See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/326811665

Assessment of poultry eggs freshness using FTIR spectroscopy combined with

HCA and PCA methods

Article · June 2018

DOI: 10.5604/01.3001.0012.1414

CITATIONS READS

0 23

2 authors, including:

Rafał Kurczab
Polish Academy of Sciences
85 PUBLICATIONS   368 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

low basicity 5-HT7 agonists View project

Quantum methods used in molecular modelling View project

All content following this page was uploaded by Rafał Kurczab on 11 August 2018.

The user has requested enhancement of the downloaded file.

Assessment of poultry eggs freshness using FTIR
spectroscopy combined with HCA and PCA methods
Ewelina Michalczyka, Rafał Kurczaba,*
a
State Higher Vocational School in Tarnów, Mickiewicza 8, 33-100 Tarnów, Poland

Article history: Abstract

Received 6 June 2018 The main aim of this study was to investigate the use of Attenuated Total Reflection Fourier
Received in revised form Transform Infrared Spectroscopy (ATR FTIR) and selected chemometric methods to classify
21 June 2018 eggs in terms of the laying hen farming method, as well as to identify changes in the individual
Accepted 23 June 2018 egg compositions during storage. In total, 50 eggs were used for the study; 10 eggs per classes:
Available online 27 June 2018 0, 1, 2, 3 and rural. Eggs were stored by 29 days period, which was divided on the 10 measuring
days in which one egg from each class was tested by recording two FTIR spectra for the shell,
albumen and egg yolk. The chemometric analysis, including Hierarchical Cluster Analysis
(HCA) and the Principal Component Analysis (PCA), was performed based on the recorded
FTIR spectra. Changes in chemical composition during the experiment in individual egg ele-
ments were analyzed. Furthermore, by analyzing the graphs (HCA and PCA) obtained by the
chemometric analysis, it was noted that the largest changes in the chemical composition of eggs
occurred in the shell and yolk, while in the albumen it was less insignificant. The chemometric
analysis of the recorded spectra also showed that combination of chemometric methods and
FTIR spectroscopy can potentially be used to develop a non-destructive method for classifying
eggs in terms of the hen culture method and to monitor of their freshness.

Key words: egg freshness, FTIR spectroscopy, chemometrics

Introduction pounds. The fact that this parameter shows high repeatability,
low natural variability in fresh eggs and independence from the
Freshness and quality of eggs are very important for the health above-mentioned factors allows for an objective assessment of
of consumers [1]. Changes occurring in eggshell, egg yolk and the quality and freshness of eggs [2].
egg white (albumen) during storage are very complex and have Non-destructive methods are very important for the industry,
a  significant impact on the functional properties of eggs [2]. which allow for unambiguous and quick assessment of the fresh-
They are caused by gas exchange occurring through the pores of ness and quality of eggs, without damaging them. The quality of
the shell and by the osmotic exchange taking place through the eggs can be examined by the use of infrared spectroscopy. The
yolk membrane between the protein and yolk [3]. advantage of using this method is that it enables non-destruc-
The decreasing quality of eggs is evidenced by weakening of tive and multi-parameter measurement in a short time [2]. An
the shell [1], weight loss, increase in the height of the air cell, important method allowing to test egg quality and freshness is
increase of the pH of the protein and its liquefaction, etc. [3]. Fourier transform infrared spectroscopy (FTIR - Fourier Trans-
The egg freshness can be assessed by destructive and/or non-de- form Infrared Spectroscopy) [1]. It allows getting information
structive methods [4]. Among destructive methods the sensory about the structure of proteins, the interaction between them and
evaluation (involving determine the quality of eggs based on other compounds. Another type of spectroscopy which can be
the senses of sight, taste, touch and smell) [2], the Haugh Units used to assess the quality of eggs is fluorescence spectroscopy,
method (which combine the weight of an egg with the height of which has a higher sensitivity compared to other spectroscop-
protein after it breaks down on a flat surface) [4], and by meas- ic methods. The fluorescent properties of aromatic amino ac-
uring the pH (for freshly laid eggs is in the range of 7.6–8.5 and ids that form proteins can be used to determine the structure of
increases over time) can be distinguished. An indicator of egg proteins and the interactions that occur between them. Infrared
freshness, independent of the weight of eggs, the relative humid- spectroscopy and fluorescence spectroscopy have the greatest
ity of storage and the age of the hen, is present in the furosine potential to assess the freshness of eggs. Based on the data ob-
protein, produced during the acid hydrolysis of Amadori com- tained through the application of these methods, it is possible to
create a mathematical predictive model of freshness prediction
*Corresponding author: r_kurczab@pwsztar.edu.pl of an unknown batch of eggs [2].

