AN ABSTRACT OF THE THESIS OF
Youling Xiong
Food
for the degree of
Master of Science
in
Science and Technology presented on January 14, 1985
Title: Gel Electrophoretic Analysis of the Protein
Changes in Ground Beef Stored at 2 C
Abstract approved:
„
Dr. Allen F. Angleprier
A
study was completed to determine the extent of the
protein
changes
occurring
in
ground
beef
stored
at
20C for 10 days.
Sections
immediately
Each
of
after
section
which
semitendinosus
muscle
the
of three beef animals.
slaughter
were
obtained
was divided into two equal portions, one of
was ground and the other remained intact (control).
All
samples
were
handled
and
stored
under
aseptic
conditions.
Grinding
manifested
during
this
the
markedly
by
samples.
glycolysis
as
the rapid pH decline in the ground samples
initial 24 hours of postmortem storage. After
storage
difference
accelerated
in
interval,
pH
values
however,
between
there
was
little
the ground and intact
Sodium
dodecyl
electrophoresis
changes
in
extracted
3,
6,
visually
sulfate-polyacrylamide
(SDS-PAGE)
was
myofibrillar
employed
and
to
gel
monitor
sarcoplasmic
proteins
from at-death muscles and samples stored for 1,
and
10
and
days
at
2 C. The gels were examined
scanned densitometrically to detect protein
changes.
The
principal
myofibrillar
gradual
and
the
electrophoretic
proteins
of
the
disappearance
of
nebulin
gradual
30,000-dalton
progressive
appearance
polypeptides.
increase
in
55,000
daltons
and
showed
similar
changes
except
that
of
the
the
of
In
samples
in
were the
and desmin components
110,000-,
95,000-, and
addition,
there
was
a
the content of a protein around
myosin light chain-3. Intact muscles
to
those
of the ground samples
the latter had a faster initial rate in some
changes, notably the disappearance of nebulin and
appearance of the 30,000-dalton polypeptide. It seems
probable
Ca
that
from
grinding
the
activated the Ca
ground
muscles.
polypeptide
an
early
reticulum,
release
of
which
-activated proteinase (CAF).
changes
in sarcoplasmic proteins of
samples closely resembled those of the intact
The
included
caused
sarcoplasmic
Electrophoretic
the
ground
changes
major alterations in both muscle treatments
the
and
gradual
three
appearance
of
a
100,000-dalton
proteins having molecular weights
(M.W.)
between
progressive
proteins.
and
from
and
disappearance
The
the
500,000
of
1,000,000
daltons, and the
300,000- and 24,000-dalton
appearance of a 100,000-dalton polypeptide
three
myofibrils
large M.W. proteins presumably originated
since they appeared to be related to the
changes in myofibrillar proteins.
Results
if
of
any,
microbial testing indicated very little,
sample
microorganisms.
Thus,
contamination
microbial
by
psychrotrophic
proteolysis
was
not a
factor in this study.
It
effect
was
on
concluded
the
changes in pH.
that
grinding
had no pronounced
protein changes of beef muscle other than
Gel Electrophoretic Analysis of the Protein Changes in
Ground Beef Stored at 20C
by
Youling Xiong
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed January 14, 1985
Commencement June 1985
APPROVED:
Professor of Food Science & Techrnology
hftolc
in charge of major
Head of department of Food Science & Technology
Dean of Graduate Scho^
Date thesis is presented
Typed by author for
January 14, 1985
Youling Xiong
ACKNOWLEDGEMENTS
I
wish
professor.
to
express my sincere gratitude to my major
Dr.
Allen
F. Anglemier, for all his guidance
and
advice
throughout
this
study and for his help with
the
preparation of this manuscript. His trust and concern
have given me great encouragement and confidence.
I
wish
commitee,
to
thank
Drs.
the
Robert
other members of my graduate
R. Becker, Daniel P. Selivonchick
and Roger G. Petersen.
Thanks
also
go
to
Don Marino and Jan Williams for
their assistance in conducting gel electrophoresis.
Great
for
his
appreciation
help
with
is
the
extended
word
to Visith Chavasit
processor
during
thesis
preparation.
I
Huai-Jen
would
like
Tsai
for
microbiological
to
thank
their
Dr. William E. Sandine and
technical
experiments
and
assistance
providing
in
the
proper
equipment and laboratory space.
I
would
also
like
to
thank Robert L. Dickson for
supplying beef samples for this study.
I
brothers
owe gratitude to my mother and father, sisters and
for
their concern and support during the course
of the thesis work.
Further
and
special
thanks
are
extended
Chinese government who sponsored my scholarship.
to
the
TABLE OF CONTENTS
Page
INTRODUCTION
1
LITERATURE REVIEW
Muscle Structure
Muscle Proteins
Sarcoplasmic Proteins
Myofibrillar Proteins
Stroma Proteins
Postmortem Changes
Postmortem Glycolysis
Postmortem Protein Degradation
Proteolytic Enzymes and Their Actions
Lysosomal Proteinases
Neutral and Alkaline Proteinases
The Ca -activated Proteinase
Microbial Proteinases
Glycolysis and Proteolysis in Ground Muscle
5
5
7
9
10
16
17
17
18
21
22
24
25
28
29
MATERIALS AND METHODS
Muscle Sample Preparation
Measurement of pH
Preparation of Myofibrillar and Sarcoplasmic
Proteins
Sodium Dodecyl Sulfate-Polyacrylamide
Gel Electrophoresis
Interpretation of Gels
Densitometric Tracings of Gels
Psychrotrophic Bacteria Inspection
Statistical Analysis
33
33
34
34
35
38
38
39
39
RESULTS AND DISCUSSION
Postmortem Muscle pH Changes
pH Changes in Ground and Intact Muscles
pH Change in Animal No.3 Muscle
Postmortem Muscle Protein Changes
Myofibrillar Proteins
Gel Electrophoresis
Densitometric Tracings
Causal Factors
Sarcoplasmic Proteins
Psychrotrophic Bacteria Inspection
41
41
41
47
51
51
51
58
61
69
77
CONCLUSIONS
79
BIBLIOGRAPHY
81
LIST OF FIGURES
Figure
Page
Structure of skeletal muscle at several
different levels of organization ranging
from an entire muscle (top of fig.) to
the molecular architecture of myofilaments
(bottom of fig.). (From Anthony and
Thibodeau, 1983)
6
Effect of grinding and postmortem storage
at 2 C on pH change in the bovine
semitendinosus muscles (from three
animals). (A) within 24 hours; (B) within
10 days
43
Effect of grinding and postmortem storage
at 2 C on pH change in the bovine
semitendinosus muscle (from animal No.3).
(A) within 24 hours; (B) within 10 days
49
SDS-polyacrylamide gel electrophoresis of
myofibrils isolated from ground(A) and
intact(B) semitendinosus of bovine muscle
stored at 20C. Gels 0, 1, 3, 6, and 10
represent myofibrils isolated from the
muscle after 0, 1, 3, 6, and 10 days of
postmortem storage. Gel S is the protein
standards (note: number X 1000 = molecular
weight). Arrows a, b, c, d, e, f, g, and
h indicate the major changes
52
Densitometer tracings of electrophoretograms
of myofibrils isolated from ground(A) and
intact(B) bovine semitendinosus muscle
stored at 2 C for 0, 1, 3, 6, and 10 days
postmortem. Letters a, b, c, d, e, f, g,
and h correspond to the changes indicated
in electrophoretograms
59
Figure
Page
SDS-polyacrylamide gel electrophoresis
of sarcoplasmic proteins extracted
from ground(A) and intact(B) bovine
semitendinosus muscle stored at 2 C.
Gels 0, 1, 3, 6, and 10 represent
sarcoplasmic proteins extracted from
the muscle after 0, 1, 3, 6, and 10 days
of postmortem storage. Gel S is the
protein standards (number X 1000 =
molecular weight). Arrows a, b, c, d, e,
f, g, and h show the major changes
70
Densitometer tracings of electrophoretograms
of sarcoplasmic proteins extracted from
ground(A) and intact(B) bovine semitendinosus
muscle stored at 2 C for 0, 1, 3, 6, and
10 days postmortem. Letters a, b, c, d, e,
ff g, and h correspond to the changes
indicated in electrophoretograms
72
LIST OF TABLES
Table
Page
Major skeletal muscle proteins and their
locations
8
Preparation of purified myofibrillar and
sarcoplasmic proteins using differential
centrifugation (modified Goll's procedure)
36
Postmortem pH changes in ground and intact
semitendinosus of bovine muscles stored
at 20C
42
Postmortem pH changes in ground and intact
animal No. 3 semitendinosus muscle stored
at 20C
48
GEL ELECTROPHORETIC ANALYSIS OF THE PROTEIN CHANGES IN
GROUND BEEF STORED AT 20C
INTRODUCTION
Ground
product.
per
beef
and
popular,
widely
consumed
meat
the demand is increasing yearly (Raccach
Henrickson,
consumption
person
a
Current consumption of this item is about 18.2kg
capita
and
is
is
in
1978).
highly
the
This
level
significant
of
because
ground
beef
the average
United States consumes approximately 36kg
of beef per year.
Ground
superior
beef
to
that
palatability
and
and
juiciness
found
to
term
prepared
from hot-boned beef muscle is
prepared
from
chilled
muscle
in
cooking properties including tenderness
(Cross et al., 1979). However, it has been
develop
storage.
characteristic off-flavors during long
This
defect may be associated with muscle
protein degradation postmortem.
It
is
myofibrillar
endogenous
connective
storage
1983).
have
generally accepted that both sarcoplasmic and
proteins
proteolytic
tissue
are
are
quite
vulnerable
to
muscle
enzymes whereas proteins from the
very
stable
during
postmortem
or aging (Lawrie, 1974; Penny, 1980; Goll et al.,
Although
been
made,
exhaustive
studies
on
muscle proteins
it remains controversial at the present
time
which proteins undergo proteolysis and which enzymes
are
the
causal
agents
for the changes occurring during
the postmortem storage of meat.
Sarcoplasmic
one-third
They
of
are
proteins
total
the
of
muscle
contain
up
to
muscle proteins (Goll et al., 1970).
most
labile
of
the
muscle
proteins.
Postmortem
alterations of sarcoplasmic proteins have been
documented
(Neelin
1969;
et
Hay
Some
of
and
Ecobichon, 1966; Parrish et al.,
al., 1973b; Harrington and Henahan, 1982).
the
proteolytic
changes
are
caused
by
acid
cathepsins (Drabikowski et al., 1977).
The
50%
of
myofibrillar
degradation
limited
but
not
muscle.
The
major
disruption
1980)
and
30,000
et
et
(Dayton
extensive
is
proteolysis
alterations
evidenced
in
the
by
intact
occurring postmortem are
(Olson et al., 1977; Penny, 1980; Parrish
1981).
changes
These
within
changes, along with several other
the
myofibrils, are thought to be
for
the
increased
(MacBride
and
Parrish, 1977; Penny, 1980; Parrish
al.,
(CAF)
myofibrils
the gradual appearance of a new polypeptide of
responsible
aging
of
of the Z-disks (Goll et al., 1970; Penny,
daltons
al.,
minor
fraction constitutes over
the total proteins in muscle (Goll et al., 1970).
Postmortem
the
protein
1981).
Although
Ca
meat
tenderness
-activated
during
proteinase
seems
to
be a strong participant in these changes
et
al.,
1975; Olson et al., 1977; Penny, 1980),
evidence
has
cathepsins
Robbins
been
are
et
1979;
In
alkaline
have
been
that
the
lysosomal
important (Schwarts and Bird, 1977;
1981).
and
proteins
also
al.,
Etherington,
neutral
accumulated
Matsukura
addition,
proteinases
et
high
al.,
1981;
activities
against
of
myofibrillar
reported (Murakami and Uchida, 1979;
Goll et al.,1983).
While
muscle
postmortem
have
knowledge
about
the
the
become
the
grinding
stimulates
glycolysis
changes
in
bovine
in
ground
beef.
can rupture the cell wall and
It
reticulum,
which
catheptic
protein
myofibrils.
sarcoplasmic
Ca
alterations
been studied extensively, there is a lack of
Ostensibly,
damage
protein
may
also
resulting
muscle
in
disrupt
a
the
release
of
ATP-ase to accelerate
(Hamm, 1977) , and rupture lysosomes to release
enzymes. In addition, proteins of ground muscle
more
susceptible
to
proteolytic bacteria due to
more exposed surface area and destruction of the cell
wall
(Tarrant et al., 1971; Dainty et al., 1975; Yada and
Skura, 1981) .
Although
Harrington
sarcoplasmic
beef,
it
influence
ground
protein
is
the
not
temperatures.
Henahan
changes
known
proteins
at-death
and
and
of
in
frozen-stored
whether
the
(1982) found some
the
bovine
subsequently
ground
grinding
would
muscle which is
stored
at
low
4
The
investigate
(made
sodium
current
study
protein
at-death)
electrophoresis.
changes
during
dodecyl
was
therefore
occurring
in
initiated
ground
to
beef
postmortem storage at 2 C by
sulfate-polyacrylamide
Postmortem
glycolysis
contamination were also monitored.
and
gel
microbial
LITERATURE REVIEW
Muscle Structure
The
fiber
mucscle.
