Validation of Apple Cider Pasteurization Treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes

1679 Journal of Food Protection, Vol. 64, No. 11, 2001, Pages 1679 1689 Copyright, International Association for Food Protection Validation of Apple Cider Pasteurization Treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes PEGGY P. MAK, BARBARA H. INGHAM, AND STEVEN C. INGHAM Department of Food Science, University of Wisconsin Madison, 1605 Linden Drive, Madison, Wisconsin 53706, USA MS 01-136: Received 10 April 2001/Accepted 2 June 2001 ABSTRACT Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in ph- and 8Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380-94) or strains engineered to contain the gene for green uorescent protein (pgfp ATCC 43894 and pgfp ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778, CDC F2833, and CDC HO662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of ph 3.3 or 4.1 and 11 or 148Brix was heated under conditions ranging from 608C for 14 s to 71.18C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.18C for 14 s. Lower temperatures, or less time at 68.18C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 68C for 14 s for Salmonella spp. L. monocytogenes survived 68.18C for 14 s, but survivors died in cider within 24 h at 48C. Laboratory results were validated with a surrogate E. coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.18C for 14 s (Wisconsin recommendations) and at 71.18C for 6 s (New York recommendations) was not signi cantly different. Hence, we conclude that 68.18C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider. Apple cider is traditionally de ned as the sweet, pulpcontaining, unfermented juice squeezed from apples. Unpasteurized apple cider has been associated with a number of outbreaks of foodborne illness (18). Pathogens involved include Escherichia coli O157:H7, Salmonella spp., and Cryptosporidium parvum. Contamination of apple cider by Listeria monocytogenes is also possible (17). The survival of pathogenic bacteria in apple cider varies widely, with E. coli O157:H7 surviving extended refrigerated storage (14, 25) and L. monocytogenes dying rapidly in cider (16). U.S. Food and Drug Administration regulations require apple cider and other juices to be treated to reduce numbers of a target pathogen by 100,000-fold (or 5 logs) relative to untreated cider (5). For practical purposes, E. coli O157:H7 has been considered the target pathogen for which a 5-log population decrease must be achieved. Thermal pasteurization has been considered the most likely way that cider makers would achieve the speci ed pathogen reduction. Nonthermal alternatives that have been studied for potential use in reducing numbers of pathogens in cider include high-pressure processing (13), supercritical uid processing (12), and UV light treatment (3, 24). The latter method, although approved by the Food and Drug Administration (4) and commercially available, may vary in effectiveness depending on the equipment used (24). The apple cider industry in Wisconsin is characterized Author for correspondence. Tel: 608-263-7383; Fax: 608-262-6872; E-mail: bhingham@facstaff.wisc.edu. by small operations, with 93% of cider mills producing less than 20,000 gal per year. Approximately 88% of the mills do not heat pasteurize their product, citing reasons of cost or anticipated changes in the quality of juice on pasteurization (21). In research done by Kozempel et al. (8), the cost of pasteurizing apple cider using a plate heat exchanger was estimated at $0.0017 per liter for a plant that processes 107 liters per min and $0.0064 per liter for a plant that processes 30 liters per min. Despite this cost, thermal pasteurization, either onsite or at a cooperating dairy plant, remains a reliable way for small apple cider operations to produce safe apple cider. Plate heat exchangers can readily be adapted to cider pasteurization, but alternative heat pasteurization equipment may also be adequate. New York State currently recommends pasteurization of apple cider at 71.18C for 6 s for most varieties of apples (15). Alternatively, milk pasteurization conditions of 71.78C for 15 s (22) have been suggested. A survey of apple growers in Wisconsin indicated that cider pasteurization treatments ranged from 68.18C/14 s to 80.98C/14 s, with shorter hold times noted at 728C (6 s) (Table 1). Speci c objectives of this study were (i) to validate time-temperature pasteurization conditions in laboratory experiments, using temperature(s) lower than 71.78C, to produce safe apple cider (a 5-log reduction of E. coli O157: H7); (ii) to con rm the lethality of these treatments against Salmonella spp. and L. monocytogenes; (iii) to investigate the effect of ph and 8Brix on the survival of E. coli O157:

1680 MAK ET AL. J. Food Prot., Vol. 64, No. 11 TABLE 1. Pasteurization parameters used by select Wisconsin cider makers Orchard Volume a Location b Time Temp Cider storage c A B C D E F G 284 liters/h 47.5 liters/h 882 liters/h 757 liters/h 2,271 liters/h 53 liters/h 474 liters/h Orchard Dairy Orchard Dairy Dairy Orchard Orchard 14 s 14 s 6 s 14 16 s 14 16 s Continuous 14 s 68.18C 80.98C 72 74.88C 88C 88C 70.98C 73.78C Freezer Refrigerated transport truck Cooler No answer Refrigerated transport truck Cooler (2 h) Cooler (24 h) a Volume of cider produced per hour. b Location where cider was pasteurized. c Storage conditions between pressing and pasteurization. H7 and acid-adapted E. coli O157:H7 in apple cider; (iv) to validate lab results using a surrogate E. coli with a bench-top plate heat-exchange pasteurizer; and (v) to investigate consumer acceptance of heat-pasteurized apple cider. MATERIALS AND METHODS Survey of cider producers. In February 2000, seven cider producers in Wisconsin and Illinois were surveyed by phone to determine parameters used for cider