Microbiological and color changes during aging of beef


Ledward, D.A.; Nicol, D.J.; Shaw, M.K.

Food Technology in Australia 23(1): 30-32

1971


AIFST
CONVENTION
PAPER,
MELBOURNE,
1970
MICROBIOLOGICAL
AND
COLOUR
CHANGES
DURING
AGEING
OF
BEEF
By
D.
A.
LEDWARD,
D.
J.
NICOL
and
M.
K.
SHAW,
CSIRO
Division
of
Food
Preservation,
Meat
Research
Laboratory,
Cannon
Hill,
Queensland,
4170
Ageing
of
beef
promotes
improve-
ments
in
tenderness
and
flavour
dur-
ing
storage.
The
storage
periods
re-
quired
to
produce
a
significant
in-
crease
in
tenderness
frequently
ex-
ceed
the
maximum
period
for
which
the
meat
can
be
held
in
air
without
bacterial
spoilage.
Means
of
extend-
ing
storage
without
weight
loss,
colour
deterioration,
or
excessive
bacterial
growth
are
discussed.
Weight
losses
during
ageing
can
be
kept
to
a
minimum
either
by
pack-
aging
the
meat
in
a
moisture
im-
permeable
film
or
by
holding
it
in
an
environment
where
the
relative
humidity
is
close
to
saturation,
i.e.
99.3
per
cent.
The
two
factors
then
of
interest
are
bacterial
growth
and
colour
deterioration.
All
work
re-
ported
here
has
been
on
beef
semi-
tendinosus
(eye
round)
muscles.
BACTERIAL
GROWTH
Some
of
the
factors
affecting
bac-
terial
growth
are
temperature,
avail-
able
moisture,
pH,
and
composition
of
the
atmosphere.
When
meat
is
stored
in
moist
air
at
chiller
tem-
peratures
(e.g.
36°F,
2°C),
spoilage
may
occur
at
about
12
days,
usually
by
Pseudomonas
sp.
(Haines,
1933),
whereas
the
ageing
period
required
is
normally
21
days.
How
can
the
meat
be
stored
for
longer
than
12
days
without
bacterial
spoilage?
Spoilage
organisms
occurring
on
chilled
meat
include
species
of
Pseudomonas
and
Microbacterium,
and
organisms
resembling
Lacto-
bacillus
(Shaw
and
Nicol,
1969).
Pseudomonas
sp.
predominate
on
meat
stored
at
low
temperatures
in
air,
as
at
32°F
(0°C)
they
have
a
generation
time
of
about
12
hr,
whereas
Microbacterium
and
the
Lactobacillus-type
organisms
have
generation
times
of
24
and
40
hr
re-
spectively
at
this
temperature.
Ex-
tension
of
the
storage
life
depends,
therefore,
on
inhibition
or
retarda-
tion
of
the
most
rapidly
growing
organisms,
i.e.
the
pseudomonads.
Effect
of
Oxygen
Pseudomonas
sp.
are
aerobic,
i.e.
they
need
oxygen
(0._)
for
growth,
but
the
0,
concentration
has
to
be
drastically
reduced
before
growth
is
inhibited.
Thus
0.8
per
cent
0.,
caused
negligible
inhibition
of
the
growth
of
Pseudomonas
1482
at
41'F
(5°C),
whilst
0.2
per
cent
0
2
caused
75
per
cent
inhibition,
and
the
com-
plete
absence
of
0„
caused
total
in-
hibition.
The
Microbacterium
22,
however,
grew
at
its
maximum
rate
down
to
0.2
per
cent
0,,
but
grew
for
only
a
short
period
(an
increase
in
population
from
103
to
10
5
/cm
2
)
in
the
complete
absence
of
0,,
whilst
the
Lactobacillus
type
58
was
indif-
ferent
to
the
presence
of
0,
(Shaw
and
Nicol,
1969).
