Synergistic Effect of Chlorine Dioxide and Drying Treatments for Inactivating Escherichia coli O157:H7 on Radish Seeds

Transcription

1 1225 Journal of Food Protection, Vol. 73, No. 7, 2010, Pages Copyright G, International Association for Food Protection Synergistic Effect of Chlorine Dioxide and Drying Treatments for Inactivating Escherichia coli O157:H7 on Radish Seeds HOIKYUNG KIM, 1 HAEYOUNG KIM, 2 JIHYUN BANG, 2 LARRY R. BEUCHAT, 3 AND JEE-HOON RYU 2 * 1 Division of Human Environmental Sciences, Wonkwang University, Shinyong-dong, Iksan, Jeonbuk , Republic of Korea; 2 Graduate School of Life Sciences and Biotechnology, Korea University, Anam-dong, Sungbuk-ku, Seoul , Republic of Korea; and 3 Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, Georgia , USA MS : Received 4 January 2010/Accepted 12 March 2010 ABSTRACT Studies were done to determine whether calcium hypochlorite (Ca(OCl) 2 ) and chlorine dioxide (ClO 2 ) treatment followed by drying had a synergistic killing effect on microorganisms on radish seeds intended for sprout production. Uninoculated radish seeds and seeds inoculated with Escherichia coli O157:H7 were treated with water, Ca(OCl) 2 (free chlorine concentrations of 50 or 200 mg/ml), or ClO 2 (50 or 200 mg/ml) for 5 min and subsequently dried at 25uC for up to 24 h. Populations of total aerobic bacteria (TAB), molds and yeasts (MY), and E. coli O157:H7 on the seeds treated with Ca(OCl) 2 were not significantly different (P ~ 0.05) than populations on seeds treated with ClO 2 at the same concentrations. However, populations of microorganisms on seeds treated with ClO 2 decreased more rapidly during drying. Treatment with ClO 2 (200 mg/ml) followed by drying caused reductions in TAB, MY, and E. coli O157:H7 of 3.1, 2.0, and 3.8 log CFU/g, respectively. When seeds were treated with water, Ca(OCl) 2 (50 or 200 mg/ml), and ClO 2 (50 mg/ml) and subsequently dried, reductions in TAB, MY, and E. coli O157:H7 were 0.2 to 2.0, 0.4 to 2.0, and 1.4 to 2.2 log CFU/g, respectively. Results indicate that inactivation of E. coli O157:H7 on radish seeds is greater after treatment with ClO 2 followed by drying than after treatment with Ca(OCl) 2 followed by drying, thus providing a synergistic treatment combination for reducing the safety risk associated with sprouts produced from these seeds. The consumption of vegetable seed sprouts has increased in many countries in last decade because sprouts are relatively easy to produce and are perceived to have a high nutritional value. Concurrent with increased consumption, the number of outbreaks of foodborne illnesses associated with sprouts also has increased. Consumption of vegetable sprouts has been reported to have caused at least 37 outbreaks of foodborne diseases in several countries (9). Alfalfa, radish, and mung bean sprouts are the sprout types most often implicated in outbreaks (20), and the most frequently associated causative agents are Salmonella and Escherichia coli O157:H7. Most sprout-associated outbreaks have been linked to seeds that were contaminated with pathogenic microorganisms (16) rather than to contamination of sprouts after production. Populations of pathogenic bacteria on seeds can dramatically increase during sprout production. Thus, pathogens on seeds should be eliminated before sprouting to reduce the risk of outbreaks of foodborne illnesses associated with consumption of sprouts. Several researchers have investigated various sanitizers for their effectiveness in inactivating pathogenic bacteria on seeds intended for sprout production (1, 6, 7, 22, 25). However, most single-sanitizer treatments of artificially * Author for correspondence. Tel: ; Fax: ; escheri@korea.ac.kr. inoculated seeds do not result in more than a 3-log reduction in foodborne pathogens (27), with some exceptions (3, 13). Many reasons may exist for the minimal lethal effects of sanitizers on microorganisms on seeds. One reason for the lack of efficacy may be that the pathogens are located in areas of seeds inaccessible to sanitizers (7, 20). Fett (4) reported that foodborne pathogens on alfalfa