooked ground beef patties. Follow-
ing the interior color measurements, freshly cut cooked patty
interior was used to measure MRA as described in the previous
section for raw patties. Only modification in the procedure was
Figure 2–Effects of packaging and temperature on
internal redness (a∗ ) of ground beef patties. a–c,
least squares means with different superscripts are
different (P < 0.05).
b
Beef reducing activity and cooked color . . .

following vacuum packaging, samples were incubated at 30 °C via increased absorbance at 340 nm recorded for 120 s during
for 4 h. A longer post-incubation time was selected based on the the initial linear phase of the reaction using a Shimadzu UV-
preliminary studies to provide more time for reduction. 2600 UV-Vis spectrophotometer (Shimadzu Inc., Columbia, Md.,
U.S.A.). An increase in absorbance was utilized to calculate LDH
Objective 2: Thermal stability of LDH, NADH-dependent activity and expressed as μM/min/mg. Protein concentration was
reductase, and myoglobin determined by bicinchoninic acid method.
Thermal stability of LDH. LDH from bovine heart muscle Thermal stability of NADH-dependent reductase.
(Sigma-Aldrich) was dialyzed against 50 mM phosphate buffer at NADH-dependent reductase was isolated from bovine longis-
pH 5.6 (represents mean pH of normal meat) or 6.4 (indicates simus lumborum muscle (postmortem age of muscle was 7 d, pH
mean pH of dark-cutting beef) for an hour with a buffer change 5.6) according to Hagler and others (1979) and Faustman and
at 30 min to remove suspended ammonium sulfate. The dialyzed others (1988). Briefly, 200 g muscle devoid of visible fat and
LDH (9.45 mg/mL) was transferred to glass test tubes and incu- connective tissue was homogenized in cold distilled water for
bated in a continuous heat increment (3.5 °C/min) water bath. At 1 min using a Waring blender. The pH of the homogenate
desired endpoint temperatures (65, 71, 77, and 84 °C), test tubes was adjusted to 7.5 with 2 N ammonium hydroxide. The ho-
containing LDH solutions were withdrawn. mogenates were centrifuged at 13700 x g for 20 min and fil-
LDH activity was determined by measuring the generation of trated through a double-layer cheese cloth. The supernatant was

Food Chemistry
NADH as described in the following equation: brought to 40% ammonium sulfate saturation and centrifuged
at 13700 x g for 20 min and filtrated through a double-layer
L − Lactate + NAD+ ↔ Pyruvate + NADH+H+ cheese cloth. The resulting supernatant was made 70% ammo-
nium sulfate saturation and centrifuged at 13700 x g for 20 min.
Briefly, to initiate the reaction, 20 μL LDH (0.2 mM) was The pellet was resuspended in distilled water and dialyzed against
added in a polystyrene disposable cuvette (0.8 mL semi-micro 20 mM sodium phosphate buffer (pH 6.0) with 3 changes of
BRAND GMBH, Wertheim, Germany) containing 700 μL of 10 volumes each. The solution was centrifuged at 26000 x g for
50 mM citrate buffer (pH 7.4), 50 μL NAD+ (0.2 mM), and 30 min and resulting supernatant was applied to carboxymethyl
50 μL l-lactate (50 mM). The NADH formation was monitored cation exchange column (4 × 5 cm; GE Healthcare BS, Uppsala,

35 Figure 3–Effects of packaging and temperature on

chroma of the internal color of cooked ground beef
c patties. a–c, least squares means with different
superscripts are different (P < 0.05).

