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jfs_227 584..

598

DOI: 10.1111/j.1745-4565.2010.00227.x

EFFECT OF STORAGE TIME ON THE EFFICACY OF CHEMICAL


AND IRRADIATION TREATMENTS TO INACTIVATE
SALMONELLA ON CILANTRO (CORIANDRUM SATIVUM L.)

NAAXIÉLLI SERNA VILLAGÓMEZ1, ERIKA A. NERI HERRERA1,


LEOPOLDO OROZCO R.1, GRACIELA WILD-PADUA2 and
MONTSERRAT H. ITURRIAGA1,3
1
Departamento de Investigación y Posgrado en Alimentos
Facultad de Química
Universidad Autónoma de Querétaro
Centro Universitario
Cerro de las Campanas s/n
Col. Las Campanas, C. P. 76010, Querétaro, Qro., México
2
Department of Food Science and Human Nutrition
University of Illinois at Urbana-Champaign
Urbana, IL 61801, USA

Accepted for Publication October 4, 2009

ABSTRACT

In this work, the efficacy of decontamination treatments to inactivate


Salmonella enterica on cilantro throughout storage under different conditions
of temperature and relative humidity (RH) was evaluated. Bunches of cilantro
(10 g) were inoculated with a rifampicin-resistant S. enterica strain (ca.
106 cfu/mL) and held at 22C/60 min. Inoculated cilantro (ca. 5.8 log cfu/g)
was then stored at: (1) 22C for 2 days under 100 or 30% RH; (2) 5C for up to
8 days under 100 or 60% RH. Periodically, inoculated cilantro was treated
with water, chlorine (200 mg/L), peracetic acid (80 mg/L) or gamma rays (0.5,
1 and 2 kGy). At time 0, reductions of Salmonella with chlorine, peracetic acid
or water were 2.6, 2.1 and 1.0 log cfu/g, respectively. At 22C, the effectiveness
of disinfection treatments to remove or kill Salmonella cells from cilantro
significantly decreased as the storage time elapsed (P < 0.05). A similar trend
was observed at 5C. On day 0, irradiation of cilantro at a dose of 0.5, 1 or
2 kGy reduced the pathogen by 3.5, 4.5 and 6 log cfu/g, respectively. At 22C,
the efficacy of irradiation decreased (P < 0.05), while at 5C, it remained
constant regardless of RH.

3
Corresponding author. TEL: (52) 442- 192-1200 ext. 5507; FAX: (52) 442- 192-1304; EMAIL:
montshi@uaq.mx

Journal of Food Safety 30 (2010) 584–598.


584 © 2010 Wiley Periodicals, Inc.
DECONTAMINATION TREATMENTS ON CILANTRO 585

PRACTICAL APPLICATIONS

This study demonstrates how the efficacy of decontamination treatments


is modified during the storage of cilantro under constant temperature and
relative humidity. The decrease in treatment efficacy suggests that microor-
ganisms colonizing the cilantro surface may produce biofilms that provide
protection; the level of naturally occurring microflora may also have an influ-
ence on its effectiveness. The results reinforce the concept that once cilantro is
contaminated with Salmonella, the pathogen can survive chemical disinfection
or irradiation, and eventually multiply on cilantro throughout its shelf life.

