131
Journal of Food Protection, Vol. 53, No. 2, Pages 131-140 (February 1990)
Copyright© International Association of Milk, Food and Environmental Sanitarians
Predicting the Safe Storage of Fresh Fish Under Modified
Atmospheres with Respect to Clostridium botulinum
Toxigenesis by Modeling Length of the Lag Phase of Growth
DAVID A. BAKER* and CONSTANTIN GENIGEORGIS
Department of Epidemiology and Preventive Medicine, School of Veterinary Medicine, University of California, Davis, CA 95616
(Received for Publication June 12, 1989)
ABSTRACT
The toxigenesis of non-proteolytic C. botulinum, types B,
E, and F, in a variety of fresh fish stored between 4 and 30°C
for up to 60 days was evaluated in a five year study. Muscle
homogenates and fillets were inoculated with 10* -lO 2 spores
from multiple strain pools, then modified atmosphere (MA)
packaged under vacuum, 100% C0 2 , or 70% C0 2 and 30% N2.
Our conclusions are based on all data generated in 927 experi-
ments comprising 18,700 samples. The earliest lag times (LT),
i.e. sampling period prior to observation of toxicity, among all
experiments conducted at 30, 20, 16, 12, 8, and 4°C, were 0.5,
1, 2, 3, 3, and 18 d, respectively. The LT across experiments
was significantly affected by temperature (p<0.00001), inocu-
lum level (p<0.00001), fish type (p<0.0001), spore pool (0.0001),
and MA (p<0.02). The LT was not affected by homogenization
of the fish muscle. We established a general formula that pro-
vides the most conservative model for the prediction of LT. This
formula yielded calculated LT's that were very close to ob-
served data and models based on individual experiments. Tem-
perature explained 74.6% of experimental variation in the final
multiple linear regression model (r2=0.883). The spore inoculum
level accounted for 7.4%, while other factors explained <3%
together. The utility of the model is demonstrated by its ability
to predict the time before toxigenesis in qualitative studies of C.
botulinum toxicity in inoculated fish stored under different MA's,
reported in the international literature.
The beneficial effect of modified atmospheres (MA)
to extend the shelflife of fresh fish has been demonstrated
(13,14,29,53,54,60,72). Commercial use of MA to extend
the shelflife of fishery products has been limited by the
potential of Clostridium botulinum growth and toxin pro-
duction in refrigerated, MA packed fish, without overt
sensory evidence of spoilage (18,23,25,27). Despite these
concerns, fillets of fresh fish packaged under MA and
stored continuously at temperatures continuously below
3°C have appeared in European supermarkets. No cases of
botulism have been associated with the consumption of
such products thus far. A provisional marketing approval
has been issued in the United States (3).
Qualitative studies to assess the risk from C. botu-
linum toxigenesis in a variety of MA packaged fish stored
JOURNAL OF FOOD PROTECTION, VOL. 53. FEBRUARY 1990
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at low and abusive temperatures have been conducted
(10,12,13,14,19,50,51,59,65,70). Studies to quantify the safe
storage limits for various fish types, based upon load of C.
botulinum spores, temperature, and MA packaging condi-
tions have been reported recently by our group
(6,24,25,44,48). Our studies have demonstrated the utility
of predictive models to estimate the length of the lag time
prior to C. botulinum toxigenesis (LT), and the probability
(P) of growth of one non-proteolytic type B, E, and F
spore as single or multiple type pool in different types of
fish, under a variety of storage temperatures and MA's.
Since, presence of sensory spoilage could not be regarded
as an accurate warning signal for toxicity, we considered
determination of the LT and P as better indicators of the
risk of C. botulinum growth and toxigenesis.
The statistical analysis and conclusions of all the data
generated in previously reported studies over a five year
period are discussed here. While P was determined in many
experiments, only LT will be considered here, since it is
the most conservative quantitative estimate of the risk of
C. botulinum growth and toxigenesis. A mathematical model
for LT prediction will be shown to agree well with toxic-
ity data reported by others. This model allows comparison
of results presented in the reported studies.
MATERIALS AND METHODS
Experimental design
Seven experiments were arranged in a factorial fashion which
included 7 tenfold inoculum levels of 104-10~2 spores from pools
made of individual or multiple, non-proteolytic types B, E, and
F C. botulinum. Other continuous variables included up to 7
incubation temperatures in the range 4-30°C, the initial level of
fish microbial flora which was kept to a minimum by aseptic
filleting of extremely fresh fish from a local processor, and
multiple sampling during storage for up to 60 d. Experimental
designs differed from one another in their combinations of discrete
variables, these included 2 spore inocula (equal levels of non-
proteolytic B, E, & F pools, or an E pool), 3 MA (vacuum,
100% C0 2 or 70% CO, & 30% N2), 3 fish species (Rockfish,
Salmon, or Sole), and 2 tissue preparations (muscle homogenate
or fillets).
132 BAKER AND GENIGEORGIS

Preparation and counting of C. botulinum spore inocula
Single type spore inocula were prepared by mixing equal
levels of spores of 4-5 strains. Pools containing several C. botu-
linum types were prepared by mixing equal levels of each pool
type. Production of spore crops, counting, preparation of final
inoculum, as well as the strains and their origins, have been de-
scribed before (6,24,25,44,48). Detailed methodologies for the
following topics may also be found in these references.
Preparation of fish substrate, inoculation and packaging
Fish substrates used were either fillets or whole fish muscle
homogenates. Homogenate samples (2-3 g) were placed in 24
well tissue culture templates providing triplicate samples at each
inoculum level. The inoculum was delivered to the center of
LT, while describing the P of a single spore to initiate
toxigenesis.
Results of a single multiple linear regression analysis
inclusive for all log LT data generated in the previous
studies are presented in Table 2. Discrete factors of fish
species and spore pool (type of C. botulinum inoculum)
were highly significant (P<0.0001), while MA (vacuum or
C0 2 /N 2 gas backflush) were less significant (P<0.02).
Whether fish tissue was prepared as a fillet or as a
homogenate had no significant effects of LT prediction
(P>0.05). Continuous factors of temperature and spore
inoculum were also highly significant (P<0.0001). The
reciprocal of temperature transformation was included in

