Shell eggs storage and prevention of growth and multiplication of Salmonella
Prompt refrigeration to temperatures capable of restricting microbial growth has been recommended as an approach to reducing the likelihood that contaminated eggs will transmit S. Enteritidis to humans (Gast and Holt 2000).
In 2000, FDA published a final rule in the Federal Register (65 FR 76092), which states that a proposed maximum ambient temperature of 7.2 °C (45 °F) would extend the effectiveness of the egg's natural defenses against S. Enteritidis and would slow the growth rate of this foodborne pathogen (FDA 2000). In the Final Egg Rule (FDA 2009a), this proposition is maintained, as it is specified that this maximum value needs to be applied not only during storage, but also during transport, beginning 36 h after the time of lay. As an exception, shell eggs may be stored at ambient temperature values (above 7.2o C) if they are directed to a following step of processing, but not for more than 36 h. However, refrigeration must be kept even when using ionizing radiation (which results in only 2 to 3 logs reduction of S. Enteritidis), as this procedure is not regarded as efficient as the use of pasteurization (which ensures a 5 log reduction of S. Enteritidis) (FDA 2009b).
In Canada, shell eggs and those sent to a processing station must be kept under refrigeration, or stored for a maximum of 6 d at storage temperatures of 20 °C or less, or stored for a maximum of 2 d at temperatures between 20 °C and 30 °C (Health Canada 2011).
Concerning cold storage of eggs in the E.U., EC Commission Regulation 589/2008 specifies that “[…] eggs should be stored and transported at a constant temperature, and should in general not be refrigerated before sale to the final consumer […]” (EC 2008). Nevertheless, it has been shown that Salmonella spp. growth inside the egg is influenced by storage temperature.
Research in this field has proved that ambient temperatures are not proper for the storage of shell eggs, especially since the risk of S. Enteritidis horizontal transmission has increased, and further on, due to its capacity of growth and multiplication inside the shell eggs. Martelli and Davies (2012) suggested that the temperature values for shell eggs storage should not exceed 20 °C. In egg albumen, Salmonella spp. can grow at 20 °C, while unable to grow at temperatures below 10 o C, therefore showing that a temperature value for optimal storage of eggs should not exceed this last value.
Foodborne pathogens such as S. Enteritidis can grow in the contents of naturally contaminated eggs at room temperature (20 °C) and it does not lead to changes in the color, smell and consistency of the egg contents. However, the multiplication of S. Enteritidis in the stored eggs appears to be associated with alteration of the yolk membrane, which allowed the bacterium to either invade the yolk or obtain nutrients from it (Humphrey and Whitehead 1993).
Cogan and others (2001) reported S. Enteritidis growth after 8 d at 20 °C in 7% of the whole eggs inoculated in the albumen near the shell with as few as 2 CFU. If the inoculum equaled or exceeded 25 CFU/egg when eggs were subsequently stored at 20 °C, or 250 CFU/egg when eggs were stored at 30 °C, high levels of growth of Salmonella in the egg occurred significantly more frequently than when the inoculum dose was smaller (Cogan and others 2001).
Chen and others (2005) compared the storage of table eggs at 4 °C, 10 °C, and 22 °C. The albumen was inoculated with 102, 104, and 106 S. Enteritidis cells. At 22 °C, for all examined concentrations of inoculum, S. Enteritidis was able to grow, while at 4 °C and 10 °C, its growth was inhibited, regardless of the initial concentrations used. The authors believe that storage at 4 °C and 10 °C postponed the aging process of the eggs, preserving the antimicrobial agents of the albumen, and maintaining the integrity of the vitelline membrane.
Gast and Holt (2000) determined the extent to which small numbers of S. Enteritidis could grow to more dangerous levels at different temperatures over a period up to 3 d. Their intention was to stimulate the potential opportunities for S. Enteritidis multiplication following oviposition and prior to the achievement of internal temperatures able to prevent further microbial growth in eggs. Their results showed that extensive multiplication of S. Enteritidis was less frequently observed at lower inoculum dose (0.1 mL containing 15 CFU of S. Enteritidis), shorter storage time (1 d) and lower temperatures (10 °C and 17.5 °C). At 25 °C, with higher inoculum dose (0.1 mL containing 150 CFU of S. Enteritidis) and longer storage time (2 and 3 d), a rapid and substantial multiplication of the foodborne pathogen occurred. The inoculation site influenced in a great extent S. Enteritidis multiplication, since they used 4 types of samples: yolks (internally inoculated), albumens, whole egg, inoculated at the albumen edge and whole eggs inoculated at the yolk surface. In the yolk, multiplication occurred rapidly, with S. Enteritidis numbers reaching 8.7 log10/mL, at 25 °C, after only 2 d of storage. On the contrary, it was confirmed that the albumen is not a good growth medium for bacteria, since the S. Enteritidis levels suffered only a slight change during storage. The whole eggs inoculated at the yolk surface presented increasing levels of S. Enteritidis, during storage (no matter the dose and the storage time and temperature), while the other category of whole eggs (inoculated on the albumen edge) revealed only a slight change in these levels (Gast and Holt 2000).
It is believed that Salmonella cells that are deposited in the albumen are able to migrate to and penetrate through the vitelline membrane, in the egg, postlay, in order to reach the yolk and thus gain access to a pool of nutrients that are necessary for its survival and growth (Baron and others 1997; Gantois and others 2009).
Braun and Fehlhaber (1995) studied the migration of S. Enteritidis from the albumen into the egg yolk. Different doses of S. Enteritidis PT 4 were inoculated on the albumen (10 to 200 bacterial cells/albumen). Storage took place at 7, 12, 20, and 30 °C for 4 wk. The results showed that S. Enteritidis was able to migrate from the albumen into the egg yolk during storage. The risk of egg yolk penetration was relatively low at 7 and 12 °C. However, after 14 d, at 7 °C, the 1st positive egg yolk was found. At 20 and 30 °C, the 1st positive egg yolks were already present after 1 or 2 d. Schoeni and others (1995) also observed that the temperature values of < 10 °C will not allow but a sporadic growth of S. Enteritidis, S. Heidelberg and S. Typhimurium in the inoculated eggs, no matter the inoculum size.
