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LSU Historical Dissertations and Theses
Graduate School
1994
Effects of Heat, Lactic Acid, and Modified
Atmosphere Packaging on Listeria Monocytogenes
on Cooked Crawfish Tail Meat.
Warren Joseph Dorsa
Louisiana State University and Agricultural & Mechanical College
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Dorsa, Warren Joseph, "Effects of Heat, Lactic Acid, and Modified Atmosphere Packaging on Listeria Monocytogenes on Cooked
Crawfish Tail Meat." (1994). LSU Historical Dissertations and Theses. 5692.
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E ffects o f h ea t, lactic acid, and m odified atm osp here packaging
o n Listeria monocytogenes on cooked craw fish ta il m eat
Dorsa, Warren Joseph, Ph.D.
The Louisiana State University and Agricultural and Mechanical Col., 1994
UMI
300 N. ZeebRd.
Ann Arbor, MI 48106
EFFECTS OF HEAT, LACTIC ACID, AND MODIFIED ATMOSPHERE
PACKAGING ON LISTERIA MONOCYTOGENES
ON COOKED CRAWFISH TAIL MEAT
A Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Department of Food Science
by
Warren Joseph Dorsa
B.S., Nicholls State University, 1978
M.S., Mississippi State University, 1981
May 1994
ACKNOWLEDGEMENTS
The author would like to express gratitude to Dr.
Douglas
L.
Marshall
for his guidance while
chairman of his major professor.
thought
provoking
atmosphere
he
challenges
needed
to
Dr. Marshall's constant
provided
conduct
conducive to doctorate work.
serving as
him
research
with
in
a
the
manor
He would also like to thank
Dr. Robert M. Grodner, Dr. Michael W. Moody, Dr. John H.
Wells,
and Dr.
Paul W. Wilson for their guidance while
serving on his graduate committee.
Special thanks are extend to Harold Bennett and the
Louisiana Crawfish Promotion and Research Board for aid in
acquisition of product used in conducting my research.
He wishes to thank his friends and fiance Jessica, for
their constant support, love, and encouragement during this
undertaking.
Finally, he wishes to dedicate this work to
his parents Ted and Nancy, for without their unfaltering
support, encouragement, and love throughout the years, his
life would not be the joyous event it has become during the
completion of this task.
ii
TABLE OP CONTENTS
ACKNOWLEDGEMENTS.....................................
LIST OF T A B L E S .....
page
ii
V
LIST OF FIGURES.....................................
vi
LIST OF ABBREVIATIONS AND SYMBOLS..................... viii
ABSTRACT...........................................
ix
INTRODUCTION...................
1
REVIEW OF LITERATURE.....
9
General Microbiology of L . monocytogenes.......
Infection Characteristics.......................
Foodborne Listeriosis ..........................
Presence in Seafood............................
Thermal Tolerance.............................
Affects of Organic Acids on L.monocytogens .....
Citric A c i d ....................................
Lactic A c i d ....................................
Sorbic A c i d .......... .........................
Modified Atmosphere Packaging (MAP).............
9
10
12
13
15
18
21
23
25
29
MATERIALS AND METHODS ...............................
33
Sample Preparation .............................
Preparation of Inocula .........................
Thermal Inactivation Studies ...................
Three-neck Flask Method (TNF) ...............
Sample preparation
..................
Thermal treatment .........................
Enumeration of L. monocytogenes......
D value determination .....................
Polyethylene Bag Method (PB) ................
Sample inoculation................
Preparation & application of lactic acid ...
Introduction of modified atmospheres .......
Thermal treatment and enumeration
......
D value determination .....................
Cellulose Casing Method (CC) ................
Sample inoculation.............
Sensory Evaluation .............................
33
33
35
35
35
36
36
37
38
38
39
41
41
44
46
46
47
iii
page
Storage Studies...............................
Effect of Storage at 0, 6, & 12° C ..........
Preparation of inoculated samples ........
Enumeration of bacteria ...................
Calculation of Generation T i m e ................
Effect of Citric Acid & Potassium Sorbate on
Growth of L. monocytogenes.................
Preparation of inoculated samples .........
Effect of Lactic Acid and Atmospheres on
Growth of L. monocytogenes.................
Preparation of inoculated samples ........
Enumeration of bacteria ...................
Experimental protocol .....................
48
48
48
49
50
RESULTS AND DISCUSSION .............................
Low Temperature Growth.........................
Storage Studies Using Citric Acid & Potassium
Sorbate.............
Thermal Inactivation ..........................
Thermal Inactivation with Lactic Acid .........
Sensory Evaluation ............................
Heat and Modified Atmospheres..................
4°C Storage Under Various Atmospheres & 1% LA ...
56
56
SUMMARY AND CONCLUSIONS
91
...........................
51
51
52
52
53
53
61
64
71
75
78
83
REFERENCES.........................................
94
V I T A ...............................................
108
iv
LIST OP TABLES
Table
Page
1.
Treatment combinations for inoculated crawfish
samples ........................................ 40
2.
Gas composition as determined immediately after
introduction to sample packs and after 60°C
heat treatment..................................43
3.
Gas content
at 4°C of
tail meat
acid (LA)
4.
Generation times, least squares regression factors
and goodness of fit for Listeria monocytogenes
in cooked crawfish tail meat treated with citric
63
acid (CA) or potassium sorbate (PS) ..........
before and after 21 d storage
package atmospheres and pH of crawfish
treated with or without lactic
.................................... 54
5. Thermal death times, least squares regression
factors and goodness of fit for D value
determinations using TNF methodology for L.
monocytogenes in crawfish tail m e a t ...........
65
6. Thermal death times, least squares regression
factors and goodness of fit for D60 values
determinations of L. monocytogenes using
various methods in crawfish tail m e a t .........
68
7. Least squares regression factors and goodness of
fit for D-value determinations at 60°C for
different levels of lactic acid or HCl adjusted
crawfish.......................................72
8. Taste panel results of crawfish samples packed
under air and stored at 4°C. Values of 5.0 =
standard quality, >5.0 = less favorable quality,
and <5.0 = more favorable quality............. 77
9.
Least squares regression factors and goodness of
fit for D-value determinations after 60°C
thermal treatment of crawfish tail meat treated
with or without 1% LA and packed in different
modified atmospheres with head space .......... 80
v
LIST OF FIGURES
Figure
Page
1. Inside dimensions of polybag used in both thermal
and storage studies .................
2. Center temperature for come-up and cool-down times
for 10 g samples of crawfish tail meat in
polybags (PB) and polybags with head space
(PB-a) and cellulose casings (CC) .............
3.
42
45
Growth of L. monocytogenes on crawfish tail meat
stored at 0, 4, 6, and 12 °C ...................
57
4. Generation times for L. monocytogenes in
different food products at 4°C ...............
60
5.
Growth of L. monocytogenes on crawfish tail meat
at 4°C and treated with 0.03 g/kg Citric Acid
(CA) or Potassium Sorbate (PS)............ 62
6.
Thermal death curve for L. monocytogenes at 60°C
using various levels of spray applied
lactic acid ..............................
73
Linear regression comparing the effect of 1%
lactic acid and the equivalent HC1 adjusted pH,
on lethality of 60°C toward L . monocytogenes on
crawfish tail meat ......................
74
7.
8.
9.
10.
Thermal death curve for L. monocytogenes on
crawfish tail meat at 60°C packed under
various atmospheres ..........................
79
Thermal death curve for L. monocytogenes on
crawfish tail meat at 60°C spray treated with 1%
lactic acid and packed under various
atmospheres
..........................
Growth of L . monocytogenes stored under 4
different atmospheres at 4°C for 21 d ....
82
84
11.
Growth of L . monocytogenes on crawfish tail meat
stored under Air and N2 alone and in combination
with 1% LA at 4°C for 21 d .................... 87
12.
Growth of L. monocytogenes stored under C02 and 02
alone and in combination with 1% LA at 4°C for
21 d .........................................
vi
88
Destruction of L. monocytogenes stored under Air,
N2, C02 and 02 in combination with 1% LA at 4°C
for 21 d ...... ..............................
vii
LIST OF ABBREVIATIONS AND SYMBOLS
AcH
—
pediocin (form of)
mL
- milliliter(s)
ANOVA
-
analysis of variance
MPa
-
atm
-
atmosphere(s)
P
- probability
BPB
- buterfield phosphate
buffer
°C
-
degrees Celsius
PB
CA
-
citric acid
PB-a - polybag method
(with head space)
CC
- cellulose casing
method
PH
CFU
- colony forming units
PKa
-
co2
-
carbon dioxide
PMF
-
proton motive force
CAT
--
catalase
PS
-
potassium sorbate
d
-
day(s)
sp
-
species (singular)
^60
-
D value at 60°C
spp
- species (plural)
op
-
degrees Fahrenheit
SOD
-
superoxide dismutase
g
-
gram(s)
TDT
-
thermal death time
h
-
hour(s)
TNRF
-
HCl
- hydrochloric acid
TSA
-
kg
-
L
- liter
USFDA- U.S. Food and Drug
Administration
LA
- lactic acid
Wt
-
LOA
-
listeria oxoid
agar
w/w
- weight to weight
MAP
-
modified atmosphere
packaging
%
min
-
minute(s)
kilograms
millibar per
atmosphere
PA-1 - pediocin (form of)
-
polybag method
- (-) log of hydrogen
ions
dissociation
constant
3 neck reaction
flask method
tryptic soy agar
USDA - U.S. Department
of Agriculture
weight
percent
viii
ABSTRACT
The generation time of L. monocytogenes was calculated
to be 72.2, 28.5, 17.0, and 6.9 h on crawfish tail meat held
at 0, 4, 6, and 12°C, respectively.
Potassium sorbate (PS)
(0.3%) spray treatments on crawfish held at 4°C, extended
the lag phase of the bacterium.
However, once exponential
growth began, a generation time not significantly different
(P>0.05)
than that of untreated
crawfish was observed.
Samples sprayed with 0.3% citric acid (CA) and held at 4°C
supported growth of the bacterium equally well as that of
untreated samples.
Thermal inactivation of L. monocytogenes using a three
neck reactionary flask method yielded D556065 values of 10.23,
1.98, and 0.19 min, respectively.
calculated.
Polybag
and
A z value of 5.5°C was
cellulose
casing
methodology
yielded significantly higher (P> 0.05) D60 values of 4.68 and
3.84 respectively,
for the bacterium.
D60 values for L.
monocytogenes decreased as the percent lactic acid (LA)
spray
treatments
sensitivity
to
reduction of pH.
packed
in
air,
levels
heat
with
were
increased.
LA was
not
Increasing
solely
due
to
a
D60 values for the bacterium on samples
02, C02, or
N2 were
not
significantly
different (P>0.05), however, D60 of samples packed under 02
were lowest.
ix
Generation times for heat damaged L . monocytogenes on
crawfish meat stored at 4°C and packed under air, 02, C02/ or
N2 was determined.
Twenty percent of the cell population
was damaged when thermally treated at 60°C for 12.5 min.
The
bacterium consistently grew
slowest
in C02 and 02.
Samples receiving a treatment of 1% LA, packaging under
either of the 4 different atmospheres, and storage at 4°C,
were lethal to the bacterium.
These
studies
indicate
that
post-packaging
heat
treatments of 60°C or greater in the presence of C02 or 02,
and the addition of 1% LA sprays, would be detrimental to
the survival of L . monocytogenes on crawfish meat.
x
INTRODUCTION
Increased public
awareness
and perception
of
food
quality and* safety has escalated government monitoring of
seafood products.
As a result, development of more safety
focused processing techniques is critical.
Employment of
multi-barrier methodology through modification of presently
used processes would be an economical way to accomplish this
goal.
Outbreaks of foodborne listeriosis involving a variety
of
foods
important
has
established Listeria
foodborne
pathogen
monocytogenes
(Fleming
et
as
al.,
an
1985;
Kvenberg, 1988; Linnan et al., 1988; Sclech et al., 1983).
The association of L. monocytogenes with seafood products
has
also
seafood
been
established
products
such
(McLauchlin,
as
shrimp,
1987).
crabmeat,
Frozen
lobster,
langostinos, scallops, squid, and surimi have been shown to
contain L. monocytogenes when samples were obtained from
several different countries (Weagant et al., 1988).
The
study found that 26% of the products sampled contained L.
monocytogenes.
Farber (1991) found 13% of fish products at
the wholesale level in the U.S.A., Britain, and Taiwan, to
be positive for L. monocytogenes.
He also found ready-to-
eat shrimp, crab, and salmon to contain the bacterium.
L.
monocytogenes flourishes well in aquatic environments of
1
decaying vegetative matter (Gray and Killinger, 1966) , which
is the typical environment in a crawfish pond.
As a result
L. monocytogenes can usually be found on live crawfish
(crayfish) entering processing plants.
Crawfish tail meat is hand picked from parboiled whole
crawfish and usually packed for retail sale in one pound,
low oxygen permeable polybags, that are sometimes vacuum
sealed.
to
kill
While boiling temperatures (100°C) are sufficient
L.
monocytogenes
(Fain
et
al.,
1991),
cross
contamination from workers and food contact surfaces may
occur.
Postprocessing contamination with L . monocytogenes
has been identified as a major source of contamination for
many food products (Farber and Peterkin, 1991).
Low numbers
of L . monocytogenes have been observed in selected frozen
crawfish tail meat (Dorsa and Marshall, 1993).
One pound
bags of crawfish tail meat are typically sold unfrozen on
ice.
Harrison et al.
(1991) found that L. monocytogenes
populations on vacuum packed iced shrimp did not increase.
Despite this finding,
it is important to consider that
evenly distributed product in ice-packs or 0°C conditions
are not always practical during product transportation or
during retail display of packaged crawfish tail meat.