pasteurization (Table 1). Processors were identi ed in a previous survey (21) as producing pasteurized cider. Based on survey responses, pasteurization timetemperature combinations were selected for lethality testing against E. coli O157:H7, L. monocytogenes, and Salmonella spp. Bacterial strains and culture conditions. A cocktail of six E. coli O157:H7 strains was used in this study. Strains ATCC 43894 and ATCC 43895 were obtained from American Type Culture Collection (Manassas, Va.). The former strain was isolated from the stools of an infected patient, and the latter was isolated from ground beef implicated in an outbreak. Strains C7927 and USDA-FSIS-380-94 were obtained from Dr. John Luchansky (Food Research Institute [FRI], University of Wisconsin Madison). Strain C7927 was originally isolated from a patient in an outbreak linked to apple cider. Strain USDA-FSIS-380-94 was isolated from salami linked to an outbreak. Two strains, pgfp ATCC 43894 and pgfp ATCC 43889, were obtained from Dr. Randy Worobo (Cornell University, Geneva, N.Y.). These strains were modi cations of the American Type Culture Collection strains, engineered to contain a plasmid-borne gene for green uorescent protein (pgfp) for selective enumeration. A three-strain Salmonella spp. cocktail and a three-strain L. monocytogenes cocktail were also used in this study. Salmonella spp. strains CDC 0778 (Salmonella Hartford), CDC F2833 (Salmonella Rubislaw), and CDC H0662 (Salmonella Gaminara) were provided by Lorrie Friedrich (Citrus Research & Education Center, Lake Alfred, Fla.). Strains H0222, F8027, and F8369 were L. monocytogenes strains isolated from raw potato, celery, and corn, respectively. E. coli strains ATCC 4351, ATCC 25922, FRIK 185, and FRIK 859 were potential surrogates for use in experiments with a bench-top plate heat-exchange pasteurizer located at a local dairy. ATCC 4351 was isolated from bovine diarrhea, and ATCC 25922 was a clinical isolate. Strain FRIK 185 was a K-12 strain of E. coli obtained from Dr. John Luchansky (FRI). FRIK 859 was isolated by Susan Linden (FRI) from Muenster cheese purchased at a local store. Cultures were maintained at 2208C in brain heart infusion (Difco Laboratories, Franklin Lakes, N.J.) with 10% (vol/vol) added glycerol (Sigma Chemical Co., St. Louis, Mo.). Each strain was cultured on nutrient agar (NA; Difco) twice at 358C before use in experiments. Inocula were prepared by transferring a colony from NA to 10 ml of nutrient broth (Difco). Each culture was in stationary phase (ca. 8 log 10 CFU/100 l) after 18 h of incubation at 358C and was then transferred to a 95-ml centrifuge tube and cells spun down using a Marathon 21K centrifuge (Fisher Scienti c, Pittsburgh, Pa.) at 5,000 3 g (8,000 rpm) for 8 min. The supernatant was decanted, and the pellet was resuspended in 1 ml of apple cider to make the inoculum level 9 log 10 CFU/100 l. Resuspended cells for each strain were combined, and inoculum level was determined by dilution in Butter eld s phosphate buffer (Nutramax Products, Inc., Gloucester, Mass.) before spread plating and microplating on NA. The log 10 CFU/ml was calculated by counting colonies on spread plates. Microplating involved plating three 10- l microspots of one dilution on a plate. The log 10 most probable number (MPN)/100 l was determined by counting the number of microspots with growth in three serial dilutions and multiplying the MPN value by the appropriate reciprocal dilution factor (9). Plates were incubated at 358C for 18 h. The log 10 MPN/ 100 l for the two pgfp strains was determined by counting microspots with uorescent colonies under UV light in three serial dilutions and performing the appropriate calculation. Apple cider for thermotolerance studies. Apple cider (not pasteurized, no preservatives) was purchased frozen at a local orchard in late 1999. The ph was measured with an Accumet Basic AB15 ph meter (Fisher Scienti c, Chicago, Ill.) for each gallon (3.785 liters) of cider. The 8Brix was measured with a temperaturecompensated hand-held refractometer (Leica Inc., Buffalo, N.Y.). Ciders were dispensed into 250-ml screw-capped plastic bottles (Nalgene, Nalge Nunc Int., Rochester, N.Y.), frozen at 2208C, and then thawed at 48C for use in laboratory studies. Twenty milliliters of thawed apple cider was dispensed into each of four sterile 50-ml polypropylene screw-capped tubes (Becton Dickinson Labware, Franklin Lakes, N.J.). The ph and 8Brix were adjusted with L(2) malic acid (Sigma) and D(2) fructose (Sigma) as follows: cider A (ph 3.3, 118Brix), cider B (ph 3.3, 148Brix), cider C (ph 4.1, 118Brix), and cider D (ph 4.1, 148Brix). The ph and 8Brix values were chosen based on research with apple cider indicating an average ph of 3.7 with a range of 3.3 to 4.1 and an average 8Brix of 12.6 (10). Survival of E. coli O157:H7 in apple cider. Thawed tubes of cider (ph and 8Brix adjusted) were kept on ice until use. For a given cider, 180 l was dispensed into a sterile 10- by 75-mm disposable culture tube (Fisher) and heated in a circulating water bath (Haake W15 DC3, Germany) at the target temperature for 15 min to reduce come-up time. The water level was kept at ap-

