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Survival of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes on Raw Peanut and Pecan Kernels Stored at −24, 4, and 22°C

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Cocktails of lawn-collected cells were used to determine the survival of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes on the surface of raw peanut and pecan kernels. Kernels were inoculated with mixtures of four to five strains at 3 or 6 log CFU/g, dried at room temperature, and then stored at –24 ± 1, 4 ± 2, and 22 ± 1°C for 28 or 365 days. In most cases, rates of decline of the pathogens did not differ significantly between the two inoculum concentrations in the 28-day study. At 6 log CFU/g, populations of all pathogens were reduced by 0.5 to 1.6 log CFU/g during an initial 3-day drying period on both peanuts and pecans. The moisture content of peanuts and pecans remained stable at –24 ± 1 and 22 ± 1°C; at 4 ± 2°C, the moisture content increased from 3.8 to 5.6% on peanuts and from 2.6 to 3% on pecans over 365 days. Pathogen populations were stable on pecans stored under frozen and refrigerated conditions, except for L. monocytogenes, which declined at a rate of 0.03 log CFU/g/30 days at 4 ± 2°C. Salmonella populations were stable on peanuts stored at –24 ± 1 and 4 ± 2°C, but E. coli O157:H7 and L. monocytogenes declined at rates of 0.03 to 0.12 log CFU/g/30 days. At 22 ± 1°C, Salmonella, E. coli O157:H7, and L. monocytogenes declined at a rate of 0.22, 0.37, and 0.59 log CFU/g/30 days, respectively, on peanuts, and at 0.15, 0.34, and 1.17 log CFU/g/30 days, respectively, on pecans. Salmonella counts were above the limit of detection (0.30 log CFU/g) throughout the study. In most cases during storage, counts obtained from pecans were higher than from peanuts.
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Survival of Salmonella, Escherichia coli O157:H7, and Listeria
monocytogenes on Raw Peanut and Pecan Kernels Stored
at 224, 4, and 22uC
PARDEEPINDER K. BRAR,
1
LISSETH G. PROANO,
1
LORETTA M. FRIEDRICH,
1
LINDA J. HARRIS,
2
AND
MICHELLE D. DANYLUK
1
*
1
Department of Food Science and Human Nutrition, Citrus Research and Education Center, Institute of Food and Agricultural Sciences,
University of Florida, 700 Experiment Station Road, Lake Alfred, Florida 33850; and
2
Department of Food Science and Technology, University of
California, Davis, One Shields Avenue, Davis, California 95616, USA
MS 14-327: Received 10 July 2014/Accepted 28 September 2014
ABSTRACT
Cocktails of lawn-collected cells were used to determine the survival of Salmonella, Escherichia coli O157:H7, and Listeria
monocytogenes on the surface of raw peanut and pecan kernels. Kernels were inoculated with mixtures of four to five strains at 3
or 6 log CFU/g, dried at room temperature, and then stored at 224 ¡1, 4 ¡2, and 22 ¡1uC for 28 or 365 days. In most cases,
rates of decline of the pathogens did not differ significantly between the two inoculum concentrations in the 28-day study. At 6
log CFU/g, populations of all pathogens were reduced by 0.5 to 1.6 log CFU/g during an initial 3-day drying period on both
peanuts and pecans. The moisture content of peanuts and pecans remained stable at 224 ¡1 and 22 ¡1uC; at 4 ¡2uC, the
moisture content increased from 3.8 to 5.6%on peanuts and from 2.6 to 3%on pecans over 365 days. Pathogen populations were
stable on pecans stored under frozen and refrigerated conditions, except for L. monocytogenes, which declined at a rate of 0.03
log CFU/g/30 days at 4 ¡2uC. Salmonella populations were stable on peanuts stored at 224 ¡1 and 4 ¡2uC, but E. coli
O157:H7 and L. monocytogenes declined at rates of 0.03 to 0.12 log CFU/g/30 days. At 22 ¡1uC, Salmonella, E. coli O157:H7,
and L. monocytogenes declined at a rate of 0.22, 0.37, and 0.59 log CFU/g/30 days, respectively, on peanuts, and at 0.15, 0.34,
and 1.17 log CFU/g/30 days, respectively, on pecans. Salmonella counts were above the limit of detection (0.30 log CFU/g)
throughout the study. In most cases during storage, counts obtained from pecans were higher than from peanuts.
Almonds, cashews, coconut, hazelnuts, pine nuts,
pecans, pistachios, sesame seeds, and peanuts, as well as
several processed nut products, have been associated with
foodborne outbreaks and/or recalls after isolation of
foodborne pathogens (19, 28). The majority of these
outbreaks and recalls have been associated with Salmonella,
and many of the outbreaks have lasted for months and have
included cases from multiple states in the United States (10–
12, 14–17) and/or from other countries (9, 20, 22, 24).
Outbreaks and recalls due to Escherichia coli O157:H7 (8,
13) and recalls due to Listeria monocytogenes contamina-
tion (35) also are documented for some nuts and nut
products. The low water activity of nuts and nut products
prevents multiplication of microorganisms (4); however, the
survival of Salmonella, E. coli O157:H7, and L. monocy-
togenes has been documented on almond kernels for at least
365 to 550 days at 219, 4, 24, and 35uC(21), on pistachios
for at least 365 days at 219, 4, and 24uC(21), and on
walnut kernels for at least 365 days at 220, 4, and 23uC(5).
Salmonella can survive in peanut butter and peanut butter
spreads for at least 168 days at 5 and 21uC(6). At 220, 4,
21, and 37uC, Salmonella survived on pecan halves and
pieces for 365 days and on in-shell pecans for 550 days (3).
The shelf life of peanuts and pecans is influenced by
initial moisture as well as humidity and temperature of
storage. Pecan handlers generally store bulk raw product
under controlled conditions to maintain quality. The common
storage temperatures (and suggested storage times) for
pecans, both in-shell and kernels, include 218uC(forupto
6 to 8 years), 0uC (for about 12 to 18 months), or ambient (for
about 3 to 6 months) (30). Common storage temperatures
(and suggested times) for raw peanuts, both in-shell and
kernels, include 218uC (for 2 to 10 years), 1 to 5uC (for
approximately 1 year), or ambient (up to 6 months) (26).
After processing and packaging, both raw and roasted nuts
are typically stored under ambient conditions at the retail
level; consumers may store nuts under ambient, refrigerated,
or freezer conditions for extended periods (25). In general,
Salmonella survives on nut surfaces at lower temperatures
(i.e., refrigeration or freezer conditions) without significant
declines in populations over time, whereas slow, but
significant, reductions are typically observed at ambient
temperatures, 21 to 25uC(3, 5, 21, 33).
Data on the behavior of foodborne pathogens on peanut
and pecan kernels at common storage temperatures are
* Author for correspondence. Tel: 863-956-8654; Fax: 863-956-4631;
E-mail: mddanyluk@ufl.edu.
323
Journal of Food Protection, Vol. 78, No. 2, 2015, Pages 323–332
doi:10.4315/0362-028X.JFP-14-327
Copyright G, International Association for Food Protection
limited. The objectives of this study were to determine the
survival of Salmonella, E. coli O157:H7, and L. monocy-
togenes on inoculated peanut and pecan kernels during
ambient, refrigerated, and frozen storage.
MATERIALS AND METHODS
Nuts. Raw pecan (Carya illinoinensis) halves and peanut
(Arachis hypogaea L. hypogaea) kernels were obtained from nut
shellers in the southeastern United States. Raw pecan halves were
mammoth size (14 to 15 halves per 28 g); raw peanut kernels were
medium runner type splits (40 to 50 kernels per 28 g). All nuts
were stored at 4uC, for no more than 2 weeks before use.
Bacterial cultures. The strains used by Kimber et al. (21) for
a similar study on almonds and pistachios were used in the current
study to inoculate peanuts and pecans. Five serotypes of
Salmonella enterica were used: Anatum, strain 1715, isolated
from an almond survey; Enteritidis PT 30, strain ATCC BAA-
1045, isolated from raw almonds associated with an outbreak;
Enteritidis PT 9c, strain RM4635, a clinical isolate from an
almond-associated outbreak; Oranienburg, strain 1839, isolated
from pecans (provided by Dr. Beuchat, University of Georgia); and
Tennessee, strain K4643, a clinical isolate from a peanut butter–
associated outbreak. The five strains of E. coli O157:H7 used were
all clinical isolates from patients in the associated foodborne
outbreaks: Odwalla strain 223 (apple juice); CDC 658 (canta-
loupe); H1730 (lettuce); F4546 (alfalfa sprouts); and EC4042
(spinach). Four strains of L. monocytogenes were used: 101M
(serotype 4b), isolated from beef from a beef-associated outbreak;
Scott A (serotype 4b), a clinical isolate from a milk-associated
outbreak; V7 (serotype 1/2a), isolated from milk associated with an
outbreak; and LCDC81-861 (serotype 4b), isolated from raw
cabbage associated with an outbreak. The moisture of peanuts and
pecans was determined during storage on nuts that were inoculated
with E. coli K-12, a nonpathogenic organism.