As a
result, it is possible for the product to be subjected to
periods of warming, potentially resulting in proliferation
of L . monocytogenes.
L. monocytogenes has been shown to grow well on many
food products stored under refrigeration.
The generation
times for the bacterium has been determined for red meats,
vegetables,
and seafood products
(Berrang et al.,
Marshall et al., 1991; Shelef, 1989).
for
the bacterium
on
1989;
The generation time
crawfish tail meat has
not
been
determined, however.
Most information available for the heat resistance of
L. monocytogenes in foods has concentrated on milk and red
muscle products.
L. monocytogenes has been shown to survive
improper heat treatments in red meats and chicken (Farber,
1989; Harrison and Carpenter, 1989).
Little is known about
the thermal resistance of L. monocytogenes in seafoods,
although D-values for some products such as crab and lobster
have been determined (Budu-Amoako et al., 1992; Harrison &
Huang, 1990).
that
Presently, there are few crawfish processors
subject crawfish tail meat to post-packaging heat
treatments.
Since post-packaging heat treatments might be
beneficial in reducing post-processing contamination and
insure product safety, there is a need to study the heat
resistance of L. monocytogenes on this product.
L. monocytogenes has been detected in 53% of vacuum
packed processed meats obtained from retail stores (Grau and
Vanderlinde
1992).
Several
studies
with
modified
atmospheres indicate that the bacterium can survive low
temperature
conditions
in
reduced
conditions.
Marshall
et
al.
oxygen
(1991,
or
1992)
anaerobic
found
that
atmospheres lower in oxygen than air, reduced growth of the
bacterium at low temperatures, but were not lethal.
Manu-
Tawiah et al. (1993) determined that atmospheres containing
C02 concentrations normally used for commercial packaging of
fresh red meats are unlikely to reduce the risk factor for
listeriosis in ready-to-eat pork chops. Additional studies
have determined that if L. monocytogenes is heat injured
prior to incubation, exposure to oxygen will decrease its
chances for recovery (Knabel et al., 1990).
These findings
agree with earlier researchers who theorized that severely
heat injured cells acquire a strict anaerobic nature (McCord
et al., 1971).
Dallmier and Martin (1988) reported that
catalase (CAT) and superoxide dismutase (SOD) are rapidly
inactivated when the bacterium is heated.
The subsequent
buildup of toxic 02 radicals due to inactivation of these
enzymes converts L. monocytogenes into an obligate anaerobe
(Knabel et al., 1990).
modified
package
treatments
to
Consequently, the use of various
atmospheres
control
or
in
combination
with
heat
eliminate L . monocytognes
on
crawfish tail meat warrants investigation.
Studies have shown that organic acids and their salts
inhibit the growth of L. monocytogenes.
Ahamad and Marth
(1989) determined that citric and lactic acids, with large
dissociation
constants,
caused
cell
damage
to
L.
monocytogenes. El-Shenawy and Marth (1988) observed that L.
monocytogenes was susceptible to sorbic acid.
Chung and Lee
(1981) found that potassium sorbate on English sole delayed
the onset of logarithmic growth of Pseudomonas sp.
Organic
acids are effective for controlling several bacteria in
broth media and some food products.
Continued investigation
into the use of organic acids on other food products is
warranted.
with
other
The impact of combining organic acid treatments
barriers,
in
an
attempt
to
enhance
their
effectiveness in controlling L. monocytogenes on crawfish
tail meat, remains unknown.
More specifically, Wei et al. (1991) determined that
decreasing the pH of a Listeria spp. suspension for 2 h
using 0.1 N HC1 was not effective in killing the bacterium
below detectible levels.
Ita and Hutkins (1991) suggested
that inhibition of L. monocytogenes by acids may not be
caused by a decrease in the intracellular pH, but rather by
some
specific
effect
of
undissociated
acid
metabolic or other physiological activities.
species
on
The inhibitory
effects of LA and other organic acids can be correlated with
their dissociation constants or pKa values.
In general,
weak acids having higher pKa values are thought to be more
inhibitory to L. monocytogenes at a given pH than strong
acids at the same pH
(Ita and Hutckins,
1991) .
These
studies allow the development of a working hypotheses where
weak organic acids should be effective for controlling L.
monocytogenes on crawfish tail meat.
The bacterial properties of lactic acid (LA) are well
documented
(Ingram et al.,
1956; Van Netten and Mossel,
1980; Woolthuis and Smulders, 1985).
Ockerman et al. (1974)
found that spraying 12% LA on hot sheep carcasses reduced
the surface aerobic colony counts by about 1 log10. Similar
reductions with only 1.0% LA on hot beef carcasses were
achieved by Snijders et al. (1979) . Woolthuis and Smulders
(1985)
determined
that
1.25%
LA
solutions
resulted
noticeable decontamination of calf carcasses.
in
Ahamad and
Marth (1989) observed that tryptose broth acidified with 0.3
or 0.5% LA was lethal to L . monocytogenes at 7, 13, 21, and
35°C.
They also found the rate of inactivation to be much
greater at 35°C than at lower temperatures.
Considering the
previous research relating to the use of LA to control L .
monocytogenes
on
red
meat
carcasses,
LA
treatment
on
crawfish tail meat might prove effective for controlling the
bacterium.
Roberts
(1989)
noted that except where one or two
factors limit microbial growth, our understanding of the
relative contributions of multiple factors to give safe and
shelf-stable food products
result,
is surprisingly poor.
As a
there is an inadequate data base with which to
develop microbiologically stable and safe foods that have a
long shelf life.
Roberts (1989) stated that there is an
urgent need for a better understanding of the effects that
combinations
of
factors
survival in foods.
have
on
Chung et al.
microbial
(1982)
growth
and
found that the
presence of an organic acid magnified the effect of heat
injury of several microorganisms
isolated from seafood.
Thus heat treatments in combination with an organic acid
could form the basis of a multifactorial
(multibarrier)
processing technique to improve crawfish tail meat safety.
The overall objective of the present study was to
determine the impact of heat,
lactic acid,
and various
gasses when used independently and in combination against L.
monocytogenes in cooked crawfish tail meat.
A multibarrier
approach was postulated to more aggressively destroy L.
monocytogenes; however,
such
methodology
could
not
be
considered independent of desirable sensory qualities of
crawfish tail meat.
The specific objectives of this work
were:
1. To determine the generation time for L. monocytogenes
on crawfish tail meat at 0, 4, 6, and 12°C.
2. To develop and evaluate the effect of several thermal
destruction methods used for D value determination of
L. monocytogenes on crawfish tail meat.
To determine the lethal effects of heat, lactic acid
and modified atmosphere packaging (MAP) applied both
separately and in combination on L. monocytogenes on
crawfish tail meat.
To determine the effects resulting from process
deviations of lactic acid in combination with MAP on
survivability of heat injured L. monocytogenes at
4°C on crawfish tail meat.
To measure the sensory effect of lactic acid during
refrigerated storage of crawfish tail meat.
REVIEW OF LITERATURE
General Microbiology of L. monocytogenes
L. monocytogenes is a foodborne pathogen that infects
humans, domesticated animals, wildlife and various aquatic
species.
It is considered an environmental contaminant
widely present in plant, soil, and surface water samples
(Moore, 1988; Weis and Seeliger, 1975).
found
in
silage,
sewage,
It also has been
slaughterhouse
solid
waste,
effluent of abattoirs and poultry packing plants, milk of
normal and mastitic cows, pond reared rainbow trout, and
human and animal feces (Gray and Killinger, 1966; McCarthy,
1990; Watkins and Sleath, 1981) .
preferences of the bacterium,
Because of environmental
both pond raised and wild
stock crawfish (Crayfish, Procambarus clarkii) are natural
hosts.
The bacterium was first described by Murray et al.
(1926) .
The pathogenic form of L.
monocytogenes
is a
smooth, small gram-positive, nonsporeforming, facultatively
anaerobic, non-acid fast, diphtheroid-like rod with rounded
ends measuring 1.0 to 2.0 microns by 0.5 microns (Gray and
Killinger,
1966).
It is catalase positive and oxidase
negative and expresses a beta-hemolysin which produces zones
of clearing on blood agar (Farber and Peterkin, 1991) . The
bacterium possesses peritrichous flagella, which give it a
9
10
characteristic tumbling motility, occurring only in a narrow
temperature range.
When grown at temperatures between 20
and 25°C, flagellin is both produced and assembled at the
cell surface, but at 37°C flagellin production is markedly
reduced (Peel et al., 1988).
demonstrate
a
Colonies of the bacterium
characteristic
blue-green
sheen
under
obliquely transmitted light (Henry, 1933).
Infection Characteristics
In humans, the greatest impact of L. monocytogenes is
to the elderly, immunosuppressed, and to pregnant women and
their fetuses.
listeriosis,
infection,
The symptoms of the resulting infection,
include meningitis,
stillbirths,
central
abortions,
septicemia (Health and Welfare, 1990).
nervous
premature
system
labor
and
There are, however,
instances when apparently normal healthy individuals have
become ill with listeriosis in both foodborne epidemics
(Schultz, 1945) and sporadic cases (Azadian et al., 1989).
Most cases of human listeriosis appear to be sporadic,
although
a
portion
of
these
cases
unrecognized common-source clusters
may
be
previously
(Ciesielski
et al.,
1988) .
Serovar 4b of L. monocytogenes is the most virulent in
human and animal disease.
This serovar is only rarely
isolated from foodstuffs, in contrast to the more common
11
serovars
l/2a and
l/2c
(Menudier
et
al.,
1991).
The
existence of avirulent serovars l/2a strains may account for
the infrequent involvement of this serovar in human disease.
Serovar l/2c has been found to be less virulent than serovar
4b, which may help explain its relatively low involvement in
human listeriosis (McLauchlin, 1990).
The infective route of orally ingested cells appears to
be via the Peyer patches of the intestine and then into
phagocytes, passing into the mesenteric lymph nodes by way
of the lymphatic pathways (Farber and Peterkin, 1991).
infected orally have shown variable responses,
Mice
with 50%
infectious doses ranging from 1.7 X 103 to 9.9 X 106/g cells
(Audurier et al., 1980; Golnazarian et al., 1989).
Other
studies on oral feeding of mice with L. monocytogenes have
shown that very high
(> 2.5 X 108/g cells)
numbers are
required to cause invasion of the Peyer's patches or death
in normal mice (MacDonald and Carter, 1980).
Miller and
Burns (1970) found that a dose of >4 X 107/g cells caused
fetal death in 6 of 10 pregnant mice.
Feeding trials in
which nonhuman primates were used showed that only animals
receiving a dose of 109/g I#, monocytogenes cells became
noticeably ill.
Smaller numbers of cells did not result in
noticeable symptoms of disease (Farber et al., 1990).
The
minimum number of pathogenic L. monocytogenes that must be
ingested by humans to cause illness in either normal or
12
susceptible individuals is not known.
Farber & Peterkin
(1991) compiled a list of cases in which the numbers causing
illness were approximated for humans.
In most of these
cases the approximated number of cells that caused illness
was 106/g of food.
Foodborne Listeriosis
Not until the late 1970's was foodborne listeriosis
documented in North America and Europe.
In 1986 the Council
of State and Territorial Epidemiologists recommended that
listeriosis become a reportable disease in the United States
(Gellin and Broome, 1989).
But as early as 1979, the first
outbreak of listeriosis occurred in Boston.
Lettuce, celery
and tomatoes were implicated and 23 cases were reported with
5 fatalities (Ho et al., 1986).
Coleslaw was the cause of
a listeriosis outbreak in Nova Scotia in 1981 resulting in
18 deaths from 41 cases (Schlech et al., 1983).
Pasteurized
milk was implicated as the transmitter of a listeriosis
outbreak in Massachusetts during
1983, where 49 cases were
reported with 14 fatalities (Fleming et al., 1985).
The
largest outbreak occurred in southern California in 1985
where 142 cases were reported causing 48 deaths and was
traced to Mexican
style
cheese
(Linnan
et al.,
1988).
Farber and Losos (1988) reported that all of these outbreaks
were caused by L. monocytogenes serotype 4b.
In 1980 an epidemic of perinatal listeriosis in New
Zealand suggested a possible link to the consumption of
shellfish and raw fish, resulting in 22 reported cases with
5 deaths (Lennon et al., 1984) . To date, there have been no
reported
cases
crawfish.
of
listeriosis
consumption
of
However, since crawfish are crustaceans that are
grown in static pond water,
loads,
from the
the
potential
for
typically high in detritus
contamination
of
hand
picked
crawfish tail meat is omnipresent.
Presence in Seafood
Listeria is considered an environmental contaminant and
can be found in waters of rivers, lakes, ponds and canals
(Moore, 1988; Watkins & Sleath, 1981).
In 1966, Gray and
Killinger reported L. monocytogenes to be present in pond
raised rainbow trout.
Bracket (1988) hypothesized a cycle
of infection for Listeria indicating that contaminated water
infects fish and shellfish which in turn infect humans
through consumption of the contaminated food product.
Since
crawfish are boiled as part of the peeling process, the
packaged
tail
meat
is
considered
cooked.
Cross
contamination during peeling is a possible avenue of product
inoculation with L. monocytogenes.
It is conceivable that
consumers might mistakenly consider crawfish tail meat
14
ready-to-eat
from
the
retail
package,
resulting
in
a
possible infection with the bacterium in consumers.
A Class I recall was made by the U.S. Food and Drug
Administration (USFDA) on vacuum and air packed smoked shad
because of the presence of L. monocytogenes
1992).
(Anonymous,
Several surveys have detected various levels of
Listeria spp.
in seafood products
from retail markets.
