Maillard Browning Reaction

Maillard Browning Reaction
of Glucose-Lysine Model System

in a Microwave Field
by
Biwen Kristi Xing
B.Sc. (Food Science and Technology), The Ohio State University, 1994
A THESIS SUBMITTED IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES

(Food Science)
We accept this thesis as conforming

to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
December 2002
© Biwen Kristi Xing, 2002

In presenting this thesis in partial fulfilment of the requirements for an advanced
degree at the University of British Columbia, I agree that the Library shall make it
freely available for reference and study. I further agree that permission for extensive
copying of this thesis for scholarly purposes may be granted by the head of my
department or by his or her representatives. It is understood that copying or
publication of this thesis for financial gain shall not be allowed without my written
permission.
Department
The University of British Columbia

Vancouver, Canada
7^
DE-6 (2/88)
11
ABSTRACT
The impact of microwave heating on the formation of Maillard reaction products
(MRPs) was investigated and compared with the effect of conventional convective
heating, using glucose-lysine as a model system. Two different microwave treatments
were used to compare with conventional heating in a heated water bath: a 700 W, 2450
MHz vacuum microwave and, an Ethos Synth microwave reactor at 2450 MHz, which
dropped the microwave power level from 700 W to below 100 W after reaching the
desired temperature. Experiments were carried out at controlled temperatures of 63°C up
to 120°C, and Maillard reaction rate, UV/Visible spectral characteristics of MRPs,
dielectric properties of MRPs, matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS) analysis of MRPs, Fourier transform infrared
(FTIR) spectroscopy analysis of MRPs and cell toxicity test of MRPs were studied.
Comparison with control samples treated by conventional heating showed a significant
rate enhancement of the studied reactions during vacuum microwave heating. Results of
UV/visible spectral patterns and dielectric properties indicated that different compounds
or different concentrations of compounds were produced during vacuum microwave
treatment. However, these differences were not observed during the Ethos Synth
microwave heating. Ethos Synth microwave heating produced similar MRPs as
conventional heating. MALDI-TOF MS analysis of 81°C MRPs and FTIR analysis of
90°C MRPs also indicated the major chemical components of MRPs from different
treatments were the same, however, the concentrations of compounds were different. In
addition, the MRPs at 90°C derived from vacuum microwave heating of glucose-lysine
showed a higher cytotoxic effect when compared with MRPs derived from water bath or
Ethos Synth microwave reactor heating at lower dose concentration (0.5 mg/ml). These
differences are possibly due to the higher microwave power in vacuum microwave.
iv
T A B L E OF CONTENTS
ABSTRACT ...ii
TABLE OF CONTENTS iv
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF SYMBOLS AND ABBREVIATIONS xi
ACKNOWLEDGEMENT xiii
1 INTRODUCTION 1
2 LITERATURE REVIEW 3
2.1 Discovery of Maillard Browning Reaction 3

2.1.1 Initial Stage of the Maillard Reaction 4
2.1.2 Intermediate Stage of Maillard Reaction 8
2.1.3 Final Stage of Maillard Reaction 12
2.1.4 A Conceptual Approach of
Classification of Maillard Reaction 12
2.2 Factors Affecting the Maillard Browning Reaction 13
2.2.1 Nature and Molar Ratio of Reactants 14
2.2.2 Moisture Content of the Food 16
2.2.3 Temperature and Duration of Heating 17
2.2.4 Effect of pH 18
2.2.5 Other Miscellaneous Factors Affecting
the Rate of Maillard Reaction 19
2.3 Kinetics of Maillard Reaction 20
2.4 Brown Pigment Measurement of MRPs 21
2.5 Significance of Maillard Reaction in Foods 22
2.6 Toxicological Aspects of Maillard Browning 24
2.6.1 In vivo Gross Toxicological Effects of MRPs 24
2.6.2 In vitro Toxicological Effects of MRPs 26
2.6.3 Acrylamide: a Carcinogen Formed in
the Maillard Reaction. 27
2.7 Microwave Characteristics 27
2.7.1 Electrical and Physical Properties 29
2.7.2 Heating Patterns 31
2.7.3 Microbial Inactivation 32
2.7.4 Human Safety 33
V
2.7.5 Nutritional and Toxicological Effects of

Microwave-Treated Foods 34
2.8 Comparison of Maillard Reaction in
Microwave and Conventional Heating 35
3 THESIS DEVELOPMENT 38
4 MATERIALS AND METHODS 39

4.1 Materials 39
4.2 Overview of Methods 42
4.2.1 Sample Preparation 42
4.2.2 Heat Treatments 43
4.2.3 Maillard Reaction Rate Determination. 45
4.2.4 Comparison of UV/Visible
Spectral Characteristics of MRPs 46
4.2.5 Dielectric Property Measurements 47
4.2.6 MALDI-TOF MS Analysis of MRPs of
Glucose-Lysine Model System 48
4.2.7 FTIR Analysis 49
4.2.8 Cell Toxicity Test 50
4.2.9 Kinetic Studies 52
4.2.10 Statistical Analysis 53
5 RESULTS AND DISCUSSION 54

5.1 Maillard Reaction Rate Constants and
Activation Energies 54
5.2 Comparison of UV/Visible
Spectral Characteristics of MRPs 64
5.3 Dielectric Property Measurements 77
5.4 MALDI-TOF MS Analysis of MRPs of
Glucose-Lysine Model System 86
5.5 FTIR Analysis 95
5.6 Cell Toxicity Test 103
6 CONCLUSION 106
7 REFERENCES 108
8 APPENDIX 122
vi
LIST OF T A B L E S
Table 5.1 Temperature dependence of the rate constant and activation

energy for water bath, vacuum microwave and Ethos Synth
microwave reactor at different temperatures 61
Table 5.2 Relative intensities (height) of peaks obtained from the

MALDI-TOF spectra of MRPs (81°C) 94
Table 5.3 Functional groups and vibrational modes obtained from

the FTIR spectra of MRPs 102
Table 5.4 Effects of MRPs (90°C) on viability (% control) of Caco-2 cells 104
vii
LIST OF FIGURES
Figure 2.1 Review of Maillard reaction pathway 5
Figure 2.2 Initial stage of the Maillard reaction 7
Figure 2.3 Condensation of carbonyl compounds with amino compounds 7
Figure 2.4 A) Formation of sugar fragmentary products. B) Two possible

Pathways (I) and (II) of free radical formation 9
Figure 2.5 Two possible pathways of Amadori compound degradation.

Pathway I via 1,2 enolization and Pathway II via
2,3 enolization 11
Figure 2.6 Deoxyosone degradation via Strecker degradation Pathway III 11
Figure 4.1 The schematic of a vacuum microwave apparatus 40
Figure 4.2 Schematic diagram of the Ethos Synth microwave system 41
Figure 5.1 Log of Maillard browning rates of 0.05 M glucose and

0.05 M lysine as a function of temperature and heat treatment 56
Figure 5.2 A shorter time period of log of Maillard browning rates of

0.05 M glucose and 0.05 M lysine as a function of
temperature and heat treatment 57
Figure 5.3 Dependence of D value on temperature. Plot of log D

versus temperature for water bath, vacuum microwave
and Ethos Synth microwave systems 62
Figure 5.4 Dependence of rate constant, k, on temperature. Plot of

In k versus 1/T for water bath, vacuum microwave and
Ethos Synth microwave systems 63
Figure 5.5 UV/vis spectral patterns comparison (220-600 nm) of Maillard

Browning samples, which had the same absorbance values at
420 nm (beginning of the process), of vacuum microwave,
water bath and Ethos Synth microwave system at 63°C. 68

420 nm (middle of the process), of vacuum microwave,
water bath and Ethos Synth microwave system at 63°C 69
viii

420 nm (the end of the process), of vacuum microwave,






Figure 5.14 Dielectric constant of collected Maillard browning samples

(63°C) as a function of vacuum microwave, water bath and
Ethos Synth microwave heating 80
Figure 5.15 Loss factor of collected Maillard browning samples

ix




Figure 5.20 MALDI-TOF mass spectrum of heated water bath sample (81°C) 90
Figure 5.21 MALDI-TOF mass spectrum of vacuum microwave sample (81°C) 91
Figure 5.22 MALDI-TOF mass spectrum (high-mass expansion)

of heated water bath sample (81°C) 92
Figure 5.23 MALDI-TOF mass spectrum (high-mass expansion)

of vacuum microwave sample (81°C) 93
Figure 5.24 FTIR overlay spectra of MRPs, from a glucose-lysine

mixture in water bath, vacuum microwave and
Ethos Synth microwave reactor at 90°C 98
Figure 5.25 FTIR stack spectra of MRPs, from a glucose-lysine

mixture in water bath, vacuum microwave and
Ethos Synth microwave reactor at 90°C 99
Figure 5.26 FTIR overlay spectra of water bath MRP (90°C) and
non-treated mixture of glucose-lysine model 100
Figure 5.27 FTIR stack spectra of water bath MRP (90°C) and
non-treated mixture of glucose-lysine model 101
Figure 8.1 MALDI-TOF CCA matrix background 122
Figure 8.2 FTIR original spectrum of D-glucose 123
Figure 8.3 FTIR original spectrum of L-lysine 124
Figure 8.4 FTIR original spectrum of SCB buffer 125

X
Figure 8.5 FTIR original spectrum of non-treated mixture

of glucose-lysine model 126
Figure 8.6 FTIR original spectrum of MRPs from a

glucose-lysine mixture in water bath at 90°C 127

glucose-lysine mixture in vacuum microwave at 90°C 128

glucose-lysine mixture in Ethos Synth
microwave reactor at 90°C 129
xi
LIST OF S Y M B O L S AND ABBREVIATIONS
Symbols
°c Degrees Celsius
op Degrees Fahrenheit
a.i. Arbitary intensity
B Measurement of browning in absorbance
D Time required to change the concentration of reactants or products
by 90%
E' Dielectric constant
8" Loss factor
Ea Activation energy
K Kelvin
k Rate of browning
log logarithm
M mole/liter
mM millimole/liter
ml milliliter
u\ microliter
cm centimeter
nm nanometer
m/z mass/charge
MHz Megahertz
min minute
hr hour
t Heating time
W Watts
z Temperature change required to change the decimal reduction
time by a factor of 10
Abbreviations
ANOVA Analysis of Variance

CCA 4-hydroxy-alpha-cyanocinnamic acid
DHB 2,5-dihydroxybenzoic acid
DMEM10 Dulbecco's modified eagle medium containing 10% fetal
bovine serum
EDTA Ethylenediaminetetraaceticacid
FTIR Fourier transform infrared spectroscopy
MALDI-TOF MS Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
MR Maillard reaction
MRP Maillard reaction product
MRPs Maillard reaction products
PBS Phosphate buffer saline
SCB Sodium carbonate/bicarbonate
UV Ultraviolet
Vis Visible
xiii
Acknowledgement
First of all, I have to express my greatest gratitude to Dr. Tim Durance, my
supervisor, for his guidance, advice, encouragement, and appreciation of this work. I
would also like to thank the other members of the supervisory committee, Dr. David
Kitts, Dr. Eunice Li-Chan, Dr. Chris Seaman, for their constructive input and valuable
suggestions on improving this work.
My sincere appreciation is also extended to Mr. Sherman Yee and Ms. Valerie
Skura for their technical assistance and invaluable advice during the course of this study.
Grateful thanks are due to Hao Jing for helping me out on the cell toxicity test, and to
Guangtao Meng, for her help on FTIR analysis and her valuable advice. Analytical
guidance provided by Mr. Marshall Lapawa, Department of Chemistry, during MALDI-
TOF MS analysis is deeply appreciated.
I wish to thank Bo Lei and Parastoo Yaghmaee for always being there while I was
having problems and helping me to clean the mess I made. I am also greatly indebted to
Esmond Kan, for his graphic assistance and for helping me solve computer problems no
matter how busy he was.
Finally, a very special thank-you to Mona Chow, without her help I couldn't
finish all my lab works! Without her caring, I probably gave up on myself! Many thanks
to Min Lau, for his networking expertise and his encouragement, patience, understanding
throughout difficult times, and especially for accepting me just as I am.

1
1. INTRODUCTION
The Maillard reaction plays an important role in food chemistry because of its
significant effects in developing color and flavor properties of foods (Feather, 1989;
Ames, 1990; Bailey et al., 1995; Arnoldi et al., 1997). The Maillard reaction refers to the
interaction initiated between the carbonyl group of a reducing sugar and a free amino
group of an amino acid or a protein. A cascade of complex reactions follows the
formation of the initial intermediates, which include Amadori and Heyn's products
(Hodge, 1953; Namiki, 1988; Yaylayan, 1997). The complex Maillard reaction consists
of both early and advanced Maillard reactions, which can be characterized by absorption
spectra and molecular weights (Cuzzoni et al., 1988; Ames, 1992; Wijewickreme et al.,
1997). Early Maillard reaction corresponds to well-defined chemical steps without
browning and progress by fragmentation and polymerization to volatile or soluble
substances, and finally to insoluble brown polymers (Mauron, 1981).
Microwave heating systems have been widely used in the food manufacturing
industry within the last decade because of its convenience, economy and time saving
(Mingos and Baghurst, 1997; Oruna-Concha et al., 2002). The principle of heating with
microwaves is very different from that of conventional heating by conduction or
convection. It is caused by molecular friction of electrical dipoles under an oscillating
electric field of specific frequency (EFT, 1989; Fakhouri and Ramaswamy, 1993; Finot
and Merabet, 1993). In addition, the question of the existence of a possible microwave
athermal (non-thermal) effect, was raised very early (Jacobs et al., 1950). Since then,
numerous studies have been reported and so far, no clear experimental evidence
supporting a 'per se' effect of microwave in different reaction models has been presented
2
(Jahngen et al., 1990; Laurent et al., 1992; Stuerga and Gaillard, 1996a;b). Therefore, the
generation of color and flavor during microwave cooking is of interest to the food
industry and the consumers. Until now very few studies have been reported comparing
the formation of Maillard products in microwave cooking versus conventional cooking
(Fay et al, 1991; Meifmer and Erbersdobler, 1996), with the exception of reports about
flavor development of Maillard reaction compounds (Whorton and Reineccius, 1989;
Yeo and Shibamato, 1991; Yu et al., 1993; Oruna-Concha et al., 2002).
The aim of the present study was to compare the Maillard browning reaction in
microwave fields with that in conventional heating system under controlled conditions.
For this reason, it was necessary to hold exact temperatures to get comparable results
among the heating methods. A Maillard model system, consisting of D-glucose and L-
lysine, was used in this investigation. The study was designed to compare the Maillard
reaction rate, the compositions of the MRPs and the cytotoxicity effects of the MRPs,
using three heat treatments - vacuum microwave, Ethos Synth microwave and water
bath. The information provided by this study may help us to better understand the effects
of microwave heating on Maillard reactions and to extend the use of microwave heating
to the production of superior quality Maillard browning products.

3
2. L I T E R A T U R E R E V I E W
2.1 Discovery of Maillard Browning Reaction
The Maillard reaction is a type of non-enzymatic browning reaction that involves
the reaction of carbonyl compounds, especially reducing sugars, with compounds that
possess a free amino group, such as amino acids and proteins. The reaction products are
significant in foods because they are responsible for flavor and color changes, which may
be desirable or undesirable depending on the type of foods (Ames, 1990).
Despite its name, the reaction was actually discovered by an Englishman named
Ling who, in 1908, first noted that the color produced in the brewing process came from a
reaction between sugars and proteins (Ling, 1908). However, it was Louis Camille
Maillard, who, in 1912, first described the reaction systematically while observing the
evolution of a mixed solution of glucose and glycine subjected to heating (Maillard,
1912). As a result, Maillard found marked development of color and flavoring (Maillard,
1912; Kawamura, 1983).
Since that time these reactions, often called Maillard reactions, continue to be a
significant focus of attention in the food chemistry. During the past 90 years, numerous
studies on the Maillard reactions have been published. As summarized and reviewed by
Hodge (1953), the interest of researchers was at first limited to the browning and
flavoring of foods. Later, studies on the effects of the reactions on nutritional and
physiological properties of foods, as well as changes in the physicochemical properties of
proteins and antioxidative activity, began to increase in number (O'Brien and Morrissey,
1989; Ames, 1992; Brands et al., 2000).

The Maillard reaction, which is actually a complex network of chemical reactions
(Figure 2.1), is traditionally divided into three stages as follows (Hodge, 1953):
I. Initial stage (colorless, no absorption in the near-UV)
A. Sugar-amine condensation
B. Amadori rearrangement
II. Intermediate stage (colorless or yellow, with strong absorption in the near-
UV)
C. Sugar dehydration
D. Sugar fragmentation
E. Amino acid degradation
III. Final stage (highly colored)
F. Aldol condensation
G. Aldehyde-amine polymerization; formation of heterocyclic nitrogen
compounds
2.1.1 Initial Stage of the Maillard Reaction
The initial stage of the Maillard reaction is presented in Figure 2.2 (Hodge, 1967).
The first step in the overall reaction is a simple condensation between the carbonyl group
(aldehyde form of the reducing sugar) and the free amino acid giving rise to a N-
substituted glycosylamine compound and followed by the reversible formation of the
Schiff base derivative.

aldose sugar + amino compound
N-substituled glycosylamine + HjO
Amadori rearrangement^
I - amino -I- deoxy -2- ketose

(1,2 enol form)
Amadori compound
(Q + amino acid (E)
-3H 0
r
2
-2H,0 (Q Strecker degradation

Schiffbaseof (D)
Reductones
HMF or furfural
Fission products
[£<h\
-amino compound
+2H (acetol, pyruvaldehyde, \aldehyde
-2H
diacetyl, etc)
HMF or furfural dchydro reductones
+ amino cdmpd.
(G) (F)
+ amino compound with or without (F) + amino compd (G)
amino compound (F)
Aldimines
Aldimines
(F) Aldols and
N-free polymers Aldimines
or ketimines
+ amino compd (G) (G)

(G)
(G)\
MELANOIDINS
Figure 2.1. Review of Maillard reaction pathway (Hodge, 1953).