INTRODUCTION

Produce, including green salads, have been associated with foodborne


outbreaks (Sivapalasingam et al. 2004). Among green leafy vegetables,
parsley and cilantro were linked to shigellosis and salmonellosis outbreaks,
respectively (CDC 1999; Campbell et al. 2001). Recently, in the 2008 Salmo-
nella Saintpaul Outbreak associated with consumption of raw jalapeño
peppers, cilantro was one of the suspected items during the epidemiological
investigation (CDC 2008). The risks of foodborne outbreaks is increased
because cilantro is widely used in the cuisines of México, South America,
China, India and Southeast Asia, and usually in minor quantities in salsas or as
a dish garnish.
Preventive measures to avoid pathogen contamination remain the most
important step to safeguard the microbial safety of produce (Beuchat and Ryu
1997). Nevertheless, complementary actions to prevent survival and growth
of pathogenic bacteria on fruits and vegetables are generally recommended.
Among such practices, refrigeration and disinfection have long been employed
by the food industry and at home (Beuchat 1998, 2002). There are several
factors that affect the efficacy of the disinfection treatments on produce,
including the treated surface, the type of disinfectant, pathogen concentration,
temperature and time of exposure (Beuchat 1998). However, gamma irradia-
tion has become a more definitive intervention step to protect the microbial
safety of food. Since 1986, irradiation at 1.0 kGy has been approved in the
U.S.A. for sprouting inhibition and insect disinfestation on fruits and veg-
etables, and in 2008, the use of ionizing radiation was approved by the Food
and Drug Administration (FDA) for control of foodborne pathogens, and
extension of shelf life, in fresh iceberg lettuce and fresh spinach at a dose of up
to 4.0 kGy (FDA 2008). A dose of 1.0 kGy is highly effective to inactivate
foodborne pathogens and parasites in various foods, like apples, cauliflower,
strawberries and cilantro (Tauxe 2001).
586 N.S. VILLAGÓMEZ ET AL.

Generally, evaluations of disinfection treatments have been carried out


immediately after inoculation (Wright et al. 2000; Hellstrom et al. 2006).
However, after contamination, produce may undergo prolonged periods of
storage under diverse conditions until disinfection is performed either at
packing sheds, restaurants or at home. To the best of our knowledge, there are
no published studies that investigate the effects of storage on the efficacy of
decontamination methods on cilantro. The aim of this work was to evaluate the
effect of storage conditions on the efficacy of disinfection treatment and
gamma irradiation for cilantro inoculated with Salmonella.

MATERIALS AND METHODS

Source of Cilantro
Bunches of fresh cilantro (Coriandrum sativum L.) were obtained from
local supermarkets in Urbana-Champaign, IL, for the irradiation treatments
and in Querétaro, Qro., México, for the chemical disinfection studies.
Preparation of Inoculum
Salmonella Thompson ATCC 8391 and Salmonella Montevideo ATCC
8387 were maintained at -72C in tryptic soy broth (TSB, Difco Laboratories,
Sparks, MD) containing 10% glycerol (Karal S.A. de C.V., León, Gto.,
México) before the start of this study. For the present studies, resistance to
rifampicin (Sigma Chemical Co., St. Louis, MO) was induced in the strains
(Kaspar and Tamplin 1993). To summarize the method briefly, 10 mL of 24-h
culture was centrifuged and the pellet was resuspended in 1 mL, then cell
suspension was surface plated (0.1 mL per plate) on tryptic soy agar (Difco
Laboratories) supplemented with rifampicin (100 mg/L; TSAR) and incubated
for up to 72 h. Rifampicin-resistant strains were grown individually by three
successive loop transfers into TSB supplemented with rifampicin (100 mg/L)
and incubated at 35C for 24-h intervals before cells from a 20-h culture were
harvested by centrifugation (3500 ¥ g, 15 min, 22C). Cells were washed twice
with saline solution (0.85%), and then, following the final wash, equal
volumes of each Salmonella strain were combined. One milliliter of the pooled
cell suspension was added to 1 L of sterile distilled water to yield a final
concentration of ca. 6 log cfu/mL. The final cell concentration of the dip
suspension (inoculum) was determined by pour plating onto TSAR. Plates
were incubated at 35C for 24 h before the colonies were counted.
Inoculation Procedure
Bunches of cilantro free of defective leaves and stems were weighed into
10-g samples. Inoculation was performed by immersing each cilantro bunch
DECONTAMINATION TREATMENTS ON CILANTRO 587

into the dip suspension for 60 s with manual shaking. After dipping, the
cilantro was shaken gently to remove excess of inoculum and allowed to stand
in a laminar flow hood at 25C for 60 min.

Storage Conditions
Inoculated cilantro samples were placed inside clear plastic containers
that were covered and hermetically sealed with high-vacuum grease (Dow
Corning Corp., Midland, MI) and stored at 22 and 5 ⫾ 1C for 2 and 8 days,
respectively. Relative humidity (RH) for containers stored at both storage
temperatures had been previously equilibrated at 100% RH (by placing sterile
water in the bottom of the containers) or without controlling RH (reaching
30% RH at 22C, and 60% RH when stored at 5C). Atmospheric RH was
monitored by introducing a RH/temperature meter (Control, Company,
Friendswood, TX) inside each container.

