217

Journal of Food Protection, Vol. 82, No. 2, 2019, Pages 217–225

doi:10.4315/0362-028X.JFP-18-351
Published 2019 by the International Association for Food Protection
This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/)

Research Paper

Growth of Salmonella on Inoculated Inhull Pistachios during

Postharvest Handling
MAHTA MOUSSAVI,† VANESSA LIEBERMAN, CHRIS THEOFEL, JAVAD BAROUEI†, AND LINDA J. HARRIS

Department of Food Science and Technology, University of California, One Shields Avenue, Davis, California 95616, USA
(ORCID http://orcid.org/0000-0002-1911-752X [L.J.H.])
MS 18-351: Received 25 July 2018/Accepted 23 September 2018/Published Online 23 January 2019

ABSTRACT
Salmonella has been isolated from dried pistachios in both postharvest and retail surveys. The source of Salmonella in
pistachios is unknown, but introduction is possible at points during production, harvest, and postharvest activities. To examine
the behavior of Salmonella on pistachios during simulated postharvest conditions, early-, mid-, and late-season inhull pistachios
were collected from two commercial processors over five different harvests. Pistachios were inoculated with cocktails of
nalidixic acid– or rifampin-resistant Salmonella at 0.64 to 1.59 log CFU/g (low) or 2.73 to 3.27 or 4.29 to 4.31 log CFU/g (high)
and were incubated for up to 30 h under commercially relevant conditions (23, 35, or 378C and 50 or 90% relative humidity
[RH]). Populations of Salmonella were measured by plating onto tryptic soy agar and CHROMagar Salmonella with added
nalidixic acid or rifampin. Individual growth curves at the same temperature and RH differed significantly among different lots
of pistachios. Except for a single late-season lot in which no significant growth was observed, Salmonella multiplied under all
storage conditions. In the first 3 h after inoculation, insignificant (most cases) to small (0.41 to 0.67 log CFU/g) but significant (P
, 0.05) mean increases in Salmonella populations were measured; the mean predicted time to achieve maximum populations (5
to 8 log CFU/g) was 16 6 4 h. In paired samples, longer lag phases, lower growth rates, and lower maximum increases were
observed with inoculated inhull pistachios incubated at 238C and 50% RH compared with 35 or 378C and 90% RH. Similar
growth curves were observed at the low and high inoculum levels; throughout the 30 h of incubation, Salmonella populations
were consistently ~1 to 2 log CFU/g lower on pistachios inoculated at the low inoculum level. Managing the time between
harvesting and hulling will reduce the potential for growth of Salmonella on pistachios during postharvest handling.

