Reduction of Microbial Pathogens during Apple Cider

766 Journal of Food Protection, Vol. 67, No. 4, 2004, Pages 766–771 Copyright Q , International Association for Food Protection

Reduction of Microbial Pathogens during Apple Cider Production Using Sodium Hypochlorite, Copper Ion, and Sonication STEPHANIE L. RODGERS1,2 1 Department

AND

ELLIOT T. RYSER 1*

of Food Science and Human Nutrition, 2108 South Anthony Hall, Michigan State University, East Lansing, Michigan 48824-1224; and 2Abbott Laboratories, Ross Products Division 625 Cleveland Avenue, Columbus, Ohio 43215-1724, USA MS 03-54: Received 14 February 2003/Accepted 24 October 2003

ABSTRACT Sodium hypochlorite (100 ppm), copper ion water (1 ppm), and sonication (22 to 44 kHz and 44 to 48 kHz) were assessed individually and in combination for their ability to reduce populations of Escherichia coli O157:H7 and Listeria monocytogenes on apples and in apple cider. Commercial unpasteurized cider was inoculated to contain approximately 106 CFU/ml of either pathogen and then sonicated at 44 to 48 kHz, with aliquots removed at intervals of 30 to 60 s for up to 5 min and plated to determine numbers of survivors. Subsequently, whole apples were inoculated by dipping to contain approximately 106 CFU/ g E. coli O157:H7 or L. monocytogenes, held overnight, and then submerged in 1 ppm copper ion water with or without 100 ppm sodium hypochlorite for 3 min with or without sonication at 22 to 44 kHz and examined for survivors. Treated apples were also juiced, with the resulting cider sonicated for 3 min. Populations of both pathogens decreased 1 to 2 log CFU/ml in inoculated cider following 3 min of sonication. Copper ion water alone did not signi cantly reduce populations of either pathogen on inoculated apples. However, when used in combination with sodium hypochlorite, pathogen levels decreased approximately 2.3 log CFU/g on apples. Sonication of this copper ion–sodium hypochlorite solution at 22 to 44 kHz did not further improve pathogen reduction on apples. Numbers of either pathogen in the juice fraction were approximately 1.2 log CFU/ml lower after being juiced, with sonication (44 to 48 kHz) of the expressed juice decreasing L. monocytogenes and E. coli O157:H7 populations an additional 2 log. Hence, a 5-log reduction was achievable for both pathogens with the use of copper ion water in combination with sodium hypochlorite followed by juicing and sonication at 44 to 48 kHz.

During the last 10 years, the microbiological safety of fresh apple cider has come under close scrutiny, with outbreaks of Escherichia coli O157:H7 infection continuing to pose signi cant challenges to both cider producers and regulatory agencies. Since 1990, at least six documented outbreaks of E. coli O157:H7 infection in North America were associated with unpasteurized apple cider, with ve of these outbreaks occurring in the United States over a 3-year period (3, 8, 22). Another potential contaminant of apples is Listeria monocytogenes. This pathogen is commonly found on decaying vegetation and in soil, animal feces, sewage, silage, and water (4). The acid tolerance and psychrotrophic nature of L. monocytogenes makes this pathogen another likely (23, 28), but as yet unidenti ed, threat to the cider industry. The increased frequency of outbreaks has heightened the need for regulations to enhance the safety of apple cider. Consequently, the U.S. Food and Drug Administration (FDA) published the Juice Hazard Analysis and Critical Control Points (HACCP) Final Rule on 19 January 2001 requiring fresh juice manufacturers to adopt procedures, such as thermal (23) or ultraviolet pasteurization (31) or other treatments (12), that will achieve at least a 5-log reduction in bacterial levels in the nal product (11). How* Author for correspondence. Tel: 517-355-7713, Ext 185; Fax: 517-3531676; E-mail: [email protected].

