Matrix Effects on the Detection of Milk and Peanut Residues by Enzyme-Linked Immunosorbent

162 Pages·2017·1.13 MB·English

by  Abigail S. Burrows

University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
Dissertations, Theses, & Student Research in Food
Food Science and Technology Department
Science and Technology
8-2016
Matrix Effects on the Detection of Milk and Peanut
Residues by Enzyme-Linked Immunosorbent
Assays (ELISA)
Abigail S. Burrows
University of Nebraska-Lincoln, [email protected]
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Burrows, Abigail S., "Matrix Effects on the Detection of Milk and Peanut Residues by Enzyme-Linked Immunosorbent Assays
(ELISA)" (2016).Dissertations, Theses, & Student Research in Food Science and Technology. 74.
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MATRIX EFFECTS ON THE DETECTION OF MILK AND PEANUT RESIDUES BY
ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA)

by

Abigail S. Burrows

A THESIS

Presented to the Faculty of
The Graduate College at the University of Nebraska
In Partial Fulfillment of Requirements
For the Degree of Master of Science

Major: Food Science and Technology

Under the Supervision of Professors Steve L. Taylor and Joseph L. Baumert

Lincoln, Nebraska

August, 2016
MATRIX EFFECTS ON THE DETECTION OF MILK AND PEANUT RESIDUES BY
ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA)
Abigail S. Burrows, M.S.
University of Nebraska, 2016
Advisors: Steve L. Taylor and Joseph L. Baumert

Food matrices are complex systems of lipids, carbohydrates, and proteins in which
interactions between matrix components and allergenic proteins are known to have negative
effects on the recovery of allergens when analyzed by ELISA. The purpose of this study was to
first evaluate the recovery of milk and peanut residues from multiple food matrices and mixes
and to secondly evaluate the use of a modified extraction protocol, sequential extractions, on the
recovery of milk and peanut allergens.
Pastry dough matrices and pastry dough mixes incurred with milk were prepared at
varying concentrations of flour and evaluated for recovery of NFDM. Secondly, a series of
samples were prepared with increasing flour concentrations (wheat, corn, rice, soy flour) while
maintaining a constant allergen (milk or peanut) concentration. Evaluation of sequential
extractions was done on pastry matrices (wheat, corn, or rice flour) incurred with milk or peanut.
Two matrix types, raw dough and baked matrices were analyzed for recovery.
Recovery of NFDM was reduced in wet pastry dough matrices in comparison to dry
pastry dough mixes, indicating that the formation of a food matrix influences the detection of
allergens. In concentration mixes, upon the addition of each flour type, the recovery of milk
residues decreased as the concentration of flour increased whereas the recovery of peanut
residues was not affected by the increasing concentrations of flour. The implementation of
sequential extractions yielded additional soluble protein from all matrices analyzed.
Interestingly, ELISA detectable protein was only extracted from raw dough matrices. No
detectable allergenic protein was extracted from baked pastry matrices.
The formation of a food matrix reduces the detection of milk allergens and reduced
recoveries of milk allergens were observed with both glutinous and non-glutinous flour mixes.
Peanut residues are less affected in sample mixes of different flour types. The use of a modified
extraction procedure improved the recovery of soluble protein (all matrices) and allergenic
protein (in raw matrices only).
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To my ohana, for filling my life with love, laughter, inspiration, and dance parties.

"Ohana means family. Family means nobody gets left behind or forgotten."
- Lilo and Stitch
iv
ACKNOWLEDGEMENTS
First and foremost, I must thank my advisors Drs. Steve Taylor and Joe Baumert
for allowing me to continue my education within FARRP here at the University of
Nebraska – Lincoln. I am extremely grateful to have you both as advisors and for your
constant advice and support.
Thanks to my committee members: Drs. Steve Taylor, Joe Baumert, Phil Johnson,
and Randy Wehling for your guidance and suggestions throughout the duration of this
project.
I must also thank my lab mates for making the lab an enjoyable place to work.
Thanks to Lynn, Deb, Sean, and the rest of the FARRP staff for all the help provided
throughout this project. Many thanks to Julie and Samudra for your patience and
guidance when I first began this research project.
Special thanks to my parents, Bill and Patty, and siblings, Cody, Amanda, and
Libby for their love and support during my time in graduate school. I am profoundly
grateful for the work ethic and respect you taught me growing up and for nurturing my
love of science.
A very special thanks to my ohana: Fish, Buns, Nessie, Farmer, Elle, and B.
Although we may live many miles apart, your unwavering support and unconditional
love travels with me every time I leave home.
I would be remiss if I didn’t extend my deepest thanks to Sydnee, my best friend,
who never ceases to amaze me in her support for her friends and family. For whom
without, my life would much less sparkly and my phone bill would be much less
v
expensive. We transcend best friend status, and I look forward to the many more
adventures and achievements life has in store for us. Here’s to us, fabulous and all.
Lastly, many thanks to the numerous friends and family, new and old, who have
supported me throughout this journey. I am extremely grateful for each of you and your
support.
Here’s to the next adventure.
vi
GRANT INFORMATION
Funding for this research was supported by the USDA – NIFA – AFRI Grant
2011-67017-20080 and the Food Allergy Research and Resource Program at the
University of Nebraska – Lincoln.
vii
TABLE OF CONTENTS
ABSTRACT…………………………..…...………………………………………………i
DEDICATION……………………...………………………………………...………..…iii
ACKNOWLEDGEMENTS……………………………………………...……………….iv
GRANT INFORMATION…………...………………………………...…………………vi
TABLE OF CONTENTS……..……………………………...……………………….…vii
LIST OF TABLES……………………………………………………………………....xiv
LIST OF FIGURES……………………………………………………………………...xv

CHAPTER 1: LITERATURE REVIEW…………………………..…………………16
I. INTRODUCTION…………………………………………………………...16
II. FOOD SENSITIVITIES……………………………………………………..17
A. Food Intolerances………………………………………………………...18
B. Food Allergies……………………………………………………………20
C. Prevalence of Food Allergy……………………………………………...23
D. Conclusion……………………………………………………………….24
III. BOVINE MILK…………………………………………………………...…24
A. Cow’s Milk Composition……………………………………………...…25
B. Cow’s Milk Proteins……………………………………………………..26
i. Caseins………………………………………………………...…26
ii. Whey Proteins……………………………………………………28
viii
1. Major Whey Proteins………………………………….…28
2. Minor Whey Proteins…………………………………….29
iii. Heterogeneity of Milk Proteins…………………………………..30
C. Adverse Reactions to Cow’s Milk……………………………………….31
D. Conclusion……………………………………………………………….33
IV. PEANUT……………………………………………………………………..34
A. Peanut Composition…………………………………………………..….34
B. Major Allergenic Peanut Proteins………………………………………..35
i. Ara h 1……………………………………………………………35
ii. Ara h 3……………………………………………………………37
iii. Ara h 2……………………………………………………………38
iv. Ara h 6……………………………………………………………39
C. Prevalence of Peanut Allergy…………………………………………….40
D. Conclusion……………………………………………………………….41
V. ALLERGEN DETECTION METHODS…………………………………….42
A. DNA Based Methods…………………………………………………….43
B. Mass Spectrometry Methods……………………………………………..44
C. ELISA Methods………………………………………………………….45
D. Conclusion…………………………………………………………….…49
VI. PROCESSING EFFECTS ON THE DETECTION OF FOOD
ALLERGENS………………………………………………………………..50
A. Effect of Processing on Allergenicity (IgE)……………………………...51
B. Effect of Processing on Detection (IgG)…………………………………54