Reduction of Salmonella Enteritidis in Shell Eggs Using Directional ...

PROCESSING, PRODUCTS, AND FOOD SAFETY Reduction of Salmonella Enteritidis in Shell Eggs Using Directional Microwave Technology D. G. Lakins,* C. Z. Alvarado,*1 L. D. Thompson,* M. T. Brashears,† J. C. Brooks,* and M. M. Brashears* *Department of Animal and Food Science, Texas Tech University, Box 42141, Lubbock 79409; and †Department of Agricultural Education and Communication, Texas Tech University, Box 42131, Lubbock 79409 ABSTRACT Microwaves have been shown to cause thermal as well as nonthermal destruction of pathogens such as Salmonella Enteritidis, which is commonly found in shell eggs. The objective of this study was to examine the use of new directional microwave technology to reduce Salmonella Enteritidis without causing any detrimental effects on quality in white and brown eggs. Treatments were control and microwaved white and brown eggs. Applying directional microwave technology resulted in

a 2-log reduction of Salmonella Enteritidis in both the high (105 cfu/g) and low (102 cfu/g) inoculum. At d 0, there were no differences in water activities, albumen pH, and combined pH between treatments; however, there were significant changes in yolk pH. Collectively, these results indicate that applying directional microwave technology can reduce Salmonella Enteritidis in shell eggs without causing any detrimental effects to quality.

Key words: egg, Salmonella Enteritidis, directional microwave 2008 Poultry Science 87:985–991 doi:10.3382/ps.2007-00393

however, the national incidence of Salmonella Enteritidis in eggs has been estimated to be much lower (Ebel and Schlosser, 2000). Gast and Beard (1990) reported that egg contamination by Salmonella Enteritidis involved relatively small initial numbers of bacterial cells. Most of the naturally contaminated eggs contain less than 10 cells per egg (Humphrey et al., 1989). Occasionally, eggs are reported to have much larger Salmonella Enteritidis numbers (Humphrey et al., 1989). It has been previously reported that Salmonella Enteritidis can be deposited into the yolk or albumen of a developing egg by an infected hen (Humphrey et al., 1989; Gast and Beard, 1990; Clay and Board, 1991; Gast and Holt, 2000a). The albumen can support Salmonella Enteritidis; however, very little bacterial growth occurs due to the bacteriostatic and bactericidal characteristics of albumen proteins (Bradshaw et al., 1990; Clay and Board, 1991; Lock and Board, 1992; Humphrey and Whitehead, 1993; Braun and Fehlhaber, 1995; Baron et al., 1997; Gast and Holt, 2000a, 2000b). Lysozyme is effective against Gram-positive organisms but not Salmonella. Therefore, initially barriers against Salmonella Enteritidis penetration into the egg are the shell, shell membranes, and albumen, which can act as barriers against microbial growth. However, the yolk provides a nutritious medium for growth of Salmonella Enteritidis (Bradshaw et al., 1990; Humphrey et al., 1991) and extensive growth of Salmonella Enteritidis at storage temperatures above 20°C (Braun and Fehlhaber, 1995). Eggs can also be contaminated due to cross-contamination during processing or the migra-

INTRODUCTION In 2003, over 200 million cases of eggs were produced for consumption (AEB, 2005). However, the AEB reported that every 1 out of 20,000 eggs may be contaminated with Salmonella Enteritidis. It is estimated that 200,000 to 1 million cases of human Salmonella Enteritidis foodborne cases occur in the United States alone, leading to an economic loss of up to $1 billion annually (Barbour et al., 2001). The Centers for Disease Control and Prevention estimates that 75% of Salmonella Enteritidis outbreaks are associated with the consumption of raw or inadequately cooked Grade A whole shell eggs. The USDA published regulations in 1990, establishing a mandatory testing program for egg-producing breeder flocks and commercial flocks that were implicated in causing human illness (Hope, 2002). This testing program led to a reduction (12%) in cases of gastroenteritis caused by Salmonella Enteritidis contamination in Grade A whole shell eggs (Hope, 2002). Naturally contaminated eggs are apparently produced at very low frequencies by commercial laying flocks in the United States. Several reports (Kinde et al., 1996; Schlosser et al., 1999) have shown that a naturally infected flock has egg contamination frequencies below 0.03%;

©2008 Poultry Science Association Inc. Received September 20, 2007. Accepted February 8, 2008. 1 Corresponding author: [email protected]