Earlier, Hammack and others (1993) showed that the growth of S. Enteritidis on artificially inoculated shell eggs was negligible in eggs refrigerated up to 16 d. On the contrary, the population level of this food borne pathogen increased by more than 8 log10 units in unrefrigerated eggs stored for the same amount of time. Lock and Board (1992) observed that when inoculating different Salmonella serotypes, among them S. Enteritidis, S. Typhimurium and S. Infantis, on egg albumen, their persistence in vitro was different during storage at 3 different temperatures: 4, 20, and 30 °C. The majority of serotypes remained viable but did not increase in numbers at 20 and 30 °C, for 42 d. At 4 °C, many of the serotypes died. At 20 °C, upon inoculation with 39 CFU/mL−1 albumen, both S. Enteritidis and non-S. Enteritidis strains were able to grow in separated fresh albumen samples up to > 106 CFU/mL−1, during a storage period of 3 wk (Messens and others 2004; Clavijo and others 2006). It appears that the survival of S. Enteritidis in egg albumen is regulated by nucleic acid and aminoacid metabolism, and furthermore is related to genes involved in cell wall structural and functional integrity (Clavijo and others 2006). When extending the incubation time and increasing the storage temperature, the numbers of samples with pronounced growth increases further. Moreover, near room temperature (approximately 20 °C), the probability that an outgrowth takes place is much higher when the albumen of a fresh rather than a stored whole egg becomes contaminated with Salmonella. Even in the presence of a small number of S. Enteritidis cells present in the egg contents, cooling practices should be applied shortly after lay, to prevent Salmonella from growing in eggs (Messens and others 2004).
The egg yolk is a very important source of high quality nutrients, therefore fast growth of Salmonella is expected to occur in this site, when temperature will allow it. Experimentally infected laying hens often deposit S. Enteritidis on the vitelline membrane (Gast and Holt 2001; Gast and others 2007). The fast growth of S. Enteritidis occurs after a certain delay, during this period the integrity of the vitelline membrane being lost and finally resulting in a leakage of nutrients into the albumen. This enhances further migration and multiplication of S. Enteritidis in the yolk (Humphrey and Whitehead 1993). The initial growth phase potentially involves the use of iron reserves. This appears to be sufficient to support 4 generations, but once these reserves are depleted, Salmonella cells would enter in a lag phase, further on translated as a stagnation in the number of bacterial cells (Gantois and others 2009). The site of deposition of S. Enteritidis in the shell egg could influence the extent to which this pathogen multiplies before the refrigeration would achieve growth-inhibiting internal temperature values (Gast and Holt 2001). When 102 CFU of S. Enteritidis was inoculated onto the exterior surface of intact egg yolk (the vitelline membrane), multiplication within the interior egg yolk contents occurred in 10% of the samples after 6 h of incubation, in 75% of the samples after 24 h at 25 °C (reaching mean levels of 104 CFU/mL) and in only 20% of the samples incubated for 72 h at 15 °C (Gast and others 2001). Further on, Gast and others (2006) tested the effect of refrigeration on the frequency of in vitro S. Enteritidis penetration of the egg yolk membrane. After inoculating intact exterior surface of the egg yolks with a suspension of 0.1 mL containing approximately 100 CFU, samples were held 5 min at room temperature (24 °C) to facilitate bacterial attachment to the vitelline membrane. Further on, these samples were kept at 30 °C for different periods of time (2 h, 6 h, and 24 h), followed by refrigeration at 7 °C for 18 to 22 h. S. Enteritidis penetrated inside the egg yolk contents in 4% of contaminated egg samples refrigerated after 2 h at 30 °C, 15% of samples refrigerated after 6 h of storage at 30 °C and 40% of samples stored at 30 °C for 24 h, followed by refrigeration. Lublin and Sela (2008) showed that from an initial concentration of 3.65 log CFU of S. Enteritidis inoculated into the egg yolk, the concentration increased by 1 log during the 1st 2 wk postinoculation at 6 °C, after which it remained constant, at around 4 logs, for up to 8 wk. At 25 °C, the bacterial concentrations increased to 5 logs by week 4 postinoculation and remained at 8 to 9 logs until the end.
In different European countries, cold storage of eggs is banned on the market place. The reason is related to the concept that the eggs kept in cold storage can no longer be regarded as “fresh.” On the other hand, consumers are advised to keep purchased table eggs in the refrigerator until consumption (FDA 2009b). The practical aspects of this situation are different from one country to another. However, the scientific data clearly prove that refrigeration reduces in a great extent the risk of contaminated table eggs to become a vehicle for S. Enteritidis, a main worldwide cause of foodborne human salmonellosis.
Decontamination methods for reducing the risk of Salmonella spp. penetration through the eggshell and further contamination of the egg content
In the last decades, different methods have been studied for microbial decontamination of shell eggs, with a focus on Salmonella. We can distinguish the procedures tested for on shell decontamination from those, more limited, also active inside the shell. Moreover, concerning the 1st category, the procedures can be classified into 3 classes: the chemical, the physical and the biological procedures (Table 3).
Table 3. Methods of shell eggs surface decontamination as postharvest control procedures for reducing the risk of salmonellosis due to Salmonella contaminated eggs
|Chemical methods|| |
|Washing (use of sanitizers)|| |
|Hydrogen peroxide|| |
|Electrolyzed water|| |
|Physical methods|| |
|Microwave technology|| |
|Ultraviolet light technology|| |
|Pulsed light technology|| |
|Gas plasma technology|| |
|Biological methods|| |
|Plant extracts|| |
Several procedures are currently approved by the FDA or USDA in the U.S.A. and also, commercially available for shell eggs processing facilities. As none of them is perfect, they are continuously improved, as new procedures may emerge as well. However, the need for improvement is continuous, and research should focus more on the effects on sensory, rheological, and functional properties of eggs and their acceptability by the consumers, once the decontamination was performed. Moreover, when a new method emerges, research will still be a necessity before an efficient application on a full-scale production would take place.
In the E.U., egg washing is currently banned (with same exceptions—see further on) but this subject is always under a rigorous debate (Nys and Van Immerseel 2009). In this chapter, we will review different procedures that have the main objective of reducing or eliminating Salmonella spp. In order to be considered efficient, a decontamination procedure must lead to a reduction of at least 5 log CFU/eggshell−1, otherwise the resulting shell eggs being regarded as inappropriate for egg safety point of view (FDA 2009b).
