Food Protection Trends - November/December 2017 - 410

the food under the conditions in which the food is stored,
distributed, retailed and held by the user (22). For acidified
foods, thermal processing is normally a mild heat treatment,
such as steam pasteurization or an inverted hot-fill hold that,
for low pH products, is generally sufficient to guarantee
safety (2).
In the manufacture of acidified canned foods, acidification
is most often accomplished by addition of organic acids such
as acetic, lactic or citric acid, singly or in combination, and
employed to control microbial growth, improve sensory
attributes, and reduce microbial spoilage of foods (1, 9, 15).
In a review of 179 tomato-based acidified food products
found in local grocery stores, we noted that citric and acetic
acids were the most commonly used acidulants, usually in the
form of lime juice or distilled vinegar. Citric acid has been
shown to inactivate Salmonella Typhimurium in tahini (1),
while acetic acid has been shown to inactivate Escherichia coli
O157:H7 in simulated pickle products (4) and apple-carrot
juice blends (23). Acetic acid was also effective at inactivating
Salmonella, E. coli O157:H7, and L. monocytogenes to various
degrees in asparagus purée (20). Acetic and citric acids have
been shown to inactivate E. coli O157:H7, Salmonella, and
L. monocytogenes on fresh apples and lettuce (18) and in
cucumber purée (3).
Processors of acidified canned foods are required to register their manufacturing facility and file scheduled processes
for their products with the Food and Drug Administration
(FDA) (22). FDA filings for acidified canned foods must be
supported by research that validates destruction of important foodborne pathogens, and E. coli O157:H7, S. enterica,
and L. monocytogenes are considered targets for thermal processing and acidification (5-7). The work of Breidt and colleagues used a re-parameterized Weibull model to estimate
thermal processing conditions necessary to achieve a 5-log
reduction of target pathogens in an acidified cucumber
juice medium (5-7). Recent studies in our laboratory have
confirmed the appropriateness of the non-linear Weibull
approach for calculating accurate thermal processing D- and
z-values (10).
The process authority, in using laboratory data to establish
a scheduled process for an acidified food, must rely on an
understanding of heat penetration in a particular food matrix,
the impact of formulation on microbial inactivation, and
heat tolerance of spoilage organisms and target pathogens.
It is in the best interest of manufacturers to have appropriate evidence to validate thermal processing conditions that
produce a safe but not over-processed product, leading to a
balance between food safety and food quality. In the absence of current recommendations developed with use of a
tomato-based medium, this study was conducted to calculate
thermal processing parameters that would inactivate vegetative cells of the target pathogens E. coli O157:H7, S. enterica
and L. monocytogenes in tomato purée. This study expanded
on previous work (10) and examined the effect of acidifica-

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Food Protection Trends November/December

tion with citric and acetic acids on thermal inactivation of
these vegetative pathogens in tomato purée at 54°C. Time/
temperature processing conditions were estimated that would
ensure a minimum 5-log pathogen reduction of vegetative
pathogens and could be used to support safe manufacture of
tomato-based acidified canned foods.
MATERIALS AND METHODS
Strain selection and maintenance
Strains of E. coli O157:H7, S. enterica, and L. monocytogenes
were used in this study (Table 1). With one exception, these
strains were the ones used in work by Breidt et al. (5-7);
E. coli O157:H7 strain ATCC 43895 (American Type
Culture Collection, Manassas, VA) replaced strain ATCC
43888 used in the prior work. Stock cultures of each strain
were maintained in Tryptic Soy Broth at pH 7.1 (TSB;
Difco, Becton, Dickinson and Company, Sparks, MD)
containing 10% (v/v) glycerol (Fisher Scientific, Itasca,
IL) and stored frozen at -20°C. Bacterial strain identity
and purity were assessed and confirmed by Gram reaction,
cell and colony morphology, and responses to biochemical
profiling kits (bioMerieux, Durham, NC) for E. coli
O157:H7 and S. enterica (API 20E), and L. monocytogenes
(API Listeria). Working cultures were prepared monthly
by streaking for isolation from partially thawed stock
cultures onto appropriate selective plating medium: L.
monocytogenes on Listeria Selective agar (LSA; Oxoid LTD,
Basingstoke, Hampshire, England) with added Listeria
Selective Supplement (Oxoid), and S. enterica and E. coli
O157:H7 on modified Levine's Eosin Methylene Blue agar
(mLEMB; Difco). mLEMB was prepared from Levine's
Eosin Methylene Blue agar, with addition of D-sorbitol
(10 g/liter; Fisher) and NaCl (5 g/liter, Fisher). Working
culture plates were incubated at 35°C for 24 h for S. enterica
and E. coli O157:H7, or 48 h for L. monocytogenes, followed
by storage at 4°C for ≤ 40 days. E. coli O157:H7 colonies
appear colorless to pale pink on mLEMB agar, S. enterica
colonies appear dark red to iridescent green on mLEMB agar,
and L. monocytogenes colonies appear pale yellow or grey
surrounded by a black halo on LSA.
Inoculum preparation
For each trial, a 5-strain single-pathogen cocktail was
prepared. Inocula were prepared by the selection, for each
strain, of a single colony from a working culture plate, the
cells of which were suspended into 9 ml of TSB +1% added
glucose (Fisher) and statically incubated at 35°C for 24 ± 2
h to obtain stationary-phase cells (108-109 CFU/ml) and
expose growing cells to acid, as previously described (5, 8).
Contents of all five tubes for a species were combined in one
conical 50 ml centrifuge tube and harvested by centrifugation
(4,500 g, 7 min, 21°C, Marathon 21K, Fisher). The supernatant was discarded and the pellet was re-suspended in 4.5
ml Butterfield's Phosphate Diluent (BPD; Nelson Jameson,

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