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tion of bacteria through the pore of the shell (Braun and Fehlhaber, 1995). It has been recently estimated that 3.2 million eggs contaminated with Salmonella Enteritidis can be produced annually in the United States (Ebel and Schlosser, 2000). During transportation and storage of shell eggs, the use of refrigeration has been proposed and instituted as a means of preventing small initial numbers of Salmonella Enteritidis contaminants from multiplying to levels that are more likely to cause human illness (USDA, 1998; President’s Council on Food Safety, 1999). Because Salmonella Enteritidis is more likely to contaminate the nutrient-rich yolk or attach to the exterior vitelline membrane surface, refrigeration can control an increase in numbers of bacteria within or on the surface of yolks (Gast and Holt, 2001). Gast and Holt (2000a) have determined that Salmonella Enteritidis grew more rapidly at temperatures of 17.5°C and 25°C in the yolk, but growth was attenuated at 10°C. Penetration of Salmonella Enteritidis bacteria into the yolk membrane from cross-contamination occurs slowly. However, once penetration of the yolk occurs, the nutrient-dense yolk provides an excellent source of nutrients for growth for Salmonella Enteritidis. Therefore, there is a direct positive correlation with rapid refrigeration of eggs to minimize rapid bacteria multiplication using yolk nutrients (Gast and Holt, 2001). Previous studies involving Salmonella Enteritidis prevention have been conducted to evaluate the effects of pasteurization and dry heat treatments on intact shell eggs (Jeng et al., 1987; Hou et al., 1996; FDA, 2000; Barbour et al., 2001; James et al., 2002; Ferroni et al., 2003; Froning et al., 2002). Barbour et al. (2001) subjected eggs to internal inoculation of low and high levels of a 5-strain Salmonella Enteritidis cocktail and then treated the eggs for 25 min at 57°C in a water bath followed by 57 min at 55°C in a hot oven. This treatment resulted in a 6-log reduction of Salmonella Enteritidis with no effect on the overall functionality of the eggs (Barbour et al., 2001). In addition, James et al. (2002) suggested that a time-temperature relationship was responsible for the destruction of Salmonella Enteritidis on the shell by steam, hot air, hot water, and infrared light. These authors (James et al., 2002) concluded that shell eggs subjected to steam for 2 s at 100°C achieved significant reductions in bacterial numbers on the shell without increasing the interior temperature of the egg contents, which could result in decreased egg protein functionality. Whole egg pasteurization requirements in other countries are as follows: China (63.3°C for 2.5 min), Australia (62°C for 2.5 min), and Denmark (65°C for 90 to 180 s; Cunningham, 1995). However, the USDA pasteurization requirements indicate that whole eggs must reach a minimum temperature of 60°C for 3.5 min (USDA, 1980). Other pasteurization methods such as hot water, hot air, and the combination of both hot water and hot air, have been used with some success in reducing pathogen loads in shell eggs (Jeng et al., 1987; Vanlith et al., 1995). In 2000, the FDA approved ionizing radiation for the reduction of Salmonella in fresh eggs. Treating eggs with irradiation has

proven to be an effective method of reducing Salmonella in eggs; however, consumer concerns with irradiation of foods have prevented the widespread use of this practice. Microwaves are electromagnetic waves that can be used to reduce Salmonella Enteritidis in shell eggs (Jeng et al., 1987; Ferroni et al., 2003). The standard frequency used in domestic microwaves is 2.45 GHz. The frequency for microwaves ranges from 300 MHz to 300 GHz, whereas the wavelength ranges from 1 mm to 1 m. Jeng et al. (1987) suggested that the electromagnetic energy in the microwave region (223 MHz to 3.45 GHz) could be an alternative source of energy for sterilization. Microwaves interact with dielectric materials to generate heat by the agitation of molecules in an alternating electromagnetic field (FDA-CFSAN, 2000). Water, carbon, and matter with high water or carbon contents are good microwave absorbers, thus leading to a higher amount of microwave absorption in food systems, which can result in a more rapid temperature increase (FDA-CFSAN, 2000). The effect of microwaves are influenced by many factors including the characteristics of foods such as humidity, ionic content, density, and weight; however, the characteristic of the device (i.e., shape of cavity, power of microwave wave supply, and product movements underneath the field) are also crucial factors that determine the potential effects of microwaves (Ferroni et al., 2003). The effect of microwaves on pathogens can be generally expressed in 2 forms, thermal and nonthermal destruction of pathogens. Thermal destruction of pathogens is caused by heating of the product during the microwave process. If temperatures reach pathogen destruction levels, then the bacterial cells are destroyed. Other researchers have also concluded that destruction of microorganisms may be caused by nonthermal effects. Four theories have been used to explain the nonthermal inactivation by microwaves or cold pasteurization: selective heating, electroporation, cell membrane rupture, and magnetic field coupling (Kozempel et al., 1998). Selective heating theory states that microwaves heat solid microorganisms more effectively than by the surrounding medium, and this causes a more rapid killing of the organism (Kozempel et al., 1998). Electroporation is caused when an electrical potential crosses the membrane of the microorganism, causing the formation of pores in the membrane that results in leakage of cellular contents. Cell membrane rupture is related to the voltage drop across a membrane resulting in the rupture of the cell membrane. Magnetic field coupling causes a disruption in the internal components of the cell, which leads to cell lysis. This allows bacteria to be destroyed at lower temperatures than using heat alone. Olsen et al. (1966) observed that the nonthermal effect of microwaves played a vital role in the inactivation of the microorganism in suspension causing the formation of hydrogen peroxide and other chemical transformations of small molecules, which resulted in cleavage of chemical bonds. The innovative directional microwave technology used at Texas Tech University is very different from commercial or household microwaves and was developed by an

