Research Note
The roles of the actin-myosin interaction and proteolysis in tenderization
during the aging of chicken muscle
S. Li, X. Xu,1 and G. Zhou
Key Laboratory of Meat Processing and Quality Control, Ministry of Education, College of Food Science
and Technology, Nanjing Agricultural University, Nanjing 210095, China
ABSTRACT The objective of this study was to investigate the contribution of the changes in the actinmyosin interaction and proteolysis on meat tenderization during postmortem storage. Following slaughter,
chicken breast muscles were removed and stored at 4°C.
Changes in the actin-myosin interaction over 48 h of
aging were determined by monitoring the Mg2+- and
Ca2+-ATPase activities. Shear force values, pH, protein
degradation, calpain activities, and myofibrillar ultrastructures were also investigated. Results showed that
the initial weak actin-myosin interaction strengthened
at 12 h postmortem followed by a gradual weakening,
which was supported by a decrease in Mg2+-ATPase
activities and a lengthening of the sarcomeres. According to SDS-PAGE and Western blotting analyses,
the 30-kDa troponin-T fragment could not be readily
detected until 12 h, whereas, at the same time, desmin had been rapidly degraded. However, there was a
gradual decline in μ-calpain activity, commencing after
about 6 h. Meanwhile, the largest decline in shear force
was observed between 12 and 24 h postmortem. These
findings suggest that weakening of the strong actin-myosin interaction formed at rigor may play a large role in
meat tenderization during the early period of storage.
It is proposed that weakening of the actin-myosin interaction results in lengthening of the sarcomeres, and
then activated calpains are more able to reach their targeted sites, enabling proteolysis. These 2 factors may
be involved in the conversion of muscle to tender meat
during postmortem storage.
Key words: actin-myosin interaction, proteolysis, postmortem, tenderization
2012 Poultry Science 91:150–160
doi:10.3382/ps.2011-01484
INTRODUCTION
tion differs considerably in different meat sources and
lasts more than 24 h in cattle and sheep (Wheeler and
Koohmaraie, 1994) and less than 6 h in chicken breast
muscle (Alvarado and Sams, 2000b; Schreurs, 2000).
Particularly, it has been reported that electrical stunning could delay the development of rigor mortis in
chickens and turkeys because of a decrease in struggling (Lee et al., 1979; Murphy et al., 1988; Alvarado
and Sams, 2000b), whereas electrical stimulation could
increase the rate of rigor development in chicken breast
muscle, shown by a more rapid decrease in pH and an
increase in the inosine:adenosine ratio (Alvarado and
Sams, 2000a). On the other hand, during the onset of
rigor and during postrigor chilled storage, meat tenderness is improved as a result of limited proteolysis of
critical myofibrillar and cytoskeletal proteins, which results in myofibril fragmentation and loss of muscle cell
integrity (Taylor et al., 1995; Koohmaraie and Geesink,
2006; Huff-Lonergan et al., 2010). Maximum tenderness
is reached after 8 d of storage in cattle. As for chicken
meat, the rapid maximum tenderness reached could be
explained by a greater activation of the calpain system
(Lee et al., 2008). However, the exact mechanism of
this tenderizing process is still a matter of dispute. Al-
Meat tenderness is considered to be the most important trait affecting consumer satisfaction (Miller et
al., 2001; Shackelford et al., 2001). However, variation
in beef and lamb tenderness still remains one of the
most critical quality problems encountered by the meat
industry (Morgan et al., 1991; Weaver et al., 2008),
and earlier deboning times and shorter aging times
have resulted in a marked increase in tough poultry
breast meat (Craig et al., 1999). Meat tenderness is
largely dependent on the state of contraction of skeletal
muscle fibers and the amount and extent of collagen
crosslinking in the surrounding extracellular connective
tissues (Koohmaraie, 1996; Huff-Lonergan et al., 2010;
Nishimura, 2010; Sikes et al., 2010). Meat toughness
accumulates as rigor mortis develops in early periods
postmortem, but the time for rigor mortis comple-
©2012 Poultry Science Association Inc.
Received March 15, 2011.
Accepted September 11, 2011.
1 Corresponding author: [email protected]
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though there is good evidence that specific myofibrillar
proteins are degraded during the immediate postmortem period, the total proportion of protein degraded
is considered to be small, whereas there are substantial reductions in toughness (Goll et al., 1995; Hopkins
and Thompson, 2001, 2002). More importantly, Goll et
al. (1997) concluded that despite the small amount of
cytoskeletal protein degradation and some changes in
the Z-disk, Z-disk and I-band integrity, and sarcomerecostamere integrity (presumably caused by proteolytic
degradation), there is no change in tenderness of unshortened muscle during the first 72 h postmortem.