30

65°C 71°C
b
25

a a
20
Chroma

15

10

0
HiOX-MAP VP
Packaging type

Vol. 82, Nr. 2, 2017 r Journal of Food Science 307

 Beef reducing activity and cooked color . . .
Sweden) previously equilibrated with 20 mM sodium phosphate
buffer (pH 6.0). The enzyme moved through the column unim-
peded while the colored heme protein adhered to the matrix.
Fractions were collected and those with absorbance at 280 nm
were pooled and saturated with 70% ammonium sulfate, cen-
trifuged at 13700 x g for 20 min. The pellet was dialyzed in
2 mM sodium phosphate (pH 7.0) to 1 mM EDTA. The solution
was centrifuged at 26000 x g for 30 min and resulting supernatant
was applied to a diethylaminoethyl anion exchange column (4 ×
5 cm) which had been previously equilibrated with 2 mM sodium
phosphate (pH 7.0) to 1 mM EDTA. Elution was carried out by
a gradient formed from use 2 mM sodium phosphate (pH 7.0) to
1 mM EDTA and 20 mM sodium phosphate (pH 7.0) to 1 mM
EDTA. The isolated NADH-dependent reductase was stored at
−80 °C until needed.
To determine NADH-dependent reductase activity, the isolated
Food Chemistry
enzyme was thawed at room temperature. The pH of the NADH-
dependent reductase was adjusted by dialyzing against 50 mM
phosphate buffer either at pH 5.6 or 6.4. Reductase activity was
measured according to the methodology of Reddy and Carpenter
(1991) in a polystyrene cuvette at 37 °C. The reaction mixture
contained 0.15 mM equine metmyoglobin, 5 mM EDTA, 3 mM
potassium ferrocyanide, 1 mM NADH, and one of the following:
purified NADH-dependent reductase heated to 37, 45, 50, 55,
65, 71, 77, or 84 °C. The reaction was initiated by adding NADH
and was monitored by the changes in absorbance at 580 nm, and
indicated by nanomoles of metmyoglobin reduced per min per mg
of protein.
Thermal stability of OxyMb. OxyMb solutions were
prepared via sodium hydrosulfite (0.01 mg/mL) mediated
40
d0 d2
35 b b
b
30
25
a
% MRA
20
15
10
0
HiOX-MAP VP
Packaging
308 Journal of Food Science r Vol. 82, Nr. 2, 2017
reduction of equine MetMb solutions (0.15 mM) followed by 10
min oxygenation (Brown and Mebine 1969). Equine myoglobin
shares 88.2% homology with bovine counterpart and has been
previously utilized to study cooked color in vitro (Hunt and others
1999). Hydrosulfite in myoglobin solutions was removed using
a PD-10 desalting column (GE Healthcare BS) precalibrated
with citrate buffer at pH 5.6 or 6.4. The desalting column is a
chromatographic gel filtration tool containing a Sephadex G-25
medium to purify proteins based on the molecular weight.
OxyMb solutions were heated in a continuous heat increment
of 3.6 °C/min in a water bath (Isotemp 2223; Fisher Scientific,
Marietta, Ohio., U.S.A.). The samples containing tubes were with-
drawn at given endpoint temperatures (65, 71, 77, or 84 °C). Heat
treated solution were then transferred to Eppendorf tubes and cen-
trifuged at 15000 x g for 5 min. The supernatant was centrifuged
for a 2nd time to remove additional turbidity. The resulting su-
pernatant solutions were transferred into a disposable polystyrene
cuvette (0.8 mL semi-micro BRAND GMBH, Germany) and
the absorbance at 525 nm was recorded using a Shimadzu UV-
2600 UV-Vis spectrophotometer (Shimadzu Inc., Columbia, Md.,
U.S.A.). Percentage of myoglobin denaturation was estimated us-
ing the equation: 
Percentage Mb denaturation = (A525 (raw OxyMb)
− A525 (heated OxyMb)) /

