Ph.D.
Egg Safety and Quality Research Unit
Richard B. Russell Research Center
Athens, GA
U.S.A.
Introduction
Hazard Analysis and Critical Control Point (HACCP) management systems are used by the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) and the U.S. Food and Drug Administration (FDA) to ensure the safety of meat, poultry, seafood, and other foods (USDA, 1996).
The effectiveness of HACCP relies heavily upon published scientific data. Currently a voluntary quality-based egg inspection system is administered by the USDA Agricultural Marketing Service (AMS) (USDA, 2001; USDA, 2003). However, FSIS is currently drafting HACCP documentation for the shell egg processing industry that will be similar to regulations already in place for meat and poultry plants (Carson, 2000). A great deal of work has been published on the effect of processing on broiler carcass contamination (Bailey et al, 1987; Berrang et al, 2000; Cox et al, 1975).
As a result, step-by-step fluctuations in various microbial populations on broiler carcasses have been determined. This has assisted researchers, industry, and regulators in developing HACCP plans in their efforts to decrease contamination of poultry meat with human pathogens (USDA, 1996). However, comparable information for shell egg processing facilities has only recently become available.
Methods
A survey was conducted of in-line egg processing facilities. Three plants were selected for sampling on three separate processing days. These plants were designated as X, Y, and Z to protect the anonymity of the participating companies. Plant X was over 20 years old with a 135,000 eggs/h production capacity. Mixed operations (in-line and off-line) were processed though only in-line eggs were being processed during collection. Plant Y was an in-line operation, less than 3 years old, and processed approximately 95,000 eggs/h. Plant Z, with only a 10,500 egg capacity also ran mixed operations but only in-line eggs were being processed when samples were collected.
Eggs were collected from commercial plants at the following points of processing: at the accumulator (A), pre-wash wetting (B), first washer (C), second washer (D), sanitizer spray (E), dryer(F), oiler (G), scales following check detection and candling (H), re-wash belt entrance (I), re-wash belt exit (J), and packaging (at two different packer lane belts, K and L). Each of these sample sites are depicted in Figure 1. Eggs were collected after the line had been operating for at least two hours and during the mid-morning break so as not to interfere with operations. Twelve eggs from each collection site were aseptically placed into foam cartons, packed into half-cases and transported back to the laboratory at ambient temperature.
Ten of the twelve eggs collected at each site were sampled using a shell rinse technique. Each egg was placed into a sterile whirl-pack bag with 10 ml of sterile phosphate buffered saline (PBS) and rinsed by shaking for 1 minute. After a rinse sample was obtained each egg was removed and transferred to a different sterile bag. Rinsates and intact eggs were then stored at 4°C overnight. On the following morning, each egg was removed from the second bag and cracked open on the edge of a sterile beaker. Egg meats were discarded and the inside of the shell was rinsed using sterile PBS to remove most of the adhering albumen. An effort was made to eliminate as much of this material as possible because of the antimicrobial components of albumen. Shell and membranes from a single egg were crushed in a gloved hand and forced into a sterile 50 ml disposable centrifuge tube. After 20 ml of sterile PBS was added, a sterile glass rod was moved vertically in and out of the tube for 1 min. This allowed for a maceration of shells and membranes as well as a thorough mixing of the sample with the diluent. Rinsate from every egg was then subjected to microbiological analyses.
Bacterial populations from individual samples obtained with this method were enumerated for total aerobes, yeasts and molds, E. coli, and Enterobacteriaceae. Aerobic populations were enumerated on plate count agar (PCA) after incubation at 35°C for 48 hours. Yeasts and mold counts were determined on dichloran rose bengal chloramphenicol (DRBC) agar plates incubated at 22-25°C for 5 days. Escherichia coli were enumerated on Petri-film plates (blue gas producing colonies), incubated at 35-37°C for 18-24 hours. Enterobacteriaceae were enumerated on violet red bile glucose gar (VRBGA) plates with overlay (purple-red colonies). Plates were incubated at 37°C for 18-24 hours. Presumptive colonies were counted and reported as log10 CFU/ml egg rinse or contents.
Salmonella enrichment was performed for each of the twelve collection sites, two pooled samples were formed by combining shell egg rinses or crushed shells and membranes from five eggs.
