Eggs are a highly nutritious, inexpensive food commodity which is accepted by most cultures. The safety of eggs has become a global issue with emergence of the pathogen Salmonella enteritidis as a major hazard associated with the consumption of raw and semi-cooked eggs. By 1993 the occurrence of Salmonella enteritidis has reached epidemic proportions in many countries. 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.
In the UK alone, there were 14500 confirmed human food poisoning cases caused by Salmonella enteritidis in 1991. In the USA, the consumption of shell eggs per annum is estimated between 50 and 65 billion eggs, of which 1 in 10000 eggs is estimated to be infected with Salmonella enteritidis resulting in a 1 in 50 chance of an individual consuming a contaminated egg in any given year. Although there has been a reduction in the occurrence of Salmonella enteritidis since 1993, due to better poultry flock management, the organism has not been eradicated from poultry flocks, especially in countries where legislative controls are not as strict.
Salmonella enteritidis is killed by heat treatment and adequate cooking of eggs will thus kill the microorganism. However, the use of raw eggs in food as well as the consumption of partially cooked eggs is a reality, particularly in catering establishments and homes. Thus, in order to remove or reduce the risk of Salmonella enteritidis food poisoning, eggs should be pasteurized before distribution. Apart from having the effect of providing eggs substantially free of the risk of causing Salmonella enteritidis food poisoning, the shelf life of pasteurized eggs will also be significantly increased.
The infections of laying hens with Salmonella enteritidis, and the resultant contamination of egg contents, have resulted in a marked increase in human salmonellosis since the mid 1980's. The route of infection for Salmonella enteritidis is transovarian, whilst the route of infection for other serovars is trans-shell.
Both mechanical and chemical inhibitors restrict the growth of Salmonella enteritidis and other trans-shell contaminants in the albumen of fresh eggs. Fresh albumen is highly viscous and organized to confer biological structure to the egg contents, holding the vulnerable yolk away from the shell and its membranes. Fresh albumen contains factors inhibitory to the growth of microorganisms. These include lysozyme, which is an antimicrobial against Gram-positive bacteria, and conalbumin (ovotransferrin), which binds metallic ions making Fe+++ unavailable to the organisms. Additional inhibitors include ovoflavoprotein, which binds riboflavin, and anti-trypsin factors such as ovoinhibitor. As the albumen ages, it becomes less viscous and less inhibitory. The deterioration of the vitelline membrane around the yolk allows leakage of yolk contents into the albumen triggering off rapid multiplication of surviving organisms in the albumen. Temperature of egg storage is a key factor in determining the rate of albumen aging.
The control of Salmonella and spoilage organisms in whole shell eggs is multi-faceted. High general Salmonella infection rates in poultry and sub-clinical ovarian infection in laying hens have been attributed to such factors as intensive farming, forced moulting practices and use of contaminated animal by-products as dietary protein supplements. About 30% of eggs are sent for breakage and processing into egg products for the food processing industry. Pasteurization practices are well established for these egg products and aim to reduce the loads of Salmonella by around 5 logs. The principle source of Salmonella on eggs for breakage is fresh fecal matter on the shells and loads can be high in newly pooled fresh egg contents. Pooled yolks, pooled whole egg and pooled albumen are pasteurized at different temperatures reflecting the differences in thermal stability of the egg products.
The pasteurization of whole shell eggs is much more problematical than the pasteurization of egg products and must balance the required reduction of target organisms against the maintenance of albumen quality.