Egg washing is currently used in the U.S.A., Canada, Australia, and Japan, in order to reduce the bacterial contamination and to prevent the penetration of bacteria in the egg contents. Moreover, in the U.S.A., egg washing is followed by cold storage.
Washing of class A-table eggs is banned in the E.U., but still under discussion following the increase in noncage egg production. Moreover, Member States which, on June 2003 authorized packing centers to wash eggs, may continue to authorize these packing centers to wash eggs, but the eggs may only be marketed in the Member States in which such an authorization has been issued (EC 2007). For example, in Sweden, several providers are allowed to perform it, as the washing practice has been used for the last 40 y and the consumers prefer washed eggs (Hutchison and others 2003).
In the U.S.A., there are not specific guidelines provided by the FDA to the specific process of egg washing. However, FDA provides general information for the Food Service and Inspection Service (FSIS) to provide to companies and local producers, as to what types of chemicals are allowed to be used during cleaning and destaining of shell eggs. Usually, the compunds included in the list of Generally Regarded As Safe (GRAS) ones can be used without any specific limits when cleaning shell eggs. These are mentioned and described in the Code of Federal Regulations (CFR), Title 21, parts 178 to 186 under the general term of “food additives.” However, for several of these so-called “food additives,” limits are mentioned, and maximum allowable concentrations are described and recommended to be followed, as allowed by the food additive regulations, especially for the indirect food substances affirmed as GRAS (CFR 2012a)
In the CFR Title 7, section 56.76, there are described the minimum facility and operating requirements for shell egg grading and packing plants, point (f) clearly specifying the shell egg cleaning operations. It is stated that the temperature of the wash water shall be maintained at 90 °F (32.2 °C) or higher, and shall be at least 20 °F (6.7 °C) warmer than the internal temperature of the eggs to be washed. These values shall be maintained throughout the entire cleaning cycle. For safety reasons, the wash water has to be changed approximately every 4 h or more often if needed, in order to maintain the sanitary conditions, and mandatory at the end of each shift. In addition, special measures have to be taken in order to avoid foaming during the egg washing operation. During the cleaning cycle, the addition of replacement water it is mandatory and has to be performed continuously. The replacement water may contain residues of chlorine or quaternary compounds, provided they are compatible with the compound used for washing. The use of iodine sanitizing water for rinse is forbidden (CFR 2012b).
Also for safety purposes, only potable water may be used during the shell eggs cleaning cycle, and it is mandatory the analysis of the iron concentration of the water supply. When the iron content exceeds 2 ppm, it has to be reduced to the maximum allowed level, and each time the water source is changed, new tests are required.
Wastewater is directly discarded, through its piping directly to the drains. Considering the type of washing procedure and equipment used, it is specified that eggs shall not be allowed to stand or soak in the water, therefore the use of immersion-type washers is forbidden. The washed eggs may be rinsed through spraying, with water having a temperature equal to, or warmer than the temperature of the wash water. It is specified that the rinse water should contain a sanitizer, approved by the national supervisor.
The main advantages of egg washing procedure are:
- - the reduction of microbial load on the shell surface, minimizing the risk associated with the presence of foodborne pathogens, especially Salmonella spp.
- - further reduction occurring after washing, since different chemicals may still be present after the washing step, continuing to exert their antibacterial effect;
- - reduced risk of cross-contamination of other foods;
- - reduced risk of contamination of the egg content, provided that the shell itself is not damaged.
The main disadvantage comes from the potential damage that this practice can cause to the physical barrier of the egg, especially to the cuticle (EFSA 2005). It is well known that the cuticle is the 1st defense against bacterial contamination (Board and Halls 1973).
Egg washing procedure uses water or solutions that involve chemicals (sanitizers) to determine an efficient decontamination. It is believed that different chemicals used to decontaminate the eggshell may interact with its physical barrier components. Depending on the types of chemicals used in the wash water, different microstructural changes may occur in the eggshell surfaces, and the more damaged eggshell surfaces are, the more they may allow bacterial penetration (Kim and Slavik 1996). In a study performed to investigate the abilities of different solutions, a quaternary ammonium compound (pH 7.5) and NaOCl (same pH value) succeeded in reducing bacterial penetration, without any changes to the eggshell, while Na2CO3 (pH 12) altered the eggshell surface which allowed bacterial recontamination (Wang and Slavik 1998). However, without using sanitizers in the washing water, it has been proven that spray washing of eggs in 15.5 °C water does not appear to increase internal shell bacterial counts (Lucore and others 1997).
Due to the concern that using a high temperature during egg washing may determine changes in egg quality, several studies have aimed to this point, evaluating as well the reduction of microbial load. Caudill and others (2010) concluded that wash water temperature did not significantly affect average Haugh Unit values, albumen height, vitelline membrane strength or aerobic bacteria in the shell matrix, but did affect average numbers of aerobic microorganisms on the exterior shell surfaces. In fact, a treatment with cool water, maintained at a pH of 10 to 12, has the potential of reducing also the internal egg temperature during and after processing, enhancing the physical qualities of the eggs, and improving their microbial quality. Using different schemes of temperature, Caudill and others (2010) obtained a reduction from 2.98 to 3.12 log CFU/mL.
Another study performed by Jones and others (2005), using 6 temperature schemes, with an exposure time of 60 s, maintaining the pH between 10.5 and 11.5, a postwashing treatment consisting of spraying a 200 ppm chlorine solution at 48.9 °C and a period of 9 wk of storage and continuous sampling, resulted in an aerobic bacterial load from 2.3 log CFU/mL to 2.87 log CFU/mL on shells and membranes, while between 53.33% and 61.8% of the samples inoculated experimentally with S. Enteritidis were negative. The conclusion is that washing shell eggs initially at 48.9 °C followed by a 2nd washing temperature of 23.9 °C or 15.6 °C led to a fewer aerobic bacteria present on the shell surface, than eggs washed in a combination of 23.9 °C and 15.6 °C.
