Allergen in Almond

147 Pages·2015·2.42 MB·English

by  Rashmi S. Tiwari

Florida State University Libraries
Electronic Theses, Treatises and Dissertations The Graduate School
2004
Effect of Food Matrix on Amandin (an
Allergen in Almond) Recovery and
Immunorecognition
Rashmi S. Tiwari
Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected]
THE FLORIDA STATE UNIVERSITY
COLLEGE OF HUMAN SCIENCES
Effect of Food Matrix on Amandin (an allergen in almond)
Recovery and Immunorecognition
By
RASHMI S. TIWARI
A thesis submitted to the
Department of Nutrition Food and Exercise Sciences
In partial fulfillment of the
Requirements for the degree of
Master of Science.
Degree Awarded:
Spring Semester, 2004
The members of the committee approve the thesis of Rashmi. S. Tiwari defended on 8th April
2004.
Shridhar K. Sathe
Professor Directing Thesis
Kenneth H. Roux
Committee Member
Cathy W. Levenson
Committee Member
Approved:
James M. Overton, Chair, Department of Nutrition Food, and Exercise Sciences
Penny A. Ralston, Dean, College of Human Science
The office of Graduate studies has verified and approved the above named committee members.
ii
This Master’s Thesis is dedicated to my parents –Tiwari, Satyanarayan and Tiwari, Naina, who,
in not consciously trying to mold in there own image, have influenced me for more than they will
ever know. They found no sacrifice too great to have me educated to this level. I also dedicate
my achievement to my sisters and brother. Their love, support and encouragement provided me
courage throughout.
Thank you!
Mom and Papa!
iii
ACKNOWLEDGEMENTS
The completion of this thesis represents the contribution of many persons. I am indebted to my
committee members, friends, and family, whose joint efforts on my behalf intersect within these
pages.
A thesis is created from the brooding, foreboding, and fearing of a student and the cajoling,
affirming, and critiquing of a committee. Dr. Shridhar. K. Sathe my major professor provided
healthy skepticism about the theory and the unconscious, but consistently affirmed the
worthiness of this study. His comments influenced the study from a rough hunch to a finished
product. He gave me clear and manageable goals, and was always incisive in his comments. My
sincere thanks to him for all his personal and professional help. I deeply acknowledge Dr.
Kenneth. H. Roux and Dr. Cathy Levenson for serving on my committee. I am also grateful to
Dr. J. Michael Overton and Dr. Moffatt for their professional advice and support.
I am also thankful to Dr. Bonnie Greenwood for providing me financial support through
Resource and Technology center.
Last but not the least I am also thankful to my fellow graduate students Mahesh, Harshal, Susan
and Erin who shared the trials of graduate work and the frustrations of research, I extend current
thanks for the friendship, especially to Mahesh. Things couldn’t have being that easier without
them. My special thanks to Mengna Su.
My family members mom, dad, sisters, brother and my relatives have always been the joy of my
life. I’d like to take this opportunity to express my love to them and complete this
acknowledgement.
My special thanks to Dr. Mary Ann Moore an educator, counselor and friend who was always
available for direction, support and encouragement.
iv
TABLE OF CONTENTS
List of tables……………………………………………………………………………..…….viii
List of figures…………………………………………………………………….………………xi
Abbreviations…………….………………………………………………………….…….……xii
Abstract………………………………………………………..…………………………….....xiii
CHAPTER I
Introduction………………………………………………………………………..……….……...1
Statement of the problem…………………………………………………….………...…...……..7
Research hypothesis…………………………………………………..……..………….…….…..8
Significance of the study…………………………………………………….……………….…..10
Limitation of the study………………………………………………………...………….……...11
CHAPTER II
Review of the literature………………………………………………………………...……...…12
Detection of allergens……………………………………………………….……...……12
Immunoassays…………………………………………………………………...………13
Applications of immunoassays……………………………………….……...………..…14
Cross-reactivity……………………………………………………………………….….16
v
Recovery studies…………………………………………………………………..……..17
CHAPTER III
Materials…………………………………………………………………………………………20
Methods………………………………………………………………………….……………….25
Preparation of defatted food matrix samples……………………………………….....…26
Preparation of amandin………………………………………………………………..…26
Protein extractions ………………………………………………………………...…….26
Protein determination……………………………………………………….…………....26
Enzyme-linked immunosorbent assay (ELISA)……………………….…………….…..27
Electrophoresis and western blotting…………………………………………..………...27
Dot blots……………………………………………………………………………….…28
Dark chocolate experiment………………………………………………………………28
Effect of extraction step………………………………………………………………….29
Effect of extraction ratio…………………………………………………………………29
Effect of extraction buffer……………………………………………………………….29
Effect of pH……………………………………………………………………………..29
Effect of spiking almond flour…………………………………………..……………….30
vi
Statistical analysis………………………………………………………………..………30
CHAPTER IV
