Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN 968-878-105-3
IMPROPER POSTHARVEST HANDLING PRACTICES MAY INCREASE THE RISK OF
STAPHYLOCOCCAL ENTEROTOXIN PRODUCTION IN FRESH AND BLANCHED
MUSHROOMS
P. A. Cheplick and R. B. Beelman
116 D, Borland Laboratory, Department of Food Science
The Pennsylvania State UniversityUniversity Park, Pennsylvania 16802, U.S.A.
<rbb6@psu.edu>
ABSTRACT
Several food poisoning outbreaks in the U. S. linked to the consumption of canned mushrooms from
the People’s Republic of China (PRC) have prompted studies to examine potential causes of this
problem. It was shown that the presence of staphylococcal enterotoxin (SE) in mushrooms from
China was responsible for the outbreaks and was most likely present in the mushrooms before
thermal processing. Two potential scenarios that could permit growth of Staphylococcus aureus to
sufficient populations to produce SE prior to canning were examined: time/temperature abuse of
fresh mushrooms during transport to the processing plant, and time/temperature abuse of blanched
mushrooms in the processing plant prior to thermal processing. Fresh mushrooms were inoculated
with enterotoxigenic type A S. aureus 743 and stored under time/temperature abuse conditions
(35ºC and 40ºC for 24 hr.) and subsequently analyzed for growth of natural microflora and S.
aureus as well as the formation of SE. Enterotoxin was detected by the mini VIDAS fluorescent
immunoassay system and SE concentration was estimated by employing a standard curve generated
using purified enterotoxin. Blanched mushrooms, held in water at 95ºC for 5 min. and cooled to
20ºC in tap water, were also examined in a similar manner. Time/temperature abuse of fresh
mushrooms permitted sufficient growth of S. aureus to allow production of SE in less than 24
hours. More rapid growth and SE production was observed earlier (12 vs. 16 hours) at 40º C than at
35º C, respectively. Growth and SE production occurred more rapidly in blanched mushrooms. SE
was detected in 8 and 9 hours at 40º C and 35ºC, respectively. Although higher S. aureus
populations and enterotoxin concentrations were found in blanched mushrooms, higher temperature
(40ºC) had more of a stimulatory effect on enterotoxin production in fresh mushrooms than in
blanched mushrooms. These results indicate that time/ temperature abuse of either fresh or
blanched mushrooms contaminated with S. aureus can permit sufficient bacterial growth to produce
SE and cause food poisoning from consumption of fresh or canned products.
INTRODUCTION
Canned mushrooms imported from the People’s Republic of China (PRC) were implicated in
several food poisoning outbreaks in 1989. The outbreaks occurred primarily in food-service
establishments that were using the mushrooms from #10 (68oz) cans as toppings or condiments for
main items (CDC 1989). The cause of the outbreaks was determined to be the presence of
staphylococcal enterotoxin (SE) in the product. As a result, the United States Food and Drug
Administration issued a mandatory hold on all imported canned mushroom products from the PRC.
Prior to the outbreaks and subsequent FDA action against the PRC, the United States had imported
about 50 million pounds of mushrooms annually (Levine et al. 1996). These outbreaks were
significant because they were the first major outbreaks of staphylococcal food poisoning in the
United States directly related to a canned food product (Beelman 1990, Hardt-English 1990). Since
then, several studies have focused on the causative factors involved in these outbreaks (Martin and
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Beelman 1996, Brunner and Wong 1992), however many questions remain to be answered in order
to fully understand this potential problem.
Staphylococcal enterotoxin is a proteinaceous toxin produced by the bacteria, Staphylococcus
aureus, and is responsible for the physiological effects of staphylococcal food poisoning.
Staphylococcal food poisoning is characterized by severe cramps, abdominal pain, nausea, diarrhea,
and vomiting. Infants, the elderly, and those with compromised immune systems are primarily
affected (Genigeorgis 1989). The enterotoxin is produced by the organism once a cell concentration
of at least 106 CFU/g food is present, which normally requires growth of the organism in a food
prior to thermal processing. While examining potential scenarios that would have lead to
staphylococcal contamination of the mushrooms in the PRC, Martin and Beelman (1996)
demonstrated that growth of S. aureus and SE production could occur in fresh mushrooms in as few
as 18 hours. Additionally, the enterotoxin has been shown to be extremely heat stable, enough so
that it can survive a typical thermal process for canned foods (Anderson et al. 1996). Therefore,
current knowledge indicates that if S. aureus grows to sufficient populations to produce SE before
thermal processing, the potential exists for staphylococcal intoxication from canned mushrooms.