www.stijournal.pl Sci, Tech. Innov., 2018, 2 (1), 7-12

8 Science, Technology and Innovation Original Research

The aim of this paper was to evaluate the possibility of us- egg to minimize the inaccuracy related to sample heterogeneity.
ing FTIR ATR spectroscopy and selected chemometric meth- Each egg was broken and placed in a  beaker. To obtain an
ods (i.e. HCA and PCA) for the problem of egg classification in albumen spectrum a small amount of sample was taken with
terms of chicken farming methods and to determine changes in a needle and syringe and placed on the ATR plate. For the egg
eggs during storage. yolk sample, a new syringe was used to avoid mutual contam-
ination of albumen and yolk samples. The spectrum of the egg
Materials and Methods shell was made by breaking off a small fragment of it and plac-
ing on the ATR plate in a way that the outer side of the shell
Samples adheres to the surface of the crystal. In total, 30 spectra were ob-
Tests were carried out on L-large grade A shell eggs purchased tained during one measurement day, while 300 spectra were re-
at the local grocery store (TESCO), representing 4 different rear- corded during the whole experiment. A SpectraGryph program
ing methods (Table 1), and one purchased at the local small-scale was used to visualize and transform the recorded spectra [5].
family farm. For each rearing method, measurements were con-
ducted on 10 samples of eggs, each after storage at constant con-
ditions (i.e. at a temperature about 20°C, and at constant humidity
and light exposition) for 1, 3, 8, 10, 12, 15, 17, 22, 24 and 29 days.

Table 1. Characterization of the tested egg classes. The origin of egg classes was determined based on the codes presented on the
shell of eggs purchased.

Rearing method Class Number Code Origin Expiration dateb

0 (organic) A 10 0-PL 26121311 świętokrzyskie, district staszowski 11.03.2017

1 (free range) A 10 1-PL 30211341 wielkopolskie, district poznański 14.03.2017

2 (barn)a A 10 2-PL 30291366 wielkopolskie, district wolsztyński 17.03.2017

3 (cage) A 10 3-PL 30251340 wielkopolskie, district średzki 24.03.2017

small-scale family farm N.A. 10 N.A. małopolskie, district dąbrowski N.A.

a 
i.e. the deep litter indoor housing,
b
the experiment started at 04.03.2017 (the first measuring day).

Data collection Chemometric analysis

A Thermo Scientific Nicolet iS5 FTIR Spectrometer was used. The data of FTIR spectra is a set of multiple variables (wav-
The equipment included an accessory with a ZnSe single-re- enumbers and absorbance), which contains overlapping infor-
flexion ATR crystal (provided an angle of incidence of 45˚). As mation. Multivariate data analysis makes possible to extract
a reference, the background spectrum of air was collected before useful information from original spectral data, eliminate much
the acquisition of the sample spectrum. After each sample, the overlapping information, and reduce the dimension of data. In
crystal was rinsed first with a 98% ethyl alcohol solution, next order to perform chemometric analysis, recorded spectra have
with distilled water (to avoid denaturing egg samples), and then been encoded according to the following algorithm: the first two
dried with a soft tissue. Spectra were recorded with a resolution symbols denoted the class of egg (i.e. k0 – class 0, k1 – class
of 2 cm-1, and 32 scans were averaged for each spectrum (scan 1, k2 – class 2, k3 – class 3, kw – from a local farm), the next
4000–650 cm-1). digit in the label means the measurement day, the next symbol
The measurement consisted of recording FTIR spectra of the indicated the type of material of egg being tested (i.e. s – egg
outer part of the egg shell, albumen and egg yolk representing shell, b – albumen, and z – yolk), and the last symbol denot-
each of the tested classes of eggs. For each egg, the spectra were ed the measurement number of the tested material (1 – the first
collected in duplicates, taking a new sample of each part of the measurement, 2 – the second measurement). The chemometric

