Research Note. MS : Received 10 October 2003/Accepted 8 January 2004 ABSTRACT

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1 1238 Journal of Food Protection, Vol. 67, No. 6, 2004, Pages Copyright q, International Association for Food Protection Research Note Ef cacy of and a Peroxyacetic Acid Sanitizer in Killing Listeria monocytogenes on Iceberg and Romaine Lettuce Using Simulated Commercial Processing Conditions LARRY R. BEUCHAT,* BARBARA B. ADLER, AND MEGAN M. LANG Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Grif n, Georgia , USA MS : Received 10 October 2003/Accepted 8 January 2004 ABSTRACT The ef cacy of chlorine (100 mg/ml) and a peroxyacetic acid sanitizer (80 mg/ml; 100) in killing Listeria monocytogenes inoculated at populations of 1 to 2, 2 to 3, and 4 to 5 log CFU/g of iceberg lettuce pieces, shredded iceberg lettuce, and Romaine lettuce pieces was determined by treatment conditions simulating those used by a commercial fresh-cut lettuce processor. The lettuce/treatment solution ratio was 1:100 (wt/vol), treatment temperature was 48C, and total treatment time was 30 s. Compared with washing in water, treatment of iceberg lettuce pieces containing all levels of inoculum and shredded iceberg lettuce containing 2 to 3 or 4 to 5 log CFU/g with chlorine or resulted in signi cant reductions (P # 0.05) of pathogen populations. Populations recovered from Romaine lettuce pieces treated with chlorine or were not signi cantly different from populations recovered from pieces washed with water, regardless of the inoculum level. Within lettuce type and inoculum level, in no instance was the number of L. monocytogenes recovered from lettuce treated with chlorine or signi cantly different. The rate of decrease in free chlorine concentration in treatment solution as affected by the weight/volume ratio (1:100, 1:10, 2:10, and 4:10) of lettuce and solution was determined. The rate of reduction increased as the ratio decreased. The overall order of magnitude of reduction was shredded iceberg lettuce. iceberg pieces. Romaine pieces. The highest reductions in free chlorine concentration in solutions used to treat shredded lettuce are attributed to the release of tissue juices, which increases the concentration of soluble organic materials available for reaction with chlorine. Blenden and Szatalowicz (4), noting that 731 cases of human listeriosis were documented in the United States between 1933 and 1966, suggested that Listeria monocytogenes might contaminate lettuce and other vegetables eaten raw and perhaps was associated with some of those cases. L. monocytogenes has since been isolated from lettuce (7, 16, 21, 27) and other salad vegetables (1, 8, 9, 13, 15, 19, 26). Outbreaks of listeriosis have been epidemiologically linked to consumption of celery, tomatoes, and lettuce in a Boston hospital (10) and of raw cabbage in Nova Scotia (17). The pathogen is able to grow on cut lettuce (3, 6, 12, 20), thereby increasing the risk of illness associated with its consumption. Several studies have described the ef cacy of chlorine and other sanitizers in killing L. monocytogenes on inoculated, cut lettuce (3, 6, 14, 22, 28). Various ratios of lettuce weight and treatment solution volume, as well as different treatment times and temperatures, have been used in these studies, making comparisons across laboratories dif cult. In some instances, treatment conditions did not mimic those used in commercial fresh-cut lettuce operations. A recent study (22) in collaboration with a commercial fresh-cut iceberg lettuce processor in Australia evaluated the effective- * Author for correspondence. Tel: ; Fax: ; lbeuchat@uga.edu. ness of chlorine and a mixture of hydrogen peroxide and peroxyacetic acid in killing L. monocytogenes on shredded lettuce. The ratio of lettuce weight to treatment solution volume was 1:19. Results of the experiment were used to demonstrate how the manufacturer could meet a food safety objective of less than 100 CFU L. monocytogenes per g of lettuce. We undertook a study to determine the ef cacy of chlorine and a peroxyacetic acid based sanitizer in killing L. monocytogenes inoculated onto iceberg lettuce pieces, shredded iceberg lettuce, and Romaine lettuce pieces with scaled-down treatment conditions simulating those used by a fresh-cut lettuce processor. The rate of reduction in concentration of free chlorine in the treatment solution as affected by the lettuce/solution ratio was measured. MATERIALS AND METHODS Strains used. L. monocytogenes strains F8027 (serotype 4b, isolated from celery), F8255 (serotype 1/2b, from peach and plum), F8369 (serotype 1/2a, from corn), G1091 (serotype 4b, from the feces of a patient in a coleslaw-associated outbreak of listeriosis), and H0222 (serotype 1/2a, from potato) were used. Stock cultures were maintained on brain heart infusion agar (BBL/ Difco, Sparks, Md.) supplemented with nalidixic acid (50 mg/ml) (BHIAN) at 48C.