Key words: Harvest; Pistachio; Postharvest; Salmonella

Low-moisture foods do not support the growth of
foodborne pathogens. However, low-moisture foods, in-
cluding several different tree nuts, have been associated
with outbreaks of salmonellosis and, more rarely, entero-
hemorrhagic Escherichia coli gastroenteritis (13). Pista-
chios have been recalled in the United States for the
presence of Salmonella (21). Consumption of raw or roasted
pistachios was linked to one case of salmonellosis in 2009
(7) and two multistate outbreaks of salmonellosis in 2013
and 2016 (8, 20). Raw inshell pistachios collected shortly
after harvest from California storage silos had a 3-year
average weighted prevalence of Salmonella of 0.61% (3,966
100-g samples) (12); geometric mean levels of Salmonella
in positive samples, as determined by a most-probable-
number (MPN) method, ranged from 0.0010 to 0.053 MPN/
g. The source of Salmonella in pistachios is unknown;
however, similar to other tree nuts, introduction is possible
at any step from production, harvest, and postharvest
* Author for correspondence. Tel: 530-754-9485; E-mail:
ljharris@ucdavis.edu.
† Present address: Cooperative Agricultural Research Center, Prairie
View A&M University, Prairie View, TX 77446, USA.
handling to final processing (10, 11). Adequately dried
pistachios (,7% moisture [approximate water activity (aw)
of ,0.70]) do not support the growth of foodborne
pathogens. The decline of Salmonella populations during
storage of dried inshell pistachios was slow (approximately
0.15 log CFU/g/month) at 238C, or insignificant over more
than a year at 48C (15, 17).
The United States leads the world in commercial
pistachio production (2), and nearly all (99%) of the total
U.S. crop is produced in California (3). Production of
pistachios in California has grown rapidly over the past two
decades, from 81 million kg in 1997 to over 408 and 272
million kg in 2016 and 2017, respectively (1). Pistachio
trees are alternate bearing, producing a greater than average
crop one year and a lower than average crop the following
year. California produces a small number of cultivars
(primarily Kerman) that have a relatively short harvest
window (18). The overall production increases and
somewhat unpredictable annual harvest size have some-
times strained the postharvest handling system during the
relatively short harvest period (approximately 6 weeks from
late August to early October), increasing the possibility for
delays in unloading harvest trailers (2, 18).
218 MOUSSAVI ET AL.
In California, with the exception of young trees (
6
years), pistachio fruits are harvested by mechanically
shaking the tree and dropping the pistachios onto a catch
frame (18). Maturation may be uneven throughout the tree,
and thus an orchard may be shaken more than one time over
a period of several weeks. The fragility of the hulls
increases with increasing maturity associated with the
second or third shake of the trees.
A conveyor system moves the fruit from the catch
frame to a plastic bin or into a small hopper. The bins or the
hoppers are moved to the edge of the orchard. For a few of
the smaller growers, the plastic bins are used to transport the
pistachios to a hulling facility. For others, either a forklift
(bins) or conveyor system (hoppers) is used to transfer
product to a bottom dump trailer. These trailers are then
transported to hulling facilities, where the pistachios are
weighed, sampled, and evaluated for quality. Trailers are
unloaded as the processing capacity of the facility allows.
After unloading, pistachios are hulled and then dried to 8 to
15% moisture using forced hot air (70 to 1058C) (11).
Further drying takes place after the pistachios are
transferred to large silos, with application of forced warm,
dry ambient air over the first few days of storage. At this
point pistachios can be held for up to 14 months before
further processing.
The time the harvested pistachios remain in the trailer
before unloading is impacted by the speed at which the
trailer is filled, the time for the trucker to retrieve the trailer
from the orchard, the distance (travel time) from the orchard
to the hulling facility, and the hold time between receiving
and unloading of the trailer (11). Harvested pistachios are
40 to 50% moisture on a fresh weight basis (18), and their
respiration can result in rapid elevation of temperature (19)
and relative humidity (RH) in the harvest trailers. Pistachio
hulls contain 3 to 4% soluble sugars (primarily glucose and
fructose) on a dry weight basis (16), providing a potential
food source for microorganisms. The objective of this study
was to examine the fate of Salmonella enterica on inhull
pistachios during simulated postharvest conditions, from the
time of shaking the tree to just before hulling.
MATERIALS AND METHODS
Temperature and humidity monitoring. Daily high and
low ambient temperatures were obtained from the nearby Stratford
weather station through the California Irrigation Management
Information System (5). Ambient conditions at the orchard were
determined by a single data logger (TempTale 4, Sensitech Inc.,
Beverly, MA) placed near the location where pistachios were
loaded into bottom dump trailers (capacity, ~25,000 kg).
Temperature and RH in loaded trailers were monitored and
recorded using data loggers. To protect the data loggers from