Samples were pre-enriched in buffered peptone water at 35°C for 18-24 hours, followed by enrichment in TT broth and Rappaport-Vassiliadis broth overnight at 42°C. Enriched samples were plated onto BG Sulfa and XLT-4 agar plates and incubated at 37°C for 24 hours. Presumptive positives were inoculated into lysine iron agar (LIA) and triple sugar iron (TSI) slants and incubated at 35°C for 18-24 hours. Those samples giving presumptive results on each of these media were confirmed using sero-grouping anti-sera.
Confirmed isolates were then streaked for purity and stocked onto agar slants and ceramic beads in cryogenic protective media. A copy of each isolate was provided to the National Veterinary Services Laboratory of the USDA's Animal and Plant Health Inspection Services in Ames, Iowa for serotyping. A sample was recorded as positive if it was confirmed and serotyped from either of the shell rinse or crushed shell and membrane composite samples.
Population data were analyzed using the general linear model of SAS (1994). Means were separated with the least-squared means option of the general linear model procedure of the SAS/STAT program using significance levels of P < 0.05 (1994). A comparison of recovery frequency was accomplished by Chi-square test of independence (1994).
Results and discussion
This study was conducted to provide an intensive analysis of the effects of each stage of processing for five microbial populations that affect shell egg quality or safety. There were some differences in microbial levels recovered from eggshells collected at different plants on different visits (replications). Each plant was visited within two weeks of each other in sequential fashion to prevent a seasonal bias.
Prior to processing, aerobic microorganisms, E. coli, and yeasts/molds were determined to be less than a log10 CFU/ml rinse different among the plants. Despite differences in age, processing capacity, and water quality, all three plants were contaminated at comparable levels for yeasts/molds, Enterobacteriaceae and E. coli at the end of processing.
For this reason, most of the data will be discussed as averages among the three plants.
Plant X was significantly (P < 0.05) more contaminated with aerobic microorganisms than Y or Z by greater than 2 log10 CFU/ml rinse for eggs that were ready to be packaged. Aerobic plate counts are a gauge of sanitary quality and adherence to good manufacturing practices (Morton, 2001). Plant X was the oldest plant with the highest production capacity, lowest average wash water pH (10.0 v. 10.3 and 11.2 for plants Y and Z), and with the least hygienic product flow. Pre-wash rinsing had less effect for all populations than was observed for the other two plants. For plant X only, none of the directly plated populations decreased by a log until eggs reached the first washer. There was also a great deal of foaming noted during the first visit to plant X. Excessive foaming is one of the wash water parameters recommended in the Agricultural Marketing Service list of guidelines (USDA, 2000). Knape et al. (2002) compared aerobic microbial counts for shell egg surfaces between in-line and off-line operations. They determined that counts were almost a log higher per egg for in-line eggs. All eggs sampled in this study were collected during in-line processing though plants X and Z also process off-line eggs. Perhaps greater contamination of equipment surfaces, wash water, and plant environment occur at plants where off-line eggs are processed. Based on surveys of commercial shell egg plants, Moats (1981) concluded that bacteria on equipment surfaces were the most important sources of egg shell contamination. Plant Z maintains the highest pH levels in their washer water (> 11). This may have allowed plant Z to decrease aerobic microbial levels equivalent with plant Y which had the lowest overall microbial contamination on the unprocessed eggs.
Plants X and Z re-washed sound eggs that were visibly dirty. When re-washing is incorporated into a plant's processing chain, an egg will either become visibly clean or break. This practice means that a higher proportion of visibly dirty eggs will be passed through the washers. Before parameters known to limit microbial contamination of wash water were determined, it was recommended that dirty eggs not be re-washed. It was thought that re-washing visibly dirty eggs would increase microbial counts in the wash water, increasing chances for cross-contamination, (Bruce and Dysdale, 1994). Eggs with considerable visible stains or adhering foreign matter are downgraded so re-washing eggs helps to increase profits (Zeidler, 2002). When temperatures are moderate (32-42°C) and pH levels are 9-10 microorganisms are more likely to survive the many hurdles presented by commercial shell egg processing. Plant X also had the lowest average temperatures recorded for washer 1 (39.7°C v. 44.1 and 44.5°C for plants Y and Z). Even at this plant, all populations were reduced by l log10 CFU/ml except for yeasts/molds.
