1Student Name: Student StudentStudent ID Number: uXXXXXXAssignment Name: Lab reportWord Count: 3044Title: Comparison of internal quality traits between fresh and deteriorated hen eggs2Comparison of internal quality traits between fresh and deteriorated hen eggsAbstract: Background:Internal quality traits of shell eggs are important indicators of their functionalproperties and thus their suitability for use in food production.The aims of this study were to compare internal quality traits between cold- and hot-stored eggs, with particular focus on the Haughunit value, and to investigate the relationship between temperature and time, egg storage temperatureand the formation of ferrous sulphide rings after heat treatment. Methods: Free range chicken eggswere stored at either 4°C (fresh) or 40°C (deteriorated) for 2 weeks and then broken out eggs wereassessed for internal quality trait measures including Haugh unit value, albumen index, yolk indexand thick albumen percentage. Eggs were also exposed to different heat treatments (5 for fresh eggs,1 for deteriorated eggs) and then assessed for ferrous sulphide ring formation and internal qualitytraits including yolk position and air cell size. Results: There was no significant difference ininternal quality trait measures (including Haugh unit) between fresh and deteriorated broken outeggs, except for a significantly greater percentage of thick albumen in fresh eggs (p = 0.000). Heattreated deteriorated eggs showed a greater air cell size and less-centred yolks than fresh eggs, andonly deteriorated eggs showed formation of ferrous sulphide rings on their yolks after heat treatment.Discussion: The lack of difference between some internal quality trait measures between fresh anddeteriorated eggs in this study is suspected to be a result of poor measuring technique, especiallygiven the highly significant difference in thick albumen, yolk position and air cell size between thetwo egg types. Egg storage temperature was shown to be a clear determinant of ferrous sulphide ringformation in heat treated eggs.Introduction:Eggs are a widely consumed and highly nutritious food source for humans. They are a high qualityprotein source, containing the complete array of essential amino acids in a well-balanced proportion,and also contain other important nutrients like essential fatty acids, choline, vitamin A and vitaminB12, (1). The unique and varied properties of egg yolks and egg whites have made them essential tothe food industry for a range of functions. Egg yolks contain proteins and various lipids, includingtriglycerides, cholesterol and phospholipids such as lecithin, which is well known in the foodindustry for its emulsifying properties (2). Egg whites are composed almost entirely of water andproteins, with the most abundant proteins being ovalbumin (1).3While there is generally no nutritional difference between fresh and old eggs, the functionalproperties of fresh eggs are known to be superior to those of old eggs (3). Therefore assessment ofegg quality is important in the food industry in order to develop optimum storage and handlingpractices to maintain the functional properties of eggs for as long as possible. High quality eggs arepreferred by consumers (4) and are essential for many processes used in food production. Forexample, egg white foams are central to many baked products and albumen foaming capacity andstability have been shown to decrease considerably with age (5). The interior quality of eggs can beaffected by numerous factors including temperature, humidity, air movement and storage time (4).Moisture and CO2 loss through the porous egg shell results in decreased egg weight and reducedquality, also leading to increased air cell size between the inner and outer shell membranes (3).Storage temperature is considered the most important factor for preserving egg quality; the fact thatgases are more soluble in cold liquids than hot liquids means that less CO2 is released through theegg shell at colder temperatures (6).Egg albumen is an important protein-based foaming, whipping and thickening agent in the foodindustry and its properties are important indicators of egg quality (7). Egg whites are composed ofthree layers of albumen and the chalazae, twisted cord-like strands of albumen that keep the yolkcentred (4). The thick albumen, a viscous gel fraction which makes up about half of the white of afreshly laid egg, is located between two layers of thin albumen (8). Thick albumen has a more rigidstructure due to the higher content of the fibrous protein ovomucin (3). As an egg ages there is littlechange in the viscosity of thin