International Journal of Food Microbiology 207 (2015) 71–76

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Effectiveness of levulinic acid and sodium dodecyl sulfate employed as a

sanitizer during harvest or packing of cantaloupes contaminated with
Salmonella Poona
Cathy C. Webb ⁎, Marilyn C. Erickson, Lindsey E. Davey, Michael P. Doyle
Center for Food Safety, Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223, USA

a r t i c l e i n f o a b s t r a c t

Article history: Freshly harvested Eastern variety cantaloupes (Cucumis melo L. var. reticulatus cv. Athena) were subjected to
Received 22 January 2015 three different harvest and wash treatments to examine conditions under which the efficacy of the sanitizer,
Received in revised form 15 April 2015 levulinic acid (LV) plus sodium dodecyl sulfate (SDS), could be enhanced to reduce Salmonella contamination.
Accepted 25 April 2015
In treatment set one, cantaloupes were spot inoculated with Salmonella enterica serovar Poona (prepared from
Available online 2 May 2015
solid or liquid media cultures) before or after a 1-min dip treatment in LV (2.5, 5.0, 7.5, or 10%) and 2.5% SDS.
Keywords:
S. Poona initial populations on rind tissue (4.26–5.04 log CFU/sample) were reduced to detection by enrichment
Levulinic acid culture when cantaloupes were subsequently exposed to any of the LV/SDS solutions. When S. Poona was intro-
Sodium dodecyl sulfate duced after cantaloupes had been dip-treated, greater decreases in pathogen populations at the stem scar were
Cantaloupes observed when cantaloupes were treated with increasing concentrations of LV. In treatment set two, the
Stem scar response of S. Poona dip-treated with 5% LV/2.5% SDS was compared to a simulated commercial dump tank treat-
Salmonella ment incorporating 200 ppm chlorine as well as a two-stage treatment employing both the chlorine tank and LV/
SDS dip treatments. S. Poona levels (log CFU/sample or # positive by enrichment culture/# analyzed) after treat-
ments were 5.25, 3.07, 7/10, 5/10 (stem scar) and 3.90, 25/40, 28/40, 20/40 (rind) for non-treated, chlorine tank,
LV/SDS dip, and tank plus dip treatments, respectively. In treatment set three, freshly harvested cantaloupes were
first treated in the field using a needle-free stem scar injection (200 μl, 7.5% LV/1.0% SDS, 60 psi) and a cantaloupe
spray (30 ml, 7.5% LV/0.5% SDS). Cantaloupe stem scar and rind tissue were then spot-inoculated with S. Poona
using either a liquid or soil-based medium followed by a simulated dump tank treatment incorporating either
200 ppm chlorine or 5% LV/2% SDS. S. Poona inoculated on field-treated cantaloupe rind decreased by 4.7 and
5.31 (liquid) and 3.27 and 3.36 (soil) log CFU/sample after simulated chlorine and LV/SDS tank treatments,
respectively. In the case of stem scar tissue, S. Poona populations exhibited a 1.0 log greater reduction when can-
taloupes were treated with LV/SDS compared to chlorine in the dump tank (P b 0.05). Based on this study, appli-
cation of multiple hurdles is warranted, as additional decreases in S. Poona populations were obtained when
cantaloupes were subjected to a chlorine dump tank followed by a LV/SDS dip treatment.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction created guidance documents to aid growers in the production of safe

Salmonella spp. contamination of cantaloupes continues to be a sig-
nificant concern for the produce industry. The Centers for Disease Con-
trol and Prevention has reported 9 outbreaks of salmonellosis caused by
contaminated cantaloupes from 1990 to 2014 which resulted in almost
500 reported illnesses and 5 deaths (CDC, 2002, 2014). A Salmonella
outbreak in 2012 associated with cantaloupes grown by Chamberlain
Farms was significant in that 127 people were hospitalized and 3 died
from consuming the contaminated fruit. As a result of these Salmonella
outbreaks and another in 2011 involving Listeria monocytogenes con-
taminated cantaloupes (McCollum et al., 2013), several groups have
⁎ Corresponding author. Tel.: +1 770 228 7284; fax: +1 770 229 3216.
E-mail address: ccwebb@uga.edu (C.C. Webb).
melons (Eastern Cantaloupe Growers Association, 2013; National
Cantaloupe Guidance, 2014). These guidance documents recommend
the use of antimicrobials in post-harvest packing processes to prevent
cross contamination of wash water. Such processes could involve
dump tanks, pools, flumes, bar sprayers, and hydro-coolers. Although
banned in some countries, chlorine is commonly used for minimally
processed produce commodities to prevent cross contamination of
wash water; however, high pH and the presence of organic material in
chlorine wash systems can lead to decreased effectiveness and exposure
to hazardous chemicals (Gil et al., 2009; Richardson et al., 1998).
A number of sanitizers have been tested on contaminated canta-
loupes as potential antimicrobials for processing. For example, chlorine
(200 ppm) reduced Salmonella populations on cantaloupe rind by 1.8
log CFU/melon after a 60 s soak (Parnell et al., 2005). Similar decreases

