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An Investigation of Effects of Microwave Treatment on the Structure,Enzymatic Hydrolysis, and Nutraceutical Properties of β-Lactoglobulin

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An Investigation of Effects
of Microwave Treatment on the Structure, Enzymatic Hydrolysis, and
Nutraceutical Properties of -Lactoglobulin
By
Ahmed I. Gomaa
Department of Food Science and Agricultural Chemistry
Macdonald Campus of McGill University
December, 2010
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment
of the requirements of the degree of Doctor of Philosophy
©Ahmed Gomaa, 2010
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ABSTRACT
Whey proteins are used extensively by the food industry in such products as
infant formulas and dietetic and health foods owing to the high nutritional value and
useful functional properties of these milk-derived proteins. In addition, in recent years,
the nutraceutical properties of native or predigested whey proteins have become the
focus of much research. Both the functional and nutraceutical properties of whey
proteins can be enhanced by exposing them to various non-denaturing physicochemical
conditions that modify their native structure. It is within this context that the present
investigation of the effects of microwave treatments (MW) on the structure, degree of
enzymatic hydrolysis, and nutraceutical properties of �-lactoglobulin (�-Lg), the
predominant whey protein in cow’s milk, was undertaken.
Microwave treatment of �-Lg in D2O solution under various conditions was
monitored by Fourier transform mid infrared (mid-FTIR) spectroscopy. At sub-ambient
and ambient temperatures, no microwave-induced changes in the conformation of the
protein were detected. Microwave heating of the �-Lg solutions to temperatures in the
range of 40-60 ºC resulted in a marked increase in the rate of hydrogen-deuterium (H-D)
exchange by comparison with conventional heating (CH) at the same temperature. At
heating temperatures in the range of 70-90 ºC, the microwave-heated solutions exhibited
more extensive protein aggregation than conventionally heated solutions. Application of
two-dimensional (2D) correlation analysis to the Fourier self-deconvolved FTIR spectra
recorded as a function of number of cycles of microwave or conventional heating
revealed that the unfolding pathway of β-Lg was different in these two temperature
ranges but was not altered by microwave heating as compared to conventional heating.
Kinetic analysis of the FTIR data revealed that the accelerated rate of protein unfolding
observed in microwave-heated samples is attributable to a lower energy of activation and
supported the existence of non-thermal effects of microwaves. Circular dichroism (CD),
fluorescence spectroscopy, and 2D 1H NMR spectroscopy were used to confirm and
complement the results obtained by FTIR spectroscopy.
i
The effects of microwave and conventional heat treatments at three temperatures
(40, 60, and 90 oC) on the enzymatic hydrolysis of β-Lg and on the peptide profiles of the
hydrolysates were also investigated. The degree of hydrolysis of β-Lg by pepsin, trypsin
and chymotrypsin as well as in a two-stage hydrolysis with pepsin followed by trypsin
and
chymotrypsin,
simulating
gastrointestinal
conditions,
was
determined.
Microwave-treated samples exhibited more extensive enzymatic hydrolysis than
conventionally heated samples under all hydrolysis conditions, with the greatest degree
of hydrolysis being found following microwave treatment at 60 °C, and hydrolysates of
microwave-treated β-Lg exhibited both enhanced angiotensin-converting enzyme (ACE)
inhibition and enhanced antioxidant activity. LC-ESI-MS and MS/MS in conjunction
with the use of advanced bioinformatics software were employed in the identification of
the peptide profiles of the hydrolysates and revealed the presence of unique peptides in
hydrolysates of microwave-treated samples, suggesting that the microwave treatment
may have exposed new cleavage sites within the interior of the protein. Overall, the
findings of these studies indicate that microwave heat treatments may be an attractive
approach to enhance the nutraceutical properties of whey and other proteins.
ii
RÉSUMÉ
Les protéines laitières sont utilisées en grande partie par l'industrie alimentaire
dans des produits tels que les préparations pour nourrissons et des aliments diététiques et
de santé en raison de la valeur nutritive élevée et des propriétés fonctionnelles utiles de
ces protéines laitières. Les propriétés nutraceutiques des protéines laitières indigènes ou
prédigérées ont fait ces dernières années l'objet de beaucoup de recherches. Les
propriétés fonctionnelles et nutraceutiques des protéines laitières peuvent être améliorées
en les soumettant à diverses conditions physico-chimiques de non-dénaturation qui
modifient leur structure native. Dans cette recherche portant sur les effets de traitements
micro-ondes de la structures, le degré d'hydrolyse enzymatique, et les propriétés
nutraceutiques de la �-lactoglobuline (�-Lg), la protéine laitière primordiale dans le lait
de vache, a été entrepris.
L´irradiation micro-ondes de �-Lg dans la solution D2O sous des conditions
différentes a été suivie par la spectroscopie infrarouge à transformée de Fourier (IRTF).
À températures ambiantes et sous-ambiantes, aucun changement induit par les
micro-ondes n'a été détecté dans la conformation de la protéine. Le chauffage à
micro-ondes des solutions �-Lg à des températures de l'ordre de 40 à 60 ºC a entraîné une
augmentation prononcée du taux d´échange d'hydrogène-deutérium (H-D) par rapport au
chauffage classique à la même température. Aux températures de chauffage de l'ordre de
70 à 90 ºC, les solutions chauffées à micro-ondes ont montré plus d´agrégation des
protéines que les solutions chauffées classiquement.
L´application de l´analyse de corrélation à deux dimensions (2D) aux spectres d’IRTF
auto-déconvolués enregistrés comme une fonction du nombre de cycles de chauffage à
micro-ondes ou de chauffage classique a révélé que la voie de déroulement de la β-lg
était différente dans ces deux intervalles de température. Cependant cette voie n'a pas été
altérée par le chauffage à micro-ondes par rapport au chauffage classique. L'analyse
cinétique des données IRTF a révélé que l'accélération du taux de déroulement des
protéines observée dans les échantillons chauffés à micro-ondes est attribuable à une
énergie d'activation faible et a soutenu l'existence d'effets non thermiques des
iii
micro-ondes. Le dichroïsme circulaire (CD), la spectroscopie de fluorescence et la
spectroscopie RMN 1H 2D ont été utilisés pour confirmer les résultats obtenus par la
spectroscopie IRTF.
Les effets de traitements à micro-ondes et de chauffage classique à trois
températures (40, 60 et 90 oC) sur l'hydrolyse enzymatique de β-Lg et sur les profils
peptidiques des hydrolysats ont également été étudiés. Le degré d'hydrolyse de β-Lg par
la pepsine, la trypsine et la chymotrypsine, ainsi que dans une hydrolyse en deux étapes
avec la pepsine suivie par la trypsine et la chymotrypsine, en simulant les conditions
gastro-intestinales, a été résolu. Les échantillons traités à micro-ondes présentaient une
hydrolyse enzymatique plus étendue que les échantillons traités au chauffage classique
sous toutes les conditions d'hydrolyse; le plus grand degré d'hydrolyse est trouvé lors du
traitement à micro-ondes à 60° C, et les hydrolysats de la β-Lg traités à micro-ondes
présentaient à la fois une amélioration de l'inhibition de la conversion angiotensine de
l'enzyme de (ACE) et celle de l'activité antioxydant. LC-ESI-MS et MS / MS, en lien
avec l'utilisation de logiciels de pointe de bioinformatique ont été utilisés pour identifier
les profils peptidiques des hydrolysats et ont révélé la présence des peptides uniques dans
les hydrolysats des échantillons traités à micro-ondes, ce qui suggère que le traitement à
micro-ondes peuvent avoir exposé des nouvelles sites de clivage à l'intérieur de la
protéine. En conclusion, les traitements thermiques à micro-ondes peuvent être une
approche intéressante pour améliorer les propriétés nutraceutiques des protéines laitières
et d'autres protéines.
iv
ACKNOWLEDGEMENTS
First of all I express my sincere thanks to my supervisor Dr. Ashraf Ismail for his
professional guidelines, unrelenting support, and brilliant input throughout my research
project. During the course of my doctoral studies, he was more than helpful to me
whenever I needed it. His sagacious counseling always bore me nice fruitions.
I also owe a debt of gratitude to Dr. Jacqueline Sedman for her excellent editorial
help in my dissertation write-up and timely help in paper presentations. I would like to
extend my thanks to Dr. Joanne Turnbull for giving me access to CD and fluorescence
facilities in addition to her valuable help in providing access to the software for data
analysis.
At this moment of extreme happiness, I should not forget the mammoth help of
the Egyptian government in the form of financial support in the form of a postgraduate
scholarship.
I’d also like to thank all of my lab mates for their friendship and for making my
hours in the lab more enjoyable in pursuance of my whole doctoral studies.
Finally, very sincere and special thanks to my family in Egypt and my wife Afaf,
son Mohammed and daughter Mariam without whose co-operation I could not have
accomplished this task.
v
CONTRIBUTION OF AUTHORS
Chapter 3-6 are the text of papers being prepared for publication. The author of
the thesis is responsible for developing the ideas and concepts presented and for the
analysis of all the data in the study. Dr. Ashraf A. Ismail, the thesis supervisor, provided
support and guidance throughout the course of this research. Dr. Jacqueline Sedman
provided valuable advice and editing for this research.
Chapter 3: Investigation of the effect of microwave treatment on the structure and
unfolding pathways of β-lactoglobulin by FTIR spectroscopy with the application of
2-dimensional correlation analysis.
Chapter 4: Investigation of the effect of microwave heating treatment on the structure of
β-lactoglobulin by circular dichroism and fluorescence spectroscopy.
Chapter 5: Study of the effect of microwave heating on the enzymatic hydrolysis of
β-lactoglobulin.
Chapter 6: Examination of the nutraceutical properties of whey protein hydrolysates
subjected to conventional and microwave heating.
vi
TABLE OF CONTENTS
ABSTRACT .................................................................................................. i
RÉSUMÉ .................................................................................................... iii
ACKNOWLEDGEMENTS.......................................................................... v
CONTRIBUTION OF AUTHORS ............................................................. vi
TABLE OF FIGURES ................................................................................ x
LIST OF TABLES .................................................................................... xv
CHAPTER 1: GENERAL INTRODUCTION ............................................ 1
CHAPTER 2: LITERATURE REVIEW .................................................... 4
1. Structure and biological functions of whey proteins ............................................ 4
1.1. Structure and biological activity of individual whey proteins ....................... 6
1.1.1. β-Lactoglobulin ............................................................................................ 6
1.1.2. α-Lactalbumin ............................................................................................ 10
1.1.3. Immunoglobulins ....................................................................................... 12
1.1.4. Bovine serum albumin ............................................................................... 13
1.1.5. Proteose peptones ....................................................................................... 14
1.1.6. Bioactive proteins ....................................................................................... 14
1.2. The benefits of dietary whey protein on the immune response and health. 15
1.2.1. Anti-cancer effect of WPC ......................................................................... 15
1.2.2. Effects on immune associated factors ........................................................ 16
1.2.3. Angiotensin I-converting enzyme inhibition activity ................................ 18
1.2.4. Antioxidant activity.................................................................................... 20
1.2.5. Health benefits of whey proteins for active people ................................... 21
2. Effects of Microwave treatment on proteins....................................................... 22
2.1. Thermal and non-thermal effects of microwaves ........................................ 22
2.1.1. Thermal effects (microwave heating) ........................................................ 23
2.1.2. Non-thermal effects of microwaves ........................................................... 24
2.2. Microwave Effects on Protein ...................................................................... 26
2.2.1. Microwave Effects on Protein Structure ................................................... 26
vii
2.2.2. Effect of microwave on enzymes ................................................................ 29
Chapter 3. An investigation of the effect of microwave treatment on the
structure and unfolding pathways of β-lactoglobulin using FTIR
spectroscopy with the application of two-dimensional correlation
spectroscopy (2D-COS). ............................................................................ 33
INTRODUCTION ................................................................................................... 33
MATERIALS AND METHODS............................................................................. 37
RESULTS AND DISCUSSION............................................................................... 40
Chapter 4: Investigation of the effect of microwave treatment on
the structure of
-lactoglobulin by circular dichroism and
fluorescence spectroscopy...................................................................... 72
INTRODUCTION ................................................................................................... 72
MATERIALS AND METHODS............................................................................. 75
RESULTS AND DISCUSSION............................................................................... 77
CD in the far-UV region ...................................................................................... 77
CD in the near-UV region ................................................................................... 82
Fluorescence ......................................................................................................... 89
Chapter 5: Study of the effect of microwave treatment on the
enzymatic hydrolysis of
lactoglobulin ............................................ 100
INTRODUCTION ................................................................................................. 100
MATERIALS AND METHODS........................................................................... 102
RESULTS AND DISCUSSION............................................................................. 106
Hydrolysis of β-Lg ............................................................................................. 106
Kinetic parameters ............................................................................................ 115
Chapter 6: Examination of the nutraceutical properties of hydrolysates
derived from conventionally- and microwave- treated β-lactoglobulin
solutions .................................................................................................. 123
INTRODUCTION ................................................................................................. 123
MATERIALS AND METHODS........................................................................... 127
viii
RESULTS AND DISCUSSION............................................................................. 132
ACE inhibition ................................................................................................... 132
Antioxidant activity ........................................................................................... 137
Peptide identification ......................................................................................... 142
SUMMARY AND CONCLUDING REMARKS ...................................... 152
REFERENCES ....................................................................................... 155
ix
TABLE OF FIGURES
Figure 2.1: The crystal structure of β-lactoglobulin (Brownlow et al., 1997). ................... 9
Figure 2.2: The crystal structure of α-lactalbumin (Mueller-Dieckmann et al., 2007) ..... 11
Figure 2.3: Proposed mechanism of microwave heating (source: Buffler, 1993). ........... 24
Figure 3.1: The crystal structure of a single subunit of bovine β-Lg (Brownlow et al.,
1997). ............................................................................................................ 41
Figure 3.2: The amide I′ region of the FTIR spectrum of β-lactoglobulin in D2O (5% w/v)
at 25˚C after Fourier self-deconvolution (bandwidth = 22 cm-1 and k factor of
2.4)................................................................................................................ 42
Figure 3.3: The amide I region of FTIR spectra of β-Lg at different pH values. ............. 43
Figure 3.4: Overlaid FTIR spectra in the amide I′ absorption region of β-Lg solutions in
D2O subjected to 1-10 cycles of microwave irradiation at 40oC. .................... 47
Figure 3.5: Bar graphs illustrating the decrease in the peak height of the 1692 cm-1 band
in the FTIR spectrum of β-Lg in D2O at pH 2, 4, 7 and 9 as a function of the
number of cycles of microwave irradiation (blue) and conventional heating
(red) at 40°C. ................................................................................................. 48
Figure 3.6: Bar graphs illustrating the decrease in the peak height of the 1692 cm-1 band
in the FTIR spectrum of β-Lg n 0.5 M NaCl and 2 M NaCl in D2O at 40°C as a
function of the number of cycles of microwave irradiation (blue) and
conventional heating (red) at 40oC. ................................................................ 49
Figure 3.7: Overlaid FTIR spectra in the amide I’ absorption region of β-Lg solutions
subjected to microwave irradiation and conventional heating at different
temperatures ranging from 40oC to 60oC........................................................ 54
Figure 3.8: Overlaid FTIR spectra in the amide I’ absorption region of β-Lg solutions
subjected to microwave irradiation and conventional heating at different
temperatures ranging from 70oC to 90oC........................................................ 55
Figure 3.9: Plot of the decrease in the peak height of the band at 1692 cm-1 in the FTIR
spec:tra of β-Lg in D2O as a function of the number of cycles of microwave
irradiation or conventional heating at the indicated temperatures. .................. 56
x
Figure 3.10: Bar graphs illustrating the decrease in the peak height of the 1692 cm-1 band
in the FTIR spectrum of β-Lg in D2O (pH ~6) as a function of the number of
cycles of microwave irradiation or conventional heating. at 40°C and 50°C. .. 57
Figure 3.11: Plot of the change in the peak height of the band at 1612 cm-1 in the FTIR
spectra of β-Lg in D2O as a function of the number of cycles of microwave
irradiation or conventional heating at temperatures ranging from 60 to 90°C. 58
Figure 3.12: The synchronous and asynchronous 2D IR spectra generated from the
difference spectra obtained during microwave irradiation treatment at 50 oC. . 60
Figure 3.13: The synchronous and asynchronous 2D IR spectra generated from the
difference spectra obtained during conventional heating at 50oC.................... 61
Figure 3.14: The synchronous and asynchronous 2D IR spectra generated from the
difference spectra obtained during microwave irradiation treatment at 90 oC. . 62
Figure 3.15: The synchronous and asynchronous 2D IR spectra generated from the
difference spectra obtained during conventional heating at 90oC.................... 63
Figure 3.16: Arrhenius plot for 5% β-lactoglobulin in D2O heated at 40, 45, 50 and 55°C
for 10 conventional heating and microwave cycles analyzed in triplicate by
FTIR spectroscopy. Error bars represent one standard deviation. ................... 67
Figure 4.1: The 3D structure and amino acid sequence of bovine β-Lg. ......................... 74
Figure 4.2: Far-UV CD spectra (200-250 nm) of β-Lg after (A) conventional heating and
(B) microwave treatment at 40 and 50 oC. ...................................................... 79
Figure 4.3: Far-UV CD spectra (200-250 nm) of β-Lg after (A) conventional heating and
(B) microwave treatment at 60 and 70 oC. ...................................................... 80
Figure 4.4:Far-UV CD spectra (200-250 nm) of β-Lg after (A) conventional heating and
(B) microwave treatment at 80 and 90oC. ...................................................... 81
Figure 4.5: α-Helix content estimated from far-UV spectra of β-Lg subjected to
microwave irradiation and conventional heating at various temperatures ....... 84
Figure 4.6: �-Sheet content estimated from far-UV spectra of β-Lg subjected to
microwave irradiation and conventional heating at various temperatures ....... 85
xi
Figure 4.7: Comparison of near-UV CD spectra of heat-treated and microwave-treated
β-Lg (treated at temperatures of 40 and 50ºC) with the spectrum of native
β-Lg. ............................................................................................................. 86
Figure 4.8: Comparison of near-UV CD spectra of heat-treated and microwave-treated
β-Lg (treated at temperatures of 60 and 70ºC) with the spectrum of native
β-Lg. ............................................................................................................. 87
Figure 4.9: Comparison of near-UV CD spectra of heat-treated and microwave-treated
β-Lg (treated at temperatures of 80 and 90ºC) with the spectrum of native
β-Lg. ............................................................................................................. 88
Figure 4.10: Fluorescence intensity of Trp residues for β-Lg samples subjected to
microwave irradiation and conventional heating at 40, 60 and 90ºC. The upper
trace in each panel shows the fluorescence intensity measured for native β-Lg.
...................................................................................................................... 90
Figure 4.11: TOCSY spectrum of untreated β-Lg in 100% D2O (pH 2, 25°C) at 500
MHz. ............................................................................................................. 94
Figure 4.12:(Hn, Hα) cross peaks from TOCSY spectra of β-Lg, (pH 2.0, 25°C) after
microwave treatment at 50°C......................................................................... 96
Figure 4. 13: (Hn, Hα) cross peaks from TOCSY spectra of β-Lg, (pH 2.0, 25°C)
showing the non-exchanged resonances that persist through conventional heat
treatment (light to dark color) at 50°C (a) and 60°C (b). ................................ 97
Figure 5.1: Degree of hydrolysis determined by the o-phthaldialdehyde method in
enzymatic hydrolysates from β-Lg. Error bars represent SE of triplicate
experiments. See Materials and Methods for designations of sample
pretreatments. .............................................................................................. 108
Figure 5.2: Degree of hydrolysis determined by the o-phthaldialdehyde assay method as
a function of time for the enzymatic hydrolysis of β-Lg by simulated gastric
digestion. ..................................................................................................... 110
Figure 5.3: The amino acid sequence of β-Lg and its secondary structure (Ragona et al.,
1999). .......................................................................................................... 113
xii
Figure 5.4: Amino acid sequence of β-lactoglobulin A and B. Arrows indicate theoretical
cleavage points for tryptic digestion. Shaded portions indicate residues
involved in �-sheet secondary structure, and the single open portion, residues
130-140, indicates the lone �-helix. (Turula et al., 1997) ............................. 114
Figure 5.5: Lineweaver-Burk double-reciprocal plots for hydrolysis of β-lactoglobulin
by trypsin at 40ºC. ....................................................................................... 118
Figure 5.6: Lineweaver-Burk double-reciprocal plots for hydrolysis of β-lactoglobulin
by trypsin at 60 and 90 ºC . .......................................................................... 119
Figure 5.7: Lineweaver-Burk double-reciprocal plots for hydrolysis of β-lactoglobulin
by α-chymotrypsin....................................................................................... 120
Figure 5.8: Lineweaver-Burk double-reciprocal plots for hydrolysis of β-lactoglobulin
by α-chymotrypsin at 60 and 90 ºC. ............................................................. 121
Figure 6.1: IC50 values (mg mL-1) for ACE inhibition by β-Lg hydrolysates obtained by
pepsin and trypsin hydrolysis of untreated, microwave-treated, and
conventionally heated β-Lg; within each panel, treatments with the same letter
have no significant differences (P > 0.05). ................................................... 133
Figure 6.2: IC50 values (mg mL-1) for ACE inhibition by β-Lg hydrolysates obtained by
chymotrypsin
and
two-stage
enzymatic
hydrolysis
of
untreated,
microwave-treated, and conventionally heated β-Lg; within each panel,
treatments with the same letter have no significant differences (P > 0.05). ... 134
Figure 6.3: Antioxidant activity of the �-Lg hydrolysates obtained by pepsin and trypsin
hydrolysis of untreated, microwave-treated, and conventionally heated β-Lg,
determined by DPPH assay; within each panel, treatments with the same letter
have no significant differences (P > 0.05). ................................................... 139
Figure 6.4: (A) UV-chromatogram of the MW60 tryptic β-Lg hydrolysate (B) Mass
spectrum of the selected chromatographic peak in (A). (C) MS/MS spectrum
of the doubly charged ion m/z 838.5. Following sequence interpretation and
database searching, the MS/MS spectrum was matched to β-Lg f(142–148).143
Figure 6.5: The 3D structure and amino acid sequence of bovine β-Lg. ....................... 146
xiii
Figure 6.6: Secondary structure of β-Lg: (a) Selected peaks in Table 6.6 are highlighted
in yellow; (b) sequences of peptides unique to microwave treatment obtained
by MSMS analysis are highlighted in gray. .................................................. 149
xiv
LIST OF TABLES
Table 2.1: Composition and biological functions of whey proteins in bovine milk (Sadler,
R., 1992). ........................................................................................................ 5
Table 3.1: The sequence of unfolding events upon microwave irradiation or conventional
heating of β-Lg at 50ºC. ................................................................................ 64
Table 3.2: The sequence of unfolding events upon microwave irradiation of β-Lg at 90
ºC. ................................................................................................................. 65
Table 3.3: The sequence of unfolding events upon conventional heating of β-Lg at 90ºC.
...................................................................................................................... 65
Table 3.4: Kinetic parameters for thermal unfolding of β-Lg (pH 6.5) under
non-denaturing conditions ............................................................................. 68
Table 4.1: β-Sheet and α-helix contents estimated from far-UV spectra of β-Lg subjected
to microwave irradiation and conventional heating at various temperaturesa .. 84
Table 4.2: Peak assignments of TOCSY spectrum of untreated β-Lg in D2O (pH 2, 25°C)
...................................................................................................................... 95
Table 5.1: Degree of hydrolysis in enzymatic hydrolysates from β-Lg, as determined by
the o-phthaldialdehyde method. ................................................................... 107
Table 5.2: Kinetic parametersa for trypsin hydrolysis of native β-lactoglobulin and of
β-lactoglobulin samples that had been subjected to conventional and
microwave heating pretreatments ................................................................ 117
Table 6.1: Overview of identified bioactive peptides derived from milk proteins
(Pihlanto-Leppala, 2001) ............................................................................. 124
Table 6.2: IC50 values for ACE inhibition by β-Lg hydrolysates obtained by enzymatic
hydrolysis of untreated, microwave-treated and conventionally heated β-Lg
samples ....................................................................................................... 132
xv
Table 6.3: Antioxidant activity of the β-Lg hydrolysates determined by DPPH assay .. 138
Table 6.4: Peptides identified by RP-HPLC–MS/MS in β-Lg chymotrypsin hydrolysates
.................................................................................................................... 147
Table 6.5: Peptides identified by RP-HPLC–MS/MS in β-Lg trypsin hydrolysates ...... 147
Table 6.6: Peptides identified by RP-HPLC–MS/MS in β-Lg hydrolysates from twostage enzymatic hydrolysis .......................................................................... 148
xvi
CHAPTER 1: GENERAL INTRODUCTION
Bovine milk provides nutrition, promotes growth, and protects the immune
system of the young calf. Since prehistoric times, bovine milk has been consumed
by humans either as fluid milk or in dairy products. Indeed, in the last century, the
production of milk in western countries far exceeded overall consumer demand.
The surplus bovine milk was utilized as a source of functional and nutritional
ingredients for new food products. The worldwide production of skim milk
powder is estimated at 2.3 million tons (Riedel, 1994) and that of whey solids is
estimated at 7 million tons (ZMP Europamarkt Dauermilch. 1996). Annual global
production of whey is estimated to be in excess of 100 million metric tons.
Whey protein ingredients come in different forms depending on their
composition and the preparation procedures employed in their manufacture, which
also largely determine their nutritional, sensory and functional properties (Morr &
Foegeding, 1990; Morr & Ha, 1993). For instance, new developments in
fractionation, modification, and preservation techniques of milk protein products,
including whey products, have improved their functional and nutritional
properties (Jensen, 1995, Murphy and Fox, 1991). Consequently, in recent years,
interest has grown in utilizing whey proteins in infant formula and in dietetic and
health foods, using either native or predigested proteins (De Wit, 1998; James,
1993). More recently, there is a growing trend of modulating the native structure
of whey proteins through exposure to varying non-denaturing physicochemical
conditions. Processing techniques employing mild heating or exposure to high
1
hydrostatic pressure were found to impart new functional and nutraceutical
properties (Penas et al., 2006). Microwave treatment has been claimed to induce
changes in the composition of whey proteins in whole milk, and microwave
treatment of cow’s milk appeared to modify the antigenicity properties of whey
proteins by reducing their reactivity towards specific antibodies (IgG) (Kaddouri
et al., 2006).
The use of controlled microwave heating to alter protein structure and
functionality is an area of interest in our laboratory. Along these lines, the work
outlined in this thesis aims to examine the thermal and non-thermal effects of
microwave treatment on the structure, functionality and nutraceutical properties
(anti-oxidant and anti-hypertensive effects) of whey proteins. In this work a
systematic investigation of the effect of microwave treatment on �-lactoglobulin
will be undertaken with the following global objectives:
1. To evaluate changes in protein conformation as a function of microwave
treatment (MW) by mid-FTIR, circular dichroism, fluorescence, and 1H
NMR spectroscopy.
2. To study the effect of microwave treatment on the enzymatic hydrolysis
efficiency and peptide profiles of β-Lg hydrolysates and search for unique
protein hydrolysates (compared to native and conventionally heated (CH)
proteins) and then proceed to assess the nutraceutical properties of β-Lg
hydrolysates from microwave treated β-Lg by employing ACE enzyme
inhibition assay and antioxidant activity.
Chapter 3 will deal with the investigation of the effect of microwave treatment on
the structure and unfolding pathways of β-lactoglobulin by FTIR spectroscopy with
2
the application of two-dimensional correlation spectroscopy (2D-COS). Chapter 4
will address the effect of microwave treatment on the structure and unfolding
pathways of β-Lg by circular dichroism, fluorescence, and 1H NMR spectroscopy.
The effect of microwave treatment of β-Lg on the enzymatic hydrolysis efficiency
will be investigated in Chapter 5, while in Chapter 6 the examination of the
nutraceutical properties of whey protein subjected to microwave heating will be
undertaken.
3
CHAPTER 2: LITERATURE REVIEW
1. Structure and biological functions of whey proteins
In cow’s milk, roughly 20% of the total protein content is whey proteins.
Whey proteins are obtained from the liquid left over after clotting skim milk by
rennet coagulation or acidification. After the whey is spray-dried, it consists of:
lactose (73-74%), minerals (12%) and a protein component (11-12%), which in
turn is comprised of β-lactoglobulin (~47%), α-lactalbumin (~13%), serum
albumin (~4.6%), and various immunoglobulins (~12.7%). Whey protein isolates
may contain different protein ratios, with the most common being: β-lactoglobulin
(55%), α-lactalbumin (24%), serum albumin (5%), and immunoglobulins (15%)
(Swaisgood, 1996). Other proteins in minor amounts include: partially hydrolyzed
caseins, lactoperoxidase, lysozyme, lactoferrin and lactallin (Bottomley et al.,
1990).
