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Effect Of Carvacrol-Loaded Nanoemulsions On ABioluminescent Strain Of Escherichia ColiO157:H7Clara Maria Vasquez MejiaPurdue University

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Recommended CitationVasquez Mejia, Clara Maria, "Effect Of Carvacrol-Loaded Nanoemulsions On A Bioluminescent Strain Of Escherichia Coli O157:H7"(2014). Open Access Theses. 276.https://docs.lib.purdue.edu/open_access_theses/276

01 14

PURDUE UNIVERSITY GRADUATE SCHOOL

Thesis/Dissertation Acceptance

Thesis/Dissertation Agreement.Publication Delay, and Certification/Disclaimer (Graduate School Form 32)adheres to the provisions of

Department

CLARA MARIA VASQUEZ MEJIA

EFFECT OF CARVACROL-LOADED NANOEMULSIONS ON A BIOLUMINESCENT STRAINOF ESCHERICHIA COLI O157:H7

Master of Science

BRUCE APPLEGATE

MARIA F. SAN MARTIN-GONZALEZ

LESLIE N. CSONKA

BRUCE APPLEGATE

MARIA F. SAN MARTIN-GONZALEZ

BRIAN E. FARKAS 04/03/2014

i

EFFECT OF CARVACROL-LOADED NANOEMULSIONS ON A BIOLUMINESCENT STRAIN OF ESCHERICHIA COLI O157:H7

A Thesis

Submitted to the Faculty

of

Purdue University

by

Clara Maria Vasquez Mejia

In Partial Fulfillment of the

Requirements for the Degree

of

Master of Science

May 2014

Purdue University

West Lafayette, Indiana

ii

Para mi familia, con todo mi amor y esfuerzo, y para Papá Dios, por guiarme y

fortalecerme siempre, en especial a lo largo de ese proceso.

iii

ACKNOWLEDGEMENTS

I want to thank God for giving me the strength I needed during hard moments and

being my friend at all times. Thanks to Dr. Bruce Applegate and Dr. Fernanda San Martin,

for giving me the opportunity in science, believing in me and being my guidance

throughout this process. I also want to thank Dr. Laszlo Csonka for being part of my

committee for all his support and remarkable suggestions. I am grateful for my lab mates

and office mates, for their help, support, and sharing their experience, specially Eileen

Duarte, Veronica Rodriguez, Kyle Parker, Tengliang Zhu, Sydney Moser, and Aaron

Pleitner. I have no words to express my gratitude to my family, they have been my

inspiration and happiness. Special thanks to Alejandro Salazar for all his truthful love and

company and to my good friends Milena Leon, Randol Rodriguez, Alejandra Mencia,

Natalia Jaramillo and Sofia Perez for their genuine friendship over time.

LIST OF T

LIST OF F

ABSTRAC

CHAPTER

CHAPTER

2.1 

2.2 

2.2.1 

2.3 

2.3.1 

2.3.1.

2.3.1.2

2.3.1.3

2.3.1.4

2.3.2 

2.4 

2.4.1 

2.5 

2.5.1 

2.5.2 

2.6 

2.6.1 

2.6.2 

TABLES ....

FIGURES ...

CT ....

R 1.  INTRO

R 2.  LITER

Escherich

Biolumin

Bact

Nanoemu

Nano

  Gravita1

  Ostwal2

  Drople3

  Bioacti4

Nano

Antimicr

Carvacro

Emulsifie

Lecithin

Tween 20

Oil Phase

Coco

Palm

TAB

....................

....................

....................

ODUCTION

RATURE RE

hia coli O15

nescent repor

erial lucifera

ulsions .........

oemulsion st

ational separ

ld ripening...

et aggregatio

ive solubility

oemulsions a

obial spectru

ol ..................

ers ...............

....................

0 ..................

e ..................

onut Oil .......

m Stearin ......

LE OF CON

....................

....................

....................

N ...................

EVIEW ........

57:H7 ...........

rters ............

ase ...............

....................

tability.........

ration ...........

....................

n .................

y ..................

as natural an

um of herbs

....................

....................

....................

....................

....................

....................

....................

NTENTS

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

ntimicrobial

and spices ..

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

....................

vehicles ......

....................

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....................

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iv

Page .... vii

..... ix

.... xii

...... 1 

...... 2 

.......2 

.......6 

.......8 

.....10 

.....12 

.... 12 

.... 13 

.... 13 

.... 14 

.....14 

.....16

.....21 

.....23

.....26

.....26

.....27 

.....28 

.....28 

CHAPTER

BIOLUM

3.1

3.2 

3.3 

3.3.1 

3.3.2 

3.3.3 

3.3.4 

3.3.4.

3.3.5 

3.3.6 

3.4 

3.4.1 

3.4.2 

3.4.2.

3.4.3 

3.4.3.

3.4.3.2

3.4.3.3

3.4.4 

CHAPTER

LIST OF R

APPENDI

Appendix A

Appendix B

Appendix C

Appendix D

Appendix E

R 3.  EFFEC

MINESCENT

Abs

Introduct

Materials

Nano

Bact

Chem

In viv

  Dose-r1

Effec

ATP

Results a

Evalu

In viv

  Dose-r1

Effec

  Revers1

  2,4-Din2

  Additio3

ATP

R 4.  CONC

REFERENC

ICES

A Nanoemu

B  Non-norm

C  Biolumin

D  ATP Swa

E  Minimal

CT OF CARV

T STRAIN O

stract ...........

tion ..............

s and method

oemulsions f

erial strain a

mostat setup

vo biolumin

esponse and

ct of carvacr

Assay ........

and discussio

uation of cel

vo biolumin

esponse (BA

ct of carvacr

sibility of car

nitrophenol a

on of n-deca

Swab Hygi

CLUSIONS A

CES ..............

ulsions effec

malized biolu

nescence resp

ab Hygiene t

Inhibitory C

VACROL-L

OF ESCHERI

....................

....................

ds ................

formulation .

and media ....

...................

escence mon

d bactericidal

rol on biolum

....................

on ................

ll viability ...

escence mon

A50) ..............

rol on biolum

rvacrol effec

and carvacro

anal ..............

ene Test ......

AND FUTU

....................

ct on bacteria

uminescent

ponse to slow

test ..............

Concentration

LOADED NA

RICHIA COL

....................

....................

....................

....................

....................

....................

nitoring and

l activities (B

minescence ..

....................

....................

....................

nitoring .......

....................

minescence ..

ct .................

ol compariso

....................

....................

URE WORK .

....................

al luminesce

results .........

w addition o

....................

n .................

ANOEMUL

LI O157:H7 .

....................

....................

....................

....................

....................

....................

cell viabilit

BA50 values)

....................

....................

....................

....................

....................

....................

....................

....................

ons...............

....................

....................

....................

....................

ence product

....................

of emulsion .

....................

....................

LSIONS ON

....................

....................

....................

....................

....................

....................

....................

ty .................

) ..................

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....................

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tion ..............

....................

....................

....................

....................

v

Page

N A

.... 30

.... 30

.....31 

.....33 

.....33 

.....34 

.....34 

.....36 

.... 38 

.....38 

.....39 

.....40 

.....40 

.....48 

.... 54 

.....59 

.... 59 

.... 62 

.... 65 

.....68 

.... 72 

.... 74

.....84 

.....93

.....96

.....97 

.....99 

vi

Page

Well Diffusion Agar ...................................................................................................99 

Disk Diffusion Agar ...................................................................................................99 

Appendix F  Statistical output ....................................................................................102 

vii

LIST OF TABLES Table .............................................................................................................................. Page

Table 1. Escherichia coli O157:H7 outbreaks. ................................................................... 4 

Table 2. Comparison of thermodynamic stability and physicochemical properties of

colloidal dispersions prepared with oil, water and emulsifier. ................................... 12 

Table 3. Antimicrobial composition of different EO. ....................................................... 20 

Table 4. Functionality of surfactants in some foods ......................................................... 25 

Table 5. Bacterial enumeration in CFU/mL after treatment with carvacrol-loaded

nanoemulsions............................................................................................................. 42 

Table 6. Nanoemulsions that completely inactivated bacterial growth. ........................... 44 

Table 7. General Linear Model: Analysis of Variance for bacterial enumeration. ........... 46 

Table 8. LSMEANS separation of carvacrol concentrations ............................................ 46 

Table 9. LSMEANS separation of antimicrobial dilution. ............................................... 46

Table 10. Non-normalized bioluminescent values of PS|Lu|C%. ..................................... 53 

Table 11. Microbial enumeration at three different dilutions of emulsions containing 2.5%

carvacrol. ..................................................................................................................... 55

Appendix Table

Table 12. Non-normalized bioluminescent values of CO|Lu|C%. .................................... 93 

Table 13. Non-normalized bioluminescent values of CO|Tw|C%. ................................... 94

Table 14. Non-normalized bioluminescent values of PS|Tw|C%. .................................... 95 

Table 15. MIC values emulsions diluted to 500 ppm. .................................................... 100 

viii

Appendix Table Page

Table 16. MIC values emulsions diluted to 750 ppm. .................................................... 100 

Table 17. MIC values emulsions diluted to 1000 ppm. .................................................. 101 

Table 18. One-way ANOVA (p<0.05).. ......................................................................... 102

ix

LIST OF FIGURES Figure ............................................................................................................................. Page

Figure 1. Graphic representation of timeline for reporting cases ....................................... 3 

Figure 2. Chemical Structure of the bacterial luciferin (FMNH2). ..................................... 8 

Figure 3. Biochemical pathway for bacterial luminescence . ............................................. 9 

Figure 4. Expression of the lux gene cassette (luxCDABE) in bacterial luciferase .......... 10 

Figure 5. Chemical structure of carvacrol and thymol ..................................................... 21 

Figure 6 Nomenclature used for different formulations of nanoemulsion. ...................... 33 

Figure 7. Chemostat setup................................................................................................. 35 

Figure 8. Photo Multiplier Tube System for real-time bioluminescence monitoring. ...... 37 

Figure 9. Effect of nanoemulsions containing Palm Stearin (PS), Lecithin (Lu) and 1% of

carvacrol at different dilutions: 500, 750 and 1000 ppm. ........................................... 50 

Figure 10. Effect of nanoemulsions containing Palm Stearin (PS), Lecithin (Lu) and 2%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ....................................... 51 

Figure 11. Effect of nanoemulsions containing Palm Stearin (PS), Lecithin (Lu) and 2.5%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ....................................... 52 

Figure 12. Bioluminescent response of Escherichia coli O157:H7 lux to emulsions

containing 2.5% of carvacrol and diluted to 250 and 350 ppm. ................................. 56

Figure 13. Bactericidal activities (BA50) of all nanoemulsions containing 2.5% of

carvacrol. ..................................................................................................................... 58

x

Figure Page

Figure 14. Light response of Escherichia coli O157:H7 in presence and removal of

PS|Lu|C0% and PS|Lu|C2% at 500 ppm ..................................................................... 61 

Figure 15. Addition of sub-lethal doses of 2,4-DNP (0.1 mM, 0.5 mM, and 1mM) to

Escherichia coli O157:H7 lux. ................................................................................... 64 

Figure 16. Addition of n-decanal to Escherichia coli O157:H7 lux in presence of

carvacrol-containing nanoemulsions .......................................................................... 67 

Figure 17. ATP Standard Curve........................................................................................ 69 

Figure 18. ATP values of Escherichia coli O157:H7 in presence and absence of

carvacrol-loaded emulsions. ....................................................................................... 70

Appendix Figure

Figure 19. Effect of nanoemulsions containing Coconut oil (CO), Lecithin (Lu) and 1%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ....................................... 84