Effect
of
Carbon
Dioxide
Growth
of
Pseudomonas
sp.
was
inhibited
at
elevated
levels
of
CO.,
whereas
that
of
Microbacterium
and
the
Lactobacillus
types
was
not.
Thus
at
36°F
(2°C)
10
per
cent
CO,
caused
44
per
cent
inhibition
of
the
growth
of
Pseudomonas
1482,
and
25
per
cent
CO.,
caused
75
per
cent
inhibition.
Ogilvy
and
Ayres
(1951)
found
that
the
inhibition
by
CO,
increased
with
decreasing
temperature,
e.g.
in
our
experience
50
per
cent
inhibition
was
achieved
at
36°F
(2°C)
with
10
per
cent
CO,,
but
65
per
cent
inhibition
at
32°F
(0°C).
To
achieve
comparable
inhibition
of
Pseudomonas
1482
to
that
caused
by
10
per
cent
CO.
)
,
the
0,
concen-
tration
had
to
be
decreased
to
about
0.4
per
cent.
It
is
much
easier
under
industrial
conditions
to
increase
the
concentration
of
CO
2
to
10
per
cent
than
to
decrease
that
of
0
2
to
0.4
per
cent.
Thus
if
conditions
of
extremely
low
O.,
concentration
and/or
elevat-
ed
CO,
concentration
can
be
estab-
lished
around
the
meat,
storage
times
will
be
increased
since
the
Pseudomonas
types
are
inhibited
and
the
slower
growing
Lactobacillus
and
Microbacterium
types
predominate
in
the
spoilage
flora.
Effect
of
pH
Most
bacteria
grow
over
a
range
of
pH
from
4.8
to
8.5,
with
optimum
growth
usually
near
pH
7.0.
Muscle,
when
rigor
is
complete,
usually
has
a
pH
of
5.5-5.8.
If
glycogen
reserves
are
depleted
prior
to
slaughter
the
pH
may
remain
above
6.0.
The
stor-
age
period
is
then
reduced
since
the
spoilage
bacteria,
such
as
Pseud-
omonas
sp.,
grow
at
about
twice
the
rate
at
pH
6.2
as
at
pH
5.7.
Selection
of
Storage
Conditions
A
storage
temperature
of
36°F
(2°C)
was
chosen
for
four
reasons:
the
effect
of
CO.,
increases
as
the
temperature
is
lowered;
the
organ-
isms
which
are
not
inhibited
by
CO„
grow
poorly
at
36°F
(2°C)
and
be-
low;
although
an
increase
in
tem-
perature
from
36°F
to
43°F
(2°C
to
6°C)
approximately
halves
the
ageing
period
there
is
a
four-
to
six-fold
in-
crease
in
bacterial
growth
rate;
and
food
poisoning
organisms,
such
as
Salmonella,
grow
on
meat
even
under
conditions
of
low
0,
or
high
CO,
concentration
at
temperatures
abOve
45°F
(7.2°C)
(Shaw
and
Nicol,
1969).
30
FOOD
TECHNOLOGY
IN
AUSTRALIA,
JANUARY,
1971
20
10
ds
0
7
B
9
5
3
a
5
2
z
6
0
0
4
Mb
PURPLE
RED
GREEN,
FADED
OXIDISED
PORPHYRINS
At
36°F
(2°C)
alteration
of
the
gaseous
environment
can
give
the
desired
storage
life
(greater
than
21
days).
The
gaseous
environment
may
be
adjusted
in
two
ways:
by
vacuum-
packing
meat
in
a
gas-impermeable
film
(e.g.
Cryovac
S),
or
by
packing
the
meat
in
a
gas-permeable
film
and
storing
in
a
controlled
atmosphere
in
a
gas-tight
chiller
(Bate,
1968).
Vacuum
Packaging
It
has
been
known
for
some
time
that
meat
vacuum-packed
in
gas
im-
permeable
films
keeps
longer
than
meat
stored
in
air
(Ayres,
1960;
Hal-
leck
et
a!,
1958;
Wells
et
al,
1958).