6
To initiate this condensation reaction, a nucleophilic amino nitrogen interacts with
an unshared electron pair on the carbonyl carbon. Once the carbonyl group is protonated,
its reactivity to the nucleophilic reagent is enhanced, while protonation of the nitrogen of
the amino group inhibits the attack on the carbonyl carbon (Figure 2.3).
A Schiff base is an intermediate step in the Maillard reaction since it rapidly
cyclizes to a N-substituted glycosylamine. Up to this step, the reaction is reversible
because the glycosylamine can be hydrolyzed to its parent compounds in aqueous
solutions. Aldose sugars react with amino groups to form aldosylamines, where as the
keto sugars react with amino groups to form ketosylamines. Both aldosylamines and
ketosylamines are highly reactive and are converted quickly to Amadori compounds (1-
amino-l-deoxy-2-ketose) and Heyns products (1-amino-l-deoxy-2-aldose), respectively
(O'Brian and Morrissey, 1989). Since amino acids serve as their own acid catalysts, the
reaction is rapid even in the absence of added acids (Namiki, 1988).
It is also important to note that the neutral or alkaline pH conditions which
promote the browning reaction are not conducive to the initial stage of the MRPs, which
is favored by acidic conditions (Namiki, 1988). Another important observation in this
early stage is the fact that free radical formation occurs in the process of sugar
fragmentation (Mitsuda et al., 1965; Namiki et al., 1973; Namiki and Hayashi, 1981,
1983). Stable free radicals have also been observed in alkaline solutions of reducing
sugars (Lagercrantz, 1964) and in amino-carbonyl reactions of ninhydrin with amino
acids (Yuferov et al., 1970). These studies suggest that Maillard reaction may also
involve a free radical process or produce some form of free radical compounds through a
HOO RNH RN RNH RNH
| +RNH I -HjO
^H,
2
(CHOH). <=> QHOH o
CHfiH (CHOH)* (CHOH). (CHOH).., 0 C=0
CH/)H CUfiH JKj 1 (H(^OH)n
CHjOH CHfiH
Aldose in Addition Schiffbase N-substituted N-substituted

aldehyde form compound glycosylamine l-amino-l-deoxy-2-ketose
Amadori compound
Figure 2.2. Initial Stage of the Maillard reaction (Hodge, 1967).
©
/ NH/t
:NH} -R
^OH
e
< NHjR
OH
NHR -HjO
+HjO
>ON+
R
/ti
\
-t©
" >C=N-R
Figure 2.3. Condensation of carbonyl compounds with amino compounds
(Namiki, 1988).
8
similar process that occurs in the above mentioned compounds. A free radical formation
mechanism through sugar fragmentation as proposed by Namiki (1988) is presented in
Figure 2.4.
2.1.2 Intermediate Stage of Maillard Reaction
Both Amadori and Heyns compounds are initiators of the intermediate step in
Maillard reaction and play important roles in physical, nutritive and physiological
properties of proteins (Adrian, 1974; Mauron, 1981; Mester et ah, 1981; Monnier and
Cerami, 1983; Baynes et al., 1986; Erbersdobler, 1986). The main process of color and
flavor formation through Amadori or Heyns steps involve degradation reactions
associated with the formation of deoxyosones and Strecker degradation (Mauron, 1981).
In the pH range between 4 and 7, Amadori compounds and Heyns products are
predominantly degraded to deoxyosones (Beck et al., 1988; Huber et al., 1988). Two
major pathways of Amadori rearrangement product (ARP) degradation and deoxyosone
production which ultimately produce color and flavor products are shown in Figure 2.5
(Namiki, 1988).
Pathway I occurs via 1,2-enolization, followed by elimination of the hydroxyl
group at C-3 and deamination at C-l, thus yielding 3-deoxyhexasone, a reactive carbonyl
product and a hydroxyl-methyl furfural (HMF). This pathway is favored under acidic
conditions.
Pathway II occurs via 2,3-enolization of the Amadori reaction products, followed
by elimination of the amino group from C - l which gives rise to a 1-deoxydicarbonyl

9
A)
H
CHO CH=NR HONR HC-NR
I ICHOH
CHOH + RNHj { HC-OH II
I > I ~ ~* 2-CHC-OH
fragmentary
product
CHOH -HjO CHOH
R' R'
CHO
sugar Schiff base reverse aldol reaction
I
R' x
B)
m H
Urownutg
HC-NR
II
R
HC-OH condensation
u — • • a
N
R
H
H3C-NR
HC=0
glycoaldehyde dialkyl-dihydro dialkyl dialkyl
pyrazine pyrazine pyrazinium
radical compound
ID
H
HC-NR HONR HONR • Hzf4 HONR
II HOO
+ H2O
HC-OH H3C-OH oxidation I * HONR

I • •2RNHj ' I
glycolaldehyde HOO +RNHj glyoxal HOO
alkylimine glyoxal
mono-alkylimine di-alkylimine glyoxal
Figure 2.4. A) Formation of sugar fragmentary products. B) Two possible pathways (I)
and (II) of free radical formation (Namiki, 1988).

10
intermediate. This compound further reacts to produce methylglyoxal, diacetyl, and other
intermediates (Hodge, 1967). This pathway is favored under alkaline conditions.
The products of deoxyosones are important since these intermediate products of
Maillard reaction lead to a number of further reactions, such as ring formation,
enolization, dehydration, condensation and C-C fission reactions of Amadori and thus a
multiplicity of MRPs are formed. In addition to the number of different MRPs, the yield
will also vary, depending on the reaction conditions (eg. type of sugars, amines, reaction
times, temperatures, water activity and pH). Consequently, the compositions of MRPs
differ, which in turn result in changes in both sensory (e.g., flavor, color and aroma), as
well as non-sensory (e.g., antioxidative properties, mutagenic activities) attributes.
Strecker degradation is the third pathway in the formation of brown colored
compounds from Amadori rearrangement products (Namiki, 1988). This series of
reactions involves the oxidative degradation of amino acids by a-dicarbonyl compounds
that are produced from the breakdown products of Amadori compounds in pathway I and
II. In the Strecker degradation (Figure 2.6), oc-amino acids react with a-dicarbonyl
compounds to form Schiff bases which in turn enolize to form amino acid derivatives that
are easily decarboxylated. The new Schiff base with one carbon atom less is then easily
split hydrolytically into the amine and the aldehyde which in turn correspond to the
original amino acid with one less carbon atom. The net result of the reaction is a
transamination which could be an important reaction for the incorporation of nitrogen
into melanoidins. Most of the CO2 released during the Maillard reaction is produced
during the Strecker degradation step (Stadtman et al., 1952; Wolfrom and Rooney, 1953).
11
HC-N< HOfT< HOC HOO
C\-OH IC\OH L
do ho
LowpH •H(OH) CHfOH) CH(OH) -famine

f
CH(OH) C\H(OH) ):H(OH)
I I I
H CfN<
(3-deoxy osone)
2
intermediate
V
I Amadon
CH(OH) c m p d
CH (i
1(OH)
reddIctones
c\f{com { U )
OOH OOH O O CHrCO-CHO

HighpH
jJ(W • j>=0 CHrCO-COCH,
CH(OH) CHOH HOCHtCO-CO-CH,
CH(OH) CH(OH)
r m
\
etc.
/
CH(OH)
Figure 2.5. Two possible pathways of Amadori compound degradation. Pathway I
via 1,2 enolization and Pathway II via 2,3 enolization (Namiki, 1988).
Figure 2.6. Deoxyosone degradation via Strecker degradation Pathway III
(Namiki, 1988)
12
2.1.3 Final Stage of Maillard Reaction
In the final stage of the reaction, where most of the color is produced, high
molecular weight brown pigments, referred to as melanoidins, are formed. These
compounds are believed to form as a result of polymerization of many highly reactive
compounds formed during the intermediate stage in the Maillard reaction (Reynolds,
1965). Melanoidins have different solubilities. Some are readily soluble in water while
others are slightly soluble in water, or completely insoluble in water (Mauron, 1981). It
is believed that there is a repeating unit for many melanoidins (Kato and Tsuchida, 1981;
Cammerer and Kroh, 1995). Namiki (1988) emphasized that even small quantity of a
product isolated from a reaction mixture, may be important in melanoidin formation. It
was also noted that the more reactive the product, the more difficult it is to isolate and
identify, thus conveying the complexity and ongoing dynamics of this reaction under
different conditions.
2.1.4 A Conceptual Approach of Classification of Maillard Reaction
According to Yaylayan (1997), it was too general and simplistic to define and
classify the Maillard reaction into initial, intermediate and final stages. A conceptual
representation of the processes occurring during the reaction was developed in which the
Maillard reaction was described as proceeding by the formation and interaction of
'chemical pools'. A collection of products arising from the decomposition of well
defined precursors was termed a fragmentation pool, and a group of related chemical
reactions and their products was termed an interaction pool. The pools that were formed
from the decomposition of the sugar, amino acid and Amadori or Heyn's products were
termed primary fragmentation pools. Further reactions among the populations of these
13
pools led to 'interaction pools'. Three types of pool interactions were identified: 'self-
interaction', 'secondary interaction' and 'multiple-interaction'. In addition, the
fundamental reactions that underlay the pool interactions were classified according to the
type of chemical transformation effected. Yaylayan (1997) claimed that such
classification could be used to predict the formation of Maillard reaction products in
model systems.
2.2 Factors Affecting the Maillard Browning Reaction
The formation of brown pigments and related browning aromas can be considered
positive or negative, depending on the product. Examples of products that benefit from
the reaction are roasted coffee, meat, baked bread, cereals, and processed maple syrup.
On the negative side is formation of brown pigments in nonfat dried milk powder or
dehydrated potatoes, leading to off-flavors and undesirable darkening of color. In dried
milk, lactose reacts with the e-amino group of lysine exposed from the casein micelle,
resulting in severe reduction of the nutritional value of the product. Labuza and
Saltmarch (1981), as well as many others, have studied this loss of protein quality in
different foods.
The Maillard reaction is strongly influenced by the reaction conditions (Mauron,
1981; Ashoor and Zent, 1984). A number of factors influencing Maillard reaction are
discussed below.
14
2.2.1 Nature and Molar Ratio of Reactants
Low molecular weight compounds are considered more reactive than high
molecular weight compounds. As such, aldopentoses are generally more reactive than
aldohexoses (Spark, 1969), and monosaccharides are more reactive than di- or oligo-
saccharides. Aldoses have been reported to be more reactive than ketoses (O'Brien and
Morrissey, 1989). Lewis and Lea (1959) reported that sugars when placed in descending
order of reactivity have the following sequence: xylose > arabinose > glucose > lactose >
maltose > fructose. As a result, Finot et al. (1981) showed that replacement of lactose by
fructose in milk samples resulted in a decreased lysine destruction in the order of 0-2%.
Furthermore lysine destruction was decreased up to 50% when such samples were spray
dried.
The ratio of reducing sugar to amino acid has been suggested as an important
factor in determining the rate of Maillard reaction and has important implications both
from a food formulation standpoint and in vivo glycation of proteins (Baisier and Labuza,
1992). Generally, an excess of reducing sugar to that of an amino compound accelerates
Maillard reaction (O'Brien and Morrissey, 1989). Lea and Hannan (1951) observed a
maximum browning rate at a 3:1 molar ratio in a glucosexasein system. Warmbier et al.
(1976) reported that the rate of browning increased as the molar ratio of glucose:glycine
increased from 2:1 to 3:1 in an intermediate moisture model system. Moreover, studies
performed by Wolfrom et al. (1974) indicated color development in D-glucose-glycine
mixtures, containing 65% water and stored at 65°C, was markedly dependent on the
relative proportion of the amino acid to sugar ratio. These results indicated that color
production was relatively modest at a D-glucose-glycine molar ratio of 2:1 to 10:1, but
15
that the development of color rapidly accelerated at a molar ratio of 1:1 to 5:1. The
degree of browning, therefore, appears to be maximum when sugar is present in excess.
An optimal molar ratio of 3:2 has been reported for a glucose:methionine system
(Dworschak and Orsi, 1977). However, an opposite trend of the sugar-amino acid molar
ratio on browning was reported by Warmbier et al. (1976). The reason for the opposite
trend observed above remains unclear; perhaps it may have stemmed from the lower
water activity (0.52) used by these scientists compared to other work done in solution
(Kannane andLabuza, 1989).
The chemical nature of the amino acid also affects the rate of Maillard reaction
formation. Amino acids with e- and end terminal amino groups are the most reactive in
Maillard reaction. For example, the amino acid lysine which possesses two amino groups
on the molecule, is more reactive than other amino acids which have no available amino
side chains. Therefore, the concentration of lysine in a protein is closely related to the
degree of browning that takes place in a particular food following processing and storage.
It is also known that amino acids affect the pH of the system and, therefore, have an
influence on the degree of browning.
Ashoor and Zent (1984) reported that amino acids and amides might be classified
into three groups according to their Maillard reactivity when heated in the presence of
common reducing sugars: (i) the four amino acids classified as the most reactive group
were lysine, glycine, tryptophan and tyrosine; (ii) an intermediately reactive group
including proline, leucine, iso-leucine, alanine, hydroxy proline, phenylalanine,
methionine, valine and the amides glutamine and asparagine; (iii) histidine, threonine,
aspartic acid, arginine, glutamic acid and cysteine were considered as the least reactive
16
group. This classification is not in agreement with the results reported by Massaro and
Labuza (1990) who observed that lysine produced the least browning when compared to
tryptophan and cysteine. In another study, Fry and Stegink (1982) found that proline and
amino acids with hydrophobic side chains reacted more slowly than amino acids without
these side chains. These conflicting results may be explained by the pH dependence of
different amino acids in the Maillard reaction. As such, it may be expected that the
amino acids that react similarly at a certain pH range may not necessarily respond the
same way under other pH ranges.
Wolfrom et al. (1974) designed a comprehensive study to determine the effect of
amino acid type on the extent of browning in a D-glucose-amino acid mixture. The study
involved using amino acids that were commonly found in orange juice and other foods.
The results of those studies indicated that both L-arginine and aminobutyric acid, for
example, produced the most intense color, followed by glycine, alanine, serine, and L-
proline. Aspartic acid, L-glutamic acid and L-glutamine showed a behavior similar to
that of glycine.
2.2.2 Moisture Content of the Food
Maillard reaction in foods and model systems occur over a wide range of water
activities. Water activity exerts a primary influence by controlling the liquid phase
viscosity by dissolution, concentration, or by dilution of the reactants (O'Brien and
Morrissey, 1989). Maximum browning occurs in most foods with a water activity in the
range of 0.3 to 0.7 (Labuza et al., 1970). The reaction rate increases exponentially with
increasing moisture content up to a maximum in the intermediate moisture range due to
increased mobility of reactants. A further increase in water activity decreases the

17
reaction rate due to a dilution effect. However, the position of maximum reaction
kinetics depends on the physicochemical properties of the respective food system
(Supplee, 1926). For example, an amorphous food system absorbs more moisture than a
crystalline food. Eichner and Karel (1972) studied the isolated effect of water content
and that of water activity similarly on the browning rate. They reported that a decrease in
water content would increase the rate of browning except in those systems where water
mobility was limited by high viscosity. In essence, the extent of browning was
determined to be influenced by the amount of available water and by the state of water
binding in the system.
2.2.3 Temperature and Duration of Heating
Many scientists have observed that the rate of browning increases with the
reaction temperature and the reaction time. Cuzzoni et al. (1988) showed that the
reaction temperature, absorbance at 420 nm and 280 nm, mutagenicity and the level of
furfurals were all correlated at temperatures between 120-140°C. Baxter (1995) reported
that the reaction rate increases also with an increase in pH, temperature, and reducing
sugar content. The importance of reaction temperature and duration of heating was
further illustrated by Hurrell and Carpenter (1974) who demonstrated the similarity in the
amount of e-aminolysine groups reacted in an albumin-glucose system in low
temperature, long duration experiments (30 days at 37°C) and with those of high
temperature, short duration experiments (15 minutes at 121°C).

18
2.2.4 Effect ofpH
Numerous research projects have been undertaken to investigate the effect of pH
on browning reactions in a variety of foods (Underwood et al., 1959; Kato et al., 1969;
Wolfrom et al., 1974; Ashoor and Zent, 1984). Ashoor and Zent (1984) reported that
Maillard reaction increased as the pH of the system increased up to a maximum of 10,
and that the reaction rate then declined above a pH value of 10. Although the reason for
a decrease in the rate of browning at pH values above 10 is unknown, two mechanisms
have been proposed to explain the effect of pH on the increased rate of browning for
those pH values below 10. These mechanisms can be summarized as follows: (i)
mechanism I: the availability of an unprotonated amino group is a prerequisite for the
occurrence of Maillard reaction. Therefore, an increase in the reaction medium pH
ultimately drives the amino group from a protonated form to an unprotonated form,
which increases the rate of browning. The protonated and unprotonated forms of the
amino group are shown by the following:
-NH 3
+
( H>pKa)
P -NH + H
2
+
(Eq.2.1)
According to equation 2.1, at pH values higher than the pKa of the amino acid, the amino
groups do not possess a positive charge thus an increase in the extent of browning occurs;
(ii) mechanism II: the amount of acyclic form, or the reducing form, of sugar also affects
the rate of browning and is pH dependent. With an increase in medium pH, the
concentration of acyclic form of reducing sugar increases, which in turn increases the rate
of Maillard reaction (Kannane and Labuza, 1985).
In addition to the two above mentioned mechanisms, sugar fragmentation also
plays an important role in Maillard reaction kinetics. Higher pH values at the beginning
19
of the reaction increase the degree of sugar fragmentation leading to increased reaction
rates due to the greater reactivity of smaller compounds. More detailed information on
the effect of pH on Maillard reaction can be found in an extensive review on the control
of browning reaction in foods conducted by Labuza and Schmidl (1986).
2.2.5 Other Miscellaneous Factors Affecting the Rate of Maillard Reaction
Certain metal ions are known to affect Maillard reaction kinetics. Copper and
iron salts appear to accelerate the Maillard reaction while tin and manganese salts appear
to inhibit the reaction (Bohart and Carson, 1955; Kato et al., 1981). Iron has been
suggested to catalyze the browning of soy sauce and also has been identified to be a
constituent of the melanoidin chromophore (Hashiba et al., 1981). To a certain extent,
the effect of metal ions on browning is known to result from the pH changes occurring in
the medium due to the presence of metal salts.
Another set of compounds that affect Maillard reaction are phosphates and
sulphites. Phosphates promote the Maillard reaction and chromophore development
(Reynolds, 1959). However, the mechanism is not known. Sulfur dioxide and sulfites
have long been known to inhibit the Maillard reaction (Modderman, 1986; Taylor and
Bush, 1986; Wedzicha and Kaputo, 1987). Although the chemical basis for this effect is
not completely understood, sulfur dioxide and sulfites appear to react with reducing
sugars and carbonyl products of the Maillard reaction to produce sulfuric acid derivatives
which have a diminished tendency to brown.

