NANOENCAPSULATION STRATEGIES FOR ANTIMICROBIAL CONTROLLED RELEASE TO ...

214 Pages·2014·3.71 MB·English

NANOENCAPSULATION STRATEGIES FOR ANTIMICROBIAL
CONTROLLED RELEASE TO ENHANCE FRESH AND FRESH-CUT
PRODUCE SAFETY

A Dissertation
by
LAURA ELLEN HILL

Submitted to the Office of Graduate & Professional Studies of
Texas A&M University
in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Chair of Committee, Carmen Gomes
Committee Members, Rosana Moreira
Elena Castell
Sandun Fernando
T. Matthew Taylor
Head of Department, Stephen Searcy

May 2014

Major Subject: Biological and Agricultural Engineering

Copyright 2014 Laura Ellen Hill
ABSTRACT

Spice essential oils and their constituents are powerful antimicrobials against
foodborne pathogens. However, their low sensory threshold and low aqueous solubility
make their application to fresh produce a challenge. Encapsulation within a
biocompatible material has the potential to mask sensory attributes and increase aqueous
solubility of the oils, thereby improving their applicability as antimicrobials onto fresh
produce. Cinnamon bark extract (CBE), trans-cinnamaldehyde, clove bud extract, and
eugenol were encapsulated in β-cyclodextrin (BCD), poly(DL-lactide-co-glycolide)
(PLGA), alginate, chitosan, and poly(N-isopropylacrylamide) (PNIPAAM) singly and in
combination. All essential oil capsules were characterized for particle size and
morphology, polydispersity index, entrapment efficiency, phase-solubility, and
controlled release profile. Following physical and chemical characterization, the oils
and their nanocapsules were analyzed for their antimicrobial activity against Salmonella
enterica serovar Typhymirium LT2 and Listeria spp. using a microbroth dilution assay
to determine minimum inhibitory and bactericidal concentrations at 35°C. The most
efficacious antimicrobial nanocapsules during in vitro testing were BCD-CBE, PLGA-
CBE, and chitosan-PNIPAAM-CBE, which were applied to fresh-cut romaine lettuce,
along with free CBE, to determine their efficiency against L. monocytogenes in a food
system. The chitosan-PNIPAAM-CBE yielded the greatest bacterial inhibition (P<0.05);
therefore, it was subjected to a shelf-life study to determine if there were any effects of
the particles on fresh-cut romaine lettuce quality over the course of storage. The
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antimicrobial nanoparticles did not significantly affect (P>0.05) overall product quality,
making encapsulated essential oils a viable treatment for improving food safety without
negatively impacting the product’s key attributes. This research project developed
several natural antimicrobial delivery systems that each exhibited unique release
properties and mechanisms, which improved the antimicrobial efficacy (P<0.05) of
essential oils and their active compounds. This study sought to characterize and compare
different nanoencapsulation systems based on their performance as controlled delivery
systems for natural antimicrobials against foodborne pathogens, which has not been
previously reported.
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DEDICATION

I dedicate this to my older brother, Austin, who was the first person to challenge
me and push me further than I ever imagined I was capable. Also to my parents, who
always picked me up when I fell, never let me give up, and loved me unconditionally
through it all.

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ACKNOWLEDGEMENTS

I would like to thank all of my committee members for all of their help and
guidance throughout my studies. Thank you to Dr. Elena Castell for allowing me to be
her teaching assistant and for being an excellent mentor in the classroom. And thank you
to Dr. Rosana Moreira for her excellent and honest advice on my research as well as for
my career. I would especially like to thank my advisor, Dr. Carmen Gomes, for all of the
time she spent guiding me through my experiments and studies. Her advice helped me
overcome countless technical hurdles and her willingness to help was always
appreciated.
I would also like to thank all of my labmates in the food engineering group that
provided not only help in the lab, but were also excellent company as we worked
together. I would especially like to thank Alex Puerta for constantly offering great
advice, Paulo Silva for his guidance, and also Kate Hills, Cassie Giacobassi, and Andre
Loquercio for providing tremendous amounts of assistance with my experiments and
work in the lab. I greatly appreciate the assistance of Dr. Sandun Fernando and his
students for allowing me to use their lab and providing technical support. I would also
like to acknowledge Dr. Matthew Taylor and the food microbiology group for
welcoming me into their lab to perform extensive experiments and a special thank you to
Keila Perez, to whom I owe a tremendous debt of gratitude for her years of patience,
help, guidance, and cheerful attitude in the microbiology lab.
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Finally, I would like to thank my family and friends—near and far. My friends in
College Station and beyond kept me sane the past several years and dragged me out of
the lab whenever they could, for which I am eternally grateful. My family provided me
with unwavering support and even when I felt discouraged, they always believed in me.
Thank you to my mom, who was always adept at getting me to see the positive side of
things and who I can always count on to be rooting for me. Her constant encouragement
kept me motivated throughout my Ph.D. Thank you also to my dad and step mom, who
helped me focus on my studies and research by allowing me to live with them and taking
great care of me. I never had to wonder if they believed in me, as they were constantly
supporting me, encouraging me, and fighting for me. My dad, especially, has been my
greatest supporter throughout my education and always believed in me. He never
allowed me to settle for anything less than my absolute best. Lastly, thank you to my
younger brothers for providing constant entertainment and my older brother for
providing ample advice on every area of my life—both solicited and not.