Figure 20. Effect of nanoemulsions containing Coconut oil (CO), Lecithin (Lu) and 2%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ........................................ 85 

Figure 21. Effect of nanoemulsions containing Coconut oil (CO), Lecithin (Lu) and 2.5%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ....................................... 86 

Figure 22. Effect of nanoemulsions containing Palm Stearin (PS), Tween 20 (Tw) and 1%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ....................................... 87 

Figure 23. Effect of nanoemulsions containing Palm Stearin (PS), Tween 20 (Tw) and 2%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ....................................... 88

Figure 24. Effect of nanoemulsions containing Palm Stearin (PS), Tween 20 (Tw) and 2.5%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ....................................... 89 

xi

Appendix Figure Page

Figure 25. Effect of nanoemulsions containing Coconut oil (CO), Tween 20 (Tw) and 1%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ....................................... 90 

Figure 26. Effect of nanoemulsions containing Coconut oil (CO), Tween 20 (Tw) and 2%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ....................................... 91 

Figure 27. Effect of nanoemulsions containing Coconut oil (CO), Tween 20 (Tw) and 2.5%

of carvacrol at different dilutions: 500, 750 and 1000 ppm. ....................................... 92 

Figure 28. Slow addition of nanoemulsion to Escherichia coli O157:H7 lux. ................. 96 

Figure 29. ATP values of Escherichia coli O157:H7 in presence and absence of

carvacrol-loaded emulsions ........................................................................................ 97 

Figure 30. ATP values of Escherichia coli O157:H7 in presence and absence of

carvacrol-loaded emulsions. ....................................................................................... 98 

Figure 31. Zones of bacterial inhibition of carvacrol-loaded nanoemulsions on E. coli

O157:H7.. .................................................................................................................. 101

xii

ABSTRACT

Vasquez Mejia, Clara M. M.S., Purdue University, May 2014. Effect of carvacrol-loaded nanoemulsions on a bioluminescent strain of Escherichia coli O157:H7. Major Professors: Maria Fernanda San Martin-Gonzalez and Bruce M. Applegate.

Nanoemulsions have been shown to be effective delivery vehicles for natural

antimicrobials that are poorly soluble in water. In this study the antimicrobial activity of

carvacrol (5-isopropyl-2-methylphenol) nanoemulsions against a bioluminescent

Escherichia coli O157:H7 was investigated using light emission as an indicator of cell

viability. Different emulsifiers (Ultralec Lecithin and Tween 20), oils (Palm stearin and

Coconut oil) and various carvacrol concentrations (0, 1, 2 and 2.5%) were evaluated.

Bioluminescence was monitored in situ using a Hamamatsu (photo multiplier tube)

sensor module integrated with a Programmable Logic Controller interfaced with a PC for

data acquisition. Bioluminescence decreased rapidly with the addition of emulsions

containing increasing concentrations of carvacrol (250ppm-1000ppm). However when

cells were assayed for viability, plate counts showed there was not a correlation of

bioluminescence to cell inactivation. Bioluminescence was able to recover after the

removal of carvacrol from the surrounding media. The same bioluminescent pattern was

also observed with sub-lethal doses of the oxidative uncoupler 2,4-dinitrophenol and the

supplementary addition of the luciferase reaction substrate recovered the light emission in

presence of carvacrol.

xiii

These results suggest that carvacrol and 2,4-DNP uncouple oxidative

phosphorylation reducing the ATP available for the biosynthesis of the aldehyde

substrate. Previous reports suggested the mechanism of inactivation of carvacrol was

membrane damage resulting in loss of cellular contents and viability. However the

results of this work suggest that the mechanism of carvacrol inactivation is due to the

uncoupling of oxidative phosphorylation.

1

CHAPTER 1. INTRODUCTION

The main objectives of this work are: 1) assess the in vitro and in vivo antimicrobial

efficiency of nanoemulsions containing carvacrol against a bioluminescent strain of

Escherichia coli O157:H7. 2) Investigate the antimicrobial mechanism of action of

carvacrol using the bioluminescent bioreporter Escherichia coli O157:H7 lux as a target.

The following section is divided into four main subdivisions: Escherichia coli

O157:H7, bioluminescence, nanoemulsions and natural antimicrobials. An extensive

literature review was conducted on each topic in order to build an appropriate

background and achieve the objectives of this work.

The section of Escherichia coli O157:H7 covers the taxonomy of this pathogen and

recent outbreaks on leafy greens in the United States. The second section explains in

detail bioluminescent reporters and bacterial luciferase. The third section is dedicated to

nanoemulsions and their functional properties, including their role as natural

antimicrobial vehicles. The natural antimicrobial section covers in more detail their

properties and specifically, is focused on the antimicrobial effect of carvacrol.

2

CHAPTER 2. LITERATURE REVIEW

2.1 Escherichia coli O157:H7

Nonpathogenic Escherichia coli is naturally found in the gastrointestinal flora.

However, some Escherichia coli strains can cause gastrointestinal, urinary or central

nervous system diseases during infection. Escherichia coli are serotyped based on their

surface antigen profile: Somatic (O), flagellar (H) and capsular (H) (Nataro and Kaper

1998). In 1982, Escherichia coli O157:H7 was first recognized as a pathogen, after being

found in 58% of patients with Hemolytic Uremic Syndrome (HUS) (Neill et al. 1987).

HUS symptoms include decreased frequency of urination (renal insufficiency), tiredness,

and loss of pink color in cheeks and inside the lower eyelids (known as hemolytic anemia

and thrombocytopenia respectively). Strains with the ability to produce shiga toxin (Stx)

are called STEC such as Escherichia coli O157:H7 (now and on referred as E. coli

O157:H7) (Neill et al. 1987). Shiga toxin is composed by two subunits (A-B), where A

contains the enzymatically active molecule and B has the ability to bind with the

holotoxin receptor to the target eukaryotic cell (Neill et al. 1987). The eae (E. coli

attaching and effacing) gene has been found to be essential for the production of Shiga-

toxin, and the “attaching and effacing” (A/E) effect (Yu and Kaper 1992). The A/E effect

is the intimate attachment of the bacterial cells to the intestinal epithelial by the

d

1

il

re

fo

id

co

Fwapco

in

ou

th

T

estruction o

991, Yu and

An outb

llness resulti

eporting cas

ollowed are

dentification

onfirmation

Figure 1. Grwhere the perpproximatelyonsidered pr

The C

nfection are p

utbreaks we

hese outbrea

The primary i

f microvilli

d Kaper 1992

break is def

ing from th

ses of E. co

e: ingestion

n of E. coli

of case (Fig

aphic reprerson gets ill ty 2-3 weeksreliminary an

CDC has repo

pre-packed l

re related to

ks was repor

intervention

and disrupt

2, Chen et al

fined as an i

e ingestion

li O157:H7

of contam

i O157:H7,

gure 1) (CDC

esentation oto the confir

s. Therefore, nd must be a

orted severa

leafy greens

consumptio

rted due to f

by the Food

tion of the c

l. 2013).

ncident in w

of a comm

takes from

inated food

verification

C 2011).

f timeline formation of becase counts

analyzed in t

l E. coli O15

(Table 1). S

on of ground

food safety in

d Safety and

cellular cyto

which two o

mon food (C

m 6 to 23 da

d, patient b

n by a Pub

or reportingeing part of

s during an othe same con

57:H7 outbre

Since 1997 a

d beef. Howe

ntervention

d Inspection S

oskeleton (G

r more peop

DC 1997).

ays and the

ecomes ill,

blic Health

g cases. Thean outbreak

outbreak inventext. (CDC

eaks where t

all previous E

ever in 2003

efforts with

Service (FSI

Griffin and T

ple experien

The timelin

e order of

stool samp

Laboratory

e time from takes

estigation ar2011)

the sources o

E. coli O157

, a decrease

meat produc

IS) was the

3

Tauxe

ce an

ne for

steps

pling,

y and

re

of

7:H7

in

cers.

4

release of a notice to all raw ground beef producers to reassess their HACCP plans and no

distribution of meat should be done unless it tested negative for this pathogen (CDC 2003)

Table 1. Escherichia coli O157:H7 outbreaks. The CDC has reported four E. coli O157:H7 outbreaks related with pre-packed leafy greens.

Year Number of cases reported

Hospitalization cases

Deaths HUS cases

Source

2006 199 102 3 31 Fresh Bagged Spinach

2011 60 45 0 2 Packaged Romaine Lettuce

2012 33 15 2 2 Pre-packed leafy greens

2013 33 11 0 2 Ready-to-eat Salads

On 2006, a multistate (Wisconsin, Oregon and New Mexico) outbreak of E. coli

O157:H7 occurred by the consumption of bagged fresh spinach where a total of 199

people were infected, 51% were hospitalized, 15% had Hemolytic Uremic Syndrome

(HUS) and three people died (CDC 2006). The most frequently reported symptoms

among affected individuals included diarrhea (96%), abdominal cramps (96%), bloody

diarrhea (88%), fatigue (80%), watery diarrhea (63%), and chills (57%). After the

outbreak was announced by the CDC, an environmental investigation conducted jointly

by the FDA and the California Department of Health Services, Food and Drug Branch

(CDHS) reported river water, cattle feces, and wild pig feces as possible sources of the

pathogen (Wendel et al. 2009). Washing pre-packed spinach before consumption might

not decrease the risk of illness because E. coli O157:H7 could be spread from one

infected plant to the rest of the product and allow internalization in the spinach leaves

(Solomon et al. 2003).

5

Three different multistate outbreaks of E. coli O157:H7 were reported in 2011,

2012, and 2013. They were linked to romaine lettuce, pre-packaged leafy greens, and

ready-to-eat salads respectively. Unfortunately, records and traceability were not

sufficient to understand how the leafy greens growing fields became contaminated

(Slayton et al. 2013). It remains unclear if the produce was infected in the field, during

harvesting, or throughout processing as initially proposed by (Brandl 2008).

Produce contamination can occur during agricultural production (irrigation, soil,

via animals, dirty equipment, and human handling), harvesting, processing (cutting,

washing, incorrect hygiene practices, and shredding), packaging, transportation, and

distribution (Francis et al. 2012). Brandl (2008) tested the susceptibility of mechanically

damaged tissues (cut and shredded) and lesions caused by diseases (soft rot and tip burn)

to be infected by E. coli O157:H7 and concluded that this pathogen can replicate on

damaged tissues by 11-fold in just 4 h.

Therefore, the ability of E. coli O157:H7 to colonize cut tissues can be related to

its numerous outbreaks in minimally processed leafy greens and how small numbers of

pathogens can increase to minimal infection doses if post-harvest and processing is not

conducted with precaution.

