465292

DETERMINATION OF LIQUID EGG QUALITY AND
EFFECTS OF HEAT TREATMENT ON EGG PROTEINS
SIVI YUMURTA KALİTESİNİN VE ISIL İŞLEMİN YUMURTA

PROTEİNLERİ ÜZERİNE ETKİLERİNİN BELİRLENMESİ
REYHAN SELİN UYSAL
PROF. İSMAİL HAKKI BOYACI

Supervisor
Submitted to Graduate School of Science and Engineering of Hacettepe University

as a Partial Fulfillment to the Requirements
for the Award of the Degree of Doctor of Philosophy
in Food Engineering
2017
Every challenging work needs self-efforts as well as
encouragement, remarkable patience, emotional support by person
who makes sense of your life
I dedicate this thesis to my love and husband,
who makes sense of my life,
Engin Afacan,
ABSTRACT
DETERMINATION OF LIQUID EGG QUALITY AND EFFECTS OF

HEAT TREATMENT ON EGG PROTEINS
REYHAN SELİN UYSAL

Doctor of Philosophy, Department of Food Engineering
Supervisor: Prof. İsmail Hakkı BOYACI
March 2017, 132 pages
Egg is one of the fundamental food that has been consumed by human beings for
many years due to its high nutritional value. Besides high nutrient content, it
contributes to textural and sensory properties of food thanks to its functional
properties (foaming, emulsifying, and gelling). Determination of the egg quality is
an important concern for food industry because of the valuable position of the
liquid egg in food production. There are two major phenomena that determine the
liquid egg quality: adulteration and being damaged of the functional properties of
egg proteins by heat treatment. In this context, the first aim of this study is
detection of liquid egg adulteration with water. The other aim is to monitor the
effect of heat treatment on egg proteins, and to demonstrate the effect of liquid
egg heat-treated at different parameters on the final product quality.
As the first step of adulteration detection, yolk-white ratio of the liquid eggs was
determined by SDS-PAGE technique. Depending on the yolk-white ratio, it has
been revealed whether egg sample is adulterated or not. As the second step,
compositional analysis of liquid egg was performed by ATR-FTIR spectroscopy.
Protein, lipid, moisture, and total soluble solid contents of many egg samples were
determined. The actual values of the components were detected by reference
methods. Data analysis was performed via PLS. The actual values of the
components were compared to predicted values by the models, and determination
of coefficient values (R2) for protein, lipid, moisture, and total soluble solid were
obtained as 0.95, 0.992, 0.994, and 0.972, respectively. At last, liquid egg
adulteration with water was also analyzed by ATR-FTIR and NIR spectroscopies.
The analysis was performed using both liquid and dry samples. Data analysis was
performed via PCA. Adulterated dry samples were classified by ATR-FTIR while
both liquid and dry samples were classified by NIR spectroscopy.
i
The effect of heat treatment on egg proteins was monitored by capillary
electrophoresis and UV-VIS spectroscopy. Electrophoresis data were analyzed by
PCA. Untreated and heat-treated egg samples were separated successfully from
each other. Then, heat-treated samples were classified according to their heat
treatment parameters. As the last part, sponge cake, was selected as a final
product, and was analyzed to demonstrate the effect of heat-treated liquid eggs on
the final product quality. Rheological behavior of the cake batter prepared using
heat-treated liquid egg was investigated. It was observed that cake batter exhibits
a pseudo-plastic behavior. Rheological measurement values were fitted to a
power-law model. An increase in consistency index (K) was observed as the
extent of heat treatment was increased. Heat treatment has an effect on flow
behavior index (n) whereas any effect depending heat treatment parameters was
not observed. Fundamental cake quality analyses (moisture loss, color, porosity,
texture analysis (hardness, cohesiveness, and gumminess), and specific volume)
were performed. The baked cakes prepared with heat-treated liquid eggs were
found to have less porous and lower specific volume than untreated cakes. Heat-
treated liquid eggs resulted in the formation of harder, sticky, and gumminess
cakes.
Keywords: liquid egg, liquid egg adulteration, SDS-PAGE, ATR-FTIR

spectroscopy, NIR spectroscopy, pasteurization of egg, CE, cake quality.
ii
ÖZET
SIVI YUMURTA KALİTESİNİN VE ISIL İŞLEMİN YUMURTA

PROTEİNLERİ ÜZERİNE ETKİLERİNİN BELİRLENMESİ
REYHAN SELİN UYSAL

Doktora, Gıda Mühendisliği Bölümü
Tez Danışmanı: Prof. Dr. İsmail Hakkı BOYACI
Mart 2017, 132 sayfa
Yumurta, içerdiği yüksek besin değeri ile insanların uzun yıllar boyunca tükettiği
temel gıdalardan birisidir. Yüksek kalitede besin içeriğinin yanı sıra; fonksiyonel
özellikleri sayesinde (köpükleşme, emülsifiye edici, jelleşme gibi) pek çok gıdanın
dokusal ve duyusal özelliklerine katkı sağlamaktadır. Sıvı yumurtanın gıda
üretimindeki bu değerli konumu nedeniyle yumurta kalitesinin belirlenmesi gıda
sektörü için önemli bir ihtiyaçtır. Sıvı yumurta kalitesini etkileyebilecek nedenlerden
birisi yumurtanın tağşiş edilmesidir. Bunun yanı sıra, ısıl işlem uygulamasıyla
yumurta proteinlerinin fonksiyonel özelliklerinin zarar görmesi sıvı yumurta
kalitesini etkileyen bir diğer önemli etkendir. Bu bağlamda, bu çalışmanın ilk
amacı, sıvı yumurtanın suyla tağşişini farklı teknikler kullanarak tespit etmektir.
Diğer bir amaç ise, ısıl işlemin yumurta proteinleri üzerindeki etkisini gözlemlemek
ve farklı parametrelerde ısıl işlem görmüş yumurtaların son ürün kalitesi üzerindeki
etkisini göstermektir.
Tağşiş belirlemede ilk olarak, SDS-PAGE tekniği ile sıvı yumurtaların sarı-beyaz
oranları belirlenmiştir. Elde edilen sarı-beyaz oranları kullanılarak örneğin suyla
tağşiş edilip edilmediği ortaya çıkarılmıştır. İkinci adımda, sıvı yumurtanın
bileşimsel analizi ATR-FTIR spektroskopisi ile yapılmıştır. Çok sayıda sıvı yumurta
örneğinin protein, yağ, su ve toplam çözünür kuru madde miktarı tespit edilmiştir.
Bileşenlerin gerçek değerleri referans yöntemlerle belirlenmiştir.
iii
Veri analizi PLS ile yapılmıştır. Bileşenlerin gerçek değerleri ile modellerin tahmin
ettiği değerler kıyaslanmıştır ve protein, yağ, su ve toplam çözünür kuru madde
için belirleme katsayıları (R2) sırasıyla 0.95, 0.992, 0.994, 0.972 olarak elde
edilmiştir. Son olarak, suyla tağşiş edilmiş sıvı yumurta örnekleri ATR-FTIR ve NIR
spektroskopileri ile analiz edilmiştir. Analiz hem sıvı hem de kuru örnekler ile
gerçekleştirilmiştir. Veri analizi PCA ile yapılmıştır.
ATR-FTIR tekniği ile kuru örneklerle yapılan analizde tağşiş olmuş örnekler
başarıyla ayrıştırılmıştır. NIR spektroskopisi ile yapılan analizde ise hem sıvı hem
de kuru örneklerde tağşişli örneklerin ayrımı sağlanmıştır.
Sıvı yumurtaya farklı parametrelerde uygulanan ısıl işlemin yumurta proteinleri
üzerindeki etkileri kapiler elektroforez ve UV-VIS spektroskopisi ile takip edilmiştir.
Kapiler elektroforezden elde edilen veriler PCA ile analiz edilmiştir. Isıl işlem
görmemiş ve görmüş yumurta örnekleri başarıyla birbirinden ayrılmıştır. Isıl işlem
görmüş örneklerde ise ısıl işlem parametresine bağlı olarak ayrım
gerçekleştirilmiştir. Bu çalışmanın son aşamasında, farklı parametrelerde ısıl işlem
görmüş yumurtaların son ürün kalitesine olan etkisini incelemek için sünger kek
örnek olarak seçilmiş ve analiz edilmiştir. Bu amaçla ilk olarak, ısıl işlem görmüş
yumurtalar kullanılarak elde edilen kek hamurunun reolojik davranışı incelenmiş
olup hamurun psödoplastik akış davranışı sergilediği gözlenmiştir. Reolojik ölçüm
değerleri Power-law modeline uydurulmuştur. Isıl işlem derecesinin artmasıyla
birlikte hamurun kıvam indeksinde (K) artış gözlenmiştir. Akış davranış indeksinde
(n) ise ısıl işlemin etkisi görülmüş olup, farklı ısıl işlem derecelerinin bir etkisi
gözlenmemiştir. Isıl işlem görmüş yumurtaların kekin kalitesi üzerindeki etkileri ise
kek analizleri ile incelenmiştir. Bu analizler, kekin pişme sürecindeki nem kaybı,
renk, gözeneklilik, yapısal dağılım analizi (sertlik, yapışkanlık ve sakızımsılık) ve
özgül hacimdir. Isıl işlem görmüş yumurtalarla pişirilen keklerde ısıl işlem
görmemişlere oranla daha az gözenekli ve daha düşük özgül hacime sahip kekler
elde edilmiştir. Isıl işlemli yumurtalar daha sert, yapışkan ve sakızımsılık değerine
sahip keklerin oluşmasına neden olmuştur.
Anahtar Kelimeler: sıvı yumurta, sıvı yumurtada tağşiş, SDS-PAGE, ATR-FTIR

spektroskopisi, NIR spektroskopisi, yumurta pastörizasyonu, CE, kek kalitesi.
iv
ACKNOWLEDGEMENT
Undertaking this PhD has been a truly life-changing experience for me. During this
long period, many people supported me and I believe that I could never succeed
without their guidance and support. First, I would like to express my deepest
feelings to my husband whose endless confidence, guidance, and remarkable
patience always encouraged me. Thank you my love!
The other first gratitude goes to my lovely parents, my mother and father
Tülin&Sedat Uysal, who have always loved, cared, believed, and supported me
without any expectation during my all life. Actually, any word cannot express my
feelings. I just say that I owe them forever for everything that I have succeeded!
My brother Eren, my sister-in-law Esra, and my nephew Çınar! Thank you very
much for your support and love.
Foremost, I sincerely appreciate my supervisor Prof. İsmail Hakkı BOYACI, who
has supported and guided me with his vast knowledge and experience from the
beginning to the end. He provided all sorts of possibilities during my PhD story. I
learned lots of things from him, which will also contribute to my career in future.
I would like to thank Prof. Gülüm Şumnu, Assoc. Prof. Ali Topcu, Prof. Uğur
Tamer, and Prof. Nusret Ertaş for their help, guidence and valuable contributions. I
also thank Assist. Prof. F. Ceyda Dudak Şeker for being in my thesis jury. I am
grateful to Dr. Esra Acar Soykut, Dr. Özay Menteş Yılmaz, and Assist. Prof. Haslet
Ekşi Koçak for their valuable help and recommendations during my study.
At last, my lab colleagues, Şebnem and Ezgi, you have always been my little
sisters; thank you for your support and confidence in me. My colleagues, Kübra,
Elif, Banu, Zeynep, and Nurdan! Thank you very much girls for your sisterhood
and camaraderie. Your positive energy always motivated me during tedious
laboratory days (actually everyday). I will miss coffee breaks and tittle-tattle we
made with you. I thank also Efe Geniş for his support until this time and his effort
during our mutual work.
I would like to thank TÜBİTAK Department of Science Fellowships and Grant
Programs (BİDEB) thanks to its support within the scope of the 2211-Domestic
Doctoral Scholarship Program.
v
TABLE OF CONTENTS
Page
ABSTRACT .............................................................................................................. i
ÖZET ....................................................................................................................... iii
ACKNOWLEDGEMENT .......................................................................................... v
TABLE OF CONTENTS ..........................................................................................vi
TABLES ...................................................................................................................ix
FIGURES ................................................................................................................xi
SYMBOLS AND ABBREVIATIONS ...................................................................... xiv
1. INTRODUCTION ................................................................................................. 1
2. LITERATURE REVIEW ....................................................................................... 5
2.1. Structure and Chemical Composition of Eggs.................................................. 5
2.1.1. Egg Shell ....................................................................................................... 6
2.1.2. Egg White ...................................................................................................... 8
2.1.3. Egg Yolk ...................................................................................................... 11
2.2. Functional Characteristics of Egg in Food Systems ....................................... 16
2.2.1. Egg Functionary in Foam Systems ............................................................. 16
2.2.2. Coagulation/Gelation of Egg ....................................................................... 18
2.2.3. Egg Constituents in Emulsion System ........................................................ 18
2.2.4. Egg Importance in Baked Products ............................................................. 19
2.3. Liquid Egg Products ....................................................................................... 20
2.3.1. Pasteurization of LEPs ................................................................................ 21
2.3.2. Effects of Pasteurization on Functional Properties of LEPs ........................ 21
2.4. Analyses of Egg Components ........................................................................ 23
2.4.1. Infrared Spectroscopy ................................................................................ 24
2.4.1.1. NIR Spectroscopy .................................................................................... 25
2.4.1.2. ATR-FTIR Spectroscopy .......................................................................... 27
2.4.2. SDS-PAGE .................................................................................................. 29
2.4.3. Capillary Electrophoresis............................................................................. 31
3. MATERIAL AND METHOD ............................................................................... 33
3.1. Determination of LWE Quality ........................................................................ 33
3.1.1. Determination of Yolk-White Ratio in Liquid Egg by SDS-PAGE ................ 33
3.1.1.1. Chemicals and SDS-PAGE Solutions ...................................................... 34
vi
3.1.1.2. Preparation of Samples ............................................................................ 35
3.1.1.3. Electrophoresis ........................................................................................ 35
3.1.1.4. Analysis of Protein Bands ........................................................................ 36
3.1.2. Determination of Egg Components by ATR-FTIR Spectroscopy ................ 36
3.1.2.1. Chemicals ................................................................................................ 36
3.1.2.2. Analyses of Egg Components by Reference Methods ............................. 37
3.1.2.3. ATR-FTIR Spectroscopy Measurements ................................................. 39
3.1.2.4. Data Analysis ........................................................................................... 39
3.1.3. Detection of Egg Adulteration by ATR-FTIR and NIR Spectrometer........... 41
3.1.3.1. Preparation of Samples ............................................................................ 41
3.1.3.2. ATR-FTIR Spectroscopy Measurements ................................................. 41
3.1.3.3. NIR Spectroscopy Measurements ........................................................... 42
3.1.3.4. Data Analysis of ATR-FTIR and NIR measurements ............................... 42
3.2. Analysis of Heat Treated LWE by CE ............................................................ 43
3.2.1. Chemicals.................................................................................................... 43
3.2.2. Preparation of Samples ............................................................................... 43
3.2.3. UV-VIS Spectrophotometer ......................................................................... 44
3.2.4. CE Apparatus and Conditions ..................................................................... 44
3.2.5. Data Analysis .............................................................................................. 45
3.2.6. Statistical Analysis....................................................................................... 46
3.3. Effect of Heat Treated LWE on the Cake Quality ........................................... 46
3.3.1. Preparation of Heat Treated LWE ............................................................... 46
3.3.2. Analysis of Cake Ingredient......................................................................... 46
3.3.2.1. Protein Analysis ....................................................................................... 46
3.3.2.2. Moisture Analysis ..................................................................................... 47
3.3.2.3. The Other Analyses by NIR Spectroscopy ............................................... 47
3.3.3. Cake Batter Preparation and Baking Procedure ......................................... 47
3.3.4. Rheological Analysis of Cake Batter ........................................................... 48
3.3.5. Cake Analysis.............................................................................................. 48
3.3.5.1. Moisture Loss ........................................................................................... 48
3.3.5.2. Color Analysis .......................................................................................... 49
3.3.5.3. Porosity Analysis ...................................................................................... 49
3.3.5.4. Texture Analysis ....................................................................................... 50
3.3.5.5. Specific Volume ....................................................................................... 50
vii
3.3.5.6. Statistical Analysis ................................................................................... 51
4. RESULTS AND DISCUSSIONS ....................................................................... 52
4.1. Determination of LWE Quality ........................................................................ 52
4.1.1. Determination of Yolk-White Ratio in LWE by SDS-PAGE ......................... 52
4.1.1.1. Determination of Yolk-White Ratio in LWE Containing Egg White ........... 58
4.1.1.2. Determination of Yolk-White Ratio in LWE Containing Water .................. 60
4.1.2. Determination of Egg Components by ATR-FTIR Spectroscopy ................ 63
4.1.2.1. ATR-FTIR Spectra of Samples ................................................................ 63
4.1.2.2. Results of Reference Analyses ................................................................ 65
4.1.2.3. Data Analysis ........................................................................................... 67
4.1.3. Detection of Egg Adulteration by ATR-FTIR and NIR Spectroscopy .......... 75
4.1.3.1. ATR-FTIR Spectroscopy Analysis ............................................................ 75
4.1.3.2. NIR Spectroscopy Analysis ...................................................................... 80
4.2. Analysis of Heat Treated LWE by CE ............................................................ 87
4.2.1. UV-VIS Measurements................................................................................ 87
4.2.2. CE Electropherograms of Samples ............................................................. 90
4.2.3. PCA Analysis............................................................................................... 96
4.3. Effect of Heat Treated LWE on the Cake Quality ........................................... 99
4.3.1. Analysis of Cake Ingredient......................................................................... 99
4.3.2. Rheology of Cake Batter ........................................................................... 100
4.3.3. Cake Analysis............................................................................................ 104
4.3.3.1. Moisture Loss ......................................................................................... 104
4.3.3.2. Color Analysis ........................................................................................ 105
4.3.3.3. Porosity Analysis .................................................................................... 106
4.3.3.4. Texture Analysis ..................................................................................... 107
4.3.3.5. Specific Volume ..................................................................................... 111
4.3.3.6. Discussion of Rheology and Cake Analyses .......................................... 112
5. CONCLUSION ................................................................................................ 114
REFERENCES .................................................................................................... 117
CURRICULUM VITAE ......................................................................................... 129
viii
TABLES
Page
Table 2.1. Components of eggshell ........................................................................ 7
Table 2.2. Components of egg white ...................................................................... 9
Table 2.3. Mineral composition of the albumen .................................................... 11
Table 2.4. Vitamin composition of the albumen ................................................... 11
Table 2.5. Components of the yolk ....................................................................... 12
Table 2.6. Mineral composition of the yolk ........................................................... 15
Table 2.7. Vitamin composition of the yolk ........................................................... 16
Table 4.1. Identification of the yolk peptides from SDS-PAGE profile according to
different studies ..................................................................................................... 53
Table 4.2. Band volume of apovitellenin Va (120kDa) and ovalbumin (45kDa)
proteins .................................................................................................................. 56
Table 4.3. Actual and predicted concentration of the yolk in liquid egg samples .. 58
Table 4.4. Band volume (intensity) of apovitellenin Va and ovalbumin proteins
(120kDa and 45kDa) ............................................................................................. 59
Table 4.5. Predicted concentration of yolk in the samples containing egg white .. 60
(120kDa and 45kDa) ............................................................................................. 61
Table 4.7. Predicted concentration of the yolk in the samples containing egg white
.............................................................................................................................. 62
Table 4.8. Wavenumbers of bands of the egg samples with the assigned functional
groups and the modes of vibration ........................................................................ 64
Table 4.9. A lower, average, and upper actual content of protein (%, w/w) in LWE,
LEW, and LEY samples ........................................................................................ 65
Table 4.10. A lower, average, and upper actual content of lipid (%, w/w) in LWE,
Table 4.11. A lower, average, and upper actual content of moisture (%, w/w) in
LWE, LEW, and LEY samples............................................................................... 66
Table 4.12. A lower, average, and upper actual content of TSS (%, w/w) in LWE,
Table 4.13. The regression statistics of the calibration and validation data sets of
protein analysis and LOD-LOQ values of the method ........................................... 69
ix
lipid analysis and LOD-LOQ values of the method ............................................... 70
moisture analysis and LOD-LOQ values of the method ........................................ 72
TSS analysis and LOD-LOQ values of the method ............................................... 73
Table 4.17. The cumulative variance, RMSEC and RMSECV values of the liquid
samples PCA models ............................................................................................ 78
Table 4.18. The cumulative variance, RMSEC and RMSECV values of the models
.............................................................................................................................. 80
Table 4.19. Functional groups and mode of vibrations in the NIR spectrum of the
egg samples .......................................................................................................... 82
Table 4.20. The cumulative variance of the PCs and the error values of the PCA
models formed using five-three liquid sample groups ........................................... 85
models formed using five-three dry sample groups .............................................. 87
Table 4.22 Comparison of means of samples treated at different temperatures .. 90
Table 4.23. Protein (dry basis), moisture, ash, wet gluten, and sedimentation
values of the flour sample ..................................................................................... 99
Table 4.24. Consistency (K) and flow behavior indices (n) of cake batters including
untreated and LWE heat-treated at different parameters ................................... 103
x
FIGURES
Page
Figure 2.1. Schematic representation of cross-section of an egg ........................... 6
Figure 2.2. Structure of egg shell ........................................................................... 7
Figure 2.3. Fractionation of egg yolk into granules and plasma ............................ 13
Figure 2.4. Schematic representation of yolk LDL ............................................... 14
Figure 2.5. Energy states in molecule vibrational levels ...................................... 24
Figure 2.6. Principal types of NIR absorption bands and their locations .............. 26
Figure 2.7. Schematic representation of the ATR-FTIR sensor ............................ 29
Figure 2.8. A schematic representation of capillary electrophoresis system......... 32
Figure 3.1. Dry samples of LWE, LEW, LEY, and liquid whole egg containing egg
white and water ..................................................................................................... 41
Figure 3.2. Baked cakes........................................................................................ 48
Figure 4.1. SDS-PAGE gel image of protein bands of liquid egg samples. 1. LWE,
2. LWE, 3. LWE, 4. 100%Y, 5. 90%-Y, 6. 80%-Y, 7. 70%-Y, 8. 60%-Y, 9. 50%-Y,
10. 40%-Y, 11. 30%-Y, 12. 20%-Y, 13. 10%-Y, 14. 0%-Y, 15. marker. Y: Yolk,
LWE: Liquid whole egg.......................................................................................... 52
Figure 4.2. An image of protein bands detected by image analysis software ....... 54
Figure 4.3. Pixel position and intensity of the bands in the lane ........................... 55
Figure 4.4. The calibration curve of the concentration of the yolk ......................... 56
Figure 4.5. SDS-PAGE gel image of the protein bands of eleven liquid egg
samples ................................................................................................................ 57
Figure 4.6. SDS-PAGE gel image of protein bands of liquid egg samples
containing egg white. 1. Marker, 2. 65:35 - EW: LWE, 3. 55:45 – EW: LWE, 4.
40:60 – EW: LWE, 5. 30:70 – EW: LWE, 6. 20:80 – EW: LWE, 7. 10:90 – EW:
LWE, 8. 5:95 – EW: LWE, 9. LWE All concentration is given as percentage (%).
EW: Egg white, LWE: Liquid whole egg ................................................................ 58
containing water. 1. Marker, 2. LWE, 3. 5:95 – W: LWE, 4. 10:90 – W: LWE, 5.
20:80 – W: LWE, 6. 30:70 – W: LWE, 7. 40:60 – W: LWE, 8. 55:45 – W: LWE, 9.
65:35 – W: LWE All concentration is given as the percentage (%). W: Water, LWE:
Liquid whole egg ................................................................................................... 60
Figure 4.8. ATR-FTIR spectra of the LEY, LWE, and LEW................................... 63
xi
Figure 4.9. Correlation between the actual and predicted values of protein, (a)
Calibration data set, (b) Validation data set .......................................................... 68
Figure 4.10. Correlation between the actual and predicted values of lipid, (a)
Figure 4.11. Correlation between the actual and predicted values of moisture, (a)
Figure 4.12. Correlation between the actual and predicted values of total soluble
solid (TSS), (a) Calibration data set, (b) Validation data set ................................. 73
Figure 4.13. The ATR-FTIR spectra of (a) LEW, LWE (brix adjusted to 21 by
adding egg white, BXEW), LWE (brix adjusted to 21 by adding water, BXW), LWE,
and LEY; (b) dry egg white (DEW), dry whole egg (brix adjusted to 21 using egg
white, BXEW), dry whole egg (brix adjusted to 21 using water, BXW), dry whole
egg (DWE), and dry egg yolk (DEY) ..................................................................... 76
Figure 4.14. PCA score plot of (a) five liquid sample groups (LWE, LEY, LEW,
BXEW, and BXW) and (b) three liquid sample groups (LWE, BXEW, BXW) ........ 77
Figure 4.15. PCA score plot of (a) five dry sample groups (DWE, DEY, DEW,
BXEW, and BXW) and (b) three dry sample groups (DWE, BXEW, BXW)........... 79
Figure 4.16. NIR spectra of the (a) LEY, LWE, BXW, BXEW, and LEW, (b) DEY,
DWE, BXW, DEW, and BXEW .............................................................................. 81
Figure 4.17. The PCA score plot of (a) the five liquid sample groups (LWE, LEY,
LEW, BXEW, and BXW) and (b) the three liquid sample groups (LWE, BXEW, and
BXW) ..................................................................................................................... 84
Figure 4.18. The PCA score plot of (a) the five dry sample groups (DWE, DEY,
DEW, BXEW, and BXW) and (b) the three dry sample groups (DWE, BXEW, and
BXW) ..................................................................................................................... 86
Figure 4.19. UV-VIS absorbance measurements of untreated and treated LWE
samples at (a) 60 °C, (b) 64 °C, (c) 68 °C for five min with a one-minute interval
and the statistically differences of measurements within one temperature.
Significant differences (p<0.05) between means are indicated by different letters 89
Figure 4.20. Electropherograms of (a) liquid egg white (LEW), liquid egg yolk
(LEY), egg yolk supernatant (EYS) and egg yolk granule (EYG); (b) egg yolk
supernatant and granule added liquid whole egg (LWE), LWE; (c) lysozyme,
conalbumin, ovomucoid, ovalbumin added LWE, and LWE.................................. 93
xii
Figure 4.21. Electropherograms of untreated and treated LWE samples at (a) 60
°C, (b) 64 °C, (c) 68 °C for five minutes with a one-minute interval ...................... 96
Figure 4.22. PCA score plot of untreated and treated LWE samples (60 °C, 64 °C,
68 °C for five minutes with a one-minute interval .................................................. 97
Figure 4.23. PCA score plot of treated LWE samples at 60 °C, 64 °C, 68 °C for five
minutes with a one-minute interval ........................................................................ 98
Figure 4.24. An apparent viscosity versus shear rate of different batter
formulations including untreated and heat-treated LWE for two and five min at (a)
60 °C, (b) 64 °C, and (c) 68 °C. Lines represent the power-law model ............... 102
Figure 4.25. The moisture loss (%) of the baked cakes prepared using untreated
(control) and heat-treated LWE for two and five min at 60 °C, 64 °C, 68 °C ....... 105
Figure 4.26. ∆E values (%) of the baked cakes prepared using untreated (control)
and heat-treated LWE for two and five min at 60 °C, 64 °C, 68 °C .................... 106
Figure 4.27. The porosity values (%) of the baked cakes prepared using untreated
(control) and heat-treated LWE for two and five min at 60 °C, 64 °C, 68 °C ....... 107
Figure 4.28. The hardness values (N) of the baked cakes prepared using
untreated (control) and heat-treated LWE for two and five min at 60 °C, 64 °C, 68
°C ........................................................................................................................ 108
Figure 4.29. The cohesiveness values (ratio) of the baked cakes prepared using
°C ........................................................................................................................ 109
Figure 4.30. The gumminess values (N) of the baked cakes prepared using
°C ........................................................................................................................ 110
Figure 4.31. The specific volume values (cm3/g) of the baked cakes prepared
using untreated (control) and heat-treated LWE for two and five min at 60 °C, 64
°C, 68 °C ............................................................................................................ 111
Figure 4.32. Correlation between the hardness and porosity values .................. 112
Figure 4.33. Correlation between the hardness and specific volume values ...... 113
xiii
SYMBOLS AND ABBREVIATIONS
Symbols
a Red/Green Opponent Colors
b Yellow/Blue Opponent Colors
K Consistency Index
L Lightness
n Flow Behavior Index
R2 Determination of Coefficient
α Alpha
β Beta
γ Gamma
𝛾 Shear Rate
𝜏 Shear Stress
Abbreviations
ANOVA Analysis of Variance
AOAC Association of Analytical Communities
AOTF Acoustic Optic Tunable Filter
AP Ammonium Persulphate
ATR Attenuated Total Reflectance
ATR-FTIR Attenuated Total Reflectance Fourier Transform Infrared
BGE Background Electrolyte
CCD Charge Coupled Devices
CD Circular Dichroism
CE Capillary Electrophoresis
CIE International Commission on Illumination
DSC Differential Scanning Calorimetry
FSIS Food Safety and Inspection Service
FT Fourier Transform
FTIR Fourier Transform Infrared
FT-NIR Fourier Transform Near Infrared
xiv
GPC Gel Permeation Chromatography
HDL High-Density Lipoproteins
HPLC High-Pressure Liquid Chromatography
IGY Immunoglobulin Y
LDL Low-Density Lipoproteins
LEPs Liquid Egg Products
LOD Limit of Detection
LOQ Limit of Quantification
LVs Latent Variables
LEW Liquid Egg White
LEY Liquid Egg Yolk
LPC Lysophosphatidylcholine
LWE Liquid Whole Egg
MALDI-TOF Matrix-assisted Laser Desorption/Ionization - Time of Flight
MLR Multiple Linear Regression
NIR Near Infrared
PC Phosphatidylcholine
PCs Principal Components
PE Phosphatidylethanolamine
PCA Principal Component Analysis
PLS Partial Least Square
RMSEC Root Mean Square Error of Calibration
RMSEP Root Mean Square Error of Prediction
RMSECV Root Mean Square Error of Cross Validation
RP-HPLC Reversed Phase High-Performance Liquid Chromatography
SDS Sodium Dodecyl Sulfate
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
TEMED Tetramethylethylenediamine
TNF Tumor Necrosis Factor
TSS Total Soluble Solid
USDA United States Department of Agriculture
UV Ultraviolet
UV-VIS Ultraviolet Visible
xv
1. INTRODUCTION
Food quality and safety have become more pronounced in recent years, and it
seems that they will maintain to be a significant debate not only in food industry,
but also in public. Therefore, academic research has become more focused on
these problems in order to come with solutions providing considerable and more
importantly consistent improvements in food production. The answer to the
question, why food quality and safety problems have become more controversial is
not trivial, rather complicated since several factors have effect on these problems.
However, one should consider that the human being is the most dominant factor
among various components of quality and safety problems in food.
Recently, the doubts over the food safety and quality have increased.
Furthermore, people has become more interested in all processes of food
production from the farm level to the end consumer, in other words, they want to
have a knowledge of all the processes behind the food production. Especially,
consumers in developed regions have become more sensitive, more demanding,
and more fractioned in their eating habits [1]. As a result, providing quality control
in food production has become one of the important issues in food industry.
Obviously, quality in food production highly depends on the use of quality raw
materials. Therefore, analysis of raw material on its quality parameters or
adulteration has been the first step in the quality control of production, which
provides a comprehensive characterization of food quality to the producer. Beside
the quality of raw materials, food processing has also a substantial effect on the
quality food production. Due to the side effects of processes, such as heating, food
can be damaged in terms of nutritional and functional properties, which result in
lower-quality products. Therefore, having the knowledge of the impact of
processing on food is very critical in order to guarantee a certain quality in food
industry.
In food industry, there are some major raw materials are used in many products.
One of these materials is egg. Egg is comprehensively used in many food sectors
as a main ingredient thanks to its exceptional functional properties and techno-
unique features [2,3].
1
Food producers commonly prefer to use liquid egg products (LEPs) instead of
shell eggs due its ease of use and microbial reliability. However, further problems
may arise at this point. At first, it is critical for producers to have information about
the content of the egg that is used in the product. If the producers can have a
control on the egg quality before the processing, they would overcome the
problems (such as adulteration in egg) that may originate from the eggs in the next
steps. Another important step is the heat treatment applied to LEPs. The heat
treatment process may have some harmful side effects on the functional
properties of egg proteins, which lead to commercially undesirable results [4].
Therefore, it is highly critical to know what the consequences of heat treatment in
order to take precautions.
Egg components (protein, lipid, and water) have been analyzed by reference
methods in the literature. Conventionally, Kjeldahl method is utilized for the protein
analysis while; official methods proposed by association of analytical communities
(AOAC) are commonly employed to analyze lipid and water in egg. However,
these analyses are highly complicated and very time consuming. Therefore, more
simplified and immediate-responded approaches are required to determine the
egg components. In addition to that, any analytical method detecting liquid egg
adulteration has not been proposed and the effect of heat treatment on proteins in
liquid whole egg (LWE) has not been demonstrated by chromatographic
techniques in the literature. At last, demonstrating the effect of heat-treated LWE
on quality of final product is required to reveal the importance of egg quality in
products. However, the effect of heat-treated LWE on the rheological properties of
cake batter and on cake physical quality has not been demonstrated in the
literature.
The first objective of this study is to determine of LWE quality by rapid analysis of
the whole egg components and detection of a possible LWE adulteration. The
second aim is to observe the effect of the heat treatment on egg proteins. At last,
examination of the effect of the heat-treated LWE on the food quality is aimed.
Sponge cake was chosen as the model product since it contains a high ratio of the
whole egg; thus, the effect of the heat-treated LWE on the final product quality can
easily be observed.
2
This study can be divided into three sections. As the first part of the study, egg
yolk-white ratio of the LWE was detected by SDS-PAGE. LWE adulteration by
addition of water instead of egg white was determined by using SDS-PAGE.
Calibration model was constructed using different concentrations of the yolk-white
ratio by mixing a range from 0% yolk to 100% (w/w). Concentration of the egg yolk
in the adulterated samples was detected according to calibration model.
Furthermore, components of the LWE; protein, lipid, water, and total soluble solid
were determined simultaneously by attenuated total reflectance Fourier transform
infrared (ATR-FTIR) spectrophotometer. Calibration equations were developed for
each component, where component values (protein, lipid, moisture, and total
soluble solid) were detected by reference methods in order to obtain a calibration
model. Data analysis was performed using partial least square regression (PLS
regression). In addition, LWE and dry whole egg adulteration by addition of water
were determined by using both near infrared (NIR) and ATR-FTIR spectroscopy,
and data analysis was carried out by principal component analysis (PCA).
As the second part, the effect of the heat treatment on the egg proteins was
analyzed by capillary electrophoresis (CE). A number of different heat treatment
parameters were applied to the LWE samples. The heat-treated samples were
analyzed under suitable conditions of CE. Data analysis was performed using
PCA.
In the last part, the effect of using the heat-treated LWE on sponge cake quality
was demonstrated. First cake batter rheology was analyzed by rheometer, which
was followed by analyses performed on a number of baked cakes in order to
measure the cake quality. The effect of the heat-treated LWE on the cake
structure was reported and discussed at the end of the study.
Main contributions of this thesis can be summarized as follows;
! ATR-FTIR spectroscopy was used to perform compositional analysis of

