A study on effect of immersion heat treatment on viability of Salmonella enteritidis in table eggs.

a study on effect of immersion heat treatment on viability of salmonella enteritidis in table eggs.

BY

ZIENAB I.SOLIMAN and AZZA A. EL-TABIY

Animal Health Research Institute, dokki

Port Said Laboratory for Food Hygiene.

دراسة تأثير المعالجة الحرارية بالغمر على حيوية ميکروب

 السالمونيلا انتريتدس  فى بيض المائدة

زينب إبراهيم سليمان‘ عزة على حسين التابعى

يعتبر البيض من أهم العناصر الغذائية التي تدخل في العديد من أصناف الطعام والحلويات0 ولما کان البيض النيئ يستخدم في تحضير بعض أصناف من الطعام مثل المايونيز والمارنج والأيس کريم والذي قد يؤدى  إلى  العدوى بميکروب السالمونيلاانتريتدس وخاصة أن معظم الدراسات تشير أن هناک ارتباط وثيق بين تناول البيض النيئ والغير مطهو جيدا والإصابة بذلک الميکروب0 لذلک يهدف هذا البحث لدراسة تأثير غمر البيض فى حمام مائى بدرجات حرارة مختلفة على حيوية ميکروب السالمونيلا انتريتدس  0 لذا تم حقن البيض بميکروب السالمونيلا انتريتدس (⁷10 خلية/ جم ) قرب المح وتم استبيان اثر غمر البيض فى حمام مائى بدرجات حرارة تتراوح بين 57-59 درجة مئوية على حيوية الميکروب وذلک على فترات أثناء الغمر0 وقد تبين أن هناک نقص تدريجيا في العدد الکلى لميکروب السالمونيلا انتريتدس أثناء الغمر ويتوقف ذلک على عاملي درجة الحرارة ومدة الغمر0 هذا وقد تم القضاء على الميکروب کليا بعد 70و55 و  45  دقيقة من الغمر فى درجات حرارة 57و 58 و59 درجة مئوية على التوالى ولم يکن هناک تغير ملحوظ ظاهريا فى الشکل الخارجى للبيض المعالج بالغمر عن البيض الذى لم يتم غمره  0 کذلک تم دراسة اثر التعرض لمختلف درجات الحرارة أثناء المعالجة الحرارية  بالغمر على الخصائص الطبيعية والوظيفية لزلال البيض وقد تبين من الدراسة أن المعالجة الحرارية بالغمر عند درجة 57 درجة مئوية کان له تأثير اقل فى أحداث تغير في الخصائص الطبيعية لزلال البيض مقارنة بالمعالجة  فى درجات حرارة 58 و59 درجة مئوية0 کذلک وجد انه يلزم زيادة الوقت اللازم لخفق بياض البيض للحصول على المارنج فى البيض المعالج بالغمر  0 هذا وتعتبر طريقة المعالجة الحرارية بالغمر طريقة للتخلص من ميکروب السالمونيلا مما يشکل استخدام امن للبيض النيئى الذي يدخل فى العديد من الأطعمة وما له من اثر على الصحة العامة 0  هذا وقد تم مناقشة الأهمية الصحية وکذلک الاحتياطات التى يجب مراعاتها للحد من خطورة ميکروب السالمونيلا.   

summary

Eggs are a highly nutritious, inexpensive food commodity which is accepted by most people. The safety of eggs has become a global issue with emergence of the pathogen Salmonella enteritidis as a major health hazard associated with the consumption of raw and semi-cooked eggs. the effects of water – bath heat treatments on the inactivation of Salmonella enteritidis within eggs were evaluated. Salmonella enteritidis (10⁷ cfu/g, inoculated near the centre of the yolk) was completely eliminated within 70 min at a bath temperature of 57⁰C, within 55 min at a bath temperature of 58⁰C and within 45 min at a bath temperature of 59⁰C. No significance difference in the visual appearance between the control and immersion heated eggs meaning that heated eggs will be seen as normal eggs.In addition, effect of immersion heat treatments on the functional properties of egg white was evaluated. Evidence from the current study confirms that prolonged egg heating at 57⁰C  for 70 min was effectively eliminate  Salmonella and produce Salmonella free eggs with an acceptable quality. There is no coagulation, or loss in functionality of egg components. Albumen turbidity and functionality were significantlyaffected by thermal treatments in eggs treated at 58 and 59⁰C and was less affected at 57⁰C. extended whip time would require for meringue preparation using immersion heated egg white. Immersion heattreatment could provide Salmonella enteritidis free egg for preparation of food receive little or no heat treatment prior to consumption and thereby combat the risk of salmonella.