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.04.041
0168-1605/© 2015 Elsevier B.V. All rights reserved.
72 C.C. Webb et al. / International Journal of Food Microbiology 207 (2015) 71–76
(1.5 log CFU/g) of S. Poona on cantaloupe rind were reported for acidi-
fied calcium sulfate (1.2%), acidified sodium chlorite (1000 ppm) and
peroxyacetic acid (80 ppm) (Fan et al., 2009). Slightly greater log reduc-
tions of 2.5 and 2.3 of Salmonella on cantaloupe rind were obtained with
lactic acid (2% for 2 min) and ozone (30 ppm for 5 min), respectively
(Vadlamudi et al., 2012), whereas hydrogen peroxide (5% at 70 °C,
1 min) reduced Salmonella on cantaloupe rind by 3.8 log CFU/cm2
(Ukuku et al., 2004). Chlorine dioxide (5 ppm) in both aqueous and gas-
eous forms decreased S. Poona populations by ca. 5 log CFU/g on whole
cantaloupes (Rodgers et al., 2004; Mahmoud et al., 2008), but this amount ex-
ceeds the maximum level (3 ppm) allowed for application to whole produce
(CFR, 2013). Hence, physical interventions have also been evaluated for their
efficacy for inactivating Salmonella on cantaloupes. For example, hot water
treatment of whole cantaloupes at 92 °C for 90 s decreased S. Poona levels
by 5 log CFU/g on rind (Annous et al., 2013). The maintenance of high
temperature wash water and the potential of contamination on the pro-
cessing line after hot water treatment, however, are potential drawbacks
to this type of treatment (Akins et al., 2008). Therefore, to date, most of
the chemical and physical treatments do not sufficiently reduce patho-
genic bacteria on cantaloupe surfaces, or they employ non-allowable or
impractical procedures for the cantaloupe industry.
One chemical treatment that in recent years has shown considerable
promise as an antimicrobial intervention for produce is a levulinic acid
and sodium dodecyl sulfate (LV/SDS) aqueous solution. This mixture
has been used to reduce pathogenic bacteria on lettuce, alfalfa seeds,
and tomatoes (Zhao et al., 2009, 2010, 2011a,b). Benefits for the use of
LV and SDS include their being recognized as safe food additives for spe-
cific applications by the FDA, as well as a potential for LV to be produced
in large quantities at low cost (Bozell et al., 2000; Fang and Hanna,
2002). When applied to cantaloupe wash water, 2% LV and 0.2% SDS
reduced S. Poona populations by 3.4 and 4.5 log CFU/g on netted rind tis-
sue after a 6 min tank or tank treatment with brushing, respectively,
compared to 1.6 and 2.5 log CFU/g reductions after the same process
treatments with 120 ppm chlorine (Webb et al., 2013). Unfortunately,
neither the 2% LV/0.2% SDS nor the 120 ppm chlorine treatment sub-
stantially reduced S. Poona on stem scar tissue (Webb et al., 2013).
The objectives of this study were to determine whether the efficacy of
LV plus SDS for inactivating S. Poona could be enhanced by (1) increasing
concentrations of LV plus 2.5% SDS; (2) applying LV and SDS to stem scar
and rind tissue prior to their inoculation and treatment in a dump tank;
and (3) incorporating dual chlorine dump tank and LV/SDS dip treatments.
2. Materials and methods
2.1. Bacterial strains
Four strains of Salmonella enterica serovar Poona isolated from pa-
tients who consumed contaminated cantaloupe, 01A4754, 00A3279,
01A242 and 00A3208, were obtained from Larry Beuchat at the University
of Georgia, Center for Food Safety. Each strain was transformed with plas-
mid pDsRed-Express2 (Clonetech, Mountain View, CA) containing genes
to produce Discosoma sp. red fluorescent protein (DsRed) and ampicillin
resistance. The resulting strains produced bright red fluorescence when
exposed to 500 nm using a Dark Reader trans-illuminator (Clare Chemi-
cal, Dolores, CO). The S. Poona pDsRed-labeled strains SD50 (01A4754),
SD51 (00A3279), SD52 (01A242), and SD53 (00A3208) were stored at
−80 °C in tryptic soy broth (TSB) (Neogen, Lansing, MI) with 25% glycer-
ol. Strains from frozen stock were streaked on to tryptic soy agar (TSA)
(Neogen) supplemented with 100 μg/ml ampicillin (Amp) (Thermo
Fisher Scientific, Inc., Waltham, MA) and incubated at 37 °C for 18–21 h.
2.2. Cantaloupes
Freshly harvested, untreated, Eastern variety cantaloupes (Cucumis
melo L. var. reticulatus cv. Athena) were obtained from a grower in
Tifton, GA. The melons were chosen from the transport trailers to be
of similar maturity, size, degree of netting, and free of any visible
blemishes.
2.3. Inoculum preparation
One or two, bright red-glowing colonies from the second passage on
TSA-Amp were chosen from each of the Salmonella strains to inoculate
50 ml of TSB-Amp (liquid media) or 3-TSA-Amp plates per strain
(solid media). The liquid media culture was incubated at 37 °C with ag-
itation (150 rpm) for 21–24 h and the solid media plates incubated at
37 °C for 24 h. Colonies were gently removed from solid media plates
with 4 ml of 0.1% peptone water (Difco, Sparks, MD) by a glass spreader
and collected in a 50-ml centrifuge tube. Remaining colonies were
dislodged from plates with an additional 4 ml of 0.1% peptone water.
Both types of cultures were individually sedimented by centrifugation
(4193 ×g for 15 min at 4 °C), washed two times in sterile 0.1% peptone
water, and resuspended in 3 ml of 0.1% peptone water. Individual
strains from each preparation were combined in equal proportions to
make a mixture of ca. 10 log CFU/ml. Preliminary cultivation trials had
