2657 Journal of Food Protection, Vol. 67, No. 12, 2004, Pages 2657–2660 Copyright Q, International Association for Food Protection
Variations in External and Internal Microbial Populations in Shell Eggs during Extended Storage D. R. JONES,* M. T. MUSGROVE,
AND
J. K. NORTHCUTT
Russell Research Center, Poultry Processing and Meat Quality Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia 30605, USA MS 04-34: Received 24 January 2004/Accepted 19 July 2004
ABSTRACT The current project was conducted to determine the microbial quality of commercially processed shell eggs during extended storage. Unwashed eggs were collected at the accumulator before entering the processing line. Washed eggs were retrieved after placement in flats. All eggs were stored on pulp flats at 48C for 10 weeks. Twelve eggs from each treatment were rinsed on the day of collection and during each week of storage. After rinsing, eggs were sanitized in ethanol, and contents were aseptically collected. Total aerobes, yeasts and molds, Enterobacteriaceae, and pseudomonads were enumerated from shell rinses and pooled egg contents. During storage, no differences were found between unwashed and washed eggs for Enterobacteriaceae and pseudomonads in either shell rinses or contents. No differences were found between treatments for population levels of total aerobes or yeasts and molds in the egg contents throughout the storage period. Significant differences between treatments were found at each week of storage for external shell contamination by total aerobes. The highest unwashed egg contamination occurred at week 8 of storage and the lowest was at weeks 0 and 1 of storage. The highest shell contamination with aerobic bacteria on the washed eggs was found at week 0 of storage and the lowest was at week 7. Yeast and mold contamination determined by shell rinses was also significantly different between treatments at each week of storage. Commercially washed eggs were significantly less contaminated than were unwashed eggs for the populations monitored.
Shell eggs are a unique agricultural commodity because when they reach the consumer they are still in the same packaging as when they left the hen. Most eggs contain little to no bacteria when they are laid, and contamination usually occurs after oviposition (16). The egg has many natural defenses against microbial contamination, but the shell membranes have been identified by many as the primary barrier (6, 9, 10, 14, 16). Many of the natural defenses present in the egg degrade over time. Board (2) summarized a collection of previous research and determined there was a 20-day lag between shell penetration and the contamination of the egg contents. Stored or aged eggs are more easily infected than fresh eggs when inoculated (5). In another study, the infection rate after inoculation was greater for eggs with moderate weight loss, which is common during storage (15). The washing of shell eggs for retail sale has not always been a common industry practice. Visibly clean eggs usually have fewer microorganisms on the surface than do dirty eggs (3). Garibaldi and Bayne (8) postulated that one reason for the trend toward washing eggs was that it took less time to wash all eggs than to sort clean from dirty eggs. As washing became more common and eventually regulated, research emphasis shifted to the role of specific steps during processing and of new processing technology on the microbiological safety of eggs. In recent years, authors have found equipment-cleaning practices in egg packing (with* Author for correspondence. Tel: 706-546-3486; Fax: 706-546-3633; E-mail:
[email protected].
out egg washing) and egg processing (with egg washing) to be less than completely effective (4, 13). This study was undertaken to determine what effect current shell egg processing technologies have on the microbial quality of eggs during prolonged storage. MATERIALS AND METHODS Egg collection. Eggs were collected over three consecutive days (replications) from a single in-line processing facility equipped with a dual-tank washer. All eggs processed in this facility are manufactured under U.S. Department of Agriculture Agricultural Marketing Services requirements for shielded shell eggs. One hundred eighty unwashed eggs were randomly selected from the accumulator during each replicate. Eggs were aseptically placed on new pulp flats. One hundred eighty washed eggs were collected after passing the packer head (where they are placed into the new pulp flats). The processing line randomly assigns eggs of the appropriate size to a packer head; therefore, these eggs were a random assortment of large size eggs. All flats of large eggs were placed into cardboard half cases (15 dozen) according to treatments and stored in the same 48C refrigerator for the extent of the study. Egg sampling. A dozen eggs from each treatment were rinsed according to the methods of Jones et al. (12) utilizing sterile phosphate-buffered saline on the day of collection and once during each of the subsequent 10 weeks of storage. After rinsing, eggs were aseptically placed on clean plastic flats and submersed in 95% ethanol and then allowed to dry to sanitize the shell surface. The egg contents were collected by aseptically cracking the egg on the rim of a sterilized glass beaker. The contents of three eggs were pooled in a sterile sample bag (Whirl-Pak, Fort Atkinson,
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FIGURE 1. Effects of washing and extended storage on total aerobic bacteria concentrations on external egg shells (n 5 36 eggs). Significant differences were found between unwashed and washed eggs (****P , 0.0001).
