Determining the Efficacy of Antimicrobials for the Inhibition and Inactivation of Listeria

124 Pages·2017·0.81 MB·English

University of Connecticut
OpenCommons@UConn
Master's Theses University of Connecticut Graduate School
12-14-2016
Determining the Efficacy of Antimicrobials for the
Inhibition and Inactivation of Listeria
monocytogenes in Broth, Whole Milk, and Fresh
Cheese.
Sarah Kozak
University of Connecticut - Storrs, [email protected]
Recommended Citation
Kozak, Sarah, "Determining the Efficacy of Antimicrobials for the Inhibition and Inactivation of Listeria monocytogenes in Broth,
Whole Milk, and Fresh Cheese." (2016).Master's Theses. 1039.
https://opencommons.uconn.edu/gs_theses/1039
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Determining the Efficacy of Antimicrobials for the Inhibition and Inactivation of Listeria
monocytogenes in Broth, Whole Milk, and Fresh Cheese.

Sarah Marlene Kozak
B.S., Cornell University, 2015

A Thesis
Submitted in Partial Fulfillment of the

Requirements for the Degree of
Master of Science
At the
University of Connecticut
2016
Copyright by
Sarah Marlene Kozak
2016

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APPROVAL PAGE
Masters of Science Thesis
Determining the Efficacy of Antimicrobials for the Inhibition and Inactivation of Listeria
monocytogenes in Broth, Whole Milk, and Fresh Cheese.

Presented by
Sarah Marlene Kozak, B.S.

Major Advisor________________________________________________________________
Dr. Dennis J. D’Amico
Associate Advisor_____________________________________________________________
Dr. Mary Anne Amalaradjou
AssociateAdvisor_____________________________________________________________
Dr. Kumar Venkitanarayanan

University of Connecticut
2016

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Dedication
This thesis is hereby dedicated to my beautiful little babies, especially Mr. Meatball, may
he rest in peace. Of all the things in life that are precious and good, Meaty reigned superior. Blessed
to have the honor of being his mama, I can say with my whole heart that his hairlessness and
wrinkles are just a few of his unique qualities that made him the most beautiful creature in the
world. Kind, caring, and loving, Meaty never failed to comfort me throughout the best and worst
of times. His snuggles were the warmest, his purrs the loudest, and his naps the longest. After a
long day in the lab, being able to count on the sound of his tiny feet running to the door was what
I looked forward to most. If I asked him to nap with me, he never said no, and did not get up from
the nap for food until I was also ready for food. This work would not have been possible without
his unconditional love and support, and for that I am forever grateful. His brother (Doom) and
sisters (Tinky and Shadow Girl) have assisted with snuggles and always know how to put a smile
on my face, especially in the time since Meatball’s passing. An infinite number of thank-yous to
these four impeccable creatures, my babies.

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Acknowledgements
First and foremost, I would like to thank my major advisor Dr. Dennis D’Amico and our
lab group. As a whole, the D’Amico lab group has been instrumental in my success. My fellow
graduate student, Stephanie Barnes, has provided me with every form of support a person could
need. Whether it be helping me study, providing helpful tips from her own Master’s education
experience, watching my cats, or relaxing after a hard week, Stephanie’s shoulder is always there
to lean on and I would not have been able to complete my education without her continuous
support. Our lab group has been blessed with some wonderful undergraduate assistants, all of
whom I would like to thank, especially Yustyna Bobak and Emily Forauer, two bright young
scientists who will certainly be lifelong friends.
I would like to thank the Department of Animal Science for having me and supporting my
education, especially my committee members Dr. Kumar Venkitanarayanan and Dr. Mary Anne
Amalaradjou, not only for advising me on my research and thesis but for adding a diverse range
of students and research to the department from which I have learned an immeasurable amount.
Furthermore, to Dr. Steven Zinn, Tina Burnham, and the rest of the main office members for
providing technical support, friendly faces, and financial assistance. A very special thank you to
Bill Sciturro and the UConn Creamery for the production of the cheese used in experiments.
Aside from faculty and students at UConn, my success is largely attributed to the support
of my friends and family. My family has always supported and encouraged my endeavors,
especially my mom, dad, and brothers Alex and Kyle; graduate school was no exception.
Voicemails, long phone calls, and care packages from my Grammie became my constant
connection to home and my solace in times of stress. A material scientist, physicist, and
cheerleader, Alexis Ernst has been an absolute inspiration, and conveniently located across the
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hall. Always putting her well-being first, she exemplifies the most important kind of love, self-
love, and for allowing me to learn from her I am forever grateful and hope that I will continue to
keep learning after my time at UConn has ended. My many friends from Rochester, Irondequoit,
and Cornell, I do not have the space to list, but you know who you are (especially you, Katy
Zuchowski) and you are so very important to me. To Dr. Anne-Lise Smith, thank you for seeing
me, hearing me, and allowing me to grow in ways that have forever changed me; I will carry the
lessons you have taught me throughout my entire life.
Lastly, I would like to thank my husband, Ryan Kozak-Weaver as well as Lucy, Thomas,
and Julia for being the most open-hearted, loving, and awesome children. Words cannot begin to
express how important Ryan been throughout this process. Encouraging, patient, and caring, he is
my most valuable person, and I hope he knows how much I love him from the bottom of my heart
(of course he does).

