C. B. MICHALSKI, R. E. BRACKETT,* Y.-C. HUNG, AND G. O. I. EZEIKE

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1 11 Journal of Food Protection, Vol. 6, No., 1999, Pages Copyright, International Association of Milk, Food and Environmental Sanitarians Use of Capillary Tubes and Plate Heat Exchanger to Validate U.S. Department of Agriculture Pasteurization Protocols for Elimination of Salmonella Enteritidis from Liquid Egg Products C. B. MICHALSKI, R. E. BRACKETT,* Y.-C. HUNG, AND G. O. I. EZEIKE Center for Food Safety and Quality Enhancement, University of Georgia, Griffin, Georgia 303, USA MS : Received 9 April 1998/Accepted October 1998 ABSTRACT D values for a five-strain cocktail of Salmonella Enteritidis in five different liquid egg products (whole egg, egg yolk, egg white, egg yolk 5% sucrose 5% NaCl, and egg yolk 10% NaCl) were determined using 100- l capillary tubes. The egg products were inoculated with approximately organisms/ml and heated in capillary tubes to temperatures ranging from 51 to 68 C for various time intervals. Using a pilot scale plate heat exchanger, the U.S. Department of Agriculture (USDA) protocols for pasteurization were also evaluated using egg products inoculated with approximately Salmonella Enteritidis/ml. Results of experiments with capillary tubes suggested that almost all processes would result in less than the 9D process recommended by the USDA. However, when the egg products were pasteurized using the plate heat exchanger, a greater than 9D process was achieved for Salmonella Enteritidis in all products except egg yolk containing 5% sucrose 5% NaCl, which received approximately a 4D process. The demand for safer egg products by consumers and food-processing plants resulted in the number of eggs used for pasteurized egg products to increase from 8.7 billion eggs (13.8%) in 1980 to 15 billion eggs (4.6%) in 199 (14). Pasteurization of liquid egg products became mandatory in the United States on 1 June 1966 in response to the presence of Salmonella species in liquid egg products. The U.S. Department of Agriculture (USDA) subsequently recommended a 9D process to provide a wide safety margin. More recently, the emergence of Salmonella Enteritidis, a more heat-resistant Salmonella strain, and the presence of Listeria monocytogenes with its ability to grow at refrigeration temperatures in liquid egg products has caused some concern. In fact, at least one source has predicted a major outbreak of Salmonella due to pasteurized egg products since a recent survey of pasteurized eggs found % positive for Salmonella (18). However, there have thus far been no foodborne illnesses attributed to pasteurized egg products. Salmonellosis cases caused by Salmonella Enteritidis have dramatically increased since 1976 when 5% of salmonellosis cases reported to the Center for Disease Control (CDC) were Salmonella Enteritidis compared to 1994, when 6% of the salmonellosis cases were attributed to Salmonella Enteritidis (). The major source of these infections was properly sanitized grade A table eggs that had been served raw or lightly cooked (16, 19). Many studies have established that Salmonella Enteritidis contaminate eggs when the organism is passed from the infected repro- * Author for correspondence. Tel: ; Fax: ; rbracke@cfsqe.griffin.peachnet.edu. ductive tissue of hens, rather than the shell, to the contents of contaminated eggs (1, 8, 1, 1). The number of liquid whole egg samples positive for Salmonella ranged from 4. to 56%, with a higher incidence in summer and fall than in winter and spring (7, 13). In a later study, 15% of unpasteurized liquid whole eggs were positive for Salmonella Enteritidis, with no seasonal differences (5). Even though the percent positive was high, the populations of salmonellae in these samples were low. One USDA investigation found a maximum of 5.3 salmonellae/g while another study found a maximum of 110 salmonellae/g. The two studies combined found that less than 3 salmonellae/g were present in 96% of the positive samples (7). A summary of three investigations reported by Elliott and Hobbs (6) states that 93% of the samples that were positive for Salmonella contained less than 1 salmonellae/g and none contained more than 110 salmonellae/g. Salmonella Enteritidis is considered a heat-sensitive organism. Based on published data, Salmonella Enteritidis is most resistant in egg yolk, followed by whole eggs, and egg white. This variability in thermal inactivation of Salmonella in different egg products is due to the products different ph, absorbance in water (a w ), and nature of constituents. Addition of solutes such as NaCl and sucrose to egg products increases Salmonella Enteritidis heat resistance in that product. Even though original populations of Salmonella Enteritidis in liquid egg are low, there is the potential for these populations to increase to levels capable of causing disease. Temperature abuse of the egg product can lead to higher numbers of organisms that may not be completely eliminated by current pasteurization protocols.

