ANTIMICROBIAL ACTION OF THE PEPSIN HYDROLYSATE OF LACTOFERRIN (LFH) ON ...

160 Pages·2009·2.13 MB·English

ANTIMICROBIAL ACTION OF THE PEPSIN HYDROLYSATE OF
LACTOFERRIN (LFH) ON ESCHERICHIA COLI O157:H7
by
CHRISTOPHER A. MURDOCK

A Dissertation submitted to the
Graduate School-New Brunswick
Rutgers, The State University of New Jersey
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
Graduate Program in Food Science
written under the direction of
Karl Matthews, PhD
and approved by

New Brunswick, New Jersey
May, 2009
ABSTRACT OF THE DISSERTATION

ANTIMICROBIAL ACTION OF THE PEPSIN HYDROLYSATE OF
LACTOFERRIN (LFH) ON ESCHERICHIA COLI O157:H7

By Christopher A. Murdock

Dissertation Director:
Karl Matthews, Ph. D.

Foodborne illnesses are a significant problem and a major public health concern in the
United States and throughout the world. The control of microbial pathogens in foods is a
significant concern and numerous methods have been employed to control or prevent the
growth of pathogenic microorganisms in food, including the use of synthetic and natural
antimicrobial agents. There exist a plethora of literature on “natural” antimicrobial
compounds (e.g. nisin, lactoferrin) and their possible use in food systems to eliminate or
control the growth of pathogenic microorganisms. The actual antimicrobial mechanism
of action for some antimicrobials has been extensively studied and well documented but
for other potential natural biopreservatives, such as lactoferrin, the actual mechanism of
action is not well defined. Lactoferrin is a 78 kilo Dalton cationic iron-binding
antimicrobial glycoprotein produced in many mammalian secretions, including milk,
tears, saliva, and serum. Previous research has focused on iron starvation and cell
membrane damage. However, treatment with pepsin yields a peptide fragment, termed
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lactoferricin that lacks the iron binding sites and is still antimicrobial. It has also been
hypothesized that the peptide, due to its small size, in comparison to the whole molecule,
might be able to penetrate the outer membrane or that the smaller size of lactoferricin
may facilitate its access to microbial cell surface components. The peptide and pepsin
hydrolysate have been shown to depolarize the outer membrane of E. coli, however, this
is likely not the mechanism of action. The data presented in this study demonstrate that
the pepsin hydrolysate of lactoferrin (LfH) exerts its antimicrobial action on the inner
membrane of E. coli O157:H7 by forming pores in the membrane. This membrane
damage results in a loss of energy and ion balance (potassium ion (K+) efflux and
decreases in intracellular ATP concentrations coupled with increases in extracellular ATP
concentrations) leading to a collapse of membrane potential (∆Ψ) and a loss of cell
viability.
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ACKNOWLEDGEMENTS

I thank Dr. Matthews for his willingness to take me in as a student, coach and guide me
to the success we have reached together. Your understanding of a part-time students
need to balance work, family and school was key.

I would like to express my sincere deepest appreciation to my committee members, Dr.
Mikhail Chikindas, Dr. Stanley Katz and Dr. Thomas Montville for their guidance and
consistent motivation to achieve this goal. Each in their own way gave me the “right
guidance” at the “right time” and challenged me when I needed to think harder to reach
the solution to any problem. I truly thank each of you for success may not have happened
without you.

I would also like to thank all my friends in our labs as their assistance, even with the
smallest of tasks helped me in many ways to accomplish this research.

And last but by no means last, I thank my wife, Kim, and our children, Christopher and
Alexis, who have endured missing a husband and father on many a day or night so that I
could complete this daunting task. Their love and support never wavered.
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DEDICATION

To the memory of my father, Dr. Wade Thomas Murdock, MD and my father in law
Frank Antonacci. You were both taken from us too soon but your love and support will
always be with me.
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TABLE OF CONTENTS

Abstract ii
Acknowledgements iv
Dedication v
Table of Contents vi
List of Tables xi
List of Illustrations xiii
List of Abbreviations xvi

Chapter 1 1
I. Introduction 1
A. Background and Significance 1
II. Evaluation of the Potential Mechanisms 4
A. Elimination of Iron Starvation as Potential Mechanism 4
B. Other Potential Mechanisms for Lactoferrin and Lactoferricin Activity 7
III. Tables and Illustrations 12
IV. Bibliography 21

Chapter 2 23
I. Hypothesis 23
II. Dissertation Statement 23
III. Objectives 24
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Chapter 3 25
I. Comprehensive Materials and Methods 25
A. Bacterial Strains 25
B. Lactoferrin and Lactoferrin Hydrolysate preparation 26
C. Minimum Inhibitory – Minimum Lethal Concentration Assays (PYG) 28
D. Assay for activity in Ultra-high temperature (UHT) milk with or without
EDTA 29
E. Assay for activity in Ultra-high temperature (UHT) milk with pH
Neutralization or pH acidification 30
F. Assay for antimicrobial activity in liquid egg product 31
G. Development of spheroplast cells from E. coli O157:H7 32
H. Determination of the leakage of cellular constituents (e.g. Potassium (K+))
in LfH treated E. coli O157:H7 33
I. Determination of the of the intracellular, extracellular and total ATP levels
of LfH treated E. coli O157:H7 35
J. Determination of the transmembrane potential in LfH treated
E. coli O157:H7 38
K. Viability Assays in LfH treated E. coli O157:H7 39
II. Illustrations 43
II. Bibliography 46

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Chapter 4 48
I. Abstract 50
II. Introduction 51
III. Methods and Materials 53
A. Bacteria 53
B. Lactoferrin and lactoferrin hydrolysate 54
C. Assays for antimicrobial activity in peptone yeast extract glucose 55
D. Assays for antimicrobial activity in ultra-high temperature (UHT) milk
and pH-neutralized or acidified UHT milk 56
IV. Results and Discussion 56
V. Acknowledgements 61
VI. Tables and Illustrations 62
VII. Bibliography 70

Chapter 5 72
I. Abstract 74
II. Introduction 75
III. Methods and Materials 78
A. Bacteria 78
B. Antimicrobials 78
C. Microtitre Plate Assay 79
D. Fractional Inhibitory Concentration (FIC) 80
IV. Results and Discussion 80
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V. Acknowledgements 84
VI. Tables and Illustrations 85
VII. Bibliography 90

Chapter 6 93
I. Abstract 94
II. Introduction 95
III. Methods and Materials 98
A. Bacteria 98
B. Preparation of Lactoferrin and lactoferrin hydrolysate 99
C. Lf and LfH Sensitivity Assay 100
D. Development of spheroplast cells from E. coli O157:H7 101
E. Potassium efflux (K+) assay 103
F. ATP assay 104
G. Transmembrane potential assay 106
H. Cell viability assay 108
IV. Results and Discussion 110
V. Conclusion 117
VI. Acknowledgements 118
VII. Tables and Illustrations 119
VIII. Bibliography 126

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Chapter 7
I. Discussion 131
II. Future Work 137
III. Bibliography 139

Curriculum Vita 143
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