Fibers
is
the
are
essential
long,
structural
unit
of
narrow, multinucleated cells
stretching
from a few to as long as 34 cm with a diameter
of
urn
10-100
(Walls,
parallel
fashion
make
a
by
up
a
to
1960).
Fibers
are
arranged
in
form bundles, and groups of bundles
muscle. Each of the above units is surrounded
specific
endomysium
sheath of connective tissue: fiber by the
through
sarcolemma,
fiber
bundle
by
the
perimysium, and finally muscle by epimysium (Figure 1).
Myofibrils
muscle
dark
the
basic
structural
unit
of the
fiber. They are composed of light bands (I-bands),
bands
myofibrils
1).
are
The
(A-bands)
into
light
and transverse Z-disks that divide
regular
units called sarcomeres (Figure
band consists of thin filaments while the
dark
band contains thick filaments each surrounded by six
thin
filaments
microscope
1972) .
of
The
which
can
be
cross-sections
seen through the electron
of the myofibrils (Huxley,
interdigitating thick and thin filament array
has
enormous implications for all phases of meat science,
and
a
strong
discovery
of
case
can
be
made
for the proposal that
this structure is the single most important
finding ever made in meat science (Goll et al., 1977).
Muscle
Eplmyiium
Perimyiium
Endomyiium
/
Myofibrll
Sorcoplasmic
rrtirulum and
T lubules
forming triad
■
(bond
1
A bond
L/lrH'onen
Aclin
•
• •
•*
Myoiin and oclin
•I: in
Ad
/
Figure 1.
Structure of skeletal muscle at several different levels of
organization ranging from an entire muscle (top of fig.) to
the mDlecular architecture of rryofilaments (bottom of fig.).
(From Anthony and Thibodeau, 1983)
7
As
indicated
cross-striated
T-tubules
in
and
and
Figure
contain
sarcolemma.
mitochondria,
lysosomes,
granules
also
are
1
muscle
cells
sarcoplasmic
are
reticulum,
Other cell organelles such as
nuclei,
ribosomes and glycogen
present in muscle cells (Goll et al.,
1974) .
Muscle Proteins
In
found
general,
in
proteins
which
are
bind
them
which
muscle
proteins
refer to all proteins
muscle cells (Goll et al., 1977). This includes
capable
of
rapid
surrounded
into
supply
by
place,
the
contraction
relaxation
a connective tissue network to
and
muscle
and
a complex system of enzymes
elements with energy (Bendall,
1964).
Muscle
which
proteins
are
soluble
(sarcoplasmic
concentrated
those
in
water
proteins),
salt
be
broadly divided into those
or
those
diluted salt solutions
which
are
soluble
in
solutions (myofibrillar proteins), and
which are insoluble (stroma proteins or proteins of
connective
1974;
can
Goll
abundance
Table 1.
tissue
et
and
al.,
other formed structures)
1977).
Distribution
and
(Lawrie,
relative
of these skeletal muscle proteins are listed in
Table 1.
Major skeletal muscle proteins and their
locations
protein
location
I. myofibrillar
( 60 )
myosin
50
actin
20
connectin(titin)
5.5
tropomyosin
5
II
III
troponin T,I & C
nebulin
M-protein
C-protein
a-actinin
g-actinin
desmin
5
4. 5
4
2
2
1
0. 35
others
0. 65
sarcoplasmic
( 30 )
glyceraldehyde phos.
dehydrogenase 22
aldolase
11
enolase
9
creatine kinase
9
lactate dehydro/
genase
pyruvate kinase
5. 5
phosphorylase b
4..5
1
myoglobin
extracellular
8
others
23
stroma
(
collagen
elastin
reticulin
mitochondrial
other particulate
10 )
47
2.,5
25
25
thick filaments
thin filaments
gap filaments
thin filaments and
Z-disks
thin filaments
N2-lines
M-lines
thick filaments
Z-disks
thin filaments
intermediate filaments (Z-disk)
sarcoplasm
outside muscle cell
sarcoplasm
connective tissue,
sarcoplasmic reticula, sarcolemma.etc
mitochondria
Sources are from Scopes (1970); Lawrie (1974); Dayton
et al. (1975); Wang and Ramirez-Mitchell (1983); and
Stanley (1983) .
'Percentage is approximation. Data in parenthesis
represent the percent of the total muscle proteins;
data without parenthesis indicate the percentage of
each component within the group.
Sarcoplasmic Proteins
The
in
sarcoplasmic
cytoplasm
strength
of
of
the
0.1
or
sarcoplasmic
contain
less
protein
Exact
protein
and
at
are
soluble at ionic
neutral
fraction
has
pH.
been
The
entire
estimated
to
protein
composition
of
the
sarcoplasmic
fraction is influenced considerably by conditions
used
during
and
extent
extraction,
solvent,
soluble
extraction
of
pH
and
of
the
extract
the
tissue
before
force
used
to
separate
the
protein fraction from unsolubilized
different
sarcoplasmic
(Scopes,
sucrose
of
subcellular organelles (Goll et al., 1970).
of
iso-osmotic
may vary depending on speed
extraction, nature of extraction
centrifugal
and
number
and
homogenization
sarcoplasmic
proteins
water
muscle
are those proteins found
at least 100-200 different proteins (Goll et al.f
1977).
A
proteins
proteins, including the use of pure
1964;
Harrington
glycerol
solution
procedures have been employed to
(Hay
and Henahan, 1982), 3%
solution
(Scopes,
et
1973a,
al.,
1966),
b),
0.25M
and
0.03M
potassium
phosphate (Goll et al., 1964) as the extracting
solvents.
However, under most extraction conditions used,
the
sarcoplasmic
enzymes
protein
associated
with
fraction
glycolysis
contains
and
most
all
the
of
the
enzymes
involved
with carbohydrate and protein synthesis
because
these processes occur largely in the cytoplasm of
10
muscle
at
cells. These enzymes, in addition to being soluble
low
when
ionic
the
strength, are free and readily solubilized
muscle
sarcoplasmic
enzymes.
listed
the
in
extract
several
Table
sarcoplasmic
1
consists
hundred
make
protein
sarcoplasmic
of
glycolytic
enzymes thought to be
because
sarcoplasmic
lysosomes
(Reville
and
more
fractions
proteins
color
up
fraction
protein
homogenization
meat
ruptured. At least 80% of the
the sarcoplasm, the six most abundant enzymes
in
enzymes
is
protein
Of
present
cell
(Scopes,
contain
are
et
than one-half of the
some
labile
al.,
1970).
Most
lysosomal
to
muscle
1976). Other important
are myoglobin which contributes to
the
calcium
activated
factor (CAF), a
proteinase which attacks myofibrils.
Although
the
to
texture
the
sarcoplasmic
of
muscle, they do contribute significantly
development
Moreover,
nutritive
proteins contribute little to
of
sarcoplasmic
the
characteristic meat flavor.
proteins
are
of
significant
value since they account for 30% or more of the
total protein in skeletal muscle (Goll et al., 1970).
Myofibrillar Proteins
Myofibrillar
those
muscle
ionic
strength
proteins
proteins
(u
<
that
0.2),
are
are
frequently
defined
as
not extractable at low
but are solubilized at high
11
ionic
strength
myofibrils
strength
must
is
completely
required
interactions
disrupted,
to
destroy
among
However,
high
ionic
the intimate
protein
molecules
some of the recently
myofibrillar proteins can be extracted at very
ionic
strength
of
(u
<
0.01)
upon
disruption
or
the muscle tissue. Once extracted, most of
myofibrillar
from
(Goll et al.f 1970). Since the
myofilaments.
maceration
the
be
and
the
discovered
low
> 0.4)
usually
associations
within
(u
proteins are soluble at ionic strengths
0 to 1.5 (Goll et al., 1970). Therefore, it has been
suggested
proteins
that
that
myofibrillar proteins be defined as those
constitute
the
myofibrils
(Goll et al.,
1970; Goll et al., 1977).
The
structure
extensively.
which
Unlike
contains
of
including
several
thin
an
only
the
regulate
(tropomyosin,
assist
in
organized
has
sarcoplasmic
about
are
of
in_ vitro
of
the
myofibril
a
dozen
discovered
actin
filaments
Most
to
and
the
been studied
protein
fraction
many different proteins, the myofibril is
composed
Myosin
of
the
different
proteins
in recent years (Table 1).
main components of thick and
the myofibril. Both are necessary for
contraction response (Goll et al., 1977).
other myofibrillar proteins function either
or
control
troponin,
assembly
of
the
and
actin-myosin
possibly
interaction
a-actinin)
or to
actin and myosin into the highly
three-dimensional
structure
in
myofibrils
12
(C-protein,
a-actinin,
paramyosin)
(Goll
include
several
nebulin,
and
myofibrils
et
al.,
newly
desmin)
in
8-actinin,
the
1977).
The
discovered
which
form
of
M-protein,
and
latter may also
proteins
have
been
fine
filaments
(titin,
observed in the
(Wang
and
Ramirez-Mitchell, 1983).
Myosin
is
constituting
fraction.
and
insoluble
ability
both
about
It
heavy
the
major
protein
found in myofibrils,
50% of the total myofibrillar protein
is composed of two types of subunits, light
meromyosins.
and
has
while
no
heavy
The
light
meromyosin
is water
ATP-ase activity or actin-binding
meromyosin is water soluble and has
ATP-ase activity and actin-binding ability (Lowey et
al., 1969) .
Actin
is
the
protein
accounting
weight.
It
exist
in
(fibrillar).
which
form
the
second
for
contains
two
G-actin
globular
some
only
forms,
most
abundant
20%
of
the
myofibrillar
myofibril by
one polypeptide chain and can
G-actin
will
(globular)
polymerize
units
are
into
and
F-actin
F-actin
in
aggregated end-to-end to
a double-stranded filament (Murray and Weber, 1974).
F-actin
combines
actomyosin
of
with
myosin
active
or
to
form
pre-rigor
the contractile
muscle
and
the
inextensible actomyosin of muscle in rigor mortis.
Other
small
contractile proteins are present in relatively
quantity.
Tropomyosin
molecules
are
aggregated
13
end-to-end
near
filaments.
In vertebrate muscle tropomyosin is capable of
contributing
whereas
the
groove of the double-stranded actin
mechanical stability to the muscle filaments
paramyosin
invertebrate
which is tropomyosin found in certain
muscles
is
capable
of tetanic contraction
(Poglazov, 1966) .
Troponin
is
another regulatory protein. Greaser and
Gergely
(1971)
molecule
contains
intimately
subunit
binds
demonstrated
three
concerned
that
dissimilar
in
the
subunits
troponin
which are
muscle contraction. The largest
is called troponin T (T for tropomyosin) since it
strongly
troponin
I
inhibit
the
(I
preventing
for
first
to
tropomyosin.
The
for
inhibition),
binds to actin and can
ATP-ase
the
activity
actin-myosin
of
smaller
subunit,
actomyosin,
interaction.
thus
Troponin C (C
calcium) is the smallest subunit which binds calcium.
Troponin
C
troponin
I
important
to
associated
form
with
troponin
implication
relaxation
in
both
complex,
muscle
troponin
which
T
and
has very
contraction
and
(Margossian and Cohen, 1973). Cohen (1975) has
illustrated
among
is
and
the
described
three
in
troponin
detail
subunits
the
and
interactions
with
other
contractile components.
a-Actinin
a-Actinin
Masaki
is
and
located
6-actinin
in
are
structural proteins.
the Z-disks (Goll et al., 1967;
et al., 1967). It promotes the lateral association
14
of
F-actin.
appears
B-Actinin
to
maintain
inhibit
the
is
found
network
physical
in
thin filaments. It
formation
state
of
F-actin
of I-filaments
and
in
vivo
the
muscle
(Maruyama, 1965).
The
three-dimensional
myofibril
(line).
the
is
also
However,
structure
of
maintained by C-protein and M-protein
the
significance
of their presence to
integrity of the myofibril remains obscure. C-protein
exists
in
filament.
like
bands
It
staves
together
completely
encircle
the
thick
has been suggested that these rings may act
around
a
barrel to hold the thick filament
during tension development (Offer et al.f 1973).
M-protein
nearest
which
appears
to
neighbours
link myosin filaments to their six
in order to keep the myosin filaments
centrally aligned in the sarcomere.
Recently,
myofibrillar
titin
and
high
myofibrillar
Initially,
suggest
that
daltons)
of
major
1981a).
a
been
skeletal
discovered
1981).
Titin
molecular
weight
(>
titin
et
have
large
(Wang,
proteins
Maruyama
al.,
extremely
proteins
nebulin
uncommonly
by
two
was
al.
muscle
and
is
a
700,000
named
pair of
daltons)
(Wang and Ramirez-Mitchell, 1979).
confused with connectin discovered
(1977a, b). However, recent studies
high molecular weight doublet (> 800,000
connectin is identical to titin (Maruyama et
It has been suggested that titin may be the
cytoskeletal
protein
constituting
a
set of very
15
thin,
longitudinal
filaments,
1979,
in
running elastic filaments, called gap
the
1983).
sarcomere (Wang and Ramirez-Mitchell,
The gap filaments are parallel to the fiber
axis and provide intracellular tensile strength.