2 J. Food Prot., Vol. 6, No. ELIMINATION OF SALMONELLA ENTERITIDIS FROM LIQUID EGG PRODUCTS 113 TABLE 1. Some selected quality attributes of unpasteurized and pasteurized liquid egg products Product Liquid whole egg Liquid egg white Liquid egg yolk Liquid egg yolk 5% salt 5% sugar ph Solids (%) Viscosity a (MPa s) a Viscosity measurements were determined on eggs at 0 C. Many previous thermal inactivation studies used 9-ml test tubes to determine D values. Such procedures may overestimate the true D values. For example, one study involving Aeromonas hydrophila in liquid whole egg found that capped test tubes resulted in a tailing effect and that D values were significantly higher than those using the submerged capillary tube method (0). In the study described in this paper, a submerged capillary tube method was used to achieve instantaneous heating to get a better estimate of the D values of Salmonella Enteritidis in egg products. In addition, a plate heat exchanger was used to verify current USDA protocols for liquid egg products and compare to values obtained with capillary tubes. MATERIALS AND METHODS Culture and culture media. For all studies using submerged capillary tubes and plate heat exchanger, a five-strain cocktail of Salmonella Enteritidis was used. The five Salmonella Enteritidis strains and their sources of isolation were E (tuna salad), E (human), E (egg), SE (human), and E 194 (human, egg outbreak) from Dr. Tong Zhao in our laboratory. All cultures were maintained on tryptic soy agar (Difco, Detroit, Mich.) slants at 4 C. For in vitro heating studies, the Salmonella Enteritidis strains were individually grown in 10 ml of tryptic soy broth (Difco) at 35 C. After 4 h, the strains were transferred to new 10-ml tubes of tryptic soy broth for an additional 4 h at 35 C. The cultures were then centrifuged (1,100 g for 15 min), resuspended in ml of 0.1% peptone water. The 10 ml of cocktail were then added to 90 ml of liquid egg product in a stomacher bag, and mixed in a stomacher (Seward Medical, London, UK) for 1 min. The inoculated egg product was then kept on ice for a maximum of 3 h before using. For the study involving the plate heat exchanger, cultures were grown in 10 ml of tryptic soy broth for 4 h before being transferred to 50 ml tryptic soy broth for an additional 4 h at 35 C. The cultures were centrifuged (,000 g for 15 min at 10 C), resuspended in 0 ml of 0.1% peptone water, mixed in a stomacher bag, and 100 ml of cocktail added to 4 liters of liquid egg product. Inoculated product was refrigerated at 7 C, for a maximum of 10 h, until it was pasteurized. Heating menstruum. Commercially broken, raw whole egg, egg yolk, egg white, and egg yolks containing 10% NaCl were delivered from a local egg processor within 4 h of breaking and kept at 4 C in a cold room for a maximum of 30 h before using. Egg yolk containing 5% sucrose 5% NaCl was prepared at the laboratory from the plain egg yolk. Before heating, the ph, soluble solids, and viscosity of egg products were determined and these data are listed in Table 1. The viscosity of the liquid egg products was measured at 4, 7, and 0 C with a Brookfield viscometer as described by Hamid-Samini et al. (10). Total dissolved solids was determined as described by Guerrero and Alzamora (9) using a Bausch and Lomb refractometer (Kernco Instruments Co., Horizon City Industrial Park, El Paso, Texas). The ph of egg products was measured with a Fisher Acccumet ph meter model 15 (Fisher Scientific Co., Pittsburgh, Pa.). Thermal inactivation of Salmonella Enteritidis in capillary tubes. Ninety milliliters of liquid egg sample were inoculated with 10 ml of the cocktail described previously, resulting in 10 9 to CFU/ml in the unheated product. Inoculated and uninoculated egg product were serially diluted, 10 0 to 10 9, as appropriate, in 9.0 ml