LWE which is the first application based on ATR-FTIR in the literature.
! Quantitative analysis of protein, lipid, moisture, and total soluble solid
contents of LWE was performed by combination of ATR-FTIR spectroscopy
and PLS.
3
! A method via both ATR-FTIR and NIR spectroscopies was carried out for
the first time to detect LWE adulteration with water.
! Determination of yolk-white ratio of LWE was performed by SDS-PAGE
analysis that detected LWE adulteration with water.
! Capillary electrophoresis was used to monitor the effects of heat treatment
parameters on LWE proteins that is the first analysis based on
chromatographic technique in the literature.
! The effect of heat-treated LWE on rheological behavior of cake batter was
examined for the first time.
! The effect of heat-treated LWE on the quality of sponge cake was also
investigated at first time in the literature.
The remainder of this thesis is as follows: In Chapter 2, a fundamental background

is provided, where physical and functional properties of egg and analysis
techniques utilized in the study are explained in detail, respectively. Material and
methods are comprehensively explained in Chapter 3. Experimental results are
provided and discussed in Chapter 4. Finally, Chapter 5 concludes this study,
where the future work is also discussed.
4
2. LITERATURE REVIEW
Egg is one of the fundamental human food that has been consumed since the
ancient times. It is one of the perfect protein sources, which contains almost every
essential amino acid. Furthermore, it also contains other high quality nutrients
such as lipids, minerals, and vitamins [3,5,6]. Eggs can be easily digested and can
supply an important part of the nutrients required daily for growth and care of body
tissues [5]. In addition to its nutritional values, it serves as a crucial ingredient in
products thank to its multifunctional properties, such as foaming, emulsifying,
gelling, thickening, coloring, and flavoring, which contribute to the texture and
sensory characteristics of food [7].
2.1. Structure and Chemical Composition of Eggs

Egg consists of three main components; the eggshell, the egg white, and the yolk.
Average composition of egg contains 10.3% eggshell, 56.9% egg white, and
32.8% egg yolk. The average weight of the whole egg is 58 g. Its major ingredients
are water (74%), protein (12%), and lipids (11%). The carbohydrate content in egg
is very low (~1%). It also includes all necessary vitamins (except vitamin C) and
minerals [5,7].
A schematic illustration of the egg is given in Figure 2.1. Egg is covered by a

calcareous and porous shell. The interior of the shell is coated with inner and outer
membranes that adhered closely to each other. These membranes are separated
at the end of the egg to create an air space called air cell. Its size is around 5 mm
in diameter for fresh eggs. This dimension increases during the storage, and is
used to determine the age of egg. The median part of the egg is the egg white
(albumen). It is aqueous and gel-like liquid. Interior portion of the egg is the yolk
surrounded by the albumen. A thin layer of albumen encircles the yolk and it
divaricates on reverse sides of the yolk into two chalazae, which extend into the
thick albumen. Chalazae keeps the yolk in the center of egg. The germinal disc
has a position at the top of a club-shaped latebra on one part of the yolk [5].
5
Figure 2.1. Schematic representation of cross-section of an egg
2.1.1. Egg Shell

The thickness of the eggshell is around 0.2-0.4 mm. Its color varies from white-
yellow to brown. The shell structure of an egg is visually illustrated in Figure 2.2,
where the shell is composed of five different layers from the inside to the outside:
Inner shell membrane, outer shell membrane, inner calcified layer, palisade layer,
and cuticle layer [5,8]. Major components of eggshell are presented in Table 2.1.
6
Figure 2.2. Structure of egg shell [10]
Table 2.1. Components of eggshell [11]

Constituent Major components (relative %, w/w)
Egg shell Inorganic salts (91.87)
Calcium carbonate (98.4)
Magnesium carbonate (0.8)
Tricalcium phosphate (0.8)
Proteins (6.4)
Water (1.7)
Lipids (0.03)
The inner shell membrane (20 µm) is directly contacted the albumen. The outer
shell membrane (50 µm) is located between the inner membrane and the calcified
layer. These membranes consist of organic fibers including interwoven network of
protein-polysaccharide fibers. This structure is very critical in protecting to
microorganism penetration. There have been tiny pore canals (7000 -17,000 per
egg) that extend throughout the shell. The cuticle proteins partially seal the pores
that allow the gas passage while restricting microorganism penetration [5,8].
7
The inner calcified layer (cone or mammillary layer) is located between the
palisade layer and the outer membrane. It has a thickness of 70 µm and
composed of calcified columns and cones, which penetrate into the outer
membrane. There are some organic cores (mammillary knobs) on the surface of
the outer membrane. These cores are the sites including calcium carbonate
crystals deposition formed by a spherulitic deposition of microcrystals of calcite
(the calcium carbonate polymorph of the shell) around the mammillary knobs [9].
The palisade (spongy, calcareous) layer is located under the thin cuticle. This
layer has a 200µm thickness representing two-third of the shell thickness. It is
made of calcite crystals. It has the pores that allow exchange of gas between
inside and outside. The palisade layer extends to the vertical crystal layer that is a
narrow band of crystals aligned vertically to the shell surface [5,8].
The outermost layer is cuticle (bloom). It is a very thin (10 µm) organic layer
including the majority of shell pigments. It is transparent, mucilaginous layer, and
has a thin film of hydroxyapatite crystals in the inner side of the cuticle. It prevents
the microbial penetration by clothing the shell and plugging the pores. Due to the
drying, it has a cracked appearance that enables gas transfer between the pores
[5,8].
2.1.2. Egg White

The weight of the egg white (albumen) is about 60% of the total egg weight. The
components of the albumen are presented in Table 2.2. Its main components are
water and protein. The difference in the structure between thick (gel-like) and thin
albumen stems from the content of ovomucin that is four-fold more in the thick
albumen.
The pH of the albumen is around 7.6-7.9 for freshly laid eggs, but it rises up to 9.7
during the storage because of diffusion of solubilized CO2 via the shell. The pH
rise depends on temperature and time [5,10].
8
Table 2.2. Components of egg white [11]
Egg white Proteins (9.7-10.6):
Ovalbumin (54)
Ovotransferrin (12)
Ovomucoid (11)
Ovomucin (3.5)
Lysozyme (3.4)
G2 globulin (4.0)
G3 globulin (4.0)
Ovoinhibitor (1.5)
Ovoglycoprotein (1.0)
Ovoflavoprotein (0.8)
Ovomacroglobulin (ovostatin) (0.5)
Cystatin (0.05)
Avidin (0.05)
Lipids (0.03)
Carbohydrates (0.4-0.9)
Ash (0.5-0.6)
A number of different types of proteins are present in the aqueous solution of the
albumen. Most of these proteins exhibit a biological activity. It may be concerned
with protection of the egg against microbial spoilage. The main protein of the egg
white is ovalbumin, which is a glycophospho-protein with 3.2% carbohydrates. The
ovalbumin composes of a peptide chain including 385 amino acid residues, and
has a molecular weight of 44.5kDa. S-ovalbumin (coagulation temperature 92.5
°C), a more heat-stable form of ovalbumin (coagulation temperature 84.5 °C), is
formed during the storage of eggs. Fresh eggs have 5% S-ovalbumin, but the
content of S-ovalbumin rises to 81% in the eggs stored at cold temperatures for 6
months. The ovalbumin can be readily denatured by whipping or shaking its
aqueous solution. It is an interphase denaturation that happens through unfolding
and aggregation of protein molecules. The ovalbumin is an important protein in
terms of its biological activity.
It has been known that peptides generated by the enzymatic digestion of the
ovalbumin, exhibit an antibacterial activity against to some bacteria. The
ovalbumin has also an immunomodulatory activity inducing the release of tumor
necrosis factor (TNF) α in a dose-dependent manner in vitro, when modified with
dicarbonyl methylglyoxyl [5,10].
9
The other abundant protein in the albumen is conalbumin (ovotransferrin). This
protein coagulates at lower temperatures (61.5 °C), but not denatured at the
interphase. It is composed of a peptide chain, and includes one oligosaccharide
unit. The conalbumin can bind with metal ions (2 moles Fe3+, Cu2+ or Zn2+ perm
ole of protein) that ability is a unique feature. It can also retard the microorganism
growth by preventing the microorganism from exploiting iron. It has been shown
that the conalbumin also has an antibacterial activity against a wide spectrum of
bacteria [5,10].
Ovomucoid is the third most common protein in the albumen. It has 2 or 3 different
forms, where the amount of sialic acid varies in each form. It is a heat-stable
protein (denaturation at 70 °C) thanks to including 9 disulfide bonds. The
ovomucoid, a serine protease inhibitor, hinders digestive enzymes like trypsin and
α-chymotrypsin. Thus, it is suitable for the oral delivery of protein/peptide
therapeutics [5,10].
Lysozyme (ovoglobulin G1) is an N-acetylmuramidase enzyme that hydrolizes the

structural component of gram-positive bacteria cell walls. It has been used as a
natural food preservative. The lysozyme includes four disulfide bonds, and has a
high denaturation temperature (75 °C). On the other hand, ovoglobulins (G2 and
G3) are known as a good foam builders [5,10].
Ovomucin can constitute fibrillar structure that is responsible for the rising in the
viscosity of albumen, especially for the thick albumen. Thus, the spread of
microorganism has been prevented. It is a heat-stable protein, and can form a
water-insoluble complex with lysozyme depending of the ambient pH. This protein
also exhibits an antiviral activity [5,10].
Ovoinhibitor is very similar to ovomucoid, as a proteinase inhibitor. It prevents the

activities of trypsin, chymotrypsin, and some proteinases of microbial origin.
Avidin, a glycoprotein, specifically binds with the water-soluble vitamin biotin.
It inhibits the growth of bacteria and yeasts that require biotin. The avidin also
exhibits an antibacterial property by binding to various gram-positive and gram-
negative bacteria. Cystatin, an inhibitor, inhibits cysteine proteases having an
significant role in cancer invasion [5,10].
10
The content of other components (carbohydrates, lipids, vitamins, and minerals) is
very low in the albumen. Despite the low content of carbohydrates, most of the
carbohydrates in eggs are present in the albumen. Some of them (~0.5%) are
bound to the proteins; on the other hand, the other (~0.4-0.5%) is free. The
mineral content of the albumen is around 0.6%, whose composition is provided in
Table 2.3. The vitamins found in the albumen are presented in Table 2.4 [5,7].
Table 2.3. Mineral composition of the albumen [5]

Mineral Egg white (ppm)
Sulfur 1950
Phosphorus 150-300
Sodium 1610-1690
Potassium 1450-1670
Magnesium 90
Calcium 80-200
Iron 1-2
Table 2.4. Vitamin composition of the albumen [5]

Vitamin Egg white (ppm)
Retinol (A) 0
Thiamine 0.22
Riboflavin 2.7
Niacin 1
Pyridoxine (B6) 0.12
Pantothenic acid 1.4
Biotin 0.07
Folic acid 0.09
Tocopherols 0
2.1.3. Egg Yolk

Egg yolk weight is approximately 30-35% of the whole egg. The dry matter of the
yolk is around 50%, depending on the duration of the storage. During the storage
of eggs, a portion of water in the albumen passes through the yolk. The
components of the yolk are listed in Table 2.5.
11
The yolk is a fat-in-water emulsion, and comprises 62.5% lipids, 33% proteins, 1%
carbohydrates, vitamins and 3.5% minerals on the basis of its dry matter. The pH
of the fresh yolk is 6.0 and, unlike the albumen it can rise up to 6.4-6.9 even after
extended storage [5,8].
Table 2.5. Components of the yolk [11]