Chemical Disinfection
On the day of the experiment, solutions of free chlorine (200 mg/L,
pH 6.5) (J. T. Baker, Xalostoc, México) and peracetic acid (80 mg/L) (Oxok-
leen, Sanox, Co., Tultitlan, Mexico) were prepared in distilled water. Sani-
tizers (200 mL) were distributed in resealable polyethylene bags
(17.8 ¥ 23.3 cm). A water treatment was also included. Samples of inoculated
cilantro stored at 22C were taken out of the containers on days 0, 1 and 2. At
5C, samples were removed on days 0, 2, 4, 6 and 8. All samples were
submerged into each disinfectant solution and water for 5 min without agita-
tion. Immediately after treatment, cilantro was removed from the solutions
with sterile stainless steel tongs and placed into another polyethylene bag
containing 90 mL of Dey and Engley neutralizer broth (pH 7.6) (Difco Labo-
ratories) for homogenization in the Stomacher 400 (Seward, Norfolk, UK) at
medium speed for 1 min. Untreated, inoculated cilantro samples were used as
control at each sampling time.

Irradiation
As with the disinfection treatments, after inoculation of cilantro with
Salmonella, irradiation treatments were performed periodically. On scheduled
sampling days (at 22C on days 0, 1 and 2; at 5C on days 0, 2, 4, 6 and 8),
inoculated cilantro was irradiated with 0.5, 1 or 2 kGy. The samples were
irradiated with a Co-60 gamma radiator (Gammacell®, 220 Excel, MDS
Nordion, Ottawa, Canada) in the facilities of the Nuclear Radiation Labora-
tory, University of Illinois at Urbana-Champaign. In all cases, the irradiation
was conducted at 5C. Irradiated samples were then homogenized with 90 mL
588 N.S. VILLAGÓMEZ ET AL.

of 0.1% peptone water as described above. Untreated, inoculated cilantro


samples were used as control at each sampling time.

Bacterial Enumeration
Homogenates of disinfected and irradiated samples were serially diluted
in 0.1% peptone water. Total mesophilic populations were estimated from the
aerobic plate count (APC) by plating onto plate count agar (Difco Laborato-
ries). Salmonella was counted by pour plating onto TSAR. Plates were incu-
bated at 35C for 48 h; presumptive Salmonella colonies were randomly
selected from each sample and subjected to confirmation by biochemical tests
and agglutination using commercial antiserum (Difco Laboratories) (Andrews
et al. 1992). Salmonella counts (cfu/g) were subtracted from the APC to obtain
the aerobic microbial enumeration on cilantro.

Scanning Electron Micrographic Examination


At each sampling day, one cilantro sample was prepared for scanning
electron microscopy (SEM), according to the procedure described by Getz
et al. (1983), with some modifications. Sections of cilantro leaves (5 ¥
5 ¥ 1 mm) were fixed for 3 h in 3% glutaraldehyde, followed by a treatment
for 3 h in 1% osmium tetroxide. Both solutions were prepared in 0.1 M sodium
cacodylate (pH 7.36). Fixed tissues were dehydrated in a graded series of
ethanol concentrations at 4C. Following dehydration, tissues were critical-
point dried, mounted, sputter coated with gold and examined with a Zeiss
DSM-950 (Carl Zeiss, Jena, Germany) scanning electron microscope operat-
ing at 15–20 kV.

Statistical Analysis
The experiments were performed in duplicate, and each replicate con-
sisted of three bunches of cilantro, making a total of six cilantro samples per
treatment. Data (log cfu/g) were subjected to analysis of variance and Tukey’s
test using the JMP 5.0 software (SAS Institute, Cary, NC).