20
2.3 Kinetics of Maillard Reaction
A number of kinetic studies have been carried out on the Maillard reaction. Two
approaches with respect to Maillard reaction kinetic studies have been proposed in the
literature. The first approach focuses on the rate of browning, and the other relates to the
rate of loss of sugar and amino acids. General kinetic models for Maillard reaction have
been proposed by various research groups (Baisier and Labuza, 1992; Vernin et al., 1992;
Yaylayan and Forage, 1992; Bell, 1996). Most of these models have been based on a
general scheme in which the reducing sugar reacts with the amine to produce a Schiff
base that undergoes the Amadori rearrangement to produce the Amadori product.
Extensive reviews on the kinetics of Maillard reaction (Baisier and Labuza, 1992) and
that of Amadori rearrangement products (Yaylayan and Huyghues-Despointes, 1994)
have been reported.
Baisier and Labuza (1992) reported that although the overall kinetics of Maillard
reaction are more complex than the individual loss of sugar or amino acid, the initial
stage of the reaction follows pseudo-first order kinetics. After the initial first order
period, the loss of reactants tapers off into a phase with little reactant disappearance (no
loss period) which can be explained by means of steady state kinetics (Massaro and
Labuza, 1990). The first report on the absence of reactant loss during initial Maillard
reaction phase was published by Wolf et al. (1977). Massaro and Labuza (1990) were the
first two scientists to show a first-order period followed by steady state kinetics on
glucose and lysine loss during Maillard reaction. Most of the other published reports in
the literature do not seem to be based on enough data to quantitatively establish the
occurrence of a steady state kinetic phase.

21
2.4 Brown Pigment Measurement of MRPs
Spectrophotometer in the ultraviolet-visible (UV-Vis) range is one of the most
commonly methods used to measure the brown pigment of MRPs. Electromagnetic
radiation in the UV-Vis portion of the spectrum ranges in wavelength from
approximately 200 to 700 nm. The UV range runs from 200 to 350 nm and the Vis range
from 350 to 700 nm. The UV range is colorless to the human eye, while different
wavelengths in the visible range each have a characteristic color, ranging from violet at
the short wavelength end of the spectrum to red at the long wavelength end of the
spectrum (Penner, 1994).
It has been known for quite some time that colored substances owe their color to
the presence of one or more unsaturated linkages. These linkages or groups conferring
color on substances are called chromophores. Some groups which by themselves do not
confer color on a substance are found to increase the coloring power of a chromophore.
Such groups are called auxochromes. Typical examples of chromophores would be,
C=C, C=0, N=N etc., and of auxochromes, C-Br, C-OH, C-NH etc. 2 In general,
saturated organic molecules do not exhibit any absorption in the near UV-Vis region.
Introduction of an auxochrome to a saturated system usually shifts the absorption
maximum to a longer wavelength. However, the presence of a chromophore, i.e. a
multiple bond, generally causes absorption in the 200-700 nm region (Rao, 1975).
It has also been known that the Maillard reaction leads to the formation of a large
number of products including low molecular weight carbonyl compounds, heterocyclic
compounds, high molecular weight compounds such as polymers and complex brown
pigments (pre-melanoidins and melanoidins (Cuzzoni et al., 1988). Therefore, Maillard

22
reaction product (MRP) mixtures are a composite of several discrete chromophores that
exhibit reduced absorbance capacity with increased wavelength (O'Brien and Morissey,
1989). The first stage of glucose-lysine MRP formation is characterized by the
appearance of an absorption maximum at 220 nm which can be related to the presence of
carbonyl compounds, and an absorption around 280 nm, which has been partly attributed
to the presence of heterocyclic derivatives. As the reaction proceeds, absorbance values
at 280 nm progressively decline along with the appearance of more complex soluble pre-
melanoidins possessing an absorption around 320 nm. The single wavelength
measurement of browned solutions at 420 nm is frequently used to measure the rate and
extent of formation of the colored stage of the Maillard reaction (Cuzzoni et al., 1988;
Wijewickreme et al., 1997). Color formation is likely due both to the formation of low
molecular weight compounds and to the presence of melanoidins with high molecular
weight (Ames, 1992).
2.5 Significance of Maillard Reaction in Foods
The treatment of foods rich in reducing sugars and free amino acids results in the
production of MRPs. Therefore, heat treatments such as frying, baking, broiling and
stewing have an integral role in the quality of browning which in turn will influence the
sensory and nutritional composition of the food. For example, browning reactions are an
integral part of the production of caramel, coffee, bread and maple syrup. In the case of
chocolate, the cocoa beans are fermented and roasted to develop the chocolate flavor.
Unfermented cocoa beans, which contain sucrose, have little flavor. However, during the
fermentation process, the sucrose is converted to glucose and fructose, and the
concentration of free amino acids increases by 3-4 fold (Danehy and Wolnak, 1983).
23
This sets the stage for the development of the chocolate aroma which is produced during
subsequent roasting of fermented cocoa beans by the interaction of free amino acids with
reducing sugars through Strecker degradation reaction (Rohan and Stewart, 1965). More
than 350 cocoa volatiles have been identified and the routes for the formation of many of
these compounds have been elucidated (Hoskin and Dimick, 1984; Danehy, 1985).
MRPs are also added to foods and beverages (e.g. soft drinks, beers and sake) as
ammonia caramel or ammonia sulphite caramel to impart desirable colors, aromas and
flavors (O'Brien and Morrissey, 1989). Fermented oriental foods, notably soy sauce and
miso (bean paste), which are composed of amino acids, peptides, proteins and sugars,
also contain MRPs (Hashiba et al., 1981). Soy sauce is prepared by digesting mixtures of
soy beans and wheat with enzymes produced by Koji molds followed by fermentation by
yeasts in the presence of 17-18% sodium chloride. Hashiba et al. (1981) found six
Amadori compounds in soy sauce, miso, white wine and Sake, and explained the
important role of Amadori compounds in oxidative browning. Since soy sauce contains
large amounts of amino acids, peptides and sugars, and is brewed over a period of 6-12
months to obtain the required ageing, intermediate browning products accumulated
within this food system ultimately result in the presence of brown pigments.
The most important influence of Maillard reaction in food is its ability to
transform a great variety of food and raw materials into palatable food products with
pleasant aromas and flavors (Mauron, 1981). In most traditional food processing
operations, such as frying, roasting and baking, the development of color and flavor
formation is controlled by long term practical experience. However, in new technologies,
such as microwave, infra-red and extrusion cooking technologies a new basis for the
24
control of product quality has to be developed (Lingert, 1990). Therefore, understanding
the reaction conditions for generating MRPs may provide an important way of improving
the control of this reaction in different processed food systems.
2.6 Toxicological Aspects of Maillard Browning
Since the food constituents participating in Maillard reactions in foods represent
major dietary constituents, the safety evaluation of the products formed presents special
problems. It is particularly difficult to incorporate the same margins for dosage in animal
experiments that can be employed in the safety evaluation of environmental chemicals or
drugs. In addition, the separation of nutritional from purely toxicological effects is
difficult due to the high doses administered in such diets to animals (O'Brien and
Morrissey, 1989). This review also summarized some of the potential difficulties that
have impeded the examination of nutritional and toxicological aspects of MRPs: 1) the
difficulty of isolating and purifying many individual MRP compounds; 2) the difficulty
of purifying a sufficient quantity of pure MRPs to conduct meaningful studies. Ideally, a
proper examination of MRPs would require identification and isolation of all products
from the crude MRP mixtures and detailed study of isolated pure fractions.
2.6.1 In vivo Gross Toxicological Effects of MRPs
The reduction in growth rate associated with feeding MRPs to weanling rats has
long been known. While the separation of toxicological effects per se from purely
nutritional effects is difficult, such reductions in body weight are not entirely explained
by the destruction of essential amino acids. This is suggested by the failure of added
essential amino acids to completely restore growth rates to control values in rat
experiments (Rao et al., 1963; Adrian, 1982).

25
Sgarbieri et al. (1973) reported cecal enlargement in rats fed diets containing
fructose-leucine or fructose-tryptophan. This condition is generally regarded as having
no toxicological significance per se, but merely represents an adaptive change in response
to undigested material reaching the lower gastrointestinal tract (O'Brien and Morrissey,
1989).
The toxicity of several MRP compounds was tabulated by Mauron (1981). A
review of the reported toxicities of MRPs indicated that the LD50 values of heated
glucose-amino acid and heated glucose-nucleic acid mixtures were 50% lower than the
toxicity of free amino acids and nucleic acids used to prepare them.
Kimiagar et al. (1980) studied the effects of feeding a browned egg albumin diet
to rats and, reported increases in relative heart, lung, cecum, spleen, and stomach weights
after 12 months on the diet. These workers attributed the increase in stomach weight to a
reduced rate of stomach emptying in rats fed the browned egg albumin, since the
stomachs of rats on the brown diet were still full after 18 hours of fasting.
Lee and Chichester (1983) also studied the toxicity in rats of browned egg
albumin over a 18-month feeding period. Results indicated that no pattern of significant
changes was detected in clinical, biochemical and pathological analysis. Therefore, the
Maillard browned protein was proved to have no cytotoxic effect in rats. In addition,
Hayase and Kato (1994) reported the MRPs prepared from the glycine-glucose mixture
caused no abnormality of gross anatomic findings in rats through long-term experiments.
It appears that some animals fed browned egg albumin diets may also develop
vacuolated hepatocytes, hepatomegaly, and deposition of fat in the liver cells (Lee et al,
1981). However, this effect was detected only after 3 months of feeding and appears to
26
represent an intermediate or long-term consequence of feeding such diets. Furthermore,
the causative fraction has not been identified, and it is, therefore, difficult to say whether
the effect was a toxic one or merely the result of a long-term nutrient imbalance.
2.6.2 In vitro Toxicological Effects of MRPs
In vitro studies, Maillard reaction products have been reported to have cytotoxic
effect on both rat and human cells (Wang et al., 1987; Vagnarelli et al., 1991; Jing and
Kitts, 2000). The whole mixtures of MRPs, made from model systems of glucose-, and
fructose-lysine, have been shown to produce a cytotoxic effect on rat glioma cells (Wang
et al., 1987) and mouse embryonic fibroblast cells (C3H/10T1/2 cells) (Wijewickreme,
1997) when cells were exposed to different concentration of MRPs. Cytotoxicity of the
ribose-lysine MRPs on FT cells (Hela clone) was also reported (Vagnarelli et al., 1991).
The Caco-2 cell represents a useful model for human intestine-food nutrient
interactions. Numerous workers have used the Caco-2 cell line to study absorption of
nutrients, and nutrient-induced effects on Caco-2 cell growth and differentiation (Finley
and Monroe, 1998; Glahn et al., 1998; Jing and Kitts, 2000). Therefore, assessment of
toxicity potential of MRPs using Caco-2 cells is an important in vitro test in evaluating
MRP toxicity in human. Jing and Kitts (2000) studied the cytotoxic effect of different
fractions generated from whole mixtures of model MRPs made from glucose-, and
fructose-lysine, and results showed that the MRP made from fructose-lysine model
system was more toxic than MRP derived from glucose-lysine. In addition, the greater
cytotoxicity observed for both glucose-lysine and fructose-lysine precipitates was
specific to the higher molecular weight fractions. Finally, concentration of MRPs and the
27
incubation time used for reacting Caco-2 cells with MRPs were important factors in the
expression of toxicity.
2.6.3 A c r y l a m i d e : a C a r c i n o g e n F o r m e d i n the M a i l l a r d Reaction
Recent reports of the presence of high amounts of acrylamide in various common
fried and oven-cooked foods (Mottram et al., 2002; Rosen and Hellenas, 2002; Tareke et
al., 2002) have attracted worldwide attention because this compound has been classified
as 'probably carcinogenic to humans' (Group 2A) by the International Agency for
Research on Cancer (IARC, 1994). Acrylamide can be generated by the thermal
treatment of certain amino acids (asparagine, for example), particularly in combination
with reducing sugars, and of early Maillard reaction products (N-glycosides) (Mottram et
al., 2002; Stadler et al., 2002). Significant quantities of acrylamide (221 mg per mol of
amino acid) were found when an equimolar mixture of asparagine and glucose was
reacted at 185°C in phosphate buffer in a sealed glass tube (Mottram et al., 2002).
Findings indicate that the Maillard-driven generation of flavor and color in thermally
processed foods under particular conditions can be linked to the formation of acrylamide,
and might explain the increased concentrations of acrylamide in certain cooked plant-
based foods, such as cereals and potato, which are rich in asparagine.
2.7 M i c r o w a v e Characteristics
Microwave heating is an alternative to conventional conductive heating for
introducing energy into reactions. The principle of heating with microwave is very
different from that of conventional heating by conduction or convection, so it is
technically difficult to measure and standardize the influencing parameters. The
microwave dielectric heating effect uses the ability of some liquids and solids to
28
transform electromagnetic energy into heat and thereby drive chemical reactions. This in
situ mode of energy conversion has many attractions to the chemist, because its
magnitude depends on the properties of the molecules. This allows some control of the
material's properties and may lead to reaction selectivity (Finot and Merabet, 1993;
Mingos and Baghurst, 1997).
A reliable device for generating fixed frequency microwaves was designed by
Randall and Booth at the University of Birmingham as part of the development of
RADAR during the Second World War. This device, the magnetron, was produced in
large numbers during the war with the aid of United States industrial expertise. Even in
those early days it was recognized that microwaves could heat water in a dramatic
fashion, and domestic and commercial appliances for heating and cooking foodstuffs
began to appear in the United States in the 1950s. The widespread domestic use of
microwave ovens occurred during the 1970s and 1980s as a result of effective Japanese
technology transfer and global marketing (Mingos and Baghurst, 1997). With the
increasing use of microwave heating in industrial food processes, an understanding of
microwave characteristics is helpful for developing appealing microwavable food
products and better applications of microwave ovens in food industrial processing. Some
of the characteristics that are important in the development and use of microwave
processes and products are electrical and physical properties, heating patterns, microbial
inactivation, and safety (IFT, 1989).

29
2.7.1 Electrical and Physical Properties
Microwaves are generated by a magnetron - a device that converts electrical
energy at low frequencies into an electromagnetic field with centers of positive and
negative charge that change direction billions of times each second. Penetration and
heating of foods by microwave energy sources are instantaneous. In contrast,
conventional heating methods transfer thermal energy from product surfaces toward their
center much slower. As the microwaves enter the product, they interact with regions of
positive and negative charges on water molecules, (electrical dipoles) that rotate the
molecules in the electrical field by forces of attraction and repulsion between oppositely
charged regions of the field and the dipoles. This results in the disruption of hydrogen
bonds between neighboring water molecules and generates heat by "molecular friction"
(IFT, 1989).
Positive and negative ions of dissolved salts in foods, such as common table salt,
sodium chloride, also interact with an electrical field by migrating toward oppositely
charged regions of the electrical field and disrupt hydrogen bonds with water to generate
additional heat. Magnetic field interactions are negligible, since foods contain only trace
amounts of magnetic minerals such as nickel, cobalt and iron. The heat generated within
a product by the electrical interactions is then transferred throughout the product by
conventional thermal conduction. Microwave penetration depths within the product are
determined by its electrical and physical properties and can vary significantly with
chemical composition and temperature of the product and the processing frequency
(Buffler, 1993).
30
The electrical and physical properties which determine microwave penetration
depth, conventional heat transfer, and overall heating rate are the dielectric constant and
loss factor, heat capacity (specific heat), density, and thermal conductivity. Dielectric
properties determine the response of a material to an electromagnetic field, such as in a
microwave oven. The inability of the molecules to instantaneously align with the applied
electromagnetic field leads to dissipation of electromagnetic energy. The dielectric
properties refer to a complex number consisting of a real portion (the dielectric constant)
and an imaginary portion (the dielectric loss factor). The dielectric constant is an
indication of the polarizability of the molecules and their ability to store electrical energy.
The dielectric loss factor is related to the energy absorption and dissipation of
electromagnetic energy from the field (Mudgett, 1986). The dielectric properties and
some related electrical properties which affect the transmission and absorption of
microwave energy are rigorously defined in classic references on the nature of
electromagnetic waves and applications of dielectric materials (Von Hippel, 1954a;b).
Values of the dielectric properties for a large number of agricultural and food products
are available in a bibliography written by Kent (1987).
In food samples, water and salt are the two major ingredients which influence
dielectric properties. Other food components usually have a minor influence on the
dielectric properties. The electromagnetic field frequency and sample temperature affect
dielectric properties as well. Water and salt content, frequency and temperature are
commonly included in predictive equations, such as those of Calay et al. (1995) and Sun
et al. (1995). The physical state of the food is not included in these equations, though it
may influence the mobility of water and salt, which in turn may affect the dielectric
31
properties. It has been reported that changes in physical state affect dielectric properties.
Effect of microwave and conventional heating on dielectric properties of starch and
wheat gluten mixtures was investigated (Umbach et al., 1992). Measurements were made
for the individual powders and for each of the 15 combinations of starch, vital wheat
gluten and water before heating, after conventional heating and after microwave heating.
Results indicated that no differences were found in dielectric properties for dry powders.
Dielectric properties were affected by heating method; the dielectric constant was more
dependent on moisture content than was dielectric loss, which showed very little
variation over different moisture contents. The dielectric loss factor of whey protein
solutions increases after whey protein denaturation (Barringer et al., 1995). Dielectric
properties are dependent on whether the electromagnetic field is oriented perpendicular
or parallel to meat fibers (Bengtsson et al., 1963). When starch gelatinizes the dielectric
loss factor increases but the dielectric constant shows no change (Miller et al., 1991).
2.7.2 Heating Patterns
Energy absorption and transmission in foods are determined by mutual
interactions between the microwave power source and the food product. Some energy
may be reflected from product surfaces without being absorbed, causing standing wave
patterns of nodes and antinodes. These cause high and low incident power levels in the
microwave cavity that result in uneven energy distribution at product surfaces and hot
and cold spots within the product. This problem can be minimized by using wave stirrers
which distribute microwaves more uniformly at product surfaces or by rotating the
product in the microwave oven. More uniform heating can also be obtained in ovens
with multiple feed systems (Buffler, 1993; Finot and Merabet, 1993). Power transfer is
32
efficient for high-moisture products, but less efficient for low-moisture products.
Unreflected, i.e., transmitted, power is absorbed by interaction with active food
constituents and decreases within the product with increasing penetration. Power
penetration in the product is about 2-3 times greater at 915 MHz than 2,450 MHz (the
two microwave frequencies most commonly used in the United States), although at
higher temperatures the differences in some cases may be insignificant. In general,
product thickness must be limited to power penetration depth, i.e., the depth at which
about 67% of transmitted power is absorbed, to obtain uniform heating (Buffler, 1993).
The power level and, consequently, the heating rate needed for uniform heating
depend very much on the product's composition, temperature, shape, structure, size and
on the microwave frequency. In terms of composition, moisture, solids, and salt contents
determine microwave penetration depth and, therefore, heating uniformity. Products
which have high solids and low moisture or salt contents sustain larger penetration depths
and thus may be processed in larger thicknesses than products of diverse composition at
either 2,450 or 915 MHz (IFT, 1989).
2.7.3 Microbial Inactivation
Microwave energy is believed to inactivate microorganisms by conventional
thermal mechanisms, e.g., thermal denaturation of proteins and nucleic acids. This is
supported by experiments comparing the survival of vegetative cells and spores exposed
to microwaves with survival under conventional heating methods (Goldblith and Wang,
1967; Carroll and Lopez, 1969; Lechowich et al., 1969). Microbial inactivation by
microwaves depends on the same time/temperature relationships as in conventional