2 J. Food Prot., Vol. 67, No. 6 CHLORINE AND PEROXYACETIC ACID SANITIZERS ON LETTUCE 1239 Preparation of inocula. Stock cultures were inoculated into 10 ml of brain heart infusion broth (BBL/Difco) supplemented with nalidixic acid (50 mg/ml) (BHIBN) and incubated at 378C for 24 h. Cultures were transferred twice at 24-h intervals with an inoculating loop. A third transfer of 1.0 ml of 24-h culture to 100 ml of BHIBN in a 250-ml Erlenmeyer ask and incubation at 378C for 24 h was done immediately before cells were harvested to prepare inoculum for lettuce. Cells of each strain were collected by centrifugation (4,000 3 g, 15 min, 48C), washed in 50 ml of sterile deionized water, collected again by centrifugation, and resuspended in 50 ml of sterile deionized water. Suspensions (2 ml) of each strain were combined to give a ve-strain mixture of L. monocytogenes containing approximately equal populations of each strain. The suspension was diluted in sterile deionized water to give populations of 1 to 2 log CFU/g of lettuce (low inoculum), 2 to 3 log CFU/g of lettuce (medium inoculum), and 4 to 5 log CFU/g of lettuce (high inoculum) on application of 100 ml on 10 g of lettuce. Preparation of lettuce. Iceberg lettuce (Lactuca sativa var. capita) and Romaine lettuce (L. sativa var. longifolia) were purchased at a supermarket in Grif n, Ga. The core and two to four outer leaves of iceberg lettuce were removed and discarded. Internal leaves were cut into pieces measuring 3.8 by 3.8 cm (ca. 29 cm 2 surface area on each piece) with a sterile stainless steel template and scalpel. Care was taken to minimize surface tissue damage. A sample unit (10 g) consisted of seven or eight pieces. Shredded iceberg lettuce was also evaluated. Internal leaves were cut in pieces (ca. 0.6 by 4 cm) with a stainless steel knife. A sample unit consisted of 10 g. Pieces (3.8 by 3.8 cm) of Romaine lettuce were prepared by the same procedure described for iceberg lettuce, except both outer and inner leaves were included in each 10-g sample (12 pieces). Procedure for inoculation. Each 10-g sample of lettuce at 48C was spot inoculated with 100 ml of the ve-strain suspension of L. monocytogenes and placed in a forced-air incubator at 48C for 24 h to facilitate evaporation of the water in the inoculum. A total of 100 ml of inoculum was deposited at 5 to 10 locations on each 10-g sample to facilitate drying. Treatment of lettuce. To each 10-g sample of inoculated lettuce placed in a 1-gallon (3.79-liter) Ziploc (S. C. Johnson, Racine, Wis.) bag, l liter of sterile deionized water (control), chlorinated water (100 mg/ml), or 100 (80 mg/ml) (Ecolab, St. Paul, Minn.), all at 38C to 48C, was added. The chlorine solution was prepared by combining NaOCl (Aldrich, Milwaukee, Wis.) with 0.05 M potassium phosphate buffer (ph 6.8, C). Free chlorine concentration was determined with an amperometric titrator (Hach, Ames, Iowa) immediately before use. The bag containing lettuce and sanitizer solution or water was vigorously shaken by hand for 15 s to simulate a commercial wash operation, and solutions or water treatments were decanted. A second 15-s treatment with 1 liter of water, chlorinated water, or 100 water was done. All treatment solutions were at 38C to 48C. and sanitizer were decanted, and the drained lettuce was immediately placed in a Stomacher 400 bag (Seward Medical, Ltd., London, UK) containing 90 ml of Listeria enrichment broth (Oxoid, Basingstoke, UK) supplemented with nalidixic acid (50 mg/ml) and sodium pyruvate (0.1%) (LEBNP), which in preliminary experiments was shown to neutralize residual sanitizer remaining on the lettuce after treatment. Microbiological analysis. Stomacher bags containing 10 g of lettuce treated with water or sanitizer and 90 ml of LEBNP were pummeled by a Stomacher 400 homogenizer (Seward Medical, Ltd.) for 1 min at normal speed. A sample (1 ml) of undiluted homogenate from each sample of lettuce inoculated with a population of 1 to 2 log CFU/g was