It is widely acknowledged that the structure of whey proteins has an
impact on their physicochemical characteristics and functional properties. Much is
known about the size, shape and amino acid composition of the major milk
proteins, and it is of interest to evaluate how these characteristics are related to
their biological functions (De Wit, 1998.). The composition and main biological
functions of the proteins in the whey fraction of milk are summarized in Table
2.1.
4
Table 2.1: Composition and biological functions of whey proteins in bovine milk
(Sadler, R., 1992).
Weight
Biological function
contribution
Whey protein
(g/L of milk)
Major
--For the calf-(Pro)vitamin A transfer
3.2
β-Lg
Lactose synthesis
1.2
α-Lac
Fatty acid transfer
0.4
BSA
Passive immunity
0.8
IgG
Bioactive
--General--
1
Bacteriostatic agents
0.2
LF1
Antibacterial agent
0.03
LP
Health indicators
0.03
Enzymes (>50)
Opioid activity
≥1
Proteose-peptones
LF = lactoferrin; LP = lactoperoxidase.
5
1.1. Structure and biological activity of individual whey proteins
1.1.1. β-Lactoglobulin
β-Lactoglobulin (β-Lg) is the major whey protein of ruminant species, and
its properties have been regularly reviewed (e.g., Tilley, 1960; Lyster, 1972;
Kinsella and Whitehead, 1989; Hambling et al., 1992; Sawyer, 2003). It is the
dominant non-casein protein in bovine milk and is found in the milk of most
ruminants but has generally been reported to be absent from human breast milk
(Bottomley et al., 1990), although some reports have suggested that minor amounts
do occur in human milk (Hambraeus & Lonnerdal, 2003).
β-Lg is heat labile and denatures at a temperature, depending on the pH, of
roughly 80°C. It is largely responsible for aggregation and gelation of whey
proteins (Boye et al., 1996). Of the ten genetic variants of bovine β-Lg identified
so far, the most abundant in North America are the A and B forms (Eigel et al.,
1984). The primary structures of these two variants differ in positions 64 and 118,
where the aspartic acid and valine of β-LgA are replaced by glycine and alanine in
β-LgB (Boye et al., 2004).
The structure
β-Lg is a relatively small protein that exists as a dimer in dilute salt
solutions at physiological pH. Each monomer consists of 162 amino acid residues
(Mr ~18,400) that fold into an 8-stranded, antiparallel β-barrel with a 3-turn αhelix on the outer surface and a ninth β-strand flanking the first strand. It is this
strand that forms a significant part of the dimer interface in the bovine proteins
(Figure 2.1). The secondary structure of β-Lg is roughly 50% β-sheet, 10-15% αhelix, and 20% turns, the remainder being random structures (Boye et al., 1996;
Qin et al., 1998). The protein has two disulfide bridges, one between residues 106
and 119, and the other between residues 60 and 160. There is also a free thiol
group at position 121 which can potentially form disulfide linkages with other
proteins or within its own protein upon denaturation (Bottomley et al., 1990).
6
Furthermore, the proximity of the thiol group to the disulfide bonds facilitates
disulfide exchange, causing β-Lg to denature easily (Barraquio et al., 1988).
β-Lg exists as a dimer at physiological pH. However, if the pH is brought
down to 4.5, the protein agglomerates, forming an octamer. If the pH is further
reduced to 3.5, then the molecule exists as a monomer and conformational
changes slowly occur (Hambling et al., 1992). If the pH is increased above pH
8.5, there is reversible dissociation of the dimer, and above pH 9.0, irreversible
dissociation and aggregation begin to occur. The latter aggregation is thought to
occur as a result of sulfhydryl (disulfide) breakdown, exposing the free thiol
groups and hydrophobic residues to solvent, leading to aggregate formation
(Bottomley et al., 1990). Also, as the pH becomes more alkaline, the free thiol
group begins to oxidize and becomes more reactive, producing intermolecular
disulfide interchanges (Hambling et al., 1992). From pH 8 to pH 11, the
secondary structure begins to unfold, specifically the β-strands and α-helices.
Above pH 11, the β-sheets also unfold and the structure becomes completely
random (Casal et al., 1988).
The eight antiparallel β-strands are associated with each other and are
assigned letters A-H (starting from the carboxyl end), with β-strands A-D forming
one sheet, and strands E-H forming a second. The two sheets form a central cavity
or calyx. Strand A bends through a right angle such that the C-terminal end forms
an antiparallel strand with H; strands D and E also form a less significant
interaction, thereby completely closing the central cavity. It is this central cavity,
the calyx, that is thought to provide a ligand-binding site. β-Lg binds a variety of
hydrophobic ligands and it has been assumed that the biological function of β-Lg
is the transportation of retinol or fatty acids (Kontopidis et al., 2004; Konuma et
al., 2007). On the outer surface of the β-barrel, between strands G and H, is the 3turn α-helix. The loops that connect the β-strands at the closed end of the calyx,
BC, DE, and FG, are generally quite short, whereas those at the open end are
significantly longer and more flexible. In particular, the EF loop acts as a gate
over the binding site. At low pH (3-5), the “gate” is in the “closed” position, and
binding is inhibited, whereas at high pH (6-8) it is open, allowing ligands to
7
penetrate into the hydrophobic binding site. The “latch” for this gate is Glu89, the
residue implicated in the Tanford transition (a conformational change of bovine
β-Lg occurring at around pH 7) that was identified originally on the basis of
optical rotatory dispersion and the accessibility of a thiol group (Sakurai and
Goto, 2006), although not identified as having an abnormally high pKa (Tanford
et al., 1959; Brownlow et al., 1997; Qin et al., 1998). The free Cys121, with its
reactive thiol group, has a pH-dependent activity that parallels that of the Tanford
transition and its involvement in the denaturation and aggregation behavior is now
firmly established (Havea et al., 2001). This residue is situated on the outer
surface of the β-barrel on strand H, under the α-helix, and quite some distance
from the EF loop. The effect of this is to mask its accessibility to solvent,
particularly at low pH. Reaction with heavy metals like Hg2+ or Cd2+ does occur at
most pH values and leads to dissociation of the dimer. Indeed, reaction of the Cys
with almost any thiol reducing reagent appears to enhance dissociation into
monomers (McKenzie and Sawyer, 1967; Hambling et al., 1992).
Many groups have examined the thermal stability of β-Lg. At neutral pH,
dimer dissociation into monomers is observed with increasing temperature
between 30 and 55°C (Bottomley et al., 1990); above 55°C, the thiol group begins
to oxidize and the molecule begins to unfold and aggregate (Bottomley et al.,
1990). The mechanism of thermal denaturation appears to be an unfolding of a βtype structure in the interior of the protein followed by the unfolding of α-helical
structures and finally by the formation of intermolecular β-sheet structures
corresponding to aggregation (Boye et al., 1996). The denaturation temperature of
β-Lg depends on the pH. At pH 6.5, denaturation occurs at 80°C, and at pH 8.0,
denaturation occurs at 60°C. Lower pH stabilizes β-Lg against heat denaturation
due to an increase in hydrogen bonding (Hambling et al., 1992) and shielding of
the thiol group. An increase in concentration of β-Lg decreases thermal
denaturation (Singh and Creamer, 1992) while an increase in salt concentration
increases the thermal stability of β-Lg (Boye et al., 1996). Besides alkaline and
thermal denaturation, β-Lg can be denatured upon addition of heavy metals (Ag,
8
Hg, Cu, etc.) and organic compounds such as urea, guanidium hydrochloride,
SDS, etc. (Hambling et al., 1992).
The thermal unfolding of bovine β-LgA in 50mM phosphate buffer at pH
3.0 was followed by nuclear magnetic resonance (NMR) hydrogen/deuterium
(H/D) exchange experiments. The exchange behavior of a large number of
backbone amide protons (HNs) was monitored at temperatures between 37°C and
80°C to determine the relative thermal stability of different elements of the native
protein structure. The H/D exchange was rapid at 37°C for the HNs on the
residues in loops and in the terminal regions of the native protein. Further H/D
exchange was promoted by heat treatment at temperatures up to 80°C and was
completed for the identifiable HNs of the various � and � structural elements in
the following order: D-E strand (55–60°C); C-D strand and α-helix (60–65°C); AB, A-I and E-F strands (65–70°C); and A-H, B-C and F-G strands (75–80°C). At
80°C, the only identifiable HN signal indicated that the G-H pair of disulfidelinked strands formed the most heat-resistant feature of the β-Lg structure. The
effect of heating to 80°C was shown to be largely reversible by subsequent D/H
exchange and sodium dodecyl sulfate polyacrylamide gel electrophoresis
(Edwards et al., 2002).
Figure 2.1: The crystal structure of β-lactoglobulin (Brownlow et al., 1997).
9
Biological activity of β-Lg
The globular structure of β-Lg is remarkably stable against acids and
proteolytic enzymes present in the stomach (Papiz et al., 1986). This stability is
relevant to the biological function of β-Lg as a carrier of retinol (a provitamin A)
from the cow to the young calf. This biological function appears to be less
important for human babies, which may explain why β-Lg does not occur in
human milk. β-Lg is a rich source of cysteine, an essential amino acid that appears
to stimulate synthesis of glutathione, an anticarcinogenic tripeptide produced by
the liver that protects against intestinal tumors (Mcintoshet al., 1995).
1.1.2. α-Lactalbumin
α-Lactalbumin is the second largest component of whey proteins. It is a
very compact, spherically shaped globulin (Barraquio et al., 1988). Although its
thermal denaturation temperature (~60°C )is less than that of β-Lg, it is
considered more heat stable due to its renaturation characteristics (Jelen, 1991).
The structure
The structure of α-lactalbumin is stabilized by four disulfide bonds and
about 10% of bovine α-lactalbumin is glycosylated (Brew et al., 1992). X-ray
crystallography has shown that the protein is structurally homologous to lysozyme
(Bottomley et al., 1990). The secondary structure has four α-helices, several
regions of 310 helices, and an antiparallel β-sheet separated by irregular β-turns
(Brew et al., 1992). Circular dichroism studies have shown that α-lactalbumin is
26% α-helix, 14% β-structure and 60% unordered structure (Figure 2.2)
(Bottomley et al., 1990). α-Lactalbumin has a strong binding site for calcium
called the “elbow” formed from a helix-turn-helix motif separated by an irregular
β-turn. This site has five aspartic acid residues that bind calcium (Brew et al.,
1992). There is also a cleft region that separates the molecule into two halves
which is responsible for hydrolytic activity in lysozyme but has no known
function in α-lactalbumin (Brew et al., 1992). There are also metal binding sites
that are not well characterized, especially for manganese (Brew et al., 1992).
10
α-Lactalbumin has a very stable conformation between pH 5.4 and 9.0.
Below pH 4.0 and above pH 9.0, there are conformational changes in αlactalbumin (Bottomley et al., 1990). These conformational changes affect the
metal binding properties but do not cause any secondary structure changes (Brew
et al., 1992). Upon heating (at physiological pH), between 35-40°C the molecule
undergoes changes in its tertiary structure but not its secondary structure. Above
65°C, at physiological pH, there is a breakdown of the 310-helix and an increase in
turns (Boye et al., 1996). However, this is reversible and upon cooling 80-90% of
the protein renatures, making α-lactalbumin the most heat stable of whey proteins.
This is due to calcium ion dissociation and reassociation (Singh et al., 1992).
Above pH 9 and below pH 6, partial irreversible aggregation does occur (Boye et
al., 1997). Increasing salt concentration stabilizes α-lactalbumin (Boye et al.,
1997) but with an increasing concentration of α-lactalbumin, an increase in
denaturation is observed (Singh et al., 1992).
Figure 2.2: The crystal structure of α-lactalbumin (Mueller-Dieckmann et al., 2007)
11
Biological activity of α-lactalbumin
The biological function of α-lactalbumin is to support the biosynthesis of
lactose, which is an important source of energy for the newborn. At pH ≤4.0, αlactalbumin unfolds and is susceptible to digestion by pepsin in the stomach
(Miranda, 1983). The function of α-lactalbumin in lactose synthesis is well
known. It forms a complex with galactosyl transferase to form lactose synthase,
which catalyzes the synthesis of lactose from UDP galactose and glucose. αLactalbumin is the regulatory component of lactose synthase and it promotes
binding of glucose to galactosyl transferase during lactose synthesis (Brew et al.,
1992).
1.1.3. Immunoglobulins
There are many classes of immunoglobulins known as isotypes. Bovine
milk contains three classes of immunoglobulins in significant amounts, IgG, 1gM
and IgA (Larson and Fox, 1992). IgG comprises about 80% of the
immunoglobulins in whey protein of which IgG1 is the most abundant. IgG2 is
also present in appreciable amounts (Bottomley et al., 1990). There are two light
polypeptide chains and two heavy polypeptide chains per immunoglobulin that are
linked by four disulfide bonds. There are two kinds of light chains (κ and λ) with
a constant region and a variable region. Each light chain is approximately 20 kDa
(Bottomley et al., 1990; Larson and Fox, 1992). There are several types of heavy
chains with Mr ~ 50-70 kDa in which the heavy chains possess variable and
constant regions (Bottomley et al., 1990; Larson and Fox, 1992). The variable
regions of the antibodies are at the N-terminus of the light and heavy chains and it
is this domain that binds antigen. The constant regions at the C-terminal end
determine the class of immunoglobulins (Bottomley et al., 1990). IgG exists as a
monomer and is approximately 150 kDa. IgG also contains about 3%
carbohydrate (Larson and Fox, 1992; Bottomley et al., 1990). IgA exists as a
dimer linked by a polypeptide known as the J-component and IgM exists as a
pentamer
also
linked
by the
J-component
12
(Bottomley et
al.,
1990).
Immunoglobulins are thermally labile and will fully denature upon heating for 30
minutes at 70°C (Larson and Fox, 1992).
Biological activity of IgG
The
function of immunoglobulins in immunity is well known.
Immunoglobulins, known as antibodies, recognize foreign molecules, known as
antigens, and produce an immune response against them. The dominant species of
immunoglobulins in bovine milk, IgG, is identical to blood serum IgG in virtually
all mammalian species (Larson, 1989). IgG is the carrier of passive immunity to
the newborn.
1.1.4. Bovine serum albumin
Bovine serum albumin (BSA) is the largest single polypeptide chain
among the whey proteins. Its function is to transport fatty acids in the circulatory
system (Bottomley et al., 1990). It has 582 amino acid residues with a molecular
weight of 66,267 daltons. The protein is globular and has 17 disulfide bonds and
one free sulfhydryl group at position 34. The secondary structure is roughly 54%
α-helix and 40% β-structure and contains three domains specific for metal-ion,
lipid and nucleotide binding) (Boye et al., 1996).
Bovine serum albumin is very heat labile and denatures at ~ 66°C (Jelen,
1991). Heat denaturation proceeds with a decrease in α-helical content and an
increase in β-sheet and random coil structures followed by aggregation (Boye et
al., 1996). Below pH 4.0, the molecule undergoes denaturation because of charge
repulsion (Bottomley et al., 1990). Bovine serum albumin has maximum thermal
stability at a pH of ~5. Addition of salt and sugars stabilizes BSA against thermal
denaturation (Boye et al., 1996).
Biological activity of BSA
Bovine serum albumin is identical to blood serum albumin and is
transported into the milk by leaky junctions of the blood vessels in the mammary
gland (Harzer and Haschke, 1989.). Bovine serum albumin binds insoluble free
13
fatty acids for transportation in the blood and is probably a significant source of
glutathione production in the liver.
1.1.5. Proteose peptones
Proteose peptones are polypeptide fragments derived from various
sources. Most of them are fragments of casein that have been digested by
indigenous milk enzymes (Bottomley et al., 1990). They are very heat stable
(Jelen, 1991). Their effects on the properties of whey protein concentrates and
isolates are still not well known (Bottomley et al., 1990).
The proteose-peptone (PP) fraction, often regarded as part of the whey
protein fraction, is composed mainly of polypeptides derived from β-casein
through actions of proteinases, particularly plasmin. The origin and storage time
of fluid milk may be responsible for the highly variable PP content, ranging from
1 to 3 g/L. Phosphopeptides in the PP fraction are thought to enhance
gastrointestinal absorption of calcium (Kitts and Yuan, 1992). Other peptides in
the PP fraction have been shown to have opioid activity (known as βcasomorphins) (Schlimme et al., 1988).
1.1.6. Bioactive proteins
Well-known
bioactive
proteins
in
milk
are
lactoferrin
(LF),
lactoperoxidase (LP), and endogenous enzymes, each of which is present in minor
quantities. Two main biological functions have been assigned to LF (an ironcontaining protein): antibacterial activity in the mammary gland, and the
nutritional activity of making iron more available for absorption in the gut
(Ribadeau-Dumas and Grappin, 1989). Lactoperoxidase functions biologically in
the mammary gland and in the digestive tract of the calf as part of a bactericidal
system (LP system) that is active against a number of enteric bacterial strains.
Both LF and LP have been reported to have beneficial actions in reducing the
incidence of chronic diarrhea (Lonnerdal and Atkinson, 1995).
14
1.2. The benefits of dietary whey protein on the immune response and health
1.2.1. Anti-cancer effect of WPC
Whey proteins have been implicated in providing protection against cancer
in animal models when delivered orally. Such activity has been investigated in
order to establish the role of these proteins in disease prevention and to contribute
to a basis for their inclusion as ingredients in functional foods.
Several studies evaluated the possible role of whey protein in the
prevention and/or treatment of cancers. The influence of formula diets containing
20 g protein/100 g diet of either whey protein concentrate (WPC), casein or
Purina mouse chow on dimethylhydrazine (DMH) induced carcinogenesis in mice
was investigated (Sadler, 1992). After 20 weeks of DMH treatment, the number of
antibody-forming cells in the spleen following intravenous inoculation with sheep
red blood cells (SRBCs) was nearly three times greater in the WPC-fed group
than in the casein-fed mice, although both values were substantially below
normal. After 24 weeks of DMH treatment, both the size and incidence of tumors
were substantially less in WPC-fed mice than in mice fed either the casein or
Purina diets. The body weight curves were similar in all the feed groups (i.e. same
nutritional efficiency).
Putative anti-cancer activity of whey proteins has been investigated by
McIntosh et al. (1998). Animal feeding trials have compared the efficacy of dietary
whey proteins in. compared with other dietary proteins (meat, soy). Dairy proteins,
in particular whey protein, were found to be efficacious in retarding chemically
induced colon cancer in a rat model of the disease retardation of colon cancer in
young rats. The influence of dietary whey protein on development of colon cancer
in mature rats has also been examined. Results similar to those with younger
animals have been demonstrated, a finding that suggests age does not significantly
alter the outcome. Efficacy of whey protein fractions has also been assessed. They
suggest that diets supplemented with lactoferrin or β-lactoglobulin enhance
15
protection against the development of putative tumor precursors in the hind gut
wall.
Xiao et al. evaluated partially hydrolyzed whey protein (WPH) for
inhibitory effects on the development of colon aberrant crypt foci (ACF) and
intestinal tumors in azoxymethane (AOM)-treated rats. At 6 and 23 weeks,
post-AOM, WPH-fed rats had fewer ACF than did casein-fed rats. Intestinal tumors
were most frequent at 23 weeks, post-AOM. At this time, differences in colon
tumor incidence with diet were not observed; however, WPH-fed rats had fewer
tumors in the small intestine (7.6% vs. 26% incidence) (Xiao et al., 2006).
The mechanism behind the apparent anti-cancer activity of dietary whey
proteins in these studies may be related to their sulfur amino acid content, for which
there is a high requirement in the rat, and a hypothesized role in protecting DNA in
its methylated form.
1.2.2. Effects on immune associated factors
Investigations (Bounous et al., 1989) revealed increased levels of
glutathione in the spleens, livers and lymphocytes of mice on concentrated whey
protein diets. In light of this discovery, a change in the functional responsiveness
of the lymphocytes due to the protective effect of glutathione was deemed most
likely. Adequate levels of glutathione are necessary for lymphocyte proliferation
in the development of the immune response. Glutathione is known to function
directly or indirectly in many important biological phenomena such as protein and
DNA synthesis, regulation of enzyme activity, immune response, protection of the
cell against reactive oxygen compounds and free radicals, aging, intermediary
metabolism and drug metabolism (Moister and Anderson, 1983). In addition to
this, there are differential effects of glutathione depletion on the various T-cell
subsets (Gmunder and Droge, 1991).
Glutathione also has a role in detoxifying processes and is also the source
of the cysteine utilized in the biosynthesis of mercaptouric acid conjugates of N-
16
acetylcysteine, end-products of a process which serves to detoxify a variety of
harmful compounds and xenobiotics (Megaw, 1984). Induction and increased
activity of detoxifying hepatic microsomal enzymes by dietary supplementation
with glutathione has been reported (Nyandieka et al., 1989). Dietary
supplementation of glutathione resulted in inhibition of aflatoxin B1 induced liver
cancer.
Another aspect of human health that a dietary whey protein diet may
benefit is the decrease in both intracellular glutathione levels and response to
mitogenic stimulation in lymphocytes associated with increasing age (Makinodan
and Kay, 1980). In addition, depletion of glutathione has also been implicated in a
number of degenerative diseases including osteoarthritis, the formation of
cataracts associated with aging, Alzheimer’s disease and arteriosclerosis
(Bounous and Gold, 1991). The glutathione enhancing property of WPC implies a
vital role for this protein in the promotion of health and well-being.
In vivo immune activity was assessed after 4- or 8-week dietary regimens.
After 8 weeks of dietary intake, splenic lymphocytes derived from mice fed with
IMUCARE WPC showed significantly elevated (P < 0.05) proliferative responses
to a T-cell mitogen (concanavalin A) compared with those from soy-fed mice;
cheese WPC did not affect lymphocyte proliferation significantly. Mice fed with
whey proteins tended to exhibit elevated mean intestinal tract antibody responses
to orally administered ovalbumin and cholera toxin antigens, whereas soybean
protein did not affect antibody responses. This study confirms that WPCs are able
to modulate some aspects of the immune response (Rutherfurd and Gill, 2005),
Low et al. (2003) investigated the effects of feeding WPC to mice on
specific antibody responses to several orally or parenterally administered antigens,
including influenza vaccine, diphtheria and tetanus toxoids, poliomyelitis vaccine,
ovalbumin and cholera toxin sub-unit. WPC-fed mice produced elevated levels of
antigen-specific intestinal tract and serum antibodies against all tested antigens,
compared to mice that were fed a standard chow diet. Both primary and secondary
intestinal tract antibody responses were elevated by WPC feeding, while only
secondary serum responses were increased in WPC-fed mice. Significant up-
17
regulation of intestinal tract antibody was observed within 2 weeks of primary
oral immunizations. A period of prefeeding with WPC, prior to commencement of
immunization, did not alter the kinetics or magnitude of immune enhancement.
On the basis of these results, Low et al. (2003) identified bovine WPC as a
potentially important dietary protein supplement, capable of enhancing humoral
immune responses to a range of heterologous antigens.
1.2.3. Angiotensin I-converting enzyme inhibition activity
Angiotensin I-converting enzyme (ACE, peptidyl dipeptide hydrolase, EC
3.4.15.1) has been classically associated with the renin-angiotensin system, which
regulates peripheral blood pressure. ACE raises blood pressure by converting
angiotensin I released from angiotensinogen by renin into the potent
vasoconstrictor angiotensin II. ACE also degrades vasodilative bradykinin and
stimulates the release of aldosterone in the adrenal cortex. Consequently, ACEinhibitors may exert an inhibitory effect (Petrillo and Ondetti, 1982). ACE is an
exopeptidase, which cleaves dipeptides from the C-terminal of various peptide
substrates. ACE is an unusual zincmetallopeptidase, as it is activated by chloride
and lacks in vitro substrate specificity (Ondetti and Cushman, 1984).
Ferreira et al. (2007) studied whey protein hydrolysates with tryptic
hydrolysis and assessed their application as ingredients with ACE inhibitory action.
The levels of α-lactalbumin (α-Lac) and β-lactoglobulin (β-Lg) remaining after
hydrolysis were quantified. Peptides were separated by RP-HPLC, and
Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR), a potent β-Lg-derived ACE-inhibitory
peptide, was monitored. A correlation curve was established for the production of
this peptide as a function of hydrolysis time, and it was found that extensive
hydrolysis was required to obtain peptides suitable for functional ingredients
having ACE-inhibitory effect.
The influence of heat and enzymatic treatments on the hypotensive activity
of hydrolysates derived from whey protein isolate (WPI) was examined by la Costa
et al. (2007). WPI was heat-denatured and hydrolyzed using the enzymes Alcalase,
18
α-chymotrypsin or Proteomix. The hydrolysates thus obtained were characterized
and studied with regard to their ACE inhibitory activity and hypotensive activity in
spontaneously hypertensive rats (SHR). The enzyme α-chymotrypsin was found to
produce hydrolysates with the highest ACE inhibitory activity. The hydrolysate
that most effectively reduced blood pressure in SHR was obtained from WPI
previously denatured at 65 °C and treated with the enzyme Alcalase. The
hydrolysate with the highest ACE inhibitory activity was able to reduce the arterial
blood pressure of the animals only after intraperitoneal administration, suggesting
an interference of gastrointestinal enzymes in the absorption of active peptides
from this hydrolysate. (La Costa et al., 2007)
Hernandez-Ledesma et al. (2006) measured the ACE-inhibitory activity of the 3000
Da-permeates obtained from �-Lg A hydrolysis with thermolysin. All their samples
showed notable ACE-inhibitory activity ranging from 7.3 to 13.2 µg/mL. The
lowest IC50 values corresponded to the hydrolysates produced at 60 °C where they
were 45% lower than �-Lg A hydrolysates obtained at 37°C.
Ortiz-Chao et al. (2009) hydrolyzed β-Lg using Protease N amano at two different
temperatures (45 and 55°C). They reported that temperature was found to be a key
factor in generating the different peptide profiles. For example, peptide profiles
obtained by heating the samples at 45 and 55 °C are different after 360 min of
digestion despite having reached similar levels of β-Lg hydrolysis (75% at 45 °C
and 82% at 55 °C). They also reported a novel heptapeptide SAPLRVY as a result
of hydrolysis with protease N amano at 55 oC. The peptide corresponded to β-Lg
f(36–42) and had an IC50 value of 8 µM, making it by far the most potent ACE
inhibitory peptide derived from bovine β-Lg reported so date.
Van der Ven et al. (2002), using WPI and pancreatic enzymes, obtained
hydrolysates with IC50 values between 0.16 and 0.84 mg/mL. The lower activities
corresponded to hydrolysates with a degree of hydrolysis of 10%.
19
1.2.4. Antioxidant activity
The identification and development of food-derived peptides with
antioxidant properties has attracted increased attention due to heightened safety
concerns over the use of synthetic antioxidants and the consumer’s preference for
naturally derived products (Sakai et al., 1997; Shahidi and Zhong, 2008). Once
proven safe and effective, naturally derived antioxidant peptides could have utility
in the food industry through their incorporation into fat-rich meat products,
replacing synthetic antioxidants such as BHA to inhibit lipid oxidation and increase
shelf life. Interest in the development of peptides as alternative antioxidants stems
from studies in which a variety of food proteins, such as milk, soy, zein, canola,
capelin, and porcine collagen, were found to inhibit lipid oxidation in commonly
consumed foods and in in vitro models (Amarowicz & Shahidi, 1997; Cervato et
al., 1999; Chen et al., 1996; Cumby et al., 2008; Li et al., 2007). These antioxidant
properties are believed to be due to unique amino acid sequences inherent in the
protein structures (Giovanna et al., 1999; Wanget al., 1991). For example, clear
evidence of the antioxidant activities associated with specific peptides was
demonstrated in oil-in-water emulsions, beef homogenates, liposomal suspensions,
and �-carotene/linoleate model systems (Kong & Xiong, 2006; Shahidi &
Amarowicz, 1996). Certain hydroxyl- or sulfate-bearing amino acids such as Tyr,
Met, His, Lys, and Trp have been shown to possess some degree of antioxidant
properties (Chen et al., 1996; Saito et al., 2003). Furthermore, specific peptides
have been reported to have enhanced antioxidant activities when compared to
constituent amino acid mixtures (Guo et al., 2009). Consequently, it has been
suggested that the synergistic effect of specific amino acid sequences in peptides,
molecular structures, and functional groups are all major factors that contribute to
the antioxidant potential of peptides.