J. Food Prot., Vol. 64, No. 11 VALIDATION OF CIDER PASTEURIZATION 1681 TABLE 2. Pathogens utilized in thermotolerance studies and heat treatments evaluated in ph- and 8Brix-adjusted apple cider Temp/Time Cider a : Escherichia coli O157:H7 b A, B, C, D Salmonella spp. c A, B, C, D Listeria monocytogenes d A, B, C, D Acid-adapted E. coli O157:H7 e A, B, C, D Potential surrogate E. coli f A, D Surrogate E. coli g (lab-scale) past. fresh h 608C/14 s 62.18C/14 s 68C/14 s 68.18/3 s 68.18C/7 s 68.18C/10 s 68.18C/12 s 68.18C/14 s 71.18C/3 s 71.18C/6 s 71.18C/11 s 71.18C/14 s a A (ph 3.3, 118Brix), B (ph 3.3, 148Brix), C (ph 4.1, 118Brix), D (ph 4.1, 148Brix). b E. coli O157:H7 cocktail USDA-FSIS-380-94, C7927, ATCC 43894, ATCC 43895, pgfp ATCC 43894, and pgfp ATCC 43889. c Salmonella spp. cocktail CDC 0778, CDC F2833, and CDC H0662. d L. monocytogenes cocktail H0222, F8027, and F8369. e Acid-adapted 4 or 24 h at 48C. f E. coli strains ATCC 4351, ATCC 25922, FRIK 185, and FRIK 859. g E. coli strain FRIK 859. h Fresh cider collected fall 2000. proximately 60 mm above the sample level. A blank (cider with no inoculum) containing an Atkins Series 396K digital thermocouple with a hypo needle-tip probe (49122-K) (All QA Products, Belmont, N.C.) was placed in the water bath throughout the experiment to calibrate the water bath temperature. Next, 18 l of E. coli O157:H7 inoculum was added to the prewarmed cider with an extra long pipette tip (Molecular Bio-Products, Inc., San Diego, Calif.) to avoid touching the sides of the tube, and the mixture was heated (without agitation) as shown in Table 2. Timing of heat treatments began when the cider-inoculum mixture returned to the target temperature (1 to 2 s). After heating, tubes were immediately cooled by swirling in ice with water for 30 s. Contents were pipetted out with an extra long pipette tip into a 2-ml Eppendorf centrifuge tube (Brinkmann Instruments, Westbury, N.Y.). The mixture was diluted with Butter eld s phosphate buffer and microplated on NA and sorbitol MacConkey agar (SMac; Difco). Plates were incubated at 358C for 18 h. The MPN/100 l was determined from microspots with growth on each agar. For pgfp strains, uorescent microspots were counted as described earlier, and MPN values were calculated. This procedure was carried out in triplicate for ciders A through D. From each pasteurization treatment, one colony was picked from the SMac plates and plated on Levine eosin-methylene blue agar (Oxoid Ltd., Ogdensburg, N.Y.). After incubation at 358C for 18 h, a metallic green colony was picked and then tested using Gram stain, oxidase test (Difco), and API 20E System kits (biomérieux Inc., Hazelwood, Mo.) to con rm the identity of each isolate as E. coli O157:H7. Survival of Salmonella spp. and L. monocytogenes in apple cider. Survival of Salmonella spp. and L. monocytogenes in ph- and 8Brix-adjusted apple cider was evaluated for selected time-temperature combinations (Table 2). Xylose-lysine-deoxycholate agar (Oxoid) and Listeria-selective agar (Oxoid) replaced SMac for enumeration of the Salmonella spp. cocktail and L. monocytogenes cocktail, respectively, with concurrent plating on NA as before to evaluate extent of injury. Gram stain and API Listeria system kits (biomérieux) were used to con rm L. monocytogenes survivors. If L. monocytogenes survived initial heat treatment, the heat-treated inoculated cider was stored overnight at 58C, and survivors were enumerated the following day. The mean log reduction was calculated by subtracting the MPN/100 l determined after the heating process from the log 10 CFU/100 l of inoculated cider for each replicate and then determining the mean of the resulting values. Effect of acid adaptation on thermotolerance of E. coli O157:H7 in apple cider. E. coli O157:H7 cocktail was acid adapted in each of the ph- and 8Brix-adjusted ciders (A through D) by storing at 48C for 4 or 24 h. Following acid adaptation, 18 l of acid-adapted cocktail (room temperature) was added to 180 l apple cider as described and subjected to selected time-temperature thermal treatments (Table 2). Selection of a surrogate E. coli for bench-top plate heatexchange pasteurization experiments. A surrogate E. coli strain was needed for use with a bench-top plate heat-exchange pasteurizer at a local dairy. E. coli strains ATCC 4351, ATCC 25922, FRIK 185, and FRIK 859 were evaluated as potential surrogates. Each of these four nonpathogenicstrains was individually exposed to selected time-temperature combinations (Table 2) in ciders A and D using methods previously described. The strain with the log 10 reduction most comparable to that of the six-strain E. coli O157:H7 cocktail (same thermotolerance or greater) was further tested for acid tolerance. To do this, 1.0 ml of the E. coli O157: H7 cocktail and 1.0 ml of the potential surrogate (FRIK 859) were individually inoculated into 10 ml of ciders A and D, then incubated in a 248C water bath. Surviving cells were enumerated by spreadplating on both NA and SMac at 0, 12, 24, and 48 h. Log 10 reductions of the surrogate and cocktail were compared. Survival of E. coli surrogate in apple cider during benchtop plate heat-exchange pasteurization. Survival of nonpatho-

1682 MAK ET AL. J. Food Prot., Vol. 64, No. 11 TABLE 3. Fresh apple cider used in lab validation of thermotolerance and pulp analysis Cider a Date ph 8Brix Lab validation b Dry wt. pulp (g) Time to 68.18C (s) c E-1 c K-2 N-3 E-4 K-5 N-6 N-7 K-8 K-9 13 Sep 00 14 Sep 00 14 Sep 00 28 Sep 00 28 Sep 00 28 Sep 00 12 Oct 00 12 Oct 00 26 Oct 00 3.4 3.8 3.7 3.4 3.6 3.6 3.6 3.6 3.7 10.0 10.3 10.4 11.5 11.5 11.0 11.5 11.5 12.5 0.0127 0.0122 0.0077 0.0154 0.0064 0.0101 0.0078 0.0077 75.33 70.33 58.33 71.00 70.33 53.33 60.67 67 a Orchard E, K, or N and sample number i.e., E-1 was the rst sample tested and was from orchard E. b Heat treatment of E. coli O157:H7 cocktail: 68.18C/14 s. c Average time to reach 68.18C from 0.08C (s). genic E. coli surrogate (FRIK 859) in apple cider was evaluated using an Arm eld FT43A plate heat-exchange pasteurizer (Arm- eld, Hampshire, UK) located at a local dairy. Three trials were carried out on 29 September, 13 October, and 3 November 2000. Freshly pressed cider was collected at a local orchard 1 day before each experiment and transported on ice to the laboratory. The ph and 8Brix were measured, and the cider was stored at 48C. Background ora were enumerated on NA and SMac spreadplates and Petri lm E. coli-coliform count plates (3M Microbiology Products, St. Paul, Minn.). To prepare inocula, the