Taken together, it is conceivable that there are other
processes involved in postmortem tenderization.
It has been proposed that changes in the actin-myosin interaction are involved in postmortem tenderization (Goll et al., 1995). Evidences for a potential effect
of changes in the actin-myosin interaction on meat tenderness during postmortem aging are provided by numerous investigators, indicating that actomyosin from
aged muscle was in a weaker bonding state compared
with that from rigor muscle (Fujimaki et al., 1965; Goll
and Robson, 1967; Takahashi et al., 1981). Unfortunately, these studies have not attempted to link changes in actin-myosin and tenderization. However, Hopkins
and Thompson (2001) reported that in their study with
lamp M. longissimus thoracis et lumborum that there
was no apparent effect of dissociation of actomyosin on
tenderness.
To our knowledge, this area of research linking changes in the actin-myosin interaction with meat tenderization during postmortem storage has received little
attention. Therefore, the objective of this study was
to investigate the role of changes in the actin-myosin
interaction and proteolysis on the tenderization of meat
during postmortem storage.
MATERIALS AND METHODS
Birds and Sample Preparation
Four yellow-feathered chickens, a Chinese native
breed (male, 40 d, 2.0–2.5 kg) from the animal experimental station of Nanjing Agricultural University (China) were cared for and slaughtered as outlined in the
guide for the care and use of experimental animals (Animal Experimental Special Committee of NAU, Nanjing,
China). The birds were held for 12 h without feed and 3
h without water before slaughter. The birds were killed
directly by bleeding from a unilateral neck cut severing
the left carotid artery and jugular vein (without stunning). Postmortem time started after a 2-min bleeding
period, and then the birds were skinned immediately
and chicken breast muscles (M. pectoralis superficialis)
were rapidly excised from carcasses and stored at 4°C.
All of the birds were slaughtered on the same day. At
the following times postmortem, shear force values and
pH values were determined individually; samples from
Mg2+
and Ca2+each chicken for the determination of
ATPase activity of myofibrils, SDS-PAGE, Western
blot, and casein zymography were taken and snap-frozen in liquid nitrogen and then stored at −80°C before
subsequent analysis. Samples for transmission electron
microscopy were also taken individually.
Measurement of Shear Force Value and pH
Muscle samples were subjected to shear force measurements after storage at 4°C for 4, 12, 24, and 48 h.
The breast fillets wrapped in plastic bags were cooked
in a water bath at 80°C to an internal temperature of
75°C. Cooked fillets in plastic bags were stored overnight at 4°C and then cut into 6 strips (cross-sectional
area of 1 cm2, cut parallel to the longitudinal orientation of the muscle fibers) that were measured perpendicularly to the meat strips using a texture analyzer (TAXT2i, Stable Micro Systems Ltd., Godalming,
UK). Peak shear force values were recorded.
For the measurement of pH at 1, 2, 4, 6, 12, 24, 48
h postmortem, 2 g of each muscle was homogenized in
16 mL of chilled deionized water (0°C) using a homogenizer (S10, Ningbo, China) at 720 × g for 30 s. The
pH values of the homogenates were then determined using a pH meter (pH211, Hanna Ltd., Póvoa de Varzim,
Portugal). All of these procedures were conducted at
4°C in a refrigerator to inhibit prerigor enzymatic reactions. Enzymatic reactions occur very slowly or not at
all at low temperatures, such as 4°C or below (Solomon
et al., 2001; Shanmugam and Sathishkumar, 2009). The
data collected in this research should not be compared
with other data because iodoacetate was not used during pH measurement on prerigor meat. The observed
changes in pH could be due to metabolic changes that
occur during prerigor. Three determinations were made
for each sample.
Measurement of Actin-Myosin Interaction
Changes in the actin-myosin interaction were measured by determining myofibrillar Mg2+- and Ca2+ATPase activity and the thermal inactivation of myosin. The method used was previously described by
Benjakul et al. (2007).