ever, producers of small batches of cider, who sell only at retail, are exempt from these regulations, with these individuals able to incorporate other microbial reduction strategies, such as short-term storage at an elevated temperature, freezing and thawing, or addition of organic acids and preservatives either alone or in combination (10, 26, 27), in lieu of pasteurization to enhance the safety of their cider. Commercially, apples are most commonly washed in chlorine-based sanitizers and less frequently ozonated water before pressing. However, both of these treatments have yielded only a 2- to 3-log maximum microbial reduction on apples (1, 5, 30), with survivors being reported in the stem and calyx regions (2, 13) as well as within cracks of the waxy cuticle (13). Furthermore, chlorine-based sanitizers can react to produce trace amounts of carcinogenic organochlorine compounds and are rapidly inactivated by organic material inherent to the surface of incoming unwashed apples. Alternative microbial reduction strategies that are effective against a wide range of bacteria, do not produce harmful by-products, and are unaffected by the presence of organic matter are gaining interest. One such option is sonication, a process that involves emission of high-frequency sound waves through a liquid medium to dislodge organic debris and mechanically disrupt bacterial cells. Although routinely used to clean equipment in medical and dental

J. Food Prot., Vol. 67, No. 4

MICROBIAL REDUCTION ON APPLES USING CHLORINE, COPPER ION, AND SONICATION

of ces (24, 29), sonication has not yet seen widespread use in the food industry, with investigations generally limited to uid milk (25, 33), eggs (33), and poultry chill water (21). At least one study indicated that the ef cacy of sonication was enhanced in the presence of a chlorinated sanitizer (20). Although sonicating a Salmonella Typhimurium cell suspension (108 cells per ml of peptone) at 20 kHz for 55 min reportedly decreased the pathogen to nondeductible levels, this same study showed a 1- to 1.5-log reduction of Salmonella populations attached to broiler skin by sonicating in peptone water at 20 kHz for 30 min, a less than 1log reduction by chlorine alone, and a 2.4- to 4-log reduction by sonicating in a solution containing 0.5 ppm free residual chlorine. Copper ion has been reported to possess antibacterial activity against a wide range of organisms, including E. coli, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Legionella pneumophila, at concentrations of less than 1 ppm, with the ef cacy of copper ion synergistically enhanced when used in combination with chlorinated sanitizers (14–16). The antibacterial action of copper ion has been attributed to interference with cellular respiration (9) and binding to speci c DNA sites (18, 19). In addition, copper ion is nonvolatile; is noncorrosive to processing equipment, even at high temperatures; and does not produce any known off-odors, off-tastes, or toxic by-products. Although information concerning the use of copper ion for decreasing microbial loads on fresh produce is scarce, several in vitro studies indicate that copper ion might be potentially useful as a produce sanitizer, with Kutz et al. (14) reporting a 4.2-log reduction in numbers of E. coli O157:H7 after 1 min of exposure to 0.4 ppm copper ion. The objective of this study was to assess the individual and combined use of copper ion, sodium hypochlorite, juicing, and sonication (low and high frequency) to reduce populations of E. coli O157:H7 and L. monocytogenes during apple cider production, with such a treatment providing an economically viable alternative for producers of small batches of cider to enhance the microbial safety of their product. MATERIALS AND METHODS Bacterial strains. Three strains of E. coli O157:H7 (AR [acid-resistant], AD 305, AD 317 originally obtained from A. Datta) and L. monocytogenes (CWD 17, CWD 95, CWD 246 originally isolated from silage or raw milk) were obtained from C. W. Donnelly (Department of Nutrition and Food Sciences, University of Vermont, Burlington). Stock cultures were maintained at 2708C in Trypticase soy broth (TSB; Difco Laboratories, Becton Dickinson, Sparks, Md.) containing 10% (vol/vol) glycerol (Sigma Chemical Co., St. Louis, Mo.) and were subcultured twice in TSB (9 ml) containing 0.6% (wt/vol) yeast extract (TSBYE; Difco) at 358C for 18 to 24 h before use. Culture preparation. Individual cultures were centrifuged at 10,000 3 g for 15 min at 48C to obtain a pellet, which was resuspended in 30 ml of sterile tap water to simulate commercial practices. These suspensions served as inocula for commercial