physical damage, a stainless steel coupon (5 by 10 cm, 32-mm
thick) was placed on each side. The data loggers were then placed
inside steel mesh envelopes. On one occasion, a single data logger
was suspended on metal chains in the approximate center of each
of six different trailers before they were loaded with pistachios;
when the trailer was full, the data logger was covered with
pistachios to a depth of ~1.2 m. On a separate day, seven data
loggers were suspended along the length of a single trailer at the
approximate midpoint of the trailer's width at a depth between 0.3
and 1.0 m from the top of the loaded trailer. Temperature and RH
J. Food Prot., Vol. 82, No. 2
were recorded every 2.5 min for up to 13 h, from before loading,
through loading, transportation, and unloading. The trailers were
purposely held at the hulling facility to facilitate measurements
over a longer time.
Pistachios. Inhull pistachios (Pistacia vera) were collected
during five commercial harvests (2010, 2011, 2014, 2016, and
2017) from two different processors in the Central Valley of
California. Samples were collected during the ‘‘early season’’
(week 1 or 2), ‘‘midseason’’ (week 3 or 4), or ‘‘late season’’ (week
5) of the harvest. Inhull pistachios were collected as they were
unloaded from harvest trailers or bins (receiving pit). The
pistachios were placed into polyethylene bags (30.5 by 30.5 cm;
Bitran, Com-Pac International, Carbondale, IL), which were
sealed and then transported (~4 h) on ice to the laboratory. The
sealed bags were then transferred to lidded plastic tubs and stored
at 48C for up to 12 h before inoculation.
Bacterial cultures. Nalidixic acid–resistant (in 2010 and
2011) or rifampin-resistant variants of the following strains were
used: Salmonella enterica Enteritidis phage type 30 (ATCC BAA-
1045), isolated from raw almonds associated with a 2000 to 2001
outbreak (14); Salmonella Enteritidis phage type 9c (RM4635), a
clinical isolate from a 2004 outbreak associated with raw almonds
(6) (provided by Dr. Robert Mandrell, U.S. Department of
Agriculture, Agricultural Research Service); Salmonella Anatum
LJH1242, isolated from an almond survey (9); Salmonella
Tennessee (K4643), a clinical isolate from a 2006 to 2007
outbreak associated with peanut butter (provided by Dr. Larry R.
Beuchat, University of Georgia, Griffin); Salmonella Montevideo
(GRC1), isolated from pistachios (7) (provided by U.S. Food and
Drug Administration); and, in some cases, Salmonella Oranien-
burg (1839; used in 2010 and 2011 cocktails in addition to the
Salmonella strains listed above), isolated from pecans (provided
by Dr. Larry R. Beuchat). Isolates were stored at 808C in tryptic
soy broth (TSB; BD, Franklin Lakes, NJ) supplemented with 15%
glycerol.
Preparation of inocula. Unless otherwise specified, all
media were obtained from BD. The preparation of inocula and
inoculation procedure were based on the method described by
Kimber et al. (15). Briefly, before each experiment, frozen stock
cultures of each strain were streaked onto tryptic soy agar (TSA)
supplemented with 50 μg/mL nalidixic acid (TSAN) or 75 to 100
μg/mL rifampin (TSAR) and incubated at 37 6 28C for 24 6 4 h.
From each plate, a single isolated colony was transferred into 10
mL of TSB and incubated at 37 6 28C for 24 6 4 h. A loopful
(~10 μL) of each culture was then transferred into fresh TSB and
incubated at 37 6 28C overnight. An aliquot (1 mL) of each
overnight culture was spread over a large TSAN or TSAR plate
(150 by 15 mm; Fisher Scientific, Pittsburgh, PA) and incubated at
37 6 28C for 24 6 1 h. After incubation, the resulting bacterial
lawn was collected by adding 9 mL of 0.1% peptone to each
TSAN or TSAR plate and then scraping the slurry on the plate
surface with a sterile spreader. A multistrain cocktail was prepared
by combining equal volumes (5 mL) of the cell suspensions of
each Salmonella strain, resulting in levels of 11 log CFU/mL. The
cocktail was then diluted with sterile ultrapure water (Milli-Q
Advantage A10, MilliporeSigma, Burlington, MA) to the desired
final target inoculum level.
Inoculation procedure. Before inoculation, pistachios were
removed from refrigerated storage and held at room temperature
for ~1 h. Preliminary data indicated that the temperature of the
J. Food Prot., Vol. 82, No. 2 GROWTH OF SALMONELLA ON INOCULATED INHULL PISTACHIOS
pistachios approached ambient temperature during this time.
Inhull pistachio samples (400 g) were weighed into polyethylene
bags (30.5 by 30.5 cm; Com-Pac International), and 25 mL of
inoculum was added. Each bag was sealed and then shaken
continuously by hand for 2 min to ensure a thorough coating of the
nuts with the inoculum. The inoculated nuts were then spread onto
two sheets (46 by 57 cm) of filter paper (Qualitative P5, Fisher
Scientific) that were folded in half and placed in a biosafety
cabinet for 30 min (to a point where they were visibly dry).
Incubation of inhull pistachio samples. In initial experi-
ments (2010 and 2011), different humidity levels were evaluated:
after the 30-min drying period the inoculated pistachios were
placed into large weigh boats (50% RH or ambient humidity) or in
zippered plastic bags (.90% RH) and held for 24 h on a
laboratory bench at ambient conditions (238C) or in an incubator at
358C. In later experiments (2014 to 2017), inoculated pistachios
