Chemical Composition and Protein Antigenicity â•fi Almond (Prunus Dulcis)

181 Pages·2015·6.65 MB·English

by  Erin Kelly Monaghan

Florida State University Libraries
Electronic Theses, Treatises and Dissertations The Graduate School
2008
Chemical Composition and Protein
Antigenicity Almond (Prunus Dulcis) and
Macadamia Nut (Macadamia Integrifolia)
Seeds
Erin Kelly Monaghan
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FLORIDA STATE UNIVERSITY

COLLEGE OF HUMAN SCIENCES

CHEMICAL COMPOSITION AND PROTEIN ANTIGENICITY – ALMOND (Prunus
dulcis) AND MACADAMIA NUT (Macadamia integrifolia) SEEDS

By

Erin Kelly Monaghan

A Dissertation submitted to the
Department of Nutrition, Food and Exercise Sciences
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy

Degree Awarded:
Summer Semester, 2008
The members of the Committee approve the dissertation of Erin Kelly Monaghan defended on
June 24, 2008.

________________________________
Bahram Arjmandi
Professor Directing Dissertation

________________________________
Kenneth H. Roux
Outside Committee Member

________________________________
Yun-Hwa Peggy Hsieh
Committee Member

Approved:
________________________________________________________________
Bahram Arjmandi
(Chair, Department of Nutrition, Food, and Exercise Sciences)

________________________________________________________________
Billie Collier
(Dean, College of Human Sciences)

The Office of Graduate Studies has verified and approved the above named Committee
members.

ii
ACKNOWLEDGMENTS

I would like to express my deepest appreciation to my committee members, Dr. Bahram
Arjmandi, Dr. Kenneth Roux, and Dr. Peggy Hsieh for their unwavering support and
encouragement throughout my graduate program. Without their wisdom and guidance this
dissertation would never have come to fruition.

I owe a special thanks to my major advisor Dr. Bahram Arjmandi for his willingness to
“adopt” me and help me reach the finish line. His infectious enthusiasm and dedication to his
students defines a true and trustworthy mentor.

A special thanks to Dr. Mary Ann Moore, a wise and caring advisor, who gave me much-
needed reassurance and guidance.

I would like to offer my heartfelt gratitude to Dr. Jason Robotham and Dr. Kenneth Roux.
I am indebted to them for their assistance and guidance. They are extraordinary individuals who I
admire tremendously.

I would like to thank all faculty and staff from the Department of Nutrition, Food, and
Exercise Science for their assistance, guidance, and encouragement over the years.

A special thanks to Margaret Seavy for her continuous support, compassion, and
understanding.

I am thankful for all my past and present lab mates for making everyday life in the
laboratory enjoyable. It has been a true pleasure to work with each and every one of you and I
wish you all the best.

I have been very blessed in my life, particularly in my friendships. In all of the ups and
downs that came my way during the “dissertation years”, I knew I always had the support and
love of friends. In particular, I am grateful to Terry Love, April Hambrecht, Jennifer Hura,
Debbie Iarossi, Gina Pitisci, Molly Carey, Jenny Hansen, Brent Stoney, David Krum, Mary
Susan Gerber, and Elizabeth Ibbotson for always being there when I needed support. A very
special thanks to Grant Cotner for being incredibly supportive, patient, and constantly bringing a
smile to my face.

iii
I would like to thank my grandparents, aunts, uncles, cousins, and close family friends for
their enthusiasm and support throughout my graduate education.

I am forever grateful to Mallary and Rayleigh, my loyal fans and my greatest comforts,
who have blessed my life with their presence and brought it so much joy and pleasure.

I am extremely thankful to Ian Monaghan, who is not only my big brother but also my
oldest and closest friend. His support and humor has enriched my life more than he will ever
know.

Finally, I dedicate this dissertation to my parents, Patrick and Mary Monaghan. Words
alone cannot express the thanks I owe to my parents who have guided me, inspired me, and
encouraged me throughout my entire life. Their unwavering faith and confidence in me has
shaped me into the woman I am today. I am eternally grateful and blessed to have two of the
greatest people I have even known as my parents.