To enumerate pathogens in the presence of relatively high
background microbial populations on the raw nuts, we used a
stepwise procedure (29) to isolate mutants of all strains that were
able to grow on media supplemented with nalidixic acid (Sigma-
Aldrich, St. Louis, MO). Unless otherwise specified, all media
were from Difco, BD (Franklin Lakes, NJ) and were supplemented
with nalidixic acid (N) at 50 mg/ml. The isolates were stored at
280uC in tryptic soy broth (TSB) supplemented with 15%
glycerol.
Inoculum preparation. Frozen cultures of Salmonella and E.
coli O157:H7 were streaked onto tryptic soy agar (TSAN;
nonselective media), and L. monocytogenes cultures were streaked
onto brain heart infusion (BHIN) agar (nonselective media); all
plates were incubated at 37 ¡2uC for 24 h. Each strain was
subcultured twice in TSB supplemented with nalidixic acid at
50 mg/ml (Salmonella and E. coli O157:H7) or BHIN broth (L.
monocytogenes) and was incubated at 37 ¡2uC for 24 h. After
overnight growth, 1 ml of culture was spread over large TSAN or
BHIN plates (150 by 15 mm) and was incubated at 37 ¡2uC for
24 h to produce a bacterial lawn. Sterile 0.1%peptone water (9 ml)
was added to each plate, and the bacterial lawn was loosened with
a sterile spreader. Cells were collected from three plates for each
strain. To prepare Salmonella, E. coli O157:H7, or L. monocyto-
genes inocula, 25 ml of each strain was combined for each
pathogen in a 200-ml sterile bottle, and the three separate cocktails
were mixed for 1 min on a stir plate. To achieve the target
concentrations, pathogen cocktails were serially diluted in 0.1%
peptone water. Pathogen populations from cocktails were deter-
mined by plating onto nonselective media, namely TSAN
(Salmonella and E. coli O157:H7) or BHIN (L. monocytogenes),
at 37 ¡2uC for 24 h and by plating onto selective media, namely
bismuth sulfite agar (BSAN) for Salmonella, sorbitol MacConkey
agar (SMACN) for E. coli O157:H7, and modified Oxford agar
(MOXN) for L. monocytogenes, at 37 ¡2uC for 48 h.
Inoculation and storage of peanuts and pecans. Nuts were
inoculated as described by Uesugi et al. (33) for almonds, with a
ratio of 25 ml of inoculum for 400 g of peanut or pecan kernels.
Inoculation with each pathogen cocktail was carried out separately.
Nuts (400 g) were weighed into a plastic bag (30.5 by 30.5 cm;
Bitran Com-Pac International, Carbondale, IL), 25 ml of inoculum
cocktail was added, and the bag was sealed. Each bag was shaken
and massaged by hand for 1 min to ensure that the nuts were
evenly coated with the inoculum. Inoculated nuts were spread onto
four layers of filter paper (two sheets folded in half; Qualitative P8
Grade sheets, Fisher Scientific, Pittsburgh, PA) placed on a metal
rack inside a large lidded plastic container; the lid was left ajar to
allow the nuts to dry at ambient temperature and relative humidity
(RH) conditions in the laboratory (,22uC, 56%RH). After the
drying period, the inoculated nuts were transferred into sterile
plastic bags (30.5 by 30.5 cm), which were sealed using the built-in
‘‘zipper’’ before the nuts were placed into storage.
The survival of pathogens was investigated during short-term
(28 days) and long-term (365 or 550 days) storage. Vacuum or
modified atmosphere storage was not considered in the study
because it is not a common practice for bulk storage of raw nuts.
The short-term storage study was carried out to evaluate the
influence of inoculum levels on pathogen survival. Nuts were
inoculated with the Salmonella, E. coli O157:H7, or L.
monocytogenes cocktail at inoculum levels of 6 log CFU/g (high)
and 3 log CFU/g (low) and then were dried for 24 h at ambient
conditions. For the long-term storage study, nuts were inoculated
with the Salmonella, E. coli O157:H7, or L. monocytogenes
cocktail at 6 log CFU/g and were dried for 3 days at ambient
conditions. These dried nuts were then held in sealed zipper-top
bags for an additional 4 days at room temperature to ensure
moisture equilibration, for the long-term study only. Inoculated,
uninoculated, and control nuts in sealed bags were stored at 224
¡1, 4 ¡2, or 22 ¡1uC. Temperature and RH (%) in each
storage area were monitored with data loggers (TempTale 4,
Sensitech Inc., Beverly, MA) throughout the storage periods.
Moisture analysis. The moisture content of inoculated nuts
was determined monthly throughout storage at each temperature
for up to 12 months. The nuts used for moisture analysis were
inoculated with E. coli K-12 at 6 log CFU/g, dried at ambient
conditions for 3 days, and held for an additional 4 days before
storage. Peanut or pecan kernels (,10 g) were finely ground in a
food processor for 30 s and then were placed into foil pans,
weighed, and dried in an oven at 100uC for 24 ¡2 h. The moisture
content was determined by measuring the weight lost as a result of
heating.
Recovery and enumeration. Nuts were sampled weekly for
the short-term study and monthly for the long-term study. At each
sampling time, three 10-g subsamples from each of two replicates of
inoculated nuts (n~6) were added to 207-ml Whirl-Pak bags
(Nasco, Fort Atkinson, WI) with 20 ml of 0.1%peptone and then
were macerated (Smasher, AES Chemunex, Cranbury, NJ) for 2 min.
Serial dilutions were prepared with 0.1%peptone. Appropriate
dilutions were plated (0.1 ml) in duplicate onto nonselective media
324 BRAR ET AL. J. Food Prot., Vol. 78, No. 2
(TSAN for Salmonella and E. coli O157:H7 and BHIN for L.
monocytogenes) and selective media (BSAN for Salmonella,
SMACN for E. coli O157:H7, and MOXN for L. monocytogenes).
To improve the limit of detection, 1 ml of the lowest dilution was
plated onto four plates (0.25 ml per plate). TSAN, BHIN, and
SMACN plates were incubated at 37 ¡2uC for 24 h; BSAN and
MOXN plates were incubated at 37 ¡2uC for 48 h. Colonies were
counted, and bacterial populations were determined.
When the counts fell below the limit of detection (0.3 log
CFU/g), enrichment for pathogens was conducted according to
standard enrichment protocols (34), with some modifications. For
E. coli O157:H7, samples were enriched by the addition of 20 ml
of double-strength modified buffered peptone water with pyruvate
(Fisher Bioreagents, Fair Lawn, NJ) and incubation at 42uC for 18
to 24 h. Enrichments were streaked onto SMACN supplemented
with cycloheximide (CYC) at 50 mg/ml to inhibit molds
(SMACNzCYC). Plates were incubated at 37 ¡2uC for 24 h
and then were examined for typical E. coli O157:H7 colonies.
For L. monocytogenes, 20 ml of double-strength University of
Vermont–modified Listeria enrichment broth was added to each
sample, and these were incubated at 30uC for 48 h. Enrichments
were streaked onto MOX supplemented with CYC at 50 mg/ml
(MOXzCYC); plates were incubated at 37 ¡2uC for 48 h and
then were examined for the growth of typical colonies.
Statistical analysis. All experiments were replicated twice
with three samples per replication (n~6), and pathogen
populations were analyzed by analysis of variance (ANOVA)
and Tukey-Kramer tests with JMP 8 software (SAS Institute Inc.,
Cary, NC). Differences between mean values were considered
significant at P,0.05. The best-fit models were generated with
the aid of DMFit and were selected on the basis of R
2
values (2),
and pathogen reductions per month were calculated from the
curves obtained. The decline rates from high and low inoculums
observed during the 28-day study were analyzed by linear
regression with an interaction term in the model. The decline rate
was considered significant at P,0.05.
RESULTS
Temperature and RH during storage. The median
temperature and RH values at the three storage conditions
were as follows: 224 ¡1uC and 69%¡5%RH in frozen
storage, 4 ¡2uC and 94%¡9%RH in refrigerated
storage, and 22 ¡1uC and 56%¡8%RH in ambient
storage. Fluctuations in humidity were greater than
fluctuations in temperature, with maximum fluctuations
observed at ambient storage (Fig. 1). Climatic conditions
can impact laboratory humidity during events such as
rainfall (21). Rainfall is common in central Florida; hot
temperatures and high humidity are typical, and air
conditioning is used to mediate temperature and humidity.