Generally, bacterial identification methods include morphological evaluation of

the microorganism as well as selective enrichment exploiting the pathogens ability to

grow in several media under diverse conditions (Ivnitski et al. 1999). Traditional methods,

such as colony counting for enumerating bacteria, are often really time consuming and

non-rapid, since the development of a colony containing 106 organisms will take between

18 and 24 h (Ivnitski et al. 1999). However, over the last three decades, numerous

im

th

(D

te

It

d

(H

lu

ph

ox

(P

re

en

2

2

h

mmunologic

hese are: Enz

Dwivedi and

echniques ar

t is clear that

evelop new

Biolum

Hastings and

uciferin, is o

hoton (Hasti

xygen into th

P*) with eno

eaction can b

Biorep

nvironment

004) wherea

006). An oft

ave been gen

al and molec

zyme-linked

d Jaykus 201

re coupled w

t the field of

concepts and

minescence is

d Nealson 19

xidized by th

ings and Nea

he substrate

ough energy

be summariz

porters are li

by displayin

as the signal

ten used bior

netically mo

cular-based

d immunosor

1). Howeve

with a prelimi

f food microb

d techniques

2.2 B

s the product

977, Shimom

he action of

alson 1977).

and forming

to emit a ph

zed as:

iving microo

ng a specific

produced is

reporter are

odified to con

rapid assays

rbent assay (

r it is import

inary enrichm

bial detectio

s is widely o

ioluminesce

tion and emi

mura 2012). I

f the luciferas

. All lucifera

g a molecule

hoton (Hastin

organisms th

and easily m

proportiona

bioluminesc

ntain the lux

s have been d

(ELISA), DN

tant to note t

ment which

on is very dy

open.

ent reporters

ission of ligh

In biolumine

se enzyme, r

ases are oxyg

e (P) in an el

ngs 1983). T

E

hat are susce

measurable s

al to the anal

cence-based,

x genes whic

developed. E

NA hybridiz

that all of th

can last from

ynamic and th

ht by a living

escent reacti

resulting in e

genases, inco

lectronically

The general b

Equation 1

eptible to cha

signal (Van D

lyte concentr

, in which m

ch when expr

Examples of

zation, and P

he above

m 6 to 24 ho

he opportun

g organism

ions a substr

emission of

orporating

y excited stat

bioluminesce

anges in the

Der Meer et

ration (Lei e

microorganism

ressed in a v

6

f

CR

ours.

nity to

rate,

a

te

ent

al.

et al.

ms

viable

7

microorganism results in bioluminescence (D'Souza 2001). A successful bioreporter

consists of the appropriate selection of a microorganism that can survive the

environmental conditions under study and the bioluminescent genes must be genetically

incorporated into the target microorganisms without compromising essential cellular

functions (Simpson et al. 1998).

Bioreporters have tremendous applications in food safety. Ripp et al. (2008)

developed a bacteriophage which is able to detect extremely low levels (1 CFU/ mL) of

Escherichia coli O157:H7 in artificially contaminated food samples such as apple juice,

tap water and spinach leaf rinsates. Also, the development of a bioluminescent strain of

Pseudomonas fluorescens 5RL (King et al. 1990) allowed the in situ monitoring of

gaseous chlorine dioxide disinfection whereas the higher the levels of ClO2, the faster

decrease in detectable luminescence (del Busto-Ramos et al. 2008). Another application

is to determine the survival of microbial populations as well as information on the

bacterial spatial distribution on/in a specific food sample. This application of bacterial

luminescence will help to identify hot-spots for contamination based on light emission

intensity (Morrissey et al. 2013). With this being said, bioluminescence-based assays

offer a sensitive, real-time, accurate, and spatial methodology for monitoring the presence

and behavior of bacterial population in food samples (D'Souza 2001, Morrissey et al.

2013).

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11

diameter range from 0.1 µm to 100 µm. These emulsions are usually optically turbid or

opaque because their droplet sizes have similar dimensions to the wavelength of light (r ≈

λ) and so they can scatter light strongly (McClements and Rao 2011). Nowadays, due to

the ultra-high-pressure technologies, emulsions with particles sizes in the nano-size range

(r: 10- 100 nm) can be manufactured. These are called nanoemulsions and they have

shown better stability to gravitational separation and aggregation than traditional

emulsions due to their smaller particle size. However, these systems are still

thermodynamically unstable and phases will tend to separate over time (McClements and

Rao 2011) Nanoemulsions differ from conventional emulsions in their appearance,

physicochemical stability, and texture, making them more suitable for delivering

encapsulated bioactive compounds (Weiss et al. 2009). It is worth noting that there is no

specific critical particle size dependence on the functional properties of nanoemulsions

(Weiss et al. 2009). However, emulsions appearance are highly dependent on particle size

as they become translucent when the diameter is below 90nm. In the food industry, this

physical property is crucial when manufactures need to add antimicrobials or flavors to a

beverage without compromising transparency (Weiss et al. 2009) (Table 2)

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008). Theref

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bial vehicles

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e compounds

e tested on or

microbial grow

ul with 5g/L

14

with

bilty.

e

by

le

ty

ials

ed

s

range

wth

15

terpene (Donsì et al. 2011). The mechanism by which antimicrobials loaded in

nanoemulsions successfully inhibit antimicrobial growth depends on the nature of the

encapsulated component (e.g. essential oil, protein, surfactant), and also, on the

characteristics of the nanoemulsions themselves (e.g. size, charge, composition)

(McClements and Rao 2011). For example, the lipid phase of the nanoemulsion can be

loaded with a non-polar antimicrobial for further delivery by mass transport of the

antimicrobial from the inside of the emulsion through the aqueous phase of the food

system to the membrane of the pathogen (Weiss et al. 2009). Basically, these delivery

systems must be antimicrobial reservoirs ensuring constant concentrations in the aqueous

phase during time. This function is extremely important based on the lower solubility of

essential oils, therefore, the amount of the antimicrobial in a certain water volume would

be very limited (Donsì et al. 2011). According to McClements et al (2011) when droplet

sizes are smaller in oil-in-water (O/W) nanoemulsions, the oil-water surface area

increases leaving more lipid phase exposed to the surrounding aqueous phase; therefore,

they are more susceptible to chemical degradation due to oxidation or hydrolysis of the

antimicrobial. It is recommended to store the nanoemulsions in a dark place because

chemical degradation can be accelerated by UV or visible light which can penetrate them

more easily due to their small size.

Likewise, it is essential to choose an antimicrobial that will be capable of

exhibiting its desired functional properties under the conditions of the nanoemulsion

production, storage, and usage (McClements 1999). However, the biggest disadvantage

of using nanoemulsions to encapsulate bioactive compounds is that the equipment

16

required (homogenizer, microfluidizer, and ultrasonicator) is expensive, therefore a large

amount of initial monetary infusion is necessary (Kumar and Singh 2012).

2.4 Antimicrobial spectrum of herbs and spices

An interest in using natural antimicrobials to prevent food-borne outbreaks is

growing rapidly due to the emerging consumer demand for non-artificial food products,

and as a consequence, food manufacturers are putting efforts into replacing chemical

preservatives with natural and label-friendly antimicrobials extracted from herbs, spices

and/or plants while maintaining microbiological safety (McClements 1999, Smid and

Gorris 1999, Nazer et al. 2005, Tajkarimi et al. 2010).

The main purpose of using spices and herbs in the food industry is usually

flavoring. Their functional properties (e.g. flavor and aroma compounds, and

antimicrobial activity) are found in the essential oils (EOs) which are aromatic oily

liquids, commonly obtained by steam distillation of plant material (Burt 2004).

Antimicrobial agents originally from plants, can be phytoalexins, isothiocyanates,

alliin, plant pigments and phenolic compounds from herbs and spices (Cutter 2000).

Phytoalexins are antimicrobials with low molecular weight, broad-spectrum, and host

synthesized compounds, which are produced when the plant is injured. Isothiacyanates

are stored in the cell vacuoles of plants and their release occurs when tissue is disrupted.

Alliin, is a broad-spectrum antimicrobial and precursor of allicin, it is produced when

plant or bulb tissues are injured (Cutter 2000). Many factors have influence on the

antimicrobial activity of phyto-phenols, such as: chemical composition, geographic origin

17

and crop to crop variations (Moyler 1994) as well as characteristics of the target

microorganism such as genus, species, strain, and stage of growth (Naidu 2010).

The effect of natural antimicrobials against food-borne pathogens have been widely

investigated. The antimicrobial effect of the essential oil of eucalyptus (40.5% piperitone,

17.4% α-phellandrene) was tested against Gram positive and Gram negative

microorganisms using the agar diffusion method whereas Staphylococus aureus (Gram

positive) was the most sensitive with an inhibition zone of 52.3 ± 5.6 mm, and

Pseudomona aeruginosa (Gram negative) was the most resistant strain with an inhibition

zone of 9.1 ± 0.3 when 10 µl of the essential oil was added (Gilles et al. 2010). Donsi and

colleagues research has focused on the effect of encapsulating two essential oils, a

mixture of terpenes from Melaleuca alternifolia (commonly known as Narrow-leaved

Tea-tree, which have been used for medical purposes since the colonization of Australia

at the end of the 18th century), and D-limonene into nanoemulsion to enhance their

antimicrobial activity and improve the delivery system and have published 2 reports on

this topic. In their 2011 study, they concluded that all minimal inhibitory concentrations

(MIC) against E. coli, L. delbrueckii and S. cerevisae where lower when the antimicrobial

was encapsulated in comparison to the non-encapsulated compound. They also

investigated the antimicrobial activity of an encapsulated mixture of terpenes against L.

delbrueckii in pear and orange juices. Results showed that 1.0g/L of terpenes was needed

to inhibit microbial growth and 5.0g/L was needed to completely inactivate the

microorganisms without changing the organoleptic properties of the fruit juices (Donsì et

al. 2011). Based on these results, Donsi et al. (2012) investigated the antimicrobial effect

18

of nanoencapsulating carvacrol, limonene and cinnamaldehyde into sunflower oil

droplets stabilized with different emulsifiers (lecithin, pea proteins, sugar ester, and

combination of Tween 20 and glycerol monooleate). The results proposed that the

efficiency of the antimicrobials is positively related with its concentration in the aqueous

phase which is ruled by the effect of the emulsifier, whereas emulsions with Tween 20

and glycerol monooleate has a higher bactericidal activity in less than 2h (Donsi et al.

2012). The antimicrobial activity of the essential oil of lemon myrtle, mainly formed by

citral, was tested against fifteen different types of microorganisms including yeasts, fungi,

Gram positive and Gram negative bacteria. Lemon myrtle samples were prepared in

different ways to test differences in antimicrobial activities: 1) tea was infused in boiling

water, 2) tea was infused in water at 45°C to prevent loss of any antimicrobial activity

through evaporation, 3) tea was prepared ten times more concentrated than manufactures

recommendation in boiling water (10X= 5g of tea leaf in 25 mL of water), and 4) the

effect of citral alone was conducted as a control. The concentrated sample was an

effective antimicrobial against all microorganisms and no differences between Gram

positive and Gram negative were identified (Wilkinson et al. 2003).

Although the comparison of results concerning the antimicrobial activity of natural

herbs and spices is challenging due to the differences in methods, bacterial strains,

antimicrobial concentrations and contact time (Burt 2004), it has been proved that the

amount of essential oil needed to effectively inactivate microorganisms in vitro is much

lower than the quantity required to achieve the same results in a food system (Burt 2004,

Devlieghere et al. 2004, Gutierrez et al. 2008). In addition, gram positive strains have

shown to be more susceptible to the antimicrobial effect of essential oils than gram

19

negatives (Tassou and Nychas 1995, Mendoza-Yepes et al. 1997, Lambert et al. 2001,

Gilles et al. 2010).

Burt (2004) conducted an extensive review on essential oils and their antibacterial

properties with various food systems and concluded the following ranking of essential

oils from higher bactericidal effect to lower: oregano/ clove/ coriander/ cinnamon>

thyme> mint> rosemary> mustard> clinatro/ sage. The components of the essential oils

were also a ranked and reported as to their bactericidal effect as well: eugenol> carvacrol/

cinnamic acid > basil methyl chavicol> cinnamaldehyde> citral/ geraniol. Table 3

exhibits the antimicrobial composition of several EO.

