Application of ultra high hydrostatic pressure for investigating the binding of flavor compounds to ß-lactoglobulin via headspace solid phase microextraction-gas chromatography

APPLICATION OF ULTRA HIGH HYDROSTATIC PRESSURE FOR
INVESTIGATING THE BINDING OF FLAVOR COMPOUNDS
TO β-LACTOGLOBULIN VIA HEADSPACE SOLID PHASE

MICROEXTRACTION-GAS CHROMATOGRAPHY

By
TINYEE ARDEN HOANG

A dissertation submitted in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY
Department of Food Science & Human Nutrition
DECEMBER 2006
© Copyright by TINYEE ARDEN HOANG, 2006
All Rights Reserved

© Copyright by TINYEE ARDEN HOANG, 2006
All Rights Reserved

To the Faculty of Washington State University:
The members of the Committee appointed to examine the dissertation of
TINYEE ARDEN HOANG find it satisfactory and recommend that it be
accepted.

___________________________________
Chair
___________________________________

___________________________________

___________________________________

ii

ACKNOWLEDGMENT
I would like to express my sincere gratitude to my major advisor, Dr. Barry G.
Swanson. His straightforwardness and ability to tear up a dissertation like no other make
him very special, and I hope that I can continue to be his “unofficial student” as I become
part of the “real” world.
Great appreciation is also extended to other committee members Dr. Joseph R.
Powers and Dr. John K. Fellman for their time, patience, guidance, support, advice, and
encouragement from the beginning of my Ph. D. program to the completion of my
dissertation.
Words cannot express how thankful I am to have Dr. Stephanie Clark as a last
minute addition to my committee, but also as an advisor for the Food Product
Development Team and a friend. Her knowledge and charisma make her a tremendous
asset for students like me.
I want to thank Dr. Herbert H. Hill, Jr. for giving me guidance on special areas of
analytical chemistry and support for my “unofficial” minor in chemistry. I want to thank
Dr. Carolyn Ross for her acceptance as a substitute for Dr. Fellman’s absence in my
defense. I am especially thankful to my committee as a whole for guiding me in the right
direction and assisting my research activities throughout my Ph. D. program.
I would like to express my appreciation to Frank L. Younce for his assistance
with the use of the high hydrostatic pressure equipment in the pilot plant; Karen Weller
and Vaughn Sweet for their help with the use of the laboratory facilities; and Scott
Mattinson for his tremendous help, knowledge, expertise, and advice on gas
chromatography.
iii

I want to express my thanks to my dearest friends, Hamzah Al-Qadiri, Esteban
Mejia-Meza, Subba Rao Gurram, Seung-Yong Lim, Coco Umiker, Shantanu Agarwal,
Jaydeep Chauhan, and Todd Yee, as well as other friends for their advice and
encouragement. I will miss you guys.
I would also like to thank all faculty, staff, and colleagues who have been
involved at all stages of my education. Special thanks go to Dr. Boon Chew, for his
encouragement, support, and friendship. Their support and encouragement contributed to
my academic achievements and success.
Great appreciation is also due to my parents Tuan Hoang and Mysi Hoang for
their constant encouragement, some financial aid, and support throughout my academic
career; my sister Tinya for her jokes and sense of humor to get me through difficult
times; and my fiancée Huajing “Jing” Xing for her love and support throughout my Ph.
D. program.
Finally, I am most grateful to USDA for their financial support throughout my
Ph. D. study at Washington State University.

iv

APPLICATION OF ULTRA HIGH HYDROSTATIC PRESSURE FOR
INVESTIGATING THE BINDING OF FLAVOR COMPOUNDS
TO β-LACTOGLOBULIN VIA HEADSPACE SOLID PHASE

MICROEXTRACTION-GAS CHROMATOGRAPHY

Abstract

by Tinyee Arden Hoang, Ph.D.
Washington State University
December 2006

Chair: Barry G. Swanson
Fluorescence spectroscopy and headspace-solid phase microextraction (HS-
SPME) gas chromatography analysis were used to evaluate the effects of ultra high
hydrostatic pressure (UHP) (600 MPa) and pH (3.0-9.0) on β-lactoglobulin (BLG)
surface hydrophobicity and binding of selected flavor compounds. An increase in
tryptophan intrinsic fluorescence intensity of BLG was observed after UHP of 16 min,
which suggested that the tryptophan residues were exposed during the unfolding of BLG.
A 2 nm red-shift in tryptophan emission wavelength was observed after UHP come-up
time, indicating changes in the polarity of tryptophan residues from a less polar to a more
polar microenvironment. After UHP treatment come-up time of BLG at pH 9.0, there was
an increase in BLG surface hydrophobicity, suggesting a flexible molecular structure due
to surface denaturation of BLG at alkaline pH. UHP treatment of BLG solutions at pH
3.0, 5.0, and 9.0 resulted in decreases in the number of binding sites for the nonpolar
fluorescence probe 6-propionyl-2-(dimethylamino)-naphthalene (PRODAN). UHP