This combination of factors likely resulted in the greater
fluctuations in RH we observed at ambient conditions.
Influence of inoculum level on pathogen survival.
Pathogen cocktails were inoculated at 6 log CFU/g (high
level) and 3 log CFU/g (low level) onto peanuts and
pecans; nuts were stored for 28 days under frozen,
refrigerated, and ambient conditions. Salmonella popula-
tions enumerated on BSAN (selective) and TSAN
(nonselective) did not differ significantly for peanuts and
pecans at any of the storage temperatures or at either
inoculum level (P.0.05) (data not shown). E. coli
O157:H7 populations enumerated on TSAN were signif-
icantly higher than on SMACN (selective) for both nut
types during storage and for both inoculum levels (P,
0.05), with two exceptions: at ambient storage for peanuts
inoculated at the low level and for pecans inoculated at the
high level (data not shown). L. monocytogenes populations
enumerated on BHIN (selective) and MOXN (nonselective)
were not significantly different for peanuts (P.0.05), but
counts on MOXN were significantly higher than on BHIN
for pecans (P,0.05) for both inoculum levels (data not
shown). Significant differences in counts on selective and
nonselective media were independent of initial inoculum
concentration.
In most cases, the rates of pathogen population decline
on both nut types were not significantly different between
the two inoculum levels (P,0.05) (Tables 1 and 2).
Significantly slower rates of decline were observed with
peanuts that were inoculated at log 3 CFU/g with
Salmonella and L. monocytogenes at storage temperatures
of 4 ¡2 and 22 ¡1uC, respectively. Significantly greater
rates of decline were observed with pecans that were
inoculated at log 3 CFU/g with Salmonella and E. coli
O157:H7 and stored at 224 ¡1uC. We used the high
inoculum concentration (6 log CFU/g) for the long-term
study and believe that this approach provided a conservative
FIGURE 1. (A) Temperature (
u
C) and (B) relative humidity (%)
in each storage condition as recorded monthly by data loggers
over 365 days. Whiskers denote maximum and minimum values,
the box denotes the 75th and 25th percentile, the solid line in the
middle denotes the median, and the dotted line denotes the mean.
J. Food Prot., Vol. 78, No. 2 SURVIVAL OF FOODBORNE PATHOGENS ON PEANUTS AND PECANS 325
prediction of the behavior of pathogens that may be present
on nuts at lower concentrations.
Moisture content of inoculated nuts during storage.
Kimber et al. (21) found very minor differences in moisture
content between inoculated and uninoculated pistachios
and almonds; here, the changes in moisture content of
inoculated peanuts and pecans during long-term storage are
shown in Figure 2, whereas those of the uninoculated nuts
were not recorded. The initial moisture content of the
peanuts and pecans used in this study was 3.8 and 2.0%,
respectively. Immediately after inoculation, the moisture
content increased to 8.0%in peanuts and to 6.5%in
pecans; after the 3-day inoculum-drying period, the
moisture content decreased to 3.8 and 2.6%in peanuts
and pecans, respectively. During storage at 4 ¡2uC, the
TABLE 1. Calculated rates of decline for inoculated pathogen populations on peanuts during short-term storage
a
Pathogen cocktail Storage temp (uC) Inoculum level
Initial (day 1) population
level (log CFU/g)
Final (day 28) population
level (log CFU/g)
Rate of change
(log CFU/g/30 days) R
2
Salmonella 224 ¡1 H 4.5 ¡0.1 4.5 ¡0.1 20.01 0.00
L 2.6 ¡0.1 2.6 ¡0.1 20.01 0.00
4¡2 H 4.7 ¡0.1 4.4 ¡0.1 20.33 A0.47
L 2.7 ¡0.1 2.6 ¡0.1 20.02 B0.00
22 ¡1 H 4.7 ¡0.0 4.2 ¡0.2 20.49 0.51
L 2.6 ¡0.1 2.3 ¡0.1 20.56 0.51
E. coli O157:H7 224 ¡1 H 3.2 ¡0.2 3.0 ¡0.1 20.28 0.26
L 1.2 ¡0.3 1.3 ¡0.2 20.28 0.02
4¡2 H 3.3 ¡0.1 3.2 ¡0.3 20.43 0.25
L 1.4 ¡0.2 1.2 ¡0.2 20.51 0.14
22 ¡1 H 3.1 ¡0.1 2.0 ¡0.3 21.1 0.71
L 1.1 ¡0.2 0.3 ¡0.0 20.92 0.53
L. monocytogenes 224 ¡1 H 5.5 ¡0.2 5.3 ¡0.2 20.20 0.15
L 3.9 ¡0.1 3.7 ¡0.1 20.29 0.26
4¡2 H 5.5 ¡0.1 5.3 ¡0.1 20.29 0.28
L 4.0 ¡0.1 3.7 ¡0.2 20.43 0.22
22 ¡1 H 5.6 ¡0.2 4.4 ¡0.2 21.3 A0.90
L 3.8 ¡0.3 3.0 ¡0.2 21.2 B0.29
a
Short-term storage lasted 28 days. H, high inoculum level (6 log CFU/g); L, low inoculum level (3 log CFU/g) before 24 h of drying.
Population level values are mean ¡standard deviation, n~6; values shown represent enumeration on nonselective media only.
Different letters represent significant differences between the rates of decline at high and low inoculum (P,0.05).
TABLE 2. Calculated rates of decline for inoculated pathogen populations on pecans during short-term storage
a
Pathogen cocktail Storage temp (uC) Inoculum level
Initial (day 1) population
level (log CFU/g)
Final (day 28) population
level (log CFU/g)
Rate of change
(log CFU/g/30 days) R
2
Salmonella 224 ¡1 H 4.8 ¡0.1 5.1 ¡0.1 20.26 A0.08
L 2.9 ¡0.2 3.2 ¡0.2 0.40 B0.14
4¡2 H 4.9 ¡0.1 5.0 ¡0.1 0.13 0.06
L 2.9 ¡0.2 3.1 ¡0.1 0.28 0.18
22 ¡1 H 5.0 ¡0.1 4.8 ¡0.1 20.27 0.17
L 2.8 ¡0.2 2.6 ¡0.1 20.25 0.13
E. coli O157:H7 224 ¡1 H 4.5 ¡0.2 4.9 ¡0.2 0.33 A0.11
L 2.4 ¡0.1 2.7 ¡0.1 0.51 B0.00
4¡2 H 4.7 ¡0.1 4.9 ¡0.1 0.09 0.00
L 2.5 ¡0.2 2.6 ¡0.1 20.03 0.00
22 ¡1 H 4.7 ¡0.3 4.1 ¡0.1 20.62 0.45
L 2.6 ¡0.1 2.0 ¡0.1 20.64 0.67
L. monocytogenes 224 ¡1 H 6.9 ¡0.1 6.6 ¡0.1 20.31 0.33
L 5.2 ¡0.1 4.5 ¡0.2 20.60 0.21
4¡2 H 7.0 ¡0.2 6.8 ¡0.1 20.38 0.29
L 5.3 ¡0.1 4.8 ¡0.2 20.79 0.36
22 ¡1 H 7.2 ¡0.1 6.2 ¡0.3 20.98 0.77
L 5.5 ¡0.1 4.5 ¡0.1 20.88 0.69
a
Short-term storage lasted 28 days. H, high inoculum level (6 log CFU/g); L, low inoculum level (3 log CFU/g) before 24 h of drying.
Population values are mean ¡standard deviation, n~6; values shown represent enumeration on nonselective media only. Different
letters represent significant differences between the rates of decline at high and low inoculum (P,0.05).
326 BRAR ET AL. J. Food Prot., Vol. 78, No. 2
moisture content increased to 5.8%in peanuts (Fig. 2A)
and to 3.0%in pecans (Fig. 2B) over 365 days. In contrast,
at 224 ¡1or22¡1uC storage, the moisture content of
peanuts and pecans remained steady at 3.8 and 2.6%,
respectively, over 365 days. The increase in the moisture
content at 4 ¡2uC is likely due to the high RH in
refrigerated storage.