surface plated (0.25 ml per plate) on modi ed Oxford medium (MOX, ph 7.0; Oxoid) supplemented with nalidixic acid (50 mg/ml) and sodium pyruvate (0.1%) (MOXNP). Samples (0.25 ml in quadruplicate and 0.1 ml in duplicate) of undiluted homogenate from lettuce inoculated with L. monocytogenes at populations of 2 to 3 or 4 to 5 log CFU/g were also plated on MOXNP. Plates were incubated at 378C for h before presumptive L. monocytogenes colonies were counted. Colonies (one to ve, depending on the number isolated) from each sample were subjected to con rmation testing with an API Listeria diagnostic kit (biomérieux Vitek, Inc., Hazelwood, Mo.) Homogenates of lettuce and LEBNP were incubated at 378C for 48 h then streaked on MOXNP. Plates were incubated at 378C for 48 h and examined for presumptive colonies of L. monocytogenes, which were con rmed by API Listeria diagnostic kits. Uninoculated lettuce and inoculated lettuce not subjected to treatment with water or sanitizers were analyzed for the presence and populations of L. monocytogenes. Uninoculated lettuce (10 g) was combined with 90 ml of LEBNP in a stomacher bag, pummeled for 1 min, and incubated at 378C for 48 h. Enriched samples were streaked on MOXNP and incubated at 378C for 48 h before examining for presumptive L. monocytogenes colonies. Inoculated lettuce (10 g) was combined with 90 ml of LEBNP and pummeled for 1 min. Undiluted homogenate (0.25 ml in quadruplicate and 0.1 ml in duplicate), serially diluted homogenate (0.1 ml in duplicate), or both, depending on the inoculum population, were surface plated on MOXNP and incubated at 378C for 48 h before counting presumptive L. monocytogenes colonies and subjecting isolates to con rmation tests. Change in free chlorine concentration. The reduction of free chlorine concentration in treatment solution as affected by the lettuce/solution ratio (wt/vol) was determined. Iceberg lettuce pieces, shredded iceberg lettuce, and Romaine lettuce pieces were prepared as described above. Lettuce samples (10, 100, 200, and 400 g) were deposited in 3.79-liter Ziploc bags with 1,000 ml of water (48C) containing free chlorine at concentrations of mg/ml. containing no added NaOCl was used as a control. The mixture was vigorously agitated for 1, 2, 5, or 10 min before withdrawing samples of chlorinated water for analysis of free chlorine content with an amperometric titrator. Statistical analysis. All experiments were replicated three times, and four samples subjected to each treatment were analyzed in each replicate. Within each type of lettuce and inoculum population, data were analyzed by SAS (Statistical Analysis Systems Institute, Cary, N.C.) for analysis of variance and Duncan s multiple range tests to determine signi cant differences (P ) between populations of L. monocytogenes recovered from lettuce treated with water, 100 mg/ml chlorine, or 80 mg/ml 100. Within the type and cut of lettuce and treatment time, signi cant differences between concentrations of free chlorine present in solution after treating various weights of lettuce in 1 liter of chlorinated water were determined. RESULTS AND DISCUSSION Populations of L. monocytogenes (CFU/g) inoculated onto lettuce are listed in Table 1. Within inoculum level, populations applied to each type and cut of lettuce were not signi cantly different (P. 0.05). The populations shown in Table 2 are L. monocytogenes recovered from inoculated treated and untreated let-

