Rapid Communication Estimation of egg freshness using S-ovalbumin as an indicator Q. Huang,*†1 N. Qiu,†1 M. H. Ma,†2 Y. G. Jin,† H. Yang,† F. Geng,† and S. H. Sun*† *Institute of Food Science, Jishou University, Jishou 416000, China; and †National R&D Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China ABSTRACT The aim of this research was to study Sovalbumin as a reference index for the freshness of commercial shell eggs in terms of equivalent egg age. The Sovalbumin content, yolk index, albumen pH, and Haugh units were determined at the storage temperature of 25 and 37°C, respectively, using 85 fresh-laid eggs. A correlation analysis showed a high correlation coefficient of S-ovalbumin content to storage time as well as to the
3 frequently used freshness indices (Haugh unit, yolk index, and albumen pH). Furthermore, a prediction model of equivalent egg age at 25°C was established using S-ovalbumin content as an index on the basis of the correlation analysis. This study confirmed the possibility of using S-ovalbumin as a reference index to express commercial shell egg freshness as equivalent egg age.
Key words: S-ovalbumin, egg freshness, equivalent egg age, Haugh unit 2012 Poultry Science 91:739–743 http://dx.doi.org/10.3382/ps.2011-01639
INTRODUCTION
leneuve, 1994) and albumen pH that is determined by the release of carbon dioxide (Dutta et al., 2003). Some successful studies, such as furosine analysis, viscosity measurements, yolk biogenic, visible and near infrared, and spectroscopy, had also been used as new estimation indices (Hidalgo et al., 2006; Karoui et al., 2006; Nicolas et al., 2009; Ramos et al., 2009). Ovalbumin, which constitutes about 55% of the total proteins in a newly deposited egg, is the most abundant protein component in shell eggs (Nakamura and Ishimaru, 1981; Huntington et al., 1995; Mellet et al., 1996). During storage, ovalbumin is gradually converted into S-ovalbumin, an irreversible and extremely heat-stable form compared with ovalbumin, determined by a thermogram of the differential scanning calorimeter (Smith and Back, 1965; Huntington et al., 1995). The formation of S-ovalbumin is affected by both pH and temperature. The S-ovalbumin content increases from 18% of the egg white to 86% after 6 mo of refrigerated storage (Smith and Nguyen, 1984; Alleoni and Antunes, 2004). Some most commonly used quality parameters are closely related to breed, hen age, and nutritional status, and vary with the change of these factors (Scott and Silversides, 2000; Karoui et al., 2006). But S-ovalbumin, as an independent characteristic not varied from these factors and keeping to similar levels after the egg was laid, showed high repeatability and low natural variability in fresh eggs. Hence, it is a promising and significant shell egg freshness index when determined in albumen (Masaaki, 2005). The objective of this study was to evaluate S-ovalbumin as a reference index for the quantitative estimation of the shell egg freshness in terms of equivalent egg age,
The chicken egg is one of the most important sources of protein in the human diet because it is affordable and nutritious. The internal quality, when related to the functional characteristics of an egg, declines with storage time. The degradation in quality results in some unsatisfactory results associated with the chemical, functional, nutritional, and hygienic changes due to storage (Scott and Silversides, 2000; Silversides and Scott, 2001). These complex changes that occur in shell eggs during storage include egg-white thinning, increasing pH, weakening and stretching of the vitelline membrane, increasing of water content in the egg yolk, as well as changes in protein conformation (Hisil and Ötles, 1997; Hammershoj et al., 2002; Silversides and Budgell, 2004; Hidalgo et al., 2006; Karoui et al., 2006). Most shell eggs in the Chinese market are fresh eggs without any treatments and must be sold and used by 21 and 28 d, respectively, after being laid. Thus, the estimation of egg freshness is particularly important, given that the egg quality cannot be simply defined only by egg age because it varies with time as a function of storage temperature and humidity. The most commonly used indices for evaluating egg freshness are Haugh unit (HU) that is influenced by hen age (Williams, 1992; Silversides et al., 1993; Silversides and Vil-
©2012 Poultry Science Association Inc. Received May 28, 2011. Accepted September 26, 2011. 1 Both authors contributed equally to this paper. 2 Corresponding author:
[email protected]
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and thus, to establish a more ideal method for freshness estimation of commercial shell eggs.
MATERIALS AND METHODS Experimental Design Two experiments were carried out on hen eggs. In total, 85 newly laid eggs of Hy-Line Brown Plus hens of the same flock, weighing between 54 and 62 g, were selected for these experiments. In experiment 1, 50 collected eggs were stored under controlled conditions at 25°C and 65% RH for 27 d to observe the variation trends of HU, yolk index, albumin pH, and S-ovalbumin formation. Five eggs were analyzed per 3 d, and any sample was tested in triplicate. In experiment 2, another 35 collected eggs were stored at 37°C and 55% RH for 12 d. Five eggs were analyzed per 2 d.
