Food Protection Trends - July/August 2021 - 410

reported (1, 2, 10, 16, 27, 28). Antimicrobials serve a critical
role in preventing cross-contamination of noncontaminated
produce by contaminated produce in wash water systems
(13). Chlorine is commonly used as a sanitizer for produce
wash water systems; however, the efficacy of chlorine for
reducing pathogen populations on produce and wash water
is affected by multiple water- and produce-related parameters
(28, 32). Organic matter can impact the use of chlorine
by lowering the residual concentration, thus reducing the
ability of the chlorine to eliminate indicator and pathogenic
microorganisms (1). Peroxyacetic acid (PAA) has been
suggested as an alternative to chlorine-based sanitizers
because it is less affected by organic matter (1).
PAA is composed of acetic acid and hydrogen peroxide
(22, 44). The production of reactive oxygen species is
responsible for the primary antimicrobial effect of PAA on
bacterial cells (39, 44). These reactive oxygen species damage
bacterial lipids and DNA (39, 44). PAA denatures enzymes
and proteins and increases permeability of the cell wall (18,
39, 44) and disturbs cell membranes and blocks transport
and enzymatic systems (24, 44).
When added to water, bisulfate of soda (BS) dissociates
into ions of sodium, hydrogen, and sulfate, lowering the pH
and creating an osmotic effect (23) that stresses bacterial
cells. Slight declines in pH require enteric microorganisms
to expend energy to regulate cytoplasmic pH to maintain
conditions that are near neutral; however, this process
stresses bacterial cells and will often lead to cell death (19,
25). In 1998, the U.S. Food and Drug Administration (41,
42) identified BS as generally recognized as safe (GRAS), and
BS is used as a food additive for many applications, including
the prevention of browning in fresh-cut produce (41).
A wash water solution containing PAA and BS would expose
microorganisms to multiple mechanisms of antimicrobial
action, which could result in microbial population reductions
on produce greater than those achieved by traditional chlorine
postharvest washes. Kim et al. (21) combined PAA and BS as
a synergistic hurdle intervention to reduce Listeria innocua on
whole apples postharvest. The efficacy of PAA and BS blends
was compared with that of a water control and 150 ppm of
chlorine at pH 6.5. The treatment with 3% BS and 60 ppm of
PAA resulting in a 5.56-log reduction in L. innocua after 14
days of storage (21). The reductions achieved by the blend
of PAA and BS against L. innocua on apples suggested that
more research investigating the efficacy of this blend in other
postharvest applications was warranted. Therefore, the present
study was conducted to quantify reductions of Salmonella
populations on fresh-cut spinach after use of a blend of PAA
and BS as a postharvest wash solution.
MATERIALS AND METHODS
Culture preparation
The inoculation cocktail was prepared from frozen stock
cultures of S. enterica subsp. enterica serovars Anatum
410 Food Protection Trends July/August
(Kansas State University TX 2006 C20), Montevideo (TX
2006 C7), Newport (TX 2006 F10), and Typhimurium
(ATCC 14028) that had been stored in tryptic soy broth
(TSB; Difco, BD, Franklin Lakes, NJ) with 15% glycerol
at −80°C. The isolates of Salmonella serovars Anatum,
Montevideo, and Newport were originally recovered from
cattle at Kansas State University. All Salmonella isolates were
revived from frozen by streaking onto tryptic soy agar (TSA;
Difco, BD) at 37°C for 24 h and selecting a colony to transfer
in 9 mL of TSB at 37°C for 24 h. Two tubes were prepared for
each serotype (18 mL total). Following 24 h of incubation,
18 mL of each serotype was centrifuged at 5,200 × g for 15
min at 4°C (Allegra X-30R, Beckman Coulter, Brea, CA).
The supernatant was discarded, and pellets were resuspended
in 18 mL of buffered peptone water (BPW; Difco, BD).
Resuspended cultures were combined in equal proportions
to prepare an inoculum cocktail (75.33 mL), which was
diluted in 8.7 L of BPW to achieve a starting titer of 7.0 log
CFU/mL.
Spinach preparation and inoculation
Fresh, unwashed mature spinach (Spinacia oleracea) was
purchased from a local wholesale produce supplier. Stems
remaining from harvest were trimmed with sterile tools to
represent fresh-cut spinach, and the product was held at 4°C
until inoculation. Spinach was inoculated via complete product
submersion as previously described (14, 15) to simulate a
worst case contamination scenario that occurs from exposure
to contaminated wash water. Postharvest washing may lead to
pathogen internalization (8), and this inoculation approach
simulates such a contamination event, which allows for
treatment efficacy to be tested under extreme conditions. The
spinach (1,000 g) was completely submerged in 8.7 L of the
Salmonella inoculum (ca. 7.0 log CFU/mL) inside a biosafety
cabinet. Spinach leaves were stirred into the inoculum with a
spatula, soaked for 30 min, then placed on four stainless steel
trays with grate overlays in the biosafety cabinet for 30 min to
facilitate drying. Spinach leaves were then turned over and left
to dry for an additional 30 min, for a total 1 h of drying time.
Although quality was not specifically assessed in this study,
wilting and other product quality defects were not observed
during this process.
Preparation of wash treatments
Three gallons (11.4 L) of each water washing treatment
was prepared: a tap water wash containing BS and PAA (SP),
a sodium hypochlorite (chlorine) wash, and tap water. SP
was prepared by adding 4.6 mL of a commercial produce
wash (Tsunami 100, Ecolab, St. Paul, MN) containing 15.2%
PAA and 56.78 g of BS (0.50% [w/v] target concentration
of BS; pHase, Jones-Hamilton Co., Walbridge, OH). A target
concentration of 80 ppm of PAA was determined with the
manufacturer's test kit (Ecolab). Kim et al. (21) evaluated
1 and 3% concentrations of BS with 60 ppm of PAA on
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