A variety of methods have been employed with the purpose of generating
potentially functional peptides. For example, food-derived peptides with biological
activities are primarily produced through enzymatic hydrolysis, fermentation, and
chemical or enzymatic synthesis (Shahidi & Zhong, 2008). Amongst these
processes, enzymatic hydrolysis is often the method of choice in which common
20
digestive enzymes are exploited, such as trypsin and pepsin, to generate peptides
with potential antioxidant properties (Jiang et al., 2007). During hydrolysis the
protease specificity is essential because it dictates the amino acid sequence of the
resultant peptides and their bio-functional properties (Murray and FitzGerald,
2007). Use of commercial proteases of microbial or plant origin has gained
increased popularity due to their unique functions and cost-effectiveness. For
instance, a commercially developed and marketed Bacillus protease enzyme,
Protamex by Novozyme, was used to generate catfish protein hydrolysates with
notable antioxidant activities (Theodore et al., 2008). Furthermore, the plant
derived papain enzyme has been attributed to utility in the production of a variety of
protein hydrolysates with different functions in food science (Kong & Xiong,
2006). Additional enzyme hydrolysis research is merited due to the fact that there is
a general recognition that the type of enzyme used in the hydrolysis process will
impact the potency of bioactive properties such as antioxidant capacity.
1.2.5. Health benefits of whey proteins for active people
Whey proteins have a strong position in the sports nutrition market as a
potential means to enhance lean body mass in conjunction with appropriate
training based on the purported “quality” of proteins. Recent studies employing
stable isotope methodology demonstrate the ability of whey proteins or amino
acid mixtures of similar composition to promote whole body and muscle protein
synthesis (Ha and Zemel, 2003). Calcium and nonfat dry milk have been shown to
regulate nutrient partitioning, adiposity, and body composition, suggesting
another manner by which whey and whey components may optimize body
composition (Moister and Anderson, 1983; Zemel, 2003).
Other developing avenues of research explore health benefits of whey that
extend beyond protein and basic nutrition. Many bioactive components derived
from whey are under study for their ability to offer specific health benefits. These
functions are being investigated predominantly in tissue culture systems and
animal models. The capacity of these compounds to modulate adiposity and to
enhance immune function and antioxidant activity presents new applications
21
potentially suited to the needs of those individuals with active lifestyles (Ha and
Zemel, 2003). Recent findings suggest that benefits of whey may extend beyond
muscle anabolism. The calcium and mineral mix found in certain whey products
can potentially mediate body composition by shifting nutrient partitioning from
adipose to lean tissue. Individual amino acids and bioactive compounds isolated
from whey may also improve immune function and gastrointestinal health
although many of these functions are just now being defined in model systems
(Ha and Zemel, 2003).
2. Effects of Microwave treatment on proteins
The research presented in this thesis focuses on the effects of microwave
heating on the structure and enzymatic hydrolysis of WPI and its principal
components, β-lactoglobulin and α-lactalbumin. Selected hydrolysates with
unique amino acid sequences (compared with conventional hydrolysates of native
and heat-treated WPI and its principal components, β-lactoglobulin and αlactalbumin) are sought and their nutraceutical properties such as ACE inhibitor
activity will be examined. Accordingly, a brief discussion of the principles of
microwave treatment and the thermal and non-thermal effects of such treatment
will be presented in the following sections.
2.1. Thermal and non-thermal effects of microwaves
The microwave ovens traditionally employed for heating food employ
wavelengths of ~1 cm corresponding to a frequency of ~ 2.5 GHz. Microwave
treatment does not have sufficient energy to break any chemical bonds (E = 1
J/mole ) and for that reason belongs to the group of non-ionizing forms of radiation
(Anantheswaran and Ramaswamy, 2001). According to the literature, two types of
effects can be ascribed to microwaves, referred to as thermal and non-thermal (or
athermal) effects (Marani and Feirabend, 1994; Kirschvink, 1996). Microwaves
have the ability to penetrate the food and generate heat by friction resulting from
the oscillation of water dipoles as they try to align their dipole with the microwave
22
field, resulting in friction with other food constituents; the resulting increase in
temperature within the food is the macroscopic thermal effect of microwave
treatment. Non-thermal effects under the application of electromagnetic radiation
refer to observable changes that do not involve a significant rise in temperature.
According to Tong, microwave non-thermal effects are defined as effects that
cannot be explained by heat alone (Tong, 1996). Risman (1996) proposed that any
non-thermal effect must not be explicable by macroscopic changes in temperature
or time-temperature histories or gradients.
2.1.1. Thermal effects (microwave heating)
Microwave heating is also referred to as “radio frequency” or “electronic”
heating and is related not merely to the dielectric properties of foods, but to their
electrical transmission properties as well (Decareau, 1985). Microwave heating or
dielectric heating refers to the heating that occurs in a nonconductor due to
polarization effects at frequencies between 300 MHz and 300 GHz. Essential
differences between capacitive (macrowave) and microwave heating are the
frequencies used (1 to 300 MHz in macrowave heating) and the compartment in
which heating is carried out. In microwave heating a closed cavity or oven is often
used, whereas in capacitive heating the material is usually placed between
electrodes and treated with frequencies between 1 MHz and 300 MHz) (Decareau,
1985).
Microwaves in themselves are not heat; it is the material absorbing
microwaves that converts the energy to heat. In food systems, the heat is generated
by interaction between microwaves and polar molecules or ions.
Water is the ubiquitous polar molecule in the majority of foods. Since water
molecules have partial negative and positive ends, in the presence of a microwave
electric field these charges attempt to line up with the electric field. As the
microwave field reverses its polarity billions of times per second, the water
molecule, constrained by the natural structure of the food of which it is a part,
begins doing a flip-flop movement billions of times per second. In doing so, weak
hydrogen bonds are disrupted and heat is generated by the molecular friction and is
dissipated rapidly throughout the food. Because the molecules are forced to rotate
23
first, there is a slight delay between the absorption of microwave energy and the
development of linear momentum, or heat. Other constituents such as salts also
contribute to heat generation by either friction or rapid electrophoretic migration in
the electric field. Charged ions are influenced by the microwave electric field, so
they migrate first in one direction then in the other direction as the electric field is
reversed (Figure 2.3). There are also some secondary effects of microwaves,
including ionic conduction, which are negligible in producing heat.
Alternating electric
field
�+
H
E
+
Na
�O
Cl
Rotation
�+
H
Ionic Interaction
Dipolar Interaction
Figure 2.3: Proposed mechanism of microwave heating (source: Buffler, 1993).
2.1.2. Non-thermal effects of microwaves
Very little is known about the molecular mechanisms involved in the
presumed non-thermal effects of microwave heating. It has been postulated that
they might involve direct energy transfer from the electromagnetic field to the
vibrational modes of macromolecules, altering their conformation (Taylor, 1981).
Khalil and Villota (1989) studied the relative effectiveness of conventional and
microwave heating for inactivation of microorganisms by comparing the injury and
recovery of Staphylococcus aureus during microwave and conventional heating
and concluded that microwave-heated cells suffered a larger injury as well as
greater membrane damage.
24
There are also some non-thermal effects of microwave heating supported by
microwave specific activations which enhance reaction rates and are summarized
as under;
1. The reaction rate enhancement due to "hot spots"/localized heating effects may
be the result of the reactive polar molecules as they absorb microwaves selectively
enhancing the reaction quickly. Similarly, under microwave treatment, secondary
amines absorb microwave energy causing inversion of the molecule similar to
primary amines, and the reactivities of the two amino groups become identical.
Therefore, the enhanced cross-linking occurs due to accelerated reaction of the
secondary amine group. So, the microwave activation could be due to hot spots
generated by dielectric relaxation on the molecular scale. Furthermore, the
acceleration of the reaction by microwave treatment may be either due to the
superheating effects of the presence of a large number of ions or superheating
effects at the boundary between non-miscible liquids along with efficient mixing of
reactants, and rapid achievement of the reaction temperature caused by microwave
dielectric effects (Lewis et al., 1992; Marnard et al., 1992).
2. Reaction rate enhancement due to molecular agitation where microwaves cause
rapid shift of the dipoles found in the molecules of a compound. The intermolecular
bonds hinder the rotation of the dipoles causing a lag in the dipoles following the
electromagnetic radiation. This might be the reason for the heating effect observed
on treating a compound with microwaves (Chen et al., 1991).
3. Reaction rate enhancement due to improved transport properties of the
molecules, as the slowest step in solid state reactions is the diffusion of reactants
towards one another through an unreactive medium. Since the rate of a reaction is
controlled by the slowest step, any process which could enhance the diffusion of
reactants can lead to a significant rate enhancement (Jacob et al., 1995).
4. Reaction rate enhancement due to other reasons, as the extent of a chemical
reaction on treating with microwaves is not related purely to the temperature rise in
the sample. (Bose et al., 1990).
5. In the dry organic reactions, product selectivity due to microwave treatment
25
where microwave treatment improves the sample productions compared to
conventional heating (Alloum et al., 1989).
6. Superior mechanical properties on microwave treatment seem to have focused
largely on the reaction rate enhancement and there are very few reports comparing
the morphological and mechanical properties of the microwaved sample with the
conventionally treated sample (Jacob et al., 1995).
These enhanced effects of microwave are already proven in the organic and
chemical synthesis system, however it is still debatable in the food system.
One of the clear examples of the enhanced effects of the microwave is the
efficiency of microwave treatment at low temperature for glycosylations. Although
oligosaccharide synthesis usually requires reactive donors for glycosylations,
which have leaving groups, the suitable donors in the microwave supported
synthesis of Lewis X oligosaccharide were very stable acetate derivatives.
Synthesis of Lewis X derivatives was achieved only with microwave treatment at
low temperatures. Without microwave treatment, only byproducts were obtained
and none of the designed product at any reaction temperature (Shimizu et al., 2008).
2.2. Microwave Effects on Protein
2.2.1. Microwave Effects on Protein Structure
Microwave treatment has been reported to alter protein tertiary structure.
Tertiary structure refers to the complete three-dimensional structure of the
polypeptide units of a given protein. Included in this description is the spatial
relationship of different secondary structures to one another within a polypeptide
chain
and
how
these
secondary structures themselves
fold
into
the
three-dimensional structure of the native protein. Secondary structures of proteins
often constitute distinct domains. Therefore, tertiary structure also describes the
relationship of different domains to one another within a protein. The interactions
of different domains are governed by several forces. These include: hydrogen
bonding, hydrophobic interactions, electrostatic interactions and van der Waals
26
forces.
Surrounding water molecules exposed to hydrophobic regions on protein
molecules adopt hydrogen-bonded pentagonal structures. An increase in the
temperature of the water can have significant consequences for protein
conformation.
The
breaking
of
hydrogen
bonds
within
the
water
pentagonal-structure may cause it to give way to the bulk water state
(Chattopadhyay et al., 1997). As a result, hydrophobic groups on the surface of the
protein may be able to interact with each other to a greater extent and the protein
may adopt a different conformation.
Bohr reported that at low temperatures, the re-folding of cold-denatured
�-lactoglobulin was enhanced by microwave treatment, while at elevated
temperatures the denaturation of �-lactoglobulin from its folded state was enhanced
by microwave treatment. In the second case, a negative temperature gradient is
needed for the denaturation process, suggesting that the effects of the microwave
treatment are non-thermal (Bohr and Bohr, 2000). Also, there is experimental
evidence that microwave treatment could speed up rates of folding and unfolding of
other globular proteins in solution (Bohr and Bohr, 2000). Larger proteins,
chemically modified proteins, protein complexes and membrane proteins, which
together comprise most of nature’s proteins, remain largely uncharacterized.
Therefore, protein folding may be assisted by chaperones, challenged by stochastic
events such as aggregation, and modulated by the cellular status at any moment in
time (Bartlett & Radford, 2009).
Exposure to microwave treatment was found to increases the aggregation of
bovine serum albumin in vitro in a time- and temperature-dependent manner (de
Pomerai et al., 2003). The same group also claimed that microwave treatment can
induce amyloid fibril formation in vitro, at least under the non-physiological
conditions used in the experiment (de Pomerai et al., 2003).
Al-Jundi (2004) studied the non-thermal effects induced upon treatment of
protein solutions with microwave energy by employing Fourier transform infrared
27
spectroscopy, circular dichroism, and fluorescence spectroscopy to elucidate the
unfolding pathways of hemoglobin and β-lactoglobulin (5% in D2O) subjected to
either microwave treatment (2.45 GHz) or conventional heating. The microwave
treatments were performed for different times and numbers of cycles at selected
temperatures to investigate the influence of these parameters. At 54˚C, hemoglobin
aggregated faster upon microwave treatment than with conventional heating (water
bath) under the same conditions. The �-helix and 310-helix contents of hemoglobin
decreased after microwave treatment compared to conventional heating at the same
temperature, and the hydrophobic interior of the protein was more exposed to
solvent in the microwave treatment samples. In the case of β-lactoglobulin, higher
rates of H-D exchange occurred during microwave treatment of D2O solutions at
35oC by comparison with conventional heating at the same temperature. At 70°C
(close to the aggregation temperature), �-lactoglobulin aggregated faster when
exposed to microwave treatment compared to conventional heating. The
experimental data provides evidence for the existence of a non-thermal effect of
microwave treatment on protein unfolding. A possible mechanism was postulated
to involve the direct absorption of microwave energy by the protein backbone
leading to an increase in the inter-domain motion, which may result in the
loosening of the tertiary structure and enhanced protein aggregation close to the
denaturation temperature of the proteins.
Although the mechanism of the putative non-thermal effects of microwaves
on protein tertiary structure is unknown, violent motion of dipoles in molecules in a
microwave field may destabilize the structure of polyatomic molecules such as
protein and phospholipids. Microwaves either cause ions to accelerate and collide
with other molecules or cause dipoles to try to rotate and line up with rapidly
alternating electrical field at 2.450 billion oscillations per second (Sato et al., 1996).
Bohr suggested that the initiation of protein folding is a resonance phenomenon and
that protein folding takes place when the amplitude of the resonance mode exceeds
a certain threshold. Folded structures become stabilized by van der Waals
interactions, hydrogen bonding, salt bridges, disulfide bonds, and even in some
28
cases by dynamic forces (Bohr, 1997). Wring modes will divide a protein chain into
regions, which are subject to large and small torsional amplitudes. An amino acid
substitution in a region with high torsional amplitude is more likely to have a
modifying effect on the folding of the protein than a substitution in a region of low
torsional amplitudes. A phase transformation in proteins can be initiated by
long-range collective wring modes of the backbone. Bohr suggested that this is an
important part of the mechanism underlying the transformation of a protein from
the unfolded to the folded structure. It is also assumed that as a wring mode is being
pumped to levels of higher energy and higher amplitude, eventually this wring
mode becomes unstable in favor of curvature. The nature of such a transition is
better characterized as being catastrophic than entropic. Therefore, the primary
reason for the transition is not a change in entropy; rather, a resonator is responsible
for pumping the twist mode to a higher and higher level. Folded proteins can also
maintain wring modes, and it is the disappearance of these wring modes upon
lowering of the temperature that leads to cold denaturation of certain proteins. Only
contributions to the change in free energy, when going from an unfolded to a
folded, and from a folded to an unfolded protein, are caused by their differences in
wring activity. Proteins can sustain eigenmodes involving a wringing, and the
eigenfrequency of the wring mode may be as high as values typical for excitation of
molecular structures. This points to the intriguing possibility that the transition
from the unfolded to the folded state of a protein can occur when a wring mode of
the protein backbone becomes unstable to curvature (Bohr, 1997). Furthermore,
when the proteins become larger, their folding reactions become multi-state and/or
irreversible because the consistency is lost and local minima are generated on the
folding energy surface (Sakurai et al., 2009).
2.2.2. Effect of microwave on enzymes
Henderson et al. (1975) studied inactivation of horseradish peroxidase using
2.45 GHz microwaves by circulating carbon tetrachloride as a coolant to control the
temperature of the sample at 25oC. These researchers reported significant enzyme
inactivation at high power and reasoned that protein denaturation had occurred,
29
perhaps due to local heat generation within the sample (Henderson et al., 1975).
The inactivation of commercial soybean lipoxygenase was studied at
various temperatures using conventional and microwave heating by Kermasha et al.
(1993) Lipoxygenase inactivation was assessed using first-order reaction kinetics.
This experiment showed that microwave heating resulted in a more rapid
inactivation of the enzyme. Higher enzyme inactivation rates under microwave
heating conditions were ascribed to possible non-thermal effects (Kermasha et al.,
1992).
The effect of microwave treatment at 10.4 GHz on a thermostable enzyme
(ß-galactosidase) was experimentally tested by La Cara et al. (1999) to study the
effects of microwaves on protein stability. They observed that microwave treatment
induced an irreversible inactivation of the enzyme at 70˚C compared to
conventional heating at the same temperature. This inactivation was dependent on
the intensity of the microwave treatment as well as the protein concentration.
Inactivation was not observed following conventional heating at the same
temperature (La Cara et al., 1999).
Inactivation kinetics of an α-amylase enzymatic time-temperature
integrator (TTI) from Bacillus subtilis (BAA) under continuous-flow microwave
and conventional hold conditions were evaluated and compared by Tong et al.
(2002). Comparing the D-values between continuous-flow microwave and thermal
holding, enzyme destruction occurred much faster under continuous-flow
microwave heating. Hence, there is evidence that microbial lethality under
microwave heating conditions cannot be fully accommodated by conventional
models employing thermal kinetic data. The results indicated further the possible
existence of non-thermal or enhanced thermal microwave effects (Tong et al.,
2002).
The effects of hydrostatic high pressure, microwave treatment, and
conventional heating on hydrolysis of bovine β-lactoglobulin AB by pronase and
α-chymotrypsin were studied by Izquierdo et al. (2005). The high-pressure
treatments were performed between 100 and 300 MPa, and the microwave
treatment was applied at powers of 30 W (pronase) and 15 W (α-chymotrypsin ).
30
All enzymatic reactions were carried out for 10 and 20 min, at 40 °C and the
products were analyzed by HPLC. The initial rate of hydrolysis of protein by
pronase and α-chymotrypsin was increased by using physical treatment during the
enzymatic digestion. The microwave-assisted digestion by pronase was more
effective than digestion performed under conventional heating. In the case of
protein digestion by α-chymotrypsin, the only apparent microwave-induced
difference was a lower relative concentration of the peak eluting at 24.5 min and the
conversion of this fraction into final peaks eluting from 24.5 to 28.5 min (F1
fraction) after 20 min of incubation (Izquierdo et al., 2005).
Exposure of two thermophilic and thermostable enzymes to microwave
treatment resulted in a non-thermal, unalterable and time-dependent inactivation of
both enzymes below their optimal operating temperatures. The inactivation rate
was correlated to the energy absorbed and was sensitive to the enzyme
concentration,
suggesting
that
microwaves
stimulate
protein
structural
rearrangements that are not related to temperature (Porcelli et al., 1997). In the
same experiment, the effect of salts on the enzyme inactivation was studied by
subjecting AdoHcy hydrolase and MTA phosphorylase to 10.4 GHz microwave
treatment at 90oC in the presence of 250 mM KCl or 250 mM KH2PO4. The two
thermophilic enzymes showed different behavior. The addition of KCl or KH2PO4
to the enzymatic solution exposed to microwave treatment did not have any effect
on microwave inactivation of AdoHcy hydrolase. On the other hand, KH2PO4
exerted a moderate protection towards microwave inactivation of MTA
phosphorylase while KCl enhanced the inactivation process. Furthermore, a similar
experiment performed with 250 mM NaCl and 250 mM Na2SO4 revealed that after
1 hour of microwave treatment of MTA phosphorylase at 90°C, Na2SO4 exerted a
moderate protection (76% residual activity), while NaCl caused an increase in the
inactivation of the enzyme (34% residual activity).
The protection against
microwave inactivation exerted by phosphate or its analog sulfate could be ascribed
to their role as substrates of MTA phosphorylase.
31
More recently, Shazman et al. (2007) challenged the existence of all
non-thermal microwave effects. They examined the possibility of athermal
(non-thermal) effects due to microwave treatment in a number of chemical,
biochemical and microbial systems. Athermal effects were not detected in any of
the tested systems. They attributed the putative non-thermal effects to lack of
adequate control of the temperature during thermal treatment. For their studies, a
special flow-through set-up was built in order to eliminate, as much as possible,
experimental artifacts (Scheme 1). The protein solution was circulated during
microwave treatment and the temperature was measured in the microwave cavity as
well as the optical path. The extent of structural changes was determined by
changes in the turbidity of the solution.
Scheme 1. Experimental set up employed by Shazman et al. (2007).
(TC1 and TC2 are the microwave cavity outlet and inlet, respectively).
It is of interest to note that the turbidometeric method employed is far less
sensitive than other spectroscopic methods employed by previous researchers.
Accordingly, it is appropriate to repeat the experiments carried out employing more
sensitive spectroscopic methods that can affirm or challenge these results.
32
Chapter 3. An investigation of the effect of microwave
treatment on the structure and unfolding pathways of
β­lactoglobulin using FTIR spectroscopy with the
application of two­dimensional correlation spectroscopy
(2D­COS).
INTRODUCTION
β-Lactoglobulin (β-Lg) is the major whey protein of ruminant species, and
its properties have been regularly reviewed (e.g., Tilley, 1960; Lyster, 1972;
Kinsella and Whitehead, 1989; Hambling et al., 1992; Sawyer, 2003). As β-Lg is
largely responsible for the aggregation and gelation of whey proteins (Boye et al.,
1996), the thermal behavior of this protein has been extensively studied (Bottomley
et al., 1990; Hambling et al., 1992; Singh and Creamer, 1992; Boye et al., 1996).
The vast majority of such studies have involved conventional heat treatments, but a
few of them have examined the response of β-Lg to microwave treatments (Bohr et
al., 1997; Bohr and Bohr, 2000a,b; Al-Jundi, 2004). The latter studies have not only
indicated that the effects of microwave treatment on the structure of β-Lg are not
equivalent to those of conventional thermal treatments but have also been
suggested to support the putative non-thermal effects of microwaves. However,
these investigations have been fairly limited in scope, and further examination of
their findings through more detailed studies appears warranted. Furthermore, the
focus of these studies has been on fundamental questions regarding the nature of the
interactions of microwave treatment with proteins, which remain controversial,
rather than on the possible implications of the findings in relation to the
conformational stability and hence functional and nutraceutical properties of β-Lg.
Microwaves are part of the electromagnetic spectrum with a frequency
range of 300 MHz to 300 GHz, corresponding to a wavelength range of 1 m down
to 1 mm. The thermal effects of microwave treatment are related to the heat
generated following the absorption of microwave energy by water, organic
molecules or ions (Porcelli et al., 1997). Microwave-enhanced effects, however,
refer to observable phenomena which cannot be explained by only the rise in
33
temperature. A body of scientific evidence attributes the microwave-enhanced
effects to the existence of non-thermal effects of microwave treatment (Bohr et al.,
1997; Bohr and Bohr, 2000; de Pomerai et al., 2003; Jacob et al., 1995; Porcelli et
al., 1997). For their studies of microwave effects, Bohr et al. (1997; 2000a,b)
employed β-Lg as a model protein as it is well characterized and has a relatively low
molecular mass. These authors reported that microwave treatment of
cold-denatured β-Lg enhanced the re-folding of the protein. Furthermore, when the
temperature was raised to 48°C during microwave treatment, the denaturation of
the protein from its folded state was enhanced, relative to conventional heating at
the same temperature. The authors proposed that the microwave effect is
non-thermal and that protein unfolding takes place when the amplitude of a wring
excitation of the protein polypeptide becomes so large that bending of the protein
backbone becomes energetically favorable. In contrast, other authors have argued
that only thermal effects of microwave treatment should be considered because
microwave fields are incapable of inducing effects beyond the heating that results
from the absorption of microwave energy by water, organic molecules or ions
(Banik et al., 2003). For example, Gedye (1997) tested a series of organic reactions
that were previously reported to be significantly accelerated by microwave
treatment. In his experiments, which had a very careful temperature control, Gedye
did not detect any microwave athermal effects. More recently, Shazman et al.
(2007) also challenged the existence of non-thermal microwave effects. They
employed a special flow-through set-up (constructed to eliminate, as much as
possible, experimental artifacts) to examine the possibility of non-thermal effects
due to microwave treatment in a number of chemical, biochemical and microbial
systems and concluded that the putative non-thermal effects reported by other
authors were attributable to inadequate temperature control during thermal
treatment.
During the past three decades, FTIR spectroscopy has been developed as a
powerful tool to characterize structural changes in proteins, particularly in the
determination of their secondary structure (Arrondo et al., 1993; Dong et al., 1990,
1996; Surewicz and Mantsch, 1988; Surewicz et al., 1993; Qi et al., 1997). The
34
technique does not appear to have been utilized to study the effects of microwave
treatment on protein structure, except in preliminary studies conducted in our
laboratory (Al-Jundi, 2004), but has proved very useful in studying the secondary
structural changes in proteins induced by the following parameters: temperature
(Gorne-Tschelnokow et al., 1993; Ismail et al., 1992; Qi et al., 1997; Surewicz et
al., 1987a,b), pH (Casal et al., 1988), chemicals (Jackson and Mantsch, 1991) and
genetic variation (Dong et al., 1996). Since a protein usually contains different
secondary structural elements such as α-helix, β-sheet, β-turn and random coil, the
amide I band is a composite band constituting overlapping signals. However, the
application of band narrowing techniques coupled with computerized data handling
enables one to resolve the bands, which can then be attributed to individual
secondary structural elements. This also makes quantification of protein secondary
structure possible, provided unambiguous assignment of the bands is possible
(Surewicz et al., 1993).
FTIR spectroscopy has been used to study β-lactoglobulin by Casal et al.
(1988), who examined the conformational changes induced by changes in pH and
temperature. Dong et al. (1996) studied the subtle structural differences between
β-lactoglobulin A and B variants using this technique. Qi et al. (1997) examined the
possibility of the existence of a molten globule state of β-lactoglobulin at higher
temperatures by FTIR spectroscopy. FTIR spectroscopy was also employed to
monitor changes in the secondary structure and thermal stability of β-lactoglobulin
A and B in the presence of sodium dodecyl sulfate (SDS), N-ethylmaleimide
(NEM), urea and cysteine (Boye et al., 2004). The technique has also been
employed to investigate the secondary structural changes of β-lactoglobulin in
water/ethanol mixtures at two different pH levels and at high protein concentrations
(Dufour et al., 1994). Also, the effects of pH, temperature, and adsorption to the
oil–water interface on β-lactoglobulin have been examined (Fang and Dalgleish,
1997).
In the past decade, two-dimensional correlation spectroscopy (2DCOS) has
been established as a tool for extracting useful but obscured information from a
complex spectral dataset, Various types of perturbations (e.g. thermal, mechanical,
35
and chemical) have been utilized to obtain 2D correlation spectra using various
electromagnetic probes, such as IR, UV, Raman, and fluorescence. Recently,
applications of 2D correlation analysis to spectra affected by a simple static
physical factor, such as temperature, pressure, or composition, have gained
considerable interest (Noda, 2000), and there are numerous examples of
applications of 2DCOS in studies of the folding/unfolding processes of proteins
(Noda and Ozaki, 2004). Accordingly, this technique can potentially contribute to
our understanding of the effects of microwave treatment on proteins.
In this research work, FTIR spectroscopy is employed to compare the
effects of microwave treatment and conventional heat treatments on the secondary
and tertiary structure elements of β-lactoglobulin under varying physicochemical
conditions. The mechanism of protein unfolding and aggregate formation has been
delineated by the application of two-dimensional IR correlation spectroscopy to the
FTIR spectra acquired as a function of increasing temperature and heating cycles.
36
MATERIALS AND METHODS
Bovine β-lactoglobulin (β-Lg) composed of mixtures of genetic variants, A
and B, was a generous gift from Davisco Foods International (Eden Prairie, MN,
USA). The purity of the protein was confirmed by ESI-MS. D2O was obtained from
Aldrich (Milwaukee, WI, USA).
Two sets of β-Lg solutions were prepared in triplicate at a concentration of
5% (w/v) in D2O (pH ~ 6.8). These two sets were employed in the microwave
treatment and conventional heating studies, respectively.
Microwave Treatments
The β-Lg solutions prepared as described above were subjected to
microwave treatment using a focused microwave Synthewave 402 (PROLABO,
Fontenay-Sous-Bois, France), operating at a frequency of 2.45 GHz (λ = 12 cm),
with adjustable power between 15 and 300 W. A special equipment set-up was built
in order to have very good temperature control in both the microwave and
conventional thermal treatments. The temperature of the solution was measured at
the microwave cavity prior, during, and immediately after treatment. The
temperature of the solution was also measured prior to FTIR analysis. The accuracy
of the temperature measurements was within +0.5oC.
Sub-ambient temperature samples
Microwave experiments at sub-ambient temperatures were carried out by
circulating pre-cooled (-20oC) toluene through the outer jacket of a glass tube. The
β-Lg solution was placed in the inner tube. With this setup the β-Lg solution was
maintained at 4 ±0.5°C.
pre-cooled toluene
samples
37
With the use of this apparatus, microwave treatment was applied to β-Lg solutions
for 1-10 cycles and from 1-100 cycles; each cycle constituted 10 seconds of
treatment with the power set to 15 W (5 % of the total power). After each cycle, the
sample was immediately placed in a cold water bath until it reached 4°C.
Ambient temperature samples
Microwave treatment (15 W) was applied to β-Lg solutions for 1-10 cycles.
