Effects Of Select Phenolic Compounds, Sugars And Thermal Processing On Immunoreactivity Of

90 Pages·2015·1.8 MB·English

by  Ying Zhang

Florida State University Libraries
Electronic Theses, Treatises and Dissertations The Graduate School
2012
Effects of Select Phenolic Compounds,
Sugars and Thermal Processing on
Immunoreactivity of Almond (Prunus Dulcis
L.) Proteins
Ying Zhang
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THE FLORIDA STATE UNIVERSITY

COLLEGE OF HUMAN SCIENCES

EFFECTS OF SELECT PHENOLIC COMPOUNDS, SUGARS AND THERMAL
PROCESSING ON IMMUNOREACTIVITY OF ALMOND (PRUNUS DULCIS L.)
PROTEINS

By

YING ZHANG

A Thesis submitted to the
Department of Nutrition, Food and Exercise
in partial fulfillment of the
requirements for the degree of
Master of Science

Degree Awarded:
Fall Semester, 2012
Ying Zhang defended this thesis on October 15th, 2012.
The members of the supervisory committee were:

Shridhar K. Sathe
Professor Directing Thesis

Yun-Hwa Peggy Hsieh
Committee Member

Mary Ann Moore
Committee Member

The Graduate School has verified and approved the above-named committee members, and
certifies that the thesis has been approved in accordance with university requirements
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ACKNOWLEDGEMENTS
First of all, I am very grateful to my major professor, Dr. Shridhar K. Sathe, for providing all the
high quality facilities, materials and methods in our lab in order for me to conduct and finish all
the experiments, for providing suggestions and ideas to help me with my experiment design, for
spending so much time, with patience, to assist me analyzing my data and complete my thesis
writing, and for providing financial support. I also want to thank Dr. Yun-Hwa Peggy Hsieh for
serving as my committee member and teaching me useful knowledge about immunoassays. I also
want to thank Dr. Mary Ann Moore for willing to serving as my committee member and
providing suggestions and answering questions about the academics during my study in Florida
State University. Moreover, I am thankful to Dr. Bahram H. Arjmandi and Dr. Yun-Hwa Peggy
Hsieh for allowing me to use their laboratory instruments.

I would like to express my gratitude to all those staff who helped me during my study here at
FSU, especially Tara Hartman, Ann Smith, David Parish, and Joseph Carroll.

My laboratory mates, Mengna, Changqi, Sahil, Guneet, Jasamrit, Dr. Misra, and Sophie in Dr.
Hsieh’s laboratory made my life in laboratory more joyful. Special thanks to should be given to
Dr. Mengna Su who spent lots of time teaching me key laboratory skills and giving me
suggestions for my experiments. I am also thankful to Changqi Liu for his advice for my
experiments and my thesis writing. Moreover, I want to thank Dr. Ofori in Dr. Hsieh’s lab who
helped me with my autoclaving sample preparation.

I want to thank my parents and all family members such as my grandparents, uncle and aunt for
financial support to my study in the US. Finally, I want to say I love you, mom and dad. Without
your support and love, I could not have come this far.