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homogenate samples in 20 uf aliquots. Inoculum (20 uf) was the regression analysis to allow C. botulinum LT to be-
placed between 2 fillets (50 g each). Homogenates and fillets come asymptotic as incubation temperatures approach growth
were MA packaged (Table 1) in barrier bags (Cryovac type B
limits (Figure 1). Fresh whole fishes, used aseptically to
540, W. R. Grace & Co., Hayward, CA).
prepare muscle homogenate and fillet samples had APC<10 2
TABLE 1. Number of inoculated pack experimental settings' cfu/g. Thus, the APC's were less significant (P<0.0179),
conducted under modified atmospheres with non-proteolytic as the influence of competitive bacteria on LT was mini-
Clostridium botulinum types B, E, and F, and stored between 4- mized to increase the probability of even a single C. botu-
30° C for 0.25 - 60 d. linum spore to produce toxigenesis (24).
Fish Tissue Spores Atmospheres
TABLE 2. Multiple linear regression" coefficients and model of
Vacuum 100% 70% C0 2
CO, 30% N,
lag time prior to C. botulinum toxigenesis in raw fish being
stored under modified atmospheres between 4-30'C for up to 60
Rockfish Homogenate BEF 200 32 35
d.
Rockfish Homogenate E 217 42
Parameter Estimate (B) P>F
Rockfish Fillet BEF 17 20 16
Salmon 35 68 Intercept .453
Homogenate BEF 35
Salmon Homogenate E 35 35
Salmon 16 20
Discrete Factors
Fillett BEF 20
Fish 0.0001
Sole Homogenate E 42 42
Rockfish -0.205
Totals 562 226 139
Salmon -0.149
'Each experiment with homogenate represents a 3 x 8 (24-sample)
Sole 0.000
most probable number analysis. Spore Pool 0.0001
Toxicity testing and lag phase estimation B, E & F -0.200
Spore inocula magnitude were determined by the most probable E 0.000
number (MPN) technique in both the fish muscle homogenate MA Package 0.0205
type to be used experimentally, and BHI broth (Difco, Detroit, Vacuum -0.072
MI) media, incubated under vacuum, at 30°C for 3 d. The greatest 100% CO, -0.063
viable spore inoculum level yielded by either media was used 70% CO,,' 30% N, 0.000
analytically. The first occurrence of one mouse minimum lethal Tissue NS
dose (MLD) of C. botulinum toxin at each spore level was
determined according to The Centers for Disease Control (15) Continuous Factors
protocol. Lag time was defined by the incubation time up to the Temperature -0.042 0.0000
sampling period prior to first observation of C. botulinum toxin. 1/Temp °C 2.741 0.0001
Thus, if a sample was toxic on the 9th d, though negative on the Spore Inoculum (I) 0.091 0.0000
6th d, then 6 d would be considered to be the observed LT. Aerobic Plate Count 0.035 0.0179
Statistical analysis "Multiple r2 = 0.883.
Data for LT, the dependent variable, was pooled for a total NS not significant.
of 927 experimental settings comprised of approximately 18,700
The variables entering into the model presented were
inoculated fish samples. The BMDP2V program for analysis of
variance (17) was used to determine significance of differences able to account for 88.3% of the total variation (r2) within
in LT in relation to the continuous and discrete variables in re- discrete factors and among all variables entered into the
spect to predicting lag time was obtained using the QLM pro- model. Although a significant variable may enter a predic-
gram for multiple linear regression (62). tive equation, its importance is reflected in the amount of
experimental variation it is able to explain. Expression of
RESULTS AND DISCUSSION the proportion of explained experimental variation due to
Development of a general formula each variable as a relative height, allows quantification
In previous reports (6,24,25,28,44,48), we have devel- and pictorial presentation of the "Hurdle" principle (47),
oped separate models able to incorporate the prediction of (Fig. 2).