of 0.1% peptone water. One-tenth milliliter of the serial dilutions were deposited on the surface of duplicate plates of tryptic soy agar (TSA) and xylose, lactose, desoxycholate (XLD) agar (Difco) and uniformly spread with a sterile bent glass rod. All plates were incubated at 35 C for 4 h before counting Salmonella Enteritidis colonies. One-hundred-microliter capillary tubes (Fisher brand, borosilicate glass) were prepared by sealing one end with a flame and autoclaving at 11 C for 15 min. Using an 18-gauge needle and a 1-cc syringe, 100 l of inoculated liquid egg was injected into the capillary tube, the open end flame sealed, and the tube placed in an ice-water bath (maximum 3 h) until used. The capillary tubes were heated in a controlled-temperature water bath (B. Braun, Thermomix 1480). Temperatures inside the capillary tubes were monitored using a thermocouple (Omega, 176A digital thermometer) inserted inside an open capillary tube filled with 100 l of uninoculated liquid egg. The time required for the egg products to reach pasteurization temperature was approximately 4 s. At each of four equal time intervals, triplicate capillary tubes were removed from the water bath and placed in an ice-water bath (maximum 1.5 h) until microbiological analysis. Liquid whole egg and egg yolk were heated in capillary tubes at 55 C for 8, 16, 4, and 3 min, 58 C for.5, 5, 7.5, and 10 min, 61 C for, 44, 66, and 88 s, and 64 C for 3, 6, 9, and 1 s. Egg yolk 10% NaCl was heated in capillary tubes at 55 C for 8, 16, 4, and 3 min, 58 C for.5, 5, 7.5, and 10 min, 61 C for 40, 80, 10, and 160 s, and 64 C for 10, 0, 30, and 40 s. Egg yolk 5% NaCl 5% sucrose was heated in capillary tubes at 58 C for, 44, 66, and 88 min, 60 C for 9.8, 19.6, 9.4, and 39. min, 6 C for 4, 8, 1, and 16 min, and 64 C for 54, 108, 16, and 16 s. Egg white was heated in capillary tubes at 51 C for 8, 16, 4, and 3 min, 53 C for, 4, 6, and 8 min, 55 C for 53, 106, 159, and 1 s, and 57 C for 4, 48, 7, and 96 s. Before microbiological analysis, tubes were immersed in 70% ethyl alcohol and allowed to dry. The tube was then placed in a sterile test tube containing 9.9 ml of 0.1% peptone water and crushed with a sterile glass rod. Suspensions were serially diluted in 0.1% peptone water and 0.1 ml was spread-plated with a sterile bent glass rod, in duplicate, on TSA. The TSA plates were incubated at 35 C for 48 h, colonies counted, and countable plates (30 to 300 colonies) replica plated () onto XLD agar. The XLD plates were then incubated for 4 h at 35 C before counting Salmonella Enteritidis colonies. Survival of Salmonella Enteritidis in egg products. This set of experiments was done to determine the reason for a rapid decrease ( log units) in Salmonella Enteritidis populations observed in the salted (NaCl) and sugared (sucrose) egg yolk products. The Salmonella Enteritidis five-strain cocktail was prepared as stated above for the heat-inactivation studies. Twenty-five milliliters of the cocktail was added to 50 ml of either egg yolk 10% NaCl or egg yolk 5% NaCl 5% sucrose and put in a stomacher for min. The inoculated egg was stored at 4 C, and

3 114 MICHALSKI ET AL. J. Food Prot., Vol. 6, No. TABLE. D values (minutes) of Salmonella Enteritidis in liquid egg products heated in capillary tubes Egg product z values ( C) Temperature ( C) Whole egg Egg white Egg yolk Egg yolk 10% salt Egg yolk 5% salt 5% sugar a sample was taken every h for 1 h, then every 4 h for 7 h. Each sample was plated on TSA and XLD agars in duplicate and incubated at 35 C for 4 h before being counted. Validation of USDA protocol with Salmonella Enteritidis. This validation study was performed to determine if current USDA pasteurization protocols for liquid egg products eliminate Salmonella Enteritidis from the product and provided a margin of safety. Twenty-four liters of