Jemmi (1990) detected L. monocytogenes in 12% of smoked and
marinated fish from 377 samples tested.
products such as shrimp, crabmeat,
Frozen seafood
lobster,
langostinos,
scallops, squid, and surimi have been shown to contain L.
monocytogenes in samples obtained from several different
countries (Weagant et al., 1988).
The study found that 26%
of the products sampled contained L . monocytogenes.
and Sirvas
(1991)
Fuchs
surveyed the incidence of Listeria in
ceviche products bought from markets in Lima and Callao,
Peru.
Approximately,
contained
Listeria
75%
of
innocua
the
and
samples
9%
they
tested
contained
L.
monocytogenes.
Noah
et
al.
(1991)
isolated
Listeria
from
frozen
lobster tails, shrimp, prawn, breaded shrimp, whole fish,
and fish fillets.
Listeria spp.
Of 211 composites, 28% were positive for
Wong et al. (1990) conducted a local market
survey in Taiwan and found L. monocytogenes in 10.5% of
seafoods containing fish and squid.
Farber (1991) found 13%
15
of ready-to-eat shrimp, crab, and salmon at the wholesale
level in the U.S.A., Britain and Taiwan to be positive for
L.
monocytogenes.
No
surveys
conducted
included peeled crawfish tail meat.
to
date
have
Since live crawfish are
raised in a potentially hazardous environment,
it would
warrant the inclusion of this product in future surveys for
L . monocytogenes.
Thermal Tolerance
Following
the
1983
Massachusetts
outbreak
of
listeriosis from pasteurized milk, the thermal tolerance of
L. monocytogenes was questioned
(Fleming et al.,
1985).
Since this time, Bradshaw et al. (1985) determined the D633
value to range from 13.4 to 28.4 s.
did
similar
between
studies and calculated D62 values
6 and 24 s.
intracellular
Donnelly et al. (1987)
to range
The suggestion was made that an
positioning
of
the
bacterium
protective milieu (Bunning et al., 1988).
provided
a
However, after an
extensive study involving sterile whole milk in which L.
monocytogenes was either freely suspended or located within
bovine milk phagocytes, Bunning et al. (1988) reported the
intracellular
position
of
the
bacterium
did
not
significantly increase thermal resistance.
Recent studies conducted on thermal resistance of
monocytogenes
in
red
meats
have
resulted
in
xj .
several
interesting observations.
enhance
The addition of beef fat does not
the heat resistance
of the bacterium,
but the
presence of curing salt substantially increases it (Farber
et al., 1989b; Mackey et al., 1990).
However, work by Fain
et al. (1991) with L. monocytogenes in lean and fatty turkey
meat indicated that the bacterium was more resistant to heat
when in fatty meat than in lean turkey meat.
Crawfish tail
meat is typically packaged with fat remaining.
Since the
present state of knowledge is inconclusive, the presence of
fat in crawfish tail meat should be a consideration when
conducting any thermal studies with L. monocytogenes on
crawfish tail meat.
Additionally, the heat shock phenomenon must be taken
into account when heating solids that heat much slower than
liquids.
Heat shock in this case would be defined as any
sublethal exposure to heat that alters the ability of a
bacterium to function or survive normally.
heat rapidly,
Liquids, which
do not allow bacteria acclimation time to
temperature induced stresses as do solids, in which some
limited survival may result from a bacterial adjustment to
environmental changes.
This is especially true for meats,
which heat slowly to a final internal temperature (Farber
and Brown, 1990) as is the case for 1 lb crawfish tail meat
packages.
Little research has been performed to determine the
thermal resistance of L. monocytogenes in seafoods.
Thermal
death times for L. monocytogenes in crab meat and lobster
meat have been evaluated (Budu-Amoako et al. , 1992; Harrison
and Huang,
1990) .
No research
has
been
conducted
to
determine the effect of heat on the bacterium on crawfish
tail meat.
Harrison and Huang (1990) determined D values at
55 and 60°C to be 12.00 and 2.61 min, respectively for crab
meat.
Budu-Amoako et al. (1992) determined the D60 value on
lobster for L. monocytogenes was 2.39 min.
Interestingly,
Harrison and Huang's (1990) D55.60 values ranged from 40.43 to
2.61 min when using non selective tryptic soy agar (TSA) as
the plating medium,
but when using a selective medium,
modified Vogel-Johnson agar, the D55.60 values were reduced to
34.48 to 1.31 min.
This implies that injured cells may have
been inhibited on the selective medium.
McCarthy et al.
(1990) tested the recovery of heat
stressed L. monocytogenes from artificially and naturally
contaminated shrimp.
No L. monocytogenes were recovered
from naturally contaminated shrimp boiled at 1, 3, or 5 min.
However, L . monocytogenes were detected in experimentally
internally-infected shrimp when boiled up to 5 min.
This
study also revealed the affects of freezing on heat stressed
cells
in
frozen
cooked
shrimp.
They
found
no
L.
monocytogenes cells survived the cook-freeze-thaw process.
This indicates that the combination effect of the multi­
barriers, heat then cold, can be lethal to the bacterium.
The increase in lethality observed by McCarthy et al. (1990)
resulting from exposure of the bacterium to multi-barriers
does
not
always
hold
for
other
combinations,
however.
Pickett and Murano (1993) determined that heat shock did not
result
in any significant difference in survival of L.
monocytogenes when incubated at 37°C and exposed to the
minimum
inhibitory
concentrations
of
chlorine-base,
iodophor, phosphoric acid, or guaternary ammonium compounds
or citric, lactic, or propionic acids.
The addition of a
third barrier, however, could possibly change the effect of
any one barrier, a facet the present study will investigate.
Affects of Organic Acids on L. monocytogenes
L.
monocytogenes
is more
acid
tolerant
than
most
foodborne pathogens and can grow or survive in broth media
with pH values as low as 4.3-5.2 (Ahamad and Marth, 1989;
Conner et al. 1986; El-Shenaway and Marth, 1988; Farber et
al., 1989a; Parish & Higgins, 1989).
Marth (1989) and Farber et al.
Studies by Ahamad and
(1989) indicated that the
effects of various organic acids differ.
These studies
determined that acetic acid is more detrimental to Listeria
spp.
than
Acetic
either
acid
acts
lactic,
mainly
citric
by
or
hydrochloric
lowering
the
pH
of
acid.
the
environment to the point where microorganisms can no longer
grow (Liewen and Marth, 1985).
form
of
acetic
acid
has
Although the undissociated
antimicrobial
action,
it
is
primarily the hydrogen ion and the resulting decrease in pH
that
inhibits
Fellers,
microorganisms
1939).
(Cowles,
1941;
Levine
&
Subsequent studies by Buchanan et al.
(1993) support these findings at low pH values.
However,
researchers determined that at higher pH values LA was more
effective than acetic acid.
The pH of crawfish tail meat
was determined in the present study to be 7.6, supporting
the use of LA rather than acetic acid, as a controlling
agent for L. monocytogenes.
Also the rate at which lactic
and acetic acids inactivate L. monocytogenes is dependent on
the identity of the acid, the concentration of the acid, and
the pH of the system.
The inhibitory effects of citric,
lactic and sorbic
acids can be correlated with their dissociation constants or
pKa values (Ita and Hutkins, 1991).
In general, weak acids
having higher pKa values are thought to be more inhibitory
to L. monocytogenes, at a given pH, than strong acids, at
the same pH (Ita and Hutkins,
1991) .
The amount of the
molecule in the undissociated form is determined by pH.
As
a result, this along with solubility properties, determines
the foods in which these acids are most effective (Ray and
Bullerman,
1982).
For
example
the
antimicrobial
20
effectiveness of sorbic acid increases as the pH value
approaches its pKa value of 4.75, the upper pH limit for
activity being 6.0-6.5.
Many weak acids, in their undissociated or protonated
form, have the ability to penetrate the cell membrane and
accumulate within the cell
1991).
cytoplasm
(Ita and Hutkins,
If the interior of the cell is more alkaline than
the pKa of the acid,
more of the acid will dissociate,
releasing a proton and acidifying the cytoplasm of the cell
(Booth, 1985).
These events are thought to result in a
variety of detrimental
effects
(Kashket,
1987) .
As
a
defense, many bacteria possess proton pumps or proton/cation
exchange systems to deal with the influx of protons and to
maintain the cytoplasm near neutral.
regulatory
systems
are unable
to
However, if these pH
function
sufficiently
(i.e., if the proton concentration is too great), then the
pH gradient (the difference between the intracellular and
the
extracellular
pH)
will
collapse.
Intracellular
acidification will then result in loss of cell viability or
cell destruction.
The acidified cell is forced to consume
a great
of
energy
It
has
deal
homeostasis.
internal
or
in
been
cytoplasmic
pH
an
attempt
further
is
the
to maintain
suggested
relevant
that
pH
pH
the
which
ultimately affects cell metabolic activities (Booth, 1985).
It is also established that undissociated and uncharged
weak lipophilic acids, such as acetic acid, are permeable
through the cell membrane (Kashket, 1987).
In contrast,
lactic and citric acids are generally unable to passively
diffuse across cell membranes; uptake of these acids by
cells is instead thought to be carrier mediated (Kashket,
1987) .
Ita and Hutkins (1991) determined that acetic acid
(pKa = 4.76) accumulated in the cells of L. monocytogenes at
a higher concentration than lactic (pKa = 3.86) or citric
(pKa = 3.13) acid.
Their work was consistent with other
reports showing that L. monocytogenes was most inhibited by
acetic acid, followed by lactic then citric acid (Ahamad &
Marth,
1992).
1989;
Sorrells et al.,
1989;
Young & Foegeding,
Since organic acids have been shown effective for
inhibiting the growth of L. monocytogenes,
studies that
would evaluate their effectiveness as one part in a system
of barriers to control the bacterium would be of interest.
Citric Acid
Citric acid
recognized
as
(CA)(molecular wt 192.12)
safe
(GRAS)
for use
regulations
(Food & Drug Admin.,
limits
imposed
are
Standards of Identity.
for
foods
in
1990).
not
is generally
foods
under
FDA
Thus no upper
covered
by
Federal
CA is presently used in the seafood
22
industry primarily for flavor enhancement and to prevent
discoloration.
Because CA is a weak acid having a higher dissociation
constant (pKa = 3.13) than strong acids, it's ability to
inhibit the growth of L. monocytogenes has been studied.
Ahamad and Marth (1989) determined that citric and lactic
acids,
with smaller pK„'s, were
less detrimental
monocytogenes than was acetic acid.
done by Ahamad and Marth
though
acetic
monocytogenes,
acid
(1990)
caused
to L.
In subsequent studies
it was found that even
greater
inactivation
CA caused the greatest degree of
followed by lactic and acetic acid.
of
L.
injury
CA treated cells
incurred more initial inactivation than the other two acids,
however eventually acetic acid produced more total cell
inactivation.
Ita and Hutkins (1991) observed that both citric and
lactic acids were more effective in lowering intracellular
pH of the pathogen than acetic acid.
When citric and lactic
acid-treated cells at pH values of 3.5, 4.0, and 4.5 were
incubated for 24 h, both the pH gradient (intracellular pHexternal pH)
and the intracellular pH values decreased.
They noted, however, that although the total amount of added
LA reached 160 mM, the amount of undissociated acid was less
than 50 mM (pKa of lactic acid = 3.86).
23
Lactic Acid
LA (molecular wt 90.08) is listed as GRAS in the United
States (Food and Drug Admin., 1990).
Similarly, in Europe
it is considered a harmless constituent of food
1980).
(Lueck,
The bacterial properties of LA are well documented
(Ingram et al., 1956; Van Netten and Mossel, 1980; Woolthuis
and Smulders, 1985).
Ryser and Marth (1988) studied the effect of acetic and
lactic acid on L . monocytogenes in cold-packed cheese.
They
observed the degree of dissociation related to the pKa of
acidic (4.76) and lactic acid (3.86).
They noted that at pH
5.0, 36.0 and 5.9% of acetic and lactic acid, respectively,
will be undissociated.
Therefore, the higher proportion of
undissociated acetic as compared to LA in cheese food at pH
5.0 would most likely account for the increased capability
of acetic acid to decrease a viable Listeria population.
Buchanan, et al. (1993) determined that at pH levels <6.0
lactic
acid
was
a
more
effective
bacterium than acetic acid.
inactivator
of
the
Models produced from their
studies also indicated that LA was more effective than
acetic acid for inactivating the bacterium when expressed in
terms of concentration of undissociated acid.
Ockerman et al. (1974) found that spraying 12% LA on
hot
sheep carcasses
reduced the surface aerobic colony
counts by about 1 log10 CFU/cm2 and did not bleach the meat.
Smijders et al. (1979) achieved similar reductions with only
1% LA on hot beef carcasses, however they reported that some
bleaching of product did occur.
(1985)
Woolthuis and Smulders
determined that LA solutions of
bactericidal qualities.
1.25% exhibited
When measured 24 h postmortem,
aerobic colony counts (3 d, 30°C) were reduced as much as
1.3 log10 CFU/cm2 on calf carcasses.
Additionally, 1.25% LA
solutions did not produce commercially unacceptable calf
carcass discoloration.
Ahamad and Marth (1989) observed that in tryptose broth
(TB) acidified with 0.05% LA, L . monocytogenes proliferated
under a wide range of temperatures (7, 13, 21, 35°C). This
also was the case when 0.1% LA was employed except at 7°C
where lag times were longer.
They further determined that
strain V7 of L. monocytogenes showed more resistance to LA
than strain CA.
while
no
In addition, the same study indicated that
growth
of
the
bacterium
concentration was 0.2%, it did survive.
was lethal to the bacterium.
occurred
when
acid
At 0.3 and 0.5%, LA
The rate of inactivation was
much greater at 35°C than at lower temperatures.
