Effects of Maillard Reaction on the Immunoreactivity of Almond Major Protein in the Food Matrices

87 Pages·2015·2.26 MB·English

by  Guneet Singh Chhabra

Florida State University Libraries
Electronic Theses, Treatises and Dissertations The Graduate School
2013
Effects of Maillard Reaction on the
Immunoreactivity of Almond Major Protein
in the Food Matrices
Guneet Singh Chhabra
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FLORIDA STATE UNIVERSITY
COLLEGE OF HUMAN SCIENCES

EFFECTS OF MAILLARD REACTION ON THE IMMUNOREACTIVITY OF ALMOND
MAJOR PROTEIN IN THE FOOD MATRICES

By
GUNEET SINGH CHHABRA

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

Degree Awarded
Fall Semester, 2013
Guneet Singh Chhabra defended this thesis on October 18, 2013.
The members of the supervisory committee were:

Shridhar K. Sathe
Professor Directing Thesis

Yun-Hwa Peggy Hsieh
Committee Member

John G. Dorsey
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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ACKNOWLEDGMENTS

This master’s thesis is dedicated to my niece, Noor who has come in my life as a blessing
of the almighty. Your smile is my remedy. I wish to see you grow all your life and live all your
dreams, for that will make my one dream come true, to see you happy. God bless you always. I
love you!
The journey that has lead to the completion of this Thesis has been long and challenging
and could not have been accomplished without several people. Thank you first to Dr. Sathe, who
gave me a chance to come back and supported me to finish my master’s degree. Sir, you literally
gave me a new beginning in life and when I needed it the most. I owe my life to my family and
friends, whose moral support kept me going when I got discouraged or frustrated. The
encouragement I received outside of the academic world overwhelms me.
To perhaps my biggest supporter throughout my life, the almighty, I call him Waheguru,
whose blessings carried me through many tough times and kept me going when the end was not
insight, thank you. Thank you to my Mom, Harpreet Kaur and Dad, Jasbir Singh, who got me
where I am today and always have stood with me in my decisions. To my sister, Gursimran Kaur
and brother-in-law, Harpreet Singh for being the best advisors all the time and bringing the angel
of my life, Kulpreet Kaur aka Noor, who is most precious to me. To my brother, Mandeep Singh,
my best friend for ever, thank you for always being there with all your love.
Thank you so much to my committee members, Dr. Hsieh and Dr. Dorsey, who provided
valuable suggestions and guidance in this research and otherwise. I could not have completed
this Thesis without the academic and moral support of my lab mates. Coming to lab everyday
was so easy when you visited an environment of learning, thinking, and encouragement. I was so
lucky to be a part of a lab with so many caring and gifted people that have become dear friends.
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They have taught me not only about protein research, but have taught me the value of peer
support. Thank you to Changqi Liu “Ben”, who always helped me selflessly and gave me
valuable suggestions during this research. There is so much to learn from you and you are a good
man my friend. Thank you to Sahil Gupta for being a friend and a colleague and always being
there to celebrate good and bad days. I would take this opportunity to thank Ying Zhang for
being an inspiration to work hard. Thank you to my colleagues Jasamrit Bakshi and Sepideh
Alasvand. I will cherish all of you.
Finally, thank you again to the person who has had the biggest impact on me through the
process of completing my Master’s Thesis, my major professor, Dr. Shridhar Sathe. I cannot
thank you enough for everything that you have done for me. Dr. Sathe loves research, and he
loves his graduate students. Thank you for giving me the finest education that one could receive
in this area of research and for always finding a way to support me financially and giving me the
opportunity to teach. Thank you for serving as my major professor for the past three years, for
being a mentor and a friend. Most of all, thank you for believing in me, even when I did not. You
have instilled in me a confidence that is worth more than I can begin to explain.
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TABLE OF CONTENTS
List of Tables................................................................................................................................vii
List of Figures.............................................................................................................................viii
Abstract..........................................................................................................................................ix
INTRODUCTION..........................................................................................................................1
Statement of the Problem................................................................................................................5
Research Hypothesis.......................................................................................................................5
Limitations of the Study…..............................................................................................................6
Significance of the Study................................................................................................................6
Definitions.......................................................................................................................................7
REVIEW OF LITERATURE.......................................................................................................10
Maillard Reaction Mechanism......................................................................................................11
MATERIALS AND METHODS..................................................................................................16
Isolation of Almond Major Protein (AMP)...................................................................................21
Protein Extraction..........................................................................................................................22
HunterLab LabScan XE Spectrophotometer.................................................................................22
Water Activity (Aw)….................................................................................................................22
Soluble Protein Content................................................................................................................23
Immunoassay.................................................................................................................................24
Statistical Analysis........................................................................................................................28
RESULTS AND DISCUSSION...................................................................................................29
Water Activity (Aw) of Food Matrices.........................................................................................29
Color of Food Matrices.................................................................................................................29
Effect of Maillard Reaction and Food Matrix on the Immunoreactivity of Amandin.................33
Sandwich ELISAs.........................................................................................................................35
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Effect of Maillard Reaction on Immunoreactivity of Food Matrices............................................49
Dot Blots........................................................................................................................................50
Western Blots.................................................................................................................................51
Conclusion.....................................................................................................................................59
APPENDIX: The Animal Care and Use Committee Approval Memorandum.............................61
REFERENCES..............................................................................................................................64
BIOGRAPHICAL SKETCH.........................................................................................................74

