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Keywords:

  • eggs;
  • food safety;
  • heat;
  • ozone;
  • Salmonella

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To assess the contribution of ozone to lethality of Salmonella enterica serovar Enteritidis in experimentally inoculated whole shell eggs that are sequentially treated with heat and gaseous ozone in pilot-scale equipment.

Methods and Results:  Whole shell eggs were inoculated with small populations of Salmonella Enteritidis (8·5 × 104–2·4 × 105 CFU per egg) near the egg vitelline membrane. Eggs were subjected to immersion heating (57°C for 21 min), ozone treatment (vacuum at 67·5 kPa, followed by ozonation at a maximum concentration of approx. 140 g ozone m−3 and 184–198 kPa for 40 min) or a combination of both treatments. Survivors were detected after an enrichment process or enumerated using modified most probable number technique. Ozone, heat and combination treatments inactivated 0·11, 3·1 and 4·2 log Salmonella Enteritidis per egg, respectively.

Conclusions:  Sequential application of heat and gaseous ozone was significantly more effective than either heat or ozone alone. The demonstrated synergy between these treatment steps should produce safer shell eggs than the heat treatment alone.

Significance and Impact of the Study:  Shell eggs are the most common vehicle for human infection by Salmonella Enteritidis. Many cases of egg-related salmonellosis are reported annually despite efforts to reduce contamination, including thermal pasteurization of shell eggs and egg products. Treatment with ozone-based combination should produce shell eggs safer than those treated with heat alone.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Salmonella is one of the most prevalent pathogens associated with foodborne illness. It is the cause of an estimated 1·4 million illnesses annually in USA alone (Lynch et al. 2006). In recent decades, proportion of salmonellosis attributed to Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis), relative to other Salmonella serovars, has increased. According to a 2001 report (Guard-Petter 2001), Enteritidis is the most prevalent serovar implicated in verified cases of salmonellosis worldwide. The most common food source of Salm. Enteritidis is eggs. Shell eggs are commonly contaminated in a vertical manner during egg formation in colonized hen oviduct, or horizontally via migration of salmonellae through the egg shell after contact with contaminated substances such as hen faeces (Guard-Petter 2001; Centers for Disease Control and Prevention (CDC) 2003; Grijspeerdt et al. 2005; Lynch et al. 2006). Internally contaminated eggs, obtained from naturally infected hens, may contain ≤10 Salm. Enteritidis per egg and the pathogen is most likely localized outside the vitelline membrane or in the albumen surrounding it (Humphrey 1994; Humphrey et al. 1991; Poppe et al. 1992). Consumption of eggs that are raw or undercooked and pooling of eggs (a common practice in restaurants and institutional settings) can increase risk of illness (Centers for Disease Control and Prevention (CDC) 2003; Lynch et al. 2006).

The widespread risk of salmonellosis has led to the introduction of numerous strategies to decrease egg contamination and resulting illness. These strategies include consumer education regarding safe egg handling and cooking practices and implementation of quality assurance programmes in egg production facilities (Mumma et al. 2004). While these efforts have succeeded in reducing the number of cases of salmonellosis from the peak of 3·9 per 100 000 people reached in 1994, data for 2003 showed 1·7 cases per 100 000 people, indicating that a serious risk remains (Braden 2006).

Ozone is a colourless gas consisting of three oxygen atoms. It is a strong oxidizing sanitizer and has been approved by the US Food and Drug Administration (FDA) for use in foods [United States Food and Drug Administration (US-FDA) 2006]. Application of ozone either in gaseous form or via oxidized water has shown promise as an antimicrobial agent against a number of pathogens in several food systems including lettuce and beef (Novak and Yuan 2004; Selma et al. 2007). The efficacy of gaseous ozone against Salm. Enteritidis on the surface of whole eggs has also been demonstrated, as has ozone’s ability to penetrate intact egg shells (Rodriguez-Romo and Yousef 2004; Rodriguez-Romo et al. 2007). The present research investigates the possibility of reducing or potentially eradicating Salm. Enteritidis in whole shell eggs via treatment with a novel process involving immersion heating and application of high levels of ozone under pressure. Previous research has been aimed at assessing the effectiveness of combination processes and defining process parameters (Rodriguez-Romo 2004). In the current study, emphasis has been placed on possible industrial application of this technology by simulating conditions of natural contamination through use of lower inoculum level and scaled-up treatment setup.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Egg preparation