Several years ago, Hutchison and others (2004) had undertaken a study on the effects of spray washing under various processing conditions to shell surface counts of Salmonella spp. and the presence of bacteria in egg contents. Experiments mimicked the natural conditions: they were carried out over a complete laying cycle, the eggs were contaminated with S. Enteritidis PT4 and S. Typhimurium DT104 before cuticle hardening. They used a standardized set of best washing guidelines as recommended by the equipment manufacturer and within the ranges discussed by Hutchison and others (2003). They used 2 different wash chemicals a chlorine based sanitizing agent in a concentration of 3 g/L and a quaternary ammonium based sanitizing agent in a concentration of 25mL/L, with 3 different steps in the egg washing process: prewash at 44 °C, with water flow pressure of 138 kPa; wash at 44 °C and water flow pressure of 262 kPa and rinse at 48 °C, with a water flow pressure of 262 kPa, followed by a final step consisting of air drying at 42 °C for 2 min. The used water was soft, potable and had an iron concentration of 1.4 ppb. In addition, the belt speed was 111 cm/min.
In another study that aimed to investigate the effects of different chemicals used in solution as egg disinfectants, a 1st commercial disinfecting product (pH 6.6) was used in water at 43.3 °C for 5 min, determining a 4.27 log reduction of microbial aerobic flora on eggshell. A 2nd one (pH 7.56) was used in the water at 25 °C for 10 min, determining a 3.11 log reduction. A 3rd solution of sodium hypochlorite (containing 100 ppm free chlorine, pH 8.74) used at 25 °C for 10 min, determined a log reduction of 3.08. A combination of sodium hypochlorite and 2nd solution (pH 8.4) used at 25 °C for 10 min resulted in a log reduction of 2.38. Considering side effects, the 1st compound determined a cuticle erosion, showing also an increased pore size, while in the case of the 2nd, the inside layer of the shell presented a great number of fissures and pores (Favier and others 2000).
In order to assess the food safety implications of washing table eggs under a deviation from the standard set of procedures for washing, several parameters have been modified. The results of their study have shown that when undertaken according to a strictly controlled set of best practices conditions, washing eggs that have been contaminated with Salmonella spp. resulted in a reduction of more than 5 log of Salmonella spp. counts from the shell surface. In addition, this does not lead to contamination of egg contents with the foodborne pathogen.
The concentration of the washing chemical compounds, the length of the washing period, the lowered pressure of the water flow and the age of the laying hens do not appear to influence the contamination of the egg contents. However, if the wash and rinse water temperatures are allowed to drop under 34 °C, the risk of content contamination is increased.
In commercial processing, eggs are most frequently rinsed with chlorine and chlorine-containing compounds that act as antimicrobial agents. In addition, they are widely available, have a relatively low cost and a high efficacy. Zeidler (2001a) observed that under optimal parameters, commercial egg washing can lead to a reduction of the bacterial load on the shell of 2 to 3 log10. A high level of chlorine can be detrimental for the quality of eggs (Bialka and others 2004) due to remaining residues deposited on the eggshell.
Hydrogen peroxide (H2O2) is responsible for bactericidal effects in biological systems. Its toxicity is apparently due to its capacity to generate more reactive and cytotoxic oxygen species such as the radical hydroxyl (-OH), that can initiate biomolecules' oxidation. The conversion of the H2O2 into these toxic compounds may be potentiated by reducing agents and by paroxydases (Juven and Pierson 1996).
Padron (1995) successfully used H2O2 for the decontamination of hatching eggs in a challenge involving S. Typhimurium. The eggs were treated by double dipping in H2O2 at a concentration of 6%.
Cox and others (2000) reported that S. Typhimurium contaminated shell eggs were treated with H2O2 (1.4%) by immersion in a solution containing a surfactant and submitted further on to a vacuum of 12 to 13 in Hg (0.4 bar) applied for 4 min. This treatment maximized the elimination of salmonellae on fertile hatching eggs, without adversely affecting the hatchability or the early chick mortality. These results demonstrated the difficulty in killing salmonellae that had already penetrated the shell egg.
Such a treatment could be extended to table eggs, with the difference considering the immersion. This latter process should be replaced by spray washing to enhance the practicability at industrial scale.
Water electrolysis technology was 1st used around 1900 in the soda industry, including in the production of sodium hypochlorite, being now applied in various fields and regarded as a promising nonthermal treatment for hygiene control (Al-Haq and others 2005). Electrolyzed oxidizing water (EOW) is produced by passing a diluted salt solution through an electrolytic cell, within which the anode and cathode are separated by a membrane, obtaining an acidic and an alkaline component (Huang and others 2008; Howard and others 2012). The acidic EOW may have a pH of 2 to 3, an oxidation reduction potential (ORP) of 1.150 mV and a free available chlorine concentration of up to 50 ppm, while the alkaline EOW may reach a pH of 6.8 to 11.6 and an ORP of 795 mV at the maximum value of pH (Mukhopadhyay and Ramaswamy 2012). Many studies conducted for the evaluation of the bactericidal activity of EOW have proved that it possesses antimicrobial activity on a variety of microorganisms: Staphylococcus aureus (Park and others 2002b), E. coli O157:H7 (Kim and others 2000a, 2000b), Salmonella Enteritidis (Venkitanarayanan and others 1999), S. Typhimurium and Listeria monocytogenes (Fabrizio and Cutter 2003), Campylobacter jejuni (Park and others 2002a) and others.
The antimicrobial effect of EOW is attributed mainly to pH, ORP, and HOCl (Mukhopadhyay and Ramaswamy 2012). Aerobic bacteria grow mostly at ORP range of +200 to 800 mV, while anaerobic bacteria grow well at –700 to +200 mV. The high ORP in the EOW could cause the modification of metabolic fluxes and ATP production, probably due to the change in the electron flow in cells. In general, bacteria grow in a pH range of 4 to 9. A low pH may sensitize the outer membrane of bacterial cells to the entry of HOCl, the most active of chlorine compounds. The latter appears to kill the microbial cell through inhibiting glucose oxidation by chlorine-oxidizing sulfhydryl groups of certain enzymes important in carbohydrate metabolism. Other modes of chlorine action that have been proposed are: disruption of protein synthesis; oxidative decarboxylation of amino acids to nitrites and aldehydes; reactions with nucleic acids, purines, and pyrimidines; unbalanced metabolism after the destruction of key enzymes; induction of deoxyribonucleic acid (DNA) lesions with the accompanying loss of DNA-transforming ability; inhibition of oxygen uptake and oxidative phosphorylation, coupled with leakage of some macromolecules; formation of toxic N-chlor derivatives of cytosine; and creation of chromosomal aberrations (Marriott and Gravani 2006; Huang and others 2008).