JOURNAL OF FOOD PROTECTION, VOL. 53, FEBRUARY 1990

 PREDICTING SAFE STORAGE OF FRESH FISH
Temperature is the most important variable contribut-
ing the greatest effect to C. botulinum growth and toxi-
genesis, and accounting for 74.6% of the increase in the
multiple r2. The initial spore inoculum accounted for 7.4%,
demonstrating that increased contamination shortens the
length of LT and increases the risk of C. botulinum.
Differences between spore type, fish species, and MA
composition together accounted for only a 2.3% increase
in r2. Thus, it became apparent that temperature alone was
the only significant growth hurdle in these studies, and
MA, fish species, etc, refine the predictive capabilities of
these LT models.
Temperature WC
Figure 1. Predicted lag time in rockfish, inoculated with nonpro-
teolytic Clostridium botulinum type B, E, and F, as affected by
spore inoculum and storage temperature, and stored under vacuum.
Log Spore Load Clostridium botulinum
0.074*
p<0.0000 Growth "Hurdes"
Temperature
pO.OOOO Spore Type
T p<0.0001
Microbial Competitor!
p<0.0179
Fish Type
p<0.0001
Atmosphere
p.0.0205
0.746
/cToosN /fToo5N / ^ o o > \ /fTor^N
Low Microbial Flora in Al Samples
2 on spoilage vs. toxicity in vacuum packaged raw fish in-
Increase in multiple R (estimate)
Figure 2. Pictorial representation of the effect' of six variables
(temperature, spore load, microbial competition, modified at-
mosphere, fish species, and spore type) on the lengths of lag
time of Clostridium botulinum inoculated in fresh fish and stored
under modified atmospheres.
1. Based upon a multiple regression model for the shortest lag
time derived from pooled data generated in 927 experiments
(Table 5).
2. Hurdles (barriers) prevent or inhibit bacterial growth either
alone, additively, or synergistically.
JOURNAL OF FOOD PROTECTION. VOL. 53, FEBRUARY 1990
133
The general model (GM) using all experimental data
(Table 2) was able to predict in most situations shorter
LT's than those observed or predicted in each individual
experiment (6,24,25,28,44,48). Observed LT's and LT
predictions from formulae using regression coefficients from
Table 2 are presented in Tables 3 and 4 for experiments
inoculated with C. botulinum type B, E, and F and type E
spore pools, respectively. Figure 3 presents predicted LT's
calculated from the GM (Tables 3-4) for salmon homoge-
nate inoculated with type E and stored, under vacuum
(Fig. 3a), and under C0 2 (Fig. 3b). The GM graphs for
salmon homogenate inoculated with spore types B, E, and
F and stored under vacuum (Fig. 3c), and sole homogenate
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inoculated with type E spores and stored under vacuum
(Fig. 3d), are also presented. The relation of the LT sur-
faces among these examples are as expected from the co-
efficients presented in Table 2. The LT surface for Salmon
homogenate inoculated with types B, E, and F spores (Fig.
3d) is beneath those inoculated with type E, when correct-
ing for spore inoculum. The LT surface for vacuum pack-
aged salmon (Fig. 3a) was slightly beneath that of the C0 2
MA (Fig. 3b), while the LT surface for the sole (Fig. 3d)
was far above the others.
The most conservative model for prediction of LT
was developed by substituting those regression coefficients
for each categorical variable in Table 2, which caused the
greatest shortening of the length of LT, into a new model
of the dependent variable (Table 2). Thus, rockfish (coef-
ficient, -0.205), non-proteolytic B, E, and F C. botulinum
spore pool (coefficient, -0.200), and vacuum packaging
(coefficient, -0.072) were entered into the following equa-
tion: log LT = 0.974 - 0.042(Temp. °C) + (2.74/Temp.°C)
-0.091 (log spore inoculum) + 0.035(initial log APC). This
general formula (GF) yields predictions for the safest storage
of MA packaged fresh fish within the limits of the experi-
mental design. Prediction of LT (in days) based on GF,
temperature of storage between 3.3-30 °C, and inocula
levels between 1-10,000 spores are presented in Table 5
for quick reference.
Spoilage in comparison to C. botulinum toxigenesis
Hobbs (37) suggested toxin production in fish stored
below 10°C to be usually so slow that spoilage is organo-
leptically apparent before detectable amounts of toxin are
produced, thus providing consumer protection against botu-
lism. Eyles and Warths (23) summarizing literature data
oculated with C. botulinum type E stated that spoilage was
"usually" more rapid than development of toxicity in raw
fish held below 10°C. Eklund and Pysky (21), suggested
that storage temperatures below 8°C presented a margin of
safety, since "only one" sample of haddock inoculated
with 106 spores/g contained toxin after 14 d, 4 d prior to
detection of spoilage by sensory (organoleptic) indicators.
Banner (8) is often cited for publishing, "There was no
Bot-toxin before the product became so absolutely unac-
ceptable that no one would possibly ever eat it," in regard
to vacuum package salmon inoculated with C. botulinum
134 BAKER AND GENIGEORGIS

TABLE 3. Observed and predicted! lag times (d) in 2 fish species inoculated with increasing levels of non-proteolytic Clostridium
botulinum. types B, E, and F2 spores, and stored under vacuum or 100% CO, atmospheres, at temperatures from 4 - 30°C.
Log Sp>ore Inoculum
0 1 2 3 4 0 I 2 3 4 0 1 2 3 4
°C
Salmon General Model Observed Data 1 Observed Data 4
4 38.4 31.1 25.2 20.5 16.6 60.0 30.0 18.0 18.0 12.0 60.0 60.0 60.0 60.0 60.0
Vacuum 8 11.8 9.6 7.8 6.3 5.1 15.0 9.0 9.0 9.0 9.0 6.0 6.0 6.0 6.0 6.0
12 6.2 5.0 4.1 3.3 2.7 6.0 6.0 3.0 3.0 2.0 2.0 2.0 2.0 2.0 2.0
16 3.7 3.0 2.4 2.0 1.6 3.0 3.0 2.0 1.0 1.0
30 0.8 0.6 0.5 0.4 0.3 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

4 39.2 31.8 25.8 20.9 17.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0
100% C 0 2 8 12.1 9.8 8.0 6.5 5.2 15.0 9.0 9.0 6.0 6.0 15.0 9.0 6.0 12.0 6.0

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12 6.3 5.1 4.2 3.4 2.7 9.0 6.0 3.0 3.0 3.0 6.0 6.0 6.0 6.0 6.0
16 3.8 3.1 2.5 2.0 1.6 3.0 2.0 2.0 1.0 1.0
30 0.8 0.7 0.5 0.4 0.3 0.5 0.5 0.5 0.5 0.5 2.0 0.5 0.5 0.5 0.5

Rockfish Observed Data 5 Observed Data 6

4 33.7 27.4 22.2 18.0 14.6 21.0 21.0 19.0 15.0 15.0 21.0 21.0 21.0 21.0 21.0
Vacuum 8 10.4 8.4 6.8 5.6 4.5 15.0 15.0 12.0 12.0 9.0 19.0 15.0 15.0 12.0 9.0
12 5.4 4.4 3.6 2.9 2.4 9.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0
16 3.2 2.6 2.1 1.7 1.4 3.0 3.0 3.0 3.0 3.0 3.0 3.0 <3.0 <3.0 <3.0
30 0.7 0.6 0.5 0.4 0.3 1.0 1.0 0.5 0.5 0.5 1.0 0.5 0.5 0.5 0.5