Egg yolk Proteins (15.7-16.6):
Lipovitellenin (I-VI) (37.3)
Lipovitellin apoproteins (40.0)
α-lipovitellin
β-lipovitellin
Livetins (9.3)
α-livetin
β-livetin
γ-livetin
Phosvitin (13.4)
Biotin-binding protein (trace)
Lipids (32.0-35.0)
Triglycerol (66)
Phosphatidylcholine (PC) (24)
Phosphatidylethanolamine (PE) (2.8)
Lysophosphatidylcholine (LPC) (0.6)
Sphingomyelin (0.6)
Cholesterol (5.0)
Others (1.0)
Carbohydrates (0.2-1.0)
Ash (1.1)
The lipids are the main components of the yolk structure. They form 32-35% of the
yolk. The lipids are present as in the form of lipoproteins when they are combined
with the proteins existing in the yolk. The dry yolk includes around 60% lipids of
which approximately 65% is triglyceride, 28% is phospholipid, and 5% is
cholesterol. Phospholipids are lipids, which include phosphate and phosphate-
glycerol backbone. Cholesterol (96%) is the main component of sterols existing in
the yolk [5,10].
Lipids and proteins in the yolk are present mostly in the form of lipoprotein.
Proteins are present as either apoproteins (included in lipoprotein structure) or free
proteins. Lipoproteins contain both lipids and proteins.
12
The yolk can be separated into two fractions (supernatant and sediment)
according to the method proposed by McBee and Cotterill [12]. Fractionation of the
yolk is presented in Figure 2.3. The sediment, a pale pellet, is a granule fraction
(19-23% of yolk dry matter) and the supernatant, a dark orange part, is a plasma
fraction (77-81% of yolk dry matter). The granules include α and β lipovitellins
(70% high-density lipoproteins, HDL), phosvitin (16%), and low-density
lipoproteins (12% LDLs). The dry matter content of granules is approximately 44%,
and they include 50% of the yolk proteins and 7% of the yolk lipids. The plasma
contains LDL (85%) and livetins (85%), which are free proteins. It is an aqueous
phase, where yolk particles are in suspension. It contains 90% of the yolk lipids
(including almost carotenoids) and 50% of the yolk proteins [8,10].
Figure 2.3. Fractionation of egg yolk into granules and plasma
Low-density lipoproteins (LDLs) and lipovitellenin are spherical particles, and are
composed of a lipid core in a liquid state (triglycerides and cholesterol esters),
which are covered by a monofilm of phospholipid and protein as visually illustrated
in Figure 2.4.
13
They are soluble in aqueous solution depending on their relatively low density.
LDLs consist of 11-17% protein and 83-89% lipid. LDL includes six major
apoproteins. These apoproteins are amphiphilic and flexible molecules. LDLs have
a crucial role in emulsifying properties of the yolk due the interaction of amphiphilic
apoproteins with phospholipids. The coalescence of apoproteins and
phospholipids lets transport of these insoluble amphiphilic structures through the
aqueous phase until the interface (oil-water) is reached. They form the interfacial
film of LDL that leads to a decrease in the interfacial tension, and resistance to
rupture. As a result, it can be pointed out that LDLs have a key role in techno
functional properties of the yolk in food [9].
Figure 2.4. Schematic representation of yolk LDL [9]
The other group of lipoproteins is high-density lipoproteins (HDLs). The HDLs,

lipovitellin, have a globular structure. They are linked to phosvitin via
phosphocalcic bridges. The HDLs are composed of 75-80% protein and 20-25%
lipids. Therefore, their density is close to that of proteins.
14
The HDLs are separated into two sub-groups as α-HDL and β-HDL. These sub-
groups have a similar chemical composition except the content of sialic acid and
phosphorus. The HDLs are heat stable molecules, however, they lose that
property when the lipids are separated [5,8].
Phosvitin, a free protein, is a phosphoglycoprotein including relatively high amount

of phosphoric acid bonded to serine residues. It comprises two polypeptides; α-
phosvitin and β-phosvitin. Each component has four or five sub-units. Phosvitin is
soluble in pure water, and is a relatively heat stable protein even at 110 °C for 10
min. Phosvitin has a very strong affinity to metal ions thanks to its high content of
phosphorous. 95% of iron in the yolk is bonded to phosvitin. The high iron binding
ability of phosvitin gives rise to bactericidal effect and antioxidant activity.
Phosvitin can also capture the other heavy metal ions; thus, it serves as
antioxidants [5,8]. Livetin, a free protein, is separated into three fractions; α-livetin,
β-livetin, and γ-livetin, respectively. All of which are water-soluble globular
proteins. α-livetin exhibits allergic properties. γ-livetin is also known as
immunoglobulin Y (IgY). IgY has an important role in the immune system thanks to
exhibiting an antimicrobial activity against a number of bacteria and viruses. It can
bind and inhibit the infection and disease symptoms of gastrointestinal pathogens
[5,8]. The other components of the yolk are carbohydrates, minerals, vitamins, and
colorants. Amounts of minerals and vitamins in the yolk are listed in Table 2.6. and
2.7., respectively.
Table 2.6. Mineral composition of the yolk [5]

Mineral Egg yolk (ppm)
Sulfur 160
Phosphorus 5430-9800
Sodium 260-860
Potassium 1120-3600
Magnesium 160
Calcium 1210-2620
Iron 53-110
15
Table 2.7. Vitamin composition of the yolk [5]
Vitamin Egg yolk (ppm)
Retinol (A) 11.2
Thiamine 2.9
Riboflavin 4.4
Niacin 0.65
Pyridoxine (B6) 3
Pantothenic acid 37.2
Biotin 0.53
Folic acid 1.5
Tocopherols 65
α-Tocopherol 54
Vitamin D 0.056
2.2. Functional Characteristics of Egg in Food Systems

In various food products and food industries, egg is extensively used as an
essential ingredient thanks its exceptional functional properties [3]. Egg not only
enhances food nutritional value, but also improves sensual characteristics of the
product (e.q, color and flavor). Furthermore, egg enhances the product’s
foaming/whipping, emulsifying, and coagulation/gelation properties. Hence, it can
be referred as multifunctional food product included into a range of food systems.
Formation and physicochemical stability of many food products such as cakes,
meringue, custard, and salad dressings are related to the presence of egg
constituents functioning as gel network builder, foam agents, and emulsifiers. Egg
plays an effective role in the improvement of rheological and sensory
characteristics of products. The ability of egg serving as a functional ingredient in
food structure stems from their protein fractions. In food systems, eggs have three
major roles: foam builder, emulsifying agents, and gel former [13].
2.2.1. Egg Functionary in Foam Systems

A large number of egg based food products such as, cake batter, meringue, and
soufflé are in the foam form. Foam is a dispersion system including a whole of
small air bubbles in aqueous continuous phase. The egg white proteins provide air
entrapment and bubble stabilization against drainage or coalescence. However,
the yolk has poor foam formation ability because of its lipid content.
16
Lipids can be adsorbed at the air/water interface and replace the protein
molecules, resulting in poor interfacial phase [13].
Foam is a two-phase system, in which air bubbles form the dispersed phase
surrounded by a continuous liquid phase. A thin layer of the denatured proteins is
located at the interfacial film of the foam. Formation of the foam is based on the
surface activity and the ability of specific proteins that form the interfacial film
[13,14]. The egg white proteins are mechanically denaturalized by whipping.
Proteins have both hydrophilic and hydrophobic parts, where hydrophilic part is
attached to the liquid (continuous) phase, on the other hand, the hydrophobic one
is linked to air (dispersed) phase [15]. Hydrophobic part exists at the interface,
which lead to a decrease in the surface energy and tension. Formation and
stability of the foam against mechanical shock, aging, or shearing depends on a
strong cohesive interfacial film that has an ability to rapidly entrapping and
retaining air [13,14].
The main characteristics of protein to be a good foam builder, are the capability of
rapidly adsorbing at the air/water interface during whipping, rapid changing in
conformational and rearrangement at the interface, and form a cohesive elastic
film through intermolecular interactions [16]. A number of globular proteins are
responsible for egg whites being the exceptional foam agent. Among albumen
proteins, globulins are the excellent proteins as foam builder. Protein interactions
of ovomucin-lysozyme and a lesser extent of ovalbumin, ovotransferrin, and
ovomucoid contribute to the foam formation and stabilization [17]. Ovomucin
increases foam stability by supporting the formation of a thick membrane at the
interphase. Prevention of thinning of the interfacial film and drainage (that leads to
foam rupture) is dependent on the formation a thick membrane. Ovalbumin is
adsorbed at the air/water interface and unfolded. It is reorganized to orientate its
hydrophobic areas towards the oil phase. Then, the adsorbed molecules interact
via both disulfide bridges and non-covalent bonds in order to form a cohesive and
elastic interfacial film [13]. A variety of factors are effective on foaming properties
of albumen proteins. These are; hen age, storage time of egg, storage conditions,
speed and time of whipping, egg temperature, homogenisation, centrifugation,
pasteurisation, pH, dry matter, sugar, presence of egg yolk or oil, salt, stabilisers
and surface active compounds, metal ions, and proteolytic enzymes [14,16].
17
2.2.2. Coagulation/Gelation of Egg
Gelation is a phenomenon resulted in formation of a three-dimensional matrix via
inter-protein bonding [18]. Gel is an intermediate form between liquid and solid.
This structure can immobilize the water and avoid losses [19]. Heat treatment of
egg causes to denaturation (unfolded) and coagulation/gelation of proteins.
Thermal setting of cake batters, custards and puddings is based on the presence
of egg proteins that unfold and interact together to form the gel networks. Protein
gel networks are formed via hydrophobic interactions of denatured proteins with
heating. When heating above 70 ºC, both albumen and yolk can form gel networks
which are thermally irreversible [12,19].
Appearance of albumen changes to a milky white color with an elastic gel

coagulum. Textural qualities of some products such as meringue and angel cake
can be gotten by thermo-coagulation of the unique albumen proteins [16]. While
coagulation of albumen starts around at 60 ºC related with denaturation of
conalbumin, formation of gel structure is completed approx. at 85 ºC because of
ovalbumin denaturation [13]. Ovalbumin has a major effect on gelling process due
to its high content in egg white [20]. Yolk gel has a crumbly texture and low
strength. Plasma proteins (livetins and apolipovitellenins) in yolk are predominant
constituents in the formation of yolk gel. Granular constituents are less important
in gel process [13].
2.2.3. Egg Constituents in Emulsion System

Egg yolk is an excellent emulsifying agent in food products such as mayonnaise,
salad dressings, cakes or egg-based creams. The emulsifiying properties of yolk
are originated from phospholipids, cholesterol, lipoproteins and proteins (phosvitin
and livetin). LDL is especially considered as the main factor in the emulsifiying
characteristic of yolk [21]. The main function of the emulsifiying agents is to form
the tiny oil droplets, via adsorption and reduction in interfacial tension, and then to
stabilize the droplets against coalescence during storage [13].
The albumen proteins are generally referred as a foam and gelation agent. They
may also behave as an emulsifier when they are acting on their own. However,
albumen proteins cannot be adsorbed in the presence of yolk and are ejected from
the oil/water interface by more flexible lipoproteins.
18
Non-adsorbed white proteins may cause deterioration of stability and a change in
the rheological properties of the structure [13].
2.2.4. Egg Importance in Baked Products

The mission of egg or egg fractions in the baked products is to not only increase
their nutritional value but also improve the functional properties of the ingredients
to provide a food with acceptable quality characteristics and extended shelf life. In
some foods such as cookies, biscuits and pastry, egg may be an optional
ingredient, which modifies product characteristics like texture, flavor or color.
When these products are produced without egg, an important adverse effect on
their quality should not be observed. However, the preparation of products such as
various types of cakes can not be obtained without using egg because of its
multifunctional role during several stages of the baking process which can not
progress using other cake ingredients (sugar, flour, milk or artificial emulsifiers)
[13].
Depending on the type of cake, usage of egg parts that are whole egg and
albumen may change [13]. The mission of egg in cakes also changes based on
the type of the products. Egg foam cake types, sponge cake is made up of whole
egg that is responsibled for aerating and coagulation/gelation functions throughout
the preparation of the product. However, yellow layer cake includes high
proportions of fat which must be dispersed and emulsified during the mixing stage,
this function is performed by yolk lipoproteins [12,21].
The process of cake making comprises of three main stages, which are
preparation of cake batter, intermediate and structure development. In the first
stage, an aerated dispersion system is obtained with water constituting the
continuous phase in which flour and fat are dispersed. The relative importance of
the egg white and yolk in assisting air entrapment and stabilization of air bubbles
against coalescence or rupture has continued debate. However, it is approved that
yolk constituents are in charge of dispersing the fat and stabilizing the fat globules.
Lipovitellenin components are considered that aid in aeration and foaming, while
high density lipovitellin components in granules inhibit air incorporation but assist
in the retention of air presented in whipped system by the low density lipovitellenin
and egg white [13].
19
During the intermediate stage of baking, stabilizing impact of egg constituents on
the air and fat dispersions continues to be active. Heating above 70 °C, the
process of egg protein coagulation induces a gel network development. After egg
white and yolk protein denaturation and starch gelatinization, an alteration at this
stage occurs via bubble coalescence followed by channeling that lets escape
vapor and air from the cake structure. The alteration is critical in terms of avoiding
collapse event and transforming the foam into a sponge. The final cake structure is
solid foam that includes cell walls of high brittleness and low elasticity.
Presumably, this is because of the egg proteins in the system that prevent broad
gluten network formation throughout the cake structure but locally form a
composite gel matrix supported by gelatinized starch granules [13].
Disruption of the thiol-disulfide interactions that are in charge of gluten network

development may stem from interactions between egg and gluten proteins. When
the egg white and yolk proteins are denatured upon 70 °C, they can include into
disulfide bond bridges. In addition, when the whole egg is used in cake system,
both yolk and white proteins are considered contributing to structure development
in the form of producing a phase-separated gel network structure. The final cake
structure composes of a mixed gel network system depending gluten
development, but considerably modulated by the heat-setting ability of egg
proteins, leading to producing a product with higher brittleness and lower elasticity
than bread [13].
2.3. Liquid Egg Products

Egg products are produced from whole egg, egg white and egg yolk in the form of
liquid, frozen or dried [5]. In recent years, the food producers have preferred to use
liquid egg products (LEPs) obtained via shelling and pasteurization of shell eggs
[22,23]. Following shelling, pasteurization of eggs is necessary for microbial
safety.
20
2.3.1. Pasteurization of LEPs
Although the interior of freshly laid eggs is generally sterile, the shell surface may
involve many bacteria contaminated via intestinal tract of the hen or other
materials contacted after laying. Some of bacteria can be contaminated into LEPs
during shelling. Bacteria can also penetrate into the egg through pores in shell
[24,25]. For this reason, pasteurization is required to destroy the pathogens in
LEPs. Heat pasteurization is considered the best formula to eliminate pathogens
[26]. It is important to eradicate the pathogens and especially Salmonella spp. that
have different resistances to heat. S. senftenberg, S. oranienburg and S. paratyphi
B are the most resistant species. Sufficiency of pasteurization can be controlled
according to inactivation of α-amylase because the temperature for inactivation
and destroying of S. senftenberg is close. The extent of heat treatments may
change for different LEPs [5]. It is stated by EU legislation that egg products shall
not include Salmonella (in 25 g), and can contain Enterobacteriaceae to a
maximum of 100 cfu/g after heat process [27].
The pasteurization treatments are organized by regulations and they can vary
according to countries [28]. Heat treatment of egg white is proposed at 57.5 ºC for
2.5 minutes by the Food Safety and Inspection Service (FSIS) of United States
Department of Agriculture (USDA) [26], since coagulation of albumen proteins will
start at temperatures above 57 ºC. On the other hand, albumen has inherent
protectors against microbial growth and these protectors contribute to
pasteurization efficiency at low temperatures. Pasteurization of egg yolk can be
performed above 60 ºC due to heat resistance of the yolk [29]. Heat treatment of
liquid egg yolk is generally applied between the ranges of 60 to 68 ºC for 3.5 to 4.5
minutes [30]. Required minimum treatment of liquid whole eggs is also determined
by regulation of 60 ºC-3.5 minutes and 64 ºC-2.5 minutes, respectively, in U.S.
and UK [32,31].
2.3.2. Effects of Pasteurization on Functional Properties of LEPs

The extent of heat treatments (temperature and duration of treatment) for LEPs is
considered by egg processors for not only ensuring microbiological safety but also
performing its functions satisfactorily.
21
Severe heat processing can provide almost microbial safety and increase shelf life
of LEPs, whereas it can cause a destructive effect on the functional and
technological properties of egg proteins leading to commercially undesirable final
products [28]. Heat treatment causes a different effect on the functional properties
of the yolk and the albumen proteins.
Pasteurization parameters of egg yolk from 60 ºC to 68 ºC for 3.5 to 4.5 minutes

do not provide total elimination of microbial flora, in which causes to have a short
shelf life of pasteurized liquid yolk. Therefore, some egg users and processors
prefer to apply further heat treatments to increase shelf life of pasteurized yolk.
However, these treatments can damage egg yolk proteins, and its functional and
physical properties [30]. Egg yolk proteins are crucial for emulsifying properties.
According to Tsutsui [33], emulsifying activity of LDL gradually decreased while
the heat treatment temperature increased. Moreover, emulsifying capacity and
stability of LDL decreased when extensive heat treatment applied to liquid egg. On
the other hand, foaming properties of LDL, which may be play a role as a foaming
agent in madeira cake, was investigated. It was obtained that foaming capacity
and foam stability of LDL decreased through heat treatment of the yolk above 77.5
ºC.
Heat treatment of egg white may cause a defect of its components, non-enzymatic
browning, and coagulation of proteins. Denatured proteins form a gel and
aggregate through association of the unfolded chains. This aggregation resulted in
changes in the functional and physico-chemical properties of albumen proteins [2].
According to Hatta [34], pasteurization of egg white decreased the foaming ability
that caused to drop in the quality and volume of angel cake. The reason is that
denaturation of conalbumin through pasteurization at 53 ºC. Another study
reported that a longer whipping time for pasteurized albumen was required to
acquire a close value for specific gravity of foam of unpasteurized albumen. This is
because of irreversibly denatured ovomucin-lysozyme network. If it is eliminated,
albumen recovers its normal foaming ability [35].
22
2.4. Analyses of Egg Components
Liquid egg quality can be measured in terms of egg components. Up till now, many
egg components have been analyzed using different techniques such as
spectroscopy, electrophoresis, circular dichroism (CD) and chromatographic.
Compositional analysis of LEPs was performed by infrared transmission
spectroscopy [36], egg freshness was analyzed via visible transmission
spectroscopy [37]. The influence of raw egg hygienic quality on functional
properties of LEPs was studied [7]. LDLs in egg yolk were characterized by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and CD
[38], yolk fractions (granules, lipidic paste and watery fraction) were characterized
by SDS-PAGE, differential scanning calorimetry (DSC) and analyzed in terms of
rheologically and emulsifying properties [39]. Egg white proteins were analyzed
with cellulose-cation exchanger [40], thermal denaturation parameters of albumen
proteins were identified using DSC [41]. Egg proteins were also identified by 2D
gel electrophoresis with MALDI-TOF mass spectrometry and characterized by
capillary electrophoresis (CE) [39,40]. Egg white was fractionated via ion
exchange chromatography for further studies on biological activities of egg white
[44]. Egg white was also analyzed by gel-permeation chromatography, reversed
phase high-performance liquid chromatography (RP-HPLC) and HPLC on an
anion exchange column [45].
Besides analyzing of non-treated liquid egg quality, there have been many studies
about effects of heat treatment on egg components. Heat-denatured egg white
proteins were analyzed by SDS-PAGE and CD [46]. Effect of heat on physico-
chemical properties of the albumen was studied by NATIVE-PAGE [2]. Thermally
pasteurized LEW was analyzed on functional, physical, and microbiological
properties. Functional properties were investigated with a cake product [4].
Changes in heat-treated liquid egg yolk (LEY) products were analyzed by PAGE
and diethylaminoethyl-cellulose ion-exchange chromatography [47]. Functional
properties of LDLs in heat-treated LEY were searched with spectrophotometer and
viscometer [33]. Following pasteurization, secondary structures of yolk proteins
were analyzed by Fourier transform infrared (FTIR) and rheology [48]. Effects of
heat treatment on emulsifying properties, viscosity, and denaturation of yolk
proteins were studied [49].
23
Texture profile analysis of heated egg yolks was also performed [50]. Heat stability
and emulsifying property of whole egg added sugar and salt were studied [28].
Effects of pasteurization parameters and frozen storage on the functionality and
composition of LWE was analyzed [51]. As mentioned above, many studies about
egg quality and functionality of egg components were performed by a variety of
techniques. However, there has been no study on determining of adulteration of
LEPs with water. In this study, the techniques of attenuated total reflectance FTIR
(ATR-FTIR) spectroscopy, SDS-PAGE and near infrared (NIR) spectroscopy were
used to detect liquid egg quality based on egg components. LEPs were analyzed
based on the quantitative determination of protein, lipid, water and total soluble
solid by ATR-FTIR. In addition, adulterated samples were determined by SDS-
PAGE, ATR-FTIR and NIR techniques. CE was also used to reveal the effects of
heat treatment on egg proteins.
2.4.1. Infrared Spectroscopy

Infrared spectroscopy, a type of vibrational spectroscopy, is related to vibrations of
the atoms of a molecule [52]. Vibration of chemical bonds between atoms occurs
behaving as a simple harmonic motion. The vibration frequency is based on the
masses of the two atoms, m1 and m2, and the force constant of the bond k [53].
The majority of the molecules is located in their fundamental vibrational energy
state at ambient temperature [54]. When infrared radiation passes through a
sample, a part of its at a particular energy is absorbed from analytes [52].
Following absorbing the infrared radiation, the molecule is promoted to a higher
vibrational state as illustrated in Figure 2.5.
Figure 2.5. Energy states in molecule vibrational levels [55]
24
Specific features are necessary for a molecule to indicate its infrared absorptions.
First, the correspondence must be provided between the frequency of radiation
that is absorbed and vibration frequency of the molecule. Second, the molecule
must be an infrared-active, a heteronuclear diatomic molecule. This means that
the molecule shows changes in the dipole moment during vibration [52].
An infrared spectrum is obtained by measuring a net transfer of energy from

radiation to molecule versus wavelength. Molecules can just place certain energy
levels characterized by whole numbers 0, 1, 2,.. etc. Most of the molecules in
samples are generally promoted from lowest energy state 0 to 1, named
fundamental. Transitions from energy state 0 to 2, 0 to 3 etc. can also occur and
these are named as first, second overtones having twice or three times more
frequency than fundamental [53].
A broad range of radiation in the infrared region that is 4000 – 200 cm-1 (2.5 – 50
µm) is used to obtain vibrational excitation [55]. İnfrared region in electromagnetic
spectrum is separated into three spectral regions that are near, mid, and far varied
depending on their wavenumbers [56].
2.4.1.1. NIR Spectroscopy

NIR radiation was explored by Friedrich Wilhelm Herschel in 1800 [57]. NIR
spectroscopy is a technique that absorbs the electromagnetic radiation at
wavelengths between the range of 780 to 2500 nm (Figure 2.6) corresponded to
photon energy (hv) in the range of 2.65 × 10-19 to 7.96 × 10-20 J [51,52]. This
energy range is higher than required to raise molecules to their lowest excited
vibrational modes and lower than amounts required for electron excitation in
molecules [54]. When NIR radiation interacts a sample, incident radiation could be
absorbed, reflected or transmitted depending on the chemical constitution and
physical characteristics of the sample [57]. NIR region posses the frequencies of
many overtone bands. Another band arising in the NIR region is combination
bands originated from interaction of two or more vibrations occurring
simultaneously [53]. These combination bands seem between 1900 nm and 2500
nm. The intensity of NIR bands is related to change in dipole moment of the bond.
Overtone bands also seem between 780 nm and 2000 nm, based on the overtone
order and bond strength [58].
25
Figure 2.6. Principal types of NIR absorption bands and their locations [53]
NIR instrumentation can combine several devices based upon the features of the
sample, analytical conditions, and requirements (speed, environmental conditions
and sample complexity) [58]. A NIR spectrophotometer comprises of a light
source, monochromator, sample presentation accessory, detector, and other
optical components, such as beam splitters, lenses, collimators, optical fibers and
integrating spheres. A halogen lamp is generally used as a light source [57].
NIR spectrophotometers can be classified into two types in point of wavelength

selection. These are discrete wavelength and whole spectrum. Filters or light-
emitted diodes can be used for discrete spectrophotometers. Diffraction grating or
Fourier transform (FT-NIR) could be used for whole-spectrum NIR instruments. A
new one of wavelength selection is also acoustic optic tunable filter (AOTF) [58].
Devices including semiconductors (PbS or InGaAs) are used as a single channel

detector in spectrophotometer. In multi-channel detectors, diode arrays or charged
coupled devices (CCD) are utilized to record many wavelengths at once [58].
26
The first application of NIR spectroscopy was performed in agricultural field for
measurement moisture in grain [59]. From that time, it has been used for rapid
analysis of protein, fat, and moisture content of various food and agricultural foods
[57]. In recent years, NIR technology is also used other fields such as textiles,
pharmaceuticals, cosmetics, medicine, and others [60]. Analysis of food
ingredients and final products in terms of compositional, functional and sensory is
carried out by NIR spectroscopy. One of the advantages is that sample
preparation is not required and so the analysis is very fast and simple. In addition,
the analysis can be monitored on-line [53].
2.4.1.2. ATR-FTIR Spectroscopy

The principle of ATR-FTIR spectroscopy is based upon absorption in the mid-
infrared region [61]. This region is between the range of 4000 – 400 cm-1 in
electromagnetic spectrum [56]. The mid-infrared spectroscopy has been widely
used for the qualitative analysis and structural characterization of the “fingerprint”
of organic compounds. This is because some groups of atoms show characteristic
vibrational absorption frequencies in this infrared region. In addition, mid-infrared
spectroscopy can be performed for quantitative analysis, since the intensities of
the bands in the spectrum are proportional to the concentration of their individual
functional groups.
Food can be analyzed using this technique because of its compounds such as
lipids, proteins, carbohydrates and water, which display characteristic absorption
bands in this region. These bands belong to vibration of the carbonyl group and
absorptions of the lipid hydrocarbon backbone, the amino group of proteins, the
aldehyde group of carbohydrate, and absorptions of water [56].
Dispersive mid-infrared spectroscopy has been performed for food analyses with
some difficulties in sample preparation, handling and the dispersive tools are not
equipped with the convenient data acquisition and processing systems to obtain
quantitative information in an easily usable form. Since the 1970s, FTIR
spectroscopy has replaced the dispersive tools and has been extended to record
infrared spectra [56].
27
Fourier transform (FT) spectrometers have some advantages over dispersive
instruments. These are less measuring time and enhanced light throughput and so
a better signal-to-noise ratio. FT spectrometer is a Michelson interferometer where
the spectrum is reconstructed using a FT of the interference pattern of the
measured sample. A FT instrument provides to measure all wavelengths at once
while in a dispersive instrument; a monochromatic beam changes its wavelength
over time. Therefore the whole measuring time is shorter in a FT spectrometer
than a dispersive instrument [61].
There has been many studies on different groups of foods by mid-infrared

spectroscopy, and FTIR technology has notable potential as a quantitative quality
control instrument in food industry [56]. Quality controls of a variety of food such
as butter for fat and moisture determination [62], for milk analyses [63],
determination of ethanol in beer and alcoholic beverages [62,63], determination of
fat and protein content in raw meat [66] and analysis of edible fats and oils [65,66]
were analyzed with FTIR spectroscopy.
In the field of FTIR spectroscopy a different technique, ATR, has been developed
for surface analysis that is particularly useful for the research on most liquids [56].
ATR spectroscopy is based on the existence of an evanescent field in an optically
rarer medium (the sample) in contact with an optically denser medium (the crystal)
within which radiation is propagated due to internal reflection [69]. This
phenomenon is shown in Figure 2.7.
Denser medium must be permeable to infrared and of high refractive index [61].
The radiation interacts with the surrounding sample at each point of reflection and
then goes to the detector [56]. ATR technique is a useful tool for the samples that
transmits the radiation insufficiently and when preparation of sample is difficult via
traditional methods or when the sample has to be analyzed without modification.
The samples such as flexible and smooth films, plastics, coatings and gums can
be analyzed very well by ATR [56].
28
Figure 2.7. Schematic representation of the ATR-FTIR sensor
ATR-FTIR spectroscopy can be performed for quantitative analysis of foods.