The D-
value of 0.3% citric acid (49.5 h) was also determined to be
higher than that of 0.3% LA (29.53 h) at 35°C. At 0.5% acid
the D-value for citric was 32.47 h and lactic was 14.6 h at
the same temperature.
Bruno and Montville (19S3) determined the mechanisms by
which bacteriocins from lactic acid bacteria inhibit L.
monocytogenes.
They found that the dissipation of the
proton motive force (PMF) is accomplished by pediocin PA-1,
AcH, leuconocin S and nisin.
These antimicrobial proteins
are classified as colicins and human defensins.
They are
characterized according to their energy-dependent or energyindependent mode of PMF depletion.
They determined that
pediocin PA-1, AcH and leuconocin S dissipated PMF in an
energy-independent fashion,
energy-dependent.
whereas action of nisin was
Morille et al.
(1993) determined that
nisin in a neutral-pH pudding product was limited as an
antilisterial agent.
However, in systems acidified to pH
>5.0, nisin inhibited the growth of the bacterium at 6, 25,
and 37°C.
Sorbic Acid
Sorbic acid (pKa = 4.75) is GRAS under regulations of
the U.S. FDA (Food & Drug Admin., 1990).
As a result no
upper limits are imposed for foods not covered by Federal
Standards
of
Identity.
In natural
cheese
that
has
a
standard of identity for example, the maximum quantity of
sorbic acid may not exceed 0.3% by weight.
The upper limits
are normally set at 0.1 and 0.2% in the U.S. in foods
26
covered by Federal Standards of Identity (Liewen & Marth,
1985).
Sorbic acid is among the most thoroughly studied food
preservatives.
Since sorbic acid is only slightly soluble
in water it is commonly solubilized with sodium or potassium
hydroxide.
The most widely used forms are sorbic acid and
its potassium salt.
These compounds are collectively known
as sorbates (Liewen and Marth, 1985) .
It has been found
non-toxic in numerous acute, subchronic and chronic toxicity
tests
(Gaunt et al.,
1975; Luck, 1976).
Sorbic acid is
metabolized in the body like other fatty acids and has a
half-life of 40-110 min, depending on the dosage (Liewen &
Marth,
1985).
As a result,
sorbic acid,
which occurs
naturally in mountain cish berries or can be chemically
synthesized
(Windholz
et
al.,
1976),
is
used
as
an
antimicrobial agent in a variety of foods (Sofos and Busta,
1981).
The acid serves to extend the storage life of such
products as butter, cheese, meats, cereals and bakery items
(Anonymous,
1979; Kaul et al., 1979), some fruits, berry
products, vegetable products and other foods
1978; Moline et al., 1963).
acid
against
various
(Anonymous,
The effectiveness of sorbic
microorganisms
has
been
reviewed
(Liewen & Marth, 1985; Sofos & Busta, 1981) .
Work
by
Ryser
monocytogenes was
and
Marth
(1988)
showed
that
L.
inactivated more rapidly in cold-pack
27
cheese food containing 0.3% sorbic acid than when it was
absent.
El-Shenawy and Marth (1988) observed the effect of
sorbic
acid
on
L.
monocytogenes
in
tryptose
broth
supplemented with potassium sorbate ranging from 0 to 0.3%.
They adjusted the pH of the broth to
5.6
or
5.0
and
incubated it at several temperatures ranging from 4 to 35°C.
The bacterium grew in potassium sorbate-free controls under
all conditions except 4°C and pH 5.0.
combinations,
as
percent
potassium
In all treatment
sorbate
inhibition of the bacterium increased.
increased,
This indicates an
inhibitory effect of sorbic acid toward the bacterium.
The
study also indicates that the behavior of the bacterium in
the
presence
of
different
concentrations
of
potassium
sorbate is affected by incubation temperature and pH of the
substrate.
Chung and Lee
(1981)
noted that potassium sorbate
delayed the onset of microbial logarithmic growth in English
sole but did not alter the rate of microbial growth nor
ultimate predominance by Pseudomonas sp. in fish stored at
1.1°C.
Cytophaga
Other
sp.,
microorganisms
such
Arthrobacter,
as
Flavobacterium-
Acinetobacter,
and
Staphylococcus disappeared from samples treated with sorbic
acid during 1.1°C storage.
Chung and Lee (1982) determined
the effect of potassium sorbate on sub-lethally injured
bacteria found on English sole.
They found that even though
28
Pseudomonas
sp.
was
relatively
resistant
to
potassium
sorbate and was not affected by 0.3% potassium sorbate after
freeze-thaw
treatment,
it
showed
delayed
onset
of
logarithmic growth for up to 20 h after heating at 50°C for
5 min.
The process by which sorbate inhibits microbial growth
is not clear and a variety of mechanisms
involved in its antimicrobial action
1985).
Melnick et al.
are probably
(Liewen and Marth,
(1954) proposed that sorbic acid
inhibits certain dehydrogenase enzymes which are involved in
the J3-oxidation of fatty acids.
proposed
that
sorbic
phosphorylation
by
Troller
proposed
(1965)
York and Vaughn
acid
inhibiting
that
uncouples
(1964)
oxidative
enzymes within
the
the
action
inhibitory
cell.
of
sorbate resulted from inhibiting catalase, which would cause
an increase of toxic hydrogen peroxide within the cell.
Freese et al.
(1973) have proposed that sorbate prevents
microbial growth by inhibiting the transport of substrate
molecules into cells.
Yousef and Marth (1983) concluded
that sorbate inhibited aflatoxin biosynthesis in the same
manner.
Inhibition of transport of carbohydrates has also
been suggested as the inhibitory mechanism of sorbic acid
(Deak and Novak, 1972).
Several investigators have shown that C02 and sorbate
in combination will
effectively
inhibit microorganisms.
Elliot et al.
(1981,
1982)
showed that C02 and sorbate
acting synergistically inhibited Salmonella enteriditis and
Staphylococcus aureus.
Carbon dioxide and sorbate will
inhibit mold spoilage of grains (Danzinger et al., 1973).
Sorbate will delay the growth of psychrotrophic bacteria in
vacuum-packed pork
sorbates
have
loins
proven
(Myers et
successful
al.,
for
1983).
Since
controlling
many
different bacteria in a variety of foods, its effectiveness
in controlling L. monocytogenes in crawfish tail meat should
be evaluated.
Modified Atmosphere Packaging (MAP)
The use of MAP to extend shelf-life of foods and
suppress the growth of bacteria has been studied extensively
(Gill
and
Tan,
1979;
Hintlian
and
Hotchkiss,
1986).
Killeffer (1930) conducted some of the earliest studies with
C02 and seafood products.
His work with cod was later
corroborated by Coyne (1932, 1933) using fish held under
high levels of C02 and by Stansby and Griffiths (193 5) who
worked with haddock.
Several subsequent studies have been
conducted showing the beneficial effects of MAP to extend
the shelf life of fresh fish (Gray et al., 1983; Mokhele et
al. 1983; Molin et al., 1983).
Przybylski et al.
(1989)
noted that while microbiological counts for catfish packaged
in C02/air (80:20) atmosphere were significantly lower than
30
those packaged in 100% air, growth did progress.
researchers have
levels
of
observed reductions
muscle
atmospheres
products
(Banks et al.,
Other
in microbiological
packaged
in
various
C02
1980; Mokhele et al,. 1983;
Parkins et al., 1981; Wang & Ogrydziak, 1986).
Studies using C02 dissolved
in refrigerated brines
improved quality of rockfish and chum
(Barnett et al.,
1971) , silver hake (Hiltz et al. , 1976) , and pink shrimp
(Bullard and Collins, 1978; Barnett et al., 1978). Brown et
al.
(1980)
stored
at
demonstrated that rockfish and silver salmon
4.5°C,
under
high
levels
of
C02, had
lower
microbial counts and less undesirable sensory changes than
controls.
They
trimethylamine
samples
held
Gerdes et al.
(TMA)
also
determined
that
levels
of
and ammonia were markedly lower in
in atmospheres
containing
20
(1989) and Wang and Brown
or
(1980)
40%
C02.
observed
these same trends for TMA and ammonia in crawfish held under
atmospheres of high C02.
Although MAP has been shown to suppress the growth of
spoilage bacteria the subsequent increased potential for the
growth
of
several
pathogenic
bacteria,
such
monocytogenes, has been questioned (LaBell, 1986).
al.
as
L.
Wei et
(1991) determined that L. monocytogenes suspended in
distilled water was completely killed after C02 treatments
at 6.18 MPa (61.2 atm) and 35°C for 2 h.
They observed a
31
similar affect of C02 on L. monocytogenes in spiked shrimp.
Garcia et al.
(1987)
and Garcia and Genigeorgis
(1987)
determined that when salmon fillets were stored at 4°C,
under several modified atmospheres (vacuum, 100% C02/ and
70% C02+30% air), Clostridium botulinum did not produce
detectable toxin for 60 d.
Toxin detection coincided with
spoilage at 30°C, but preceded spoilage at 8 and 12°C.
In studies done by Marshall et al.
monocytogenes on chicken was moderately
(1991, 1992), L.
inhibited under
refrigeration temperatures by MAP (76 and 80% C02) compared
with storage in air.
They also observed that growth was not
completely retarded nor was the organism killed by the
applied storage environments.
Kallander et al. (1991) determined that spiked cabbage
stored at 25°C under 70% C02 yielded a slightly lower count
(>1 log/g) of L. monocytogenes after 24 h of storage than
did cabbage stored in normal atmosphere (>2 logs/g).
They
also observed that during subsequent storage at 25°C, L.
monocytogenes counts decreased to undetectable levels (<20
CFU/g)
at 6 d of storage under both atmospheres.
decline,
however,
The
was more dramatic for the cabbage in
modified than in normal atmosphere between days 3 and 4 of
storage.
Ingham (1990) working with crawfish determined under
MAP (80% C02/balanced propriety) and refrigeration, growth
of Aeromaonas hydrophila and Plesiomonas shigelloides was
effectively inhibited.
Since researchers have used MAP to
successfully control L. monocytogenes
in various muscle
foods and MAP has proved inhibitory to pathogens found on
crawfish,
its application to control L. monocytogens on
crawfish warrants investigation.
MATERIALS AMD METHODS
Sample Preparation
For all studies conducted, frozen crawfish tail meat
(Procambarus clarkii) was purchased from local suppliers, in
one pound heat sealed retail packs.
The frozen meat was
stored in these retail packs at -10°C for less than 6 months
until needed.
Before using,
the frozen package was thawed under
refrigeration for 24 h.
After thawing, a mercury-in-glass
thermometer was placed
in the geometric
package through
the top
submerged
boiling
into
temperature
of
90°C
background microflora.
of the
water and
center of the
bag.The bagwasthen
heated
(approximately
10
min)
to a
to
core
reduce
The package of crawfish meat was
immediately removed and placed into an ice bath until a core
temperature of approximately 20°C was achieved.
Preparation of Inocula
L.
monocytogenes
strains
Scott
A
(Economics
Laboratories, St. Paul, MN), F5069, and S4b (C. Donnelly,
Univ. of Vermont, Burlington, VT) were
maintainedas stock
cultures through
trypticase soy 0.6%
monthly transfers on
yeast extract agar slants (TSYEA) (BBL Microbiology Systems,
Cockeysville, MD) and stored at 4°C.
33
All L . monocytogenes
34
cultures were verified to species using the API Listeria
system (bioMerieux,
La Balme-les-Grottes,
system consists of the following 10 tests to
France).
This
differentiate
between L. innocua and L . monocytogenes: presence or absence
of arylamidase (DIM test), hydrolysis of esculin, presence
of a-mannosidase, and acid production from D-arabitol, Dxylose, L-rhamnose, a-methyl-D-glucoside, D-ribose, glucose1-phosphate, and D-tagatose.
A study conducted by Bille et
al. (1992) determined that with this system, 97.7% (252 of
258) of the L. monocytogenes strains tested were correctly
identified and differentiated from 99.4% (175 of 176) of the
L.
innocua
strains tested.
included by Bille et al.
Lactobacillus
spp.,
Other gram-positive genera
(1992) were Enterococcus spp.,
Carnobacterium
spp.,
Erysipelothrix
spp., Oerskovia spp., Corynebacterium spp., and Rhodococcus
spp.
The 3 biochemical characteristics that are positive
for Listeria isolates include: hydrolysis of esculin and
acid production from D-arabitol and a-methyl-D-glucoside.
These tests easily eliminate the above listed non-Listeria
cultures.
Individual strains of L. monocytogenes were subcultured
by quiescent incubation for 20-22 h in trypticase soy 0.6%
yeast extract broth at 30°C.
Ten mL of each subcultured
strain were mixed into a sterile centrifuge tube.
The mixed
strain cells were harvested by centrifugation (3000 X g) for
10 min at 4°C
(RC5C,
Sorval Instruments,
Norwalk,
CT) ,
washed twice in sterile Butterfield Phosphate Buffer (BPB),
and resuspended to give desired target levels.
Samples were
spread plated on TSA to verify target levels.
It is known
that a particular species of bacterium grown strictly under
the same conditions will always reach approximately the same
maximum number of viable cells in a given time period
(Nickerson and Sinskey,
criterium,
1972).
Using this established
subsequent inoculation target levels for all
experiments were successfully replicated under the same
strict conditions.
Thermal Inactivation Studies
Three-neck Flask Method (TNF)
Sample preparation
The
first method employed to determine
the
lethal
effects of heat on L . monocytogenes was TNF, as described by
the Food Processors Institute (1988).