A525 (raw OxyMb) × 100
Statistical analysis. For the objective 1, the experimental de-
sign was a completely randomized block design. Each chub served
as a block. For the objective 2, the experimental design was a
Figure 4–Effects of packaging and storage time on
internal MRA of raw ground beef patties. a–b, least
squares means with different superscripts are different (P
< 0.05). %MRA = [%reflectance at
630 nm − %reflectance at 580 nm]. A greater number
indicates more MRA.
Beef reducing activity and cooked color . . .
completely randomized. Both objectives were replicated 6 times.
The data were analyzed using the Mixed Procedure of SAS 9.3.
For the objective 1, least squares means were generated to assess
the effects of packaging, temperature, storage time, and their inter-
actions for raw surface and internal color, lipid oxidation, MRA,
and cooked color. For the objective 2, least squares means were
generated to assess the effect of temperature, pH, and their inter-
action on thermal stability of LDH, NADH-dependent reductase,
and OxyMb. For all analyses, protected F-test was used to separate
least squares means using the PDIFF option in SAS (P < 0.05).
Results
Objective 1: Effects of packaging and temperature on MRA
of cooked beef patties
pH and proximate composition. The average pH of ground
beef was 5.73 ± 0.02. Moisture, protein, and fat content in per-
centage were 62.87 ± 0.88, 18.61 ± 0.21, and 16.88 ± 0.73,
respectively.
Effects of packaging and time on raw surface and inter-
nal color. There was a significant effect of packaging and storage
time on surface redness (Table 1; P < 0.05). Ground beef pat-
ties in HiOX-MAP had greater a∗ values and more intense red
color (chroma; P < 0.05) compared with VP ground beef pat-
ties at both 2 and 48 h. Vacuum-packaged ground beef patties
had a lower K/S474 ÷ K/S525 value (indicating more DeoxyMb;
P < 0.05) than HiOX-MAP at 2 and 48 h. Patties in HiOX-MAP
had a lower (P < 0.05) K/S610 ÷ K/S525 ratio, indicating more
OxyMb on the surface than VP beef patties.
Patties packaged in VP had greater internal redness (P < 0.05)
than HiOX-MAP patties. This is probably due to the presence of
DeoxyMb in the interior and also its greater affinity to oxygen
following exposure to air (time delay between cutting and taking
reading was approximately 90 s).
20
HiOx VP
c least squares means with different superscripts are
18
16
ab
14
a
12
% MRA
10
0
65°C 71°C
Temperature
Effects of packaging and storage time on lipid oxida-
tion. There was a significant effect of packaging x time interac-
tion observed for lipid oxidation (Figure 1). HiOX-MAP patties
had greater (P < 0.05) TBARS values than VP patties. Storage
time significantly increased TBARS values in HiOX-MAP patties
(P < 0.05) but had no effect on patties packaged in vacuum (P >
0.05).
Effects of packaging and temperature on cooked inter-
nal color. There was a significant packaging x temperature inter-
action for cooked internal meat color (a∗ values and chroma). Beef
patties packaged in HiOX-MAP had lower (P < 0.05) a∗ values
(indicative of well-done appearance) than VP patties irrespective
of the temperature (Figure 2). There was no difference in internal
cooked meat color (a∗ values) between HiOX-MAP ground beef
patties at 65 and 71 °C (P > 0.05). The intensity of the internal
color (chroma; Figure 3) of ground beef patties followed the same
Food Chemistry
pattern as that of internal a∗ values. Vacuum-packaged ground
beef patties retained pinkish red color (P < 0.05) at 65 than at
71 °C as indicated by greater a∗ and chroma values.
Effects of packaging and temperature on MRA. Packag-
ing affected MRA of both raw and cooked ground beef (Figure 4
and 5; P < 0.05). Ground beef patties packaged in HiOX-MAP
had lower (P < 0.05) raw and cooked meat MRA compared with
VP beef patties at the conclusion of 48 h. There was no difference
(P > 0.05) in cooked patty MRA for HiOX-MAP between 65 and
71 °C. Cooked patty MRA decreased (P < 0.05) as temperature
increased for VP patties.
Objective 2: Effects of temperature and pH on thermal
stability of LDH, NADH-dependent reductase, and OxyMb
Activity of LDH. There was a significant pH × temperature
interaction for LDH activity (P < 0.05; Figure 6). Up to 77 °C,
LDH activity at pH 6.4 was almost 2-fold greater than that of LDH
Figure 5–Effects of packaging and temperature on