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unpasteurized apple cider. Equal volumes (10 ml) of these cultures were also combined to produce three-strain cocktails of E. coli O157:H7 and L. monocytogenes for use on whole apples. These cocktails were similarly centrifuged and resuspended in 30 ml of sterile tap water. Inoculation of cider. Commercially prepared unpasteurized apple cider was obtained locally, stored at 48C, and then tempered to room temperature (;258C) before being inoculated with the three strains of E. coli O157:H7 and L. monocytogenes at 106 CFU/ml. Inoculation of apples. Unwaxed, blemish-free Golden Delicious apples of uniform size and shape (2½- to 2¾-in. [6.4- to 7.0-cm] diameter, weighing ;40 g each) were obtained from a local cider mill and stored at 48C. Six to eight apples were placed in sterile polyethylene bags (25 by 20 cm) containing 300 ml of sterile distilled water that was previously inoculated from the three-strain 18- to 24-h-old cocktails of E. coli O157:H7 or L. monocytogenes. The apples in each bag were agitated with a sterile glass rod for 20 min to ensure even inoculation at a level of about 106 CFU/g. Thereafter, the apples were removed from the bag and air dried in a laminar ow hood for 18 to 24 h at 248C in an attempt to obtain attached cells before being subjected to the various treatments. Sonication of cider. Inoculated cider samples (1.5 ml) were placed in sterile microcentrifuge tubes and sonicated at 44 to 48 kHz for 5 min at 20 to 258C in a water-jacketed sonicator (Vibra cell 600-W ultrasonic processor; Sonics and Materials, Inc., Newtown, Conn.) to determine optimal conditions for destruction of the three individual strains of E. coli O157:H7 and L. monocytogenes. Initially and at 30- to 60-s intervals, the microcentrifuge tubes were removed from the sonicator over a period of 5 min, and 1-ml samples were serially diluted in 0.1% peptone and spiral plated (400 Autoplate Automated Spiral Plater, Spiral Biotech, Inc., Bethesda, Md.) on sorbitol MacConkey agar (SMAC) (Difco) or modi ed Oxford agar (MOX; Difco) for enumeration of E. coli O157:H7 and L. monocytogenes, respectively. All plates were counted after 24 h of incubation at 378C, with all treatments performed in triplicate. Preparation of sanitizer solutions. A sanitizer solution containing 100 ppm total residual chlorine was prepared by adding 1.13 g of sodium hypochlorite (Johnson Wax Professional, Racine, Wis.) to 1 liter of sterile distilled water. Total residual chlorine was measured by a chlorine colorimetric test kit (Hach Co., Ames, Iowa). Copper ion water was generated with a pilot plant–sized copper ion generator that pumped electrolytically generated copper ions into a stream of sterile distilled water (Superior Water Systems Inc., Fort Wayne, Ind.). Copper ion concentration was determined before treatment by a colorimetric copper ion test kit (model EC-20; La Motte Chemical Products Co., Inc., Chestertown, Md.). Sodium thiosulfate (0.1 N) stock solution was prepared by dissolving 25 g of sodium thiosulfate (Sigma) in 1 liter of sterile distilled water. This solution was used to neutralize residual chlorine, with 1-ml aliquots added to the rst tube for serial dilution. Apple processing. Populations of E. coli O157:H7 and L. monocytogenes were determined in the following samples: (i) inoculated apples, (ii) inoculated apples after sonication and sanitizer treatment, (iii) apple pulp after juicing, (iv) apple cider, and (v) apple cider after sonication. Inoculated apples were drained, weighed (;40 g each), placed individually in bags containing 100 ml of 0.1% peptone, shaken vigorously for 5 min, and

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FIGURE 1. Average reductions of E. coli O157:H7 and L. monocytogenes in apple cider during 5 min of sonication at 44 to 48 kHz (n 5 3).