To determine HU, each egg was weighed (± 0.01 mg) and broken carefully on a flat surface. The albumen height was measured using a mounted digital Vernier Caliper (Marathon Watch Company Ltd., Richmond Hill, ON, Canada), and the HU was determined using the following formula: HU = 100 log(h − 1.7w 0.37 + 7.6), [1]
where h is the observed height of albumen in millimeters and w is the weight of the egg in grams. The yolk index was determined by measuring the width of the yolk with dial calipers and the height of the yolk with a standard tripod micrometer. The measurements were taken with the yolk in the natural position when the egg was broken out:
Yolk index = height of yolk/width of yolk.
Statistical Analyses Descriptive statistics, 2-way ANOVA, and exponential equation analysis were all performed with SPSS for Windows, v. 15.0 (SPSS, Chicago IL).
RESULTS AND DISCUSSION Formation Kinetics of S-Ovalbumin
Analytical Methods
After resting for 10 min, the tubes were centrifuged at 10,733 × g for 5 min at 4°C and the supernatant was obtained. Two milliliters of the supernatant was placed in a test tube with 4 mL of Biuret solution added. This supernatant aliquot was left to rest for 30 min, and the absorbance was determined at 540 nm using a Beckman DU-70 spectrophotometer (Fullerton, CA). The results are expressed as a percentage of S-ovalbumin in the total amount of ovalbumin.
[2]
After separating the albumen from the yolk, the albumen pH was determined using a pH meter (Accumet Basic AB15 pH meter, Fisher Scientific, Hampton, NH). The pH meter was calibrated using buffer solutions at pH 7 and 10. The S-ovalbumin content of the egg white was measured using the method described by the literature (Smith and Nguyen, 1984; Alleoni and Antunes, 2004). Five grams of egg whites was placed in a 100-mL beaker, and 25 mL of 0.5 mol/L phosphate buffer (pH 7.5) was added; the mixture was agitated for 5 min with a magnetic stirrer. Afterward, 5 mL of the suspension was placed into 2 test tubes; one of the tubes was heated at 75°C for 30 min. Upon cooling, 5 mL of the precipitating solution was added in each test tube, and the solution was transferred to centrifuge tubes with the addition of another 5 mL of precipitating solution.
Rather than a chemically modified derivative, S-ovalbumin is a conformational isomer of native ovalbumin. Spectroscopic studies indicated that the conformational change is very limited and involves a small alteration in the secondary structure, without a major alteration in the overall folding of the protein (Huntington et al., 1995; de Groot et al., 2007). Because ovalbumin is transformed into S-ovalbumin irreversibly during prolonged storage of shelled eggs and the formation velocity is only affected by pH and temperature, it was chosen as the reference index to express the freshness of the commercial shell eggs in the equivalent age (Smith and Back, 1965; Masaaki, 2005; de Groot et al., 2007). In this study, S-ovalbumin formation kinetics at 25 and 37°C were computed respectively by interpolation of the S-ovalbumin content obtained in 2 different storage assays using 2 lots of shell eggs laid by hens of the same age and breed. It can be seen from Figure 1 that there was a confirmed rapid increase in S-ovalbumin content after storage at both 25 and 37°C, and the formation velocity at 37°C was significantly higher than that at 25°C. The initial average value of S-ovalbumin content in all examples was 14.42% in the albumen. During storage, the S-ovalbumin content reached up to 91.86% after 27 d at 25°C and 91.24% after 12 d at 37°C, and henceforth remained stable. The result indicated that high temperature could accelerate the S-ovalbumin formation, which was in agreement with the former report (Alleoni and Antunes, 2004). Remarkably, these results showed a high correlation coefficient between S-ovalbumin content and storage time at a given temperature, and Rsquare values were 0.9477 and 0.9060 at 25°C and 37°C, respectively. A significant difference (P ≤ 0.05) was observed in these 2 experiments. Hence, S-ovalbumin was considered as a feasible novel indicator to evaluate shell egg freshness.
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Figure 1. Relationship between storage time and S-ovalbumin content from experiment 1 and experiment 2 eggs stored at 25 and 37°C, respectively (linear regression with 95.00% individual prediction interval; R2 = 0.9477 for experiment 1 and 0.9060 for experiment 2). Color version available in the online PDF.
Figure 2. Relationship between S-ovalbumin content and yolk index from experiment 1 and experiment 2 eggs stored at 25 and 37°C, respectively (linear regression with 95.00% individual prediction interval; R2 = −0.9160 for experiment 1 and −0.9538 for experiment 2). Color version available in the online PDF.
Correlation Analysis
at 25°C in storage (R-square = 0.97531). Because the Pearson correlation indicates the prediction capacity of one variable from the other by using a linear equation (Wilkinson et al., 1992), the best correlation coefficient observed for S-ovalbumin may be a consequence of the linear kinetics patterns of these variables. All of the other freshness parameters (yolk index, HU, and albumen pH), which have curvilinear kinetics, are strongly correlated to each other. The results and analysis from the present investigation demonstrated that it was feasible to use S-ovalbumin as a novel egg freshness indicator.
