Volume 63, No. 2, 1998—JOURNAL OF FOOD SCIENCE 359 MICROBIOLOGY Challenge Studies with Selected Pathogenic Bacteria on Freshly Peeled Hamlin Orange STEVEN PAO, G. ELDON BROWN and KEITH R. SCHNEIDER ABSTRACT The survival and growth of Salmonella spp., Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on peeled Hamlin orange were examined. Fruits were peeled by infusing the peel with water to assist hand removal. The peeled oranges had an average pH of 6.0–6.5 at the sur- face and 3.8 in the juice. After surface inoculation, peeled fruits were incubated for up to 14 days. Growth was observed with all tested pathogens only at the abusive storage tem- perature (24°C). Refrigeration (4 or 8°C) effectively inhibited the growth of all pathogens and caused population reduc- tion of Salmonella spp. and S. aureus. Key Words: oranges, fresh peeled, Salmonella, Listeria, E. coli INTRODUCTION FRUITS AND THEIR PRODUCTS CAN SERVE AS VEHICLES FOR MI- croorganisms that cause human diseases under certain conditions (Brackett, 1994; Beuchat, 1996). The peel of citrus fruits serves as a natural protectant that prevents microbiological contamination of the interior flesh. Removing the peel eliminates this protective layer and subjects the edible portion to potential microbial invasion and spoil- age. The primary concern with undesirable microorganisms in citrus products has historically been with their capability to generate off- flavors (Parish, 1991). The growth of human pathogens in citrus prod- ucts has been presumed to be prevented because of the acidity of the juice (Redd et al., 1986; Kimball, 1991). In addition, commercial citrus juice is processed under strict sanitation to prevent contami- nation, and is often treated with sufficient heat during processing to eliminate most spoilage organisms and all human pathogens (Carter, 1990). The safety of fresh-squeezed unpasteurized juice, however, relies mainly on processing sanitation and juice pH which usually ranges from 2.8–4.0 (Banwart 1989). Accordingly, a combination of inappropriate plant hygiene and high juice pH (3.9–4.3) probably contributed to an uncommon outbreak of salmonellosis (Parish, 1996). Fresh-cut citrus has received much attention because of its large potential market (Garbner, 1996). Pao and Petracek (1997) reported that the surface pH of peeled oranges was relatively high (pH 5.5– 6.0). They isolated nonacid-tolerant spoilage bacteria (Enterobacter and Pseudomonas) that had not been previously considered as spoil- age agents of citrus products. No foodborne pathogenic microorgan- isms have been isolated from peeled citrus, but introduced patho- gens may potentially grow on the surface of peeled citrus. Our objective was to assess such potential. Peeled oranges were challenged with selected pathogenic bacteria, including three meso- philes (Salmonella spp., Staphylococcus aureus, and Escherichia coli O157:H7), and one psychrotroph (Listeria monocytogenes) at refrig- eration and abusive storage temperatures. METHODS & MATERIALS Fruit preparation Mature Hamlin oranges (Citrus sinensis L.) were purchased from a local packinghouse before washing and waxing. °Brix and %acid of the fruits were measured, according to methods described by Kim- ball (1991), and they were about 10.1 and 0.61, respectively. Fruits were washed on roller brushes with FMC Fruit Cleaner 395 (FMC Corporation, Lakeland, FL) and rinsed with potable water on the day of peeling. No attempt was made to sterilize the fruit surface. Fruits peels were scored with six radial cuts from the stem end to blossom end to penetrate the flavedo and thus permit infusion of solution into the inner part of the peel (Baker and Bruemmer, 1989). Fruits were submerged into a vacuum chamber containing deionized water (Pao et al., 1996). Fruits were infused by evacuating the vacu- um chamber to about 3 kPa (Bruemmer, 1981), held for 1 min, then gradually released over a 3 min interval. Peel and fibers were then removed manually from the infused fruit immediately after infusion. Fruits were individually packed in perforated plastic containers to allow fruit respiration during storage (Pao et al., 1996). Packed fruits were refrigerated at 4°C for about 24h before inoculation. Analyses of pH Juice pH was measured by a pH meter (Model HI 9219, Hanna Instruments, Woonsocket, RI). Juice was extracted from ten fruits for each of the triplicate tests. The surface pH of three peeled fruits was measured by contacting pH test papers (Full Range pH Kit, Fisher Scientific, Atlanta, GA) to the exposed segment membrane. Inoculum Cultures consisted of pooled inocula of two species or strains of a particular genus. Cultures were: Salmonella typhimurium ATCC 14028 and gaminara ATCC 8324 (ATCC, Rockville, MD); Listeria monocytogenes strains 1B and Scott A-4B (obtained from S. Madzo, FDA, Cincinnati, OH); Staphylococcus aureus ATCC 8095 and 6538 (ATCC, Rockville, MD); and Escherichia coli O157:H7 strains A9124-1 (clinical isolate) and MF6707A (beef isolate) (Obtained from B.E. Rose, Food Safety and Inspection Service, Beltsville, MD). Cultures were grown and propagated in Tryptic Soy broth at 37°C for 24h under static condition. Each inoculum was harvested by cen- trifugation at 10,000  g for 10 min. The supernatant was discarded, and the pellet was washed twice with sterile Butterfield’s phosphate buffer (BPB, pH 7.2) which is buffered dilution water containing 0.3 mM KH 2 PO 4 (FDA, 1995). Inoculation The cells were resuspended in BPB to provide an inoculum level of about 8–9 log CFU/mL for each microorganism. Two 1:10 dilu- tions were made in BPB yielding an estimated final inoculum level of 6–7 log CFU/mL. To each orange, 20 L of inoculum was distrib- uted over about one-eighth of the total surface area with a sterilized L-shape glass stick. Inoculated fruit (at 4°C) were then individually packed in perforated plastic containers and incubated at 4, 8 or 24°C. Microbial analyses After incubation for 0, 0.5, 1, 2, 3, 7 and 14 days, approximately Authors Pao and Brown are affiliated with the Florida Department of Citrus, Citrus Research & Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850. Author Schneider is affiliated with ABC Research Corporation, 3437 S.W. 24th Av- enue, Gainesville, FL 32607.