been performed to confirm that this high concentration of S. Poona
could be achieved from both solid and liquid media-grown cultures of
all four isolates. In addition, the stock cultures were enumerated to con-
firm the target population in each replicate trial. Liquid and solid media-
grown stocks were diluted in 0.1% peptone water to 8 and 9 log CFU/ml
for spot inoculation of stem scar and rind tissue, respectively.
Soil was collected from the cantaloupe farm and sifted to remove
rocks and other large debris. A portion (200 g) was placed in a 3.07-l
Glad container (The Clorox Company, Oakland, CA) and 4 ml of a 10
log CFU/ml S. Poona pDsRed mixture was applied in a fine mist spray.
The inoculated soil was mixed for 1 min with a spoon, covered, and
held overnight in the dark for pathogen acclimation.
2.4. Preparation of sanitizing solutions
Levulinic acid (98%, Acros Organics, Fair Lawn, NJ) and sodium dodecyl
sulfate (20%, Acros Organics) were combined with sterile deionized water
to make dip treatments comprised of either 3 or 4 l of 2.5, 5.0, 7.5, or 10.0%
LV and 2.5% SDS with pH values of 3.1, 2.9, 2.7, and 2.7, respectively. Solu-
tions were also made for cantaloupe spray (2-l, 7.5% LV/0.5% SDS, pH 2.7),
stem scar injection (200 ml, 7.5% LV/0.5% SDS, pH 2.71), and dump tank
(10-l 5% LV/2.0% SDS, pH 2.8) treatments. The dip, spray, stem scar injec-
tion, and dump tank solutions of LV/SDS were prepared fresh for each
replicate trial. Chlorine (200 ppm) was prepared by adding approximate-
ly 40 ml of sodium hypochlorite solution containing 5% available chlorine
(Ricca Chemical Company, Arlington, TX) to 10-l of sterile de-ionized
water. The chlorine solution was adjusted to pH 7.0 with sulfuric acid
(Sigma-Aldrich, St. Louis, MO). Free chlorine was determined by the
Hach digital titrator using the DPD-ferrous ethylenediammonium sulfate
titration cartridge (Hach Co., Loveland, CO). Chlorine solutions were pre-
pared fresh for each replicate trial.
2.5. Treatment of cantaloupes by dip treatment with different concentrations
of levulinic acid and 2.5% SDS
Cantaloupes held at room temperature (~22 °C) for 5 or 9 h (replicates
1 and 4, respectively), 24 h (replicate 2), or 48 h (replicate 3) after harvest
were spot-inoculated either prior to or after sanitizer treatments. In either
case, 10 μl of a 9-log CFU of S. Poona pDsRed/ml inoculum or 10 μl of a 8-
log CFU/ml inoculum, prepared from cultures grown either in liquid or on
solid media, was applied within 2-cm diameter templates marked on net-
ted rind or to stem scar tissue, respectively. Melons inoculated pre-
treatment were held for 18 h at 22 °C. Cantaloupes that were dip-
treated involved submersion for 1 min in 11.35-l pails (33.02 cm diame-
ter, 26.04 cm height; Walmart, Bentonville, AR) containing 3-l of sanitizer
solution (2.5, 5.0, 7.5, or 10% LV and 2.5% SDS). After treatment, melons
were placed in clean 354-ml foam bowls (Walmart, Bentonville, AR)
 C.C. Webb et al. / International Journal of Food Microbiology 207 (2015) 71–76
and held for 1 h before sample analysis (pre-inoculated melons) or inoc-
ulation (post-inoculated melons). Post-inoculated melons were held for
an additional 1 h at 22 °C after inoculation before sample analysis. Treat-
ment and inoculation procedures were staggered to maintain equal time
intervals before analysis of each melon. Four replicate trials were per-
formed for this experimental study.
2.6. Exposure of cantaloupes to sanitizers via dip, dump tank, and dump
tank followed by dip treatment
Cantaloupes held at room temperature (~22 °C) for 7 or 24 h after
harvest (replicate 1 and 2, respectively) were spot-inoculated with
10 μl of S. Poona pDsRed (liquid media culture) on 2-cm diameter tem-
plates marked on netted rind (9 log CFU/ml) or stem scar tissue (8 log
CFU/ml). Inoculated cantaloupes were then held for 16 h at 22 °C before
treatment. Dip-treated cantaloupes were individually submerged in
11.35-l pails containing 4-l of 5.0% LV/2.5% SDS for 1 min and placed
in a foam bowl. Dump tank treatment involved holding for 10 min in a
53-l (60.7 by 40.4 by 31 cm) storage tub (Roughneck, Rubbermaid
Home Products, Fairlawn, OH) up to 4 melons in 10-l of 200 ppm chlo-
rine, pH 7.0, with inoculation zones submerged. Cantaloupes designated
for both tank and dip treatments, received the 10-min tank immersion
in chlorine immediately followed by the 1-min dip in LV/SDS. All treated
cantaloupes were then held for 1 h in a clean foam bowl prior to sam-
pling. Treatment and inoculation procedures were staggered to main-
tain equal time intervals before analysis of each melon. Two replicate
trials were performed for this experimental study.
2.7. Stem scar injection and spray treatment of cantaloupes with LV/SDS
(pre-inoculation) followed by dump tank treatment in different sanitizers
(post-inoculation)
Cantaloupes held at ~22 °C for 20–24 h after harvest were injected at
the stem scar with 0.2 ml of 7.5% LV/0.5% SDS at 60 psi with a P50
Microdose needle free injector fitted with a 3-stream nozzle (Pulse
NeedleFree Systems, Lenexa, KS) and a compressed carbon dioxide can-
ister. The stem scar-injected melons were immediately sprayed for
10 sec with 30 ml of 7.5% LV/0.5% SDS. Stem scar- and spray-treated can-
taloupes were then placed in foam bowls and held at 22 °C for 2 h before
S. Poona pDsRed spot inoculation on the netted rind with 100 μl of a 9-