Unfertilized, unwashed eggs were obtained from the farms of Hemmelgarn & Sons, Inc. (Coldwater, OH). Eggs were stored at 4°C and were used within 2 weeks of laying. Eggs were measured with Vernier calipers (Manostat Co., Merenschwand, Switzerland) and only those with a width of 45–46 mm were used for experimental treatments. Selected eggs were stored at room temperature for approx. 2 h before being scrubbed individually with a plastic brush under cool running tap water. Washed eggs were soaked in ethanol (70% v/v) for 30 min to sanitize shells. Eggs were then placed in previously sterilized egg trays and allowed to dry at room temperature for 40 min, approximately, prior to inoculation.

Culture preparation

Salmonella Enteritidis ODA 99-30581-13, isolated from an egg source, was provided by the Ohio Department of Agriculture (Reynoldsburg, OH, USA). The pathogen was cultured in tryptic soy broth (TSB) (Criterion, Hardy Diagnostics, Santa Clara, CA, USA) and incubated at 37°C for 24 h. Thereafter, 0·15 ml of overnight culture was transferred to 150 ml MacConkey broth (Alpha Biosciences, Baltimore, MD, USA), and incubated for additional 24 h in an orbital shaker (Lab-Line, Mumbai, India) set at 37°C and 175 rev min−1. Duplicate 45-ml aliquots of this culture were centrifuged at 4°C and 3020 g for 10 min. Cell pellets were resuspended in 2·5 ml chilled phosphate buffer (0·2 g mol−1, pH 7) and combined for a final concentration of approx. 1011 CFU ml−1; this cell suspension was diluted as needed to achieve proper cell concentration for desired inoculum level.

Inoculation

Shell eggs were inoculated with Salmonella as described earlier (Rodriguez-Romo 2004). Briefly, sanitized eggs were punctured in the approximate centre of the blunt side using a sterile 1·5-inch/20-gauge needle attached to a disposable 1-ml syringe (Becton Dickinson, Franklin Lakes, NJ, USA). Salmonella Enteritidis cell suspension (10 μl, approx. 107 CFU ml−1, verified by direct plating) was introduced into eggs near the vitelline membrane using a repeating pipette (Yellow Springs Instrument Co., Yellow Springs, OH, USA) with rubber stopper affixed 5 mm from needle point. Inoculation site was wiped with ethanol (70% v/v) and allowed to dry for one minute. Holes were then sealed using Teflon tape (Cole-Parmer, Vernon, IL, USA). Internal inoculation level of Salm. Enteritidis was 8·5 × 104–2·4 × 105 CFU per egg.

Heat treatment

Inoculated eggs were placed in a wire basket and heat treated by submersion in a circulating waterbath (Thermo Corporation, Waltham, MA, USA) set to 57°C. Total treatment time was 21 min. Internal egg temperature was monitored using an uninoculated egg with thermocouple (Fluke, Everett, WA, USA) wire inserted to depth of placed inoculum. The change in internal temperature with heating time is displayed in Fig. 1. Immediately after treatment, eggs were either stored at 4°C (for heat alone samples) or transferred to ozonation vessel for further treatment.

image

Figure 1.  Internal egg temperature and water bath temperature during heat treatment of shell eggs. (bsl00041) Egg interior; (bsl00066) water bath.