RESULTS

Disinfection Treatments
The behavior of Salmonella cells inoculated onto cilantro throughout the
storage time for all combinations of temperature and RH was determined on
samples that were not subjected to any treatment (control). The purpose was to
calculate the actual reduction because of the disinfection treatments regardless
DECONTAMINATION TREATMENTS ON CILANTRO 589

of the population changes caused by storage. During the storage at 5C, as it


was expected, Salmonella did not grow but survived up to 8 days: population
decreased by 1.5 and 2.2 log cfu/g in samples stored at 100 and 60% RH,
respectively (Table 1). At 22C and 100% RH, the pathogen increased by 1.3
log cfu/g; in contrast, when the RH was not controlled (~30%), a reduction of
1.0 log cfu/g was observed.
The efficacy of chlorine and peracetic acid to inactivate Salmonella on
cilantro was similar along the study (P > 0.05). In general, regardless of
temperature or RH, when cilantro was treated with water, chlorine or peracetic
acid, the number of Salmonella cells that were removed or inactivated from
cilantro significantly decreased as the storage time elapsed (P < 0.05)
(Table 1). On day 0, reductions after treatment with water for all storage
conditions were less than 1 log, and by the end of the study, there was not any
detectable reduction in Salmonella size on cilantro. A similar trend was
observed when chlorine was applied to cilantro stored at 5C: On day 0, the
reduction was 2.6 log cfu/g, and after 8 days, reductions were 2.2 and 1.4 log
cfu/g in samples kept at 100 or 60% RH, respectively. The decrease in efficacy
of the chlorine treatment was more pronounced for samples stored at 22C: At
day 0, Salmonella population decreased by 2.1 log cfu/g; after 2 days, reduc-
tions were only 1.8 and 0.8 log cfu/g at 100 and 30% RH, respectively. Results
for peracetic acid showed a trend similar to those observed with chlorine.
The changes in APC populations on cilantro during the storage are shown
in Table 2. On day 0, APC in control samples was 6.9 log cfu/g at 22C and 6.6
log cfu/g at 5C. At 5C and both RH (100 and 60%), an increment was observed
(P < 0.05); however, the growth was higher during the storage at 22C, showing
an increase of 2.9 and 1.2 log cfu/g after 2 days of storage at 100 and 30% RH,
respectively. Soaking cilantro in chlorine or peracetic acid only reduced the
counts of APC of approximately 1 log in all storage conditions (Table 2).
Comparable results were observed when water was applied, suggesting that
the reduction in counts may be due only to mechanical removal.
SEM revealed bacterial cells on the surface of cilantro leaves after treat-
ment with water or disinfectant solutions (Fig. 1). On samples stored at 100%
RH, the presence of bacteria embedded in a matrix was evident, whereas at
30% RH, the occurrence of an amorphous material was common.
Irradiation Treatments
On inoculated samples not subjected to irradiation (control), the fate of
Salmonella was similar to that observed for the control in the chemical disin-
fection experiments (Table 3). The inoculated pathogen grew on cilantro
stored at 100% RH and 22C (<1 log cfu/g) but decreased on the remaining
storage conditions: 0.1, 2.0, 0.9 log cfu/g at 5C–100% RH, 5C–60% RH and
22C–30% RH, respectively.
590

TABLE 1.
POPULATION OF SALMONELLA ENTERICA RECOVERED FROM CILANTRO AFTER APPLYING DECONTAMINATION TREATMENTS
DURING STORAGE AT DIFFERENT TEMPERATURE AND RH CONDITIONS

Temperature RH (%) Storage Population recovered (log cfu/g)*


(C) time (days)
Control Water Chlorine (200 ppm) PA (80 ppm)

5 100 0 5.80 ⫾ 0.12 A 5.06 ⫾ 0.14 A 3.20 ⫾ 0.19 A 3.39 ⫾ 0.42 A


2 5.86 ⫾ 0.29 A 4.92 ⫾ 0.15 A 2.80 ⫾ 0.49 AB 3.01 ⫾ 0.35 AB
4 5.49 ⫾ 0.16 A 4.95 ⫾ 0.09 A 2.37 ⫾ 0.34 BC 2.75 ⫾ 0.19 BC
6 4.77 ⫾ 0.36 B 4.78 ⫾ 0.18 AB 2.26 ⫾ 0.41 BC 2.44 ⫾ 0.29 C
8 4.27 ⫾ 0.36 BC 4.46 ⫾ 0.35 B 2.03 ⫾ 0.47 C 2.57 ⫾ 0.14 BC
60 0 5.80 ⫾ 0.12 A 5.06 ⫾ 0.14 A 3.20 ⫾ 0.19 A 3.39 ⫾ 0.42 A
2 4.81 ⫾ 0.24 B 4.15 ⫾ 0.31 B 2.60 ⫾ 0.47 AB 2.54 ⫾ 0.29 BC
4 4.49 ⫾ 0.24 BC 4.04 ⫾ 0.35 B 2.49 ⫾ 0.30 BC 2.73 ⫾ 0.61 AB
6 4.05 ⫾ 0.40 CD 3.87 ⫾ 0.26 B 1.94 ⫾ 0.35 C 1.91 ⫾ 0.50 C
8 3.61 ⫾ 0.29 D 3.59 ⫾ 0.54 B 2.20 ⫾ 0.42 BC 1.80 ⫾ 0.40 C
22 100 0
N.S. VILLAGÓMEZ ET AL.