The goal of this study was to model the potential abusive handling and storage conditions that fresh
and blanched mushrooms may have been exposed to in the PRC, thereby resulting in food
poisoning outbreaks in the United States. In addition, the parameters used in previous studies
(Martin and Beelman 1996) were expanded upon to provide a more complete understanding of the
relationship time and temperature has in regard to S. aureus growth and SE production. Several
potential scenarios that could permit growth of S. aureus to sufficient populations to produce SE
prior to canning were examined: 1) time/temperature abuse of fresh mushrooms during transport to
the processing plant, and 2) time/temperature abuse of blanched mushrooms in the processing plant
prior to thermal processing. The goal of this study was to provide the domestic mushroom and
mushroom-product industry with detailed information in regard to safe handling and processing of
their products in order to reduce the risk of staphylococcal food poisoning.
MATERIALS AND METHODS
Staphylococcus aureus Culture
Staphylococcus aureus 743 (isolated from a food poisoning outbreak) was grown in Brain Heart
Infusion broth (BHI) at 37C to a final concentration of approx. 2 x 109 CFU/ml. This strain
produces staphylococcal enterotoxin Type A (SEA), which is the enterotoxin serotype most
commonly associated with food poisoning.
Mushrooms
All mushrooms used in this study were a hybrid off-white (U-1) strain of Agaricus bisporus and
were obtained from a University-operated mushroom growing facility. In all cases, the mushrooms
were stored for approx. 24 hr in 4C immediately after harvesting, prior to inoculation and storage.
Inoculation of Fresh Mushrooms
An overnight culture of Staphylococcus aureus 743 was inoculated onto fresh mushrooms using a
stock suspension at approximately 2 x 105 CFU/ml. To obtain the desired concentration of cells on
each mushroom, a 10µl disposable loop was used to spread the diluted stock suspension over the
cap of each mushroom. The mushrooms were then transferred aseptically into 8-oz polystyrene tills
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which were subsequently overwrapped with a commercial plasticized polyvinyl chloride (PVC)
film. Upon being overwrapped, two, 2-mm holes were made in the film at opposite corners to
prevent development of an anaerobic environment within the till. Uninoculated, control mushrooms
were prepared in the same manner, and all tills were then incubated at the appropriate temperature.
Inoculation of Blanched Mushrooms
Mushroom stipes were trimmed to within 5mm of the cap and an appropriate amount was then
blanched by submersion in boiling water for 5 min. Immediately following blanching, the
mushrooms were transferred to one of two cooling water buckets and held for 24hr at 4C. The
cooling water contained either sterile water (control) or water inoculated with S. aureus 743 at a
final cell concentration of ~ 104 CFU/g mushroom. Mushrooms were then transferred to sterile
storage containers, covered with aluminum foil, and placed into the appropriate incubator.
Sampling of Mushrooms
Fresh and blanched mushrooms that were held at 35C were sampled every 6 hr while mushrooms
held at 40C were sampled every 4 hr. At the appropriate time interval, one control and one
inoculated till of fresh mushrooms was removed from the incubator and used to enumerate S.
aureus cells and background microflora. The mushrooms were transferred into sterile blender jars
to which an equal weight of sterile 0.25M TRIS buffer (pH 8) was added (TRIS: Fisher Biotech,
Fair Lawn, NJ). The mushrooms were then blended for 2 min using a Waring blender and
appropriate serial dilutions were made using 0.1% peptone dilution blanks. Following dilution,
0.1ml of each dilution was spread plated in duplicate onto Tryptic Soy agar and Baird-Parker agar
for the enumeration of background microflora and S. aureus, respectively. The plates were
incubated at 35C for 48 hr prior to counting. An aliquot of each mushroom blend was set aside for
use in determining the presence of SE.
Detection of Staphylococcal Enterotoxin
After the bacterial enumeration procedures of the mushrooms were completed, 100g samples of the
mushroom blend was carefully transferred to sterile centrifuge bottles and centrifuged for 20 min /
4C / 10,000g. It was assumed that if any staphylococcal enterotoxin was present in the sample
blend, it was present in the supernatant. A 2ml aliquot of the supernatant was removed and stored
in cryogenic Nalgene tubes at -70C until needed for further analysis. Similarly, 2ml aliquots of the
BHI broth samples were removed and stored, however the broth cultures were not centrifuged prior
to this removal.
The miniVIDAS (bioMerieux Vitek, Inc., Hazelwood, MI) system was used to detect for
staphylococcal enterotoxin in samples. This system is a diagnostic assay system which is used to
perform an automated qualitative enzyme-linked fluorescent immunoassay for the detection of
staphylococcal enterotoxins in food ingredients and products. The intensity of fluorescence is
related to the amount of SE present in a sample, and is printed as a Test Value (TV) after analysis
by the system’s on-board computer. Any TV < 0.13 was interpreted as a negative sample while any
sample with a TV  0.13 was considered positive for staphylococcal enterotoxin. Final SE
concentrations were estimated using a standard curve generated using highly purified (96%)
staphylococcal enterotoxin A and plotting TV versus known SE concentrations (data not shown).