"I believe that everything happens for a reason. People change so that you can learn to
let go, things go wrong so that you learn to appreciate them when they're right. You believe lies
so you eventually learn to trust no one but yourself...and sometimes good things fall apart so
better things can fall together."
~ Marilyn Monroe ~

“Everyone who has even done a kind deed for us, or spoken one word of encouragement
to us, has entered into the make-up of our character and of our thoughts, as well as our success.”
~ George Burton Adams ~

“The future belongs to those who believe in the beauty of their dreams.”
~ Eleanor Roosevelt ~

“People grow through experience if they meet life honestly and courageously. This is how
character is built.”
~ Eleanor Roosevelt ~

iv
TABLE OF CONTENTS

List of Tables vii
List of Figures ix
Abbreviations xii
Abstract xiv

INTRODUCTION 1
Background 1
Statement of the Problem 4
Research Hypothesis 8
Significance of the Study 9
Limitations of the Study 9

REVIEW OF LITERATURE 10
Almond Cultivars 10
Macadamia Nut Cultivars 10
Physical Seed Characteristics 13
Chemical Composition 14
Tree Nut Allergies 23
Detection Methods for Tree Nut Allergens 25
Almond and Macadamia Nut Allergens 25
Cross-Reactivity 26
Allergen Stability 27

MATERIALS AND METHODS 29
Materials 29
Methods 30
Physical Seed Characteristics 30
Individual seed weight 30
Seed dimension 30
Preparation of Nut Seed Flours 30
Chemical Composition 30
Moisture 30
Lipid 30
Protein 31
Amino Acid Composition 31
Total amino acids 31
Free amino acids 32
Ash 33
Total soluble sugars 33
Tannins 33
Soluble Protein Estimation 34
SDS-PAGE 34
Polyclonal Antibody Production and Characterization 35
Monoclonal Antibody 4C10 Production and Characterization 35
Protein G Purification of Rabbit anti-Macadamia Nut Protein IgG 36
v
Competitive Inhibition ELISA Development 36
Almond Protein Detection 36
Macadamia Nut Protein Detection 36
Sandwich ELISA Development 38
Western Blot Analysis 38
Dot Blot Analysis 39
Densitometric Quantification 40
Cross-Reactivity Studies 40
Thermal Processing of Almond and Macadamia Nut Seeds 41
Autoclaving 41
Blanching 41
Dry Roasting 41
Frying 41
Microwave Heating 41
pH Treatments 42
Spiking Studies 42
Data and Statistical Analysis Procedures 42

RESULTS AND DISCUSSION 49
Environmental Conditions 49
Physical Seed Characteristics 49
Individual seed weight 49
Seed dimension 50
Chemical Composition 51
Moisture 51
Lipid 52
Protein 53
Ash 54
Total soluble sugars 54
Tannins 55
Amino Acid Composition 60
Total amino acids 60
Free amino acids 61
Electrophoretic Analysis 82
ELISA 82
Western and Dot Blotting 92
Cross-Reactivity Studies 92
Stability Studies 96
Spiking Studies 96

CONCLUSION 97
APPENDIX A 105
ANOVA Tables 105
APPENDIX B 142
Animal Research Approval 142
REFERENCES 145
BIOGRAPHICAL SKETCH 164
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LIST OF TABLES

Table 1. Proximate composition of almond and macadamia nut seeds 18

Table 2. Lipid composition of almond and macadamia nut seeds 19

Table 3. Mineral and vitamin content of almond and macadamia nut seeds 20

Table 4. Amino acid composition of almond and macadamia nut seeds 21

Table 5. Immunoassays for tree nut protein detection 28

Table 6. Cross-reactivity testing matrices 40

Table 7. Almond cultivars and history of origin 44

Table 8. Average climatic conditions in select California counties during 46
2003/2004 and 2005/2006 almond seasons

Table 9. Cultivar, geographic location, and harvest year variation on the 56
physical characteristics and chemical composition of select almond
seeds

Table 10. Interaction of cultivar and geographic location on the physical 57
characteristics and chemical composition of select almond seeds

Table 11. Physical characteristics and chemical composition of select 59
macadamia nut seeds

Table 12. Cultivar, geographic location, and harvest year variation on the 63
essential amino acid composition of select almond seeds

Table 13. Cultivar, geographic location, and harvest year variation on the 64
non-essential amino acid composition of select almond seeds

Table 14. Interaction of cultivar and geographic location on the essential 65
amino acid composition of select almond seeds

Table 15. Interaction of cultivar and geographic location on the non-essential 67
amino acid composition of select almond seeds

