is likely that some botulism outbreaks caused by semipreserved (salted, pickled) fish have been due to botulinum toxin formed in the raw material before ...
International Journal of Food Microbiology, 3 (1986) 167-181 Elsevier
167
JFM 00098
Probability of growth and toxin production by nonproteolytic Clostridium botulinum in rockfish stored under modified atmospheres Seppo
E. L i n d r o t h *
and Constantin A. Genigeorgis
Department of Epidemiology and Preventive Medicine, University of California, Davis, CA 95616, U.S.A. (Received 5 August 1985; accepted 4 April 1986)
The potential risk of C botulinum growth in fresh fish stored under modified atmospheres remains unclear. Few studies have identified qualitatively certain conditions leading to toxigenesis. This paper is the first of a series attempting to quantify the effect of a variety of parameters on the probability (P) of toxigenesis by one spore in fish. The factorial design experiments included red snapper tissue homogenate inoculated with a pool of nonproteolytic spores (5 type E, 4 type B and 4 type F strains) at 7 levels (104-10 -2 per 3 g sample) and incubated at 4, 8, 12, 17 and 30°C under 3 modified atmospheres (vacuum, 100% CO2 and 70% CO 2 +30% air) for up to 21 days. At the 10 ° spore/sample level the earliest time to detect toxin production at 4, 8, 12, 17 and 30°C under all modified atmospheres was > 21, 12, 9, 6 and 2 days, respectively. At the 101 spores/sample level the earliest times for the same temperatures were > 21, 9, 6, 3-6 and 1-2 days, respectively. The probability of toxigenesis was affected significantly (P < 0.005) by temperature, storage time, atmosphere × temperature, and temperature × time but not by atmosphere (P > 0.1). Using linear and logistic regression models, equations were derived which can predict the P of 1 spore initiating growth and toxigenesis by a particular day and at a particular temperature of storage. Studies involving other fish substrates are in progress. Key words: Clostridium botulinum; Modified atmospheres; Probability; Risk assessment; Risk evaluation; Predictive model; Toxigenesis
Introduction A l t h o u g h q u a l i t y d e t e r i o r a t i o n of flesh s e a f o o d is the r e s u l t of a n u m b e r of d i f f e r e n t factors, m i c r o b i a l activity is b y far the m o s t i m p o r t a n t factor. T h e r e f o r e c o n t r o l of m i c r o b i a l g r o w t h is the key to the e x t e n s i o n o f p r o d u c t shelf-life d u r i n g t r a n s i t a n d s u b s e q u e n t storage. M o d i f i e d a t m o s p h e r e ( M A ) storage u s u a l l y i n v o l v e s elevated CO 2 concentrations combined with refrigeration temperature. Elevated levels of C O 2 selectively i n h i b i t the g r o w t h of G r a m - n e g a t i v e s p o i l a g e b a c t e r i a (Pseudomonas, Acinetobacter, Moraxella a n d r e l a t e d p s y c h o t r o p h s ) w h i c h g r o w r a p i d l y a n d c a u s e o f f - o d o r s a n d -flavors a n d t h u s spoilage, while G r a m - p o s i t i v e
* Present address and author to whom correspondence should be sent: Food Research Laboratory, Technical Research Centre of Finland, Biologinkuja 1, SF-02150 Espoo, Finland.