v

treatment did not show significant influences in the apparent dissociation constant of
PRODAN.
An extraction time of 10 min was used for HS-SPME of δ-decalactone, 2-
methylbutyraldehyde, ethyl lactate, and diacetyl. Although the CAR/PDMS fiber was
able to detect diacetyl, ethyl lactate, and 2-methylbutyraldehyde, the PDMS/DVB SPME
fiber was selected for the extraction of selected flavor compounds due to reproducible

2

and linear (RP

P

> 0.954) calibration plots, and its semi-polar nature to extract δ-
decalactone.
As observed by fluorescence quenching, there is no linear relationship between
UHP treatment of BLG at 600 MPa and the number of binding sites for diacetyl, 2-
methylbutyraldehyde, δ-decalactone, and ethyl lactate. BLG has low binding affinity for
the selected flavor compounds with polar groups, and UHP treatment of BLG did not
influence binding of 2-methylbutyraldehyde as observed by fluorescence quenching
experiments. Headspace analysis of UHP–treated BLG resulted in significant increases (p
< 0.05) in flavor retention over native BLG. In addition, a short UHP treatment time
(come-up time) may be adequate for flavor retention.

vi

TABLE OF CONTENTS

Page
ACKNOWLEDGEMENTS................................................................................................ iii
ABSTRACT..........................................................................................................................v
LIST OF TABLES...............................................................................................................ix
LISTOF FIGURES ...............................................................................................................x
CHAPTER

1. INTRODUCTION .................................................................................................1
2. SURFACE HYDROPHOBICITY OF ULTRA HIGH PRESSURE TREATED

β-LACTOGLOBULIN – PRODAN FLUORESCENT PROBE.........................29

Abstract..........................................................................................................30
Introduction....................................................................................................31
Materials and methods ...................................................................................33
Results and discussion ...................................................................................37
Conclusions....................................................................................................45
References......................................................................................................46
3. APPLICATION OF SOLID-PHASE MICROEXTRACTION WITH

HEADSPACE GAS CHROMATOGRAPHY TO THE ANALYSIS OF
DIACETYL, 2-METHYLBUTYRALDEHYDE, ETHYL LACTATE, AND δ-
DECALACTONE IN MODEL BUFFER SOLUTION ......................................85

Abstract..........................................................................................................86
Introduction....................................................................................................87
Materials and methods ...................................................................................91
Results and discussion ...................................................................................94
Conclusions....................................................................................................96
References......................................................................................................97

vii

4. APPLICATION OF SOLID-PHASE MICROEXTRACTION WITH
HEADSPACE GAS CHROMATOGRAPHY TO THE ANALYSIS OF
DIACETYL, 2-METHYLBUTYRALDEHYDE, ETHYL LACTATE, AND δ-
DECALACTONE IN ULTRA HIGH PRESSURE-TREATED β-
LACTOGLOBULIN SOLUTIONS...................................................................103

Abstract........................................................................................................104
Introduction..................................................................................................105
Materials and methods .................................................................................109
Results and discussion .................................................................................112
Conclusion ...................................................................................................116
References....................................................................................................117
5. CONCLUSIONS..................................................................................................129

viii

LIST OF TABLES

Chapter 2: Surface hydrophobicity of ultra high pressure treated β-lactoglobulin –
PRODAN fluorescent probe

1. Apparent dissociation constant (K’d) and the number of binding sites (n)
of BLG for PRODAN after UHP treatment (600 MPa) for selected holding
times of 0 to 32 min (UHP0-UHP32) at pH 3.0-9.0 calculated using the
method by Cogan and others (1976).......................................................................84
Chapter 3: Application of solid-phase microextraction with headspace gas
chromatography to the analysis of diacetyl, 2-methylbutyraldehyde, ethyl lactate,
and δ-decalactone in model buffer solution

1. Volatile compounds identified using HS-SPME GC-FID....................................102
Chapter 4: Application of solid-phase microextraction with headspace gas
chromatography to the analysis of diacetyl, 2-methylbutyraldehyde, ethyl lactate,
and d-decalactone in ultra high pressure-treated β-lactoglobulin solutions

1. Static quenching constate (KSV), fractional number of binding sites (n), and
dissociation constate (K’d) of BLG for selected flavor compounds as a
function of UHP holding times (0, 8, 32 min) ......................................................124

ix