Considering shell eggs alone, a study was undertaken to compare EOW treatment with a commercial detergent-sanitizer treatment, both in vitro, for the decontamination of shell eggs, artificially inoculated with S. Enteritidis. For this in vitro study, eggs were soaked in alkaline EOW followed by soaking in acidic EOW at various temperatures. Treated eggs showed a reduction in population between ≥ 0.6 and ≥ 2.6 log10 CFU/g of shell S. Enteritidis. The log10 reduction of 1.7 for S. Enteritidis was observed for typical commercial detergent-sanitizer treatments, whereas log10 reduction of ≥ 2.1 for S. Enteritidis was achieved using the EOW treatment (Bialka and others 2004).
In a study conducted on shell eggs, performed in order to determine the effect of EOW applied using electrostatic spraying (in 4 different repetitions) on S. Typhimurium and other pathogenic bacterial species after applying the inoculum onto the shell eggs and allowed the bacteria to attach for 1 h, EOW completely eliminated all S. Typhymurium on 3, 7, 1, and 8 out of 15 eggs in 4 different treatment repetitions, respectively, even when high inoculations were used (Russell 2003).
In another study, the authors (Cao and others 2009) observed that acidic EOW is effective in reducing the populations of pathogenic microorganisms on the surface of shell eggs (aiming at S. Enteritidis), but its use is limited when low pH values are observed (≤2.7), because dissolved Cl2 gas can be rapidly lost due to volatilization, decreasing the bactericidal activity of the solution with time. On the other side, slightly acidic electrolyzed water (produced by electrolysis of a dilute hydrochloric acid in a chamber without a membrane), minimizes the safety issues for human health, regarding Cl2 off-gassing. At the same time, slightly EOW reduces the corrosion of the surfaces, and because at a pH of 5.0 to 6.5, its effective form of chlorine is the HOCl, this type of EOW may result in a stronger antimicrobial activity, in comparison to acidic EOW. The same authors proved that the bactericidal efficiency of slightly acidic EOW increases with temperature, the reduction for log10 CFU/mL at 45 °C, after 1 min reaching less than 1.0, after an initial value of 8.0 to 8.4 log10 CFU/mL. After 2 min, using temperatures of 4 °C, 20 °C, and 45 °C, S. Enteritidis was killed (Cao and others 2009). In conclusion, the last study shows that slightly acidic oxidized water can efficiently act as a promising disinfectant agent for the shell egg washing process and the reduction or inactivation of S. Enteritidis inoculated on the surface of shell eggs, without environmental damages.
On the other side, Bialka and others (2004) have shown that acidic electrolyzed water did not significantly affect albumen height or eggshell strength but there were significant effects on cuticle presence. It must be mentioned that the processing parameters of their study has much more severe effects in comparison to the slightly acidic oxidizing water processing parameters, mentioned above.
Ozone is one of the most potent sanitizers known, active against all forms of microorganisms at relatively low concentrations (Khadre and others 2001). Due to its low stability, ozone cannot be stored, being produced on demand. At commercial level, corona discharge method is usually used. In corona discharge, 2 electrodes, one of which is the high-tension electrode and the other one the low-tension electrode (ground electrode) are separated by a ceramic dielectric medium, providing a narrow discharge gap. When the electrons have sufficient kinetic energy to dissociate the oxygen molecule, a certain fraction of these collisions occurs and a molecule of ozone is formed from each oxygen atom (Guzel-Seydim and others 2004a). Ozone destruction of bacteria is accomplished by attacking on the bacterial membrane glycoproteins and/or glycolipids, resulting in cellular components leakage and followed by cell death, through the progressive oxidation of vital cellular components, reaching the nucleic material and causing DNA-strand breaks (Guzel-Seydim and others 2004b; Perry and Yousef 2011). In addition to its bactericidal effectiveness, ozone decomposes spontaneously to O2, hence having the advantage of being a nonpolluting sanitizer for shell eggs.
Ozone is a strong microbial agent that effectively inactivates Salmonella in shell eggs, its efficacy in aqueous phase being demonstrated. Salmonella Enteritidis was effectively inactivated ≥ 5 log units on the surfaces of shell eggs by high ozone concentrations (12% to 14% wt/wt O3 in O2 mix) (Rodriguez-Romo and others 2007). In another study involving the same serotype of S. enterica, ozone treatment of shell eggs, at atmospheric pressure for 3 min significantly (P < 0.05) reduced S. Enteritidis on eggshell by 3.1 log units compared with the untreated control, while longer times (up to 8 min) did not cause additional inactivation. Application of pressurized gaseous ozone for up to 20 min resulted in nonlinear inactivation of the microorganism, a trend similar to that observed when ozone was applied at atmospheric pressure. Populations of Salmonella Enteritidis decreased significantly (P < 0.05) on shell eggs treated with pressurized ozone. The 10-min treatment inactivated 4.5 and 5.9 log units or more, and the 20-min treatment inactivated 3.7 and 5.7 log units or more compared with the untreated controls (Rodriguez-Romo and Yousef 2005). On the same subject, Perry and others (2008) applied sequentially and in combination heat and ozone to shell eggs, in order to assess the log reduction of Salmonella Enteritidis on eggshells. 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 reduction in a many of the samples. 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. Also, in an attempt to differentiate the various treatments involving ozone on table eggs, Davies and Breslin (2003a) used dry and moist ozonated air, the results showing that 23 of 24 (95.8%) eggs remained contaminated after treatment compared with 11 of 12 (91.7%) controls, for the 1st one, and 4 of 12 treated eggs were contaminated compared with 9 of 12 (75.0%) control eggs for the 2nd. Therefore, the application of ozone in either type of environment was only partially effective.
For food irradiation, currently there are 3 types of ionizing radiation that are allowed to be used for sanitation: radiation from high-energy gamma rays, X-rays and accelerated electrons (Codex Alimentarius Commission 2003).