Soluble solid content, individual sugars and organic acids in apricot slurry [70],
water-soluble proteins as B1, B2, B6 and niacin in synthetic mixtures [71], sugar
and organic acid contents in tomatoes [72] were determined by using ATR-FTIR
spectroscopy. Protein, lipid, and water contents of LEPs were detected with
infrared transmission spectroscopy [36].
2.4.2. SDS-PAGE
Electrophoresis is described as the migration of charged molecules in a solution
via an electrical field. The most widespread type of is zonal electrophoresis in
which proteins are distinguished from complex mixture into bands by migration in
buffers via a solid polymer matrix called gel. Among from polymer matrix,
polyacrylamide gels are the most common matrix for zonal electrophoresis of
proteins.
29
Electrophoresis can be carried out in two forms that non-denaturing (native form)
electrophoresis, in which proteins are separated in their native form or denaturing
electrophoresis [73].
SDS-PAGE separates polypeptide chains of proteins according to their molecular

weights. SDS breaks down proteins into their individual polypeptide chains [74].
SDS (CH3-(CH2)10-CH2OSO3-Na+) is an anionic detergent. When the proteins are
interacted with SDS in the presence of a reducing agent such as β-
mercaptoethanol, SDS binds to hydrophobic regions of protein molecule and the
molecule resulted in the negative charging. The binding of SDS with per-unit-
length of protein molecule is nearly constant even for large number of proteins so
the ratio of charge to mass of proteins is constant [75]. The electrophoretic motion
of molecule in gel depends on the friction of the protein in the matrix and charge of
the proteins. The mobility is defined by the following equation:
𝐴𝑝𝑝𝑙𝑖𝑒𝑑 𝑣𝑜𝑙𝑡𝑎𝑔𝑒 (𝑁𝑒𝑡 𝑐ℎ𝑎𝑟𝑔𝑒 𝑜𝑛 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒)

𝑀𝑜𝑏𝑖𝑙𝑖𝑡𝑦 =
𝐹𝑟𝑖𝑐𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒
The size of the charge and applied voltage has an effect on determining of how far
a protein will migrate in the electrical field. Migration is supported with the higher
voltage and the stronger charge on protein. When the molecular friction increases,
mobility of proteins decreases; thus high molecular weight proteins migrate slower
through the gel matrix [73].
A power supply, polyacrylamide gel matrix and buffer reservoir are required to
create an electrophoresis system. The power supply makes the electrical field by
providing a source of constant voltage, current or power. The electrode buffer
conducts the current via gel matrix and controls the pH to supply an appropriate
charge on the protein. A polyacrylamide gel matrix is formed by polymerizing
acrylamide and a small amount of N,N’-methylenebisacrylamide, in the presence
of a catalyst tetramethylethylenediamine (TEMED), and source of free radicals,
ammonium persulphate (AP). A discontinuous gel matrix is generally used to
enhance resolution of proteins in complex mixture [73]. In discontinuous matrix,
stacking gel (large pore gel) is layered over separating gel (small pore gel).
30
When electrophoresis starts, proteins, ions from stacking gel (leading ion) and ions
from buffer tank (trailing ion) proceed to move into stacking gel. Protein molecules
are moved until reaching separating gel between leading ions and trailing ions in
the form of a thin zone referred as stack [75]. Separating gel (resolving gel) at
different pH leads to separation of proteins into discrete bands [73]. The size of the
analyzed protein can be determined by comparing its electrophoretic mobility with
the mobility of a marker protein well-characterized in terms of molecular weight
[74].
SDS-PAGE is an easy and rapid method, and needs only inexpensive equipment.
The required sample quantity is approximately at the microgram level. It is reliable
and reproducible, and the results can be commented easily [74]. It has been
commonly used for analyzing of protein mixtures. It is a convenient technique to
observe the purification steps of proteins since the method depends on the
separation of proteins according to size, and it is utilized to identify the relative
molecular mass of proteins [75]. There have been many studies related with SDS-
PAGE and analysis of food proteins. Using SDS-PAGE technique, allergen protein
in peach was detected [76], sesame seed proteins caused allergenity were
identified [77], hen egg white proteins caused to allergy are separated [78], and
hen egg white and yolk proteins were identified [42].
2.4.3. Capillary Electrophoresis

CE has the similar principles for the separation of proteins as the conventional
electrophoresis techniques. Proteins can also be separated based on charge or
size in an electric field. The difference in CE is that capillary tubing is used instead
of acrylamide gel cast between slabs [73].
A CE system composes of a capillary column, two buffer reservoirs, power supply

and detector. A schematic representation of this system is presented in Figure 2.8.
The sample is placed in the inlet side of the capillary tube by replacing the inlet
buffer reservoir with the sample solution. Low voltage or pressure is applied
across the capillary until the desired amount of the sample is loaded onto the
column. Capillaries comprise fused silica with narrow internal diameters in the
range 25 – 100µm. Column length can be in this range a few centimeters to 100
cm.
31
High electric fields (100 – 500 V/cm) can be applied because narrow columns can
spread heat effectually, leading to short run times of 10-30 minutes. Proteins
bands are determined on the column as they goes through a detector. The most
common detector used in CE is ultraviolet-visible (UV-VIS) detector, though
conductivity and fluorescence detectors are available [73].
Figure 2.8. A schematic representation of capillary electrophoresis system
CE has been used for characterization of many food proteins. Among these
studies; milk proteins [79], fish muscle sarcoplasmic proteins [80], and peanut
proteins [81] were characterized by CE. Egg white proteins that are conalbumin,
lysozyme and ovalbumin were separated with CE [82]. Ovalbumin glycoforms
were also separated by using CE [83]. Major proteins in hen eggs and cow milk
were characterized by CE [43].
32
3. MATERIAL AND METHOD
Experimental and analytical analyses utilized in this thesis are introduced and
comprehensively explained in this chapter, which can be categorized under three
main sections:
! Determination of LWE quality by SDS-PAGE, ATR-FTIR and NIR

spectroscopy
! Analysis of heat-treated LWE by UV-VIS spectroscopy and CE
! Effect of heat-treated LWE on cake batter rheology and baked cake quality
3.1. Determination of LWE Quality

To determine the LWE quality, a number of different approaches were utilized,
which are; determination of yolk-white ratio and adulteration of egg with water by
SDS-PAGE, determination of egg components by ATR-FTIR spectroscopy, and
detection of egg adulteration by ATR-FTIR and NIR spectroscopy, respectively.
All experiments were carried out using different shell eggs, including grade A-large
and medium eggs obtained from the commercial channel. Samples of LWE, LEW,
LEY, and LWE containing egg white or water were analyzed in this section. LEW
and LEY samples were prepared manually. Eggs were broken and separated as
white and yolk. The eggs for LWE samples were also manually broken. On the
other hand, LWE samples, containing extra egg white or water, were prepared by
the addition of egg white or water at different concentrations. The as-prepared
samples were homogenized (IKA T-18 Ultra Turrax Digital Homogenizer,
Deutschland, Germany) separately at 6500 rpm for 3 minutes.
3.1.1. Determination of Yolk-White Ratio in Liquid Egg by SDS-PAGE

The yolk-white ratio and adulteration of liquid egg with water were determined by
SDS-Page, where a similar procedure proposed by Laemmli [84] was followed
with applying minor changes in solutions.
33
3.1.1.1. Chemicals and SDS-PAGE Solutions
Glycine, tris, glycerol, TCA, and potassium hydroxide (KOH) were purchased from
Merck Co. (Darmstadt, Germany); SDS was supplied from Sigma (St. Louis, MO);
methanol was purchased from Sigma Aldrich (Steinheim, Germany), and β-
mercaptoethanol, bisacrylamide, AP, and TEMED were purchased from
AppliChem (Darmstadt, Germany). Egg yolk and white proteins were separated
according to their molecule weights by SDS-Page (Bio-Rad, Hercules, CA).
Stacking (5%) and separation (12.5%) gels were prepared from a stock solution of
30% by weight of acrylamide: N,N’-methylenebisacrylamide (29:1). The prepared
solutions are described below.
Electrophoresis Buffer (5X) 1000 ml
Glycine 94 g
Tris 15.1 g
SDS (10%) 50 ml
Sample Buffer 20 ml
0.5 M TrisCl (pH 6.8) 2.5 ml

Glycerol 2 ml
SDS (10%) 4 ml
β-mercaptoethanol 1 ml
Bromophenol blue (0.05%) 0.5 ml
Deionized water 8 ml
5% Stacking Gel 20 ml
Deionized water 13.6 ml

30% Acrylamide 3.3 ml
1 M TrisCl (pH 6.8) 2.5 ml
SDS (10%) 200 µl
AP (10%) 200 µl
TEMED 20 µl
34
12.5% Separation Gel 50 ml

30% Acrylamide 16.7 ml
1.5 M TrisCl (pH 8.6) 12.5 ml
SDS (10%) 500 µl
AP (10%) 500 µl
TEMED 40 µl
Fixing Solution 250 ml

Methanol 82.5 ml
TCA (100%) 25 ml
Dye Solution 192.5 ml
Coomassie brilliant blue 150 ml

TCA (100%) 25 ml
KOH (10 N) 17.5 ml
3.1.1.2. Preparation of Samples

To construct a calibration curve, egg mixtures (yolk-white) were prepared by
mixing the pure yolk and white at different concentrations from 0 to 100%, w/w.
The LWE and liquid whole eggs with different ratios of egg white and water (65,
55, 40, 30, 20, 10, 5%, w/w) were also prepared. All samples were diluted twenty
times with deionized water. Diluted samples (100 µl) were mixed with sample
buffer (250 µl) and kept in boiling water bath for five minutes. Following the boiling
process, the samples were cooled immediately.
3.1.1.3. Electrophoresis
Approximately 10 µl of each cooled samples were loaded into a sample well of the
polyacrylamide gel. Electrophoresis was employed arranging a current of 30 mA
per gel until the bromophenol blue marker reached the bottom of the gel.
35
Following electrophoresis, gels were kept in a fixation solution overnight in order to
fix proteins. These gels were stained with the dye solution in order to detect
proteins about 3 hours and then coomassie-stained gels were washed with water
[84].
3.1.1.4. Analysis of Protein Bands

Stained protein bands in gels were analyzed by using image analysis software
(Phoretix 1D Pro, Totallab Inc., Warwickshire, UK). Protein band intensity was
measured by removing background intensity. The calibration curve was formed
using two protein bands having the highest intensity values at 45 and 120 kDa
molecular weight.
3.1.2. Determination of Egg Components by ATR-FTIR Spectroscopy

The aim of this analysis is detection of egg components quantitatively within a very
short time, where spectroscopic method was employed instead of reference
methods. Therefore, ATR-FTIR spectroscopy was used in order to quantitate
protein, lipid, water, and total soluble solid (TSS, °Brix) contents in the liquid egg
samples. The actual values of the components obtained from reference methods
were correlated with ATR-FTIR spectrum, and calibration models were
constructed to predict the components values.
3.1.2.1. Chemicals
Potassium sulphate (K2SO4), iron (II) sulfate heptahydrate (FeSO4*7H2O), copper
(II) sulfate pentahydrate (CuSO4*5H2O), salicylic acid (C7H6O3), sulfuric acid
(H2SO4), boric acid (H3BO3), and technique sodium hydroxide (NAOH) were
purchased from Merck Co. (Darmstadt, Germany) for Kjeldahl method. Chloroform
(CHCl3) and absolute alcohol were also purchased from Merck Co. (Darmstadt,
Germany) in order to use in total lipid analysis.
36
3.1.2.2. Analyses of Egg Components by Reference Methods
LWE, LEW, LEY, and LWE including egg white or water (65, 55, 40, 30, 20, 10,
5%, w/w) samples were prepared for protein (n=63), total lipid (n=67), total solid
(n=85), and total soluble solid (n=83) analyses.
As a reference method, Kjeldahl analysis was performed according to AOAC [85].

To calculate the percentage of protein in the samples, the nitrogen percent is
multiplied by a conversion factor of 6.25. Reagents of analysis: catalyst mixture
(K2SO4: FeSO4*7H2O:CuSO4*5H2O, 20:2:1), acid solution (salicylic – sulfuric acid,
3.5%), NaOH solution (33%), and boric acid indicator (pH 5). Approximately 1 g of
sample was used for each Kjeldahl flask. Experiments were carried out in
triplicate. The protein content of samples was calculated according to Equation 3.1
and 3.2.
!"##$%&$' !"#$ !"#$%& !" ! ! !""

%N = 𝑁 𝐻! 𝑆𝑂! ∗ ! !" !"#$%&
× !"#
× !"""
Eq. 3.1
Corrected acid volume = (mL std. acid for sample) - (mL std. acid for blank)
%N × 6.25 = %𝑝𝑟𝑜𝑡𝑒𝑖𝑛 Eq. 3.2
Total lipid content of samples was determined as described in the Official Methods
of Analysis of AOAC [86]. A well-mixed sample (4 g) was weighed into 100 mL
volumetric flask, and 25 mL mixed solvent (chloroform and absolute alcohol) was
included very slowly and shaking constantly until proteins coagulate, and are then
thoroughly broken down. 60-65 mL mixed solvent was added and stood
periodically shaked in every 5 min during 1 hour. To achieve the final volume, the
solution was filled up with the solvent, and waited until it becomes clear. 50 mL
aliquot was transferred to the beaker and evaporated in the water bath for drying
at 100 °C. Following evaporation, dry extract was dissolved in 5-10 mL chloroform,
filtered into weighed beaker through a Whatman #1 filter paper packed into stem
of funnel, and transferred all solution extract from bottom and sides of the beaker
with chloroform.
37
Finally, funnel and stem tip were washed. As expected, the filtrate was clear.
Chloroform was evaporated from the beaker in water bath. The beaker and the
contents were dried in the oven at 100 °C to achieve a constant weight. The
beaker was kept in air for 30 min for achieving constant weight, and then the first
weigh was measured. Experiments were carried out in triplicate. Total lipid content
was calculated according to Equation 3.3.
!!" !!!
% 𝑇𝑜𝑡𝑎𝑙 𝐿𝑖𝑝𝑖𝑑 = × 100 Eq. 3.3
!!
WS: Weight of sample
WB: Weight of beaker
WLB: Weight of lipid + WB
Moisture content of samples was analyzed by using AOAC method [85]. A well-
mixed egg sample (4 g) was accurately weighed into dried weighing bottle.
Weighing sample was kept in the oven at 110 °C for 22 hours for drying. After
drying process, the samples were cooled in a desiccator and reweighed until a
constant weight is achieved. Experiments were carried out in triplicate. Moisture
content of the samples reached constant weight was calculated according to
Equation 3.4.
!! !(!!" !!! )
% 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 = × 100 Eq. 3.4
!!
WB: Weight of bottles
WSB: Weight of solid + WB
38
TSS content (°Brix value) of samples was measured by an optical refractometer
(RFM 330, Bellingham + Stanley Ltd., Kent, UK). A 1-ml sample was poured into
refractometer glass prism and measurements were taken at 25 °C. The observed
data was recorded as °Brix value of sample.
3.1.2.3. ATR-FTIR Spectroscopy Measurements

The ATR-FTIR measurements were performed by using a Thermo Nicolet iS50
FTIR (Thermo Fisher Scientific Co., Waltham, MA) spectrometer equipped with a
single-bounce diamond crystal and a deuterated triglycine sulphate (DTGS)
detector. XT-KBr was utilized as the beamsplitter. A 1-ml of sample was used for
the measurements. Experiments were performed at ambient temperature. The
ATR-FTIR spectra of all egg samples were observed to be in the range of 400 –
-1 -1
4000 cm with a resolution of 8 cm . Each spectrum was collected by subtracting
the background from all 16 scans in the absorbance format. All measurements
were performed in triplicate.
3.1.2.4. Data Analysis

Data analysis was performed using PLS with Stand-alone Chemometrics Software
(Version PLS_Toolbox 8.0 for Windows 7, Eigenvector Research Inc., Wenatchee,
WA). ATR-FTIR spectrum contains 3734 points in total. Through simultaneous
determination of specific analytes in a complex media, working with
spectrophotometric techniques is difficult highly without manipulation due a huge
amount of data. Therefore, partial least squares (PLS) regression was used for
quantitative analysis of the egg components. Protein, lipid, moisture, and TSS
content of the egg samples were determined separately by using PLS models.
Utilizing the PLS method, ATR-FTIR spectra were correlated to the results of
reference methods performed for protein, lipid, moisture, and TSS analysis.
The progression of PLS regression involves; creating calibration and validation

data, pre-processing to calibration data before development of the model,
development of the model using pre-processed calibration data, and evaluation of
validation data. To analyze protein, a calibration model was constructed with 43
ATR-FTIR spectra. 20 spectra of the egg samples were also used for validation.
39
To improve the model performance, autoscale preprocessing was applied in order
to calibrate the data. Calibration and validation data sets of lipid analysis were
formed using 30 and 34 spectra, respectively. Mean center and smoothing
(Savitsky-Golay) preprocessing were applied in order to increase the model
efficiency. A calibration model was also constructed with 40 spectra for moisture
analysis. 45 spectra from samples were used in validation data set. Autoscale and
baseline (Automatic Whittaker Filter; lambda indicating baseline curvature, 100; p
indicating asymmetry to use with Whittaker filter, 0.001) preprocessing were
applied in order to improve the model performance. For TSS analysis, calibration
and validation data sets were formed using 40 and 43 spectra, respectively.
Smoothing and autoscale preprocessing were used to improve of model
performance.
The optimum number of latent variables (LVs) was selected depending on the
performance of the models that were evaluated by the statistic RMSEC (root mean
square error of calibration), RMSECV (root mean square error of cross validation),
RMSEP (root mean square error of prediction) values, a coefficient of
determination (R2) value for calibration and validation, and the percentage
cumulative variance captured by model. In PLS model, Venetian Blinds cross-
validation was employed. Limit of detection (LOD) and limit of quantitation (LOQ)
values were also estimated for protein, lipid, moisture, and TSS models.
!
− 𝑝𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑)!
!!!(𝑎𝑐𝑡𝑢𝑎𝑙
𝑅𝑀𝑆𝐸𝐶 =
𝑀−1
!
!!!(𝑎𝑐𝑡𝑢𝑎𝑙 − 𝑝𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑)!
𝑅𝑀𝑆𝐸𝑃 =
𝑁
The term “actual” refers true amount of the selected data set, whereas, the
“predicted” refers to a value computed by the model using spectral data, where M
is the number of samples used in the calibration data set and N is the number of
samples used in the validation data set.
40
3.1.3. Detection of Egg Adulteration by ATR-FTIR and NIR Spectrometer
The purpose of this study is to detect adulteration in the production of LWE by
addition of water using ATR-FTIR and NIR spectroscopy.
3.1.3.1. Preparation of Samples

Liquid (n=50) and dry (n=50) samples of LWE, LEY, LEW, and liquid whole egg
containing egg white and water (°Brix adjusted to 21) were prepared. During the
preparation of dry samples, A 5 gr of liquid samples was dried in the vacuum oven
for 12 hours at 50 °C. Dry samples were pestled in a mortar until they become
homogeneous. Dry samples are presented in Figure 3.1.
Figure 3.1. Dry samples of LWE, LEW, LEY, and liquid whole egg containing egg
white and water
3.1.3.2. ATR-FTIR Spectroscopy Measurements

The instrument mentioned in the section (3.1.2.3) was used also in this analysis.
The ATR-FTIR measurements were carried out for both liquid and dry samples. A
1-ml of liquid and 1g of dry samples were used for the measurements.
Experiments were performed at ambient temperature.
41
The ATR-FTIR spectra of all egg samples were observed as to be in the range of
-1 -1
400 – 4000 cm with a resolution of 4cm . Each spectrum was obtained by
collecting 32 scans in the absorbance format while the background was subtracted
from all scans. Experiments were performed in triplicate.
3.1.3.3. NIR Spectroscopy Measurements

The NIR measurements of both liquid and dry samples were performed by using a
Thermo Nicolet iS50 NIR module and InGaAs detector (Thermo Fisher Scientific
Co., Waltham, MA). XT-KBr was utilizes as the beamsplitter. A 2 ml of liquid and
2g of dry samples were used for the analysis. Experiments were performed at
ambient temperature. The NIR spectra of all egg samples were observed as to be
-1 -1
in the range of 4000 – 10000 cm with a resolution of 8cm . Each spectrum was
collected by taking 32 scans in the reflectance, where the background was also
subtracted from each scan. Dry samples were placed in a solid sample spinner
accessory, which is equipped with a motor that allows spinning. The
measurements of the dry samples were carried out in the sample spinner.
Experiments were performed in triplicate.
3.1.3.4. Data Analysis of ATR-FTIR and NIR measurements

Data analysis was performed using principal component analysis (PCA) with
Stand-alone Chemometrics Software (Version PLS_Toolbox 8.0 for Windows 7,
Eigenvector Research Inc., Wenatchee, WA). Data obtained from 50 liquid and 50
dry samples were used to construct a PCA model. Several pre-processing steps
were applied to the data while constructing the model. During the ATR-FTIR
measurements, baseline, autoscale, and the first derivative (Column-wise
Derivative; polynomial order 2) were applied in order to create a PCA cluster of
five liquid sample groups (LWE, LEY, LEW, and liquid whole egg containing egg
white and water). Autoscale and the first derivative were applied to the data
obtained from 50 dry samples. After classification of five sample groups, a second
PCA model was constructed with 30 liquid and dry samples (LWE and liquid whole
egg containing egg white and water) in order to employ a better classification of
samples that were adulterated with water.
42
Autoscale and smoothing were applied to data obtained from 30 liquid samples.
Similarly, baseline, autoscale, and the first derivative were utilized for pre-
processing of 30 dry samples.
During the NIR measurements, baseline and autoscale were applied in order to
create a PCA cluster of five liquid sample groups (LWE, LEY, LEW, and liquid
whole egg containing egg white and water) as well. Baseline, autoscale, and the
first derivative were also applied to the data obtained from 50 dry samples. After
the classification of five sample groups, a second PCA model was constructed
using both 30 liquid and dry samples (LWE and liquid whole egg containing egg
white and water). Similar to the former measurements, baseline and autoscale
were applied to the data obtained from 30 liquid samples. Baseline, autoscale and
first derivative were also used in order to pre-process 30 dry samples. The
performance of the models and the optimum number of principal components
(PCs) were determined based on RMSEC, RMSECV values and the explained
variance. In PCA model Venetian Blinds cross-validation was employed.
3.2. Analysis of Heat Treated LWE by CE