then rubbed and massaged for an additional 10 min. Triplicate samples of apple wash water were then serially diluted, spiral plated on SMAC or MOX, and incubated for 24 h at 378C to determine numbers of E. coli O157:H7 and L. monocytogenes, respectively. Inoculated whole apples were placed in sterile polyethylene bags (25 by 20 cm) containing approximately 300 ml of water, 1 ppm copper ion water or 1 ppm copper ion water with 100 ppm sodium hypochlorite. These bags were then submerged in a 1.4liter-capacity sonicating water bath (22 to 44 kHz; Fisher Ultrasonic Cleaner model FS140, Hanover Park, Ill.) and sonicated for 3 min. After sonication, apples were removed from the bags, drained, weighed, placed in individual Whirl-Pack bags containing 100 ml of 0.1% peptone (Difco) or 0.1% sodium thiosulfate (for samples containing chlorine) and vigorously shaken for 5 min followed by 10 min of rubbing. Triplicate samples of apple wash water were serially diluted in 0.1% peptone, spiral plated on SMAC and MOX, and then incubated for 24 h at 378C to quantify E. coli O157:H7 and L. monocytogenes, respectively. Apples previously sonicated in copper ion water, sodium hypochlorite, or copper ion water containing sodium hypochlorite were removed from the sonicating water bath, drained, weighed, and then individually processed in a juicerator (Omega OM 9000, Omega Products, Inc., Harrisburg, Pa.) for 5 min to obtain the cider and pulp fractions. Pulp samples were weighed, placed in individual sterile polyethylene bags containing 100 ml of 0.1% peptone, and homogenized in a stomacher (model SD-45, Tekmar Co., Cincinnati, Ohio) for 2 min. Triplicate samples of apple pulp were serially diluted, spiral plated on SMAC and MOX, and incubated for 24 h at 378C to quantify E. coli O157:H7 and L. monocytogenes, respectively. Samples (1 ml) of the cider fraction were serially diluted in 0.1% peptone, spiral plated on SMAC and MOX, and incubated for 24 h at 378C to enumerate E. coli O157:H7 and L. monocytogenes, respectively. All treatments were performed in triplicate. Finally, samples (1.5 ml) of the cider fraction were placed in microcentrifuge tubes and sonicated at 44 to 48 kHz for 3 min. Onemilliliter samples were serially diluted and spiral plated on SMAC and MOX and incubated for 24 h at 378C to enumerate E. coli O157:H7 and L. monocytogenes, respectively. All treatments were performed in triplicate. Statistical analysis. Microbial data obtained from triplicate trials were analyzed by a factorial analysis of variance on duplicate samples at a signi cance level of P , 0.05. Statistical results

were subjected to a Bonferroni adjustment for conservative analysis.

RESULTS Initial concentrations of E. coli O157:H7 and L. monocytogenes in inoculated cider ranged from 6.3 to 6.4 log CFU/ml (Fig. 1). Sonication reduced E. coli O157:H7 and L. monocytogenes populations 0.8 to 1.9 and 1.0 log, respectively, after 3 min, with no additional reductions observed after 5 min. Log reductions for the individual L. monocytogenes and E. coli O157:H7 strains were not signi cantly different from each other during sonication. When inoculated apples containing 6.3 to 6.4 log CFU/ g E. coli O157:H7 were treated with water (control) or 1 ppm copper ion water, no signi cant difference in reduction of E. coli O157:H7 was observed (Fig. 2). However, treatment with 100 ppm sodium hypochlorite as well as 1 ppm copper ion and 100 ppm sodium hypochlorite signi cantly (P , 0.05) reduced E. coli O157:H7 populations by 1.5 and 2.3 log, respectively. Juicing apples treated with 1 ppm copper ion water or 1 ppm copper ion water with 100 ppm sodium hypochlorite led to fractionation of E. coli O157: H7 between the cider and pulp. Populations of E. coli O157:H7 on apples treated with 1 ppm copper ion water were 4.68 and 1.25 log CFU/g or ml in the cider and pulp fractions, respectively, after juicing. Sonication of the remaining cider for 3 min decreased E. coli O157:H7 populations an additional 2 log. With the use of 100 ppm sodium hypochlorite–treated apples, 3.5 and 1.3 log CFU/g of E. coli O157:H7 were detected in the cider and pulp fractions, respectively, after juicing. Sonicating the resulting juice yielded an additional 1.6-log reduction. Juicing of apples following combined treatment with 1 ppm copper ion water and 100 ppm sodium hypochlorite yielded E. coli O157:H7 populations of 2.9 and 1.1 log CFU/g or ml in the cider and pulp, respectively. Further sonication of the resulting cider fraction decreased E. coli O157:H7 an additional 1.8 log. Inactivation of L. monocytogenes on apples and in cider with the use of sonication and 1 ppm copper ion or 100 ppm chlorine is depicted in Figure 3. Treatment with water