Gamma rays are produced by radioactive substances, called radioisotopes, among them the allowed ones being: cobalt-60 (60Co) and cesium-137 (137Cs). Their energy content arrives up to 1.17 to 1.33 megaelectronvolts (MeV) (60Co) and 0.662 MeV (137Cs). The accelerated electrons (or the electron beams) have a maximum quantum energy that does not exceed 10 MeV, being produced in linear accelerators at nearly the speed of light. X-rays, called also decelerating rays, are also produced in accelerators, their quantum energy of the electrons not exceeding 5 MeV (Riganakos 2010). The mechanism of microorganisms' inactivation is explained by the fact that ionizing rays (gamma rays) are picking electrons from the atoms of the treated product, therefore the free electrons can take part further on in the chemical reactions, also being able to destroy the DNA molecules from the living microorganisms (Riganakos 2010).
In comparison to the latter, electron beams (ionizing electrons) are more often easily accepted because there are no radioactive substances in the process (Riganakos 2010). By the acceleration to the speed if light, the electron beam gun subsequently passes the high-energy electrons onto the product, resulting in microbial activation. Electron-beam processing does not alter the temperature of the processed food and permits the application of high dose rates (103 to 105 Gy/s in comparison to only 0.01 to 1 Gy/s for gamma radiation) (Tahergorabi and others 2012). However, the depth of penetration is only 8 to 10 cm, for typical food products, therefore before irradiation of food products, the size has to be considered prior processing (Jaczynsky and Park 2003).
The X-rays are generated by interposing a metal target between the electron beam and the food product. This way, the high-energy electrons produced by the accelerator will impinge upon the metal target and produce the X-ray. The energy level is lower than in the case of electron beams, but the depth of penetration is higher (Tahergorabi and others 2012).
The scientific literature shows different attempts on shell eggs, in order to prove the efficacy of the Salmonella spp. inactivation.
Fresh shell eggs were inoculated with 108 CFU of S. Enteritidis with the aim of testing the effect of 3 doses of gamma-irradiation (1, 2, and 3 kGy). After the irradiation treatment, the eggs were kept at 4 °C for 42 h. The irradiation dose of 1 kGy determined a reduction of 3.9 logCFU for detectable S. Enteritidis on the shell. Further on, the higher used doses determined a reduction of bacterial contamination to nondetectable levels on the shell, proving the efficacy of this treatment for shell eggs surface decontamination (Tellez and others 1995).
Serrano and others (1997) tested the irradiation sensitivity of 5 S. Enteritidis isolates inoculated either on the surface (a level of 106 CFU/mL) or inside the shell eggs (by injecting 1 mL of 108 cells/mL). The inoculated samples were subjected to irradiation doses of 0, 0.5, 1.0, and 1.5 kGy. A minimal dose of 0.5 kGy was considered sufficient for the elimination of all the isolates from the surface. However, the same isolates showed a greater resistance when inoculated in the contents, and in this case, only the maximum dose included in the test was able to reduce S. Enteritidis counts by approximately 4 log10 in the contents.
In 2003, Wong and Kitts used low doses of electron beam irradiation (2, 3, and 4 kGy) to examine the antimicrobial effects on shell eggs inoculated with a 0.5 mL suspension of L. monocytogenes, E. coli, and S. Typhimurium, at a dose of 109 cells/mL−1. After holding the inoculated samples at 20 °C for 24 h, the irradiation treatment was conducted, using the doses mentioned above. The doses of electron beam irradiation of 3 and 4 kGy determined the reduction of the 3 pathogens to undetectable levels, with S. Typhimurium showing a higher resistance to irradiation, the counts decreasing slower than on the case of the other 2 species.
Using an inoculum of 107 to 108 CFU/egg, shell eggs were artificially contaminated with reference strains of S. Typhimurium, S. Enteritidis, Campylobacter coli, and C. jejuni. The range of irradiation doses for the determination of D values (the values of heat resistance for microorganisms) was 0.2 to 1 kGy for Salmonella spp. and 0.2 to 0.7 kGy for Campylobacter spp. The gamma irradiation doses were included in the range 0.5 to 5 kGy. The D values varied between 0.31 and 0.26 and 0.20 and 0.19 kGy for S. Typhimurium and S. Enteritidis, respectively, and between 0.21 and 0.18 kGy and 0.07 and 0.09 kGy for C. coli and C. jejuni for the eggshell (Cabo Verde and others 2004).
Al-Bachir and Zeinou (2006) performed another study on the irradiation of shell eggs. Using a suspension of 107 CFU/mL of Salmonella spp. the shell eggs were inoculated and subjected further on to doses of gamma irradiation from 500 to 3000 Gy, with the estimation of survival curves. The radiation dose required to reduce the Salmonella spp. load one log cycle (D10) was 448 Gy.
Yun and others (2012) suggested another approach. They aimed to predict the optimal conditions to minimize quality deterioration while maximizing safety and functional properties of irradiated eggs, by combining different concentrations of chitosan coatings and different ionizing radiation doses. In a 1st step, eggs were coated with chitosan, using concentrations of: 0.0%, 0.5%, 1.0%, 1.5%, and 2.0%. The 2nd step consisted in the inoculation of the shell eggs, through dipping, with S. Typhimurium and further on subjected to an irradiation treatment, using doses of: 0.0, 0.5, 1.0, 1.5, and 2.0 kGy. The results showed that using doses of more than 0.5 kGy, in combination with concentrations of more than 1% chitosan, S. Typhimurium was successfully removed from the eggshell. Moreover, foam stability, foaming capacity and Haugh units are not negatively affected when using a 0.45 kGy irradiation dose and a concentration of 0.525% chitosan coating.
Microwaves are oscillating electromagnetic waves with frequencies in the 300 MHz to 300 GHz range.
The effects of microwaves on pathogens can be generally expressed in 2 forms: thermal and nonthermal. Thermal inactivation is caused by heating during the microwave application process, involving changes such as denaturation of enzymes, proteins, nucleic acids or other vital components as well as disruption of membranes. Nonthermal effects have been classified in 4 categories:
- selective heating, explained by the fact that microwaves heat solid microorganisms more effectively than by the surrounding medium, causing a more rapid killing of the organism;
- electroporation, caused when an electrical potential crosses the membrane of the microorganism, determining the formation of pores in the membrane, and a further leakage of cellular components;
- cell membrane rupture, due to the voltage drop across a membrane;
- magnetic field coupling, caused by a disruption in internal components of the cell, leading further on to cell lysis (Datta and Davidson 2000; Leonelli and Mason 2010).