Pathogen survival on inoculated nuts during drying
and holding periods. Before long-term storage, kernels
were inoculated (8.0 ¡0.0, 7.3 ¡0.4, and 8.2 ¡0.1 log
CFU/ml for Salmonella, E. coli O157:H7, and L. monocy-
togenes, respectively) to 5.4 ¡0.3, 5.3 ¡0.2, and 5.7 ¡
0.2 log CFU/g on pecans and 5.4 ¡0.1, 5.4 ¡0.4, and 5.4
¡0.4 log CFU/g on peanuts, for Salmonella, E. coli
O157:H7, and L. monocytogenes, respectively. Nuts were
dried at ambient temperature for 3 days. During the drying
period, Salmonella, E. coli O157:H7, and L. monocytogenes
populations decreased significantly (P,0.05) to 4.7 ¡0.1,
4.7 ¡0.9, and 5.0 ¡0.8 log CFU/g on pecans and 4.5 ¡
0.1, 3.9 ¡0.2, and 3.8 ¡0.5 log CFU/g on peanuts,
respectively. Nuts were held in sealed bags at ambient
temperature for 4 days, and no significant changes in
pathogen populations (,0.5 log CFU/g) occurred on either
nut type during the holding period (P.0.05). Populations
of Salmonella, E. coli O157:H7, and L. monocytogenes at
the initiation of storage were 5.1 ¡0.1, 4.7 ¡0.6, and 5.3
¡0.2 log CFU/g on pecans, respectively, and 4.4 ¡0.3,
3.6 ¡0.1, and 4.1 ¡0.2 log CFU/g on peanuts,
respectively.
Salmonella survival during storage. Salmonella
populations enumerated on BSAN were significantly lower
(P,0.05, up to 0.3 log CFU/g) than those on TSAN for
peanuts and pecans stored at 224 ¡1 and 4 ¡2uC
(Fig. 3); there were no significant differences in Salmonella
populations enumerated on TSAN and BSAN for the nuts
stored at 22 ¡1uC (Fig. 3). Salmonella populations
declined slightly but not significantly (P.0.05) on peanuts
and pecans at 224 ¡1 and 4 ¡2uC (Fig. 3): reductions
were 0.3 and 0.4 log CFU/g on peanuts and 0.5 and 0.1 log
CFU/g on pecans, respectively, over 365 days.
Salmonella populations on peanuts declined by 2.8 log
CFU/g over 365 days of storage at 22 ¡1uC (Fig. 3A).
After being fit to a best-fit model, levels declined linearly at
the rate of 0.22 log CFU/g/30 days (R
2
~0.88; Table 3).
On pecans, Salmonella populations declined by 2.0 log
CFU/g over 365 days (Fig. 3B); a linear decline at the rate
of 0.15 log CFU/g/30 days (R
2
~0.90; Table 3) was
calculated from the best-fit model.
Salmonella levels remained above the limit of detection
(0.3 log CFU/g) throughout 365 days on both nut types. Cell
counts obtained from the peanuts and pecans were not
significantly different at 224 ¡1 and 4 ¡2uC(P.0.05),
but significantly higher counts were obtained for pecans
FIGURE 2. Moisture (%) of inoculated (A) raw peanut kernels
and (B) raw pecan halves during storage at 224
¡
1
u
C (triangle),
4
¡
2
u
C (square), and 22
¡
1
u
C (diamond) over 365 days.
FIGURE 3. Survival of Salmonella on inoculated (A) raw peanut
kernels and (B) pecan halves stored at 224
¡
1
u
C (triangle), 4
¡
2
u
C (square), and 22
¡
1
u
C (diamond). Counts were determined
on TSAN (closed symbol) and BSAN (open symbol). Values are the
average of six replicates (n ~6), with standard deviation shown.
The limit of detection was 0.3 log CFU/g (solid line).
J. Food Prot., Vol. 78, No. 2 SURVIVAL OF FOODBORNE PATHOGENS ON PEANUTS AND PECANS 327
than for peanuts at the ambient storage condition (P,
0.05).
E. coli O157:H7 survival during storage. E.
coli O157:H7 populations enumerated on SMACN were
significantly lower (P,0.05;upto1.2logCFU/g)than
those on TSAN for peanut and pecans at the three storage
temperatures (Fig. 4), with one exception for pecans stored
at 22 ¡1uC (Fig. 4B). E. coli O157:H7 populations
declined significantly (P,0.05) by 0.6 and 0.7 log CFU/g
on peanuts at 224 ¡1and4¡2uC over 365 days
(Fig. 4A), at a rate of 0.03 (R
2
~0.42) and 0.12 (R
2
~
0.71) log CFU/g/30 days, respectively (Table 3). E. coli
O157:H7 populations did not decline significantly on
pecans at 224 ¡1and4¡2uC (Table 3); reductions of
0.5 and 0.2 log CFU/g were observed over 331 and
365 days, respectively (Fig. 4B).
At 22 ¡1uC, E. coli O157:H7 populations declined by
3.2 log CFU/g over 272 days on peanuts (Fig. 4A); when
data were fit to the no-lag model, a decline rate of 0.37 log
CFU/g/30 days (R
2
~0.93) was calculated (Table 3). On
pecans, E. coli O157:H7 populations declined by 4.3 log
CFU/g over 365 days (Fig. 4B) at a linear rate of 0.34 log
CFU/g/30 days (R
2
~0.89) (Table 3).
E. coli O157:H7 levels on SMACN were below the
limit of detection for peanuts and pecans after 211 and
331 days of storage, respectively, at 22 ¡1uC. Population
levels on TSAN were not below the limit of detection at
any time at all three temperatures for both nut types.
Significantly higher population counts were obtained from
the pecans stored at 224 ¡1 and 4 ¡2uC(P,0.05), but
no significant differences in counts between peanuts and
pecans were observed at 22 ¡1uC(P.0.05).
L. monocytogenes survival during storage. L.
monocytogenes populations enumerated on MOXN were
significantly lower (P,0.05; up to 0. 8 log CFU/g) than
those on BHIN for peanuts and pecans stored at 224 ¡1
TABLE 3. Calculated rates of decline for inoculated pathogen populations on raw peanut kernels and pecan halves during storage
a
Pathogen cocktail Nut Storage temp (uC) ANOVA Pvalue Model
b
Rate of change
(log CFU/g/day)
Rate of change
(log CFU/g/30 days) R
2
Salmonella Peanut 224 ¡1 0.88 ND
4¡2 0.09 ND
22 ¡1,0.0001 Linear 20.007 20.22 0.88
Pecan 224 ¡1 0.09 ND
4¡2 0.06 ND
22 ¡1,0.0001 Linear 20.005 20.15 0.90
E. coli O157:H7 Peanut 224 ¡1 0.001 Linear 20.001 20.03 0.42
4¡2,0.0001 No lag 20.004 20.12 0.71
22 ¡1,0.0001 No lag 20.012 20.37 0.93
Pecan 224 ¡1 0.59 ND
c
4¡2 0.06 ND
22 ¡1,0.0001 Linear 20.011 20.34 0.89
L. monocytogenes Peanut 224 ¡1,0.0001 Linear 20.002 20.06 0.91
4¡2,0.0001 Linear 20.002 20.06 0.75
22 ¡1,0.0001 No lag 20.019 20.59 0.93
Pecan 224 ¡1 0.10 ND
4¡2,0.0001 Linear 20.001 20.03 0.06
22 ¡1,0.0001 No lag 20.038 21.17 0.98
a
Peanuts and pecans were stored at 224 ¡1, 4 ¡2, or 22 ¡1uC for 365 days. ND, not done; there was no significant change in
population levels over the storage period.
b
Model (DMfit) was chosen based on R
2
value and the shape of the curve.
c
Counts obtained from last sampling day were not included in the analysis.
FIGURE 4. Survival of Escherichia coli O157:H7 on inoculated (A)
raw peanut kernels and (B) pecan halves stored at 224
¡
1
u
C
(triangle), 4
¡
2
u
C (square), and 22
¡
1
u
C (diamond). Counts were
determined on TSAN (closed symbol) and SMACN (open symbol).
Values are the average of six replicates (n
~
6), with standard
deviation shown. The limit of detection was 0.3 log CFU/g (solid line).
328 BRAR ET AL. J. Food Prot., Vol. 78, No. 2
and 4 ¡2uC (Fig. 5); there were no significant differences
in colony counts on the two media for both nut types stored
at 22 ¡1uC. L. monocytogenes populations declined by 0.7
and 0.8 log CFU/g on peanuts at 224 ¡1 and 4 ¡2uC,
respectively, over 365 days (Fig. 5A). The linear rate of
decline on peanuts was 0.06 log CFU/g/30 days at 224 ¡
1uC(R
2
~0.91) and 4 ¡2uC(R
2
~0.75) (P,0.05;
Table 3). On pecans, L. monocytogenes populations re-
mained stable at 224 ¡1uC, with reductions of 0.3 log
CFU/g over 365 days (Fig. 5B). At 4 ¡2uC, the
populations on pecans declined by 0.6 log CFU/g over
364 days; the linear rate of decline was 0.03 log CFU/g/
30 days (R
2
~0.06) (Table 3).
At 22 ¡1uC, L. monocytogenes declined by 3.4 log
CFU/g over the first 181 days and by 3.8 log CFU/g over
365 days on peanut kernels (Fig. 5A). According to the
best-fit model for the initial slope, the rate of decline was
0.59 log CFU/g/30 days over 181 days (R
2
~0.93;
Table 3). L. monocytogenes populations on peanuts fell
below the limit of detection on the last sampling day; all
samples were positive upon enrichment (n~6) (Fig. 5A).