albumen; however, viscosity and height of the thick albumen begin todecline immediately after laying (9). This happens because of loss of CO2, an increase in albumenpH and degradation of ovomucin, resulting in the breakdown of albumen structure (3). As a resultthe ratio of thick to thin albumen in the egg decreases. This process is further accelerated by storageat incorrect temperature and humidity (3).Egg yolks are also important in the food industry and are frequently used as lipoproteins for formingand stabilising emulsions (7). Egg yolks have been shown to increase in size and flatten with age.This is due to the increased albumen pH which weakens the vitelline membrane, or the thin sac thatencloses the yolk, and allows water from the albumen to cross into the yolk (3). There are variousmethods of assessing egg quality based on both egg yolks and egg whites. A common measure ofegg yolk quality is the yolk index, defined as the height of the egg yolk divided by the diameter (10).Yolk index decreases with increased temperature and storage time of eggs (11, 12).4The Haugh unit was developed in 1937 (13) and is a widely accepted standard for measuringalbumen quality of hen eggs (14). It is defined as a log-transformed measurement of the height ofthe thick albumen with an adjustment for egg weight (14). The Haugh unit value is easy to measureand is well correlated with the freshness of an egg. It has been shown to decrease with storage time,increased storage temperature and increased age of the laying bird (11, 15, 16). Nonetheless, thereare several criticisms against the method (8), particularly criticisms of the validity of the correctionfor egg weight (17), and some researchers suggest using albumen height alone as an alternative (17,18). Another frequently used quality measure is the albumen index, defined as albumen heightdivided by albumen diameter of a broken out egg (10). Although there are no prescribed Australianstandards for grading eggs, the US Department of Agriculture standards have developed a qualityrating system for commercially sold eggs based on the Haugh unit value. Grade AA eggs must havea clear, firm white with a Haugh unit value of 72 or higher, grade A eggs must have a reasonablyfirm white and a Haugh unit value from 60-71 and grade B eggs may have weak and watery whitesand a Haugh unit value below 60 (19).Heat treatment or cooking of eggs results in denaturation and coagulation of proteins (2). In a hardcooked egg the positioning of the egg yolk within the white can be used as an indirect measure of theratio of thick to thin albumen (2). An egg with a relatively large proportion of thick albumen willhave a well-centred yolk, whereas an egg with a larger proportion of thin white will have its yolklocated against the shell. Hard cooking also allows easy assessment of air cell size once the egg ispeeled, but this can also be determined by candling (4), which has the advantage of not requiringcooking or destruction of the egg. If eggs are cooked at too high a temperature or for too long, a greyring can form on the exterior of the yolk (20). This occurs due to a chemical reaction between iron inthe yolk and hydrogen sulphide released from sulphur-containing proteins in the white, whichcombine to form dark-coloured ferrous sulphide. Formation of ferrous sulphide rings occurs morefrequently in old or deteriorated eggs because the increased albumen pH increases the production ofhydrogen sulphide in the white (20).The objectives of this research were two-fold. The first aim was to assess and compare the quality ofshelled eggs after storage at cold and hot temperatures using the Haugh unit value and other standardinternal quality measures including yolk index, albumen index and relative proportion of thick andthin albumen. The second aim was to determine the relationship between time, temperature, eggstorage temperature and the formation of ferrous sulphide rings in eggs.5Methods:The methods for this research were followed according to the University of Canberra Food Sciencelaboratory instructions (21). The eggs used were free range hen eggs with a best-before date of 18thSeptember 2014 (experiments were conducted on the 19th August 2014). For 2 weeks prior totesting, fresh eggs were refrigerated (~4°C) and deteriorated eggs were incubated at 40°C. Fresheggs were weighed and then carefully cracked onto large flat ceramic plates. Measurements werethen performed on the eggs using Vernier callipers. The diameter and height of the egg yolk andthick white were measured to calculate the yolk index and albumen index. The egg whites were thenpassed through a strainer