The
reason
for
this
is
apparent
when
one
considers
what
occurs
inside
the
bag
just
after
packaging.
CO,
is
evolved
so
that
the
concentration
in
the
headspace
within
4
hr
after
pack-
ing
is
about
10-20
per
cent,
with
a
corresponding
reduction
in
0.,
con-
centration
(Fig.
1).
When
an
animal
is
slaughtered
the
blood
flow
ceases,
and
the
0.,
trapped
in
the
tissue
is
converted
to
CO.,
which
is
released
on
slicing
and
exposure
to
air.
The
initial
rapid
CO.,
evolution
appears
to
be
temperature
independent
over
the
range
32-99°F
(0-37°C),
but
final
equilibrium
CO,
levels
increase
with
increasing
temperature,
probably
0
10
20
30
40
TIME
(146
A:
CO.;
B:
O..;
C:
Total;
D:
Gram
-i
-;
E:
Gram
—.
FIG.
1
Selection
of
slower-growing,
gram-positive
bacteria
at
68°F
(20°C)
In
closed
meat
packs.
owing
to
the
effect
of
temperature
on
the
solubilities
of
the
gases.
Simi-
lar
results
have
been
obtained
by
Urbin
and
Wilson
(1961)
and
Gard-
ner
et
al
(1967).
Bacteria
do
not
sig-
nificantly
influence
the
level
of
CO.,
at
temperatures
of
45°F
(7°C)
and
below.
The final
equilibrium
0,
level
is
usually
between
1
and
3
per
cent.
It
appears,
then,
selection
of
slow-
er-growing
types
and
inhibition
of
growth
of
Pseudomonas
occur
be-
cause
of
the
evolution
of
CO.,
from
the
meat.
Fig.
1
shows
that
selec-
tion
of
the
more
slowly
growing
gram
positive
types
began
when
the
0,
level
was
still
about
10
per
cent.
On
meat
stored
at
36°F
(2°C)
in
Cryovac
bags
(which
contained
equilibrium
concentrations
15-25
per
cent
CO.,
and
1-2
per
cent
0
2
)
the
predominant
spoilage
organism
was
Microbacterium.
Controlled
Atmosphere
Storage
Instead
of
allowing
the
meat
to
establish
its
own
altered
environ-
E
0
5
10
15
20
25
30
TIME
(Days)
A:
Air;
B:
0.6%
0„,
10%
CO,;
C:
Air;
10%
CO,.
D:
Air,
25%
CO„;
E:
0.2%,
25%
CO,
FIG.
2
Growth
of
bacteria
on
meat
stored
under
different
controlled
atmospheres
at
2°C
(36°F).
ment,
controlled
atmosphere
storage
was
investigated.
Meat
was
packed
in
a
gas-permeable,
moisture-im-
permeable
film
(MSADT-80
or
poly-
thene)
and
stored
in
gas-tight
con-
tainers
filled
with
various
gas
mix-
tures.
Fig.
2
shows
the
effects
of
these
atmospheres
on
bacterial
growth
at
36°F
(2°C).
A
mixture
of
0.6
per
cent
O.,
and
10
per
cent
CO.,
(remainder
nitrogen)
gave
a
similar
result
to
10
per
cent
CO
2
in
air,
indi-
cating
that
0.6
per
cent
0,
is
not
inhibitory.
Increasing
the
CO.,
level
from
10
per
cent
to
25
per
cent
gave
greater
inhibition.
When
the
0.,
level
was
reduced
to
about
0.2
per
cent
in
the
presence
of
25
per
cent
CO.,
the
greatest
inhibition
was
obtained,
owing
to
the
additive
effect
of
high
CO.,
with
low
0.,
concentration.