For this method 25 g
of heat treated crawfish tail meat was placed into a sterile
stomacher bag with approximately 225 mL of sterile BPB to
make a 1:10 dilution.
The sample was blended for 2 min
using a stomacher (Model STO, Tekmar, Cincinnati, OH) and
aseptically transferred to a 3 neck 1000 mL sterile reaction
flask.
36
Thermal treatment
An initial uninoculated sample was taken from the flask
to verify product sterility.
The flask containing the
sample was secured into a swirling water bath that was
maintained at the targeted study temperature.
Twenty-five
mL of a mixed cell suspension of approximately 109 cells per
mL, were aseptically pipetted into the sample slurry.
The
flask was swirled in the water bath, allowing the sample
slurry and inoculum to mix.
When the sample reached the
study temperature a zero time sample was taken.
Sample
temperature was monitored using a high precision mercury-inglass thermometer (Sargent-Welch, Skokie, IL) placed from
the top of the flask into the middle of the sample slurry.
The sample slurry remained in motion and completely immersed
during the heat treatment.
The swirling motion was stopped
for several seconds during each sampling period.
Five mL
aliquots from all 3 test temperatures (55, 60, and 65°C)
were removed at designated time intervals and placed into
sterile tubes.
Tubes were rapidly cooled by swirling in an
ice slurry, then iced for up to 1 h until analyzed.
Enumeration of L. monocytogenes
After cooling,
transferred
thoroughly.
to
9
The
1-mL portions of
mL
sterile
resulting
BPB
1:10
iced
samples were
diluent
dilution
and
was
mixed
further
37
serially
diluted
as
required.
Enumeration
of
the
L.
monocytogenes mix as conducted by spread plating 0.1 ml
dilutions on Tryptic Soy Agar (TSA) modified with 0.6% yeast
extract and 1.0% sodium pyruvate (Fain et al., 1991; Smith
and Hunter, 1988).
ambient
Plates were allowed to stand for 4 h at
temperature
(approximately
25°C)
after
spread
plating to enable recovery of injured cells (Smith, 1990).
After this period, approximately 10 mL of Listeria Oxford
Agar (LOA) (Unipath Limited, Hampshire, England) was used as
an overlay.
that
when
A study by Xavier and Ingham (1993) suggest
determining
the
survival
of
facultatively
anaerobic micro-organisms in foods that have been subjected
to heat-shock,
an anaerobic enumeration method produces
higher counts.
Their work showed that an overlay method
could
be
used
as
an
alternative
to
a total
anaerobic
enumeration method.
After being overlaid, plates were incubated at 27°C for
48 h before enumeration.
L. monocytogenes were enumerated
using standard microbial
count methods
(Messer et al.,
1985).
D value determination
A thermal death time
(TDT) plot was established by
plotting logi0 number of survivors against time for each
heating temperature (Nickerson and Sinskey, 1972).
The time
38
required to bring about a 90% reduction in the number of
survivors
(i.e.
the
D
value
for
each
treatment)
was
calculated from the slope of the best fit line representing
data on the TDT plot.
The log,0 of D value (min) was plotted
versus temperature (°C) to determine the z value.
Lotus
Freelance 3.01 Software (Lotus Corp., Orem, UT) was used to
determine slope, y intercept and goodness of fit values
(e.g., R2) for the best fit line by least squares linear
regression of the thermal death time (TDT) plot.
Polyethylene Bag Method (PB)
Sample inoculation
The second method employed 3-mil polyethylene bags (PB)
(Koch, Kansas City, MO) that have a gas transmission rate of
9 cc/m2/24 h at 0°C.
This type of PB is normally used to
commercially pack crawfish meat.
For this method, samples
were inoculated by blending 1 mL of the mixed culture (109
cells per mL) with 100 g of crawfish tail meat, prepared as
described previously,
homologous
composite
in a Waring blender to produce a
sample.
Inoculation
levels
approximately 107 CFU/g were consistently achieved.
separate
100
g
inoculated
samples
were
of
Two
aseptically
transferred to a 4000 mL collection beaker then hand mixed
under a laminar flow hood (Labconco, Kansas City, MO) for 5
39
min, using a sterile spatula.
this
homogeneous
enumerated
Inoculated
to
composite
verify
At least 2 10-g subsamples of
sample
beginning
samples were either
had L.
monocytogenes
inoculation
levels.
left untreated or spray
treated with LA as described in the following procedure.
Treatment combinations are given in Table (1).
Preparation & application of lactic acid
A stock solution of 10% LA was prepared and filter
sterilized.
Appropriate amounts of the stock solution to
produce desired concentrations of 0.5, 0.75, or 1.0% w/w
were sprayed onto inoculated crawfish using an autoclavable,
forced air, clinical atomizer.
These concentrations were
chosen because they cover the common range of usage for this
preservative.
indicated
that
Additionally,
the
a preliminary sensory study
highest
concentration
did
not
significantly alter the sensorial quality of crawfish tail
meat.
The pH of the crawfish tail meat was measured using
an Orion model 420A pH meter (Orion Research Inc., Boston,
MA).
To determine if the effect of LA was attributable to
a reduction in pH caused by the addition of LA, the pH of
additional crawfish samples was adjusted using 10% HC1 to
values approximating that of 0.5 and 1% LA.
Approximately
3.5 and 7.5 mL of HCl per 100 g of crawfish meat was
required to achieve pH values of 6.5 and 5.4, respectively.
40
Table 1.
Treatment combinations for inoculated crawfish
samples
GAS TYPE
TREATMENT
°C
CONCENTRATION
% LA
AIR
60
0.00
0.50
4
1.00
100% C02
100% 02
100% N2
AIR
100% C02
100 % 02
100% N.2
0.75
1.00
41
Treated crawfish were segregated into 10-g samples and
placed into polyethylene bags (Figure 1) .
The bags were
then heat sealed using a standard industrial heat sealer,
model LS (Rennco, Homer, MI) after forcing as much air from
the head space as possible without applying a vacuum.
Introduction of modified atmospheres
The polyethylene bags with samples were vacuum packed
using
a
Multivac
Model
A300/22
vacuum-packing
machine
(Multivac SEPP Haggenmuller K.G., Germany) and held under
vacuum for approximately 15 min.
Bags were then opened and
resealed using several methods.
For the heating trials
studying LA levels alone, bags were heat sealed with no head
space using a standard industrial heat sealer, Model LS
(Rennco, Homer, MI).
If the sample bags were to be given a
headspace of 100% air, 100% 02, 100% C02, or 100% N2 they
were backflushed with the appropriate gas using the Multivac
vacuum-packing machine.
Verification of 02 and C02 levels in
the packs was done before and after heat treatments using a
Servomex Food Package Analyzer, Series 1400 (Servomex, Inc.,
Croroborough, Sussex, England)
(Table 2).
Thermal treatment and enumeration
For all thermal treatments the sample bags were clipped
to a stainless steel test tube rack and completely submerged
in a 60°C swirling water bath.
Sample temperature was
42
HEAD SPACE
4.0 c m
Figure
1.
16.5 c m
Inside dimensions of polybag used in both
thermal and storage studies.
43
Table 2.
Gas composition as determined immediately after
introduction to sample packs and after 60°C heat
treatment
Package
Gas
Before heat
O
CO
After heat
O
CO
2
2
2
2
Air
21.6
0.6
21.1
0.3
02
99 .0
0.1
99 .2
0.1
C02
0.0
99.6
0.4
99.5
N2
0.0
0.1
0.2
0.1
44
monitored
and
thermocouples
come
up
placed
time
in
uninoculated sample bags.
recorded
the
(Figure
geometric
2)
center
using
of
2
Sample bags remained in motion
and completely immersed during the entire heat treatment.
Bags were removed at designated time intervals and agitated
in an ice bath for rapid cooling until analysis.
After cooling, a zero time sample was immediately taken
by aseptically removing it from the heat sealed bag and
placing it into a stomacher bag to which a sufficient volume
of sterile BPB was added to achieve an initial dilution of
1:10.
The diluted sample was homogenized by stomaching
(Tekmar) for 2 min and additional decimal platings were made
by removing 0.1-mL from serially diluted samples.
procedure was repeated for all samples.
This
Enumeration of L.
monocytogenes was done as previously described.
D value determination
D values (min) for the polybag method were determined
as described previously for the TNF method.
D value means
for thermal treatments of different LA concentrations and
all atmospheres were subjected to ANOVA for determination of
significant differences at the 5% probability level using
Minitab Statistical Software (Minitab, Inc., PA).
Student's
t-test was used to quantify significant (P<0.05) differences
between means of each individual analysis.
Temperature i°C)
45
Time (min)
Figure 2.
Center temperature for come-up and cool-down
times for 10 g samples of crawfish tail meat in
polybags (PB) and polybags with head space (PB-a)
and cellulose casings (CC).
46
Cellulose Casing Method (CC)
Sample inoculation
A third heat treating method was utilized to determine
a D60 value for L. monocytogenes in crawfish tail meat.
This
method utilized 18 mm EZ Peel Nojax cellulose casings (CC)
(Viskase Corp.,
Chicago,
IL) .
Cellophane and cellulose
casings as a vessel for ground or emulsified food products
inoculated with a variety of bacteria are used extensively
for D value determinations (Zaika et al., 1990).
For this method crawfish tail meat was prepared and
inoculated as described
inoculated
product
was
for the PB method.
aseptically
stuffed,
sterilized glass funnel, into individual casings.
Ten g of
using
a
Casings
were then sealed by tieing off before being secured to test
tube racks and placed
treatment.
into the water bath for thermal
Cell enumeration and D value determination was
accomplished as described for the PB method.
No additional
treatments other than heat were applied to the crawfish tail
meat using this method.
D60 value means for all three thermal treatment methods
(TNF, PB, and CC) were subjected to ANOVA for determination
of significant differences at the 5% probability level using
Minitab Statistical Software (Minitab, Inc. PA).
47
Sensory Evaluation
A trained sensory panel of 15 judges was used to
evaluate all LA treatment levels.
A single training session
that involved group sampling of crawfish tail meat treated
with known levels of LA was conducted.
During the training
session open discussion of any observed sensory alterations
due to LA on crawfish meat was discussed among panelist.
All levels of LA (0.5, 0.75 and 1.0%) that would be tested
for by the taste panel were introduced to the panelist
during the training
session.
Since these
levels were
relatively low and difficult to detect, samples treated with
10% LA also were used to sensitize the panel to the taste of
LA in crawfish tail meat.
Uninoculated crawfish tail meat samples were spray
treated with 0.0, 0.5, 0.75, and 1.0% LA then sealed in
heatable polyethylene bags (Koch, Kansas City, MO).
They
were heat treated at 60°C for 10 min in a swirling water
bath
as
described
previously.
Samples
were
tested
immediately after treatment then held at 4°C and tested
after 2 and 5 days of storage.
given to the sensory panel,
Immediately prior to being
the samples were placed in
boiling water for 10 min then cooled to ambient temperature
to assure product safety.
Samples were presented to panelists at room temperature
under
white
fluorescent
lighting.
All
panelist
were
48
isolated from one another during sampling periods.
For each
LA treatment level, 2 segregated 20-g samples were used to
evaluate four attributes.
The samples were evaluated on a
10-point descriptive scale for odor (10=clean odor, l=strong
odor),
color
look),
(10=bright
taste
excellent
(10=clean
look,
desirable
l=graying dull
flavor,
l=strong
acid/undesirable flavor), and texture (10=tender, l=tough).
Mean scores of each attribute for all LA treatment
levels
were
subjected
to
ANOVA.
Determination
of
significant differences was done at the 5% probability level
using Minitab Statistical Software (Minitab, Inc., PA).
Storage Studies
Effect of Storage at 0, 6, & 12°C
Preparation of inoculated samples
One hundred g of heat treated crawfish tail meat was
placed into a Waring blender (Waring Products, Co., Winsted,
CT) prior to introduction of the inoculum.
One mL of the
diluted mixed L. monocytogenes culture (107 cells per mL)
was added to the 100-g sample and blended to obtain a
uniform distribution of cells.
Each 100-g portion was
aseptically transferred to a 4000 mL collection beaker after
inoculation.
The inoculation procedure was repeated 6 times
in the same beaker until 600 g of crawfish tail meat was
uniformly
inoculated.
The
total
inoculated
600
g
of
49
crawfish meat in the collection beaker was then hand mixed
aseptically using a sterile spatula, under a laminar flow
hood
(Labconco,
Kansas
City,
MO)
for
5
min.
This
homogeneous composite sample contained approximately 10s
CFU/g
inoculum.
Negative
control
samples
were
left
uninoculated to test for sterility.
After inoculating the crawfish tail meat, 25-g samples
were aseptically placed into sterile, 6-oz whirl pack bags
and randomly assigned to 3 storage treatments of 0, 6, and
12°C for 20 d.
Additionally, negative controls of 25-g
portions were distributed to storage treatments.
Enumeration of bacteria
Two replicate experiments were conducted with each
replicate consisting of 3 storage temperatures (0, 6, and
12°C). Samplings were made at 2-d intervals for 0 and 6°C,
and at 1-d intervals for 12°C stored samples.
For each
sampling period, 2 25-g samples were analyzed to enumerate
L.
monocytogenes.
In
addition,
a
25-g
portion
of
uninoculated meat was used to verify the sterility of the
initial uninoculated crawfish tail meat.
aseptically
individually
removed
into
from
a
previously described.
a
"whirl
stomacher
bag
Each sample was
pack"
and
and
placed
stomached
as
50
Enumeration of L. monocytogenes was accomplished by
duplicate surface platings of serially diluted samples made
on LOA (Unipath)•
Counting of the bacterium was conducted
using standard microbial count methods (Messer et al., 1985)
Inoculated plates were incubated at 27°C for 48 h prior to
counting.