168 lactic acid bacteria (Lactobacillus and Streptococcus) are less affected and often become the dominant flora on fresh foods held in CO2-enriched atmospheres (Eklund, 1982; Genigeorgis, 1985; Mokhele et al., 1983; Oberlender et al., 1983). Lactic acid bacteria grow more slowly and produce less-noticeable and less-offensive sensory changes than the normal aerobic spoilage flora. By using modified atmosphere storage at refrigeration temperatures the microbial stability of fresh seafood has been extended for at least 7 to 14 days (Brown et al., 1980; Lannelongue et al., 1982a; Mokhele et al., 1983; Molin et al., 1983; Parkin and Brown, 1982). However, with respect to fish, use of MA may increase the risk for Clostridium botulinum growth and toxigenesis. The prevalence of C. botulinum spores in fresh and salt water fish is very high (Sakaguchi, 1979). The available data also suggests that it is very difficult to raise fish in the Northern Hemisphere without the risk of these fish being contaminated with C. botulinum (Huss, 1981). Type E is the type most often encountered in fish. Type E and nonproteolytic types B and F are able to grow and produce toxin at temperatures as low as 3.3°C (Eklund, 1982; Hobbs, 1976). A maximum spore load of 17 spores/100 g has been found in fillets of haddock caught from open waters (Eyles and Warth, 1981), and 5.3 spores/g in farmed trout (Huss et al., 1974). Increased amounts of CO 2 around the fish do not cause increased toxin production (Huss et al., 1979). However, reduced O 2 pressure, extended keeping time, reduced competition with spoilage flora and change in spoilage pattern may render fish toxic before spoilage is obvious. A possibility that the residential microbial flora of fish may enhance C. botulinum growth through oxygen consumption and decrease of E h cannot be ignored (Huss et al., 1979). Keeping fish continuously below 3°C would eliminate the C. botulinum hazard. However, with existing refrigeration equipment and practices a maximum temperature of 3°C throughout distribution and sale cannot be guaranteed (Hobbs, 1976). Studies in regard to whether sensory spoilage of fish will precede toxin formation (Cann et al., 1980; Eklund, 1982; Eyles and Warth, 1981; Lee and Solberg, 1983; Lindsay, 1983) have indicated that as the storage temperature is increased from 0 to 10°C, the time interval between obvious spoilage and detectable toxin production in fish stored in MA shortens. Below 10°C, it seems that spoilage usually precedes toxigenesis. However, toxin production before obvious spoilage at 10°C or below has been reported (Eklund, 1982; Lee and Solberg, 1983). A safeguard against poisoning is the fact that raw fish are normally adequately cooked before being eaten. Yet, reliance cannot be placed on the consumer's ability to detect signs of spoilage as a guarantee against consuming toxic foods. Solberg (1983) found that absolute rejection of fish coincided or always followed toxin detection. Moreover, it is likely that some botulism outbreaks caused by semipreserved (salted, pickled) fish have been due to botulinum toxin formed in the raw material before processing which has prevented the growth of C. botulinum but preserved the toxin (Huss, 1981). Presently we lack ways to predict the fate of C. botulinum spores found in fresh fish when the fish is stored under MA. Using factorial design experiments and regression analysis Genigeorgis (Genigeorgis et al., 1977; Metaxopoulos et al., 1981;
169 Riemann et al., 1972) has developed practical models for prediction of the fate of selected bacterial pathogens when exposed to various product formulations or processes. While similar approaches have been used successfully by the canning industry for decades (Licciardello, 1983), it has only been very recently that their application to other types of food preservation has been recommended (Broughall and Brown, 1984; Broughall et al., 1983; Roberts et al., 1981; Schultz, 1983). The purpose of this work was to develop a prediction model for C. botulinum growth and toxigenesis in red snapper inoculated with spores of 13 nonproteolytic strains and stored under 3 MA at 4°C to 30°C for up to 21 days. Materials and Methods
Fish
Fresh dressed red snapper (Sebastes paucispinis) was purchased from a wholesale market. Most (approx. 75%) of the rockfish coming to the wholesaler had been caught some 30 miles off the Californian coast. The rest had been caught somewhere else along the west coast. Fish had been iced on boats. After landing of the vessels fish had been transported during the same day under refrigeration to the wholesaler where it was gutted and kept at 0-2.2°C (32-36°F). Fish used for experiments was purchased less than 24 h from landing of fishing boats. Dressed, iced fish was taken to the laboratory, rinsed in cold tap water, drained and filleted using aseptic technique and sterile equipment. Fillets were ground in a sterile mechanical grinder, put in a pan, mixed gently and divided into approx. 225 g portions in zip lock plastic bags. Fish bags were frozen until used. Presence and M P N counts of residential C. botulinum in fish
Portions of ground fish were placed in a tissue culture plate (sterile, 24 well, 16 mm diameter, tissue culture clusters with cover, Mark II; Costar, Cambridge, MA) wells. Plates were incubated anaerobically at 30°C for 5 days. Contents of 6 to 12 wells (15-30 g) were combined and analyzed for toxin. Three tube MPN method with sample sizes of 10.0, 1.0, 0.1 and 0.01 g of ground fish was used for counting. Cooked meat medium (30°C 6 days) and tryptone-peptone-ghicose-yeast extract with trypsin (20°C 13 days) were used as cultivation broths. After incubation, tubes were checked for turbidity and gas production and analyzed for botulinum toxins. MPN value was derived from a 3-tube MPN table (Mayou, 1976). p H measurement
pH was determined without any dilution from ground fish with a pH meter. Aerobic plate count (APC)
25 g of original fish or 1.0 g of fish used in experiments was homogenized with nine volumes of 0.1% (w/v) peptone-water. Homogenate and 10-fold dilutions in
170 peptone-water were used for plating on tryptone-peptone-yeast extract-glucose-salt agar (Lee and Pfeifer, 1974) and incubated at 20°C for 4 days (Liston a3ad Matches, 1976).