introduction

table eggs are one of the most economic and balanced sources of protein available with cost per kg to consumers lower than chicken or meat in most countries. In addition, eggs contain unsaturated fatty acids, iron, phosphorus, trace minerals, and vitamins (Stadelman, 1995 and Watkins, 1995). Due to its exceptional nutritive value, eggs remain a potential host for pathogens like Salmonella enteritidis. Gast and Beard (1992) suggested that human-salmonellosis outbreaks, related to consumption of eggs, occurred as a consequence of three independent events; (i) contamination of eggs with Salmonella enteritidis by infected hens, (ii) improper handling of eggs or egg products allowing proliferation of the microorganism to infectious levels, and (iii) ingestion of raw or undercooked contaminated eggs.  it is apparent from the voluminous reports that Salmonella enteritidis was more oftenly associated with human foodborne disease outbreaks than other Salmonella serotypes particularly those associated with egg and egg products (Brackett et al., 2001 and Shirota et al., 2001). Unlike others of the 2000 serovars of Salmonella, this organism infects the egg before the egg is laid, with the organism being transmitted to the ova or the albumen before the formation of the shell of the egg (Humphrey, 1999).More than 90 percent of food borne Salmonellosis, caused by Salmonella enteritidis is through the shell eggs (Woodward et al., 1997 and Schroeder et al., 2005). The probability of fresh eggs having Salmonella varies from 0.005 % to 1 %, depending on various factors involved in the egg production (Mermelstein, 2001). Food borne salmonellae are estimated to cause 1.3 million illnesses, 15,000 hospitalizations, and 500 deaths per year (Schroeder et al., 2005). Likewise,  gastroenteritis is the most common clinical manifestation of the human Salmonella enteritidis infection (Bennasar et al., 2000).  

Generally, Salmonella enteritidis is the causing agent of human gastroenteritis, an infection that results in a clinical syndrome generally known as salmonellosis. Symptoms of gastroenteritic salmonellosis may include severe abdominal pain, non-bloody diarrhea, myalgia, chills, nausea, headache, fever, vomiting, and prostration. In addition, other medical conditions such as pericarditis, neurological and neuromuscular diseases and reactive arthritis may result in some individuals after the infection (D’Aoust, 1989). Symptoms occur 12-72 h after consumption of Salmonella enteritidis contaminated food, and the infective microbial dose, necessary to cause foodborne illness, varies from 100 cells in high fat foods to 10⁵ cells in lower lipid content foods (Bell and Kyrikiades, 2002). The microorganism multiplies and colonizes the small intestine, produces an enterotoxin that causes inflammatory reaction and diarrhea, and in some cases it can invade the blood stream to cause more severe illness (Poppe, 1999; D’Aoust, 2001and Bell and Kyrikiades, 2002). Duration of gastroenteritis syndrome generally varies from 4 to 10 days; during this time, microbial invasion of the small intestine and colon could affect absorption of nutrients in the patient (Poppe, 1999). moreover, susceptibility of humans to Salmonella infections depends on a series of factors that include the dose of the pathogen, the type of contaminated food, and the age and immune condition of the host (D’Aoust, 1989 and Poppe, 1999). The newborn, elderly, and individuals with immune deficiencies are more susceptible than the rest of the population to infection by Salmonella enteritidis. In these groups at risk, salmonellosis could result in serious systemic infections with sporadic cases of death (D’Aoust, 2001). on the other hand, healthy individuals rarely die from salmonellosis, and they normally recover from the disease after treatment with fluid and electrolyte replacement, while antibiotic therapy is not usually recommended in developed countries (Bell and Kyrikiades, 2002).