seeds that had been immersed in culture suspensions were harbored deep within the hilum, micropyle, or cracks. Attachment of cells and the formation of biofilms by microorganisms on the seed surface are additional factors that may increase resistance of microorganisms to sanitizers. Attached cells and cells in biofilms are more resistant to sanitizers than are planktonic cells (5, 8, 19, 24). Rapid neutralization of sanitizers by organic constituents in surface tissues of seeds also may limit lethal activity. To achieve greater reductions in numbers of microorganisms on seeds intended for production of sprouts, two or more types of treatments, e.g., chlorine-based sanitizers, organic acids, heat, and high pressure, applied in combination either simultaneously or sequentially are more effective than single treatments (2, 10, 14, 17, 21, 28). Although in most studies populations of foodborne pathogens on seeds treated with multiple stressors decreased to a greater extent than did populations on seeds receiving a single stress treatment, elimination of pathogens on seeds without decreasing the germination percentage is difficult. A

2 1226 KIM ET AL. J. Food Prot., Vol. 73, No. 7 combination of treatments to maximize lethality to microorganisms without adversely affecting seed germination has not been optimized. To maximize lethality of a combination of stress agents, it is first necessary to determine whether there are additive or synergistic effects from simultaneous or sequential application of treatments. Appropriate combinations of stress agents may lead to enhanced efficacy of treatments to inactivate pathogenic microorganisms on seeds intended for sprout production. According to previous research (12, 18), spores of Bacillus cereus treated with chlorine dioxide (ClO 2 ) were more sensitive to wet heat than to treatment with chlorine. In this study, the synergistic effects of ClO 2 plus drying for killing spoilage microorganisms and E. coli O157:H7 were investigated. The combined effect of calcium hypochlorite (Ca(OCl) 2 ) plus drying also was tested because Ca(OCl) 2 is recommended by the National Advisory Committee on Microbiological Criteria for Food for decontamination of sprout seeds (16). Therefore, the objective of this study was to determine the effectiveness of chlorine (from Ca(OCl) 2 ) and ClO 2 sanitizers at various concentrations followed by drying for killing total aerobic bacteria (TAB), molds and yeasts (MY), and E. coli O157:H7 on radish seeds. MATERIALS AND METHODS Preparation of inoculum. Five strains of E. coli O157:H7 were used: ATCC (from hamburger), E0018 (from bovine feces), F4546 (from a patient in an outbreak associated with alfalfa sprouts), H1730 (from a lettuce-associated outbreak), and 932 (from a patient with hemorrhagic colitis). Laboratory stock cultures were activated by transferring loop inocula into 10 ml of tryptic soy broth (TSB; BBL, Becton Dickinson, Sparks, MD) and then adapted to grow in TSB supplemented with nalidixic acid (50 mg/ ml) (TSBN) at 37uC for 24 h. After three consecutive transfers of ca. 10 ml of culture into TSBN (10 ml) at 24-h intervals, 2 ml aliquots of each strain culture were combined to give 10 ml of a five-strain mixture containing approximately equal populations of each strain. Cultures were collected by centrifugation (2,000 g for 15 min at 25uC), and cells were resuspended in 10 ml of sterile distilled water. The culture suspension was diluted in an appropriate volume of sterile distilled water as needed to achieve a population of 8 log CFU/ml. Inoculation of radish seeds with E. coli O157:H7. Radish seeds were obtained from a retail market (Saessakmart, Seoul, Korea). Seeds (350 g) were immersed in 1,050 ml of the E. coli O157:H7 suspension (ca. 8 log CFU/ml) with gentle swirling for 5 min at room temperature (25 1uC). The inoculum suspension was decanted, and seeds were placed on a sterile sieve (203 mm in diameter by 41 mm deep; 600-mm pore size) and dried for 2 h at 25 1uC in a laminar flow biosafety hood before use in experiments. Preparation of sanitizers. Ca(OCl) 2 solutions (free chlorine concentrations of 50 and 200 mg/ml, ph 6.8) and ClO 2 solutions (50 and 200 mg/ml, ph 3.8) were prepared. To prepare Ca(OCl) 2 solutions, Ca(OCl) 2 (Sigma-Aldrich, Milwaukee, WI) was combined with potassium phosphate buffer (0.05 M, ph 6.8) and held at 25 1uC for 1 h. The Ca(OCl) 2 solutions were diluted in potassium phosphate buffer to obtain free chlorine concentrations of 50 and 200 mg/ml. A ClO 2 solution was prepared by adding 1 N HCl to 200 ml of sodium chlorite solution (10,000 mg/ml). This solution was held at 25 1uC for 1 h and then diluted in sterile distilled water to give ClO 2 concentrations of 50 and 200 mg/ml. All sanitizers were used in the experiments immediately after preparation. Free chlorine concentrations from Ca(OCl) 2 and ClO 2 concentrations were measured using a chlorine colorimeter (model Dr/820, Hatch, Loveland, CO). Treatment of radish seeds with sanitizers and drying. Radish seeds (65 g; not inoculated or inoculated with E. coli O157:H7) were placed in a sterile glass bottle, and 195 ml of sterile water, chlorine (50 and 200 mg/ml) solution, or ClO 2 (50 and 200 mg/ml) solution was added. The mixtures (seeds plus water or sanitizer) were incubated at 25 1uC for 5 min with intermittent swirling. After treatment, water or sanitizer was decanted, and the seeds were rinsed twice in 195 ml of sterile distilled water. The treated seeds were placed on a sterile sieve and dried for up to 24 h at 25 1uC in a laminar flow biosafety hood. The relative humidity in the biosafety hood was 40 2% during drying. Populations of TAB were determined on uninoculated seeds, and populations of MY and E. coli O157:H7 were determined on inoculated seeds before treatment with water or sanitizer (0 h), after treatment for 5 min, and after drying for 1, 3, 6, 12, and 24 h. Microbiological analyses. To determine the TAB on radish seeds, 10 g of uninoculated seeds before and after treatment with water or sanitizer was combined with 100 ml of TSB in a 400-ml polyolefin stomacher bag (Interscience, St. Nom La Breteche, France) and pummeled for 1 min. Undiluted TSB rinsate (0.25 ml in quadruplicate and 0.1 ml in duplicate) and rinsate serially diluted in sterile 0.1% peptone water (0.1 ml in duplicate) were surface plated on tryptic soy agar (BBL, Becton Dickinson). Plates were incubated at 37uC for 48 h, and then colonies were counted. To determine populations of E. coli O157:H7 on inoculated seeds, 10 g of seeds was combined with 100 ml of TSB in a 400-ml stomacher bag and pummeled for 1 min, and undiluted and serially diluted samples of TSB rinsate were spread plated on MacConkey sorbitol agar (BBL, Becton Dickinson) supplemented with nalidixic acid (50 mg/ml) (MSAN). Samples from the TSB rinsate also were plated on dichloran rose bengal chloramphenicol agar (DRBC; Becton Dickinson) to determine MY counts. MSAN plates were incubated at 37uC for 24 h and DRBC plates were incubated at 25uC for 5 days before colonies were counted. Five presumptive colonies of E. coli O157:H7 from randomly selected MSAN plates were subjected to the E. coli O157:H7 latex agglutination test (Oxoid, Basingstoke, UK) for confirmation. Determination of germination of radish seeds. Radish seeds (n ~ 100) treated with water or sanitizer and dried for 24 h at 25 1uC in a laminar flow biosafety hood were placed on sterile cheesecloth in a commercial sprout cultivator (225 by 325 by 150 mm; Shinhan Innovation & Creative, Suwon, Republic of Korea) containing sterile distilled water and incubated at 25uC for 5 days. The number of seeds that germinated was counted, and the germination percentage was calculated. Statistical analyses. All experiments were performed in triplicate. Data were analyzed using the general linear model of the Statistical Analysis Systems procedure (version 9.1; SAS Institute, Cary, NC). Combined effects of water or sanitizer treatment plus drying were determined by comparing the number of surviving microorganisms using Fisher s least significant difference test. Significant differences are presented at a 95% confidence level (P # 0.05).