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List of Tables and Figures
Figure Title Page Number
Chapter III
Minimum inhibitory concentrations (MIC) of antimicrobial
Table 1. 60
compounds in BHI broth adjusted to pH 7.4 and 5.5.
Minimum bactericidal concentrations (MBC) of antimicrobial
Table 2. 61
compounds in BHI broth adjusted to pH 7.4 and 5.5.
Synergistic and additive inhibitory antimicrobial
Table 3. 62
combinations in BHI broth adjusted to pH 7.4.
Synergistic and additive bactericidal antimicrobial
Table 4. 63
combinations in BHI broth adjusted to pH 7.4.
Synergistic and additive inhibitory antimicrobial
Table 5. 64
combinations in BHI broth adjusted to pH 5.5.
Synergistic and additive bactericidal antimicrobial
Table 6. 65
combinations in BHI broth adjusted to pH 5.5.

Chapter IV
Change in Listeria monocytogenes counts in UHT milk with
Figure 1. 80
caprylic acid over 21 days of storage at 7˚C.
Change in Listeria monocytogenes counts in UHT milk with
Figure 2. 81
sodium caprylate over 21 days of storage at 7˚C.
Change in Listeria monocytogenes counts in UHT milk with
Figure 3. 82
lauric arginate over 21 days of storage at 7˚C.
Change in Listeria monocytogenes counts in UHT milk with
Figure 4. 83
-polylysine over 21 days of storage at 7˚C.
Change in Listeria monocytogenes counts in UHT milk with
Figure 5. 84
hydrogen peroxide over 21 days of storage at 7˚C.
pH of ultra-high-temperature pasteurized whole milk
Table 1. containing antimicrobials at varying concentrations on day of 85
inoculation.

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Chapter V
Table 1. Composition of Queso Fresco. 100
Approximate concentration of antimicrobials applied to
Table 2. 101
Queso Fresco.
Changes in Listeria monocytogenes counts on Queso Fresco
Figure 1. during storage at 7˚C following dip application of hydrogen 102
peroxide.

Changes in Listeria monocytogenes counts on Queso Fresco
during storage at 7˚C following dip application of -
Figure 2. 103
polylysine, lauric arginate ethyl ester, and both antimicrobials
sequentially.

Changes in Listeria monocytogenes counts on Queso Fresco
during storage at 7˚C following dip application of -
Figure 3. 104
polylysine, lauric arginate ethyl ester, and both antimicrobials
sequentially.
Changes in Listeria monocytogenes counts on Queso Fresco
during storage at 7˚C following dip application of aqueous
Figure 4. 105
solutions of -polylysine, sodium caprylate, and both
antimicrobials sequentially.
Changes in Listeria monocytogenes counts on Queso Fresco
during storage at 7˚C following dip application of lauric
Figure 5. 106
arginate ethyl ester, sodium caprylate, and both antimicrobials
sequentially.
Changes in Listeria monocytogenes counts on Queso Fresco
during storage at 7˚C following dip application of acidified
Figure 6. 107
calcium sulfate with lactic acid, sodium caprylate, and both
antimicrobials sequentially.

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List of Abbreviations

ACSL Acidified Calcium Sulfate with Lactic Acid
ACSP Acidified Calcium Sulfate with Propionic Acid
BR Beta-Resorcylic Acid
BHI Brain Heart Infusion
BLEB Buffered Listeria Enrichment Broth
BPB Butterfield's Phosphate Buffer
CA Caprylic Acid
CDC Centers for Disease Control and Prevention
CFR Code of Federal Regulations
CFU Colony Forming Units
CCP Critical Control Point
DE Dey-Engley Broth
DMSO Dimethyl Sulfoxide
EAFUS Everything Added to Food in the United States
EPL -Polylysine
FDA Food and Drug Administration
FBC Fractional Bactericidal Concentration
FBC Fractional Bactericidal Concentration Index
I
FIC Fractional Inhibitory Concentration
FIC Fractional Inhibitory Concentration Index
I
GRAS Generally Recognized as Safe
GMP Good Manufacturing Practices
HP Hydrogen Peroxide
LAE Lauric Arginate Ethyl Ester
MBC Minimum Bactericidal Concentration
MIC Minimum Inhibitory Concentration
MOX Modified Oxford Agar
NIFA National Institute of Food and Agriculture
PDA Plant-derived Antimicrobials
QF Queso Fresco
RTE Ready-to-Eat
SC Sodium caprylate
SDW Sterile Deionized Water
TSB Tryptic Soy Broth
TPB Tryptose Phosphate Broth
UHT Ultra-high-temperature Pasteurized
USDA United States Department of Agriculture

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