Sci, Tech. Innov., 2018, 2 (1), 7-12 www.stijournal.pl

E. Michalczyk, R. Kurczab | Assessment of poultry eggs freshness using FTIR spectroscopy combined with… 9

analysis was performed using the RStudio [6] environment, by bands appeared at the 2900 and 2850 cm-1 wavenumbers can be
means of the ChemoSpec library [7]. As a result of the analysis, associated to the stretching vibrations of the C-H group in lipid
the Hierarchical Clustering Analysis (HCA) and Principal Com- molecules. The band at the region of 1750 cm-1 can be connected
ponent Analysis (PCA) plots were obtained. The HCA analysis with stretching vibrations of the C= O group of saturated aliphat-
was performed using the complete clustering method and Eu- ic esters. In the areas of ​​1650 and 1550 cm-1, there are two amide
clidean distance measure. bands (respectively: amide I and II) associated with vibrations
of peptide bonds of proteins. During the experiment, the largest
Results and Discussion changes in spectra are visible in the area of the stretching vibra-
tions of O-H group and amide A band. Changes in the intensity
Spectra analysis of the amide I and II bands may indicate a breakdown of peptide
The aligned FTIR spectra of egg shell, albumen and yolk for all bonds, and thus an increase of free amino acids in egg yolks.
studied egg classes on day 1 were shown in Figure 1. The egg
shell spectra (Figure 1A) are characterized by the presence of
a wide and intense band in the 3700–3000 cm-1 range originating
from the O-H stretching and N-H stretching vibrations of the
peptide bond (amide A band). The two bands at 1650 and 1550
cm-1 wavenumbers are connected with vibrations of the amide
I  protein groups (combination of C=O  stretching, N-H defor-
mation, and C-N deformation vibrations) and amide II (C-N
stretching, N-H deformation), respectively. The band appearing
at approximately 1400 cm-1 is associated with the presence of
carbonate groups in the egg shell (in calcium carbonate). The
band in the 1170–990 cm-1 region may come from the polysac-
charides contained in the cuticle covering the egg shell. During
the experiment, the largest changes in FTIR spectra of egg shells
of all classes occurred in the areas of (Figure 2): O-H stretching
group (mainly from water), an amide
​​ A, amide I, amide II, and
bands coming from vibrations of carbonate and polysaccharides
groups. Changes in the intensity of the peak originating from
the O-H stretching vibrations in water indicated that over time
the water content in the egg shell decreases which is related to
gas exchange processes and storage conditions. Changes in the
intensity of the remaining bands may result from the degrada-
tion or formation of the compounds forming the shell, as well as
from the inhomogeneity of the material tested.
The FTIR spectra of albumen contain three bands at the wav-
enumbers identical to water spectra [8]. In the range 1550–1500
cm-1 of the FTIR spectra of albumen (Figure 1), a narrow band
with low intensity, and partially covered by a water band is vis- Figure 1. Alignment of the FTIR ATR spectra of all egg classes measured
ible. Due to the fact that in an egg albumin, apart from a water, at the beginning of the experiment (day 1) for egg shells, albumen, and yolk,
respectively
contains proteins, therefore it can be assumed that this band
comes from C-N stretching vibrations and bending of N-H pep-
tide bond (amide II band) [9]. Moreover, it can be concluded that Evaluation of egg classification possibilities based
on spectroscopic measurements
during the storage the water content in the albumen decreases,
which can be caused by gas exchange through the porous shell, The chemometric analysis was performed using the full FTIR
as well as the diffusion of water into the yolk through the vitel- range (4000–650 cm-1). At first, the analysis was focused on dis-
line membrane. crimination properties for egg classes using FTIR spectra of an
The FTIR spectra of egg yolk (Figure 1) also show many sim- individual part of the eggs (i.e. shell, albumen, and yolk) at the
ilarities between the studied egg classes. The wide band in the beginning of the experiment (i.e. day 1). The second analysis
3700–3000 cm-1 range comes from the O-H stretching vibration was performed using FTIR spectra recorded for all measure-
of water molecules, and also from the N-H stretching vibrations ment days and was aimed to illustrate the dynamics of changes
of peptide bonds (amide A  band) [9]. In the yolk spectrum, of individual egg samples during aging.