The purpose of this analysis is to demonstrate the effect of heat treatment on egg
proteins and to observe the changes in egg proteins by UV-VIS spectroscopy and
CE.
3.2.1. Chemicals
Boric acid and ammonium sulphate were supplied from Merck Co. (Darmstadt,
Germany); sodium dodecyl sulphate and sodium chloride were purchased from
Sigma Aldrich (St. Louis, MO), and sodium hydroxide was supplied from J.T.
Baker (Deventer, Holland). Pure proteins; ovalbumin, conalbumin, ovomucoid, and
lysozyme were supplied from Sigma Aldrich (St. Louis, MO).
3.2.2. Preparation of Samples

The samples (n=54) of LEW, LEY and LWE were prepared. LWE (approximately
20 mL) was heat-treated at 60 ± 0.2 °C, 64 ± 0.2 °C, and 68 ± 0.2 °C for five
minutes with a one-minute interval under laboratory conditions.
43
Samples stirred with a magnetic stirrer were heat-treated in a glass constant
temperature jacket connected to a water bath. LEW, LEY, and the treated LWE
samples were mixed with 1 M sodium chloride and 4.06 M ammonium sulphate
(1:1:2) and shaken for 1 minute. Mixed samples of LEW, LEY, and the treated
LWE were centrifuged at 21500g for 20 minutes at 4 °C in order to precipitate
proteins denatured by heating. After the centrifugation process, supernatants of
LEW and LEY were obtained and diluted fifteen times for CE measurement. The
supernatant of the LWE was taken and diluted twenty times and five times for UV-
VIS spectroscopy and CE measurements, respectively. Supernatants were diluted
with 100 mM borate buffer at pH 9.2 for measurement of CE and pure water for
UV-VIS spectroscopy.
To separate the egg yolk into two parts (granules and supernatant), it was diluted
at a ratio of 1:1 with 1% sodium chloride. The dilution was centrifuged at 4 °C with
10000g for 10 minutes [12]. After the centrifugation process, firm pellet of granules
on the bottom and an orange supernatant were obtained and kept until the
analysis. Supernatant was diluted, and granules were dissolved in buffer.
3.2.3. UV-VIS Spectrophotometer

Absorption spectra were measured by using an Agilent 8453 UV-VIS
spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA). Measurements
of centrifuged samples were carried out at 280 nm as triplicate. Pure water was
used as the blank sample.
3.2.4. CE Apparatus and Conditions

Experiments were performed using an Agilent G1600A model 3-D CE system
(Waldbronn, Germany) equipped with a 1200 series diode array detector (model
G1315B), an automatic sample injector, a temperature controller, and a high-
voltage power supply. The process was controlled with ChemStation software
(Agilent Technologies, Waldbronn, Germany). Separations were carried out in an
uncoated fused-silica capillary (Agilent Technologies, USA) of 50 µm id and 31.5
cm total length with effective length to the detector of 23 cm. Capillary was
inserted into the standard HP cassette.
44
The detector signal was recorded at 200 nm. Injections were performed at the
anodic end of the capillary, while detection was performed at the cathodic end.
Before optimum conditions were decided, the applied voltage (10, 15 kV) and 200,
250, 300 mM borate buffer were also tested. To achieve optimum resolution and
efficiency, capillary temperature was reserved at 25 °C, where a separation
voltage of 10 kV and a background electrolyte (BGE, 300 mM borate buffer at pH
9.2 containing 25 mM SDS) were used. The analytes were diluted with 100 mM
borate buffer at pH 9.2 and injected for 5 s at 40 mbar. A 23 cm effective capillary
length was used in order to obtain a satisfied resolution for the all peaks within
acceptable analysis duration by using this BGE composition. The current was
measured 88 µA under these conditions.
New capillaries were flushed with 1 M sodium hydroxide (30 minutes), DI water
(15 minutes), and then with the BGE for 35 minutes. To prevent adhesion of
protein on capillary, 20kV (20 s) voltage was applied to the capillary, and it was
flushed with 1 M sodium hydroxide (2 minutes), DI water (3 minutes), and then
BGE for 8 minutes between each run. The capillary was flushed at the end of the
working session via 1 M sodium hydroxide (10 minutes) and DI water (20
minutes).
3.2.5. Data Analysis

PCA performed in the section (3.1.3.4) was used during the classification of the
LWE samples for either untreated or treated ones. For treated ones, they were
also classified according to their treatment parameters. The data obtained from 54
samples in total were used to construct a PCA model. During PCA model
construction, several pre-processing steps were applied to the data. Baseline, the
first derivative, class centroid centering, and scaling were applied in order to
classify the samples as untreated or treated. Smoothing, the first derivative, and
mean center were also applied to create a PCA cluster of treated samples at
different treatment parameters. The model criteria assessed in the section
(3.1.3.4) was also considered for the evaluation of model performance.
45
3.2.6. Statistical Analysis
IBM SPSS Statistics software version 23.0 (SPSS Inc., Chicago, IL) was utilized
for statistical analysis. One-way analysis of variance (ANOVA) test was used to
examine the significance of within one temperature and between temperatures
differences of UV-VIS measurements. Duncan test was applied for the post-hoc
test, where the effect is commonly considered significant in the case of the
resulting p-value is small (0.05 is taken as threshold).
3.3. Effect of Heat Treated LWE on the Cake Quality

The aim of this analysis is to reveal the effect of heat-treated LWE on cake quality.
In this context, rheological properties of cake batter and the quality of baked cake
were analyzed, respectively.
3.3.1. Preparation of Heat Treated LWE

The LWE was heat-treated for two and five minutes at 60 ± 0.2 °C, 64 ± 0.2 °C,
and 68 ± 0.2 °C under laboratory conditions. Samples stirred with a magnetic
stirrer were heat-treated in a glass constant temperature jacket connected to water
bath. After the heating process, the samples were immediately cooled down to
ambient temperature.
3.3.2. Analysis of Cake Ingredient

Flour and sugar, the main components of cake, were purchased from commercial
channel (Ankara, Turkey). Protein, moisture, ash, wet gluten, and sedimentation
analyses were performed for estimation of quality of flour samples.
3.3.2.1. Protein Analysis

The nitrogen content of the flour sample was determined by means of the DUMAS
principle (Flash 4000, Thermo Scientific, Waltham, MA). Before analyzing, the
flour samples were dried at 135 °C for 2 hours. A 500 mg of dry sample was
wrapped and tightly pelleted into tin foil. After sample combustion, the percentage
of protein was calculated by multiplying the percent nitrogen by 5.7.
46
3.3.2.2. Moisture Analysis
Moisture content of the flour sample was determined according to a similar
procedure reported by Manley [87]. A 2 g of flour sample was weighed into dried
weighing bottle. The sample was kept in oven at 135 °C for 2 hours.
Drying of samples was continued until achieving a constant weight. Experiments

were carried out in triplicate. Moisture content of the samples was calculated
according to Equation 3.5.
!! ! (!!" !!!)
% 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 = × 100 Eq. 3.5
!!
WB: Weight of bottle
WSB: Weight of solid + WB
3.3.2.3. The Other Analyses by NIR Spectroscopy

Ash, wet gluten, and sedimentation values of flour sample were detected using a
TANIRPRO Lab NIR spectroscopy (Nanosens A.Ş., Ankara). Experiments were
performed at ambient temperature. The NIR spectrum of flour sample was
-1
observed as to be in the range of 5882 – 11111 cm . All measurements were
taken in reflectance format. Measurements were obtained in triplicate.
3.3.3. Cake Batter Preparation and Baking Procedure

A standard sponge cake batter was prepared following the recipe described by
Bent et al. [88]. Wheat flour, sugar, and egg are the main ingredients in sponge
cakes. The sponge cakes were prepared with 100% wheat flour, 100% sugar, and
125% LWE (all percentages are given on flour weight basis). Orthodox procedure
was used during preparation of the sponge cake. According to this method, egg
and sugar were whisked together to obtain stable foam, and then flour is folded in
carefully to avoid any air loss.
47
As the first step, the heat-treated LWE and sugar were mixed for 4 minutes at
speed 6 with Kitchen Aid Professional mixer (St. Joseph, MI). Then, flour was
mixed for 1 minute at speed 4. A 60 gr of cake batter was placed into 250 ml a
glass beaker. Four samples were weighed from the same batter and cakes were
baked for 25 minutes at 175 °C in a conventional oven (Arçelik A.Ş., Istanbul,
Turkey). The baked cakes are shown in Figure 3.2.
Figure 3.2. Baked cakes
3.3.4. Rheological Analysis of Cake Batter

The rheological measurements were conducted by a rheometer (Malvern
Instruments – Kinexus Lab+, Malvern, UK). All measurements were carried out at
25 °C using a parallel plate geometry (20 mm diameter). The batter sample was
placed between the plates, where the gap adjusted to 1 mm and the outside
portion of the plate was carefully trimmed with a spatula. The experiments were
carried out under steady-shear conditions, in which the shear rate was ranged
-1 -1
from 0.1s to 100s in 2 minutes. The data of shear rate-viscosity and shear rate-
shear stress were collected throughout the experiment. The experiments were
performed in duplicate.
3.3.5. Cake Analysis
3.3.5.1. Moisture Loss

Moisture loss of the cakes during baking was calculated by measuring the weights
of batter samples and cake samples after baking [89].
48
The results of weight loss were expressed as the percentage of the initial value.
Weight loss of four cakes was measured and the average value was obtained from
one experiment. The experiments were performed in duplicate. The moisture loss
(%) was calculated according to Equation 3.6.
!"#$!!!"##$% !!"#$!!!"#$
𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑙𝑜𝑠𝑠 % = !"#$!!!"##$%
×100 Eq. 3.6
Weightbatter : Weight of cake batter (g) before baking
Weightcake : Weight of baked cake (g) measured immediately after removed from
the oven
3.3.5.2. Color Analysis

The surface color of the baked cakes was measured using a Minolta CM-5
spectrophotometer (Osaka, Japan). Measurements were performed in reflectance
format. CIE L* (lightness), a* (red), b* (yellow) color values were recorded. At the
beginning of the experiment, white calibration was performed by using the built-in
white calibration plate of the instrument. Measurements were taken as the final
format value of L*, a*, b*. Two readings of each cake were taken from different
region of surface and the average value was recorded. Color of two cake samples
was measured and the average value was obtained from one experiment. The
experiments were carried out in duplicate. Color change (∆E) was calculated
according to Equation 3.7.
∆𝐸 = [ 𝐿∗ − 𝐿! !
+ (𝑎∗ − 𝑎! )! + (𝑏 ∗ − 𝑏! )! ]!/! Eq. 3.7
3.3.5.3. Porosity Analysis

Porosity analysis was performed mechanically by compacting of baked cake to
eliminate all the pores [90]. After the baking process, the interior of the cake
sample was analyzed immediately. Sample was placed into a cylindrical container
of diameter 4.7 cm. The sample was pressed under 974 g loads for 5 minutes.
49
Initial height of the sample before the pressing and the final height of the sample
after pressing were recorded, assuming that all the pores inside the cake were
compressed. The experiments were taken in duplicate. Porosity (%) was
calculated according to Equation 3.8.
!! !!!
𝑃𝑜𝑟𝑜𝑠𝑖𝑡𝑦 % = ×100 Eq. 3.8
!!
Hi : Initial height (cm) of baked cake sample
Hf : Final height (cm) of baked cake sample
3.3.5.4. Texture Analysis

Cohesiveness, and hardness of cakes were measured by using a Texture
Analyzer (TA Plus, Lloyd Instruments, West Sussex, UK). Measurements were
taken at 25 °C after 1 hour cooling to room temperature. Samples were cut into a
cubic shape with a 25 x 25 x 25 mm dimension from the center of cakes. The
samples were compressed at a rate of 100 mm/min to 27% of its initial height with
a cylindrical probe (10 mm diameter) under the influence of 0.1 N forces. Two
cake samples were measured and the average value was obtained from one
experiment. The experiments were taken in duplicate. Gumminess of cakes was
also calculated as the function of hardness x cohesiveness in Equation 3.9 [91].
𝐺𝑢𝑚𝑚𝑖𝑛𝑒𝑠𝑠 = 𝐻𝑎𝑟𝑑𝑛𝑒𝑠𝑠 × 𝐶𝑜ℎ𝑒𝑠𝑖𝑣𝑒𝑛𝑒𝑠𝑠 Eq. 3.9
3.3.5.5. Specific Volume

Specific volume of cakes was determined by a rapeseed displacement method
(AACC Method 74-09) [92]. Analysis was performed at 25 °C after 1 hour cooling
to room temperature. Before analyzing, cake weight was recorded. Cake was
placed into a glass beaker with known of weight and volume. Then, the beaker
was tapped via rapeseed with known of density. The surface was smoothed with a
ruler and the beaker was again tapped with rapeseed.
50
Tapping and smoothing were continued until constant weight was achieved
between the sequential measurements. The total weight of the beaker (sample
and rapeseed) was recorded. Specific volume was calculated according to
Equation 3.10, 3.11, 3.12, and 3.13.
𝑊!""# = 𝑊! − (𝑊! + 𝑊! ) Eq. 3.10

!!""#
V!""# = !!""#
Eq. 3.11
𝑉!"#$ = 𝑉! − 𝑉!""! Eq. 3.12

!!"#$
𝑆𝑉!"#$ = !!
Eq. 3.13
Wseed : Weight of rapeseed (g) in beaker
Wt : Total weight of beaker (g) containing sample and rapeseed
Wc : Weight of cake (g)
Wb : Weight of beaker (g)
Vseed : Volume of rapeseed (cm3)
ρseed : Density of rapeseed (g/cm3)
Vcake : Volume of cake (cm3)
Vb : Volume of beaker (cm3)
SVcake : Specific volume of cake (cm3/g)
3.3.5.6. Statistical Analysis

Statistical analysis performed in the section (3.2.6) was applied using IBM SPSS
Statistics software version 23.0 (SPSS Inc., Chicago, IL) to examine the
significance differences in moisture loss, porosity, color, texture and specific
volume measurements of the baked cakes prepared with untreated and heat-
treated LWE at 60 °C, 64 °C and 68 °C for 2 and 5 minutes.
51
4. RESULTS AND DISCUSSIONS
4.1. Determination of LWE Quality
In this section, methodologies for determination of LWE quality are presented. In
this manner, three different approaches are utilized; SDS-PAGE, ATR-FTIR
spectroscopy, and ATR-FTIR & NIR spectroscopy, respectively. Proposed
techniques are demonstrated through experiments and results are discussed in
detail.
4.1.1. Determination of Yolk-White Ratio in LWE by SDS-PAGE

An SDS-PAGE analysis was performed using liquid egg samples including yolk-
white at different concentrations from 0% to 100%, with an interval of 10%. The
SDS-PAGE gel image of protein bands of the samples is presented in Figure 4.1.
Figure 4.1. SDS-PAGE gel image of protein bands of liquid egg samples. 1. LWE,
2. LWE, 3. LWE, 4. 100%Y, 5. 90%-Y, 6. 80%-Y, 7. 70%-Y, 8. 60%-Y, 9. 50%-Y,
10. 40%-Y, 11. 30%-Y, 12. 20%-Y, 13. 10%-Y, 14. 0%-Y, 15. marker. Y: Yolk,
LWE: Liquid whole egg
52
The molecular weights of proteins of the egg white and the yolk were determined
according to marker proteins (lane 15) previously identified as molecular weights.
Both the egg white and the yolk proteins were sorted according to their molecular
weights from the top to the bottom. Ovalbumin and conalbumin in the albumen
(lane 14) were determined at molecular weights of 45kDa and 79kDa, respectively.
The measured molecular weights are consistent with the results presented in [45].
Since ovalbumin (54%) and conalbumin (12%) are the most abundant proteins in
the albumen, they can be easily monitored. On the other hand, the amount of
other albumen proteins (ovomucin 3.5%, lysozyme 3.4%, etc.) is considerably low
compared to the ovalbumin and conalbumin, so it is not practical to determine
them in the gel image. However, much more proteins could be described in the
egg yolk compared to the albumen. As can be seen from the gel image, the egg
yolk (lane 4) is composed of many polypeptides with a variety of molecular weights
from 20kDa to over 200kDa. To identify these polypeptides in the gel image,
results provided by different studies in the literature were utilized, which were
listed in Table 4.1. While the LDL apoproteins were determined at 120kDa
(apovitellenin Va), 60-70kDa, 50-60kDa (apovitellenin III), and 20kDa
(apovitellenin II), HDL apoproteins were identified at 79kDa and 30kDa. The other
free proteins; α-livetin, α-, β- livetins, and phosvitin were observed at 50-60kDa,
38-40kDa, and 45kDa, respectively.
Table 4.1. Identification of the yolk peptides from SDS-PAGE profile according to
different studies
Molecular weight (kDa) Identification References
120 apovitellenin Va [93]
79 apo-HDL [94]
60-70 apo-LDL [95], [96]
50-60 α-livetin, apovitellenin III [93], [97]
45 Phosvitin [94]
38-40 α-, β- livetins [98]
30 apo-HDL [94], [97]
20 apovitellenin II [97]
Considering the gel image, protein band intensity changes depending on the yolk-
white concentration. Namely, while the ratio of the yolk in liquid egg is high, the
band intensity of the yolk proteins is also high.
53
On the other hand, when the ratio of the egg white in liquid egg is high, the band
intensity of the albumen proteins is high. It can be concluded that these bands
intensities are directly related to the concentration of the proteins. Thus, the band
intensities can be used to detect the egg yolk-white ratio in the liquid egg. To
determine the yolk-white ratio in the liquid egg, one protein band representing the
concentration of egg white and another protein band representing the
concentration of the yolk were both selected to construct the calibration curve. The
ratio of the band intensities was used in order to eliminate the error in the case of
using single protein band intensity in the gel image. Ovalbumin (45kDa) and
apovitellenin Va (120kDa) were selected for the representation of the albumen and
the yolk, respectively. They were preferred due to their high intense bands in the
albumen (lane 14) and the yolk (lane 4) gel profiles. Furthermore, there is no
protein band at 120kDa in the albumen lane (14), so this band represents only the
yolk concentration. Although ovalbumin and phosvitin bands are present at 45kDa,
phosvitin (lane 4) can be neglected because of its low band intensity. As seen in
Figure 4.1, as the albumen concentration increases in the liquid egg, ovalbumin
band intensity (45kDa) also increases. Similarly, an increase in the yolk
concentration results in increase of apovitellenin Va band intensity (120kDa).
Figure 4.2. An image of protein bands detected by image analysis software
54
Band volume intensity of the egg proteins was determined by using an image
analysis program. The SDS-PAGE image (Figure 4.1) was converted to another
image (Figure 4.2), which explicitly indicates the detected protein bands. A protein
band graph (Figure 4.3) of each lane was formed with pixel position of the bands
versus the intensity. Then, the band volume intensity of each protein in
corresponding lane was determined.
Figure 4.3. Pixel position and intensity of the bands in the lane
Band volume intensities of the ovalbumin and apovitellenin Va proteins were

selected according to their pixel position in the lane. The measured band volume
intensities of these proteins between the lanes from 14 to 4 are presented in Table
4.2. These lanes used in the construction of the calibration curve contain liquid
egg with the concentration of yolk from 0% (lane 14) to 100% (lane 4). As can be
seen in the Table 4.2, a decrease in the band volume intensity of ovalbumin was
observed depending on the increase in the yolk concentration in the liquid egg.
There was also an increase in the band volume intensity of apovitellenin Va,
however, the band volume intensity of apovitellenin Va (120kDa) in the albumen
sample was not detected as seen in the Figure 4.1 (lane 14). Therefore, the
intensity value was set as 0. On the other hand, the band volume intensity at
45kDa in the yolk sample (lane 4) could be measured thanks to the presence of
phosvitin protein.
55
Table 4.2. Band volume of apovitellenin Va (120kDa) and ovalbumin (45kDa)
proteins
Lane (% yolk Band volume Band volume
concentration) (intensity, 120kDa) (intensity, 45kDa)
14 (0% Y) 0 4.41×105
13 (10% Y) 0.49×105 4.26×105
12 (20% Y) 0.88×105 4.11×105
11 (30% Y) 1.24×105 4.17×105
10 (40% Y) 1.67×105 3.79×105
9 (50% Y) 2.19×105 3.65×105
8 (60% Y) 2.42×105 3.23×105
7 (70% Y) 2.79×105 2.85×105
6 (80% Y) 3.13×105 2.49×105
5 (90% Y) 3.20×105 1.85×105
4 (100% Y) 3.09×105 1.73×105
The ratio of band volume intensities (120/45kDa) of apovitellenin Va and

ovalbumin proteins was determined using the intensity values provided in Table
4.2. Then, the calibration curve given in Figure 4.4 was constructed using the
ratios of the band volume (120/45kDa) and the concentration of the yolk (from 0%
to 100%) in the liquid egg samples. A high coefficient of determination value (R2 =
0.992) was obtained from the model. An increasing trend in the ratio values of the
band volume was observed by increasing in the yolk concentration.
Figure 4.4. The calibration curve of the concentration of the yolk
56
The accuracy of the model was validated using different liquid egg samples known
actual yolk-white ratio (n=11, including different shell eggs in the size of small,
medium, and large) to use the model in the prediction of the yolk-white ratio in
different LWE samples. For that purpose, SDS-PAGE analysis of these new
samples was performed, where SDS-PAGE image of these liquid egg samples is
showed in Figure 4.5. Similar to the previous (Figure 4.1) analysis, the band
volume intensities of the proteins for each lane (from 2 to 12) in the Figure 4.5
were also obtained using the image analysis program. Then, the ratio of the band
volume intensity of apovitellenin Va and ovalbumin proteins (120/45kDa) of each
sample was calculated. By substituting the ratio of band volume intensities of
these proteins in the model, concentration of the yolk (%) in the samples was
predicted. The actual and the predicted concentration of the yolk (%) in eleven
liquid egg samples are listed in Table 4.3. Concentration results demonstrate that
the predicted values are consistent with the actual values.
The relative error of the results for almost all samples are less than 10%, which
make the model quite accurate to be used for the determination of the yolk
concentration in different LWE samples.
Figure 4.5. SDS-PAGE gel image of the protein bands of eleven liquid egg
samples (from lane 2 to 12)
57
Table 4.3. Actual and predicted concentration of the yolk in liquid egg samples
Actual concentration Predicted concentration
Lane REa (%)
of the yolk (%) of the yolk (%)
2 28.0 24.0 14.0
3 30.0 27.4 8.7
4 29.5 27.1 8.0
5 25.7 25.7 0.0
6 29.6 30.4 2.8
7 30.1 28.0 7.0
8 32.9 29.8 9.4
9 29.3 30.3 3.3
10 29.0 29.4 1.3
11 35.5 35.8 0.9
12 31.4 29.7 5.3
a
Relative error
4.1.1.1. Determination of Yolk-White Ratio in LWE Containing Egg White

An SDS-PAGE analysis was performed using the samples of liquid whole egg
(LWE) containing the egg white. Egg white at different concentrations of 65, 55,
40, 30, 20, 10, and 5% was added to LWE. The SDS-PAGE image of these
samples is presented in Figure 4.6.
containing egg white. 1. Marker, 2. 65:35 - EW: LWE, 3. 55:45 – EW: LWE, 4.
40:60 – EW: LWE, 5. 30:70 – EW: LWE, 6. 20:80 – EW: LWE, 7. 10:90 – EW:
LWE, 8. 5:95 – EW: LWE, 9. LWE All concentration is given as percentage (%).
EW: Egg white, LWE: Liquid whole egg
58
As can be seen in the figure, while the albumen concentration increases (from
lane 9 to 2) in the liquid egg, the band intensity of apovitellenin Va (120kDa)
decreases due to the decrease in the yolk concentration. However, any
considerable change in the band intensities of ovalbumin and phosvitin proteins
(45kDa) was not observed. The band volume intensity of the proteins for each lane
(from 2 to 9) was determined by the image analysis program. The measured band
volume intensities of apovitellenin (120kDa) and ovalbumin (45kDa) are listed in
Table 4.4. As presented in the table, the band volume intensity of apovitellenin Va
increases (from lane 2 to 9) due to the decrease in the albumen concentration.
Considering the band at 45kDa, similar values in the band intensity volume were
obtained.