Nebulin
daltons.
It
that
a
is
(Wang,
Np-line
may
steers
a
is
the
molecular
major
transverse
region
"
has
1981) .
weight
of
about 500,000
component of the N_-line
structure
located
in the I-band
Wang (1981) has suggested that the
be involved in a dynamic mechanism which
" actin filaments to a more favorable alignment
to optimize interaction with myosin thick filaments.
In
the
filament
that
promote
of
a
Z-disk
area
functions
there
exists
another type of
to connect adjacent Z-disks and
lateral registration. These filamens are composed
smaller contractile protein called desmin (Stanley,
1983) .
It
interact
has
with
been
gap
proposed
that
these filaments may
filaments in the region of the Z-disk
to
form a three-dimensional network that holds myofibrils
in
place and thus provides an ordering of the contractile
mechanism
(Stanley,
has
documented
been
1983).
through
The
presence of the network
transmission
and scanning
electron
microscopic studies by Wang and Ramirez-Mitchell
(1983).
In view of above findings, it is clear that these
newly
discovered
thin
filaments have a very significant
implication to meat toughness.
It
is
possible that other yet unidentified proteins
16
may
also
exist
within the contractile apparatus and may
be discovered with advances in analytical techniques.
Stroma Proteins
The
stroma
proteins
insoluble
proteins
of
salt-soluble
all
lipoproteins
surfaces
and
(e.g.
mitichondrial
constitute
alone
epimysial,
capacity
to
include
cell
membranes and
sarcoplasmic
reticular and
perimysial
and
released
into
(Brostrom
et
introcellular
they
and
endomysial
in
to
reduce
al.,
1971;
proteinases
1976).
the
toughness. Besides, due to the
the
sarcoplasmic
sarcolemmal membranes, Ca
cytoplasm
al.,
tend
of meat (Goll et al., 1977). They
meat
alterations
mitochondrial
et
from
They
proteins have a significant impact on
because
contribute
Reville
proteins.
surrounding the muscle cell. Collagen
Stroma
quality
postmortem
muscle
up about half of the stroma protein fraction
water-holding
also
as the
after exaustive extraction
sarcolemmal,
tissues
1).
meat
remaining
measured
membranes) in addition to the proteins that
makes
(Table
usually
mucoproteins
the
connective
are
to
Hamm,
accelerate
1977)
(Dayton
Thus,
et
membrane
far-reaching effects on meat quality.
or
reticular,
may be
glycolysis
activate the
al.,
1976a,
proteins
b;
have
17
Postmortem Changes
Postmortem Glycolysis
Postmortem
of
muscle.
The
glycolysis
same
glycolysis
sequence
as
in
temporarily
energy
of
chemical
steps
by
which
is converted to lactic acid is essentially the
postmortem
becomes
reflects the basic metabolism
in
the
vivo
inadequate
muscle;
when the oxygen supply
for
the
provision
of
but it proceeds further (Lawrie,
1974) .
Glycolysis
animal
is
6.9
conversion
phosphate,
The
the
the
supply
glycogen
final
pH
is
ATP turnover. When the
no
longer
produced via
Consequently, at the point of
to ATP at the expense of creatine
when the latter is exausted the ATP level
of
ATP
triggers
the
anaerobic
glycogen
to
lactic acid. This conversion
until
pH
is reached where the enzymes
a
breakdown
production
of
determines
ADP
depletion
continue
affecting
Since
of
of
with
- 7.2) the muscle ATP level is maintained
but
conversion
will
ATP
phosphorylation.
death • (pH
falls.
coupled
slaughtered
oxidative
by
is
of lactic acid is dependent on the
glycogen,
the
content
because
the
muscle
is
are inactivated (Lawrie, 1974).
level
ultimate
pH.
of
this
But
the
component
initial
not the sole factor determining the
approximately 10% of the total drop in
18
pH
postmortem
hydrolysis
is
of
due
ATP
to
protons
liberated
by
the
present in the tissue at death (Hamm,
1977) .
In
pH
is
typical
about
extent
of
when
postmortem
pH
animals,
administration
the
of
temperature
muscle
postmortem
muscle
temperature
et
5.5. However, both the rate and the
between
environmental
faster
-
fall
are
influenced
by
factors such as species, the type of muscle and
variability
as
5.4
the
intrinsic
such
postmortem mammalian muscle the ultimate
and
drugs
by
extrinsic factors
pre-slaughter and the
(Lawrie, 1974). For example, a
pH
decline
has been observed
is subjected to increased environmental
(Marsh,
1954) , electric stimulation (Sonaiya
al., 1982), pressurization (Elgasim et al., 1983), and
grinding (Hamm, 1977).
Postmortem Protein Degradation
Proteolysis
muscle.
in
and
is
an
important
postmortem
change in
Postmortem protein breakdown affects meat quality
various
aspects, including tenderness, functionality,
the characteristic flavor and color of meat. Although
numerous
studies
controversial
at
have
the
undergo
proteolysis
factors
for
the
been
conducted,
it
remains
present time which muscle proteins
and
which
enzymes
are
the causal
changes occurring during the meat aging
19
process.
Generally,
few
proteins
myofibrils
and
extensive
degradation
temperatures
have
storage
muscle
during
are
the
tissue,
subjected to
aging
period
at
above the freezing point. Many early studies
measurements
(Locker,
al.,
to
absent
even
of
1960;
1969).
resistant
is
of
connective
shown that no large changes were detected by various
chemical
et
sarcoplasm
of
proteolysis
during postmortem
Davey and Gilbert, 1966; Parrish
Connective tissue proteins are generally
proteolytic
enzymes in muscle. Proteolysis
in collagen and elastin of fresh sterile meat,
storage of 1 year at 370C (Lawrie, 1974).
after
Clearly,
very
little
degradation
of
collagen
occurs
postmortem (Goll et al., 1970; Etherington, 1981).
Sarcoplasmic
in
proteins
may disintegrate and dissolve
a very concentrated solution
most
labile
degradation
postmortem
of
of
the
iri
vivo . They are the
muscle proteins. Slight proteolytic
sarcoplasmic
proteins
may
occur during
storage. Sharp (1963) demonstrated that during
storage
at
soluble
protein nitrogen declined from 28% of the initial
nitrogen
of
to
37 C
Parrish
acids
et
sarcoplasmic
sterile
beef
muscle the total
13% after 20 days. The lowered concentration
sarcoplasmic
amino
of
and
proteins was due to their proteolysis to
not
al.
protein
to
(1969)
precipitation
have
also
(Lawrie, 1974).
shown
that
the
fraction yields an increase in free
20
amino
acids and non-protein nitrogen in postmortem bovine
muscle.
Although
difference
and
al.
(1970)
muscle
of
the
shown
a
significant
proteins
in
al.,
1973b)
have
sarcoplasmic
1966;
et
found little
in the sarcoplasmic proteins between pre-rigor
post-rigor
workers
Whitaker
Hay
et
rabbit
and
pig,
other
degradation
of
chicken (Neelin and Ecobichon,
and
bovine
(Harrington and
Henahan, 1982) muscles during postmortem storage.
Because
of
contraction
they
are
changes
and
observed
have
largely
by
1983;
in
been
electrophoresis
Olson
et
postmortem
storage
(Hay
et
1977;
al.,
al.,
are
1973b; Penny,
Bechtel and Parrish,
1984).
occurring
in
The
proteolytic
myofibrils
appreciable.
alterations
in
gel
muscle
during
The
major
observed
during
a slow loss of the M-line, broadening and loss
fibrillar
seen
between
Goll
et
structure
in the Z-disks, and the fracture
Z-disk
and
I-band (Fukazawa et al., 1969;
1970;
Hay
et
al.,
Paralleling
consistent
sulfate-polyacrylamide
al.,
et
changes
of
postmortem muscle, the myofibrillar
dodecyl
Koohmaraie
are
muscle
the subject of many careful studies,
degradative
aging
in
it has long been suspected that
(SDS-PAGE)
ultrastructural
importance
responsible for many of the physical
sodium
1980;
obvious
because
primarily
proteins
1974,
their
the
and
breakdown
noticeable
al.,
of
1973a; Penny, 1980).
troponin
change
that
T,
the
occurs
most
in
21
myofibrils
during
appearance
of
migrating
30,000
three
storage
closely
is
spaced
the
gradual
protein
bands
at rates corresponding to approximately 34,000,
and
Parrish,
et
postmortem
27,000
1977;
al.,
daltons
Olson
1981).
30,000-dalton
troponin
in
SDS-PAGE
(MacBride
and
et al., 1977; Penny, 1980; Parrish
It
seems
component
highly
arises
possible
from
that
the
the degradation of
T (Olson et al., 1977; Penny, 1980; Goll et al.,
1983).
Recently,
newly
discovered
nebulin,
are
Lusby
et
al.
(1983) reported that two
myofibrillar
gradually
degraded
proteins,
during
titin
and
the postmortem
storage of bovine muscle.
Nevertheless,
pronounced
proteins,
aging
no evidence thus far has indicated any
postmortem
myosin
change
in
the
major contractile
and actin, especially during the normal
period. Most other contractile components also seem
unaltered during the aging process.
Proteolytic Enzymes and Their Actions
Numerous
evidence
protein
the
of
studies
have
proteolytic
degradation
been made in attempt to find
activity responsible for muscle
during
postmortem storage. However,
involvement of the exact enzymes remains enigmatic. A
number
of
endogenous
from
skeletal
proteinases
muscles.
These
have
been discovered
include
lysosomal
22
proteinases
(cathepsins),
proteinases,
and
Several
Ca
excellent
available
neutral
-activated
reviews
and
alkaline
proteinase
about
these
(CAP).
enzymes
are
(Etherington, 1981; Goll et al., 1983; Dahlmann
et al., 1984).
Lysosomal Proteinases
Lysosomal
cathepsins.
in
the
of
muscle
largely
of
of the cathepsins have optimum activity
cells.
H,
Of
the
thirteen
reported
lysosomal
L
and lysosomal carboxypeptidase B, have been
to exist inside skeletal muscle cells (Goll et al.,
1983) .
Among
cathepsins
of
consist
hydrolases, however, only seven, cathepsins A, B,
D,
shown
Most
enzymes
acidic pH range and are associated with lysosomes
peptide
C,
proteolytic
these
enzymes
only
the
endopeptidases,
B, D, H and L are the most important. All four
these
enzymes
possess
activity
against
myosin and
actin
(Goll
et al., 1983). Schwartz and Bird (1977) have
shown
that,
at
major
fragment
degrades
107,000
at
of 150,000 daltons. At pH 4.0 cathepsin D
myosin
cathepsin
B,
pH 5.2, cathepsin B degrades myosin into
more
forming
daltons
and
rapidly
and more extensively than
two major fragments of 110,000 and
several smaller fragments. However,
pH values above 6.0, neither cathepsin B nor D has any
appreciable
effect
on
myosin (Schwartz and Bird, 1977).
23
These
researchers
degrade
the
thin
et
Tropomyosin, the other major component of
filament,
al.,
that
D
actin.
also found that both cathepsin B and D
1984).
is degraded by cathepsin B (Dahlmann
Further,
Robbins et al.
(1979) reported
the Z-disks were substantially degraded by cathepsin
at pH 5.2 to 5.3. Cathepsin L resembles cathepsin B, so
its
in
activity may not have been separated from cathepsin B
many
of
Cathepsin
chains
al.,
the
L
of
degrades
myosin
1981).
myosin.
and
earlier studies (Okitani et al., 1980).
actin
very
et
H
al.
both
quickly
Cathepsin
Goll
and
also
heavy
and light
at pH 5.0 (Matsukura et
has
a high affinity for
(1983) suggested that cathepsins L
H each have far greater myosin-degrading ability than
cathepsins
B
and D combined. Dahlmann et al.
indicated
that
cathepsins
(1984) have
B, D and L degrade troponin T
and troponin I in vitro.
Some
of the lysosomal enzymes possess collagenolytic
activity,
1981)
particularly
and
cathepsins
B, L, N (Etherington,
G (Starkey, 1977). But neither cathepsin G nor
cathepsin N has been detected in skeletal muscle.
At
the
present
activity
of
proteins
although
proteins
can
et
the
time,
catheptic
there
was
data
are
lacking about the
enzymes against sarcoplasmic
a report that sarcoplasmic
be degraded by acid cathepsins (Drabikowski
al., 1977). It is possible that this activity could be
present during the postmortem storage of muscle.
24
Neutral and Alkaline Proteinases
Enzymes
the
in
this
group have proteolytic activity in
neutral and alkaline pH range. Many of them have been
detected
in
isolated
neutral or alkaline proteinases are localized in
mast
skeletal
muscle
tissue.