raw egg product were inoculated to contain 10 7 to 10 8 log CFU/ml as described previously. This was confirmed by serially diluting inoculated and uninoculated eggs in 0.1% peptone water, spread plating 0.1 ml of appropriate dilutions on duplicate plates of TSA and XLD agar (Difco) with a sterile glass rod, and incubating at 35 C for 4 h. A pilot-scale continuous flow heat exchanger (Armfield Ltd., West Street, Hampshire, UK) was used to pasteurize egg products. To achieve appropriate holding times at pasteurization temperatures, a holding tube was added after heating sections of the heat exchanger to meet the required holding time. Temperature in the holding tube was maintained at pasteurization temperatures by immersing it in a water bath controlled with a resistance temperature detector (RTD) temperature controller with direct current pulse and solid-state relay (Omega Engineering Inc., Stanford, Conn.). Water was run through the system until the temperature stabilized at the desired temperature, at which time the egg product was added to the system and again the temperature was allowed to stabilize. To test USDA protocols, all products were heated in the plate heat exchanger for min at 60 C (whole egg), 56.7 C (egg white), 61.1 C (egg yolk), or 63.3 C (NaCl and sucrose added yolk). At a steady-state flow rate, two samples each were taken to determine physicochemical characteristics of egg products. The inoculated product was then passed through the pasteurizer and again the system was allowed to stabilize to the established process temperature, at which time samples were taken for microbiological evaluations. Two replications were done with two samples taken at each sampling time. In addition, two control (unpasteurized) samples from each replication were also taken for quality evaluation and to determine physicochemical characteristics of the products. Samples were kept on ice (maximum 30 min) until microbiological analysis. Samples were serially diluted, 10 0 to 10 8, in 9.0-ml tubes of 0.1% peptone water. From appropriate dilutions, 0.1 ml was spread uniformly, in duplicate, on TSA, and incubated at 35 C for 48 h. Plates with 30 to 300 colonies were replica plated on XLD agar and incubated an additional 4 h at 35 C. Data analysis (thermal inactivation in capillary tubes). Populations were converted to log 10 values and plotted versus time (minutes or seconds). All log values are averages of three trials. A commercial spreadsheet program (Microsoft Excel version 7.0) was used to perform linear regression, and D values were calculated as the negative reciprocal of the slope of the thermal inactivation curve. The correlation coefficient (r ) was for all Salmonella Enteritidis trials, indicating that the inactivation curves (log number versus time) were linear and D values could be calculated directly from them. RESULTS The D values (in minutes) calculated from the thermal inactivation curves for the five strains of Salmonella Enteritidis in the five different liquid egg products at various temperatures can be seen in Table. As expected, D values decreased with increasing temperature. D values were similar for whole egg and egg yolk across the entire temperature range tested. D values for egg white were 10-fold lower than those in whole egg and egg yolk. The addition of 10% NaCl to egg yolk increased D values 4-fold. In contrast, the addition of 5% NaCl 5% sucrose to egg yolk raised the D values as much as 0-fold. Based on our calculated D values using capillary tubes, USDA pasteurization requirements would result in an approximately 5.5D reduction of Salmonella Enteritidis in liquid whole egg, a 7.5D reduction in egg white, a 13D reduction in egg yolk, an 8D reduction in egg yolk 10% NaCl, and a 1D reduction in egg yolk 5% NaCl 5% sucrose. In experiments using capillary tubes, populations