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TABLE OF CONTENTS

Page
ABSTRACT .......................................................................................................................ii
DEDICATION .................................................................................................................. iv
ACKNOWLEDGEMENTS ............................................................................................... v
TABLE OF CONTENTS .................................................................................................vii
LIST OF FIGURES ............................................................................................................ x
LIST OF TABLES .......................................................................................................... xiv
CHAPTER I INTRODUCTION ....................................................................................... 1
CHAPTER II LITERATURE REVIEW ........................................................................... 5
2.1 Foodborne Illness Linked to Fresh Produce ............................................................. 5
2.2 The Mechanism of Essential Oils as Natural Antimicrobials .................................. 6
2.3 Nanoencapsulation for Controlled Release Applications....................................... 12
2.4 Beta-Cyclodextrin .................................................................................................. 13
2.5 Poly-D,L-Lactide-co-Glycolide (PLGA) ............................................................... 16
2.6 Stimuli-Responsive Polymers ................................................................................ 18
2.7 Summary ................................................................................................................ 24
CHAPTER III CHARACTERIZATION OF BETA-CYCLODEXTRIN
INCLUSION COMPLEXES CONTAINING ESSENTIAL OILS (TRANS-
CINNAMALDEHYDE, EUGENOL, CINNAMON BARK, AND CLOVE
BUD EXTRACTS) FOR ANTIMICROBIAL DELIVERY APPLICATIONS ....... 26
3.1 Overview ................................................................................................................ 26
3.2 Introduction ............................................................................................................ 27
3.3 Materials & Methods .............................................................................................. 30
3.3.1 Materials .......................................................................................................... 30
3.3.2 Preparation of beta-Cyclodextrin Inclusion Complexes ................................. 30
3.3.3 Particle Characterization ................................................................................. 31
3.3.4 Minimum Inhibitory and Bactericidal Concentration ..................................... 34
3.3.5 Statistical Analysis .......................................................................................... 36
3.4 Results and Discussion ........................................................................................... 37
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3.4.1 Particle Characterization ................................................................................. 37
3.4.2 Minimum Inhibitory and Bactericidal Concentration ..................................... 49
3.5 Conclusions ............................................................................................................ 53
CHAPTER IV ANTIMICROBIAL EFFICACY OF POLY (DL-LACTIDE-CO-
GLYCOLIDE) (PLGA) NANOPARTICLES WITH ENTRAPPED
CINNAMON BARK EXTRACT AGAINST LISTERIA MONOCYTOGENES
AND SALMONELLA TYPHIMURIUM .................................................................. 55
4.1 Overview ................................................................................................................ 55
4.2 Introduction ............................................................................................................ 56
4.3 Materials & Methods .............................................................................................. 58
4.3.1 Materials .......................................................................................................... 58
4.3.2 Nanoparticle Synthesis .................................................................................... 58
4.3.3 Particle Size Analysis and Morphology .......................................................... 59
4.3.4 Entrapment Efficiency ..................................................................................... 60
4.3.5 Controlled Release .......................................................................................... 60
4.3.6 Minimum Inhibitory and Bactericidal Concentration ..................................... 61
4.3.7 Statistical Analysis .......................................................................................... 64
4.4 Results & Discussion ............................................................................................. 64
4.4.1 Particle Size Analysis and Morphology .......................................................... 64
4.4.2 Entrapment Efficiency ..................................................................................... 67
4.4.3 Controlled Release .......................................................................................... 68
4.4.4 Minimum Inhibitory and Bactericidal Concentration ..................................... 72
4.5 Conclusions ............................................................................................................ 75
CHAPTER V OPTIMIZATION OF A SYNTHESIS PROCEDURE FOR
THERMALLY-RESPONSIVE POLY-N-ISOPROPYLACRYLAMIDE
NANOPARTICLES FOR THE ENTRAPMENT OF HYDROPHOBIC SPICE
EXTRACTS .............................................................................................................. 77
5.1 Overview ................................................................................................................ 77
5.2 Introduction ............................................................................................................ 78
5.3 Materials & Methods .............................................................................................. 80
5.3.1 Materials .......................................................................................................... 80
5.3.2 Particle Synthesis ............................................................................................ 81
5.3.3 Particle Characterization ................................................................................. 84
5.3.4 Minimum Inhibitory and Bactericidal Concentration (MIC and MBC) ......... 88
5.3.5 Statistical Analysis .......................................................................................... 90
5.4 Results and Discussion ........................................................................................... 91
5.4.1 Particle Characterization ................................................................................. 91
5.4.2 Minimum Inhibitory and Bactericidal Concentration (MIC) and (MBC) ..... 104
5.5 Conclusions .......................................................................................................... 106
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CHAPTER VI CHARACTERIZATION OF TEMPERATURE AND PH-
RESPONSIVE POLY-N-ISOPROPYLACRYLAMIDE-CO-POLYMER
NANOPARTICLES FOR THE RELEASE OF CINNAMON EXTRACT ........... 108
6.1 Overview .............................................................................................................. 108
6.2 Introduction .......................................................................................................... 109
6.3 Materials & Methods ............................................................................................ 112
6.3.1 Materials ........................................................................................................ 112
6.3.2 Particle Synthesis .......................................................................................... 113
6.3.3 Particle Characterization ............................................................................... 117
6.3.4 Minimum Inhibitory and Bactericidal Concentration (MIC and MBC) ....... 120
6.3.5 Statistical Analysis ........................................................................................ 122
6.4 Results & Discussion ........................................................................................... 123
6.4.1 Particle Characterization ............................................................................... 123
6.4.2 Minimum Inhibitory and Bactericidal Concentration (MIC and MBC) ....... 137
6.5 Conclusions .......................................................................................................... 139
CHAPTER VII EFFECTS OF THE APPLICATION OF NANOENCAPSULATED
CINNAMON EXTRACT ON MICROBIAL SAFETY AND QUALITY OF
FRESH-CUT ROMAINE LETTUCE .................................................................... 140
7.1 Overview .............................................................................................................. 140
7.2 Introduction .......................................................................................................... 141
7.3 Materials & Methods ............................................................................................ 144
7.3.1 Materials ........................................................................................................ 144
7.3.2 Nanoparticle Synthesis .................................................................................. 144
7.3.3 Challenge Study ............................................................................................ 147
7.3.4 Shelf Life Study ............................................................................................. 150
7.3.5 Statistical Analysis ........................................................................................ 154
7.4 Results & Discussion ........................................................................................... 154
7.4.3 Challenge Study ............................................................................................ 154
7.4.4 Shelf Life Study ............................................................................................. 162
7.5 Conclusions .......................................................................................................... 179
CHAPTER VIII CONCLUSIONS ................................................................................ 181
CHAPTER IX RECOMMENDATIONS FOR FURTHER STUDY ........................... 184
REFERENCES ............................................................................................................... 185

