Food Protection Trends - May/June 2019 - 202

not observed in the inoculated product. As a result, a study
of acid adaptation of E. coli was not pursued for chili con
carne with beans. After incubation, the 25-ml culture tubes
were centrifuged at 5,000 × g for 15 minutes at 4°C. The
supernatant was discarded, and the pellets were re-suspended
in 25 ml of 0.1% BD Bacto™ Peptone Water (PW; Fisher
Scientific, Waltham, MA), after which all four strains were
combined in a sterile 100-ml container, resulting in a master
inoculum cocktail containing approximately 109 CFU/ml. To
achieve a target concentration of 104 CFU/g of product, the
inoculum for each pan was prepared in 0.1% PW based on
the weight of food product within each pan, with the liquid
of the inoculum comprising no more than 1% of the food
product (14).
Food product preparation
All food products met the nutritional standards for Child
Nutrition Programs (22). Food products and ingredients
were ordered from a foodservice distributor. Pre-cooked,
frozen taco meat was thawed in a commercial refrigerator at
4°C for several days prior to each experimental replication.
On day 0 of each experimental replication, five-pound bags
were placed in 2-inch stainless steel steam table pans and
heated in commercial steamers (Electrolux Air-o-Steam
Touchline Combi Oven and Cleveland SteamChef Electric
Countertop Steamer) to 73.9°C, which was determined by
use of a Taylor 9842FDA waterproof digital thermometer
(Taylor Precision Products, Oak Brook, IL) at several
locations throughout the product. Canned, low-sodium
marinara sauce was cooked to 73.9°C in a commercial tilt
skillet (Cleveland Tilt Skillet). Chili was prepared according
to a recipe used by a local school nutrition program following
USDA guidelines and was cooked to 73.9°C in the same
commercial tilt skillet as used for the marinara sauce. After
the food products were reheated or cooked, they were
portioned in 2- and 3-inch depths in 2½- and 4-inch deep
stainless steel steam table pans. Products portioned to 2-inch
depths were placed into pans with dimensions of (W × L ×
D) 30.5 cm × 50.8 cm × 6.4 cm (12-inch × 20-inch × 2.5inch), while products portioned to 3-inch depths were in
pans with dimensions of (W × L × D) 30.5 cm × 50.8 cm ×
10.2 cm (12-inch × 20-inch × 4-inch).
Food product inoculation
The temperature of all products was monitored with a
Taylor 9842FDA waterproof digital thermometer (Taylor
Precision Products, Oak Brook, IL), and all food products
were stirred and allowed to cool to 60°C ± 5°C prior to
inoculation. After the calculated volume of inoculum
was added to each pan, food was manually stirred for
approximately 2 minutes to distribute the bacterial cells.
Inoculation times were recorded for each pan upon
completion of stirring, and time points were set accordingly.

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Food Protection Trends May/June

Sampling
After inoculation, samples were collected from each pan
after 0, 4, 8, 12, and 24 hours of cooling. At each time point, a
composite sample was obtained by using a plastic disposable
tablespoon to gather food from four or five randomly chosen
sub-surface areas within each pan, where the food was likely
warmer than at the product surface and therefore more at risk
for microbial growth. Collecting food from five locations in
every pan at each sampling point was the goal. If pans stored
in the freezer began to solidify, making it difficult to collect
food during sampling, at least four random locations were
sampled. Samples were collected at random to account for
the possibility that, following stirring of the product, pockets
of elevated temperature may have remained.
Composite samples were homogenized by hand mixing,
after which a 25-g sample from each composite was diluted
with 225 ml of BPW and stomached for one minute at
230 rpm (Stomacher® 400 Circulator; Seward, Bohemia,
NY). Serial dilutions of the samples were then prepared in
9-ml tubes of BPW, and dilutions were spread plated onto
MacConkey (MAC; Remel, Lenexa, KS) agar. MAC plates
were incubated at 37°C for 18 to 24 hours, and representative
colonies were counted.
Treatments and cooling
After the food products were inoculated and time point 0
samples obtained, each pan was fitted with a Lascar EL-USB-2LCD USB temperature data logger (Lascar Electronics,
Erie, PA) in the center of the pan, as previously described
by Roberts et al. (19), to record the product temperature
every 60 seconds for 24 hours. Pans were then prepared with
three cover types: uncovered, covered with a single layer of
aluminum foil over the top of the pan, or double covered
to restrict air exposure. Double-covered pans had one layer
of plastic wrap (for marinara sauce product, because of its
acidity) or aluminum foil (pre-cooked taco meat and chili
con carne with beans) directly contacting the top surface of
the food product and another layer of aluminum foil over
the top of the pan (air space present between layers). Each
cover type was applied to pans with both a 2- and 3-inch food
product depth and prepared in duplicate so that one pan could
be stored at 4°C (Standard Deviation: 1.34°C) in a walk-in
refrigerator and the duplicate pan could be stored at -20°C
(Standard Deviation: 3.83°C) in a walk-in freezer. Pans in the
refrigerator were situated into ice baths as suggested in the
FDA Food Code (28). The ice baths were prepared by filling
4- and 6-inch stainless steel steam table pans three-quarters
full with crescent-shaped ice (for use with the 2½- and 4-inch
depth pans, respectively). Thus, six pans of each inoculated
food product were stored for cooling and sampling in each of
the storage locations (refrigerator or freezer). To prevent food
products from becoming completely frozen and thus unable to
be sampled, pans in the freezer were transferred to a shelf in the
refrigerator immediately after the 8-hour sampling.

Food Protection Trends - May/June 2019

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