Reduction of Eggshell Aerobic Plate Counts by Ultraviolet Irradiation C. Chavez, K. D. Knape, C. D. Coufal, and J. B. Carey1 Poultry Science Department, 101 Kleberg Center, Texas A&M University, College Station, Texas 77843-2472 experiment, UV lights were placed in a chamber equipped with a commercial-style egg conveyor. A UV treatment of 7.5 mW/cm2 and time intervals of 0, 12, 36, and 48 s were used. Three eggs were placed consecutively on the conveyor and passed through the chamber. The center egg was selected for APC evaluation. Sample size, dilution, plating, and incubation procedures were used as described for the first experiment. A significant 1 to 2 log10 reduction in colony-forming units per egg between the eggs treated 48 s to the untreated eggs was detected. The results of these studies show that UV light treatment at high intensities and low time intervals has the potential to reduce aerobic plate counts of eggshells.
(Key words: eggshell, ultraviolet irradiation, aerobic plate count) 2002 Poultry Science 81:1132–1135
INTRODUCTION Horizontal contamination of eggs can be a major route of infection in the poultry industry (Pardon, 1995). Eggs that come from breeder farms could contain high numbers of bacteria that could affect other eggs at the hatchery, which in turn leads to poor chick quality. Therefore, lesser quality birds will be delivered to the processing plant. Even visibly clean eggs may harbor considerable numbers of bacteria on the shell surfaces due to contamination from feces, infested nests, transfer belts, and air and packaging materials (Mayes and Takeballi, 1983). These bacteria can penetrate the shell and contaminate internal contents of the egg (Board, 1964). Bacteria that have entered the egg could cause damage or death to embryonic development. To increase survivability of an embryo, the egg surface must remain free of contaminants (Kuo et al., 1997a). Previous research has shown that contaminated hatching eggs result in reduced hatchability, spread of diseases, early chick mortality, and impairment of bird performance (Quarles et al., 1970; Ernst et al., 1980; Gardner et al., 1980; Cox et al., 1990). An effective sanitation method is needed that maintains the cuticle on
2002 Poultry Science Association, Inc. Received for publication November 2, 2001. Accepted for publication March 1, 2002. 1 To whom correspondence should be addressed: jcarey@poultry. tamu.edu.
the egg to keep bacteria from causing internal contamination (Peebles and Brake, 1986). The cuticle is an important natural physical defense involved in protection of egg contents from invading organisms. The cuticle acts to interfere with bacterial invasion by closing the pores resulting in a reduction in the permeability of the shell (Fromm, 1963). Once the cuticle is removed, bacteria gain the ability to cross the shell more rapidly. With these considerations, ultraviolet (UV) radiation is a potentially favorable alternative to keep the cuticle on the egg (Kuo et al., 1997b). UV light has been shown to be effective in reducing various bacterial populations on eggshell surfaces (Goerzen and Scott, 1995; Gao et al., 1997). These reductions in bacteria populations could give the embryo greater survivability during incubation. Past research has used a variety of UV intensities and exposure times on eggshell surfaces (Goerzen and Scott, 1995; Gao et al., 1997). However, an effective UV treatment to maximize exposure of the entire eggshell surface has not been thoroughly investigated (Kuo et al., 1997a). Kuo et al. (1997a) showed that using higher UV intensities and rotating the eggs at one revolution per minute to give the eggshell surface a more complete exposure to UV light reduced bacterial populations significantly. The objectives of this study were to use greater UV intensities, rotate the eggs at more revolutions during UV
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Abbreviation Key: APC= aerobic plate count, UV= ultraviolet light.
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ABSTRACT The effects of 254 nm ultraviolet light (UV) radiation on aerobic plate count (APC) of egg shells were investigated. In the first experiment, eggs were exposed to UV treatment (7.35 mW/cm2) for 0, 15, 30, and 60 s. Three eggs from each treatment were aseptically collected and placed into sterile plastic bags containing 50 mL of sterile phosphate-buffered solution. Serial dilutions of the phosphate-buffered solution were plated on aerobic plate count agar and incubated at 37 C for 48 h. Exposure of eggshells to 30 and 60 s UV significantly reduced aerobic plate counts compared to untreated eggs. Exposure to 60 s of UV resulted in a 2 to 3 log10 cfu/egg APC reduction and reduced counts below detectable levels. In the second
EGG SANITATION BY ULTRAVIOLET LIGHT
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FIGURE 1. Diagram of hand-operated egg roller. Eggs are placed aseptically on hand-operated egg roller, which is enclosed in a chamber surrounded by eight 91.5-cm ultraviolet (UV) lights with an intensity of 7.35 mW/cm2.