Microwaves can be used to reduce the load of different bacteria found on the eggshell, among them S. Enteritidis, as Lakins and others (2008) already showed. Using a new directional microwave technology (ITACA New Tech, Brescia, Italy), the eggs were exposed to 2.45 GHz, corresponding to 12.2 cm wavelength, for 20 s. A CO2 treatment for 30 s was performed at the end of the microwave processing. The maximum reduction of S. Enteritidis on the eggshell was of approximately 2 log cycles, this value being considered by the authors as appropriate to eliminate S. Enteritidis in most naturally contaminated eggs. However, further studies are required to reach a minimum reduction of 3 to 4 log10.
Ultraviolet light technology
Ultraviolet (UV) light occupies a wide band of wavelengths in the nonionizing region of the electromagnetic spectrum between X-rays (200 nm) and visible light (400 nm), but only UV in the range of 250 to 260 nm (short-wave UV radiation, or UVC) may be lethal to most microorganisms. Among its practical applications may be mentioned: inhibition of microorganisms on surfaces, destruction of microorganisms in the air and sterilization of liquids (Bintsis and others 2000). UV radiation inactivates microorganisms by inducing a cross-linking between pyrimidine nucleotide bases in the DNA, this resulting in inhibition of DNA transcription and replication mechanisms, leading eventually to microbial cell death. In addition, it has been demonstrated that UV radiation affects cell membrane integrity, inducing protein modifications and inhibiting oxidative phosphorylation (Rodriguez-Romo and Yousef 2005).
Using UV pulsed light (3 times per second, each pulse's duration 360 μs) of 3800 V input voltage, Keklik and others (2009) generated 1.27 J/cm2/pulse of radiant energy at 1.5 cm below the lamp surface. Samples consisting of shell-eggs artificially contaminated with S. Enteritidis were subjected to different treatment periods and different distances were also used (1, 3, 5, 10, 15, 20, and 30 s at 9.5 and 14.5 cm). Results showed that at a treatment distance of 9.5 cm from the UV-strobe, the reduction was between 2.0 and 5.3 CFU/cm2 and the visual appearance of samples did not show any difference after treatments. Treatments for 3, 5, and 10 s were not significantly different (P < 0.05), while 10 s treatment was not significantly different from 15 s treatment (P > 0.05). The results for 20 s and 30 s were significantly different from other treatments (P < 0.05), and considering the distances, the treatments at 9.5 and 14.5 cm were not significantly different (P > 0.05) regardless of the treatment times. The treatment with the shortest time that resulted in negative enrichments was the one comprising the distance of 9.5 cm.
Treatment of Salmonella-contaminated shell eggs with UV radiation (100 μW/cm2) for 2 and 4 min significantly (P < 0.05) decreased S. Enteritidis population by 2.6 and 2.0 log units, respectively, compared with the untreated controls. In the same study, but another trial, Salmonella-contaminated shell eggs were treated with higher UV radiation intensity (1500 to 2500 μW/cm2) for up to 5 min; this treatment resulted in significant (P < 0.05) microbial reductions; UV treatments for 1, 3, and 5 min decreased Salmonella populations by 3.4, 3.0, and 4.3 log units, respectively, compared with the untreated controls; no significant difference (P > 0.05) was observed when reductions in Salmonella populations after 1, 3, and 5 min of irradiation were compared (Rodriguez-Romo and Yousef 2005).
Using a hand-operated egg roller, an UV treatment consisting of 254 nm light, at 7.35 mW/cm2, for 0, 15, 30 and 60 s, was applied to shell eggs, finally assessing APC, in order to observe the reduction of microbial load on the eggshells. In all 30-s UV exposure trials, there was a significant reduction of 1 to 2 log10 CFU/egg, compared to the controls. Eggs rotated for 60 s had significantly greater reductions of APC than the other time intervals of exposure (a 2 to 3 log10 CFU/egg of aerobic microorganism compared to controls was observed after 60 s of exposure to UV radiation) (Chavez and others 2002).
Using an UVC (254 nm) dose rate of 10 mW/cm2/s at a 20 cm distance from the bulbs and irradiation by 90o rotation 4 times during exposure, Sommers and others (2010) obtained different log reduction per J/cm2 of Salmonella spp. on shell eggs: 0.43 ± 0.21 at 0.5 J/cm2; 0.31 ± 0.2 at 1 J/cm2; 0.53 ± 0.52 at 2 J/cm2 and 0.98 ± 0.55 at 4 J/cm2.
Pulsed light technology
Pulsed light (PL) treatment is a nonthermal technology that consists of the application of short duration pulses of an intense broad spectrum light (200 to 1000 nm). This part of the spectrum is mainly responsible for the lethal effect of the PL, through photochemical and/or photothermal mechanisms. The photochemical damage produced on the bacteria is induced mainly on DNA, by the UV-C region of the spectrum (200 to 290 nm), while photothermal damage is due by the absorption of light by microorganisms, which causes a temporary overheating leading to the vaporization of water inside the cell and the rupture of the membrane (Wekhof and others 2001; Woodling and Moraru 2005). Hierro and others (2009) showed that the inactivation of S. Enteritidis by using PL delivered in 100 μs, with 30% of the spectral output corresponding to UV light, is possible. For this, they used washed and unwashed eggs, in order to observe also the effect of the absence/presence of the cuticle. Dipping unwashed eggs into the culture provided an initial contamination of 4.5 log units in the shell. For this category, the PL treatment determined a reduction of 3.6 logCFU/egg in 24% to 80% of the eggs. For washed eggs, the inoculation determined an initial contamination of 6.3 log units, while the maximum reduction obtained was of only 1.8 logCFU/ egg. This method does not pose any risk for the egg quality, as the maximum temperature increase recorded in the eggs was 3 °C when 12 J/cm2 were applied. The lower contamination obtained in washed eggs supports the hypothesis that the state of the cuticle influences the utility of the treatment. Therefore, any circumstance that causes the loss of integrity of the cuticle reduces the efficacy of PL treatment.
Using also unwashed eggs, by inoculation with S. Enteritidis and treatment with 8 flashes of 0.5 J/cm2, an 8 log reduction was observed on the surface of the shell eggs. The same author observed than when using an inoculum solution colder than the egg, a deeper penetration of the microorganisms into the shell was enhanced, while the inactivation yielded 2 to 4 folds lower log reductions, in comparison to the 1st experiment (Dunn 1996).