surrogate (FRIK 859) was inoculated into 22 tubes of NA and incubated at 358C for 18 h. A total of 21 cell pellets were prepared by centrifugation as described previously. Pellets and cider were kept on ice until used in the experiment. Immediately before each pasteurization trial, pelleted surrogate inoculum (FRIK 859) was resuspended in 1,000 ml of fresh cider, resulting in an inoculum of 6 log 10 CFU/ml. Inoculum level was determined once for each time-temperature combination tested by dilution in Butter eld s phosphate buffer and spreadplating on NA. Inoculated cider was pasteurized using selected time-temperature combinations (Table 2). Each time-temperature combination was tested using three separate lots of inoculated cider for each of three plant trials. The Arm eld pasteurizer was adjusted to selected time-temperature pasteurization conditions according to directions in the equipment manual. Settings were veri ed using a thermocouple to check temperature and uninoculated cider to test product ow rate. Uninoculated cider owed through the unit between trials. For each trial, the rst 250 to 300 ml of pasteurized inoculated cider was discarded, and the next 250 ml of product was collected in sterile screw-capped plastic bottles (Nalgene, Nalge Nunc). The nal 300 ml of pasteurized product was also discarded. Sample collected was plated to evaluate lethality of each heat treatment. To improve recovery of heat-injured cells, the ph of the cider collected was increased to values from 5.5 to 5.7 by the addition of 3 N NaOH, and the undiluted cider was spreadplated. Three plates each (0.3, 0.3, and 0.4 ml) of NA and SMac were prepared. The plates were incubated at 358C for 18 h. Cider pasteurized at 68C/14 s, 68.18C/7 s, 68.18C/10 s, and 68.18C/14 s was also plated on NA and SMac after the cider had been held at 48C for 4 and 24 h in order to evaluate recovery of sublethally injured inoculum cells. Enumeration of the surrogate E. coli FRIK 859 was based on its ability to ferment sucrose or myo-inositol. Small, round, white colonies on NA plates were counted and spotted on SMac. Red colonies on SMac were counted and spotted on NA with 10 g sucrose (Sigma), 10 g myo-inositol (Sigma), and 4 ml of 1% Brom Cresol Purple (Sigma) per liter. Plates were again incubated at 358C for 18 h. Purple colonies on Brom Cresol Purple NA were counted and identi ed according to Gram-stain reaction, cell morphology, and API 20E (biomérieux) biochemical pro le. The log 10 CFU/ml was calculated using the number of colonies that had the same SMac, Brom Cresol Purple NA, and API 20E results as the surrogate. Lab validation with fresh apple cider. Fresh apple cider was used to verify the thermotolerance of E. coli O157:H7 as determined in previously frozen cider (Table 3). Sampling was designed to correspond with early (mid-september), middle (early October), and late (late October to early November) season cider processing. Due to warm weather in the summer and early fall and an early frost, apples ripened early, and cider processing at the three orchards ended earlier than anticipated. Cider was collected immediately after pressing, transported on ice to the laboratory, stored at 48C, and used within 2 days. The ph and 8Brix were measured on each cider, and the thermotolerance was established with the E. coli O157:H7 cocktail at 68.18C/14 s as previously described. This treatment was chosen because it was determined to be the least severe heat treatment that resulted in a 5-log reduction of the E. coli O157:H7 cocktail in previously frozen ph- and 8Brix-adjusted cider. Effect of pulp on thermotolerance of E. coli O157:H7 in fresh apple cider. Experiments by Ingham and Uljas (7) with ltered apple cider showed that much of the major difference in thermotolerance between cells of E. coli O157:H7 strain ATCC 43894 in apple cider and in ltered apple cider at 618C was attributable to the apple pulp. Estimated D-values for this strain ranged from 37.0 to 45.5 s at 618C for apple cider and ltered cider, respectively. We analyzed the effect of pulp on thermotoleranceof a cocktail of E. coli O157:H7 at 68.18C/14 s or 68C/14 s: heat treatments that met (68.18C/14 s) and failed to meet (68C/14 s) the minimum 5-log reduction in the target pathogen. The thermotolerance of E. coli O157:H7 in ltered apple cider (juice) was determined using fresh cider collected over the fall 2000 growing season (Table 3). Filtered cider (juice) was prepared from the fresh product as described below. The thermotolerance of the E. coli O157:H7 cocktail in the ltered cider (juice) was determined at 68C/14 s and 68.18C/14 s as previously described. To prepare the ltered cider (juice) and to estimate the weight of pulp, 10 ml of apple cider was ltered with a disposable lter

J. Food Prot., Vol. 64, No. 11 VALIDATION OF CIDER PASTEURIZATION 1683 funnel (Whatman) that had been predried (958C overnight), cooled, and weighed. A plastic 1-liter Erlenmeyer ask with side arm (Nalgene), plastic tubing, and lab-bench vacuum source was used to lter the cider and collect the ltrate. The ltration process took approximately 25 s. The difference in weight of the Erlenmeyer ask before and after ltration was the weight of ltered cider (juice). The lter with apple pulp was dried in an oven at 958C overnight, cooled in a desiccator, and weighed. Pulp and juice weights were recorded for the eight fresh ciders collected over the course of the season. The dry weight of pulp was calculated using the following equation: Dry Weight of Pulp (g/g) 5 (Weight of Filter with Dry Pulp) 2 Weight of Filter) Weight of Filtered Cider Association of E. coli O157:H7 with pulp and juice in ltered cider. To determine whether E. coli O157:H7 was predominantly associated with pulp, 2 ml of the E. coli O157:H7 cocktail was added to 20 ml of thawed cider (48C). The inoculated cider