Tarea 2.Effect of Storage Time on the Efficacy of Chemical Treatments

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jfs_227 584..598

EFFECT OF STORAGE TIME ON THE EFFICACY OF CHEMICAL

AND IRRADIATION TREATMENTS TO INACTIVATE

SALMONELLA ON CILANTRO (CORIANDRUM SATIVUM L.)

NAAXILLI SERNA VILLAGMEZ1

, ERIKA A. NERI HERRERA1

,LEOPOLDO OROZCO R.1, GRACIELA WILD-PADUA2 andMONTSERRAT H. ITURRIAGA1,3

1Departamento de Investigacin y Posgrado en AlimentosFacultad de Qumica

Universidad Autnoma de QuertaroCentro Universitario

Cerro de las Campanas s/nCol. Las Campanas, C. P. 76010, Quertaro, Qro., Mxico

2Department of Food Science and Human NutritionUniversity 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 inactivateSalmonella enterica on cilantro throughout storage under different conditionsof 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 ofSalmonella with chlorine, peracetic acidor water were 2.6, 2.1 and 1.0 log cfu/g, respectively. At 22C, the effectivenessof disinfection treatments to remove or kill Salmonella cells from cilantrosignificantly decreased as the storage time elapsed (P < 0.05). A similar trendwas 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 remainedconstant regardless of RH.

3 Corresponding author. TEL: (52) 442- 192-1200 ext. 5507; FAX: (52) 442- 192-1304; EMAIL:[email protected]

DOI: 10.1111/j.1745-4565.2010.00227.x

Journal of Food Safety 30 (2010) 584598.584 2010 Wiley Periodicals, Inc.


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PRACTICAL APPLICATIONS

This study demonstrates how the efficacy of decontamination treatmentsis modified during the storage of cilantro under constant temperature andrelative humidity. The decrease in treatment efficacy suggests that microor-ganisms colonizing the cilantro surface may produce biofilms that provideprotection; the level of naturally occurring microflora may also have an influ-ence on its effectiveness. The results reinforce the concept that once cilantro iscontaminated with Salmonella, the pathogen can survive chemical disinfectionor irradiation, and eventually multiply on cilantro throughout its shelf life.

INTRODUCTION

Produce, including green salads, have been associated with foodborneoutbreaks (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 jalapeopeppers, cilantro was one of the suspected items during the epidemiologicalinvestigation (CDC 2008). The risks of foodborne outbreaks is increasedbecause cilantro is widely used in the cuisines of Mxico, South America,China, India and Southeast Asia, and usually in minor quantities in salsas or asa dish garnish.

Preventive measures to avoid pathogen contamination remain the mostimportant step to safeguard the microbial safety of produce (Beuchat and Ryu1997). Nevertheless, complementary actions to prevent survival and growthof pathogenic bacteria on fruits and vegetables are generally recommended.Among such practices, refrigeration and disinfection have long been employedby the food industry and at home (Beuchat 1998, 2002). There are severalfactors 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 microbialsafety of food. Since 1986, irradiation at 1.0 kGy has been approved in theU.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 Foodand Drug Administration (FDA) for control of foodborne pathogens, andextension of shelf life, in fresh iceberg lettuce and fresh spinach at a dose of upto 4.0 kGy (FDA 2008). A dose of 1.0 kGy is highly effective to inactivatefoodborne pathogens and parasites in various foods, like apples, cauliflower,strawberries and cilantro (Tauxe 2001).

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Generally, evaluations of disinfection treatments have been carried outimmediately after inoculation (Wright et al. 2000; Hellstrom et al. 2006).However, after contamination, produce may undergo prolonged periods ofstorage under diverse conditions until disinfection is performed either atpacking sheds, restaurants or at home. To the best of our knowledge, there areno published studies that investigate the effects of storage on the efficacy ofdecontamination methods on cilantro. The aim of this work was to evaluate theeffect of storage conditions on the efficacy of disinfection treatment andgamma irradiation for cilantro inoculated with Salmonella.

MATERIALS AND METHODS

Source of Cilantro

Bunches of fresh cilantro (Coriandrum sativum L.) were obtained fromlocal supermarkets in Urbana-Champaign, IL, for the irradiation treatmentsand in Quertaro, Qro., Mxico, for the chemical disinfection studies.