5.71 ⫾ 0.54 A 4.89 ⫾ 0.09 B 3.60 ⫾ 0.11 B 3.62 ⫾ 0.38 C


1 5.95 ⫾ 0.10 B 5.27 ⫾ 0.11 B 4.15 ⫾ 0.45 B 5.00 ⫾ 0.46 B
2 7.00 ⫾ 0.45 B 6.01 ⫾ 0.41 A 5.18 ⫾ 0.57 A 6.13 ⫾ 0.29 A
30 0 5.85 ⫾ 0.45 A 4.89 ⫾ 0.09 B 3.60 ⫾ 0.11 BC 3.52 ⫾ 0.31 B
1 4.74 ⫾ 0.54 B 4.42 ⫾ 0.42 C 3.40 ⫾ 0.28 C 3.28 ⫾ 0.51 B
2 4.87 ⫾ 0.41 B 5.21 ⫾ 0.27 AB 4.06 ⫾ 0.40 AB 4.50 ⫾ 0.47 A

Means in the same column within temperature and RH followed by different letters are significantly different (P < 0.05).
* Results are reported as means ⫾ standard deviations (n = 6).
PA, peracetic acid; RH, relative humidity.
TABLE 2.
POPULATION OF TOTAL PLATE COUNTS RECOVERED FROM CILANTRO AFTER APPLYING DECONTAMINATION TREATMENTS
DURING STORAGE AT DIFFERENT TEMPERATURE AND RH CONDITIONS

Temperature RH (%) Storage Population recovered (log cfu/g)*


(C) time (days)
Control Water Chlorine (200 ppm) PA (80 ppm)

5 100 0 6.55 ⫾ 0.18 C 6.64 ⫾ 0.33 B 6.04 ⫾ 1.01 A 5.20 ⫾ 0.30 B


2 6.73 ⫾ 0.15 C 7.04 ⫾ 0.07 AB 5.34 ⫾ 0.19 A 7.22 ⫾ 0.21 A
4 7.30 ⫾ 0.28 B 6.68 ⫾ 0.19 B 6.75 ⫾ 0.23 A 6.36 ⫾ 0.47 A
6 8.08 ⫾ 0.09 A 7.00 ⫾ 0.07 AB 5.66 ⫾ 0.33 A 6.65 ⫾ 0.46 A
8 7.51 ⫾ 0.07 AB 7.79 ⫾ 0.63 A 6.76 ⫾ 0.51 A 7.44 ⫾ 0.48 A
60 0 6.55 ⫾ 0.18 B 6.34 ⫾ 0.33 AB 6.04 ⫾ 1.01 A 5.20 ⫾ 0.30 B
2 6.68 ⫾ 0.05 AB 6.13 ⫾ 0.11 B 5.83 ⫾ 0.52 A 6.89 ⫾ 0.70 A
4 6.65 ⫾ 0.21 AB 6.62 ⫾ 0.09 AB 6.46 ⫾ 0.71 A 6.27 ⫾ 0.48 AB
6 7.07 ⫾ 0.36 AB 7.28 ⫾ 0.48 A 5.47 ⫾ 0.54 A 5.76 ⫾ 0.23 AB
8 7.23 ⫾ 0.01 A 6.76 ⫾ 0.20 AB 7.19 ⫾ 0.07 A 6.70 ⫾ 0.06 A
22 100 0 6.88 ⫾ 0.27 C 6.66 ⫾ 0.53 C 6.81 ⫾ 0.05 C 6.12 ⫾ 0.11 B
1 8.46 ⫾ 0.37 B 7.84 ⫾ 0.19 B 7.33 ⫾ 0.15 B 7.78 ⫾ 0.18 A
2 9.12 ⫾ 0.74 AB 8.88 ⫾ 0.29 A 8.11 ⫾ 0.10 A 7.86 ⫾ 0.22 A
30 0 6.88 ⫾ 0.27 B 7.02 ⫾ 0.31 C 6.81 ⫾ 0.05 B 6.12 ⫾ 0.11 B
1 7.12 ⫾ 0.32 B 7.40 ⫾ 0.16 BC 7.20 ⫾ 0.34 B 6.55 ⫾ 0.25 B
DECONTAMINATION TREATMENTS ON CILANTRO