was mixed and ltered with a disposable lter funnel (Whatman) and glass-micro ber lter (Whatman). The ltered juice was diluted with Butter eld s phosphate buffer, then spread plated and microplated on NA and SMac. The glass lter with pulp was put into a stomacher bag (Seward, London, UK) with 20 ml of Butter eld s phosphate buffer and mixed for 5 min (Seward stomacher). The pulp mixture was diluted with Butter eld s phosphate buffer, then spread plated and microplated on NA and SMac. Effect of pulp on heat-transfer properties of cider. Preliminary experiments were conducted to investigate whether the presence of pulp in cider would affect heat-transfer properties during pasteurization. One milliliter of fresh cider collected during the fall season (Table 3) was placed into a 10- by 75-mm disposable culture tube (Fisher); the cider level was 18.5 mm above the tube bottom. A thermocouple was placed into the tube to record cider temperature. The tube was set into an ice bath until cider temperature reached 0.08C, then transferred immediately to a 68.18C water bath with approximately 65 mm of the tube immersed in water. The time required for the cider to reach 68.18C was recorded. This experiment was done in triplicate. Consumer panel. A taste panel was held at Babcock Dairy Store (Babcock Hall, University of Wisconsin Madison) on 20 October 2000 with two kinds of pasteurized apple cider. Unpasteurized cider was purchased and pasteurized using the bench-top plate heat-exchange pasteurizer. Half of the cider was pasteurized at 68.18C for 14 s (our recommendations) and the other at 71.18C for 6 s (New York State recommendations). Unscreened panelists (n 5 192) evaluated the two ciders for general preference using two structured seven-point hedonic scales (1). Each panelist was given approximately 45 ml of each refrigerated apple cider in a cup with a three-digit random number label and a sample ballot. After evaluating samples for preference, panelists were asked to respond to the question Which sample do you prefer the most? and were asked to comment on color, avor, sweetness, smell, mouth feel, and tartness. Panelists were also asked to indicate the type of apple cider they usually buy; options were pasteurized, unpasteurized, and don t know. For calculation of overall preference scores, a code value of 1.0 was assigned to the category of dislike very much and a value of 7.0 to the category of like very much, with appropriate whole-number code values assigned to the intermediate categories. The coded values from the panel session were subjected to analysis of variance appropriate for a randomized complete block design (20). For the overall preference attribute, statistical analysis provided the mean score for each sample, the F-value, and the least signi cant difference for making sample comparisons. RESULTS AND DISCUSSION Survey of cider producers. Results of a phone survey of select Wisconsin cider makers are shown in Table 1. Among the seven cider producers, four of them pasteurized cider at their orchards, and three pasteurized cider at a dairy plant. Pasteurization treatments ranged from 68.18C (1558F) for 14 s to 88C (1908F) for 16 s. Only the three producers that pasteurized cider at a dairy plant used a temperature higher than 808C. All cider to be pasteurized was stored after pressing, and before heat treatment, either refrigerated (cooler or refrigerated transport truck) or frozen for 2 h, 24 h, or an unstated time. Results of the survey of cider producers showed that they currently use a wide range of pasteurization conditions. Based on this survey and knowledge of state recommendations for pasteurization of apple cider, several conditions were chosen for evaluation of thermotolerance of E. coli O157:H7, Salmonella spp., and L. monocytogenes cocktails in apple cider. Both New York State and Wisconsin currently recommend a pasteurization time of 1608F (71.18C) for 6 s to ensure safety of cider made from more than one variety of apple. An exception to this is cider produced from Red Delicious apples, which requires a temperature of 1608F for 11 s or 1708F (76.78C) for 2 s (15, 23). The higher temperature and longer time required for cider made from Red Delicious apples are due to the higher ph associated with the juice of this cultivar (10). Five temperatures (71.1, 68.1, 6, 62.1, and 608C) and various times (3 to 14 s) were set for laboratory trials, including 71.18C/14 s (recommended temperature-sur vey time), 68.18C/14 s (survey time-temperature), and 68C/14 s (potentially acceptable lower temperature-survey time) (Table 2). Both ph and 8Brix may have an effect on the survival of pathogens in apple cider (19), so thermotolerance was rst evaluated in ph- and 8Brix-adjusted ciders: cider A (ph 3.3, 118Brix); cider B (ph 3.3, 148Brix); cider C (ph 4.1, 118Brix); and cider D (ph 4.1, 148Brix). Thermotolerance of pathogens in apple cider. The thermotolerance of E. coli O157:H7, Salmonella spp., and L. monocytogenes was investigated, and results for E. coli O157:H7 and L. monocytogenes are shown in Tables 4 and 5, respectively. The lowest heat treatment that resulted in a 5-log reduction of E. coli O157:H7 for all ciders was 68.18C for 14 s (Table 4). Pasteurization at 71.18C for 3 s was also suf cient to obtain a 5-log reduction of E. coli O157:H7, as determined using both plating media. Mazzotta (11) evaluated the heat resistance of stationary-phase and acid-adapted E. coli O157:H7, Salmonella spp., and L. monocytogenes in single-strength apple, orange, and white grape juices adjusted to ph 3.9. While the pathogens varied in thermotolerance, Mazzotta calculated that a process of 71.18C for 3 s would ensure safety (a 5-log reduction) from all three pathogens. Our work con rms that of Mazzotta while also validating a lower temperature process (68.18C/14 s) for cider processors.