Chemical oxygen demand (COD) is a measure of organic material in water (Northcutt et al, 2005). Plant Z had COD values twice those recorded for the other two plants. This possibly indicates that there were more eggs breaking in the washers of plant Z. This was the only one of the plants that did not oil eggs. About 30% of commercially processed and graded shell eggs are covered with a thin layer of odourless, tasteless mineral oil to occlude pores and minimize water and gas exchange (Northcutt, 2005). Oiling helps to prolong internal quality and can be very important in warmer climates or for eggs to be imported. Immersing eggs in warm water can decrease shell strength (Froning, 1973). Oiling may contribute to shell strength (Ball et al, 1976). Plant Z had the highest wash water pH (> 11 in both washers) as well as the most buffering capacity. Perhaps very alkaline wash water in combination with no oiling resulted in weaker shells. Egg solids and other organic materials reduce the efficacy of detergents in wash water (Moats, 1979; Moats, 1981). However, counts from plant Z were at the lowest levels recorded for aerobic microorganisms, yeasts/molds, Enterobacteriaceae, and E. coli despite highest pre-processing levels for all populations but yeasts/molds.
Despite plant differences, the way shell eggs were washed, graded, and sorted were similar. Regardless of plant or microbial population, highest bacterial and fungal counts were observed at the accumulator or the re-wash belts. These are visibly dirty or unwashed eggs. In fact, wash water at plant Z was harsh enough that all populations were decreased by greater than a log10 CFU/ml at the pre-wash rinse. Plant Y achieved the same result except for aerobic microorganisms, which were reduced in washer 1. Plant X achieved a log reduction in washer 1 for the four directly plated populations only after eggs reached the first washer.
Data for each population was averaged for the three plants and separated by sample site. Aerobes reached the lowest levels by the dryer while Enterobacteriaceae and E. coli were reduced to the lowest levels by washer 1. Yeasts/molds were reduced at pre-wash rinse but increased again at oiling. Oiling follows drying, accomplished by forcing warm air over the eggs as they emerge from the sanitizer rinse. A survey of air quality in shell egg processing plants indicated poorest yeast/mold air quality near the dryers and washers (Nortcutt et al, 2004). De Reu et al. (2005) compared aerobic shell populations on eggs collected from production through retail from cage and organic production systems. Their results indicated that air quality affected shell counts regardless of production system. However, by the end of the processing chain, all microbial populations determined in our study were significantly reduced compared to pre-processing levels.
Sanitizing rinse application is just one of the hurdles designed to diminish microbial egg shell contaminants. In a 1979 study, Moats (Moats, 1979) visited commercial facilities in Maryland and Pennsylvania that used different combinations of washing compounds and sanitizing or water rinses. Microbial populations on shell eggs in plants using sanitizer rinses were very low (<50 cells/shell), and significantly lower than one plant using an unsupplemented water rinse. However, when sanitizer rinse was temporarily cut off in plants that employed this type of rinse, populations on the shell did not change.
Moats concluded that a lack of significant change in egg shell bacterial numbers indicated that sanitizer rinse was at most an indirect effect. In a separate study, Moats (1981) obtained population data from equipment surfaces, wash water, and eggs. Based on correlations, he concluded that the sanitizer rinse was of no use. Our data supports his conclusion. AMS guidelines specify that sanitizer rinses must be compatible with detergents and of a strength equivalent to 50-200 ppm chlorine. Chlorine compounds perform optimally between pH 6.5-7.5 (Curtis and Johnston, 1998), much lower than that measured for wash water in this study. Other compounds have been analyzed to replace chlorine but none have been as effective (Kuo et al, 1997).
Once eggs were introduced into the washer, microbial populations were reduced and biologically significant increases were not observed through the remainder of the processing chain. Sanitation affects microbial populations during shell egg processing. Certain sections of the equipment are not water proof (scales), are difficult to reach (re-wash belt), or are difficult to remove and clean regularly (packer head brushes). However, contact with these surfaces did not result in significant increases in counts.
Salmonella is considered the most important human enteropathogen associated with shell eggs (Baker and Bruce, 1995; Ricke et al, 2001; USDA, 1998). S. Enteritidis is the serotype most often implicated in egg-borne outbreaks of salmonellosis though product temperature abuse followed by consumption of raw or undercooked eggs are usually factors. This serotype occurs at a low frequency (1 in 20,000 eggs) even when flocks are known to be S. Enteritidis colonized. However, all serotypes of Salmonella enterica are potential human pathogens and their presence on eggshells is of interest (USDA, 1996).