In each cycle the samples were subjected to microwave treatment until they reached
the desired temperature of 37oC, after which the samples were immediately placed
in a cold water bath until they cooled to 4°C.
Microwave-heated samples
β-Lg solutions were subjected to microwave treatment in order to attain
targeted temperatures in the range of 40-90°C (inclusive) at 10-degree intervals.
The treatment power was selected in order to achieve the desired sample
temperature within 5 minutes. For purposes of comparison, β-Lg solutions were
heated in a water bath to the same temperatures as targeted in the microwave
experiments. The desired temperature was attained within 5 minutes.
Effect of pH of microwave treatment samples
To study the effect of pH on the response of β-Lg solutions subjected to
microwave treatment, β-Lg (5% w/v) was dissolved in 0.2 M deuterated phosphate
buffers of the following pHs: 2, 4, 7 and 9. In this work, pH is employed in place of
pD and calculated from pD = pH + 0.4 (Boye et al., 1995). β-Lg (5% w/v) solutions
were also prepared in D2O containing 0.5 M and 2 M NaCl. Two sets of each of
these β-Lg solutions were prepared in triplicate, one of which was employed in the
microwave treatment studies while the other was subjected to conventional heating
under the same conditions. The temperature measurements were within +0.5oC.
38
Fourier Transform Infrared (FTIR) Spectroscopy
FTIR spectra were recorded (512 scans at 4 cm-¹) on a Magna 550 FTIR
spectrometer (Nicolet, Madison, WI) equipped with a deuterated triglycine sulfate
(DTGS) detector and purged with dry air. A sample volume of 8 μl was placed in an
IR cell consisting of two CaF2 windows separated by a 50-�m Teflon spacer. The
temperature of the sample was regulated by placing the IR cell in a
temperature–controlled holder. The reported temperatures are accurate to within
+0.1 ˚C.
The various steps in the FTIR data analysis are summarized in the flow
diagram presented below. Resolution enhancement of the amide I′ band in the
spectra was achieved with the use of the Fourier self-deconvolution (FSD) function
in OMNIC 7.3 (Nicolet, Thermo Electron Cooperation) employing a bandwidth of
22 cm-¹ and enhancement factor (k) of 2.4 as described by Kauppinen et al. (1981).
The FSD spectra were baseline-corrected between 1700 and 1600 cm-1 and
normalized by dividing the absorbance value at each wavenumber in this range by
the integrated area over this range. 2D correlation analysis was performed using the
KG2D software written by Y. Wang.
All samples were measured in triplicate and statistically analyzed using
Origin (Microcal Software Inc., Northhampton, MA). The standard error between
triplicate measurements was ~3%.
FTIR data analysis and 2D correlation procedure
Raw Data
(spectra)
Fourier self-deconvolution
(FSD)
Baseline
correction
Region (1700-1600) cm-1
Synchronous &
Asynchronous
maps
Sequence of
unfolding events
2D
correlation
39
Normalization
RESULTS AND DISCUSSION
FTIR Spectrum of -Lactoglobulin
The crystal structure of β-lactoglobulin (β-Lg) (Figure 3.1) reveals the
existence of nine antiparallel ß-strands and one α–helix (Papiz et al., 1986; Forge et
al., 2000). The Fourier self-deconvolved FTIR spectrum of β-lactoglobulin in D2O
in the amide I′ absorption region between 1700 and 1600 cm-1 at 25˚C is shown in
Figure 3.2. Six bands can be clearly discerned and have been previously assigned
by Boye et al. (1996) as follows. The most intense band, located at ~1632 cm-1, is
assigned to the low-frequency component of the amide I′ absorption of antiparallel
β-sheets and reflects the large proportion of β-sheets in this protein. A shoulder on
this at ~1623 cm-1 is attributed to extended β-sheet structure (Jackson and Mantsch,
1992; Fabian and Schultz, 1993). Sawyer et al. (1971) reported that β-Lg at pH 6.6
exists as a dimer at room temperature, and Casal et al. (1988) and Lefevre and
Subirade (1999) reported that the presence of the two components at ~1632 and
1623 cm-1 is characteristic of β-Lg in its dimeric form, while the presence of only
one component located at ~1630 cm-1 is associated with the monomeric form. The
bands at 1682 and 1676 cm-1 are assigned to high-frequency components of the
amide I′ absorption of antiparallel β-sheets that have undergone significant H-D
exchange. The band at 1662 cm-1 is assigned to turn structures, while the band at
1647 cm-1 represents the overlap of absorptions of α-helical and random coil
structures. Finally, the 1692 cm-1 band is assigned to β-sheet structure that has not
undergone H-D exchange. The rates at which H-D exchange of amide linkages in
proteins occurs when a protein is dissolved in D2O depend on whether or not the
amide N-H moieties are accessible to the solvent. Thus, the 1692 cm-1 band in the
FTIR spectrum of β-lactoglobulin has been assigned to amide groups of antiparallel
β-sheet(s) that are hidden in the interior of the protein and undergo very slow H-D
exchange at ambient temperatures (Boye et al., 1996). Exposure of the interior of
the protein to D2O (e.g. as a result of protein unfolding) results in the disappearance
of the 1692 cm-1 band, and accordingly this band can be an effective probe for the
propensity of �-Lg to unfold under various physicochemical conditions. More
40
specifically, the intensity of the 1692 cm-1 band has been found to decrease at
temperatures well below the denaturation temperature of β-lactoglobulin (Boye et
al., 1996), and accordingly changes in the intensity of this band serve as a probe of
changes in the tertiary structure that allow D2O to enter the interior of the protein.
Beyond the disappearance of the 1692 cm-1 band, the thermal behavior of
�-Lg is marked by progressive disappearance of the band at ~1623 cm-1, indicating
the dissociation of dimers into monomers upon heating (Lefevre and Subirade,
1999). Each of the other components of the amide I′ band broadens as the
temperature increases, especially at and above 70°C as a result of protein unfolding.
At 85°C the spectrum is characterized by two new components located near 1612
and 1680 cm-1, which are associated with the formation of intermolecular
hydrogen-bonded anti-parallel β-sheets resulting from protein aggregation (Clark et
al., 1981).
Figure 3.1: The crystal structure of a single subunit of bovine β-Lg (Brownlow et
al., 1997).
41
Antiparallel
β-sheet
β-Sheet
Absorbance
α-Helix
β-Turns
Antiparallel
β-sheet
Antiparallel
β-structure
0.0
1700
1680
1660
1640
1620
Wavenumbers (cm-1)
Figure 3.2: The amide I′ region of the FTIR spectrum of β-lactoglobulin in D2O
(5% w/v) at 25˚C after Fourier self-deconvolution (bandwidth = 22 cm-1 and k
factor of 2.4).
The pH dependence of the FTIR spectrum of β-Lg in D2O-phosphate buffer
(pH 2, 4, 7 and 9) in the amide I′ absorption region is shown in Figure 3.3. β-Lg is
known to undergo several local and global structural transitions between pH 2 and
pH 13. β-Lg exists as a dimer at physiological pH. However, if the pH is brought
down to 4.5, the protein agglomerates, forming an octamer. If the pH is further
reduced to 3.5, then the molecule exists as a monomer and conformational changes
slowly occur (Hambling et al., 1992). If the pH is increased above pH 8.5, there is
reversible dissociation of the dimer, and above pH 9.0, irreversible dissociation and
aggregation begin to occur. In general, the native-like β-barrel conformation
remains relatively unaffected between pH 2 and pH 9 (Blanch et al., 1999), whereas
a number of structural differences in the main α-helix (residues 129 to 142) have
42
been observed in X-ray crystallographic (Qin et al., 1998) and NMR data (Fogolari
et al., 1998). The FTIR spectrum recorded at pH 2 (Figure 3.3) shows bands similar
to those discussed for the spectrum at pH 7, although there are differences in the
bands at 1623 cm-1 and 1632 cm-1 indicative of an increase in β-strands and a
decrease in β-sheets, respectively. Papiz et al. (1986) found that one exposed
β-strand on each monomer is involved in the formation of the β-Lg dimer (between
pH 4 and pH 5) by forming an anti-parallel sheet. The increase in the intensity of
the 1632 cm-1 band relative to the 1623 cm-1 band may therefore be attributed to the
formation of this β-sheet structure at the expense of the β-strands involved, as
mentioned by Casal et al. (1988). Above pH 7, β-Lg has been reported to possess
increased reactivity which induces a unique phenomenon of intra- or intermolecular
thiol-disulfide interchange reactions (De Wit, 1989). This may explain the sharp
increase in the band at 1632 cm-1 relative to the band at 1623 cm-1.
Absorbance
pH
2
4
7
9
1700
1680
1640
1660
1620
-1
Wavenumbers (cm )
Figure 3.3: The amide I region of FTIR spectra of β-Lg at different pH values.
43
FTIR spectra of microwave-treated β-Lg
The response of β-Lg in D2O solution to microwave treatment was
monitored by acquiring FTIR spectra as a function of the number of treatment
cycles, and detailed examination of the Fourier self-deconvolved amide I′ band
profile in. these spectra was conducted to discern the effects of microwave
treatment on the protein’s structure. When β-Lg solutions were maintained at
sub-ambient temperature during microwave treatment, no changes in the amide I′
band were observed with increasing number of treatment cycles, up to 100 cycles.
On the other hand, microwave treatment of β-Lg solutions at 40oC resulted in a
progressive drop in the intensity of the 1692 cm-1 band with increasing number of
treatment cycles, together with the appearance of a new band at 1682 cm-1, a slight
increase in the 1645 cm-1 band, and a shift of the 1632 cm-1 band to 1630 cm-1
(Figure 3.4).
The above experiments were repeated with solutions of β-Lg in
D2O-phosphate buffer (pH 2, 4, 7 and 9) to examine the effect of solution pH on the
observed phenomena. In parallel, a replicate set of these solutions was subjected to
conventional heating cycles under identical temperature/time conditions and
monitored by FTIR spectroscopy. Irrespective of the pH and the type of heat
treatment applied (microwave vs. conventional), a decrease in the peak height of
the 1692 cm-1 band was observed as the number of heating cycles increased.
However, the magnitude of this decrease was strongly dependent on both the type
of heat treatment and the solution pH. Thus, irrespective of pH, the 1692 cm-1 band
exhibited a larger and more significant rapid drop in intensity in spectra of samples
that had been microwave treatment than in those of samples subjected to
conventional heat treatment. Similarly, irrespective of the type of heat treatment,
the magnitude of the drop in intensity of the 1692 cm-1 band increased with
increasing pH. A similar pH dependence was previously observed by Boye et al.
(1996), who examined the effect of temperature on β-Lg at different pH values.
They reported that the 1692 cm-1 band disappeared when the β -Lg solutions were
heated to 72, 59, 51, and 47°C at pH 3, 5, 7, and 9 respectively. They attributed the
44
increase in H-D exchange with increasing pH to a more relaxed state of β -Lg at pH
9 and a more compact state at lower pH values.
The combined effects of type of heating and pH resulted in a large variation
in the percent decrease in the peak height of the 1692 cm-1 band, as illustrated in
Figure 3.5. Thus, at the lowest pH examined (pH 2), the 1692 cm-1 band lost ~26%
of its intensity after 10 cycles of microwave treatment as compared to ~7% after 10
cycles of conventional heating while the corresponding values at the other end of
the pH range examined (pH 9) were 72% and 37% for the microwave and
conventionally heated samples, respectively. At neutral pH, the 1692 cm-1 band lost
more than half its intensity (57%) after 10 cycles of microwave treatment but only
one-quarter of its intensity after a matching conventional heat treatment.
The influence of ionic strength also was examined by conducting a similar
set of experiments using 0.5 M and 2 M NaCl solutions of �-Lg in D2O (pH ~6).As
shown in Figure 3.6., there was only a slight decrease of the 1692 cm-1 band after
conventional heating of these solutions whereas 10 cycles of microwave treatment
caused a substantial drop in the intensity of this band with this microwave-induced
effect being slightly more pronounced for the lower ionic strength solution The
latter difference may be attributable to the lower availability of D2O molecules for
protein solvation at higher salt concentrations.
In this context, the possible role of NaCl in stabilizing �-Lg dimers may be
noted. In studying the effect of temperature on the structure of β-Lg at different
ionic strengths using fluorescence spectroscopy, Albright and Williams (1968).
suggested the existence of a NaCl- dependent conformational change (step I) in the
β-Lg monomer prior to dimerization (step II):
Thus, if the presence of sodium chloride favors the formation of β*, the extent of
dimerization will be increased. Sedimentation equilibrium studies of the
association of β-Lg B showed that increasing the NaCl concentration from 0.05 to
0.10 M at pH 2.5 increased the extent of association of the protein. In addition,
45
Kella and Kinsella (1988) suggested, from their studies on the effects of urea and
heat on the association properties and structure of β-Lg AB at pH 6.85, that chloride
ions decreased the extent of dimer dissociation. Finally, FTIR and differential
scanning calorimetry studies of the effects of heat and sodium chloride
concentration on β-Lg showed that the presence of sodium chloride stabilized the
protein against heat denaturation (Boye et al., 1996). The authors suggested that
since the first step in heat denaturation of β-Lg is the dissociation into monomers,
the stabilization of β-Lg resulting from the presence of sodium chloride may be due
to reduced dissociation of dimers.
46
1.0
2.0
1.8
1.6
* B lac
* B l ac
* B l ac
* B l ac
* B l ac
* B l ac
* B l ac
* B l ac
* B l ac
* B l ac
cont rol
1cy cle MW 30C
2cy cle MW 30C
3cy cle MW 30C
5cy cle MW 30C
6cy cle MW 30C
7cy cle MW 30C
8cy cle MW 30C
9cy cle MW 30C
10cy cle M W 30C
Ab so r b ance
Absorbance
1.4
1.2
0.5
1.0
0.8
0.6
1692
0.4
0.2
0.0
0.0
1700
1700
1690
1680
1680
1670
1660
1650
1660
Wavenumbers (cm-1)
Wavenumbers
1640
1640
1630
1620
1620
Figure 3.4: Overlaid FTIR spectra in the amide I′ absorption region of β-Lg
solutions in D2O subjected to 1-10 cycles of microwave treatment at 40oC.
47
1610
100.0
80.0
60.0
pH2
40.0
20.0
0.0
N1
1
2
3
4
5
6
7
8
9
10
N2
N1
1
2
3
4
5
6
7
8
9
10
N2
N1
1
2
3
4
5
6
7
8
9
10
N2
N1
1
2
3
4
5
6
7
8
9
10
N2
100.0
80.0
60.0
pH4
40.0
20.0
0.0
100
80
pH7
60
40
20
0
100
80
pH9
60
40
20
0
Number of cycles
Figure 3.5: Bar graphs illustrating the decrease in the peak height of the 1692 cm-1
band in the FTIR spectrum of β-Lg in D2O at pH 2, 4, 7 and 9 as a function of the
number of cycles of microwave treatment (blue) and conventional heating (red) at
40°C.
N1 (set as 100%) corresponds to the peak height of the band in the spectrum of
native (untreated) �-Lg in D2O at 25oC; N2 represents the intensity observed when
this �-Lg solution was left at 25oC for 9 hours, which is the total time required to
complete the heating or microwave treatment experiments.
48
100.0
80.0
0.5M
NaCl
60.0
40.0
20.0
0.0
N1
1
2
3
4
5
6
7
8
9
10
N2
N1
1
2
3
4
5
6
7
8
9
10
N2
100.0
80.0
2M
NaCl
60.0
40.0
20.0
0.0
Number of cycles
Figure 3.6: Bar graphs illustrating the decrease of the peak height of the 1692 cm-1
band in the FTIR spectrum of β-Lg n 0.5 M NaCl and 2 M NaCl in D2O at 40°C as
a function of the number of cycles of microwave treatment (blue) and conventional
heating (red) at 40oC.
49
Effect of microwave and conventional heating at different temperatures on
-Lg at pH 7
The FTIR spectra in the amide I′ absorption region of �-Lg in D2O
subjected to microwave treatment and conventional heating at different
temperatures between 40 and 90oC are shown as a function of the number of
heating cycles (1 to 10) in Figures 3.7 and 3.8. Various changes indicative of
protein denaturation and aggregation are observed in the spectra recorded above
40oC, and these will be discussed in detail below, following a description of the
effect of temperature on the behavior of the 1692 cm-1 band. Figure 3.9 shows plots
of the peak height of this band as a function of the number of heating cycles for
microwave and conventional heating at 40, 50, 60, and 70oC. At each of the two
lower temperatures, microwave treatment had a more pronounced effect on the
band intensity than conventional heating, while no comparisons could be made at
higher temperatures because the 1692 cm-1 band disappeared almost completely
after the first cycle of either microwave or conventional heating. The bar graphs
presented in Figure 3.10 show the decrease in the peak height of the 1692 cm-1 band
with increasing number of microwave or conventional heating cycles at 40 and 50
o
C. At 40°C, the decrease following microwave treatment was more than double
that following conventional heating for the same number of cycles (e.g., a 34% vs.
15% decrease after the 4th heating cycle and a 54% vs. 25% decrease after the 10th
heating cycle). At 50°C, the 1692 cm-1 band dropped in intensity by 81% after the
4th microwave heating cycle and completely disappeared after the 5th, whereas in
the case of conventional heat treatment, the band dropped to 90% of its initial
intensity after the 10th cycle but did not completely disappear even after the
conventional heat treatment was extended to 15 cycles.
As noted earlier, decreasing intensity of the 1692 cm-1 band is attributed to
H-D exchange of amide groups that are buried within the interior of the protein in
its native state and is indicative of a loss of tertiary structure. Thus, the results
provide presented above provide evidence that the disruption of the tertiary
structure of �-Lg is enhanced by the use of microwave heating as compared to
conventional heating. Beyond this apparent effect on tertiary structure, the FTIR
50
spectra in Figures 3.7 and 3.8 reveal other microwave-induced differences in the
thermal behavior of �-Lg over the temperature range of 40-90oC, as described in
the following paragraphs.
1. At 40 ºC
An increase in the intensity of the band at 1676 cm-1 (assigned to the
high-frequency component of the amide I′ absorption of anti-parallel β-sheets)
together with a shift to 1674 cm-1 was observed with increasing cycles of
microwave treatment. A new band at 1682 cm-1 started to appear and increased in
intensity with increasing cycles of microwave treatment at the expense of the 1692
cm-1 band, which decreased. The band at 1647cm-1, which has been attributed to
α-helical structure or random coil (Casal et al., 1988), showed a very slight decrease
with increasing cycles of conventional and microwave treatment treatments. A
decrease in the intensity of the 1633 cm-1 band (assigned to the low-frequency
component of the amide I′ absorption of intramolecular anti-parallel β-sheets) was
observed with increasing microwave heating cycles. By comparison, β-Lg in D2O
subjected to conventional heating at 40°C under the same conditions showed very
little change in secondary structure (Figure 3.7).
2. At 50 ºC
An increase in the intensity of the 1676 cm-1 band (assigned to anti-parallel
β-sheets) was observed with a shift to 1674 cm-1 with increasing cycles of
microwave treatment. The 1682 cm-1 band started to appear from the 1st cycle of
microwave treatment and increased with increasing cycles of microwave treatment,
paralleling the decrease in the 1692 cm-1 band. The band at 1647 cm-1 shifted to
1645 cm-1 with a slight increase in intensity from the 1st microwave treatment cycle
to the 5th cycle and was not affected further with increasing cycles of microwave
treatment. The overall decrease during the first 5 cycles of conventional heating
was equivalent to the changes observed in the 1st cycle of microwave treatment. A
decrease in the intensity of the band at 1633 cm-1 was observed with a shift from
1633 to 1631 cm-1(Figure 3.7).
51
3. At 60 ºC
The 1682 cm-1 band markedly increased in intensity starting from the 1 st
cycle of microwave treatment and continued to increase until the 5th cycle,
paralleling the decrease in the 1692 cm-1 band. A slight increase in the intensity of
the 1676 cm-1 band after the 1st cycle was observed together with a shift to 1674
cm-1. The band at 1647 cm-1 shifted to 1645 cm-1 and decreased in intensity after the
1st cycle, then increased slightly with increasing microwave treatment cycles. The
band at 1633 cm-1 decreased and gradually shifted from 1633 to 1631 cm-1 as a
function of microwave treatment. A parallel rise of the band at 1625 cm-1 was
observed with increasing treatment cycles (Figure 3.7). In comparison, when β-Lg
in D2O was subjected to conventional heating up to a temperature of 60°C under
conditions similar to the microwave treatment experiments, the band at 1682 cm-1
increased in a similar manner to that observed during the microwave heat treatment,
albeit at lower intensity. A slight increase in the intensity of the 1676 cm-1 band
after the 1st cycle was observed with a shift to 1674 cm-1. The band at 1647 cm-1
decreased and shifted to 1645 cm-1 after the 1st cycle, then increased slightly with
increasing cycles of conventional heating. The band at 1633 cm-1 was also
observed, but shifted to 1632 cm-1 and then decreased after the 1st cycle.
Nevertheless, this band decreased slightly as the band at 1625 cm-1 increased in
intensity for all heating cycles. The magnitude of these changes was lower for
conventional heat treatment than for microwave treatment treatment, for all heating
cycles (Figure 3.7).
4. At 70 and 80ºC
A substantial drop in the intensities of the amide I′ bands occurred with the
concomitant appearance of new bands assigned to the formation of aggregate
structures after the 1st cycle of microwave treatment treatment. In comparison, β-Lg
in D2O subjected to conventional heating at 70°C under conditions similar to the
microwave treatment experiment showed the same trends as those observed for
conventional heating at 60oC, but with more intense changes. At 80oC the changes
in the amide I′ band were similar to those for the 70oC treatment in both microwave
52
and conventional heating, but the magnitude of the changes was significantly
higher (Figure 3.8).
5. At 90 ºC
The band at 1647 cm-1 shifted to 1644 cm-1 and decreased after the 1st cycle
of microwave treatment and disappeared after 3 cycles. The band at 1632 cm-1
decreased dramatically after the 1st cycle, shifted to 1628 cm-1 after the 2nd cycle
and almost disappeared after the 4th cycle. There was no significant difference in
the spectra acquired for the microwave treatment at 80ºC and 90ºC. The same
trends were observed for the conventional heating.
The microwave heating
treatments, however, had more pronounced effects and, as discussed below,
resulted in more aggregation of β-Lg than during conventional heating (Figure 3.8).
Effect of increasing temperature on the denaturation and aggregation of -Lg
Figure 3.11 shows the change in the peak height of the band at 1612 cm-1 ,
which is assigned to intermolecular anti-parallel �-sheets resulting from the
denaturation and aggregate formation of �-Lg at elevated temperatures (higher than
55oC) as a function of the number of conventional heating and microwave
treatment cycles. Heating at 60°C by either microwave treatment or conventional
heating did not result in aggregation of �-Lg. The 1612 cm-1 band associated with
aggregate formation was first observed after the 7th cycle of microwave treatment at
70 °C; in the case of conventional heating, this band was not observed at 70°C. At
80ºC, the 1612 cm-1 band was observed after the 4th cycle in the case of microwave
heat treatment, and after the 6th cycle in the case of conventional heating. At 90ºC
the 1612 cm-1 band appeared after the 2nd cycle in the case of microwave heat
treatment and after the 3rd cycle in the case of conventional heating. Thus, at all
temperatures, the extent of aggregation of β-Lg was higher during microwave
treatment than during conventional heating.
53
Conventional Heating
Microwave Heating
3.2
3.2
40
3.0
2.8
2.8
2.6
2.4
2.4
2.2
Absorbance
Absorbance
2.2
2.0
2.0
1.8
Abs orbance
Abs orbance
1.8
1.6
1.4
1.2
1.6
1.4
1.2
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
-0.2
-0.2
1700
1680
1700
1660
1640
Wavenumbers (cm-1)
Wavenumbers
1680
1660
1640
1700
1700
1620
1620
3.2
50
3.2
3.0
3.0
2.8
2.8
2.6
2.6
2.4
2.4
2.2
1660
1640
1660
1640
Wavenumbers (cm-1)
Wavenumbers
MW50-0
MW50-1
MW50-2
MW50-3
MW50-4
MW50-5
MW50-6
MW50-7
MW50-8
MW50-9
MW50-10
1620
1620
1600
50
Absorbance
2.0
2.0
1.8
Abs orbance
1.8
Abs orbance
1680
1680
Absorbance
2.2
1.6
1.4
1.2
1.6
1.4
1.2
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
-0.0
-0.2
-0.2
-0.4
-0.4
1700
1700
3.2
1680
1680
1660
1640
Wavenumbers (cm-1)
Wavenumbers
1660
1640
1620
1620
1600
1700
1700
2.8
1680
1660
1680
1660
Wavenumbers
(cm-1)
Wavenumbers
3.2
60
3.0
1640
1640
1620
1620
60
3.0
2.8
2.6
2.6
2.4
2.4
2.2
Absorbance
Absorbance
2.2
2.0
2.0
1.8
Absorbance
1.8
Absorbance
40
3.0
2.6
1.6
1.4
1.2
1.6
1.4
1.2
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
-0.2
-0.2
-0.4
-0.4
1720
1700
1700
1680
1660
1640
1680
1660
1640
Wavenumbers (cm-1)
Wavenumbers
1620
1620
1600
1700
1700
1680
1680
1660
1640
1660
1640
Wavenumbers (cm-1)
Wavenumbers
Figure 3.7: Overlaid FTIR spectra in the amide I’ absorption region of β-Lg
solutions subjected to microwave treatment and conventional heating at different
temperatures ranging from 40oC to 60oC.
54
1620
1620
Conventional Heating
Microwave Heating
3.2
3.2
70
3.0
2.8
2.8
2.4
2.2
2.2
Absorbance
2.6
2.4
Absorbance
2.6
2.0
2.0
1.8
Absorbance
1.8
Absorbance
70
3.0
1.6
1.4
1.2
1.6
1.4
1.2
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
-0.2
-0.2
-0.4
-0.4
1700
1700
1680
1680
1660
1640
Wavenumbers (cm-1)
Wavenumbers
1660
1640
1620
1620
1600
1700
1700
1680
1660
1640
1680
1660
1640
Wavenumbers (cm-1)
Wavenumbers
1620
1620
3.2
80
3.0
2.8
3.0
2.6
2.4
2.4
2.2
2.2
Absorbance
Absorbance
2.0
2.0
1.8
1.6
Absorbance
Absorbance
1.8
1.4
1.2
1.0
1.6
1.4
1.2
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
-0.0
0.0
-0.2
-0.2
-0.4
-0.4
1700
1700
1680
1680
1660
1640
1660
1640
Wavenumbers (cm-1)
Wavenumbers
1620
1620
1700
1700
1600
3.2
1680
1660
1640
1680
1660
1640
Wavenumbers (cm-1)
Wavenumbers
1620
1620
1600
3.2
90
3.0
2.8
90
3.0
2.8
2.6
2.4
2.4
2.2
2.2
Absorbance
Absorbance
2.6
2.0
2.0
1.8
Absorbance
1.8
Absorbance
80
2.8
2.6
1.6
1.4
1.2
1.6
1.4
1.2
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
-0.2
-0.2
-0.4
-0.4
1700
1700
1680
1680
1660
1640
1660
1640
Wavenumbers (cm-1)
Wavenumbers
1620
1620
1700
1700
1600
1680
1680
1660
1640
1660
1640
Wavenumbers (cm-1)
Wavenumbers
1620
1620
Figure 3.8: Overlaid FTIR spectra in the amide I’ absorption region of β-Lg
solutions subjected to microwave treatment and conventional heating at different
temperatures ranging from 70oC to 90oC.
55
1600
Peak hight of 1692 cm-1 band
100
90
80
70
60
50
40
30
20
10
0
CH
70
60
50
40
0
2
4
6
8
10
12
Peak hight of 1692 cm-1 band
Cycles
100
90
80
70
60
50
40
30
20
10
0
MW
70
60
50
40
0
2
4
6
8
10
12
Cycles
Figure 3.9: Plot of the decrease in the peak height of the band at 1692 cm-1 in the
FTIR spec:tra of β-Lg in D2O as a function of the number of cycles of microwave
treatment or conventional heating at the indicated temperatures.
56
Peak hight of 1612 cm-1 band
40 oC
120
100
80
60
40
MW
20
CH
0
0
1
2
3
4
5
6
7
8
9
10
Cycles
Decrease in 1692 cm-1 band
50 oC
120
100
80
60
40
MW
20
CH
0
0
1
2
3
4
5
6
7
8
9
10
Cycles
Figure 3.10: Bar graphs illustrating the decrease in the peak height of the 1692 cm-1
band in the FTIR spectrum of β-Lg in D2O (pH ~6) as a function of the number of
cycles of microwave treatment or conventional heating. at 40°C and 50°C.