Preparation of Myofibrils
Myofibrils were prepared from breast muscles at 4, 8,
12, 24, and 48 h postmortem according to the method
of Johnston et al. (1972) with some modifications. The
sliced muscle was homogenized in 7.5 volumes of buffer
containing 0.1 M KCl, 2 mM EDTA, and 20 mM TrisHCl (pH 7.0) at a speed of 720 × g for 45 s, and then
the homogenate was centrifuged at 1,000 × g for 10 min
at 4°C. The pellet was suspended in 0.1 M KCl and 20
mM Tris-HCl (pH 7.0) and was washed twice using the
same buffer. The washed myofibrils were then treated
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Li et al.
with 1% Triton X-100 for 30 min, and then they were
washed 3 times in 0.1 M KCl and 20 mM Tris-HCl (pH
7.0). The final pellet was suspended in 0.6 M KCl and
20 mM Tris-HCl (pH 7.0). The protein concentration
was determined by the Bradford method using BSA as
a standard (Kruger, 1996) and diluted to 4 mg/mL.
Myofibril preparations were then analyzed for Mg2+and Ca2+-ATPase activity.
Thermal Inactivation of Myosin
The principle of this method is that actin denaturation (by high salt) would release myosin from the actomyosin complex. The existence of free myosin in the
treated myofibril can be detected from its subsequent
thermal inactivation profile at 60°C. If the myofibril
contains free myosin, the inactivation profile will exhibit a rapid fall during the early phase, followed by a slow
decreasing phase. The former and the latter represent
inactivation of free myosin and remaining actomyosin,
respectively (Kawakami et al., 1971).
The myofibril samples prepared as above were incubated at 60°C for different times (0, 15, and 30 min). At
the designated time, the samples were cooled rapidly in
iced water and assayed for Ca2+-ATPase activity.
Determination of Ca2+- and Mg2+-ATPase
Activity of Myofibrils
Determination of Mg2+- and Ca2+-ATPase was based
on the method described by Benjakul et al. (2007).
To 0.5 mL of myofibrils (4 mg/mL), 0.25 mL of 0.5
M Tris-maleate (pH 7.0), 0.25 mL of 0.1 M MgCl2 or
CaCl2, and 3.75 mL of deionized water were added.
The reaction was initiated by adding 0.25 mL of 20
mM adenosine triphosphate (ATP). It was conducted
for 10 min at 25°C and stopped by the addition of 2.5
mL of 15% chilled trichloroacetic acid. The reaction
mixture was subjected to centrifugation at 3,000 × g
for 5 min at 4°C. The inorganic phosphate liberated in
the supernatant was measured by the method of Fiske
and Subbarow (1925). Specific activity was expressed
as micrograms of inorganic phosphate released per milligrams of protein per minute. A blank was performed
by adding the chilled trichloroacetic acid before the addition of ATP.
Preparation of Sarcoplasmic
and Myofibrillar Proteins
Snap-frozen samples at 0, 6, 12, 24, and 48 h postmortem were used to prepare sarcoplasmic and myofibrillar proteins. Sarcoplasmic proteins were extracted
according to the procedure reported by Veiseth et al.
(2001) with modifications. Finely minced meat samples
(1 g) were homogenized in 3 volumes (wt/vol) of extraction buffer containing 10 mM EDTA, 0.1% (vol/
vol) β-mercaptoethanol, and 100 mM Tris-HCl (pH
8.3) with a polytron (S10, Ningbo, China) at a speed
of 1,620 × g for 15 s, twice, with a 15-s cooling period
interval between bursts. The homogenate was centrifuged at 15,000 × g for 30 min at 4°C, and the protein
concentration of the supernatant was determined by
the Bradford method using BSA as a standard (Kruger, 1996). One volume of supernatant was combined
with one volume of tracking dye solution [20% (vol/vol)
glycerol, 0.75% (vol/vol) β-mercaptoethanol, 0.05%
(wt/vol) bromophenol blue, and 150 mM Tris-HCl (pH
6.8)], and then it was stored at −80°C for subsequent
analysis by casein zymography.
The extraction of myofibrillar proteins from muscle
tissues was performed as described by Huang et al.
(2009). The protein concentration of the supernatant
was determined by the method of Bradford (Kruger,
1996).