log inoculum or on the stem scar with an 8-log CFU/ml inoculum, the in-
ocula in each case being prepared from a solid media-grown culture.
Soil inoculation involved pressing ca. 2.5 g of S. Poona-contaminated
soil, prepared from a solid culture-grown inoculum, on the stem scar
or within a 2-cm diameter circle template marked on the netted rind
tissue. Inoculated cantaloupes were then held for 2 h at 22 °C prior to
a 10-min treatment of melons in a dump tank, as described above, con-
taining either 10-l of 200 ppm chlorine or 5% LV/2.0% SDS. The treated
cantaloupes were subsequently held in a clean foam bowl for 2 h at
22 °C prior to analysis. Treatment and inoculation procedures were
staggered to maintain equal time intervals before analysis of each
melon. Four replicate trials were performed for this experimental study.
2.8. Sample collection and analysis of cantaloupe stem scar and rind tissue
Cantaloupes were placed on a sterile cutting board and cut with a
sterile, stainless steel knife to separate inoculated stem scar and netted
rind tissue. Cantaloupe rind and flesh directly under the inoculated site
were individually excised to 3.13 cm3 (2.5 cm by 2.5 cm by 0.5 cm)
pieces. Each piece constituted a “sample” and was placed in sterile
Whirl-Pak bags (Nasco, Fort Atkinson, WI). Residual LV or chlorine on
cantaloupe pieces was neutralized by addition of 25 ml of 0.1% buffered
peptone water (Neogen) or 0.1% of peptone water supplemented with
0.01 g/l sodium thiosulfate (Sigma-Aldrich), respectively. The samples
were macerated in a Stomacher 400C (Seward Laboratory Systems, Inc.,
Port Saint Lucie, FL) for 1 min at 260 rpm. Portions of the macerated
73
sample (250 μl) were either directly plated or a serial dilution of the sam-
ple in 0.1% peptone water was plated for enumeration on TSA-Amp
plates supplemented with 100 μg/ml sodium pyruvate (Fisher
Bioreagents, Fair Lawn, NJ). The limit of detection for directly-plated
samples was 2 log CFU/sample. Double-strength TSB-Amp (25 ml) was
added to the remaining sample, and incubated with agitation
(150 rpm) for 21–24 h at 37 °C. The enriched cultures were plated on
TSA-Amp and incubated at 37 °C for 21–24 h. The limit of detection by
the enrichment culture method was 1 CFU/sample. Positive control
(not treated) and negative control samples (non-inoculated canta-
loupes) were analyzed for each replicate trial. No S. Poona pDsRed was
detected by enrichment culture for any negative control samples during
any of the replicate trials (data not shown).
2.9. Statistical analysis
Data were analyzed by analysis of variance using StatGraphics Centuri-
on XVI statistical software package (Statpoint, Inc., Herndon, VA). The least
significant difference test was used to determine significant differences be-
tween log reductions of Salmonella Poona in samples treated with the dif-
ferent sanitizer solutions. The level of significance (P) was set at 0.05.
3. Results and discussion
3.1. Effectiveness of different concentrations of LV applied to Salmonella-
contaminated cantaloupes
Based on previous studies, the conditions in which a bacterial inocu-
lum is prepared may affect its survival on food surfaces. For example,
populations of Salmonella Enteritidis grown in broth cultures, had great-
er reductions on raw almonds after drying than did cultures grown on
agar (Uesugi et al., 2006). In another example, Escherichia coli O157:
H7 cultures harvested from agar plates were more hardy than those
grown from broth cultures for inoculation of lettuce leaves (Theofel
and Harris, 2009). Hence, initial studies were performed concurrently
to determine (1) if inoculum prepared on nutrient-rich solid or liquid
media would have an effect on S. Poona survival on cantaloupes and
(2) if increasing concentrations of levulinic acid combined with 2.5%
SDS would improve sanitizer efficacy when applied before or after ex-
posure to S. Poona in a dip treatment rather than in a dump tank. It is
envisioned that a dip treatment would involve substantially less volume
of the antimicrobial solution than would be required in a dump tank and
hence would be more cost effective.
To simulate a field contamination event, solid or liquid media-
prepared inoculum was applied to cantaloupe netted rind tissue only
as the stem would still have been attached to the melon at that time.
The melons were held for 18 h prior to sanitizer treatment to provide
ample time for pathogen drying, adaptation, and possible biofilm
Table 1
Survival of Salmonella Poona when cantaloupe rind was inoculated with cultures grown on
solid or in liquid media and held for 18 h at 22 °C prior to exposure to 2.5% SDS and
different levels of LV in a dip treatment.
LV concentration Salmonella Poona (# positive by enrichment
(%)b culture/total # samplesa analyzed)
Solid mediac Liquid mediad
2.5 12/12 9/12
5.0 7/12 9/12
7.5 9/12 9/12
10.0 8/12 7/12
a
Each sample consisted of a 3.13-cm3 excised piece of rind.
b
Whole cantaloupes were submerged for 1 min in solutions containing 2.5% SDS and
various concentrations of LV.
c
Salmonella Poona inoculum prepared from solid media (TSA-Amp) culture and initial
levels on rind prior to treatment were 5.04 ± 0.28 log CFU/sample.
d
Salmonella Poona inoculum prepared from liquid media (TSB-Amp) culture and initial
levels on rind prior to treatment were 4.26 ± 0.39 log CFU/sample.
74 C.C. Webb et al. / International Journal of Food Microbiology 207 (2015) 71–76