3 J. Food Prot., Vol. 73, No. 7 SYNERGISTIC LETHAL EFFECT BETWEEN CHLORINE DIOXIDE AND DRYING 1227 RESULTS AND DISCUSSION The hypothesis tested in this study was that when sanitizer treatment of radish seeds is followed by drying to kill microorganisms, the extent of lethality caused by drying is influenced by the type and concentration of sanitizer. Support for this hypothesis would suggest that the proper selection and concentration of sanitizer are important factors for optimizing the lethal activity of the combined sanitizer and drying treatments. Table 1 shows the TAB on radish seeds before and after treatment with water, chlorine (50 or 200 mg/ml), or ClO 2 (50 or 200 mg/ml) plus drying at 25 1uC for up to 24 h. The initial population of TAB on radish seeds was 4.1 to 4.4 log CFU/g. A similar population of TAB was found on radish seeds by Kim et al. (11), who profiled the microbiological quality of sprouts and seeds sold at retail shops in Seoul, Korea. These authors reported that radish seed TAB populations were 4.08 log CFU/g. After treatment of the seeds with water or with chlorine or ClO 2 at 50 mg/ml for 5 min, populations of TAB did not decrease significantly (P ~ 0.05). However, when seeds were treated with chlorine or ClO 2 at 200 mg/ml, the TAB population decreased significantly, by 1.4 to 1.5 log CFU/g. To minimize the residual effects of sanitizer during drying, radish seeds treated with sanitizers were rinsed with sterile water twice for 1 min. After the second rinsing, the concentrations of chlorine and ClO 2 on seeds were to and to mg/g of seed, respectively. During drying for 24 h, TAB populations on radish seeds that had been treated with water, chlorine at 50 or 200 mg/ml, or ClO 2 at 50 mg/ml did not decrease significantly. However, the TAB populations on radish seeds treated with ClO 2 at 200 mg/ml decreased significantly within 6 h, followed by a further significant decrease between 6 and 24 h. A 2.4-log reduction occurred as a result of drying seeds for 24 h. This finding indicates that chlorine and ClO 2 at 200 mg/ml had similar initial lethal activities, but with prolonged drying only ClO 2 at 200 mg/ml caused additional lethality. Overall, the combined effects of ClO 2 (200 mg/ml) treatment plus drying caused a.3.1-log reduction in the TAB population on radish seeds, whereas water, chlorine at 50 or 200 mg/ml, and ClO 2 at 50 mg/ml plus the drying treatment caused 0.9-, 0.2-, 2.0-, and 1.8-log reductions, respectively. Changes in populations of MY on radish seeds treated with water, chlorine, or ClO 2 plus drying for 24 h were monitored because MY populations also can affect the quality of sprouts (Table 2). The initial MY population on untreated seeds was 3.2 log CFU/g. In a previous study, the MY count on radish seeds obtained from commercial sources was 2.4 log CFU/g (11). After treatment with ClO 2 at 200 mg/ml, the MY count was significantly lower (1.6 log CFU/g). Other treatments did not cause a significant reduction. After drying for 24 h, the MY population on radish seeds treated with water or chlorine at 50 or 200 mg/ ml did not change significantly. However, the MY population on radish seeds treated with ClO 2 (50 mg/ml) decreased significantly, from 2.3 to,1.2 log CFU/g during TABLE 1. Populations of total aerobic bacteria on uninoculated radish seeds treated with water or sanitizer (chlorine or ClO2) and dried at 25uC for up to 24 h Population (log CFU/g) a Treatment time (min): Drying time (h): concn (mg/ml) Water 0 A a A abc A abc A ab A bc A bc AB c Chlorine 50 A a A a AB a AB a A a AB a A a 200 A a B b AB b BC b AB b CD b CD b ClO 2 50 A a AB abc AB ab BC bc A abc BC bc BC c 200 A a B b B bc C bcd B cd D cd D,1.0 b 0.0 d a Within a row, values followed by the same lowercase letter are not significantly different (P. 0.05). Within a column, values preceded by the same uppercase letter are not significantly different (P. 0.05). b Number of colonies was below the detection limit (1 log CFU/g) for at least one of three replicates.