4 39.0 32.0 26.0 21.0 17.0 19.0 19.0 19.0 19.0 19.0 21.0 21.0 21.0 21.0 21.0
100% CO, 8 12.1 9.8 8.0 6.5 5.2 15.0 9.0 9.0 9.0 9.0 21.0 21.0 12.0 9.0 9.0
12 6.3 5.1 4.2 3.4 2.7 9.0 3.0 3.0 3.0 3.0 6.0 3.0 3.0 3.0 3.0
16 3.8 3.1 2.5 2.0 1.6 3.0 3.0 <3.0 <3.0 <3.0
30 0.8 0.7 0.5 0.4 0.3 1.0 1.0 1.0 0.5 0.5 1.0 0.5 0.5 0.5 0.5
'General model predictions are based upon lag time data pooled from 927 experiments (Table 2).
2
Spore pool comprised of equal portions of types B, E, & F, 4-5 strains of each.
^Garcia, G., and C. Genigeorgis. 1987. L Food Prot. 50:390-397.
4
Garcia G„ et al. 1987. J. Food Prot. 50:330-336.
5
Lindroth, S., and C. Genigeorgis. 1986. Int. .1. Food Microbiol. 3:167-181. Samples stored up to 21 d at 4-30°C.
6
Ikawa, J., and C. Genigeorgis. 1987. Int. L Food Microbiol. 4:167-181. Samples stored up to 21 d at 4-30°C.
type E and stored at 7.2°C for up to 30 d. linum cells is not detected until one mouse MLD is present.
However, C. botulinum toxigenesis in fish can pre- The correlation between the mouse MLD and human in-
cede production of organoleptic spoilage indicators as shown toxication is not well understood (69). Researchers should
by a number of investigators including our group not accept a threshold level (no matter how low) as repre-
(18,21,25,37,51,59,70). Based on the above, spoilage may senting a safe limit of botulinal toxin in foods. Rather there
not be used as a criterion to assess the risk of C. botulinum should be zero tolerance with the most sensitive test util-
toxin presence in fresh fish stored under MA. Predicting ized to detect preformed botulinum toxin. The limits of
the length of LT for various storage conditions and then detection in inoculated pack studies should guide food
deciding whether to accept or reject a temperature and/or processors develop their hazard analysis of critical control
time abused product is considered a safer assessment of points (HACCP) plan for marketing MA package foods
the risk of presence of botulinal toxins. capable of supporting C. botulinum growth.

Experimental design for modeling C. botulinum toxigene- The effect of temperature

sis
A conservative model for the study of food safety
seeks to incorporate worst case scenarios of events lead-
ing to the earliest growth of pathogens and/or appearance
of toxins. In the development of models to predict the safe
storage time for MA packaged fresh fish, our experimen-
tal designs included methodologies and variables to favor
the growth of C. botulinum and the detection of preformed
toxin. Basing LT on the sampling interval prior to the de-
velopment of one mouse MLD makes the dependent vari-
able, i.e. LT, inherently conservative. Growth of C. botu-
Non-proteolytic C. botulinum strains grow at tempera-
tures as low as 3.3°C (38°F) (19,20,52,57,63,64). C. botu-
linum type E did not grow in chopped meat medium after
170 d at 3°C (2). Vacuum packaged herring inoculated
with C. botulinum type E (106 spores/lOOg flesh) devel-
oped toxin after 21 d at 3.3°C (11). Stier et al. (70), stated
that commercial transporters of MA packaged salmon would
consider 4.4"C storage temperature abusive, although no
toxin was detected in salmon stored at this temperature for
up to 57 d.
The effect of spore concentration Shorter times to C.

JOURNAL OF FOOD PROTECTION, VOL. 53. FEBRUARY 1990

 PREDICTING SAFE STORAGE OF FRESH FISH 135

TABLE 4. Observed and predicted] lag times (d) in 3 fish species inoculated with increasing levels of Clostridium botulinum, type
E2 spores, and stored under vacuum or 100% CO? atmospheres, at temperatures from 4 - 30° C.
Log Spore Inoculum
4 0 3

Salmon General Model1 Observed Data3

4 60.8 49.3 40.0 32.5 26.3 60.0 60.0 60.0 60.0 60.0
8 18.8 15.2 12.3 10.0 8.1 15.0 9.0 9.0 9.0 6.0
Vacuum 16 5.8 4.7 3.8 3.1 2.5 6.0 3.0 3.0 2.0 1.0
20 3.7 3.0 2.4 2.0 1.6 3.0 2.0 1.0 1.0 1.0
30 1.3 1.0 0.8 0.7 0.5 0.5 0.5 0.3 0.3 0.3

4 62.1 50.4 40.9 33.1 26.9 60.0 60.0 60.0 60.0 60.0
8 19.2 15.5 12.6 10.2 8.3 15.0 12.0 12.0 12.0 9.0
100% C0 2 16 6.0 5.0 4.0 3.0 3.0 3.0 3.0 3.0 3.0 2.0

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20 3.7 3.0 2.5 2.0 1.6 2.0 2.0 1.0 1.0 1.0
30 1.3 1.0 0.8 0.7 0.6 3.0 0.5 0.5 0.5 0.3

Rockfish Observed Data4

4 53.5 43.4 35.2 28.5 23.1 60.0 60.0 60.0 60.0 60.0
8 16.5 13.4 10.9 8.8 7.1 21.0 21.0 21.0 18.0 18.0
12 8.6 7.0 5.7 4.6 3.7 15.0 9.0 9.0 9.0 6.0
Vacuum 16 5.1 4.2 3.4 2.7 2.2 9.0 9.0 9.0 3.0 3.0
20 3.2 2.6 2.1 1.7 1.4 6.0 6.0 3.0 3.0 2.0
30 1.1 0.9 0.7 0.6 0.5 2.0 1.0 1.0 1.0 1.0