33
processing, with rates of inactivation that can be evaluated from kinetic parameters
available in the literature (Stumbo, 1973; Lund, 1977).
However, the question of the existence of a possible microwave 'per se' effect,
different from the thermal effect, was raised very early (Jacobs et al., 1950). A critical
review of the literature on the athermal (non-thermal) effects of microwaves on the
viability of microorganisms has been published by Fung and Cunningham (1980). It
appeared that even though most of the changes observed in living systems exposed to
microwaves may be explained by the induced thermal effects, some modifications
nevertheless require recourse to non-thermal concepts to be fully understandable. Since
then, numerous studies have been published, but so far, no clear experimental evidence
supporting a specific effect of microwaves in different reaction models has been
presented. Hence, there is still an ongoing debate about whether there are athermal (non-
thermal) as well as thermal effects associated with electromagnetic energy, and whether
these effects can be applied in the food industry to enhance microbial inactivation
(Jahngen et al., 1990; Laurent et al., 1992; Stuerga and Gaillard, 1996a;b). The question
remains open.
2.7.4 Human Safety
Concerns about the safety of microwaves are a persistent consideration. Although
athermal effects of microwave have been suggested, it is generally recognized that
microwave interactions are solely due to thermal effects (IFT, 1989; Mertens and Knorr,
1992). Based on whole-body research, the American National Standards Institute has
established a standard that whole-body exposures of 10 mW/cm are safe levels for
2
human exposure of unlimited time periods under normal exposure conditions (Decareau,
34
1985). However, to provide an additional margin of safety, the Radiation Control for
Health and Safety Act (Public Law 90-602) limits leakage from consumer and industrial
microwave ovens to 1 mW/cm at a distance of 5 cm from oven surfaces prior to sale and
2
5 mW/cm during a lifetime of use. In addition, home and industrial oven doors are also
2
doubly interlocked, door seals are carefully designed to avoid accidental exposure to high
oven power levels, and a monitor suspends microwave generation when the door is
opened (Decareau, 1992).
2.7.5 Nutritional and Toxicological Effects of Microwave-Treated Foods
Since the first introduction of microwave ovens, a large number of studies have
been carried out on the nutritional and toxicological aspects of microwave heating in
foods. Microwave-treated foods are characterized by the diffusion of water from the core
to the outside with the migration of the water-soluble nutrients, such as vitamins, free
amino acids and minerals, which are recovered in higher quantities in the drippings
(Baldwin and Tettambel, 1974). Lipids and proteins are therefore retained in higher
proportion in the food as a consequence of this water loss, resulting in changes in its
sensory properties rather than major changes in nutritional properties.
Mutagens formation has been measured in microwave-treated meats. The amount
of volatile nitrosamines has been found to be lower in bacon when fried by microwaves
than when fried in a pan (Miller et al., 1989; Osterdahl and Alriksson, 1990) and the level
of heterocyclic amines decreased during the microwave treatment of meat when
compared to using conventional heating methods (Felton et al., 1992).
In addition, the effect of microwave treatments on the nutritional value of food
proteins has been studied: the amino acid composition of vegetables, peas, meat and fish
35
(Bodwell and Womack, 1978); rat bioassays on casein (Jonker and Penninks, 1992);
chick bioassay on soya (Hafez et al., 1983). The nutritional effects of microwave
treatments in foods on amino acids (Fay et al., 1991; Marchelli et al., 1992), lipids (Pikul
et al., 1985; Wong et al., 1991), vitamins (Goldblith et al., 1968; Sigman-Grant et al.,
1992) and minerals (Toma et al., 1978) have also been studied. Microwave treatment
does not impair the nutritional value of foods very differently from that of the
conventional cooking conditions and does not induce more toxic effects than
conventional heat treatments. Very often, microwave treatment has an advantage as the
temperatures are generally lower and the cooking time shorter.
2.8 Comparison of Maillard Reaction in Microwave and Conventional Heating
Microwave processing can offer several distinct advantages when compared to
conventional heating methods. These advantages include speed of operation, energy
savings, precise process control, and faster start-up and shutdown times (Decareau,
1985). Speed of operation is the primary advantage. Since microwaves penetrate within
a food and not just at the surface, heating occurs more rapidly. Microwave processing
can also help control energy costs, since heating takes place only in the food material
being processed and not in the surrounding medium. However, microwave heating is not
without problems. One of the major problems associated with microwave cooking is the
lack of desired color as well as flavors (Risch, 1989). The short cooking time and low
temperatures common to microwave processing usually do not promote the Maillard
reaction, which is responsible for the production of many flavor and colored compounds
(Yeo and Shibamoto, 1991).

36
Until now there have been only a few comparative reports about the formation of
Maillard products in microwave cooking versus conventional cooking (Fay et al., 1991;
Mei(3ner and Erbersdobler, 1996), with the exception of reports about flavor development
of Maillard reaction compounds (Whorton and Reineccius, 1989; Yeo and Shibamato,
1991; Yu et al., 1993; Oruna-Concha et al., 2002). Whorton and Reineccius (1989)
demonstrated that a cake mix gave more flavor compounds derived from the Maillard
reaction after baking conventionally compared to using microwave irradiation.
According to Yeo and Shibamato (1991), flavors produced by microwave irradiation and
thermal heating differ both sensorially and chemically. They reviewed the chemical
differences between the flavor compounds of microwaved and conventionally heated
Maillard model systems, meat, baked products and vegetables. Studies with the Maillard
model system have shown that microwaved samples possess characteristic off-flavors
attributable to the presence of 2-methylpyridine and 4,5-dimethyloxazole. Pyrazine
derivatives were formed in lower amounts in the microwaved samples, reflected by less
intense 'popcorn', roasted and meaty flavors. Studies with baked batter demonstrated a
lack of nutty, brown and caramel-type aromas in microwaved cakes, which were
dominated by 'green vegetable' flavors. Compounds present in conventionally baked
cake and not in microwaved cake include furan and pyrazine derivatives. Studies also
point out that both the food temperature and the length of time the food is heated play
important roles in the development of flavor compounds in microwaved systems. Beef
samples cooked to equal degrees of doneness produced lower amounts of compounds
with low rates of formation when cooked in a microwave oven, because the short
irradiation time does not allow flavor to develop fully. However, when samples were
37
subjected to equal cooking times, higher amounts of volatiles were obtained from
microwaved beef samples, due to the rapid initial rise in temperature that occurs during
microwave cooking (Yeo and Shibamoto, 1991). Yu et al. (1993) and Oruna-Concha et
al. (2002) also reported the chemical differences between the volatile compounds of
microwaved and conventionally heated garlic slices and potato, respectively.
Mei(3ner and Erbersdobler (1996) studied the impact of microwave cooking on
the formation of early Maillard products in milk compared with the effect of conventional
cooking. Results showed that none of the reaction products were significantly different
between the microwave heated and conventional cooked milk, and also confirmed the
statements of other authors (Fay et al., 1991) that there were no microwave-specific
changes in food proteins. The heating time and temperature seemed to be more important
than the source of the heat treatment for food damage.

38
3. THESIS D E V E L O P M E N T
3.1. Thesis Hypotheses
The hypotheses of this research are as following:
• Non-enzymatic browning occurs at a different rate during microwave heating
compared to conduction/convection heating at the same temperature,
• Different compounds or different concentrations of MRP compounds are produced
during microwave heating compared to conduction/convection heating at the same
temperature.
3.2. Thesis Objectives
Long-term objectives
The long-term objectives of this research are to provide information that may help us to
better understand the effects of microwave heating on Maillard browning reactions and to
extend the use of microwave to the economic production of superior quality Maillard
browning products.
Objectives for this thesis
The specific objectives for this thesis project are:
• To compare the reaction rates of the Maillard browning in microwave fields with that
in a water bath heating system under controlled temperature condition,
• To compare the compositions of the MRPs produced under microwave fields with
that provided in a water bath heating system under controlled temperature condition,
• To study the cytotoxicity effects of the MRPs produced by microwave and water bath
treatments.
39
4. M A T E R I A L S A N D M E T H O D S
4.1 Materials
D-glucose, L-lysine, 4-hydroxy-alpha-cyanocinnamic acid (CCA) and MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) were purchased from
Sigma Chemical Co. (St. Louis, MO). Sodium bicarbonate, sodium carbonate and
potassium bromide were purchased from Fisher Scientific Co. (Fair Lawn, NJ).
Dulbecco's modified eagle medium containing 10% fetal bovine serum (DMEM10) was
purchased from Invitrogen Canada Inc. (Burlington, ON). The Caco-2 cells (ATCC No.
ETTB-37) were purchased from American Type Tissue Collection (Manassas, VA).
40
Apparatus 1. The apparatus consisted of a modified microwave oven (General Electric
Company, Canada; model No. JE435) rated 700 W, 2450 MHz, within which a special
glass vessel was placed. The glass vessel was designed with an internal volume of
approximately 1000 ml. It was connected to a pump (Cole Parmer Instrument Company,
WA, USA; model No. 2630) allowing the circulation of the solution out of and back into
the vessel. Thermocouples were placed in the circulation tube to record the temperature.
Low pressure was achieved by connecting the glass chamber to a vacuum pump. The
circulating pump was adjusted to give a flow rate of 3 ml/sec. A schematic of the
apparatus is shown in Figure 4.1.
G j___H
Vacuum
Pump Computer Recording
Temperature
BB Sample Port
I Circulating Pump
Microwave Oven
Figure 4.1. The schematic of a vacuum microwave apparatus

41
Apparatus 2. The apparatus consisted of an Ethos Synth 2450 MHz microwave reactor
with a temperature/pressure control system which was operated by a computer software
program. A schematic of the apparatus is shown in Figure 4.2. The microwave reaction
vessel was connected to a peristaltic pump (Mandel Scientific Company Ltd., Bel,
France; Model No. M312) to allow filling the vessel through a Teflon tube. The vessel
was designed with an internal volume of approximately 400 ml, which was a completely
sealed mechanical mixing system with a temperature sensor, cooling finger, safety valve
and tubing. The Ethos Synth had a built-in magnetic stirring device that was located
underneath the bottom plate of the Ethos Synth microwave cavity. Motor-driven
powerful magnets rotated below the microwave cavity, generating a rotating magnetic
field. By placing a magnetic spin bar in the vessel, a stirring effect can be obtained in the
same, wherever positioned. Efficient stirring guaranteed the uniform heating of sample
solution.
Ethos Synth
Microwave
Reactor System
Computer
Figure 4.2. Schematic diagram of the Ethos Synth microwave system.

42
4.2 Overview of Methods
4.2.1 Sample Preparation
D-glucose (0.05M) and L-lysine (0.05M) were prepared by dissolving the
appropriate quantities of glucose or lysine in 0.05M sodium carbonate/bicarbonate (SCB)
buffer. The SCB buffer was prepared by mixing 0.1M sodium carbonate and 0.1M
sodium bicarbonate in the correct proportions to give pH 9.95. It has been stated that the
glucose-lysine model has the highest browning rate between pH 9 and 10 (Ashoor and
Zent, 1984). The pH of the medium was measured using a Fisher Accumet model 620
pH meter.
43
4.2.2 Heat Treatments
Water Bath. Samples without lysine were obtained by pouring 5 ml of 0.05M
glucose solution into each Bausch & Lomb round cuvette that was then immersed in a
heated water bath (Blue M Electric Company, Blue Island, IL). The water bath was set at
63°C, 81°C or 90°C. Two thermocouples were positioned into the round cuvettes and one
was positioned in the water bath. The cuvettes were capped to eliminate solution loss due
to evaporation. Agitating the water bath at 2 cycle/sec provided uniform temperature
during the experiment. Upon reaching the desired temperature, one of the glucose
solutions was chosen as the blank sample by the addition of 0.26 ml of 0.05 M SCB
buffer. A 0.26 ml aliquot of 0.05 M lysine solution was added to each of the remaining
cuvettes.
Vacuum Microwave. A 950 ml aliquot of 0.05M glucose solution was re-
circulated within a vacuum chamber equipped with a sampling port and a temperature
monitor. The solutions were filled into the chamber and heated to different boiling points
at different pressures. The vacuum pump was adjusted to control the boiling point and
equilibrium temperature of the solution. Upon achievement of equilibrium, the
circulating pump was set at a flow rate of 3 ml/sec and the microwave was programmed
at the desired power level (700 W). When the target temperature (63°C, 81°C or 90°C)
was reached, a 5 ml of glucose solution was collected then mixed with 0.26 ml of 0.05 M
SCB buffer to use as the spectrophotometer blank. The Maillard reaction was initiated by
injection of 0.05M lysine (50 ml) solution into the glucose solution through the sampling
port after equilibrium temperature was achieved. Since the rate of water evaporation in
the vacuum microwave was measured to be llml/min, to compensate for water

44
evaporation and maintain concentration levels of MRPs, 11 ml of distilled water was
injected every minute via the sampling port throughout the process.
Ethos Synth Microwave Reactor System. A 400 ml aliquot of 0.05M glucose
solution was placed in the reaction vessel of the Ethos Synth microwave reactor which
has a temperature/pressure control system operated by a computer software program.
Temperature was controlled in this system by adjusting the microwave power output of
oven. When the target temperature (63°C, 81°C, 90°C, 100°C or 120°C) was reached a
blank sample was taken as described above, then the Maillard reaction in the glucose
solution was initiated by pumping in 0.05M lysine (21 ml) solution through the valve.
Generally, an excess of reducing sugar to that of an amino compound accelerates
Maillard reaction (O'Brien and Morrissey, 1989), hence, the molar ratio of 19:1 was
chosen for the glucose:lysine system to accelerate the Maillard reaction and to surely
follow the pseudo-first-order reaction.
90°C or Above Blank Samples. For 90°C or above treatments, all blank samples
were kept at water bath preset at 90°C during the process. This is to minimize the
difference caused by occurrence of caramelization at high temperatures.

45
4.2.3 Maillard Reaction Rate Determination
The reaction rate was determined by means of Spectronic 20 colorimeter-
spectrophotometer (Bausch & Lomb Inc.) preset at 420 nm. The absorbance level was
recorded e^very 5 minutes (63°C and 81°C) or every other minute (90°C) for water bath
trials, every other minute for all vacuum microwave trials, and every 5 minutes (63°C),
every other minute (81°C, 90°C and 100°C), or every minute (120°C) for Ethos Synth
microwave system, at holding times from 30 min up to 200 min. However, due to the
difficulty of collecting the first sample at time 0, the first samples of all treatments were
collected at 15 seconds instead. Samples displaying absorbance readings above 0.6 were
diluted with SCB buffer and the final absorbance was then multiplied by the dilution
factor. Each experiment was replicated three times.

46
4.2.4 Comparison of UV/Visible Spectral Characteristics of MRPs
Three temperatures were tested (63°C, 81°C and 90°C). For each heat treatment,
three samples were collected during each experiment period at each temperature for each
heat treatment. Three samples of vacuum microwave heating at each temperature were
first collected. The first sample was collected at 15 seconds for all temperatures, the
second sample was collected at 30 min (63°C and 81°C) or 14 min (90°C), and the third
sample was collected at 60 min (63°C), 50 min (81°C) or 32 min (90°C). These three
samples of each temperature were classified as sample at the beginning, middle and the
end of the process. The absorbance of the collected sample was first measured by ATI
Unicam UV2-100 UV/visible spectrophotometer preset at 420nm, then the UV/visible
absorption spectrum of the same sample was recorded by the same Unicam
spectrophotometer (data was collected from 220 to 600 nm). For heated water bath
samples or Ethos Synth microwave samples, samples were collected and absorbance were
measured by the ATI Unicam UV2-100 UV/visible spectrophotometer preset at 420 nm
till absorbance reached the exact same reading as vacuum microwave sample. The
UV/visible absorption spectra of these samples were then recorded by the Unicam
spectrophotometer. Therefore, samples were selected based on the absorbance, not the
process time, in this study. In addition, samples displaying absorbance readings at 420
nm above 0.6 were diluted with SCB buffer and the final absorbance was then multiplied
by the dilution factor. Each experiment was replicated three times. All the collected
samples were cooled in crushed ice to minimize further Maillard reaction and held for
further uses.
47
4.2.5 Dielectric Property Measurements
Samples from the above study were pulled from the ice bath and held in a test
tube rack till they reached ambient temperature (25°C), and their dielectric properties
were measured with the Open-Ended Probe system (Hewlett Packard Corp., Santa Clara,
CA). The test probe consisted of an open ended coaxial probe (HP 85070) operating at
2.45 GHz. The probe was calibrated with air, a metal fitting that provided an electrical
short, and deionized distilled water before each set of experiments, then checked
afterwards to insure the calibration was stable. The system software automatically
calculated the dielectric parameters from the phase and amplitude of the reflected signal
at the interface between the open-ended coaxial line and the sample to be analyzed. The
dielectric properties were determined by a Hewlett-Packard 85070 Network Analyzer.
Each sample had a volume of 12 ml and a depth of 12 mm. Care was taken to ensure
proper contact between the test probe and the sample. The dielectric constant (e') and
loss factor (e") were recorded to define dielectric properties of each sample.
48
4.2.6 MALDI-TOF MS Analysis of MRPs of Glucose-Lysine Model System
One vacuum microwave MRP sample and one water bath MRP sample at 81°C
were collected. A 0.01 M SCB buffer (pH 9.95) was used instead of 0.05 M SCB buffer
(pH 9.95) in this case. The sample from vacuum microwave heating was first collected.
The absorbance of collected sample was measured by ATI Unicam UV2-100 UV/visible
spectrophotometer preset at 420nm, then the UV/visible absorption spectra of the same
sample was measured and recorded by the same Unicam spectrophotometer (data was
collected from 220 to 600 nm). Next a water bath sample was collected and the
absorbance and spectrum collected in a similar manner. Once again, samples were
selected based on the absorbance not the process time. Both collected samples had the
same absorbance at 420 nm. The mass/charge of both samples was determined by
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-
TOF MS) (Department of Chemistry, UBC) using 4-hydroxy-alpha-cyanocinnamic acid
(CCA) as the matrix. The molecular weights of original compounds could then be
identified based on the mass/charge obtained. Samples were first diluted 1:10 in 0.1%
trifluoroacetyl with de-ionized distilled water. The matrix (1 u\) was deposited on the
probe tip and vacuum dried to obtain a thin-film matrix layer, subsequently 1 wl of 1:10
diluted samples were applied and vacuum dried. The molecular masses of samples were
analyzed in the positive ion mode on a Bruker Biflex instrument with a nitrogen laser at
337 nm. For each acquisition, 200 laser shots were fired and the resulting spectra were
averaged.
49
4.2.7 FTIR Analysis
The final MRPs at 90°C of the three heat treatments from the previous study
(comparison of UV/visible spectral characteristics of MRPs) were used for FTIR
analysis. A Thermo Nicolet Nexus 670 (Waltham, MA, USA) spectrometer equipped
with a DTGS KBr detector was used for FTIR analysis. All three collected samples were
freeze-dried, then pelleted in potassium bromide (KBr) at 1:100 ratio. 32 scans were
taken on each sample at a resolution of 4 cm" . Single beam spectra (4000-400 cm") of
1 1
the samples were obtained to present the spectra in absorbance units. Baseline correction
was applied to each spectrum, and then the spectra were normalised on the integrated
spectral area (height) over the peak at 833 cm" which was from glucose.
1
50
4.2.8 Cell Toxicity Test
Caco-2 cells (a human colon adenocarcinoma cell line) were purchased from the
American Type Culture Collection (Manassas, VA). Cells were cultured in 75 cm
polyester plastic flasks (SARSTEDT Inc., Montreal, PQ). Dulbecco's modified eagle
medium containing 10% fetal bovine serum (DMEM10) was used as a basal growth
medium. The cells were cultured in an incubator (37°C) under an atmosphere of 5% CO2
with 90% humidity. The culture medium was changed every 2-3 days. Prior to reaching
confluence, the monolayer cells were detached and separated with trypsin (0.25%)-
ethylenediaminetetraaceticacid (EDTA) (1 mM) and aspirated to make a suspension of
single cells. The cells were plated at 0.75xl0 cells/ml (100 id/well) in a 96-well cell
3
culture plate with DMEM10 and maintained overnight at 37°C in the CO2 incubator. The
cell culture medium was replaced with phosphate buffer saline (PBS, pH 7.3) before
adding freeze-dried final MRPs of the three heat treatments at 90°C from the previous
study. After 3 hours incubation with MRPs, PBS media was replaced with DMEM10,
and treated cells were cultured for further 72 hours before assessing the cell viability with
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). In this study,
PBS buffer without adding final MRPs was used as control.
The MTT assay was performed according to Mosmann (1983). The cleavage of
the tetrazolium salt MTT into a blue colored product (formazan) by the mitochondrial
enzyme succinate-dehydrogenase was used for assaying cell survival and proliferation.
The conversion took place only in living cells and the amount of formazan produced was
proportional to the number of cells present (Mosmann, 1983). Culture media of
monolayer cells were removed from the cell culture plates, and 70 ul of MTT solution
51
(0.5 mg/ml) was added to each well. Cultures were incubated at 37°C for 3 hours then
the untransformed MTT was removed before adding 90 ul of 0.04 N HCl-isopropanol to
each well. The plate was vigorously shaken for 5 minutes on a vortex and the absorbance
of each cell was measured at 570 nm, using a microplate reader (Bio-Rad model 550).
MTT assay was replicated three times for each sample.