Eggs are one among the major animals foods mostly marketed raw and frequently consumed raw. Many of the dishes like Caesar salad, mayonnaise, eggnog, mousse, home made ice cream and etc., which form an important part of meals, contain raw eggs as an essential ingredient. These dishes are not heated up to the FDA recommended temperatures of 155ºF for at least 15 seconds (Mermelstein, 2001). Although there are several methods of microbial destruction like rapid chilling and ultrasonic treatments to destroy Salmonella, they are not effective on the Salmonella present inside shell eggs (Hou et al., 1996). so control of Salmonella enteritidis within shell eggs has been attempted with a limited number of procedures that include the use of gamma & X-ray radiation and thermal pasteurization (Tellez et al., 1995; Schuman et al., 1997 and Serrano et al., 1997). Public health concerns regarding contamination of fresh eggs with Salmonella enteritidis prompted approval of a thermal process to eliminate this microorganism in shell eggs (USDA, 1997). Pasteurization of eggs (in shell pasteurization) is a commercially available process, which consists of extended heating of shell eggs by immersion in water baths at 55-60ºC or by hot air in convection ovens (Schuman et al., 1997; Brackett et al., 2001 and Zeidler, 2001). Salmonella enteritidis has been in fact used as sentinel organism for the establishment of pasteurization schedules of processed liquid egg products (Ponce et al., 1999). Previous investigators indicated that immersion heat treatments of eggs at 57⁰C or above are required to effectively inactivate Salmonella enteritidis inside shell eggs (Hou et al., 1996; Schuman et al., 1997 and Stadelman et al., 1996).

The heat treatment of whole shell eggs required balance between reduction of target organisms and the maintenance of albumen quality. The physical properties like whip ability, foam ability and foam stability, affecting the functional properties, which make the eggs an inevitable ingredient of various food products are severely affected by high temperatures (Iesel et al., 2006).  Whip volume is a measure which gives a clear picture of the quantity of air incorporated in the egg white foam. This is an important factor that tells about the aerating properties of the egg white in its food applications. The commercial use of egg white is highly dependent on its foam ability in many of its applications in the food industry (McDonnell et al. 1955).

Therefore, this study investigates the effects of water bathimmersion heat treatment at 57⁰C, 58⁰C and 59⁰C on viability of Salmonella enteritidis within table eggs. In addition, the impact of the treatment on functional properties of the egg white was assessed.

Materials and methods

Bacterial culture:

Salmonella enteritidis, isolated from eggs, was kindly provided by animal Health research Institute, dokki, Giza. Stock culture was transferred to brain heart infusion (BHI) broth and incubated at 37⁰C for 24 h. This concentrated suspension was used to prepare working cell suspension in phosphate buffer. the cell suspension was serially diluted to a concentration of 10⁷ cfu/ml.

Preparation of eggs:

Fresh and unfertilized hen shell eggs obtained from local farm, proved to be free from Salmonella enteritidis according to methods carried out by fda (1998), were kept at 4ºC and used within one week of laying. Selected eggs were transferred to trays and then held at 22-25ºC for 2 h. Individual shell eggs were washed with tap water and gently scrubbed with a plastic brush. Clean eggs were submerged in ethanol (70% vol/vol) for 30 min as described previously by Hammack et al.(1993). Sanitized shell eggs were immediately transferred to sterile container and permitted to dry at 22-25°C for 40 min before inoculation with Salmonellaenteritidis.

Inoculation of shell eggs with Salmonella enteritidis:

sanitized eggs were carefully drilled in the approximate center of pointed end of shell eggs (opposite to the air cell) with 5-6 mm sterile needle, placement in close proximity to the yolk (Chantarapanont et al., 2000). Salmonella enteritidis cell suspension (10⁷ cfu/g) was inoculated into yolk. After inoculation, the hole in the shell was then sealed with a small piece of sterile aluminum foil and super glue. Internally contaminated eggs were immediately placed on sterile trays and incubated at 37⁰C for 30 min. After incubation, contaminated eggs were held at 22-25°C for approximately 10 min before heat inactivation trial.