were transferred to a metal tray inside a plastic bin, and the open
bins were placed in an environmental chamber (Percival, Geneva
Scientific LLC, Fontana, WI) and incubated for up to 30 h at 378C
and 90% RH. In 2017, inoculated samples were incubated at both
23 and 378C and 90% RH. Uninoculated control pistachios were
held under the same conditions. Temperature and RH were
monitored and recorded using data loggers (Sensitech Inc.) during
the incubation period.
Enumeration of inoculated cells and background micro-
biota. At each sampling time, inhull pistachios were mixed, and
10 g (2010 and 2011) or 40 g was combined with 20 or 80 mL of
0.1% peptone, respectively, in a two-chamber filter bag (Nasco,
Modesto, CA). Each sample was processed by shaking for 30 s,
rubbing for 15 s, and shaking for an additional 30 s. The liquid
portion in each bag was serially diluted in 0.1% peptone.
Appropriate dilutions were plated in duplicate using spot plating
(20 μL of each dilution), spread plating (100 μL per plate), or a
spiral plater (Autoplate, Advanced Instruments, Inc., Norwood,
MA) onto TSAN or TSAR supplemented with 50 μg/mL
cycloheximide to suppress molds, and onto bismuth sulfite agar
(2010 and 2011) or CHROMagar Salmonella (CHROMagar, Paris,
France) supplemented with 75 μg/mL rifampin. Samples were
incubated at 37 6 28C for 24 6 2 h. Colonies were counted
manually or with a ProtoCOL 2 colony counter (Synbiosis,
Frederick, MD). Because the pistachios were not liquefied during
mixing, the calculated CFU per milliliter of diluent multiplied by
the ratio of the volume of diluent to weight of the sample was
considered equivalent to the CFU per gram of pistachios.
The background microbiota for uninoculated pistachios was
determined by plating appropriate dilutions onto TSA for aerobic
plate count (APC) and, for 2016 samples, onto CHROMagar ECC
for Escherichia coli–coliform counts (ECCs), and incubating at 37
6 28C for 24 6 2 h. All colonies visible on TSA (APC), and all
pink (presumptive coliform) and all blue (presumptive E. coli)
colonies on CHROMagar ECC were counted. In most cases,
uninoculated samples were also plated onto TSAR and CHRO-
Magar Salmonella supplemented with rifampin (75 μg/mL) and
incubated at 37 6 28C for 24 6 2 h.
Characterization of pistachio samples. In some cases,
inhull pistachios, as obtained from the hulling facility, were
characterized on the basis of the weight of different fractions of a
~1-kg sample. Each sample was weighed, placed in a tray, and
then sorted into six fractions: sticks, loose material (leaves, hulls,
and unidentified debris), inhull (with hull or unhulled pistachios),
inshell (hull-free inshell pistachios), shells (loose), and kernels
219
(loose). Each fraction was weighed, and the weight percentage for
each of the components was calculated.
pH of the hull extract. In 2016, hull materials were removed
from early-season (two samples), midseason (one sample), and
late-season (one sample) pistachios, and each sample was blended
for 30 s in a blender (Waring, Torrington, CT). The hull extract
was filtered through cheesecloth, and the pH of three subsamples
of the resulting filtrate was determined with a digital pH meter
(Thermo Fisher Scientific, Waltham, MA).
Moisture content and aw. Moisture content and aw were
determined for uninoculated pistachio hulls or kernels at the
beginning and end of the incubation period. Inhull pistachios were
hulled and shelled. Hulls or kernels (40 g) were processed for 20 s
in a commercial food processor (Waring). The aw and percent
moisture were determined for triplicate subsamples of the ground
material with a water activity meter (Aqualab model 4TE,
Decagon Devices, Pullman, WA) and moisture analyzer (3.6- to
4.0-g samples; model HG63, Mettler-Toledo, Columbus, OH),
respectively.
Statistical analysis. Pistachios collected from a single
processor on a single day were considered an independent lot.
For each incubation condition or inoculum level evaluated for that
lot, two separate sublots of 1,000 g of pistachios were inoculated
with the same inoculum preparation. At each sampling time, the
pistachios were mixed and three samples were randomly taken
from each of the two sublots (n ¼ 6). Duplicate, uninoculated
control sublots (1,000 g) were also stored under the same
conditions as inoculated sublots. One random sample of
uninoculated pistachios from each of the two separate sublots
was plated onto selective and nonselective agars at each sampling
time (n ¼ 2). Three random uninoculated samples were used to
determine moisture content and aw at the beginning and end of the
incubation period. Data were analyzed using Microsoft Excel 2010
and Prism 6 software (GraphPad Software, Inc., La Jolla, CA).
Analysis of variance, Tukey's multiple comparisons test, and t test
were performed. Differences between the mean values were
considered significant at P , 0.05. Individual growth curves for
Salmonella on inoculated inhull pistachios were fit using the
DMFit version 3.5 add-in to Microsoft Excel (http://www.
combase.cc/) (4). The lag time, growth rate, and maximum
population changes (the difference between the mean initial
inoculated and mean highest concentrations) of Salmonella were
determined.
RESULTS AND DISCUSSION
Temperature and humidity in pistachio harvest
trailers. Most pistachios are harvested between dawn and
dusk, but some commercial harvesters are equipped to