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Statistical Analysis
All statistical analysis was performed using StatView Version 4.5 (Brainpower, Inc., Berkeley,
CA). ANOVA tables and Fisher’s Protected Mean Separation procedure were used to determine
significant differences between temperatures and other treatments in regard to S. aureus growth and
enterotoxin production. Additionally, StatView was used to calculate standard error of the mean
which was used as the basis for Y-error bars found in the graphs of the results section.
RESULTS AND DISCUSSION
Fresh Mushrooms
When fresh mushrooms were inoculated with S. aureus and held at 35C and 40C, rapid growth
and enterotoxin production occurred (Figure 1). Fresh mushrooms stored at 40C allowed increased
growth, and much more rapid and extensive production of enterotoxin than at 35C. SE was
detected earlier at 12 hours at 40C, as compared to 16 hours at 35C (Figure 1). By 24 hours of
storage, the difference in SE production was highly significant and considerably more dramatic than
the difference in growth (Figure 1B). Holding temperatures of 40C could easily occur under
Chinese processing conditions where refrigeration is lacking and mushrooms are often transported
long distances from farms to canning factories. Hence, the results of this experiment present a
realistic scenario of how SE could be present in mushrooms canned in China. In addition it is likely
they could cause staphylococcal food poisoning, since it is now known that thermal processing does
not destroy SE (Anderson et al. 1996). Martin and Beelman (1996) previously demonstrated that
growth of S. aureus and SE production in fresh mushrooms could occur within 18 hours at 35C.
However, they did not evaluate higher temperatures on growth and SE production of S. aureus and
they did not quantitate enterotoxin produced.
Interestingly, Martin and Beelman (1996) found that growth of the natural microflora in fresh
mushrooms was suppressed when stored at 35C compared to 25C and 30C. They suggested that
this suppression might have been responsible for the more rapid growth of S. aureus at 35C.
Hence, it was assumed that the natural microflora would be suppressed even more in this study
when mushrooms were stored at 40C and this might allow more rapid growth of S. aureus.
However this was not observed; the rate of natural microflora growth was similar at both 35C and
40C (Figure 2). In the present study, the initial natural microflora populations on the fresh
mushrooms were approximately 106 CFU/g and populations increased slowly and at the same rate
over the 24 hr incubation period at both temperatures. Although not identified in this work, the
majority of the background microflora were most likely fluorescent Pseudomonas spp. Fluorescent
Pseudomonas spp. are the most prevalent microbes native to fresh mushrooms (Doores et al. 1986).
Blanched Mushrooms
Results indicated that rapid growth of S. aureus and subsequent SE production occurred within 24
hr when blanched mushrooms were stored at 35C and 40C. SE was detected as soon as 8 hr after
incubation at 40C and as soon as 9 hr at 35C (Figure 3). These temperatures were used to
simulate abusive conditions blanched mushrooms may have been subjected to at Chinese mushroom
processing facilities prior to thermal processing. Although blanched mushrooms held at both 35C
and 40C permitted rapid growth and SE production, there were no significant differences between
temperatures in regard to these parameters. The rapid growth and SE production was most likely
caused by the absence of competing microflora due to the blanching process.
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Comparing Growth and SE Production in Fresh vs. Blanched Mushrooms Stored at Elevated
Temperatures
At 35ºC, SE was detected earlier, 9 vs. 16 hr, in blanched mushrooms compared to fresh
mushrooms. The amount of enterotoxin produced by S. aureus in blanched mushrooms after 24 hr
of storage was significantly greater at 175.07 ng/ml, compared to 18.67 ng/ml in fresh mushrooms.
Similar results were observed at 40ºC. Significantly more rapid growth and SE production occurred
in blanched mushrooms compared to fresh mushrooms (Figure 5). SE was again detected earlier in
blanched mushrooms as compared to fresh mushrooms (8 vs. 12 hr, respectively). The production
of increased amounts of SE occurred over the 24 hr storage period although the difference between
fresh and blanched mushrooms was not as dramatic as that found at 35ºC. This was likely caused
by the significant increase in SE production that occurred in fresh mushrooms held at 40ºC rather
than a decrease in SE production in blanched mushrooms.
Blanched mushrooms appear to provide a better environment for growth of S. aureus due to
disruption of the cell membranes within the mushroom tissue during the blanching process that
releases nutrients to become available for bacterial growth. Additionally, in blanched mushrooms,
there is less competitive inhibition of the S. aureus by other microorganisms, as found in fresh
mushrooms. Also, the blanched mushrooms have more moisture on their surface, aiding in the
growth of S. aureus.