Table 1 provides some information on the composition and characteristics of egg albumen and Table 2 provides more information on the characteristics of the various proteins in egg albumen:
TABLE 1DESCRIPTION AND CHARACTERISTICS OF PROTEINS IN EGG ALBUMENAlbumenIsoelectricMolecularDenaturationProteinProteins (%)PointsWeightTemperature1Characteristicsi.Ovalbumin544.545 00084.0Phosphoglycoproteinii.Ovotransferrin126.176 00061.0Binds metallic ionsiii.Ovomucoid114.128 00079.0Inhibits trypsiniv.Ovomucin3.54.5-5.05.5-8.3 × 106—Sialoprotein, viscousv.Lysozyme (G1)3.410.714 30075.0Lyses some bacteriavi.G2 globulin4.05.53.0-4.5 × 10492.5—vii.G3A globulin4.04.8—55.0 at pH 9—viii.Ovoinhibitor1.55.149 000—Inhibits serineproteasesix.Ovoflavoprotein0.84.032 000—x.Ovomacroglobulin0.54.57.69 × 105—Strongly antigenicxi.Cystatin0.055.112 700Inhibits thiolproteasesxii.Avidin0.051063 300Binds biotin1In water or buffer
TABLE 2DESCRIPTION OF THE CHARACTERISTICS OF EGG ALBUMEN PROTEINSProteinDescription(i) OvalbuminThis is the predominant protein in albumen. It is a phosphoglycoprotein and the onlyalbumen protein to contain free sulfhydryl group. Ovalbumin is readily denatured bymechanical stress (e.g., shaking). This is referred to as surface denaturation. Ovalbuminis thermally resistant. Ovalbumin is converted to S albumin, a more heat stable protein,during egg storage. The conversion is prevented by oiling the eggs. The denaturationtemperature of native ovalbumin and S ovalbumin are reported 84.5 and 92.5° C.(ii) ConalbuminThis is a glycoprotein that contains no phosphorus or free sulfhydryl groups. Fifteen(Ovotransferrin)disulphide bonds maintain the quaternary structure. Conalbumin is more heat sensitivethan ovalbumin but less susceptible to surface denaturation. It is reported to be stable at57° C. for 10 minutes at pH 9. Di and trivalent metallic ions are bound by conalbumin.Two atoms of Fe+++, Al+++, Cu++ and Zn++ per molecule of protein form stablecomplexes with conalbumin above pH 6. These complexes are red, colourless, yellowand colourless respectively. Complexes of conalbumin with metallic ions are resistant tothermal denaturation and proteolytic attack. The iron binding properties of conalbuminare suggested to be responsible for its anti-microbial properties.(iii) OvomucoidOvomucoid is a heat resistant glycoprotein that contains no tryptophan. Ovomucoid is atrypsin inhibitor. Chicken ovomucoid is classified as a single headed inhibitor, i.e., eachmolecule of ovomucoid binds with only one molecule of trypsin (serine proteinase).Turkey ovomucoid is double headed and duck ovomucoid triple headed. The protein isdenatured at 80° C. at pH 9.(iv) OvomucinOvomucin is a sulphated glycoprotein that may contribute to the gel like structure ofthick white in the form of flexible fibres visible under electron microscopy. The amountof ovomucin in the thick white is four times greater than in thin white. Ovomucin insolution is resistant to heat between pH 7.1 and 9.0 at 90° C. for two hours. Ovomucinand lysozyme in solution can interact to form a water-insoluble complex. Much of theinterest in ovomucin has arisen from its possible roles in maintenance of the gelstructure of thick egg white and in the process of egg white thinning. Contradictoryhypotheses have been advanced for the egg white thinning mechanisms.(v) LysozymeLysozyme is an albumin enzyme that has a lytic action on bacterial cell walls,particularly Gram positives. Lysozyme hydrolyses the Beta (1-4) linkages between N-acetylneuraminic acid and N-acetylglucosamine. In egg white, the enzyme is 50 timesmore heat sensitive than in phosphate buffer. In egg white heated to 63° C. for 10minutes, lysozyme is inactivated as the pH rises above pH 7. It has been suggested thatits role in egg white may be more important in maintaining gel structure than as an anti-microbial.(vi) OvoglobulinsThe globulin fraction consists of three proteins, G1, G2 and G3. G1 is lysozyme. G1 andG3 are glycoproteins with excellent foaming properties. G3 is also the most heatsensitive of all the albumen proteins and foaming properties of pasteurized eggs is acritical quality parameter.(vii) OvoinhibitorOvoinhibitor is a proteolytic enzyme inhibitor capable of inhibiting trypsin andchymotrypsin as well as a variety of fungal and bacterial proteases.(viii) OvoglycoproteinThis glycoprotein is an acidic glycoprotein that comprises 1% of the egg white proteins.Little is known in respect of its functionality in egg albumen.(ix) OvoflavoproteinAll the riboflavin in egg albumen is bound in the flavoprotein in a 1:1 ratio.Ovoflavoprotein is sometimes referred to as the riboflavin binding protein. The majorfunction of the riboflavin binding protein is presumably to ensure transfer of theriboflavin from the blood serum to the albumen.(x) OvomacroglobulinOvomacroglobulin is a glycoprotein with inhibitory activity against diverse proteolyticenzymes such as trypsin, papain and thermolysin.(xi) CystatinCystatin is an inhibitor of thiol proteinases such as ficin and papain. It is highly stableagainst denaturation. Since the isolation of cystatin from chicken egg white, a “superfamily” of cystatins have been isolated and characterized from mammalian tissues. It issuggested that certain cystatins may have not only the general role of protecting cellsagainst uncontrolled activity of their own proteinases, but may have a more specificaction against virus infections. This has led to research on cystatins, including eggcystatin, as possible agents for anti-viral chemotherapy.(xii) AvidinAvidin is a glycoprotein that combines with biotin to form a stable complex incapable ofabsorption by the intestinal tract of animals. In addition to its physiological importanceas a possible anti-nutrient due to its strong affinity for biotin, avidin has also beensuggested to play a role as an anti-microbial. Inhibition of microbial growth may arisenot only from the unavailability of avidin-bound biotin, but also from the ability to bindvarious Gram-negative and Gram positive bacteria.