into a measuring cylinder to separate the thick and thin albumen andmeasure their volumes. The Haugh unit value was calculated for each egg using the equation H =100 x log (h – 1.7(W0.37) + 7.6) (21). Duplicates of fresh eggs were subjected to five different heattreatments (as indicated in table 3), and duplicates of deteriorated eggs were simmered in water at95°C for 20 minutes. Each treatment was performed by a different group of three-four students inthe 8251/8252 Food Science class. After treatment the eggs were immediately cooled under coldrunning water. Eggs were then peeled, noting air-space between the white and the shell, and cut inhalf lengthwise. After egg yolk position was recorded the yolks were carefully removed using aspoon in order to assess the colour of the curved side of the egg yolk. Where applicable results foregg quality traits were compared between fresh and deteriorated eggs using mean ± standard error ofmean (SEM). Unpaired student’s t tests were performed using SPSS (22) to determine whether meanquality measurements were significantly different between fresh and deteriorated eggs.Results:Measures of internal quality traits for fresh and deteriorated broken out eggs are presented in tables 1and 2. There was no significant difference (t7.56 = -0.15, p = 0.88) in average yolk index betweenfresh eggs (0.43 ± 0.08, n=10) and deteriorated eggs (0.44 ± 0.05, n=2) (table 1). The albumen indexwas slightly lower in the fresh eggs (0.054 ± 0.013) than the deteriorated eggs (0.062 ± 0.007), butagain the difference was not significant (t8.19 = -0.59, p = 0.58). There was a very large spread ofresults for measurements on the fresh eggs (table 1), indicated by high standard deviation values(data not shown). The mean Haugh unit value was slightly higher for fresh eggs (56.0 ± 6.4) than fordeteriorated eggs (54.0 ± 4.3) (table 2), but the difference was non-significant (t8.53 = 0.28, p = 0.79).Even after the elimination of an anomalously small albumen height measurement and itscorresponding negative Haugh unit value (egg 1.1, table 2), the large standard deviation of results6(SD = 19.1) indicated an extremely large spread of Haugh unit values among the fresh eggs. Theslightly greater average albumen height of fresh eggs (4.16 ± 0.63) compared to deteriorated eggs(3.49 ± 0.37) was also a non-significant difference (t7.38= 0.92, p = 0.39).Table 1: Yolk and albumen measurements of shelled fresh and deteriorated hen eggs. Deterioratedeggs are shown in the shaded rows. Eggs are numbered to indicate the lab group that performedmeasurements (eggs 1.1 and 1.2 were measured by group 1, eggs 2.1 and 2.2 were measured bygroup 2, etc.). EggYolk diameter(mm)Yolk height(mm)YolkindexAlbumendiameter (mm)Albumenheight (mm)Albumenindex1.143.117.00.3981.10.64*0.008*1.241.917.70.4281.23.340.0418.104.22.168.4958.17.580.1302.238.919.50.5065.26.850.1053.130.931.91.0390.92.970.0333.239.915.20.38150.02.070.0144.143.818.80.4370.73.770.0534.246.617.30.3794.34.710.0505.149.45.50.11101.33.090.0315.252.08.30.1699.83.030.030Fresh eggs(n=10)mean (±SEM)42.7(±1.9)17.1(±1.7)0.43(±0.08)89.3(±8.16)4.16(±0.63)0.054(±0.013)22.214.171.124.4055.93.120.056126.96.36.199.4955.93.860.069Deterioratedeggs (n=2)mean (±SEM)40.2(±1.1)17.8(±1.5)0.44(±0.05)55.9(±0.0)3.49(±0.37)0.062(±0.007) *This anomalous result was excluded from mean and SEM calculations.The average percentage of thick white in the fresh eggs (63.2% ± 2.1, n = 10) was considerablyhigher than in the deteriorated eggs (45.0% ± 1.0, n = 2), with the difference being highly significant(t9.16 = 7.95, p = 0.000) (figure 1). The measurements of thick and thin white volume appeared moresimilar between eggs measured by the same group compared to eggs measured by different groups(figure 1). The same is true of the other internal egg quality trait measures (tables 1 and 2).7Table 2: Haugh unit values of fresh and deteriorated eggs calculated from egg weight and albumenheight. Fresh eggs are shown in unshaded rows, while deteriorated eggs are shown in shaded rows. EggEgg weight (g)†WAlbumen height (mm)hHaugh unit valueH1.1–0.64*-34.6*1.2–3.3449.82.1–7.5886.92.2–6.8582.43.1–2.9744.43.2–2.07188.8.131.523.7755.44.264.964.7165.55.1–3.0946.35.2–3.0345.3Fresh eggs (n=9)mean (±SEM)4.16(±0.63)56.0(±6.4)6.154.943.1251.06.260.183.8657.0Deteriorated eggs(n=2)mean (±SEM)3.49(±0.37)54.0(±3.0) *This anomalous result was excluded from mean and SEM calculations.