MATERIALS AND METHODS Hand Operated Egg Study Eggs were placed on a hand-operated egg roller (Figure 1) rotated at nine revolutions per minute and exposed to (7.35 mW/cm2) of UV radiation for 0, 15, 30, or 60 s. All UV intensities were measured by a UV meter2 at the closest point between the eggshell and the UV lamps.3 Visibly clean, unwashed eggs were collected in a random sample 1 h before the experiments were run. After UV treatments, eggs were aseptically transferred to sterile plastic bags (one egg per bag) and rinsed with 50 mL of sterile PBS, pH 7.2. After 5 s of gentle hand massage, 1 mL of the rinse solution was used to estimate the viable microorganisms. Aerobic microorganisms were counted by plating the serially diluted rinse solution on plate count agar4 and incubating the plates at 37 C for 48 h before counting viable colonies. Plate count agar was used to enumerate all aerobic bacteria found on the surface of the eggshell. The colony counts were multiplied by 50 (50 mL PBS) to estimate the total microbial load recovered from the eggshell and transformed to log10 colony-forming units per eggshell (log10 cfu/egg). The lowest detection level for this procedure was 1.7 log10 cfu/egg (log10 50). Zero colonies is equivalent to <1.7 log10 cfu/egg. For statistical analysis, 0.85 was used to represent <1.7 log10 cfu/egg. With three eggs per treatment and three trials, a total of 36 eggs were sampled in this experiment.
2
Model UVX-25, UVP, Inc., San Gabriel, CA. G30T8 Germicidal lamps, General Electric Company, Cleveland, OH. 4 Difco Laboratories, Sparks, MD. 5 G30T8 lights, General Electric Company, Cleveland, OH. 6 G8T5 lights, General Electric Company, Cleveland, OH. 3
FIGURE 2. Diagram of prototype machine. Eggs are placed aseptically on the egg conveyor belt of prototype machine. The belt carries the egg through an ultraviolet (UV) chamber, which contains eight 91.5cm (long lamps) UV lights with an intensity of 7.5 mW/cm2 in front of the chamber, and eight 30.5-cm (short lamps) UV lights with an intensity of 6.55 mW/cm2 at the rear of the chamber.
Prototype Machine Egg Study The second experiment tested a prototype machine (Figure 2) to commercially implement the UV sanitation method. The prototype machine contained a chamber equipped with UV lights and an egg conveyor system that rotated eggs as they passed through the chamber. Eight long UV lights5 (91.5 cm) with a UV intensity of 7.5mW/cm2 were placed in the front of the chamber. Eight short lights6 (30.5 cm) with a UV intensity of 6.55mW/cm2 were placed in the back of the chamber. Eggs were exposed to four different UV intensity combinations: no lights, short lights, long lights, and both lights. These treatments resulted in exposure of eggs to four different intervals and rotation combinations. The short light treatment exposed eggs for 12 s and 3 revolutions, long lights for 36 s and 9 revolutions, and both lights for 48 s and 12 revolutions. Three eggs were placed consecutively on the conveyor to simulate egg collection methods at a fertile egg farm, and the center egg was selected for aerobic plate count (APC). Three sets of eggs were treated for each UV intensity treatment and control. Twelve eggs were sampled for each trial, and three trials were con-
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exposure, and use shorter intervals. Such improvements in UV application could lead to an effective, economical eggshell-sanitizing method for commercial application.
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CHAVEZ ET AL. 1
TABLE 1. Effects of hand-operated egg roller ultraviolet (UV) treatments on eggshell aerobic plate counts Treatment
Trial2 1
Trial2 2
Trial2 3
TABLE 2. Effects of prototype machine ultraviolet (UV) treatment on eggshell aerobic plate counts Treatment
Trial1 1
(log10 cfu/egg) Control 15 s 30 s 60 s SEM3
3.96a 3.21ab 2.73b 1.90c 0.48
4.17a 3.64ab 3.38b 2.15c 0.34
Statistical Analysis Data were subjected to analysis of variance using the general linear models procedure of SAS software.7 Mean differences were separated by the PDIFF option (pairwise t-tests) Statistical significance for all data was considered at P < 0.05.