DIRECTIONAL MICROWAVE APPLICATION ON SHELL EGGS

Italian company (ITACA New Tech, Brescia, Italy). In general, this technology differs from traditional/home electromagnetic pasteurization technology because of the following factors: • This equipment uses a horizontal and rotary movement. Traditional microwave ovens only have a rotary movement. In this way, food exposure to directional microwave is more uniform. • This equipment has several sources of microwaves: horizontal and vertical. With this procedure, the power can vary over a wider range of values and provide a more homogeneous distribution of power within the chamber. Traditional microwaves ovens have only 1 source. • In addition to heating, this equipment utilizes fast cooling using CO2. The ability to cool product quickly after microwaving is important in preserving quality of the product, because heat can denature proteins. Based on these differences, this directional microwave technology available at Texas Tech University is able to destroy pathogens at a lower temperature and without affecting the quality of the food. The directional microwave frequency used for the treatments is 2.45 GHz (corresponding to a 12.2-cm wavelength), which is allowed in the United States. Microwaves dissipate rapidly a short distance from their source, eliminating issues associated with microwave leakage. This equipment is manufactured based on international safety codes and procedures. Limited research has been conducted on the use of directed microwave technology on shell eggs as a method to reduce Salmonella Enteritidis. Thus, the major objectives of this study were to determine whether microwave technology can reduce Salmonella Enteritidis in shell eggs.

MATERIALS AND METHODS Directional Microwaves United States Department of Agriculture grade AA eggs were obtained from a local grocery store. All eggs were candled upon arrival to ensure AA quality egg. Three eggs at a time were placed in a plastic container (Figure 1), which was positioned in the chamber of the microwave. This directional microwave technology has several sources of microwaves (magnetrons), namely horizontal and vertical, which allows one to vary the power over a wider range of values and provide a more homogeneous distribution of power within the chamber. Traditional microwave ovens have only 1 source of microwaves. Eggs were placed in the microwave for 20 s (2.45 GHz; 12.2-cm wavelength; 80% magnetron power), 2-piston oscillation, and 30 s of CO2 applied at the end of the treatment. Temperature of the interior of the egg was verified with a calibrated probe thermometer after treatment was completed to ensure that the eggs reached a temperature of 45 to 50°C. This temperature range was chosen because Salmonella Enteriditis destruction occurs at 60°C for 3.5 min; however, because thermal and nonthermal destruc-

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tion occurs with microwave heating, a reduction in temperature may have the same destructive effect on Salmonella Enteritidis. Preliminary data indicated that achieving a temperature of 45 to 50°C for 20 s at 80% power would achieve a 2-log reduction. Twelve eggs of each egg type (brown and white) were exposed to either control (no treatment) or directional microwaves for 20 s. Three randomly selected eggs were used as controls to determine if naturally occurring Salmonella was present in the selected eggs.

Preparation of Salmonella Enteriditis Salmonella enterica ssp. Enterica serovar Enteritidis was obtained from America Type Culture Collection (ATCC #BAA-708). The strain used in this study was originally isolated from a human patient with salmonellosis associated with an egg outbreak. The culture was grown overnight in tryptic soy broth (MP Biomedicals Inc., Irvine, CA) at 37°C. Two inoculation doses were used, 1 high dose (105 cfu/mL) to measure actual log reductions and a low dose (102 cfu/mL) to observe Salmonella presence by mimicking conditions that would be observed in naturally contaminated eggs.