57
Peak height of 1612 cm-1 band
MW
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
90
80
70
60
10
9
8
7
6
5
4
3
2
1
Cycles
Peak height of 1612 cm-1 band
CH
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
90
80
70
60
10
9
8
7
6
5
4
3
2
1
Cycles
Figure 3.11: Plot of the change in the peak height of the band at 1612 cm-1 in the
FTIR spectra of β-Lg in D2O as a function of the number of cycles of microwave
treatment or conventional heating at temperatures ranging from 60 to 90°C.
58
Elucidation of the unfolding mechanism of
-Lg as a function of heat
treatment
In order to ascertain the effect of microwave treatment on the sequence of
β-Lg unfolding, the Fourier self-deconvolved spectra of β-Lg solutions recorded as
a function of treatment cycles (Figures 3.7 and 3.8) were analyzed by 2D IR
correlation spectroscopy (Noda, 1993; Ismoyo et al., 2000). The synchronous and
asynchronous 2D IR spectra are plotted in Figures 3.12-3.15. Interpretation of the
upper diagonal of the synchronous and asynchronous maps led to the generation of
the sequence of events presented in Tables 3.1-3.3. Briefly, the first sign in any row
or column in the table is taken from the synchronous map. When this sign is
positive it means that the two correlated peaks on the X and Y axis are changing in
intensity in the same direction (either up or down); when the first sign is negative,
that means the two correlated peaks are changing in intensity in different directions.
When the two signs in any row or column are the same (either two positives or two
negatives) it means that the changes in the peak on the X axis take place before the
changes on the Y axis. When the two signs in any row or column are different
(either negative-positive or positive-negative), it means that the changes in the peak
on the Y axis take place before those on the X axis.
59
Synchronous
Asynchronous
Figure 3.12: The synchronous and asynchronous 2D IR spectra generated from the
difference spectra obtained during microwave treatment at 50oC.
60
Synchronous
Asynchronous
Figure 3.13: The synchronous and asynchronous 2D IR spectra generated from the
difference spectra obtained during conventional heating at 50oC.
61
Synchronous
Asynchronous
Figure 3.14: The synchronous and asynchronous 2D IR spectra generated from the
difference spectra obtained during microwave treatment treatment at 90oC.
62
Synchronous
Asynchronous
Figure 3.15: The synchronous and asynchronous 2D IR spectra generated from the
difference spectra obtained during conventional heating at 90oC.
63
Table 3.1: The sequence of unfolding events upon microwave treatment or
conventional heating of β-Lg at 50ºC.
cm-1
1621
1692
+-
1682
-+
1676
--
1647
--
1632
+-
1626
-+
+
++
++
--
1632
+-
-+
--
--
1647
-+
+-
++
1676
-+
+-
1682
-+
1626
-+
1621
The sequence of events of partial protein unfolding leading to enhanced
H-D exchange (prior to onset of aggregate formation) was found to be the same for
�-Lg during either conventional heating or microwave treatment at 50oC based on
the 2D maps in Figure 3.12 and 3.13. These changes included:
(i) an initial drop in the intensity of the 1692 cm-1 band accompanied by
simultaneous increases in the 1682 and 1626 cm-1 bands, assigned to anti-parallel
�-sheets exposed to solvent.
(ii) a drop in the population of extended anti-parallel β-sheets (1632 cm-1)
and β-strands (1621 cm-1) (the portion of the �-sheet structure initially shielded
from D2O), an increase in C=O…D-N hydrogen bonding of �-helix structure or
random coil (initially at 1647 cm-1, then shifts to 1645 cm-1) and, finally, an
increase in β-structure (1676 cm-1),.
64
Table 3.2: The sequence of unfolding events upon microwave treatment of β-Lg at
90 ºC.
cm-1
1692
1682
1676
1647
1640
1632
1626
1621
1612
--
++
--
--
++
--
++
--
1621
++
-
++
+
--
+
--
1626
-
+-
-+
-+
+
-+
1632
++
-
+
+-
-+
1640
--
++
+
1647
++
-
+
1676
++
-
1682
--
Table 3.3: The sequence of unfolding events upon conventional heating of β-Lg at
90ºC.
cm-1
1692
1682
1676
1647
1640
1632
1626
1621
1612
--
++
--
--
++
--
++
--
1621
++
--
++
++
--
++
--
1626
-
+
-+
-+
+-
-+
1632
++
--
+-
+
-+
1640
--
+
1647
++
--
1676
++
--
1682
--
65
The sequence of unfolding events of β-Lg under conditions of aggregate
formation (70 to 90 ºC) for both microwave and conventionally heated samples,
based on the 2D analysis of the results presented in Figures 3.14 and 3.15, is as
follows:
(i) an initial drop in the intensity of the 1692 cm-1 band accompanied by
simultaneous increases in the 1682 and 1626 cm-1 bands, assigned to anti-parallel
�-sheets exposed to solvent; this indicates that H-D exchange takes place via the
same route as at lower temperatures.
(ii) substantial loss of anti-parallel �-sheets (1632 cm-1), followed by an
increase in random structure (1640 cm-1) at the expense of �-structure (1676 cm-1),
followed by a loss of helical structure (1647 cm-1); substantial loss of β-strands
(1621 cm-1) above and beyond that observed upon H-D exchange, with the
formation and increase of intermolecular anti-parallel �-sheets (1612 cm-1),
indicative of aggregate formation. It should be noted that the amount of aggregation
was significantly higher in the microwave- treated samples. Therefore, microwave
treatment does not alter the unfolding pathway but accelerates the unfolding
process.
66
Kinetic parameters of β-Lg thermal unfolding
Measurements of the changes in the 1692 cm-1 band in the FSD FTIR
spectra of 5% β-Lg solutions in D2O recorded as a function of the number of cycles
of microwave and conventional heating under non-denaturing conditions were used
to determine the kinetic parameters for thermal unfolding of β-Lg. First-order rate
constants (k) at 40, 45, 50 and 55 °C were obtained by plotting ln(A/A0), where A is
the absorbance at 1692 cm-1, against the number of heating cycles. Figure 3.16
shows the Arrhenius plots, generated by plotting ln(k) against the inverse of the
temperature (in Kelvins), and their slopes and intercepts obtained by linear
least-squares fits, from which the energy of activation (Ea) and the pre-exponential
factor, respectively, were determined. The kinetic parameters obtained in this
manner for thermal unfolding of β-Lg subjected to microwave treatment and to
conventional heating are presented in Table 3.4.
Figure 3.16: Arrhenius plot for 5% β-lactoglobulin in D2O heated at 40, 45, 50 and
55°C for 10 conventional heating and microwave cycles analyzed in
triplicate by FTIR spectroscopy. Error bars represent one standard
deviation.
67
Table 3.4: Kinetic parameters for thermal unfolding of β-Lg (pH 6.5) under
non-denaturing conditions
Conventional
Heating
Microwave
Heating
Energy of activation
(kJ/mol)
207
150
Pre-exponential factor
75.3
56.1
Table 3.4 indicates that microwave treatment substantially reduces the
energy of activation for thermal unfolding of �-Lg. The Ea value obtained for
conventional heating, which is 207 kJ/mol higher than that for microwave
treatment, is in agreement with values reported in the literature for the thermal
denaturation of β-Lg. Kella and Kinsella (1988) measured the thermal unfolding of
β-Lg at pH 6.5 via a two-state model (native and folded) and obtained an Ea value of
224 kJ/mol. Ruegg et al. (1977) obtained an Ea value of 227 kJ/mol for the thermal
unfolding of β-Lg at pH 6.66, and De Wit and Swinkels (1980) reported a very
similar Ea value (230±15 kJ/mol). Le et al. (2004) obtained an Ea value of 206±12
kJ/mol for thermal unfolding of β-Lg at temperatures ranging from 65 to 80°C, and
van Teeffelen et al. (2005) reported an Ea value of 200 kJ/mol for thermal
denaturation in the presence of urea at concentrations ranging from 0.2 to 2.4 M. It
is noteworthy that these literature values are all significantly higher than the E a
value obtained in the present study for thermal unfolding of β-Lg subjected to
microwave treatment (Table 3.4).
Because the pre-exponential factor for microwave treatment in Table 3.4 is
lower than that for conventional heating, the enhanced rate of thermal unfolding
associated with microwave treatment can be ascribed solely to the reduction in the
energy of activation. In this regard, it may be noted that several authors have
discussed the possible role of the pre-exponential factor, related to collision
efficiency, in so-called microwave-enhanced effects. As explained by Perreux and
Loupy (2001), the collision efficiency can be effectively influenced by mutual
68
orientation of polar molecules involved in the reaction, as might occur in a
microwave field. Binner et al. (1995) found that the increased reaction rates
observed during the microwave synthesis of titanium carbide was attributable to an
increase in the pre-exponential factor. On the other hand, Lewis et al. (1992)
reported pre-exponential factors for an imidization reaction of 24±4 and 13±1 for
thermal and microwave treatments, respectively.
Given the possibility that
microwave-induced effects may result from localized microwave-induced heating
(“hot spots”), these authors employed their kinetic data to calculate an “effective
temperature” at the reaction site through use of the following equation:
where Kmicrowave and Kthermal are the rate constants measured at the observed
temperature (Tobserved) and Ea is the energy of activation determined from the kinetic
data for the thermal reaction. The “effective temperature” of the microwave
reaction is then given by Tobserved + ΔT. The experimental and effective
temperatures calculated in this manner from the kinetic data for the microwave and
conventional heat treatments of �-Lg are presented below:
Experimental
Temperature
(oC)
40
ΔT
(oC)
6.0
Effective
Temperature
(oC)
46
45
4.9
49.5
50
2.6
52.6
55
2.0
57.0
69
The ΔT values obtained are relatively small, as would be anticipated given that the
microwave treatment enhances the rate of unfolding by only a factor of ~2. It may
be noted that the value of ΔT decreases with increasing experimental temperature,
which does not seem consistent with the possibility of localized hot spots.
70
CONNECTING STATEMENT
In the previous chapter the effect of microwave heating on the structure of �-Lg
was examined by FTIR. The unfolding pathways of �-Lg were also elucidated by
two-dimensional (2D) correlation spectroscopy. The results of the FTIR
spectroscopic investigation demonstrated that microwave treatment had a
significant effect on �-Lg structure as compared to conventional heating to the
same temperature.
In the next chapter, CD, fluorescence, and 2D 1H NMR
spectroscopy were applied to extend the spectroscopic studies in order to confirm
and complement the results reported in the previous chapter.
71
Chapter 4: Investigation of the effect of microwave
treatment on the structure of
-lactoglobulin by
circular dichroism and fluorescence spectroscopy
INTRODUCTION
Experiments in which the responses of �-lactoglobulin (�-Lg) to various
microwave treatments were monitored by FTIR spectroscopy have provided
evidence that microwave-induced heating has substantial effects on the
conformational stability of the protein (Chapter 3). These effects were delineated
by comparison of the amide I′ band profile in the spectra of �-Lg solutions in D2O
subjected to microwave treatment and conventional heat treatments under the same
conditions. A key finding was that microwave treatment at temperatures in the
range of 40-60 oC significantly increased the rate of hydrogen-deuterium (H-D)
exchange of amide groups initially buried within the protein, indicating the loss of
tertiary structure.
Various spectroscopic techniques provide valuable insight into the effects
of varying physicochemical conditions on the secondary and tertiary structures of
proteins and may thus be employed to corroborate and complement the results of
these FTIR studies. For the elucidation of protein secondary structure in solution,
far-UV circular dichroism (CD) and FTIR spectroscopy provide complementary
information. For instance, antiparallel β-sheets and unordered structures are
generally more reliably estimated from FTIR spectra, whereas CD spectra are more
reliable for the prediction of �-helical content. Near-UV CD and fluorescence
spectroscopy are valuable techniques for monitoring changes in protein tertiary
structure resulting from changes in physicochemical conditions. Proteins display
near-UV CD and intrinsic fluorescence due to the presence of aromatic amino acids
such as tryptophan, tyrosine, and phenylalanine. The main advantages of
fluorescence are its exceptional sensitivity and the ability to collect dynamic
information from specific sites within the protein (Ladokhin, 2000). The changes in
72
the emission spectra of tryptophan, which is the dominant fluorophore, can provide
tertiary structural information, as the fluorescence of tryptophan is highly sensitive
to its local environment. However, the interpretation of the spectrum is not always
easy in the case of multi-tryptophan proteins, as the emission spectrum is the result
of the sum of the emissions from each residue and they overlap at most usable
wavelengths (Lakowicz, 1999). Manderson et al. (1999b) studied the effect of heat
treatment on bovine β-Lg using thiol availability and fluorescence spectroscopy.
The results showed that heat treatment of β-Lg resulted in detectable changes in the
structure of β-Lg, with the initial underlying structural change resulting from loss of
the hydrophobicity of the Trp19 environment (see Figure 4.1).
For the detailed examination of H-D exchange in proteins, 1H NMR
spectroscopy may be considered the technique of choice. The amide protons (HNs)
of unstructured peptides dissolved in D2O tend to exchange in minutes or hours
even under acidic conditions. On the other hand, amide protons that are buried
within the interior of a protein and thus hidden from contact with the solvent tend to
persist for many days or weeks. Therefore, 1H NMR spectra of freshly prepared
D2O solutions of proteins show peaks for solvent-exposed HNs that quickly
disappear, leaving behind the more persistent signals from HNs that are protected to
a greater or lesser extent from H/D exchange. Subsequent changes in
physicochemical conditions that increase the exposure of the HNs, resulting in
increased H-D exchange, which will be accompanied by a concomitant loss of their
signals in the 1H NMR spectra (Molday et al., 1972).
Although one-dimensional (1D) 1H NMR can be used to determine whether
a protein is folded or denatured and to determine whether exchangeable protons
have exchanged with solvent deuterium, 2D NMR affords higher resolution by
spreading the resonances across a second dimension (Belloque and Smith, 1998).
1
H 2D total correlation spectroscopy (TOCSY) (Braunschweiler and Ernst, 1983) is
well suited to H/D exchange measurements when working with non-isotopically
labeled proteins. The fingerprint region of a 2D TOCSY spectrum includes a single
peak for each residue (except prolines) of a protein’s sequence at coordinates that
correspond to the chemical shifts of the HNs, and peptide Cα hydrogen atoms (Hαs).
If the HNs of certain residues are exchanged for deuterium, then the HN/Hα
correlation peaks disappear from the spectrum.
73
Figure 4.1: The 3D structure and amino acid sequence of bovine β-Lg.
(A) Ribbon diagram of a single subunit of bovine β-Lg lattice X, whose pdb code is
1BEB (Sakurai, et al., 2009). The β-strands are labeled. Trp residues are
represented as balls and sticks. The diagram was produced using the program
MolFeat (FiatLux, Tokyo, Japan). (B) A schematic representation of the amino acid
residues of the β-Lg sequence. Residues making up the α-helix, β-sheet, and loop
are represented by hexagons in red, squares in blue, and circles in grey,
respectively. Green lines indicate the positions of disulfide bonds. Β-Lg can be seen
to have two β-sheets; The B–D strands and N-terminal half of the A strand (denoted
AN) consist of one and the E–H strands and the C-terminal half of the A strand
(denoted AC) represents the other.
74
Therefore, if the HN/Hα peaks in the fingerprint region can be assigned to their
respective residues, the persistence of structural features can be monitored by the
perpetuation of these TOCSY peaks from the protein in D2O solution as segments
of the protein backbone become increasingly exposed to the D2O solvent (Edwards
et al., 2002).
In this work, conformational changes of bovine β-Lg as a function of
microwave and conventional heat treatment were tracked by CD and fluorescence
spectroscopy to complement the results obtained from the FTIR studies described
above. In addition,1H 2D NMR TOCSY was employed to compare the extent of
H-D exchange in microwave-treated and conventionally heated β-Lg as well as to
identify, where possible, the specific amino acid residues undergoing H-D
exchange.
MATERIALS AND METHODS
β-Lg was subjected to conventional and microwave heating from 40°C to
90°C in 10oC increments, as previously described in Chapter 3. Following heating
treatments, the samples were analyzed by CD and fluorescence spectroscopy.
Circular Dichroism
CD spectra were recorded using a J-710 spectropolarimeter (Japan
Spectroscopic, Tokyo, Japan), operating under Jasco software. Protein solutions of
50 mg/mL that had been subjected to microwave treatment or conventional thermal
treatments were diluted to a concentration of 0.1 mg/mL (in 20 mM phosphate
buffer) for far-UV CD studies or 1 mg/mL for near-UV CD studies. A cylindrical
cell with a 2 mm path length was used for far-UV experiments while a cylindrical
cell of 10 mm path length was used for near-UV experiments. Typical instrumental
parameters were: bandwidth = 1.0 nm, sensitivity = 50 mdeg (for far UV) and 20
75
mdeg (for near UV), time constant = 2 s, step resolution = 0.2 nm/data, scan speed
= 20 nm/min, and number of accumulations = 5. The far-UV range scanned was
200–250 nm and the near-UV range scanned was 250–300 nm. All CD spectra were
recorded at room temperature (25°C).
Fluorescence Spectroscopy
Protein solutions (0.1 mg/mL) were prepared by a 500-fold dilution of the
50 mg/mL solutions (in H2O or 0.02 M phosphate buffer) that had been subjected to
microwave treatment or conventional thermal treatments. Samples was placed in a
1 cm quartz cuvette and scanned using an Aminco Bowman series II
fluorophotometer (Rochester, NY) in the wavelength range between 300 and 400
nm. Excitation parameters: step size: 1 nm, wavelength: 295 nm or 280 nm,
bandpass: 8 nm. Emission parameters: step size: 1 nm, wavelength: 320 nm,
bandpass: 4 nm. Emission scan: 300-400 nm, scan rate 1.00 nm/sec
NMR Spectroscopy
Microwave-treated β-Lg at 50ºC or thermally treated β-Lg at 50ºC or 60ºC
as well as non-treated β-Lg samples were selected for study by 2D 1H NMR
spectroscopy. To prepare samples for the NMR analysis, β-Lg solutions (1% in
D2O, pH 2) were microwave-treated at 50°C for 10 microwave cycles as described
previously (Chapter 3) or conventionally heated for up to 14 hours at 50°C or 60°C.
All microwave or conventionally heated samples as well as non treated samples
were stored frozen at -20°C for subsequent analysis by NMR.
All samples were examined using a 500-MHz Varian INOVA NMR
spectrometer equipped with a 5-mm triple-resonance H{C} cold probe with z-axis
gradients. One-dimensional proton spectra were recorded with a sweep width of
76
8000 Hz, 16 transients, 0.8 s acquisition time and a recycle delay of 2 s. Spectra
were processed using VNMRJ 2.2D. A cosine-squared apodization function was
applied and spectra were zero-filled prior to Fourier transformation. All spectra
were referenced to internal DSS at 0 ppm. TOCSY spectra with WATERGATE
(Piotto et al., 1992) water suppression to remove residual HOD signal and a 50 ms
mixing time were recorded with a sweep width of 8000 Hz in the direct dimension
and 6496 Hz in the indirect dimension. For each spectrum, 200-256 indirect points
were collected; each row contained 833-1024 complex points, with a recycle delay
of 0.9-1 s. The number of transients collected was 16 for pH 2 samples, TOCSY
spectra were processed using NMRPipe (Delaglio et al., 1995). Cosine-squared
apodization functions were applied in both dimensions, followed by zero-filling
and Fourier transformation. TOCSY spectra were visualized with NMRView
(Johnson, 2004), and assignments were made based on comparison to reported
chemical shifts (Uhrínováet al., 1998).
RESULTS AND DISCUSSION
CD in the far-UV region
CD spectroscopy in the far-UV region (200-250 nm) is frequently
employed to monitor changes in secondary structure (Schmid, 1989). Changes in
the β-Lg secondary structure before and after microwave or conventional heating
were investigated by CD measurements. Figures 4.2, 4.3 and 4.4 show far-UV CD
spectra of β-Lg subsequent to microwave and conventional heating from 40ºC to
90ºC in 10oC increments for 10 heating cycles at each temperature.
77
The native conformation of ��-Lg exhibits a minimum ellipticity at 213 and
216 nm in the CD spectrum, attributable to β-sheet structures (Yang et al., 1986).
The β-Lg subjected to conventional heating at 40 and 50°C possessed the same CD
spectrum as the native protein. On the other hand, microwave-treated β-Lg
exhibited a slight increase in ellipticity with a tendency to shift towards shorter
wavelength (a shift from 213 to 206 nm) typical of α-helical structure. In all cases
microwave-treated β-Lg solutions had a higher ellipticity and a shift towards
shorter wavelength than those subjected to conventional heat treatment. The
maximum ellipticity was -60 mg deg, achieved by microwave heating of ��Lg at
90 ºC for 2 cycles. The same effect was achieved with conventional heating after 10
cycles.
The CD spectra were analyzed with the use of DichroWeb software
(Whitmore and Wallace, 2004) to estimate the secondary structure content of the
protein as a function of increasing temperature. The output of the structure
prediction software is shown in Table 4.1. The effect of microwave and
conventional heat treatment on the secondary structure (as predicted by the
software) is plotted in Figures 4.5 and 4.6. Table 4.1 shows that the native structure
of �-Lg contains 15.1% α-helix and 50.8% β-sheet. No change was observed when
β-Lg was heated at 40°C by conventional means, whereas in the case of microwave
heating the β-sheet content decreased to 47.6%.
78
At 40° C:
A
B
30
30
20
20
0
0
CD[mdeg] -20
CD[mdeg] -20
-40
-40
-60
200
210
220
230
240
250
-60
200
210
Wavelength [nm]
220
230
240
250
240
250
Wavelength [nm]
At 50° C:
CD[mdeg]
20
20
0
0
-20
CD[mdeg]
-40
-60
200
-20
-40
210
220
230
240
250
-60
200
Wavelength [nm]
210
220
230
Wavelength [nm]
Figure 4.2: Far-UV CD spectra (200-250 nm) of β-Lg after (A) conventional
heating and (B) microwave treatment at 40 and 50 oC.
79
At 60° C:
B
A
CD[mdeg]
20
20
0
0
-20
CD[mdeg]
-40
-40
-60
200
-20
210
220
230
240
250
-60
200
210
220
230
240
250
240
250
Wavelength [nm]
Wavelength [nm]
At 70° C:
20
10
0
0
CD[mdeg]
-20
-20
CD[mdeg]
-40
-40
-60
200
210
220
230
240
250
Wavelength [nm]
-60
200
210
220
230
Wavelength [nm]
Figure 4.3: Far-UV CD spectra (200-250 nm) of β-Lg after (A) conventional
heating and (B) microwave treatment at 60 and 70 oC.
80
At 80° C:
B
A
20
20
0
0
CD[mdeg]
CD[mdeg]
-20
-40
-40
-60
200
-20
210
220
230
240
-60
200
250
210
220
230
240
250
Wavelength [nm]
Wavelength [nm]
At 90° C
30
30
20
20
0
0
CD[mdeg]
CD[mdeg]
-20
-40
-40
-60
-60
-70
200
-20
210
220
230
240
250
Wavelength [nm]
-70
200
210
220
230
Wavelength [nm]
Figure 4.4: Far-UV CD spectra (200-250 nm) of β-Lg after (A) conventional
heating and (B) microwave treatment at 80 and 90oC.
81
240
250
For both microwave and conventional heat treatments at temperatures of ≥50oC,
decreases of both �-sheet and �-helical content were observed with increasing
temperature (Table 4.1), with larger changes in the case of microwave treatment.
Finally, at 90ºC, the β-sheet and �-helical content in the case of microwave
treatment decreased to 28.7% and 8.3%, respectively, as compared to 34.4% and
10.5% in the case of conventional heating. These trends are largely in agreement
with our FTIR results (Chapter 3), which also indicate decreases in (intramolecular)
�-sheet and �-helical content over the temperature range of 50-90oC. In contrast,
other authors have reported that the CD spectra of β-Lg A at elevated temperatures
show that a decrease in �-sheet content is accompanied by an increase in �-helical
content (Griffin and Griffin, 1993; Matsuura and Manning, 1994); however, their
CD measurements were made under different experimental conditions (e.g., protein
concentration, ionic strength) from those employed in the present study.
CD in the near-UV region
The near-UV CD spectra of conventional and microwave heat-treated
samples of β-Lg are shown in Figures 4.7, 4.8 and 4.9. The near-UV CD spectra of
native β-Lg were similar to those previously reported in the literature, showing
negative bands at 266, 285 and 293 nm associated with the rigid tertiary structure of
native β-Lg (Kuwayima et al., 1996; Manderson et al., 1999; Considine et al.,
2007). The conventional and microwave heat treatments resulted in a decrease in
the intensity of the 285 and 293 nm bands with increasing temperature. Samples
treated with microwave and conventional heating exhibited a significant decrease
in the magnitude of negative bands in the near-UV CD spectra, an indication of loss
of aromatic dichroism (Matsuura and Manning, 1994; Ptitsyn et al., 1995). At all
temperatures, a substantial reduction in the intensity of bands was observed in the
case of microwave- treated samples compared to conventionally heated samples.
Figures 4.7, 4.8 and 4.9 show that microwave treatment resulted in a substantial
enhancement in the loss of native �-Lg structure compared to conventional heating.
82
At 40 and 50ºC the effect was less pronounced, but at 60ºC a significant difference
was observed between conventional and microwave heating.
In a previous study Manderson et al. (1999a) showed a strong correlation between
the CD signal at 293 nm and the quantity of native �-Lg in a heated sample,
indicating that the loss of chirality was an index of the availability of a free thiol
(Manderson et al., 1999b) and was probably linked to the interaction of Cys121
with Cys106:Cys119 (Creamer et al., 2004).
A decrease in the protein's dichroism signals indicates that an increasing number of
aromatic side-chains (Trp, Tyr, and Phe) were exposed to a modified environment;
these changes are related to the loss of native-like structure (Chang and Yang,
1978). Our results with conventional heating were in strong agreement with the
literature. Many authors have reported that thermal treatment of �-Lg caused a
reduction in the intensity of the bands ascribed to tryptophan (288 and 293 nm)
(Manderson et al., 1999; de la Hoz, and Netto, 2008). Microwave-heated �-Lg
solutions also exhibited a more pronounced decrease in their near-UV CD spectra,
suggesting a more dramatic change in the tertiary structure than in the case of
conventional heating and supporting the results obtained in our FTIR spectroscopic
studies.
83
Table 4.1: β-Sheet and α-helix contents estimated from far-UV spectra of β-Lg
subjected to microwave treatment and conventional heating at various
temperaturesa
β-Sheet
α-Helix
Temp.
Microwave
Conventional
Microwave
Conventional
ºC
treatment
heating
treatment
heating
-
a
50.8%
15.1%
40
47.6%
50.8%
16.4%
15.1%
50
42.9%
46.1 %
17.5%
16.1%
60
39.6%
43.0 %
15.6%
14.3%
70
36.8%
41.5%
10.7%
12.1%
80
33.5%
38.5%
9.9%
11.2%
90
28.7%
34.4%
8.3%
10.5%
As estimated by DichroWeb software.
Figure 4.5: α-Helix content estimated from far-UV spectra of β-Lg subjected to
microwave treatment and conventional heating at various temperatures
84
Figure 4.6: �-Sheet content estimated from far-UV spectra of β-Lg subjected to
microwave treatment and conventional heating at various temperatures
85
At 40°C:
-2
-4
MW
-6
CD[mdeg]
CH
-8
Native
-10
-11
250
260
270
280
290
300
Wavelength [nm]
At 50° C:
-2
-4
MW
-6
CH
CD[mdeg]
-8
Native
-10
-11
250
260
270
280
290
300
Wavelength [nm]
Figure 4.7: Comparison of near-UV CD spectra of heat-treated and
microwave-treated β-Lg (treated at temperatures of 40 and 50ºC) with the spectrum
of native β-Lg.
86
At 60° C:
-2
MW
-4
CH
Native
-6
CD[mdeg]
-8
-10
250
260
270
280
290
300
Wavelength [nm]
At 70° C:
-1
-2
MW
CH
-4
CD[mdeg] -6
Native
-8
-10
250
260
270
280
290
300
Wavelength [nm]
Figure 4.8: Comparison of near-UV CD spectra of heat-treated and
microwave-treated β-Lg (treated at temperatures of 60 and 70ºC) with the spectrum
of native β-Lg.
87
At 80° C:
0
MW
CH
-5
CD[mdeg]
Native
-10
-11
250
260
270
280
290
300
Wavelength [nm]
At 90° C:
-1
MW
-2
CH
-4
Native
CD[mdeg]
-6
-8
-9
250
260
270
280
290
300
Wavelength [nm]
Figure 4.9: Comparison of near-UV CD spectra of heat-treated and
microwave-treated β-Lg (treated at temperatures of 80 and 90ºC) with the spectrum
of native β-Lg.