Table 2 concentration of LV did lead to statistically greater reductions (P b 0.05)

Reduction of Salmonella Poona on rind and stem scar samples when cantaloupes were of S. Poona on pre-treated stem scar tissue, with 7.5% considered to be
dipped in LV/SDS sanitizers prior to being inoculated with the pathogen and held for an
additional two hours.
the most cost-effective concentration.

LV concentration (%)c Salmonella Poona reduction, [Log

CFU/samplea (Mean ± SD)b] 3.3. Effectiveness of disinfectants applied to cantaloupes in simulated dump
Stem scar d
Netted rind e tank and/or dip treatments

2.5 0.53 ± 0.64 A 2.19 ± 0.78 A

5.0 1.08 ± 0.33 AB 2.11 ± 0.52 A
7.5 1.72 ± 1.17 BC 2.54 ± 0.84 A
10.0 2.29 ± 1.10 C 2.78 ± 0.88 A
a
Each sample consisted of a 3.13-cm3 excised piece of tissue.
b
Values within a column followed by different letters are significantly different
(P b 0.05)
c
2.5% SDS included with each LV dip treatment
d
Initial levels on stem scar tissue were 5.36 ± 0.55 log CFU/sample. Stem scar tissue was exposed
to S. Poona prepared from either a culture grown on solid or liquid media. No significant differences
were observed in the response of the pathogen to the presence of different concentrations of LV (P N
0.05). Hence, the data were merged (n = 6).
e
Initial levels on rind tissue were 5.08 ± 0.78 log CFU/sample. Rind tissue was exposed
to S. Poona prepared from either a culture grown on solid or liquid media. No significant
differences in the response of the pathogen to the presence of different concentrations
of LV (P N 0.05). Hence, the data were merged (n = 24).
formation. After this time, initial levels on rind were 0.78 log higher
when the inoculum was prepared from solid media than on rind inocu-
lated from the liquid media preparation; however; melons dipped for
1 min at each concentration of LV and 2.5% SDS were all reduced to de-
tection by enrichment culture for both types of culture preparations
(Table 1). Increasing concentrations of LV in the sanitizer applied to can-
taloupe, however, were not associated with major changes in preva-
lence of S. Poona on rind tissue when using either an inoculum
prepared from solid or liquid culture. These results contrast to those
from studies conducted by Uesegi et al. (2006) and Theofel and Harris
(2009) and could be attributed to differences in the ability of cells cul-
tured on solid media to resist a physical stress (desiccation) versus a
chemical stress (LV).
3.2. Effectiveness of different concentrations of levulinic acid applied to
cantaloupes prior to Salmonella contamination
To evaluate the effectiveness of sanitizer concentration in dip treat-
ments applied to cantaloupes before contamination events, cantaloupes
were first exposed to solutions containing 2.5% SDS and varying
levels of LV prior to application of an inoculum prepared from either a
liquid or solid culture. As was the case in the previous experiment
when LV/SDS was applied after the contamination event, preparation of
the inoculum (solid versus liquid culture) had no effect on the fate of
Salmonella on cantaloupes treated with LV/SDS before contamination.
Hence, these data were merged prior to examining the effect of LV/SDS
concentrations on Salmonella inactivation at the two cantaloupe sites
(Table 2). No significant reductions were observed on the rind tissue
when LV concentrations were increased. In contrast, increasing the
To determine whether a multiple hurdle approach would be more
effective in reducing Salmonella contamination than application of indi-
vidual treatments, cantaloupes were subjected to either a 10-min dump
tank treatment incorporating 200 ppm chlorine, a 1-min dip treatment
incorporating 5% LV/2.5% SDS, or a dump tank treatment followed by a
dip treatment. The chlorine tank treatment (200 ppm) reduced Salmo-
nella on the rind to levels that could only be detected by enrichment cul-
ture (Table 3). Similar levels of efficacy (~3 log) have been reported for
Salmonella on cantaloupe rind exposed for 5 min to 200 ppm chlorine
(Magnone et al., 2013), whereas when rind was exposed to lower con-
centrations of chlorine (120 ppm), reductions of Salmonella of only 2 log
CFU/g were observed (Webb et al., 2013). In agreement with results of
previous studies (Webb et al., 2013), the efficacy of chlorine to inactivate Sal-
monella in stem scar tissue was much less than it was for cantaloupe rind,
whereas the 1-min LV/SDS dip treatment succeeded in reducing Salmonella
populations in both rind and stem scar-contaminated samples to levels that
could only be detected by enrichment culture (Table 3). Furthermore, when
cantaloupes were subjected to both the chlorine tank and LV/SDS dip treat-
ments, this double hurdle approach resulted in further reductions of Salmonella
prevalence, with 50% of the rind and stem scar samples being negative for the
pathogen after enrichment culture. The increased efficacy is likely due to differ-
ent mechanisms by which the disinfectants inactivate pathogens. However,
given that cantaloupes are not submerged completely in dump tanks, incorpo-
rating another hurdle such as a dip tank where the cantaloupe is completely
submerged, would ensure that all surfaces are exposed to a disinfectant.
3.4. Effectiveness of LV/SDS treatments applied in both field and packing
plant simulations
To prevent cross-contamination of freshly harvested cantaloupes
from contaminated field debris or contaminated transport and packing
shed surfaces, application of disinfectant to cantaloupes immediately
after removal from the vine in the field may be beneficial. Hence, prior
to their inoculation with Salmonella, cantaloupes were treated with
7.5% LV/0.5% SDS by injecting the disinfectant into the stem scar tissue
with air pressure and spraying the disinfectant over the entire canta-
loupe surface. After applying this simulated field treatment, the canta-
loupes were spot (to represent splash from contaminated liquids) or
soil-inoculated (to represent cross-contamination by soil on transport
trailers or in packing sheds). Cantaloupes were then exposed to a
sanitizer tank treatment of 200 ppm chlorine (commonly used by can-
taloupe growers in packing houses) or 5% LV/2% SDS prior to pathogen
analysis of stem scar or netted rind tissue.