operate around the clock. Pistachio temperature at the time
of harvest depends on the ambient temperature and location
of the nut within the tree canopy. Temperature and RH in
multiple trailers loaded with pistachios were determined on
a single day (27 September 2011) beginning at midday, with
single data loggers placed in six different loaded trailers
(Fig. 1A). Average ambient conditions at the start of loading
were 278C and 54% RH; high and low ambient temperatures
reported from the nearby Stratford weather station for that
day were 30.7 and 13.28C, respectively. Temperature and
RH in a single loaded trailer with seven data loggers were
220 MOUSSAVI ET AL.
FIGURE 1. Mean temperature (solid line) and relative humidity (dashed line) in (A) six loaded pistachio trailers, each measured with one
data logger, or in (B) one loaded pistachio trailer measured with seven data loggers. Time zero is the point at which the temperature
recording device was covered with pistachios. *, number of data loggers used to calculate average and standard deviation for time and
relative humidity.
determined on a different day (11 October 2011) beginning
at midday (Fig. 1B). Average ambient conditions at the start
of loading were 288C and 52% RH; high and low weather
station temperatures were 28.5 and 14.98C, respectively, for
that day. The initial temperatures recorded by the metal-
encased data loggers on the two test days (40 and 348C,
respectively) reflect direct exposure to sunlight before and
while the trailer was being filled. During the first 2 h after
the data loggers were completely covered with pistachios,
the average RH in the loaded trailers increased to over 90 or
88% (Fig. 1A or 1B, respectively); temperatures decreased
to 30 or 288C (Fig. 1A or 1B, respectively). Subsequent
increases in temperature up to 378C were observed in
trailers that were held for longer times (e.g., ~12 h). In a
previous study, initial load temperatures for eight different
trailers were close to the air temperature at the time of
harvest, ranging from 22 to 348C (19); maximum temper-
atures at unloading (6.8 to 14.2 h after loading) ranged from
30 to 418C. On the basis of these data, the present
experiments were conducted at 23, 35, or 378C and 50 or
90% RH to reflect a range of conditions up to a worst-case
but commercially relevant scenario. Longer trailer hold
times correlate with decreased product quality (e.g., shell
staining), especially at higher temperatures (18, 19). At
258C, significant increases in shell staining were sometimes
noted at 40 h of holding (19). At 30 and 408C, staining
damage significantly increased at holding times of between
16 to 24 h.
Background microbiota. Colonies were not detected
when the lowest dilution of uninoculated inhull pistachios
was plated onto TSAN or TSAR and bismuth sulfite agar, or
CHROMagar Salmonella. E. coli colonies were not detected
on CHROMagar ECC in any sample (,1.3 log CFU/g).
Initial background populations for uninoculated early-
season and midseason inhull pistachios ranged from 3.00
to 3.84 log CFU/g (APC) and 0.90 to 2.80 log CFU/g
(presumptive coliform) (Fig. 2). Initial APC and presump-
tive coliform counts were significantly higher in one lot
J. Food Prot., Vol. 82, No. 2
(2016) of late-season inhull pistachios (7.42 and 7.38 log
CFU/g, respectively). APC and presumptive coliform
counts increased within 12 h by 2.19 to 3.04 log CFU/g
in early-season and by ~1 log CFU/g in late-season
harvested inhull pistachios, and they either decreased by
0.55 log CFU/g or increased by 1.99 to 4.48 log CFU/g in
midseason inhull pistachios (Fig. 2).
Growth of inoculated Salmonella on inhull pista-
chios. For all experiments in which Salmonella was
inoculated onto pistachios, Salmonella counts on TSAN or
TSAR and on bismuth sulfite agar or CHROMagar
Salmonella for individual pistachio samples were not
significantly different at any time (P . 0.05); thus, only
data from TSAN or TSAR are presented. However,
individual growth curves at the same temperature and RH
differed significantly among different pistachio samples,
and thus each lot of pistachios collected within or among
different years was treated separately.
In initial experiments conducted in 2010 and 2011,
inhull pistachios were collected from a single huller during
late-season commercial harvest, inoculated with Salmonella
at 4.29 to 4.31 log CFU/g, and held at either 23 or 358C and
50 or 90% RH (Fig. 3). During the first 12 h of incubation at
238C, maximum mean increases in the population of
Salmonella ranged from 0.77 to 0.96 log CFU/g at 50%
RH (Fig. 3A) and from 1.90 to 1.92 log CFU/g at 90% RH
(Fig. 3B). At 358C, maximum mean increases in the
population of Salmonella in the first 12 h ranged from 0.77
to 2.59 log CFU/g at 50% RH (Fig. 3D) and from 2.13 to
3.53 log CFU/g at 90% RH (Fig. 3E). For all paired samples
(same lot of pistachios, same inoculum preparation,
different incubation conditions), consistently higher maxi-
mum population increases were observed at higher humidity
and higher temperature (Fig. 3). Similar observations were
made in 2017 when inoculated pistachios were incubated at
23 or 378C (Fig. 3C and 3F).
For subsequent experiments, inhull pistachios were
collected from one of two hullers on different days during
J. Food Prot., Vol. 82, No. 2 GROWTH OF SALMONELLA ON INOCULATED INHULL PISTACHIOS 221