4 54.6 44.3 35.9 29.1 23.6 60.0 60.0 60.0 60.0 60.0
8 16.9 13.7 11.1 9.0 7.3 21.0 21.0 21.0 21.0 21.0
12 8.8 7.1 5.8 4.7 3.8 9.0 9.0 9.0 9.0 9.0
100% C0 2 16 5.2 4.2 3.4 2.8 2.3 12.0 6.0 3.0 3.0 3.0
20 3.3 2.7 2.2 1.8 1.4 9.0 3.0 3.0 3.0 3.0
30 1.1 0.9 0.7 0.6 0.5 2.0 1.0 0.8 0.8 0.5

Sole Observed Data3

4 85.8 69.5 56.4 45.7 37.1 60.0 60.0 60.0 60.0 60.0
8 26.5 21.5 17.4 14.1 11.4 24.0 24.0 24.0 12.0 9.0
12 14.0 11.0 9.0 7.0 6.0 21.0 21.0 21.0 21.0 12.0
Vacuum 16 8.2 6.7 5.4 4.4 3.6 12.0 12.0 12.0 9.0 9.0
20 5.2 4.2 3.4 2.8 2.2 6.0 3.0 3.0 3.0 1.0
30 1.8 1.4 1.2 0.9 0.8 2.0 2.0 2.0 2.0 2.0

87.5 71.0 57.6 46.7 37.9 60.0 60.0 60.0 60.0 60.0
27.0 21.9 17.8 14.4 11.7 24.0 24.0 24.0 24.0 24.0
12 14.1 11.4 9.3 7.5 6.1 21.0 21.0 21.0 18.0 12.0
100% CO, 16 8.4 6.8 5.5 4.5 3.6 12.0 12.0 12.0 9.0 9.0
20 5.3 4.3 3.5 2.8 2.3 2.0 2.0 2.0 2.0 3.0
30 1.8 1.5 1.2 1.0 0.8 1.0 1.0 1.0 1.0 1.0
'General model predictions are base upon lag time data pooled from 927 experiments (Table 2).
2
Spore pool comprised of equal portions of 4 type E strains.
'Baker, D. A. 1989. Ph.D. Thesis. Univ. of Calif., Davis, CA.
"Glover, J. M. T. 1987. MPVM Report. Univ. of Calif., Davis, CA.

botulinum toxigenesis have been attributed to increases in
spore inoculum (18,23,30,49). These observations are only
as accurate as the sampling intervals used in each study.
Herring inoculated with C. botulinum type E spores be-
tween lO'-IO 6 spores/pk showed little difference in time of
first toxic sample determined at 10 and 20°C, while at 5°C
contradictive results were presented. The 106 inoculum
became toxic at 11 d, the 102 sample became toxic after
15 d, while all inoculum levels in between first became
toxic at 32 d (10). Flounder fillets incubated at 10°C and
packed under MA (100% C 0 2 & 70% C 0 2 & 30% air),
became toxic after 6 d at all spore levels leading to the
conclusion that spore concentration had little effect on
toxigenesis (10).
Subsequent studies supported the contention that given
a conducive environment, the spore inoculum will have
little effect on time to toxigenesis (41). However, when
less favorable conditions are presented, increasing num-
bers of spores will help C. botulinum overcome the intrin-
sic hurdles and allow earlier toxigenesis (26,30,61).

JOURNAL OF FOOD PROTECTION, VOL. 53, FEBRUARY 1990

136 BAKER AND GENIGEORGIS

TABLE 5. Predicted time in d before C. botulinum toxicity occurs linum spores in 3/6 herring examined; 1 contained 1 spores/
in model fish systems as a function" of initial spore inoculum 16 g, and 2 contained 1 spores/200 g. The incidence of C.
and incubation temperature. botulinum type E was up to 100% in fish from Scandi-
Temp. Time in D at each Log]() Spore Level navian coastal waters and the Baltic Sea (37). The C.
"C botulinum type E incidence rate in Scandinavian, farmed,
0 1 2 3 4
freshwater fish was also 100% (37). The highest spore
49.57 40.20 32.60 26.44 21.44
load found in any food so far seems to be 5.3 spores/g
33.10 26.84 21.77 17.65 14.32
21.87 17.74 14.38 11.67 9.46
shown in Danish farmed trout (39). On the basis of avail-
16.06 13.02 10.56 8.56 6.94 able information, all fishes should be assumed to be carrying
12.52 10.15 8.23 6.68 5.41 C. botulinum spores, able to allow cross-contamination of
10.13 8.22 6.66 5.40 4.38 spores from the gut or skin to the muscle surface or in
8.41 6.82 5.53 4.48 3.64 punctures of the final product for MA packaging.
7.10 5.76 4.67 3.79 3.07 On the basis of viable spore counts from Scandinavian
6.07 4.93 3.99 3.24 2.63 waters, where mud samples contained 50-60 C. botulinum