Production and counting of C. botulinum spore suspensions Thirteen nonproteolytic type B, E and F strains were used (Table I). Spore crops were produced either in 5% trypticase, 0.5% peptone, 0.1% sodium thioglycollate, 0.8% glucose medium (Jensen, 1984) or in fresh cooked meat medium (Eklund et al., 1967). Spores were washed with cold sterile distilled water. Vegetative cells were killed by letting them stand in 50% (w/v) sterile ethanol at room temperature for an hour. Washing and centrifuging cycle was repeated at least four times. Clean spores were suspended in a small volume of cold sterile distilled water and stored at or below 4°C. The spores were counted microscopically using Petroff-Hausser counting chambers. Based on spore counts in each stock suspension approriate volume of suspensions of the same type were mixed and diluted with water to make the desired spore concentration with equal number of spores of each strain. The concentrations of the type pools were verified by microscopic counting. Nonproteolytic pool was prepared by mixing approriate volumes of the type pools to make the mix having equal number of spores of each strain. The presence of spores in singles in the pools was accomplished by glass beads and verified by microscopy.
TABLE I Nonproteolytic Clostridiumbotulinum strains used for inoculation of rockfish Type
Strain
Origin
Source
B B B B E E E E
2 17 194 706 211 250 4062 KA-2
Eklund a Eklund Crowther b Hatheway c Hatheway Crowther Hatheway Riemann d
E F F F F
Beluga 187 202 3194 56KA172
Marine sediment Marine sediment Herring Salted salmon F.P. Dolman strain Canned salmon Muktuk (fermented whale blubber) Seola Creek strain used as test toxin source F.P. Dolman strain Herring Marine sediment Venisonjerky Salmon
a Eklund, M.W., National Marine Fisheries Service, Seattle, WA, U.S.A. b Crowther, J.C., Unilever Research, Bedford, England. c Hatheway, C.C., Centers for Disease Control, Atlanta,'GA, U.S.A. a Riemann, H.P., University of California, Davis, CA, U.S.A.
Eklund Crowther Eklund Hatheway Hatheway
171
Inoculation offish samples Defrosted (overnight at 4°C) ground fish was packed in tissue culture plates (approx. 1.5 g/well, 24 wells/plate). 20/tl of spore dilutions in water was pipetted with micropipette (Finnpipette, Labsystems, Finland) in triplicate into fish to give 10-fold dilutions of spores. Additional (1.0 g/well) homogenate was added to each well, and the plates were covered with the lid. Experiments with type E pool were carried out with eight 10-fold dilutions of spores starting from log 3.5-6.5 spores/ sample, depending on the storage temperature of fish samples. Final spore concentrations per fish sample in the nonproteolytic pool test where 0 (control) and log 4, 3, 2, 1, 0, - 1 and - 2 . Inclusion of 10 -1 and 10 -2 spore concentrations was done for a more precise measurement of MPN by avoiding situations where all samples would show growth and toxigenesis. Spores were kept iced during inoculation. Concentration of inoculum was also checked by MPN counting in brain heart infusion + 0.1% L-cysteine (BHIC) tubes incubated anaerobically at 30°C.