On pecan halves, L. monocytogenes populations declined by
4.9 log CFU/g over the first 150 days (Fig. 5B). L.
monocytogenes populations on pecans reached the limit of
detection after 150 days of storage at 22 ¡1uC and
occasionally fell below the limit of detection during the
remaining storage period up to 365 days; upon enrichment,
all samples were positive at each time point (n~6)
(Fig. 5B). The rate of decline was 1.17 log CFU/g (R
2
~
0.98), and the curve was best described by the no-lag model
(Table 3). Populations of L. monocytogenes were signifi-
cantly higher on pecans than on peanuts stored at 224 ¡1
and 4 ¡2uC; at 22 ¡1uC storage, counts obtained from
peanuts were significantly higher (P,0.05) than on
pecans.
Differences in pathogen survival during long-term
storage. On peanut kernels at 224 ¡1, 4 ¡2, and 22 ¡
1uC, E. coli O157:H7 and L. monocytogenes populations
declined significantly (P,0.05), whereas Salmonella
populations were stable. There was no significant difference
in the decline of populations of E. coli O157:H7 and L.
monocytogenes on peanut kernels at 224 ¡1uC(P.
0.05). At 4 ¡2uC, E. coli O157:H7 declined significantly
more than L. monocytogenes on peanut kernels; and, at 22
¡1uC, L. monocytogenes declined significantly more than
E. coli O157:H7 (P,0.05).
On pecan halves stored at 224 ¡1uC, all pathogen
populations were stable for the duration of the study. At 4
¡2uC, L. monocytogenes declined more than Salmonella
on pecans (P,0.05), but no significant differences in the
declines of Salmonella and E. coli O157:H7 or E. coli
O157:H7 and L. monocytogenes populations were observed
(P.0.05). At 22 ¡1uC, L. monocytogenes populations
declined significantly more than both E. coli O157:H7 and
Salmonella on pecans (P,0.05), and E. coli O157:H7
populations declined significantly more than Salmonella (P
,0.05).
All the data in the study were collected in the presence
of background microflora, as would be typical under
natural contamination events of peanuts or pecans. No
population changes in background microflora occurred,
and it is unlikely that these organisms affect pathogen
survival.
DISCUSSION
Peanuts and pecans can become contaminated at any
number of points during production or processing. Peanuts
grow underground, and at harvest the peanut plants are
lifted from the soil and inverted into windrows to allow the
nuts to dry (36). The close proximity of peanuts to soil can
increase the opportunity for contamination of peanuts (18).
At harvest, pecans are shaken from the trees onto the
orchard floor, swept up, and then transported to holding
locations for drying. Pecans have a thick outer covering, or
husk, which is removed prior to shelling (1). Pecans on the
ground can absorb moisture from free-standing water during
events like rainfall, and the rate of water infiltration into in-
shell pecans depends upon the variety of pecan and the
degree of shell damage or cracking (3).
Since 1986, peanuts (primarily peanut butter) have been
associated with several outbreaks of salmonellosis around
the world (19). Pecans have not been associated with
recorded outbreaks, but several recalls have been reported in
FIGURE 5. Survival of Listeria monocytogenes on inoculated (A)
raw peanut kernels and (B) pecan halves stored at 224
¡
1
u
C
(triangle), 4
¡
2
u
C (square), and 22
¡
1
u
C (diamond). Counts
were determined on BHIN (closed symbol) and MOXN (open
symbol). Values are the average of six replicates (n
~
6), with
standard deviation shown. The limit of detection was 0.3 log CFU/
g (solid line).
J. Food Prot., Vol. 78, No. 2 SURVIVAL OF FOODBORNE PATHOGENS ON PEANUTS AND PECANS 329
the United States since 2001 due to isolation of Salmonella
and Listeria during routine sampling (28). The prevalence of
Salmonella naturally present on raw shelled peanuts in the
United States was 2.3%of 944 peanut samples (375 g each)
from three crop years (7), and the corresponding concen-
tration of Salmonella on peanuts as determined by a most-
probable-number (MPN) assay was ,0.03 to 2.4 MPN/g.
Another study of raw shelled peanuts found Salmonella and
enterohemorrhagic E. coli in 0.67 and 0.03%of 10,162
peanut samples (350 g each), respectively, averaged over
three crop years (27); the calculated Salmonella levels were
0.74 to 5.25 MPN/350 g (0.002 to 0.015 MPN/g). To our
knowledge, no published research has reported the natural
contamination levels of L. monocytogenes on peanuts and
pecans.
Microbiological analysis of previously unopened jars of
peanut butter from product involved in several cases of
salmonellosis revealed the presence of Salmonella at three
cells per gram (31). Salmonella levels recovered from the
contaminated peanut butter were well below the inoculum
levels commonly used in the long-term survival studies of
other nuts (3, 5, 21, 33). However, the short-term survival
experiment conducted in the current study confirmed the use
of a high-level inoculum (6 log CFU/g) as a suitable
predictor of pathogen behavior at low levels, as seen in
similar studies on almonds (21, 33), pecans (3), and
pistachios (21). Pathogen decline rates were different
between the short-term and the long-term studies, likely
due to methodology differences in drying the inoculated
nuts: ambient conditions for 24 h in the short-term study
versus a total of 7 days for the long-term study. Longer
drying periods before the beginning of storage bring the
moisture content and water activity closer to the original
levels, whereas shorter drying periods may not adequately
reduce these levels, subsequently affecting the decline rates
calculated for pathogens.
In the current study, peanuts and pecans were stored
under various conditions typically used by handlers and
consumers. In frozen and refrigerated storage, Salmonella
populations did not decline significantly with time on
peanuts or pecans, E. coli O157:H7 populations declined
significantly on peanuts but did not decline significantly on
pecans, and L. monocytogenes populations declined signif-
icantly with time, except on pecans stored under the freezer
conditions. Similarly, no significant decline of Salmonella
was observed for over 12 months for almonds or in-shell
pistachios inoculated with the same Salmonella cocktail
used in the current study (21) or with a single strain of
Salmonella (ATCC BAA-1045) (33). In contrast, a previous
study conducted on pecan halves documented small declines
(0.48 to 0.69 log CFU/g) in Salmonella levels at frozen and
refrigerated storage over 9 months (3).
A significant but slow decline of E. coli O157:H7
inoculated on peanuts occurred under refrigeration condi-
tions, which is in agreement with the observations for
E. coli O157:H7 inoculated on almonds (21). No significant
declines in E. coli O157:H7 populations were observed on
pecans in the current study or on in-shell pistachios under
either refrigerated or frozen storage (21). The slow declines
in L. monocytogenes populations observed on peanuts
(in refrigerated and frozen storage) and on pecans (in
refrigerated storage, R
2
~0.06) were not documented for
almonds and pistachios under either condition (21).
Our observations on the survival of Salmonella at
ambient conditions are in agreement with similar studies on
raw almonds and pistachios (21, 33). The rates of decline
of Salmonella at ambient conditions on raw peanuts (0.22
log CFU/g/30 days) and raw pecans (0.15 log CFU/g/
30 days) are comparable to the rates of decline reported for
almonds (average 0.24 log CFU/g/mo over eight studies)
(23) and pistachios (0.15 log CFU/g/mo) (21) but are
higher than for walnut kernels (0.05 to 0.1 log CFU/g/mo)
(5). The reduction of Salmonella (2.0logCFU/g)onpecan
halves after 365 days of ambient storage, as observed in the
current study, is similar to the reduction (2.1 log CFU/g)
that was observed on immersion-inoculated pecan halves
(3). For immersion inoculation, pecan halves were
immersed in Salmonella cocktail for 30 s and then dried
in a forced-air oven at 30uC for 20 to 27 h (3), whereas in
the current study, nuts were dried at room temperature for
3 days and then held for another 4 days at ambient
conditions. In both studies, Salmonella counts were above
the limit of detection throughout the storage period.
The decline rates for E. coli O157:H7 on peanuts (0.37
log CFU/g/30 days) and pecans (0.34 log CFU/g/30 days)
were higher than for Salmonella on these nuts, similar to
those on pistachios (0.35 log CFU/g/mo) and walnuts (0.21
log CFU/g/mo), and were lower than those previously
reported on almonds (0.60 log CFU/g/mo). Decline rates of
L. monocytogenes on peanuts (0.59 log CFU/g/30 days),
pecans (1.17 log CFU/g/30 days), almonds (0.71 log CFU/
g/mo), pistachios (0.86 log CFU/g/mo), and walnuts (1.2
log CFU/g/mo) were the highest among all three pathogens
in each of the three studies.