4 1228 KIM ET AL. J. Food Prot., Vol. 73, No. 7 TABLE 2. Populations of molds and yeasts on inoculated radish seeds treated with water or sanitizer (chlorine or ClO2) and dried at 25uC for up to 24 h Population (log CFU/g) a Treatment time (min): Drying time (h): concn (mg/ml) Water 0 A a A ab AB ab A ab A ab AB b A ab Chlorine 50 A ab AB b A a A b A ab A ab A ab 200 A a AB ab B ab A,2.3 b 1.2 ab A ab B b A ab ClO2 50 A a AB bc AB ab A c B cd C, d B, d 200 A a B b C b A, b C, b C, b B, b a Within a row, values followed by the same lowercase letter are not significantly different (P. 0.05). Within a column, values preceded by the same uppercase letter are not significantly different (P. 0.05). b Number of colonies was below the detection limit (1 log CFU/g) for at least one of three replicates. the 24-h drying period. Thus, treatment with ClO 2 at 50 mg/ ml resulted in increased lethality for MY during the drying treatment. The MY population on radish seeds treated with ClO 2 at 200 mg/ml decreased to,1.0 log CFU/g within 6 h. Overall, the combined effects of ClO 2 (50 or 200 mg/ml) and drying treatment caused a 2.0-log reduction in the MY population on radish seeds, whereas treatment with water or chlorine at 50 or 200 mg/ml plus drying caused 0.4-, 0.4-, and 0.5-log reductions, respectively. Table 3 lists the surviving E. coli O157:H7 populations on radish seeds after treatment with water, chlorine (50 or 200 mg/ml), or ClO 2 (50 or 200 mg/ml) followed by drying for up to 24 h. The initial population was 6.1 log CFU/g of radish seeds. Seeds treated with water had the highest population of E. coli O157:H7 before drying, although the population was not significantly different from that on seeds treated with chlorine. The E. coli O157:H7 population on seeds after treatment with ClO 2 was significantly lower than that on seeds after treatment with water but not after treatment with chlorine. Similar reductions have been reported in other studies. Taormina and Beuchat (26) found that on alfalfa seeds inoculated with E. coli O157:H7 and treated with chlorine (200 mg/ml) for 3 and 10 min, there was a,0.5-log reduction in the pathogen. Singh et al. (23) found a 1.14-log reduction in the E. coli O157:H7 population after treatment of alfalfa seeds with ClO 2 (50 mg/ml) for 5 min. In another study (15), a,1-log reduction in E. coli O157:H7 was observed in radish seeds treated with ClO 2 (200 mg/ml) for 5 min. In our study, the E. coli O157:H7 populations on radish seeds decreased significantly during drying regardless of the type of sanitizer used to treat seeds before drying. The E. coli O157:H7 population on seeds treated with water gradually decreased by 1.1 log CFU/g during drying for 24 h. This finding led to an assessment of the possible additive effects of sanitizer and drying treatments for killing E. coli O157:H7 on radish seeds. The E. coli O157:H7 population on seeds treated with water, chlorine at 50 mg/ml, and chlorine at 200 mg/ml decreased by 1.1, 0.8, and 0.7 log CFU/g, respectively, after seeds were dried for 24 h. Thus, when compared with the water treatment followed by drying, the chlorine treatment plus the drying treatment did not have a synergistic killing effect on E. coli O157:H7. However, seeds treated with ClO 2 (50 or 200 mg/ml) and dried for 24 h had significantly lower E. coli O157:H7 populations compared with seeds treated with water or chlorine and then dried. The E. coli O157:H7 population on radish seeds treated with ClO 2 at 200 mg/ml decreased from an initial 5.0 log CFU/g to 2.3 log CFU/g after drying for 24 h. Overall, combined treatments of ClO 2 (200 mg/ml) plus drying caused a 3.8-log reduction in the population of E. coli O157:H7 on radish seeds, whereas treatment with water, chlorine at 50 and 200 mg/ml, and ClO 2 at 50 mg/ml combined with drying caused reductions of only 1.5, 1.4, 1.5, and 2.2 log CFU/g, respectively. Table 4 lists the germination percentages for radish seeds after treatment with water or sanitizer for 5 min followed by drying for 24 h. Regardless of the type or concentration of sanitizer, the germination percentage was not significantly different from that of seeds treated with water.