Modified atmosphere packing and incubation offish plates Type E inoculated fish plates were incubated in anaerobic jars flushed three times with 25 : 75 CO 2 : N 2 gas mixture. Nonproteolytic pool inoculated fish plates were inserted in 20 × 25 cm plastic bags [Barrier bag, type B 540, Cryovac Division, W.R. Grace & Co., Hayward, California; 02 transmission 30-50 cc/m2/24 h/1 atm/22.8°C (73°F)]. Bags were placed in Multivac A 300 Model 22 (Sepp Haggenmi~ller KG, F.R.G.) packing machine, and vacuum was created. Bags were either left in vacuum or flushed with 100% CO 2 (99.8: CO 2, Liquid Carbonic, San Carlos, CA) or 70% CO2 balanced with air, to produce 5:1 gas:fish volume ratio, and sealed. MA packed fish plates were incubated at 4, 8, 12, 17 and 30°C. Incubation period for type E pool samples was 6 to 60 days depending on storage temperature. Samples inoculated with the nonproteolytic pool were incubated for up to 21 days. One fish plate per each test condition was taken for analysis at regular intervals.
Gas composition in fish packs C O 2 and 0 2 concentrations were analyzed from C O 2 flushed fish packs immediately after packing and at each analysis time. Gas samples were drawn into 10 ml syringes and analyzed with a Carle 8000 gas chromatograph (Hach Carle, Loveland, Colorado) using Porapak N (80-100 mesh, 8 ft, diam. 1/8 inch) and Molecular Sieve 5 A (45-60 mesh, 6 ft, diam. 1/8 inch) columns. Injection volume was 1.0 ml. Carrier gas was helium and flow rate 18 ml/min. Column temperature was set at 70°C. Detection was through thermal conductivity.
Volume measurement offish packs Two extra fish packs were prepared for each atmosphere and temperature. Total volume of these sample bags was assayed after packing, after 12 h, and at each analysis time by water displacement in a large plastic cylinder.
172
Toxicity testing The Centers for Disease Control, USA (CDC, 1979) protocol with slight modifications was used. Samples (2.5 g each) were extracted overnight with 5 ml of gel-phosphate, p H 6.2, buffer. The mixture was centrifuged at 27,000 x g for 20 min and the supernatant collected. If the pH of the supernatant was equal to or more than 7.0 it was adjusted to pH 6.2-6.5 with 1 N HC1. 2 / 5 of CDC protocol samples volumes were used for trypsinization and neutralization testing. O n l y one mouse, instead of two, was used per test. Type A antiserum, produced in our laboratory, was often used to neutralize possible toxin produced by residential type A C. botulinum in samples, before testing for toxicity of other types. Type B, E, F and poly antisera were from CDC (Atlanta, GA). When several 10-fold inoculum levels showed toxicity, neutralization test was performed only with the lowest toxic inoculum level sample extracts.
Calculation of probabifity Based on 10-fold dilutions of spore inoculum and triplicate samples the number of toxic samples was converted to MPN of spores able to initiate growth and produce toxin in each set of conditions. MPN table was derived from Fisher and Yates (1957). Probability ( P ) of one spore to initiate growth and produce toxin was defined as P (%) = (MPN X 100)/inoculum. When not a single sample was toxic the P was defined as 10-3%.
Analysis of variance ANOVA was run on toxicity and aerobic plate count data to see the impact of time, temperature and atmosphere. The BMDP2V (Dixon et al., 1981) program was used.
Prediction model fitting (1) The longest storage period when no toxin was detected in any sample (lag period) was described as a log-linear function of temperature. Data for all three atmospheres were combined. Time for 30°C was defined as 0.04 days (1 h) and for 17°C, when required, as 0.5 days. BMDP1R was used to derive the best fit equation. (2) Logistic regression model (BMDPLR) was used to describe the P curves after lag periods. For this purpose the log P scale of - 3 to 2 was converted to 0 to 1. (3) The final model was a combination of lag period and logistic regression. Results and Discussion
Residentml C. botufinum All seven lots of fresh fish (1.36 kg (3 lbs) to 29 kg (64 lbs) each) analyzed contained residential type A C. botufinum at levels of 0.09 to 2.4 organisms/g. One
173 lot had type A and E (0.09/g) organisms. The high presence of C. botulinum types A, B, E and F in mud samples and fish from the Pacific Coast of the U.S. has been reported before (Craig and Pilcher, 1967; Eklund and Pousky, 1967) with type E being the most prevalent. Llobrera (1983) did not find any C. botulinum in rockfish harvested off the Pacific Coast. The highest M P N count of 2.5 s p o r e s / g found in this study is higher than levels reported before for fish harvested from the open marine waters (Eklund, 1982) but similar to the m a x i m u m of 5.3 s p o r e s / g recorded for farmed trout (Huss et al., 1974). Spore build-up in the products during processing in the factories is possible (Eyles and Warth, 1981).