†Egg weights were only recorded for four eggs in total. The average of the two fresh egg weights (61.18) wasused in all other Haugh unit calculations for fresh eggs.All cooking treatments used on the fresh eggs resulted in yolks with a yellow-coloured exterior, but agrey exterior was observed upon heat treatment of the deteriorated eggs (table 3). The centres of allegg yolks remained yellow after heat treatment. Yolks were positioned slightly off-centre in thefresh eggs treated by boiling, but centred in the remaining fresh eggs. The yolks of the deterioratedeggs were very off-centre, positioned right against the side of the eggshell (table 3). A large air cellwas present in the deteriorated eggs, and no air cell was recorded as being present in any of the fresheggs except those boiled for 6 min, which had a very small air cell (table 3).8Figure 1: Percentage of thick and thin egg white volume measured in fresh or deteriorated shelledeggs by six lab groups. Graphed values show the mean percentage thick/thin white ± SEM (n=2).Table 3: Evaluation of fresh and deteriorated eggs cooked in shell. Each treatment was performedon two eggs by a different lab group. Yolk colour refers to the colour of the exterior. Egg typeEgg treatmentYolk colourYolk positionAir cellFreshSimmered 85°C; 30 minBright yellowCentredNoneFreshSimmered 95°C; 30 minPale yellowCentredNoneFreshBoiled 100°C; 13 minPale yellowOff-centreNoneFreshBoiled 100°C; 6 minBright yellowOff-centreVery smallFreshSteamed; 13 minBright yellowCentredNoneDeterioratedSimmered 95°C; 20 minGreyVery off-centreLarge 010203040506070801fresh2fresh3fresh4fresh5fresh6deterioratedPercentage of total egg white volumeLab group and egg typeThick and thin egg white volumeThin whiteThick white9Discussion:The first objective of this research was to compare internal quality traits between fresh anddeteriorated hen eggs, with particular focus on the Haugh unit. There was no significant differencein Haugh unit values between fresh and deteriorated eggs (p = 0.79, table 2). This result iscontradictory to the published literature which consistently shows a significant decrease in Haughunit value with increased storage temperature (11, 12, 23). Previous studies also show a decline inalbumen height, albumen index and yolk index with increased storage temperature (11, 12, 23) whichwas not observed in this study. In fact the only measure of quality on broken out eggs that wassignificantly different between the two egg types was ratio of thick to thin egg albumen, withpercentage of thick albumen significantly higher in the fresh eggs (t9.16 = 7.95, p = 0.000, figure 1).This result is in agreement with previous studies (10). The differences in quality trait measuresbetween fresh and deteriorated heat-treated eggs, namely less centred yolks and increased air cellsize in deteriorated eggs (table 3), are also consistent with published literature. However, theobservation of differently centred yolks between fresh and deteriorated eggs is inconsistent with thesimilar albumen height, albumen index and Haugh unit value observed between the two egg types.This suggests a problem with some of the measurement techniques in this study, which is furtherdiscussed below.The Haugh unit was specifically formulated to include a correction for egg weight (14). Thereforethe fact that egg weights were only collected for four of twelve eggs in this study makes the use ofthe Haugh unit somewhat redundant. Some researchers have suggested that the correction for eggweight in the Haugh unit is invalid or unnecessary and instead suggest that the albumen height (17)or albumin index (24) should be used as the standard measure of egg quality. But as expected giventhe reliance of each method on measurement of albumen height, the difference in albumen height andindex between fresh and deteriorated eggs was also non-significant (p = 0.39 and p = 0.58, table 2).The second objective of this research was to investigate the relationship between heat treatmenttemperature and timing on formation of ferrous sulphide rings in fresh and deteriorated eggs. Onlythe deteriorated eggs showed ferrous sulphide ring formation after heat treatment, with noneobserved in the fresh eggs regardless of heat treatment (table 3). Given that ferrous sulphideformation is known to increase with increased temperature and longer cooking times (2), the 13minutes at 100°C and 30 minutes at 95°C might be expected to result in some ferrous sulphideformation even among the fresh eggs, yet this was not the case. The eggs sourced for this study werevery fresh, with a best-before date of 30 days from the date of the experiment (or 44 days including10the 2 week incubation period). According to the Australian Egg Corporation’s voluntary code ofpractice, ‘best before’ dates on eggs should be no more than six weeks from the date of packing (andeggs are to be delivered to packing stations within 96 hours of lay) (25), suggesting the eggs werelikely to have been packed just before they were sourced. Ferrous sulphide ring formation occursless easily in fresh eggs, and at least one previous study (20) has shown no formation of ferroussulphide in eggs boiled at 100°C for 15 min (a longer time than the 13 min used in this study),provided that the egg was cooled in cold water (as was done for all eggs tested in this study). On theother hand ferrous sulphide rings did form when eggs were boiled for 30 min, even when the eggswere cooled in cold water after heat treatment (20). In further experiments use of longer cookingtimes would allow more successful evaluation of the relationship between heating time andtemperature and the formation of ferrous sulphide in fresh eggs.Although egg to egg variability has been noted in previous studies (7), it is not usually seen to thesame extent as in the present study. Variability can be affected by hen health, hen age, nutrition,management practices and other factors (7). However, it is strongly suspected that the largevariability in fresh eggs in this study was largely a result of inconsistent measuring techniquesbetween lab groups or perhaps even some data transcription errors. One impossibly low thickalbumen height was excluded from analysis (tables 1 and 2), and several other data points appearhighly suspect. Hen egg albumen heights 30 mm are unheard of in the literature, but all of these were recorded in the current study.Measurements on eggs appeared more similar within the same group than between groups suggestingthat skill and technique using the Vernier callipers had a considerable effect on results. To eliminatethis ‘researcher skill’ effect the eggs should all be measured by the same (well trained) individual.Alternatively a Haugh unit device such as a tripod micrometer to measure albumen height, or anelectronic method such the Technical Services and Supplies QDC Egg Quality System thatautomatically measures and calculates Haugh units (10) could be used. These automated methodshave the added advantage of making results more comparable between different laboratories andmaking the process of egg quality assessment much faster in commercial settings.Several other important indicators of egg quality exist that could be included in future research, suchas the strength of the vitelline membrane, the sac enclosing the yolk. Vitelline membrane strengthdecreases during storage (15), and poor vitelline membrane strength makes clean separation of yolksand whites very difficult (3). This has become especially important given the growth of the eggbreaking industry, where yolks and whites are separated for use in different food productionprocesses (26). Even the slightest contamination of egg white with egg yolk can have serious11consequences for foaming capacity (27), which is a major problem given that it is currentlypractically impossible to produce entirely yolk-free white on a large-scale commercial basis (26).Viscosity of albumen is another important indicator of egg quality since it is directly related to thefunctional characteristics of the albumen and determines whipping, emulsifying and gellingproperties (8). Albumen pH has been shown to increase rapidly with storage time, even at 5°C (12),so this is another measure of quality that could be compared between eggs stored at differenttemperatures.In conclusion, the Haugh unit value, yolk index, albumen index and albumen height were notsignificantly different between eggs stored for two weeks at 4°C and 40°C. There was, however, asignificantly higher percentage of thick albumen, a smaller air cell and a more centred yolk in therefrigerated eggs, indicating that there was indeed a decrease in quality of the eggs stored at thehigher temperature. These inconsistent results between experiments combined with a large numberof suspect data points suggested that results may have been heavily influenced by inconsistent andincorrect measurement techniques. Ferrous sulphide ring formation was only observed in the 40°Cstored eggs, with all cold-stored eggs showing yellow yolk exteriors regardless of heat treatment.This suggests that egg storage temperature is a strong determinant of ferrous sulphide formation inheat-treated eggs.12References:1. Iannotti LL, Lutter CK, Bunn DA, Stewart CP. Eggs: The uncracked potential for improvingmaternal and young child nutrition among the world’s poor. Nutrition Reviews.2014;72(6):355-68.2. McWilliams M. Foods Experimental Perspectives (7th ed.). 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