1684 MAK ET AL. J. Food Prot., Vol. 64, No. 11 TABLE 4. Mean log reduction 6 (standard deviation) in E. coli O157:H7 cocktail or acid-adapted E. coli O157:H7 cocktail in ph- and 8Brix-adjusted cider following heat treatment; NA, nutrient agar; SMac, sorbitol MacConkey agar Log reduction for E. coli O157:H7 cocktail Cider: A (ph 3.3, 8Brix 11) B (ph 3.3, 8Brix 14) C (ph 4.1, 8Brix 11) D (ph 4.1, 8Brix 14) Media: NA SMac NA SMac NA SMac NA SMac 608C/14 s 62.18C/14 s Acid-4 b Acid-24 c 68C/14 s Acid-4 b Acid-24 c 68.18C/7 s 68.18C/10 s Acid-4 b 68.18C/12 s 68.18C/14 s Acid-4 b Acid-24 c 71.18C/3 s 71.18C/6 s 71.18C/14 s a 3.7 3.8 (0.5) 3.7 (0.3) 4.2 (0.5) 5.1 (0.6) 4.6 (0.5) (0.4) 2.5 (0.5) 3.6 (0.1) 2.2 (0.2) (0.4) 5.1 d (0.4) 4.8 (0.2) 5.3 (0.4) (0.1) 2.1 (0.2) 3.9 (0.7) 3.3 3.4 3.5 (0.2) 4.5 (0.2) 4.1 (0.2) 5.5 (0.1) 5.3 (0.5) 6.3 (0.5) 3.4 (1.2) (0.4) 3.1 (0.1) 3.2 (0.4) 5.7 4.7 (0.8) 4.9 5.4 (0.2) (0.1) 4.1 (0.5) 3.1 (0.3) 3.2 (0.3) 3.3 (0.2) 4.7 3.4 (0.2) 4.8 (0.2) 6.4 (0.2) 4.3 (0.4) 5.1 3.1 (0.5) 1.7 (0.2) (0.2) 4.9 (0.5) 4.6 (0.5) 4.7 (0.7) 4.5 (0.5) (0.9) 4.0 (0.6) 2.6 2.8 (0.2) 3.7 (0.9) 4.9 (0.7) 3.1 (0.4) 4.7 (0.4) 4.3 (0.4) 5.9 (1.1) 2.5 (0.2) 3.7 (0.2) 2.1 (0.2) 5.1 4.6 (0.4) 4.6 (0.4) 5.5 (0.1) 4.7 (0.6) 5.9 (1.1) a Absence of standard deviation indicates same microspot MPN value obtained on all three trials. b Acid adapted in appropriate cider (A, B, C, D) for 4 h at 48C before thermal treatment listed directly above. c Acid adapted in appropriate cider for 24 h at 48C before thermal treatment. d Bold numbers indicate that a 5-log reduction was achieved.

J. Food Prot., Vol. 64, No. 11 VALIDATION OF CIDER PASTEURIZATION 1685 TABLE 5. Mean log reduction 6 (standard deviation) of L. monocytogenes cocktail in ph- and 8Brix-adjusted cider following heat treatment; NA, nutrient agar; LSA, Listeria-selective agar Log reduction for L. monocytogenes cocktail Cider: A (ph 3.3, 8Brix 11) B (ph 3.3, 8Brix 14) C (ph 4.1, 8Brix 11) D (ph 4.1, 8Brix 14) Media: NA LSA NA LSA NA LSA NA LSA 4.0 1.5 (0.2) 3.6 (0.2) 3.9 (0.2) 1.5 (0.2) 3.6 (0.2) 4.9 3.0 4.9 a 68C/14 s 68.18C/14 s 71.18C/11 s a Absence of standard deviation indicates same microspot MPN value obtained on all three trials. Survival of pgfp-e. coli O157:H7 strains was determined separately by counting uorescent colonies on NA and SMac under UV light. The two pgfp strains were less thermotolerant than the three other E. coli O157:H7 strains in the cocktail. A higher log reduction was observed compared to the overall E. coli O157:H7 cocktail for each pasteurization treatment, with a 5-log reduction obtained at 68.18C for 12 s (data not shown). Thus, these strains would not be appropriate markers of successful pasteurization of apple cider. For Salmonella spp., a 5-log reduction was obtained for each cider type, as determined using both media, with any heat treatment 68C for 14 s (data not shown). Thus, Salmonella spp. were destroyed with a less severe heat treatment than the least severe heat treatment that resulted in a 5-log reduction of E. coli O157:H7. While Goverd et al. (6) showed that salmonellae could survive and perhaps grow in ph 3.6 apple juice, Mazzotta (11) showed that the D-value of stationary-phase acid-adapted and nonacid-adapted Salmonella spp. in ph 3.9 apple juice at 628C ranged from 5.4 to 11.4 s. Given the calculated z-values, a 5-log reduction could be obtained on heating Salmonella spp. for 2.5 to 5.7 s at 67.7 to 688C. Our work con rms the ndings of Mazzotta (11) that Salmonella is not an appropriate target organism to calculate lethality of an apple cider pasteurization treatment. There was substantial survival of L. monocytogenes in all ciders heated at 68C for 14 s, with injury noted in all cases (Table 5). Survival of L. monocytogenes was greater than for E. coli O157:H7 (Table 4) at this time-temperature combination. The L. monocytogenes cocktail was more thermotolerant than the E. coli O157:H7 cocktail in ph 4.1 ciders at 68.18C/14 s and 71.18C/11 s (Tables 4 and 5). Up to 99% of surviving L. monocytogenes cells were injured after pasteurization treatments in these ciders (Table 5). These results appear to con rm Mazzotta s speculation (11) that L. monocytogenes is more heat resistant than E. coli O157:H7 at typical juice-processing conditions. However, 68.18C for 14 s is still considered a validated pasteurization treatment for apple cider because L. monocytogenes survivors died within 24 h when stored in apple cider at 58C (data not shown). Roering et al. (16) also found that L. monocytogenes survived very poorly when stored in preservative-free apple cider stored at 4 or 108C. The situation in which L. monocytogenes might prove harmful in apple cider is if the organism were present in freshly pressed cider that was consumed with no heat treatment and no intervening hold. However, no outbreaks involving L. monocytogenes in fruit juices have been reported, and survey results of Uljas and Ingham (21) show that Wisconsin processors routinely hold freshly pressed (unpasteurized) cider for 1 day before sale. Overall, these results clearly con rm that E. coli O157:H7 is the appropriate target organism for evaluating the lethality of pasteurization treatments for apple cider. Effect of ph, Brix, and acid adaptation. In the present study, there was no consistent effect of ph on thermotolerance of E. coli O157:H7 (Table 4; NA data). Plating

