Food Protection Trends - July/August 2021 - 425

products is illegal. To demonstrate that L. monocytogenes
is not present in a RTE product, meat processors must
demonstrate that L. monocytogenes cells are not able to grow
at any point throughout the shelf life of the product (41).
Lastly, the USDA-FSIS requires that Campylobacter spp. be
addressed in HACCP documentation for meat products
containing poultry and that a 5-log CFU reduction of
Campylobacter spp. be achieved in RTE poultry products
(40). To demonstrate those reductions in a duck salami,
an experiment was designed to validate the bacterial safety
of a process used to manufacture a fermented and dried
duck salami, utilizing Salmonella spp., L. monocytogenes, and
Campylobacter spp. as pathogens of concern.
MATERIALS AND METHODS
Culture selection and growth
The following cultures were received or purchased from
American Type Culture Collection (ATCC; Manassas, VA),
CDC, or USDA: Salmonella enterica subsp. enterica serovar
Typhimurium (ATCC 14028, isolated from chicken organs),
S. enterica subsp. enterica serovar Montevideo isolate SMvo13
(CDC), S. enterica subsp. enterica serovar Derby (ATCC
7378, human isolate), L. monocytogenes strain Scott A, L.
monocytogenes serotype 1/2a isolate FSL R2-603 (deli meats
outbreak), L. monocytogenes serotype 4b isolate H3396 (hot
dog outbreak), Campylobacter jejuni isolate PSU99 (isolated
from chicken in Pennsylvania, species confirmed by USDAAgricultural
Research Service), C. jejuni (ATCC 29428,
human isolate), and Campylobacter coli (ATCC 33559, pig
isolate). These cultures were chosen for their role in human
illness or association with meat and food products.
Overnight cultures of each Salmonella serovar and L.
monocytogenes strain were grown from laboratory stocks by
adding a loopful of culture to 10 mL of tryptic soy broth
(TSB; BD, Sparks, MD) and incubating for 24 h at 37°C
under aerobic conditions. To ensure working stocks were
not contaminated, the following procedure was used. Each
bacterium was streaked for isolation from the overnight
culture on selective media. Xylose lysine deoxycholate agar
(BD) was used for Salmonella spp., and modified Oxford
agar (BD) was used for L. monocytogenes. Salmonella species
and Listeria latex confirmation tests were performed using
an isolated colony for each strain of bacteria (Microgen
Bioproducts, Camberley, UK). A single colony of each
Salmonella serovar and L. monocytogenes strain was added
to 10 mL of fresh TSB (BD) and incubated under the same
conditions to achieve a concentration of approximately 8 log
CFU/ml.
Campylobacter spp. cultures were grown from agar slants
received from ATCC or the USDA Agricultural Research Service.
A loopful of culture for each strain was inoculated into
10 mL of Brucella broth (Hi Media Laboratories, Mumbai,
India) and grown for 48 h at 37°C under microaerophilic
conditions (10% CO2
and 90% N2). Strains were streaked
for isolation on charcoal cefoperazone deoxycholate agar
(CCDA; Oxoid Ltd., Basingstoke, England) and incubated
for 48 h under the same conditions. Campylobacter spp. latex
confirmation tests (Microgen Bioproducts) were performed
for each strain using an isolated colony. An isolated colony
was also inoculated into 10 mL of fresh Brucella broth (Hi
Media Laboratories) for 48 h under the same conditions to
achieve an approximate concentration of 8 log CFU/ml.
An inoculation bath was created for the inoculation of
raw duck and pork belly. Fresh 10-mL overnight cultures of
each of the nine strains of Salmonella spp., L. monocytogenes,
and Campylobacter spp. were incubated in either TSB (BD)
or Brucella broth (Hi Media Laboratories). The overnight
cultures were then added to a fresh 340-mL amount of either
TSB (BD) or Brucella broth (Hi Media Laboratories) and
incubated again for the same length of time. After incubation,
the 350-mL broth cultures were centrifuged for 5 min at 21°C
at approximately 11,000 × g (Avanti J-26 CPI; Beckman
Coulter, Inc., Pasadena, CA). After centrifugation, the
supernatant was decanted and disposed of, and the cultures
were resuspended in 1× phosphate buffered saline (Alfa
Aesar, Tewksbury, MA). The resuspended cultures of all nine
strains were combined in a sterilized metal bin to make an
inoculation bath approximating 3.2 L.
Duck salami production
Duck trim, pork belly, seasonings, and casings were
provided separately for each of the three independent
replications by a salami company in the northeastern United
States. For a single replication, duck trim and pork belly were
thawed upon arrival (4°C), cut into pieces of approximately
5 cm3
, and weighed into a 13.6-kg meat block containing
70% duck and 30% pork belly. On the first day of production,
the duck trim and pork belly pieces were submerged in the
inoculation bath for 30 min in a biological safety cabinet,
with stirring every 5 min, so that the pathogens could attach
to the meat surfaces. After 30 min of submersion in the
inoculation bath, the meat was scooped onto a draining
cart and allowed to remain undisturbed for 30 min in
the biological safety cabinet. After drying, the meat was
transferred to a clean plastic container, treated with 1 L
of 2.5% Beefxide solution (Birko Corp.) that contained a
proprietary blend of lactic acid, citric acid, and potassium
hydroxide, and manually tumbled with a large, sterile spoon
for 15 min. When tumbling was completed, the meat was
moved to a different clean plastic container and placed into
a walk-in cooler (4°C) overnight. Approximately 24 h after
tumbling, the duck and pork meat was ground once through
a 6-mm grinding plate using a size 22 grinder (Avantco
Equipment MG22, Clark Associates, Inc., Lancaster, PA)
and then mixed with salt (2.5%; w/w), 0.24% cure #2
(92.75% salt, 6.25% sodium nitrite [150 ppm in-going],
1% sodium nitrate [24 ppm in-going], and dextrose [0.5%;
w/w]), coriander (0.35%; w/w), garlic (0.2%; w/w), black
July/August Food Protection Trends 425
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