20

Table 3. Antimicrobial composition of different EO. Essential oil Latin name of plant

source Major

component Approximate

% composition Cilantro Coriandrum sativum

(immature leaves) Linalool 26%

E-2-decanal 20% Coriander Coriandrum sativum

(seeds) Linalool 70%

E-2-decanal --- Cinnamon Cinnamomum

zeylandicum Trans-

cinnamaldehyde 65%

Oregano Origanum vulgare Carvacrol Trace-80% Thymol Trace-64% ϒ-terpiene 2-52% p-Cymene Trace-52%

Rosemary Rosmarinus officinalis α-pinene 2-25% Bornyl acetate 0-17%

Camphor 2-14% 1,8-cineole 3-89%

Sage Salvia officinalis L. Camphor 6-15% α-pinene 4-5% β-pinene 2-10%

1,8-cineole 6-14% α-tujone 20-42%

Clove Syzygium aromaticum Eugenol 75-85% Eugenyl acetate 8-15%

Thyme Thymus vulgaris Thymol 10-64% Carvacrol 2-11% ϒ-terpiene 2-31% p-Cymene 10-56%

Mint (Govindarajan et al.

2012)

Mentha spicata Carvone 48.60% cis-Carveol 21.30% Limonene 11.30% 1,8-cineole 2.55%

Mustard (Kirk et al. 1964)

Brassica juncea Allyl isothiocyanate

90.50%

Allyl thiocyanate

9.50%

Source: (Burt 2004)

1

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Figure 5. Chmonoterpenic

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be

22

The efficiency of carvacrol as food antimicrobial has been tested (Lambert et al.

2001, Ultee et al. 2002, Ben Arfa et al. 2006). Arfa et al. (2006) compared the

antimicrobial activity of five different aroma compounds (carvacrol, carvacrol methyl

ester, carvacryl acetate, eugenol, menthol) concluding that the chemical structure of

carvacrol is benefited by the hydroxyl group, which made carvacrol more efficient than

the other compounds. These results are comparable with the study conducted by Ultee et

al. (2002) when testing carvacrol, thymol and menthol against Bacillus cereus. The

activity of thymol (having a hydroxyl group in the meta position) was effective at

minimal concentrations of 0.75 mM and was similar to carvacrol. Menthol, on the other

hand, that does not have a hydroxyl methyl group was approximately 10 times less

effective than carvacrol. However, when carvacrol was tested in fish samples, the

antimicrobial activity was lower due to interactions with the food matrix (Kim et al.

1995). A partially synergistic effect against oral bacterial strains of Streptococcus and

Prevotella was observed when combinations of carvacrol with thymol and, carvacrol with

eugenol, resulted in Fractionary Inhibitory Concentrations (FIC) between 0.5 and 0.75.

The FIC ranking is divided in sections: FIC ≤ 0.5 (total synergism), 0.5 < FIC ≤ 0.75

(partial synergism), 0.75 < FIC ≤ 2.00 (no effect), and FIC > 2 (antagonism) (Didry et al.

1994). In contrast, the synergistic effect of carvacrol and p-cymene (0.30mg/g and

0.27mg/g respectively) against B. cereus, was reduced when salt was added to rice

samples (1.25 g salt/L of rice) (Ultee et al. 2000).

The following two sections are divided in emulsifiers and lipids, which are crucial

components of nanoemulsions. Particularly, the emulsifier section will be focused on

Lecithin and Tween 20 which are used in the present study. On the other hand, the lipid

23

piece includes a description of coconut oil and palm stearin since they are utilized in this

study.

2.5 Emulsifiers

A strong interfacial tension exists between both the water and the oil phase,

therefore, nanoemulsions are thermodynamically unstable and must be stabilized by

adding emulsifiers which adsorbs at the interfaces and reduce the interfacial tension (Mao

et al. 2010). As a result, association colloids (or micelles) are formed where the

unfavorable contact area between the non-polar tails of the surfactant and water is

minimized. Consequently, addition of emulsifiers is essential to improve emulsion

formation, enhance stability against coalescence or flocculation, and reduce the particle

size needed to create nanoemulsions (Weiss et al. 2009).

Emulsifiers are molecules which consist of hydrophilic/lipophobic and

lipophilic/hydrophobic parts. They are categorized mainly by their hydrophilic/lipophilic

balance (HLB) (Whitehurst 2008) which is summarized in the equation below:

HLB = 20* (M0/M) Equation 3

Where M0 is the molecular weight of the hydrophilic part of the emulsifier

molecule and M is the total molecular weight. There are different uses depending on the

HLB value: 0-3: antifoaming agents, 4-6: water-in-oil emulsifier, 7-9: wetting agent, 8-18:

water-in-oil emulsifier, 13-15: detergents, and 10-18: solubilizes (Whitehurst 2008).

Table 4 shows what type of emulsifier should be used in different food products based on

24

their functionality. Before a food emulsifier is legally allowed for use in food products it

has to be tested in several toxicological studies. Emulsifiers are regulated by the Joint

Expert Committee on Food Additives (JECFA) of the Food and Agricultural

Organization/World Health Organization (FAO/WHO) (Friberg et al. 2003).

25

Table 4. Functionality of surfactants in some foods. The hydrophilic/lipophilic balance (HLB) of an emulsifier will determine their functionality in food products Functionality Surfactant Food examples

Foam aeration/stabilization Propylene glucol esters Cakes, whipped toppings

Dispersion stabilization Mono/diglycerides Peanut butter

Dough strengthening DATEM Bread, rolls

Starch comlexation (anti-

staling)

SSL, CSL Bread, other baked goods

Clouding (weighting) Polyglycerol esters, SAIB Citrus beverages

Crystal inhibition Polyglycerol esters,

oxystearin

Salad oils

Antisticking Lecithin Candies, grill shortenings

Viscosity modification Lecithin Chocolate

Controlled fat

agglomeration

Polysorbate 80,

polyglycerol esters

Ice cream, whipped

toppings

Freeze-thaw stabilization SSL, polysorbate 60 Whipped toppings, coffee

whiteners

Gloss enhancement Sorbitan monoestearate,

polyglycerol esters

Confectionary coatings,

canned and moist pet

foods

Source:(Hasenhuettl 2008)

26

2.5.1 Lecithin

Lecithin is commercially isolated from either soybean or sunflower by solvent

extraction and subsequent precipitation. The hydrophilic component of lecithin is the

phosphatidyl group, whereas two fatty acid chains are the hydrophobic side (Hasenhuettl

2008). Lecithins are considered zwitterionic surfactants since they have both positively

and negatively charged groups on the same molecule. In many cases, lecithin is an ideal

biological emulsifier because it is biodegradable, acquired GRAS status, and researchers

agree that addition of lecithin decreases emulsion particle size (Mao et al. 2010, Donsi et

al. 2012). There is a variety applications of lecithin nanoemulsions. In pharmaceutics,

lecithin increases skin hydration penetration due to the greater partitioning degree into the

stratum corneum (top layer of epidermis) (Zhou et al. 2009). In the food industry, the use

of marine lecithin nanoemulsions increases considerably the stability of salmon oil

against oxidation (Belhaj et al. 2010). The addition of lecithin to chocolates and coatings

reduces noticeably the viscosity due to its hydrophilic sugar crystal surface (Hasenhuettl

2008).

2.5.2 Tween 20

Tween emulsifiers have a polysorbate structure, are amphipathic, nonionic and they

acquire a number on their nomenclature depending on the type of fatty acid ester

associated with the polyoxyethylene sorbitan. For example, Polysorbate 20

(polyoxyethylene sorbitan monolaurate) and Polysorbate 80 (polyoxyethylene sorbitan

monooleate) (Kerwin 2008). Non-ionic surfactants are commonly chosen because of their

27

stability to pH and changes in ionic strength (Azeem et al. 2009). According to Donsi et

al. (2012) the combined use of Tween 20 and glycerol monooleate as surfactants, causes

the measured aqueous-phase concentration of carvacrol, limonene and cinnamaldehyde to

be 4 times, 20 times and 4-to-6 folds higher than its water solubility respectively. Also,

Tween 20 (1-2% wt) combined with caseinate (1-2% wt) have the ability to reach

sufficient surface-active material to saturate the droplet interface without inducing

flocculation by excess of protein and emulsifier (Dickinson et al. 1999). It has been

reported that an increase in Tween 20 concentration resulted in a lineal improvement on

curcumin solubility reaching an upper limit of 294 µM curcumin with 0.05 w/v% Tween

20 (O’Toole et al. 2012). Since Tween 20 is a nonionic surfactant, its binding with

compounds such as nisin and zein weakens hydrophobic attraction and improves the

release of nisin from capsules (Xiao et al. 2011).

2.6 Oil Phase

The stability and formation of nanoemulsions depend on the physicochemical

characteristics of the oil phase such as: polarity, water-solubility, interfacial tension,

refractive index, viscosity, density, phase behavior, chemical stability (McClements and

Rao 2011), and good solvent capacity (Sanghi and Singh 2012). Several non-polar

compounds can be used to prepare nanoemulsions, e.g. triacyglycerols (TAG),

diacyglycerols, monoacyglycerols, free fatty acid, flavor oils, essential oils, and mineral

oils, among others. (McClements and Rao 2011). The oil phase can sometimes act as a

solvent for various important food components, including oil-soluble vitamins,

28

antioxidants, preservatives and essential oils (McClements 1999, Azeem et al. 2009). In

food emulsions, the oil phase plays a critical role on texture. For example, “spreadability”

on margarines is determined by a three-dimensional network formation of fat crystals in

the continuous phase, which provides mechanical rigidity (McClements 1999).

2.6.1 Coconut Oil

Coconut oil (CO) is one of the most widely used confectionary fats with a simple

TAG composition of 90% saturated fatty acids. (Chaleepa and Ulrich 2011). It is the most

important oil crop in tropical regions and is extensively used for food and industrial

purposes (Sriamornsak et al. 2012). CO has been successfully applied to chitosan edible

composite films at a CO/chitosan ratio of 0.5 to 1, above which phase separation was

observed during the drying process (Binsi et al. 2013). Also, CO provided the smallest

particle size (100-500 nm) when tested against sunflower and castor oils in a system

where the emulsifier was a combination of polysorbate 80 and sorbitan monooleate 80

(Sriamornsak et al. 2012). Due to its high fatty acid content, particles interfaces acquired

a multi-lamellar structure (Lee et al. 2011).

2.6.2 Palm Stearin

Palm stearin (PS) is obtained by fractionation of palm oil which results in

approximately 62.7% of palmitic acid content which makes it suitable for several

industry applications: the addition of PS to hydrogenate palm kernel oil (HPKO) in a

ratio of 66:34 HPKO:PO increases the performance and stability of whipping cream

29

(Nesaretnam et al. 1993). Also, by combining PS with palm olein the solid fat content

(SFC) profile improved resulting in smoother margarines and spreads (Wassell and

Young 2007). PS have diverse melting, crystallization, and polymorphic transformation

temperatures which increase confounds the understanding of its polymorphism in

comparison to simple TGA (Sonoda et al. 2004). However, the crystallization and

polymorphic transformation of PS in nanoemulsions can be modified by using

polyglycerine fatty-acid esters (Sonoda et al. 2006).

30

CHAPTER 3. EFFECT OF CARVACROL-LOADED NANOEMULSIONS ON A BIOLUMINESCENT STRAIN OF ESCHERICHIA COLI O157:H7

3.1 Abstract

Nanoemulsions have been shown to be effective delivery vehicles for natural

antimicrobials that are poorly soluble in water. The antimicrobial activity of carvacrol (5-

isopropyl-2-methylphenol) nanoemulsions against a bioluminescent Escherichia coli

O157:H7 was investigated using light emission as an indicator of cell viability. Different

emulsifiers (Ultralec Lecithin and Tween 20), oils (Palm stearin and Coconut oil) and

various carvacrol emulsion concentrations (0, 1, 2 and 2.5%) were evaluated.