Table 3
Reduction of Salmonella Poona in stem scar and rind tissue samples following treatment of cantaloupes with sanitizers.

Salmonella Poona [Log CFU/samplea (Mean ±

S.D.), or # positive by enrichment culture/#
samples analyzed]

Sanitizer application method Sanitizer Stem scar Netted rind

None None 5.25 ± 0.18 3.90 ± 0.20

Simulated dump tankb 200 ppm chlorine, pH 7.0 3.07 ± 0.82 25/40
Dip tankc 5% LV/2.5% SDS 7/10 28/40
Sequential dump and dip tanksd 200 ppm chlorine in tank and 5% LV/2.5% SDS in dip 5/10 20/40
a
Each sample consisted of a 3.13-cm3 excised piece of tissue.
b
Cantaloupes were exposed to sanitizer for 10 min.
c
Cantaloupes were exposed to sanitizer for 1 min.
d
Cantaloupes were exposed for 10 min to the sanitizer in the dump tank and immediately thereafter exposed for 1 min to the sanitizer in the dip tank.
 C.C. Webb et al. / International Journal of Food Microbiology 207 (2015) 71–76
Table 4
Reduction of Salmonella Poona on stem scar and rind tissue when cantaloupes were first treated with LV/SDS sanitizer via injection and spray, respectively, inoculated 1 h later, held for an
additional 2 hours at 22 °C, and then treated with different sanitizers in a simulated dump tank.
Treatment prior to inoculation Treatment after inoculation
Injectione and sprayf 200 ppm chlorine, pH 7.0
5%LV/2% SDS
a
Each sample consisted of a 3.13-cm3 excised piece of tissue.
b
Values within each tissue type followed by a different letter are significantly different (P b 0.05).
c
Initial levels by spot and soil inoculation were 6.54 ± 0.22 and 5.37 ± 0.33 log CFU/sample, respectively. There were no significant differences in the response of S. Poona to the combi-
nation treatments (P N 0.05) on stem scar tissue inoculated by either spot or soil inoculation. Hence, the data were merged (n = 40).
d
Initial levels by spot (n = 20) and soil (n = 20) inoculation were 6.97 ± 0.30 and 4.54 ± 0.64 log CFU/sample, respectively.
e
Stem scar tissue was injected with 200 μl of 7.5% LV/1.0% SDS via a 3-stream nozzle air pressure injector.
f
Cantaloupe surfaces were sprayed with 30 ml of 7.5% LV/0.5% SDS.
Analysis of the data revealed no significant differences in the effect of
the combined field and packing house treatments on the reduction of S.
Poona in stem scar tissue when this pathogen had been applied either
through spot-or soil-inoculation, hence the data were merged for each
of the treatment combinations (Table 4). In contrast, the mode of inoc-
ulation did affect the pathogen's response on rind tissues subjected to
either of the field/packing house treatments, with reductions of S.
Poona for spot-inoculated samples being 1.5- to 2-log greater than re-
ductions occurring for soil-inoculated samples. Soil particles that were
ground into the rind may have shielded adhering pathogens from the
disinfectants. Field application of sanitizer did not completely eliminate
S. Poona from subsequent spot or soil contamination events as the path-
ogen remained on the rind tissue at 1.18 to 2.23 log CFU/sample after
the combination treatment.
Reductions of S. Poona populations on inoculated stem scar samples
were 1 to 3 log less than rind samples, even though the former tissue
had been injected with a 7.5% LV/0.5% SDS solution prior to pathogen in-
oculation (Table 4). These results are consistent with those of a previous
study that revealed a 1.5- to 2-log difference in reduction of S. Poona
populations on stem scar and rind tissues when cantaloupes were treat-
ed with chlorine or LV/SDS post-inoculation (Webb et al., 2013). One
possible explanation for the decreased effectiveness of disinfectants at
the stem scar may be related to its porosity (Richards and Beuchat,
2004) whereby pathogens can infiltrate internally into the tissue
(Webb et al., 2013). With that premise and the reported enhancement
of Salmonella inactivation by vacuum perfusion of disinfectants into to-
mato stem scars (Gurtler et al., 2012), it was hypothesized that injection
at the cantaloupe's stem scar with LV/SDS would similarly enhance
pathogen inactivation. However, reductions of S. Poona in stem scar tis-
sue of cantaloupes that had been injected with LV/SDS and held in a
chlorine dump tank (1.4 log/sample, Table 4) were less than had oc-