52
4.2.9 Kinetic Studies
Numerous studies have applied a pseudo-first-order model to successfully
describe the rate of browning in a food or in a model system when reactant
concentrations are not limiting for the rate of formation of brown pigment (Mizrahi et al.,
1970; Warmbier et al., 1976; Waletzko and Labuza, 1976; Franzen et al., 1990), as
dBldt = kb = rate of browning (1)
where B is a measurement of browning in absorbance and t is time. Assuming no
induction period, a plot of B versus time (t) should give a straight line where the slope is
equal to k .
b
The Maillard browning reaction, as with other chemical reactions, has been
successfully described with respect to temperature dependence by the Arrhenius
relationship
-Ea/RT
k =k e
b h0 (2)
where k\> is the rate constant at a temperature T, &t,o is the rate constant at a reference
temperature To, E is the activation energy (kJ/mol), R is the gas constant (8.314 J/mol
a
K), and Tis the absolute temperature (K) (Labuza and Saltmarch, 1981). In a plot of ln k
versus IIT, the slope is equal to -EJR.
The D value or decimal reduction time (i.e., the time required to change the
concentration of reactants or products by 90%) can be obtained from the semi-logarithm
curve as the time taken to traverse one log cycle and are related by Equation (3):
D = 2.303/k (3)
53
The TDT approach is based on the assumption that the D value follows a semi-
logarithmic relationship with temperature as illustrated in Equation (4):
Log (D,/D ) = (T -T,)/z

2 2 (4)
where Di = decimal reduction time at Ti; D = decimal reduction at T , and z = negative

2 2
reciprocal slope of the D value curve, i.e. temperature change required to change the
decimal reduction time by a factor of 10.
4.2.10 Statistical Analysis
One-way analysis of variance (ANOVA), followed by Tukey multiple range test
(Minitab Inc., State College, PA) was used for data analysis. The level of confidence
required for significance was selected as p < 0.05. Each experiment was replicated three
times.
54
5. R E S U L T S A N D DISCUSSION
5.1 Maillard Reaction Rate Constants and Activation Energies
The absorbance of MRP solutions at 420 nm in this study was used to measure the
Maillard reaction rate and extent of formation of the colored stage of the Maillard
reaction - measurement of color changes due to the presence of soluble pre-melanoidins.
According to Ames (1992), color formation is likely due both to the formation of low
molecular weight compounds and to the presence of melanoidins with high molecular
weight. The extent of browning (as measured by absorbance at 420 nm) as a function of
heating time at different temperatures of three heat treatments is shown in Figure 5.1 and
5.2. An induction period was observed for all three heating treatments and decreased
with increasing temperature. After the induction period, the browning rate at each
temperature then followed pseudo-first-order reaction kinetics, i.e. the log of absorbance
increased apparently linearly with time, till it reached a plateau, as shown by the linear
plot of log of absorbance versus time for all three testing methods. Similar phenomena,
with respect to induction period and reaction order, have been shown by many
researchers (Mizrahi et al., 1970; Warmbier et al., 1976; Singh et al., 1983; Franzen et al.,
1990). Rate constant was calculated from the linear portion after the induction period.
The slope of these lines was calculated using the least-squares fit method, and results
(Table 5.1) showed that the slope for vacuum microwave was greater than that obtained
with the water bath method and Ethos Synth microwave system for any of the
temperatures. In addition, the biggest difference of slopes between vacuum microwave,
water bath and Ethos Synth microwave reactor was seen at 63°C compared to 81°C and
55
90°C. This indicated that the vacuum microwave permitted the Maillard reaction to take
place at lower temperature.
It was noticed that temperature influenced the induction period. The lag time
decreased as temperature increased, as also reported by Warmbier et al. (1976). It has
been suggested that at higher temperatures the induction period should decrease or
disappear altogether (Labuza and Baisier, 1992). In fact, Nelson (1948) reported that at
temperatures of 121°C or higher, an induction period could not be detected. In this study,
typical curves consisted of an induction period of decreasing slope, followed by a linear
portion, which then followed by a portion of falling rate that eventually became a plateau
of constant absorbance at 420 nm. The linear portion was of interest for this study. The
induction period was considered to be finished when five consecutive points had an R-
squared value of greater than 0.908. The linear portion was considered to be finished
when addition of another point reduced the slope by more than 15% (Figure 5.1 and 5.2).
From Figure 5.1 and 5.2, an induction period was seen at temperatures up to 120°C.
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Comparing the reaction rate of the three heating methods at the same
temperature, the reaction rate achieved in the heated water bath or the Ethos Synth
microwave system was markedly lower than in the vacuum microwave (Table 5.1). It
was also clear (Table 5.1) that the Maillard reaction rate increased with increasing
temperature. For vacuum microwave, the Maillard reaction rate was determined for
700W power level at different temperatures (Table 5.1). It was observed that the reaction
rate also increased with increasing temperature. The Maillard browning reaction is
known to be a heat sensitive reaction that increases proportionally with the temperature.
This was demonstrated as the absorbance of reaction mixtures increased with increasing
temperature. In contrast to the reaction rate in the heated water bath or Ethos Synth
microwave system, a temperature increase from 63°C to 81°C resulted in a significant
difference (p<0.05) in the reaction rate by vacuum microwave heating.
Despite the fact that similar experimental conditions were used for all three
treatments, the browning reaction obtained was much faster with the vacuum microwave
heating. From these results it can be inferred that the microwave power greatly promotes
the Maillard browning rate (Table 5.1). At the same temperature, the browning rate of
Ethos Synth microwave system at 63°C, 81°C or 90°C was almost the same (p<0.05) as
the heated water bath method. This is presumably because as the temperature reaches the
desired temperature in the Ethos Synth microwave system, microwave power drops to
below 100 W. The measurements of browning reaction rates at 100°C and 120°C in
Ethos Synth microwave system show the advantage of using Ethos Synth microwave
system at high temperatures since it is difficult for the other two heat treatments to reach
temperatures above 100°C.

59
The temperature sensitivity of chemical reactions is dependent upon the activation
energy Ea. Ea is the extra energy that molecules require in order to react (Petrucci,
1989). The larger the Ea, the smaller the fraction of the activated molecules and the
slower a reaction proceeds. Chemical reactions tend to go faster at higher temperatures.
Increasing the temperature increases the fraction of the molecules sharing the Ea,
therefore the k decreases and reaction rate increases. D values of these three heat
treatments were obtained from known k values, and the negative reciprocal slopes of the
regressed straight line of log D values at different temperatures gave the corresponding z
values (Figure 5.3). In this experiment the Arrhenius equation was used to explain the
relationship between reaction rate and temperature. Table 5.1 summarizes the findings
for water bath, vacuum microwave and Ethos Synth microwave trials.
The Arrhenius equation when applied to vacuum microwave, heated water bath
and Ethos Synth microwave reactor indicated that the reaction rates for the vacuum
microwave were much higher (p<0.05) than the other two (Table 5.1 and Figure 5.4).
The greater reaction rate achieved by vacuum microwave system could be explained by
the superheating effect. Superheating occurs as the temperature of a solution in a
microwave rises rapidly, reaching very high temperatures for a very short time interval
(1/10 sec). Therefore the reaction rate might increase quickly during those fluctuations.
It was also noticed that there was greater superheating effect at lower temperature, hence,
the superheating caused the change of the slope of Arrhenius curve and the change of Ea.
Greater superheating at lower temperatures might be caused by the difficulty of
controlling vacuum pump at lower temperature which led to higher temperature
fluctuations. In this study, the temperature fluctuation range dropped from +2.1°C at
63°C to ±0.9°C at 81°C, and finally dropped to ±0.5°C at 90°C. Unfortunately, the
superheating phenomenon could not be exactly recorded in this experiment as
superheating was very fast and difficult to measure.

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5.2 Comparison of UV/Visible Spectral Characteristics of MRPs
The typical brown color obtained in a model system, or a food subjected to heat
treatment, usually indicates some degree of occurrence of the Maillard reaction (Spingarn
and Garvie, 1979). Therefore, measurement of color quality in a reaction solution was
found to be valuable in objectively predicting the extent of browning reactions.
The Maillard reaction leads to the formation of a large number of products
including low molecular weight carbonyl compounds, heterocyclic compounds, high
molecular weight compounds such as polymers and complex brown pigments (pre-
melanoidins and melanoidins) (Cuzzoni et al, 1988). Numerous studies (Cuzzoni et al,
1988; Wijewickreme et al., 1997; Hill et al., 1999) have reported that the formation of the
MRPs of glucose-lysine model system is greatly influenced by the reaction conditions.
However, none of them investigated the effects of microwave irradiation on the
composition of the Maillard reaction products of glucose-lysine model system. The
comparison of spectral patterns between 220 and 600 nm for vacuum microwave, heated
water bath and Ethos Synth microwave system at 63°C, 81°C and 90°C at different stages
are shown in Figure 5.5-5.7, Figure 5.8-5.10 and Figure 5.11-5.13, respectively.
Results indicated that at the same temperature and the same stage, even though
the absorbance of the collected samples at wavelength 420 nm for all three methods were
the same, they were not the same at other wavelengths. However, the difference between
water bath sample and Ethos Synth microwave sample was much smaller than the
difference between vacuum microwave sample and water bath sample or Ethos Synth
microwave sample. The biggest difference of absorbance between vacuum microwave
and the other two heating treatment samples was seen at the second spectral pattern of
65
63°C. However, the reason is not clear at this point. In addition, the difference of
absorbance values between vacuum microwave, water bath and Ethos Synth microwave
samples was more obvious at wavelengths lower than 420 nm (220-420 nm) than at
wavelengths higher than 420 nm (420-600 nm). It was also observed that as the heating
process progressed the peaks of the spectral patterns increased for all three heating
methods at all temperatures.
It has been reported that MRP mixtures are a composite of several discrete
chromophores that exhibit decreased absorbance with increasing wavelengths (O'Brien
and Morrissey, 1989). According to Cuzzoni et al. (1988), simple structure compounds,
produced during the early stages of the Maillard reaction, are related to absorbance in the
UV region (200-350 nm). Therefore, the analysis of the spectra of the browned solutions
produced by different treatments and temperatures used, allowed the detection of changes
related to subsequent stages of the Maillard reaction. It has been reported that, the first
stage of glucose-lysine MRP formation is characterized by the appearance of an
absorption around 280 nm that can be related to heterocyclic derivatives. As the reaction
proceeds, this absorbing material becomes progressively less concentrated due to the
appearance of more complex compounds which are known as soluble pre-melanoidins
having an absorbance maximum at 320 nm (Cuzzoni et al.,1988; Wijewickreme et al.,
1997). An absorption maximum at 220 nm, due to carbonyl compounds produced in the
first stage of the Maillard reaction, is also detected in the glucose-lysine system by
Cuzzoni et al. (1988). In this study, even though 220 nm was the cut-off wavelength for
the MRP spectral pattern measurements, we could still consider there was one absorption
shoulder at around 225 nm (Figure 5.5-5.13). Therefore, except the first spectral pattern
66
of 63°C, all other spectral patterns had at least two absorption shoulders, one at around
225 nm and the other around 290 nm. The first spectral pattern of 63°C also had one
absorption shoulder at around 225 nm for all three treatment samples, however, the
absorption shoulder at around 290 nm only appeared in water bath and Ethos Synth
microwave samples. The vacuum microwave sample had one absorption shoulder at
around 270 nm instead. As such, the shoulders detected in this study were an indication
of the presence of simple structure compounds, carbonyl compounds and heterocyclic
compounds, produced during the early stages of Maillard reaction. In addition, at 63°C
(Figure 5.6-5.7), the shoulders of vacuum microwave samples were lower than the
shoulders of water bath and Ethos Synth microwave samples, however, this situation was
reversed at temperature 81°C and 90°C (Figure 5.8-5.10 and Figure 5.11-5.13,
respectively). Higher fluctuation at lower temperatures in vacuum microwave treatment
might be the reason that caused the slower rate of simple structure compounds produced
in vacuum microwave MRPs at lower temperatures.
The comparison of the spectra of browning reaction solutions treated under
different treatments showed that the vacuum microwave treatment produced either
different Maillard reaction compounds or compounds present in different concentrations
from the water bath and Ethos Synth treatments. The differences might be mostly caused
by the differences of the low molecular weight carbonyl compounds and heterocyclic
compounds that were produced during different heating treatments since differences were
more obvious in the UV region. However, due to the lack of previous study in
comparison of the Maillard reaction products between microwave field and conventional
67
convective heat transfer, further analysis was needed to prove this, such as MALDI-TOF
MS or FTIR analysis.
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5.3 Dielectric Property Measurements
Because microwave radiation is part of the electromagnetic spectrum, it behaves
very much like the more familiar visible light irradiation we experience daily. Materials
interact with microwaves in three ways (Buffler, 1993). First, they reflect microwave
radiation impinging on them. Second, they transmit microwaves that have entered into
them. Finally, they absorb some of the microwave energy being transmitted through
them. The dielectric constant (e') and the loss factor (e") are the two important
parameters to measure the dielectric properties of materials. Dielectric constant is a
measure of the ability of a material to couple with microwaves while the loss factor is a
measure of the ability of a material to heat by absorbing microwave energy. The loss
factor actually refers to effective loss factor which also includes the direct current
(ohmic) conductivity effect. The power dissipated inside a material is proportional to e"
(Buffler, 1993) and the ratio, e'Ve', termed the loss tangent, is thus an indicator of the
material's ability to generate heat (Mudgett, 1990). It is also known the more microwave
absorptive a material is (i.e., the higher the loss factor e"), the less deep microwave
energy will penetrate into that material. It is very instructive to define a parameter that
indicates how far into a material a microwave penetrates before it is reduced to a certain
fraction of its initial value. This parameter, defined as penetration depth, is a function of
both dielectric properties of a material, £' and e". Knowledge of penetration depth serves
as a guideline to the heating effectivity of a material and is therefore an important
parameter that must be known in the design of a microwave experiment (Buffler, 1993).
A relationship for penetration depth d is given by:

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78
where X is the wavelength radiation. At the microwave frequency of 2450 MHz, the
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In this study, the open-ended probe system was used to measure the dielectric
properties of collected MRPs of these three heat treatments, and non-treated mixture of
glucose-lysine. The purpose of measuring the dielectric properties of non-treated
glucose-lysine mixture was to calculate the approximate penetration depth. In this case,
the penetration depth was 1.81 cm, which was within the penetration depth range of most
liquid products (Buffler, 1993). The dielectric constant (£') and loss factor (e") of the
collected Maillard reaction samples at 63°C as a function of vacuum microwave, water
bath and Ethos Synth microwave heating are shown in Figure 5.14 and 5.15, respectively.
The dielectric constant and loss factor both were affected by the heating method. It can
also be seen from the figures that as the heating process progressed, the loss factors for
water bath and Ethos Synth microwave samples stayed almost the same, but increased
considerably (p<0.05) for vacuum microwave samples. However, the changes of
dielectric constant over time of all three methods samples can be considered negligible
0?>0.05). Similar results were also found for the dielectric constant (e') and loss factor
(e") of the collected Maillard reaction samples at 81°C and 90°C (Figure 5.16, 5.17 and
Figure 5.18, 5.19, respectively). The biggest difference in loss factor changes over time
between vacuum microwave, water bath and Ethos Synth microwave samples was seen at
63°C compared to 81°C and 90°C samples. Once again, reasons remain unknown.
It has been reported that in food samples, water and salt are the two major
ingredients which influence dielectric properties. Other components usually have a
minor influence on the dielectric properties. The electromagnetic field frequency and
79
sample temperature affect dielectric properties as well (JET, 1989; Bircan and Barringer,
2002). According to Yaghmaee and Durance (2002), a 10% change of sucrose in
concentration only led to a 0.1 unit change in loss factor, however, a 0.2% change of
sodium chloride in concentration led to a 1.83 unit change in loss factor. Galema (1997)
also reported that the presence of salts in a solution does not influence the dielectric
constant greatly, but has a marked effect on the dielectric loss factor. The dielectric loss
factor is elevated above that of pure water because of the electrophoretic migration of
dissolved salt, which is also directly related to the size and charge of the dissolved ions.
In this study, the differences of loss factor between vacuum microwave, water bath and
Ethos Synth microwave reactor ranged from 3.53 units at 63°C, 2.91 units at 81°C to 1.63
units at 90°C, however, a 0.2% change of salt in sample concentration takes a significant
change in the sample volume. In addition, all the compared samples were collected at the
same absorbance at 420 nm under controlled conditions, hence, the significant change in
loss factor of vacuum microwave MRPs in this study was not caused by the possibility of
change of concentration of solution, the difference of the dielectric properties among
these heat treatments samples must be due to different charged compounds or different
concentrations of charged compounds in the Maillard reaction products. Apparently the
high microwave field strength of the vacuum microwave system resulted in differences
from the conduction/convection heating while the low microwave field strength of the
Ethos Synth microwave system had little impact.