SDS-PAGE and Western Blotting Analysis
Sodium dodecyl sulfate-PAGE was performed on
a 4% stacking gel [acrylamide: N,N′-bis-methylene
acrylamide = 37.5:1 (wt/wt), 0.1% (wt/vol) SDS,
0.125% (vol/vol) N′N′N′N′-tetramethylethylenediamine
(TEMED), 0.075% (wt/vol) ammonium persulfate
(APS), and 0.125 M Tris-HCl (pH 6.8)] and a 12.5%
separating gel [acrylamide: N,N′-bis-methylene acrylamide = 37.5:1 (wt/wt), 0.1% (wt/vol) SDS, 0.05%
(vol/vol) TEMED, 0.05% (wt/vol) APS, and 0.5 M
Tris-HCl (pH 8.8)]. Gels (9 cm wide, 8 cm tall, and
1 mm thick) were run on mini slab electrophoresis
units (Bio-Rad Laboratories, Hercules, CA). Gels were
loaded with 20 μg of protein/well for troponin-T and
desmin, respectively, and were run at a constant voltage of 80 V for about 45 min and then at 120 V for
approximately 1.5 h. The running buffer contained 25
mM Tris, 192 mM glycine, and 0.1% (wt/vol) SDS (pH
8.3). Gels for the detection of troponin-T were stained
in a solution containing 0.1% (wt/vol) Coomassie Brilliant Blue R-250, 45% (vol/vol) methanol, and 10%
(vol/vol) glacial acetic acid, and they were subsequently destained overnight with a solution of 10% (vol/vol)
methanol and 10% (vol/vol) glacial acetic acid. Desmin
degradation was analyzed by Western blotting following the protocol described by Huang et al. (2009). Gels
were immediately transferred to polyvinylidene fluoride
membranes (Millipore, Temecula, CA) using a MiniProtean II system (Bio-Rad Laboratories) at a constant current of 200 mA for 1 h at 4°C. The transfer
buffer consisted of 25 mM Tris, 192 mM glycine, and
10% (vol/vol) methanol (pH 8.3). The membrane was
then blocked for 2 h in blocking buffer [0.05% Tween
20, 20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 5 mM
KCl, and 5% skim milk powder] at room temperature.
After blocking, the membrane was incubated overnight
at 4°C with polyclonal rabbit antibody raised against
the chicken skeletal muscle protein desmin at a dilution
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of 1:500 in blocking buffer. The antibody (a generous
gift) was prepared in the meat laboratory, Hokkaido
University, Japan, and it appeared as a single immunoreactive band by Western blotting (data not shown).
After 4 washes with blocking buffer for 5 min each,
the membrane was incubated with goat anti-rabbit IgG
HRP-conjugated second antibody (Chemicon, Tmemecula, CA) at a dilution of 5,000 in blocking buffer.
After 4 more washes, the membrane was stained with
a Sigma fast 3,3′-diaminobenzidine tablet set (SigmaAldrich, St. Louis, MO).
The gels and membranes were photographed with a
Gel Doc XRTM System (Bio-Rad Laboratories). Then
the intensities of the gels and Western blotting bands
were quantified by Quantity One software (Bio-Rad
Laboratories) within the calibration range.
Casein Zymography
Casein zymography was based on the protocol described by Veiseth et al. (2001) with slight modifications.
Samples were thawed and loaded immediately onto
nondenaturing PAGE casein gels [12.5% separating gel:
acrylamide:N,N′-bis-methylene acrylamide = 70:1 (wt/
wt), 0.05% (vol/vol) TEMED, 0.05% (wt/vol) APS, casein (2.1 mg/mL), and 375 mM Tris-HCl (pH8.8); and
4% stacking gel: acrylamide: N,N′-bis-methylene acrylamide = 37.5:1 (wt/wt), 0.125% (vol/vol) TEMED,
0.075% (wt/vol) APS, and 125 mM Tris-HCl (pH 6.8)].
The casein mini-gels (9 cm wide, 8 cm tall, and 0.75
mm thick) were run on mini slab electrophoresis units
(Bio-Rad Laboratories). Gels were prerun at 100 V for
15 min at 4°C with a running buffer containing 25 mM
Tris-HCl (pH 8.3), 0.05% β-mercaptoethanol, 192 mM
glycine, and 1 mM EDTA before samples were loaded
into the wells. Following sample loading, the gels were
run at 100 V for about 7 h at 4°C, and then they were
incubated at room temperature in 50 mM Tris-HCl (pH
7.5), 0.05% β-mercaptoethanol, and 4 mM CaCl2 with
slow shaking for 1 h (3 changes of buffer), followed by
a 16-h incubation in the same buffer at room temperature. Gels were stained in a solution containing 0.1%
(wt/vol) Coomassie Brilliant Blue R-250, 45% (vol/vol)
methanol, and 10% (vol/vol) glacial acetic acid for approximately 2 h and subsequently destained overnight
using 10% (vol/vol) methanol and 10% (vol/vol) glacial acetic acid. Calpain activity was indicated by clear
bands in the stained gels.
buffer for 2 h, after which they were rinsed briefly in
2% sodium acetate. A negative stain with 2% uranyl
acetate in water was applied for 1 h in a foil-covered
bottle in a fume hood. Samples were then dehydrated
in ethanol using the following sequence: 50% for 10
min, 75% for 10 min, 95% for 10 min, and 100% (dry)
for 10 min. Ultrastructural changes in myofibrils were
observed and photographed using a transmission electron microscope H-7650 (Hitachi Ltd., Tokyo, Japan)
operated at 80 kV.