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TABLE OF CONTENTS
List of Tables ............................................................................................................................. vi
List of Figures ...........................................................................................................................vii
List of Abbreviations............................................................................................................... viii
Abstract ...................................................................................................................................... x
1. INTRODUCTION .............................................................................................................. 1
Background ......................................................................................................................... 1
Statement of the Problem..................................................................................................... 4
Research Hypothesis ........................................................................................................... 6
Specific Aims ...................................................................................................................... 6
2. REVIEW OF LITERATURE .............................................................................................. 8
Food Allergies ..................................................................................................................... 8
Peanut Allergy and Tree Nut Allergies ......................................................................... 9
Almond ............................................................................................................................. 10
Almond and Almond Allergy ..................................................................................... 10
Almond Allergens...................................................................................................... 11
Phenolic Compounds ......................................................................................................... 14
Phenolic Compounds in Food and Dietary Intake ....................................................... 15
Bioavailability of Phenolic Compounds ..................................................................... 16
Disease Prevention .................................................................................................... 17
Allergic Diseases ....................................................................................................... 18
Interactions between Proteins and Polyphenols ................................................................. 20
Influence of Protein Structure .................................................................................... 21
Influence of Polyphenol Structure .............................................................................. 21
Influence of Protein and Polyphenol Concentration .................................................... 22
Binding Force between Proteins and Polyphenols ...................................................... 23
Covalent Interactions ................................................................................................. 23
Food Processing ................................................................................................................ 24
Effects of Food Processing on Food Allergens ........................................................... 24
Maillard Reaction ...................................................................................................... 25
3. MATERIALS AND METHODS....................................................................................... 27
Materials ........................................................................................................................... 27
Methods ............................................................................................................................ 28
Preparation of Defatted and Full Fat Almond Flour .................................................... 28
Protein Extract Preparation ........................................................................................ 28
Sample Preparation .................................................................................................... 29
Liquid System .................................................................................................... 29
Solid System ...................................................................................................... 29
Protein Determination ................................................................................................ 30
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis ................................... 30
Western Blot .............................................................................................................. 31
Dot Blot. .................................................................................................................... 31
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Enzyme Linked Immunosorbent Assay ...................................................................... 32
Data Analysis and Statistical Procedures .................................................................... 32
4. RESULTS AND DISCUSION .......................................................................................... 33
ELISA Results ................................................................................................................... 33
Liquid System............................................................................................................ 33
Room Temperature Incubation ........................................................................... 33
Water Bath (100ºC) ............................................................................................ 34
Solid System .............................................................................................................. 34
Dry Roasting with Sugars................................................................................... 34
Dry Roasting ...................................................................................................... 34
Microwaving ...................................................................................................... 35
Autoclaving and Blanching ................................................................................ 35
Immunoblotting Results .................................................................................................... 35
Liquid System............................................................................................................ 35
Room Temperature Incubation ........................................................................... 35
Water Bath (100ºC) ............................................................................................ 36
Solid System .............................................................................................................. 37
Dry Roasting with Sugars................................................................................... 37
Dry Roasting ...................................................................................................... 37
Microwaving ...................................................................................................... 37
Autoclaving and Blanching ................................................................................ 37
Discussion ......................................................................................................................... 38
Effect of Assay Formats and Polyphenols .................................................................. 38
Room Temperature Incubation ........................................................................... 38
Water Bath (100ºC) ............................................................................................ 38
Effect of Processing Methods .................................................................................... 40
Effect of Sugars ......................................................................................................... 40
Effect of Phenolic Compounds................................................................................... 41
Conclusion ........................................................................................................................ 43

APPENDIX .............................................................................................................................. 62
A. THE ANIMAL CARE AND USE COMMITTEE APPROVAL MEMORANDUM .......... 62
REFERENCES ......................................................................................................................... 63
BIOGRAPHICAL SKETCH ..................................................................................................... 77

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LIST OF TABLES
1 Polyphenols content in different foods and beverages .................................................... 17

2 Effect on immunoreactivity (R)a of almond proteins incubated with indicated polyphenols
at room temperature for 1 h ....................................................................................................... 44

3 Effect on immunoreactivity (R)a of almond proteins incubated with indicated polyphenols
in 100ºC water bath for 1 h ........................................................................................................ 45

4 Effect of dry roasting (170ºC 20 min) with sugar and polyphenols on the
immunoreactivity (R)a of almond proteins ................................................................................. 46

5 Effect of dry roasting (170ºC 20 min) with corn syrup and polyphenols on the
immunoreactivity (R)a of almond proteins ................................................................................. 47

6 Effect of different dry roasting temperature and polyphenols on the immunoreactivity
(R)a of almond proteins ............................................................................................................. 48