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86
5.4 MALDI-TOF MS Analysis of MRPs of Glucose-Lysine Model System
Recent advances in MS techniques have allowed for quantitative analysis.
MALDI-TOF is a new MS technique which is gaining popularity as instrumentation
improves (Abell and Sporns, 1996).
MALDI-TOF MS was first introduced in 1987 and was originally developed for
large biomolecules (Karas et al., 1987). It can readily be used with compounds of up to
350 kDa (Stahl et al., 1991). Analysis can be performed with picomole quantities of
samples with reported sensitivity in the femtomole range. While the use of MALDI-TOF
MS with high molecular weight compounds is of great value, it also has potential for
quantification of both high and low molecular weight compounds (Gusev et al., 1995).
MALDI-TOF uses low molecular weight UV-absorbing compounds to act as a matrix
vehicle for desorption and ionization of molecules of interest. The sample of interest is
cocrystallized with excess matrix and then pulsed with an UV laser to desorb and ionize
the matrix. The matrix absorbs energy from the laser and transfers it to the analyte. This
soft ionization method provides intact molecules in the gas phase, principally as cations
(Harvey, 1994). Typically, the MALDI is linked to a TOF instrument for mass
determination. TOF analysis works well in conjunction with MALDI as it does not have
an upper limit for high m/z ions and works well with the pulsed laser used in MALDI
(Siuzdak, 1994). Both the matrix and the sample will appear on the spectrum, therefore,
matrices must be low molecular weight or the sample must be of sufficient mass to
prevent overlap with the matrix peaks (Abell and Sporns, 1996).
MALDI-TOF MS has advantages over other methodologies including speed of
analysis, high sensitivity, wide applicability combined with a good tolerance toward
87
contaminants, and the ability to analyze complex mixtures (Karas, 1996). However,
simple MALDI-TOF MS instruments cannot tell the difference between isomers, which
have identical mass. The potential application of MALDI-TOF MS in food systems
allows for analysis of most molecules (Wang and Sporns, 2000).
Even though MALDI-TOF has been used to quite an extent for analyzing
proteins, its use in carbohydrate chemistry has been developed for only a few years (Stahl
et al., 1994). Until now, only a few reports related to Maillard reaction have used this
spectroscopic method (Wijewickreme et al., 1997; Kim et al., 1997; Yeboah and
Yaylayan, 2001; Humeny et al., 2002). Among these reports, only one report studied the
glucose-lysine MRP mixture (Wijewickreme et al., 1997), the other three reports
analyzed the protein glycation. In this study, even though the biggest difference between
vacuum microwave and water bath MRPs from previous experiments (Maillard reaction
rate determination and UV/visible spectral patterns) was seen at 63°C, however, the
absorbance at 420 nm of collected samples of 63°C were very low, i.e., the concentrations
of MRP mixtures were low, which might have been difficult for MALDI-TOF MS to
analyze. Therefore, MRPs of vacuum microwave and water bath treatments at 81°C were
collected. Although MALDI-TOF is comparatively tolerant to the presence of
contaminants such as buffer salts and detergents, these compounds can influence the
intensity and quality of the signal (Mock et al., 1992). Therefore, 0.01 M SCB buffer
was used instead of 0.05 M in this case to reduce the influence from buffer salts. Prior to
using MALDI-TOF MS as an analytical technique, a matrix in which the sample of
interest desorbs and ionized well must be found. Compounds of similar structure will
typically produce similar results with a given matrix. A number of common matrices
88
were tested, including sinapic acid, 2,5-dihydroxybenzoic acid (DUB) and 4-hydroxy-
alpha-cyanocinnamic acid (CCA). The use of CCA as matrix gave only one extra peak at
m/z of 642 daltons (Appendix 8.1), consequently CCA was the chosen matrix. MALDI-
TOF, since its introduction by Karas et al. (1987), has been used mainly for molecular
weight determination. However, recently a number of studies have shown that the
method can be also applied for quantification of compounds (Harvey, 1993; Naven and
Harvey, 1996; Wang et al., 1999). MALDI-TOF mass spectra of MRP mixtures of
vacuum microwave and heated water bath are given in Figure 5.20-5.23. Both mixtures
contained eight principal peaks: a major one with a m / z of approximately 656 daltons,
and the other seven peaks with m / z of 580 daltons, 625 daltons, 642 daltons, 867
daltons, 1078 daltons, 1289 daltons and 1501 daltons (the first three numbers were not
shown on spectra) (Figure 5.20 and Figure 5.21). The CCA matrix itself gave the signal
at m / z of 642 daltons (Appendix 8.1). The spectra showed the MRP compounds
produced in this study were all low molecular weight compounds. Once again, MALDI-
TOF results agreed with the results of UV/visible spectral patterns comparison.
Accompanying these eight peaks were other smaller peaks indicating the presence of
other trace compounds produced in the crude MRP mixtures during different stages of the
Maillard reaction. Both spectra indicated that the MRP compounds of vacuum
microwave and heated water bath treatments were identical since no different peaks were
found between these two mass spectrums. Similar results were also seen in Figures 5.22
and 5.23, which represented the high-mass expansion spectra of the two samples.
Relative intensities of peaks (Table 5.2), i.e. the ratios between peaks, were used to
compare the differences between these two samples. Results indicated the concentrations
89
of compounds might be different between these two samples since some of the relative
intensities of peaks between these two samples were different (Table 5.2). It was difficult
to identify the compounds in the samples through MALDI-TOF MS analysis due to lack
of standard Maillard reaction samples. However, based on the identification of
compounds from glucose-lysine model system reported by other scientists (D'Agostina et
al, 1998; Hill et al., 1999; Bailey et al., 2000), suggestions of compounds formed in this
study could be given. Bailey et al. (2000) studied the glucose-lysine MRPs, which were
isolated by combinations of solvent extraction and semipreparative HPLC, prior to
identification by NMR and mass spectrometry. Two compounds were identified in their
study as e-[2-formyl-5-(hydroxymethyl)pyrrole-l-yl]-L-norleucine (pyrraline) ( M i ) with
molecular weight of 260, and l-(5-carboxy-5-aminopentyl)-2-formyl-3-(l,2,2-
trihydroxypropyl)pyrrole (M ) with molecular weight of 315. Bailey et al. (2000) also

2
reported adducts of these two compounds as (M + H - H 0 - CHOH), (M + H - CHOH

2
- C0 ), (M + H - H 0 - CHOH - C0 ), (M + FT), (2M + Na ) and (2M + O + Na ).

2 2 2
+ +
Therefore, in this study, peaks at 580 daltons, 625 daltons and 1078 daltons have the
possibility to be (2M, + 3Na ), (2M, + 20 + 3Na ) and (4Mi + Na ), respectively. In

+ + +
addition, peaks at 656 daltons, 867 daltons, 1289 daltons and 1501 daltons have the
possibility to be (2M + Na ), (3M + H - CHOH - C0 ), (4M + Na ), and (5M + H -

2
+
2 2 2
+
2
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2 Since results in this study showed the concentration of
compounds was different between water bath and vacuum microwave samples, FTIR
analysis was used to further support this inference.

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5.5 FTIR Analysis
Mid-infrared (IR) spectroscopy is a well-established technique for the
identification and structural analysis of chemical compounds. The peaks in the IR
spectrum of a sample represent the excitation of vibrational modes of the molecules in the
sample and thus are associated with the various chemical bonds and functional groups
present in the molecules. Thus, the JR. spectrum of a compound is one of its most
characteristic physical properties and can be regarded as its "fingerprint". Infrared
spectroscopy is also a powerful tool for quantitative analysis as the amount of infrared
energy absorbed by a compound is proportional to its concentration (Ismail, 1997).
The development of FTIR spectroscopy during the past two decades has
revitalized the field of IR spectroscopy. This is due not only to the superior performance
of FTIR spectrophotometers by comparison to that of the dispersive IR
spectrophotometers that they have virtually replaced, but also to a number of other
factors. First, the development of FTIR spectrophotometers has been paralleled by
advances in sample handling techniques. Second, the dedicated computer that is an
integral component of all FTIR systems has been extensively exploited in the
development of sophisticated data handling and data analysis routines (Ismail et al.,
1997).
In this analysis, final MRPs of the three heat treatments at 90°C were selected
instead of final MRPs at 63°C, which was based on the same reasoning as the MALDI-
TOF MS sample selection. The same reasoning was applied to cell toxicity sample
selection. Freeze-dried solid samples were used instead of aqueous solutions to
overcome the broad bands that were caused by the overlap of the vibrational bands of
96
water and moisture with the solutes. In appendix, Figures 8.2-8.8 are the original spectra
of the MRP samples and reactants. Baseline correction of each spectrum was done to
bring it down to a baseline of zero absorbance which made it easier to compare the
relative sizes of peaks. To compare differences between the three treatments,
normalization of all the spectra based on one peak was also done, which helped to
compare the relative intensity (height) of the other peaks in the normalized spectra among
the three treatments. Figure 5.24 shows the overlay spectra of MRPs of vacuum
microwave, water bath and Ethos Synth microwave reactor heat treatments, which
provided an overview of the difference between treatments. Figure 5.25 shows the stack
spectra of MRPs of three heat treatments, which made it easier to see the peaks of each
spectrum. In addition, Figures 5.26 and 5.27 compare the spectrum of the water bath
MRP with the non-treated mixture of glucose-lysine model. Figures 5.24 and 5.25
indicate that the locations (frequency values) of peaks were quite similar among all
MRPs from three different heat treatments, i.e. all MRPs from these three heat treatments
should have the same functional groups. Figures 5.26 and 5.27 indicate that the non-
treated mixture had quite different peaks than the water bath MRP, and seemed to be
some combinations of the spectra of the glucose, lysine and SCB buffer. Therefore, the
peaks in the MRP spectra are from the MRP samples not from the reactants. The spectra
of the three MRPs showed absorbance bands at 3421, 1669, 1623, 1404, 1077, 1046, 833
and 703 cm" . These bands are representative of the chemical or functional groups of
1
components present in the samples. Therefore, according to Ismail et al. (1997) and
Keyhani and Yaylayan (1997), FTIR analysis indicated that all three MRPs had the
presence of phenolic, carbonyl and alkyl functional groups. The 700-1500 cm" region
1
97
corresponds to the absorption zones of glucose. The bands in the 900-1100 cm' region 1
are assigned to C-0 and C-C stretching modes, and those around 1300-1500 cm" are due 1
to the bending modes of O-C-H, C-C-H and C-O-H angles. Those around 1618-1670 cm"
1
are due to the stretching modes of C=0 and C=N. The broad, strong N H 3
+
stretching
band in the 2000-2950 cm" region can be related to lysine, and the bands in the 3404-
1
3421 cm" region corresponds to stretching mode of O-H (H-bonded). Table 5.3 shows
1
the functional groups along with the corresponding modes of vibration obtained from the
FTIR spectra of MRPs. The peak at 703 cm" may be due to C-H stretching of
1
carbohydrates, whereas the peaks observed at 1046 and 1077 cm" may be due to C-O
1
stretching in the C-OH group as well as C-C stretching in the carbohydrate structure. In
addition, it may be due to C-N stretching. The peak at 1404 cm" may be due to the O-H
1
bending of the C-OH group, whereas the peaks at 1623 and 1669 cm" may be due to
1
C=N stretching as well as the C=0 stretching. Finally, the peak at 3421 cm" may be 1
mainly due to O-H (H-bonded) stretching vibration. However, FTIR analysis only
indicates the functional groups of glucose-lysine MRPs, it is not suitable for measuring
the compound compositions of MRPs.
In this study, the absorbance levels were different among samples, which
indicated the quantity of each functional group was different among samples. This was
especially apparent when vacuum microwave sample versus water bath and Ethos Synth
microwave reactor samples. For example, the absorbance of the 3421 cm" band for 1
vacuum microwave MRP was around 3.0, which was much higher than the absorbance of
2.6 for water bath MRP, or 2.4 for Ethos Synth microwave MRP (Figure5.24-5.25).
Once again, the spectral results confirmed the results from previous experiments.
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5.6 Cell Toxicity Test
MRPs have been reported to have cytotoxic effects on both rat and human cells
(Wang et al., 1987; Vagnarelli et al., 1991). The whole mixtures of MRPs, made from
model systems of glucose-, and fructose-lysine, have been shown to produce a cytotoxic
effect on rat glioma cells (Wang et al., 1987). Since Caco-2 cells represents a useful
model for human intestine-food nutrient interactions, numerous workers have used the
Caco-2 cell line to study absorption of nutrients, and nutrient-induced effects on Caco-2
cell growth and differentiation (Finley and Monroe, 1998; Glahn et al., 1998).
Assessment of toxicity potential of MRPs using Caco-2 cells could be an important in
vitro test in evaluating MRP toxicity in humans. Table 5.4 showed the toxicity of
glucose-lysine MRPs (90°C) on Caco-2 cells over different concentration ranges (0.5-2
mg/ml) after 72 hours incubation. Results indicated at MRP concentration of 0.5 mg/ml,
the cell viability of Caco-2 cells exposed to MRPs produced by the vacuum microwave
treatment was much less than the other two heat treatments. Significant (p< 0.05)
toxicity was observed for each MRP at all concentrations in comparison with the control
(PBS buffer).
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Our data confirmed the reports of others (Wang et al., 1987; Vagnarelli et al.,
1991), that MRPs have a cytotoxic effect, however, the underlying mechanism of MRP-
induced cytotoxicity is not clear at this time. In addition, MRP made from vacuum
microwave heating system was more toxic than MRP derived from water bath and Ethos
Synth microwave reactor systems at lower dose concentration (0.5 mg/ml). As the dose
concentration increased, the viability of Caco-2 cells treated by MRPs of all three heat
treatments dropped to very low levels (Table 5.4), such that no significant toxicity
difference was found at dose concentrations of 1 mg/ml and 2 mg/ml. Significant
toxicity difference (p<0.05) was found between vacuum microwave and, water bath or
Ethos Synth microwave treatments at dose concentration of 0.5 mg/ml, however, no
significant toxicity difference was found between water bath and Ethos Synth microwave
treatments at this dose concentration. Therefore, concentration of MRPs used for
reacting Caco-2 cells with MRPs was an important factor in the expression of toxicity by
different MRPs, which agreed with the report of Jing and Kitts (2000). Once again,
results of lower dose concentration (0.5 mg/ml) showed a difference in vacuum
microwave MRPs over water bath and Ethos Synth microwave generated MRPs.
106
6. C O N C L U S I O N
A comparison was made of the formation of Maillard products in two microwave
heating treatments (vacuum microwave and Ethos Synth microwave reactor) and a
conventional heating treatment (water bath). Notable differences in the UV/visible
absorbance spectra between vacuum microwave MRPs and heated water bath MRPs or
Ethos Synth microwave reactor MRPs at three tested temperatures (63°C, 81°C and 90°C)
denoted differences in the chemical components of MRPs or differences in the
concentrations of MRP compounds from the three heat treatments. The study of
dielectric properties confirmed that the chemical components of MRPs or the
concentrations of MRP compounds from the three heat treatments were different.
Further, MALDI-TOF MS and FTIR analysis of MRPs at 81°C or 90°C indicated the
major chemical components of MRPs of higher temperatures from different treatments
were the same, however, the concentrations of compounds were different. In addition,
the MRPs of water bath and Ethos Synth microwave reactor seemed to be quite similar.
The differences of MRPs resulted in variations in biochemical activities in cytotoxic
activity. A comparison of toxicities of the MRPs (90°C) from the three heat treatments
indicated that the MRPs from vacuum microwave had greater toxicity on Caco-2 cells,
compared to the MRPs from the other two heat treatments. The browning rates obtained
in a vacuum microwave were greater than the rates obtained in a heated water bath or an
Ethos Synth microwave reactor at similar temperatures. The vacuum microwave
decreased the Ea, which corresponded to increased reaction rates. Therefore, the vacuum
microwave system permitted the Maillard reaction to take place at a lower temperature.
The browning rates obtained in the water bath were similar (p>0.05) to the rates obtained
107
in the Ethos Synth microwave reactor. Apparently, reaction rates increased
proportionally to the microwave power used. As the microwave power dropped from 700
W to below 100 W, the browning reaction rate of the microwave treatment approached
that of the conventional heating. In this study, higher microwave power also produced
different concentrations of compounds of MRPs in comparison with conventional heating
and microwave of very low power. The information provided by this study helps us to
better understand the reasons for the differences in Maillard reaction caused by vacuum
microwave when compared to conventional heating methods. This study also points to
advantages (e.g. faster Maillard reaction) and disadvantages (e.g. greater toxicity) of
using vacuum microwave for Maillard browning reaction. However, due to the technical
difficulty, it was not feasible for us to study the minor components of the MRP mixtures.
This study only included the early stages of the Maillard reaction; future studies are
needed to determine the advanced Maillard reaction in vacuum microwave treatment. In
addition, study of the Maillard browning reaction of asparagine and reducing sugar in a
microwave field might be valuable since the presence of high amounts of acrylamide
(221 mg per mol of amino acid) in various common fried and oven-cooked foods has
attracted worldwide attention.

108
7. R E F E R E N C E S
Abell, D.C. and Sporns, P. 1996. Rapid quantitation of potato glycoalkaloids by matrix-
assisted laser desorption/ionization time-of-flight mass spectrometry. J. Agric.
Food Chem. 44:2292-2296.
Adrian, J. 1974. Nutritional and physiological consequences of Maillard reaction. World

Rev. Nutri. Diet. 19:71-122.
Adrian, J. 1982. The Maillard reaction, in Handbook of Nutritive Value of Processed

Food, vol.1. Food for Human Use. Rechcigl, M. (Ed.), CRC Press, Boca Raton,
FL. p.529-563.
Ames, J.M. 1990. Control of the Maillard reaction in food systems. Trends Food Sci.
Technol. 150-154.
Ames, J.M. 1992. The Maillard reaction, in Biochemistry of Food Proteins.