Results……………………………………………………………………………………………31
ELISA………………………………………………………………………….………...31
Western blotting…………………………………………………………….…...….……48
Cross-reactivity……………………………………………………………..……..……..48
Qualitative detection of amandin……………………………………………………..….58
Dot blotting………………………………………………………………………...……63
LOWRY………………………………………………………………………….………65
CHAPTER V
Discussions……………………………………………………………………..….…….………88
Conclusions………………………………………………………………………….……….…..93
Appendix………………………………………………………………………………….…..….94
References………………………………………………………………………...……….……124
Biographical sketch………………………………………………………………..……..…..…131
vii
LIST OF TABLES
1. Examples of food allergens homologous to pathogenesis related proteins…………….....2
2. Selected ELISA applications for the detection of food allergens………..………….…...18
3. List of food matrices…………………………………………………………………..…21
4. Experimental design…………………………………………………………..…………25
5. Detection of amandin in 100 mg of food samples spiked with different levels of
amandin………………………………………………………………………...……..….32
6. The following tables shows the LSD values (p = 0.05) for comparing the means of
recoveries within the category…………………………..…...…………………..………37
7. Category effect on amandin detection………………………………….…..………....…39
8. The following tables list the categories that are significantly different from each other at
three spike levels…………………………………………………………………….…..40
9. The ratio of IC values of amandin (spike level 100 µg/100 mg of food)…………..…..42
50
10. The regression equation used for molecular weight determination……..……………….48
11. The molecular weights (kDa) of the protein bands identified in western blots
(pAb)………………………….……………………………………………………..…...54
12. Amandin detection using Lowry assay…..……………………………………………....65
13. List of cross-reactive food matrices……………………………………………………...70
14. Detection of amandin (almond major protein) in dark chocolate (100 mg) at different
spike levels..……………………………………………………………………………...74
15. The ratio of IC of amandin in dark chocolate…………………………..……………...75
50
16. Effect of spiking amandin (100 µg) before and after extraction on
recovery…………………………………………………………………………………..76
17. The ratio of IC values of amandin when spiked either before or after
50
extraction……………………………………………………………………...………….77
18. Effect of ratio (food matrix: extraction buffer) on amandin (100 µg spike level) recovery.
The two ratios tested are 1:10 and 1:20……………………………………………...…..79
19. The ratio of IC values of amandin extracted with different extraction
50
ratios……………………………………………………………………………………..80
20. Recovery of amandin (at 100 µg spike level) from different extraction
buffers…………………………………………….………………………………….…..81
21. The ratio of IC values of amandin when spiked in different buffers………………..…82
50
viii
22. Recovery of amandin from food matrices in different buffers (spike level= 100
µg)………………………………………………………………………………………..82
23. The IC values of amandin from food matrices in different buffers (spike level= 100
50
µg)……………………………………………………………………………….…….…83
24. Recovery of amandin from BSB (pH of 0.1 M BSB adjusted with 1 M HCl)………..…85
25. The ratio of IC values of amandin in BSB (pH adjusted with 0.1M HCl)…...………...85
50
26. Effect of pH on amandin detection……………………………………………..………..86
27. Detection of amandin in foods spiked with almond flour…….……………………….…87
28. One-Way ANOVA for detection of amandin in 100 mg of food samples spiked with
different levels of amandin…………………………………………...…..………….…..94
29. One-way ANOVA for comparing mean within the category of food
matrices…………………………………………………………………….….……….103
30. One-Way ANOVA for detection of amandin in 100 mg spiked foods to compare the
recoveries across the food matrices……………………………………………….…...107
31. One-Way ANOVA for detection of amandin in different food categories spiked with
different levels of amandin………………………...……………………………...……108
32. One-Way ANOVA for detection of amandin to compare the recoveries across the food
categories……………………………………………………………………………….109
33. One-Way ANOVA for detection of amandin in dark chocolate (100 mg) at different
spike………………………………………………………………………………….…109
34. One-Way ANOVA for recovery of amandin (at 100 µg spike level) from different
extraction buffers……………………………………………………………………….109
35. One-Way ANOVA for recovery of amandin from BSB (pH of 0.1 M BSB adjusted with
1 M HCl).……………………………..…………………………………………...……109
36. One-Way ANOVA for effect of spiking amandin (100 µg) before and after extraction on
recovery…………………………………………………………………………….…..110
37. One-Way ANOVA for effect of ratio (food matrix: extraction buffer) on amandin (100
µg spike level) recovery. The two ratios tested are 1:10 and
1:20………………………………………………………………………….………….111
38. One-Way ANOVA for recovery of amandin from food matrices in different buffers
(spike level-100 µg)…………………………………………………………………….112
39. One-Way ANOVA for recovery of amandin from food matrices using Lowry assay
(spike level-100 µg)……………………..……………………………………..……….112
40. One-way ANOVA for IC values……………………………………………………...113
50
41. One-way ANOVA for IC values……………………………………..……………….120
50
ix