Eight of the nine
cells rather than in skeletal muscle cells, and some
are
yet of unknown origin (Goll et al., 1983). Initially,
identification
recent
have
of
these
enzymes
immunohistochemical
been
resolved.
proteinase,
alkaline
myofibrillar
proteinase
all
tests
Thus,
alkaline
was confusing but with
the
it
some
seems
of
the problems
likely
myofibrillar
that
the
proteinase,
the
proteinase and the myosin-cleaving
originate
from
the same enzyme (Goll et
al.,
1983). Libby and Goldberg (1980) have suggested that
most,
if not all, proteolytic activity having an alkaline
pH
mast
optimum
in
crude
cells
in
the original tissue and not from striated
muscle cells
The
for
specificity
myosin,
rupture
se .
neutral
important
example,
per
muscle homogenates originates from
and
muscle
against
neutral
alkaline
proteinases
may
be
protein turnover because they have
certain myofibrillar components. For
serine
proteinase
can
cleave
actin,
a-actinin, tropomyosin, troponin T and I, and can
the
myosin-cleaving
Z-disks
(Goll
proteinase
et
is
al.,
capable
1983)
of
while
degrading
25
C-protein,
myosin,
tropomyosin
However,
could
it
act
proteins
muscle
and
Z-disks
is
on
M-protein,
and
T,
I,
Uchida,
and
C,
1979).
difficult to conceive that these enzymes
directly
proteinases
(Murakami
myofibrils
cells.
troponin
and other intracellular muscle
since
The
they
role
and
are
the
not
located
inside
significance of these
to muscle postmortem proteolysis need further
investigation.
The Ca
The
was
presence
purified
optimum
structure
by
Ca
-activated protinase (CAF)
usually
muscle
by
Dayton
electron
et
al
microscopy.
(1976a).
It was later
Although
CAF
has
activity between pH 7.0 and 7.5 (Penny, 1980), it
proteinases
the
of
discovered by Busch (1972) during the study of rabbit
muscle
is
-activated proteinase
not
group because it is localized inside skeletal
cells
at
cytoplasmic
Schollmeyer,
classified into the neutral and alkaline
the level of the Z-disk and adjacent to
face
1981).
characteristic,
the
of the plasma membrane (Dayton and
Since
it
properties
has
a unique proteolytic
of
CAF has been studied
extensively in the past several years.
CAF
et
al.
effect
has a very unusual and limited specificity. Goll
(1983)
have
indicated
that CAF has very little
on the sarcoplasmic protein fraction from skeletal
26
muscle
cells
these
but it degrades the contractile proteins in
cells
in
Ultrastructurally,
from
and
myofibrils
degrades
ability
of
proteins
1975) .
but
selectively
limited
removes
way.
the Z-disks
(Busch et al., 1972; Olson et al., 1977)
the
CAF
This
M-line
(Dayton
et
al.,
1976b). The
to break down certain other myofibrillar
includes
the
degradation
of
tropomyosin,
T, troponin I and C-protein, but myosin, actin,
tx-actinin
and
troponin
Pemrick
et
myosin
heavy
al.
evidenced
C
(1980)
chain
unphosphorylated.
al.
CAF
specific
has been thoroughly investigated (Dayton et al.,
troponin
et
a
have
if
That
(Maruyama
were
not
However,
found that CAF may degrade
the
CAF
affected.
LC_
light
chain
is
can degrade nebulin was also
et al., 1981b). More recently, Zeece
(1983) reported that CAF can break down the newly
discovered myofibrillar protein, titin.
There
low-Ca++
CAF
is
free
CAF
(Goll
forms
et
al.,
much
completely
of CAF, high-Ca
CAF and
et al., 1983). The high-Ca++
active at pH 7.0 to 8.0 as long as the
concentrations
requires
range
1976b).
lower
between
The low-Ca
Ca
1
CAF, however,
concentration.
active at 70 uM of Ca
to 5 mM
It
is
and is active even
10 uM (Dayton et al., 1981). The latter suggested that
low-Ca
of
two
maximally
Ca
(Dayton
at
are
pH
CAF
values
is active over a slightly broader range
than
high-Ca
CAF and it may retain
27
significant
proteolytic
activity
at pH values as low as
5.5.
Since
high-Ca
concentration
of
intracellular
free
uM,
Goll
low-Ca
and
et
it
muscle
be
the
1
to
5
Ca
requires
mM for optimum activity while
level rarely rises above 10
al.
(1983)
suggested
may
be
responsible
degradation
cells.
of
for
that
only
the
most of the postmortem
myofibrillar proteins inside
They also suggested that high-Ca
be a storage form of the enzyme
converted
to
regulation
assumed
plasma
and
Ca
form of CAF is active in living muscle cells
proteolytic
may
CAF
the
of
in
vivo
more active low-Ca
CAF
that high-Ca
inhibitor.
CAF
which can
CAF under
Alternatively, it is
CAF is possibly located at the
membrane or in the connective tissue matrix (Barth
Elce,
millimolar
Low-Ca
1981) ,
where
levels of Ca
CAF
it
could
be
activated
by
that exist extracellularly.
may be localized at the Z-disk (Goll et
al., 1983).
Indeed,
degradation
quality
are
the
leading
changes
necessary
achieved.
precise
to
meat
remains
before
role
a
of CAF to muscle protein
tenderness
and other meat
unknown.
Clearly, more studies
thorough
understanding
can be
28
Microbial Proteinases
Certain
elaborate
digest
bacteria,
extracellular
muscle
Pseudomonas
chill
pH
growing
proteolytic
on
meat,
can
that
may
enzymes
proteins. If microorganisms are involved,
bacteria
normally
predominate
on
meat at
temperatures, and these organisms are unaffected by
in
the
Newton,
range
enzymes;
It
is
now
species
are
able
P^
fragi
al., 1971),
P.
that normally occurs in meat (Gill and
1982).
Pseudomonas
et
when
P^
fluorescens
recognized
to
excrete
that
some
proteolytic
(Borton et al., 1970a, b; Tarrant
perolens
(Buckley et al., 1974) and
(Gill and Penney, 1977) are examples of
these microorganisms.
Observations
bacterial
proteolysis
researchers
Shelef,
(Jay,
to
have
1967;
the
been
importance
of
contradictory.
Ockerman
Some
et al., 1969; Jay and
1976) believe that bacterial proteolysis plays an
insignificant
role
others
(Borton
Dainty
et
et
al.,
significant
that
storage
of
myofibrillar
in
muscle
al.,
1975;
protein
reported
observed
pertinent
1970a,
Yada
P^
at
proteins
breakdown
while
b; Tarrant et al. 1971;
and Skura, 1981) have shown
degradation.
when
pork
protein
fragi
2-10 C
occurred.
Borton
et al. (1970a)
was present during the
some
proteolysis
Tarrant
et
of
al. (1971)
considerable salt-soluble protein degradation in
29
pork
muscle by
fragi
was
P^
also
fragi
shown
in 20 days at 10 C.
to
alter
P.
sarcoplasmic proteins
extensively (Hasegawa et al., 1970).
Gill
and
penetrate
happen
Penney (1977) have found that bacteria may
meat
upon
proteolysis
although
it
will not
until the bacteria have reached their maximum cell
density.
The
effect
proteins
depends
generally
meat
proteins
a
approximately
which
the
critical
muscle
extent of contamination. It is
no
place
stage
of
significant
until
of
degradation of
spoilage
has
become
spoilage. Yada and Skura
sarcoplasmic,
proteins
SDS-PAGE.
number
proteolysis
on
P^
fragi
and found that a count of
10 10 /cm 2 is the minimum level at
tissue
by
proteinases
protein breakdown can be detected only
breakdown
connective
detectable
that
advanced
studied
bacterial
the
takes
Clearly,
very
(1981)
on
accepted
evident.
at
of
of
It
bacteria
by
has
may
P^
been
be
myofibrillar and
fragi
became
suggested that a
required
before
can be detected (Dainty et al., 1975; Jay and
Shelef, 1976; Yada and Skura, 1981).
Glycolysis and Proteolysis in Ground Muscle
Grinding
muscle,
i.e.,
causes
muscle
two large structural changes of the
surface increase and muscle tissue
30
damage.
The
enhanced
other
more
exposed
substrate
hand
surface
availability
muscle
area
results
in an
for the enzymes. On the
tissue particles can be ruptured when
there has been extensive tissue damage.
In
than
ground
muscle
metabolism
is faster
in intact muscle. It has been observed that grinding
pre-rigor
bovine
turnover
manifested
of
postmortem
ATP
and
(Newbold
glycogen
Scopes,
the
rate
of
ATP
a faster depletion in the levels
an
1971;
accelerated
Hamm,
1977).
drop
of
pH
Hamm
(1977)
the acceleration of postmortem metabolism
by a faster depletion in the levels of ATP and
and
an
accelerated
drop
of
pH
(Newbold and
1971; Hamm, 1977). Hamm (1977) suggested that the
acceleration
of
results
from
muscle
cells,
activates
content
that
increases
and
Scopes,
that
manifested
by
glycogen
and
suggested
muscle
postmortem
damage
causing
myosin
triggers
the
to
a
of
sarcoplasmic
release
of
to grinding
reticulum of
Ca
activity.
lactic
(Hamm,
phosphorylase
due
The
which
lowered
ATP
the activation of glycolytic enzymes so
phosphofructokinase
acid
and pH decline are
1977).
(PFK)
and,
Two
to
a
enzymes,
lesser
extent,
(PP) play a dominant role in the control of
metabolic
levels
(Dalrymple
and
there
no
was
the
ATP-ase
formation
accelerated
metabolism
of
Hamm,
glycolysis
1975).
Hamm
in
ground
muscle
(1977) reported that
major difference between intact and ground
31
tissue
except
changes
such
destruction
influence
as
the
of
difference
of
metabolism.
rate
a
glycolysis.
the
pattern
Recently,
glycolysis
muscle
temperature
support
to
ground
the
muscle
in
the
He
concluded
the
can
not
by
but
the rate of postmortem
only
can
be
falls
observation
grinding
that
membranes
Etherington
because
rate of postmortem
(1981) mentioned that
markedly increased when the
below
12 C.
of
faster
This
pH
lends
decrease in
it cools more rapidly than intact
muscle.
There
is very little information about the effect of
grinding
Several
and
on
chemical
thawing,
have
been
al.,
1964).
and
protein
degradation
postmortem.
and physical factors, e.g. pH, freezing
thermal activation and detergent treatment,
implicated
in lysosomal disruption (Sawant et
Indeed, lysosomal membranes are very fragile
can be ruptured during electrical stimulation (Dutson
et
al.,
of
muscle.
similar
a
muscle
low
It
and pressurization (Elgasim et al., 1983)
is
highly likely that grinding may exert
effects, especially when muscle pH has dropped to
value (Lawrie, 1974). It is possible that a faster
muscle
proteolysis
release
This
1980)
of
of
take
place
due
to the early
catheptic enzymes from lysosomes by grinding.
possible
release
could
Ca
acceleration
fa
sarcoplasmic reticulum.
may be triggered also by the
co-factor
of
CAP,
from the
32
During
structurally
proteolytic
that
ground
the
grinding
and
enzymes.
muscle
may
process myofibrils are damaged
become
While
has
an
there
more
has
accelerated
vulnerable
to
been no evidence
proteolysis of
myofibrils,
Harrington and Henahan (1982) have found some
differences
between
ground
beef
and intact beef in the
postmortem degradation of sarcoplasmic proteins.
33
MATERIALS AND METHODS
Muscle Sample Preparation
Semitendinosus
were
448
obtained
-
497
according
Science
kg.
from
equal
80%
and
food
had
was
was
subjected
to
The
cutting board, was sanitized
knife
was
flame
sterilized.
dried rubber gloves were always used
and
in
Muscle
was
ground
in
a
machine (Model 308) after the
free
minced.
paper
of
The
external
ground
toweling
fat
and
muscle was
soaked
in 0.1M
and then wrapped in plastic film and enclosed in
plastic
recommendations
bags
and
trimmed
wrapped
section
was held intact. All equipment,
preparation
tissues
immediately
fraction
preparation.
been
muscle
and was cut immediately into
One
grinder
sample
two-pound
other
alcohol.
connective
Ziploc
good quality heifers weighing
carcass
the
the
Moulinex
a
fractions.
Alcohol-treated
NaN-.
this study
Oregon State University. At about 25
each
and
including
during
throughout
The animals were slaughtered and dressed
postmortem,
grinding
muscle
three
Laboratory,
removed
with
from
used
to normal slaughter practices at the Clark Meat
minutes
two
muscles
were
approximately
storage
bag
according
to
the
of Bechtel and Parrish (1983) . The sample
stored
30
at
grams
2 C.
Each
of
ground
sample
muscle.
contained
The
intact
34
muscle
(3
X
7 X 15 cm) was treated similarly and stored
at 20C.
Measurement of pH
Measurements
procedure
muscle
1:10
of
adjusted
inner
a
5mM
were
(1982)
Waring
made
after
according
to
the
homogenization
of
blender for 30 seconds in a
iodoacetate
solution which had been
pH 7.0. Samples were taken from the cores or
parts
samples
in
of
to
surfaces.
pH
Petersen
sample
ratio
of
of
the wrapped meat trimmed free of exposed
The pH measurements of ground and intact muscle
were
obtained
simultaneously at the hours of 1,
2,
4, 8, 12, 16, 20 and 24, and the days of 0 (at death),
1,
3,
6
Corning
glass
4.0
and
Digital
postmortem.
pH
electrodes.
and
7.0
temperature
were
10
measurement.