7 Effect of microwaving and polyphenols on the immunoreactivity (R)a
of almond proteins .................................................................................................................... 49

8 Effect of autoclaving and polyphenols on the immunoreactivity (R)a
of almond proteins .................................................................................................................... 49

9 Effect of blanching and polyphenols on the immunoreactivity (R)a of almond seed
proteins ..................................................................................................................................... 50

10 Summary of ELISAs, western blots and dot blots results in liquid system ...................... 61

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LIST OF FIGURES
1 Category of selected phenolic compounds in foods ........................................................ 15

2 Interference by polyphenols for the A under the same conditions used for the murine
405nm
mAb 4C10-based standardized sandwich ELISA ....................................................................... 50

3 Western blot (without BME) (A) and Ponceau S staining (B) of polyphenol treatment
samples in room temperature ..................................................................................................... 51

4 Dot blot for polyphenol treatment samples in room temperature .................................... 52

5 Western blot (without BME) (A and B) and Ponceau S staining (C) of polyphenol
treatment samples in 100ºC water bath for 1 h ........................................................................... 53

6 Dot blot for polyphenol treatment samples in 100ºC water bath for 1 h .......................... 54

7 Western and dot blot (A) and Ponceau S staining (B) of effect of dry roasting with
phenolic compounds and sugar on immunoreactivity of almond ................................................ 55

8 Western blot and dot blot (A) and Ponceau S staining (B) of dry roasting with phenolic
compounds and corn syrup on immunoreactivity of almond ...................................................... 56

9 Western blot and dot blot (A) and Ponceau S staining (B) of different dry roasting
temperature and phenolic compounds on the immunoreactivity of almond ................................ 57

10 Western blot and dot blot (A) and Ponceau S staining (B) of microwaving and phenolic
compounds on the immunoreactivity of almond ........................................................................ 58

11 Western blot and dot blot (A) and Ponceau S staining (B) of autoclaving and phenolic
compounds on the immunoreactivity of almond ........................................................................ 59

12 Western blot and dot blot (A) and Ponceau S staining (B) of blanching and phenolic
compounds on the immunoreactivity of almond ........................................................................ 60

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LIST OF ABBREVIATIONS
AD Atopic Dermatitis
AMP Almond Major Protein
AP Alkaline Phosphatase
ANOVA Analysis of Variance
a Water Activity
w
BME (cid:533)-Mercaptoethanol
BSA Bovine Serum Albumin
BSB Buffer Saline Borate
ºC Degree Celsius
CV Coefficient Variation
DI water Distilled water
EGCG Epigallocatechin Gallate
ELISA Enzyme Linked Immunosorbent Assay
HRP Horse Radish Peroxidase
50% signal Concentration of the analyte require to generate 50% signal
in ELISA
IgE Immunoglobulin E
IgG Immunoglobulin G
IKK IкBα Kinase
IL-6 Interleukin 6
kDa Kilo Dalton
LOD Limit of Detection
LSD Fisher’s Least Significant Difference
mAb Monoclonal Antibody
MHC Major Histocompatibility Complex
MW Molecular Weight
ND Not Detected
NFDM Nonfat Dried Milk
NFκB Nuclear Factor Kappa B
viii
Nrf2 Nuclear factor-erythroid-2-related factor 2
nsLTP non-specific Lipid Transfer Protein
pAbs Polyclonal Antibodies
PPO Polyphenol Oxidase
PR Pathogenesis-Related
PRPs Proline-Rich Proteins
RNS Reactive Nitrogen Species
ROS Reactive Oxygen Species
rPru 1 Recombinant Prunin 1
RT Room Temperature
SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
SEM Standard Error of Mean
TBS-T Tris-Buffered Saline-Tween
TH cells T Helper 2 cells
2
TLP Thaumatin-Like Protein
TNF-α Tumor Necrosis Factor-alpha
v/v Volume by Volume
WA Whole Almond
w/v Weight by Volume
w/w Weight by Weight

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