RESULTS AND DISCUSSION Hand Operated Egg Study Aerobic microorganism populations were significantly reduced by the combination of high UV radiation and roller treatment. In Trials 1 and 2, the 15-s exposure treatment when compared to control eggs was not significantly different. However, in Trial 3 there was a significant reduction of 0.5 log10 cfu/egg (Table 1). In all 30-s UV exposure trials, there was a significant reduction of 1 to 2 log10 cfu/egg (Table 1) compared to the controls. Eggs rotated for 60 s had significantly greater reductions of APC than the other time intervals of exposure. A 2 to 3 log10 cfu/egg of aerobic microorganisms compared to controls was observed after 60 s of exposure to UV radiation (Table 1). Kuo et al. (1997b) reported a significant reduction as UV exposure time increased. However, the exposures in their study were 0, 5, 10, and 15 min at 4.35 mW/cm2 UV. By using a 15-min exposure treatment, there was a 2.8 log10 cfu/egg reduction compared to the control. In this study, we were able to achieve the same amount of log10 cfu/egg reduction but at a shorter interval (30 and 60 s) using a higher UV intensity (7.35 mW/cm2). These results were in agreement with Kuo et al. (1997b) that higher UV intensities and longer exposure times resulted
Control Short lights2 Long lights3 Both lights SEM4
4.61a 4.32ab 3.80b 2.62c 0.16
4.48a 4.27a 3.85b 3.10c 0.12
4.63a 4.28ab 3.78b 2.38d 0.31
a-d Values within a column with different superscripts differ significantly (P < 0.05). 1 Number of observations per trial = 12 (3 eggs per treatment). 2 UV intensity of 6.55 mW/cm2, 12 s, three revolutions. 3 UV intensity of 7.5 mW/cm2, 36 s, nine revolutions. 4 Pooled standard error of the mean.
in higher bactericidal effects. These results reinforced the theory that an apparatus could be built to apply this UV sanitation method in commercial applications.
Prototype Machine Egg Study In the second experiment, aerobic microbial populations were significantly reduced by exposure to greater intensity (7.5 mW/cm2) of UV radiation in the prototype machine. This increase was 0.15 mW/cm2 UV intensity of that in first experiment. In all three trials, there was no significant difference between short lights (12 s at 6.55 mW/cm2) and no lights (control eggs). Eggs that were exposed to long lights (36 s at 7.5 mW/cm2) had a 0.5 to 0.9 log10 cfu/egg reduction compared to controls. Exposure to both lights resulted in a significant 1 to 2 log10 cfu/egg reduction in APC compared to controls (Table 2). Aerobic microbial populations were significantly reduced more in the first experiments (hand operated) than in the second (prototype machine). Two reasons could account for these differences. The second experiment used less exposure time, which was 48 s compared to 60 s. Another difference between experiments was the rate of revolutions. The second study used up to 12 revolutions for 48 s of UV treatment, whereas six revolutions for 60 s were used in the first experiment. This finding may suggest that fewer revolutions at longer intervals could give more complete exposure of the egg to UV radiation. In conclusion, the results of these studies indicate that higher UV intensity at short intervals with egg rotations can significantly reduce aerobic microbial populations. The data show that UV light could significantly reduce aerobic microbial populations, allowing fewer bacteria to penetrate and contaminate the inside of the eggshell. This decreased contamination would result in fewer bacteria that could cause damage or death to the embryo during incubation. These data seem promising for implementing a UV sanitation method at commercial breeder farms. Fertile hatching eggs would be treated on the egg conveyor belt before collection. Such methodology could allow eggs to be treated before they arrive at the hatchery, thereby leading to better hatchability, better chick quality, and
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ducted, for a total of 36 eggs in this experiment. APC enumeration methods and statistical procedures were as described for the previous experiment.
SAS Version 6.11, SAS Institute, Cary, NC.
Trial1 3
(log10 cfu/egg)
4.11a 3.60b 2.07c 0.80d 0.36
a-d Values within a column with different superscripts differ significantly (P < 0.05). 1 UV intensity of 7.35 mW/cm2. 2 Number of observations per trial = 12 (3 eggs per treatment). 3 Pooled standard error of the mean.
7
Trial1 2
EGG SANITATION BY ULTRAVIOLET LIGHT
ultimately greater yield. Therefore, a better product will be delivered to the consumer. For future studies, UV sanitation method needs to be evaluated for its effectiveness against pathogenic organisms such as Salmonella and Escherichia coli on the shells of broiler hatching eggs and resulting effects on hatchability.
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