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5.25 4.25 3.45 2.80 2.27 type E spores/g, Cann et al. (//) determined that inocu-
4.56 3.70 3.00 2.43 1.97 lated pack studies should be conducted with 104 spores/g
3.99 3.24 2.63 2.13 1.73 fish to allow an adequate safety margin. Spores building
3.51 2.85 2.31 1.87 1.52
up in processing plant micro-environments may be sources
3.10 2.51 2.04 1.65 1.34
2.74 2.22 1.80 1.46 1.19
of C. botulinum capable of the above contamination lev-
2.43 1.97 1.60 1.30 1.05 els, (34).
2.16 1.76 1.42 1.15 0.94 The upper level of inoculations in our experiments
1.93 1.56 1.27 1.03 0.83 ranged from 104-105 spores/sample (2-3 g for tissue
1.72 1.40 1.13 0.92 0.74 homogenates, 60 g for fillets). If the C. botulinum con-
1.54 1.25 1.01 0.82 0.67 tamination level found in Danish trout (5.3 spores/g) is
1.38 1.12 0.91 0.73 0.60 assumed to be representative of a naturally contaminated,
1.23 1.00 0.81 0.66 0.53 high quality fresh food, and a pack size of 1000 g, one
1.10 0.90 0.73 0.59 0.48 could justify 5.3 x 103 spores/pack as the upper limit of
0.99 0.80 0.65 0.53 0.43 inoculation for challenge studies. Increasing this inoculum
0.89 0.72 0.59 0.47 0.38
5 times for additional safety (104 spores/pack) and de-
0.80 0.65 0.53 0.43 0.35
0.72 0.58 0.47 0.38 0.31
creasing the pack size to 100 g or less, should provide
0.65 0.52 0.43 0.34 0.28 sufficient initial number of C. botulinum spores to yield
a
LogLt = 0.974-0.042 (Temp) + (2.741/Temp) - 0.091 (Log
minimal LT's in challenge studies establishing safety of
Inoculum) + 0.035 (Log Initial APC) b . MA packaged products.
b
Discrete factors: Rockfish, BEF pool, Vacuum; APC assumed
to be 10 cfu/g. Effect of spore type
c
Estimate outside range of regression model. C. botulinum type E is noted for its ability to grow at
temperatures including 3.3°C, and frequent isolation from
In this study, greater spore inoculum lead to shorter fish, and has therefore been commonly used in inoculated
predicted length of LT, as demonstrated by its contribu- pack studies. Non-proteolytic C. botulinum type B (20)
tion to the prediction formulas (Fig. 1, Table 2). Figure and Type (79) isolated from pacific coast mud are also
1 demonstrates that increased spore loads have the great- able to grow at 3.3°C. Inoculated pack studies with non-
est effect on shortening the length of LT at the lower re- proteolytic B and F strains should not behave significantly
frigeration temperatures, i.e. below 8°C. As temperatures different than those conducted with type E according to
become more optimal the effect of spore inoculum Eklund and Poysky (21). Only toxin of C. botulinum type
diminishes, and the P of growth by a single C. botulinum B was detected when pools containing equal number of
spore increases (Fig. 1). non-proteolytic type B, E, and F were inoculated in MA
C. botulinum contamination of the gut is most com- packaged fish (24,25,44). Our studies indicated that chal-
mon in bottom-feeding fish, while surface contamination lenge tests involving MA package fish should include both
of pelagic fish is expected (40). A high prevalence of C. non-proteolytic C. botulinum type B and E spores. When
botulinum spores have been found in the gut contents of C. botulinum type F spore pools were inoculated repeat-
herring harvested close to land while feeding. C. botu- edly in rockfish homogenate and fillets, no toxin was detected
linum spores are widely distributed in coastal waters where in the fish stored at 4-30°C for up to 60 d (6). Spore types
they are washed from land and marine carrion (33, 38). which grow poorly in a particular media should be deleted
This is a probable explanation for the high prevalence of from the spore pool inoculum in C. botulinum growth
C. botulinum spores found in herring gut contents. Zale- challenge studies, since they would effectively reduce the
ski (74) reported that the average contamination of Baltic overall challenge inoculation by reducing the total number
herring gut contents did not exceed 100 C. botulinum of spores capable outgrowth and production of detectable
type E spores/g. While, Cann et al. (11) found C. botu- toxin.

JOURNAL OF FOOD PROTECTION. VOL. 53, FEBRUARY 1990

 PREDICTING SAFE STORAGE OF FRESH FISH 137

Log Time (d)

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10.5

Temperature (C)
Initial Spore Inocula (Log)

Figure 3. Comparison of Clostridium botulinum lag times predicted from a multiple linear regression, derived from pooled data
generated in 927 experiments, as effected by temperatue and spore inocula, are presented between salmon homogenate inoculated with
type E. and stored under vacuum (Fig. 3a), and under C02 (Fig. 3b), salmon homogenate inoculated with non-proteolytic types B, E,
and F and stored under vacuum (Fig. 3c), and sole homogenate inoculated with type E and stored under vacuum (Fig. 3d).

and potential microbial competition. Thus, the GF reflects

Effect of competitive microflora
Microflora of fish differs with season, location (66),
fish anatomy and physiology (67). Shewan (66) noted that
some tropical species of fish have a substantially greater
refrigerated shelflife probably because the number and
diversity of contaminating pyschrotrophic bacteria are small.
This effect must be considered if MA packaging technol-
ogy is readily exported or utilized on fish from non-tem-
perate water since all the safety research has been con-
ducted in temperate environments.
In most studies reported in the literature, fish obtained
were cleaned and filleted at the wholesale or retail level
allowing normal contamination of the experimental tissue.
Few studies accounted for initial microbial flora. Even
less attention has been paid to differences between studies
of vacuum and C0 2 MA in regard to initial bacterial type,
number, and selection for spoilage types and rates. Garcia
and Geingeorgis (24), demonstrated increased LT for C.
botulinum as the initial contamination level of fish was
increased prior to inoculation and storage.
The wholesaler who provided fish used in each ex-
periment, assured us that they had been caught within the
last 24 h. Fish were sanitized prior to aseptic filleting,
minimizing the initial microbial load of the fish muscle
a small contribution of competitive microbial flora in the
determination of the length of C. botulinum LT, making
LT predictions more conservative.
Effect of packaging
During aerobic cold storage, psychrotrophic spoilage
organisms grow rapidly and cause the characteristic dete-
rioration of fresh fish as determined by sensor (organolep-
tic) indicators. Their rapid growth at low temperatures
may slow down the growth of C. botulinum, and the
production of proteolytic enzymes may even inactivate
botulinal toxin formed (18). When atmospheric 0 2 is limited
as in MA packaged fish, growth of aerobic microbial flora
may rapidly create reduced environments and significantly
lower Eh, thus enhancing the chance for C. botulinum
growth (18). Post et al. (59) reported that fish packaged
with 2 or 4% 0 2 became toxic prior to those packaged
under vacuum and other MA without 0 2 .
Storage of fish under abusive time/temperature condi-
tions can lead to toxigenesis regardless of packaging
(1,10,12,45,59). Cann et al. (12) references several studies
on vacuum packaged smoked fish which demonstrated
vacuum packaging to only slightly increase the toxicity
rate in contaminated fish. Time for C. botulinum spores to