2 8.17 ⫾ 0.58 A 7.89 ⫾ 0.36 AB 7.71 ⫾ 0.76 AB 7.95 ⫾ 0.11 A

Means in the same column within temperature and RH followed by different letters are significantly different (P < 0.05).
* Results are reported as means ⫾ standard deviations (n = 6).
PA, peracetic acid; RH, relative humidity.
591
592 N.S. VILLAGÓMEZ ET AL.

FIG. 1. SCANNING ELECTRON MICROGRAPHS OF CILANTRO INOCULATED WITH


SALMONELLA ENTERICA AND STORED AT 22C FOR 2 DAYS
(A) Day 0, cilantro not inoculated (control). (B) Cilantro inoculated with S. enterica. (C) Enlargement
from panel B. (D) Cilantro stored at 100% RH, treated with water. (E) Cilantro stored at 100% RH,
treated with chlorine; well-structured biofilms are shown with embedded bacteria. (F) Enlargement
from panel E (arrow). RH, relative humidity.
DECONTAMINATION TREATMENTS ON CILANTRO 593

TABLE 3.
POPULATION OF SALMONELLA ENTERICA RECOVERED FROM CILANTRO AFTER
APPLYING IONIZING RADIATION DURING STORAGE AT DIFFERENT TEMPERATURE
AND RH CONDITIONS

Temperature RH Storage Population recovered (log cfu/g)*


(C) (%) time (days)
0 kGy (control) 0.5 kGy 1 kGy 2 kGy

5 100 0 5.84 ⫾ 0.28 A 2.07 ⫾ 0.57 B 0.33 ⫾ 0.52 B ND


2 5.67 ⫾ 0.22 AB 2.53 ⫾ 0.25 AB 0.17 ⫾ 0.41 B ND
4 5.77 ⫾ 0.10 A 2.05 ⫾ 0.21 B 0.17 ⫾ 0.17 B ND
6 6.07 ⫾ 0.05 A 2.15 ⫾ 0.23 B 1.03 ⫾ 0.67 AB ND
8 5.70 ⫾ 0.24 AB 2.41 ⫾ 0.40 AB 0.50 ⫾ 0.70 B ND
60 0 5.92 ⫾ 0.17 A 2.30 ⫾ 0.48 A 0.40 ⫾ 0.55 B ND
2 5.43 ⫾ 0.33 AB 1.49 ⫾ 0.90 ABC 1.49 ⫾ 0.43 A ND
4 5.03 ⫾ 0.16 BCD 1.51 ⫾ 0.93 ABC <1 ND
6 4.69 ⫾ 0.16 CD 0.50 ⫾ 0.54 C 0.17 ⫾ 0.41 B ND
8 3.98 ⫾ 0.41 E 0.77 ⫾ 0.61 BC <1 ND
22 100 0 6.04 ⫾ 0.09 B 2.22 ⫾ 0.41 B 1.54 ⫾ 0.20 B ND
1 6.41 ⫾ 0.27 AB 2.84 ⫾ 0.61 B 3.43 ⫾ 0.30 A ND
2 6.84 ⫾ 0.24 A 4.74 ⫾ 0.07 A 3.91 ⫾ 0.84 A ND
30 0 5.83 ⫾ 0.15 A 2.75 ⫾ 0.11 A 0.33 ⫾ 0.52 B ND
1 5.18 ⫾ 0.51 B 2.11 ⫾ 0.09 B 1.48 ⫾ 0.44 A ND
2 4.90 ⫾ 0.32 B 2.55 ⫾ 0.09 A 1.60 ⫾ 0.61 A ND

Means in the same column within temperature and RH followed by different letters are significantly
different (P < 0.05).
* Results are reported as means ⫾ standard deviations (n = 6).
ND, none detected (detection limit was <10 cfu/g); RH, relative humidity.