internal MRA of cooked ground beef patties. a–c,
different (P < 0.05). %MRA = [% reflectance at
630 nm − %reflectance at 580 nm]. A greater
number indicates more MRA.
b
Vol. 82, Nr. 2, 2017 r Journal of Food Science 309
 Beef reducing activity and cooked color . . .
at pH 5.6 (P < 0.05). The activity of LDH at both pHs increased
concomitantly as temperature increased until 77 °C, and then it
dropped at 84 °C. The activity of LDH at 84 °C was similar for
both pHs. Nevertheless, this study indicates that LDH was able to
regenerate NADH at 84 °C.
Activity of NADH-dependent reductase. There was a
pH × temperature interaction for NADH-dependent reductase
activity (Figure 7; P < 0.05). A greater temperature decreased
NADH-dependent reductase activity. In general, NADH-
dependent reductase demonstrated more resistance to temperature
(P < 0.05) at pH 6.4 than pH 5.6. This was shown by a greater en-
zyme activity exhibited across the range of temperatures at 6.4 than
at pH 5.6. Maximum activity was recorded at 45 °C compared
with other endpoint temperatures. At 65 °C, NADH-dependent
reductase did not show any activity at both pHs.
OxyMb denaturation. A pH × temperature interaction was
Food Chemistry
significant for OxyMb denaturation (Figure 8; P < 0.05). For
both pHs, the increase in temperature resulted in more myoglobin
denaturation (P < 0.05). At 84 °C, both pHs showed maximum
denaturation. Denaturation of OxyMb was lower (P < 0.05) at
pH 6.4 than pH 5.6 when OxyMb solutions heated at 71 and
77 °C.
Discussion
As expected, in this study packaging had a significant effect
on cooked meat color development. HiOX-MAP made patties
very susceptible to PMB compared with PVC and VP. This is
primarily due to differences in myoglobin forms present within
the interior of patties before cooking (Hunt and others 1999; John
and others 2004, 2005; Seyfert and others 2004). In this study,
patties packaged in HiOX-MAP had a lower internal meat color
compared with VP. Suman and others (2009) indicated that the
interior of steaks packaged in HiOX-MAP had lower a∗ values at
20
18
pH5.6 pH6.4
16
14
d
LDH activity (μM/min/mg)
12
10
8 b b
6
a
4
0
65°C 71°C
Temperature
310 Journal of Food Science r Vol. 82, Nr. 2, 2017
65 °C than VP steaks cooked at the same temperature. However,
limited studies have determined the role of MRA of cooked patties
in cooked meat color development.
In this study, we determined nitric oxide-induced MRA of pat-
ties packaged in HiOX-MAP and VP at different temperatures.
Patties packaged in VP had greater reducing activity than those
in HiOX-MAP. A greater reducing activity indicates that MetMb
reducing system retain some activity at cooking temperatures. Os-
born and others (2003) demonstrated heat-induced MetMb for-
mation is reversible using cooked meat extracts and NADH. They
proposed that either a modified NADH-dependent reductase or
a new protein system with a ferryl myoglobin component was
responsible for the reduction observed under heating conditions.
Ferryl myoglobin has been known to undergo a 2-electron reduc-
tion by NADH to form OxyMb (Mikkelsen and Skibsted 1995).
Various factors can influence red color in cooked patties (Corn-
forth and others 1991). Van Laack and others (1996) reported
that incomplete denaturation of myoglobin was responsible for
pink color in ground beef patties cooked to 71 °C. Similarly,
Osborn and others (2003) observed that heated MetMb was re-
duced to a red pigment that had a similar spectrum as that of
OxyMb. They also demonstrated that reacting reducing or oxi-
dizing agents with heated myoglobin yielded characteristic spectra
of DeoxyMb or MetMb, respectively. A reducing system capable of
reducing MetMb extract at cooking temperatures was responsible
for the red color (Osborn and others 2003). In support, Bernofsky
and others (1959) noted that 95% of cooked ground beef extract
was constituted by OxyMb. This suggests that reduction of meat
pigments can occur in cooked meats. High temperature has been
shown to cause MetMb formation (Brooks 1935) due to release
of oxygen from the oxygenated pigment and also due to oxida-
tion. This study is the 1st to report differences in MRA of cooked
patties in situ.
e Figure 6–Effects of pH and temperature on LDH
activity. a–e, least squares means with different
superscripts are different (P < 0.05).
c
b b
77°C 84°C
Beef reducing activity and cooked color . . .