88
Fluorescence
Intrinsic fluorescence spectra of β-Lg solutions heated conventionally and
by microwave treatment were acquired. Figure 4.10 shows the fluorescence spectra
of native as well as microwave- and conventionally-heated β-Lg samples at 40, 60
and 90ºC. According to the literature, �-Lg exhibits λmax at 333 nm, which is
attributed to tryptophan residues. The λmax of tryptophan shifts to a longer
wavelength (red shift) and the intensity of λmax decreases as the polarity of the
solvent increases (de la Hoz and Netto, 2008). Phenylalanine and tyrosine have
weak fluorescence and are normally not observed. At 40ºC, microwave-treated and
conventionally-heated β-Lg did not show any red-shift but only a slight decrease in
the intensity of λmax at 333 nm, with a more significant decrease in the case of
microwave heating (Figure 4.10). At 60ºC, �-Lg showed λmax values at 339 nm
(red-shift of 6 nm) and 343 nm (red-shift of 10 nm), in the cases of conventional
and microwave heating respectively, with a larger decrease in the peak in the case
of microwave-heated samples (Figure 4.10). After both conventional and
microwave treatments at 90ºC, �-Lg showed a λmax value of 343 nm (red-shift of 10
nm), with a larger decrease in the peak occurring in the case of microwave-treated
samples (Figure 4.10). A red-shift is indicative of increased exposure of Trp
residues to the solvent (Lakowicz, 1999), signaling a loss of tertiary structure or
denaturation of protein (Chen & Barkley, 1998). Some red-shift values reported for
chemically denatured �-Lg are 13 nm in the presence of 8 M urea (Yang et al.,
2001), 18 nm in 9.5 M urea (Creamer, 1995) and 20 nm in 2.5 M guanidine
hydrochloride (Subramaniam et al., 1996). Red-shift values of 4 and 8 nm were
reported for �-Lg subjected to high-pressure of 600 MPa (Yang et al., 2001) or
heating to 85ºC (Manderson et al., 1999b), respectively. Al-Jundi (2004) reported a
red shift from 331 nm to 333 nm for microwave treated hæmoglobulin at 48ºC with
no change in the case of conventional heating. At 54ºC a red shift of 7 nm was
observed for microwave-heated samples as compared to only a 2-nm shift in the
case of conventionally heated samples.
89
Β-Lg
Β-Lg
CH CH
MWMW
Emission
750
500
40 ºC
250
0
300
320
340
Wavelength (nm)
360
Emission
750
380
Β-Lg
Β-Lg
CH
CH
MW
MW
500
60 ºC
250
0
300
320
340
Wavelength (nm)
360
380
750
Emission
Β-Lg
Β-Lg
500
90 ºC
CH
CH
MW
MW
250
0
300
320
340
Wavelength (nm)
360
380
Figure 4.10: Fluorescence intensity of Trp residues for β-Lg samples subjected to
microwave treatment and conventional heating at 40, 60 and 90ºC. The upper trace
in each panel shows the fluorescence intensity measured for native β-Lg.
90
NMR Spectroscopy
The NMR study of large proteins or protein aggregates is problematic
because of congestion of the spectra and broadening of the signals. This has meant
that all detailed NMR studies of native bovine β-Lg structure have been performed
to date at low pH where β-Lg is monomeric (McKenzie and Sawyer, 1967). β-Lg is
unusually stable to acid denaturation, and circular dichroism studies in the near-UV
region have shown that the structure of β-Lg at pH 2 is essentially the same as that
at neutral pH (Molinari et al., 1996). Accordingly, the present study was carried out
at pH 2 as the protein is in the monomeric form at this pH. The TOCSY spectrum of
β-Lg in D2O at pH 2 is shown in Figure 4.11. The expanded region in the bottom
panel shows the (HN, Hα) cross peaks, which correspond to amide protons that have
not undergone H-D exchange with the D2O solvent and hence belong to residues
that are protected from the solvent. The presence of such a large number of
unexchanged amide groups confirms the conservation of the secondary structure at
pH 2, in agreement with previous studies (Molinari et al., 1996). Assignments of
the backbone HN and Hα peaks for 44 of the 76 amide peaks observed in Figure 4.11
were made by reference to the assignments for a 15N- and 13C-labelled recombinant
form of β-Lg A (Uhrínováet al., 1998) and are presented in Table 4.2. These
assignments could be confirmed as additional TOCSY cross peaks, usually H �,
were observed for all of them. The locations of the identified residues within the
secondary structure of β-Lg may be seen by examination of Figure 4.1.
The extent of further H-D exchange induced by microwave treatment of
�-Lg at 50oC as well as by conventional heating at 50oC and 60oC was assessed by
detailed examination of the TOCSY spectra acquired from D2O solutions (pH 2.0,
25°C) of samples that had been subjected to these pretreatments. No changes were
observed in the aliphatic region of any of these spectra, indicating that there was no
major conformational change or degradation of the protein during these treatments.
Figure 4.12 shows the (Hn, Hα) cross peaks in the TOCSY spectrum of the
microwave-treated β-Lg. By comparison with Figure 4.11, the number of cross
peaks is markedly reduced, indicating that microwave treatment at 50°C resulted in
91
a substantial increase in H-D exchange. Indeed, only 10 cross peaks were observed
in this TOCSY spectrum: based on Table 4.2, these are assigned to isoleucine-56,
leucine-57, and leucine-58, which are located in strand C, threonine-102,
leucine-104, phenylalanine-105, and methionine-107 which are located in strand G,
and valine-118, leucine-122, and valine-123, which are located in strand H. It is
interesting to note that this list encompasses all six of the residues (namely,
isoleucine-56, leucine-57, threonine-102, leucine-104, phenylalanine-105, and
methionine-107) reported by Belloque and Smith (1998) to be thermally resistant to
H-D exchange up to 75°C, which was the highest temperature investigated in their
experiments. In a subsequent study, Edwards et al. (2002) reported H-D exchange
of these residues between 75°C and 80°C, except for phenylalanine-105. Based on
the observation that the peak assigned to the latter was the only identifiable HN
signal at 80°C, Edwards et al. (2002) indicated that the G–H pair of disulfide-linked
strands forms the most heat-resistant feature of the β-Lg structure. This structural
feature also appears to be resistant to microwave-induced heating, given that the
cross peak assigned to phenylalanine-105 is among the small number of cross peaks
observed in the TOCSY spectrum of �-Lg acquired after microwave treatment at
50oC.
In contrast to the above findings, the pattern of (Hn, Hα) cross peaks in the
TOCSY spectrum of �-Lg that had been conventionally heated at 50oC (Figure
4.13a ) was similar to that obtained for untreated β-Lg, with the loss of only seven
cross peaks. Among the latter, four could not be assigned to specific residues while
the other three were assigned to glutamic-44, which is located in strand B,
lysine-47, which is located in strand B, and lysine-100, which is located in the loop
between strands F and G. These results are consistent with those obtained by
Edwards et al. (2002) in a 2D NMR study, in which lysine-47 and lysine-100 were
found to undergo H-D exchange at 45°C and 37°C, respectively. When the
conventional heating pretreatment was carried out at 60oC (Figure 4.13b), there was
an additional loss of 12 cross peaks that were present in the TOCSY spectrum
acquired after conventional heating at 50oC. Seven of these could be assigned to the
following residues based on the assignments in Table 4.2: alanine-26, which is the
92
last residue of strand A, tyrosine-42, which is the first residue in strand B,
glutamic-74 which is located in the loop between strands D and E, leucine-32,
which is located in the loop between strand A and strand B, alanine-80, which is
located at the end of the loop between strands D and E, and lysine-138 and
alanine-139, which are both located in the loop between the α-helix and strand F.
Many of these cross peaks are reported in the literature to undergo H-D exchange
around 60°C. Alanine-26 has been reported to undergo H-D exchange at 55°C
(Belloque and Smith, 1998) or at 60°C (Edwards et al., (2002), and tyrosine-42 and
leucine-32 have been reported to undergo H-D exchange above 45°C.
Overall, the results obtained for the conventionally heated samples are in
agreement with the results reported by Edwards et al., (2002), who studied the
thermal unfolding of the A variant of bovine β-Lg, in 50 mM phosphate buffer at
pH 3.0, by 2D NMR examination of H-D exchange at temperatures between 37°C
and 80°C. They reported that residues in loops and in the terminal regions rapidly
undergo H-D exchange when the protein is dissolved in D2O. Further H/D
exchange was promoted by heat treatment above 55°C and attributed to residues in
the D and E strands, indicating that the hydrogen bonds between the D and E
strands had been broken at ~55°C. Also, they reported that A–B and B-C strands
undergo H-D exchange at temperatures between 65 and 70°C and between 75 and
80°C, respectively. In the present study, these regions did not undergo H-D
exchange under conventional heating up to 60°C as expected. However, the
microwave heated samples exhibited complete H-D exchange in these regions at
50°C. In addition, Belloque and Smith (1998) reported that H-D exchange in the
α-helix occurred at 75°C by conventional heating, whereas H-D exchange of
α-helix amide protons was seen at 50°C in the TOCSY spectra of microwave
treated β-Lg.
93
Figure 4.11: TOCSY spectrum of untreated β-Lg in 100% D2O (pH 2, 25°C) at 500
MHz.
94
Table 4.2: Peak assignments of TOCSY spectrum of untreated β-Lg in D2O (pH 2,
25°C)
HN
Residue
Hα
Intensity
Number
number
(ppm) (ppm)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
{15.HA}
{19.HA}
{20.HA}
{21.HA}
{23.HA}
{25.HA}
{26.HA}
{32.HA}
{41.HA}
{42.HA}
{44.HA}
{46.HA}
{47.HA}
{54.HA}
{56.HA}
{57.HA}
{58.HA}
{71.HA}
{73.HA}
{74.HA}
{81.HA}
{82.HA}
{83.HA}
{90.HA}
{91.HA}
{92.HA}
{93.HA}
{95.HA}
{100.HA}
{102.HA}
{103.HA}
{104.HA}
{105.HA}
{106.HA}
{107.HA}
{118.HA}
{119.HA}
{121.HA}
{122.HA}
{123.HA}
{138.HA}
{139.HA}
{140.HA}
{149.HA}
4.86
4.82
5.23
4.90
5.23
5.33
5.45
4.70
4.70
4.87
5.15
5.19
5.16
4.72
4.40
5.17
5.46
4.52
5.11
4.65
5.01
5.28
5.07
5.74
5.62
4.88
5.40
4.34
4.37
6.59
4.45
5.79
5.85
5.76
5.74
5.25
5.70
4.69
5.98
6.18
4.01
4.14
3.95
4.76
95
7.43
7.50
9.08
9.84
7.71
9.30
8.36
7.10
6.94
8.61
8.95
8.67
9.16
8.77
9.27
8.51
9.09
8.93
9.27
9.45
7.33
9.54
9.46
9.09
8.59
9.07
9.42
8.42
8.98
8.72
9.20
9.30
8.91
9.65
9.91
8.81
9.42
7.92
9.73
9.49
7.91
7.85
7.49
9.14
0.0323
0.003
0.0075
0.0016
0.0063
0.006
0.0055
0.0061
0.009
0.0071
0.0061
0.0162
0.0118
0.0108
0.0122
0.014
0.0211
0.0239
0.01
0.0074
0.013
0.0089
0.0129
0.0101
0.0154
0.0137
0.0118
0.0093
0.0078
0.0176
0.0069
0.0098
0.0176
0.0037
0.0075
0.0097
0.005
0.0065
0.0077
0.0181
0.0064
0.0035
0.0073
0.0103
Figure 4.12:(Hn, Hα) cross peaks from TOCSY spectra of β-Lg, (pH 2.0, 25°C) after
microwave treatment at 50°C.
96
Figure 4. 13: (Hn, Hα) cross peaks from TOCSY spectra of β-Lg, (pH 2.0, 25°C)
showing the non-exchanged resonances that persist through conventional heat
treatment (light to dark color) at 50°C (a) 60°C (b).
97
CONCLUSION
The far-UV CD spectra (190-260 nm) of β-Lg subjected to microwave or
conventional heating from 40ºC to 90ºC for 10 temperature cycles showed
minimum ellipticity at 213 and 216 nm. The β-Lg subjected to conventional heating
at 40 and 50 °C presented the same CD spectrum as the native protein while the
microwave-treated β-Lg exhibited a slight increase in ellipticity. In all cases,
microwave-treated β-Lg solutions had a higher ellipticity and a shift towards a
lower wavelength than the conventional heat-treated solutions. Although no change
was observed in the native structure of β-Lg conventionally heated to 40°C, a
decrease in the content of β-sheets and a slight increase in the α-helical content
occurred with microwave heating. At temperatures of ≥50°C, a decrease in both
β-sheet and α-helical content occurred with both microwave and conventional
heating, with a higher decrease in both structures being observed for microwave
heating. These results are in agreement with FTIR results, which showed that the
decrease in native β-sheet content was accompanied by a decrease in the �-helical
content and a substantial increase in intermolecular antiparallel β-sheet content. On
this basis, it is likely that the CD prediction algorithms are an inadequate tool for
predicting the intermolecular �-sheet content. Accordingly, it may be worthwhile to
develop calibration algorithms employing CD spectra from samples of varying
intermolecular �-sheet content to compensate for this limitation. The near-UV CD
spectra of β-Lg provided evidence of a significant loss of aromatic dichroism. A
substantial reduction of the intensity of bands in the near-UV CD spectra was
observed in the case of microwave vs. conventional heating at all temperatures. The
increased solvent exposure of tryptophan residues under microwave vs.
conventional heating of β-Lg solutions was shown by the substantial decrease in the
fluorescence intensity and the red shift of the emission maximum in the case of
microwave treatment, at temperatures up to 90ºC. Finally, the results of 1D 1H
NMR TOCSY experiments showed that much more extensive H-D exchange had
occurred in �-Lg solutions in D2O (pH 2) that had been subjected to microwave
treatment at 50oC than in solutions that had been conventionally heated at either 50
or 60oC. Thus, the results of all the various spectroscopic studies conducted in this
work support a substantial enhancing effect of microwave vs. conventional heating
on the thermal unfolding of �-Lg, in agreement with the results previously obtained
by FTIR spectroscopic studies.
98
CONNECTING STATEMENT
In the previous two chapters the effect of microwave heating on the
structure of �-Lg was examined by FTIR, CD, fluorescence, and 1H NMR
spectroscopy. The unfolding pathways of �-Lg were also elucidated by
two-dimensional (2D) correlation spectroscopy. The results of the spectroscopic
investigation demonstrated that microwave treatment had a significant effect on
�-Lg structure as compared to conventional heating to the same temperature. In the
next chapter a study of the effect of microwave treatment on the efficiency of the
enzymatic hydrolysis of �-Lg is presented.
99
Chapter 5: Study of the effect of microwave treatment
on the enzymatic hydrolysis of
lactoglobulin
INTRODUCTION
The enzymatic hydrolysis of �-lactoglobulin (�-Lg) is of substantial interest
in relation to the production of both infant formulas and nutraceuticals. With
respect to the first of these, although breast-feeding is the best form of nutrition for
neonates, mother’s milk may be replaced or supplemented with infant formulas
when breast-feeding alone is not possible throughout the early months of life. Most
formulas employ bovine milk protein hydrolysates to meet infant nutritional
requirements as they provide an excellent source of essential amino acids
(Hernandez-Ledesma et al., 2004) and show greater intestinal absorption due to
their increased solubility (Ziegler et al., 1998). In addition, allergy to cow’s milk is
one of the most common allergies among children under 2 years of age; indeed,
1–2% of newborns show an allergenic response to cow’s milk (Svenning et al.,
2000). Accordingly, the enzymatic proteolysis of whey protein is normally used in
the production of partially or extensively hydrolyzed formulas to lower the content
of intact β-Lg, which is responsible for most of the antigenicity of milk proteins.
Partial protein hydrolysis should be sufficient to remove epitope structures and is
preferred over extensive hydrolysis, which makes the product less palatable (bitter)
and can also reduce functional properties such as emulsifying activity and emulsion
stability. Therefore, a balance between elimination of allergenicity and functional
and nutritional attributes must be achieved. However, residual allergenicity has
been reported in several commercial preparations (Restani et al., 1995; Van
Beresteijn et al., 1995), which could be due to inaccessibility of some sequential
epitopes to proteases, even in the denatured protein
Milk proteins, in addition to their nutritional properties, also represent a rich
source of biologically active peptides, ranging in activity from mineral binding
(Toba et al., 2000), to opioid (Bitri, 2004) and inmunomodulatory effects (Dutta,
100
2002). The nutraceutical properties of whey proteins include a lowering of blood
pressure, attributed to the inhibition of the angiotensin converting enzyme (ACE),
as well as increases in blood and tissue glutathione (GSH) concentrations, which in
turn promote detoxification of free radicals produced by the metabolism of
carcinogenic compounds (Bounous, 2000). The bioactivities of several milk
proteins are latent, either absent or incomplete in the native protein. Only during
proteolytic digestion of the protein are the active peptides released from the native
protein.
As such, enzymatic hydrolysis of �-Lg can provide a route to the
identification and production of bioactive peptides, and it is primarily from this
nutraceutical perspective that the research presented in this and the next chapter
was undertaken.
The overall objectives of the work described in this chapter were to: (i)
investigate the extent of enzymatic hydrolysis of β-Lg following various
microwave and conventional heating treatments and (ii) to interpret the results in
the light of the structural studies described in Chapters 3 and 4. As described in the
latter chapters, heating at 40ºC resulted in minor changes in the structure of �-Lg in
solution, while at 60ºC the tertiary structure was altered, and at 90ºC considerable
loss of secondary and tertiary structures occurred. Based on these results, the
heating temperatures selected for this work were 40ºC, 60ºC and 90ºC. The
experimental approach involved the following:
1. Microwave treatment and conventional thermal treatment of �-Lg at the
three selected temperatures as described in Chapter 3.
2. Enzymatic hydrolysis by means of in vitro digestion with enzymes
currently employed in generating hydrolysates from �-Lg (pepsin, trypsin
and chymotrypsin) in addition to a two-stage enzymatic hydrolysis process
simulating the human gastrointestinal digestion process.
3. Measuring the degree of hydrolysis and the kinetic parameters of the
substrate as a function of the pretreatment applied.
101
MATERIALS AND METHODS
-Lg samples
Samples of �-Lg that had been microwave- or thermally-treated at 40ºC,
60ºC or 90ºC, as well as native �-Lg were examined. These samples are designated
MW40 and CH40, MW60 and CH60, and MW90 and CH90, respectively.
Enzymes
The following enzymes were employed in these studies and were obtained
from Sigma-Aldrich: pepsin (EC 3.4.23.1) from porcine gastric mucosa;
�-chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) from bovine pancreas.
Digestion of β-Lg
In the enzymatic hydrolysis of microwave- and conventionally-heated
�-Lg, control solutions were prepared under the same conditions but without the
addition of enzyme. All experiments were performed in triplicate.
1. Pepsin hydrolysis:
�-Lg (10 mg) was solubilized in 1 mL of 0.1 M HCl. The solution was then
incubated at 37°C and the pH adjusted to 2 using 1.0 M NaOH. The hydrolysis
reaction was initiated by the addition of a pepsin solution (10 mg/mL) at an
enzyme: substrate (E:S) ratio of 1:20. Following gentle stirring for 6 h, the reaction
was stopped by heating the solution to 80°C for 15 min, and the pH was then
adjusted to 7.0.
2. Chymotrypsin and trypsin hydrolysis:
�-Lg (10 mg) was solubilized in 1 mL of 0.1 M sodium phosphate buffer
(pH 8.0) and the resultant solution was incubated at 37°C. The hydrolysis reaction
was initiated by the addition of a chymotrypsin or trypsin solution (10 mg/mL) at an
E:S ratio of 1:100. Following gentle stirring for 6 h, the reaction was stopped in the
same manner as for pepsin hydrolysis.
102
3. Two-stage hydrolysis (simulated gastrointestinal digestion):
A two-stage hydrolysis process was carried out according to the method
described by Vercruysse et al. (2005) whereby consecutive hydrolysis of �-Lg with
pepsin, trypsin and α-chymotrypsin took place. First, the samples were acidified by
lowering the pH to 2 with HCl (4 M), pepsin was added (E/S: 1:250), and the
samples were incubated for 2 h at 37°C to mimic digestion in the stomach. Second,
incubation with trypsin (E/S: 1:250) and �-chymotrypsin (E/S: 1:250) at pH 6.5
(pH adjusted with 10 M NaOH) for 2.5 h at 37°C was carried out to simulate
digestion in the small intestine.
Aliquots were removed during the course of the hydrolysis in 30- min
increments. During the trypsin, chymotrypsin and two stages hydrolysis, the pH
was maintained by the addition of 2N NaOH:KOH (1:2) according to the pH-stat
technique of Adler-Nissen (1977), while in the case of the pepsin hydrolysis, the pH
was maintained by the addition of 1M NaOH hydrolysis. When the hydrolysis
reaction was complete (confirmed by no further change in the pH of the reaction
mixture), the reaction mixture was heated to 95°C for 10 min, in a water bath,
followed by cooling to room temperature. Samples were stored at −20 °C for
subsequent analysis. All hydrolysis reactions were performed in triplicate.
Measuring the degree of hydrolysis
The extent of hydrolysis was assayed directly by quantification of cleaved
peptide bonds using the o-phthaldialdehyde (OPA) spectrophotometric assay
described by Church et al. (1985). The α-amino groups released by hydrolysis react
with OPA and β-mercaptoethanol to form an adduct that absorbs strongly at 340
nm. The OPA reagent also reacts with ε-amino groups, and the resulting absorbance
represents the blank for proteolytic assays.
The OPA solution was prepared by combining 25 mL of 100 mM sodium
tetraborate, and 2.5 mL of 20% SDS and bringing the total volume to 50 mL by
addition of a solution containing 40 mg of OPA in 1 mL of methanol and 100 mL of
β-mercaptoethanol. To determine the degree of proteolysis, 100 μL of the
103
hydrolysate was directly added to 2 mL of OPA solution. The solution was swirled
by inversion and then incubated for 2 min at room temperature, and its absorbance
at 340 nm was then measured in a spectrophotometer (Shimadzu, UV-1601). The
number of amino groups released, n, was determined from the relationship:
� A340 � d E
P
n
M
(5.1)
where
�A340 is the experimentally observed change in absorbance
d is the assay dilution factor
E is the extinction coefficient (= 6000 M-1 cm-1) ,
P is protein concentration (mg mL-1), and
M is the molecular weight of the protein substrate (Da).
The degree of hydrolysis (DH, as %) was expressed as:
DH �
n
� 100
N
(5.2)
where N is the total number of amino acids in β-Lg (= 162).
Determination of kinetic parameters
Kinetic parameters were determined for the enzymatic hydrolysis of �-Lg
that had been subjected to conventional and microwave heating according to
Izquierdo et al. (2007). Km and Vmax for trypsin, chymotrypsin, and pepsin
hydrolysis of �-Lg at concentrations ranging from 0.17 to 0.85 mM were
determined. The initial velocity (V0) of liberation of NH2 groups was calculated and
plotted versus the �-Lg concentrations, and the plots generated followed
Michaelis-Menten kinetics. A regression analysis using Lineweaver–Burk doublereciprocal plots was subsequently performed. The Michaelis–Menten constant (Km)
and maximum reaction rate (Vmax) were derived from the slope (Km/Vmax) and
y-intercept (1/Vmax) of the straight lines obtained. Trpsin, chymotrypsin and pepsin
have apparent molecular masses of 23,300, 25,000 and 35,000 Da, respectively,
104
and these values were used for the calculation of kcat. Kinetic parameters were
calculated by GraphPad Prism, v. 5.02 (Graph-Pad Software, Inc., San Diego CA,
USA). The catalytic efficiency of the enzymes was expressed as kcat/Km.
Statistical analysis
Data were analyzed using SAS for Windows (version 9.1) following an
analysis of variance (ANOVA) one-way linear model. Mean comparisons were
performed using the Duncan test (Duncan, 1955), and the significance level was
established, where P� 0.05 was considered to indicate significance.
105
RESULTS AND DISCUSSION
Hydrolysis of β-Lg
The extent of �-Lg hydrolysis at 37ºC by pepsin, trypsin, and chymotrypsin
and by a simulated gastrointestinal enzymatic hydrolysis was determined during 6
hours of hydrolysis, in 30- min increments, by measuring the amount of α-amino
groups released in the enzymatic reaction, using the o-phthaldialdehyde (OPA)
reaction. Table 5.1 and Figure 5.1 show the degree of hydrolysis of β-Lg after each
enzyme treatment.
The values are the mean and standard error of triplicate
experiments. Simulated gastrointestinal enzymatic hydrolysis was the most
effective �-Lg hydrolysis method, followed by trypsin, chymotrypsin and pepsin
hydrolysis, respectively, irrespective of the sample pretreatment (Table 5.1, Figure
5.1).
A plot of the degree of hydrolysis of β-Lg in a two-stage enzymatic digestion as a
function of time (Figure 5.2) shows that the ultimate extent of hydrolysis ranged
from 18 % in the case of native β-Lg to 25% in the case of the samples subjected to
the MW60 pretreatment. Both microwave and conventional heat pretreatments of
β-Lg significantly (P�0.05) enhanced hydrolysis when compared to the control,
with one exception: there was no significant difference (P>0.05) between the
degree of hydrolysis following the CH40 pretreatment and that of the native β-Lg.
Other conditions being the same, the degree of hydrolysis of β-Lg was significantly
enhanced (P�0.05) when digestion took place after microwave as opposed to
conventional heating pretreatments with exceptions. There was no significant
difference between the degree of hydrolysis of CH90 and MW90 for two stages
hydrolysis. Also, in case of pepsin hydrolysis, there was no significant difference
between the degree of hydrolysis of microwave and conventional heating at 40°C
and 90°C.
106
Table 5.1: Degree of hydrolysis in enzymatic hydrolysates from β-Lg, as
determined by the o-phthaldialdehyde method.
Degree of Hydrolysis (%)b
Sample
Two-stage
pretreatmenta
Pepsin
Trypsin
Chymotrypsin
Untreated β-Lg
1.4±0.05 c
7.9±0.38 d
5.4±0.15 e
18.0±0.49 d
CH40
1.4±0.11 c
7.9±0.41 d
5.4±0.21 e
18.4±0.50 d
MW40
1.5±0.15 c
10.4±0.49 bc
7.2±0.23 cd
21.0±0.55 c
CH60
1.8±0.05 b
11.1±0.51 b
7.6±0.22 b
23.0±0.56 b
MW60
2.0±0.10 a
13.1±0.48 a
9.0±0.35 a
25.0±0.54 a
CH90
1.5±0.15 c
10.4±0.28 b
7.0±0.19 c
21.5±0.59 c
MW90
1.5±0.17 c
9.8±0.18 c
6.5±0.21 d
20.5±0.49 c
a
hydrolysis
See Materials and Methods for designations of sample pretreatments.
Values are means ± SE of triplicate experiments.
b
107
Figure 5.1: Degree of hydrolysis determined by the o-phthaldialdehyde method in
enzymatic hydrolysates from β-Lg. Error bars represent SE of triplicate
experiments. See Materials and Methods for designations of sample pretreatments.
108
For both the MW and the CH samples, those pretreated at 60ºC showed a
significantly (P�0.05) higher degree of hydrolysis than those pretreated at 90ºC; in
turn, the latter showed significantly greater hydrolysis than native β-Lg or samples
pretreated at 40ºC. The decrease in the degree of hydrolysis following pretreatment
at 90ºC relative to 60ºC, which was more pronounced in the case of the
microwave-treated samples, may be attributed to the protein aggregation that
occurred at 90ºC, rendering the protein less accessible to the enzyme action.
Similar trends were obtained for hydrolysis of β-Lg by trypsin and
chymotrypsin, although the degrees of hydrolysis were uniformly lower than the
corresponding values for the two-stage hydrolysis. For example, the greatest
degrees of hydrolysis, which again occurred in the MW60 samples, were 13.1%
and 9.0% with trypsin and chymotrypsin, respectively, while the values obtained
for hydrolysis of untreated β-Lg. with trypsin and chymotrypsin were 7.9% and
5.4%, respectively. In the case of hydrolysis by pepsin, the degree of hydrolysis of
β-Lg was very limited for all samples, ranging from 1.4% for untreated β-Lg to
1.8% and 2% for the CH60 and MW60 samples, respectively, with no significant
differences (P>0.05) being observed among any of the other pepsin hydrolysates. It
may be noted that the relative degrees of hydrolysis obtained with trypsin,
chymotrypsin and pepsin in this study concur with the findings of Maynard et al.
(1998) and Reddy and Kinsella, (1988), who reported native β-Lg to be very
resistant to proteolysis by chymotrypsin and pepsin but more susceptible to trypsin.
In contrast, Leppalla et al. (1991) reported a greater degree of hydrolysis with
chymotrypsin than with trypsin (6.0 vs. 4.9%, respectively, after a 24-h incubation).