Sarcomere length was measured with an Image-Pro
Plus (Media Cybernetics Inc., Silver Spring, MD). For
each specific time, at least 40 sarcomeres were determined to obtain the mean value of sarcomere length.
Statistical Analysis
The data from 4 replicates were analyzed using the
PROC ANOVA procedures of SAS (SAS Institute Inc.,
Cary, NC). Differences among the individual means
were compared by Duncan’s multiple range tests, with
P < 0.05 as the level for significance.
RESULTS
Shear Force and pH Measurements
The tenderness of the chicken breast muscles was
evaluated by the shear force value. As shown in Figure
1, shear force value decreased gradually during postmortem storage and reached a very low level at 48 h,
accounting for 32.3% of the 4-h samples. Furthermore,
no obvious difference (P < 0.05) was observed between
4 and 12 h. Aging from 12 to 24 h postmortem resulted
in a 57.3% decline, which was the largest increase in
tenderness.
Transmission Electron Microscopy
At 0, 6, 12, 24, 48 h postmortem, thin strips of muscle were excised from the breast, and all specimens were
cut into 1 × 1 × 2 mm sections and fixed with 2.5%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at
4°C overnight. Muscle samples were then washed for
several hours in sodium cacodylate buffer (0.1 M, pH
7.4) and then fixed in 2% osmium tetroxide in the same
Figure 1. Peak shear force values of cooked chicken breast muscles
at various times postmortem. Means and SD are shown in the inset.
Different letters indicate significant differences (P < 0.05).
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Li et al.
Figure 2. The pH values of chicken breast muscles at various times
of postmortem storage. Means and SD are shown in the inset. Different
letters indicate significant differences (P < 0.05).
A pH decline of chicken breast muscles during postmortem aging is shown in Figure 2. The pH value decreased sharply through 6 h postmortem and reached
the normal ultimate pH at 6 h. Then, it increased
slightly from 6 to 48 h, but there was no significant difference among 12, 24, and 48 h.
Actin-Myosin Interaction Measurements
Changes in the actin-myosin interaction were measured by determining myofibrillar Mg2+- and Ca2+ATPase activities (Figure 3a,b) and the thermal inactivation of myosin (Figure 3c). Myofibrillar ATPase
activity has been widely used as a measure of actinmyosin interaction (Ouali and Valin, 1981; Roura et
al., 1990). The Mg2+-ATPase activities are particularly
indicative of the integrity of the actin-myosin complex
(Ouali and Valin, 1981; Roura et al., 1990), and any decrease in Mg2+-ATPase can be used as an indicator for
weakening of the actin-myosin interaction. As shown
in Figure 3a, at a high ionic strength (0.6 M KCl), the
activity of myofibrillar Mg2+-ATPase increased significantly (P < 0.05) by 36.5% from 4 to 12 h postmortem, and then gradually it decreased back to the 4-h
level after 48 h of aging. The present results imply that
the initial weak bonding state of actin-myosin became
stronger during the first 12 h postmortem, and it was
then followed by a gradual weakening of the bonding
state. Additionally, the decrease observed in myofibrillar Mg2+-ATPase activity between 12 and 24 h was
31% larger than that between 24 and 48 h, indicating
that the weakening phase mainly took place between 12
and 24 h postmortem.
Meanwhile, Ca2+-ATPase activity, which is a good
indicator of the integrity of the myosin molecule (Watabe et al., 1989), changed in the opposite direction
(Figure 3b). There was an 18% decrease between 4 and
12 h, followed by a large increase up to 24 h and then a
slight increase from 24 to 48 h. This suggests that the
changes in myofibrillar Mg2+-ATPase activity observed
were not because of the changes in myosin postmortem.
Consequently, the reduction in the Mg2+-ATPase activity could be accounted for by the weakening of the
actin-myosin interaction.