1-immersion heat treatment inactivation of Salmonella enteritidis trails in shell eggs :

Contaminated eggs were placed inside aluminum baskets. Water bath was previously calibrated to attain 57, 58, or 59ºC. eggs inside basket were heat treated by immersion in water at 57, 58 or 59⁰C for 5-70 min.  contaminated untreated shell eggs were used as controls in all experiments. During heat treatments, level of water above shell eggs was 4 cm.  Experiments were performed with three shell eggs per time- temperature trial. Three eggs per experimental condition in duplicate series were taken from water bath at selected intervals. eggs were placed in 2-liter glass beakers containing 1.5 liters of sterile distilled water at 22⁰C. Eggs were held for 10 min in water for cooling. Cooled eggs were gently dry-wiped with clean paper tissue.

1.1- Enumeration of Salmonella enteritidis:

Treated or control shell eggs were carefully broken from outside with the blunt end of a clean knife’s blade. Egg contents (albumen and yolk) were blended and diluted 1:10 (w/w) with chilled sterile lactose broth. serial dilutions of this homogenate were prepared in peptone water and 0.1 ml of diluted samples was plated onto pre-poured tryptic soya agar (TSA) plates. plates were held at room temperature for 3 h and overlaid with tempered (45⁰C) xylose lysine desoxycholate agar (XLD; Difco) as described by Strantz and Zottola (1989). the overlay was permitted to solidify at room temperature and plates were incubated at 37⁰C for 48h. black or black centered colonies were enumerated as Salmonella enteritidis, and suspected isolates were confirmed by re-isolation onto XLD agar plates (37⁰C, for 24 h) and biochemical confirmation in triple sugar iron (TSI; Difco) agar slants (37⁰C, for 24 h). at the time of plating into TSA, the remaining blended egg/ lactose broth was retained as enrichment and incubated at 37⁰C for 24 h. after mixing, 1 ml of enrichment broth was transferred to 10 ml of Rappaport^s broth (37⁰C, for 24 h). The incubated culture was streaked on XLD agar plates and incubated for 48 h at 37° C. characteristic colonial morphology of Salmonella spp. was observed.  Presumptive Salmonella isolates were selected and confirmed on TSI slants as previously described.

2-Effect of thermal treatment on albumin quality:

based on salmonella inactivation data previously recorded, the second experiment was conducted to assess the effect of such treatment on albumen quality and functionality: albumen clarity, pH, whip time and whip volume (Iesel et al., 2006).

sanitized non inoculated eggs were heat treated in water bath at 57, 58, and 59⁰C for up 70, 55 and 45 min, respectively. Appropriate controls were used in all experiments. eggs from triplicate trials with a total of ten shell eggs per experimental condition were used. All experiments were performed in duplicate with appropriate controls.  Immediately after the above mentioned heat treatments the shell eggs were immersed in cold water tub containing water at 5 ºC for 10 min.  Treated and control shell eggs were transferred to clean trays and additionally permitted to cool at 22-25⁰C for 2 h before egg quality measurements. Cooled shell eggs were gently dry- wiped with clean paper tissue. Shell eggs were carefully broken, contents were divided and egg shells and yolks were discarded. Albumen of individual eggs was recovered and placed on dishes.

2.1-turbidity measurement:

Aliquots (1 ml), containing thick and thin albumen, were transferred to cuvets. Turbidity was measured at 600 nm in spectrophotometer .Measurements were performed in duplicate using distilled water as a reference (Shimada and Matsushita, 1980).

2.2-determination of whip time, whip volumes and pH:

The functionality of blended, and non centrifuged albumen was estimated by determining whip time (min) and whip volumes (ml) as described by ball and Winn (1982). The ph of non centrifuged albumin samples was determined using ph meter (Fisher Scientific, USA).

results

table 1: Thermal inactivation of salmonella enteritidis in shell eggs

              subjected to immersion heating in a water bath at 57⁰C

              (n=3).

Survivors (log10 cfu/g): represent the mean of microbial counts in experiments with duplicate series repeats.

table 2: Thermal inactivation of salmonella enteritidis in shell eggs

              subjected to immersion heating in a water bath at 58⁰C

              (n=3).