In the present study, we obtained 39 Salmonella isolates from egg shell rinses, tap water, and wash water. Individual plant visits yielded 0 – 25 Salmonella isolates. Except for X1, 0 – 4 isolates were obtained per plant visit. Between both shell rinse and crush methodologies, 35/396 (8.8%) samples were positive for Salmonella following enrichment. Jones et al., (1995) found 8/180 (4.4%) of egg shell rinses Salmonella positive. Prior to processing there were 7.8% (7/90) Salmonella positive rinses while post-processing rinses were only 1.1% (1/90) positive. March (March, 1969) and Cox and Davis (1968) did not recover Salmonella from 3,995 and 264 individual egg samples, respectively. During X1 sampling 1/3 of the tap water samples were determined to be contaminated with Salmonella. Plant X was the oldest plant (> 20 y old) included in the study and unchlorinated well water was used for processing. Potentially some animal (insect, amphibian, reptile, or mammal) may have compromised biosecurity and contaminated the plant's well water or it may have been caused by some other random event. This phenomenon was not observed again at plant X. It was never observed at plants Y and Z. Salmonella prevalence at this plant on other visits (X2, X3) was similar to that observed for other plant-visits (Y1-3, Z1-3). Salmonella prevalence for X2 and X3 averaged 6.25% (6/96) and 4.0% (10/252) for all other plant-visits, respectively. Salmonella were recovered from egg rinses collected during pre-process (10/28) more often than from in-processing (10/42) or post-processing stages (6/27). This data is evidence that commercial processes reduce Salmonella contamination of eggshells. Plant-visits in which Salmonella was recovered from eggshell rinse samples post-process were X1, Y2, Y3, and Z2.
Wash water parameters that are thought to influence Salmonella survival are temperature, pH, organic material, and iron levels. In addition to contaminated tap water, X1 was the plant-visit with the lowest average temperature and one of the only times where wash water pH was ≤10. Jones et al. (1995) and Catalano and Knabel (1994a, 1994b), detected Salmonella in shell rinses when the wash water pH was at the lowest measure (10.19). However, lowest pH was recorded for X2 (9.1) and Y1 wash water pH was 10. Average wash water temperature for all 9 plant-visits and both washers was 42.6°C. Three of the four plant visits where Salmonella was recovered post-process had wash water at or below that figure. Highest COD values were determined for all plant Z visits and the highest total solids figure was for Z3, iron levels were over 2 ppm for X3 yet Salmonella was only recovered from post-process samples from Z2. As determined from models derived from empirical data, a combination of factors affect whether or not Salmonella will survive shell egg processing. Hurdle technology has been built into the AMS guidelines and should be considered when writing HACCP plans for shell egg washing plants (Leister, 1994).
Five different serotypes from serogroups B and C were isolated from samples collected in our project. S. Enteritidis, of serogroup E was not recovered. In a national survey, Garber et al., (2003) did not isolate this serotype from production or processing samples collected in the south eastern United States. Salmonella serotypes recovered by Jones et al. (1995) from eggshells prior to processing were S. Heidelberg and S. Montevideo. Production serotypes were identified as S. Agona, S. Typhimurium, S. Infantis, S. Derby, S. Heidelberg, S. California, S. Montevideo, S. Mbandaka, and untypeable. Poppe (1991) isolated S. Typhimurium and S. Heidelberg most often from pools of layer hatching and table eggs. S. Heidelberg was a frequent egg belt and fecal contaminant from layer houses in a separate study (Davies and Breslin, 2003). Barnhart et al. (1993) sampled ovaries from spent laying hens in the south eastern United States.
Serotypes most frequently isolated were S. Heidelberg, S. Agona, S. Kentucky, and S. Typhimurium.
These data indicate that commercial egg processing significantly reduced levels of aerobic, yeasts/molds, Enterobacteriaceae and E. coli populations recovered by shell egg rinses. Populations decrease once eggs reach the first washer and remain at low levels through packaging.
Salmonella was isolated at every sample collection site on at least one of the nine plant-visits. Pre-process shell egg rinse samples were Salmonella positive more often than in-process or post-process collected samples.
Wash water pH, temperature, and condition (potability, contamination with organic material) seemed to partially account for Salmonella's ability to survive the commercial process. S. Enteritidis was never recovered from any of the samples.
Advanced analysis of the data may reveal which of these populations is the best indicator of product quality, safety, and process sanitation.
References are available on request.
From Proceedings of the "Midwest Poultry Federation Convention", St. Paul, Minnesota, U.S.A.