www.stijournal.pl Sci, Tech. Innov., 2018, 2 (1), 7-12

10 Science, Technology and Innovation Original Research

The HCA plot (dendrogram) obtained for egg shells (Figure 2) cause they form a separate branch in the graph. In addition, it
indicated that egg class 1 and 2 were the most similar from the can be seen that class 0 had a small heterogeneity because their
remaining ones because they originate from the same branch of spectra for measurement 1 and 2 have been classified into dif-
the dendrogram. In addition, it can be seen that the egg shells of ferent branches. The fact that the branch representing egg class
class 0 and 3 are not chemically homogeneous, because the first 1 and 2 eggs is divided into two groups at a  distance slightly
and second measurement of their spectrum have been classified higher than 0 shows that the egg albumens of these classes are
into two different branches, which indicates differences in the very similar to each other. Identical conclusions can be drawn
spectra between the first and the second measurement, as well on the basis of PCA plot analysis that albumen of particular egg
as the similarity of the egg shells belonging to these classes. The classes do not show significant differences in chemical compo-
shells of eggs from the local farm showed the highest similarity sition, however, by using chemometric analysis it is possible to
to shells of class 0 and 3 tested during the second measurement. recognize the individual classes.
Similar conclusions can be drawn from the analysis of the PCA When it comes to the HCA plot generated on the FTIR spectra
plot for egg shells (Figure 2), where the five main clusters can be of egg yolks, it can be concluded that class 0 is the least simi-
indicated. The identity of the spectra obtained for egg shells of lar in chemical terms to the remaining classes (forms a separate
class 1 caused there overlap, therefore, the cluster representing branch). Interestingly, egg yolks of rural and class 3, despite
this class was illustrated as one point. the fact that they originate from one branch, were also classi-
For albumen, the dendrogram (Figure 2) showed that rural fied into separate groups, which indicates the differences in the
egg had the lowest similarity to the remaining egg classes be- chemical composition of egg yolks of these classes. The fact that

Figure 2. The HCA dendrograms (Euclidean distances are marked on the Y-axis) and PCA plots obtained for
egg shell, albumen and yolk individually based on the FTIR spectra measured at the beginning of the experiment
(i.e. day 1). For each egg fragment, two spectra were recorded