Previous studies indicated that factors influencing raw reducing
activity of meats seemingly affected cooked reducing activity (Van
Laack and others 1996; Osborn and others 2003). Vacuum packag-
ing has been associated with greater enzymatic MetMb reduction
and limited lipid oxidation (Stewart and others 1965). However,
high oxygen conditions promote lipid oxidation. Lipid oxidation
products can minimize enzyme activity and its ability to regener-
ate NADH (Ramanathan and others 2014). Thus, modification of
enzyme involved in MetMb reduction, in part, can be responsible
for the lower MRA of patties in HiOX-MAP and cooked at 65
and 71 °C.
Antioxidants and chelators have been used in meats to coun-
teract the damaging effects of lipid oxidation products (Shahidi
and others 1987; Suman and others 2011). The addition of ery-
thorbate improved total reducing activity and internal cooked
color of ground beef patties than the controls (Suman and others
2005). Similarly, other research demonstrated the effects of tricar-
boxylic acid cycle substrate such as succinate can improve redness
of cooked patties (Ramanathan and others 2013). Hence, increased
reducing activity can influence redox state of myoglobin within
the patties.

e Figure 7–Effects of pH and temperature on

e
3000 NADH-dependent reductase activity. a–e, least squares
means with different superscripts are different (P <

Food Chemistry
pH5.6 pH6.4 0.05).

2500
d
Reductase activity in nM/min/mg

d
2000

c
1500

1000
b

500 a
a
a a
0
37°C 45°C 50°C 55°C 65°C

Temperature

e Figure 8–Effects of pH and temperature on

100
d e oxymyoglobin denaturation. a–e, least squares means
with different superscripts are different (P < 0.05).
90
Percentage of myoglobin denatured