Mean values for each treatment were reported as
the average of duplicate platings of all sampling points
from a given time period.
The number of bacteria present on
the crawfish meat was determined for each sampling and
expressed as log10 CFU/g.
Calculation of Generation Time
The generation time of L. monocytogenes in the crawfish
tail meat was determined for each temperature using the
following formula (Marshall and Schmidt, 1988) . Two points
on the logarithmic growth phase of each curve were used in
the calculation.
Generation times were reported as the time
required
monocytogenes populations to double
for L.
number.
Generation Time = (0.301(T2 - TJJ/flog P2 - logP,)
Where: Ti =
T2 =
P, =
P2 =
time of Pi
time of P2
CFU/g at T!
CFU/g at T2
in
51
Effect of Citric Acid & Potassium Sorbate on Growth of L.
monocytogenes
Preparation of inoculated samples
Initial storage studies were done with crawfish samples
treated with potassium sorbate (PS) and citric acid (CA) .
Crawfish tail meat
samples were stored and prepared for
inoculation as described previously in "Crawfish tail meat
sample preparation".
also
were
"Preparation
Working cultures of L. monocytogenes
subcultured
of
as
inocula".
described
Samples
previously
were
inoculated
in
as
described previously for thermal inactivation studies using
the polyethylene bag method in "Sample inoculation".
cultures
were
serially
diluted
before
using,
inoculation levels of approximately 105 CFU/g.
Mixed
allowing
Six separate
inoculated 100 g samples were aseptically transferred to a
4000 mL collection beaker then hand mixed as described
previously.
PS
or
CA
were
prepared
as
individual
solutions and sterilized by filtration.
10%
stock
Three ml of stock
solution were sprayed onto 100 g of inoculated crawfish to
achieve a 0.03 g/kg (0.3% w/w) concentration.
The spraying
procedure employed the same method described for thermal
studies with LA.
Typical use levels in foods for PS and CA
range from 0.002 to 0.3% (Liewen and Marth, 1985).
Treated
and untreated samples were then segregated aseptically into
52
15-g sub-samples that were placed into the same type 3-mil
polyethylene bags (Koch) used for thermal studies.
Effect of Lactic
monocytogenes
Acid
& Atmospheres
on
Growth
of L.
Preparation of inoculated samples
For
storage
atmospheres,
5
studies done with
mL
of
each
LA and various
subcultured
transferred to a sterile centrifuge tube.
strain
gas
were
The crawfish tail
meat samples were then secured in a 60°C swirling water bath
and heated for 12.5 min.
This procedure yielded cell counts
of
CFU/g.
approximately
106
Of
approximately 20% were heat damaged.
this
population,
The number of heat
damaged cells was determined by the difference in number of
CFU recovered on LOA divided by the number of CFU recovered
on TSA.
Crawfish tail meat samples were inoculated as described
previously
for
thermal
polyethylene
bag
Inoculation
levels
inactivation
method
of
consistently achieved.
in
studies
"Sample
approximately
Nine separate
104
using
the
inoculation".
CFU/g
were
100 g inoculated
samples were aseptically transferred to a 4000 mL collection
beaker then hand mixed as described previously for thermal
inactivation studies.
One percent LA treated samples and
untreated controls were prepared for storage as described
above.
Treatment combinations are given in Table (1).
The
53
percentage of 02 and C02 for all package atmospheres was
determined
using
the
Servomex
Food
Package
Analyzer
(Servomex) both before and after a 21 d storage period at
4°C (Table 3).
Enumeration of bacteria
Bacteria were enumerated using standard microbial count
methods as described previously (Messer et al., 1985).
At
2-d intervals, 15-g crawfish tail meat samples treated with
PS or CA were used for L. monocytogenes enumeration.
For
samples treated with LA and various gas atmospheres, counts
were made at 3-d intervals.
Samples were prepared for
enumeration as described previously for thermal inactivation
studies of the polyethylene bag method.
Enumeration of L.
monocytogenes was accomplished by duplicate surface platings
made on LOA (Unipath). Inoculated plates were incubated at
27°C for 48 h prior tocounting.
Experimental protocol
Three replicate experiments were conducted at 4°C.
each
sampling point, one randomly
portion
of crawfish
tail
meat
selected
was
used
microbial populations for each replicate.
15 or
to
At
25-g
enumerate
In addition, a 15
or 25-g portion of uninoculated meat was used to verify the
Table 3.
Gas content before and after 21 d storage at 4°C
of package atmospheres and pH of crawfish tail
meat treated with or without lactic acid (LA).
Package
Gas
Before storage
pH
02
C02
After storage
pH
02
C02
Air
+1% LA
7.6
5.4
21.6
21.4
0.5
0.4
7.6
5.5
15.4
15.9
0.5
0.3
02
+1% LA
7.6
5.4
99.1
99. 3
0.1
0.1
7.6
5.4
98.0
97.8
0.5
0.9
C02
+1% LA
7.6
5.4
0.0
0.0
99.7
99.5
6.8
5.4
ND
ND
ND
ND
n2
+1% LA
7.6
5.4
0.1
0.0
0.0
0.0
7.6
5.4
0.2
0.0
0.5
0.0
ND - Gas content of packages back filled with 100% C02 lost
all head space during storage resulting in non-readable
head space gas content.
55
sterility of the initial, uninoculated crawfish tail meat.
Mean values for each treatment were reported as the average
of duplicate platings of all sampling points of a given time
period.
The number of bacteria present on the crawfish meat
was determined for each sampling and expressed as log10
CFU/g.
Generation time means for each treatment combination
were subjected to ANOVA for determination of significant
differences
at
the
5%
probability
level
Statistical Software (Minitab, Inc., PA).
3.01
software
(Lotus
Corp.,
Orem,
UT)
using
Minitab
Lotus Freelance
was
used
in
determining slope, intercept, and goodness of fit values
(e.g., R2) of log CFU/g versus temperature and best fit line
by least squares
linear regression,
growth phase of L. monocytogenes
1993).
of the exponential
(Dogra and Schaffner,
Slopes resulting from the exponential growth phase
of the bacterium for each treatment were also compared using
ANOVA.
RESULTS AND DISCUSSION
Low Temperature Growth
Growth of L. monocytogenes on precooked crawfish tail
meat stored at 0, 6, and 12°C, either to stationary phase or
20 d, are shown in Figure 4.
The initial population of L.
monocytogenes on inoculated crawfish samples held at 0 and
6°C was approximately 104 CFU/g.
At 0°C, less than 1 log10
CFU/g growth was observed for the entire storage time of 20
d (Figure 3).
Similar results were reported for shrimp held
on ice where no increase in L. monocytogenes populations
occurred for 21 d
(Harrison et al.,
1991).
An initial
decrease in the population of L. monocytogenes stored at 0°C
was observed (Figure 3) . Harrison et al. (1991) showed that
shrimp samples held at -2 0°C had a decrease in Listeria spp.
populations of less than 1 log when stored for 3 months.
Any inhibitory environment that the bacterium is exposed to
in cold storage seems to cause an initial decrease
population.
If
the
bacterium
can
adjust
to
the
in
0°C
environment, growth ensues.
Generation time of L. monocytogenes incubated at 0°C
was approximately 72.2 h, dramatically longer than 17.0 or
6.9 h at 6 and 12°C, respectively.
crawfish
tail
meat
is
Consequently, as long as
constantly
monocytogenes will grow poorly.
56
held
at
0°C,
L.
57
10
-
o°c
4°C
6°C
Log CFU/g
12°C
0
2
4
6
8
10
12
14
16
20
Storage Time (days)
Figure 3.
Growth of L. monocytogenes on crawfish tail meat
stored at 0, 4, 6, and 12°C.
Studies conducted by Glass and Doyle (1989) indicated
that at 4.4°C the rate of growth for L.
depended largely upon product type and pH.
storage
study,
crawfish
tail
meat
monocytogenes
In the present
inoculated
with
approximately 104 CFU/g began exponential multiplication
immediately with no observed lag phase at 6°C (Figure 3).
A 1 log increase per 2-d period was observed until 10 d, at
which time maximum population density for the bacterium on
crawfish tail meat and stationary phase was reached.
This
temperature is important when determining the growth rates
of L. monocytogenes in crawfish tail meat since 6°C (42.8°F)
is close to the temperature of many retail display cases and
home refrigerators.
Because L. monocytogenes multiplies
rapidly at this storage temperature, it may be beneficial to
further heat process precooked crawfish tail meat prior to
distribution.
At
12°C,
approximately
the
initial
105
CFU/g
L.
monocytogenes
underwent
rapid
count
of
exponential
multiplication, with no observed lag phase, for only 3 d
before maximum population density
for the bacterium on
crawfish tail meat and stationary phase was reached (Figure
3).
Even
though
refrigeration,
temperature
it
if
is
12°C
is
possible
product
transportation or storage.
not
to
considered
reach
mishandling
this
occurs
proper
abusive
during
Crawfish tail meat, stored at
59
abusive temperatures even for short periods of time, could
support rapid growth of L. monocytogenes and should be
regarded as unsafe for consumption unless it undergoes an
additional heat treatment.
In the seafood industry, a 7 to 10 d shelf life is
typical for fresh, iced, products.
Czuprynski et al. (1989)
observed that L. monocytogenes at reduced temperature (4°C)
increased in virulence in intravenously inoculated mice,
although it did not appear to affect mice that had been
infected orally.
bacterium
Thus, an increase in the virulence of the
in refrigerated foods may
be possible.
The
present storage studies indicated that L. monocytogenes can
flourish on precooked packaged crawfish tail meat when
stored under refrigeration temperatures for several days.
The generation time for L. monocytogenes on crawfish
tail
meat
products.
appears
shorter
than
for
other
non-seafood
Generation times for the bacterium at 4°C on beef
(74.1 h), beef extract (50.6 h) (Shelef, 1989), and chicken
(43.3 h) (Marshall et al., 1991) have been determined.
asparagus,
broccoli,
and
cauliflower,
the
On
bacterium
experienced good multiplication when stored at 4°C (Berrang
et al., 1989).
The generation time for the bacterium on
seafood products,
especially crustaceans,
lower than on non-seafood products
appears to be
(Figure 4) .
Farber
(1991) reported the generation time for the bacterium at 4°C
Beef S.Milk Lettuce B. Ext. Chick. Salm. CrawfishShrimp Crab Lobster
*Generationtimeforlettucewas determined at5°C
Figure 4.
Generation times for L. monocytogenes in
different food products at 4°C.
61
on crustaceans such as crab,
16.9,
15.5
directed
at
and
25.3h,
lobster, and shrimp, to be
respectively.
determining
why
the
Further
bacterium
research
grows
so
successfully on shellfish products, may unlock a key to
controlling the bacterium on all food products.
Storage Studies Using Citric Acid & Potassium Sorbate
Chung and Lee (1981) observed that PS delayed onset of
exponential growth but did not alter rate of microbial
growth nor ultimate predominance of Pseudomonas sp.
English sole stored at 1.1°C.
in
The present study notes the
same effect when 0.03 g/kg PS was sprayed onto crawfish
inoculated with L. monocytogenes and held at 4°C (Figure 5) .
The lag phase of the bacterium was extended by 2 d on
treated crawfish.
Once exponential growth began, however,
the generation time was not significantly different (P>0.05)
than the generation time for untreated crav/fish meat (Table
4).
Additional studies by Chung and Lee (1982) evaluating
the effectiveness of PS for controlling growth of several
seafood bacterial isolates (Arthrobacter sp., Moraxella sp,
and Pseudomaonas sp.) found PS treatments ineffective unless
amounts
as high
as
2.73%
were
used.
Considering
the
findings of Chung and Lee (1981, 1982) and those of the
present study, additional study of PS was not pursued.
62
7.6
7.4
7.2
Control
CA
PS
6.8
6.6
Log CFU/g
6.4
6.2
6.8
6.6
6.4
6.2
4.6
4.6
0
2
4
6
8
10
STORAGE TIME (days)
Figure 5.
Growth of L . monocytogenes on crawfish tail meat
at 4°C and treated with 0.03 g/kg Citric Acid
(CA) or Potassium Sorbate (PS).
63
Table 4. Generation times, least squares regression factors
and goodness of fit for Listeria monocytogenes in
cooked crawfish tail meat treated with citric acid
(CA) or potassium sorbate (PS).
Treatment
Generation
Time (h)
Y-intercept
(Log10 CFU)
Slope
R2
none
28.5
5.45
0.239 + 0.028
0.958
CA
29.5
5.47
0.229 ± 0.029
0.953
PS
28.1
4.30
0.261 + 0.029
0.977
Samples sprayed with 0.03 g/kg CA supported growth of
L.
monocytogenes
(Figures) .
equally
well
as
untreated
samples
The generation time of the bacterium on CA-
treated samples was not significantly different
than on untreated samples
(Table 4) .
(P>0.05)
The present study
indicated that 0.03 g/kg CA had no inhibitory effect against
L. monocytogenes on cooked crawfish meat.
These results
indicate that post processing sprays of 0.03 g/kg PS or CA
would have little or no benefit in adding any degree of
safety against L. monocytogenes on crawfish tail meat.
As
a result, additional studies using mixed barriers and CA or
PS were not perused.
However, as discussed previously other
researchers have had success controlling the growth of L.
monocytogenes
using
CA
at
higher
concentrations.
Consequently, additional research directed at the use of CA
as a barrier for controlling L. monocytogenes should not be
overlooked in the future.