Table 16. Amino acid composition of select macadamia nut seeds 69

Table 17. Correlation coefficients among various physical and chemical 70
parameters of select almond seeds

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Table 18. Correlation coefficients among various physical and chemical 71
parameters of select macadamia nut seeds

Table 19. Cultivar, geographic location, and harvest year variation on the 75
free (essential) amino acid composition of select almond seeds

Table 20. Cultivar, geographic location, and harvest year variation on the 76
free (non-essential) amino acid composition of select almond seeds

Table 21. Interaction of cultivar and geographic location on the free 77
(essential) amino acid composition of select almond seeds

Table 22. Interaction of cultivar and geographic location on the free 79
(non-essential) amino acid composition of select almond seeds

Table 23. Correlation coefficients among free amino acids of select almond 81
seeds

Table 24. Effect of geographic location on the immunoreactivity of select 86
almond cultivars assessed by ELISA

Table 25. Interaction of cultivar and geographic location on the 87
immunoreactivity of select almond cultivars assessed by ELISA

Table 26. IgG purified macadamia nut pAb titer optimization 90

Table 27. Cross-reactivity of select foods/ingredients with Protein G purified 95
rabbit anti-macadamia nut IgG assessed by inhibition ELISA

Table 28. Detection of AMP in 100 mg of food matrices spiked with 102
almond seed protein extract or defatted almond seed flour

Table 29. Detection of macadamia nut protein in 100 mg of food matrices spiked 103
with macadamia nut protein extract or defatted macadamia nut flour

Table 30. Detection of almond and macadamia nut in commercially prepared 104
foods using ELISA

viii
LIST OF FIGURES

Figure 1. L ocation of alm ond see d sampl es inclu ded in the curr ent inve stigatio n. 45

Figure 2. E stimate d timel ine for Californ ia alm ond see d produ ction. * * Seas on 47
varies according to region, variety and weather conditions.

Figure 3. C limatic condit ions in Fresn o (A), Kern ( B), an d Stani slaus ( C) 48
counties during the 2003/2004 and 2005/2006 almond season. Daily
weather measurements recorded by the California Irrigation
Management Information System (CIMIS) and the National Climatic
Data Center (NCDC) of the National Oceanic and Atmospheric
Administration (NOAA) were used to calculate county averages.
Weather stations: CIMIS #2 and #80 for Fresno County, NCDC
#0442 for Kern County, and NCDC #6168 and #5738 for Stanislaus
County. T = maximum temperature and T = minimum
max min
temperature.

Figure 4. R elation ship be tween ( A) seed weigh t and se ed leng th, and (B) se ed 72
weight and seed width in 58 almond seed samples. Relationship
between (C) seed weight and seed diameter in 7 macadamia nut seed
samples. Data are expressed in grams (seed weight) and millimeters
(length, width, and diameter).

Figure 5. R elation ship be tween (A) lip id and protein , (B) p rotein and to tal 73
soluble sugars, and (C) lipid and total soluble sugars in 58 almond
seed samples. Relationship between (D) protein and total soluble
sugars in 7 macadamia nut seed samples. Data are expressed in gram
per 100 gram edible portion.

Figure 6. R elation ship be tween (A) g lutamic acid and as partic acid, ( B) 74
glutamic acid and arginine, and (C) arginine and lysine in 58 almond
seed samples. Relationship between (D) arginine and lysine in 7
macadamia nut seed samples. Data are expressed in gram per 100
gram protein.

Figure 7. P olypept ide pro file and AMP antigen icity of select almond cultiva rs 84
assessed by (A) SDS-PAGE stained with Coomassie stain; protein
load = 30(cid:1)g, (B) Western blot probed with mAb 4C10; protein load =
30(cid:1)g, (C) Dot blot probed with mAb 4C10; protein load = 500 ng.

Figure 8. S DS-PA GE ana lysis of macad amia nu t cultiv ars. A. Cooma ssie sta in; 85
protein load = 30 (cid:1)g. B. Silver stain; protein load = 10 (cid:1)g. C.
Glycoprotein stain; protein load = 200 (cid:1)g. D. Western blot; protein
load = 30 (cid:1)g. Primary Ab = Protein G purified rabbit anti- macadamia
nut IgG (1:16,000 v/v). Secondary Ab = HRP-labeled goat anti-rabbit
pAb (1:40,000 v/v).

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