FIGURE 2. Growth of background populations, i.e., aerobic plate count (APC) (2014 [^] and 2016 [u], n ¼ 2) and coliform (2016
[*], n ¼ 2), and inoculated Salmonella (2014 [n] and 2016 [high inoculum level u and low inoculum level &], n ¼ 6) and on inhull
pistachios incubated at 378 C and 90% RH; pistachios were collected during early-, mid-, or late-season commercial harvest. Results are
mean (6SD) plate counts.

FIGURE 3. Growth of Salmonella on inoculated late-season (A, B, D, and E) and midseason (C and F) inhull pistachios incubated at
238 C (A, B, and C), 358 C (D and E), or 378 C (F) and at 50% (A and D) or 90% (B, C, E, and F) RH; pistachios were collected during
commercial harvest in 2010 (*), 2011 (*), and 2017 (u); n ¼ 6.
222 MOUSSAVI ET AL.
the early-, mid-, or late-season commercial harvests in 2014
and 2016. Pistachios were inoculated with Salmonella at
low (0.64 to 1.59 log CFU/g) or high (2.73 to 3.27 log CFU/
g) levels. On early-season and midseason inhull pistachios,
in the first 3 h after inoculation, small but significant (P ,
0.05) mean increases in Salmonella populations (0.41 to
0.67 log CFU/g) were observed at the low inoculum level,
whereas no significant (P . 0.05) increases in Salmonella
populations (0.10 to 0.40 log CFU/g) were observed at the
high inoculum level (Fig. 2). Salmonella populations
increased by over 4 log CFU/g to .5 log CFU/g (low
inoculum level) or to .7 log CFU/g (high inoculum level)
after 12 h. Thereafter, populations continued to increase,
plateaued, or decreased. Similar growth curves were
observed at both the low and high inoculum levels;
throughout the 30 h of incubation, Salmonella populations
were consistently ~1 to 2 log CFU/g lower on pistachios
inoculated at the low inoculum level. In contrast, for the
single lot of late-season inhull pistachios that were
evaluated in 2016, no significant change in populations of
Salmonella was observed during 30 h of incubation at either
the low or high inoculum levels (Fig. 2). It is possible that
high background populations observed in the 2016 late
harvest pistachios (.7 log CFU/g) depleted readily
available nutrients, thus prohibiting growth of inoculated
Salmonella (Fig. 2).
Salmonella growth parameters. Sigmoid functions
(DMFit or asymmetric sigmoidal five-parameter logistic)
were used to fit growth curves where typically a no-growth
linear phase (lag) is followed by a steep mid-phase (log) and
then a stationary phase. In most cases, similar curve fits
were derived from the two fit functions. However, owing to
an insufficient number of early time points in 2010 and high
population variability at each time point in 2011, DMFit
predicted linear curve fits for five growth curves (2010,
238C and 50% RH; 2010, 238C and 90% RH; 2011, 238C
and 50% RH; 2011, 358C and 50% RH; 2011, 358C and
90% RH). In these cases, only the asymmetric sigmoidal
five-parameter logistic function was used to construct fits
more like a classic growth curve (Table 1 and Supplemental
Figs. S1 and S2).
Longer lag phases, lower growth rates (all but one
case), smaller highest concentrations, and smaller maximum
increases were observed for inoculated inhull pistachios
incubated at suboptimal growth conditions, i.e., at a lower
incubation temperature and/or RH (238C and 50% RH),
compared with the corresponding pistachios (same lot)
incubated at higher temperature and RH combinations in the
2010, 2011, and 2017 harvest years (Table 1 and Figs. S1
and S2A and S2B). The time to highest predicted
concentration ranged from 9.6 to 23 h.
In harvest years 2014 and 2016, inoculated inhull
pistachios were incubated at 378C and 90% RH, which was
at the upper end of measured temperature and RH for
harvest trailers (Fig. 1) (19). Lag times of 2.0 to 4.4 h,
growth rates of 0.52 to 0.70 log CFU/g/h, and maximum
increases of 3.5 to 5.3 log CFU/g were predicted for early-
J. Food Prot., Vol. 82, No. 2
season and midseason harvested inhull pistachios at the high
initial inoculation level.
In laboratory-based studies, artificially high levels of
inoculum are often used to facilitate enumeration. Lag time,
growth rate, maximum populations, or time to maximum
population derived from these inoculum levels are assumed
to reflect theoretical outcomes for much lower and more
realistic contamination levels. In the current study, the
‘‘high’’ inoculum level (3 log CFU/g) might reflect a point
source contaminant, such as a localized small amount of
fecal material from a bird or rodent.
In 2016, low and high initial inoculum levels of
Salmonella were compared on the same lot of early-season
and midseason inhull pistachios. High inoculum levels were
associated with longer predicted lag phases, higher growth
rates, larger highest concentrations, and lower maximum
increases (Table 1). Highest predicted populations of
Salmonella were 0.69 to 1.49 log CFU/g higher at the high
initial inoculum level. The time to achieve the highest
predicted concentration was longer at the low inoculum
level.
Characterization of inhull pistachios. Uninoculated
inhull pistachio lots collected in 2016 were sorted into
different fractions, including the inhull nuts, loose material
(mostly free hull and leaves), the hulled inshell nuts, free
kernels, free shells, and sticks or twigs (Fig. 4). The
proportion of material in each fraction was similar for early-
season and midseason pistachios (Fig. 4); more than 98% of
the material by weight was made up of pistachios with an
intact hull. In contrast, for the late season lot, 62.8% of
pistachios had intact hulls; large amounts of loose material
(12.1%) and hulled inshell pistachios (22.9%) were also
present. At full maturation, the inshell pistachio will easily
separate from the hull when pressure is applied along the
axis of the nut (18). As maturity progresses, the hulls
become increasingly susceptible to mechanical injury. The
large percentage of inshell pistachios in the 2016 late-
season lot is indicative of over-mature pistachios.
Moisture content and aw. The initial moisture levels
of hulls and kernels from uninoculated inhull pistachios
were significantly different among samples, ranging from
75.5% (midseason, 2014) to 80.7% (midseason, 2016) for
hulls and from 34.3% (midseason, 2014) to 46.7%
(midseason, 2016) for kernels (Table 2). The initial aw
was 0.98 or 0.99 for both hulls and kernels. Decreases in
moisture content were significant, ranging from 2.0 to
13.3% (hulls) and 2.0 to 8.3% (kernels), between 1 and 30 h
of incubation; aw did not change or decreased by a
maximum of 0.02. The pH of hulls from samples collected
over 4 weeks in 2016 was 5.14, 4.95, 5.03, and 5.07
(average, 5.05 6 0.10).
The current study demonstrates that Salmonella can
multiply on inhull pistachios under commercially relevant
times and temperatures. The lag times, growth rates, and
maximum and final populations observed varied signifi-
cantly among independent replicate experiments. However,
with paired samples from the same lot of pistachios, growth
 J. Food Prot., Vol. 82, No. 2