Probability of growth initiation disregarding time element The P of growth initiation and toxin production by type E C. botulinum at various temperatures is shown in Fig. 1. Fish was stored until spoiled before toxin analysis. The P of one spore to initiate growth and toxin production was 4.6 to 100% at 8 to 30°C. At 30°C the P was 100% while at 4°C it was 0.001%. Prediction equation for P as a function of temperature, calculated by regression of best growth primary data, was log P(%) = - 8.34 + 1.72. T - 0.092-
T 2 +
0.0016-
T 3, r 2 =
0.98
where T = temperature (°C). The log P-temperature curve for red snapper is very much alike to the curve obtained in B H I C broth with E. beluga, the best growing of five strains tested (Jensen, 1984). Using the mathematical equation one can calculate e.g. that log P(%) at 6°C would be -0.986, and P = 0.103%. This means that 970 spores would be needed for growth initiation at 6°C under specified conditions.
~2"
0.1) effect for the atmosphere alone. The result confirms the report by Huss et al. (1979) that increased amounts of carbon dioxide in the atmosphere around fish do not cause increased toxin production. Modified atmosphere packed fish have been found to be marginally less botulinogenic than vacuum packed controls but the temperature of storage and site of contamination were of more significance than the composition of the gas mixes (Cann et al., 1983). With respect to the site of inoculation studies have shown that
lob~o2'
~_
'
'
'
'
I
I
I
o.
O-1.- 2 nl~ _ t t
.
.
.
.
701% C~)2 J ..I
12 0
-3 [ 0
.
.
J
• 30"C o 17"C • 12"C [] 8"C • 4°C
3
6
STORAGE
~) 1'2 1'5 1'8 21 T I M E (DAYS)
Fig. 2. Observed probability of one nonproteolytic Clostridium botulinum spore to initiate growth and produce toxin by a certain day in red snapper stored under three modified atmospheres and five temperatures. Data obtained by inoculating a pool of spores of 13 different C. botulinum strains (5 E, 4 B, 4 F) at multiple levels of 10 4 or less spores per 2.5 g sample.
175 TABLE II Lowest nonproteolytic C. botulinum spore inoculum level (lOgl0) showing toxin production in at least one of three replicate red snapper samples stored under modified atmospheres Atmosphere/ temperature
Storage time (days) 1 2
3
6
2 NT a NT NT NT
0 NT NT NT NT
0 2 _ b .
0 0 1 -
3 NT NT NT NT
0 NT NT NT NT
0 2 .
3 NT NT NT NT
0 NT NT NT NT
0 .
9
12
15
19
21
0 0 .
0 0 4
0 0 2
0 0 0
0 0 0 3
0 0 1
0 0 0
0 0 0
0 0 0 3
0 0 0
0 0 0
0 0 0
0 0 0 3
Vacuum
30°C 17°C 12°C 8°C 4°C
.
.
.
.
100% CO 2
30°C 17°C 12°C 8°C 4°C
0 0 1 .
0 0 .
.
.
70 % CO 2 + 30 % air
30°C 17°C 12°C 8°C 4°C a
NT
b
_
0 0 2 .
.
0 0 1 .
.
.
not tested. no toxin production; max inoculum 1.0 x 104/sample.
= =
deep inoculated
fish became
toxic faster than comparable
surface inoculated
fish
( C a n n e t al., 1 9 8 0 ) . I n s u r f a c e i n o c u l a t e d f i s h t o x i g e n e s i s o c c u r r e d a t a n e a r l i e r t i m e u n d e r v a c u u m p a c k a g i n g t h a n s t o r a g e i n a i r ( H u s s e t al., 1 9 7 9 ) . I n t h i s s t u d y d e e p inoculation probably created more conducive conditions for toxigenesis with minimal impact
2.
w
0
I-_
1
of the type of MA used.