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Ozone treatment

Eggs were subjected to vacuum (67·5 kPa) prior to ozone treatment. Gaseous ozone was produced from pure oxygen by an ozone generator (Ozonia, Elmwood Park, NJ, USA). The output of this generator is an ozone-oxygen mixture that will be referred to as ‘ozone gas’ or ‘gaseous ozone’ throughout this manuscript. Generator output was pumped to a custom treatment vessel to a maximum ozone concentration of 140 g m−3. Ozone concentration was monitored continually using an ultraviolet ozone detector (Mini-Hicon model; IN USA, Inc., Norwood, MA, USA). Introduction of ozone to the vessel resulted in increased pressure, with the final pressure being 184–198 kPa. Once desired ozone concentration and pressure were reached, (come-up time of 10–12 min), gas inlet was closed and eggs were subjected to static treatment for 40 min. At the end of treatment, pressure and ozone were released from the vessel and ozone was destroyed by a thermal destruct unit (Ozonia). Treated eggs were removed from the vessel and held at 4°C for 18 h to insure exhaustion of ozone residues. A schematic representation of experimental setup is shown in Fig. 2.

image

Figure 2.  Representation of experimental setup used to treat whole shell eggs with gaseous ozone under pressure.

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Enumeration of surviving cells

Eggs were analysed 18-h post-treatment for surviving Salm. Enteritidis. Untreated and ozone-only controls were plated on trypticase soy agar and incubated at 37°C for 48 h. Eggs treated with heat and ozone–heat combination were analysed for viable Salmonella using a most probable number (MPN) technique that was customized (by increasing sample volume) to detect ≥0·03 MPN g−1. Tested shell eggs were pooled in groups of two and diluted to 10−1 in TSB (original dilution). Aliquots of the original dilution (100, 10 and 1 ml, containing 10, 1, and 0·1 g egg, respectively) were used for MPN analysis. Remaining volume of original dilution was incubated at 37°C for 48 h before streaking onto xylose lysine desoxycholate agar (XLD; Alpha Biosciences, Baltimore, MD, USA) for confirmation of overall negative results, i.e. when all MPN flasks tested negative. MPN flasks were incubated at 37°C for 24 h before streaking on XLD agar to confirm presence of Salmonella. When typical Salmonella colonies on XLD were detected, egg was considered positive for the pathogen.

Statistical analysis

Data analysis was performed using sas v. 9.1.3 software (SAS Institute, Inc., Cary, NC, USA). Results were compared using an unpaired t-test with probability value <5% considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Salmonella-inoculated shell eggs were exposed sequentially to heat, vacuum and pressurized ozone gas. Waterbath temperature was maintained at 57°C (±0·5°C). Average internal temperature of eggs upon conclusion of heating step (i.e. after 21 min of egg immersion in the waterbath) was 56·2°C (Fig. 1). A vacuum (67·5 kPa) was attained after ≤5 min of closing the vessel and starting the vacuum pump. The low-pressure vessel was charged with ozone gas to attain required ozone concentration and pressure; this process was completed in 11–12 min. Pressure within the treatment vessel remained relatively stable throughout treatment (Fig. 3). A slight decrease in measured ozone concentration was observed; this may be attributed to the inherent instability of ozone molecules or reaction with organic material on egg surface. Sequential treatment of shell eggs with heat, vacuum and ozone was completed in approx. 100 min, including come-up time for various treatment steps.

image

Figure 3.  Ozone concentration and pressure inside treatment vessel during treatment of shell eggs. (bsl00041) Ozone; (bsl00066) pressure.

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Heat alone and combination treatments resulted in a significant decrease in Salm. Enteritidis, but this was not the case in eggs subjected to ozone without a prior heating step (Table 1). Treatment with ozone alone resulted in only 0·11 log reduction, a difference that is not significant from the untreated control. Heat treatment decreased Salmonella population by 3·1 log and heat–ozone combination eliminated 4·2 log. This level of inactivation represents fewer than one viable cell per gram of egg treated with heat–ozone combination. Flasks producing negative MPN results were incubated to confirm presence or absence of Salm. Enteritidis. Salmonella was recovered from all eggs treated with ozone alone and heat alone, but only 10 of 18 combination-treated eggs tested positive, indicating Salmonella eradication in a considerable portion of those samples (Table 1).