of Salmonella Enteritidis exhibited an initial rapid decrease ( log units) in the salted and sugared egg yolk product (Fig. 1). In contrast, this rapid initial inactivation was not observed with other egg products (survival in egg yolk is illustrated as an example in Fig. ). In an effort to explain the reason for this observation, survival of Salmonella Enteritidis in unheated egg yolk products stored for 7-h was investigated. Populations of Salmonella Enteritidis remained essentially unchanged in all products during the 7- h storage period. Hence, it appears that neither NaCl nor sucrose directly caused the rapid inactivation observed in the egg yolk containing 5% NaCl and 5% sucrose. The Salmonella Enteritidis log reduction achieved with the plate heat exchanger following USDA minimum pasteurization protocols is listed in Table 3. Liquid egg was inoculated with 10 7 to 10 8 cells and none were recovered after pasteurization, indicating a greater than 7- to 8-log reduction. To determine the approximate log reduction, each product was again pasteurized at the previous temperature but with only a 1- and -min holding time. The log reduction achieved with the 1- and -min holding times

4 J. Food Prot., Vol. 6, No. ELIMINATION OF SALMONELLA ENTERITIDIS FROM LIQUID EGG PRODUCTS 115 FIGURE 1. Thermal inactivation of Salmonella Enteritidis in egg yolk containing 5% NaCl and 5% sucrose heated at 56, 60, 6, and 64 C in borosilicate capillary tubes. can be seen in Table 4. From these data, it can be estimated that a 9D process is achieved with USDA minimum pasteurization protocols for whole egg, egg white and egg yolk, but not for 5% NaCl 5% sucrose egg yolk. DISCUSSION Salmonella Enteritidis survived best in 5% NaCl 5% sucrose egg yolk, followed by egg yolk 10% NaCl, egg yolk and whole egg, and lastly egg white. The increased resistance of Salmonella Enteritidis in the salted egg yolk products had been documented previously by other scientists (3, 16). The solutes exert a protective effect on the organism. The exact mechanism for this phenomenon is not known but there are many hypotheses (11). The most wellaccepted hypothesis is that the salt and sugar lower water activity in a manner similar to drying. Because proteins are more stable in the dry state, the heat resistance of bacteria increases. However, as Salmonella Enteritidis does not tolerate high salt concentrations it survived better in the 5% NaCl egg yolk rather than the 10% NaCl egg yolk product. Salmonella growth is repressed at 6 to 8% NaCl, so it is possible that the 10% NaCl product was slightly inhibitory to Salmonella Enteritidis. However, the 5% NaCl in the yolk product containing 5% NaCl 5% sucrose was not inhibitory to the microorganism and higher D values resulted. Salmonella Enteritidis was also very heat sensitive in the egg white, with a D value 10 times lower than in egg yolk or whole egg. This is probably due to Salmonella Enteritidis inability to tolerate the high phs found in egg white as opposed to the neutral or slightly acidic phs of whole egg and egg yolk (17). Salmonella Enteritidis populations unexpectedly initially decreased or more log units in the salted and sugared egg yolk products during heating in capillary tubes but then decreased more slowly through the remainder of the heating period (Fig. 1). Experiments determining the survival of Salmonella Enteritidis during holding of unheated egg product were done to determine whether the added sol- TABLE 3. Reduction of Salmonella Enteritidis populations in various egg products using a plate heat exchanger and USDA pasteurization protocols Egg product Time (min) Temperature ( C) Reduction (log 10 ) Whole egg Egg white Egg yolk Egg yolk 5% salt 5% sugar a 7.7 a 7.5 a a Plates with were inoculated with 10 7 to 10 8 cells/ml and all were eliminated after pasteurization.