80
pH 5.6 pH 6.4
70
c
60

50

40

30
b
20

10 a
a a

0
65°C 71°C 77°C 84°C

Temperature

Vol. 82, Nr. 2, 2017 r Journal of Food Science 311

 Beef reducing activity and cooked color . . .
The thermal stability of NADH-dependent reductase, LDH,
and OxyMb were determined to investigate the reducing activity
in cooked meat. NADH-dependent reductase had a lower thermal
stability when compared with LDH and OxyMb. The activity of
NADH-dependent reductase was pH and temperature dependent.
The pH/temperature combination that gave optimal reductase ac-
tivity in this study was pH 6.4/45 °C. Irrespective of pH, there was
no NADH-dependent reductase activity at 65 °C. This suggests
that NADH-dependent reductase may be destroyed at cooking
temperatures and may not be involved in MetMb reduction.
There exist other enzymatic and nonenzymatic reduction sys-
tems such as diaphorase and NAD(P)H-quinone oxidoreduc-
tase that are capable of reducing MetMb (Echevarne and others
1990; Bekhit and Faustman 2005; Elroy and others 2015). Os-
born and others (2003) suggested that partially denatured NADH-
dependent reductase may be responsible for reducing activity at
Food Chemistry
cooking temperatures. Based on this study and previous find-
ings (Echevarne and others 1990), it is less likely that denatured
NADH-dependent reductase is responsible for MetMb reduction
at elevated temperatures. However, in a complex system, inter-
action among different meat compounds may protect NADH-
dependent reductase from a complete denaturation, thus allowing
MetMb reduction. Further research is needed to elucidate the
mechanism of MRA in cooked patties.
LDH can regenerate NADH, which is further utilized to reduce
MetMb (Kim and others 2006). LDH in whole meats retained par-
tial activity at cooking temperatures (Stalder and others 1991). In
this study, we used purified LDH to determine the effects of tem-
perature and pH on its activity. Stalder and others (1991) reported
the presence of LDH activity in bovine semimembranosus slurries
heated up to 66 °C. In agreement, this study suggests that a lactate
NADH-dependent reducing system may be functional at cooking
temperatures and can be involved in MRA of cooked patties. This
system may represent one of the reducing systems involved in post
cooking myoglobin reduction.
The effects of temperature on LDH, NADH-dependent re-
ductase, and OxyMb denaturation indicated that thermal stability
is protein specific. Protein function and structural conformation
dictate their thermal stability. Proteins function is determined by
environmental conditions. Alteration of physiological conditions
in general and temperature, in particular, leads to loss of struc-
tural conformation and subsequent functional integrity (Argos
and others 1979). Further, differences in amino acid composition
and protein structure can affect thermal stability. Different forces
have been proposed to play a significant role in stabilizing proteins
against thermal stability (Yang and Honig 1993). These factors
include, but are not limited to disulfide linkages, salt bridges,
increased hydrophobicity, increased hydrogen bonding, electro-
static interactions, helical content, polar surface area, and hydrogen
bonds (Argos and others 1979; Kumar and others 2000; Pace and
others 2014). LDH is a tetramer protein containing about 30% to
48% α helices and 19% to 23% β sheets (Chen and others 1972;
Pace and others 2014) whereas NADH-cytochrome b5 reductase
contains 24.7% α helices and β sheets. Myoglobin is a helical
monomer. More than β sheets, a greater proportion of helical
structure can increase protein stability (Argos and others 1979;
Kumar and others 2000). This may explain the greater thermal
stability of LDH and myoglobin compared to NADH-dependent
reductase.
Based on instability index (Bioinformatics 2016), these 3 pro-
teins can be classified as follows: NADH-dependent reductase >
LDH > OxyMb. A greater instability index is an indicator of
312 Journal of Food Science r Vol. 82, Nr. 2, 2017
greater sensitivity to temperature. Myoglobin has the lowest index