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FIGURE 2. Average reduction of E. coli O157:H7 at various stages in apple cider production with the use of copper ion, sodium hypochlorate, and sonication (n 5 3).

(control) or 1 ppm copper ion water yielded similar reductions for L. monocytogenes on inoculated apples, whereas populations decreased 1.3 and 2.2 log after treatment with 100 ppm sodium hypochlorite and 1 ppm copper ion and with 100 ppm sodium hypochlorite, respectively. Juicing inoculated apples treated with 1 ppm copper ion water and 100 ppm sodium hypochlorite yielded L. monocytogenes populations of 3.3 log CFU/ml and 1.0 log CFU/g in the cider and pulp fractions, respectively. After juicing 1 ppm copper ion water–treated apples, L. monocytogenes populations were 4.7 log CFU/ml and 1.1 log CFU/g in the cider and pulp fractions, respectively. Further sonication of the expressed juice reduced populations approximately 2.3 log CFU/ml. However, numbers of L. monocytogenes were 4.1 and 1.2 log CFU/g or ml in cider and pulp fractions, respectively, after treatment with 100 ppm sodium hypochlorite, with sonication reducing populations in cider an additional 2.3 log. Populations of L. monocytogenes on apples treated with 1 ppm copper ion water and 100 ppm sodium hypochlorite were 3.3 and 1.0 log CFU/g or ml in the pulp and cider fractions, respectively, after juicing. Sonication of the remaining cider for 3 min reduced L. monocytogenes populations an additional 2.1 log CFU/ml. Total pathogen reductions for inoculated apples that were treated with 1 ppm copper ion and water and subsequently converted into cider were approximately 3.5 and 4 log for E. coli O157:H7 and L. monocytogenes, respectively, with these reductions not signi cantly different from each other. Sodium hypochlorite treatment produced total reductions of 4.3 and 4.5 log for E. coli O157:H7 and L. monocytogenes, respectively. With the hurdle approach, the combination of 1 ppm copper ion and 100 ppm sodium hypochlorite followed by sonication was most effective, yielding a 5.3- and 5.1-log reduction of E. coli O157:H7 and L. monocytogenes, respectively, in the nal product. DISCUSSION Sonicating cider for 3 min reduced populations of E. coli O157:H7 and L. monocytogenes by 1.5 to 2.0 and 1.0 to 1.5 log, respectively. On the basis of practicality issues

for cider producers, a sonicating time of 3 min was selected for further study. However, a longer sonication period might have enhanced microbial reductions (25), with Wrigley and Llorca (33) reporting maximum inactivation of Salmonella Typhimurium after 30 min of sonication and Lee et al. (17) achieving a 4-log reduction for Salmonella in peptone water after a 10-min ultrasonic treatment. Individual strains of E. coli O157:H7 and L. monocytogenes did not differ signi cantly in their susceptibility to sonication in apple cider. These ndings suggest that sonication should produce consistent results in the destruction of both pathogens under the conditions tested. Susceptibility of L. monocytogenes to sonication was signi cantly lower compared with all strains of E. coli O157:H7 except strain AR. However, greater inactivation of L. monocytogenes likely could have been achieved if the sonication exposure time had been increased. Most sonication studies have focused on the reduction of enteric gram-negative pathogens in poultry chill water and milk (21). Our ndings suggest that the ef cacy of sonication also can be extended to apple cider and likely to other fruit juices. In 2001, the FDA passed a regulation requiring a 5log reduction of pathogens in juices, including cider (11). Producers of small batches of cider are not required to comply with these new standards and can choose to incorporate HACCP along with alternative methods of decontamination. In this study, the hurdle concept was investigated to achieve a 5-log reduction with the use of copper ion water, sodium hypochlorite, juicing, and sonication. Treatment of E. coli O157:H7– and L. monocytogenes–inoculated apples in water or copper ion water only reduced the initial inoculum levels about 0.3 to 0.4 log. These results are similar to those of many other researchers who used water to remove pathogens from produce. For example, Wright et al. (32) found that washing E. coli O157:H7–inoculated apples with water removed 1.1 log CFU/g. Similarly, Brackett (6) reported a 1-log reduction for L. monocytogenes on Brussels sprouts dipped in water. Reductions observed in