CONCLUSIONS
These experiments demonstrate that rapid growth of S. aureus and higher enterotoxin
concentrations were produced in blanched mushrooms compared to fresh mushrooms held under the
same time/temperature abusive conditions. Higher temperature (40ºC) had a stimulatory effect on
enterotoxin production in fresh mushrooms than in blanched mushrooms. Potential contamination
with S. aureus, probably occurs during harvest and handling of fresh mushrooms. If abusive
storage temperatures are encountered during transport and storage of fresh mushrooms, the risk for
occurrence of SE production would be increased. This could have important food safety
implications where proper refrigeration and handling could be compromised. Due to insufficient
refrigeration and large distances between harvesting and processing areas, the abusive conditions
examined in this study may have occurred in China at several points during the mushroom
processing schedule. Results from this study demonstrate that these abusive conditions permit and
sometimes enhance growth and SE production by S. aureus in mushrooms. These situations may
have been responsible for the outbreaks of staphylococcal food poisoning due to canned mushrooms
in the United States in 1989. Control of both processing time and storage temperature has been
shown to be critical in ensuring safe, unadulterated product. These results should also serve as a
guide for mushrooms producers to ensure the processing and delivery of safe products to
consumers.
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log CFU/g mushroom
10
A.
*
8
*
*
6
*
35 deg.C
4
40 deg.C
2
0
4
8
12
16
20
24
Time (hr)
150
B.
125
*
Toxin (ng/ml)
100
75
50
25
*
0
0
4
8
12
Time (hr)
16
20
24
Figure 1. Growth of Staphylococcus aureus 743 (A) and enterotoxin production (B) in fresh
mushrooms at 35ºC and 40ºC.
Data points represent the mean of three replicate experiments and error bars represent standard error of the
mean. Means followed by an asterisk (*) are significantly different (p < 0.05). Arrows (A) and where curves
cross dashed line (B) represent time at which samples were positive for SE.
350
log CFU/g mushroom
10
8
6
35 deg.C
40 deg.C
4
0
4
8
12
16
20
24
Time (hr)
Figure 2. Growth of natural microflora on fresh mushrooms held at 35ºC and 40ºC.
Data points represent the mean of three replicate experiments and error bars represent standard error of the
mean. Means followed by an asterisk (*) are significantly different (p < 0.05).
351
10
A.
*
*
log CFU/g mushroom
8
6
35 deg.C
40 deg.C
4
*
*
2
0
4
8
12
16
20
24
16
20
24
Time (hr)
200
B.
175
Toxin (ng/ml)
150
125
100
75
50
25
0
0
4
8
12
Time (hr)
Figure 3. Growth of Staphylococcus aureus 743 (A) and enterotoxin production (B) in blanched
mushrooms at 35ºC and 40ºC.
Data points represent the mean of three replicate experiments and error bars represent standard error of the
mean. Means followed by an asterisk (*) are significantly different (p < 0.05). Arrows (A) and where curves
cross dashed line (B) represent time at which samples were positive for SE.
352
10
A.
*
log CFU/g mushroom
8
*
*
6
*
4
Blanched
Fresh
2
0
0
4
8
12
16
20
24
Time (hr)
200
B.
175
*
Toxin (ng/ml)
150
125
100
75
50
25
*
0
0
4
8
12
16
20
24
Time (hr)
Figure 4. Growth of Staphylococcus. aureus 743 (A) and enterotoxin production (B) in fresh and
blanched mushrooms at 35ºC.
Data points represent the mean of three replicate experiments and error bars represent standard error of the
mean. Means followed by an asterisk (*) are significantly different (p < 0.05). Arrows (A) and where curves
cross dashed line (B) represent time at which samples were positive for SE.
353
10
A.
*
*
log CFU/g mushroom
8
*
6
*
4
Blanched
Fresh
2
0
0
4
8
12
16
20
24
16
20
24
Time (hr)
200
B.
175
Toxin (ng/ml)
150
125
*
100
75
50
25
*
0
0
4
8
12
Time (hr)
Figure 5. Growth of Staphylococcus aureus 743 (A) and enterotoxin production (B) in fresh and
blanched mushrooms at 40ºC.
Data points represent the mean of three replicate experiments and error bars represent standard error of the
mean. Means followed by an asterisk (*) are significantly different (p < 0.05). Arrows (A) and where curves
cross dashed line (B) represent time at which samples were positive for SE.
354
ACKNOWLEDGEMENTS
The authors acknowledge the financial support provided by the Benjamin Franklin Partnership
Program/American Mushroom Institute Processors Committee.
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