JOURNAL OF FOOD PROTECTION, VOL. 53, FEBRUARY 1990

138 BAKER AND GENIGEORGIS
produce toxin in haddock packed in oxygen permeable
film was equal to those packaged in impermeable films
(21). Cann et al. (72), found little difference in time to
toxicity between comparably inoculated vacuum or air
package fish products. Mossel and Ingram (55) conclude
that microenvironments in compact food masses exposed
to the atmosphere may develop redox potentials able to
support C. botulinum growth. The storage temperature,
phase of growth of contaminating bacteria, and concentra-
tion of C0 2 are additive factors in the assessment of the
bacteriostatic effectiveness of MA packaging. Increase of
C0 2 partial pressure (0-25%) extends the lag phase of
spoilage bacteria and decreases their generation time,
resulting in shelf-life extension of MA packaged foods.
The average generation time of aerobic spoilage bacteria
on raw chicken packed in 25% C0 2 decreased from 6 h at
10°C to approximately 18 and 60 h at 4.4 and 1.1 °C,
respectively (56).
It has been reported that C0 2 improves the growth of
many Clostridia (35) including C. botulinum (46). This
may result from C0 2 enhancing germination of Clostridia
spores (36) and specifically C. botulinum spores (73). Carbon
dioxide stimulated C. botulinum germination at one at-
mosphere or lower (22). Reduction of competitive microflora
and demonstrations of C. botulinum growth augmentation
in MA packs flushed with C0 2 , would seem to negate
increased shelflife benefits.
However, other investigations reveal negative effects
of C0 2 on the growth of C. botulinum. Eklund (18)
demonstrated the ability of an increase in spore inoculum
to overcome the effect of C0 2 ; 103 spores (type E) pro-
duced toxin in 7 d at 10°C in 60% C0 2 , while 104 were
needed in a 90% C0 2 MA. These MA's were small hurdles,
for during the next 3 d interval all samples inoculated at
the lowest level of 102 were toxic. At highly abusive storage
temperatures (20+°C) the difference between vacuum, semi-
permeable wraps, and MA offers little practical difference
in time until toxin production (1,18,59,70). Silliker and
Wolfe (68) had similar experiences with chicken and pork
held at 27°C in a 60% C0 2 MA.
Slight LT extension are predicted for fish packaged
under C0 2 and held at low temperature (Table 2, Fig. 3a,
b). However, MA packages backflushed with C02/N2 gas
combinations had little overall effect on LT, in compari-
son to the effects of spore inoculum and storage tempera-
ture (Fig. 2). Of all MA's studied, vacuum was found to
be the most conducive to C. botulinum growth at low
temperatures (Table 2), it was incorporated in the most
conservative GF for LT prediction.
Effects of fish species and inoculation site
C. botulinum toxigenesis is affected by growth sub-
strate, extracts of cod, sole, salmon, and herring enhanced
germination rates of C. botulinum type E, while extracts
of shrimp, oyster, and crab did not (71). Cod was more
conducive medium for C. botulinum. as compared to lemon
sole and coho salmon in time to toxicity (9). Herring is
considered to be the most botuligenic of commonly har-
JOURNAL OF FOOD PROTECTION, VOL. 53. FEBRUARY 1990
vested fishes tested (1,10,58), a fact often attributed to its
high body oil content. The oil content of fish flesh has
been suggested as a criteria for evaluating the botulinisity
between fish species (1,2,10). Herring inoculated with 104
spores/g developed four times the MLD/g as halibut inocu-
lated similarly and stored at 20°C, about twice the MLD/
g at 10°C, while less than 10 MLD/g developed in each
substrate at 5°C; however the time to initial toxicity was
approximately the same for each fish regardless of incuba-
tion temperature (11).
A desire to use herring as the most conservative fish
with which to conduct inoculated pack studies proved
unfeasible in our studies. This was due to the fact that C.
botulinum type B was present in the fish homogenate (1
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spore/g), which precluded experiments based on low level
inoculations and MPN studies (6). Our studies indicated
that caution should be exerted when basing safe storage
times on fish type. Rockfish and salmon seemed to be
more conducive to C. botulinum growth and toxigenesis
than the sole tested, however these results did not account
for differences attributable to season, physiological state of
the fish, etc.
Inoculation into fish muscle produced toxin approxi-
mately 1 d prior to surface inoculation of similar vacuum
packaged samples (12). Huss et al. (41) found herring flesh
and viscera to support C. botulinum toxin production equally
well at 10, 15, and 30°C, even though the initial oxidation-
reduction potential (Eh) of the flesh was positive (+10OmV)
and the viscera negative.
lnocula (20 \x\) in our studies were always introduced
with a microsyringe into the center of the fish homogenate
of flesh where Eh and microbial competition would be the
lowest. Furthermore, the use of a small volume was con-
sidered advantageous, as the germination and growth ini-
tiation of a single spore could further reduce the Eh of the
microenvironment, thus enhancing the probability of other
spores to outgrow and reduce the LT.
In summary, fish type, physiological state, season,
location, residential spore load, and gutting process all
potentially affect the outcome of inoculated pack studies,
as does the sample preparation, i.e. size, cut, and texture,
and the initial microbial flora at the time of MA packag-
ing.
Unification of qualitative C. botulinum inoculated pack stud-
ies
Accurate food safety models should have graphs of
predicted values of similar shape to observed data, yield
conservative predictions, and agree with published findings
of similar research. Figure 4 compares the predicted length
of LT derived from the GF for four spore loads, with tabulated
results of inoculated pack studies conducted with fish reported
in the international literature (6). The total number of spores
per pack was considered to be the most appropriate com-