(15.13). LDH has an intermediary instability index (27.60) which
is much lower than that of NADH-cytochrome b5 reductase es-
timated to be 48.02. The latter is classified as an unstable protein.
As expected, high pH has a protective effect on myoglobin denat-
uration. Less denaturation was reported at pH 6.4 than at pH 5.6.
These results confirmed previous findings on the effect of pH on
myoglobin denaturation (Hunt and others 1999).
Conclusion
Raw ground beef patties with greater reducing activity can
have more predictable interior cooked color. Further, reducing
activity in cooked patties suggests that enzymes involved in MRA
are functional at cooking temperatures, and have the potential
to influence MRA. Developing strategies to improve reducing
activity in ground beef can result in predictable internal cooked
meat color.
Authors’ Contributions
B. A. Djimsa conducted research, collected data, and drafted the
manuscript; A. Abraham conducted research; G. G. Mafi critically
evaluated the manuscript; D. L. VanOverbeke conducted statistical
analysis; R. Ramanathan conceived the idea, designed experiment,
and proof read the manuscript.
References
AMSA. 2012. Meat color measurement guidelines. Champaign: American Meat Science Asso-
ciation.
Argos P, Rossmann MG, Grau UM, Zuber H, Frank G, Tratschin JD. 1979. Thermal stability
and protein structure. Biochemistry 18(25):5698–703.
Bekhit AED, Faustman C. 2005. Metmyoglobin reducing activity. Meat Sci 3:407–39.
Bernofsky C, Fox JBJ, Schweigert BS. 1959. Biochemistry of myoglobin. VII. The effect of
cooking on myoglobin in beef muscles. J Food Sci 24(4):339–43.
Bioinformatics SIo. 2016. ExPASy bioinformatics resource portal. Expasy website.
Brooks J. 1935. The oxidation of hemoglobin to methaemoglobin by oxygen. (ii) The relation
between the rate of oxidation and the partial pressure of oxygen. Proceedings of the Royal
Society of London. Series B, Biol Sci 118(811):560–77.
Brown WD, Mebine B. 1969. Autoxidation of oxymyoglobin. J Biol Chem 244(24):6696–701.
Chen Y-W, Yang JT, Martinez HM. 1972. Determination of secondary structures of proteins
by circular dicroistm and optical rotatory dispersion. Biochemistry 11(22):4120–31.
Cornforth D, Calkins CR, Faustman C. 1991. Methods for identification and prevention of pink
color in cooked meat. Reciprocal Meat Conference. Manhattan: American Meat Science
Association (AMSA). p 53–8.
Echevarne C, Renerre M, Labas R. 1990. Metmyoglobin reductase activity in bovine muscles.
Meat Sci 27(2):161–72.
Elroy NN, Rogers J, Mafi GG, VanOverbeke DL, Hartson SD, Ramanathan J. 2015. Species-
specific effects on non-enzymatic metmyoglobin reduction in vitro. Meat Sci 105:108–13.
Faustman C, Cassens RG, Greaser ML. 1988. Reduction of metmyoglobin by extracts of bovine
liver and cardiac muscle. J Food Sci 53:1065–7.
Hagler L, Coppes RLJ, Herman RH. 1979. Metmyoglobin reductase. Identification and purifica-
tion of a reduced NADH dependent enzyme from bovine heart which reduces metmyoglobin.
J Biol Chem 254(14):6505–14.
Hague MA, Warren KE, Hunt MC, Kropt DH, Kastner CL, Stroda SL, Johnson DE. 1994.
Endpoint Temperature, internal cooked color, and expressible juice color relationships in
ground beef patties. J Food Sci 59(3):465–70.
Hunt MC, Sørheim O, Slinde E. 1999. Color and heat denaturation of myoglobin forms in
ground beef. J Food Sci 64(5):847–51.
Jayasingh P, Cornforth DP, Brennand CP, Carpenter CE, Whittier DR. 2002. Sensory eval-
uation of ground beef stored in high oxygen-modified atmosphere packaging. J Food Sci
67(9):3493–6.
John L, Cornforth D, Carpenter CE, Sorheim O, Pettee BC, Whittier DR. 2004. Comparison
of color and thiobarbituric acid values of cooked hamburger patties after storage of fresh beef
chubs in modified atmosphere. J Food Sci 69(8):C608–C14.
John L, Cornforth D, Carpenter CE, Sorheim O, Pettee BC, Whittier DR. 2005. Color and
thiobarbituric acid values of cooked top sirloin steaks packaged in modified atmospheres of
80% oxygen, or 0.4% carbon monoxide, or vacuum. Meat Sci 69(3):441–9.
Killinger KM, Hunt MC, Campbell RE, Kropf DH. 2000. Factors affecting premature browning