The
buffers
at
rinsed
Meter
The instrument used was a
(Model 125) fitted with Orion
meter was standardized against pH
and
was
calibrated
for
ambient
periodic
intervals. The glass electrodes
extensively
with distilled water after each
Each muscle sample was triplicately weighed,
homogenized and measured for pH.
Preparation of Myofibrillar and Sarcoplasmic Proteins
Four
grams
of
ground
or
intact muscle were taken
35
from
the
2 C.
Protein
ground
core
and
of
the
wrapped
extractions
intact
muscle sample stored at
were
prepared
from
both
at-death muscles and muscles after 1,
3, 6 and 10 days of postmortem storage.
Myofibrils
were
procedure
described
using
extracting
(pH
et
the
an
al.
(1972)
by
the
et al. (1974) at 2-40C
Goll
buffer
(Table
substitutions
vigorous
to
XI
of lOOmM KCl, 50mM Tris-HCl
in
0.1M
in
modifications
a
Table
homogenization
of
of
2). Other modifications made were
glass
stirring
in
myofibrils
loss
by
essentially
7.6), ImM NaN3 and 5mM EDTA as suggested by Busch
during
VII
isolated
in
to
2),
and
a
Waring
steps
VII
The
suspension
blender
of
was
centrifuged
according
to
Scopes
purified
XIV).
The
to XI and XIV minimized the
from
the
first
at 35,000 X g for 45 minutes
(1964).
used
(step
supernatant
extraction
was
suspend the pellets (steps
KCl by vigorous stirring rather than
myofibrils.
centrifugation
rod for polyethylene rod
as
Supernatant
the
from
this
source of sarcoplasmic
protein (Table 2).
Protein
concentration
was
measured
by
the biuret
method (Gornall et al., 1949).
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
Extracts
of
myofibrillar
and sarcoplasmic proteins
36
Table 2.
Preparation of purified nyofibrillar and sarcoplasnrLc
proteins using differential centrifugation
(modified Goll's procedure)
Supernatant
l) pass through
cheese cloth
2) centrifuge at
35,000 x £ for
1)5 minutes
Supernatant
(saxcoplasmic
proteins)
Ground or minced muecle (1) grans)
l)Suspend in 10 volumes (v/w) 100 mM KC1,
50 mM tris-HCl, pH 7.6, 5 mM EDTA,
1 mM NaNo (standard salt solution) by homogenizing
for 10 sec in Waring Blender.
2)Centrifi'.ge at 1000 x £ for 10 min.
Sediment
(discard)
II. Sediment
l)Suspend In 6 volumes (v/w) of standard salt
solution by homogeniiing for 10 sec in Waring
blender.
2)Centrifuge at 1000 x £ for 10 min.
1
III. Sediment
Supernatant
(discard)
l)Suspend in 8 volumes (v/v) of standard salt
solution by homogenizing for 10 sec in Waring
blender.
2)PasB suspension through household nylon net strainer.
3)Centrlfuge at 1000 x g for 10 min.
1
IV. Sediment
Supernatant
(discard)
l)Su8pend in 8 volumes (v/w) of standard Bait
solution by homogenirlng for 10 sec In Wiring
blender.
2)PaBS suspension through household nylon net strainer.
3)Centrlfuge at 1000 x £ for 10 nin.
V . Sediment
Supernatant
(discard)
l)Su6pend In 6 volumes (v/w) of standard salt solution
plus l£ (v/w) Triton X-100 by homogenlilng for 10
sec In Waring Blender.
2)Centrifuge at 1500 x £ for 10 min.
VI. Sediment
Supernatant
(discard)
l)Suspend In 6 volumes (v/w) of stondard salt solutio
plus 1$ (v/w) Triton X-100 by homogenlilng for 10
sec In Waring blender.
2)Centrlfuge at IJOO x g for 10 min.
"I
VII. Sediment
Supernatant
(discard)
1(Suspend In 8 volumes (v/w) of standerfl salt solution
by stirring vigorously with glass rod.
2)Centrifuge at 1500 x g for 10 Bin.
VIII to XI. Sediment
Supernatant
(discard)
l)Repeat Step VII'four tines, tut suspending in S
volumes (v/w) of 100 mM KC1 Instead of standard
salt solutions; centrifuge at 1500 x g after each
suspension.
1
XII. Sediment
Supernatant
(discard)
IJSuspend In 8 volumes (v/w) of 100 mM KC1 by
homogenizing for 3 oer in Waring blender.
2)Centrlfuge at 1500 x £ for 10 min.
XIII. Sediment
Supernatant
(discard)
l)Sucpend in B volumes (v/w) of 100 eU KC1 by
homogenizing for 3 Bee In VJarlng blender.
2)Centriruge at 1500 x g for 10 min.
XIV. Sediment
Supernatant
(discard)
1)Suspend in 2 voliunes (v/w) of 100 mM KC1 by stlrrinf
vigarously with glass rod.
2)0o protein analysis of cuspcnslon.
Purified Myofibrils
free of membranes
37
were
analyzed
by
sulfate-polyacrylamide
procedure
described
the
gel
by
sodium
dodecyl
electrophoresis
(SDS-PAGE)
Porzio
and Pearson (1977) using
10%
acrylamide tube gels (5 X 130 mm). The major steps of
the
procedure
solutions
except
followed
APS,
included:
TEMED
the
pipet;
lOmg/ml)
and
3
gel
(4)
and APS (2%) at room temperature
(2) adding TEMED and
solution
mixing
into
(3) immediately
gel
tubes
with a
myofibril suspensions (about
sarcoplasmic protein extract (about 3mg/ml)
contained
8M
2-mercaptoethanol,
urea,
and
2.5%
lOOmM
SDS,
Tris
5mM
EDTA,
5mM
/ glycine (pH 8.8));
solubilizing proteins in sample buffer by boiling for
min
min
and then centrifuging in a Beckman Microfuge for 1
to
materials
precipitate
possible
(supernatant
was
analysis);
onto
the
gels
by
acetic
gel
(6)
gel
loading
acid
traces
in
for
Blue
with
3
undissolved
30ug
of
protein of each sample
(7) fixing the
a solution of 25% isopropanol / 10%
hours
(R250)
10%
of
utilized for electrophoretic
and runnig electrophoresis;
soaking
Coomassie
the
composite gel
and 1 volumes of sample buffer respectively (sample
buffer
3
all
mixing and degassing for 1 more min;
Pasteur
(5)
mixing
by degassing for 10-15 min;
transferring
in
(1)
for
acetic
and
then
staining
in 0.01%
12 hours; and (8) destaining
acid
/
5% methanol till gel
background became clear.
The
marker
proteins
used were the standard mixture
38
of
known
molecular weight proteins obtained from Bio-Rad
Laboratory
(Richmond,
proteins:
bovine
myosin,
CA)
containing
3-galactosidase,
the
following
phosphorylase
B,
serum albumin, carbonic anhydrase, soybean trypsin
inhibitor and lysozyme.
The
cell
electrophoresis
(Bio-Rad,
Model
was
run in gel electrophoresis
155) with a constant voltage power
supply (Buchler, Model 3-1500).
Interpretation of Gels
Myofibrillar
the
procedure
components were identified according to
of
Porzio
and
Pearson
(1977).
Protein
molecular
weight (M.W.) was estimated against the protein
standards
whose logarithm M.W. were plotted against their
migrating rates in the gels.
Densitometric Tracings of Gels
Gels
were
scanned
spectrophotometer
Beckman
cell
shape
Gel
of
the
580nm
in
a
Beckman
(Model ACTA CIII) in conjunction with a
Scanner.
containing
at
The
gel
destaining
peak
was laid in the scanning
solution.
depended
width of the corresponding band.
The
size and the
upon the intensity and the
39
Psychrotrophic Bacteria Inspection
Microbial
day)
and
Samples
at
in
the
were
2 C.
knife
growth
blender
subjected
measured
(10
at the beginning (0
days) of postmortem storage.
from cores of wrapped meat stored
core sample was mixed with a sterilized
then
for
sterilized
end
obtained
The
and
was
blended in a sterile, pre-chilled Waring
exactly
peptone
to
2
minutes using 10 volumes of 0.1%
solution.
appropriate
The
muscle suspension was
dilutions
with
0.1%
peptone
done
according
solution prior to inoculation.
The
incubation
Hartley
et
Methods
Caseinate
media
al.
procedure
(1970)
Agar
and
was
Lee
(SMCA)
(1976)
medium.
to
using Standard
The innoculated
were incubated at 7 C for 10 days, which allows
the
detection
of
proteolytic
organisms
in
total
psychrotrophic bacterial counts.
Statistical Analysis
Data
were
described
in
pH
between
group
of
(ground
were
by
analyzed
paired).
to
the
methods
Snedecor and Cochran (1982). The difference
ground muscles and intact muscles (from a
animals)
muscle
according
and
was
analyzed
intact
muscle
using
paired
t-test
from the same animal
In an individual animal (animal No.3) this
40
difference
samples.
was
interpreted
by
the
mean
test
of
two
41
RESULTS AND DISCUSSION
Postmortem Muscle pH Changes
pH Changes in Ground and Intact Muscles
Table
intact
pH
3
shows
bovine
each
sampling
changes in pH of the ground and
muscles during the postmortem storage. The
measurements
from
the
of both ground and intact muscle samples
animal
were
period.
The
determined in triplicate at each
triplicate
averaged.
The
averages
postmortem
from
all
statistically
1982) .
using
This
grinding
on
obtained
three
animals
measurements
at
the
were
same
were
time
then analyzed
paired t-test (Snedecor and Cochran,
allowed
the
bovine
muscle
detection
pH
of
the
effect
of
(Table 3). The data were
depicted graphically in Figure 2.
It
is
clearly shown that a significantly (P < 0.05)
faster
pH
intact
muscle
within
in
agreement
and
Scopes,
This
is
(Newbold
decline
the
suggests
rate
1977).
fall occurred in the ground muscle than in the
of
that
the
with
1971;
the
initial
24 hours postmortem.
other workers' observations
Hamm,
1977).
The rapid pH
grinding process accelerates
glycolysis (Dalrymple and Hamm, 1975; Hamm,
Hamm (1977) has hypothesized that the enhanced ATP
turnover observed in the ground muscle is due to a faster
42
Table 3. Postmortem pH changes in ground and intact
setnitendinosus of bovine muscles stored at 2 C
Time after
slaughter
Ground
,
Intact
Difference betw.
(mean 1" S.D. ) (mean "t S.D.) ground & intact0
1
6.50 + 0.04
6.65 + 0.05
P
<
0.05
2
6.17 + 0.03
6.55 + 0.06
P
<
0.025
4
5.97 + 0.07
6.45 + 0.13
P
<
0.025
8
5.87 + 0.03
6.27 4- 0.11
P
<
0.025
12
5.72 + 0.04
6.00 + 0.06
P
<
0.01
16
5.69 + 0.06
5.88 + 0.08
P
<
0.025
20
5.64 + 0.08
5.76 + 0.08
P
<
0.005
24
5.61 + 0.08
5.69 + 0.07
P
<
0.01
1
5.61 + 0.08
5.69 + 0.07
P
<
0.01
3
5.55 + 0.04
5.50 + 0.09
N .S.
6
5.59 + 0.05
5.56 + 0.12
N .S.
10
5.45 + 0.03
5.50 + 0.10
N .S.
hours
days
mean = average of the mean values from three animals
S.D. = standard deviation.
P = probability level from the paired t-test;
N.S. = non-significant (P > 0.05).
43
7.0
0
6.6--
•
o INTACT
• GROUND
6.2PH
5.8"
5.4--
X
+
+
8
12
16
HOURS POSTMORTEM
•
3 4
DAYS POSTMORTEM
Figure 2.
+
20
24
INTACT
• GROUND
10
Effect of grinding and postmDrtem storage at 20C on pH
change in the bovine seraLtendinosus muscles (from three
animals). (A) within 24 hours; (B) within 10 days.
44
release
of
reticulum.
it
is
Ca
ions from the damaged sarcoplasmic
Since
glycolysis is coupled with ATP turnover
possible
faster
that this faster release of Ca
depletion
of
or
ATP triggered glycogen breakdown to
enhance
the formation of lactic acid. Mitochondria may be
another
possible
(1982)
have
provided
pressurization
from
source of Ca
of
. Elgasim and Kennick
evidence
that
pre-rigor
muscle causes the release of Ca
the damaged mitochondria. It is likely that grinding
would
have
a
pressurization
similar
both
effect
seem
since
grinding
and
to have the ability to rupture
the cell organelles.
Postmortem
The
cold-induced
ground
muscle
former
released
a
faster
ground
muscle
because the
accelerated
the
glycolysis.
(1981) has reported that when the temperature
pre-rigor
contraction.
Honikel
in
intact
can be expected to occur in the
which
is
C
the
breakdown
glycolysis
initial
in
could start earlier in the
heat more rapidly than the latter. Thus,
muscle,
the
contraction
than
ATP
Etherington
of
glycolysis is also temperature sensitive.
pH
drop
contrast
muscle
markedly
et
in
drops
to
hastened
al.