Our results are also in accordance with those of Dufour et al. (1995) and
Penas et al. (2006), who reported negligible digestion of dairy whey proteins by
pepsin at atmospheric pressures. They attributed this finding to the resistance of
β-Lg to peptic digestion as a result of its conformational stability at low pH, which
in turn has been attributed to the strong stabilizing action of the two disulfide bonds
(Papiz et al., 1986; Kella and Kinsella, 1988; Iametti et al., 1995).
109
Figure 5.2: Degree of hydrolysis determined by the o-phthaldialdehyde assay
method as a function of time for the enzymatic hydrolysis of β-Lg by simulated
gastric digestion.
110
Although β-Lg is known to be very resistant to proteolysis by gastric enzymes in
the stomach (Savalle et al., 1988) and to proteolysis by pepsin in vitro (Reddy et al.,
1988; Schmidt and Poll, 1991), it has been established that pepsin makes the
molecular structure of β-Lg more susceptible to proteolysis by other proteinases,
even though pepsin digestion should theoretically eliminate some of the sites
available for hydrolysis by other proteinases. The destabilization of protein
structure that results from pepsin hydrolysis appears to be the dominant factor
causing an increase in the initial rate of hydrolysis by other enzymes (Porter et al.,
1984).
Figure 5.3 shows the amino acid sequence of β-Lg and its secondary
structure, As described by Papiz et al. (1986), the X-ray crystal structure shows that
β-Lg consists of nine anti-parallel β-sheets (Ala16-Ser27, Leu39-Glu44,
Lys47-Leu58,
Glu62-Thr76,
Ala80-Ile84,
Glu89-Thr97,
Tyr102-Asn109,
GlnlSArg124 and Met145-Ser150) and one α-helix (Asp130-Leu140). Pepsin
cleaves peptide bonds at the carboxyl side of hydrophobic and aromatic amino
acids. Although the amino acid sequence of β-Lg contains a large number of
potential cleavage sites, the present study corroborates the finding in the previously
cited studies that β-lactoglobulin is not digested by pepsin under in vitro
physiological conditions.
Most studies of β-Lg structure have shown that most of the hydrophobic and
aromatic amino acid side chains of β-Lg, which are potential cleavage sites for
enzymes, are buried in the hydrophobic core and thus are not exposed to the
enzymatic action. Apparently, the majority of the identified peptic peptides are
produced by cleavages in β-turns, loops or other unordered regions (Papiz et al.,
1986). Moreover, the native and globular tertiary structure’s stabilization by two
disulfide bonds (Cys66-Cys160, and Cys106-Cys119) explains the high resistance
of β-Lg to hydrolysis (De Wit et al., 1984). Reduction of these two disulfide bridges
by denaturing treatments destabilizes the conformation of the protein (Reddy et al.,
1988), and increases its susceptibility to proteolysis.
111
Trypsin cleaves the C-terminal lysine-X and arginine-X bonds (Turula et
al., 1997). Theoretically, the action of trypsin on β-Lg produces 19 peptide
fragments, between 1 and 26 amino acid residues in length. Comparatively, the
main
substrates
of
chymotrypsin
include
tryptophane,
tyrosine,
phenylalanine, leucine, and methionine, which are cleaved at the carboxyl terminal.
Figure 5.3 shows the amino acid sequence of β-Lg and its secondary
structure, and Figure 5.4 shows theoretical cleavage points for tryptic digestion of
β-Lg. It is clear from these figures that the potential cleavage sites for enzymes are
buried in the β-Lg core and thus are not exposed to enzymatic action. Thus it is
inferred that microwave treatment better exposes susceptible peptide bonds to the
active protease than does a conventional heat treatment.
112
Figure 5.3: The amino acid sequence of β-Lg and its secondary structure (Ragona et
al., 1999).
113
Figure 5.4: Amino acid sequence of β-lactoglobulin A and B. Arrows indicate
theoretical cleavage points for tryptic digestion. Shaded portions indicate residues
involved in �-sheet secondary structure, and the single open portion, residues
130-140, indicates the lone �-helix. (Turula et al., 1997)
114
Kinetic parameters
The values of Km and Vmax for trypsin and α-chymotrypsin hydrolysis of
β-Lg samples that had been subjected to conventional and microwave heating
pretreatments were determined by linear regression analysis of Lineweaver-Burk
double- reciprocal plots (Figures 5.5-5.8). The statistical analysis was undertaken
for each enzyme independently. The values obtained are tabulated in Tables 5.2 and
5.3.
The Km values for β-Lg hydrolysis were in the range of 0.11 -0.32 mM in
the case of trypsin hydrolysis and 0.13 -0.49 mM in the case of chymotrypsin
hydrolysis. The lowest values were for the β-Lg samples that had been
microwave-treated at 60ºC (P � 0.05). Vmax and kcat values showed no significant
differences across heat treatments.
Catalytic effectiveness (kcat/Km) values were used to compare substrate
specificity as a function of microwave vs. conventional heating (Price and Stevens,
1989). Our results show that significantly (P < 0.05) higher values were obtained
for both trypsin and α-chymotrypsin digestions performed after microwave vs.
conventional heating at 40oC and 60oC. The highest catalytic effectiveness was
obtained in the case of the MW60 samples, with kcat/Km values of 2614 and 2620
min-1 mM-1 for trypsin and chymotrypsin, respectively, compared to 1805 and 1359
min-1 mM-1 in the case of the CH60 samplers and 917 and 615 min-1 mM-1 in the
case of unheated β-Lg. As k cat values showed no significant differences across heat
treatments, the increased catalytic effectiveness of trypsin and α-chymotrypsin
following MW treatment of β-Lg compared to conventional heating at the same
temperature is attributable to the greater reduction in Km. Thus, the results suggest
that the highest substrate-enzyme affinity is achieved when β-Lg is pretreated by
microwave heating at 60ºC.
Pepsin showed very low activity for the unheated β-Lg. Accordingly, no
kinetic data were obtainable with the exception of the CH60 and MW60 samples,
for which Km values of 7.9 and 3.1 mM, respectively, were obtained along with
Vmax values of 21.8 and 9.5 µmol mL-1 min-1, kcat values of 390 and 170 min-1, and
115
kcat/Km values of 49 and 56, min-1 mM-1. These results provide evidence that MW
heating resulted in a higher population of unfolded β-Lg compared to conventional
heating.
These parameters were in agreement with those reported by Izquierdo et al.
(2007), who studied the effects of microwave treatment compared with
conventional heating on kinetic parameters for pronase, α-chymotrypsin and pepsin
hydrolysis of bovine β-lactoglobulin. They reported higher catalytic effectiveness
(Kcat Km-1) values in the pronase and α-chymotrypsin digestions performed under
MW at 40°C (7793 and 2073 min-1 mM-1, respectively) in comparison with the
values in the respective CH digestions at 40°C (1802 and 941 min-1 mM-1,
respectively). The Michaelis–Menten constant (Km) for either enzyme was reduced
under MW.
In conclusion, it seems likely that the observed effects may be extended to
the proteolysis of other globular proteins and possibly to the diversification of the
proteolytic processing of proteins with other structural folds. The peptides derived
from existing proteins may be of additional interest for their potential use as
nutraceuticals and functional ingredients.
116
Table 5.2: Kinetic parametersa for trypsin hydrolysis of native β-lactoglobulin and
of β-lactoglobulin samples that had been subjected to conventional and microwave
heating pretreatments
Km
Vmax
Sample
kcat/Km
kcat (min-1)
b
-1
-1
(min-1 mM-1)
(mM)
(µmol mL min )
pretreatment
Unheated
0.32±0.02 a
16.7±1.5 a
289±0.02 a
917
�-Lg
CH 40
0.32±0.02 a
16.9±1.3 a
289±0.02 a
917
MW40
0.25±0.03 b
17.7±1.0 a
290±0.03 a
1146
CH60
0.16±0.01 e
19.9±1.5 a
287±0.01 a
1805
MW60
0.11±0.01 f
19.7±1.5 a
282±0.01 a
2614
CH90
0.17±0.02 d
19.1±1.4 a
281±0.02 a
1601
MW90
0.18±0.01 c
20.0±1.6 a
278±0.01 a
1511
a
Values are the mean ± standard error (n = 3).
b
See Materials and Methods for designations of sample pretreatments.
Table 5.3: Kinetic parametersa for chymotrypsin hydrolysis of native
β-lactoglobulin and of β-lactoglobulin samples that had been subjected to
conventional and microwave heating pretreatments
Km
kcat/Km
Vmax
Sample
(mM)
(µmol mL-1 min-1) kcat (min-1) (min-1 mM-1)
pretreatmentb
Unheated
0.49±0.08 a
16.7 a
299±10.5 a
615
�-Lg
CH 40
0.48±0.13 a
16.9 a
301±8.1 a
637
MW40
0.36±0.03 b
17.7 a
315±10.0 a
868
CH60
0.26±0.01 e
19.9 a
356±6.2 a
1359
MW60
0.13±0.01 f
19.7 a
352±7.0 a
2620
CH90
0.29±0.03 d
19.1 a
342±15.2 a
1198
MW90
0.38±0.07 c
20.0 a
357±19.1 a
887
a
Values are the mean ± standard error (n = 3).
b
See Materials and Methods for designations of sample pretreatments.
117
0.2
0.18
0.16
1/v ( µmol ml-1 min-1)
0.14
0.12
0.1
blg
0.08
mw40
0.06
0.04
0.02
0
­6
­4
­2 ­0.02 0
2
4
6
8
1/S (mM)
Figure 5.5: Lineweaver-Burk double-reciprocal plots for hydrolysis of
β-lactoglobulin by trypsin at 40ºC.
118
0.2
0.18
0.16
1/v ( µmol ml-1 min-1)
0.14
0.12
0.1
blg
0.08
ch60
0.06
mw60
0.04
0.02
0
­10
­8
­6
­4
­2
­0.02 0
2
4
6
8
1/S (mM)
0.2
0.18
0.16
1/v ( µmol ml-1 min-1)
0.14
0.12
0.1
blg
0.08
ch90
0.06
mw90
0.04
0.02
0
­10
­5
­0.02 0
5
10
1/S (mM)
Figure 5.6: Lineweaver-Burk double-reciprocal plots for hydrolysis of
β-lactoglobulin by trypsin at 60 and 90 ºC .
119
0.3
1/v ( µmol ml-1 min-1)
0.25
0.2
0.15
blg
ch40
0.1
mw40
0.05
0
­4
­2
­0.05
0
2
4
6
8
1/S (mM)
Figure 5.7: Lineweaver-Burk double-reciprocal plots for hydrolysis of
β-lactoglobulin by α-chymotrypsin at 40ºC.
120
0.3
1/v ( µmol ml-1 min-1)
0.25
0.2
0.15
blg
ch60
0.1
mw60
0.05
0
­8
­6
­4
­2
0
2
­0.05
1/S (mM)
4
6
8
0.3
1/v ( µmol ml-1 min-1)
0.25
0.2
0.15
blg
ch90
0.1
mw90
0.05
0
­4
­2
­0.05
0
2
4
6
8
1/S (mM)
Figure 5.8: Lineweaver-Burk double-reciprocal plots for hydrolysis of
β-lactoglobulin by α-chymotrypsin at 60 and 90 ºC.
121
CONNECTING STATEMENT
In the previous chapter the effect of microwave heating on the enzymatic
hydrolysis efficiency of �-Lg was examined. Microwave treatment significantly
increased hydrolysis efficiency of �-Lg. In the next chapter the nutraceutical effects
(ACE inhibitory activity and antioxidant activity) of �-Lg hydrolysates resulting
from hydrolysis after microwave vs. conventional heating treatment were
determined and compared, and the amino acid sequence of the resulting peptides
determined.
122
Chapter 6: Examination of the nutraceutical properties
of hydrolysates derived from conventionally- and
microwave- treated β-lactoglobulin solutions
INTRODUCTION
Milk is known to be a complex mixture of molecular species, including
bioactive compounds that confer special properties for the support of infant
development and growth that extend beyond basic nutrition. It contains modulators
of digestive and gastrointestinal functions, hormones and growth factors potentially
capable of influencing the development and growth of the gastrointestinal tract and
other specific organs, immunoregulation, non-immune disease defense, and
modulation of the gut microflora population (Tome and Dehabbi, 1998). Many of
the bioactivities of milk are attributable to the proteins and peptides secreted into
milk by the mammary glands. The bioactivities of several milk proteins are latent,
either absent or incomplete in the native protein. Only during proteolytic digestion
of the protein are the active peptides released from the native protein.
Once the bioactive peptides are liberated by enzymatic proteolysis, they
may act as regulatory compounds with hormone-like activity. Bioactive peptides
usually contain 3–20 amino acid residues per molecule. The possible regulatory
effects of these peptides relate to nutrient uptake, immune defense, opioid agonist
and antihypertensive activities. Although animal as well as plant proteins contain
potential bioactive sequences, milk proteins are currently the main source of a
range of biologically active peptides which, in addition to their nutritional
properties, exhibit biological activity, such as mineral binding (Toba et al., 2000),
opioid (Bitri, 2004) and immunolomodulatory effects (Dutta, 2002). Reported
nutraceutical properties of whey proteins include lowering of blood pressure,
attributed to inhibition of angiotensin converting enzyme (ACE), and increases in
blood and tissue glutathione (GSH) concentration, which in turn increases the
123
detoxification of the free radicals produced in the metabolism of carcinogenic
compounds (Bounous, 2000).
Bioactive peptides have been isolated and their amino acid sequences
established. The investigational strategies have also included the synthesis of
peptides based on sequence similarities with peptides of known biological activity.
Most of the studies so far have been conducted in vitro. The physiological functions
of these peptides, however, remain to be established in vivo. At present, numerous
peptides exhibiting various activities, such as opioid agonist, antithrombotic, or
antihypertensive activity, immunomodulation, and mineral utilization properties
have been reported (Table 6.1).
Table 6.1: Overview of identified bioactive peptides derived from milk proteins
(Pihlanto-Leppala, 2001)
1950
1970
Bioactive peptides Protein
precursor
Bioactivity
Reference
Phophopeptides
α- and β-Casein
Mineral carrier
Mellander (1950)
Casomorphins
β-Casein
Opioid agonist
Brantl et al. (1979)
α-Casein exorphin
α-Casein
Opioid agonist
Zioudrou et al.
(1979)
Immunopeptides
α- and β-Casein
Immunomodulatory
Jolles et al. (1979)
Casokinins
α- and β-Casein
ACE-inhibitory
Casoxins
κ-Casein
Opioid antagonist
Lactorphins
α-Lactalbumin
and
β-lactoglobulin
Opioid agonist
Maruyama and
Suzuki (1982)
Yoshikawa et al.
(1986)
Chiba and
Yoshikawa (1986)
1980
Casoplatelins
Antithrombotic
Jolles et al. (1986)
Tomita et al.
(1991)
Mullally et al.
(1996)
κ-Casein
1990
Lactoferriein
Lactoferrin
Antimicrobial
Lactojinins
α-Lactalbumin
and
β-lactoglobulin
ACE-inhibitory
124
In recent years, the major whey protein components, α-lactalbumin and
β-lactoglobulin, have been shown to contain bioactive sequences. Peptides showing
opioid and angiotensin I-converting enzyme (ACE) inhibitory activity were derived
from the hydrolysis of α-lactalbumin and β-lactoglobulin. The opioid agonist
peptides α-lactorphin and β-lactorphin were liberated during in vitro proteolysis of
bovine whey proteins, and their pharmacological activity at micromolar
concentrations was observed. Whey hydrolysates also showed ACE-inhibitory
activity after proteolysis with different digestive enzymes, and several active
peptides were identified. These results demonstrated the existence of several
biologically active whey-derived peptides and hydrolysates. These findings can be
exploited in the development of foods with special health claims (e.g. treatment of
hypertension) as well as in identifying new applications of whey proteins in food.
Accordingly, there is a growing interest in the identification of peptides with
anti-hypertensive activity derived from food ingredients, so as to provide a
non-pharmacological alternative for the prevention and control of systemic arterial
hypertension, the most important risk factor for cardiovascular disease (Martin,
2003). Bovine milk whey is currently considered one of the main raw materials for
the production of such peptides. The principal means for screening peptides for
their potential anti-hypertensive activity is through the use of the angiotensin
I-converting enzyme inhibition assay. The angiotensin I-converting enzyme (ACE,
peptidyl dipeptide hydrolase, EC 3.4.15.1) has long been associated with the
renin-angiotensin system, which regulates peripheral blood pressure. The activity
of ACE raises blood pressure by converting angiotensin I released from
angiotonsinogen by renin, into the potent vasoconstrictor angiotensin II. It also
degrades vasodilative bradykinin and stimulates the release of aldosterone in the
adrenal cortex. Consequently, ACE-inhibitors may exert an inhibitory effect on
blood pressure (Petrillo et al., 1982). ACE is an exopeptidase, which cleaves
dipeptides from the C-terminal of various peptide substrates. Furthermore, it is an
unusual zinc metallopeptidase, as it is activated by chloride and lacks narrow in
vitro substrate specificity (Ondetti and Cushman, 1984). Several authors have
reported that whey protein hydrolysates showed ACE inhibitory activity as well as
125
hypotensive activity in animals and humans (Fujita et al., 2001; Van der Ven et al.,
2002; Vermeirssen et al., 2003). There is controversy in the literature as to whether
it is necessary to use native protein to obtain bioactive peptides. According to
Smithers et al. (1998) and Tirelli et al. (1997), the biological activity of a whey
protein depends on the preservation of its native structure. On the other hand,
several authors (Fitzgerald and Meisel, 1999; Takadaet al., 1996; Takada et al.,
1997) report obtaining bioactive peptides from denatured protein, albeit without
specifying its degree of denaturation.
Ortiz-Chao et al. (2009) reported the most potent ACE inhibitory peptides
derived
from
bovine
�LG
f(36–42)
with
the
sequence
Ser-Ala-Pro-Leu-Arg-Val-Tyr and had an IC50 value of 8µM, the second most
potent bovine �LG-derived peptide reported by Mullally et al. which is β-lg
f(142–148) with the sequence Ala-Leu-Pro-Met-His-Ile-Arg and produced using
trypsin. This peptide has an IC50 value of 42.6 µM (Mullally et al., 1997.
Vermeirssen et al. (2002) reported that this peptide could be transported intact
through a Caco-2 Bbe monolayer. However, recent studies show that it is degraded
after simulated gastrointestinal digestion (Roufik et al., 2006; Walsh et al., 2004).
Although some peptides were degraded into less potent ACE inhibitory peptides
after a simulated gastrointestinal digestion, most of the reported ACE inhibitory
peptides were found to decrease blood pressure when tested in spontaneously
hypertensive rats.
Reactive oxygen species (ROS) have been implicated in the etiology of
various degenerative diseases, including cardiovascular disease, cancer, diabetes,
cataracts, neurodegenerative disorders, and aging. The body has its own defense
system against ROS, including antioxidant enzymes [i.e., superoxide dismutase,
catalase, and GPx] and endogenous antioxidants (i.e., glutathione)]. Oxidative
stress occurs when ROS overload the body’s antioxidant defenses or when the
antioxidant defense system loses its capacity for response (e.g., elderly people).
Such a stress can lead to the damage of vital cellular components. Enhancement of
the body’s antioxidant defenses through dietary supplementation would seem to
provide a reasonable and practical approach to reducing the level of oxidative
126
stress, and there is a wealth of evidence to support the effectiveness of such a
strategy in vitro. This hypothesis has stimulated human intervention studies
concerning well-known dietary antioxidants (i.e., vitamins E and C) and other
less-known compounds with potential health-promoting effects (i.e., carotenoids
and polyphenols), as well as research into new food ingredients with antioxidant
potential such as whey ingredients. One means of in vitro evaluation of the
antioxidant activity of peptides is the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.
This assay is based on the reduction of DPPH, which is stable in ethanol, by a free
radical scavenging antioxidant. The reduction is quantified spectrophotometrically
by the decrease in absorbance.
In this study the ACE inhibitory activity and antioxidant activities of �-Lg
hydrolysates derived from microwave- and conventionally-heated β-Lg were
measured by an enzyme inhibition assay and a DPPH radical scavenging assay,
respectively. In addition, the identification and sequencing of bioactive peptides in
hydrolysates was undertaken by means of reversed-phase high-performance liquid
chromatography electrospray ionization mass spectrometry (RP-HPLC/ESI-MS)
and MS-MS analysis.
MATERIALS AND METHODS
Samples
Samples of �-Lg that had been microwave- or heat-treated at 40ºC, 60ºC or
90ºC, as well as native �-Lg, were examined. These samples are designated MW40
and CH40, MW60 and CH60, and MW90 and CH90, respectively. Enzymatic
hydrolysis of these samples was performed as described previously (Chapter 5),
employing the following enzymes, all of which were obtained from Sigma-Aldrich:
pepsin (EC 3.4.23.1) from porcine gastric mucosa; �-chymotrypsin (EC 3.4.21.1)
and trypsin (EC 3.4.21.4) from bovine pancreas.
127
ACE inhibition
ACE-inhibitory activity of β-Lg hydrolysates derived from the enzymatic
hydrolysis of native, microwave-treated and conventionally heated β-Lg was
measured by the spectrophotometric assay of Cushman and Cheung (1971) with
some changes. A 0.3% (w/v) solution of hippuryl-L-histidyl-L-leucine (HHL)
(Sigma H-1635) was prepared in 50 mM HEPES HCl buffer (Sigma H-7006)
containing 300 mM NaCl (pH 8.3). A 200-µL aliquot of the HHL solution was
mixed with 100 µL of each hydrolysate (10 mg mL -1).
The reaction was initiated by addition of 50 µL of rabbit ACE (Sigma, EC
3.4.15.1) dissolved in cold deionized water at 0.33 U mL-1. Samples were incubated
for 30 min at 37ºC, and the reaction was then terminated by the addition of 0.25 mL
of 1 M HCl. The hippuric acid liberated from the reaction was extracted from the
solution into 2 mL of ethyl acetate by vortex mixing for 1 min. The sample was
centrifuged (5000 � g, 2 min) and 1 mL of the ethyl acetate layer was transferred
into a clean tube and heated at 95°C for 10 min to evaporate the solvent,
re-dissolved in 3 mL of distilled water and mixed by inversion. The absorbance of
the samples was measured spectrophotometrically at 228 nm. For each tested
sample, a blank was prepared by adding 250 μl of HCL (1 M) before adding the
enzyme. The reaction of ACE in the absence of inhibitor was carried out by
replacing the protein hydrolysate by deionized distilled water. A blank was also
prepared. The activity of each sample was tested in triplicate. The amount of
hippuric acid liberated from the reaction in the absence of an inhibitor was defined
as 100% ACE activity. Captopril, a synthetic ACE inhibitor, was used as a positive
control (IC50 = 0.008 µM). The ACE activity (U mL-1) was calculated as:
ACE activity �
test
blank
( A228
nm � A228 nm ) a V h
Ve E t �
�
test
blank
( A228
nm � A228 nm ) � 2 � 3
9.8 � 30 � 0.91 � 0.05
test
blank
� 0.4485 ( A228
nm � A228 nm )
where:
a
is a conversion factor equal to 2, since the hippuric acid detected is only half
of the total amount produced in the assay.
128
(6.1)
test
A228
nm is the absorbance of the test solution at 228 nm,
blank
A228
nm is the absorbance of the blank solution at 228 nm,
E
is the extraction efficiency of ethyl acetate, and is equal to 0.91,
t
is the duration of the assay (min) and is equal to 30,
Ve
is the volume of enzyme added (mL) and is equal to 0.05,
Vh
is the total volume of the hippuric acid solution (mL) and is equal to 3.0, and
�
is the millimolar extinction coefficient of hippuric acid at 228 nm, and is
equal to 9.8.
Therefore, the ACE inhibitory activity expressed as a percentage is equal to ACE
activity expressed as a percentage subtracted from 100. Inhibitory activity was
calculated as the protein concentration (mg mL-1) needed to cause a 50% inhibition
of the original ACE activity (IC50).
Antioxidant activity
Antioxidant activity of the hydrolysates (10 mg/mL) was mexasured using
the radical scavenger 2,2-diphenyl-1-picryl-hydrazyl (DPPH) whereby reduction of
the stable DPPH radical by an antioxidant compound results in a decrease in DPPH
absorbance (Vercruysse et al., 2009). A 200-µL aliquot of hydrolysate was mixed
with 1800 µL of ethanol containing 0.002% DPPH. The mixture was shaken and
absorbance was measured at 517 nm for 60 min at 1-min intervals. A control,
containing only water, was run and the background absorbance of the samples was
evaluated by adding ethanol without DPPH. Vitamin C was used as a positive
control, as it is known to have DPPH radical scavenging activity.
Antioxidant activity was expressed as % radical scavenging:
% radical scavenging =
WED
WE
SED
SE
( A517
nm � A517 nm ) � ( A517 nm � A517 nm )
WED
WE
( A517
nm � A517 nm )
129
x100%
where:
WED
A517
nm is the absorbance value of water plus ethanol with DPPH,
WE
A517
nm
is the absorbance of water plus ethanol,
SED
is the absorbance of the sample plus ethanol with DPPH, and
A517
nm
A517SE nm
is the absorbance of the sample plus ethanol.
Peptide identification by liquid chromatography-electrospray ionization mass
spectrometry and tandem mass spectrometry
Hydrolyzed β-Lg samples were dialyzed through a 30 kDa molecular
weight cut-off, lyophilized and dissolved in 0.1 % formic acid. The hydrolysates
were subjected to RP-HPLC on a Widepore C18 column (250mm�4.6 mm)
(Bio-Rad, Richmond, CA, USA). Operating conditions were: column at ambient
temperature; flow rate, 0.8 mL min-1 ; injection volume, 5 µL; A binary gradient of
solvent B (acetonitrile:0.1 % formic acid) and solvent A (water:0.1 % formic acid)
was increased from 5 to 50% (A towards B) in 40 min. Injection volume was 5 µL.
A flow of approximately 20 µL min-1 was directed into a Waters Micromass QTOF
Ultima Global (Micromass; Manchester, UK) hybrid mass spectrometer equipped
with a nanoflow electrospray source via the electrospray interface. Operating
conditions were: positive ionization mode (+ESI) at 3.80 kV with a source
temperature of 80°C and desolvation temperature of 150°C. The TOF was operated
at an acceleration voltage of 9.1 kV, a cone voltage of 100 V and collision energy of
10 eV (for MS survey). For the MS survey mass range, m/z was 400-1990, and for
MS/MS, 50-1990, scanned continuously over the chromatographic run. Instrument
control and data analysis were carried out by MassLynx v. 6.0 software (Waters
Corporation, 2005).
130
Database analysis of the MS/MS data for sequence characterization was
done using two software packages: ProteinLynx (Waters Corporation, 2005) and
Mascot (Perkins et al., 1999).
Statistical analyses: Experimental results were recorded as a mean ± standard
error. Data were analyzed using SAS for Windows (version 9.1) following an
analysis of variance (ANOVA) one-way linear model. Mean comparisons were
performed using the Duncan test, and the significance level of P�0.05 was
considered to indicate significance.
131
RESULTS AND DISCUSSION
ACE inhibition
Enzymatic hydrolysates of bovine β-lactoglobulin subjected to microwave
and conventional heating (cf. Chapter 5) were analyzed for their ACE inhibition
activity. The unhydrolyzed substrates gave very low ACE inhibition indices (IC50
value of 8 mg mL-1). Hydrolysis of β-lactoglobulin samples by pepsin, trypsin, and
chymotrypsin and in a two-stage hydrolysis simulating gastrointestinal conditionws
resulted in higher IC50 values, ranging from 0.36 to 0.99 mg mL-1 (Table 6.2,
Figures 6.1-6.2).
Table 6.2: IC50 values for ACE inhibition by β-Lg hydrolysates obtained by
enzymatic hydrolysis of untreated, microwave-treated and conventionally heated
β-Lg samples
Sample
IC50 (mg mL-1)b
Pepsin
Trypsin
Chymotrypsin
Two-stage
hydrolysis
hydrolysis
hydrolysis
hydrolysis
Unheated �-Lg
0.90±0.01 b
0.78±0.03 c
0.68±0.01 c
0.63±0.01 c
CH 40
0.90±0.02 b
0.79±0.01 c
0.68±0.04 c
0.63±0.03 c
MW40
0.86±0.02bc
0.74±0.02 d
0.57±0.02 d
0.51±0.03 d
CH60
0.84±0.02 c
0.72±0.01 d
0.57±0.04 d
0.50±0.04 d
MW60
0.80±0.01 d
0.65±0.01 e
0.43±0.04 e
36±0.04 e
CH90
0.93±0.03 a
0.89±0.02 b
0.72±0.03 b
0.69±0.02 b
MW90
0.99±0.03 a
0.98±0.02 a
0.75±0.05 a
0.74±0.03 a
pretreatmenta
a
See Materials and Methods for designations of sample pretreatments.