TABLE 1. Predicted growth parameters of Salmonella inoculated on inhull pistachios as determined by DMFit sigmoid and/or asymmetric sigmoidal five-parameter logistic functions
Harvest Incubation RH Initial concn Lag Rate Highest concn Max increase Time to highest
Harvest yr season temp (8C) (%) (log CFU/g) (h) (log CFU/g/h) (log CFU/g) (log CFU/g)a concn (h) R2 Fit functionb

2010 Late 23 50 4.07 ~6 0.15 6.81 2.74 19.0 0.81 AS-5PL

90 4.36 ~3 0.23 7.49 3.13 17.0 0.89 AS-5PL
35 50 4.40 0.00 0.22 7.98 3.58 18.0 0.92 DMFit, AS-5PL
90 4.35 0.00 0.31 8.41 4.06 14.4 0.97 DMFit, AS-5PL
2011 Late 23 50 4.17 ~7 0.087 5.36 1.19 14.0 0.25 AS-5PL
90 4.12 6.77 0.78 6.03 1.91 9.6 0.61 DMFit, AS-5PL
35 50 4.31 ~3 0.053 5.54 1.23 20.0 0.22 AS-5PL
90 4.33 0.00 0.19 7.99 3.66 23.0 0.80 AS-5PL
2014 Early 37 90 3.29 4.42 0.70 7.21 3.92 10.5 0.80 DMFit, AS-5PL
Mid 90 3.01 3.23 0.57 6.55 3.54 10.5 0.69 DMFit, AS-5PL
2016 Early 37 90 0.46 0.00 0.38 6.29 5.83 18.0 0.95 DMFit, AS-5PL
90 2.49 1.95 0.52 7.78 5.29 13.5 0.96 DMFit, AS-5PL
Mid 37 90 0.48 0.90 0.47 6.97 6.49 16.5 0.98 DMFit, AS-5PL
90 2.65 2.47 0.65 7.66 5.01 12.0 0.98 DMFit, AS-5PL
Late 37 90 1.52 0.00 0.0050 1.67 0.15 NDc ND DMFit, AS-5PL
90 3.09 0.00 0.0011 3.05 0.03 ND ND DMFit, AS-5PL
2017 Mid 23 90 2.83 4.34 0.14 5.27 2.44 22.5 0.86 DMFit, AS-5PL
37 90 3.04 0.00 0.20 6.43 3.39 19.5 0.80 DMFit, AS-5PL
a
Maximum increase ¼ highest concentration  initial concentration.
b
AS-5PL, asymmetric sigmoidal five-parameter logistic; DMFit, DMFit version 3.5 add-in to Microsoft Excel (4).
c
ND, not determined.
GROWTH OF SALMONELLA ON INOCULATED INHULL PISTACHIOS
223
224 MOUSSAVI ET AL. J. Food Prot., Vol. 82, No. 2