(1981)
below
due
12 C
to
muscle
observed a drastic
the pre-rigor muscles stored at 0.5
to
those
stored
at
higher
temperatures.
Another
possible
cause
for the rapid pH fall could
45
arise
from
the
glycolytic
rupture
increased substrate availability for the
enzymes.
the
Grinding
glycogen
could
granules
in
possibly
damage or
the muscle cells and
make them more vulnerable to the enzymes.
The
difference
in the initial pH values between the
ground
and
intact
(Table
3).
However,
(P
>
(A)
0.05)
shows
first
2
rate
of
was
significant
(P < 0.05)
this difference was not significant
after 3 days of postmortem storage. Figure 2
that
the
hours
pH
dropped
postmortem
this
because
muscles
the
relatively
high
maintained
at
the ground muscle but the
change
decreased
initial
muscle
so
a
concentrations
in
that
high
such
as
thereafter,
probably
temperature
the
level.
high
drastically within the
enzyme
remained
activity
Elevated
was
substrate
levels of glycogen and ADP
would certainly also be the contributors to this change.
The
patterns
treatments
ground
were
beef
decreased
muscle
first
the
pH
2
pH
the
(concave
pH
changes between the two muscle
slightly different (Figure 2, A). In the
muscle
rate
the
of
pH
declined
shaped)
at
while
a
in
gradually
the
intact
value dropped the most rapidly within the
hours postmortem, and then reduced slowly during
period of 2 to 8 hours postmortem. But after that the
drop
hours
was
of
explanation
accelerated again and was retarded after 12
storage
for
(convex
this
shaped).
phenomenon
could
One
be
possible
that
the
46
conditions
such as the availability of the substrates and
co-enzymes
favored
ground
enzyme
activity
in
the
beef
but
not
in
the
intact
muscle were tightly formed, but
muscle
became
acidic
granules
when
glycolytic
the
granules
in intact beef. Initially glycogen
the
structure of these
could be loosened and disordered, which enhanced
substrate availability.
After
12
treatments
resulted
continued
and
the pH changes in both
inactivation of phosphofructokinase and
(Hamm,
after
statistical
was
storage
from the limiting content of glycogen, adenosine
phosphorylase
0.05)
of
leveled off because of the retarded glycolysis
nucleotides,
between
hours
24
1977).
hours
analysis
the
two
The
of
in
postmortem
indicates
muscles
change
that
the
pH
values
storage,
but
pH difference
was no longer significant (P >
after 3 days of storage (Table 3). This observation
consistent
Similarly,
muscles
to
that
reported
by
Hamm
(1977).
during prolonged storage the pH values of both
fluctuated
essentially
there
slightly
was
no
(Figure
significant
2,
B),
but
difference
(P >
0.05) between them (Table 3).
Nevertheless,
observation
because
fall
may
one
not
should
represent
be
aware
that
this
all the individual cases
both the rate and the extent of the postmortem pH
can
variability
be
influenced by intrinsic factors such as the
between
animals,
and
by
extrinsic factors
47
such
as
exausting
(Lawrie,
1974).
postmortem
pH
exercise
immediately
Therefore,
it
is
pre-slaughter
possible
that
the
alteration in a specific animal could vary
from this pH change pattern.
pH Change in Animal No.3 Muscle
Because
in
pH
optimum
animal
rate
muscle
could
endogenous
for
activity
proteolytic enzymes vary
and
because each specific
have a unique postmortem pH decline in both
and extent, it is necessary to monitor the pH change
while
the
Figure
muscle
muscle
3
were
thesis.
made
(animal
proteolysis
proteins
from
No.3)
pH
degrading.
data
that
presented
Each
are
in
obtained
was
the
used
following
Table 4 and
from the same
for
determining
section of the
mean value was obtained from triplicate
measurements.
Basically,
muscles
the
of
of animals (Figure 2) although there were some
variations
postmortem
significantly
muscle.
pH changes in both ground and intact
of animal No.3 followed the pattern obtained from
group
specific
the
But
insignificant
in
storage,
lower
at
(P
3
(P
days
>
the former. During the first day
pH
of
the
ground
muscle
was
< 0.005) than that of the intact
postmortem
0.05),
the
difference was
both having a pH around 5.55
(Table 4). With prolonged storage at 2 C, however.
48
Table 4. Postmortem pH changes in ground and intact
animal No.3 semitendinosus muscle stored at 2 C
Time after
slaughter
Ground ,
Intact
Difference betw.
(mean 1" S.D.)(mean "t S.D.) ground & intact0
1
6.47 + 0.02
6.66 + 0.02
P < 0.001
2
6.19 + 0.01
6.54 + 0.02
P < 0.001
4
6.10 + 0.02
6.49 + 0.03
P < 0.001
8
5.91 + 0.03
6.30 + 0.01
P
<
0.001
12
5.75 + 0.02
6.00 + 0.02
P
<
0.001
16
5.88 + 0.01
5.81 + 0.01
P < 0.001
20
5.72 + 0.02
5.68 + 0.02
24
5.64 + 0.02
5.72 + 0.01
P < 0.005
1
5.64 + 0.02
5.72 + 0.01
P < 0.005
3
5.57 + 0.01
5.54 + 0.02
IU.S.
6
5.59 + 0.01
5.63 + 0.01
P
10
5.48 + 0.03
5.54 + 0.02
P < 0.05
hours
days
P < 0.001
<
0.01
mean = mean of the triplicate measurements.
S.D. = standard deviation.
P = probability level from two sample mean test;
N.S. = non-significant (P>0.05).
49
7-0
* INTACT
6.6 ■-
•
GROUND
6.2-pH
5-8--
54-
X
+
o
8
12
16
HOURS POSTMORTEM
20
24
70
■* INTACT
«
» GROUND
PH
54-
T.
Figure 3.
I
+
+
+
34
56789
DAYS POSTMORTEM
10
Effect of grinding and postmortem storage at 20C on pH
change in the bovine semitendinosus muscle (from animal
No.3). (A) within 24 hours; (B) within 10 days.
50
these
pH
two
of
the
muscle samples, again, varied in pH value. The
the intact muscle was significantly higher than in
ground
0.05)
muscle
postmortem (Table 4). The ground muscle exhibited a
further,
(PH
significant
5.48)
in
determined
at
period
day
of
increment
value
(pH
fell
5.54).
cells
may
much
comparison
3
to
<
0.01)
back
to
day
with
the
pH
value
(5.57)
6
there was a significant pH
in the intact muscle, but this pH
the same level of 3 days postmortem
glycogen
not
or some residual sugars inside the
have been completely metabolized within a
postmortem.
prolonged
reduction (P < 0.02) at 10 days
The reason is not clear but it seems possible
muscle
days
pH
3 days postmortem. In contrast, during the
(P
that
few
at 6 days (P < 0.01) and 10 days (P <
period
slower
of
rate
Although glycolysis may last for a
time postmortem, it will occur at a
in
the
than
it
did
The
may
be
concurrent
event in which some small peptides
and
amino
acids,
nucleotides were decarboxylated by
certain
enzymes
observed
that
lactic
or
pH
stages
initially.
a
increased
later
value in the intact muscle
present in the muscle cells. Hamm (1977)
glycogen
breakdown
and
the formation of
acid continued in ground and intact bovine muscles
even after 3 days of postmortem storage at 4 C.
51
Postmortem Muscle Protein Changes
Postmortem
determined
by
changes
sodium
in
beef
muscle
proteins
were
dodecyl sulfate-polyacrylamide gel
electrophoresis
(SDS-PAGE)
muscle
from each of three beef animals stored at
20C
samples
for
animals
of
both
ground
and
intact
10 days. Since the SDS-PAGE patterns between
were
protein
very similar per sampling period per muscle
category,
presented.
These
only
data
from
animal
No.3
are
data are highly representative of those
obtained during this investigation.
Myofibrillar Proteins
Gel Electrophoresis
Figure
myofibrils
samples
4
shows
isolated
stored
protein
variations.
occurred
from
the
ground
and intact muscle
at 2 C for 10 days. Comparison of the
electrophoretograms
similar
the results of SDS-PAGE analysis of
indicated
change
Essentially,
that
patterns
the
both
treatments had
except
following
for
major
a
few
changes
in both muscle treatments: a gradual decrease in
the
intensity
a),
desmin
of
a ~500,000-dalton protein band (arrow
(arrow e) and troponin T (arrow f); a gradual
increase in the content of myosin light chain-3
52
Figure 4. SDS-polyacrylamide gel electrophoresis of myofibrils
isolated from ground (A) and intact (B) semLtendinosus
of bovine muscle stored at 2 C. Gels 0, 1, 3, 6, and
10 represent myofibrils isolated from the muscle after
0, 1, 3, 6, and 10 days of postmortem storage. Gel S
is the protein standards (note: number X 1000 =
molecular weight). Arrows a, b, c, d, e, f, g, and h
indicate the major changes.
53
ill-"
200
116
93
66
45
31
21
14
0
1
3
6 10
DAYS
Figure 4
0
1
3
6 10
S
54
(MLC3)
(arrow h) and two other polypeptides of about
110,000
and
gradual
differed
(arrow
from
(arrow
storage,
was
protein
and
b
95,000-
and
and
d); and the
30,000-dalton
g). However, the ground muscle
the
gound muscle the '-SOO,000-dalton
became
faint
after 1 day of postmortem
slightly detectable after 3 days and almost
after
6
days of storage (Figure 4, A). This
is probably nebulin which was resolved from other
myofibrillar
identified
band
of
(arrow
intact muscle in the rates of some of
In
a)
disappeared
c
the
changes.
band
daltons
appearance
components
these
55,000
components
by
arose
(arrow
Lusby
polypeptide
may
(the
contrast,
muscle
(1981). At 10 days postmortem, a new
slightly
a).
doublet
Wang
by Porzio and Pearson (1977) and
beneath
et al.
be
two
the
the
original nebulin band
(1983) have suggested that this
breakdown
product of the titin
bands above nebulin) or of nebulin. In
nebulin degraded at a slower rate in the intact
(arrow
a
in
Figure
4,
B).
Nevertheless,
intensity
and
the
the
gradually
decreased
broadening
of the band ostensibly indicate the postmortem
degradation
treatments
who
of
nebulin.
support
demonstrated
significantly
in
the
that
bovine
The
changes
findings
both
muscle
by Lusby et al.
nebulin
muscle
in
progressively
can
after
be
(1983)
degraded
1 day postmortem
storage at 2 C.
The
gradual appearance of a 95,000-dalton component,
55
which
of
was
another obvious change with postmortem storage
muscle,
this
protein
treatments
in
the
the
appeared
the gels of both muscle
in
the
intensity between the two muscle
seem to parallel the changes in nebulin. These
also seem to correlate with the subtle alteration
a-actinin
be
(the dark band between bands b and c) which
detected
Koohmaraie
et
appearance
of
muscle
in the electrophoretograms of Figure 4.
al.
the
(1984)
during
However,
workers
concluded
a-actinin.
protein
reported
the
in the intact
postmortem storage. They suggested
could
other
initially
95,000-dalton
that a -actinin
have
in
sample. The formation of this protein and
difference
bovine
seen in Figure 4 (arrow c). Although
band
ground
changes
can
be
at 3 days postmortem, it appeared more intense
treatments
in
can
be
that
the possible source of origin.
(Olson et al. 1977; Penny, 1980)
postmortem
Obviously,
no
aging has no effect on
conclusive argument can be made
about this change without further evidence.
The
most
obvious
progressive
appearance
(arrow
It
this
muscle
g).
polypeptide
at
detectable
days
muscle
can
be
change
of
a
shown
in Figure 4 is the
30,000-dalton
polypeptide
seen by careful examination that
appeared
as a faint band in the ground
1 day postmortem (Figure 4, A), but it was not
in
the
postmortem
samples.
intact
muscle (Figure 4, B). After 3
storage the band became apparent in both
Interestingly,
however,
this
band
56
appeared
slightly
in
muscle
the
storage.
product
for
T
intensity
seemed
which
(arrow
muscle
causal
postmortem
the
pH
intact
muscle
However,
most
likely
prolonged
a
degradation
T and that CAF is the causal factor
product
in bovine muscle. Indeed,
can
be
seen
f).
at 3, 6 and 10 days
from
the
reduced band
This change was appreciable in the
not in ground muscle. Likely, CAF was
because
troponin
value
other
that
is
degraded
than
during
(1977) have pointed out that the
be
but
when
treatment
to
enzyme
muscle,
believe
al.
troponin
postmortem,
3).
et
degradation
troponin
the
ground
component
of
this
intact
of
Olson
30,000-dalton
more intense in the intact muscle than
during
T
was
was
day
6
and
day
10
altering in the intact
significantly
higher in the
in ground muscle (Table 4 and Figure
workers
cathepsin
L
(Matsukura
is
the
et
al.,
1981)
causal agent for the
breakdown of troponin T to the 30,000-dalton component.
The
observation
30,000-dalton
storage
is
researchers
of
the
polypeptide
consistent
gradual
with
with
appearance of the
progressive
the
findings
postmortem
of
numerous
(Hay et al., 1973b; Olson et al. 1977; Penny,
1980; Parrish et al., 1981; Koohmaraie et al., 1984).