3 1240 BEUCHAT ET AL. J. Food Prot., Vol. 67, No. 6 TABLE 1. Populations of L. monocytogenes inoculated onto lettuce Type Iceberg Romaine Lettuce Cut Pieces Shredded Pieces Inoculum (log CFU/g) Low Medium High tuce. With one exception (inoculated, untreated iceberg pieces), the number of L. monocytogenes recovered from untreated lettuce was lower than the number applied. These decreases are attributed to cell death during drying and incomplete retrieval of viable cells that might be lodged in stomata and cut edges of lettuce tissue. Foodborne pathogens can in ltrate stomata, lenticels, broken trichomes, and cracks on the surface of raw fruits and vegetables (2, 5). Seo and Frank (18) observed viable Escherichia coli O157: H7 in stomata of inoculated iceberg lettuce. The pathogen preferentially attaches to cut edges of lettuce leaves rather than the intact surface (18, 23 25). With the exception of shredded iceberg lettuce inoculated with a low population of L. monocytogenes, washing with water signi cantly (P # 0.05) reduced the number of CFUs recovered, regardless of the type of lettuce or level of inoculum (Table 2). Further signi cant reductions, compared with populations recovered after washing with water, resulted from treatment of iceberg lettuce pieces containing low, medium, or high inoculum and shredded iceberg lettuce containing medium or high inoculum with chlorine or. In contrast, on Romaine lettuce pieces with all levels of inoculum, populations recovered after treatment with sanitizers were not signi cantly different from populations recovered after washing with water. Trends toward higher reductions on iceberg lettuce treated with compared with chlorine and Romaine lettuce treated with chlorine compared with were observed. However, within lettuce type and inoculum level, the number of L. monocytogenes recovered from samples treated with chlorine or was not signi cantly different. Reductions were generally higher as the inoculum level increased, but none of the treatments eliminated the pathogen. The lettuce/water and lettuce/sanitizer solution ratios used were 1:100 (wt/vol), and the total treatment time was 30 s. With a shredded iceberg lettuce/sanitizer solution ratio of 1:10 (80 or 120 mg/ml chlorine and 80 mg/ml of a peracetic acid hydrogen peroxide mixture) and an inoculum of approximately 4.5 log CFU/g, Szabo et al. (22) reduced populations of L. monocytogenes by less than 1 log CFU/ g within 1 min. These reductions are about 0.3- and 0.8- log less than those we observed for high-inoculum (4 to 5 log CFU/g) iceberg lettuce pieces and shredded iceberg lettuce, respectively, on which 2.30 and 3.36 log CFU/g were detected after washing with water. These differences in reduction could have resulted from differences in the lettuce/ treatment solution ratios used in the two laboratories. A 2- min treatment of cut iceberg lettuce with 120 mg/ml chlorine or 120 mg/ml of the peroxyacetic acid and hydrogen peroxide mixture decreased L. monocytogenes populations by 1.1 (60.3) and 1.4 (60.5) log CFU/g, respectively (22). The initial population inoculated onto lettuce was 5.7 log CFU/g, compared with the highest test inoculum (4.5 to 4.7 log CFU/g of iceberg lettuce) used in our study. With a cut iceberg lettuce/chlorine solution (100 mg/ml) ratio of 1:13, Delaquis et al. (6) reported a 1-log reduction in an initial population (5 log CFU/g) of L. monocytogenes after 3 min. In another study, treatment of shredded iceberg lettuce with 100 or 200 mg/ml chlorine (a 1:10 lettuce/solution ratio) for 1 min caused 0.7- and 1.2-log reductions, respectively, TABLE 2. Populations of L. monocytogenes recovered from inoculated untreated and treated iceberg and Romaine lettuce a L. monocytogenes (log CFU/g) Iceberg pieces Shredded iceberg Romaine pieces Inoculum b Treatment c Reduction d Reduction Reduction Low Medium High 1.76 A 1.54 B 0.72 C 0.51 C 2.01 A 1.64 B 1.34 C 0.95 C 2.78 A 2.30 B 1.92 C 0.95 C A 0.76 AB 0.57 BC 0.47 C 1.84 A 1.55 B 1.05 C 0.99 C 3.70 A 3.36 B 2.37 C 2.11 C A 1.21 B 0.54 B 0.85 B 2.09 A 1.43 B 1.05 B 1.18 B 3.30 A 2.25 B 1.62 B 1.67 B a Within each level of inoculum, mean values (log CFU/g) in the same column that are not followed by the same letter are signi cantly different (P # 0.05). b Populations of L. monocytogenes inoculated onto lettuce are listed in Table 1. c Inoculated lettuce was untreated (none) or treated with water (control), chlorine (100 mg/ml), or 100 (80 mg/ml). d Reduction compared with population detected on inoculated untreated lettuce.