Preparation of Inoculum

Salmonella Thompson ATCC 8391 and Salmonella Montevideo ATCC8387 were maintained at -72C in tryptic soy broth (TSB, Difco Laboratories,Sparks, MD) containing 10% glycerol (Karal S.A. de C.V., Len, Gto.,Mxico) before the start of this study. For the present studies, resistance torifampicin (Sigma Chemical Co., St. Louis, MO) was induced in the strains(Kaspar and Tamplin 1993). To summarize the method briefly, 10 mL of 24-hculture was centrifuged and the pellet was resuspended in 1 mL, then cellsuspension was surface plated (0.1 mL per plate) on tryptic soy agar (DifcoLaboratories) supplemented with rifampicin (100 mg/L; TSAR) and incubatedfor up to 72 h. Rifampicin-resistant strains were grown individually by threesuccessive 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 wereharvested by centrifugation (3500 g, 15 min, 22C). Cells were washed twicewith saline solution (0.85%), and then, following the final wash, equalvolumes of each Salmonella strain were combined. One milliliter of the pooledcell suspension was added to 1 L of sterile distilled water to yield a finalconcentration of ca. 6 log cfu/mL. The final cell concentration of the dipsuspension (inoculum) was determined by pour plating onto TSAR. Plateswere incubated at 35C for 24 h before the colonies were counted.

Inoculation Procedure

Bunches of cilantro free of defective leaves and stems were weighed into10-g samples. Inoculation was performed by immersing each cilantro bunch

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

Storage Conditions

Inoculated cilantro samples were placed inside clear plastic containersthat were covered and hermetically sealed with high-vacuum grease (DowCorning Corp., Midland, MI) and stored at 22 and 5 1C for 2 and 8 days,respectively. Relative humidity (RH) for containers stored at both storagetemperatures had been previously equilibrated at 100% RH (by placing sterile

water in the bottom of the containers) or without controlling RH (reaching30% RH at 22C, and 60% RH when stored at 5C). Atmospheric RH wasmonitored 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, Mxico) 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 inoculatedcilantro stored at 22C were taken out of the containers on days 0, 1 and 2. At5C, samples were removed on days 0, 2, 4, 6 and 8. All samples weresubmerged into each disinfectant solution and water for 5 min without agita-tion. Immediately after treatment, cilantro was removed from the solutionswith sterile stainless steel tongs and placed into another polyethylene bagcontaining 90 mL of Dey and Engley neutralizer broth (pH 7.6) (Difco Labo-

ratories) for homogenization in the Stomacher 400 (Seward, Norfolk, UK) atmedium speed for 1 min. Untreated, inoculated cilantro samples were used ascontrol at each sampling time.

Irradiation

As with the disinfection treatments, after inoculation of cilantro withSalmonella, irradiation treatments were performed periodically. On scheduledsampling 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 wereirradiated with a Co-60 gamma radiator (Gammacell, 220 Excel, MDSNordion, Ottawa, Canada) in the facilities of the Nuclear Radiation Labora-tory, University of Illinois at Urbana-Champaign. In all cases, the irradiationwas conducted at 5C. Irradiated samples were then homogenized with 90 mL

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of 0.1% peptone water as described above. Untreated, inoculated cilantrosamples were used as control at each sampling time.

Bacterial Enumeration

Homogenates of disinfected and irradiated samples were serially dilutedin 0.1% peptone water. Total mesophilic populations were estimated from theaerobic 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 randomlyselected from each sample and subjected to confirmation by biochemical tests

and agglutination using commercial antiserum (Difco Laboratories) (Andrewset al. 1992). Salmonella counts (cfu/g) were subtracted from the APC to obtainthe aerobic microbial enumeration on cilantro.

Scanning Electron Micrographic Examination

At each sampling day, one cilantro sample was prepared for scanningelectron microscopy (SEM), according to the procedure described by Getzet al. (1983), with some modifications. Sections of cilantro leaves (5 5 1 mm) were fixed for 3 h in 3% glutaraldehyde, followed by a treatmentfor 3 h in 1% osmium tetroxide. Both solutions were prepared in 0.1 M sodiumcacodylate (pH 7.36). Fixed tissues were dehydrated in a graded series ofethanol concentrations at 4C. Following dehydration, tissues were critical-point dried, mounted, sputter coated with gold and examined with a ZeissDSM-950 (Carl Zeiss, Jena, Germany) scanning electron microscope operat-ing at 1520 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 pertreatment. Data (log cfu/g) were subjected to analysis of variance and Tukeystest using the JMP 5.0 software (SAS Institute, Cary, NC).