curred in stem scar tissue of cantaloupes exposed only to the chlorine
dump tank treatment (2.2 log CFU/sample, Table 3). It is likely that
this mode of treatment proved ineffective because the disinfectant
was not distributed uniformly throughout the stem scar. Based on re-
sults of a dye added to the disinfectant solution, complete coverage of
the surface was observed; however, the depth of penetration of the dis-
infectant into sub-surface tissue ranged only from 5 to 10 mm and there
was limited diffusion laterally from the three injection paths. Hence,
there would have been numerous sub-surface areas where disinfectant
was not present whereby infiltrated Salmonella could have survived.
In summary, stem scar injection of 7.5% LV/0.5% SDS was an ineffec-
tive barrier to S. Poona contamination; however, a simulated field spray
with 7.5% LV/0.5% SDS onto the surface of melons in conjunction with a
second hurdle tank treatment of 200 ppm chlorine or 5% LV/2% SDS re-
sulted in reductions of Salmonella ranging from 3.3 to 5.3-log CFU/sam-
ple. Varying the concentration of LV from 2.5% to 10% had no effect on
the efficacy of this disinfectant when combined with SDS to inactivate
S. Poona on cantaloupe netted rind. The most effective treatment for
cantaloupes was a 10-min exposure to chlorine in a simulated dump
75
Salmonella Poona reduction, [Log CFU/samplea (Mean ± S.D)b]
Spot inoculation Soil inoculation
c d
Stem scar Rind
1.36 ± 0.41 A 4.74 ± 0.99 B 3.27 ± 0.29 A
2.45 ± 0.41 B 5.31 ± 0.55 B 3.36 ± 0.51 A
tank followed by a 1-min exposure to 5% LV/2.5% SDS in a simulated
dip treatment. Using those conditions, S. Poona was reduced to non-
detectable levels in 50% of the stem scar and rind tissue samples.
Acknowledgments
This research was funded by the Georgia Agricultural Commodity
Commission for Vegetables. We thank Bill Brim, Ed Walker, Peter
Germishuizen, Pablo A. Navia Giné, Philip Grimes, Jane Grimes, Alan
Parrish, and Lynda Glenn for providing cantaloupes and invaluable in-
formation regarding cantaloupe growing and processing practices. We
also thank Charles Hall and the Eastern Cantaloupe Growers Association.
References
Akins, E.D., Harrison, M.A., Hurst, W., 2008. Washing practices on the microflora on
Georgia-grown cantaloupes. J. Food Prot. 71, 46–51.
Annous, B.A., Burke, A., Sites, J.E., Phillips, J.G., 2013. Commercial thermal process for
inactivating Salmonella Poona on surfaces of whole fresh cantaloupes. J. Food Prot.
76, 420–428.
Bozell, J.J., Moens, L., Elliott, D.C., Wang, Y., Neuenscwander, G.G., Fitzpatrick, S.W., Bilski,
R.J., Jarnefeld, J.L., 2000. Production of levulinic acid and use as a platform chemical
for derived products. Resour. Conserv. Recycl. 28, 227–239.
CDC [Centers for Disease Control and Prevention], 2002. Multistate outbreaks of
Salmonella serotype Poona infections associated with eating cantaloupe from
Mexico—United States and Canada, 2000–2002. Morbidity and Mortality Weekly
Report 51, pp. 1044–1047.
CDC [Centers for Disease Control and Prevention], 2014. List of selected multistate
foodborne outbreak investigations. Available at: http://www.cdc.gov/foodsafety/
outbreaks/multistate-outbreaks/outbreaks-list.html (Accessed 29 August, 2014).
CFR [Code of Federal Regulations], 2013. Title 21, Part 173.300. Secondary direct food ad-
ditives permitted in food for human consumption: chlorine dioxide. Available at,
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=173.300
(Accessed 5 September, 2014).
Eastern Cantaloupe Growers Association, 2013. http://www.ecga/usa.org/uploads/1/6/1/1/
16112318/ecga_commodity_specific_food_safety_guidelines_-_4.2.2013.pdf (Accessed 29
August, 2014).
Fan, X.T., Annous, B.A., Keskinen, L.A., Mattheis, J.P., 2009. Use of chemical sanitizers to
reduce microbial populations and maintain quality of whole and fresh-cut canta-
loupe. J. Food Prot. 72, 2453–2460.
Fang, Q., Hanna, M.A., 2002. Experimental studies for levulinic acid production from
whole kernel grain sorghum. Bioresour. Technol. 81, 187–192.
Gil, M.I., Selma, M.V., López-Gálvez, F., Allende, A., 2009. Fresh-cut product sanitation and
wash water disinfection: problems and solutions. Int. J. Food Microbiol. 134, 37–45.