4 J. Food Prot., Vol. 67, No. 6 CHLORINE AND PEROXYACETIC ACID SANITIZERS ON LETTUCE 1241 was determined. Such information would be of value when predicting the extent of lethality to L. monocytogenes and other produce-borne pathogens after a given treatment time. Figure 1 shows the concentrations of free chlorine in treatment solution as affected by the lettuce/solution ratio (1: 100, 1:10, 2:10, and 4:10) and type of lettuce. The initial concentration of free chlorine in treatment solutions was mg/ml, so the change in concentration after a given treatment time for each lettuce/solution ratio and type of lettuce should be based on decreases from initial concentration rather than comparing, at a speci c treatment time, differences in concentrations in solutions containing various lettuce/solution ratios. Not unexpectedly, the rate of reduction in free chlorine concentration was increased as the lettuce/solution ratio was decreased. The overall order of reduction, particularly evident as the ratio was decreased, was shredded iceberg lettuce. iceberg lettuce pieces. Romaine lettuce pieces. At a lettuce/solution ratio of 1:100, the free chlorine concentration did not decrease signi - cantly (P. 0.05) during the 10-min treatment, regardless of lettuce type. Signi cant reductions (P # 0.05) in concentration did occur, however, at a 1:10 ratio within 2, 5, and 5 min for shredded iceberg lettuce, iceberg pieces, and Romaine pieces, respectively. The most rapid reductions in free chlorine concentration in solutions used to treat shredded lettuce are attributed to the release of tissue juices, which increases the concentration of organic materials accessible for reaction with and neutralization of chlorine. Lettuce/solution ratios less than 1:10, regardless of lettuce type or cut, result in reduced concentrations of free chlorine within a shorter treatment time, thereby reducing potential lethality to L. monocytogenes and other foodborne pathogens and spoilage microorganisms that can be present on lettuce. REFERENCES FIGURE 1. Concentration of free chlorine in treatment solution containing lettuce/solution ratios (wt/vol) of 1:100 (l), 1:10 (m), 2:10 (m ), and 4:10 (v) after treatment for up to 10 min. in L. monocytogenes compared with washing in water (28). The population retrieved from lettuce that was not treated with chlorine was 5.4 log CFU/g. Lang et al. (11) used a 1:10 iceberg lettuce pieces/chlorine solution (200 mg/ml) ratio and a 5-min treatment time to reduce the population of L. monocytogenes by 1.2 to 1.8 log CFU/g. Observations from these studies on the ef cacy of chlorine in killing L. monocytogenes indicate that iceberg lettuce/treatment solution ratios ranging from 1:10 to 1:100 do not markedly affect reductions in population, at least if initial inoculum numbers are 4 to 5 log CFU/g. The rate of decrease in free chlorine concentration in treatment solution as affected by the lettuce/solution ratio 1. Beuchat, L. R Listeria monocytogenes. Incidence on vegetables. Food Control 7: Beuchat, L. R Ecological factors in uencing survival and growth of human pathogens on raw fruits and vegetables. Microbes Infect. 4: Beuchat, L. R., and R. E. Brackett Growth of Listeria monocytogenes on lettuce as in uenced by shredding, chlorine treatment, modi ed atmosphere packaging, temperature and time. J. Food Sci. 55: , Blenden, D. C., and F. T. Szatalowicz Ecological aspects of listeriosis. J. Am. Vet. Med. Assoc. 151: Burnett, S. L., and L. R. Beuchat Human pathogens associated with raw produce and unpasteurized juices, and dif culties in decontamination. J. Ind. Microbiol. Biotechnol. 27: Delaquis, P., S. Stewart, S. Cazaux, and P. Toivonen Survival and growth of Listeria monocytogenes and Escherichia coli O157: H7 in ready-to-eat iceberg lettuce washed in warm chlorinated water. J. Food Prot. 65: Francis, G. A., C. Thomas, and D. O Beirne The microbiological safety of minimally processed vegetables. Int. J. Food Sci. Technol. 34: Harvey, J., and A. Gilmour Occurrence and characteristics of Listeria in foods produced in Northern Ireland. Int. J. Food Microbiol. 19: Heisick, J. E., D. E. Wagner, M. L. Nierman, and J. T. Peeler Listeria spp. found on fresh market produce. Appl. Environ. Microbiol. 55:

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