Inoculation of Shell Eggs High Inoculum Level. The Salmonella Enteritidis suspension prepared in the step above was used for inoculation. The Salmonella Enteritidis cells were then injected into the yolks of 12 white and 12 brown eggs (Hou et al., 1996). Briefly, the eggshell and a 100-␮L gas-tight glass syringe were sanitized using 70% ethanol. A small puncture hole was made on the blunt end of the shell egg with a sterile thumbtack, and 50 ␮L of the (105 cfu/mL) Salmonella Enteritidis suspension was injected into the yolk of each egg; the hole was then sealed with a thermosetting adhesive glue, which would not melt during heating. After drying (approximately 45 s), the eggs were treated with microwave technology or used as a control. After microwaving, the exterior of the eggs was sanitized using 70% ethanol, aseptically broken into stomacher bags, and 450 mL of buffered peptone water was added. The contents of the stomacher bag were then subjected to agitation using a circulating stomacher machine (Seward 400, Brinkmann Instruments Inc., Westbury, NY) for 3 min at 200 rpm. Finally, serial dilutions were made and surface-plated in duplicate onto XLT4 agar (Becton Dickinson, Franklin Lakes, NJ). The plates were then incubated at 37°C for 24 h, after which colony-forming units were enumerated using a Q-counter (Q count, Advanced Instruments, Norwood, MA). Cultures were direct-plated onto XLT4 agar for recovery of Salmonella Enteritidis. Low Inoculum Level. Salmonella was isolated using 2 enrichment broths, namely tetrathionate (Remel, Lenexa, KS) and Rappaport-Vassiliadis (Becton Dickson) to ensure that only viable cells were recovered. The broths were then surface-plated in duplicates onto XLT4 agar as

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Figure 1. Example of egg container and egg positioning.

described in the Bacteriological Analytical Manual (USDA-CFSAN, 2003).

pH A total of 10 randomly selected eggs from each treatment were used to measure pH of the whole egg, albumen, and yolk. The albumen and yolk were separated, and a total of 5 g of the albumen and 5 g of the yolk were placed in separate 250-mL beakers. After separation, 45 mL of distilled water was added to each beaker and mixed thoroughly using a handheld blender (Select Brands Inc., Lenexa, KS). The pH measurements were obtained for the individual yolk and albumen. After individual pH measurements, the albumen and yolk were mixed for 30 s, resulting in a 10% slurry solution (AOAC, 1990). The pH of the slurry solution was measured using a pH meter (Accument Basic AB-15, Fisher Scientific, Suwanee, GA).

Water Activity The water activity of the egg was measured in duplicate at the beginning and end points (d 0) using a water activity (Aw) meter (Aw Sprint, Novasina, Lachen, Switzerland). For each analysis, 3 g of egg (composite of freezedried yolk and albumen combination) was placed in a plastic cup and inserted into the Aw meter, which was calibrated using a saturated NaCl solution obtaining a Aw of 0 0.754 (Rahman, 1995).

Statistical Analysis Data was analyzed using the GLM procedures of SAS (2003) following a completely randomized design. White

and brown eggs were pooled, because no egg type interaction by egg type was present. Means were separated using Duncan’s multiple range test as described by SAS (2003) following a protected F-test. Probability values less than or equal to 0.05 were considered significant.

RESULTS AND DISCUSSION Reduction of Salmonella Salmonella is a major contributor of foodborne illnesses associated with eggs. Humphrey et al. (1998) indicated that naturally contaminated eggs contain less than 10 cells; however, a larger level was used to be able to quantify the reduction of Salmonella. Figure 2 indicates the reduction of Salmonella Enteritidis using a 105 cfu/mL inoculum level. In this study, the maximum reduction of Salmonella Enteritidis in shell eggs by directional microwave technology was approximately 2 log cycles. Hou et al. (1996) achieved a 3- and 5-log cycle reduction using hot water and hot air heating, respectively. However, the combination of the 2 (hot air and hot water) resulted in a 7-log cycle reduction without cooking the eggs. Even though higher reductions were observed in these other studies, the complex nature and time of exposure to hot air and a water bath may make these methods more difficult to use in egg processing facilities. Also, as previously stated, the natural contamination in eggs is low, and a 2log reduction would reduce these naturally contaminated eggs to below detection limits (Humphrey et al., 1989). Using directional microwave technology for a 20-s treatment provided rapid heating of the yolk to 48 ± 4°C depending on egg position (Table 1). No differences existed between white and brown eggs that might affect

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Figure 2. Reduction of Salmonella Enteritidis on table shell eggs subjected to directional microwaves for 20 s.