Hudson, B.J.F. (Ed.). Elsevier Applied Science, London, England, p.99-153.
Arnoldi, A., Corain, E.A., Scaglioni, L., Ames, J.M. 1997. New colored compounds from
the Maillard reaction between xylose and lysine. J. Agric. Food Chem. 45:650-
655.
Ashoor, S.H. and Zent, J.B. 1984. Maillard browning of common amino acids and sugars.
J. Food Sci. 49:1206-1207.
Bailey, R.G., Monti, S.M., Ames, J.M. 1995. Formation of colored compounds in
Maillard model systems. Current Stat. Future Trends2:269-274.
Bailey, R.G., Ames, J.M. and Mann, J. 2000. Identification of new heterocyclic nitrogen
compounds from glucose-lysine and xylose-lysine Maillard model systems. J.
Agric. Food Chem. 48:6240-6246.
Baisier, W.M. and Labuza, T.P. 1992. Maillard browning kinetics in a liquid model
system. J. Agric. Food Chem. 40:707-713.
Baldwin, R.E. and Tettambel, J.E. 1974. Nitrogen content of rib eye steaks heated by
microwaves and a conventional method. Microwave Energy Appl. Newslett. 7:
3-8.
Barringer, S.A., Fleischmann, A.M., Davis, E.A. and Gordon, J. 1995. The dielectric
properties of whey protein as indicators of change in polymer mobility. Food
Hydrocol. 9:343-348.
Baxter, J.H. 1995. Free amino acid stability in reducing sugar systems. J. Food Sci.
60:405-408.
109
Baynes, J.W., Ahmed, M.U., Fisher, C L , Hull, C.J., Lehman, T.A., Watkins, N.G. and
Thorpe, S.R. 1986. Studies on glycation of proteins and Maillard reactions of
glycated proteins under physiological conditions. Dev. Food Sci. 13:421-431.
Beck, J., Ledl., F. and Severin, T. 1988. Formation of l-deoxy-D-erythro-2,3-hexodiulose

from Amadori compounds. Carbohydr. Res. 178:240-243.
Bell, L.N. 1996. Kinetics of non-enzymatic browning in amorphous solid systems

distinguishing the effects of water activity and the glass transition. Food Res. Int.
28:591-597.
Bengtsson, E.N., Melin, J., Remi, K. and Soderlind, S. 1963. Measurements of the
dielectric properties of frozen and defrosted meat and fish in the frequency range
10-200 MHz. J. Sci. Food Agric. 14:592-604.
Bircan, C. and Barringer, S.A. 2002. Determination of protein denaturation of muscle

foods using the dielectric properties. J. Food Sci. 67:202-205.
Bodwell, CE and Womack, M. 1978. Effects of heating methods on protein nutritional

value of five fresh or frozen prepared food products. J. Food Sci. 43:1543-1549.
Bohart, G.S. and Carson, J.F. 1955. Effects of trace metals, oxygen, and light on the
glucose-lysine browning reaction. Nature 175:470-471.
Brands, C.M.J., Alink, G.M., Van Boekel, M.A.J.S. and Jongen, W.M.F. 2000.
Mutagenicity of heated sugar-casein systems: effect of the Maillard reaction. J.
Buffler, C R . 1993. Dielectric properties of foods and microwave materials, in

Microwave Cooking and Processing: Engineering Fundamentals for the Food
Scientist. Van Nostrand Reinhold, NY, NY. p.47-68.
Calay, R.K., Newborough, M., Probert, D. and Calay, P.S. 1995. Predictive equations for
the dielectric properties of foods. Int. J. Food Sci. Technol. 29:699-713.
Cammerer, B. and Kroh, L.W. 1995. Investigation of the influence of reaction conditions
on the elementary composition of melanoidin. Food Chem. 53:55-59.
Carroll, D.E. and Lopex, A. 1969. Lethality of radio-frequency energy upon

microorganisms in liquid, buffered and alcoholic food systems. J. Food Sci.
34:320-324.
110
Cuzzoni, M.T., Stoppini, G., Gazzani, G. and Mazza, P. 1988. Influence of water activity
and reaction temperature of ribose-lysine and glucose-lysine Maillard systems on
mutagenicity, absorbance and content of furfurals. Food Chem. Toxicol. 26:815-
822.
D'Agostina, A., Negroni, M. and Arnoldi, A. 1998. Autoxidation in the formation of

volatiles from glucose-lysine. J. Agric. Food Chem. 46:2554-2559.
Danehy, J.P. 1985. Maillard reactions: non-enzymatic browning in food systems with
special reference to the development of flavor. Adv. Food Res. 30:77-138.
Danehy, J.P. and Wolnak, B. 1983. Maillard technology: manufacturing applications in

food, in The Maillard Reaction in Foods and Nutrition. Waller, G.R. and Feather,
M.S. (Eds.). ACS Symp. Ser. 215:303-315.
Decareau, R.V. 1985. Microwaves in the Food Processing Industry. Academic Press, NY.
Decareau, R.V. 1992. Microwave Foods: new product development. Food & Nutri. Press,
Trumbull, Conn.
Dworschak, E. and Orsi, F. 1977. Study into the Maillard reaction occuring between
methionine and tryptophan on one hand and the glucose on the other. I. Studies in
aqueous solutions. Acta Aliment. 6:59-71.
Eichner, K. and Karel, M. 1972. The influence of water content and water activity on the
sugar-amino browning reaction in model systems under various conditions. J.
Erbersdobler, H.F. 1986. Twenty years of furosine. Better knowledge about significance
of Maillard reaction in food and nutrition. Dev. Food Sci. 13:481-491.
Fakhouri, M.O. and Ramaswamy, H.S. 1993. Temperature uniformity of microwave

heated foods as influenced by product type and composition. Food Res. Int.
26:89-95.
Fay, L., Richli, L., Liardon, R. 1991. Evidence of the absence of amino acid
isomerization in microwave-heated milk and infant formulas. J. Agric. Food
Chem. 39:1857-1859.
Feather, M.S. 1989. Sugar-derived deoxy-dicarbonyl intermediates as precursors of food

flavors and aromas, in Thermal Generation of Aromas. Parliament, T.H.,
McGorrin, R.J., Ho, C.T., ACS Symp. Ser. 409:209-216.
Felton, J.S., Knize, MG., Roper, M., Fulz, E., Shen, N.H. and Turteltaub, K.W. 1992.
Chemical analysis, prevention, and low-level dosimetry of heterocyclic amines
from cooked foods. Cancer Res. 52(Suppl.):2103s-2107s.
Ill
Finley, J.W. and Monroe, P. 1998. Manganese absorption: the use of Caco-2 cells as a
model of the intestinal epithelia. Nutri. Biochem. 8:92-101.
Finot, P.A., Deutsh, R. and Bujard, E. 1981. The extent of the Maillard reaction during
the processing of milk. Maillard Reaction in Food. Prog. Food Nutri. Sci. 5:345-
356.
Finot, P.A. and Merabet, M. 1993. Nutritional and safety aspects of microwaves. Ffistory
and critical evaluation of reported studies. Int. J. Food Sci. Nutri.
44(Suppl.l):S65-S75.
Franzen, K, Singh, R.K., Okos, M.R. 1990. Kinetics of nonenzymatic browning in dried
skim milk. J. Food Engin. 11:225-239.
Fry, L.K. and Stegink, L.D. 1982. Formation of Maillard reaction products in parenteral
alimentation solutions. J. Nutri. 112:1631-1637.
Fung, D.Y. and Cunningham, F.E. 1980. Effects of microwaves on microorganisms in

foods. J. Food Protect. 43:641-650.
Galema, S.A. 1997. Microwave chemistry. Chem. Soc. Reviews 26(3):233-238.
Glahn, R.P., Lai, C , Hsu, J., Thompson, J.F., Guo, M. and Van Campen, D.R. 1998.
Decreased citrate improves iron availability from infant formula: application of an
in vitro digestion/caco-2 cell culture model. J. Nutri. 128:257-264.
Goldblith, S.A., Tannenbaum, S.R. and Wang, D.I.C. 1968. Thermal and 2450 MHz
microwave energy effect on the destruction of thiamine. Food Technol. 22:1266-
1268.
Goldblith, S.A. and Wang, D.I.C. 1967. Effects of microwaves on Escherichia coli and
Bacillus subtilis. Appl. Microbiol. 15:1371-1373.
Gusev, A.I., Wilkinson, W.R., Proctor, A., Hercules, D.M. 1995. Improvement of signal
reproducibility and matrix/comatrix effects in MALDI analysis. Analyt. Chem.
67:1034-1041.
Hafez, Y.S., Singh, G., McLellan, M.E. and Monroe-Lord, L. 1983. Effects of microwave
heating on nutritional quality of soy-beans. Nutri. Rep. Int. 28:413-421.
Harvey, D.J. 1993. Quantitative aspects of the matrix-assisted laser desorption mass
spectrometry of complex oligosaccharides. Rapid Comm. Mass Spec. 7:614-619.
Harvey, D.J. 1994. Matrix-assisted laser desorption/ionization mass spectrometry of

oligosaccharides. Am. Lab. Dec:22-28.
112
Hashiba, H., Okuhara, A. and Iguchi, N. 1981. Oxygen dependent browning in soy sauce
and some brewed products. Prog. Food Nutri. Sci. 5:93-113.
Hayase, F. and Kato, H. 1994. Maillard reaction products: safety and physiologic effects.
Comments Agric. Food Chem. 3:111-128.
Hill, V.M., Isaacs, N.S., Ledward, D.A., Ames, J.M. 1999. Effect of high hydrostatic
pressure on the volatile components of a glucose-lysine system. J. Agric. Food
Chem. 47:3675-3681.
Hodge, J.E. 1953. Dehydrated foods. Chemistry of browning reaction in model systems.
J. Agric. Food Chem. 1:928-943.
Hodge, J.E. 1967. Origin of flavors in foods. Non-enzymatic browning reactions, in

Chemistry and Physiology of Flavors. Shula, H.W., Day, E.A. and Libbey,
L.M. (Eds.). Avi. Publ. Co., Westport, CT. p.465.
Hoskin, J.C. and Dimick, P.S. 1984. Role of non-enzymatic browning during the
processing of chocolate: a review. Process Biochem. 19:92-104.
Huber, B., Ledl, F., Severin, T., Stangl, A. and Pfleiderer, G. 1988. Formation of (2-
furoyl)-4(5)-(2-furyl)-H-Imidazole in the Maillard reaction. Carbohydr. Res.
182:301-306.
Humeny, A., Kislinger, T., Becker, C-M and Pischetsrieder, M. 2002. Qualitative
determination of specific protein glycation products by matrix-assisted laser
desorption/ionization mass spectrometry peptide mapping. J. Agric. Food Chem.
50:2153-2160.
Hurrell, R.F. and Carpenter, K.J. 1974. Mechanisms in heat damage in proteins. 4. The
reactive lysine content of heat damaged material as measured in different ways.
Br. J. Nutri. 32:589-604.
IARC. 1994. IARC monographs on the evaluation of carcinogenic risks to humans.

Int. Agency Res. Cancer 60:389-433.
JET. 1989. Microwave food processing. A scientific status summary by the IFT
expert panel on food safety & nutrition. Food Technol. 43(1): 117-126.
Ismail, A.A., van de Voort, F.R. and Sedman, J. 1997. Fourier transform infrared
spectroscopy: principles and applications, in Instrumental Methods in Food
Analysis. Pare, J.R.J, and Belanger, J.M.R. (Eds.). Elsevier Science, Amsterdam,
NY. p.93-139.
113
Jacobs, S.E., Thorney, M.J. and Maurice, P. 1950. The survival of bacteria in high
frequency voltage fields. Proc. Soc. Appl. Bacterid. 2:161-169.
Jahngen, E.G.E., Lentz, R.R., Pesheck, P.S. and Sackett, P.H. 1990. Hydrolysis of
adenosine triphosphate by conventional or microwave heating. J. Org. Chem.
55:3406-3409.
Jing, H. and Kitts, D.D. 2000. Comparison of the antioxidative and cytotoxic properties
of glucose-lysine and fructose-lysine Maillard reaction products. Food Res.
Int. 33:509-516.
Jonker, D. and Penninks, A.H. 1992. Comparative study of nutritive value of casein
heated by microwave and conventionally. J. Sci. Food Agric. 59:123-126.
Kannane, A. and Labuza, T.P. 1985. Change in available lysine loss reaction rate in fish
flour due to an a change induced by a temperatue shift. J. Food Sci. 50:582-584.
w
Kannane, A. and Labuza, T.P. 1989. The Maillard reaciton in foods, in The Maillard
Reaction in Ageing, Diabetes, and Nutrition. Baynes, J. (Ed.). A.R. Liss Press,
Inc. NY. p.323-327.
Karas, M. 1996. Matrix-assited laser desorption ionization MS: a progress report.

Biochem. Mass Spec. 24:897-900.
Karas, M., Bachmann, D., Bahr, U. 1987. Matrix-assited ultraviolet laser desorption of
nonvolatile compounds. Int. J. Mass Spec. Ion Process 78:53-68.
Kato, H. and Tsuchida, H. 1981. Estimation of melanoidin structure by pyrolysis and

oxidation. Prog. Food Nutri. Sci. 5:147-156.
Kato, Y., Watanabe, K. and Sato, Y. 1981. Effects of some metals on the Maillard
reaction of ovalbumin. J. Agric. Food Chem. 29:540-543.
Kato, H., Yamamoto, M. and Fujimaki, M. 1969. Mechanisms of browning degradation

of D-fructose in special comparison with D-glucose-glycine reaction. Agric. Biol.
Chem. 33:939-940.
Kawamura, S. 1983. Seventy years of the Maillard reaction. ACS Symp. Ser. 215:3-18.
Kent, M. 1987. Electric and Dielectric Properties of Food Materials. Science and
Technology Publ., London, England.
Keyhani, A. and Yaylayan, V.A. 1997. Glycine specific novel Maillard reaction products:
5-hydroxy-l,3-dimethyl-2(lH)-quinoxalinone and related compounds. J. Agric.
Food Chem. 45:697-702.
114
Kim, H-J, Leszyk, J. and Taub, LA. 1997. Direct observation of protein glycation by
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J.
Kimiagar, M., Lee, T-C. and Chichester, C O . 1980. Long-term feeding effects of
browned egg albumin to rats. J. Agric. Food Chem. 28:150-155.
Labuza, T.P. and Baisier, W. 1992. The kinetics of nonenzymatic browning, in Physical
Chemical of Foods. Schwartzberg, H.G. and Hartel, R.W. (Eds.). Marcel Dekker
Inc., NY. p.595-650.
Labuza, T.P. and Saltmarch, M. 1981. The nonenzymatic browning reaction as affected
by water in foods, in Water Activity Influences on Food Quality. Rockland, L.B.
and Stewart, G.F. (Eds.). Academic Press, NY. p.605-650.
Labuza, T.P. and Schmidl, M.K. 1986. Advances in the control of browning reactions in
foods, in Role of Chemistry in the Quality of Processed Foods. Fennema, O.,
Chang, W.H., Lii, C. (Eds.). Food Nutri. Press, Wesport, CT. p.85-95.
Labuza, T.P., Tannenbaum, S.R. and Karel, M. 1970. Water content and stability of low
moisture and intermediate moisture foods. Food Technol. 24(5):543-550.
Lagercrantz, C. 1964. Formation of stable free radicals in alkaline solutions of some

monosaccharides. Acta. Chem. Scand. 18:1321-1324.
Laurent, R., Laporterie, A., Dubac, J., Berlan, J., Lefeuvre, S. and Audhuy, M. 1992.
Specific activation by microwaves: myth or reality? J. Org. Chem. 57:7099-7102.
Lea, C.H. and Hannan, R.S. 1951. Studies of the reaction between protein and reducing
sugars in the dry state. Biochim. Biophys. Acta. 3:313-325.
Lechowich, R.V., Beuchat, L.R., Fox, K.I. and Webster, F.H. 1969. A procedure for
evaluating the effects of 2450 MHz microwaves upon Streptococcus faecalis and
Saccharomyces cerecisiae. Appl. Microbiol. 17:106-110.
Lee, T . C and Chichester, C O . 1983. Physiological, toxicological, and nutritional aspects

of various Maillard browned proteins. ACS Symp. Ser. 234:379-408.
Lee, T . C , Kimiagar, M., Pintauro, S.J. and Chichester, C O . 1981. Physiological and
safety aspects of Maillard browning of foods. Prog. Food Nutri. Sci. 5:243-249.
Lewis, V.M. and Lea, C.H. 1959. A note on the relative rates of reaction of several
reducing sugars and sugar derivatives with casein. Biochim. Biophys. Acta.
4:532-534.
Ling, A.R. 1908, Malting. J. Inst. Brew. 14:494-521.

115
Lingert, H. 1990. Development of the Maillard reaction during food processing, in The
Maillard reaction in Food Processing, Human Nutrition and Physiology. Finot,
P.A., Aeschbacher, H.U., Hurrell, R.F., Liardon, R. and Birkhauser-Verlag, B.
(Eds.), p.171-185.
Lund, D.B. 1977. Design of thermal processes for maximum nutrient retention. Food
Technol. 31(2):71-78.
Maillard, L.C. 1912. Action des acides amines sur les sucres: formation des
melanoidines par voie methodique. C. R. Acad. Sci. 154:66-68.
Marchelli, R., Dossena, A., Palla, G. and Audhuy-Peaudecert, M., Lefeuvre, S.,
Carnevali, P. and Freddi, M. 1992. D-amino acids in reconstituted infant formula:
a comparison between conventional and microwave heating. J. Sci. Food Agric.
59:217-226.
Massaro, S.A. and Labuza, T.P. 1990. Browning and amino acid losses in model total
parenteral nutrition systems with specific reference to cysteine. J. Food Sci.
55:821-826.
Mauron, J. 1981. The Maillard reaction in food: a critical review from the nutritional
standpoint. Prog. Food Nutri. Sci. 5:5-35.
Mei(3ner, K. and Erbersdobler, H.F. 1996. Maillard reaction in microwave cooking:

comparison of early Maillard products in conventionally and microwave-heated
milk. J. Sci. Food Agric. 70:307-310.
Mertens, B. and Knorr, D. 1992. Developments of Nonthermal processes for food

preservation. Food Technol. 46(5): 124-133.
Mester, L., Szabados, M., Mester, M. and Yadav, N. 1981. Maillard type carbonyl-amine
reactions in vivo and their physiological effects. Prog. Food Nutri. Sci. 5:295-314.
Miller, B.J., Billedcau, S.M. and Miller, D.W. 1989. Formation of N-nitrosamines in
microwaved versus skillet-fried bacon containing nitrite. Food Chem. Toxicol.
27:295-299.
Miller, L.A., Gordon, J. and Davis, E.A. 1991. Dielectric and thermal transition
properties of chemically modified starches during heating. Cereal Chem. 68:441-
448.
Mingos, D.M.P. and Baghurst, D.R. 1997. Appilications of microwave dielectric heating
effects to synthetic problems in chemistry, in Microwave-Enhanced Chemistry.
Fundamentals, Sample Preparation, and Applications. Kingston, H.M. and
Haswell, S.J. (Eds.). Am. Chem. Soc, Washington, DC. p.3-53.
116
Mitsuda, H., Yasumoto, K. and Yokoyama, K. 1965. Studies on the free radical in amino-
carbonyl reaction. Agric. Biol. Chem. 29:751-756.
Mizrahi, S., Labuza, T.P., Karel, M. 1970. Computer-aided preditions of extent of

browning in dehydrated cabbage. J. Food Sci. 799-803.
Mock, K.K., Sutton, C W . and Cottrell, J.S. 1992. Sample immobilization protocols for
matrix-assisted laser desorption mass spectrometry. Rapid Comm. Mass Spec.
6:233-238.
Modderman, J.P. 1986. Technological aspects of using sulfiting agents in food. J. Assoc.
Off. Anal. Chem. 69:1-3.
Monnier, V.M. and Cerami, A. 1983. Nonenzymatic glycosilation and browning of

proteins in vivo. ACS Symp. Ser. 215:431-449.
Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J. Immunol. 65:55-63.
Mottram, D.S., Wedzicha, B.L. and Dodson, A.T. 2002. Acrylamide is formed in the
Maillard reaction. Nature 419:448-449.
Mudgett, R.E. 1986. Electrical properties of food, in Engineering Properties of Foods.