1686 MAK ET AL. J. Food Prot., Vol. 64, No. 11 TABLE 6. Mean log reduction 6 (standard deviation) of E. coli O157:H7 cocktail after heating for selected time-temperature in fresh apple cider or in ph- and 8Brix-adjusted ltered apple cider (juice); NA, nutrient agar; SMac, sorbitol MacConkey agar Fresh cider 68.18C/14 s Cider ph/8brix NA SMac E-4 3.4/11.58 a K-5 3.6/11.58 7.1 (0.6) N-6 3.6/11.08 7.3 (0.2) 7.4 (0.3) N-7 3.6/11.58 7.4 (0.2) K-9 3.7/12.58 7.5 (0.1) Filtered cider 68C/14 s Cider ph/8brix NA SMac A 3.3/118 (0.1) B 3.3/148 5.8 (0.2) C 4.1/118 4.8 (0.2) 5.4 (0.3) D 4.1/148 4.9 (0.4) 6.0 (0.2) Filtered cider 68.18C/14 s Cider ph/8brix NA SMac A 3.3/118 7.3 (0.5) B 3.3/148 C 4.1/118 6.8 (0.2) D 4.1/148 (0.2) a Absence of standard deviation indicates same microspot MPN value obtained on all three trials. on SMac indicated that sublethal injury was consistently seen at heat treatments up to 68.18C/7 s, regardless of ph or 8Brix. As previously noted, ph 4.1 increased the number of sublethally injured L. monocytogenes recovered following heat treatment (Table 5; Listeria-selective agar data). 8Brix did not appear to affect the thermotolerance of E. coli O157:H7 and L. monocytogenes cocktails. There was not enough information to determine whether 8Brix had an effect on thermotolerance of the Salmonella spp. cocktail. Splittstoesser et al. (19) also showed that the thermotolerance of E. coli O157:H7 in apple juice was unaffected by 8Brix of 11.8 to 16.5. Subsequent analysis of the thermotolerance of E. coli O157:H7 in fresh cider with 8Brix as high as 12.5 con rmed that lethality was unaffected by 8Brix at pasteurization conditions of 68.18C/14 s (Table 6). Acid-adaptation experiments showed that the thermotolerance of the E. coli O157:H7 cocktail was unaffected by storage in apple cider for 4 or 24 h at 48C (Table 4). A 5- log reduction was obtained by a pasteurization treatment of 68.18C for 14 s after both 4 and 24 h of acid adaptation. This contradicts results by Ingham and Uljas (7), which showed that thermotolerance of E. coli O157:H7 strains ATCC 43894 and ATCC 43889 in apple juice (no pulp) was signi cantly decreased by prior storage at 48C for 24 h. These results, however, con rm work by Buchanan and Edelson (2), which showed that survival of E. coli O157:H7 grown in acidogenic and nonacidogenic medium and then heated in bottled, clari ed apple juice (ph 3.5) was not signi cantly different. However, acid-adapted cells displayed nonlinear inactivation kinetics in ph 3.5 apple juice and had greater thermotolerance if heated in apple juice with ph adjusted to 4.5, 5.5, or 6.5. Buchanan and Edelson speculated that the loss of acid-induced thermal tolerance on heating in low ph medium (,4.0 to 4.5) might be due to the elimination of acid resistance-associated cross-protection. Lab validation with fresh apple cider. Five lots of apple cider purchased from local orchards in the fall of 2000, and used fresh, served to verify the thermotolerance of a cocktail of E. coli O157:H7 at 68.18C/14 s (Table 6). This heat treatment was chosen because it was determined in ph- and 8Brix-adjusted cider to be the least severe heat treatment that resulted in a 5-log reduction of the E. coli O157:H7 cocktail. The ph of the fresh ciders ranged from 3.4 to 3.7 and the 8Brix from 11.0 to 12.5. In each case, laboratory studies with fresh cider con rmed results with previously frozen ph- and 8Brix-modi ed apple cider that a pasteurization treatment of 68.18C/14 s would be suf - cient to achieve a 5-log reduction in E. coli O157:H7. Although the sample size was not large ( ve lots of fresh cider), the samples collected were typical of cider produced in Wisconsin over the pressing season and can be assumed to represent the range of conditions that might be expected. Effect of pulp on survival of E. coli O157:H7 in apple cider. The dry weight of pulp of eight samples of fresh cider is shown in Table 3. There was considerable variation in the amount of pulp in cider from various orchards and collected over the season. There was no correlation between the amount of pulp and the amount of time needed for the aliquot of cider to heat from 0 to 68.18C, indicating that little or no pulp effect would be expected on heat transfer in a plate heat-exchange pasteurizer. E. coli O157:H7 was found to preferentially associate with apple pulp in ltered cider. Cider was inoculated with 1.1 3 10 8 CFU/ml E. coli O157:H7, mixed, and ltered. The resulting juice had an average 1.5 3 10 7 CFU/ml E. coli O157:H7 on NA and SMac. The pulp had an average 7.8 3 10 7 CFU/g E. coli O157:H7 on NA and 3.1 3 10 6 CFU/g on SMac. When accounting for the original proportions of pulp and juice, the ratio of CFU in pulp to juice was :1. Results from thermal treatments with ltered apple cider showed that E. coli O157:H7 died off more rapidly without the presence of lterable pulp, with a greater reduction in inoculum cells at 68C/14 s in juice prepared from cider with ph adjusted to 3.3 versus the non ltered beverage (Tables 4 and 6). This con rms the work of In-