Survivors (log10 cfu/g): represent the mean of microbial counts in experiments with duplicate series repeats.

table 3: Thermal inactivation of salmonella enteritidis in shell eggs   subjected to immersion heating in a water bath at 59⁰C (n=3).

Survivors (log₁₀ cfu/g): represent the mean of microbial counts in experiments with duplicate series repeats.

Table 4: Albumen quality of eggs subjected to thermal heat treatment (n= 10 eggs) in water bath at 57, 58, and 59⁰C up to complete inactivation of salmonella enteritidis.

Reading represents the mean of experiments performed in duplicate per condition.

*: significant correlation at P < 0.05, relative to control.

DISCUSSION

1- effects of immersion heat treatment on viability of salmonella enteritidis in table eggs:

Thermal treatments were performed at 57-59^oC, a range of temperature previously reported for inactivation of Salmonella in eggs by Lith et al.(1995) and Schuman et al.(1997).  Treatment of inoculated eggs at 57, 58, and 59⁰C resulted in gradual reduction of Salmonella enteritidis (tables 1, 2 and 3). inactivation in survivor occurred during the first 10 min of treatment at 57 and 58⁰C, and during first 5 min of heating at 59⁰C. Since treating Salmonella at > 50⁰C usually inactivates the pathogen, observed microbial reduction is most likely caused by heat transfer during the early phase of immersion treatments (Stadelman et al., 1996). Further heating of eggs produced gradual increase in internal egg temperature. reductionof Salmonella enteritidis by 4.7, 5, and 5.4 log cycles was observed after 30 min heating at 57, 58, and 59⁰C, respectively. the present results were compatible to that recorded by Luis (2004). in this respect, Stadelman et al. (1996) reported that internal egg temperature after heating shell eggs at 57⁰C for 20 and 25 min was 56.1 and 56.5⁰C, respectively. These temperatures are close to the critical internal 55-56⁰C, in which maximum microbial inactivation occurs. however, Lith et al. (1995) heated shell eggs, containing Salmonella enteritidis inside the yolk, in water at 57⁰C for 20-30 min and illustrated that these treatmentswere not sufficient to inactivate the microorganism.

heat treatment time/temperature conditions, aims to achieve a decrease ≤ 5 log10 in the number of viable Salmonella enteritidis, organisms. This renders the microorganisms ineffective in causing disease inactivation processes (Stadelman et al., 1996 and Schuman et al., 1997). reductionsof Salmonella enteritidis by 5 log10 and 6 log10 were recorded after 50 min heating at 57 and 58 °C, respectively. likewise, Schuman et al. (1997) heated shell eggs, inoculated in the yolk with 10⁷ Salmonella enteritidis / g in water at 57 and 58 °C and reported microbial reductions by 5.6 log10 after 55 min and 5.8 log10 after 43 min, respectively.

Water –bath heat treatments which completely eliminated salmonella enteritidis inoculum were identified. The present study revealed that immersion heating at 57⁰C for 70 min was effective in eliminating all detectable salmonella enteritidis by both plating and enrichment procedures .however, salmonella enteritidis was completely eliminated within 55 min at a bath temperature of 58⁰C and within 45 min at a bath temperature of 59⁰C. In- egg shell pasteurization procedures have been developed on the assumption that Salmonella enteritidis naturally resides inside the yolk, and extensive heat treatments must target the whole egg in order to transfer appropriate thermal energy to the center of the product to effectively inactivate the microorganism (Stadelman et al., 1996; Schuman et al., 1997 and Brackett et al., 2001). However, an increase in the processing temperature should not be recommended .Even if no damage to egg should occur, one may question the necessity for a margin of safety of this magnitude (10⁷ kill), since such high Salmonella counts have never been encountered in egg (Humphrey, 1999) .Previous investigations have reported delayed inactivation of Salmonella enteritidis inside shell eggs during immersion heat treatments (Hou et al., 1996 and Schuman et al., 1997). furthermore,persistence of Salmonella in egg after prolonged heating could be explained by clumping, protective effect of dead cells, microbial debris produced after cell destruction, and localized sites with low water activity among many other conditions (Pflug et al., 2001). in this respect, Schuman et al. (1997) found complete inactivation of Salmonella enteritidis inside shell eggs during immersion heat treatments within 50- 57.5 min at a bath temperature of 58⁰C  and within 65 -75 min at 57⁰c.