Sci, Tech. Innov., 2018, 2 (1), 7-12 www.stijournal.pl

E. Michalczyk, R. Kurczab | Assessment of poultry eggs freshness using FTIR spectroscopy combined with… 11

the branch of egg yolk of class 1 and 2 branches off at a distance occurred in egg yolk class 1–3 (points corresponding to the egg
slightly higher than 0, indicated that the egg yolks of these class- yolks of these classes also showed a clear dispersion).
es are the most chemically similar to each other. The spectra In summary, during storage of eggs, the prominent change in
of egg yolk classes 1 and 2 from measurement 1 were classi- chemical composition occurred in egg shells and yolks. The low-
fied to a different branch, then the spectra of egg yolk classes est area of ​​variable spaces occupy points representing the albu-
1 and 2 from measurement 2, which may indicate similarity men of all classes during the experiment, which indicates a small
in the chemical composition of the tested samples, as well as change in their chemical composition during the experiment.
the heterogeneity of egg yolks of these classes. The same con-
clusions can be drawn from the PCA plot for egg yolks, where Conclusions
five clusters (out of which three collected egg yolks of the same
classes) were distinguished. The egg yolks of class 1 and 2 from It can be concluded that there is a possibility of classification of
the first measurement and the egg yolks of these classes from eggs in terms of the chicken farming method using a combina-
the second measurement have been grouped into two separate tion of FTIR spectroscopy and chemometric methods. Despite
clusters located close to each other. This may indicate that the small differences in recorded spectra between individual ele-
egg yolks of these classes show a high similarity in terms of ments of all egg classes, it is possible to assign eggs to individ-
chemical composition. ual classes, both on the basis of measurements of the egg shell
To determine the dynamics of changes occurred in eggs dur- spectra as well as albumen and yolk. The chemometric analysis
ing 29 days of storage, the PCA plots for all recorded FTIR spec- used to monitor the dynamics of changes in chemical composi-
tra were generated (Figure 3). In general, the results indicated tion occurred in eggs during storage showed that the changes in
that the major changes in the chemical composition occurred for albumen are the least visible, while the largest ones occurred in
egg shells of class 0 and 3 (they showed the greatest dispersion shells and yolks. It means that a non-destructive discrimination/
in variables space). At the same time, it can be stated that the classification model of the egg freshness assessment based on
smallest changes occurred in egg shell of class 1, 2 and rural the measurement of egg shell FTIR spectra may be generated. Of
eggs – the corresponding points are closely related, only a few course, a broader experiment (e.g. containing more egg classes,
showed significant differences, which resulted from inhomoge- more spectra measurement, and application of machine learning
neity of samples. The points corresponding to the albumen of algorithms) have to be carefully planned and performed.
individual classes formed a dense group (with only a few ex- It is also worth noting that despite the fact the tested eggs of
ceptions) which indicated that during the experiment changes in class 0–3 had different expiration periods, and during the exper-
the chemical composition of albumen were not significant. This iments, no significant changes in the spectra of individual egg
conclusion is consistent with the spectra of proteins of particular fragments were noticed after exceeding the asset date of con-
classes during the experiment indicates that the largest changes sumption. This outcome indicates that duration of the further
in the chemical composition occurred in class 0 and egg yolk experiments should be extended to even 60 or more storing days
(purple points representing this class are the most dispersed in to catch any significant changes after the expiration days.
the space of variables, see Figure 3). Nevertheless, significant Moreover, the presented experiments show that the FTIR
changes in the chemical composition during the experiment also method in combination with the chemometric analysis allows

Figure 3. The PCA plots illustrating the distribution of the FTIR spectra of all five classes of eggs during the 29 days of storage under constant conditions
divided into egg shell, albumen, and yolk

www.stijournal.pl Sci, Tech. Innov., 2018, 2 (1), 7-12

 12 Science, Technology and Innovation Original Research

the classification of eggs (according to their classes) and may be 3. Giunchi A, Berardinelli A, Ragni L, Fabbri A, Silaghi FA. Non-
used as the basis for the development of more sophisticated tool -destructive freshness assessment of shell eggs using FT-NIR
based on the machine learning (i.e. artificial intelligence). spectroscopy, Journal of Food Engineering, 2008, 89, 142–148.
4. A
 bdel-Nour N, Ngadi M, Prasher S, Karimi Y, Prediction of
Acknowledgments Egg Freshness and Albumen Quality Using Visible/Near In-
frared Spectroscopy, Food and Bioprocess Technology, 2011,
This study was supported by the statutory funds of the State 4, 731–736.
Higher Vocational School in Tarnow, Poland. 5. Menges F, Spectragryph – optical spectroscopy software, Ver-
sion 1.0.7, 2017, http://www.effemm2.de/spectragryph/
References 6. R. Core Team (2017). R: A language and environment for sta-
tistical computing. R Foundation for Statistical Computing,
1. R
 odríguez-Navarro AB, Dominiguez-Gasca N, Muñoz A, Or- Vienna, Austria. URL https://www.R-project.org/.
tega-Huertas M. Change in chicken eggshell cuticle with age 7. Hanson BA. ChemoSpec: Exploratory Chemometrics for Spec-
and egg freshness, Poultry Science, 2013, 92, 3026–3035. troscopy, 2016. R package version 4.4.x.
2. Karoui R, Kemps B, Bamelis F, de Ketelaere B, Decuypere 8. https://webbook.nist.gov/cgi/cbook.cgi?ID=C7732185&Type-
E, de Baerdemaeker J. Methods to evaluate egg freshness in =IR-SPEC&Index=1
research and industry: A review, European Food Research and 9. Barth A. Infrared spectroscopy of proteins, Biochimica et Bio-
Technology, 2006, 222, 727–732. physica Acta – Bioenergetic, 2007, 9, 1073–1101.

Sci, Tech. Innov., 2018, 2 (1), 7-12 www.stijournal.pl

View publication stats