Table 1.   Inactivation of Salmonella Enteritidis in shell eggs after various treatment and total number of treated samples testing positive for surviving salmonellae
TreatmentTreatment conditionsAverage* decrease in log CFU per egg Positive/total†
  1. *Averages for 18 treated eggs; initial inoculum was 8·5 × 104–2·4 × 10CFU per egg. Averages with different superscript letters are significantly different (< 0·05).

  2. †Presence of Salmonella Enteritidis confirmed by streaking of the positive MPN tube or the extended enrichment on xylose lysine deoxycholate agar.

Inoculated and nontreatedNone0A18/18
Ozone (O3) (O3) approx. 140 g m−3 (maximum) at 184–198 kPa for 40 min0·11A18/18
Heat57°C for 21 min3·1B18/18
Heat and ozone57°C for 21 min; vacuum (67·5 kPa); (O3) approx. 140 g m−3 at 184–198 kPa for 40 min4·2C10/18

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Though progress toward safer eggs has been made in recent years, a significant risk still remains. A recent risk assessment conducted by the US Department of Agriculture, Food Safety and Inspection Service, predicted that pasteurization of all shell eggs resulting in a three log reduction of Salm. Enteritidis populations would decrease the annual number of associated illnesses from 130 000 to 41 000; a reduction of nearly 70% [United States Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) 2005]. As demonstrated, a heat–ozone combination process possesses the potential of achieving this goal. Whole egg products pasteurized by heat alone are currently available in the USA market place (National Pasteurized Eggs, Inc. 2007). Since their introduction in 2000, there have been no outbreaks associated with pasteurized eggs, but the long heating time necessary to achieve a suitable reduction in salmonellae has been observed to adversely affect the quality and clarity of albumen (Schuman 1996; Rodriguez-Romo 2004). These effects on protein quality lead to increased opacity of egg whites and decreased whip volume, which may be off-putting to consumers or restrict use in whipped products. The use of heat–ozone combination, compared with heating only, allows for a considerable reduction in overall heating time without compromising the safety of treated eggs. This reduction in treatment time may serve to minimize heating’s adverse effects on egg quality.

We propose that heating shell eggs increased permeability of their membranes to ozone gas. Therefore, application of ozone was effective against internal Salmonella only when shell eggs were subjected to heat prior to ozone treatment. Unlike a previous investigation in this laboratory (Rodriguez-Romo 2004), the current study reports the synergy between ozone and heat when a smaller population of Salmonella is introduced in eggs. Additionally, eggs were treated with ozone in a larger pilot-scale chamber to simulate industrial application.

Many studies show that when bacteria are found in a complex matrix, thermal inactivation deviates from first-order kinetic (Moore and Madden 2000; Buzrul and Alpas 2007). Diminishing bacterial lethality with extended treatment is often described as death curve ‘tailing’. Presence of resistant sub-population of the treated bacterium is believed to cause the tailing phenomenon (Moore and Madden 2000). Prior work was performed on the strain used in this study has demonstrated the presence of a significant tailing effect during egg heating (Rodriguez-Romo 2004). Based on the results of the current study, it is proposed that ozone (which was applied subsequent to thermal treatment) inactivates the most heat-resistant Salmonella sub-population. Results of this study support the feasibility of pasteurizing shell eggs using minimal heating that is followed by a carefully planned ozone treatment. Additional research is needed to maximize pathogen lethality without damaging product quality and to verify the effect of reduced heating time allowed by sequential application of ozone on egg quality.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Funding was provided by the Ohio Agricultural Research and Development Center (Ohio State University) and the Food Safety Inspection Service, US Department of Agriculture. The authors would like to thank Hemmelgarn & Sons, Inc. for supplying shell eggs and the Ohio Department of Agriculture for providing the Salmonella strain. We also thank Mustafa Vurma for equipment assistance provided throughout the duration of this project.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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