Figure
4
shows
that
desmin
postmortem
(arrow
progressively
during
almost
faded
completely
ground
and intact muscle samples. The breakdown of desmin
at
6
storage
e) diminished
days
and
the
band
postmortem in both
57
may
an
be
significant
integral
part
cross-linking
to meat tenderness because desmin is
of
the
Z-disks,
transverse
functioning
filaments
as the
(Wang
and
Ramirez-Mitchell, 1983).
Other
gradual
principal
increase
indicated
by
molecular
15,000
the
arrows
weights
heavy
(140,000
b,
intensity of three proteins as
d
(M.W.)
there
chain
daltons)
throughout
10
and
h
around
(Figure 4). They had
110,000,
55,000
and
were
seemed
some
high
possible,
then,
It
M.W.
which
could
band
the
(arrow
time.
also
any detectable change
within
the
region of the
could
possible
have
that
including
M.W.
larger
originated from
any
myosin
one
of
the
and C-protein,
than 110,000 or 55,000
be the precursors. Similarly, an original
area
of
myosin
light chain-3 (MLC, )
h) was gradually intensified with extended storage
Dayton
degrade
proteins
subunit
daltons,
in
C-proteins
proteins (> 300,000 daltons). It is
proteins,
have
and
that the increased intensity of 110,000-
is
myofibrillar
daltons)
without
alterations
55,000-dalton
nebulin.
(200,000
days postmortem storage at 2 C whereas
extremely
the
in
in the myofibrils were the
daltons, respectively. From the electrophoretogram
myosin
and
changes
et
al.
tropomyosin,
range
tropomyosin
of
(1975)
yielding
have
shown
fragments
that
CAF can
having M.W. in
13,000 to 18,000 daltons. Like troponin T,
(the unseparated band from troponin T) seemed
58
to
undergo
which
a
was
Therefore,
in
the
subtle
change
during
postmortem storage
more
apparent in intact muscle (Figure 4, B).
the
gradually increased intensity of the band
area
of MLC, (arrow h) could result from the
tropomyosin degradation, but this is still uncertain.
Changes
in
possibility
Initially,
15,000
protein
which
these
salt
myofibrils
explain
were
solution
which
be
another
the intensified bands.
proteins
probably
could
(110,000,
fairly
was
55,000 and
soluble
used
to
in
the
purify
the
(Table 2). Hence, significant amounts of these
proteins
may
postmortem
possibly
might
native
daltons)
diluted
solubility
have
storage,
due
to
been
the
washed
proteins
denaturation
away. ' Later
became
so
during
less
soluble
that high yields were
isolated by centrifugation (Table 2).
Densitometric Tracings
In
an
postmortem
the
gels
technique
attempt
to
gain
more information about the
changes involved in the myofibrillar proteins,
were
subjected
to
densitometric
scanning
(Figure 5). The labeled peaks correspond to the
changes
indicated
Figure
4
Basically,
using
the
in
the
the
same
electrophoretograms
letters
of
shown in
identification.
scanning data show the major changes that
were observed in the gel patterns. Figure 5 clearly
59
Figure 5. Densitometer tracings of electrophoretograms of
myofibrils isolated from ground (A) and intact (B) bovine
semitendinosus muscle stored at 2 C for 0, 1, 3, 6, and
10 days postmortem. Letters a, b, c, d, e, f, g, and h
correspond to the changes indicated in electrophoretograms.
60
(B)
(A)
0 day
0 day
*
J
A
iJ\V
it
%
i <i»y
XJ
3 days
uJ
f days
k
\s~XJ
10 dayi
Figiare 5
W lAj
61
reveals
a
more
rapid breakdown of nebulin in the ground
muscle
sample
faster
reduction in peak size (peak a) in the former. The
95,000-dalton
ground
than
visible
in
in
intact
daltons,
ground
at
the
1
g
sample
but
storage,
change.
attenuating
increased
peak
postmortem
which
in the
was barely
day
the
intact
intact, muscle
both
muscle.
With
this peak increased in both
Simultaneously,
in
postmortem in the
showed
troponin
a
more
T (peak f)
treatments but more rapidly in
Another
the
marked
area
of
change
was
the
MLC, (peak h).
h was not appreciable but after 10 days
storage it became a major peak on the scanning
profile.
Other
augmented
sizes
110,000-
1
in
the
around
peak
at
not
muscle.
Originally,
detected
Similarly, in the area of 30,000
however,
pronounced
intact
was
postmortem,
appeared
although
the
day
c)
muscle by showing a
electrophoretogram, but was undetectable
treatments
was
intact
(peak
muscle.
peak
prolonged
the
component
muscle
the
in
and
changes
of
noted
peaks
b
55,000-dalton
include
the
gradually
and d corresponding to the
components.
The progressive
diminution of desmin (peak e) is also evident.
Causal Factors
The
changes • of
similarity
the
between
ground
the
patterns
of
protein
and intact samples suggests that
62
possibly
similar
postmortem
small
proteinases
storage
difference
that
grinding
certain
only
muscle
cells
in
muscle
cell
walls
cell
to
can disrupt
and severely damage the myofibrils so
resulting
and
walls,
In fact, due to the fracture
proteinases
enzymes.
In
in
the
addition
to
the
former case, neutral and
existing
in mast cells which could
been disrupted during the grinding process may
attack
the
neutral
serine
cleaving
an early release of some
myofibrils would become more susceptible
proteinases
have
in
coenzymes.
extracellular
myofibrils.
proteinase
actin,
I
if
such
that
possible
has
the
enzyme
is
capability of
et
al.,
1983).
Thus,
these
enzymic
any, should only affect the myofibrils of
ground
muscle
muscle
remained
extracellular
One
myosin, a-actinin, tropomyosin, troponin
(Goll
activities,
is
it
effect
that grinding can rupture or impair some
alkaline
it
Obviously,
can
possible
cell
and
grinding
enzymes.
become more labile to proteinases. It is
intracellular
T
ways.
the activities of
proteolytic
enzymatically,
two
organelles,
also
altered
will
proteinases
of
slightly
endogenous
and
highly
during the
of both treatments. Nevertheless, the
muscle
they
involved
in the rates of these changes indicates
Structurally
that
were
while
the
cell
membranes
of the intact
intact and protected the myofibrils from
enzymes during postmortem storage. However,
unlikely that these extracellular proteinases were
63
involved
since the electrophoretic patterns (Figure 4) of
myofibrillar
proteins
muscles
were
became
acidic
inactivate
very
of
both
similar.
the
In
ground
addition,
and
intact
muscles soon
after death (Table 4), which would tend to
or
inhibit
these
neutral
and
alkaline
pH
proteinases should they be found around the myofibrils.
The
Ca
activated
CAF
-activated factor (CAF), however, may be
by
has
a
unique
components
Ishiura
grinding.
specificity against certain myofibril
(Dayton
et
al.,
contributed
at
muscle
cell
et
al.,
1979).
by
active
It has been well documented that
low-Ca
70uM
Ca++
of
structures,
1975;
Olson
I_n
vivo
CAF
which
(Goll
the
et
et al., 1977;
, this activity is
is
completely
al., 1983). Two
sarcoplasmic reticulum and
mitochondria,
regulate the sarcoplasmic level of Ca
(Etherington,
1981).
reticulum
early
have
been
ruptured,
release
of
Ca
(Hamm,
the
(Cassens,
rapid
1977).
that
mitochondria
conditions
and
resulting
1977) .
can
itself
in the
The
markedly
reticulum to sequester Ca
It is probable that grinding could also
disrupted
reticulum,
glycosis
sarcoplasmic
have
release.
ground muscle, the sarcoplasmic
could
grinding-induced
hinder
In
mitochondria.
are
under
could
be
Whiting
(1980)
has found
more labile than the sarcoplasmic
normal
the
aging
initial
An elevated sarcoplasmic Ca
and cold-shortening
agents
of
calcium
level tends to
64
promote
the
activity
of the low-Ca
could
be
partially
CAF. Moreover,
high-Ca
CAF
released
Ca
.
Hence,
a
consequently,
a
faster
protein breakdown would occur in
high
activated by the
CAF
activity,
and
the
ground muscle. However, this likelihood is challenged
by
the low pH observed in the muscle postmortem since CAF
has
optimum
ground
time
pH
and
around
7
while
the
pH values in both
intact muscle samples were below 6 during the
most
of the proteolytic alterations occurred (Table
4). Hence, other proteinases may have been involved.
Lysosomal
Similarly,
ground
the
to
intact
a
would
be
caused
by the early
promote this destruction. In the
the disruption may also take place but at
rate
due to the mild pH decline (Figure 3). At
skeletal
muscle
cells
myofibrils
Reinauer,
1984).
cathepsins
B
and
1979).
lysosomes
damaged
(Goll
have
et
Bovine
D
enzymes
structure,
been shown to be active
al.,
skeletal
(Swartz
Presumably,
these
myofibrillar
were
partially
four catheptical activities isolated from lysosomes
against
al.,
apparently faster proteolysis of
of the muscle lysosomes. The sudden fall in pH
muscle,
least
of
could
grinding
slower
are other possible causal agents.
initial
muscle
disruption
due
enzymes
1983;
Dahlmann
muscle
and
contains
and Bird, 1977; Robbins et
after
could
their
release
from
diffuse readily into the
especially
when
the myofibrils
during the grinding process. Figure 4 shows
65
that
the more pronouned proteolytic changes took place at
the
later
when
the
with
both
stage
pH
is
was
postmortem storage, during the time
maintained at about 5.5 in the muscles
treatments
cathepsins
during
of
released
this
time
(Table
from
4).
the
It
seems likely that
lysosomes would be active
because they have acidic pH optima. It
not clear, however, which of the lysosomal proteinases
could
be
the
contribution
this
more
of
enzyme
important
cathepsin
generally
D
or
more
would
requires
effective.
The
seem to be small as
a
pH
below
4.5
for
activity
(Etherington, 1981) . Even though cathepsins B, H
and
are
L
(Etherington,
were
the
1981)
myosin
myofibrillar
can
(Figure
myosin
and
activity
actin
be
However,
actin
range
uncertain
of
5.0-6.0
whether they
addition
and
seen
from
the
failure
not
Most
cathepsins
of
1984),
to detect changes in
a complete absence of
the
studies
myosin
and
the
actin were
purified
myosin
(Schwartz
and Bird, 1977; Robbins et al., 1979; Matsukura
et
1981).
and
higher
showing
at
represent
or
Reinauer,
other
the electrophoretogram
mean
against
to
conducted
al.,
ambient
in
(Dahlmann
does
activity.
of
pH
causal factors since they could also
and
hardly
the
remains
proteins
4).
lysosomal
within
it
principal
degrade
which
active
actin
Evidently,
the conditions
in
temperatures, and used
or
these
purified
conditions
myofibrils
may
not
vivo , or those present in
66
this
study,
storage
(1980)
notably
temperature
the integrity of myofibrils and the
(2 C).
Moreover, Okitani et al.
have suggested that the activity of cathepsins may
vary
from species to species. Etherington (1980) reported
that
the cysteine cathepsins (e.g. cathepsins B and L) of
the
muscle lysosomes were the more effective enzymes when
pH
CAF
is
was
below 6.0 during the main meat aging period while
was
the most effective enzyme above pH 6.0. Thus, it
likely
that these participating cathepsins may have a
substrate
preference.
significantly
proteins
affected
have
been
proteinases under
While
the
myofibrillar
is
some
Myosin
actin
may
not
be
before certain other myofibrillar
markedly degraded by these lysosdmal
in
vivo
true
conditions.
mechanism
protein
involved in the observed
alterations is still obscure, there
evidence which tends to implicate that CAF was a
possible
causal
agent.
electrophoretogram
(Figure
significantly
degraded
postmortem
the
muscle.
in
This
would
the
Ca
the
muscle
when
shown
nebulin
within
the
suggest
by
As
4),
ground
initially
al.
and
in
the
(arrow
first
24
gel
a)
was
houres
muscle
but not in the intact
that
the CAF was activated
grinding-induced
early
release
of
pH remained high. Maruyama et
(1981b) found that CAF can degrade nebulin. Catheptic
enzyme
activity
condition
during
could be excluded because of the high pH
this time. Similarly, the 30,000-dalton
67
component
muscle
that appeared electrophoretically in the ground
at
undetectable
1
day
in
the
postmortem
intact
but
muscle
indication
that the early liberated Ca
CAF
the
since
30,000-dalton
was
would
completely
also
be
an
did activate
polypeptide has been shown
repeatedly
to
(Olson
al., 1977; Penny, 1980). Other data suggesting
the
in
et
be derived from troponin T degraded by CAF
involvement
the intensity of troponin T band and concurrently, the
more
appreciable
30,000-dalton
the
during
the
main
storage
(Figure
that
muscle
was
from
myofibrillar
intact
muscle
period
Interestingly,
of
of
the
than in
postmortem
during the same
significantly
higher (P < 0.05) than
the
electrophoretic
proteins
from
analysis
of
the
other animals in this study.
one case when the pH value was significantly higher in
intact
main
muscle
storage
30,000-dalton
former.
animal
the
than
time,
in the ground sample during the
the
gradual
appearance
of
the
polypeptide seemed to be more pronounced in
The opposite pattern was observed in another
when the pH value was higher in the ground muscle.
