Romdhane Karoui · Bart Kemps · Flip Bamelis · Bart De Ketelaere · Kristof Merten · Robert Schoonheydt · Eddy Decuypere · Josse De Baerdemaeker Development of a rapid method based on front-face fluorescence spectroscopy for the monitoring of egg freshness: 2—evolution of egg yolk Received: 7 August 2005 / Revised: 6 October 2005 / Accepted: 17 October 2005 / Published online: 20 January 2006 C  Springer-Verlag 2005 Abstract This preliminary study is devoted to the application of front-face fluorescence spectroscopy to the study of egg yolks during storage. A total of 79 eggs stored for 1, 2, 3, 4, 5, 9, 10, 12, 16, 18, 23, 25 and 29 days at room temperature were analysed. The fluorescence emission spectra of tryptophan residues (excitation: 290 nm; emission: 305–430 nm) of proteins and the excitation spectra of vitamin A (emission: 410 nm; excitation: 270–350 nm) were recorded directly on egg yolk samples. Factorial discriminant analysis (FDA) was used to classify the eggs according to their date after they were laid. Using tryptophan fluorescence spectra, correct classification was observed for 57.1 and 51.9% for the calibration and the validation sets, respectively. Better classification (94.9 and 91.4% of the calibration and validation samples, re- spectively) was obtained from the vitamin A fluorescence spectra. The first five principal components (PCs) of the principal component analysis (PCA) extracted from each data set (tryptophan and vitamin A fluorescence spectra) were pooled (concatenated) into a single-matrix and analysed by FDA. Correct classifications were obtained for 97.5% of the calibration and 96.3.1% of the validation spectra. The discrimination of the investigated egg yolks according to their storage time was excellent. It was concluded that the concatenation of different fluorescence spectra might be considered as a promising indicator of shell egg freshness when they are used in egg products. R. Karoui () · B. Kemps · F. Bamelis · B. De Ketelaere · K. Merten · E. Decuypere · J. De Baerdemaeker Egg Quality and Incubation Research Group, K.U. Leuven, Kasteelpark Arenberg 30, B-3001 Leuven, Belgium e-mail: Romdhane.Karoui@biw.kuleuven.be Tel.: +32(0)16321470 Fax: +32(0)16328590 R. Schoonheydt Centre for Surface Chemistry and Catalysis, K.U Leuven, Kasteelpark Arenberg 23, B-3001 Leuven, Belgium Keywords Yolk freshness . Tryptophan . Vitamin A . Chemometry Introduction Immediately after being laid, aging processes begin in shell eggs, altering their chemical, physical, and functional char- acteristics [1]. These changes depend on the storage condi- tions time, temperature and relative humidity are the main factors influencing the change in the components of the eggs [2]. At this moment, the only quantitative parame- ter for egg freshness evaluation considered by the Euro- pean Union regulation [3] is air chamber size height as an index for the commercial classification of eggs. Taking into consideration this parameter, eggs are classified ac- cording to three categories of freshness: A extra ≤4 mm, 4 mm <A≤6 mm, 6 mm <B≤9 mm. United States grad- ing requirements [4] also consider thick albumen height, expressed as Haugh Units. However, the use of these two indices for the evaluation of egg freshness is questionable because air cell height also depends on egg weight and storage humidity [5, 6], and the Haugh Units also depend on the laying-hen’s age [5, 7–9]. Recently, some authors have proposed furosine, pyrog- lutamic acid and uridine as indices of fresh egg shell [1, 10–15], especially when they are evaluated on the albumen. However, these indices seem not to be valuable when they are used on the egg yolk. Indeed, the usage of the param- eters, the high natural variability of pyroglutamic acid and uridine in the yolk and the low level of furosine at refriger- ation temperature limit, as indices to evaluate the freshness of the raw material utilised for yolk products [13]. It is well known that the vitelline membrane that sur- rounds the yolk plays a further role in egg quality [16]. Romanoff and Romanoff [17] reported that during egg stor- age, the increase in water content of the yolk, caused by osmotic migration from the albumen, stretches the vitelline membrane and flattens the yolk. The tendency to flatten