Gas plasma technology
Plasma is constituted by particles in permanent interaction: photons, electrons, positive and negative ions, atoms, free radicals and excited and nonexcited molecules. Based on the conditions in which they were created, plasma can be thermal and nonthermal. Thermal plasmas are obtained at high pressure and need substantial power to be conserved, while nonthermal ones are obtained at lower pressure, use less power and are characterized by an electron temperature which is much higher than that of gas (Moisan and others 2001; Moreau and others 2008).
During plasma treatment, microorganisms are exposed to an intense bombardment by the radicals of OH and NO, but the mechanism of their inactivation is not entirely known. The treatment probably provokes surface lesions that the living bacterial cell cannot repair sufficiently quickly. The process involved in microorganism destruction can also be represented by the absorption of the plasma components onto the surface of microorganisms, forming volatile compounds that are then eliminated from the cells. Also, plasma induces perforations in the membranes of microorganisms and provokes a marked acidification of the medium (Laroussi and others 2003; Laroussi and Leipold 2004).
Gas plasma can represent a good opportunity for the decontamination of foods as an alternative method for those products that cannot be sanitized by conventional methods. In the European Union, washing or cleaning of shell eggs before or after grading is banned; therefore the need for alternative methods is rising. Ragni and others (2010) studied the possibility of using a nonthermal gas plasma device to decontaminate the surface of shell eggs. The device was represented by a resistive barrier discharge system, which comprises 2 electrodes. One or both of them are covered by a high resistive material, which would prevent arcing. The efficacy of the prototype for superficial decontamination was evaluated by exposing shell eggs artificially inoculated with S. Enteritidis and S. Typhimurium to gas plasma for different times: 0, 10, 20, 30, 45, 60, and 90 min. For S. Enteritidis, an exposure of 10 to 20 min resulted in a decrease of 1.0 to 1.6 log CFU/eggshell, in comparison to untreated samples. A maximum reduction of 2.2 to 2.5 log CFU/eggshell were observed following 60 to 90 min, at a relative humidity (RH) of 35%, while at RH 65%, the effectiveness of the treatments was enhanced. The efficacy of the gas plasma generator increased by increasing the treatment time, this showing a quasi-linear trend. For S. Typhimurium, a higher sensitivity was observed when using 65% RH. Also, a significant reduction of 3.5 log CFU/eggshell was observed when treated for 90 min.
Kayes and others (2007) studied the efficacy of another gas plasma generator device using one atmosphere uniform glow discharge for inactivation of foodborne pathogens, showed that the microbial load of different bacterial species (E. coli O157:H7, L. monocytogenes, Staphylococcus aureus, B. cereus, S. Enteritidis, Vibrio parahaemolyticus, Yersinia enterocolitica, Shigella flexneri) was strongly reduced during an initial exposure time of 30 to 90 s. However, no appreciable differences between Gram-positive and Gram-negative pathogens were observed, although the spore-forming B. cereus was more resistant to plasma than the non-spore-forming species (Kayes and others 2007).
Ultrasound treatment of food products is a useful tool to minimal processing, due to the fact that the transfer of acoustic energy is instantaneous and distributed throughout the whole volume of the products (Ulusoy and others 2007). The mechanism of microbial killing by ultrasonic waves is mainly due to the thinning of cell membranes, localized heating and production of free radicals (Piyasena and others 2003). Micro-mechanical shock waves are created by making and breaking microscopic bubbles induced by fluctuating pressures under the ultrasonication process; these shock waves disrupt cellular structural and functional components and lead to cell lysis (Ulusoy and others 2007). The sonication process determines microbial destruction as it follows: by creating regions of alternating compression and expansion, the longitudinal waves cause cavitation to occur, and bubbles are formed; by expansion, they reach a point where the ultrasonic energy provided is not sufficient to retain a vapour phase, and therefore, rapid condensation occurs. The condensed molecules collide violently, creating shock waves; these waves create regions of very high temperature and pressure, reaching up to 5500 °C and 50 MPa. Different combinations of this technology with other treatments have been proposed: thermosonic (heat plus sonication), manosonic (pressure plus sonication), and manothermosonic (heat plus pressure plus sonication), all of them representing highly efficient methods to inactivate microbes, as they are more energy-efficient and effective in killing microorganisms (Dolatowski and others 2007). Ultrasonic method was applied efficiently on Salmonella Enteritidis, by shell eggs treatment, in combination with thermal treatment. The parameters used were: 54 °C for 5 min, 24 kHz and 400 W at 60 μm. S. Enteritidis count was reduced (P < 0.05), from 7.78 log CFU/eggshell to 2.95 log CFU/eggshell. There was a negligible effect of thermoultrasonic treatment on the eggshell morphology and structures, while the cuticle suffered some changes in its morphology, but without effect on storage conditions and bacterial growth detected in the content of eggs (Cabeza and others 2011).
The use of plant extracts
The consumers' demand for organic and nonprocessed food products is increasing; therefore the use of plant extracts for table eggs decontamination may be considered a suitable option, from this point of view.
Recently Krittika and Gi-Hyong (2012) have published a review on the inhibitory effects of several plant extracts on Salmonella spp. According to these authors, the phenolic compounds are responsible for their bactericidal effects as they interact by permeabilizing the membrane. Their biological activity seems to depend also on the solvent used for extraction.
Currently, very few studies provided published results on this subject, especially on shell eggs. Davies and Breslin (2003a) mentioned a natural herb extract that has an inhibitory effect on Salmonella and other harmful bacteria. When eggs previously contaminated with Salmonella Enteritidis were dipped in a 2% Protecta II (Bavaria Corp. Intl., Apopka, Fla., U.S.A.) and further on air-dried at room temperature, the authors did not observe a difference in the number of eggs that remained contaminated (8/20), compared with the distilled water control (8/20).
Recently, Pohuang and others (2009) have tested the effect of an ethanolic extract of Punica granatum L. against Salmonella Enteritidis on eggshells and eggshell membranes. Using a concentration of 1.25% and one of 2.5% (w/v) of this alcoholic plant extract applied for 10 min did not lead to a complete elimination of S. Enteritidis on both eggshells and eggshell membranes.
The effectiveness of these plant extracts has not been fully demonstrated until now.