during cooking of store-purchased ground beef. J Food Sci 65(4):585–7.
Kim YH, Hunt MC, Mancini RA, Seyfert M, Loughin TM, Kropf DH, Smith JS. 2006.
Mechanism for lactate-color stabilization in injection-enhanced beef. J Agric Food Chem
54(20):7856–62.
King NJ, Whyte R. 2007. Does it look cooked? A review of factors that influence cooked meat
color. J Food Sci 71: 31–40.
Kumar S, Tsai C-J, Nussinov R. 2000. Factors enhancing protein thermostability. Protein Eng
13(3):179–91.
Lytras GN, Geilesky A, King RD, Ledward DA. 1999. Effect of muscle type, salt, and
pH on cooked meat haemoprotein formation in lamb and beef. Meat Sci 52(2):189–
94.
Beef reducing activity and cooked color . . .
Mikkelsen A, Skibsted, LH. (1995). Acid-catalyzed reduction of ferrylmyoglobin: product distri-
bution and kinetics of autoreduction and reduction by NADH. Zeitschrift für Lebensmittel-
Untersuchung und Forschung 200 3: 171–77.
Mottram DS. 1998. Flavor formation in meat and meat products: a review. Food Chem 62:415–
24.
Osborn HM, Brown H, Adams JB, Ledward DA. 2003. High temperature reduction of met-
myoglobin in aqueous muscle extracts. Meat Sci 65(1):631–7.
Pace CN, Scholtz JM, Grimsley GR. 2014. Forces stabilizing proteins. FEBS Letters
588(14):2177–84.
Ralston K, Brent CP, Starke Y, Riggins T, Lin C-TJ. 2002. Consumer food safety behavior: a
case study in hamburger cooking and ordering. Econ Res Serv/USDA:1–2.
Ramanathan R, Mancini RA, Dady GA, Van Buiten CB. 2013. Effects of succinate and pH on
cooked beef color. Meat Sci 93(4):888–92.
Ramanathan R, Mancini RA, Suman SP, Beach CM. 2014. Covalent binding of 4-hydroxy-
2-nonenal to lactate dehydrogenase decreases NADH formation and metmyoglobin reducing
activity. J Agric Food Chem 62(9): 2112–7.
Reddy IM, Carpenter CE. 1991. Determination of metmyoglobin reductase activity in bovine
skeletal muscles. J Food Sci 56(5):1161–4.
Sammel LM, Hunt MC, Kropf DH, Hachmeister KA, Johnson DE. 2002. Comparison of assays
for metmyoglobin reducing ability in beef inside and outside semimembranosus muscle. J
Food Sci 67(3):978–84.
Seyfert M, Hunt MC, Mancini RA, Kropf DH, Stroda SL. 2004. Internal premature browning
in cooked steaks from enhanced beef round muscles in High-oxygen and Ultra-low oxygen
modified atmospheres. J Food Sci 69(2):142–6.
Shahidi F, Rubin LJ, Wood DF. 1987. Control of lipid oxidation in cooked meats by combina-
tions of antioxidants and chelators. Food Chem 23(2):151–7.
Stalder JW, Smith GL, Keeton JT, Smith SB. 1991. Lactate Dehydrogenase Activity in bovine
muscle as a means of determining heating endpoint. J Food Sci 56(4):895–8.
Stewart MR, Hutchins BK, Zipser MW, Watts BM. 1965. Enzymatic reduction of metmyoglobin
by ground beef. J Food Sci 30(3):487–91.
Suman SP, Mancini RA, Ramanathan R, Konda MR. 2009. Effect of lactate-enhancement,
modified atmosphere packaging, and muscle source on the internal cooked color of beef
steaks. Meat Sci 81(4):664–70.
Suman SP, Faustman C, Lee S, Tang J, Sepe HA, Vasudevan P, Annamalai T, Manojkumar
M, Marek P, S. VK. 2005. Effect of erythorbate, storage, and high-oxygen packaging on
premature browning in ground beef. Meat Sci 69(2):363–9.
Suman SP, Mancini RA, Joseph P, Ramanathan R, Konda MK, Dady G, Yin S. 2011. Chitosan
inhibits premature browning in ground beef. Meat Sci 88(3):512–6.
USDA. 1998. Premature browning of cooked ground beef. Food Safety and inspection service
public meeting on premature browning of ground beef. Washington, D.C.: Food Safety and
Inspection Services.
Van Laack RLJM, Berry BW, Solomon MB. 1996. Variations in internal color of cooked beef
patties. J Food Sci 61(2):410–4.
Warren KE, Hunt MC, Kropf DH. 1996. Myoglobin oxidative state affects internal cooked color
development in ground beef patties. J Food Sci 61(1):513–6.
Witte VC, Krause GF, Bailey ME. 1970. A new extraction method for determining 2-
thiobarbituric acid values of pork and beef during storage. J Food Sci 35, 582–5.
Food Chemistry
Yang A-S, Honig B. 1993. On the dependence of protein stability. J Mol Biol 231:459–74.
Vol. 82, Nr. 2, 2017 r Journal of Food Science 313