At all storage conditions, Salmonella survived better
than E. coli O157:H7 and L. monocytogenes, confirming
its use as a representative organism for survival studies on
peanuts and pecans. The rates of decline of all pathogens
were slower on pecans than on peanuts in most cases in
the current study; similarly, in a previous study, slower
decline rates were recorded on pistachios than on almonds
(21). The distinct tailing of the survival curves for E. coli
O157:H7 and L. monocytogenes on peanuts and pecans
is similar to that described for E. coli O157:H7 on
almonds (21) and for both pathogens on pistachios (21)
and walnuts (5). The survival potential also partially
explains the reason for the greater number of nut
outbreaks associated with Salmonella than with other
human pathogens. Studies on the survival of human
pathogens on nuts are among the first steps for
quantitative microbial risk assessment of pathogens on
nuts. The data here contribute to a growing body of
knowledge on the survival of foodborne pathogens on
nuts. The relatively consistent data trends among nuts
suggest that, collectively, these data may have utility for
other nuts in the absence of nut-specific studies.
330 BRAR ET AL. J. Food Prot., Vol. 78, No. 2
ACKNOWLEDGMENTS
This research was supported by the U.S. Department of Agriculture
CSREES NIFSI 2009-51110-20146. We are grateful for the technical
assistance of Gwen Lundy and Luis Martinez and the editorial assistance of
Sylvia Yada.
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332 BRAR ET AL. J. Food Prot., Vol. 78, No. 2
... In addition to that massive recall, other small recalls have been linked to nut butters and to sunflower seeds. Similarly, macadamia nut (MN) contamination in 2017 triggered another large food recall involving several product types (10). Despite the fact that there has never been a documented case of listeriosis linked to a seed or nut product, the detection of L. monocytogenes in commercial lots demands a better understanding of its prevalence, survival, and thermal tolerance in these commodities. ...
... Listeria is known to be able to survive in the environment for a long time, and recent reports observed prolonged viability on dry foods during storage. The count of inoculated L. monocytogenes bacteria on pecans and peanuts remained relatively unchanged at 224 and 4°C for a year, but at 22°C, its viability declined gradually from 4 Log CFU/g to less than 1 Log CFU/g after 150 days of storage (10). Salazar et al. (13) studied the survival of L. monocytogenes in pine nuts and sesame seeds and reported viable counts of more than 4 Log CFU/g (initial inoculum of 9 Log CFU/g) after 180 days of storage at different relative humidities. ...
Article
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This project was undertaken to determine the kinetic parameters of thermal inactivation of Listeria monocytogenes on pecans, macadamia nuts, and sunflower seeds subjected to heat treatments simulating industry processes. Five strains were grown in nonselective medium, mixed, and resuspended before inoculating macadamia nuts, pecans, and sunflower seeds (6 to 9 Log CFU/g). Redried inoculated pecans and macadamia nuts were heated in an oven at a temperature range of 90 to 140°C. Unshelled sunflower seeds were heated in sunflower seed oil. The thermal inactivation was determined by measuring viable cell counts using standard microbiological methods. Average count data were fit to the log-linear model, and thermal-death kinetics were calculated. On pecans, the viable Listeria counts were reduced by 3 and 3.5 Log CFU/g after 40 min at 110°C and 8 min at 140°C, respectively. On macadamia nuts, the L. monocytogenes population was reduced by 5 Log CFU/g after 20 min at 120°C. Unshelled sunflower seeds were subjected to heat treatment via a hot-oil bath. On sunflower seeds, >7 Log CFU/g reductions were observed after 15 min at 120°C. The thermal resistance (D value) for inactivation on pecans at 140°C was 3.1 min and on macadamia nuts at 120°C was 4.4 min. The inactivation of L. monocytogenes was influenced by the kind of nut or seed. These results suggest that L. monocytogenes has a relatively high thermal tolerance. The findings from this study will contribute to the assessment of the effectiveness of heat treatment for control of this pathogen on nuts and seeds. IMPORTANCE Listeria monocytogenes is a major concern for the food industry in ready-to-eat (RTE) foods. In recent years, large-scale recalls have occurred with contaminated sunflower seeds and macadamia nuts that triggered product withdrawals. These events stress the importance of understanding Listeria's ability to survive heat treatments in these low-water activity foods. Nuts and seeds are subjected to a variety of thermal treatments typically referred as roasting. To date, no listeriosis outbreak has been linked to nuts and seeds, but the recent recognition that this pathogen can be detected in commercial products stresses the need for research on thermal treatments. The characterization of heat inactivation kinetics at temperatures typically used during roasting processes will be very beneficial for validation studies. This manuscript reports inactivation rates of L. monocytogenes strains inoculated onto macadamia nuts, sunflower seeds, and pecan halves subjected to temperatures between 90 and 140°C.
... Considering the prevalence of L. monocytogenes reported in some species of fresh (especially wild) mushrooms (Chen et al., 2018;Murugesan et al., 2015;Venturini et al., 2011) and its ability to grow in sliced mushrooms, absence of L. monocytogenes in dried mushroom recalls and outbreaks as well as the inactivation observed in the present study, it appears that L. monocytogenes is a foodborne pathogen of less concern in dried mushrooms in comparison to Salmonella and B. cereus. It should, however, not be neglected that L. monocytogenes can survive in a desiccated state for months on stainless steel surfaces (Vogel et al., 2010) and has been isolated from a variety of LMFs after storage for up to a year (Brar et al., 2015;Kimber et al., 2012;Taylor et al., 2018), demonstrating how manufacturers of LMFs still need to pay attention to L. monocytogenes. ...
Article
The historic view on low-moisture foods (LMFs) as safe due to the lack of microbial growth in these foods is challenged by an increasing number of reports of outbreaks and recalls caused by LMFs contaminated with foodborne pathogens. The objective of this study was to determine the survival of Salmonella Typhimurium, Bacillus cereus and Listeria monocytogenes on sliced Portobello mushrooms (Agaricus bisporus variant Portobello) during hot-air drying (mushroom internal temperature below 45 °C) for 8 h (h) in a small household food dehydrator (250 W) and subsequent storage of the vacuum-packed dried product for 2 months at room temperature. Hot-air drying reduced the water activity (aw) of the mushrooms to 0.17 ± 0.03 well below the limit for microbial growth. S. Typhimurium and L. monocytogenes displayed total log CFU reductions of 2.5 ± 0.4 and 2.6 ± 0.8, respectively, while B. cereus exhibited significantly (p < 0.05) lower log reductions of 1.2 ± 0.1. Storage of vacuum-packed dried mushrooms further reduced L. monocytogenes by 2 log CFU, while numbers of viable S. Typhimurium and B. cereus were not further reduced. The higher stability of S. Typhimurium and B. cereus were reflected in the number of reports in the European Rapid Alert System for Food and Feed system of the presence of these organisms in dried mushrooms. All three organisms regrew to high concentrations when dried mushrooms were soaked overnight at room temperature, simulating a scenario where mushrooms are improperly rehydrated. Combining results from hot-air drying and subsequent storage underlines that hot-air drying and prolonged storage at low aw cannot be relied on alone to reduce the microbial and pathogen load on Portobello mushroom.
... However, the specific inactivation kinetics are dependent on the target pathogen as well as the food matrix [31,32]. To date, relatively little research has been done on E. coli O157:H7 inactivation in low moisture foods [33,34] or in dehydration of plant-based foods [2]. Table 1. ...
Article
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The effect of moderate-temperature (≤60 °C) dehydration of plant-based foods on pathogen inactivation is unknown. Here, we model the reduction of E. coli O157:H7 as a function of product-matrix, aw, and temperature under isothermal conditions. Apple, kale, and tofu were each adjusted to aw 0.90, 0.95, or 0.99 and inoculated with an E. coli O157:H7 cocktail, followed by isothermal treatment at 49, 54.5, or 60.0 °C. The decimal reduction time, or D-value, is the time required at a given temperature to achieve a 1 log reduction in the target microorganism. Modified Bigelow-type models were developed to determine D-values which varied by product type and aw level, ranging from 3.0–6.7, 19.3–55.3, and 45.9–257.4 min. The relative impact of aw was product dependent and appeared to have a non-linear impact on D-values. The root mean squared errors of the isothermal-based models ranged from 0.75 to 1.54 log CFU/g. Second, we performed dynamic drying experiments. While the isothermal results suggested significant microbial inactivation might be achieved, the dehydrator studies showed that the combination of low product temperature and decreasing aw in the pilot-scale system provided minimal inactivation. Pilot-scale drying at 60 °C only achieved reductions of 3.1 ± 0.8 log in kale and 0.67 ± 0.66 log in apple after 8 h, and 0.69 ± 0.67 log in tofu after 24 h. This illustrates the potential limitations of dehydration at ≤60 °C as a microbial kill step.