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LIST OF FIGURES

FIGURE Page

2.1. Chemical structure of eugenol, the primary active compound present in clove
extract.................................................................................................................... 7

2.2. Chemical structure of trans-cinnamaldehyde, the primary active compound
present in cinnamon extract. ................................................................................. 7

2.3. Essential oil mechanism of action on microbial cell (Raybaudi-Massilia and
others 2009). ....................................................................................................... 11

2.4. Chemical structure of alpha-, beta-, and gamma-cyclodextrin molecules. ......... 14

2.5. Schematic of Poly (D,L-lactide-co-glycolide) (PLGA) and its components,
Polyglycolic acid (PGA) and Polylactic acid (PLA). ......................................... 17

2.6. N-isopropylacrylamide (NIPAAM) monomer unit chemical structure. ............. 20

2.7. Chemical structure of chitosan, a biopolymer extracted from chitin. ................. 21

2.8. Chemical structure of alginate, a polysaccharide extracted from brown
algae. ................................................................................................................... 22

3.1. Oxidative differential scanning calorimetry (DSC) thermogram of trans-
cinnamaldehyde (t-cinn), eugenol (eug), their respective BCD complexes
and a BCD complex containing a 2:1 mixture of trans-cinnamaldehyde and
eugenol ............................................................................................................... .38

3.2. Oxidative differential scanning calorimetry (DSC) thermogram of cinnamon
(cinn extract), clove extract, and their respective BCD complexes. ................... 39

3.3. Transmission electron microscope (TEM) images of beta-cyclodextrin
inclusion complexes. Images are representative of samples and depict
particles containing a 2:1 mixture of trans-cinnamaldehyde:eugenol at
100,000 times magnification (a), 50,000 times magnification (b), and trans-
cinnamaldehyde at 50,000 times magnification (c). ........................................... 43

3.4. Phase solubility of (a) trans-cinnamaldehyde and BCD (b) eugenol and BCD
in water at 25, 35, and 45°C. .............................................................................. 48
x