Bioluminescence response was monitored in situ using a Hamamatsu (photo multiplier

tube) sensor module integrated with a Programmable Logic Controller interfaced with a

PC for data acquisition. Bioluminescence decreased rapidly with the addition of

carvacrol-containing nanoemulsions. However when cells were assayed for viability,

plate counts showed the bacteria were not inactivated suggesting that the carvacrol was

interfering with the luminescence reaction. To determine if the process was reversible,

carvacrol was removed from the bacteria after five min of contact time by centrifugation

and cells were resuspended in minimal media with glucose as a carbon source. Light

values recovered to levels higher than prior to the addition of carvacrol indicating

reversibility.

31

The same bioluminescent pattern was also observed with sub lethal doses of the

oxidative uncoupler 2,4-dinitrophenol. These results suggest that carvacrol and 2,4-DNP

uncouple oxidative phosphorylation reducing the ATP available for the myristyl aldehyde

biosynthesis. Previous reports suggested that the mechanism of inactivation of carvacrol

was membrane damage resulting in loss of cellular contents and viability. However these

results suggest that the mechanism of carvacrol inactivation is due to the uncoupling of

oxidative phosphorylation.

3.2 Introduction

There is an increasing interest of consumers in natural substances which has

focused the attention on plants containing bioactive compounds such as Essential oils

(EOs) due to their antimicrobial, antioxidant and antiradical properties (Cutter 2000, Ben

Arfa et al. 2006, Chang et al. 2013). EOs are aromatic oily liquids produced in plants as

secondary plant metabolites. They are responsible for the odor, aroma and flavor of

spices and herbs (Cutter 2000). EOs are commonly extracted by steam distillation

(Bakkali et al. 2008). Besides antimicrobial activities, EOs have shown antiviral,

antimycotic, antitoxigenic, antiparasitic and insecticidal properties (Burt 2004). Some

preservatives containing EOs are currently commercially available. Mendoza-Yepes et al.

(1997) reported that ‘DMS Base Natural’ (DOMCA S.A., Alhendin, Granada, Spain) is a

food preservative that contains 50% glycerol and 50% essential oils from rosemary, sage

and citrus. Isman (2000) reported that Cinnamite TM an aphidicide, miticide and fungicide

for horticultural crops and Valero TM, a miticide and fungicide in grapes, berry crops,

32

citrus and nuts (both manufactured by Mycotech Corp. Butte, MT, USA) contain

cinnamon oil and 30% of cinnamaldehyde as the active ingredient. On the other hand,

Cutter (2000) reported that ‘Protecta one’ and ‘ Protecta two’ (Bavaria Corp. Apopka, FL,

USA) are food additives with the GRAS Status (Generally Recognized as Safe), that

contain essential oils dissolved in solutions of sodium citrate and sodium chloride,

respectively.

Carvacrol (5-isopropyl-2-methylphenol) is a naturally occurring compound,

abundant in the essential oil fraction of oregano (60 to 74% carvacrol) and thyme (45%

carvacrol) (Ultee et al. 1999). Its antimicrobial properties on foods and other systems has

been widely investigated (Ben Arfa et al. 2006, Ait-Ouazzou et al. 2011, Terjung et al.

2012). In comparison to other naturally occurring compounds, carvacrol has better

antimicrobial properties than eugenol, menthol, carvacrol methyl ether and carvacryl

acetate (Ben Arfa et al. 2006). However, the mechanism of action of this compound is

still being investigated (Ultee et al. 1999, Lambert et al. 2001). Nevertheless, some

authors have proposed that it may act through disruption of membrane integrity,

increasing proton permeability by releasing cellular constituents (Sikkema et al. 1994,

Ultee et al. 1999, Lambert et al. 2001). Nychas (1995) also proposed that phenols might

alter the physiological status of cells by changing the fatty acid composition and

phospholipid content of bacteria; disrupting the electron transport or nutrient uptake and

affecting nucleic acid synthesis and energy metabolism.

The objective of this study was to evaluate the antimicrobial activity of carvacrol-

containing nanoemulsions and to understand its mechanism of action. Nanoemulsions

were selected as an antimicrobial delivery system since they have been shown to increase

th

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33

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34

3.3.2 Bacterial strain and media

A bioluminescent, kanamycin and ampicillin resistant strain E. coli O157:H7 lux

was constructed by cloning the bioluminescent gene cassette (luxCDABE) from

Photorhabdus luminescence into a pCRII cloning vector (Invitrogen, Carlsbad, CA) by

Dr. Linda Perry. The resulting plasmid, pFSP102, was inserted into E. coli O157:H7

strainC7927, an acid tolerant strain isolated from an apple cider outbreak. The

constructed strain has the ability to emit light at 490 nm in presence of oxygen and

FMNH2. E. coli O157:H7 lux was grown in a continuous culture using minimal salt

media (MSM) (0.068% monopotassium phosphate, 2.2% dipotassium phosphate; 0.1%

magnesium phosphate; 2% ammonium nitrate, Sigma®) supplemented with glucose (500

ppm), kanamycin (50mg/mL), and trace elements (0.01w/v%) (Heitzer et al. 1990)

3.3.3 Chemostat setup

A sterile 16 liter carboy containing sterile media was connected to a previously

sterilized bioreactor using a Primary IV set (Direct Pet; 73 inch). Media was pumped into

the bioreactor (Kontes Cytolift Glass Airlift Bioreactor Cell Culture) using a multistaltic

pump (Buchler, 2-6250, New Jersey, U.S.A) at a constant rate of 1.5mL/min. The

bioreactor containing the bacteria was mixed using filter-sterilized air (Pall Life Sciences

Supor® 200, 47 mm, 0.2 µm) at 5 psi using a Fairchild Industrial regulator (Kendal, 19,

North Carolina, U.S.A) (figure 7). The bacterial concentration in the bioreactor was kept

constant at an optical density (OD) of 0.500 measured using a biophotometer (22331,

Eppendorf BioPhotometer, Hamburg, Germany) and light of approximately 17,000,000

R

G

Felbduvth

RLU/s measu

Germany).

Figure 7. Chlements (A) ioreactor conue to the conigorous mixhe bioreactor

ured using a

hemostat setis pumped bntaining Escnstant mediaed using sterr (F).

luminomete

tup. Sterile Mby a peristaltcherichia cola pumping isrile air (E). A

er (Zylux Fem

MSM media tic pump (B)li O157:H7 ls collected inA port for ba

mtomaster F

a with kanam) at a constanlux (C). An n a waste conacterial colle

FB14, D-751

mycin, glucosnt rate of 1.5excess of bantainer (D). ection is loca

73, Pforzhei

se and trace 5 mL/min toacterial cultuThe bacteriaated at the to

35

im,

o a ure a is op of

36

3.3.4 In vivo bioluminescence monitoring and cell viability

One mL of bacteria from chemostat was prepared in duplicate. Kanamycin was

removed from the bacterial culture by washing three times with MSM by centrifugation

(Eppfendorf centrifuge 5415D for 10 min at 16,100 g). The bacterial pellet was

resuspended using 10 mL of MSM. Sterilized glass vials (20 mL) were used for each

experiment containing 8.1mL of MSM and 0.9 mL of the previously prepared bacteria.

Finally, 1 mL of diluted emulsion was added to each vial to reach the desired carvacrol

concentration. Bacterial concentration in the vials was about 107 CFU/mL)

Bioluminescence was measured in situ using a photo multiplier tube (PMT)

(Hamamatsu, AC135, Iwata-gun, Japan) sensor module in a light-tight box integrated

with a programmable logic controller interfaced with a PC for data acquisition (figure 8).

Bioluminescence was monitored for ten minutes as follows: photon emission of the initial

bacterial dilution was measured during one min after which, one mL of the emulsion or

decanal was injected to the sample through an inlet tubing using a syringe and photon

emission was recorded every second during nine additional minutes. The mixture was

stirred using a magnetic stirring bar during the duration of the measurement. Immediately,

after measuring bioluminescence, an aliquot of 0.1 mL was serially diluted and plated

onto Luria Broth (LB) agar plates supplemented with kanamycin (50mg/mL) (LB kan)

using the spread plate technique. Samples were incubated overnight at 37°C before

colony counting.

Fmvinan .

Figure 8. Phomonitoring.

ial (B) whernjection. Thend covered w

oto MultiplThe PMT Pr

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37

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39

3.3.6 ATP Assay

To understand if the mechanism of action of carvacrol was interfering the ATP

production, an ATP Swab Hygiene test was performed. An ATP vs light standard curve

was constructed. The initial solution contained 10 mM of ATP (Biolabs, P0756S, Ipswich,

MA, United States) and was serially diluted from 100-10-8 using sterile-filtered distilled

cold water. 100 µL of each dilution was mixed with 100 µL of Clean-Traceᵀᴹ Surface

ATP (3M ,St.Paul, Minessota, United States) reagent (water 70-80%, non-hazardous

components 15-25%, sodium azide <0.1%, luciferase 0.0001-0.0007%, and luciferin

0.0001-0.0006%) and bioluminescence was measured using a luminometer (Zylux

Femtomaster FB14, D-75173, Pforzheim, Germany). To estimate the remaining amount

of ATP after mixing Escherichia coli O157:H7 lux and nanoemulsions an ATP assay was

conducted. Bacteria from the chemostat was washed three times by centrifugation

(Eppfendorf centrifuge 5415D for 10 min at 16,100 g) and the pellet resuspended in

MSM. After the final wash the pellet was resuspended in MSM.

Emulsions were diluted to 500, 750, 1000 ppm carvacrol using filtered-sterilized

deionized water. All assay vials were sprayed with 70% ethanol and dried to ensure a

background level free of ATP. Each sample vial was prepared by mixing 90 µL of the

prepared bacteria with 10 µL of the emulsion at different concentrations (500, 750 1000

ppm). 100 µL of the Clean-Traceᵀᴹ Surface ATP reagent was added to the sample and

bioluminescence was immediately measured using a luminometer (Zylux Femtomaster

FB14, D-75173, Pforzheim, Germany). The amount of ATP was determined from the

lineal regression equation obtained in the standard calibration curve. All experiments

were performed in duplicate.

40

3.4 Results and discussion

3.4.1 Evaluation of cell viability

In order to determine the most effective formulation to inhibit the growth of a

bioluminescent strain of E. coli O157:H7, bacteria were surface plated after ten min of

contact time with emulsions previously diluted with sterile water (500, 750, and 1000

ppm), and colonies were enumerated after overnight incubation at 37ºC (table 5). Table 6

summarizes the treatments that successfully inactivated bacteria. When control emulsions

lacking carvacrol, were added to the E. coli O157:H7 lux, no bacterial inactivation was

observed indicating that the surfactant and fat used on each system do not interfere with

bacterial growth. However, emulsions originally containing 2% or 2.5% of carvacrol, and

subsequently diluted to 1000 ppm, were able to inactivate the bacteria. Donsì et al. (2011)

designed nanoemulsions made with either palm or sunflower oil as the lipid phase, and

either lecithin or modified starch (cleargum) as the emulsifier, and tested those emulsions

as antimicrobial vehicles for a mixture of terpenes against Escherichia coli. They found

that a complete bacterial inactivation was achieved at 5000 ppm and a microbial

reduction at 1000 ppm. However, these results are hard to compare to the results that we

obtained, due to the difference in emulsifiers, oils, contact time and bacterial strain.