Article
The occurrence of disease outbreaks involving low‐water‐activity (aw) foods has gained increased prominence due in part to the fact that reducing free water in these foods is normally a measure that controls the growth and multiplication of pathogenic microorganisms. Salmonella, one of the main bacteria involved in these outbreaks, represents a major public health problem worldwide and in Brazil, which highlights the importance of good manufacturing and handling practices for food quality. The virulence of this pathogen, associated with its high ability to persist in the environment, makes Salmonella one of the main challenges for the food industry. The objectives of this article are to present the general characteristics, virulence, thermoresistance, control, and relevance of Salmonella in foodborne diseases, and describe the so‐called low‐water‐activity foods and the salmonellosis outbreaks involving them.
Article
Outbreaks and recalls associated with foods containing spices suggest a need for risk assessment of Salmonella in spices. In this study, the survivability of Salmonella Enteritidis PT 30, Salmonella cocktail (S. Enteritidis PT 30, S. Tennessee K4643 and S. Agona 447967) and Enterococcus faecium NRRL B-2354 in chili, cinnamon and black pepper at water activities (aw) 0.3 and 0.5 were evaluated during one-year storage at 21 °C. The thermal resistance of Salmonella cocktail in spices was also evaluated at 70 °C before and after storage. At aw 0.5, 3-month storage caused 5 log reduction of Salmonella cocktail in chili, while 8 months led to the same level of reduction in cinnamon. But only 3 log reduction were observed in black pepper over one year. Storage at aw 0.3 caused less reduction in Salmonella cocktail during the same storage periods. Less than 2 log reduction of E. faecium were observed over the one year storage at both aw levels, except for in chili stored at 0.5 aw. The D70°C-values for Salmonella cocktail in chili, cinnamon and black pepper of aw 0.3 before storage were 15.4, 20.8 and 36.6 min, respectively. 21–50% drops in D70°C-values were obtained after two-month of storage, but the D70°C-value in black pepper after one-year of storage was as the same as that after two-month storage. Based on these results, chili powder showed the highest antimicrobial effect, followed by cinnamon and black pepper powders during storage and isothermal treatments.
Chapter
Peanuts (Arachis hypogaea) belong to the family of legumes. Similar to other nuts, peanut is an excellent source of macronutrients and micronutrients but it is more affordable. Desirable peanutty flavor is developed during roasting, frying and other processing which enhances the palatability of peanuts. Thus, peanut has been widely consumed and used as food and feed ingredient for its nutrition value and pleasant flavor. In developing countries, peanuts have been used to combat malnutrition because peanuts are energy dense and nutrient balanced food product which provides sufficient protein, healthy lipid, dietary fiber and micronutrients. The high nutritive value of peanuts makes them a perfect substrate for fungal growth and potential aflatoxin contamination. Therefore, peanut stability includes nutrient stability, flavor stability, and microbial stability. The chemical composition changes during post-harvest curing, drying, storage, processing, and post-processing storage, thus impact the nutritional quality and flavor of peanuts. The main concern of microbial stability of peanut is mold and mycotoxin contamination which is influenced by pre-harvest practice, post-harvest handling and peanut processing. Additionally, pathogenic bacteria, particularly Salmonella has been a critical food safety issue of processed peanuts, especially, peanut butter. This chapter reviewed/discussed peanut stability during post-harvest handling, storage, processing and post-processing storage.
Article
Dry roasting can reduce Salmonella contamination on peanuts. While previous studies evaluated impact of product temperature, process humidity, product moisture, and/or product water activity on Salmonella lethality, no published study has tested multiple primary and secondary models on data collected in a real-world processing environment. We tested multiple primary and secondary models to quantify Salmonella surrogate, Enterococcus faecium, inactivation on peanuts. Shelled runner-type peanuts inoculated with E. faecium were treated at various air temperatures (121, 149, and 177°C) and air velocities (1.0 and 1.3 m/s) for treatment times from 1 to 63 minutes. Peanut surface temperature was measured during treatment. Water activity and moisture content were measured, and E. faecium were enumerated after treatment. Microbial inactivation was modeled as a function of time, product temperature, and product moisture. Parameters (Dref, zT, zaw, zMC, and/or n) were compared between model fits. The log-linear primary model combined with either the modified Bigelow-type secondary model accounting for aw or moisture content showed improved fit over the log-linear primary model combined with the traditional Bigelow-type secondary model. The Weibull primary model combined with the traditional Bigelow-type secondary model had the best fit. All parameter relative errors were less than 15%, and RMSE values ranged from 0.379 to 0.674 log CFU/g. Incorporating either aw or moisture content in the inactivation models did not make a practical difference within the range of conditions and model forms evaluated, and air velocity did not have a significant impact on inactivation. The models developed can aid processors in developing and validating pathogen reduction during peanut roasting.
Article
Background Foodborne pathogen contamination in low water activity (aw) foods is a critical problem for both the food industry and public health. Low-moisture foods and food ingredients have been implicated in numerous Salmonella outbreaks and are increasingly involved in the recall of products with possible contamination by Listeria monocytogenes. L. monocytogenes is singled out as a true environmental species and a pathogenic bacterium of concern in ready-to-eat foods including low-moisture foods. L. monocytogenes can survive in a dry environment or in low-moisture foods for an extended period with increased stability at lower storage temperatures. Adaptation to low-moisture foods enables L. monocytogenes to exhibit enhanced thermal resistance. However, compared with Salmonella in low-moisture foods, less information is available about the fate of L. monocytogenes in low-moisture foods during thermal inactivation and the factors influencing its thermotolerance. Scope and approach This review summarizes pertinent literature on the desiccation and thermal resistance of L. monocytogenes in low-moisture foods, discusses factors impacting the desiccation and thermal stability, and compares desiccation and thermal stability of L. monocytogenes with Salmonella in respective low-moisture foods. The possible mechanisms underlying the stability of L. monocytogenes in low-moisture foods are also discussed. Key findings and conclusions L. monocytogenes can survive in low-moisture foods for an extended duration and is highly thermoresistant. Thermal resistance of L. monocytogenes has an inverse relationship with aw, and is dependent on the food matrix, and other factors.
Article
Wheat flour has been connected to outbreaks of foodborne illnesses with increased frequency in recent years, specifically, outbreaks involving Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC). However, there is little information regarding the survival of these pathogens on wheat grain during long-term storage in a low-moisture environment. This study aims to evaluate the long-term survival of these enteric pathogens on wheat grain over the course of a year. Hard red spring wheat was inoculated with strains of four serovars of Salmonella (Enteritidis, Agona, Tennessee, and Montevideo) and six serotypes of EHEC (O157:H7, O26:H11, O121:H19, O45:NM, O111:H8, and O103:H2) in triplicate, sealed in Mylar bags to maintain the water activity, and stored at room temperature (22 ± 1°C). The survival of each pathogen was evaluated by plating onto differential media. Viable counts of strains from all four serovars of Salmonella (Enteritidis, Agona, Tennessee, and Montevideo) were detected on wheat grain stored at room temperature (22 ± 1°C) for the duration of the study (52 weeks). Viable counts of strains from EHEC serotypes O45:NM, O111:H8, and O26:H11 were only detected for 44 weeks, and strains from serotypes O157:H7, O121:H19, and O103:H2 were only detected for 40 weeks until they passed below the limit of detection (2.0 log CFU/g). The D-values were found to be significantly different between Salmonella and EHEC (adjusted P ≤ 0.05) with Salmonella D-values ranging from 22.9 ± 2.2 weeks to 25.2 ± 1.0 weeks and EHEC D-values ranging from 11.4 ± 0.6 weeks to 13.1 ± 1.8 weeks. There were no significant differences among the four Salmonella strains or among the six EHEC strains (adjusted P > 0.05). These observations highlight the wide range of survival capabilities of enteric pathogens in a low-moisture environment and confirm these pathogens are a food safety concern when considering the long shelf life of wheat grain and its products. HIGHLIGHTS
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Foods and food ingredients with low water activity (a(w)) have been implicated with increased frequency in recent years as vehicles for pathogens that have caused outbreaks of illnesses. Some of these foodborne pathogens can survive for several months, even years, in low-a(w) foods and in dry food processing and preparation environments. Foodborne pathogens in low-a(w) foods often exhibit an increased tolerance to heat and other treatments that are lethal to cells in high-a(w) environments. It is virtually impossible to eliminate these pathogens in many dry foods or dry food ingredients without impairing organoleptic quality. Control measures should therefore focus on preventing contamination, which is often a much greater challenge than designing efficient control measures for high-a(w) foods. The most efficient approaches to prevent contamination are based on hygienic design, zoning, and implementation of efficient cleaning and sanitation procedures in the food processing environment. Methodologies to improve the sensitivity and speed of assays to resuscitate desiccated cells of foodborne pathogens and to detect them when present in dry foods in very low numbers should be developed. The goal should be to advance our knowledge of the behavior of foodborne pathogens in low-a(w) foods and food ingredients, with the ultimate aim of developing and implementing interventions that will reduce foodborne illness associated with this food category. Presented here are some observations on survival and persistence of foodborne pathogens in low-a(w) foods, selected outbreaks of illnesses associated with consumption of these foods, and approaches to minimize safety risks.