Myofibrillar Ca2+-ATPase activities remaining at different times postmortem were determined after subjecting them to incubation at 60°C for either 15 or 30 min
(Figure 3c). As expected, all of the thermal inactivation
profiles of myofibrils exhibited a rapid loss in activity
during the first 15 min of heating, followed by a slower
loss between 15 and 30 min. According to Kawakami
et al. (1971), the former and the latter losses represent
inactivation for free myosin and actomyosin, respectively. Thus it can be determined that the free myosin
contents in the myofibrils extracted from samples at
4, 8, 12, 24, 48 h were 21.0, 11.1, 0, 13.7, and 25.4%,
respectively. It was again demonstrated that the initial
weak state of actin-myosin bonding was strengthened
by 12 h postmortem, and this was followed by a gradual
weakening of the actin-myosin interaction.
Protein Degradation
The 12.5% SDS-PAGE gels clearly showed accumulation of the 30-kDa fragment degraded from troponin-T
during postmortem aging (Figure 4a). It was not until
12 h postmortem that the 30-kDa band could be readily detected. Densitometric analysis showed that at 12,
24, and 48 h postmortem the 30-kDa band intensities
over that of actin within the same lane amounted to
6.2, 11.4, and 17.1%, respectively (Figure 4b).
Desmin degradation in the Western blot analysis is
shown in Figure 5a. It revealed that desmin was degraded extensively during postmortem aging. Moreover, Desmin was degraded rapidly during the first 12
h postmortem, and approximately 80.5% of the intact
desmin disappeared within the first 12 h postmortem
(Figure 5b). The intensity of intact desmin at 24 h, as
well as that at 48 h, was very faint.
Calpain Activity
There is strong evidence that the calpain enzymes,
especially μ-calpain, are responsible for postmortem
proteolysis observed during the early stages of postmortem storage (Koohmaraie and Geesink, 2006; Zhang et
al., 2006; Huff-Lonergan et al., 2010). In the current
study, casein zymography was used to determine the
effect of postmortem storage on the proteolytic activity of μ-calpain and m-calpain in chicken breast muscle
(Figure 6). It showed that in each lane, 2 clear bands
existed, denoting μ-calpain and m-calpain, respectively,
with the former having a higher mobility than the latter. As shown, there was a gradual decline in μ-calpain
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activity after 6 h, whereas an increase in activity of
m-calpain was detected by 48 h postmortem in storage
at 4°C. In addition, compared with μ-calpain, at each
time point, m-calpain had an appreciably lower activity
indicated by the intensity of bands.
Transmission Electron Micrographs
of Myofibrils
Transmission electron micrographs of samples at different times postmortem are shown in Figure 7. Sarcomere length was initially 1.49 μm, and it increased to
1.75 μm at 24 h postmortem. Overall, sarcomere length
tended to increase throughout the 48-h aging period.
With regard to the ultrastructure of myofibrils, there
appeared to be little disruption of the Z-disk and Mline during the whole aging period. It was evident that
the I-bands broadened with aging, which might be responsible for the observed increase of sarcomere length.
During the early hours postmortem, we did not see any
reduction in sarcomere length as report by Wheeler and
Koohmaraie (1994). Perhaps such shortening had already occurred before the 6 h, given that the original
lengths were quite short.
DISCUSSION
Figure 3. a) Myofibrillar Mg2+-ATPase activities at various times
during postmortem storage at 4°C. Different letters indicate a significant difference (P < 0.05). b) Myofibrillar Ca2+-ATPase activities
at various times during postmortem storage at 4°C. Different letters
indicate a significant difference (P < 0.05). c) Changes in thermal
inaction profiles of myofibrils at various times postmortem. Different
letters for the same time point (15 or 30 min, respectively) indicate
significant differences (P < 0.05). Myofibrils prepared from muscles
stored for different amounts of time postmortem were heated at 60°C
for either 15 or 30 min, and then the remaining Ca2+-ATPase activities were measured.
Postmortem aging of carcasses is a complex process
involving many factors. From a physiological standpoint, the development of tenderness is dependent on
the architecture, the integrity of the skeletal muscle cell,
and the activity of endogenous proteases within the
cell and the extracellular matrix (McCormick, 2009).