(120kDa and 45kDa)
Band volume (intensity, Band volume (intensity,
Lane
120kDa) 45kDa)
2 1.05×105 6.09×105
3 1.27×105 6.08×105
4 1.40×105 6.05×105
5 1.65×105 6.93×105
6 1.78×105 6.18×105
7 2.22×105 7.23×105
8 2.12×105 6.36×105
9 2.20×105 6.45×105
The ratio of the band volume intensity (120/45kDa) was determined using the
intensity values given in the Table 4.4. Yolk concentrations of the samples were
predicted utilizing the model, where ratio intensity values were used. The actual
and the predicted concentration of the yolk (%) in the samples are provided in
Table 4.5. Considering the relative error values, the model can predict the yolk
concentration with a quite accuracy (error values less than 10%) for the samples
containing additional egg white up to 30%. However, the model loses the validity
when the yolk concentration of the samples containing additional egg white (40,
55, and 60 %), where the obtained error values were higher than 10%. This may
be due to the fact that the band intensity value of apovitellenin Va (120kDa) could
not be measured correctly because of decreasing band volume (lane 2, 3, and 4).
59
Consequently, the model is more suitable in predicting yolk concentration of the
LWE samples containing more than 20% yolk concentration.
Table 4.5. Predicted concentration of yolk in the samples containing egg white
Actual
Additional egg Predicted concentration
Lane concentration of REa (%)
white (%) of the yolk (%)
the yolk (%)
2 65 10.5 15.9 50.9
3 55 13.5 19.1 41.1
4 40 18.0 21.0 16.4
5 30 21.0 21.6 2.7
6 20 24.0 25.8 7.4
7 10 27.0 27.2 0.8
8 5 28.5 29.4 3.0
9 0 30.0 30.1 0.2
a
Relative error
4.1.1.2. Determination of Yolk-White Ratio in LWE Containing Water

An SDS-PAGE analysis of LWE samples containing water was conducted. Water
at different concentrations of 65, 55, 40, 30, 20, 10, and 5% was added to LWE.
The gel image of these samples is provided in Figure 4.7.
containing water. 1. Marker, 2. LWE, 3. 5:95 – W: LWE, 4. 10:90 – W: LWE, 5.
20:80 – W: LWE, 6. 30:70 – W: LWE, 7. 40:60 – W: LWE, 8. 55:45 – W: LWE, 9.
65:35 – W: LWE All concentration is given as the percentage (%). W: Water, LWE:
Liquid whole egg
60
As can be seen in the figure, the protein band intensities of the albumen and the
yolk (from 2 to 9) decrease as water content of the samples increase. A decrease
in the band volume of apovitellenin Va and ovalbumin were also observed in the
lanes (from 2 to 9). The band volume intensity of the proteins in each lanes (from 2
to 9) was determined by the image analysis program. The measured band volume
intensities of apovitellenin (120kDa) and ovalbumin (45kDa) were listed in Table
4.6. The band volume intensity of apovitellenin Va and ovalbumin (from lane 2 to
9) decreases due to the increase in the additional amount of water.

(120kDa and 45kDa)
Band volume (intensity, Band volume (intensity,
Lane
120kDa) 45kDa)
2 1.61×105 4.43×105
3 1.57×105 4.26×105
4 1.58×105 4.19×105
5 1.22×105 3.57×105
6 1.24×105 3.53×105
7 1.05×105 3.01×105
8 0.83×105 2.55×105
9 0.61×105 2.04×105
The ratio of the band volume intensity (120/45kDa) was determined using the
intensity values given in the Table 4.6. The ratio of the band intensity does not
change over the samples (from lane 2 to 9) since a simultaneous decrease in the
band intensity was observed for both ovalbumin and apovitellenin Va proteins.
Yolk concentration of the samples was predicted by the model using the ratio
intensity values (120/45kDa). The actual and the predicted concentrations of the
yolk (%) in the samples are presented in Table 4.7. Actually, addition of water to
LWE samples caused to a decrease in the both albumen and yolk concentrations.
As a result, the actual yolk concentration decreased by addition of water.
However, any difference among the predicted yolk concentration values was not
observed since the same ratio values of the band intensity were used in the
model.
61
Table 4.7. Predicted concentration of the yolk in the samples containing egg white
Actual concentration Predicted concentration
Lane Additional water (%)
of the yolk (%) of the yolk (%)
2 0 30.0 31.9
3 5 28.5 32.1
4 10 27.0 33.0
5 20 24.0 30.2
6 30 21.0 30.9
7 40 18.0 30.6
8 55 13.5 28.8
9 65 10.5 26.8
It can be concluded that detection of adulteration with water in LWE samples can
be performed by using this SDS-PAGE analysis method. In food industry, food
producers may demand LEPs including different concentration of yolk-white, such
as LWE containing higher egg white. However, food fraudsters may make an
adulteration in the LEPs using its own non-compositional substance. In this work,
detection of LWE adulteration with water was aimed. Some food producers
demand to use of LWE including more egg white than normal LWE in their
products. However, food fraudsters may use water as a substitute in the LEPs. In
this study, it is demonstrated how the SDS-PAGE gel image changes depending
on the use of egg white or water in the LWE samples. If the LWE sample includes
water instead of egg white, the yolk concentration would be in the nominal range.
However, when the LWE sample contains egg white, the yolk concentration would
be less than the nominal range. This method is an ad hoc way of analyzing
unknown LWE sample, in which the yolk-white ratio can easily be detected.
Conventionally, SDS-PAGE is used for the characterization of the proteins and/or

purification of the proteins. A composition of yolk proteins adsorbed onto an oil-in-
water interface [21], separation and identification of proteins in egg white and yolk
[42], assessment the binding type of heat-induced aggregation of albumen protein
[46], and characterization of major allergens of egg white [78] were performed by
SDS-PAGE. A different approach is proposed in this study, which utilizes SDS-
PAGE for the quantitative analysis of yolk-white ratio in the liquid egg samples. As
a result, the quality of the LEPs can be determined by using this approach.
62
4.1.2. Determination of Egg Components by ATR-FTIR Spectroscopy
Quantitative analysis of egg components that are protein, lipid, moisture, and total
soluble solid (TSS) was conducted by ATR-FTIR spectroscopy.
4.1.2.1. ATR-FTIR Spectra of Samples

A large number of (n=85) different liquid egg samples including LWE, liquid egg
white (LEW), liquid egg yolk (LEY), LWE (containing egg white), and LWE
(containing water) were analyzed by ATR-FTIR spectroscopy. ATR-FTIR spectra
of the LEY, LWE, and LEW are shown in Figure 4.8.
Figure 4.8. ATR-FTIR spectra of the LEY, LWE, and LEW
As can be seen in the figure, a couple of well resolved bands, which are assigned
to functional groups of the lipid, protein, and water in the egg, have been
observed.
63
These bands were enumerated in the figure for the identification convenience. A
functional group and vibration mode of the bands of lipid, protein, and water
components are listed in Table 4.8.
Table 4.8. Wavenumbers of bands of the egg samples with the assigned functional
groups and the modes of vibration
No Wavenumber (cm-1) Functional group Mode of vibration
1 3000-3700 –O–H (H2O) Stretching
2 2920 –C–H (CH2) Stretching (asymmetry)
3 2849 –C–H (CH2) Stretching (symmetry)
4 1743 –C=O (ester) Stretching
5 1590-1720 –C–O, –C–N Stretching
6 1541 –N–H, –C–N Bending, stretching
7 1462 –C–H (CH2, CH3) Bending (scissoring)
8 1230 –C–O, –CH2– Stretching, bending
9 1159 –C–O, –CH2– Stretching, bending
10 1085 –C–O Stretching
In the LEY spectrum, seven bands were observed at 2920 cm-1 (2), 2849 cm-1 (3),
1743 cm-1 (4), 1462 cm-1 (7), 1230 cm-1 (8), 1159 cm-1 (9), and 1085 cm-1 (10) that
were associated with the functional groups of the lipid component of the yolk [56].
On the other hand, these bands could not be observed in the LEW spectrum since
the LEW does not contain lipid component. Even if the LWE spectrum also
includes these bands sourced from the yolk constituent, their intensities are lower
in LWE compared to LEY. The region between 1590 – 1720 cm-1 (5, amide I band
region) in LEY, LWE, and LEW spectra can be related to the secondary structure
of the proteins. Therefore, this protein band was observed in all spectrum due to
presence of the protein in both white and yolk constituents. The band appeared at
1636 cm-1 is especially characterized by the amide groups involved in the β- sheet
structure [99]. The other band approximately at 1541 cm-1 (6) is characterized by
the amide II band of the protein, so it could be seen in LEY, LEW, and LWE
spectra [100]. The last component, water band, was observed with a high intensity
in LEY, LWE, and LEW having a wide range in the spectrum between 3000 and
3700 cm-1 (1) [101]. Among all spectra, the highest water band was observed in
LEW due to including a high amount of water while the lowest water band was
seen in LEY spectra because of the low water content.
64
4.1.2.2. Results of Reference Analyses
All of 85 samples in different form of liquid eggs, which are LWE, LEW, LEY, and
LWE containing egg white and water, were analyzed by reference methods in
order to determine their actual protein, lipid, moisture, and TSS contents. The
actual protein (Kjeldahl method), lipid (AOAC method), and moisture (AOAC
method) content of these samples were detected according to Equation 3.1, 3.2,
3.3, and 3.4, respectively. The observed value in the refractometer (at 25 °C) was
recorded as the actual TSS content of the samples. The detected protein contents
in LWE, LEW, and LEY samples are listed in Table 4.9. While the highest protein
content was detected in LEY samples, the least protein amount was observed in
LEW samples. Consequently, detected values were compared with the literature,
where protein values are consistent with the reported results in [5,10].
Table 4.9. A lower, average, and upper actual content of protein (%, w/w) in LWE,
LEW, and LEY samples
Lower (%) Average (%) Upper (%)
LWE 13.1 14.0 15.7
LEW 10.7 12.5 15.1
LEY 16.7 17.9 19.0
The actual contents of the lipid component in LWE, LEW, and LEY samples are
presented in Table 4.10. As known, the highest lipid content was detected in LEY
samples. On the other hand, the lipid content of LEW was obtained relatively low,
so it can be neglected in the LEW samples. The lipid contents of LWE and LEW
were found consistent with the results in the literature. However, the detected lipid
values in LEY samples remain slightly below the results in the literature [5,10].
Table 4.10. A lower, average, and upper actual content of lipid (%, w/w) in LWE,
LWE 7.0 8.5 10.4
LEW 0.1 0.1 0.1
LEY 19.2 23.3 25.7
65
Measured moisture contents of LWE, LEW, and LEY samples are listed in Table
4.11. The least moisture content was detected in LEY samples, whereas the
highest amount of moisture was obtained in LEW samples. The measured
moisture values in LWE, LEW, and LEY are consistent with the results in the
literature [5,10].
Table 4.11. A lower, average, and upper actual content of moisture (%, w/w) in
LWE, LEW, and LEY samples
LWE 75.1 76.9 78.6
LEW 84.8 87.5 89.1
LEY 49.3 51.9 54.6
TSS (°Brix) contents of LWE, LEW, and LEY samples are given in Table 4.12. As
expected, the highest and the least TSS contents were found in LEY and LEW
samples, respectively. The TSS and total solid (according to the results in Table
4.11) contents of the samples (LWE, LEW, and LEY) were obtained close to each
other.
Table 4.12. A lower, average, and upper actual content of TSS (%, w/w) in LWE,
LWE 24.8 26.5 28.0
LEW 13.5 16.2 19.2
LEY 45.3 46.6 48.4
The protein, lipid, moisture, and TSS contents of the samples can be detected by
the reference methods with a quite accuracy. However, these methods are very
time consuming, and a preliminary preparation is required before the analysis.
Furthermore, many chemicals are unnecessarily used in the analysis. Therefore, a
better approach has been required to determine liquid egg components.
66
4.1.2.3. Data Analysis
PLS analysis was performed to determine the protein, lipid, moisture, and TSS
contents in the liquid egg samples (used in reference analyses, 4.1.2.2).
Calibration models were constructed for each component using ATR-FTIR spectra
and the actual amounts of protein, lipid, water, and TSS of the samples.
Performance of models was evaluated according to regression error values
(RMSEC, RMSECV, and RMSEP) and coefficient of determination values (R2) of
calibration and validation data sets. Moreover, LOD and LOQ values were
determined to measure the quality of the calibration models. Protein, lipid,
moisture, and TSS contents of the samples were predicted by these calibration
models. Then, the predicted values were compared to the actual values.
A comparison of the actual and predicted protein values of the calibration and
validation data sets is shown in the Figure 4.9(a) and (b), respectively.
67
Figure 4.9. Correlation between the actual and predicted values of protein, (a)
Calibration data set, (b) Validation data set
The first five latent variables (LVs) were chosen to form the model for the protein
analysis. The total explained variance of the first five LVs is 98.3%. The error
values of the model, coefficient of determination values, and LOD-LOQ values of
the method are given in Table 4.13. As can be seen in the table, low error values
(RMSEC, RMSECV, and RMSEP) are obtained with this model. In addition, high
coefficient of determination values (R2) are achieved for both calibration and
validation data sets. LOD and LOQ values are calculated as 1.8% and 5.4%,
respectively. Both high coefficient of determination values and low error values
showed that the model can be used to detect protein content in liquid egg
samples.
68
protein analysis and LOD-LOQ values of the method
RMSEC RMSECV RMSEP R2 (Cal) R2 (Val) LOD (%) LOQ (%)
0.404 0.573 1.227 0.982 0.950 1.8 5.4
The actual and the predicted lipid content of the calibration and validation data
sets are given in Figure 4.10(a) and (b). To construct the model for the lipid
analysis, the first two LVs were selected. The cumulative variance of the first two
LVs is 99.1%. The regression statistics values of the model and LOD-LOQ values
of the method are provided in Table 4.14. The model error values (RMSEC,
RMSECV, and RMSEP) are within acceptable limits. In calibration and validation
data sets, high coefficients of determination values (R2) are obtained. 2.1% and
6.2% are LOD and LOQ values of the method, respectively. According to
regression statistic values, a promising performance was obtained for the lipid
model.
69
Figure 4.10. Correlation between the actual and predicted values of lipid, (a)
lipid analysis and LOD-LOQ values of the method
0.978 1.213 1.229 0.993 0.992 2.1 6.2
The plot of the actual versus the predicted values of moisture for calibration and
validation data sets are provided in Figure 4.11(a) and (b). The model for moisture
analysis was constructed using the first four LVs. The total explained variance for
these LVs is 87.0%.
70
Figure 4.11. Correlation between the actual and predicted values of moisture, (a)
71
Table 4.15 provides the regression statistics values and LOD-LOQ values of the
method. The highest coefficients of determination values (0.997 and 0.994) among
the models are obtained for calibration and validation data sets of the moisture
analysis. Model error values (RMSEC, RMSECV, and RMSEP) were relatively
low. LOD and LOQ values are obtained 2.8% and 8.5%, respectively. A high
coefficient of determination value for calibration and validation data sets and low
model error values indicates that satisfactory of the moisture model.
moisture analysis and LOD-LOQ values of the method
0.562 0.873 0.831 0.997 0.994 2.8 8.5
Figure 4.12(a) and (b) show that the actual and the predicted amounts of TSS of
calibration and validation data sets. The first four LVs were used in the formation
of TSS model. Cumulative variance of these LVs is 97.5%.
72
Figure 4.12. Correlation between the actual and predicted values of total soluble
solid (TSS), (a) Calibration data set, (b) Validation data set
The regression error values, coefficient of determination values, and LOD-LOQ

values of the method are listed in Table 4.16. The low error values (RMSEC,
RMSECV and RMSEP) and high coefficient of determination values of calibration
and validation data sets are observed for TSS model. 4.7% and 14.3% are LOD
and LOQ values, respectively. The low error values and high R2 values validates
the model, which can be used to determine TSS content in liquid egg samples.
TSS analysis and LOD-LOQ values of the method
0.941 1.822 1.845 0.992 0.972 4.7 14.3
73
It can be concluded that protein, lipid, moisture, and TSS content of liquid egg
samples can be estimated using these improved PLS models. This analysis has
several superiorities compared to the reference methods, such as Kjeldahl
(protein) and AOAC (lipid and moisture). The whole components of egg can be
detected with a single measurement of sample. It is a preferable technique in
terms of the robustness and repeatability. The analysis can be conducted within a
very short time. The time performance is highly remarkable since the former
reference methods take a long preparation and analysis duration. The proposed
technique is quite easy to be performed compared to other reference methods.
Furthermore, the amount of sample used in the analysis is considerably low,
where no chemical or solution is required during the preparation of sample. At last,
it is an environmentally friendly technique since it does not produce any waste
after the analysis.
To determine protein, lipid, moisture, and TSS content in various food products,
several analyses utilizing infrared spectroscopy have been performed in the
literature. In [36], infrared transmission spectroscopy was used for the
measurement of protein, lipid, and total solid content of liquid egg samples. Fat,
moisture, and protein contents in salmon fillets was determined in [102] by using
NIR diffuse spectroscopy. The quantitative analysis of fat, protein, and lactose of
raw milk [103], and the analysis of fat, moisture, and solids-non-fat in butter [104]
were conducted by NIR spectroscopy.
To my best knowledge, determination of the whole components (protein, lipid,

moisture, and TSS) of the liquid egg by ATR-FTIR spectroscopy combining
chemometric has not been studied in the literature. This analysis contributes to the
literature in the field of a rapid quantitative analysis with high accuracy for the
evaluation of the liquid egg quality. The reliability of the analysis was verified
through the experiments, in which a large number of different samples were
examined. Combination of ATR-FTIR spectroscopy and chemometric (PLS
regression) can be used in different fields such as in product acceptance or quality
control laboratories since it presents convenience to manufacturers and prevents
the adulteration induced problems.
74
4.1.3. Detection of Egg Adulteration by ATR-FTIR and NIR Spectroscopy
Liquid and dry samples of whole egg (n=10), egg white (n=10), egg yolk (n=10),
whole egg containing egg white (n=10), and water (n=10) were analyzed by ATR-
FTIR and NIR spectrometer to detect of adulterated egg samples with water.
4.1.3.1. ATR-FTIR Spectroscopy Analysis

To achieve a better classification of the adulterated samples, experiments were
performed for both liquid and dry samples. The ATR-FTIR spectra of both liquid
and dry samples of whole egg, egg white, egg yolk, whole egg containing egg
white (Brix adjusted 21, BXEW), and water (Brix adjusted 21, BXW) are shown in
Figure 4.13(a) and (b), respectively. The spectrum images of liquid samples (LWE,
LEY, and LEW) are similar to the images given in the Fig. 4.8. Considering the
figures, it can be concluded that the water is the dominant band in the spectrum
since a remarkable drop was observed in the intensity of water band between the
3000 and 3700 cm-1 for all spectra in the Figure 4.13(b). In addition, many bands
in the range of 900 and 1800 cm-1 became more apparent in the spectra of the dry
samples. In this range, there are functional groups of lipid and protein components
(Section 4.1.2.1). The bands at 2847 and 2915 cm-1 also became more apparent
with the disappearance of the water band. These bands are also functional groups
of lipid component in the yolk.
75
Figure 4.13. The ATR-FTIR spectra of (a) LEW, LWE (brix adjusted to 21 by
adding egg white, BXEW), LWE (brix adjusted to 21 by adding water, BXW), LWE,
and LEY; (b) dry egg white (DEW), dry whole egg (brix adjusted to 21 using egg
white, BXEW), dry whole egg (brix adjusted to 21 using water, BXW), dry whole
egg (DWE), and dry egg yolk (DEY)
Data analyses of the liquid and dry samples were performed separately using the
PCA. PCA models were formed using both liquid and dry samples. At first, the
PCA model was constructed using the first two principal components (PCs) for the
classification of the five liquid sample groups that were LWE, LEW, LEY, LWE
containing egg white (BXEW), and water (BXW). Then, a new PCA model was
constructed with the first three PCs to better classify of the three sample groups
(especially adulterated sample group, BXW), which were LWE, BXEW, and BXW.
The PCA score plot of the five and three liquid sample groups are demonstrated in
Figure 4.14(a) and (b).
76
Figure 4.14. PCA score plot of (a) five liquid sample groups (LWE, LEY, LEW,
BXEW, and BXW) and (b) three liquid sample groups (LWE, BXEW, BXW)
77
The cumulative variance and error values of the PCA models formed for five and
three liquid sample groups are listed in Table 4.17. As can be seen from the table,
the low error values (RMSEC and RMSECV) are achieved for both models. A
classification in five group samples was obtained between the LEW and LEY
samples. On the other hand, a good separation could not be achieved between
the group of BXEW and BXW samples (Fig. 4.14(b)).
Table 4.17. The cumulative variance, RMSEC and RMSECV values of the liquid
samples PCA models
PCs Cumulative variance % RMSEC RMSECV
Liquid (5) 2 30.9 0.087 0.115
Liquid (3) 3 80.8 0.424 0.687
The PCA model was also constructed for the dry samples. Using the first three
PCs, model was formed for the classification of five dry sample groups; dry whole
egg (DWE), dry egg white (DEW), dry egg yolk (DEY), DWE containing egg white
(BXEW), and water (BXW). To achieve a better classification in three sample
groups (adulterated sample group; BXW) that were DWE, BXEW, and water, a
new PCA model was constructed using the first three PCs. The PCA score plots of
these five and three dry sample groups are presented in Figure 4.15 (a) and (b).
The cumulative variance (%), RMSEC, and RMSECV values of the PCA models
are provided in Table 4.18. As can seen from the table, the low error values
(RMSEC and RMSECV) are obtained for five and three sample group models.
Classification in five group samples was provided between the LEW and LEY
samples. Furthermore, the sample group of BXEW was separated from the BXW.
A satisfied classification between the BXEW and BXW (adulterated) sample
groups was performed in the second analysis. Considering the score plots of liquid
and dry samples, it is clearly seen that a better classification was obtained from
the dry samples. The best classification between the BXEW and BXW sample
groups was obtained with the PCA model formed using three dry sample groups. It
could be concluded that the difference in the results between liquid and dry
sample groups may be originated from the water content in the liquid samples.
This is due to the fact that all bands became more apparent with disappearance of
the water band (Fig. 4.13(b)).
78
Thus, the analysis of the dry sample data, including more distinct and intense
bands, provides a better classification compared to the liquid sample data.
Figure 4.15. PCA score plot of (a) five dry sample groups (DWE, DEY, DEW,
BXEW, and BXW) and (b) three dry sample groups (DWE, BXEW, BXW)
79
Table 4.18. The cumulative variance, RMSEC and RMSECV values of the models
Dry (5) 3 82.7 0.004 0.005
Dry (3) 3 46.7 0.075 0.119
ATR-FTIR spectroscopy has been used to evaluate food quality, and detect
adulteration. Melamine adulteration in dairy milk [105] and olive oil assessment in
edible oil blends [106] were studied by means of ATR-FTIR spectroscopy.
However, liquid egg adulteration has not been analyzed yet. Therefore, this study
will contribute to the literature in the field of assessment of the liquid egg quality.
Dry samples are preferred since a better classification can be achieved. Therefore,
samples to be analyzed are dried before the analysis, and experiment is
performed with this dry samples. Combining ATR-FTIR spectroscopy with PCA is
a rapid analysis technique to detect adulteration by water in the liquid egg
samples, where the analysis takes a very short time. These superiorities make
ATR-FTIR spectroscopy very attractive to be used in the quality control
laboratories and food industry.
4.1.3.2. NIR Spectroscopy Analysis