(Terjung et al. 2012) studied the antimicrobial effect that carvacrol-loaded nanoemulsions

had against Listeria innocua concluding that at 300 ppm a net statis (no growth and no

death) was achieved and at concentrations exceeding 400 ppm, a rapid increase in the

inactivation occurred. Likewise, in this study all nanoemulsions containing 2.5% of

41

carvacrol regardless of their nanoemulsion composition revealed a complete bacterial

inactivation with 10 min of contact time when concentrations exceeding 400 ppm were

used (table 6).

42

Table 5. Bacterial enumeration in CFU/mL after treatment with carvacrol-loaded nanoemulsions. Colonies of Escherichia coli O157:H7 lux were enumerated after being in contact for 10 min with nanoemulsions containing carvacrol (1, 2 or 2.5%) at three different concentrations (500, 750 and 1000 ppm). This table shows the result divided by treatments: a) Tween 20, Coconut oil and carvacrol. b) Tween 20, Palm Stearin and carvacrol. c) Lecithin, Coconut oil and carvacrol. d) Lecithin, Palm stearin and carvacrol. The initial bacterial concentration was about 107 CFU/mL

a)           

Emulsifier  Oil  Carvacrol  (%) 

Dilution (ppm) 

Bacterial load (CFU/mL) 

Standard error 

Tween 20  Coconut  1  500  2.60E+06  7.07E+05 

Tween 20  Coconut  1  750  1.53E+06  2.65E+05 

Tween 20  Coconut  1  1000  1.25E+06  7.07E+04 

Tween 20  Coconut  2  500  2.83E+06  1.26E+06 

Tween 20  Coconut  2  750  5.70E+03  1.77E+02 

Tween 20  Coconut  2  1000  0.00E+00  0.00E+00 

Tween 20  Coconut  2.5  500  0.00E+00  0.00E+00 

Tween 20  Coconut  2.5  750  0.00E+00  0.00E+00 

Tween 20  Coconut  2.5  1000  0.00E+00  0.00E+00 

 b) 

Emulsifier  Oil  Carvacrol (%) 

Dilution (ppm) 

Bacterial load (CFU/mL) 

Standard error 

Tween 20  Palm stearin  1  500  5.25E+06  2.12E+05 

Tween 20  Palm stearin  1  750  2.45E+06  2.83E+05 

Tween 20  Palm stearin  1  1000  2.05E+06  3.54E+04 

Tween 20  Palm stearin  2  500  2.08E+03  8.31E+02 

Tween 20  Palm stearin  2  750  0.00E+00  0.00E+00 

Tween 20  Palm stearin  2  1000  0.00E+00  0.00E+00 

Tween 20  Palm stearin  2.5  500  1.37E+04  6.29E+03 

Tween 20  Palm stearin  2.5  750  0.00E+00  0.00E+00 

Tween 20  Palm stearin  2.5  1000  0.00E+00  0.00E+00 

43

Table 5. Continued

c) Emulsifier  Oil  Carvacrol 

(%) Dilution (ppm) 

Bacterial load (CFU/mL) 

Standard error 

Lecithin   Coconut   1  500  4.50E+05  7.07E+03 

Lecithin   Coconut   1  750  6.53E+05  2.49E+05 

Lecithin   Coconut   1  1000  5.00E+04  1.06E+04 

Lecithin   Coconut   2  500  8.23E+06  2.42E+06 

Lecithin   Coconut   2  750  1.50E+05  3.54E+04 

Lecithin   Coconut   2  1000  0.00E+00  0.00E+00 

Lecithin   Coconut   2.5  500  4.08E+03  1.94E+02 

Lecithin   Coconut   2.5  750  0.00E+00  0.00E+00 

Lecithin   Coconut   2.5  1000  0.00E+00  0.00E+00 

d) Emulsifier  Oil  Carvacrol 

(%) Dilution (ppm) 

Bacterial load (CFU/mL) 

Standard error 

Lecithin  Palm stearin  1  500  8.00E+05  3.18E+05 

Lecithin  Palm stearin  1  750  5.00E+05  7.07E+04 

Lecithin  Palm stearin  1  1000  5.00E+04  0.00E+00 

Lecithin  Palm stearin  2  500  5.00E+05  0.00E+00 

Lecithin  Palm stearin  2  750  0.00E+00  0.00E+00 

Lecithin  Palm stearin  2  1000  0.00E+00  0.00E+00 

Lecithin  Palm stearin  2.5  500  0.00E+00  0.00E+00 

Lecithin  Palm stearin  2.5  750  0.00E+00  0.00E+00 

Lecithin  Palm stearin  2.5  1000  0.00E+00  0.00E+00 

44

Table 6. Nanoemulsions that completely inactivated bacterial growth. A summary of the treatments that inhibited bacterial growth after ten min of contact time. The initial bacterial concentration was about 107 CFU/mL.

Emulsifier  Oil  Carvacrol (%) 

Dilution (ppm) 

Bacterial load (CFU/mL) 

Standard error 

Tween 20  Coconut  2  1000  0.00E+00  0.00E+00 

Tween 20  Coconut  2.5  500  0.00E+00  0.00E+00 

Tween 20  Coconut  2.5  750  0.00E+00  0.00E+00 

Tween 20  Coconut  2.5  1000  0.00E+00  0.00E+00 

Tween 20  Palm stearin  2  750  0.00E+00  0.00E+00 

Tween 20  Palm stearin  2  1000  0.00E+00  0.00E+00 

Tween 20  Palm stearin  2.5  750  0.00E+00  0.00E+00 

Tween 20  Palm stearin  2.5  1000  0.00E+00  0.00E+00 

Lecithin  Coconut  2  1000  0.00E+00  0.00E+00 

Lecithin  Coconut  2.5  750  0.00E+00  0.00E+00 

Lecithin  Coconut  2.5  1000  0.00E+00  0.00E+00 

Lecithin  Palm stearin  2  750  0.00E+00  0.00E+00 

Lecithin  Palm stearin  2  1000  0.00E+00  0.00E+00 

Lecithin  Palm stearin  2.5  500  0.00E+00  0.00E+00 

Lecithin  Palm stearin  2.5  750  0.00E+00  0.00E+00 

Lecithin  Palm stearin  2.5  1000  0.00E+00  0.00E+00 

45

To clarify and have a better understanding on the effect each factor had on the

microbial inactivation, microbiological results were statistically analyzed using SAS 9.3

to find significant differences (ANOVA p<0.05) between factors (surfactant, fat,

carvacrol content and dilution) and pair-wise interactions. Differences between mean

separations were calculated using LSMEANS. Carvacrol concentrations and dilutions

were significantly different with p-values of 0.0011 and 0.0006 respectively (table 7).

Emulsions with 2.5% carvacrol content exhibited the best antimicrobial results in

comparison to those containing 1% and 2% of carvacrol (table 8). In contrast, dilutions of

1000 ppm and 750 ppm were statistically more effective than 500 ppm (table 9). The fact

that no statistical differences were found between emulsifiers is supported by Donsi et al.

(2012) who tested four types of surfactants (Soy lecithin, pea proteins, sugar ester and a

mixure of glycerol monooleate and tween 20) on carvacrol-loaded nanoemulsions against

Saccharomyces cerevisae: No significant effect was found after 2 h of contact time.

The improvement in antimicrobial activity as carvacrol content increased in the

emulsion was also found by Kim et al. (1995) who tested different concentrations of

carvacrol (0.5%, 1.5% and 3%) against fish cubes inoculated with Salmonella

typhimurium over a 4 day storage period, and it was found that a 1.5% carvacrol

concentration, the efficacy of bacterial inhibition was higher during the first two days of

storage but subsequently decreased possibly due to the interactions with food components.

46

Table 7. General Linear Model: Analysis of Variance for bacterial enumeration. Sources with p<0.05 are statistically different. Results were analyzed in SAS 9.3 Statistical Software.

Source  DF  Type I SS  Mean Square  F Value  Pr > F 

Emulsifier  1  2.41E+12  2.41E+12  1.4  0.2425 

Oil  1  2.08E+12  2.08E+12  1.21  0.2772 

Carvacrol  2  2.68E+13  1.34E+13  7.75  0.0011 

Dilution  2  2.99E+13  1.50E+13  8.67  0.0006 

Emulsifier*Oil  1  4.74E+12  4.74E+12  2.75  0.1034 

Emulsifier*Carvacrol  2  3.02E+13  1.51E+13  8.75  0.0005 

Emulsifier*Dilution  2  5.74E+11  2.87E+11  0.17  0.8473 

Oil*Carvacrol  2  2.05E+13  1.02E+13  5.94  0.0048 

Oil*Dilution  2  7.56E+12  3.78E+12  2.19  0.1222 

Carvacrol*Dilution  4  2.25E+13  5.63E+12  3.26  0.0185 

Table 8. LSMEANS separation of carvacrol concentrations. Sources with different letters are statistically different (p<0.05)

Table 9. LSMEANS separation of antimicrobial dilution. Sources with different letters are statistically different (p<0.05)

Dilution (ppm)  Bacterial LSMEAN  LSMEAN 

1000  283333.33  A 

750  440266.67  A 

500  1722483.33  B 

Carvacrol (%)  Bacterial LSMEAN  LSMEAN 

2.5  1477.08  A 

2  975647.92  B 

1  1468958.33  B 

47

Theoretically, regardless of the initial carvacrol concentration of the emulsions

(i.e. 1, 2 or 2.5%) once they are diluted for evaluation (500, 750 or 1000 ppm), all

emulsions should theoretically have the same quantity of phytophenol available and

therefore, the same antimicrobial activity. However, this hypothesis differs from the

results. There are differences in microbial activity between emulsions that originally have

different carvacrol concentrations even though they were diluted to the same

concentration (500, 750 or 1000 ppm). Terjung et al. (2012) proposed a mechanistic

explanation to these discrepancies in their antimicrobial results as differences in particle

sizes were obtained. Since the same amount of surfactant is used in the construction of

all emulsions, more surfactant will be needed to stabilize interfaces in smaller particles

sizes. Consequently, less micelles would be found in the aqueous phase, and since these

micelles are excellent solubilizers for phytophenols, much less carvacrol would be found

in the aqueous phase. Therefore, smaller nanoemulsions should be less active than larger

ones. However, in our study no differences in droplet particle sizes were found (Personal

communication Veronica Rodriguez ).

Furthermore, the binding of the essential oil in the hydrophobic region of the

micelles (in presence of the emulsifier indeed), allows the movement of the antimicrobial

to the aqueous phase thus increasing the antimicrobial effect. The increased concentration

due to micelles solubilization is due to the hydrophobic interactions between the

phytophenol and the emulsifier (Donsi et al. 2012). Hence, it is possible that

nanoemulsions formulated with higher carvacrol concentrations (i.e. 2.5%) are potentially

being solubilized more easily in the aqueous phase than emulsions with lower carvacrol

concentration (i.e. 1%) in their initial manufacture. As a consequence, a higher

48

antimicrobial activity is observed in more carvacrol concentrated emulsions regardless

the final dilution. For example: Emulsions-2.5% and emulsions-1% are diluted to 500,

750 or 1000 ppm. Despite these dilutions, emulsions-2.5% were always more effective

against E.coli O157:H7 lux than emulsions-1%.