2-Albumen quality evaluation:

Of all egg components, the egg white is most sensitive to heat; egg albumen should be used as the indicator for egg quality assessment after processing (Elliott and Hobbs, 1980). in general albumen quality, including turbidity and functionality was evaluated.

2.1-albumen turbidity:

albumen turbidity was determined from changes in its absorbance after heating eggs at three temperatures up to complete elimination of Salmonella. Turbidity is a direct measure of the extent of protein coagulation, as coagulated proteins are opaque and reduce the transmittance of light through the egg white. The amount of light absorbed (absorbance) is a function of the turbidity of a liquid (Iesel et al., 2006). prolonged heating at 57⁰C for 70 min resulted in microbial inactivation without significant affecting albumen turbidity (0.07) with respect to that measured in untreated control (0.03) (Table 4). turbidity in eggs heated at 57⁰C was lower than that of eggs heated at the other temperatures, indicated that the water bath heated shell egg  at 57⁰C had better transmittance than the water bath heated eggs at other temperatures. significant (p < 0.05) increase in albumen absorbance by 0.34, and 0.39 was observed after 55, 45 min treatments at 58, and 59⁰C, respectively. the obtained findings in this work were correlated with those recorded by Luis (2004). this increase in albumen turbidity was minimal when compared to that previously reported for effective immersion heat treatments, in which heating shell eggs at 57°C for 75 min or at 58⁰C for 65 min increased albumen absorbance by 0.7 and 1.3, respectively (Schuman et al., 1997).  in this concern, Hou et al. (1996) revealed thatalthough heat treatment alone at ambient 57⁰C for 25 min resulted in 5.2 log microbial reductions, it seems unlikely that prolonged heating at this temperature could achieve higher inactivation without affecting albumen clarity, reducing lysozyme activity, or affecting egg functionality.

2.2 - whip time and whip volumes:

The turbidity detected in the albumen has been shown to have some negative effects on egg whip ability. The immersion heat treatment at 57⁰C had a less effect on whip volume and yield more consistence whip times relative to heating at 58 and 59⁰C. No change in albumen pH was observed in treated eggs with respect to untreated control (Table 4).  Eggs are popular for the exceptional functional properties. Egg white is used as a foaming, emulsifying, gelling and/or binding agent in numerous food preparations. Extended whip time would require for meringue preparation using immersion heated egg white. similar observations have been recorded by Schuman et al. (1997).

Salmonella is easy to destroy in cooking. The problem is that we often eat eggs raw or only lightly cooked. Such foods, along with eggs, should be treated as though they were contaminated. Evidence from the current study confirms immersion-heated eggs could provide Salmonella-free ingredients for the preparation of a variety of minimally-cooked foods of interest to consumers and food service operators produce eggs with acceptable quality. The trick is to reach the minimum safe internal temperature without cooking the egg, leaving a finished product that looks, acts, and tastes like a raw egg, but without the associated risk.

in order to remove or reduce the risk of Salmonella enteritidis food poisoning, eggs should be heat treated (pasteurized) before distribution. To block Salmonella enteritidis from multiplying in the egg, eggs must be held at cool temperatures (5ºC) following packing and throughout transportation. Consumers should be informed that eating undercooked eggs may result in Salmonella infection. In addition, eggs should be refrigerated to prevent proliferation of Salmonella if present and should be cooked thoroughly to kill Salmonella. Because most serious illnesses and deaths associated with salmonellosis occur among the elderly and immuno - compromised persons, these persons in particular should not eat foods containing raw or undercooked eggs. Hospitals, nursing homes, and commercial kitchens should use pasteurized eggs for all recipes requiring raw or undercooked eggs and should refrigerate all eggs and egg products. also, wash hands, utensils, equipment, and work areas with hot soapy water before and after they come in contact with eggs and egg-containing foods. finally, pasteurization process can be used as an insurance system or safety net if an outbreak of this disease becomes a reality.

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