5 J. Food Prot., Vol. 73, No. 7 SYNERGISTIC LETHAL EFFECT BETWEEN CHLORINE DIOXIDE AND DRYING 1229 TABLE 3. Populations of E. coli O157:H7 on inoculated radish seeds treated with water or sanitizer (chlorine or ClO2) and dried at 25uC for up to 24 h Population (log CFU/g) a Treatment time (min): Drying time (h): concn (mg/ml) Water 0 A a A a A a A b A b A b A b Chlorine 50 A a AB ab AB bc AB c A c A c A c 200 A a AB bc AB ab AB cd A d A d A d ClO2 50 A a B b B b AB bc B c A c B c 200 A a B b C bc B cd C cde B de C e a Within a row, values followed by the same lowercase letter are not significantly different (P. 0.05). Within a column, values preceded by the same uppercase letter are not significantly different (P. 0.05). TABLE 4. Germination percentage for radish seeds treated with water, chlorine, or ClO 2 and dried for 24 h a concn (mg/ml) Germination (%) Water Chlorine ClO a After treatment, radish seeds (n ~ 100) were germinated in a commercial sprout cultivator at 25uC for 5 days. Synergism between sequential sanitizer and drying treatments for killing microorganisms was a focus of this research. The effect of treatment with sanitizers such as chlorine and ClO 2 on subsequent resistance of microorganisms to other types of stresses, e.g., heat, was investigated. Kreske et al. (12) found that treatment of planktonic spores of B. cereus with chlorine or ClO 2 resulted in decreased tolerance to heat treatment; spores treated with ClO 2 were more sensitive to wet heat than were spores treated with chlorine at the same concentration. Ryu and Beuchat (18) observed that B. cereus spores in biofilm that survived treatment with ClO 2 had reduced resistance to wet heat; these authors concluded that chlorine-based sanitizers may have caused injury to bacterial spores, resulting in decreased heat tolerance. Our observations on E. coli O157:H7 sequentially exposed to ClO 2 and drying are in agreement with those showing decreased tolerance of sublethally stressed microbial cells to subsequent assault with a second stress. Populations of TAB, MY, and E. coli O157:H7 on radish seeds treated with ClO 2 were not significantly different from populations from seeds treated with chlorine at the same concentration. However, the viability of cells exposed to ClO 2 decreased more rapidly than did that of cells that had been exposed to chlorine or water. The synergistic effects of ClO 2 and drying for killing microorganisms on radish seeds were more pronounced when cells were treated with ClO 2 at 200 mg/ ml compared with 50 mg/ml. The results of this study indicate that when sanitizer and drying stresses are combined to reduce the microbial population on radish seeds, ClO 2 is more efficacious than chlorine at the same concentration. Treatment with the higher concentration of ClO 2 (200 mg/ml) acted synergistically with drying for killing microorganisms. Further studies are required to optimize the concentration of ClO 2 and relative humidity to induce maximum synergistic effects without decreasing the germination of the radish seeds. Other types of stresses in combination with ClO 2 also should be considered to maximize microbial reduction. For example, a combined treatment with ClO 2 and drying at higher temperature should be evaluated for its effectiveness in reducing foodborne pathogens and other microorganisms on seeds intended for sprout production. ACKNOWLEDGMENT This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) ( F00112).

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