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distribute inoculum, it is possible that the product did not
cool evenly.
Though time had a statistically significant effect for
marinara sauce, 0.21 log10 CFU/g was the largest increase
in populations, occurring between the 0- and 8-hour time
points. Natural variation of the E. coli population within the
product, or variability introduced by plating methods, may
have been more responsible than the cooling procedure for
these population changes (Fig. 7). It could be hypothesized
that heat combined with acidity (pH 4.18; Education pH
meter; Fisher Scientific, Lenexa, KS) injured the cells,
causing them to lag initially and then recover over time.
However, this is simply a hypothesis, as evaluating sublethal
injury was beyond the scope of this study. Overall, these
results indicate that all cooling method variables suppressed
growth to the same degree, suggesting that all were effective
at controlling E. coli populations in marinara sauce.
According to performance standards outlined in Annex
3, Section 3-501.19 of the 2017 FDA Food Code, hot foods
held in the absence of temperature control should not exceed
1 log10 growth of Clostridium perfringens (C. perfringens)
and Bacillus cereus (28). A limitation of this study was the
inability to model Clostridium perfringens, another foodborne
pathogen associated with improper cooling, which is
responsible for over 965,000 foodborne illnesses annually
as well as 24 outbreaks in schools, colleges and universities
between 1998 and 2016 (4, 20). A review of the literature
indicated that evidence of a proper surrogate to model C.
perfringens under cooling conditions is lacking. The FDA
Safe Practices for Food Processes, Chapter 6: Microbiological
Challenge Testing, mentions Clostridium sporogenes as a
surrogate for Clostridium botulinum but does not contain
information regarding a surrogate for C. perfringens (10). In
order to effectively simulate food preparation and product
cooling in a school setting, it was necessary to utilize
commercial scale food preparation equipment, coolers, and
freezers. These resources were not available for use with
pathogenic microorganisms within the control of a biosafety
level II (BSL II) laboratory. Therefore, it was not possible to
model C. perfringens, a BSL II microorganism, in this study.
The E. coli strains chosen from the ATCC® MP-26™ panel
have been investigated and found to be appropriate surrogates under cooling conditions not only for Shiga-toxin
producing E. coli but also for another important enteric
pathogen, Salmonella enterica (11, 15). Therefore, the results
of this research can provide insight into the behavior of
several enteric pathogens under cooling conditions, which is
advantageous, considering that Salmonella has been identified
as one of the top pathogens implicated in foodborne illness
outbreaks in school settings (8, 29).

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The 2017 FDA Food Code also states that the performance
standard is no more than 1 log10 CFU/g growth of the
non-sporeformer, Listeria monocytogenes, in cold foods
held without temperature control (28). Although the data
presented in the current study represent the cooling of
hot foods, it is relevant to mention that E. coli, also a nonsporeformer, exhibited growth (0.21 log10 CFU/g) in only
one food product (low-sodium marinara sauce). Not only
is this growth marginal, but it is also well below the 1 log10
CFU/g of Listeria monocytogenes growth allowable in cold
food products.
SUMMARY
The methods used to cool food in this study provided
equivalent microbiological control. Data suggest that all 12
cooling combinations tested can be utilized as an effective
strategy for controlling E. coli populations in these food
products, despite the inability of some cooling methods
to meet 2017 FDA Food Code criteria (28) with regard to
temperature. While microbiological data suggests that all
methods effectively controlled populations of surrogate
E. coli, it is recommended that the cooling techniques that
satisfied 2017 FDA Food Code (28) requirements for cooling
be preferentially used by school nutrition programs and other
foodservice operations. As noted before, considering that the
food products used in this study could be prepared, cooled,
and served in a variety of foodservice operations, results are
not limited to the school nutrition environment.
It must be emphasized that these data are limited in
scope with regard to food products and microorganisms
investigated. Furthermore, because generic E. coli cultures
were used, these data merely provide an indication as to
how E. coli O157:H7 might behave under simulated cooling
conditions. More research is necessary, as this study is not
exhaustive, and more combinations should be explored in
future experimentation.
ACKNOWLEDGMENTS
This is the contribution number 18-283-J from the
Kansas Agricultural Experiment Station (Manhattan, KS).
All research activities were approved by the Kansas State
University Institutional Biosafety Committee (#1043.2).
This research was conducted by Kansas State University
on behalf of the Center for Food Safety in Child Nutrition
Programs and was funded in part by the U.S. Department of
Agriculture. The contents of this article do not necessarily
reflect the views or policies of the U.S. Department
of Agriculture, nor does the mention of trade names,
commercial products, or organizations imply endorsement
by the U.S. Government.

Food Protection Trends - May/June 2019

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