FIGURE 4. Components of harvested pistachio fruit (photograph) and weight percentage of different fractions found in one ~1-kg
sample of inhull pistachios collected during the early-, mid-, and late-season commercial harvest in 2016 (pie chart). Loose materials
include hulls.

was positively influenced at a higher temperature and
humidity. Lower initial inoculum levels resulted in lower
maximum population outcomes. In most cases, insignificant
or small but statistically significant increases in populations
were observed in the first 3 h of incubation; predicted lag
times ranged from 3 to 7 h at 238C and from 0 to 4 h at 35
and 378C. The predicted mean time to achieve maximum
population was 16 6 4 h. No growth was observed in a
single lot of late-season inhull pistachios. There were
significantly greater amounts of inshell and loose material in
this lot (Fig. 4), and the initial APC was far higher than in
other inhull pistachios evaluated (.7 log CFU/g versus 3 to
4 log CFU/g; Fig. 2), which potentially resulted in a
depletion of readily available nutrients.
These findings stress the importance of managing
postharvest handling of pistachios to minimize the time
between harvest and hulling. In all but two cases, significant
growth of Salmonella was observed at 23, 35, and 378C and
at both 50 and 90% RH when incubation times exceeded 3
to 6 h. Harvest during cooler times of day (e.g., late evening
and early morning) or under conditions that prevent or
minimize temperature and humidity increases in the trailers

TABLE 2. Moisture content and water activity of hulls and kernels from inhull pistachios collected at different times during two
commercial harvest years and stored at 378 C and 90% RH
Moisture content (%)a Water activity

Material Harvest season Harvest yr 0h 30 h 0h 30 h

Hulls Early (wk 1) 2014 80.0 6 0.23 B a 78.0 6 0.31 A b 0.99 6 0.00 A a 0.98 6 0.00 A b
2016 75.9 6 0.42 D a 73.1 6 0.52 C b 0.98 6 0.00 B a 0.98 6 0.00 A a
Mid (wk 3) 2014 75.5 6 0.37 D a 68.4 6 1.23 D b 0.98 6 0.00 B a 0.97 6 0.00 B b
2016 80.7 6 0.21 A a 74.2 6 0.36 B b 0.99 6 0.00 A a 0.98 6 0.00 A b
Late (wk 5) 2016 77.4 6 0.01 C a 64.1 6 0.27 E b 0.99 6 0.00 A a 0.97 6 0.00 B b
Kernels Early (wk 1) 2014 42.8 6 0.63 F a 40.8 6 0.20 F b 0.98 6 0.00 A a 0.98 6 0.00 A a
2016 42.0 6 0.48 F a 39.5 6 0.56 G b 0.98 6 0.00 AB a 0.98 6 0.00 A a
Mid (wk 3) 2014 34.3 6 0.62 H a 26.0 6 0.57 I b 0.98 6 0.00 B a 0.96 6 0.00 C b
2016 46.7 6 0.89 E a 41.3 6 0.56 F b 0.99 6 0.00 A a 0.98 6 0.00 A a
Late (wk 5) 2016 39.6 6 0.35 G a 32.8 6 0.53 H b 0.98 6 0.00 A a 0.97 6 0.00 B b

a
Values are means 6 standard deviations, n ¼ 3. Within columns, mean values with different uppercase letters are significantly different
(P , 0.05); within rows and within moisture content or water activity, mean values with different lowercase letters are significantly
different (P , 0.05).
J. Food Prot., Vol. 82, No. 2 GROWTH OF SALMONELLA ON INOCULATED INHULL PISTACHIOS
would reduce the potential for increases in Salmonella
populations in inhull pistachios prior to hulling. This study
focused on the upper measured temperature and humidity in
harvest trailers. Additional information on individual trailer
hold times, as well as receipt and unloading temperatures,
would further help to inform the risk from delays in
unloading trailers. Significant growth of Salmonella could
occur before quality impacts, such as shell staining, are
evident. Further work is needed to assess the impact of
hulling and drying on microbial populations in inshell
pistachios.
ACKNOWLEDGMENTS
This research was supported, in part, by the California Pistachio
Research Board, the Center for Produce Safety, and the Specialty Crop
Block Grant Program at the U.S. Department of Agriculture (USDA)
through grant 14-SCBGP-CA-0006. Its contents are solely the responsi-
bility of the authors and do not necessarily represent the official views of
the USDA. The contributions made by Dr. Anne-laure Moyne, Lillian
Khan, Ethan Morgan, and Sylvia Yada in technical support and during the
writing process are greatly appreciated.
SUPPLEMENTAL MATERIAL
Supplemental material associated with this article can be
found online at: https://doi.org/10.4315/0362-028X.JFP-18-351.
s1.
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