Gurtler, J.B., Smelser, A.M., Niemira, B.A., Jin, T.Z., Yan, X., Geveke, D.J., 2012. Inactivation of
Salmonella enterica on tomato stem scars by antimicrobial solutions and vacuum
perfusion. Int. J. Food Microbiol. 159, 84–92.
Magnone, J.P., Marek, P.J., Sulakvelidze, A., Senecal, A.G., 2013. Additive approach for inac-
tivation of Escherichia coli O157:H7, Salmonella, and Shigella spp. on contaminated
fresh fruits and vegetables using bacteriophage cocktail and produce wash. J. Food
Prot. 76, 1336–1351.
Mahmoud, B.S.M., Vaidya, N.A., Corvalan, C.M., Linton, R.H., 2008. Inactivation kinetics of
inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Poona on
whole cantaloupe by chlorine dioxide gas. Food Microbiol. 25, 857–865.
McCollum, J.T., Cronquist, A.B., Silk, B.J., Jackson, K.A., O'Connor, K.A., Cosgrove, S., Gossack,
J.P., Parachini, S.S., Jain, N.S., Ettestad, P., Ibraheem, M., Cantu, V., Joshi, M., DuVernoy,
T., Fogg Jr., N.W., Gorny, J.R., Mogen, K.M., Spires, C., Teitell, P., Joseph, L.A., Tarr, C.L.,
Imanishi, M., Neil, K.P., Tauxe, R.V., Mahon, B.E., 2013. Multistate outbreak of listerio-
sis associated with cantaloupe. N. Engl. J. Med. 369, 944–953.
76 C.C. Webb et al. / International Journal of Food Microbiology 207 (2015) 71–76
National Cantaloupe Guidance, 2014. http://cantaloupe-guidance.org/docs/national-
commodity-specific-food-safety-guidelines-cantaloupes-and-netted-melons (Accessed
29 August, 2014).
Parnell, T.L., Harris, L.J., Suslow, T.V., 2005. Reducing Salmonella on cantaloupes and hon-
eydew melons using wash practices applicable to postharvest handling, foodservice,
and consumer preparation. Int. J. Food Microbiol. 99, 59–70.
Richards, G.M., Beuchat, L.R., 2004. Attachment of Salmonella Poona to cantaloupe rind
and stem scar tissues as affected by temperature of fruit and inoculum. J. Food Prot.
67, 1359–1364.
Richardson, S.D., Thruston, A.D., Caughran, T.V., Collette, T.W., Patterson, K.S., Lykins, B.W.,
1998. Chemical by-products of chlorine and alternative disinfectants. Food Technol.
52 (4), 58–61.
Rodgers, S.L., Cash, J.N., Siddiq, M., Ryser, E.T., 2004. A comparison of different chemical
sanitizers for inactivating Escherichia coli O157: H7 and Listeria monocytogenes in so-
lution and on apples, lettuce, strawberries, and cantaloupe. J. Food Prot. 67, 721–731.
Theofel, C.G., Harris, L.J., 2009. Impact of preinoculation culture conditions on the behav-
ior of Escherichia coli O157:H7 inoculated onto Romaine lettuce (Lactuca sativa)
plants and cut leaf surfaces. J. Food Prot. 72, 1553–1559.
Uesugi, A.R., Danyluk, M.D., Harris, L.J., 2006. Survival of Salmonella enteritidis phage type
30 on inoculated almonds stored at − 20, 4, 23, and 35 °C. J. Food Prot. 69,
1851–1857.
Ukuku, D.O., Pilizota, V., Sapers, G.M., 2004. Effect of hot water and hydrogen peroxide
treatments on survival of Salmonella and microbial quality of whole and fresh-cut
cantaloupe. J. Food Prot. 67, 432–437.
Vadlamudi, S., Taylor, T.M., Blankenburg, C., Castillo, A., 2012. Effect of chemical sanitizers
on Salmonella enterica Serovar Poona on the surface of cantaloupe and pathogen con-
tamination of internal tissues as a function of cutting procedure. J. Food Prot. 75,
1766–1773.
Webb, C.C., Davey, L.E., Erickson, M.C., Doyle, M.P., 2013. Evaluation of levulinic acid and
sodium dodecyl sulfate as a sanitizer for use in processing Georgia-grown canta-
loupes. J. Food Prot. 76, 1767–1772.
Zhao, T., Zhao, P., Doyle, M.P., 2009. Inactivation of Salmonella and Escherichia coli O157:
H7 on lettuce and poultry skin by combinations of levulinic acid and sodium dodecyl
sulfate. J. Food Prot. 72, 928–936.
Zhao, T., Zhao, P., Doyle, M.P., 2010. Inactivation of Escherichia coli O157:H7 and
Salmonella typhimurium DT 104 on alfalfa seeds by levulinic acid and sodium dodecyl
sulfate. J. Food Prot. 73, 2010–2017.
Zhao, T., Zhao, P., Cannon, J.L., Doyle, M.P., 2011a. Inactivation of Salmonella in biofilms
and on chicken cages and preharvest poultry by levulinic acid and sodium dodecyl
sulfate. J. Food Prot. 74, 2024–2030.
Zhao, T., Zhao, P., Doyle, M.P., 2011b. Inactivation of foodborne pathogens on tomatoes by
levulinic acid plus sodium dodecyl sulfate. The 12th ASEAN Food Conference 2011.
BITEC Bangna, Bangkok, Thailand, pp. 416–424.