• Vacuum o 100% CO 2
'4 8 1~) 16 2qO 214 218 3'2 TEMPERATURE (°C) Fig. 3. Effect of temperature and modified atmospheres on the length of the lag period preceding initiation of growth and toxin production by one nonproteolytic CIostridium botulinum spore. Observed and calculated (best fit) data based on multiple inocula of 104 or less spores per 2.5 g red snapper sample.
176 The lag period preceding rise in probability of growth initiation (Fig. 2) seemed to have a log-linear relationship with temperature (Fig. 3). Best fit regression equation for lag period with respect to temperature was log (Day) = 1.630 - 0.102. T + 0.008; r 2 = 0.93. Combining the lag period concept and a logistic regression model for data points differing from log P = - 3 (baseline, no toxin detected), the following prediction equations were derived: ey
log P(%) = 5 ( ~ )
- 3, where
vacuum: y = - 1 . 9 3 - 0.048. T - 0.370(D - LP) + 0.110- T(D - LP) 100% CO2: y = - 3 . 2 0 + 0.014. T + 0.323(D - LP) + 0.064- T(D - LP) 70% CO2: y = - 2 . 6 0 - 0.022. T - 0.054(D - LP) + 0.087. T(D - LP) All atmospheres combined: y = - 2 . 4 7 - 0.022. T - 0.068(D - LP) + 0087. T(D - LP), where T = temperature (°C); D = storage time (days) and LP = lag period (days) = 101.630-0.102T.
Using these equations the predicted P was calculated for each time and temperature setting used in experimental study (Fig. 4). The predicted P values for toxigenesis closely resembled those observed in actual experiments (Fig. 2) and they were somewhat higher than the observed. The only exception was the case of 70% CO 2 balanced with air, where the observed toxicity somewhat preceded the predicted at 8°C. Salmon fillets or steaks inoculated with up to 10 4 type E spores per 100 g sample and stored under 60% CO 2, 90% CO 2 or vacuum became toxic in 2, 4 - 5 and 7-11 days at 20-25, 15 and 10°C, respectively (Cann et al., 1984; Eklund, 1982; Stier et al., 1981). At 5°C no toxin was detected by 21 days (Eklund, 1982) and at 4.4°C by 57 days (Stier et al., 1981). In flounder fillets inoculated with up to 10 6 type E spores/100 g no toxin was found at 4.4°C by 21 days (Llobrera, 1983). At 10°C toxin was detected after 6 days and at 26.6°C after 24 h. These reports correlate well with our data except that in one experiment we found toxic samples at the 1 × 10 4 s p o r e s / s a m p l e inoculum and incubation at 4°C for 21 days under all MA's studied but not in a replicated experiment. Using the prediction model we can get estimates for the intermediate values not included in the actual experiment. For example we can calculate that red snapper stored under modified atmospheres at 10°C has a lag period of 4 days for growth initiation. Similarly the P(%) of growth initiation in red snapper stored 6 days at 10°C would be log - 1 . 7 9 or 0.02.
177
10
Vacuum
Vacuum
lOO% c o 2
.~ 1-
i-
~o-1~-2°'3. 0 "u
2.
I I 70%CO2 . . .
I
o~ / / / "
I
I
=
-
/
I
I -
~,~:c~
2
•
4°c
, 0
3
6
9
STORAGE
12 TIME
15
18 (DAYS)
~ 10~ 7°~c°2
=
7 I
• 3°~c
5J~
•
,
4~
21
0
I
I
i
3 6 9 STORAGE
I
I
4 0.05) effects of MA x time and T x time on the total APC.
178
pH No major trends were identified with respect to the effect of atmosphere on the pH of the fish homogenate. Only at 4°C and under 70 and 100% CO 2 the pH remained at its initial level of 6.7 for the first 12 days. In all other conditions the pH increased with storage. After 21 days storage pH values were close to 8 under all atmospheres and temperatures. Often an initial drop in surface pH of fish stored under CO2 atmospheres has been observed, and the pH values of these fish have stayed lower than air controls also during storage (Lannelongue et al., 1982a; Oberlender et al., 1983; Parkin et al., 1982; Richter and Banwart, 1983). pH values higher than 7.5 in fish and buffer (Huss and Petersen, 1980; Kitamura et al., 1969) have been shown to affect the stability of type E toxin. In this study samples were taken throughout the storage period and frozen immediately. To minimize the potential pH effect on the stability of toxin, sample extracts were adjusted to pH ~