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FIGURE 3. Average reduction of E. coli O157:H7 at various stages in apple cider production with the use of copper ion, sodium hypochlorate, and sonication (n 5 3).

this study with the use of copper ion water alone can be attributed to a washing rather than an antimicrobial effect because log reductions were no greater than those observed for water alone. Chlorine concentrations of 50 to 300 ppm are most often used for reducing microbial populations on apples (7). However, E. coli O157:H7 can persist on inoculated apples in areas such as the stem and calyx, where chlorine is less able to gain access to attached bacterial cells (2). Free chlorine concentrations also decreased to ineffective levels after contact with organic material. In this study, 100 ppm chlorine reduced populations of E. coli O157:H7 and L. monocytogenes on apples 1.5 and 1.3 log CFU/g, respectively. These results are similar to those of Wright et al. (32), who examined the ef cacy of 200 ppm sodium hypochlorite for apples and found that E. coli O157:H7 populations decreased 2.1 log on the surface after a 2-min exposure. Copper ions have been used in combination with various sanitizers for synergistically inactivating bacterial cells. Although information on the use of copper ion as a microbial reduction strategy for fresh produce is scarce, several in vitro studies indicate that copper ion has potential as a produce sanitizer. Kutz et al. (14) reported a 4.2-log reduction for E. coli O157:H7 after a 1-min exposure to 0.4 ppm copper ion. In this study, copper ion water containing 100 ppm sodium hypochlorite signi cantly reduced the levels of E. coli O157:H7 and L. monocytogenes on apples by 2.3 and 2.2 log CFU/g, respectively. These results suggest that chlorine acts synergistically when used with copper ion. Because cider producers routinely use chlorinated wash water, the ef cacy of this treatment could be improved by incorporating copper ion. The process of juicing, which fractionates apples into cider and pulp, yielded pathogen populations of 1.0 to 1.3 and 2.8 to 4.7 log in the pulp and cider fractions, respectively. Hence, the decrease in numbers of E. coli O157:H7 and L. monocytogenes as a direct result of juicing should also be considered in any microbial reduction strategy.

Sonication of the cider after juicing reduced levels of E. coli O157:H7 and L. monocytogenes 1.8 to 2.0 and 2.2 to 2.6 log, respectively. For the total process, populations of E. coli O157:H7 and L. monocytogenes on apples treated with 0.1 ppm copper ion water followed by sonication of cider decreased 3.5 to 4.2 log. In contrast, reductions of 5.3 and 5.1 log were achieved for E. coli O157:H7 and L. monocytogenes, respectively, with the use of 0.1 ppm copper ion water containing 100 ppm sodium hypochlorite followed by juicing and sonication for 3 min. According to the 2001 FDA regulations, cider processors that are too small to be regulated by the FDA-required 5-log reduction would be free to choose alternative decontamination methods, such as those presented in this study, that are less costly than pasteurization. On the basis of our results, sodium hypochlorite and copper ion water treatment of apples in conjunction with sonication can reduce populations of E. coli O157:H7 and L. monocytogenes by 5 log during cider production. Therefore, this process could prove useful as part of a HACCP plan to reduce public health risks associated with unpasteurized cider. ACKNOWLEDGMENT This work was supported by the Michigan State University Agricultural Experiment Station.

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