Considering that HU, albumen pH, and yolk index are the most commonly used indices to evaluate egg freshness, the correlation coefficient between S-ovalbumin content and these egg freshness parameters was investigated for analyzing the feasibility of using S-ovalbumin as a novel egg freshness indicator. Yolk index, albumen pH, HU, and S-ovalbumin content of 2 lots of 85 fresh samples were analyzed and illustrated in Figures 2, 3, and 4, and the correlation coefficient between S-ovalbumin content and the other 3 freshness parameters is shown in Table 1. A wide variation for all 3 parameters was observed during egg storage at the same conditions and time. It is suggested that egg age, expressed as days from laying, is not sufficient to express real egg freshness. It could be concluded from the results (shown in Figure 2–4) that S-ovalbumin content had a high negative correlation with yolk index and HU, and it had a high positive correlation with albumen pH. All of the R-square values were greater than 0.9160. It can be concluded that S-ovalbumin not only had satisfactory correlation but that the variance range was also compatible with HU, implying that S-ovalbumin may be a potential indicator for evaluating commercial egg freshness. The results of the correlation analysis (Table 1) evidenced a highly significant correlation (P ≤ 0.001) between storage time and S-ovalbumin content, but there was only a significant correlation (P ≤ 0.05) between storage time and yolk index, HU, as well as albumen pH. Furthermore, a highly significant correlation (P ≤ 0.001) between S-ovalbumin content and all of the other 3 freshness parameters was also observed, especially for the S-ovalbumin content correlation vs. HU
Figure 3. Relationship between S-ovalbumin content and albumen pH from experiment 1 and experiment 2 eggs stored at 25 and 37°C, respectively (linear regression with 95.00% individual prediction interval; R2 = 0.9180 for experiment 1 and 0.9573 for experiment 2). Color version available in the online PDF.
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Huang et al. Table 1. Correlation coefficients between egg freshness parameters measured on the shell egg samples Stored condition
Index
25°C
Storage days S-ovalbumin Yolk index Albumen pH Storage days S-ovalbumin Yolk index Albumen pH
37°C
S-ovalbumin
Yolk index
Albumen pH
Haugh unit
0.95887** — — — 0.91924* — — —
−0.98660** −0.91602** — — −0.97083** −0.95377** — —
0.82371* 0.91802** −0.75275* — 0.87557* 0.95729** −0.93839* —
−0.99602** −0.97531** 0.97650** −0.84688* −0.97903** −0.96833** 0.99655** −0.94070*
*P ≤ 0.05; **P ≤ 0.001.
Estimation of Equivalent Egg Age Considering that a highly significant correlation was observed between S-ovalbumin content and storage time at 25°C, and the experimental circumstance of experiment 1 was more similar to that in Chinese markets, the data from experiment 1 was used to establish a prediction model of equivalent egg age at 25°C to estimate the freshness of commercial shell eggs stored in any conditions during its shelf life. Equivalent egg age was estimated using S-ovalbumin content as a variable through an exponential equation analysis (Figure 5). The general formula for the estimation equation was as follows:
y = ae
−
x b
+ c, [3]
where y is the equivalent egg age of a shell egg, and x is the S-ovalbumin content. The exponential equation was x
y = 1.9256e 34.58751 − 1.4208, [4]
where R = 0.96685 and P < 0.01.
Figure 4. Relationship between S-ovalbumin content and Haugh units from experiment 1 and experiment 2 eggs stored at 25 and 37°C, respectively (linear regression with 95.00% individual prediction interval; R2 = −0.9753 for experiment 1 and −0.9683 for experiment 2). Color version available in the online PDF.
The above statistical analyses showed that the prediction model of equivalent egg age was feasible and precise. The egg freshness could be approximately predicted by converting S-ovalbumin content of commercial shell egg stored in any conditions into equivalent egg age at 25°C.
Conclusion In this study, the first step was the estimation of shell egg freshness on the basis of S-ovalbumin content. To evaluate S-ovalbumin as a reference index for the prediction of the freshness of commercial shell eggs in terms of equivalent egg age, the formation kinetics of S-ovalbumin in albumen showed that there was a high correlation coefficient between S-ovalbumin content and storage time at a given temperature. A correlative analysis revealed a high negative correlation of S-ovalbumin content to yolk index and HU, and it revealed a high positive correlation of S-ovalbumin content to albumen pH. Moreover, a highly significant correlation of storage time to S-ovalbumin content, yolk index, and HU, as well as S-ovalbumin content to all of the other 3 freshness parameters were evidenced. In addition, a prediction model of equivalent egg age at 25°C was established, which could be used to predict shell egg freshness by converting S-ovalbumin content of
Figure 5. Prediction model of equivalent egg age at 25°C during shelf life. Color version available in the online PDF.
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the commercial shell eggs stored in any conditions into equivalent egg age at 25°C. These studies will contribute toward further analyses between egg content variation and freshness evaluation, which will shed light on the effect of protein conversion or degradation on the deteriorative process of shell eggs.
ACKNOWLEDGMENTS This work was financially supported by the Chinese National Natural Science Funds (Grant No. 31101366) and the earmarked fund for Modern Agro-industry Technology Research System (Project No. nycytx41-g22).
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