Rao, M.A. and Rizvi, S.S.H. (Eds.). Marcel Dekker Inc., NY. p.329-387.
Mudgett, R.E. 1990. Developments in microwave food processing, in Biotechnology and

Food Process Engineering, JET Basic Symp. Ser. Schwartzberg, H.E. and Rao,
M.A. (Eds.). Marcel Dekker Inc., NY.
Namiki, M., Hayashi, T. and Kawakishi, S. 1973. Free radicals developed in the amino-
carbonyl reaction of sugars with amino acids. Agric. Biol. Chem. 37:2935-2937.
Namiki, M. and Hayashi, T. 1981. Formation of novel free radical products in an early
stage of Maillard reaction. Prog. Food Nutri. Sci. 5:81-91.
Namiki, M. and Hayashi, T. 1983. A new mechanism of the Maillard reaction involving
sugar fragmentation and free radical formation. ACS Symp. Ser. 215:21-46.
Namiki, M. 1988. Chemistry of Maillard reactions: recent studies on the browning

reaction mechanism and the development of antioxidants and mutagens. Adv.
Food Res. 32:115-184.
117
Naven, T.J.P. and Harvey, D J . 1996. Effect of structure on the signal strength of
oligosaccharides in matrix-assisted laser desorption/ionization mass spectrometry
on time-of-flight and magnetic sector instruments. Rapid Comm. Mass Spec.
10:1361-1366.
Nelson, V. 1948. The color of evaporated milk with respect to time and temperature of
milk. J. Dairy Sci. 31:415-419.
O'Brien, J. and Morrissey, P.A. 1989. Nutritional and toxicological aspects of the
Maillard browning reaction in foods. Crit. Rev. Food Sci. Nutri. 28:211-248.
Oruna-Concha, M.J., Bakker, J. and Ames, J.M. 2002. Comparison of the volatile
components of two cultivars of potato cooked by boiling, conventional baking
and microwave baking. J. Sci. Food Agric. 82:1080-1087.
Osterdahl, B.G. and Alriksson, E. 1990. Volatile nitrosamines in microwave-cooked

bacon. Food Addit. Contam. 7:51-54.
Penner, M.H. 1994. Ultraviolet, visible, and fluorescence spectroscopy, in Introduction to

the Chemical Analysis of Foods. Nielsen, S.S. (Ed). Jones Bartlett Publ. Inc.,
London, England, p.325-340.
Petrucci, R.H. 1989. General Chemistry. Principles and Modern Applications.

MacMillian Publ., NY, NY. p.546-559.
Pikul, J., Leszezynski, D.E. and Kummerew, F.A. 1985. Oxidation products in chicken
meat after frozen storage, microwave and convection oven cooking, refrigerated
storage and reheating. Poultry Sci. 64:93-100.
Rao, C.N.R. 1975. Chromophores and transitions, in Ultra-violet and Visible

Spectroscopy. Butterworth & Co. Ltd. London, England, p.13-19.
Rao, M.N., Sreenivas, H., Swaminathan, M., Carpenter, K.J. and Morgan, C.B. 1963. The
nutritionally available lysine and methionine of heated casein-glucose mixtures. J.
Sci. Food Agric. 14:544.
Reynolds, T.H. 1965. Chemistry of non-enzymic browning II. Adv. Food Res. 14:167-
283.
Reynolds, T.H. 1959. Chemistry of non-enzymatic browning III. Effect of bi-sulfite,

phosphate, and malate on the reaction of glycine and glucose. Aust. J. Chem.
12:265-274.
Risch, S.J. 1989. Flavors for microwavable foods. Cereal Foods World 34:226.
118
Rohan, T.A. and Stewart, T. 1965. The precursors of chocolate aroma: the distribution of
free amino acids in different commercial varieties of cocoa beans. J. Food Sci.
30:416-419.
Rosen, J. and Hellenas, K-E. 2002. Analysis of acrylamide in cooked foods by liquid
chromatography tandem mass spectrometry. Analy. 127:880-882.
Sgarbieri, V.C., Amaya, J., Tanaka, M. and Chichester, C O . 1973. Nutritional

consequences of the Maillard reaction. Amino acid availability from fructose-
leucine and fructose-tryptophan in the rat. J. Nutri. 103:657-663.
Sigman-Grant, M., Bush, G. and Anantheswaran, R. 1992. Microwave heating of infant

formula: a dilemma resolved. Pediat. 90:412-415.
Singh, R.K., Lund, D.B., Buelow, F.H. 1983. Storage stability of intermediate moisture
apples: kinetics of quality change. J. Food Sci. 48:939-944.
Siuzdak, G. 1994. The emergen ve of mass spectrometry in biomedical research. Proc.

Nat. Acad. Sci. U.S.A. 91:11290-11297.
Spark, A.A. 1969. Role of amino acid in non-enzymic browning. J. Sci. Food Agric.
20:308-316.
Spingarn, N.E. and Garvie, C T . 1979. Formation of mutagens in sugar ammonia model
Systems. J. Agric. Food Chem. 27:1318-1321.
Stadler, R.H., Blank, I., Varga, N., Robert, F., Hau, J., Guy, P.A., Robert, M-C and
Riediker, S. 2002. Acrylamide from Maillard reaction products. Nature 419:449.
Stadtman, F.H., Chichester, C O . and Rooney, CS. 1952. Carbon dioxide production in
the browning reaction. J. Am. Chem. Soc. 74:3194-3196.
Stahl, B., Steup, M., Karas, M., Hillenkamp, F. 1991. Analysis of netural
oligosaccharides by matrix-assisted laser desorption/ionization mass
spectrometry. Analyt. Chem. 63:1463-1466.
Stahl, B., Thurl, S., Zeng, J., Hillenkamp, F., Steup, M. and Sawatzki, G. 1994. Oligo-
saccharides from human milk as revealed by matrix assisted laser
desorption/ionization mass spectrometry. Anal. Biochem. 223:218-226.
Stuerga, D.A.C. and Gaillard, P. 1996a. Microwave athermal effects in chemistry: a

myth's autopsy. Part I: historical background and fundamentals of wave-matter
interaction. J. Microwave Power Electromag. Energy 31:87-100.
119
Stuerga, D.A.C. and Gaillard, P. 1996b. Microwave athermal effects in chemistry: a

myth's autopsy. Part II: orienting effects and thermodynamic consequences of
electric field. J. Microwave Power Electromag. Energy 31:101-113.
Stumbo, CR. 1973. Thermobacteriology in Food Processing. 2 ed. Academic Press,

nd
NY, NY.
Sun, E., Datta, A. and Lobo, S. 1995. Composition-based prediction of dielectric

properties of foods. Int. Microwave Power Institute. 30:205-212.
Supplee, G.C 1926. Humidity equilibria of milk powders. J. Dairy Sci. 9:50-61.
Tareke, E., Rydberg, P., Karlsson, P., Eriksson, S. and Tornqvist, M. 2002. Analysis of
acrylamide, a carcinogen formed in heated foodstuffs. J. Agric. Food Chem.
50:4998-5006.
Taylor, S.L. and Bush, R.K. 1986. Sulfites as food ingredients. Food Technol. 40(6):47-
52.
Toma, R.B., Augustin, J., Orr, P.H., True, R.H., Hogan, J.M. and Shaw, R.L. 1978.
Changes in nutrient composition of potatoes during home preparation. I.
Proximate composition. Am. Potato J. 55:639-645.
Umbach, S.L., Davis, E.A., Gordon, J. and Callaghan, P.T. 1992. Water self-diffusion
coefficients and dielectric properties determined for starch-gluten-water mixtures
heated by microwave and by conventional methods. Cereal Chem. 69:637-642.
Underwood, J.C, Lento, H.G.Jr. and Willits, C O . 1959. Browning of sugar solutions.
Effect of pH on color produced in dilute glucose solutions containing amino acids
with the amino group in different positions in the molecule. Food Res. 24:181-
184.
Vagnarelli, P., Sario, A.D., Cuzzoni, M.T., Mazza, P. and Carli, L.D. 1991. Cytotoxicity
and clastogenic activity of ribose-lysine browning model system. J. Agric. Food
Chem. 39:2237-2239.
Vernin, G., Debrauwer, L., Vernin, G.M.F., Zamkotsian, R-M., Metzger, J., Larice, J.L.
and Parkanyi, C. 1992. Heterocycles by thermal degradation of Amadori
intermediates, in Off Flavors in Foods and Beverages. Charlambous, G. (Ed.).
Elsevier, Amsterdam, Netherlands, p.567.
Von Hippel, A.R. 1954a. Dielectrics and Waves. MIT Press, Cambridge, MA.
Von Hippel, A.R. 1954b. Dielectric Materials and Applications. MIT Press, Cambridge,
MA.
120
Waletzko, P. and Labuza, T.P. 1976. Accelerated shelf-life testing of an intermediate

moisture food in air and in an oxygen-free atmosphere. J. Food Sci. 41:1338-
1344.
Wang, J., Sporns, P. and Low, N.H. 1999. Analysis of food oligosaccharides using
MALDI-MS: quantification of fructooligosaccharides. J. Agric. Food Ghem.
47:1549-1557.
Wang, C.J., Tseng, T.H., Yen, G.C., Shiow, S.J. and Lin, J.K. 1987. Cytotoxic effect of
nonenzymatic products from heated lysine and monosaccharides on rat C6 glioma
cell. J. Chin. Agric. Chem. Soc. 25:204-212.
Wang, J. and Sporns, P. 2000. MALDI-TOF MS analysis of food flavonol glycosides.

J. Agric. Food Chem. 48:1657-1662.
Warmbier, H.C., Schnickels, R.A., Labuza, T.P. 1976. Effect of glycerol on

nonenzymatic browning in a solid intermediate moisture model food system.
J.Food Sci. 41:528-531.
Wedzicha, B.L. and Kaputo, M.T. 1987. Reaction of melanoidins with sulfur dioxide:
stoicheometry of the reaction. Int. J. Food Sci. Technol. 22:643-651.
Whorton, C. and Reineccius, G.A. 1989. Flavor development in a microwaved versus

a conventionally baked cake. ACS Symp. Ser. 409:526-532.
Wijewickreme, A.N. 1997. Metal chelating and oxidative behaviour of Maillard reaction
products extracted from model and food systems. Dissert. Abst. Int., -B. 58:2778-
2779.
Wijewickreme, A.N., Kitts, D.D. and Durance, T.D. 1997. Reaction conditions influence
the elementary composition and metal chelating affinity of nondialyzable model
Maillard reaction products. J. Agric. Food Chem. 45:4577- 4583.
Wolf, J.C, Thompson, D.R., Reineccius, G.A. 1977. Initial losses of available lysine in
model systems. J. Food Sci. 42:1540-1544.
Wolfrom, M.L. and Rooney, CS. 1953. Chemical interactions of amino compounds and
sugars VIII. Influence of water. J. Am. Chem. Soc. 75:5435-5436.
Wolfrom, M.L., Kashimura, N. and Horton, D. 1974. Factors affecting the Maillard
browning reaction between sugars and amino acids. Studies on the Nonenzymic
browning of dehydrated orange juice. J. Agric. Food Chem. 22:796-800.
121
Wong, J.W., Yeo, H.C.H. and Shibamoto, T. 1991. Determination of malondialdehyde

and formaldehyde formed from fatty acid ethyl esters upon microwave and
thermal heating. J. Agric. Food Chem. 39:2260-2262.
Yaghmaee, P. and Durance, T.D. 2002. Predictive equations for dielectric properties of
NaCl, D-sorbitol and sucrose solutions and surimi at 2450 MHz. J. Food Sci.
67:2207-2211.
Yaylayan, V.A. 1997. Classification of the Maillard reaction: a conceptual approach.

Trends Food Sci. Technol. 8(1): 13-18.
Yaylayan, V.A. and Forage, N. 1992. A kinetic model for the reaction of tryptophan with
glucose and mannose - the role of diglycation in the Maillard reaction. Food
Chem. 44:201-208.
Yaylayan, V.A. and Huyghues-Despointes, A. 1994. Chemistry of Amadori

rearrangement products: analysis, synthesis, kinetics, reactions and spectroscopic
properties. Critical Rev. Food Sci. Nutri. 34:329-369.
Yeboah, F.K. and Yaylayan, V.A. 2001. Analysis of glycated proteins by mass
Spectrometric techniques: qualitative and quantitative aspects. Nahrung 45:164-
171.
Yeo, H. and Shibamoto, T. 1991. Chemical comparison of flavors in microwaved and

conventionally heated foods. Trends Food Sci. Technol. 39:329-332.
Yu, T-H., Wu, C-M., Ho, C-T. 1993. Volatile compounds of deep-oil fried, microwave-
heated, and oven-baked garlic slices. J. Agric. Food Chem. 41:800-805.
Yuferov, V.P., Froncsz, W., Kharitonenkov, I.G. and Kalmanson, A.E. 1970. Electron
paramagnetic resonance study of free radical products of the reaction of ninhydrin
with amino acids, peptides, and protein. Biochim. Biophys. Acta. 200:160-167.
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Maillard Browning Reaction

of Glucose-Lysine Model System

Biwen Kristi Xing

A THESIS SUBMITTED IN PARTIAL FULFILLMENT

THE FACULTY OF GRADUATE STUDIES

We accept this thesis as conforming

THE UNIVERSITY OF BRITISH COLUMBIA

© Biwen Kristi Xing, 2002

The University of British Columbia

The impact of microwave heating on the formation of Maillard reaction products

heating, using glucose-lysine as a model system. Two different microwave treatments

desired temperature. Experiments were carried out at controlled temperatures of 63°C up

to 120°C, and Maillard reaction rate, UV/Visible spectral characteristics of MRPs,

dielectric properties of MRPs, matrix-assisted laser desorption/ionization time-of-flight

mass spectrometry (MALDI-TOF MS) analysis of MRPs, Fourier transform infrared

Comparison with control samples treated by conventional heating showed a significant

or different concentrations of compounds were produced during vacuum microwave

microwave heating. Ethos Synth microwave heating produced similar MRPs as

conventional heating. MALDI-TOF MS analysis of 81°C MRPs and FTIR analysis of

LIST OF FIGURES vii

LIST OF SYMBOLS AND ABBREVIATIONS xi

2.1 Discovery of Maillard Browning Reaction 3

2.7.5 Nutritional and Toxicological Effects of

4 MATERIALS AND METHODS 39

5 RESULTS AND DISCUSSION 54

Table 5.1 Temperature dependence of the rate constant and activation

Table 5.2 Relative intensities (height) of peaks obtained from the

Table 5.3 Functional groups and vibrational modes obtained from

Figure 2.1 Review of Maillard reaction pathway 5

Figure 2.2 Initial stage of the Maillard reaction 7

Figure 2.3 Condensation of carbonyl compounds with amino compounds 7

Figure 2.4 A) Formation of sugar fragmentary products. B) Two possible

Figure 2.5 Two possible pathways of Amadori compound degradation.

Figure 2.6 Deoxyosone degradation via Strecker degradation Pathway III 11

Figure 4.1 The schematic of a vacuum microwave apparatus 40

Figure 4.2 Schematic diagram of the Ethos Synth microwave system 41

Figure 5.1 Log of Maillard browning rates of 0.05 M glucose and

Figure 5.2 A shorter time period of log of Maillard browning rates of

Figure 5.3 Dependence of D value on temperature. Plot of log D

Figure 5.4 Dependence of rate constant, k, on temperature. Plot of

Figure 5.5 UV/vis spectral patterns comparison (220-600 nm) of Maillard

Figure 5.6 UV/vis spectral patterns comparison (220-600 nm) of Maillard

Figure 5.7 UV/vis spectral patterns comparison (220-600 nm) of Maillard

Figure 5.8 UV/vis spectral patterns comparison (220-600 nm) of Maillard

Figure 5.9 UV/vis spectral patterns comparison (220-600 nm) of Maillard

Figure 5.10 UV/vis spectral patterns comparison (220-600 nm) of Maillard

Figure 5.11 UV/vis spectral patterns comparison (220-600 nm) of Maillard

Figure 5.12 UV/vis spectral patterns comparison (220-600 nm) of Maillard

Figure 5.13 UV/vis spectral patterns comparison (220-600 nm) of Maillard

Figure 5.14 Dielectric constant of collected Maillard browning samples

Figure 5.15 Loss factor of collected Maillard browning samples

Figure 5.16 Dielectric constant of collected Maillard browning samples

Figure 5.17 Loss factor of collected Maillard browning samples

Figure 5.18 Dielectric constant of collected Maillard browning samples

Figure 5.19 Loss factor of collected Maillard browning samples

Figure 5.21 MALDI-TOF mass spectrum of vacuum microwave sample (81°C) 91

Figure 5.22 MALDI-TOF mass spectrum (high-mass expansion)

Figure 5.23 MALDI-TOF mass spectrum (high-mass expansion)

Figure 5.24 FTIR overlay spectra of MRPs, from a glucose-lysine

Figure 5.25 FTIR stack spectra of MRPs, from a glucose-lysine

non-treated mixture of glucose-lysine model 101

Figure 8.1 MALDI-TOF CCA matrix background 122

Figure 8.2 FTIR original spectrum of D-glucose 123

Figure 8.3 FTIR original spectrum of L-lysine 124