Table 1 is only a generalization as there are many factors that will influence the rate and temperature at which a specific protein fraction will denature. One of the most important factors is the pH of the medium and the ionic strength. Significant differences are found between published values mainly due to differences in the pH or the ionic strength of the medium in which the protein fraction was heated. Of particular importance is the fact that globulin G3A becomes much more sensitive to denaturation than ovotransferrin at a pH above 9.0. This is significant as the ovotransferrin is generally regarded as the most heat sensitive protein. Whole shell eggs of one to two days old usually have pH ranges of more than 9.0, which create a problem if ovotransferrin is used as the indicator protein for heat damage. During the pasteurization of liquid eggs, the pH and the ionic strength is usually changed by the addition of salts or buffers to create a more stable environment for the egg proteins. For example, it is known that at a pH of 7 the globulin G3A only starts denaturing at 60° C., while at a pH of 9, almost 50% of the globulin G3A is denatured when the contents have reached 60° C. Globulin G3A plays a major role in the turbidity that is first observed at the initial stages of heat treatment of eggs. The egg albumen is still liquid, but develops a “milky” or turbid appearance as the globulin G3A denatures starting at 55° C. Denaturation of the globulin G3A is immediate at 60° C. with 50% denaturation after 5 minutes exposure. As the target organism Salmonella enteritidis needs to be destroyed without damage to the egg proteins, it becomes clear that efficient heat transfer to the eggs is necessary in order to give maximum heat exposure to the microorganisms, but minimum heat exposure to the egg proteins.
Egg yolk proteins are generally more heat stable than albumen proteins. However, as the target microorganisms are inside the yolk, yolk temperature has to be raised high enough without damage to the albumen. This is a major problem with conventional systems or processes of which the Applicant is aware using water or steam as heating media from the outside of the eggs. By the time the yolk has reached critical temperatures to kill the Salmonella enteritidis, the albumen had been exposed for extensive periods to the heat, causing the occurrence of excessive amounts of precipitate giving the albumen a milky colour. As egg albumen is generally more heat sensitive than egg yolk, egg albumen should be used as the indicator protein for egg quality assessment after processing.
Albumen or egg white consists of four distinct layers: outer thin white, viscous or thick white, inner thin white and the chalaziferous layer. The total solids content is 11 to 13%. Protein is the major constituent with up to 1% carbohydrate. Free carbohydrate is around 0.5% as glucose, with the rest as glycoprotein (mannose and galactose). Table 3 provides information on the layer structure and moisture content of albumen:
TABLE 3LAYER STRUCTURE AND MOISTURE CONTENT OF ALBUMEN% of albumenLayerMeanRange% MoistureOuter thin white23.210-6088.8Thick white57.330 to 8087.6Inner thin white16.8 1-4086.4Chalaziferous2.784.3(including chalaza)
Table 4 provides some information on the composition of albumen, yolk and whole egg:
TABLE 4LAYER STRUCTURE AND MOISTURE CONTENT OF ALBUMENEgg ComponentProtein (%)Lipid (%)Carbohydrate (%)Ash (%)Albumen 9.7-10.60.030.4-0.90.5-0.6Yolk15.7-16.631.8-35.50.2-1.01.1Whole egg12.8-13.410.5-11.60.3-1.00.8-1.0