Thermal Inactivation
The inoculation levels (log10 CFU/ml) at time zero for
each temperature tested were 9.6 (55°C) , 9.3 (60°C) , and 8.3
(65°C). The heat resistance of L. monocytogenes in crawfish
tail meat, as designated by D values (min) determined using
TNF methodology, is shown in Table 5.
From these data, a
TDT plot was constructed showing a z value of 5.5°C.
65
Table 5. Thermal death times, least squares regression
factors and goodness of fit for D value
determinations using TNF methodology for L.
monocytogenes in crawfish tail meat.
Temperature
°C
D values
(min) ±s.d.
Y intercept
(log10)
Slope
W
55
10.23 ±0.32
9.56
-0.783
0.929
60
1.98 ±0.15
8.48
-0.550
0.952
65
0.19 ±0.01
7.91
-0.080
0.987
66
Regression analysis values for intercept, slope, and R2 of
the log10 survivors versus temperature are shown in Table
5.
D value ranges for L. monocytogenes on crawfish tail
meat determined using the TNF method were very similar to
those determined previously on crab and lobster meat (BuduAmoake et al. , 1992; Harrison and Huang, 1990).
Harrison
and Huang (1990) determined D values for the bacterium on
crab
meat
at
respectively.
55
and
60°C
to
be
Budu-Amoake et al.
12.00
(1992)
and
2.61
min,
determined a D
value for L. monocytogenes on lobster to be 2.39 min at
60°C.
They calculated a z value for the bacterium of 5°C.
D values for crawfish meat obtained using the TNF method
were slightly lower than in both of those studies.
Crawfish
tail meat is packed with adherent hepatopancreas and fat
having a total lipid content of 0.939 g/100 g(USDA, 1987).
Crab meat has a total lipid content of 1.188 g/100 g (USDA,
1987).
The higher D value observed for crab meat at 60°C
might be attributable to the protective characteristics of
fat (Mackey et al., 1990).
However, this did not appear to
be the case when comparing the D values of crawfish tail
meat to that of lobster which has a total lipid content of
0.358 g/100 g (USDA, 1987).
Other factors may have contributed to the lower values
observed in the TNF thermal study.
The previous studies
67
done with crab meat and lobster, both used non selective TSA
to enumerate L. monocytogenes.
The present study used TSA
as a base medium and allowed a 4 h
resuscitate injured cells.
then
added.
The presence
recovery time
to
An overlay of selective LOA was
of the
antimicrobial
agent,
acriflavine, in this selective medium may have inhibited the
growth of some heat damaged cells that were unable to
recover completely before being overlaid.
Inhibition would
cause the recovery of fewer cells than had actually survived
thermal treatment, thus yielding a lower D value than the
previous two studies.
When Harrison and Huang (1990) used
Modified Vogel-Johnson agar, a more selective medium than
TSA, to enumerate L . monocytogenes from crab meat, fewer
bacteria were recovered.
The D values for 55 and 60°C using
this medium were 9.18 and 1.31 min,
respectively;
both
values are lower than those observed in the present study.
Additionally,
the use of a mixed strain inoculum in the
present study, as opposed to strain Scott A singly, may have
been a contributing factor producing the reported variable
results.
D60 values for L . monocytogenes differed (P<0.05) when
various methods of determination were employed (Table 6).
The TNF method required the crawfish samples to be suspended
in an aqueous environment, which leaves the bacterium less
protected by constituents of the crawfish meat.
Therefore,
68
Table 6. Thermal death times, least squares regression
factors and goodness of fit for D60 values
determinations of L. monocytogenes using various
methods in crawfish tail meat.
Slope
Std. Err.
of slope
R
Method
D values
(min) ±s.d.
TNF
1.98 ±0.15a
-0.550
0.078
0.952
PB
4.68 ±0. 56b
-0.215
0.012
0.979
CC
3.84 ±0.23b
-0.251
0.012
0.987
* D values with similar superscripts are not significantly
different (P>0.05).
69
a lower D value can be expected when using the TNF method
over the PB or CC methods.
The D60 valuesdetermined using
the PB and CC methods did not differ (P>0.05) (Table 6).
Traditionally TDT tests to determine D values
for
bacteria in foods have utilized TDT tube, TDT can, or TNF
methods.
meats.
The use of CC method is one commonly used for
Since crawfish tail meat is typically packaged by
the industry in polyethylene bags, the present study used
this type of container and developed a new method (PB) for
assessing thermal tolerance of bacteria.
methods
yielded
monocytogenes.
similar
D
values
The CC and PB
(P>0.05)
for
L.
The D value determined using TNF method was
significantly less than both the CC and PB method (P<0.05) .
Other D value determinations discussed later, done using PB
method with head space (PB-a) yielded significantly (P<0.05)
higher D values
space.
(10.58 min)
Variations
than PB or CC without head
in D values observed with different
methods indicates that methods used for the determination of
D values for L. monocytogenes on crawfish tail meat are
critical.
Since
researchers
have
used
many
different
methods to determine D values for the bacterium, it is very
difficult to accurately compare behavior of the bacterium in
different foods.
When head space was added to the PB samples (PB-a) , the
D60 values (10.58 CFU/g) increased significantly (P<0.05).
Heating profile of PB-a samples was about 0.5 to 1.0 min
slower to reach
60°C than the PB samples (Figure 3). This
may have been caused by an air insulation effect due to the
headspace.
samples
The head space
forces
product
polyethylene bags.
in PB-a crawfish tail meat
away
from
some
walls of
the
This created a situation where less
surface area of crawfish sample was in proximity to liquid
heating medium (water), thus reducing the speed at which the
core temperature reached 60°C.
The reduction in come-up
time would allow L. monocytogenes more time to acclimate to
the rise in temperature and reduce the accumulation of
lethality units (Nickerson and Sinskey, 1972).
As a result,
fewer bacteria were killed with the PB-a method than for CC
and PB methods
under similar time
frames.
It is also
possible the head space may have allowed the thermocouples
to shift slightly during agitation in the water bath.
A
slight shift from the geometric center of the samples may
have caused the centers of the crawfish tail meat samples to
remain at a slightly cooler temperature.
This would happen
because the thermocouple would have indicated that samples
had reached the targeted treatment temperature premature to
the actual occurrence.
The resulting lower core temperature
of the samples would reduce the effectiveness of the thermal
treatment, yielding higher D values.
71
Thermal Inactivation with Lactic Acid
A
D60 value
monocytogenes
method.
heat
of
on
4.68 min
untreated
was
calculated
crawfish
tail
using
for L.
the PB
LA sprays were found to enhance the lethality of
against
L.
monocytogenes
(Table
7) .
Although
a
stepwise increase of lethality of heat against the bacterium
was observed between the control,
0.5, and 0.75% LA, no
statistically significant improvement (P<0.05) in lethality
was noted until treatments reached 1.0% LA (Table
1.0% LA treatment
7).
A
increased lethality of heat during a 14-
min thermal treatment by approximately 4 logs over the
control (Figure 6).
D60 values calculated for L. monocytogenes on crawfish
tail meat that was pH adjusted using HC1 to approximate the
pH of 0.5 and 1.0% LA were 4.92 and 3.54 min, respectively.
There was no significant difference between crawfish tail
meat controls and samples adjusted with HC1 to approximate
the pH of 0.5 or 1% LA treatments (Table 7).
Although the
D60 value (Table 7) and resulting linear regression of TDT
slope (Figure 7) for L. monocytogenes on crawfish tail meat
samples pH adjusted with HC1 to approximate that of 1.0% LA
was smaller than the D60 for controls, it was significantly
greater
(P<0.05)
than
1.0%
LA
treated
samples.
indicates that while a reduction of pH may increase
This
72
Table 7.
Least squares regression factors and goodness of
fit for D60 values for L. monocytogenes on crawfish
tail meat treated with different levels of lactic
acid or HC1.
Std. Err.
of Slope
R*
Acid
%
Acid
PH
D-value
Slope
LA
0.00
7.6
4. 68“
-0.215
0.013
0.979
LA
HC1
0.50
0.35
6.4
6.5
4 .41a
4.92s
-0.237
-0.216
0. 019
0. 026
0.962
0.923
LA
0.75
6.3
3 .46a
-0.289
0. 013
0.989
LA
HC1
1.00
0.75
5.5
5.4
2.49b
3. 54a
-0.417
-0.282
0.015
0.019
0.994
0.973
a,b D values with similar superscripts are not significantly
different when compared to the control (P>0.05).
73
9
8
7
Log CFU/g
6
5
4
0. 0 %
3
0 .5 %
0 .7 5 %
2
1 .0 %
1
0
2
4
6
8
10
12
14
TIME (min)
Figure 6.
Thermal death curve for L. monocytogenes at 60°C
using various levels of spray applied lactic
acid.
74
Log CFU/g
10
CONTROL {pH 7.6)
* - 0.215 ±
- -
HCI (pH 5.5)
* - 0.282 ±
-
2
.02
1.0% LA (pH 5.5)
* - 0.417 ±
0
.01
4
.02
6
8
10
12
14
16
Treatment Time (min)
* Slope + s.d. of least squared regressions
Figure 7.
Linear regression comparing the effect of 1%
lactic acid and the equivalent HCI adjusted pH,
on lethality of 60°C toward L. monocytogenes on
crawfish tail meat.
75
lethality of heat towards L. monocytogenes,
LA augments
lethality independent of pH.
To date, the exact mechanism that makes environments
containing
weak
acids
like
LA
more
lethal
to
L.
monocytogenes than an equivalent low pH environment altered
with strong acids has not been defined.
It has, however,
been established that weak acids in their undissociated or
protonated form have the ability to penetrate the cell
membrane and accumulate within the cell cytoplasm (Ita and
Hutkins,
1991) .
It may be that LA simply is able to
penetrate the cell more effectively than other acids.
Once
the acid penetrates the interior of the cell, it releases a
proton and acidifies the cytoplasm of the cell
1985).
(Booth,
The inability to maintain a proper pH gradient via
a proton/cation exchange system, coupled with the exhaustion
of energy needed to maintain normal metabolic processes,
results in the cells inability to survive (Kashket, 1987).
Continued
research
directed
at
establishing
the
exact
mechanism is certainly warranted.
Sensory Evaluation
Sensory analysis was conducted to determine the effect
of study levels of LA on product acceptability.
Although
the sensory panel found no significant difference (P>0.05)
for all characteristics tested between controls and any LA
76
treated samples
(Table 8) , the panel observed a slight
toughening of the product when LA at any level was added.
The panel also noted that the typical fishy odor and taste
of crawfish was reduced with application of any level of LA.
Additionally, LA treated crawfish appeared to be brighter in
color.
Many panelists found these characteristics to be a
desirable change.
While
crawfish
tail meat was
stored
at 4°C under
various gases, browning of the samples was visually observed
in packages containing 02 after 6 d, when compared to other
samples packed without 02.
Paralleling this observation,
Bentley et al. (1989) observed no surface discoloration on
beef patties packaged under 100% N2, 100% C02, or Vacuum and
stored
at 0, 4,
evaluated,
or 8°C for up to 21 d.
Of atmospheres
theydetermined that beef patties stored in a C02
environment produced the highest values for all sensory
traits evaluated, including tenderness, juiciness, flavor,
and overall desirability.
stored
in 100%
packed
in all
When crawfish tail meat samples
C02 were visually compared with
other
atmospheres, an
brightness was observed.
increase
samples
of
This occurrence in crawfish has
been previously quantified by Gerdes et al. (1989).
a
HunterLab
color
color
difference
meter
(HunterLab
Using
Assoc.,
Richmond, VA) , they documented an increase in redness color
77
Table 8.
Taste panel results of crawfish samples packed
under air and stored at 4°C. Values of 5.0 =
standard quality, >5.0 = less favorable quality,
and <5.0 = more favorable quality.
%LA
DAY
0.00
0
2
5
0.50
ODOR
COLOR
TASTE
TEXTURE
4.3±1.3
4.7±1.4
5.Oil.5
4.311.2
4.411.2
4.511.2
4.411.4
4.811.5
4.511.3
4.311.4
4.511.4
4.811.3
0
2
5
4.811.7
4.611.4
4.711.1
4.411.5
4.311.2
4.410.9
4.611.8
4.511.4
4.411.1
4.211.5
4.711.5
4.811.2
0.75
0
2
5
4.611.0
4.211.1
5.111.5
4.411.0
4.211.2
4.611.0
5.011.2
4.011.4
5.411.4
4.711.3
4.311.5
5.311.2
1.00
0
2
5
4.611.5
4.611.1
5.111.1
4.511.0
4.411.4
4.611.2
4.811.4
4.711.2
5.011.5
4.511.1
5.011.3
4.911.1
78
values when crawfish samples were stored on ice in high C02
atmospheres for 21 d.
Consumers may find this change
desirable, however, additional consumer surveys would be
required to determine this.
In general it appears that LA treatment levels used for
the present study are acceptable sensorially.
As a result
the use of these levels on crawfish tail meat could be
recommended.
Heat and Modified Atmospheres
During 60°C heat treatments of up to 20 min,
gas
measurements of package headspace revealed no meaningful
changes in C02 or 02 composition of any atmospheres studied
(Table 2).
The TDT curves for L. monocytogenes on crawfish
tail meat are shown in Figure 8.
There was no statistical
difference in D60 values for L. monocytogenes on crawfish
meat packed in any of the 4 atmospheres
(Table 9) .
D60
values for L. monocytogenes on crawfish meat packed in
atmospheres that contained 02 were lower, however than for
atmospheres which were void or limited of 02 (Table 9) .