Therefore,
in
5).
intensity
of the ground muscle (Table 4). Similar results were
obtained
the
4,
the
(6 days and 10 days postmortem), the pH value of the
intact
the
in
in
muscle
time
increase
component
ground
In
of CAF are: the more pronounced decrease
it
is
concluded that CAF was indeed involved
postmortem myofibril breakdown in both ground and
68
intact muscles.
The
not
existence
surprising.
low-Ca
of
CAF
pH
5.5
and
90%
Dayton
than
activity in low pH muscle is
et
al.
high-Ca
proteolytic
5.9,
retained
CAF
(1981)
15-25%
CAF, and it may retain
activity
of
at
pH 5.5. Between pH
the maximum activity of CAF is
(Penny, 1980). Goll et al.
or
have shown that
is active over a slightly broader range
values
significant
of
(1983) suggested that
more of the postmortem proteolytic degradation of
myofibrillar
proteins
contributed
by
CAF,
inside
muscle
cells
could
be
however, this was not obtained from
actual measurement.
In
view
of these facts, it is possible that CAF was
responsible
for
against
the
myofibrils in the initial postmortem storage
period
when
probable
most
the
pH
function
disorganize
the
about
5.5
by
as
the
period
storage,
ultimate
pH
during
by
Z-disks.
When
at
5.5),
high.
The
was
to
some
of
the
the
This
M-line and
action
was
pH value dropped to
3, 6 and 10 days postmortem,
became
when
the
as
activities
stage
digesting
the
active
myofibrillar
(about
this
such
observed
hydrolyzed
relatively
proteins
grinding.
enzymes
of
CAF
in
lysosomal
proteolytic
still
myofibrils
proteins
facilitated
the
was
of
structure-maintaining
desmin
of
and
proteins.
preferentially
During the later
the muscles had achieved their
CAF
still retained a certain
69
degree
of
however,
both
its
maximum
which
ground
activity.
It remains uncertain,
enzyme(s) actually played a major role in
and
intact
muscles
during
the postmortem
storage.
Sarcoplasmic Proteins
The
sarcoplasmic
after
storage
at 20C were also analyzed by SDS-PAGE (Figure
and
gel
similar
(arrows
while
more
two
area
one
a,
6
patterns
and
that
c)
appeared
in
days of postmortem
(Figure
7).
The
both muscle treatments were
(1)
1,000,000-dalton
intense
10
scanning
of
except
b,
the
3,
densitometric
electrophoretic
very
1,
of the ground and intact
muscles
6)
0,
proteins
three high M.W. proteins
early
in the ground muscle
component
(arrow a) became
the intact muscle during the aging;
(2)
closely spaced polypeptides gradually appeared in the
around 138,000 daltons in the ground muscle but only
occurred
in
the
intact
significant
proteolytic
disappearance
of
(arrows
d,
h)
100,000-dalton
treatments.
exhibited
the
polypeptide
Alterations
from
the
gel
(arrow
changes
300,000and
muscle
in
and
Other
included
the
24,000-dalton
gradual
(arrow
these
scanning
e).
f)
appearance
in
proteins
data
proteins
both
were
(Figure
addition, scanning showed the diminution of a
of
a
muscle
also
7). In
70
Figure 6.
SDS-polyacrylamide gel electrophoresis of sarcoplasmc
proteins extracted from ground (A) and intact (B) bovine
sendtendinosus muscle stored at 2 C. Gels 0, 1, 3, 6,
and 10 represent sarcoplasmic proteins extracted from
the muscle after 0, 1, 3, 6, and 10 days of postmortem
storage. Gel S is the protein standards (nunber X 1000
= molecular weigjnt). Arrows a, b, c, d, e, f, g, and h
show the major changes.
71
B
HI
4d4
^
200
•■
116
93
iilii "
66
Mil III!!'
45
il|l
♦ 9*W - • • •
31
••••
«
0136 10
0136
DAYS
Figure 6
10
S
21
72
Figure 7. Densitometer tracings of electrophoretograms of
sarcoplasmic proteins extracted from ground(A) and
intact (B) bovine semitendinosus muscle stored at 2 C
for 0, 1, 3, 6, and 10 days postmortem. Letters a, b,
c, d, e, f, g, and h correspond to the changes indicated
in electrophoretograms.
73
(A)
(B)
\
1 day
\
3 days
6 days
.c
Ae
1/
\.
K.
Figure 7
74
26,OOO-dalton
protein
(peak
g)
at
10
days postmortem
(Figure 7).
Changes
complex
is
in the intensity in specific bands suggest a
progression
lysosomal
proteins
It
is
mitochondrial
may
et
that
proteins
proteins
al.,
or
required
at
to
some of these
contamination
such
as
with
membrane
and
fragments of the sarcoplasmic
was involved. Goll et al.
centrifugation
1983). Therefore,
contribute
likely
non-sarcoplasmic
reticulum
(Goll
proteinases
changes.
and
proteolytic events. Although CAP
present in sarcoplasm it has very little effect on the
sarcoplasmic
is
of
(1974) suggested that
25,000-100,000 X g for 120-180 minutes
to sediment microsomes, polysomes, ribosomes
fragments
of sarcolemma. In our extraction, however,
35,000 X g for 45 minutes was employed (Table 2).
Contamination
highly
possible.
with
myofibrillar
Goll
et
degraded
al.
was also
(1970) suggested that if
myofibrils
are
components
in the sarcoplasmic protein fraction should be
expected.
Careful
of
myofibrils
the
sarcopasmic
correlations
can
the
proteins
appearance
of new protein
comparisons of the electrophoretograms
proteins
(Figure
(Figure
4)
with
those
of
the
6) reveal some interesting
between the two electrophoretic patterns. It
be seen that titin had a subtle alteration during the
postmortem
storage
(Figure
appreciable
loss
titin
of
4).
There
was
a
more
in the intact muscle because
75
the
band
became
Concomitantly,
~1,000,000
of
intense
band
(Figure
6,
in
reported
are
present
a;
of
the
that
Therefore,
it
solubilize
the
have
water.
extract
reasonable
that
among
titin
out
the
most
of
(Penny,
to
the
1980).
extract
and
myofibrils, the most
the protein molecules within the
Wang
was
with
are
from
some of them
is to disrupt the intimate associations
found
associations
can
seems
proteins
(1977) have
ionic strength buffer
myosin
Although
probable
pH8.0)
Low
except
myofibrils.
is
component
proteins are soluble in
proteins
factor
that
Goll et al.
been extracted, and
with
interactions
molecules
muscle extraction
the ~1,000,000-dalton
myofibrillar
Tris-HCl,
myofibrillar
carried
intact
myofibrils.
the
they
5mM
important
the
Figure 7, peak a). This concurrent
that
extractable
and
in
the sarcoplasmic fraction probably originated
once
(e.g.
and 10 days postmortem.
both ground and intact muscles with a more
arrow
titin
water
6
new protein of the same size as titin (
suggests
arising
from
a
at
daltons) gradually appeared in the sarcoplasmic
extraction
event
thinner
Ramirez-Mitchell
salt-insoluble,
the
strongly
that
and
fresh
muscle
their
in
(1983)
study was
which
titin
cross-linked with each other. It
during
the
aging
process
the tight
among titin molecules and the interations of
titin
molecules
some
unknown
with
the
mechanism
myofibrils became weakened by
involving
certain
endogenous
76
proteinases,
and
sarcoplasmic
titin molecules were liberated into the
protein
homogenization.
It
extracting
remains
solvent
uncertain,
during
however, whether
the two proteins are identical.
Perhaps
by
a
similar
component
indicated
from
the
lower
top
of
the
by
mechanism
arrow
titin
b
another high M.W.
(Figure 6) was removed
doublet (the second band from the
gel,
Figure
4).
The ~600,000-dalton
polypeptide
of the sarcoplasmic fraction (Figure 6, arrow
c)
an
may
be
extractable
degradative product of titin
from the myofibrils.
Similarly,
a
slight
change
(Figure
4),
appearance
100,000
(1975)
have
was
from
Penny
very
the
a-actinin
and
the
a
change
sarcoplasmic
of
postmortem
paralleled
protein
the
storage
gradual
of the same size
(Figure 6, arrow f). Reddy et al.
resistant
of
has
the
to
CAF but was readily released
myofibril when CAF is present.
been able to show that the binding of
to the Z-disk structure is weakened during aging
amount
solution
findings
it
fraction
days
carefully studied a-actinin. They found that
strength
which
3
this
daltons
Z-disk
(1980)
after
and
of
about
it
the intensity of a-actinin seemed to have
is
gradually
was
the
of
a-actinin
extracted
in
low
ionic
increases with aging. In view of these
likely
arose
that
in
the 100,000-dalton protein
the
sarcoplasmic
protein
released a-actinin from the myofibrils
77
by
CAP
during the storage period. Again, this hypothesis
should
be
treated
alterations
in
with
other
caution
cell
since
organelles
postmortem
could
also
contribute to such a change.
In
Figure
6
and Figure 7, the disappearance of the
300,000-dalton
protein
minor
in
changes
both
in
ground
this
can
that
Of
be
/
and
peak
d) and some
300,000 daltons region in
or denaturation of the proteins in
course,
excluded.
sarcoplasmic
cathepsins.
to
d
intact muscles may reflect some changes
and
region.
not
105,000
and
solubility
(arrow
the possibility of proteolysis
Drabikowski
proteins
Changes
can
et al.
be
(1977) showed
degraded
by
acid
in this region have been reported by
other workers (Hay, 1973b; Harrington and Henahan, 1982).
In
the
appeared
muscle
not
area
in
138,000
daltons
two polypeptides
the ground muscle but only one in the intact
after
clear
of
6
days of postmortem storage. The cause is
but
this indicates that grinding can slightly
influence the muscle protein alterations postmortem.
Psychrotrophic Bacteria Inspection
No
growth
during
the
either
fresh
intact
muscle
of
psychrotrophic
incubation
(0
day)
samples.
period
or
The
of
stored
same
bacteria was observed
10 days at 7 C in
(10
days) ground and
results were obtained
78
from
of
the
preliminary
2 C.
aseptic
This
is
practices
sterilized
not
surprising
were
employed,
equipment
inhibitor.
tests using incubation temperature
Therefore,
and
NaN,
the
since very strict
including
use of the
which is a microbial
possibility
of
microbial
proteolytic activity was totally excluded.
In
31 C,
the
some
However,
parallel
mesophilic
incubations
at
21 C
and
at
bacterial growth was noticed.
no attempt was made to interprete this effect on
the
muscle
grow
at
proteins
2 C
which
since
was
mesophilic bacteria will not
the
temperature used in this study.
postmortem
storage
79
CONCLUSIONS
Evidence
there
were
muscle
gained throughout this study indicates that
no
during
days.
The
extensive
the
close
postmortem
of
proteins
suggests
ground
and
that
significant
influence
alterations
postmortem.
a
pronounced
myofibrils.
However,
accelerated
the
components
storage at 2 C for 10
resemblance between the electrophoretic
patterns
render
protein changes in ground beef
intact
the
on
muscle
grinding
the
upon
the
grinding
the
of
process
certain
nebulin,
had
no
protein
did
not
breakdown of the
grinding
degradation
particularly
process
sarcoplasmic
Similarly,
effect
sarcoplasmic
but
slightly
contractile
this
effect
diminished during the prolonged storage.
It
some
is
suggested,
contractile
myofibrils
on
the
proteins
during
evidence presented, that
could
postmortem
be
released
storage
from the
and they could be
extracted with diluted salt solution.
Evidence
possible
was
presented
participation
myofibrillar
proteins
grinding
process.
release
of
reticulum
ground
also
as
muscle.
Ca
of
CAF
and
The
indicates
the
the
alterations
of
that CAF was activated by the
latter could result from an early
from
manifested
in
which
the
by
damaged
the
sarcoplasmic
hastened glycolysis in
It was unlikely that neutral and alkaline
80
proteinases
strong
were
involved.
evidence
which
would
addition,
suggest
there
a
was no
significant
activity
of
not
acted individually or in co-operation with other
CAF
enzymes
lysosomal
In
(e.g.
enzymes in this study. Whether or
cathepsins) to produce the protein changes
observed in this study remains a question.
On
the
other
significantly
decline
of
in
the
grinding
accelerated
glycolysis reflected by the drastic pH
ground muscle, probably due to the activation
ATP-ase
by
sarcoplasmic
postmortem
pH
hand,
the
Ca
reticulum
released
and
from
mitochondria.
disrupted
After
3 days
storage there was no significant difference in
between
the ground and intact muscles (P > 0.05), but
this does not cover all the individual cases.
Aseptic
followed
in
contamination
sample
preparation
and
the
use
of
NaN
this study were very effective in preventing
of
psychrotrophic
is
unlikely
in
hot-boned ground beef during storage at 2 C for up
to
10
by
grinding or the alteration of muscle endogenous enzyme
activity
that
bacteria. Therefore, it
the development of certain off-flavors
days would result from the protein changes induced
per
se
.-Other mechanisms such as microbial
contamination may be involved.
81
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