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TABLE 1. Salmonella enterica strains included in the inoculation cocktail for soaking
studies of grains, nuts, and seeds
Salmonella Serotype

Isolate Identifier

Description

Isolate Source

Enteritidis PT30

ATCC BAA-1045

Almond isolate

American Type
Culture Collection

Enteritidis PT9c

RM4635

Clinical isolate from almond
outbreak

Rob Mandrell
USDA-ARS

Montevideo

GRC1

Pistachio isolate

Food and Drug Administration

Paratyphi B var. L(+)tartrate(+)

OSPHL 15092808070

Clinical isolate from 2015
outbreak associated with raw
sprouted nut butters

Oregon State Public
Health Labs

Oranienburg

MDD317

Pecan isolate

Michelle Danyluk
University of Florida

Tennessee

MDD319

Clinical isolate from peanut
butter outbreak

Larry Beuchat
University of Georgia

onto sterile weighing boats and mixed with 2 mL of diluted
inoculum with a sterile utensil to coat. For seeds and grains,
the inoculum was applied with an atomizer (Specialty Bottle,
Seattle, WA) via spraying 1 mL of diluted inoculum directly
onto seed and grain products (20 g) spread out in weigh
boats. After coating or mixing, inoculated product was dried
in the biological safety cabinet at ambient temperature
for 24 h to return product to original moisture content.
Inoculated product (~3 log CFU/g) was used immediately
for experimentation after the drying period.
Soaking process
Inoculated product was mixed and aliquoted (20 g) into
sterile 50-mL conical bottom tubes. Sterile distilled water (20
mL; pH 6.44) was added to each tube and incubated at 22°C
for 24 h. Additionally, almond samples were soaked in salt
solutions at various concentrations (1.5, 10, 20%) for 24 h.
Uninoculated controls were prepared in the same manner to
evaluate changes in the cell density of natural microbiota, pH,
water activity, and moisture content.
Microbial analyses
Upon completion of a 24-h soak, the hydrated product
and soaking solution were mixed thoroughly by shaking the
conical tubes for 30 s. For products that thickened during
soaking (i.e., flax and hemp), the entire sample (product
and solution) was homogenized with an equal volume (1:1)
of 0.1% peptone water. Soaking solution was aseptically
transferred and serially diluted in 0.1% peptone water, and
appropriate dilutions were spread plated on Hektoen enteric
agar (inoculated samples) or TSA (uninoculated samples).

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Food Protection Trends May/June

Inoculated, unsoaked samples were mixed with 20 mL of
0.1% peptone water, mixed, serially diluted, and plated as
described for soaked samples. Plates were incubated at 37°C
for 24 to 48 h prior to enumeration.
pH, water activity, and moisture content
Samples were tested for water activity with the AquaLab
CX-2 or 4TE water activity benchtop meter (Decagon
Devices, Inc., Pullman, WA). Moisture content was measured
with the HB43-S halogen moisture analyzer using method
1530.08 (Mettler Toledo, Columbus, OH). Product samples
were finely chopped in a food processor prior to moisture
content and water activity analyses. The pH of water, soaking
solution, and product were measured with the SympHony
H10P pH meter (VWR, Radnor, PA) or the Apera SX811SS (Apera Instruments, Columbus, OH). Prior to pH
measurement, distilled water was added to product samples
(1:1) and blended to homogenize.
Statistical analysis
Biological replication at the cocktail preparation level was
performed in triplicate, and Salmonella growth experiments
for each product were performed in triplicate for each
biological replicate (n = 9). Natural microbiota growth
experiments were performed on uninoculated products on
2 days in triplicate (n = 6). Moisture content, water activity,
and pH of uninoculated unsoaked and soaked products were
measured on one sample per product per day (n = 2). Mixed
model and one-way analysis of variance with a Tukey's test
as the post hoc analysis were performed to determine the
impact of water activity, moisture content, pH, and initial

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