The results for irradiation treatments are shown in Table 3. As expected,


the efficacy of gamma irradiation to kill Salmonella on cilantro increased with
the dose level. For cilantro stored at 5C regardless of the RH, reduction of the
pathogen after treatment with 0.5 kGy was 3.7 ⫾ 0.1 log cfu/g and remained
similar as storage time passed. However, when the samples were kept at 22C
and 100% RH, the efficacy of the 0.5 kGy treatment was significantly reduced
2 days after the start of storage (P < 0.05). These trends were similarly repro-
duced when irradiation dose was increased to 1 kGy; however, the reductions
of Salmonella populations were higher. One kilogray reduced as much as 5.6
log cfu/g on cilantro stored at 5C, and even in some cases, Salmonella was not
detected (detection limit 10 cfu/g). At 22C, treatment with 1 kGy achieved
Salmonella reductions of 4.4 log cfu/g on cilantro; such levels diminished up
to day 2 (~3.0 log cfu/g). At a dose of 2 kGy, the pathogen was not recovered
by pour plating from samples stored at all combinations of temperature and
RH. Thus, the higher irradiation level was able to reduce at least 5.8 log cfu/g
of Salmonella (Table 3). The fate of APC population on control samples was
594 N.S. VILLAGÓMEZ ET AL.

similar to that observed in the disinfection experiments (data not shown). At


both temperatures and 100% RH, the reduction of APC with 0.5 or 1 kGy was
approximately 3.5 log cfu/g, whereas at the low level of RH, reductions were
higher (~4.1 log cfu/g).

DISCUSSION

In our study, it was observed that Salmonella grew on the surface of


cilantro leaves stored at 22C and 100% RH regardless of the apparent low
nutrient availability. These results are similar to those previously published by
Brandl and Mandrell (2002), who found that the population of various serovars
of Salmonella (Derby, enteritidis, Newport, Thompson) inoculated on the
surface of leaves of live cilantro plants increased by 1.5–2.0 log cfu/g after 2
days of storage at 24C in a humidity chamber. It has been mentioned that
damage on plant tissue as well as high temperatures may increase the rates of
nutrient leaching from the plant cells to the leaf surface, allowing the growth
of bacteria (Tukey 1970). Survival of Salmonella on cilantro stored at refrig-
erated temperatures was also reported by Hsu et al. (2006), who found that the
pathogen decreased by 0.47 log within the first 5 days of storage.
The efficacy of chlorine and peracetic acid to disinfect different kinds of
produce, such as fresh-cut celery, cabbage, potatoes (Sapers 2003), lettuce
(Hellstrom et al. 2006), melon (Ukuku and Sapers 2001) and cilantro (Foley
et al. 2004), has been extensively studied; the reported levels of reduction were
similar to those obtained in our study (~2 log cfu). Results also showed that the
effectiveness of disinfection treatments is affected by the time interval between
the contamination event (inoculation) and the treatment application. Some
researchers have come to similar conclusions on nonleafy green produce.
Studies conducted with cantaloupes inoculated with Salmonella Stanley for
the effectiveness of chlorine (1000 mg/L), or hydrogen peroxide (5%) were
determined following inoculation and after 72 h of storage; it was found that
reduction in populations were 3 and less than 1 log, respectively (Ukuku and
Sapers 2001).
The effect of gamma irradiation for decontamination of fresh produce
also has been reported. Foley et al. (2004) found that irradiation at 1.05 kGy
resulted in a 6.7 log reduction of Escherichia coli O157:H7 inoculated on
cilantro. At 1 kGy, inoculated Listeria monocytogenes on cabbage, tomatoes
and sprouts of broccoli and mung bean was reduced by 5.3, 4.1, 4.9 and 4.6 log
cfu/g, respectively (Bari et al. 2005). The major difference between our work
and those of other authors is the time at which the treatment is applied. Thus,
the decontamination efficacy with 1 kGy irradiation applied immediately after
pathogen inoculation is similar in our study to that described in other reports.
DECONTAMINATION TREATMENTS ON CILANTRO 595