Article
While nut consumption can contribute to a healthy diet, recently nuts have been identified as a source of Salmonella. How consumers store and use nuts can guide safe handling information and assist in the development of more accurate risk assessment models. In an online survey, 279 Californian consumers reported that if almonds, pecans, and walnuts are stored up to 6 months, they are typically held at room or refrigerator temperatures. If nuts are stored 7 months or more, freezing is the most common method of storage. Pistachios are usually stored at room temperature and eaten in a short time. Garage storage, in which temperatures can range from -18°C (0°F), to over 38°C (100°F), is rarely used. The majority of nuts are eaten as a snack, but they also are commonly used as an ingredient in foods prepared in the home. Consumers replied that they most frequently use nuts in cookies (almonds 51%, pecans 48%, pistachios 9%, walnuts 70%) or tossed in salads (almonds 50%, pecans 47%, pistachios 11%, walnuts 56%). Data on consumer practices can be used to develop more accurate risk assessment models. Consumers are aware of the nutritional benefit of consuming nuts, but at the time of this survey, few were aware that low-moisture foods such as nuts could on rare occasion be a source of foodborne illness. A majority of consumers reported that they would not change their family's use of nuts to prevent foodborne illness.
Conference Paper
Background: About 40,000 laboratory-confirmed Salmonella infections are reported annually in the U.S.; serotype Typhimurium causes ~19%. Outbreak investigations involving contaminated food ingredients are complex as products may be distributed through multiple channels and consumed in various settings over an extended period of time. We investigated a large multistate outbreak of Salmonella Typhimurium infections identified in November 2008. Methods: A case was defined as infection in a person with outbreak strain of S. Typhimurium with illness onset on or after 9/1/2008. Two case-control studies (CC1 and CC2) were performed. Controls were well persons from the community matched by age and location. Traceback and environmental investigations were conducted. Results: Among 714 cases identified in 46 states, 23% were hospitalized and nine died. In CC1, illness was associated with eating any peanut butter (PB) (matched odds ratio [mOR]=2.53, 95% confidence interval [CI]=1.26-5.31). The outbreak strain was isolated from Brand A institutional PB produced by Manufacturer A. Continuing interviews of patients not associated with institutions indicated that many patients had eaten PB-containing products. In CC2, illness was associated with eating PB crackers (mOR=9.08, CI=4.86-18.05), specifically Brand B (mOR=18.65, CI=7.59-55.07) and Brand C (mOR=4.13, CI=1.65-10.68). Major national brands of jarred PB found in grocery stores were not associated with illness. The outbreak strain was isolated from Brand B PB crackers containing peanut paste from Manufacturer A, PB flavored pet treats, and other PB-containing products linked to Manufacturer A. Traceback investigations resulted in the recall of >3,900 PB and PB-containing products. Conclusion: A large multistate outbreak caused by contaminated PB and PB-containing products from Manufacturer A used as ingredients in many widely distributed foods, resulted in one of the largest U.S. food recalls in recent history.
Article
Three major outbreaks of salmonellosis linked to consumption of peanut butter during the last 6 years have underscored the need to investigate the potential sources of Salmonella contamination in the production process flow. We conducted a study to determine the prevalence and levels of Salmonella in raw peanuts. Composite samples (1,500 g, n = 8) of raw, shelled runner peanuts representing the crop years 2009, 2010, and 2011 were drawn from 10,162 retained 22-kg lot samples of raw peanuts that were negative for aflatoxin. Subsamples (350 g) were analyzed for the presence of Salmonella and enterohemorrhagic Escherichia coli. Salmonella was found in 68 (0.67%) of 10,162 samples. The highest prevalence rate (P < 0.05) was for 2009 (1.35%) compared with 2010 (0.36%) and 2011 (0.14%). Among four runner peanut market grades (Jumbo, Medium, No. 1, and Splits), Splits had the highest prevalence (1.46%; P < 0.05). There was no difference (P > 0.05) in the prevalence by region (Eastern versus Western). Salmonella counts in positive samples (most-probable-number [MPN] method) averaged 1.05 (range, 0.74 to 5.25) MPN per 350 g. Enterohemorrhagic E. coli was found in only three samples (0.030%). Typing of Salmonella isolates showed that the same strains found in Jumbo and Splits peanuts in 2009 were also isolated from Splits in 2011. Similarly, strains isolated in 2009 were also isolated in 2010 from different peanut grades. These results indicated the persistence of environmental sources throughout the years. For five samples, multiple isolates were obtained from the same sample that had different pulsed-field gel electrophoresis types. This multistrain contamination was primarily observed in Splits peanuts, in which the integrity of the kernel is usually compromised. The information from the study can be used to develop quantitative microbial risk assessments models.
Article
Recalls and/or outbreaks associated with Salmonella contamination in peanut-containing products were reported over the past several years. There are very limited data available on the prevalence and concentration of Salmonella on raw shelled peanuts in the United States. The objectives of this study were to estimate the prevalence of Salmonella on raw shelled peanuts in the United States and to estimate that concentration of Salmonella. Samples of Runner- and Virginia-type raw shelled peanuts from the 2008, 2009, and 2010 crop years were proportionately sampled from each growing region, based on 2007 production volume. Of 944 raw shelled peanut samples (375 g each), 22 (2.33%) were positive for Salmonella by the VIDAS Salmonella assay. Salmonella serovars identified in this study included Agona, Anatum, Braenderup, Dessau, Hartford, Meleagridis, Muenchen, Rodepoort, Tennessee, and Tornow. The concentration levels of Salmonella in positive samples, as determined by a most-probable-number assay, were <0.03 to 2.4 MPN/g. These data will be useful when designing and validating processes for the reduction or elimination of Salmonella in peanuts and/or peanut-containing products.
Article
The survival of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes was determined on almonds and pistachios held at typical storage temperatures. Almond kernels and inshell pistachios were inoculated with four- to six-strain cocktails of nalidixic acid-resistant Salmonella, E. coli O157:H7, or L. monocytogenes at 6 log CFU/g and then dried for 72 h. After drying, inoculated nuts were stored at -19, 4, or 24°C for up to 12 months. During the initial drying period after inoculation, levels of all pathogens declined by 1 to -log CFU/g on both almonds and pistachios. During storage, moisture content (4.8%) and water activity (0.4) of the almonds and pistachios were consistent at -19°C; increased slowly to 6% and 0.6, respectively, at 4°C; and fluctuated from 4 to 5% and 0.3 to 0.5 at 24°C, respectively. Every 1 or 2 months, levels of each pathogen were enumerated by plating; samples were enriched when levels fell below the limit of detection. No reduction in population level was observed at -19 or 4°C for either pathogen, with the exception of E. coli O157:H7-inoculated almonds stored at 4°C (decline of 0.09 log CFU/g/month). At 24°C, initial rates of decline were 0.20, 0.60, and 0.71 log CFU/g/month on almonds and 0.15, 0.35, and 0.86 log CFU/g/month on pistachios for Salmonella, E. coli O157:H7, and L. monocytogenes, respectively, but distinct tailing of the survival curves was noted for both E. coli O157:H7 and L. monocytogenes.
Article
The survival of single strains or cocktails of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes was evaluated on walnut kernels. Kernels were separately inoculated with an aqueous preparation of the pathogens at 3 to 10 log CFU/g, dried for 7 days, and then stored at 23°C for 3 weeks to more than 1 year. A rapid decrease of 1 to greater than 4 log CFU/g was observed as the inoculum dried. In some cases, the time of storage at 23°C did not influence bacterial levels, and in other cases the calculated rates of decline for Salmonella (0.05 to 0.35 log CFU/g per month) and E. coli O157:H7 (0.21 to 0.86 log CFU/g per month) overlapped and were both lower than the range of calculated declines for L. monocytogenes (1.1 to 1.3 log CFU/g per month). In a separate study, kernels were inoculated with Salmonella Enteritidis PT 30 at 4.2 log CFU/g, dried (final level, 1.9 log CFU/g), and stored at -20, 4, and 23°C for 1 year. Salmonella Enteritidis PT 30 declined at a rate of 0.10 log CFU/g per month at 23°C; storage time did not significantly affect levels on kernels stored at -20 or 4°C. These results indicate the long-term viability of Salmonella, E. coli O157:H7, and L. monocytogenes on walnut kernels and support inclusion of these organisms in hazard assessments.