Recent research conducted on Escherichia coli as well
as
L.
monocytogenes
decreased
atmospheres
thermal
rich
may
offer
resistance
in
02.
an
of
Murano
explanation
L.
for
the
monocytogenes
in
and
Pierson
(1993)
determined D value of heat-shocked E. coli 0157:H7 to be
79
9
8
7
Log CFU/g
6
5
Air
4
CO
3
2
0
4
8
12
16
20
TREATMENT TIME (min)
Figure 8.
Thermal death curve for L. monocytogenes on
crawfish tail meat at 60°C packed under various
atmospheres.
80
Table 9.
Least squares regression factors and goodness of
fit for D-value determinations after 60°C thermal
treatment of crawfish tail meat treated with or
without 1% LA and packed in different modified
atmospheres with head space.
Atmosphere
Type
pH8
D-value
Slope
Std. Err.
of Slope
Air
+1% LA
7.6
5.4
10.58b
3.44c
-0.097
-0.360
0.003
0.031
0. 995
0.986
o2
+1% LA
7.6
5.4
9.22b
3 .88°
-0.110
-0.274
0. 007
0. 031
0.983
0.975
C02
+1% LA
6.6
5.4
11. 68b
3 .48c
-0.088
-0.313
0.007
0.037
0.975
0.973
n2
+1% LA
7.6
5.4
12 .97b
3.08°
-0.081
-0.340
0.005
0.014
0.983
0.996
a pH of samples are after heating at 60°C
b,c D values with similar superscripts are not
significantly different (P>0.05).
R2
higher when cells were incubated anaerobically than when
incubated aerobically.
It has been theorized that severely
heat-injured cells acquire a strict anaerobic nature (McCord
et
al., 1971).
Thus, any cell damage due to oxidation
reactions,
which
are
diminished
in oxygen
normally
free
enhanced
by
environments.
heat,
are
Additionally,
Dallmier and Martin (1988) showed that after 10 min at 60°C,
L. monocytogenes (Scott A) retained only 42% of its initial
catalase
(CAT) activity.
It has been hypothesized that
inactivation of enzymes like CAT and superoxide dismutase
(SOD) during heating, would interrupt the cells ability to
deactivate toxic oxygen radicals during subsequent recovery
periods.
radicals
The result would be a buildup of toxic oxygen
in an 02 rich environment.
Consequently,
the
inactivation of these enzymes converts L. monocytogenes into
an obligate anaerobe (Knabel et al., 1990).
The addition of 1% LA to crawfish tail meat caused a
significant
decrease
(P<0.05)
in
D60
values
for
L.
monocytogenes, regardless of packaging atmosphere (Table 9) .
There was,
however,
no difference
environments (Figure 9).
(P>0.05)
between gas
Presence of LA masked the affect
of an 02-free environment on thermal resistance.
D60 values
determined for L. monocytogenes on crawfish tail meat using
1% LA and head space containing molecular 02, were higher
than D60 values determined from samples packed using 1% LA
82
9
8
7
Log CFU/g
6
5
X
control
4
Air + 1 %LA
3
O , + 1 %LA
CO + 1 %LA
2
1
0
0
Figure 9.
4
8
TREATMENT TIME (min)
12
Thermal death curve for L . monocytogenes on
crawfish tail meat at 60°C spray treated with 1%
lactic acid and packed under various atmospheres.
83
and
no
head
space
(Tables
5
&
9) .
This
might
be
attributable to an inability of the bacterium to detoxify
toxic products of molecular 02, the superoxide radical (02—)
and hydrogen peroxide (H202) , after thermal destruction of
the enzymes CA and SOD.
4 °C Storage Under Various Atmospheres & 1% LA
Generation
times
at
4°C
for
heat
shocked
L.
monocytogenes on crawfish tail meat packed under Air, 100%
02, 100% C02, and 100% N2 were 26.4, 33.6, 105.1, and 26.9 h,
respectively.
100%
C02,
The most inhibitive package environment was
exhibiting
significantly
(P<0.05)
larger
generation times than all other gas treatments (Figure 10).
Carbon dioxide penetrates cells very easily (Daniels et al.,
1985) and this may facilitate its chemical effects on the
internal metabolic processes of L . monocytogenes.
It has
been long established that increased inhibition of bacteria
at lower temperatures is correlated to increased solubility
of the gas in the water phase
(Golding,
1945).
Carbon
dioxide was rapidly absorbed by crawfish tail meat in the
present study.
Approximately 30 min after packing crawfish
tail meat samples in 100% C02 with 4:1 head space (gas to
product) no head space remained in the packs.
absorption
enables
conversion
of
C02 to
This rapid
carbonic
acid.
Carbonic acid produces a rapid acidification of bacterial
84
10.00
8 .6 0
' CO
7 .2 0
Log CFU/g
V
5 .8 0
4 .4 0
3 .0 0
0
3
6
9
12
15
18
21
TREATMENT TIME (days)
Figure 10.
Growth of L. monocytogenes stored under 4
different atmospheres at 4°C for 21 d.
85
cell internal pH leading to altered metabolic activities
(Daniels et al., 1985).
During the present study, pH of
crawfish tail meat dropped approximately unit during the 21
d storage period when packed under C02 (Table 3).
These findings contribute support to a hypothesis that
as with heat,
C02 appears to exert an effect on certain
enzyme systems affecting growth of the bacterium (Foster and
Davis, 1949; Fenestil et al., 1963; King and Nagel, 1975).
Several mechanisms have been hypothesized as explanations
for the reduction of growth of a bacterium in the presence
of C02.
Stimulated mitochondrial ATPase activity and the
uncoupling effect on oxidative phosphorylation resulting in
a decreased level of energy available to the bacterium for
metabolism and growth is one (Fenestil et al., 1963).
King
and Nagel (1975) postulated that C02 concentrations above
50% inhibit the activity of isocitrate dehydrogenase, malate
dehydrogenase and inhibits certain decarboxylation enzymes
through a mass action effect.
The present study found that thermally treated L.
monocytogenes tended to recover more effectively on crawfish
tail meat packaged in N2 and air, which were free or limited
in 02, than in an environment of 100% 02 (Figure 10) . Knabel
et al.
(1990)
reported that after heat shock
in milk,
anaerobic recovery of L. monocytogenes resulted in higher
numbers
when
compared
with
recovery
under
aerobic
conditions.
Murano and Pierson (1993) found the same to be
true for Escherichia coli 0157:H7 after heat shock.
The
present study determined that 20% of the cells were injured
when thermally treated at 60°C for 12.5 min.
Martin
(1988)
reported
that
CAT
and
SOD
Dallmier and
were
rapidly
inactivated when L. monocytogenes was heated at temperatures
of 60°C.
Consequently this inactivation of CAT and SOD
during the
60°C thermal treatment and subsequent buildup of
oxygen radicals in an
oxygen rich environment may
have
caused the
bacterium to grow at
a slower rate when compared
to air and
N2. However, since a
majority of survivingcells
were not injured by the thermal treatment, no significant
difference
(P>0.05)
was
observed
in
growth
inhibition
between 100% 02, air and N2 atmospheres.
One percent LA added to crawfish tail meat followed by
packaging
under
air,
100%
02,
100%
C02 and
100%
N2
atmospheres prevented growth of L . monocytogenes (Figures 11
& 12) .
The most effective atmosphere plus LA combination
appears to be 02 + 1% LA (Figure 13) . This combination was
significantly (P<0.05) more lethal to the bacterium than the
N2 + 1% LA combination and consistently more lethal than the
C02 + 1% LA or Air + 1% LA combinations (Figure 13).
destructive
element
of
this
treatment
The
combination
is
probably largely a result of build up of oxygen radicals by
the bacterium.
It appears that when this outcome is
87
10.00
Air
8 .4 0
Air + 1 % LA
CFU/g
6.8 0
5.2 0
-=i—
3.6 0
2.00
0
3
6
9
12
15
18
21
STORAGE TIME (days)
Figure 11.
Growth of L . monocytogenes on crawfish
tail meat stored under Air and N2 alone and in
combination with 1% LA at 4°C for 21 d.
88
10.00
8 .4 0
CO
O + 1% LA
Log CFU/g
6 .8 0
5 .2 0
3 .6 0
2.00
0
Figure 12.
3
6
12
9
STORAGE TIME (days)
15
18
21
Growth of L. monocytogenes stored under
C02 and 02 alone and in combination with 1% LA at
4 °C for 21 d.
89
4.2 5
3.85
Log CFU/g
3.45
Air
3.05
CO
2.65
2.25
0
3
6
9
12
15
18
21
STORAGE TIME (days)
Figure 13.
Destruction of L. monocytogenes stored under
Air, N2, C02, and 02 in combination with 1% LA at
4°C for 21 d.
90
combined with the effects of LA, a package environment of
100% Oz + 1% LA becomes the most lethal of the treatment
combinations studied.
As discussed earlier, when LA was absent, C02 was the
most
restrictive
packaging
atmosphere
monocytogenes on crawfish tail meat.
pH
reduction
resulting
from
to
growth
of
L.
However, any effect of
carbonic
acid
production
experienced with introduction of C02 was completely masked
by the presence of 1% LA.
This was evidenced by the
significant difference (P<0.05) between C02 and the other 3
atmospheres without LA present (Figure 10) versus the lack
of significant difference
(P>0.05)
between COz and other
atmospheres when 1% LA was added as a treatment (Figure 13) .
SUMMARY AND CONCLUSIONS
Heat treatments of approximately 4 min at 60°C will
reduce L. monocytogenes by 90% on packaged crawfish tail
meat.
The
addition
of
LA
spray
lethality of heat to the bacterium.
treatments
increases
The enhanced lethality
of heat when LA was introduced was not strictly due to a
simple pH effect.
It appears incorporation of two barriers,
heat and LA, in a process could contribute a significant
degree of safety to the consumer in regards to crawfish tail
meat.
When L. monocytogenes was heat treated in an 02-free
environment D60 values were consistently higher than
treatments done in the presence of 02.
supports
the
hypothesis
that
in
This observation
absence
of
02
in
the
environment used during heat treatments of L. monocytogenes
acts
to
protect
encourage
bacterium,
greater
the
bacterium
lethal
inclusion
of
heat
from
heat
processes
sufficient
injury.
To
against
the
amounts
of
02 in
packaging environments should be considered.
Results from storage studies further strengthen the
argument that packaging of
crawfish
tail
meat and
any
subsequent heat treatment should be done in the presence of
02. As mentioned above, heat treatments administered under
a 100% 02 packing environment were the most destructive to
91
92
L. monocytogenes, compared with air, C02, and N2. Storage at
4°C of 1% LA treated crawfish tail meat packed under 100% 02
proved to be the most lethal barrier combination to heat
injured L . monocytogenes.
It would appear that methodologies employed in the
determination of thermal death times for L. monocytogenes in
various
food products need careful consideration.
The
present study indicates that methods which use no head
space, thus allowing the bacterium little or no access to
molecular 02, may yield higher D values for a given product
than similar methods which include a head space containing
some
molecular
treatments
in
02.
a
Therefore,
low-oxygen
application
environment
and
of
heat
subsequent
storage of a product in a similar environment may increase,
instead of decrease the probability of listeriosis being
contracted from a given food product.
While maximum kill of the bacterium seems to occur when
it is heated in the presence of 02, minimum recovery is
realized.
Tightly packed TDT tubes and cellulose casings
that allow very little 02 to remain during heating trials,
may yield higher D values for L . monocytogenes. Because of
the threat of toxin production from Clostridium botulinum in
an anaerobic environment, many industrial packaging methods
allow inclusion of 02. Consequently, laboratory methods for
determining D values of L. monocytogenes should be designed
93
to
allow the presence
of 02.
However,
since complete
recovery of heat injured L. monocytogenes in an aerobic
environment appears impossible, media overlays which exclude
02 or pre-reduced media should be employed for enumeration
of the bacterium.
Commercially processed crawfish are boiled for 5 to 10
min before hand peeling.
Based on heat resistance of L.
monocytogenes demonstrated in the present studies,
process
bacterium
would
provide
before
hand
sufficient
peeling
and
destruction
packaging.
of
this
the
Thus,
occurrence of the bacterium in packaged crawfish tail meat
would
be mainly due to
workers or equipment.
cross
contamination
from plant
The adoption of strict
in-plant
sanitation programs to prevent post-boil cross contamination
is
paramount.
Because
of
the
possibility
of
process
deviations, post-packaging heat treatments in the presence
of 02 and addition of 1% LA sprays should be implemented.
While more extensive investigations
into the mechanisms
which cause interactions of these barriers in crawfish tail
meat and other food products is needed,
the addition of
these steps in a crawfish packing plant would increase
safety of the product.
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VITA
The
Orleans,
author was
born
on November
8,
1955,
in New
Louisiana.
He attended Archbishop Rummel High
School, located in Metairie, Louisiana, and graduated in
May, 1973.
He entered Nicholls State University, located in
Thibodaux, Louisiana and received his Bachelor of Science
degree on May 13, 1978 in Marine Biology.
He received his
Master of Science degree from Mississippi State University
located
in
Starkville,
Mississippi,
in
the
field
of
Fisheries Management on May 15, 1981.
Following completion of the M.S.
degree he became
employed in the seafood industry for ten years serving in
both the production and processing aspects of the catfish
industry in Mississippi, Arkansas, and Louisiana.
In January of 1990 he enrolled at the University of
Louisiana and received the degree of Doctor of Philosophy in
food science with emphasis in food microbiology in 1994.
108
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate: Warren Joseph Dorsa
Major Field: Food Science
Title of Dissertation: Effects of Heat, Lactic Acid, and Modified Atmosphere
Packaging on Listeria monocytogenes on Cooked
Crawfish Tail Meat
Approved:
Major Professor and Chairman
Dean of the Graduate School
EXAMINING COMMITTEE:
fi
/ A.
Date of Examination:
December 16, 1993