the absorption of microwaves. For both egg types, only position A achieved a 1-log reduction, suggesting that microwaves may not have been evenly distributed within the chamber. However, for positions B and C, a 2-log reduction was observed. The differences due to position of the egg inside the chamber may be decreased with modeling programs available that can indicate proper positioning of the magnetrons to ensure uniform electromagnetic rays throughout the entire testing area. Future research with this system can refine this research objective. These results indicate that providing a high temperature for a short time may be an effective strategy for reducing bacterial populations in shell eggs. Indeed, previous reports have demonstrated reductions of up to 80% in Salmonella Enteritidis counts from using microwave technology (Ferroni et al., 2003). A low inoculum level was used in this study to determine realistic differences in recovery of Salmonella Enteri-

Table 1. External temperature (°C) of eggs subjected to directional microwave treatment (20 s) Position in the microwave chamber1 Egg type White Brown

A2

B2

C2

44.0bc ± 2.3 44.1a ± 2.5

50.0ab ± 2.5 50.3a ± 2.1

49.3abc ± 1.9 49.1abc ± 2.0

a–c Means with different superscripts were significantly different (P < 0.05). 1 Position B is in the middle of the chamber, position A is on the left side of the chamber, and position C is on the right side of the chamber; looking straight forward at the equipment. 2 n = 18; values are means ± standard deviation.

tidis in shell eggs. Figure 3 shows the reduction of Salmonella Enteritidis using a low inoculum of 102 cfu/mL similar to naturally occurring situations and with the application of directional microwave technology. These results indicate that egg position in the chamber influenced the amount of directional microwaves received. For both white and brown eggs, position A resulted in 6 out of 6 Salmonella Enteritidis-positive eggs, whereas positions B and C resulted in 0 out of 6 positive eggs. Therefore, positions B and C provided at least a 2-log reduction.

Quality Assessment A review of research (Stadelman and Cotterill, 1995) has indicated a positive correlation between pH and Salmonella destruction. Table 2 shows the effect of directional microwave technology on egg pH. There were no significant differences in control pH, albumen pH, or combination pH of the directional microwaved eggs or the nonmicrowaved eggs. The average pH of a whole egg is around 7.0, which is slightly lower than reported in the present study (7.37 to 7.47), which could be attributed to the loss of CO2 and H2O within the bicarbonate buffering system. The pH of albumen in a freshly laid egg ranges from 7.6 to 8.5 but increases up to 9.18 after 3 d of storage at 5°C (Stadelman and Cotterill, 1995), which is in agreement with the present study. Yolk pH of freshly laid eggs is 6.0; however, previous research has indicated that during storage, it increases from 6.4 to 6.9 (Brooks and Taylor, 1955). In this experiment, the directional microwavetreated yolk pH was significantly higher (6.53) when com-

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Figure 3. Recovery of Enteritidis in table shell eggs subjected to directional microwaves for 20 s.

pared with the controls (6.23). Changes in yolk pH of the microwave-treated egg may be attributed to the formation of microscopic yolk spots, which causes the total solids of the yolk to become more concentrated. Also, the heating that occurred with the microwave treatment may have denatured some yolk proteins. Water activity plays a critical role in microbial growth. Microbial survival and growth with limited AW has been reported to be highly dependent on several factors including pH and oxygen (Chinachoti, 2000). Most bacteria growth is inhibited at AW below 0.85. An egg has a AW of around 0.96 (determined from preliminary data), which provides an ideal environment for microbial growth. In this study, there were no significant differences in AW in

Combination pH1

Albumen pH1

Yolk pH1

Water activity2

either treated or control eggs. This indicated that directional microwaves used in this study did not cause an increase in water loss from the egg due to excessive heating. As indicated above, the eggs were cooled after treatment with a rapid burst (30 s) of CO2 into the chamber to reduce heating and loss of water and CO2 from the bicarbonate buffer system. In summary, result shows that the directional microwave technology described in the present study resulted in a 2-log reduction of Salmonella Enteritidis in shell eggs. A 2-log reduction would be appropriate to eliminate Salmonella Enteritidis in most naturally contaminated eggs; however, additional studies are required to achieve a 3to 4-log reduction. In conclusion, directional microwaves can be used to reduce Salmonella Enteritidis in shell eggs; however, quality and nutritional parameters need to be examined to determine any detrimental changes that may occur due to the treatment. Future studies regarding this innovative technology will include quality and nutritional parameters.

7.37a 7.47a

9.32a 9.36a

6.23b 6.53a

0.963a 0.966a

ACKNOWLEDGMENTS

Table 2. Water activity and pH values after directional microwave treatment (20 s) on shell eggs at d 0 Treatment Control Microwave-treated

Means with different superscripts were significantly different (P ≤ 0.05). 1 n = 10. 2 n = 2. a,b

This study was supported by ITACA New Tech, and eggs were supplied by Cal-Maine Foods (Jackson, MS). We acknowledge and thank them for their assistance.

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