mon denominator among studies, as it is used by the canning
industry. Studies which did not report their sample size,
were assumed to contain at least 10 g, thus, the actual
number of spores/pack was probably underestimated.
 PREDICTING SAFE STORAGE OF FRESH FISH
Equivalent numbers of spores/g may lead to quite different
results when the experimental unit is variable, i.e. equiva-
lent inoculation of a 1 g sample compared to one of 100
g leads to a 2 log difference in initial inoculum size per
samples. When the sample is homogenized with inoculum
the spores are expected to be evenly distributed yielding
a "true" spore/g design. This method may not be represen-
2 6 10 14 18 - 22 26 30
Temperature C
Figure 4. Comparison of first time toxicities (day) in modified
atmosphere packaged fish inoculated with Clostridium botulinum
spores reported in the literature' with the shortest C. botulinum
lag times in fish predicted from a multiple linear regression (GF
of text) for four levels (101, 102, l(f and 104 spores/pack) of in-
oculum.
A# Number of observations at plotted time and temperature.
tative of "real world" contamination where surface inocu-
lation would be most common and puncture inoculation of
many spores together would be most conducive to growth.
Reported inoculum sizes falling between any consecutive
two of the four levels in Fig. 4, were compared against the
lower of the two plotted inocula levels.
Few investigators reported their sampling intervals or
acknowledged how accurate their times to toxicity were.
Samples which were reported as being toxic either prior
to, or after the sampling time, were plotted in Fig. 4 as
being toxic at the sampling time. Thus, 31 samples were
plotted as being toxic earlier than actually occurred, while
15 data points were toxic prior to the sampling period
reported. Only 1 of 165 qualitative observations from the
literature were toxic prior to the lag time predicted by the
JOURNAL OF FOOD PROTECTION, VOL. 53. FEBRUARY 1990
139
GF. Herring injected with 104 spores and incubated at 15°C
became toxic in 1 d (41), while the GF predicted 1.58 d.
Cann et al. (11) reported that whole, vacuum packaged,
herring inoculated with 106 spores/pack and stored at 3.3
and 5°C were toxic at 21 and 9 days, respectively. Though
the observations at 3.3°C and with 100 fold greater spore
inoculations were outside the range included in our experi-
ments, the GF yielded LT predictions of 14.29 and 6.32 d
for 3.3 and 5°C, respectively. Neither analyses for residen-
tial contamination of C. botulinum, nor the initial micro-
bial flora contamination level were reported in any of these
studies.
CONCLUSION
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The derived GF demonstrated the ability to yield
conservative approximations for the length of C. botulinum
LT in inoculated fish, packaged under a variety of MA,
and stored over a range of temperatures, which were in
general agreement with independent reports made by re-
searchers from different countries.
Predictive modeling applications
Models based upon LT are most appropriate for patho-
gens with zero growth tolerance like C. botulinum. Salmo-
nella, Listeria, etc. Growth rate models are best for patho-
gens which must have significant increase in numbers to
reach an infective/toxic dose, i.e. Staphylococcus aureus,
or prediction of microbial food spoilage (7).
Modeling strategies should aim toward consolidation
of mathematical and experimental approaches, thus pro-
viding the food industry with rational guidelines for design
and routine safety testing of a product. Standardization of
factorial design experimentation will allow the refrigerated
foods industry to develop a C. botulinum LT database.
Manufacturers would then be able to design a general formula
which includes nutritional, physical, and chemical factors
pertaining to their specific product. Access to such a da-
tabase would greatly reduce the cost of conducting product
challenge studies by providing C. botulinum growth expec-
tations, and improve challenge study reliability by allow-
ing tighter bracketing of sampling times and temperatures.
Processors should consider the additional safety mar-
gin attributable to terminal heating, but never rely upon it
to destroy botulism toxin. This is especially relevant when
using the process of microwave cooking which is known
for its uneven heat distribution. Inclusion of inoculated
pack studies as part of a comprehensive HACCP quality
assurance program will allow processors to establish
maximum time/temperature storage conditions for their
products, based upon worst case scenarios of C. botulinum
spore contamination in raw product, or from in-plant product
contamination and potential consumer abuse.
The predictive model we have derived from thousands
of observations by allowing estimations of the rate of LT
change as affected by temperature may also help the
development of realistic time/temperature indicators. Indi-
cators capable of being made in such a way that their
expiration, as affected by storage, temperature, and time,
will follow biochemical or physical kinetics slightly faster
140 BAKER AND GENIGEORGIS
than the rate of C. botulinum toxin production. Ultimately,
these indicators will provide a safe warning mechanism
for both the food processor, retailer, and the consumer.
ACKNOWLEDGMENT
This work is a result of research sponsored by NOAA; National Sea
Grant College Program, Department of Commerce, under grant number
NA80AA-D-00120, project number R/F 99, through the California Sea
Grant College Program, and in part by the California State Resources
Agency. The U.S. Government is authorized to reproduce and distribute
for government purposes. The senior author expresses his gratitude to the
California Sea Grant College Program for their Traineeship award, ena-
bling graduate studies. Gratitude is also expressed to Dr. C. Franti for
statistical support, Dr. R. Price for fisheries industry insights, and the
Cryovac Co. for provision of supplies and equipment.
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