Values presented are the average of two repetitions ± standard error. Column-wise
[i.e., for each hydrolysing enzyme(s) treatment] values assigned the same letter
show no significant difference from one another (P>0.05).
b
132
IC 50 mg/mL
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
PEPSIN
B
B
BC
BLG
ch40
mw40
C
ch60
D
mw60
A
ch90
A
mw90
TRYPSIN
IC 50 mg/mL
1.00
C
C
D
D
BLG
ch40
mw40
E
B
A
0.80
0.60
0.40
0.20
0.00
ch60
mw60
ch90
mw90
Figure 6.1: IC50 values (mg mL-1) for ACE inhibition by β-Lg hydrolysates
obtained by pepsin and trypsin hydrolysis of untreated, microwave-treated, and
conventionally heated β-Lg; within each panel, treatments with the same letter have
no significant differences (P > 0.05).
133
CHYMOTRYPSIN
IC 50 mg/mL
1.00
C
C
D
D
BLG
ch40
mw40
ch60
E
B
A
0.80
0.60
0.40
0.20
0.00
mw60
ch90
mw90
B
A
ch90
mw90
Two stages hydrolysis
IC50 mg/mL
1.00
C
C
D
D
BLG
ch40
mw40
ch60
E
0.80
0.60
0.40
0.20
0.00
mw60
Figure 6.2: IC50 values (mg mL-1) for ACE inhibition by β-Lg hydrolysates
obtained by chymotrypsin and two-stage enzymatic hydrolysis of untreated,
microwave-treated, and conventionally heated β-Lg; within each panel, treatments
with the same letter have no significant differences (P > 0.05).
134
The IC50 values obtained for the CH40 hydrolysates did not differ
significantly from those of hydrolysates obtained from native β-Lg. In contrast,
except in the case of pepsin, the IC50 values of the MW40 hydrolysates were
significantly lower. In the case of samples pretreated at 60ºC, MW60 hydrolysates
had significantly lower IC50 values than CH60 hydrolysates, which in turn had
significantly lower values than native �-Lg hydrolysates for all enzyme treatments
tested. Finally, conventional or microwave heating of �-Lg to 90°C resulted in
higher IC50 values than heating to 60°C, irrespective of the protease used.
It has been argued that heat treatment and mechanical damage can
drastically reduce the biological activity of food proteins (Smithers et al., 1998), as
it may alter the profile of peptides released during gastrointestinal digestion, as the
enzymes involved could hydrolyze parts of the protein that were previously
inaccessible to enzymatic action (Korhonen et al., 1998). In the present study,
however, the best ACE inhibition results were obtained from hydrolysates of �-Lg
previously heated at 60°C, whereas hydrolysates obtained from unheated �-Lg or
�-Lg heated to 90°C showed significantly lower activity. These results concur with
those of Da Costa et al. (2007), who reported that heat treatment of whey protein
isolate at 65°C for 15 min, prior to enzymatic hydrolysis, resulted in peptides with
the highest ACE inhibition activity, particularly when the enzyme employed was
α-chymotrypsin.
The differences in ACE inhibition activity among the hydrolysates obtained
following these various sample pretreatments of �-Lg can be explained by the
structural changes induced in the protein’s structure by different microwave and
conventional heat treatments. Heating at 60°C induced a partially unfolded
conformation of �-Lg, exposing inaccessible hydrophobic sites, as evidenced by
the CD and fluorescence spectroscopic investigations presented previously
(Chapter 4). These structural changes may allow enzymes greater access to certain
sites previously inaccessible to enzyme action, resulting in peptides different from
those obtained from the native �-Lg (since the IC50 is different). The role of
135
hydrophobic groups is supported by the fact that chymotrypsin hydrolysates had
lower IC50 values than pepsin or trypsin hydrolysates. Furthermore, each enzyme’s
substrate affinity was substantially altered by heat treatment of the substrate. When
the temperature exceeds 90°C, the formation of high-molecular-weight aggregates
occurs, mainly due to hydrophobic interactions (La Fuente et al., 2002). Aggregate
formation, as well as the compact structure of the untreated �-Lg, can hamper
enzyme access to specific sites of the protein, releasing peptides different from
those liberated during the hydrolysis of partially denatured �-Lg.
136
Antioxidant activity
The antioxidant activity of �-Lg hydrolysates was evaluated in vitro using a
radical scavenging assay based on the reduction of alcohol-stable DPPH by a
free-radical- scavenging antioxidant. The reduction of DPPH was quantified
spectrophotometrically by a decrease in absorbance. Vitamin C has a comparable
radical scavenging activity, as has been previously reported (Schlesier et al., 2002).
Table 6.3 and Figures 6.4 and 6.5 show the antioxidant activity determined by the
DPPH assay for the �-Lg hydrolysates at a concentration of 5 mg/mL. The values
are expressed as means of % radical scavenging ± SE based on three repetitions.
Significant differences were determined by one-way ANOVA (P < 0.05) and are
denoted by letters a-e.
There were significant differences of % radical scavenging among all the
enzyme treatments (Table 6.3 and Figure 6.3). In the case of native �-Lg and �-Lg
treated at 90°C by microwave treatment, the products of the two-stage hydrolysis
showed the highest radical scavenging activity, 33.9% and 49.3%, respectively,
followed by 27.7% and 39.6% for chymotrypsin hydrolysates, 19.3% and 28.2%
for trypsin hydrolysates, and 15.5% and 19.3% for pepsin hydrolysates. For all
enzymatic treatments, there was no significant difference in radical scavenging
activity between the native �-Lg and the CH40 treatment. The antioxidant activity
increased with the temperature at which the �-Lg samples had been pretreated and
the highest activity was found in the hydrolysates obtained from �-Lg heated to
90ºC.
The pepsin hydrolysates of the CH40 and MW40 samples showed no
significant differences in antioxidant activity. However, the antioxidant activity of
the MW60 hydrolysate was significantly greater than that of the CH60 hydrolysate.
The results for samples pretreated at 90ºC were not significantly different from
those for samples pretreated at 60°C. Finally, for trypsin, chymotrypsin
hydrolysates and the products of the two-stage hydrolysis, the antioxidant activity
137
of hydrolysates of microwave-treated samples was significantly higher than that of
hydrolysates of conventionally heated samples for all treatment temperatures.
Table 6.3: Antioxidant activity of the β-Lg hydrolysates determined by DPPH
assay
Sample
Radical scavenging (%)b
Pepsin
Trypsin
Chymotrypsin
Two-stage
hydrolysis
hydrolysis
hydrolysis
hydrolysis
Unheated �-Lg
15.52±0.51d
19.35±0.72 e
27.75±1.2 d
33.91±0.62 d
CH 40
15.52±1.22 d
19.76±0.63 e
27.57±0.8 d
32.18±0.59 d
MW40
15.69±0.75 cd
23.39±0.9 d
31.89±1.2 c
35.65±0.71 c
CH60
17.24±0.65 bc
25.81±0.72 c
32.43±1.4 c
36.84±0.70 c
MW60
19.54±1.0 a
28.11±0.99 b
37.93±1.2 ab
42.33±0.75 b
CH90
18.39±1.4 ab
28.42±0.92 b
35.68±1.3 b
41.21±0.71 b
MW90
19.37±0.9 a
30.83±0.93 a
39.64±1.5 a
49.43±1.00 a
pretreatmenta
a
See Materials and Methods for designations of sample pretreatments.
Mean % radical scavenging ± SE (n = 3). Column-wise [i.e., for each hydrolyzing
enzyme(s) treatment] values assigned the same letter show no significant difference
from one another (P>0.05). Significant differences determined by one-way
ANOVA
b
138
D
E
D
E
DC
D
BC
C
A
B
AB
B
A
A
Figure 6.3: Antioxidant activity of the �-Lg hydrolysates obtained by pepsin and
trypsin hydrolysis of untreated, microwave-treated, and conventionally heated
β-Lg, determined by DPPH assay; within each panel, treatments with the same
letter have no significant differences (P > 0.05).
139
D
D
C
C
AB
B
A
Two stages hydrolysis
60.00
% Radical scavenging
D
D
C
C
B
B
A
50.00
40.00
30.00
20.00
10.00
0.00
BLG
CH40 MW40 CH60 MW60 CH90 MW90
Figure 6.3: Antioxidant activity of the �-Lg hydrolysates obtained by chymotrypsin
and two-stage enzymatic hydrolysis of untreated, microwave-treated, and
conventionally heated β-Lg, determined by DPPH assay; within each panel,
treatments with the same letter have no significant differences (P > 0.05).
140
Antioxidant activity is ascribed to amino acids such as tyrosine, tryptophan,
methionine, lysine, cysteine and histidine (Wang and De Mejia, 2005) and tends to
be found in peptides consisting of 5–16 amino acid residues. However, the exact
mechanism by which antioxidative peptides exert their activity and their
structure–activity relationships are not fully understood (Suetsuna et al., 2000; Kim
et al., 2001; Saito et al., 2003; Peng et al., 2009). Antioxidant peptides so far
identified in milk contain one or more residues of histidine, proline, tyrosine and
tryptophan (Vercruysse et al., 2009). Suetsuna et al. (2000) separated a peptide
having strong free-radical-scavenging activities from pepsin hydrolysates of casein
by chromatographic analyses. They determined that a Glu-Leu sequence was
important in conferring antioxidant activity. Peng et al. (2009) produced a potent
antioxidative hydrolysate from whey protein isolate by treating the substrate with
Alcalase. To elucidate the mode of action of the antioxidative hydrolysate, they
fractionated the hydrolysate into four fractions using Sephadex G-10 gel filtration
chromatography and assayed
their antioxidant activities. The fraction with
peptides of 0.1–2.8k was found to possess the strongest radical-scavenging activity.
Many reports suggest that the primary amino acid sequence also contributes to
antioxidant activity. Laakso (1984) reported the quenching of free radicals by
oxidation of amino acid residues in milk casein. Free amino acids could not
substitute for casein as the antioxidants, suggesting that the primary structure of the
casein molecule played a role. Suetsuna et al. (2000) isolated a peptide from a
pepsin digest of milk casein and determined antioxidant activity. The constituent
amino acids had no activity, and thus the characteristic amino acid sequence of the
peptide was responsible for the activity. Furthermore, Saito et al. (2003) concluded
that the antioxidant property of peptides varies depending on their structure and the
assay system. Similarly, Saiga et al. (2003) stated that a difference in activity of two
hydrolysates having similar amino acid compositions might be caused by
differences in the structure and length of the peptides in the hydrolysates.
A comparison of our results with those reported in the literature is difficult
as
various
antioxidant
screening
methods
are
employed.
However,
Hernandez-Ledesma et al. (2005) investigated the antioxidant activity of
141
hydrolysates obtained from the bovine whey proteins �-lactalbumin and
�-lactoglobulin by commercial proteases (pepsin, trypsin, chymotrypsin,
thermolysin, and Corolase PP). Among those enzymes, Corolase PP was the most
appropriate enzyme to obtain hydrolysates having antioxidant activity from
�-lactalbumin and �-lactoglobulin. They identified several antioxidant peptides
from β-lactoglobulin A hydrolysates and suggested that whey protein hydrolysates
could be suitable as natural ingredients in enhancing antioxidant properties of
functional foods and in preventing oxidation reactions in food processing. Our
results concur with their results for chymotrypsin, pepsin and trypsin hydrolysates.
Peptide identification
Hydrolysates bearing ACE inhibition activity and antioxidant activity
derived from microwave and conventional heating were subjected to
RP-HPLC-MS in order to identify the mass and primary sequence of the peptides.
The UV-chromatogram of a MW60 tryptic hydrolysate shows a complex peptidic
profile, with 15 peptide peaks (Figure 6.4(A)). The mass spectrum corresponding to
the peak selected in Figure 6.4(A) is shown in Figure 6.4(B). The MS/MS spectrum
of the doubly charged ion with m/z 838.5 and the amino acid sequence of the
identified peptide with the major fragment ions are shown in Figure 6.4(C).
142
(A)
(B)
(C)
Figure 6.4: (A) UV-chromatogram of the MW60 tryptic β-Lg hydrolysate (B) Mass
spectrum of the selected chromatographic peak in (A). (C) MS/MS spectrum of the
doubly charged ion m/z 838.5. Following sequence interpretation and database
searching, the MS/MS spectrum was matched to β-Lg f(142–148).
143
Figure 6.5 shows the 3D structure and amino acid sequence of bovine β-Lg.
From the analysis of the LC-ESI-MS spectra, a total of 7, 11 and 36 peptides were
identified for chymotrypsin, trypsin and two-stage hydrolysis, respectively. A few
detected masses and their corresponding fragmentation spectra obtained by MS/MS
could not be matched with any peptide fragment originating from bovine β-Lg
hydrolysis. These might correspond to peptide fragments connected by disulfide
bonds (heterodimers).
Table 6.4 lists the peptides identified by RP-HPLC–MS/MS of hydrolysates
obtained from native, microwave-treated and conventionally heated β-Lg samples
by proteolysis with chymotrypsin. It is clear that chymotrypsin hydrolysis failed to
produce any differences among the hydrolysates of these samples (Table 6.4).
Table 6.5 shows representative LC-ESI-MS-MS spectra for hydrolysates
obtained from native, microwave-treated and conventionally heated β-Lg samples
by proteolysis with trypsin. The results show unique peptides associated with
microwave-treated samples. For example, m/z 1245.6 assigned to f (125-135) was
identified only in the MW60 hydrolysate, while m/z 2708.4 assigned to f (15-40)
was identified in both the MW90 and CH90 hydrolysates.
Table 6.6 shows representative LC-ESI-MS-MS spectra for hydrolysates
obtained from native, microwave-treated and conventionally heated β-Lg by
combined proteolysis with pepsin, trypsin and chymotrypsin, simulating
gastrointestinal digestion. A notable number of peptides were released by this
two-stage hydrolysis. They included all the peptides resulting from the action of
trypsin or chymotrypsin individually except the peptide fragment with m/z 2675.2,
detected in trypsin hydrolysates, which may have been further hydrolyzed by other
enzymes to lower molecular weight fragments. Among the peptides released, m/z
2708.4 assigned to f(15-40), which was previously detected in trypsin hydrolysates,
m/z 973 assigned to f(109-117) and m/z 751 (unassigned fragment), were identified
only in the MW90 and CH90 hydrolysates, while m/z 804.4 assigned to f(36-42),
1069.7 assigned to f(32-41), m/z 1108.4 assigned to f(125-134) and m/z 1109
(unassigned fragment) were identified only in the MW60, MW90 and CH90
144
hydrolysates. Three m/z peaks were unique to the MW60 hydrolysate: m/z 1370
assigned to f(94-104), m/z 1245.6 assigned to f(125-135) and m/z 1147 (unassigned
fragment). Two m/z peaks were unique to the MW90 hydrolysate: 774.4 assigned
to f(76-82) and 761.0 (unassigned fragment). Finally, m/z 855.5 assigned to
f(46-53) was found only in the MW60 and MW90 hydrolysates.
Figure 6.6 presents a 3D representation of the structure of �-Lg,
highlighting selected peptides in Table 6.6 as well as the sequences of peptides that
were found uniquely in hydrolysates of microwave-treated samples at 60°C.
145
Figure 6. 5: The 3D structure and amino acid sequence of bovine β-Lg.
(A) Ribbon diagram of a single subunit of bovine β-Lg lattice X, whose pdb code is
1BEB (Sakurai, et al., 2009). The β-strands are labeled. Trp residues are
represented as balls and sticks. The diagram was produced using the program
MolFeat (FiatLux, Tokyo, Japan). (B) A schematic representation of the amino acid
residues of the β-Lg sequence. Residues making up the α-helix, β-sheet, and loop
are represented by hexagons in red, squares in blue, and circles in grey,
respectively. Green lines indicate the positions of disulfide bonds.
146
Table 6.4: Peptides identified by RP-HPLC–MS/MS in β-Lg chymotrypsin
hydrolysates
Protein
Obs.
Calc.
Mass
mass
1
600.3
600.4
123-127
2
675.4
675.3
3
700.4
4
5
6
7
fragme
Sample
Sequence
β-Lg
CH40
MW40
CH60
MW60
CH90
MW90
VRTPT
+
+
+
+
+
+
+
25-31
AASDISL
+
+
+
+
+
+
+
700.34
33-39
DAQSAPL
+
+
+
+
+
+
+
715.3
715.39
88-93
NENKVL
+
+
+
+
+
+
+
1119.4
1119.2
33.42
DAQSAPLRVY
+
+
+
+
+
+
+
813.3
813.42
32-39
LDAQSAPL
+
+
+
+
+
+
+
558.6
558.32
141-149
KALPM
+
+
+
+
+
+
+
nt
Table 6.5: Peptides identified by RP-HPLC–MS/MS in β-Lg trypsin hydrolysates
Protein
Obs.
Calc.
Mass
mass
1
674.3
674.4
78-83
2
673.4
673.4
9-14
3
1452.4
1452.7
48-60
4
573.4
573.4
71-75
5
916.5
916.5
84-91
6
2675.2
2675.2
102-124
7
836.77
837.5
142-148
8
409.2
409.2
136-138
9
331.2
331.2
139-141
10
2708.4
2707.4
15-40
11
1245.5
1245.6
125-135
fragme
Sample
Sequence
β-Lg
CH40
MW40
CH60
MW60
CH90
MW90
IPAVFK
+
+
+
+
+
+
+
GLDIQK
+
+
+
+
+
+
+
PTPEGDLEILLQK
+
+
+
+
+
+
+
IIAEK
+
+
+
+
+
+
+
VLVLDTDYKK
+
+
+
+
+
+
+
YLL…LVR
+
+
+
+
+
+
+
ALPMHIR
+
+
+
+
+
+
+
FDK
+
+
+
+
+
+
+
ALK
+
+
+
+
+
+
+
VAG……LR
-
-
-
-
-
+
+
TPPVDDEALEK
-
-
-
-
+
-
-
nt
147
Table 6.6: Peptides identified by RP-HPLC–MS/MS in β-Lg hydrolysates from
two-stage enzymatic hydrolysis
Sample
Obs.
Mass
Calc.
mass
Protein
fragment
Sequence
1
600.3
600.4
123-127
2
674.3
674.4
3
673.4
673.4
4
675.3
675.3
5
700.4
700.34
6
715.39
715.39
7
753.35
8
Β-Lg
CH40
MW40
CH60
MW60
CH90
MW90
VRTPT
+
+
+
+
+
+
+
78-83
IPAVFK
+
+
+
+
+
+
+
9-14
GLDIQK
+
+
+
+
+
+
+
25-31
AASDISL
+
+
+
+
+
+
+
33-39
DAQSAPL
+
+
+
+
+
+
+
88-93
NENKVL
+
+
+
+
+
+
+
753.35
95-100
LDTDYK
+
+
+
+
+
+
+
573.7
573.4
58-61
LQKW
+
+
+
+
+
+
+
9
1119.2
1119.2
33-42
DAQSAPLRVY
+
+
+
+
+
+
+
10
813.42
813.42
32-39
LDAQSAPL
+
+
+
+
+
+
+
11
558.32
558.32
141-145
KALPM
+
+
+
+
+
+
+
12
695.33
695.33
15-20
VAGTWY (ace)
+
+
+
+
+
+
+
13
828.3
828.3
87-93
LNENKVL
+
+
+
+
+
+
+
14
1040.6
1040.5
20-29
VEELKPTPE
+
+
+
+
+
+
+
15
1452.4
452.7
48-60
PTPEGDLEILLQK
+
+
+
+
+
+
+
16
573.7
573.4
71-75
IIAEK
+
+
+
+
+
+
+
17
916.4
916.5
84-91
VLVLDTDYKK
+
+
+
+
+
+
+
18
782.7
782.4
15-21
VAGTWYS
+
+
+
+
+
+
+
19
836.77
838.5
142-148
ALPMHIR
+
+
+
+
+
+
+
20
409.2
409.2
136-138
FDK
+
+
+
+
+
+
+
21
331.2
331.2
139-141
ALK
+
+
+
+
+
+
+
22
931.4
931.1
96-102
DTDYKKY
+
+
+
+
+
+
+
23
626.4
625.38
146-150
HIRLS
-
-
-
+
+
+
+
24
2708.4
2707.4
15-40
VAG……LVR
-
-
-
-
-
+
+
25
973.0
973.44
109-117
NSAEPEQSL
-
-
-
-
-
+
+
26
751
n/a
-
-
-
-
-
+
+
27
1374
1370
94-104
VLDTDYKYLL
-
-
-
-
+
-
-
28
1245.4
1245.6
125-135
TPEVDDEALEK
-
-
-
-
+
-
-
29
1147
n/a
-
-
-
-
+
-
-
30
804.4
804.44
36-42
SAPLRVY
-
-
-
-
+
+
+
31
1071.5
1069.7
32-41
LDAQSAPLRV
-
-
-
-
+
+
+
32
1109
n/a
-
-
-
-
+
+
+
33
1108.4
1108.4
125-134
TPPVDDEALE
-
-
-
-
+
+
+
34
775
774.4
76-82
TKIPAVF
-
-
-
-
-
-
+
35
761
n/a
-
-
-
-
-
-
+
36
856.0
LKPTPEGD
-
-
-
-
+
-
+
855.5
46-53
148
a
b
Figure 6.6: Secondary structure of β-Lg: (a) Selected peaks in Table 6.6 are
highlighted in yellow; (b) sequences of peptides unique to microwave treatment
obtained by MSMS analysis are highlighted in gray.
149
Many of the peptides listed in Tables 6.4-6.6 were previously described to
have ACE inhibition or antioxidant activity in foods. For example, �-Lg f(142-148)
contains the tetrapeptide Ala-Leu-Pro-Met, termed “β-lactosin B,” which was
found to have significant antihypertensive activity when administered orally to
spontaneously hypertensive rats (SHR) and therefore was considered to have
potential as a natural antihypertensive agent for inclusion in foods (Murakami et al.,
2004). The peptide �-Lg f(15-20), found in all the hydrolysates examined above,
was reported by Pihlanto-Leppala et al. (1998) to have ACE inhibition activity.
Although this peptide was found in all the β-Lg hydrolysates its level in the MW60
hydrolysate was significantly higher. The peptide f(146-150), identified in the
MW60, CH60, MW90, and CH90 hydrolysates obtained by the two-stage
enzymatic hydrolysis, contains the sequence β-Lg f(146–149) which was
previously termed β-lactotensin and reported to be one of the most potent inhibitory
ACE peptides in addition to having opioid activity (FitzGerald and Meisel, 1999).
Moreover, the fragment f(36-42) “SAPLRVY,” which was found only in the
MW60, MW90, and CH90 hydrolysates, has been reported to be the most potent
β-Lg-derived ACE inhibitor found to date (Ortiz-Chao et al., 2009).
Hernandez-Ledesma et al. (2002) reported obtaining the potent ACE
inhibitory tetrapeptide Leu-Gln-Lys-Trp, with an IC50 value of 34.7 mM, by the
hydrolysis of caprine β-Lg with thermolysin. This peptide was also found to
decrease blood pressure when tested in spontaneously hypertensive rats.
Hernandez-Ledesma et al. (2006) hydrolyzed �-Lg A with thermolysin under
non-denaturing and heat-denaturing conditions. The peptides released during
hydrolysis were identified by HPLC–MS/MS. They reported that inaccessible
cleavage sites localized in the most buried zones of �-Lg were found to become
available to thermolysin when the incubation temperature increased in the range of
60-80°C. Three peptides appearing after 30-min hydrolysis at these incubation
temperatures, LDA, LKPTPEGD, and LQKW, had been previously identified and
reported to have potent ACE inhibition activity. Our results showed that one of
these peptides, LQKW, f(58-61), was found in all the hydrolysates, while
LKPTPEGD, f (46-53), was found only in the MW60 and MW90 hydrolysates.
150
Finally, m/z 1370 assigned to f (94-104), which was unique to the MW60
hydrolysate obtained by the two-stage hydrolysis process contained the sequence
VLDTDYK which has been reported to have ACE inhibitory activity
(Pihlanto-Leppala, 2001).
In conclusion, heat treatments were used to increase the susceptibility of
β-Lg to enzymatic proteolysis. During the heat-denaturing treatment, some of the
cleavage sites buried in the native protein were transiently exposed and thus
accessible to digestive enzymes. In the literature, hydrolysis had been done during
heat treatment, using thermophilic enzymes. Our results showed that microwave
treatment can be used to expose inaccessible cleavage sites for hydrolysis with
selected
enzymes.
The
nutraceutical
properties
of
hydrolysates
from
microwave-treated samples were found to be superior to those of conventionally
heated �-Lg solutions. This was attributed to the presence of the unique peptides as
well as higher amounts of peptides of known ACE and antioxidant activity in the
hydrolysates obtained from microwave treatment.
151
SUMMARY AND CONCLUDING REMARKS
The effect of microwave treatments on the structure and nutraceutical
properties of β-lactoglobulin was explored by a number of techniques. The first
technique, FTIR spectroscopy, was employed to probe the unfolding mechanism of
β-lactoglobulin by monitoring the extent of H-D exchange. Although isothermal
multiple-cycle microwave treatment did not result in significant H-D exchange
below ambient temperatures as compared to conventional heating, it did result in a
significantly higher H-D exchange under non-denaturing conditions above ambient
temperatures. Heating β-lactoglobulin at and above the T m not only resulted in a
greater amount of H-D exchange, but also a more significant increase in aggregate
formation in microwave- treated samples. Two-dimensional FTIR correlation
spectroscopy revealed that microwave treatment did not alter the pathways of
β-lactoglobulin unfolding and aggregate formation but rather accelerated the
unfolding of the protein.
Circular dichroism (CD), fluorescence, and
1
H NMR spectroscopic
techniques were employed to complement and confirm the results obtained from
the FTIR experiments. The results from far-UV CD spectra of β-Lg subjected to
microwave and conventional heating showed that microwave-treated β-Lg
solutions had a higher decrease in β-sheet content at each of the temperatures under
study. Similarly, the rigidity of the tertiary structure was explored by near-UV CD
spectroscopy. A substantial loss of native β-Lg structure was observed in
microwave-treated samples compared to samples heated by conventional means at
the same temperature. Furthermore, the results of 1D
1
H NMR TOCSY
experiments showed that much more extensive H-D exchange had occurred in �-Lg
solutions in D2O (pH 2) that had been subjected to microwave treatment at 50oC
than in solutions that had been conventionally heated at either 50 or 60oC. The
combined spectroscopic data provide strong evidence that isothermal microwave
and conventional heating are not equivalent in their effect on protein unfolding and
aggregate formation.
152
Based on the above findings, it was hypothesized that microwave treatment
of �-Lg may give rise to populations of thermally unfolded �-Lg with different
conformations from those present in conventionally heated samples and that
microwave-treated samples accordingly may respond differently to enzymatic
hydrolysis treatments. In experiments conducted to investigate this hypothesis,
microwave-treated β-Lg was found to undergo more extensive enzymatic
hydrolysis than conventionally heated β-Lg. Analysis of the kinetic parameters for
each enzyme employed in the hydrolysis experiments revealed that the enzyme’s
affinity�for �-Lg was significantly enhanced following microwave treatment of the
protein at temperatures above 40oC. The greatest extent of hydrolysis was found in
the case of microwave heating at 60°C. In order to assess whether the
microwave-enhanced effect also contributed to accessibility of the enzymes to new
cleavage site(s), leading to new peptide fragments, the hydrolysates of
conventionally heated and microwave-treated �-Lg were separated and their amino
acid sequences established by RP-HPLC- MS/MS.
The peptide profiles of
microwave-treated samples were dependent on the enzyme used, however, overall,
some peptides were found to be unique to microwave heated samples.
The
nutraceutical properties of hydrolysates from microwave-treated samples were
found to be superior to those of conventionally heated �-Lg solutions. This was
attributed to the presence of the unique peptides as well as a higher amount of
peptides with known ACE and antioxidant activity in the hydrolysates derived from
microwave treatment. These latter results provide a means of generating bioactive
peptides having multifunctional bioactivity.
Many other researchers have reported microwave-enhanced effects in the
form of accelerated chemical reactions and variations in the ratio of products
obtained by conventional and microwave heating at the same temperature. Such
phenomena have been attributed to local hot spots, molecular agitation, or
improved transport properties of molecules in a microwave field, among other
explanations. Many researchers have taken great care in designing experiments in
which the product of the reaction is not sensitive to temperature variation (e.g.,
photochemically induced reactions) and have observed microwave-enhanced
153
effects, providing evidence for the existence of athermal (or non-thermal) effects of
microwaves. Nevertheless, the origin of microwave-enhanced effects remains
controversial. While we have taken great care in ensuring temperature control in all
the experiments conducted in this work, the effects of molecular agitation and
improved transport properties in a microwave field cannot be discounted as the
cause of the microwave-enhanced effects on protein conformation that we have
observed. Alternatively, direct interaction between microwaves and proteins, as
proposed by Bohr et al., leading to a different population of protein conformations
must also be considered. The answer may be found through extensive kinetic
analysis of protein unfolding as a function of microwave and conventional heat
treatments.
154
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