NIR Spectroscopy was also performed for both liquid and dry samples. The same
sample groups in the former analysis (ATR-FTIR) were used in NIR analysis,
whose liquid and dry samples spectra are shown in Figure 4.16(a) and (b),
respectively. Interpretation of the complex NIR spectra of egg samples is highly
challenging due to including a large number of bands of chemical compounds that
cause band overlapping, where one band may include different types of vibration
mode data. In the observed NIR spectrum, water, lipid, and protein components of
egg were detected.
80
Figure 4.16. NIR spectra of the (a) LEY, LWE, BXW, BXEW, and LEW, (b) DEY,
DWE, BXW, DEW, and BXEW
81
Water is a strong absorber in the IR spectrum region [107]. As can be seen in the
Figure 4.16(a) and (b), the NIR spectra of the liquid and dry samples comprise of
wide bands unlike ATR-FTIR spectra. Therefore, the spectrum of each sample can
be divided into five regions named as A, B, C, D, and E. The region A (10000 –
8330 cm-1 (1000-1200 nm)) corresponds to vibration modes of CH2 and CH3
molecules of the lipid component [108]. The second region B located between
8220 – 7015 cm-1 (1216-1425 nm) was associated with the vibration bands of
H2O, CH2, and CH3 molecules of water and lipid components [101,102]. The
region C located in the range of 6800 – 5785 cm-1 (1469-1728 nm) was described
as the vibration bands of CH3, –CH=CH– molecules of lipid and H2O molecule of
water components [102,103]. Strong water bands in regions B and C suppress the
protein band in these regions. Region D (5781 – 5226 cm-1 (1729-1913 nm))
indicates vibration mode of CH3 molecule of the lipid and H2O molecule of the
water components [101–103]. The last region E located between 5083–4072 cm-1
(1967-2455 nm) contains combination of CH2 vibrations of protein side chain, –C-
H=CH–, CH3, and CH2 vibrations of the lipid substance [102,103]. All regions in
the NIR spectrum, the wavenumber range, the functional groups, and mode of
vibrations of the molecules are provided in Table 4.19.
Table 4.19. Functional groups and mode of vibrations in the NIR spectrum of the
egg samples
Region Wavenumber (cm-1) Functional group Mode of vibration
A 10000-8330 C–H (CH2) 2nd overtone
C–H (CH3) 2nd overtone
B 8220-7015 C–H (CH2) Combination
C–H (CH3) Combination
O–H (H2O) 1st overtone
C 6800-5785 C–H (CH3) 1st overtone
C–H (–CH=CH–) 1st overtone
O–H (H2O) Combination
D 5781-5226 C–H (CH3) 1st overtone
O–H (H2O) Combination
E 5083-4072 –C–H (CH2) (Protein)
C–H (–CH=CH–) Combination
–C–H (CH3) Combination
–C–H (CH2) Combination (lipid)
82
As seen from the table, combination modes of the molecules mostly appear in the
last regions, whereas overtone modes of the molecules are more common in the
initial regions. It can be observed that each of region contains more than one
functional group resulted in wide band spectrum.
The difference between the NIR spectrum of the liquid and dry sample groups can
be seen from the Figure 4.16(a) and (b). Disappearance of the water component
after drying of the egg samples leaded to reveal of the other substances like lipid
and protein. The biggest difference was appeared in the egg white samples. The
intensity of the NIR spectrum of LEW was considerably low that it could not be
detected with other spectra. Therefore, its spectrum was showed individually at the
top of the figure for convenience. When the water is not present in the structure of
the sample, a better NIR spectrum of the dry samples are obtained. The NIR
spectrum of dry egg white (DEW) was detected among the spectra of the dry
samples, and distinctive bands of the DEW samples appeared in the spectrum.
Data analyses of the liquid and dry sample groups were carried out separately via
PCA. To classify of the five liquid sample groups (LWE, LEW, LEY, BXEW, and
BXW), PCA model was formed using first two PCs. Then, a new PCA model was
constructed using three sample groups (LWE, BXEW, and BXW) to provide a
better classification between the BXEW and adulterated (BXW) sample groups.
The PCA score plot of the five and three liquid sample groups are given in Figure
4.17(a) and (b). The cumulative variance and error values of the PCA model
formed for five and three liquid sample groups are listed in Table 4.20. As can be
seen from the table, the low error values (RMSEC and RMSECV) are obtained for
five and three sample group models. According to the PCA plot result, a
classification in five sample groups was obtained between LEW and LEY groups.
BXEW sample group was also separated from BXW sample group. In the second
PCA model (Fig. 4.17(b)), the group of BXEW and BXW samples were
successfully separated. Furthermore, the BXW and LWE sample groups were also
separated. Use of liquid sample in the analysis did not cause any problem in the
classification.
83
Figure 4.17. The PCA score plot of (a) the five liquid sample groups (LWE, LEY,
LEW, BXEW, and BXW) and (b) the three liquid sample groups (LWE, BXEW, and
BXW)
84
models formed using five-three liquid sample groups
Liquid (5) 2 66.4 0.573 0.657
Liquid (3) 2 50.3 0.693 0.802
The classification of five dry sample groups (DWE, DEY, DEW, BXEW, and BXW)
was performed utilizing PCA model with the first two PCs. To evaluate of three
sample groups (DWE, BXEW, and BXW), a second PCA model was constructed
using the first two PCs. Figure 4.18(a) and (b) presents the PCA score plot of the
five and three dry sample groups.
The total explained variance, RMSEC and RMSECV values of the PCA model
constructed for five and three liquid sample groups are given in Table 4.21. The
low RMSEC and RMSECV values are observed for both models. In the first PCA
model, LEW, LEY, and BXEW sample groups were successfully classified. In the
second PCA model, a better separation between the BXEW and BXW
(adulterated) sample group was obtained. As can be seen in the Figure 4.18(b),
DWE and BXW sample groups were located in the same region in PCA plot. Since
these samples are dry, the difference stemming from the water substance
between the whole egg and whole egg containing water sample groups was
disappeared.
In summary, developed PCA models fulfilled classification regardless of the form

of the egg samples (liquid or dry). Hence, the presence of the water substance in
the structure did not cause any problem in the classification of the samples.
Consequently, unlike ATR-FTIR spectroscopy, combination of NIR spectroscopy
with PCA can be utilized for the classification of both liquid and dry egg samples.
85
Figure 4.18. The PCA score plot of (a) the five dry sample groups (DWE, DEY,
DEW, BXEW, and BXW) and (b) the three dry sample groups (DWE, BXEW, and
BXW)
86
models formed using five-three dry sample groups
Dry (5) 2 61.72 0.048 0.059
Dry (3) 2 44.00 0.052 0.069
NIR spectroscopy has been commonly preferred to ensure food safety, quality,
and to detect adulteration. In the literature, detection of beef hamburger
adulteration [110], detection and quantification of adulteration in olive oil [111],
quantification of common adulterants in powdered milk [112], and detection of
durum wheat adulteration [113] were analyzed by NIR spectroscopy. To my best
knowledge, analyzing of the egg adulteration and performing of this analysis by
NIR spectroscopy are the first attempts in the literature. Combining NIR
spectroscopy with PCA also enables rapid detection of egg adulteration, which is
non-destructive, and can be used regardless of the form of egg samples.
Moreover, it does not require any preparation process for the liquid samples.
Considering these advantages, this technique is highly promising in determination
of liquid egg quality.
4.2. Analysis of Heat Treated LWE by CE

In this section, the effect of heat treatment on egg proteins is demonstrated
through experiments carried out with UV-VIS spectroscopy and CE.
4.2.1. UV-VIS Measurements

Untreated and treated liquid whole eggs (LWEs, n=54) were centrifuged, and
supernatant was taken for UV-VIS measurements at 280 nm. Figure 3.19 shows
the UV-VIS absorbance measurements of untreated (0 minute), treated LWEs at
(a) 60 °C, (b) 64 °C and (c) 68 °C for five minutes with a one-minute interval and
the statistical differences of measurements within one temperature.
In Fig. 4.19(a), according to results of the statistical evaluation, untreated and

treated (3, 4 and 5 minutes) samples are not significantly different. The samples
treated for 1 and 2 minutes are significantly different from the other samples.
87
While there was a decrease in UV absorbance for treatment at 1 and 2 minutes,
the change was very small between UV absorbance of the samples. Therefore, it
might be deduced that egg proteins were not substantially affected at 60 °C. As
can be seen in the Fig. 4.19(b), the untreated sample is significantly different from
the treated samples. Some treated samples (from 1 minute to 5 minutes) also
showed a statistically significant difference between treatment times, but not with a
linear decrease. Although the change was very small between UV absorbance for
the untreated and treated samples, the untreated sample was substantially
different from the other samples. Thereby, it might be deduced that heat treatment
at 64 °C has a slight effect on egg proteins. Lastly, as can be seen in Fig. 4.19(c),
the untreated sample is significantly different from the treated samples according
to statistical evaluation. There was also an important difference between treated
samples (from 1 minute to 5 minutes) at different treatment times, but not with a
linear decrease. There was an apparent decrease in UV-Vis absorbance for
untreated and treated samples; thus it can be deduced that (treatment at) 68 °C
had an enormous effect on egg proteins even in the case of one minute treatment.
88
Figure 4.19. UV-VIS absorbance measurements of untreated and treated LWE
samples at (a) 60 °C, (b) 64 °C, (c) 68 °C for five min with a one-minute interval
and the statistically differences of measurements within one temperature.
Significant differences (p<0.05) between means are indicated by different letters
89
Comparison of the means between the samples treated at different temperatures
(60 °C, 64 °C and 68 °C) is shown in Table 4.22. According to the statistical
evaluation, all UV-Vis measurements belonging to treatment at 68 °C were
significantly different from the others. However, some samples (1 and 2 minutes)
treated at 60 °C and 64 °C were not significantly different. The untreated sample
was different from the treated samples for treatment at 64 °C and 68 °C, but not at
60 °C (4 and 5 minutes). The difference between untreated and treated LWEs in
terms of the decrease in UV absorbance stemmed from heat treatment and
thereby from protein denaturation. As can be seen in Table 4.22, the highest
decrease in the UV absorbance was seen at 68 °C because of the significant
decrease in protein solubility. When the temperature increased, protein solubility
decreased due to denaturation and resulted in higher precipitation. Parallel with
these findings, Herald and Smith [51] also found that LWE protein solubility
decreased as pasteurization temperature increased.
Table 4.22 Comparison of means of samples treated at different temperatures

Time (min) 60 °C (UV abs.) 64 °C (UV abs.) 68 °C (UV abs.)
untreated 1.00a 1.00a 1.00a
1’ 0.88e 0.89de 0.64fg
e e
2’ 0.87 0.87 0.60g
3’ 0.95bc 0.94cd 0.66f
4’ 0.98ab 0.93cd 0.62fg
5’ 1.00a 0.95bc 0.60g
4.2.2. CE Electropherograms of Samples

Electropherograms of LEW, LEY, egg yolk supernatant (EYS) and egg yolk
granule (EYG) are presented in Figure 4.20(a). Egg white (albumen) is an
aqueous solution of various proteins. It mainly comprises of ovalbumin (54%),
conalbumin (ovotransferrin 12%), ovomucoid (11), ovomucin (3.5%), lysozyme
(ovoglobulin G1 3.4%), ovoglobulin G2 (4%), ovoglobulin G3 (4%). Among egg white
proteins; ovalbumin, conalbumin, ovomucoid and lysozyme were identified in the
electropherogram and interpreted in Figure 4.20(c).
90
Yolk is a fat-in-water emulsion with its approximately 50% dry weight. The main
components of yolk are low density lipoprotein (LDL, 68%), high density lipoprotein
(HDL, 16%), livetins (10%) and phosvitins (4%) [5]. After centrifugation, yolk could
be fractionated to its constituents, which are plasma (85% LDL and 15% livetin)
and granules (70% HDL, 16% phosvitin and 12% LDL) [11,108]. In the figure
4.20(b), it can be seen that proteins of supernatant and granules were attained at
different migration times. Electropherogram of yolk supernatant contains LDL and
livetins (α, β, γ). HDL, phosvitin and LDL proteins are presented in yolk granule
electropherogram. It is thought that the highest peak in the granule
electropherogram may be HDL because of its highest proportion in the granule
part.
Electropherograms of LWE, yolk supernatant and granule added LWE are

illustrated in Fig. 4.20(b). When granule part was added to LWE, the peak
absorbance in the framed region at around 18 min increased since this protein
came from granule part of yolk. Besides, it is thought that this protein may belong
to the highest peak of HDL in the granule electropherogram. By means of adding
the supernatant part to LWE, the peaks in the shady region between at 9-16 min
and framed region at 20 min increased. It can be said that these regions in LWE
have supernatant proteins.
Electropherograms of LWE and white proteins (ovalbumin, conalbumin,

ovomucoid and lysozyme) added to LWE are presented in Fig. 4.20(c). The
highest peak migrated between 16 and 17.5 min in the shady region was found as
ovalbumin. Ovalbumin has the highest proportion in both egg white and whole egg
because white and yolk comprises 60% and 30-33% proportions of an egg,
respectively [6]. Ovomucoid was observed in the framed region and its migration
time was between 5 and 7.5 min. Conalbumin and lysozyme were observed later
and their migration time is 15 min. These proteins had the same electrophoretic
mobilities under those conditions, thus they were analyzed at the same migration
time.
91
92
Figure 4.20. Electropherograms of (a) liquid egg white (LEW), liquid egg yolk
(LEY), egg yolk supernatant (EYS) and egg yolk granule (EYG); (b) egg yolk
supernatant and granule added liquid whole egg (LWE), LWE; (c) lysozyme,
conalbumin, ovomucoid, ovalbumin added LWE, and LWE.
Electropherograms of untreated and heat-treated LWE samples at different

temperatures and times are shown in Figure 4.21. Heat treatment was taken at 60
°C, 64 °C, 68 °C for five minutes with a one-minute interval. All of the temperatures
are pasteurization parameters used in egg industry. 60 °C for 3.5 minutes is
approved as minimum pasteurization parameter of eggs in the U.S. [31]. In the
UK, LWE is pasteurized at minimum 64.4 °C for at least 2.5 minutes [115]. In this
study, the change in protein composition with increasing treatment time at the
same temperature was monitored. In Figure 4.21(a), the effects of 60 °C over egg
proteins were evaluated, and as can be seen, the samples resemble each other
since there was no missing or changing protein in the electropherogram.
According to the statistical results, these results were consistent with UV
measurements. Patterns of all electropherograms for 60 °C were similar to each
other.
93
Although there was no change at that temperature, it was thought that conalbumin,
the egg white protein, might have been affected slightly because its denaturation
temperature is about 60 °C [38,138]. Electropherograms of untreated and treated
LWE at 64 °C are shown in Figure 4.21(b). It might be stated that just a few
proteins in the shady region disappeared and decreased in terms of absorbance
between untreated and treated samples. These disappearing proteins can be
stemmed from supernatant proteins of yolk or undefined white proteins. γ-livetin
might have been affected because it started to disappear after heating at 62 °C
[49]. There were no major differences between the treated electropherograms
depending on increasing time. At that temperature conalbumin could be affected.
Its denaturation temperature is about 60 °C, but it can vary depending on pH and
binding of metal ions. Association with metallic ions can increase the stability of
conalbumin even when its denaturation temperature goes up to 84 °C [7,110,111].
Finally, electropherograms of untreated and treated LWE at 68 °C are shown in

Figure 4.21(c). Analysis of the electropherograms revealed that proteins were
affected substantially at that temperature. In the shady and framed regions, some
of the proteins disappeared and the absorbance decreased due to denaturation.
That temperature was found to be very effective since it was seen that protein
pattern changes even after one-minute treatment. The change in protein patterns
continued among the treated samples depending on increment of time. Similar
results were obtained for UV measurements in terms of protein denaturation
based on statistical results. Conalbumin, lysozyme and yolk supernatant proteins
were located in the shady region. Lysozyme could have probably been affected by
that temperature because its denaturation was at 67 °C [41]. Denaturation of egg
yolk proteins was more complicated due to the structure of yolk. Proteins and
lipoproteins of plasma (livetins and LDL) are more sensitive to heat treatments
than proteins and lipoproteins of granules (phosvitins and HDL). For yolk proteins,
LDL and α-livetin started to be affected at 72 °C and γ-livetin disappeared above
69 °C [49]. In the framed region, peaks related to yolk supernatant proteins began
to decrease in the 1st minute, and disappeared after the 4-5th minutes. As a result,
it was thought that conalbumin, lysozyme and some yolk supernatant proteins
disappeared at that temperature.
94
As it can be deduced from electropherograms, ovalbumin and ovomucoid weren’t
affected by all the heating conditions because of denaturation temperatures at 78
°C and 70 °C, respectively [5,38]. The most effective temperature for both egg
white and yolk proteins was found as 68 °C. In another study, egg white and yolk
proteins were characterized through capillary electrophoresis. Untreated and heat-
treated LWE were not analyzed [43].
95
Figure 4.21. Electropherograms of untreated and treated LWE samples at (a) 60
°C, (b) 64 °C, (c) 68 °C for five minutes with a one-minute interval
4.2.3. PCA Analysis

PCA was applied to determine the LWE samples to classify them as untreated or
heat-treated and to detect the extent of heat treatment parameters. PCA score plot
of untreated and treated LWE samples is shown in Figure 4.22. The variation in
the data set can be explained by the first two PCs, and PC2 versus PC1 was used
for score plot. The first two PCs were sufficient to explain 48.05% of cumulative
variance for samples. The RMSEC and RMSECV value of PCA model are 0.714
and 0.897, respectively.
Figure 4.22 indicates that the untreated samples were separated distinctly from all
the treated samples. Separation of samples into untreated-treated cluster was the
first step of classification analysis, which would provide the estimation of unknown
samples as either untreated or treated. If the sample is found in the treated cluster,
the second step of classification analysis is performed.
96
Figure 4.22. PCA score plot of untreated and treated LWE samples (60 °C, 64 °C,
68 °C for five minutes with a one-minute interval
PCA score plot of the heat-treated LWE samples at 60, 64, 68 °C for 1-5 minutes
with a one-minute interval is presented in Figure 4.23. The variation in the data set
can be explained by the first few PCs. PC4 versus PC1 was used for score plot.
The selected PCs explained 99.78% of cumulative variance for samples. The
RMSEC and RMSECV values for PCA model are 0.0003 and 0.0006, respectively.
The model was successful to classify the samples according to their treatment
temperatures as 60, 64, 68 °C. And the samples differed distinctly from each other
according to their treatment temperatures and times. The samples treated at 60 °C
differed distinctly from the other samples and clustered in the same region, except
for some samples treated for five min. Scores of those samples were found in the
cluster of some samples treated at 64 °C. It can be explained that the effect of this
treatment (60 °C for five minutes) on LWE proteins has a similar effect as 64 °C.
97
Another cluster was formed for the treatment at 64 °C. It can be seen that these
samples also separated into two groups according to their treatment times. The
samples treated for 1 and 2 minutes are presented at the bottom of the cluster,
and the remaining samples are presented in the upper part of the cluster.
Depending on the increment in treatment times, the samples were classified
separately. The last cluster was formed the samples treated at 68 °C. These
samples differed distinctly from the other treatments samples, and they were
separated into a few groups according to their treatment times. The samples
treated for 4 and 5 minutes, in particular, were classified at a different region in the
plot. And the rest of the samples also differed from each other depending on their
treatment times. When the extent of treatments (temperature and time) gradually
increased, the effect of treatment on LWE proteins increased, as well. Therefore,
protein composition changed depending on the effect of treatment, and
differentiation of samples increased. Depending on the differentiation of protein
composition, the samples differed from each other distinctly.
Figure 4.23. PCA score plot of treated LWE samples at 60 °C, 64 °C, 68 °C for five
minutes with a one-minute interval
98
If the system has a new sample, it is tested by applying the first step. However, if
the samples are found in the treated region, the second step is performed. Results
of the second step showed that the extent of heat treatments applied to liquid egg
products could be determined. In other words, these results would give an idea
about the extent of pasteurization in LWE products, which is significant for food
industry.
The effect of heat on LWE proteins depending on the extent of treatment has not
been analyzed by using chromatographic techniques up to now. Therefore, this
study could provide a significant contribution to literature. CE combined with PCA
provided efficient classification of samples untreated or treated and treated at
different treatment parameters. In this way, the extent of heat treatment applied to
LWE was determined. With the developed method in this study, adhesion protein -
which is an important problem for CE- was overcome, and repeatable results were
obtained. Despite the complexity of matrices, high reproducibility and interference-
free electropherograms were obtained. Therefore, it can be concluded that CE is a
useful technique for analyzing and monitoring protein changes, and combined with
chemometric technique, it can allow for further analysis.
4.3. Effect of Heat Treated LWE on the Cake Quality

In this part of the study, the effect of heat-treated LWE on cake batter rheology
and baked cake quality is examined by performing rheological measurement and
cake physical analyses.
4.3.1. Analysis of Cake Ingredient

To determine flour quality used in the preparation of cake batter; protein, moisture,
ash, wet gluten and sedimentation values of the flour sample were analyzed.
Protein content was given on a dry basis. Moisture content of the sample was
calculated according to Equation 2.5. The measured protein, moisture, ash, wet
gluten, and sedimentation values of the flour sample are listed in Table 4.23.
Table 4.23. Protein (dry basis), moisture, ash, wet gluten, and sedimentation
values of the flour sample
Protein (%, db) Moisture (%) Ash (%) Wet gluten (%) Sedimentation (ml)
12.24 ± 0.18 11.43 ± 0.13 1.07 ± 0.02 26.19 ± 0.66 38.36 ± 2.42
99
4.3.2. Rheology of Cake Batter
Analysis of rheological properties of cake batter is valuable, which reveals the
quality of baked cake. Flow behavior of the cake batters was analyzed by
changing shear stress depending on the shear rate. The difference between
formulation of the cake batters stemmed from LWE ingredient, which was heat-
treated for two and five minutes at 60 °C, 64 °C, and 68 °C. The change in
apparent viscosity versus the shear rate for different batter formulations containing
heat-treated LWE at 60 °C, 64 °C, and 68 °C is showed in Figure 4.24(a), (b), and
(c), respectively. All batter formulations exhibit a non-Newtonian flow behavior
since the consistency depends on the shear rate. As can be seen in Fig. 3.24, a
decrease in the apparent viscosity is observed depending on the increase in shear
rate (pseudo-plastic (shear-thinning) behavior). The obtained measurements are
consistent with previously reported results in the literature [122–124]. As seen in
the figure, the sharp decrease in the apparent viscosity disappears after a certain
shear rate (>10 s-1). Beyond this shear rate, there is no effect of shear rate over
the apparent viscosity anymore.
According to Fig. 4.24, there is a difference between the apparent viscosities of

the control (untreated LWE) and the other cake batters (including heat-treated
LWE), which reveals that using of heat-treated LWE even at low temperatures has
a notable effect on the apparent viscosity of the cake batters. Increase of the heat
treatment parameters causes an increase in the apparent viscosity of the cake
batters. The amount of the change decreases above a certain shear rate (>15 s-1);
thus, the batters have similar apparent viscosity values to each other. The
apparent viscosity of batters for treatment at 60 °C begins at 75Pa.s, while the
apparent viscosity of batters for treatment at 64 °C and at 68 °C start
approximately at 80 and 100Pa.s.
According to these results, it can be concluded that the heat treatment parameters
for LWE has a considerable effect on the apparent viscosity of cake batters, where
an increase in the heat treatment parameters leads to an increase in the apparent
viscosity.
100
101
Figure 4.24. An apparent viscosity versus shear rate of different batter
formulations including untreated and heat-treated LWE for two and five min at (a)
60 °C, (b) 64 °C, and (c) 68 °C. Lines represent the power-law model
The shear stress and shear rate data were fitted to Power Law model to obtain
consistency index (K) and flow behavior index (n) of cake batters.
𝜏 = 𝐾. 𝛾 !
Flow behavior index indicates whether flow is pseudo-plastic (n<1) or dilatant

(shear-thickening, n>1). When the index (n) equals to 1, the flow exhibits
Newtonian behavior. A power-law model was successfully fitted using the
experimental data. K, n, and coefficient of determination (R2) values of power law
models are listed in Table 4.24. A high coefficient of determination value (R2>0.98)
was obtained for all models. As previously explained, all cake batters exhibit a
pseudo-plastic behavior since the n values of all models less than 1.
102
Statistical analysis demonstrated that there is a significant difference between the
flow behavior index of control and other batters. Therefore, it can be concluded
that there is an effect of heat-treated LWE on flow behavior of cake batter. Another
important consequence according to the statistical analysis, there is no significant
difference between the batters prepared with heat-treated LWE at different
parameters. On the other hand, variation of heat treatment parameters shows no
effect on the flow behavior of cake batter.
Table 4.24. Consistency (K) and flow behavior indices (n) of cake batters including
untreated and LWE heat-treated at different parameters (Mean of two replicates ±
standard deviation)
Heat treatment
K (Pa.sn) n R2
(LWE)
Control 37.66 ± 0.06a 0.59 ± 0.00a 0.991
60 °C 2’ 43.20 ± 2.42b 0.55 ± 0.02ab 0.989
60 °C 5’ 44.52 ± 2.79b 0.52 ± 0.01b 0.989
64 °C 2’ 45.18 ± 1.52b 0.53 ± 0.01b 0.989
64 °C 5’ 48.08 ± 4.11b 0.52 ± 0.02b 0.986
68 °C 2’ 56.51 ± 2.08c 0.51 ± 0.01b 0.986
68 °C 5’ 56.94 ± 1.61c 0.52 ± 0.01b 0.988
K value, indicating consistency of batters, was calculated in the range from 37.66
to 56.94 Pa.sn. As can be seen in Table 4.24, an increase in consistency index of
the cake batters was observed depending on the increase in heat treatment
parameter. Statistical analysis reveals that both heat-treated LWE and treatment
parameters applied to LWE affect the consistency of cake batters. A significant
difference in between K values of the control and the other batters was observed.
However, there is no consistency difference between the cake batters prepared
using heat-treated LWE at 60 °C and 64 °C. The heat treatment of LWE at 68 °C
caused a substantial change in the K value of batter. According to the statistical
analysis, an increase in heat treatment time from 2 to 5 minutes at 68 °C did not
lead to any change in the K value.
103
As a result, the rheological properties of the cake batters depend on whether egg
is heat-treated or untreated. The increase in the K values of the batters originates
from the egg proteins exposed to heat treatment.
As described in the previous section, egg proteins, especially albumen proteins