As
would
be
predicted
from
pure
cul-
ture
experiments,
under
conditions
of
low
0,
(less
than
0.2
per
cent)
and
high
CO.,
(up
to
25
per
cent)
concentration,
organisms
resembling
the
Lactobacillus
type
58
predom-
inated.
MEAT
COLOUR
All
the
considerations
so
far
have
been
microbiological.
Storage
condi-
tions
have
to
be
selected
which
will
enable
extended
storage
periods
to
be
obtained
without
colour
deterior-
ation,
assuming
negligible
moisture
loss.
When
meat
is
freshly
cut
the
sur-
face
is
purple
owing
to
the
presence
of
reduced
myoglobin
(Mb).
Upon
exposure
to
air
the
ferrous
ion
in
the
heme
pigment
reacts
rapidly
and
reversibly
with
0,
to
yield
the
desir-
able
bright
red
oxymyoglobin
(Mbo
2
).
In
the
presence
of
0.,
the
ferrous
ion
in
the
heme
tends
to
oxidize
slowly
to
the
ferric
state
yielding
the
undesirable
brown
met-
myoglobin
(Mb')
(George
and
Strat-
mann,
1952a,
1952b).
In
meat
this
autoxidation
of
the
myoglobin
is
pseudo-reversible
in
that
an
enzymic
reducing
system
is
present
that
is
capable
of
converting
metmyoglobin
back
to
one
of
the
reduced
forms
(Stewart
et
a/,
1965).
These
reac-
tions
are
summarized
in
Fig.
3.
The
net
result
of
autoxidation
and
enzy-
mic
reduction
is
the
formation
at
the
meat
surface
during
storage
of
an
equilibrium
concentration
of
Mb`
which
is
virtually
constant
between
5
and
28
days
at
36°F
(2°C)
(Led-
ward
1970a,
1970b).
During
the
age-
m130
2
BRIGHT
RED
Mb
BROWN
FIG.
3
Possible
reactions
of
myoglobin
in
fresh
meat.
ing
of
fresh
beef,
without
weight
loss,
any
colour
changes
will
be
due
to
changes
in
the
relative
propor-
tions
of
the
myogloblin
derivatives,
Mb,
Mbo
9
and
Mb',
and
possibly
also
to
changes
due
to
specific
bacterial
action.
As
already
described,
bacterial
control
during
storage
can
be
ach-
ieved
by
adjusting
the
CO
2
/0.,
levels
-J
5
0
5
(a
0
4
3
FOOD
TECHNOLOGY
IN
AUSTRALIA,
JANUARY,
1971
31
in
the
surrounding
atmosphere.
These
parameters
together
with
the
bacterial
population
may
affect
the
equilibrium
concentration
of
Mb'
de-
veloped
during
storage
(Ledward,
1970a,
1970b).
Partial
Pressure
of
Oxygen
Meat
samples
stored
at
low
par-
tial
pressures
of
0
2
show
increased
Mb•
formation
compared
with
samples
stored
in
air
(Ledward,
1970a).
The
relationship
between
the
partial
pressure
of
0.,
and
the
equil-
ibrium
concentration
of
Mb*
meas-
ured
after
12±2
days
of
storage
at
45°F
(7°C)
is
shown
in
Fig.
4.
Simi-
80
60
40
20
0
0
2
4
16
18
20
OXYGEN.
C/.)
FIG.
4
The
relationship
between
the
02
concentra-
tion
and
the
"equilibrium
concentration"
of
metmyoglohin
at
7°C
(45°F).
Redrawn
from
the
data
of
Ledward
(1970a).
lar
results
were
obtained
at
32°F
(0°C)
(Ledward,
1970a).
This
depend-
ence
of
the
Mb'
concentration
on
the
partial
pressure
of
O.,
is
similar
to
that
found
by
George
and
Stratmann
(1952b)
for
the
autoxidation
of
myo-
globin
in
pure
solution.