Although postmortem proteolysis has been extensively
studied, a careful analysis of the information available
on postmortem tenderization suggests that it may have
only a relatively small role given the large changes that
are observed in tenderness during 72 h of postmortem
aging (Goll et al., 1995, 1997; Hopkins and Thompson,
2001, 2002), implying that other factors may be involved. It has been established that there must exist at
least 2 states of actin-myosin interaction; a high- and a
low-affinity state, and depending on the environment,
these states are interchangeable (dos Remedios and
Moens, 1995). On this basis, it has been suggested by
Hopkins and Thompson (2001) that actomyosin has the
potential to exist in different binding states at rigor,
and this may affect the aging of meat and the absolute
level of tenderness ultimately achieved. Furthermore, it
is also possible that some factors may weaken the rigor
state postrigor, which then would contribute to meat
tenderization.
After bleeding, as muscle ATP concentrations fall to
very low levels, skeletal muscles undergo rigor mortis
relatively earlier during the postmortem period, and at
this stage, the myosin heads are bound to actin and form
a strongly bonded rigor complex. Consequently, an increase in meat toughness occurs. In vitro experiments,
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Li et al.
Figure 4. a) Representative electrophoresis patterns of Coomassie-stained 12.5% polyacrylamide gel depicting the appearance of 30-kDa degradation fragments at 0, 6, 12, 24, and 48 h postmortem. Lanes from left to right are molecular standards (M), 0 h (A), 6 h (B), 12 h (C), 24 h
(D), and 48 h (E). Twenty micrograms of proteins was loaded per lane. b) Densitometric analysis of the appearance of the 30-kDa degradation
fragments at 0, 6, 12, 24, and 48 h postmortem. Ratios were calculated as intensity of the 30-kDa fragment in each gel over that of the actin within
the same lane. Data are representative of 4 independent experiments and are presented as means ± SD.
at neutral pH and in the absence of ATP and Ca2+,
have shown that myofibrils can remain in this rigor
state almost indefinitely (assuming microbial action is
inhibited; Goll et al., 1995). However, the environment
in postmortem muscles differs considerably from the in
vitro situation. For instance, pH decline and accumulation of adenosine monophosphate (AMP) and inosine
monophosphate (IMP) are evident during postmortem
storage. Okitani et al. (2008) suggested that AMP and
IMP accumulating in postmortem muscles should be
added to the list of possible factors in the resolution
of rigor mortis induced by the dissociation of actomyosin. Recently, our unpublished work (S. J. Li, X. Xu,
and G. Zhou, unpublished data) had confirmed this
hypothesis, in which it was found that actin liberation
occurred when chicken breast myofibrils were incubated
RESEARCH NOTE
157
Figure 5. a) Changes in intact desmin during postmortem aging. Lanes from left to right are molecular standards (M), 0 h (A), 6 h (B), 12
h (C), 24 h (D), and 48 h (E). Twenty micrograms of proteins was loaded per lane. b) Densitometric analysis of the intact desmin at 0, 6, 12, 24,
and 48 h postmortem. Ratios were calculated as the intensity of the intact desmin over the intensity of desmin at 0 h on the same membrane.
Data are representative of 4 independent experiments and are presented as means ± SD.
with ATP, AMP, and IMP at 60°C for 20 min. Overall,
from the evidence provided here, changes in the actinmyosin interaction are likely to be involved in meat
tenderization. Unfortunately, links between changes in
the actin-myosin interaction and tenderness development during postmortem storage have received little
attention.
In the present study, myofibrillar Ca2+- and Mg2+ATPase activities together with thermal inactivation
of myosin from different times postmortem were examined. A decrease in myofibrillar Mg2+-ATPase activity
and an increase in the amount of free myosin in myofibrils were observed after 12 h postmortem, indicat-
Figure 6. Representative casein zymography gel depicting μ-calpain
and m-calpain activity in sarcoplasmic extracts of the chicken breast
muscles at various times postmortem. Lanes from left to right are 0 h
(A), 6 h (B), 12 h (C), 24 h (D), and 48 h (E). Twenty micrograms of
protein was loaded per lane.
ing a gradual weakening of the previously strong actinmyosin bonded state at the onset of rigor. Similarly, as
summarized by Goll et al. (1997), the Mg2+-ATPase
activity of myofibrils (Goll and Robson, 1967; Ouali
and Valin, 1981; Ouali, 1990) or of actomyosin (Fujimaki et al., 1965; Robson et al., 1967) prepared from
postmortem muscle increased by 20 to 50% during the
first 24 h postmortem and then decreased back to initial levels after 13 d of postmortem aging. Further evidence from our experiments showing lengthening of the
sarcomeres during aging would also suggest weakening
of the actin-myosin interaction. Strengthening of the
actin-myosin interaction during rigor mortis markedly
increases meat toughness and is usually accompanied
by sarcomere shortening. Conversely, weakening of the
actin-myosin interaction may lead to the broadening of
sarcomeres (Takahashi et al., 1981; Taylor et al., 1995).