(conalbumin and lysozyme), are affected from the heat treatment even at low
temperature. Heat-treated proteins may lose their functional ability in the formation
of foam. Some of them may denature depending on the applied heat treatment
parameters, so they do not contribute to the foam formation due to entrapping less
air bubbles. Moreover, heat treatment causes a change in the egg form, where it
becomes more intense due to the coagulation of egg proteins. The loss of the
liquid form of eggs results in an increase batter consistency.
In summary, it is demonstrated that both heat treatment and heat treatment

parameters of LWE have a considerable effect on the apparent viscosity of cake
batters, where rheological properties of batter provide a prior knowledge about the
baked cake quality. To my best knowledge, there has been no similar work
reported in the literature.
4.3.3. Cake Analysis

In this part, physical properties of baked cakes are examined through moisture
loss, porosity, color, texture, and specific volume analyses, and results are
discussed in detail.
4.3.3.1. Moisture Loss

After the baking process, the moisture loss (%) of cakes during the baking was
calculated according to Equation 3.6. The percentage of moisture loss of baked
cakes is given in Figure 4.25. As can be seen in the figure, a significant difference
in the moisture loss was not observed among the baked cakes. Either using of
heat-treated LWE or increasing heat treatment parameters for LWE did not cause
any change in moisture loss of baked cakes.
104
Figure 4.25. The moisture loss (%) of the baked cakes prepared using untreated
(control) and heat-treated LWE for two and five minutes at 60 °C, 64 °C, 68 °C.
4.3.3.2. Color Analysis

Color measurements were taken from the surface of the baked cakes. The color
change (∆E) of the baked cakes was calculated according to Equation 3.7.
Measured ∆E values of the cakes are given in Figure 4.26. Heat treatment of LWE
does not cause a considerable change in the color of the baked cakes. On the
other hand, a significant difference exists only for the cakes including LWE heat-
treated for 2 and 5 minutes at 68 °C. The ∆E values among the other cakes are
almost equivalent to each other. Using LWE heat-treated at 68 °C (for 2 and 5
minutes) causes an increase in ∆E values of the baked cakes.
105
Figure 4.26. ∆E values (%) of the baked cakes prepared using untreated (control)
and heat-treated LWE for two and five minutes at 60 °C, 64 °C, 68 °C. Significant
differences (p<0.05) between means are indicated by different letters
4.3.3.3. Porosity Analysis

Porosity is another important physical property that affects the quality of baked
cake. The porosity of baked cakes was calculated according to Equation 3.8. The
measured porosity (%) values of cakes are demonstrated in Figure 4.27. As seen
in the figure, there is a decrease in porosity value (%) in the baked cakes including
heat-treated LWE. There is a remarkable difference between in the percentage of
the porosities of control and other baked cakes, which is an unfavorable effect of
the heat treatment on LWE proteins causing formation of a less porous structure in
the baked cakes. Since the damaged proteins by the heat treatment can lose their
functional properties, they become disabled during foam formation. Therefore, a
cake batter including less air bubble causes formation of a baked cake having low
porosity. On the other hand, an increase in the heat treatment parameter of LWE
does not result in a significant difference between the cakes. The porosity values
of the baked cakes containing heat-treated LWE are close to each other.
106
Figure 4.27. The porosity values (%) of the baked cakes prepared using untreated
(control) and heat-treated LWE for two and five minutes at 60 °C, 64 °C, 68 °C.
4.3.3.4. Texture Analysis

Texture analysis provides insightful information about the internal structure of
baked cake. The analysis was performed by measuring of hardness,
cohesiveness, and gumminess of the baked cakes. The gumminess was
calculated according to Equation 3.9. Measured hardness values of the baked
cakes are showed in Figure 4.28. The results show that an increase in the heat
treatment parameters resulting in an increase in the hardness value of the baked
cakes. The baked cakes including untreated (control) and treated for 2 minutes at
60 °C LWE exhibit a similar hardness structure. Similar results were obtained for
the baked cakes containing LWE treated at 60 °C – 5 minutes, 64 °C – 2 and 5
minutes. Besides, the cakes prepared with LWE treated at 68 °C for 2 and 5
minutes have also similar results.
107
The heat treatment of LWE shows its effect on the hardness of the cakes at 60 °C
for 5 minutes, which means that heat treatment of LWE even at low parameter has
a substantial effect on the hardness of the baked cakes. The highest value of the
hardness was obtained for the cake including LWE treated for 5 minutes at 68 °C.
The underlying reason of increasing of the hardness value may be damaging of
LWE proteins due to heat treatment, in which the number of air bubbles in the
cake batter decreases, and ultimately resulting in a harder cake structure than the
cake prepared with untreated LWE.
Figure 4.28. The hardness values (N) of the baked cakes prepared using
untreated (control) and heat-treated LWE for two and five minutes at 60 °C, 64 °C,
68 °C. Significant differences (p<0.05) between means are indicated by different
letters
Another texture profile parameter for the cake analysis is cohesiveness.

Cohesiveness indicates how well the product withstands against to a second
deformation relative to cake resistance under the first deformation. Cohesiveness
values of the baked cakes are given in Figure 4.29. According to the results, as
heat treatment parameter increases, cohesiveness values of the baked cakes also
increase.
108
A significant difference occurs among the cakes especially after the heat treatment
for 2 minutes at 64 °C. Up to this treatment, baked cakes have similar
cohesiveness values with the control cakes. The highest cohesiveness value was
observed for the baked cake including LWE heat-treated for 5 minutes at 68 °C.
The reason of the increase in cohesiveness values may be similar to the increase
of the hardness value. As a conclusion, it is revealed that the heat treatment and
heat treatment parameters of LWE have a role on the cohesiveness value of the
baked cakes.
Figure 4.29. The cohesiveness values (ratio) of the baked cakes prepared using
letters
The other texture analysis parameter is gumminess, which is a function of

hardness and cohesiveness values. This parameter exhibits a similar trend with
the hardness. The gumminess values of the baked cakes are presented in Figure
4.30. As can be seen in the figure, the heat treatment of LWE significantly affects
the gumminess value of the baked cakes. An increasing trend in gumminess
occurs due to increasing of the heat treatment parameter of LWE.
109
A significant difference was also obtained for the cake including LWE heat-treated
for 2 minutes at 64 °C. The baked cakes prepared LWE untreated (control) and
treated (60 °C – 2 and 5 min) exhibits similar results. The highest gumminess
value was observed for the cake containing LWE treated for 5 minutes at 68 °C.
The reason behind the increase in the gumminess is the same with the increase in
hardness value. Namely, using of heat-treated LWE results in harder and less
fluffy cake structure, which causes formation of more gumminess baked cakes.
Consequently, heat treatment of LWE and heat treatment parameters have a
notable effect on the gumminess value of baked cakes as well as the other texture
parameters.
Figure 4.30. The gumminess values (N) of the baked cakes prepared using
letters
In the literature, the contributors of cake texture have been investigated. The effect
of using different type of gums [118], using of a surfactant as a whipping aid
(sodium lauryl sulphate) [121], and effect of batter freezing conditions and resting
time [89] on the cake texture has been examined.
110
On the other hand, there are also research about egg related with using of a
substitute instead of reduced egg or a total substitution of egg proteins in cakes
[127,128]. This study shows the effect of heat treatment and heat treatment
parameters of LWE on the texture quality of baked cakes, where the texture
analysis (hardness, cohesiveness, and gumminess) of the baked cakes prepared
with heat-treated LWE has not been studied in the literature.
4.3.3.5. Specific Volume

Specific volume values of the baked cakes were calculated according to Equations
3.10 – 3.13, which are demonstrated in Figure 4.31. As seen in the figure, heat
treatment of LWE shows a remarkable effect on the specific value of the baked
cakes. According to statistical analysis, a significant difference was observed
among the baked cakes. A significant difference begins with the treatment of LWE
at 64 °C (for 2 minutes). Unfavorable effect of heat treatment on LWE proteins
may be the reason of the difference. Damaging proteins lose their functional
properties, which causes low volume of cake batter, and finally resulting in
formation of less volume of the baked cakes.
Figure 4.31. The specific volume values (cm3/g) of the baked cakes prepared
using untreated (control) and heat-treated LWE for two and five minutes at 60 °C,
64 °C, 68 °C. Significant differences (p<0.05) between means are indicated by
different letters
111
4.3.3.6. Discussion of Rheology and Cake Analyses
Even though individual analysis of physical properties of cakes was
comprehensively discussed, interpretation of the results as a whole move the
discussion one step further to understand the compatibility of porosity, hardness,
and specific volume analyses. According to the results, porosity, hardness, and
specific volume values of the baked cakes are consistent to each other.
Correlation between the hardness – porosity and hardness – specific volume
values are demonstrated in Figure 4.32 and 4.33, respectively. The correlation
between the hardness – porosity and hardness – specific volume is verified by the
high correlation coefficients (r= 0.906 and 0.885, respectively). The correlations
appear to be statistically significant (P < 0.05). As expected, as the hardness value
increases, the porosity and specific volume values decreases, which results in
harder, less fluffy, and more compact pore of the baked cakes structure.
Figure 4.32. Correlation between the hardness and porosity values
112
Figure 4.33. Correlation between the hardness and specific volume values
Furthermore, the results of rheology and cake analyses are also consistent. As
mentioned in the section 4.3.1, rheology results give an insight of how the cake
structure will look. According to the rheology results, the cake batters prepared
with heat-treated LWE have a high consistency index (K). Furthermore, increasing
of heat treatment parameters causes a further increase in the K value. Thus, the
batters have less air bubble, less volume, and a stiffer structure. Consequently,
the baked cakes becomes harder, includes less porosity and specific volume.
In the literature, there is no study related with the effect of heat-treated LWE on
the cake batter and structure. In this part, the effect of heat-treated LWE and heat
treatment parameters of LWE on cake batter rheology was demonstrated.
Moreover, the baked cake quality was investigated through moisture loss, color
change, porosity, texture analysis (hardness, cohesiveness, and gumminess), and
specific volume analyses.
113
5. CONCLUSION
Recently, LWE has been more preferred than shell eggs in food products by
producers. There are two major reasons behind the preference of LWE. At first, it
is ready to use, which is highly important especially considering the mass
production while shell egg needs additional shelling process. Secondly, microbial
safety of LWE is considerably better than shell eggs, where ensuring the microbial
safety of eggs is a great convenience for food producers who never want to take
any microbial risk in their products. All these advantages aside, food quality is
another important measure that should be taken into account since the quality of
ingredients determines the quality of the final product. However, LWE quality has
not been discussed in the literature. There are two major problems that may
degrade the LWE quality. One of them is LWE adulteration with unknown
substance. Second, negative effects of heat treatment parameters on functional
properties of egg proteins.
The main motivation behind this study is to investigate the quality problems in
LWE, and most importantly to develop reliable and rapid analytical techniques that
determine the LWE quality. Based on this motivation, determination of LWE
adulteration, revealing the effect of heat treatment parameters on the egg proteins,
and demonstrating the effect of the heat-treated LWE on cake batter rheology and
cake physical quality were researched and discussed in detail.
Determination of LWE adulteration was performed using different techniques.

First, yolk-white ratio of LWE was determined by SDS-PAGE analysis, which
makes detection of LWE adulteration with water possible. The SDS-PAGE model,
which was constructed using liquid egg samples including different yolk
concentrations (from 0% to 100%), gives the yolk-white ratio in the adulterated
LWE. Determination of coefficient value (R2) of the model was obtained 0.992. As
a result, LWE adulteration with water can be successfully detected via this model.
Second, contents of protein, lipid, moisture, and TSS components of LWE
samples (n=85) were determined by using ATR-FTIR spectroscopy. The actual
values of the samples were obtained by reference methods. Calibration models for
each components were constructed by PLS regression.
114
The accuracy of the models was validated using a large number of LWE samples.
A high coefficient of determination values (R2) for validation data sets, 0.95, 0.992,
0.994, 0.972 were obtained for protein, lipid, moisture, and TSS, respectively. At
last, ATR-FTIR and NIR spectroscopy with PCA were used to detect the LWE
adulteration with water. A large number of (n=100) liquid and dry egg samples
were used during the analysis. PCA models were constructed for both liquid and
dry samples. The samples were classified considering whether they have been
adulterated or not. While combination of ATR-FTIR spectroscopy with PCA
provides classification of adulterated dry egg samples, NIR spectroscopy with PCA
succeeds in classifying of both liquid and dry adulterated egg samples.
The effect of heat treatment parameters on egg proteins was performed by UV-
VIS spectroscopy and CE. A number of LWE samples were heat-treated at 60 °C,
64 °C, and 68 °C for five minutes with a one-minute interval. Heat-treated samples
were centrifuged, and supernatant was used for UV-VIS spectroscopy and CE
analysis. UV measurements of the samples were taken at 280 nm and results
were evaluated considering the heat treatment parameters of LWE. An
interference-free CE electropherograms of the LWE samples were obtained.
Electropherogram data were analyzed by constructed PCA model. The model
classifies the LWE samples whether they have been untreated or treated. If they
have been treated, the model considers the heat treatment parameters.
Consequently, the extent of heat treatment applied to LWE was determined using
combination of CE with PCA.
Heat treatment parameters of LWE induced change in cake batter rheology were
investigated by measuring rheological behavior of batter. The cake batter exhibited
a pseudo-plastic flow behavior. The measured data were fitted to a power-law
model. Consistency index (K) and flow behavior index (n) were calculated using
the model. An increase in (K) values of batters was observed while heat treatment
parameters of LWE increased. Considering (n) values, a difference between
control and the other batters was obtained, whereas no difference was observed
among the batters including heat-treated LWEs. The moral of the story, the effect
of heat treatment parameters of LWE on cake batter rheology was demonstrated.
115
Furthermore, the effect of heat-treated LWE on the quality of baked cakes was
performed through a number of cake analyses (moisture loss, color change,
porosity, texture (hardness, cohesiveness, and gumminess), and specific volume).
Any difference in the moisture loss (%) among the baked cakes was not observed.
Color change was obtained for the baked cakes containing LWE treated at 68 °C
for 2 and 5 minutes. Instead of using untreated LWE, use of heat-treated LWE in
the cake batter caused baked cakes having less porosity and specific volume. An
increase in the heat treatment parameters of LWE leaded to an increase in
hardness, cohesiveness, and gumminess values. As a result, heat treatment
process of LWE caused batter having less air bubbles, which resulted in harder
and less fluffy baked cakes.
As a future work, ad-hoc solutions to eliminate the negative effects of the heat-
treated LWE on the baked cake quality can be researched. These solutions may
be on how the cake batter can be recovered by changing batter preparation
parameters or using any contributors to obtain its initial rheological properties.
116
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128
CURRICULUM VITAE
Credentials
Name, Surname : Reyhan Selin Uysal

Place of Birth : ÇORUM / Merkez
Marital Status : Married
E-mail : r.selin.uysal@gmail.com
Address : Hacettepe University Food Engineering Department Research
Laboratory 1 Beytepe ANKARA
Education
High School : ÇORUM ANATOLIAN HIGH SCHOOL

BSc. : EGE UNIVERSITY
MSc. : GAZİOSMANPAŞA UNIVERSITY
PhD. : HACETTEPE UNIVERSITY
Foreign Languages
English (Fluent)
YOKDIL Test: 85
International English Language Testing System (IELTS) Score: 5.5
Work Experience
Pınar Milk Goods Inc. (Internship – İzmir, 2008)
Kavukçu Flour Factory (Internship – Çorum, 2007)
Areas of Experiences
Food Analyses by Spectroscopic Techniques (Raman, Infrared, UV-VIS)
Food Analyses by Electrophoresis Techniques (Capillary and SDS-PAGE)
Determination of Food Adulteration
Determination of Liquid Egg Quality
Heat-treated Liquid Egg Product
Determination of Quality of Bakery (Cake) Product
129
Projects and Budgets
Project: Detection of butter adulteration by Raman spectroscopy – 19.000 TL
Hacettepe University Scientific Research Projects Coordination Unit
Publications
1. Uysal, R. S., Boyacı, İ. H., Acar Soykut, E., Ertaş, N., 2017. Effects of heat
treatment parameters on liquid whole egg proteins, Food Chemistry, 216, 201-208.
2. Uysal, R.S., Sabancı, S., Akpınar, O., Bölükbaşı, U., Yılmaz, L., 2016. Xylitol
Bioproduction from tobacco stalk, Fen ve Mühendislik Dergisi, 18(1), 89-100.
3. Uysal, R.S., Sabanci, S., Sapci B., Akpinar, O. (2015). Lignoselulozik
Materyallerden Ksilitol Üretimi ve Kullanım Alanları, Akademik Gıda, 13(2), 140-
148
4. Boyaci, I. H., Temiz, H. T., Geniş, H. E., Acar Soykut, E., Yazgan, N. N., Güven,
B., Uysal, R. S., Bozkurt, A. G., İlaslan, K., Torun, O., Dudak Şeker, F. C., 2015.
Dispersive and FT-Raman spectroscopic methods in Food Analysis, RSC
Advances, 5, 56606–56624.
5. Boyacı, I. H., Uysal, R. S., Temiz, T., Shendi, E. G., Yadegari, R. J., Rishkan,
M. M., Velioğlu, H. M., Tamer, U., Özay, D. S., Vural H., 2014. A rapid method for
determination of the origin of meat and meat products based on the extracted fat
spectra by using of Raman spectroscopy and chemometric method, Eur Food Res
Technol, 238(5), 845-852.
6. Boyacı, İ. H., Temiz, H. T., Uysal, R. S., Velioğlu, H. M., Yadegari, R. J.,
Rishkan, M. M., 2014. A novel method for discrimination of beef and horsemeat
using Raman spectroscopy, Food Chemistry, 148, 37–41.
7. Uysal, R. S., Acar, E. A., Boyaci, I. H., Topcu, A., 2013. Monitoring multiple
components in vinegar fermentation using Raman spectroscopy. Food Chemistry,
141, 4333–4343.
8. Uysal, R. S., Boyaci, I. H., Genis, H. E. and Tamer, U., 2013. Determination of
butter adulteration with margarine using Raman spectroscopy, Food Chemistry,
141, 4397–4403.
9. Akpinar, O., O. Levent, S. Sabanci, R.S. Uysal, B. Sapci. (2011). Optimization
and comparision of dilute acid pretreatment of selected agricultural residues for
recovery of xylose, Bioresource, 6(4), 4103-4116.
130
Oral and Poster Presentations
Oral presentation
1. Uysal, R. S., Boyaci, İ.H., Acar Soykut, E., Ertas, N. Monitoring of Protein
Changes in Pasteurized Liquid Egg Using Capillary Electrophoresis. Pittcon
Conference & Expo, Atlanta, March 6-10, 2016.
2. Uysal, R. S., Sabanci, S., Akpinar, O., Bolukbasi, U., Yilmaz, L. Xylitol
Bioproduction From Tobacco Stalk. VII. Bioengineering Congress, İzmir, 19-21
Nov 2015.
3. Boyaci, I. H., Temiz, H. T., Uysal, R. S., Velioglu, H. M., Tamer, U., Yadegari,
R. J., Rishkan, M. M. Discrimination of beef and horsemeat by taking the
advantage of Raman spectroscopy, 248th ACS National Meeting & Exposition,
San Francisco, CA, 2014.
4. Sabancı S., Uysal R. S., Sapci B., Usal G., Akpinar O. Ksilitolun Önemi ve
Üretimi. Türkiye 11. Gıda Kongresi, Hatay, 2012.
Poster presentation
1. Uysal, R. S., Geniş, H. E., Boyacı, İ. H., Acar, E. A. Rapid Determination of
Yolk-Albumen Ratio in Liquid Whole Egg by NIR Spectroscopy, Selectbio Food
Analysis Congress, Barcelona, 2014.
2. Uysal, R. S., Temiz, T., Boyacı, İ. H., Velioğlu, H. M., Tamer, U. Discrimination
of Meat Species Using Raman Spectroscopy and PCA, Pittcon Conference &
Expo, Chicago, 2014.
3. Uysal, R. S., Genis, H. E., Boyaci, I. H. and Tamer, U. Quantification of Butter
Adulteration With Margarine Using Raman Spectroscopy, Pittcon Conference &
Expo, Philadelphia, 2013.
4. Akpinar, Ö., R.S. Uysal, S. Sabancı and B. Sapci, “Optimization Xylitol
Production Conditions From Sunflower Stalk”, 11th ICEF International Congress
on Engineering and Food, Atina, 2011.
5. Sabanci, S., R.S., Uysal, B. Sapci, and Ö, Akpınar. “Xylitol Production From
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DETERMINATION OF LIQUID EGG QUALITY AND

EFFECTS OF HEAT TREATMENT ON EGG PROTEINS

SIVI YUMURTA KALİTESİNİN VE ISIL İŞLEMİN YUMURTA

REYHAN SELİN UYSAL

PROF. İSMAİL HAKKI BOYACI

Submitted to Graduate School of Science and Engineering of Hacettepe University

I dedicate this thesis to my love and husband,

who makes sense of my life,

DETERMINATION OF LIQUID EGG QUALITY AND EFFECTS OF

REYHAN SELİN UYSAL

Supervisor: Prof. İsmail Hakkı BOYACI

March 2017, 132 pages

Keywords: liquid egg, liquid egg adulteration, SDS-PAGE, ATR-FTIR

SIVI YUMURTA KALİTESİNİN VE ISIL İŞLEMİN YUMURTA

REYHAN SELİN UYSAL

Anahtar Kelimeler: sıvı yumurta, sıvı yumurtada tağşiş, SDS-PAGE, ATR-FTIR

Main contributions of this thesis can be summarized as follows;

! ATR-FTIR spectroscopy was used to perform compositional analysis of

The remainder of this thesis is as follows: In Chapter 2, a fundamental background

2.1. Structure and Chemical Composition of Eggs

A schematic illustration of the egg is given in Figure 2.1. Egg is covered by a

2.1.1. Egg Shell

Table 2.1. Components of eggshell [11]

2.1.2. Egg White

Lysozyme (ovoglobulin G1) is an N-acetylmuramidase enzyme that hydrolizes the

Ovoinhibitor is very similar to ovomucoid, as a proteinase inhibitor. It prevents the

Table 2.3. Mineral composition of the albumen [5]

Table 2.4. Vitamin composition of the albumen [5]

2.1.3. Egg Yolk

Table 2.5. Components of the yolk [11]

Figure 2.3. Fractionation of egg yolk into granules and plasma

Figure 2.4. Schematic representation of yolk LDL [9]

The other group of lipoproteins is high-density lipoproteins (HDLs). The HDLs,

Phosvitin, a free protein, is a phosphoglycoprotein including relatively high amount

Table 2.6. Mineral composition of the yolk [5]

2.2. Functional Characteristics of Egg in Food Systems

2.2.1. Egg Functionary in Foam Systems

Appearance of albumen changes to a milky white color with an elastic gel

2.2.3. Egg Constituents in Emulsion System

2.2.4. Egg Importance in Baked Products

Disruption of the thiol-disulfide interactions that are in charge of gluten network

2.3. Liquid Egg Products

2.3.2. Effects of Pasteurization on Functional Properties of LEPs

Pasteurization parameters of egg yolk from 60 ºC to 68 ºC for 3.5 to 4.5 minutes

2.4.1. Infrared Spectroscopy

Figure 2.5. Energy states in molecule vibrational levels [55]

An infrared spectrum is obtained by measuring a net transfer of energy from

2.4.1.1. NIR Spectroscopy

NIR spectrophotometers can be classified into two types in point of wavelength

Devices including semiconductors (PbS or InGaAs) are used as a single channel

2.4.1.2. ATR-FTIR Spectroscopy

There has been many studies on different groups of foods by mid-infrared

ATR-FTIR spectroscopy can be performed for quantitative analysis of foods.

SDS-PAGE separates polypeptide chains of proteins according to their molecular

𝐴𝑝𝑝𝑙𝑖𝑒𝑑 𝑣𝑜𝑙𝑡𝑎𝑔𝑒 (𝑁𝑒𝑡 𝑐ℎ𝑎𝑟𝑔𝑒 𝑜𝑛 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒)

2.4.3. Capillary Electrophoresis

A CE system composes of a capillary column, two buffer reservoirs, power supply

Figure 2.8. A schematic representation of capillary electrophoresis system