Wis.) and blended in a stomacher (Seward Ltd, London, UK) for 1 min at normal speed. Three pools were formed for each treatment replicate. Cracked eggs were excluded from sampling. Microbial analysis. Unless otherwise noted, rinsates were plated using a spiral plater (Spiral Biotech, Norwood, Mass.), and 0.1 ml of homogenized egg contents was spread plated onto appropriate media in duplicate for analysis. Total aerobic bacteria were cultured on plate count agar (Becton Dickinson, Sparks, Md.) and incubated at 358C for 2 days, and then colonies were counted. Total yeast and mold populations were determined by plating on potato dextrose agar (Becton Dickinson) and incubated at 268C for 5 days. Pseudomonas isolation agar (Oxoid, Ogdensburg, N.Y.) with Pseudomonas C-F-C Supplement (Oxoid) was utilized to culture pseudomonads after 2 days of incubation at 268C. Enterobacteriaceae were enumerated by pour plating 1 ml of rinsate or homogenized egg contents in violet red bile glucose agar (Oxoid) and adding an overlay. Counts were determined after 1 day of incubation at 378C. Enterobacteriaceae populations were recorded for only the first 6 weeks of storage. Statistical analysis. Statistical analysis was conducted on the log transformation of the raw counts. Plate counts from plates with no bacterial growth were recorded as zero after log transformation. The general linear model procedure of SAS (20) was utilized. Data were sorted by week of storage to determine significant differences between treatments at each week. Therefore, the main effects were treatment and replicate. Means were separated by the least squares method and were considered significantly different at P , 0.05.
RESULTS The total aerobic bacteria population on the shell surface is shown in Figure 1. During each week of storage, there was a significant difference (P , 0.0001) in number of aerobic bacteria present on unwashed and washed eggs. In all cases, washed eggs had significantly fewer bacteria. Over the course of the storage period, aerobic counts for washed eggs decreased from 2.5 log CFU/ml detected on the day of collection to approximately 1.0 log CFU/ml. For the unwashed eggs, aerobic shell counts remained at about 4.0 log CFU/ml throughout most of the storage time, increasing to 5.3 log CFU/ml at 8 weeks.
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FIGURE 2. Effects of washing and extended storage on yeast and mold concentrations on external egg shells (n 5 36 eggs). Significant differences were found between unwashed and washed eggs (****P , 0.0001).
Yeast and mold populations found on the shell surface during storage are illustrated in Figure 2. A significant difference in contamination (P , 0.0001) between unwashed and washed eggs was found at each week of storage. Yeast and mold populations remained low for washed eggs throughout the study. The highest yeast and mold concentration (0.7 log CFU/ml) was detected at 8 weeks of storage. In the unwashed eggs, the lowest yeast and mold concentration was found at 2 weeks of storage (1.3 log CFU/ ml). As storage time increased, populations grew, with the highest concentration occurring at 8 and 10 weeks (2.9 and 2.6 log CFU/ml, respectively). Low concentrations of Enterobacteriaceae were detected during the study. Significant differences (P , 0.05) were found between unwashed and washed egg shell surface counts on the day of collection and after 1 week of storage. On the day of collection, no Enterobacteriaceae were found on the surface of washed eggs. The highest concentration detected on a single unwashed egg at the time of collection was 0.6 log CFU/ml. After 1 week of storage, there were still no detectable Enterobacteriaceae on the shells of washed eggs. At 1 week of storage, only 7 of 35 unwashed eggs were positive for Enterobacteriaceae. The highest concentration recorded was 1.4 log CFU/ml for a single unwashed egg. During the 6 weeks of storage, 28 of 485 (5.7%) unwashed and washed eggs were positive for Enterobacteriaceae. A majority of the positive shell samples (27 of 28) were from unwashed egg shells. Few eggs were positive for pseudomonads on the shell surface; the incidence was 28 of 764 (3.1%) rinsed eggs for both treatments combined. Of the positive samples, 16 of 28 were from unwashed eggs. The average concentrations detected were generally less than 1 log CFU/ml. The highest concentrations of pseudomonads found in unwashed and washed eggs were 3.1 and 2.8 log CFU/ml, respectively. Each of these values were found on a single egg among the dozen rinsed at that time. Figure 3 illustrates egg contents contamination with aerobic bacteria during storage. There were no significant differences (P . 0.05) between treatments during any of the sampling times. This figure shows how aerobic bacteria
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SHELL EGG MICROBIOLOGY DURING STORAGE
FIGURE 3. Effects of washing and extended storage on total aerobic bacteria concentrations in egg contents (n 5 pools).