J. Food Prot., Vol. 64, No. 11 VALIDATION OF CIDER PASTEURIZATION 1687 FIGURE 1. Thermotolerance of E. coli O157:H7 cocktail and surrogate E. coli FRIK 859 on heating in cider A or D at 68.18C for various times. gham and Uljas (7) that lterable pulp enhances the thermotolerance of E. coli O157:H7 in un ltered versus ltered apple cider. However, adequate lethality (a 5-log reduction) was still obtained in fresh cider pasteurized at 68.18C/ 14 s, even with varying pulp content. Determination of surrogate E. coli for bench-top plate heat-exchange pasteurizer. E. coli strains ATCC 4351, ATCC 25922, FRIK 185, and FRIK 859 were evaluated as surrogates for E. coli O157:H7 to be used in thermotolerance experiments with an Arm eld bench-top plate heat-exchange pasteurizer. Strains ATCC 4351 and ATCC 25922 were eliminated in tests using ciders A and D because they were less thermotolerant than FRIK 185, FRIK 859, and the E. coli O157:H7 cocktail (data not shown). Duffy et al. (3) used ATCC 25922 to validate the ef cacy of UV pasteurization for apple cider. However, our results showed that ATCC 25922 was not an appropriate surrogate for E. coli O157:H7 in terms of thermotolerance. Of the two remaining strains, FRIK 185 was less thermotolerant, while FRIK 859 was more thermotolerant than the E. coli O157: H7 cocktail (Fig. 1). To ensure adequate safety of pasteurized cider, the more thermotolerant surrogate, FRIK 859, was selected. E. coli FRIK 859 was found to be slightly less acid tolerant (0.5 log CFU/ml greater reduction in 24 h at 248C) than the E. coli O157:H7 cocktail (data not shown). Survival of E. coli surrogate in apple cider during bench-top plate heat-exchange pasteurization. Fresh apple cider inoculated with surrogate E. coli was subjected to heat treatment in an Arm eld plate heat-exchange pasteurizer, and survivors were enumerated. Pasteurization treatments of 68.18C for 14 s and 71.18C for 6 s were suf - cient to cause a 5-log reduction in E. coli FRIK 859 (Table 7). Only results with SMac were shown for the pasteurization treatments in Table 7. Since we were only interested in the survival of the surrogate E. coli, data from SMac were suf cient to determine whether a 5-log reduction could be obtained for a particular pasteurization treatment. Enumeration after subsequent storage (48C, 4 or 24 h) indicated no repair of sublethally injured inoculum cells (Table 7). Results were consistent in all three trials. Inter- TABLE 7. Mean log reduction 6 (standard deviation) of surrogate E. coli FRIK 859 upon thermal treatment in a bench-top plate heat-exchange paseurizer Thermal treatment Trial 1 SMac 4 h a 24 h b Trial 2 SMac 4 h 24 h Trial 3 SMac 4 h 24 h 68C/14 s 68.18C/7 s 68.18C/10 s 68.18C/14 s 71.18C/6 s 71.18C/11 s 71.18C/14 s c 6.4 (0.7) 6.8 (0.2) 6.8 (0.2) 6.0 () 6.8 (0.2) 6.3 5.8 (0.5) 5.5 5.8 5.8 5.4 (0.8) 5.8 (0.3) (0.2) a Spread plating on SMac of inoculated, pasteurized cider stored 4 h at 48C. b Spread plating on SMac of inoculated, pasteurized cider stored 24 h at 48C. c Absence of standard deviation indicates same microspot MPN value obtained on all three trials.

1688 MAK ET AL. J. Food Prot., Vol. 64, No. 11 TABLE 8. Response frequency and mean scores for consumer preference evaluation of apple cider pasteurized at 68.18C/14 s or 71.18C/6 s; S, signi cant at 5% level; NS, not signi cant at 5% level Preference rating Like very much Like moderately Like slightly Neither like nor dislike Dislike slightly Dislike moderately Dislike very much Assigned numerical score 7 6 5 4 3 2 1 68.18C/14 s 66 83 29 6 7 1 0 Apple ciders Total number of responses 192 Mean score F-value 6.00 A a NS 71.18C/6 s 66 88 26 5 6 1 0 6.04 A a Mean scores in the same row with the same letter are not signi cantly different at the 5% level. estingly, a 5-log reduction in inoculum was also obtained when fresh apple cider was pasteurized at 68C for 14 s using the bench-top pasteurizer. This may have been due to additional lethality on preliminary heating in the regeneration unit before the cider entered the holding tube or to an inability to rapidly cool the product on a consistent basis. Results from the bench-top plate heat-exchange pasteurization of fresh apple cider further con rm the calculations of Mazzotta (11) in apple juice and our laboratory results that heating cider for 14 s at 68.18C is suf cient to ensure a 5-log kill of E. coli O157:H7. Consumer panel. Consumers responded favorably to apple cider pasteurized at 68.18C/14 s or 71.18C/6 s (Table 8). No signi cant difference was noted between the two pasteurization treatments at the 5% level. When asked Which product do you prefer?, 98 of 192 (51%) responded they preferred cider pasteurized at 68.18C/14 s, and 94 of 192 (49%) preferred cider heated to 71.18C/6 s. There was no difference in sample preference based on color, a- vor, sweetness, smell, mouth feel, or tartness. When asked to respond to the question What type of cider do you buy?, 70 panelists (36%) responded pasteurized, 32 panelists (17%) responded unpasteurized, 79 panelists (41%) responded don t know, and 2% each responded don t buy or either. In conclusion, our laboratory work with previously frozen ph- and 8Brix-adjusted cider, and with fresh cider, and our pilot scale studies with a plate heat-exchange pasteurizer indicate that 68.18C for 14 s is a validated pasteurization treatment for apple cider. In addition, these results clearly con rm that E. coli O157:H7 is the most appropriate target organism for evaluating the lethality of pasteurization treatments for apple cider. The ph and 8Brix were shown to have no effect on the thermotolerance of the target organism, and the presence of pulp in cider was shown not to affect the heat-transfer properties. Many cider makers in Wisconsin hold their cider for a period of time between pressing and thermal pasteurization. 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