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LIST OF TABLES
2.1 Peptides recognized by murine mAbs......................................................................................11
3.1 List of food matrices for analysis.............................................................................................17
3.2 Gradient gel recipe.................................................................................................................. 26
4.1 Water activity (Aw at 25 °C) and browning of food matrices containing almonds................30
4.2 Sandwich ELISA using 4C10..................................................................................................35
4.3 Sandwich ELISA using 4F10...................................................................................................40
4.4 Sandwich ELISA using 2A3....................................................................................................44

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LIST OF FIGURES
4.1 Relationship between the L* and the Aw of the processed food matrices.............................34
4.2 Dot blot for the food matrix samples at room temperature (25 °C)........................................50
4.3Transferred sample proteins (without (cid:533)-me) on nitrocellulose membrane and Ponceau S......51
4.4 Transferred sample proteins (with (cid:533)-me) on nitrocellulose membrane and Ponceau S..........52
4.5 Western blot (without (cid:533)-me) of selected food matrices using 4C10.......................................53
4.6 Western blot (without (cid:533)-me) of selected food matrices using 4F10.......................................54
4.7 Western blot (without (cid:533)-me) of selected food matrices using 2A3........................................55
4.8 Western blot (with (cid:533)-me) of selected food matrices using 4C10...........................................57
4.9 Western blot (with (cid:533)-me) of selected food matrices using 4F10............................................58
4.10 Western blot (with (cid:533)-me) of selected food matrices using 2A3...........................................59

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ABSTRACT
In the US, among edible tree nut seeds consumed, almonds are ranked number one.
Almonds are considered heart healthy and approved by FDA for qualified health claim. Almonds
are consumed as natural raw unprocessed or as variously processed seeds such as blanched,
roasted, and fried. Additionally, almond seeds, in various forms (such as chopped, diced,
slivered, powdered and others) are used as an ingredient in many foods including baked goods
and confectionery items, granola bars, breakfast cereals, and several snack mixes. Inclusion of
almonds in these products is valued because almonds provide desirable crunchy texture, sweet
mellow flavor, and several micro- and macronutrients. Although safely enjoyed by most,
almonds may induce adverse reactions, such as allergic reaction, in sensitive individuals.
Almond allergy has been identified as the third most frequent tree nut allergy in the US and
anaphylaxis to almond has been reported. Amandin, a globular storage protein that accounts for
~65% of the extractable proteins is the major allergen in almond seeds. Amandin is a hexameric
protein and consists of two trimers. Each trimer is composed of three polypeptides each of
which is composed of 40-4β kDa acidic chain (α) linked by a disulfide bond to β0 kDa basic
chain ((cid:533)).
Foods, containing almonds, subjected to thermal processing typically experience Maillard
reaction. Maillard reaction, chemical reaction between reducing sugars and the amino groups on
amino acids/polypeptides/proteins cause non-enzymatic browning of foods. This browning
reaction is desirable in many food products due to color and flavor formation. Maillard reaction
also may glycate food proteins. As a result of Maillard reaction, destruction of amino groups,
glycation, and/or denaturation of proteins may therefore alter amandin immunoreactivity. The
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