FIGURE 4. Effects of washing and extended storage on yeast and mold concentrations in pooled egg contents (n 5 9 pools). Significant differences were found between unwashed and washed eggs (**P , 0.01).
concentrations in the contents of washed eggs remained constant during the 10 weeks of storage. The amount of bacteria detected in the unwashed eggs during storage gradually decreased. The average bacterial concentration for either treatment was less than 1 log CFU/ml during the course of the study. The highest concentration found in a pool of egg contents was 4.2 log CFU/ml at 9 weeks of storage. Yeast and mold contamination in the contents of shell eggs during prolonged storage is shown in Figure 4. Low concentrations (,0.3 log CFU/ml) were detected and were not significantly different (P . 0.05) between unwashed and washed eggs for all but one of the weeks of storage. At week 8, 0.4 log CFU/ml was found in unwashed eggs compared with 0.2 log CFU/ml for washed eggs (P , 0.01). At week 8, the highest contamination with yeast and mold on unwashed egg shell also was detected. Only 3.2% (4 of 126) of the pooled egg contents samples were positive for Enterobacteriaceae. All of the positive samples came from washed eggs. The levels detected were all less than 1.0 log CFU/ml. There were no differences between unwashed and washed eggs for Enterobacteriaceae concentration at any of the sampling periods. Pseudomonads were found in 12.1% (24 of 198) of the pooled unwashed and washed egg contents. Most of the positive samples were found in washed eggs (16 of 24). For one replicate at week 10 of storage, all unwashed and washed egg contents pools were positive for pseudomonads. The highest concentration recorded in a pool was 4.2 log CFU/ml. DISCUSSION The shell surface microbial contamination for washed eggs was low during the course of this study. At each week of storage, aerobic bacteria and yeast and mold populations on the shell were significantly lower for the washed than for the unwashed eggs. Although Jones and colleagues (13) found that current egg processing facility sanitation practices were inadequate for reducing the number of microorganisms present on direct egg-contact surfaces, the results of the present study indicate that the washed eggs were contaminated with microorganisms at low frequency and with few cells. Furthermore, no differences were found in aerobic bacteria, Enterobacteriaceae, and pseudomonads
cultured in the pooled contents of unwashed or washed eggs. Eggs are a difficult product to accurately sample for microbial contamination, especially when attempting to evaluate the growth of organisms in specific segments of the egg (16). Complete separation of egg components without crossover contamination is almost impossible. Previous research (16) has shown that the multiplication of organisms in the contents is inhibited by viscosity of the egg white, pH, and the presence of lysozyme and conalbumin. Characteristics of the shell have also been implicated in limiting the ability of organisms to enter the egg contents. Garibaldi and Stokes (9) reported a complete cessation of bacterial penetration in vitro when the shell and both membranes were present. Others have found a linear response between shell porosity and microbial infection of the contents (15). Orel (18) found more resistance to microbial penetration and egg spoilage at room temperature when shell specific gravity was greater than 1.080. Some researchers have evaluated changes during the storage of eggs. Eggs of three different shell permeabilities had similar contamination levels until 15 days of storage, when the most permeable eggs began to spoil at a greater rate (7). In the present study, we avoided some of these issues by utilizing eggs from an in-line processing facility. Under these circumstances, eggs are always less than 24 h old when processed because in-line egg processing facilities operate 7 days per week and the hen houses are attached to the plant. The hens housed in the million-hen facility represent a variety of hen ages and stages of egg production (e.g., early lay, late lay, postmolt). Therefore, the eggs sampled represented the spectrum of physical and microbial characteristics of the eggs available to the consumer in the retail market. The shell membrane loses its effectiveness as a microbial barrier as challenges increase (10). The yeast and mold results for the shell surface and egg contents at week 8 of storage further support this finding. Miller and Crawford (17) reported that spoilage organisms were virtually 100% absent from the contents of fresh eggs. In the present study, pseudomonads in pooled egg contents were fewer than 10
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CFU/ml in samples and were detected only after 3 weeks of storage. Fluorescence from Pseudomonas fluorescens has been utilized to determine when an egg has spoiled. Visible fluorescence in the egg is not detectable until concentrations have reached 5.0 log CFU/ml (11). Board and colleagues (3) had difficulty finding fluorescent pseudomonads in shell eggs. In the present study, a single pool of egg contents reached 4.2 log CFU/ml after 9 weeks of storage. Recent research has indicated that commercial sanitation operations did not significantly reduce aerobic bacteria and Enterobacteriaceae on the direct-contact surfaces in shell egg processing facilities (13). However, in the present study bacterial populations were greatly diminished by current processing techniques. This reduction in contamination of egg shell and egg contents continued throughout storage. The storage time in the present study was much greater than the 30-day sell-by date and 19 days postprocessing, when most eggs are purchased (1, 19). Therefore, current federal guidelines for the production and processing of shell eggs appear to have a beneficial effect on the microbial quality of the eggs being produced, even during long-term storage. ACKNOWLEDGMENTS The authors thank Ms. Patsy Mason, Ms. Susan Chewning, Ms. Manju Amin, and Ms. Sherry Turner for their technical assistance during the course of this project.
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