3.4.2 In vivo bioluminescence monitoring

To investigate if cell viability could be correlated to the bioluminescence effect,

in vivo measurements were conducted on the bioluminescent bacteria in presence of

nanoemulsions. Meighen (1991) reported that the expression of lux genes in several

bacterial species, can provide a simple and sensitive way for measuring growth and

bacterial distribution. In addition, bioluminescence facilitate the understanding of the

mechanistic effect that antimicrobials have on bacterial cells prior to death. When

nanoemulsion aliquots were added to the bioluminescent strain of E.coli O157:H7, light

decreased to half the initial value immediately after nanoemulsion addition. However, the

original bioluminescence value was recovered gradually over time when emulsions with

no carvacrol were added. Nevertheless, when carvacrol-loaded nanoemulsions were

added to the sample, bioluminescence was completely inhibited regardless of the

concentration of carvacrol. Figures 9, 10 and 11 show the effect on bioluminescence after

addition of 1mL of nanoemulsion containing Palm Stearin (PS), Lecithin Ultralec (Lu)

and carvacrol with 1%, 2% and 2.5% respectively diluted at 500, 750, or 1000 ppm.

Bioluminescent values were standardized to facilitate the analysis and comparison

between graphs. However, table 10 shows the last non-standardized bioluminescent value

detected by the PMT after 10 min of contact time between E. coli O157:H7 and

nanoemulsions. The final values reported for E. coli O157:H7 lux in presence of

49

PS|Lu|C0% (no carvacrol) ranged between 1.625,100 photons/s and 4,147,972 photons/s.

However, the average final bioluminescent value of E.coli O157:H7 in presence of

emulsions (PS|Lu|C%) loaded with 1, 2, and 2.5% were 10,613 photons/s, 9,188

photons/s, and 5,522 photons/s respectively. In other words, after ten minutes of contact

time between E.coli O157:H7 and PS|Lu|C1, 2, 2.5% an average reduction of 99.57% (or

2.37 logs), 99.63% (or 2.43 log), and 99.78% (or 2.65 log) was observed respectively.

Graphs for all treatments can be found in the Appendix A and the non-normalized

values on Appendix B. Nanoemulsions containing no carvacrol were used as positive

controls and Escherichia coli O157:H7 lux with in absence of nanoemulsion was used as

negative control.

Fca

igure 9. Effearvacrol at d

ect of nanoemdifferent dilu

mulsions conutions: 500, 7

ntaining Pal750 and 100

lm Stearin (P0 ppm.

PS), Lecithin

n (Lu) and 1

50

% of

Fo

igure 10. Eff carvacrol a

ffect of nanoat different d

emulsions cdilutions: 500

ontaining Pa0, 750 and 1

alm Stearin (000 ppm.

(PS), Lecith

in (Lu) and 2

51

2%

Fo

igure 11. Eff carvacrol a

ffect of nanoat different d

emulsions cdilutions: 500

ontaining Pa0, 750 and 1

alm Stearin (000 ppm.

(PS), Lecith

in (Lu) and 2

52

2.5%

53

Table 10. Non-normalized bioluminescent values of PS|Lu|C%. The last bioluminescent value of emulsions containing Palm stearin (PS), Lecithin (Lu) and carvacrol at 1, 2, and 2.5% are compared to the same emulsion lacking of carvacrol.

Emulsion  C%  Dilution (ppm) 

Final light value  (photons/s) 

Standard Deviation 

PS|Lu|C  0  500  2511758  838048.81 

PS|Lu|C  1  500  13960  3886.26 

PS|Lu|C  0  750  1625100  879510.73 

PS|Lu|C  1  750  9866  1043.69 

PS|Lu|C  0  1000  1759863  1295157.99 

PS|Lu|C  1  1000  8014  359.21 

PS|Lu|C  0  500  4147972  0.00 

PS|Lu|C  2  500  17026  3114.10 

PS|Lu|C  0  750  3299536  0.00 

PS|Lu|C  2  750  6872  854.18 

PS|Lu|C  0  1000  2255308  0.00 

PS|Lu|C  2  1000  3668  220.62 

PS|Lu|C  0  500  2767154  193812.31 

PS|Lu|C  2.5  500  5998  229.10 

PS|Lu|C  0  750  2070672  68996.65 

PS|Lu|C  2.5  750  4206  144.25 

PS|Lu|C  0  1000  1857754  32575.00 

PS|Lu|C  2.5  1000  6362  1405.73 

These results show that microbial inactivation cannot be correlated to

bioluminescence because photon emission was completely inhibited with the addition of

carvacrol-containing nanoemulsions while at certain concentrations cells remained viable.

These results suggest interference of carvacrol with the bioluminescent pathway directly

or other metabolic pathways which provide substrates and cofactors for the

bioluminescent reactions.

3

co

H

re

ex

em

2

E

pr

lo

re

b

ap

h

Dose.4.2.1

Emuls

orresponding

However, this

egardless of

xcept for the

Dose

mulsions we

.5% of carva

Escherichia c

reviously. T

ower lumine

esulted in a h

ioluminesce

pproximately

igher the num

e-response (

sions contain

gly from low

s effect was

the final con

e CO|Lu|C2.

response exp

ere equally e

acrol were d

coli O157:H7

he results w

scence decre

higher lumin

nce emission

y a 10% dec

mber of surv

BA50)

ning 1% and

wer to higher

not noticeab

ncentration E

5% emulsion

periments w

effective at lo

diluted to 250

7 lux was m

were as expec

ease compar

nescence dec

n from 250 p

crease (figure

vivors as sho

d 2% carvacr

r concentrati

ble in emulsi

E.coli O157:

n (table 6).

were conducte

ower concen

0 and 350 pp

monitored for

cted; lower c

red to the hig

crease. The m

ppm to 350 p

e 12). As pre

own in table

rol, the antim

ions (i.e. fro

ions with 2.5

:H7 lux was

ed to unders

ntrations. All

pm and the b

r 10 min follo

concentration

gher concent

most noticea

ppm was PS

edicted, the

11.

microbial act

m 500 ppm

5% carvacro

completely

stand if the 2

l nanoemuls

bioluminesce

owed by pla

n (250 ppm)

tration (350

able change i

STwC2.5% w

lower the co

tivity increas

to 1000 ppm

ol whereas

inactivated

2.5% carvacr

sions contain

ence respons

ating as desc

) resulted in

ppm) that

in

with

oncentration

54

sed

m).

rol

ning

se of

ribed

the

55

Table 11. Microbial enumeration at three different dilutions of emulsions containing 2.5% carvacrol. Samples were diluted to: 250 ppm, 350 ppm and 500 ppm and plated after 10 min of contact time with Escherichia coli O157:H7 lux. The initial bacterial concentration was 106 CFU/mL

Dilution (ppm) 

System  250  350  500 

CO|Lu|C2.5%  7.55E+05  5.93E+05  4.08E+03 

CO|Tw|C2.5%  9.68E+05  1.05E+05  0.00E+00 

PS|Lu|C2.5%  7.90E+05  1.78E+05  0.00E+00 

PS|Tw|C2.5%  9.63E+05  3.63E+05  1.37E+04 

cm

Figure 12. containing 2monitored fo

PSLuC, ascenarios.

Biolumines2.5% of carvor ten minuteand D) PSTwHowever, it

scent responvacrol and

es in the preswC. Biolumi

decreased s

nse of Eschediluted to 2sence of fourinescence deslightly more

graph

erichia coli O250 and 350 r emulsions:ecreased to be at 350 ppm

O157:H7 luppm. Biolu

: A) COLuCbackground lm. Insets are

ux to emulsiouminescenceC, B) COTwClevels in botzooms of ea

56

ons was C, C) th ach

57

BA50 value is defined as the percentage of test compound that kills 50% of the

bacteria under the test conditions. CFUs values of each dilution (250, 350, and 500 ppm)

were matched with the average control values to estimate the percentage of killed

bacteria (equation 4). Each of the dose-response profiles was analyzed graphically and

the BA50 values were estimated by linear regression (Rojas-Graü et al. 2007). The lower

the BA50 value, the higher the antimicrobial activity.

%killed bacteria= 1-(N/N0) Equation 4

Where N is the CFU values after exposure to carvacrol-loaded nanoemulsions and N0 is

the CFU values after contact with nanoemulsions lacking carvacrol.

All emulsions resulted in similar BA50 values, and ranged from 0.0236% for

PS|Lu|C2.5% to 0.0303% for PS|Tw|C2.5%. A previous study reported carvacrol BA50

values against E.coli O157:H7 between 0.0082% and 0.0088% when tested on cloudy

apple juice at 37°C for 120 min of contact time (Friedman et al. 2004). The lower BA50

values obtained by Friedman et al. compared to the ones determined in this study might

be due to the following: this study was conducted at room temperature whereas their

study was at 37°C, also the contact time in our study was 10 min as opposed to 120 min.

Likewise, Friedman and colleagues did not encapsulate carvacrol in nanoemulsions as

antimicrobial delivery vehicles.

However, Rojas-Graü et al. (2007) studied the effect of using apple puree edible

films as carriers for 0.1% oregano oil against E.coli O157:H7. They reported BA50

values of 0.034% and 0.024% after 3 min and 30 min of contact time respectively and it

58

0.0276 0.02820.0236

0.0303

0.0000

0.0050

0.0100

0.0150

0.0200

0.0250

0.0300

0.0350

Emulsion

BA50 (%)

COLuC2.5% COTwC2.5 PSLuC2.5% PSTwC2.5%

was suggested that oregano oil may have a dual benefit by imparting antimicrobial

properties, and by enhancing the water barrier properties of the films which is key to

ensure their stability.

Figure 13. Bactericidal activities (BA50) of all nanoemulsions containing 2.5% of carvacrol. The lower the BA50 value, the more effective the emulsion is. These values were calculated using Escherichia coli O157:H7 lux with emulsions lacking carvacrol as control.

3

n

o

T

in

(e

ra

em

g

up

O

m

ce

re

w

re

in

P

in

Reve.4.3.1

The re

anoemulsion

ccurs when

Therefore, ex

n nanoemuls

emulsion wit

ate of 26,144

mulsion at th

lucose) was

p to 1,085,7

O157:H7 lux

minutes. Inter

entrifugation

eported from

with the corre

eversible at l

nvestigate th

Pseudomonas

n this study,

3.4.

ersibility of

esults show t

ns loaded wi

antimicrobia

xperiments to

sions were co

th no carvac

4 RLUs min-

he five minu

214,946 RL

53 RLU/s. O

in presence

restingly, on

n and resusp

m background

esponding in

lower concen

he effect that

s fluorescenc

luminescenc

3 Effect o

carvacrol ef

that biolumi

ith any amou

al concentrat

o understand

onducted as

crol) increase

-1 before the

ute time poin

LU/s and, aft

On the contra

of PS|Lu|C2

nce the carva

pension of th

d levels to al

nactivation d

ntrations. Du

t high hydros

ce 5RL and E

ce emission

f carvacrol o

ffect

inescence is

unt of carvac

tion in the em

d the mechan

previously d

ed its light em

addition of

nt (the last m

ter the additi

ary, the biolu

2% decrease

acrol was rem

e cells in glu

lmost 200,00

data suggests

uarte-Gómez

static pressu

Escherichia

decreased w

on biolumine

dramatically

crol. Howev

mulsions is b

nism of actio

described. Th

mission ever

glucose. Th

measurement

ion of glucos

uminescence

ed to backgro

moved from

ucose (1000

00 RLU/s (F

s the antimic

z et al. (2014

ures (69, 103

coli VF lux

when high pr

escence

y reduced in

er, cell inact

between 2%

on of carvacr

he positive c

ry minute w

he value reco

before addin

se (1000 ppm

e coming fro

ound levels i

the solution

ppm) a reco

Figure 14). T

crobial action

4) used biolu

and 138 MP

x. Similar to

ressures were

n the presenc

tivation only

% and 2.5%.

rol encapsul

control

with an avera

orded for this

ng 1 mL of

m) it increas

om E. coli

in less than f

n by

overy in ligh

This result al

n of carvacro

uminescence

Pa) have on

the results fo

e applied.