5 116 MICHALSKI ET AL. J. Food Prot., Vol. 6, No. FIGURE. Thermal inactivation of Salmonella Enteritidis in egg yolk heated at 55, 58, 61, and 64 C in borosilicate capillary tubes. utes were killing a sensitive subpopulation at the beginning of each trial. However, because populations remained constant over the 3-day period of the study, this does not appear to be the case. In fact, Salmonella Enteritidis was previously observed to survive up to 10 days in 0% NaCl. Hence, the above results are not unexpected (15). However, it is possible that the salted and sugared egg yolk products are causing sublethal injury to some cells causing them to be more heat sensitive, and it is this subpopulation that is dying so quickly at the beginning of the heat treatment. Another possibility is that some physical change is occurring in the products upon heating for a few seconds that is protecting the organisms from the heat later in the heat process, possibly affecting the heat transfer properties in the egg yolk. A third possibility that is known to produce an inactivation curve as seen here is the flocculation of bacteria due to NaCl suspended in the medium. The D values calculated from experiments using capillary tubes for Salmonella Enteritidis in whole egg, egg yolk, egg white, and egg yolk 10% NaCl were generally lower than previously published values. In the case of egg yolk 10% NaCl, the calculated D value was similar to previously published values. These lower, calculated D values are presumably the result of the capillary tube method used in these experiments that allowed almost instantaneous heating to the desired temperature. Therefore, the D values calculated in these experiments might give a more accurate albeit conservative representation of the heat sensitivity of these organisms in egg products. Results of experiments with capillary tubes indicate TABLE 4. Reduction of Salmonella Enteritidis populations in egg products using a plate heat exchanger and USDA minimum temperatures with 1- and -min holding times Egg product Time (min) Temperature ( C) Log reduction (log 10 SD) Whole egg 1 Egg white 1 Egg yolk 1 Egg yolk 5% NaCl 5% sucrose

6 J. Food Prot., Vol. 6, No. ELIMINATION OF SALMONELLA ENTERITIDIS FROM LIQUID EGG PRODUCTS 117 that the pasteurization protocols being used for liquid egg products are not providing a wide margin of safety when it comes to eliminating Salmonella Enteritidis. However, in experiments using a plate heat exchanger, a 9D process was achieved for Salmonella Enteritidis in egg white, egg yolk, and whole egg. This higher kill rate in the pasteurizer was most likely due to longer come-up and come-down times as compared to the capillary tubes that were heated and cooled almost instantaneously. Experiments with the plate pasteurizer showed that USDA egg product pasteurization protocols are indeed sufficient for eliminating Salmonella Enteritidis, as the process resulted in a greater than 9D process for egg white, egg yolk, and whole egg. The egg yolk product containing 10% NaCl was too viscous to pasteurize with the plate heat exchanger and had to be omitted from this study. However, only a 4.D process was achieved following USDA pasteurization protocols with egg yolk containing 5% NaCl 5% sucrose. This does not provide a wide margin of safety and falls below the recommended 9D process. Our data show that a 9D process is being accomplished with current USDA protocols. However, recent surveys finding pasteurized egg product contaminated with Salmonella (17) and results from our capillary tube study yielding reduced lethality shed some doubt on the effectiveness of current pasteurization protocols for some egg products. Additional measures may be needed to reduce the number of Salmonella surviving pasteurization procedures. In the case of egg white, the addition of a protein stabilizer (iron or aluminum) could be used to prevent coagulation, hence allowing higher pasteurization temperatures to be used. Additionally, procedures to increase lethality of the salmonellae in eggs might also be of value. For example, increasing the ph of egg white may help increase lethality, because Salmonella Enteritidis is more heat sensitive at high ph than at low or neutral ph. Similarly, the addition of hydrogen peroxide with an appropriate holding period may result in greater lethality than just heat alone. In the absence of means to increase lethality, however, the simplest solution may be to add the salt or sugar aseptically after the product has been pasteurized. New technologies are being developed that may produce safer and more functional egg products. One such method uses a series of short-duration, high-intensity electric field pulses on liquid products to induce extensive electroporation in the cell wall of microorganisms. The cell, unable to recover, dies (4). This is a low-temperature pasteurization process that may allow the egg to retain its original functional properties. In experiments with L. monocytogenes in milk, 6 logs were killed using only a few seconds of exposure to high-intensity electric field pulses at 55 C (4). Irradiation is another promising process that not only eliminates microorganisms but inhibits microbial growth of survivors while maintaining the functional properties of liquid egg. Finally, a third process that should be explored further is that of high hydrostatic pressure coupled with a low heat treatment. This technology may create fewer off flavors and allow the egg to retain its functional properties. ACKNOWLEDGMENTS We thank National Egg Products Co. for providing the eggs for this study and technical assistance. This study was funded by a grant from the Georgia Food Processing Advisory Board. REFERENCES 1. Bradshaw, J. G., D. B. Shah, E. Forney, and J. M. Madden Growth of Salmonella enteritidis in yolk of shell eggs from normal and seropositive hens. J. Food Prot. 53: Cassiday, P. K., L. M. Graves, and B. 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