Thus,
it
seems
likely
that
the
increased
Mb`
concentrations
found
in
meat
at
low
0.,
pressures
are
due
to
increased
rates
of
autoxidation,
any
effects
of
0,
pressure
on
the
rate
of
enzymic
reduction
being
negligible.
Upon
subsequent
storage
of
the
samples
in
air,
at
the
same
temperatures,
the
high
Mb'
concentrations
were
re-
duced
but
only
slowly,
e.g.
at
45°F
(7°C)
and
32°F
(0°C)
less
than
10
per
cent
of
the
excess
Mb'
was
reduced
over
24
hours
(Ledward,
1970a).
Thus
for
practical
purposes
the
accumula-
tion
of
Mb'
is
irreversible.
Partial
Pressure
of
Carbon
Dioxide
At
32°F
(0°C)
and
45°F
(7°C),
12
per
cent
CO,
had
no
effect
on
the
formation
or
Mb',
and
CO
2
levels
up
to
80
per
cent
also
had
no
effect
provided
the
0.,
level
was
main-
tained
above
about
5
per
cent
(Led-
ward,
1970a).
CO.,
levels
above
about
30
per
cent
were
sometimes
observed
to
cause
the
meat
to
take
on
a
slight
greyish
tinge
upon
prolonged
stor-
age.
The
decrease
in
pH
of
the
meat
at
higher
CO.,
levels
(Ledward,
1970a)
possibly
caused
some
iso-
electric
precipitation
of
water-
soluble
proteins
which
tended
to
mask
the
natural
redness
of
the
meat.
For
this
reason
atmospheres
with
CO.,
levels
greater
than
25
per
-
cent
are
unsuitable
for
the
storage
of
fresh
meat.
Bacterial
Contamination
High
levels
of
contamination
with
Pseudomonas
sp.
cause
increased
Mb'
formation
in
fresh
meat,
which
is
attributed
to
0.,
depletion
at
the
surface
due
to
0
2
utilization
by
the
bacteria
(Butler
et
al,
1953;
Robach
and
Costilow,
1961;
Ledward,
1970b).
Results
obtained
during
air
stor-
age
for
7
days
at
32°F
(0°C)
showed
that
increased
Mb'
formation
oc-
curred
only
when
the
bacterial
popu-
lation
reached
spoilage
levels
(-108/cm
2
).
If
the
0
2
supply
to
the
meat
surface
is
limited
by
the
pack-
aging
conditions
then
the
increased
Mb'
formation
will
occur
at
lower
Pseudomonas
populations.
Butler,
et
al
(1953)
found
the
increase
to
oc-
cur
at
-
,
107/cm
2
on
steaks
wrapped
in
MSAT
80.
When
meat
is
stored
in
CO3-en-
riched
atmospheres
at
32°F
(0°C)
the
bacterial
flora
is
predominantly
Microbacterium
and/or
Lactobacil-
lus,
species
which
have
little
effect
on
the
formation
of
Mb'
during
chilled
storage
(Ledward,
1970b).
Specific
bacteria
may
affect
the
colour
of
meat
in
other
ways
than
by
causing
increased
Mb'
formation.
Thus
hydrogen
sulphide-producing
bacteria
may
lead
to
the
formation
of
the
green
sulphmyoglobin
(Nicol
et
al,
1970),
whilst
hydrogen
perox-
ide-producing
bacteria
may
lead
to
the
formation
of
the
green
cholemyo-
globin
(Lawrie,
1966)
(Fig.
3.)
CONCLUSION
Adequate
bacteriological
and
colour
control
during
the
ageing
of
meat
at
chiller
temperatures
may
be
achieved
in
two
ways.
The
first
requires
O.,
levels
below
about
0.2
per
cent,
preferably
in
con-
junction
with
increased
CO
2
levels.
In
such
an
atmosphere
the
meat
will
be
purple
but
upon
exposure
to
air
it
will
rapidly
turn
red.