Our findings for APC are similar to previous reports of 1-kGy treatments
on the naturally occurring bacteria of fresh cilantro (Fan et al. 2003; Foley
et al. 2004) and alfalfa sprouts (Rajkowski and Thayer 2000). Although a
sensorial evaluation was not part of our work, no visual changes were observed
on cilantro treated with 1 kGy. However, at 2 kGy, leaves turned yellowish and
fragile. Other authors have pointed out that gamma irradiation has no signifi-
cant influence over several sensorial characteristics of cilantro at levels as high
as 3.85 kGy (Fan et al. 2003; Foley et al. 2004).
In general, chemical treatments were more effective on cilantro stored at
5C than at 22C, and the efficacy of chemical treatments diminished as the
interval of the inoculation and the sanitizer treatment increased. This effect was
more evident at 22C and, in fact, may be associated with the microbial growth
of APC on cilantro stored at the higher temperature or possibly reflecting the
formation of biofilms. The presence of biofilms on cilantro leaves along with
the occurrence of open stomata where the pathogen may be lodged could be
highly advantageous for Salmonella to avoid the effect of chemical disinfec-
tants. Numerous studies have documented the ability of Salmonella to adhere
and form biofilms on inert surfaces and on the surface of natural produce like
leaves and roots of fruits and vegetables (Ronner and Wong 1993; Wells and
Butterfield 1997). The mechanisms affording protection to cells in biofilms
against antimicrobial agents have not been fully elucidated. Reaction–diffusion
kinetics associated with specific antimicrobial agents, slower rates of cell
growth and the expression of antimicrobial-resistant phenotypes in biofilms are
among the factors that may affect the efficacy of sanitizers (Costerton et al.
1995; Ryu and Beuchat 2005). Another complicating issue is the effect of the
chemical agent on the background microbial flora (Doyle and Erickson 2008).
Although gamma rays have high penetration power, the loss of efficacy of
irradiation as storage time elapsed was also observed. Irradiation dose is one
of the factors that affect the microbial inactivation by irradiation. In general,
higher doses of ionizing radiation cause a greater destruction of microorgan-
isms. However, microbial destruction at a given irradiation dose is decreased
under anaerobic or dry conditions because of the lower rate of oxidizing
reactions that produce free radicals and toxic oxygen derivates (Mendonca
2002). As with heating, use of chemical preservatives and use of certain other
food preservation methods, microbial numbers have the same impact on the
effectiveness of irradiation: Large numbers of microorganisms reduce the
effectiveness of a given irradiation dose. Also, the type of microorganisms has
influence on irradiation. It has been reported that the effectiveness of low
(<1 kGy) and medium doses (range from 0.75 to 2.5 kGy) used to improve the
safety and shelf life of food can be limited by the survival of psychrotrophic
pathogens and psychrotrophic gram-positive spoilage bacteria (Mendonca
2002). In our experiments, we observed that the population of APC increased
596 N.S. VILLAGÓMEZ ET AL.

during the storage including refrigeration temperature, suggesting that a frac-


tion of cilantro microflora is psychrotrophic.
Although irradiation is clearly effective as a single antimicrobial treat-
ment, it is generally accepted that it should be part of an overall sanitization
strategy that uses multiple interventions or “hurdles” to eliminate pathogenic
bacteria while preserving product quality. Our results highlight the need to
control bacterial growth on cilantro before sanitizing treatments are applied.
Further studies are needed to determine the efficacy of combining chemical
treatments and ionizing irradiation as well as preservation strategies to obtain
pathogen-free produce.

ACKNOWLEDGMENTS

The authors would like to thank Armando Zepeda, Francisco Pasos


Najera and Ma. Lourdes Palma Tirado from the Universidad Nacional
Autónoma de México for conducting scanning electron microscopic exami-
nations. We also thank Stoyan A. Toshkov, who performed the irradiation
treatments at the Nuclear Radiation Laboratory, University of Illinois at
Urbana-Champaign, to whom we are gratefully indebted.

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