These 2 findings suggest that after 12 h postmortem,
the strong actin-myosin crossbridges became weakened,
with major changes occurring between 12 and 24 h
postmortem. At the same time, a noticeable increase in
tenderness of the chicken breast was observed. This result agrees with the findings of Takahashi (1996), who
suggested that chicken meat requires 1 d to achieve
maximum tenderness, but this is disputed by several
158
Li et al.
Figure 7. a) Representative transmission electron micrographs of
muscle fibers from chicken breast when stored for 0 h (A), 6 h (B), 12
h (C), 24 h (D), and 48 h (E) postmortem. b) Sarcomere lengths of
chicken breast muscles at various times postmortem. Means and SD
are shown in the inset.
papers (Dransfield, 1994; Alvarado and Sams, 2000a,b)
that reported that the major part of maximum tenderness could be reached only 0.3 d or 2 h after slaughter
in chickens. This dispute may be because of the different handling of chicken in the present study, which
includes immediate deboning of breast muscle after
bleeding and rapid chilling before the completion of
rigor mortis and no electrical stunning and scalding
to defeather. As suggested by Obanor et al. (2005),
early deboning and rapid cooling could toughen the
poultry meat because of the shortening of sarcomere
length and less extensive proteolysis. So these different
processing procedures may contribute to delaying the
postrigor tenderization period in this study. Therefore,
considering the consistency of these 2 events occurring
in our study (weakening of the actin-myosin interaction
and increasing in tenderness of chicken breast), it seems
reasonable that weakening of the actin-myosin interaction is closely related to meat tenderness.
Proteolysis of key myofibrillar proteins has been studied extensively, and it has been concluded that limited
degradation of these proteins contributed to the disruption of myofibrils and the overall structural integrity of
the muscle, resulting in increased meat tenderness. In
our present study, significant degradation of troponin-T
and desmin was observed during postmortem storage.
Because troponin-T is part of the regulatory complex
that mediates the actin-myosin interaction, it is conceivable that its postmortem degradation may lead
to weakening of the strong acitin-myosin crossbridge
bonds. In addition, our results showed that degradation of desmin occurred as early as 6 h postmortem
(perhaps earlier), whereas the 30-kDa fragments of troponin-T appeared after 12 h postmortem. Meanwhile,
it was shown by the casein zymography that calpain
began to exert its proteolysis activity at 6 h postmortem. It has been proved that the troponin-T and the
desmin intermediate filaments are located in thin filaments and around the Z-lines in myofibrils, respectively
(Huff-Lonergan et al., 2010). Considering calpains are
located at or next to the Z-disk, with small amounts
in the I-band and very little in the A-band area (Goll
et al., 2003), the differences in the initial times when
troponin-T and desmin began to degrade, as shown in
the present study, may imply that there exists a timedependent process for calpains to access the substrates
and exert their proteolytic function. Accordingly, it was
hypothesized by Weaver et al. (2008, 2009) that the
extent between thin- and thick-filament overlap may
alter the availability of substrates for degradation by
calpains. Taken together, it is suggested that, as sarcomere lengthening occurs during the postrigor period,
resulting from weakening of the actin-myosin interaction, calpains (particularly μ-calpain) are more readily
able to access the I-band and A-band areas and thus
hydrolyze associated proteins, leading to the disruption
of muscle microstructure.
In summary, the present results suggest that weakening of the strong actin-myosin interaction formed at
rigor may play a large role in meat tenderization during
the early period of storage. Meanwhile, as weakening of
the actin-myosin interaction results in lengthening of
RESEARCH NOTE
the sarcomere, activated calpains are more readily able
to access their substrates and thus hydrolyze associated proteins. These 2 factors are likely to be involved
in the conversion of muscle to meat during postmortem storage. Little attention has been devoted to the
effect of such changes in the actin-myosin interaction
on tenderness development, especially those concerned
with interaction with proteolysis. Therefore, to elucidate what factors actually influence the strength of the
actin-myosin interaction, such as pH and ATP analogs
(AMP and IMP), further experiments are essential.
ACKNOWLEDGMENTS
This research was funded by China Agricultural Research System (CARS-42). The authors thank R. Tume
(CSIRO Food and Nutritional Sciences, QLD, Australia) for his careful revising of this paper.
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