The
second
method
requires
0
2
levels
higher
than
5
per
cent
and
ele-
vated
CO.,
levels
(up
to
25
per
cent).
In
such
an
atmosphere
the
meat
re-
mains
red.
Owing
to
the
practical
difficulty
of
achieving
zero
oxygen
concentration
in
a
pack
economic-
ally,
the
second
method
appears
to
be
the
more
satisfactory
alternative.
LITERATURE
CITED
Ayres.
J.
C.
(1960).
Fd
Res.
25,
1.
Bate,
H.
G.
(1968).
Aust.
Refrig.
Air
Condit.
and
Heating,
22
(3),
49.
Butler.
0.
D..
Bratzler,
L.
J..
and
Mailmann,
W.
L.
(1953).
Fd.
Technol.
7,
397.
Gardner.
G.
A..
Carson.
A.
W..
and
Patton.
J.
(1967).
J.
appl.
Bact.
30,
321.
George.
P..
and
Stratmann.
C.
J.
(1952a).
Biochem.
J.
51,
103.
George,
P..
and
Stratmann.
C.
J.
(1952b).
Biochem.
J.
51.
418.
Haines,
R.
B.
(1933).
J.
Hyg.
33,
175.
Halleck.
F.
E..
Ball,
C.
0..
and
Stier,
E.
F.
(1958).
Fd
Technol.
12,
197.
Lawrie,
R.
A.
(1966).
"Meat
Science".
Perga-
mon
Press.
Oxford.
Ledward,
D. A.
(1970a).
J.
Fd
Sci.
35,
33.
Ledward.
D.
A.
(1970b).
J.
Fd
Sci.
(sub-
mitted).
Nicol.
D.
J.,
Shaw,
M.
K..
and
Ledward.
D.
A.
(1970).
Appl.
Microblol.
(in
press).
Ogilvy.
W.
S..
and
Ayres.
J.
C.
(1951).
Fd
Technol.
5,
97.
Robach.
D.
L..
and
Costilow.
R.
N.
(1961).
Appl.
Microbiol.
9,
529.
Shaw,
M.
K.,
and
Nicol.
D.
J.
(1939).
Effect
of
the
gaseous
environment
on
the
growth
of
some
food
poisoning
and
food
spoilage
organisms
Proc.
European
Meeting
of
Meat
Research
Workers.
15th.
Helsinki.
226.
Stewart.
M.
R..
Hutchins.
B.
K..
Zipser.
M.
W..
and
Watts,
B.
M.
(1965).
J.
Fd
Sci.
30,
487.
Urbin,
M.
C..
and
Wilson.
G.
D.
(1961).
J.
Fd
Sci.
26,
314.
Wells,
F.
E.,
Spencer.
J.
V.,
and
Stadelman,
W.
J.
(1958).
Fd
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12,
925.
Received
15.7.70
Accepted
26.9.70
TECHNICAL
LITERATURE
"THE
OXFORD
BOOK
OF
FOOD
PLANTS"
By
S.
G.
Harrison,
G.
Masefield
and
M.
Wallis.
Illustrated
by
Bar-
bara
Nicholson.
220
pages,
Clarendon
Press,
Oxford.
1969.
$A8.15.
The
sixth
volume
of
this
well-
known
series
covers
the
range
of
plants
from
all
climatic
regions
of
the
world
which
provide
man
with
food
-
grains,
vegetables,
fruits,
nuts,
and
herbs.
The
96
colour
plates
enable
the
reader
to identify
the
main
food-plants
and
their
most
im-
portant
varieties,
and
they
also
illus-
trate
botanical
details.
The
text
de-
scribes
each
plant,
and
interesting
aspects
of
its
development
and
use,
in
terms
of
botany,
cultivation,
his-
tory,
and
nutrition.
M
E
TMYOGL
OB
IN
(%
)
32
FOOD
TECHNOLOGY
IN
AUSTRALIA,
JANUARY,
1971