RESULTS

Disinfection Treatments

The behavior ofSalmonella cells inoculated onto cilantro throughout thestorage time for all combinations of temperature and RH was determined onsamples that were not subjected to any treatment (control). The purpose was tocalculate the actual reduction because of the disinfection treatments regardless

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of the population changes caused by storage. During the storage at 5C, as itwas expected, Salmonella did not grow but survived up to 8 days: populationdecreased 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 1.3log cfu/g; in contrast, when the RH was not controlled (~30%), a reduction of1.0 log cfu/g was observed.

The efficacy of chlorine and peracetic acid to inactivate Salmonella oncilantro was similar along the study (P > 0.05). In general, regardless oftemperature or RH, when cilantro was treated with water, chlorine or peraceticacid, the number of Salmonella cells that were removed or inactivated fromcilantro significantly decreased as the storage time elapsed (P < 0.05)(Table 1). On day 0, reductions after treatment with water for all storageconditions were less than 1 log, and by the end of the study, there was not anydetectable reduction in Salmonella size on cilantro. A similar trend wasobserved when chlorine was applied to cilantro stored at 5C: On day 0, thereduction was 2.6 log cfu/g, and after 8 days, reductions were 2.2 and 1.4 logcfu/g in samples kept at 100 or 60% RH, respectively. The decrease in efficacyof the chlorine treatment was more pronounced for samples stored at 22C: Atday 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. Resultsfor peracetic acid showed a trend similar to those observed with chlorine.

The changes in APC populations on cilantro during the storage are shownin Table 2. On day 0, APC in control samples was 6.9 log cfu/g at 22C and 6.6log 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, showingan 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 thecounts of APC of approximately 1 log in all storage conditions (Table 2).Comparable results were observed when water was applied, suggesting thatthe 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 at30% RH, the occurrence of an amorphous material was common.

Irradiation Treatments

On inoculated samples not subjected to irradiation (control), the fate ofSalmonella was similar to that observed for the control in the chemical disin-fection experiments (Table 3). The inoculated pathogen grew on cilantrostored at 100% RH and 22C (


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TABLE1

.

POPULAT

IONOFSALMONELLAENTERICARECOVEREDFROM

CILA

NTROAFTERAPPLYINGDEC

ONTAMINATIONTREATMENTS

DURINGST

ORAGEATDIFFERENTTEMP

ERATUREANDRHCONDITIONS

Temperature

(C)

RH(%)

Storage

time(day

s)

Populationrecovered(logcfu/g)*

Control

Water

Chlorine(200ppm)

PA(80pp

m)

5

100

0

5.80

0.12A

5.06

0.14A

3.20

0.19A

3.390.42A

2

5.86

0.29A

4.92

0.15A

2.80

0.49AB

3.010.35AB

4

5.49

0.16A

4.95

0.09A

2.37

0.34BC

2.750.19BC

6

4.77

0.36B

4.78

0.18AB

2.26

0.41BC

2.440.29C

8

4.27

0.36BC

4.46

0.35B

2.03

0.47C

2.570.14BC

60

0

5.80

0.12A

5.06

0.14A

3.20

0.19A

3.390.42A

2

4.81

0.24B

4.15

0.31B

2.60

0.47AB

2.540.29BC

4

4.49

0.24BC

4.04

0.35B

2.49

0.30BC

2.730.61AB

6

4.05

0.40CD

3.87

0.26B

1.94

0.35C

1.910.50C

8

3.61

0.29D

3.59

0.54B

2.20

0.42BC

1.800.40C

22

100

0

5.71

0.54A

4.89

0.09B

3.60

0.11B

3.620.38C

1

5.95

0.10B

5.27

0.11B

4.15

0.45B

5.000.46B

2

7.00

0.45B

6.01

0.41A

5.18

0.57A

6.130.29A

30

0

5.85

0.45A

4.89

0.09B

3.60

0.11BC

3.520.31B

1

4.74

0.54B

4.42

0.42C

3.40

0.28C

3.280.51B

2

4.87

0.41B

5.21

0.27AB

4.06

0.40AB

4.500.47A

Meansinthes

amecolumnwithintemperatureandRHfollowedbydifferentlettersaresignificantlydifferent(P

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Tarea 2.Effect of Storage Time on the Efficacy of Chemical Treatments