59

ce of

y

ated

ge

s

ed

five

ht was

long

ol is

e to

found

60

However, light recovered after the pressure was released suggesting that the action of

high pressures on bacteria was also reversible. In addition, pressure treatments above

100MPa resulted in bacterial inactivation of Pseudomonas fluorescence 5RL which is

comparable to the antimicrobial effect that higher carvacrol concentrations (emuslions

with 2.5% carvacrol) had on E. coli O157:H7 lux in the present study.

FPand

Figure 14. LiPS|Lu|C0% nd the result

difference in

ight responand PS|Lu|Ctant pellet wlight effect w

se of EscherC2% at 500as resuspend

when carvac

richia coli O0 ppm. The eded in MSMrol is presen

O157:H7 in emulsion wa

M glucose (10nt and absen

presence anas removed b000 ppm). Nnt.

nd removal by centrifug

Note the

61

of gation

ex

v

d

ag

L

w

co

re

ag

se

b

w

w

pr

si

ca

ac

3

ph

It has

xhibit a linea

iability (CFU

ependent wh

gainst Pseud

Loimaranta e

was treated w

orrelated wit

esults were f

gainst Esche

econds conta

ioluminesce

was not cumu

was hypothes

roteins but b

ince in this w

arvacrol eve

ction is due

2,4-D.4.3.2

To tes

hosphorylati

been shown

ar relationsh

U/filter) (del

hen different

domonas fluo

t al. (1998) f

with chlorhex

th biolumine

found by Au

erichia coli O

act time with

nt response

ulative in com

sized that the

by interfering

work biolum

en though cel

oxidative ph

Dinitropheno

st this hypoth

ion uncouple

n that disinfe

hip (R2 = 0.99

l Busto-Ram

t concentrati

orescences 5

found that w

xidine, and p

escence emis

uer (2009) wh

O157:H7 lux

h approxima

of E. coli O

mparison to

e mechanism

g with energ

minescence de

lls remain vi

hosphorylatio

ol and carva

hesis, 2.4-Di

er was tested

ecting substa

943) betwee

mos et al. 200

ions of ClO2

5RL (del Bu

when a biolum

penicillin the

ssion in a do

hen a lethal

x and 97.4%

ately 7-log re

157:H7 to fi

one single a

m of inactivat

gy molecules

ecreased in p

iable, it was

on uncouplin

acrol compar

initrophenol

d against Esc

ances such as

en luminesce

08). The rate

2 (0.5, 1, 1.6,

sto-Ramos e

minescent St

e bacterial lo

ose and time

dose (1.2 m

% of biolumin

eduction in b

ive sub-letha

addition of 0

ation of ClO2

s such as NA

presence of

hypothesize

ng.

risons

l (2,4-DNP),

cherichia co

s chlorine di

ence (photon

e of light dec

, and 2.1 mg

et al. 2008).

treptococcus

og reduction

dependant m

mg/L) of ClO2

nescence wa

bacterial cell

al doses of 0

0.5 mg/L of C

2 was not by

ADH and NA

emulsions lo

ed that carva

, a well-know

oli O157:H7

ioxide gas (C

ns/s) and cell

crease was d

/L) were test

Also,

s mutans stra

was easily

manner. Sim

2 was tested

as lost in just

ls. However,

.1 mg/L of C

ClO2. Thus,

oxidation o

ADPH. Ther

oaded with

acrol mode o

wn oxidative

lux. The

62

ClO2)

l

dose-

ted

ain

milar

d

t ten

, the

ClO2

it

f

refore,

of

e

63

experiment was conducted in situ using a photo multiplier tube (PMT) (Hamamatsu,

AC135, Iwata-gun, Japan) sensor module in a light-tight box integrated with a

programmable logic controller interfaced with a PC for data acquisition as previously

described. Sub-lethal doses of 2,4 DNP were added to the bioluminescent strain and

bioluminescence was monitored for 600 seconds. The relative bioluminescence of E. coli

O157:H7 lux decreased immediately by 80%, 50%, or increased 20% after addition of

1mM, 0.5mM, and 0.1mM doses of 2,4-DNP respectively. Cells were tested for viability

after ten min of contact time with this compound. However, no cell inactivation was

observed (Figure 15). This chemical is known to uncouple oxidative phosphorylation

(Gage and Neidhardt 1993, Haidinger et al. 2003, Miranda et al. 2006), thus blocking

ATP production. The bioluminescent reaction requires ATP and NADPH as cofactors for

the reduction of myristic acid to myristyl aldehyde which in presence of FMNH2, O2 and

the luciferase emits light at a wavelength of 490 nm (Figure 3). Therefore, the decrease in

light emission occurs with decreasing ATP production. As a result, the similarity on the

bioluminescent behavior and the incapacity to reduce bacterial population at sub-lethal

doses of carvacrol or 2,4-DNP, is a key observation to hypothesize that carvacrol acts in a

similar manner as 2,4-DNP by uncoupling oxidative phosphorylation.

FEd0n

Solvent 

2,4‐DNP 

2,4‐DNP 

2,4‐DNP 

Figure 15. AEscherichia c

ecreased by .1mM dosesot result in a

A

B

Concentrat(ppm) 

19 

92 

184 

ddition of scoli O157:H80%, 50%,

s of 2,4-DNPa decrease in

tion  Bini

ub-lethal doH7 lux. A) Th

and increaseP respectiveln the concent

Bacterial Log tial (CFU/mL)

6.23±0.07 

6.23±0.07 

6.23±0.07 

oses of 2,4-Dhe relative bed 20% whenly. B) Howevtration of via

)  Bacteria

DNP (0.1 mbioluminescen treated witver, the addiable cells.

al Log reductio

0.04±0.01

0.03±0.02

0.05±0.06

M, 0.5 mM,ence of E. coth 1mM, 0.5ition of this

on (CFU/mL)

1 

2 

6 

, and 1mM)oli O157:H75mM, and compound d

64

) to 7 lux

did

3

al

ch

pr

ap

o

st

re

O

to

b

n

lu

b

an

A

pr

vi

an

B

ad

Add.4.3.3

Accor

ldehyde is pr

hain aldehyd

resence of F

pplications.

f the aldehyd

train of inter

eaction. For

O157:H7 and

o the addition

ioluminesce

ative flora o

uciferase-tran

ioluminesce

nd Poppe 20

To un

ATP, n-decan

resence of th

ivo for 10 m

nd it was fol

Bioluminesce

ddition (Figu

dition of n-de

rding to the b

roduced and

de, such as n

FMNH2, O2,

The additio

de substrate

rest. This ap

r example, th

d Listeria mo

n of 10µl of

nt colonies o

f raw milk (

nsducing ɸV

nce emission

000).

nderstand if m

nal (Sigma A

he carvacrol-

minutes. Each

llowed by th

ence recover

ure 16). The

ecanal

bioluminesc

d therefore no

n-decanal, sh

and the lucif

on of n-decan

thus requirin

pproach also

he imaging o

onocytogenes

f 1% n-decan

out of the cro

Ramsaran et

V10 bacterio

n and is usef

myristyl alde

Aldrich, St L

-containing n

h nanoemuls

hree addition

ry of 25%, 14

e gradually d

ent pathway

o light is em

hould result i

ferase which

nal (in exces

ng only the i

eliminates s

of biolumine

s in Camemb

nal in the non

owed plates

t al. 1998). A

phage which

ful for the de

ehyde was n

Louis, MO) w

nanoemulsio

ion was add

ns of n-decan

4%, and 10%

decrease on b

y, when ATP

mitted. Howe

in photon em

h opens the d

ss) eliminate

insertion of

substrate lim

escent coloni

bert and Fet

n-selective m

containing t

Another case

h is depende

etection of E

not being gen

was added to

on and light

ded after one

nal at minute

% was obser

bioluminesce

P is lacking n

ever, the add

mission at 49

door to sever

es the need fo

the luxAB in

mitation for th

ies of Escher

ta cheeses w

medium to h

the starter cu

e is the const

ent to n-deca

E. coli O157

nerated due t

o the biolumi

emission wa

minute of li

es 2, 4.5, and

rved respecti

ent recovery

no myristyl

dition of a lon

90 nm in the

ral food safe

for the synthe

nto the bacte

he luciferase

richia coli

as possible d

highlight the

ulture and th

truction of a

anal for

:H7 (Wadde

to the lack o

inescent stra

as monitored

ight monitor

d 7.

ively after ea

y can be

65

ng

ety

esis

erial

e

due

he

a

ell

f

ain in

d in

ring

ach

66

explained by the lack of glucose in the solution resulting in no synthesis of FMNH2

which is a co-factor for light production. These results suggest that carvacrol was indeed

acting by uncoupling oxidative phosphorylation resulting in lack of ATP for the synthesis

of myristyl aldehyde in the bioluminescent cycle. The addition of n-decanal (substitute of

myristyl aldehyde) provided a substrate which in the presence of oxygen and FMNH2

emitted a photon by the action of luciferase, encoded by the luxAB genes.

FcaprSS(7

Figure 16. Aarvacrol-coresence of: Atearin| Tweetearin|Lecith750 ppm). A

B

ddition of nntaining naA) Palm Steaen 20| Carvahin|Carvacro

An increase in

n-decanal toanoemulsionarin|Lecithin

acrol 2% (75ol 2% (750 pn light after

D

o Escherichins. Escherichn|Carvacrol 20 ppm) and

ppm), and D)each additio

ia coli O157hia coli O152% (750 ppmn-decanal. C) Palm Stearon of n-decan

7:H7 lux in p57:H7 lux bem) and n- deC) Palm rin| Tween 2nal is observ

presence of ehavior in ecanal. B) Pa

0| Carvacrolved.

67

alm

l 2%

68

3.4.4 ATP Swab Hygiene Test

Adenosine triphosphate (ATP) is one of the cofactors in the bioluminescent

reaction cycle, and each cell contains a constant intracellular ATP pool that is effectively

regulated. Therefore, in situ light emission is a sensitive and rapid indicator of the

intracellular state of cells (Stanley 1989), and is an effective way to estimate microbial

activity and the quantity of microorganisms present (Lin et al. 2013). At least 104 cells

are required to produce a signal (de Boer and Beumer 1999). In this study, the

bioluminescence-based ATP hygiene monitoring system was used to measure the

remaining ATP after ten min of contact time between cells and nanoemulsions loaded

with carvacrol.

A standard curve was developed using an ATP standard with an original

concentration of 10mM. The range of the reaction was between 691,480.5 RLU/s and

189.5 RLU/s which corresponded to 100 nM and 0.01 nM of ATP respectively. The

resulting linear regression was:

y=0.0001x-1.5833 Equation 5

Where ‘y’ is the ATP concentration (nM) and ‘x’ is light (RLU/s) (Figure 17). The

amount of ATP detected when carvacrol-loaded nanoemulsions were tested against E.

coli O157:H7 was 335-fold less than those without carvacrol (T-test<0.05). ATP values

were between 0.2 nM and 0.8nM. However, the controls (emulsions with no carvacrol)

had ATP concentrations up to 67 nM (figure 18). Helander et al. (1998) demonstrated

that at 300 ppm of carvacrol, the ATP pool changed from 3.1 to 0.3nM/mg. ATP has

been shown to be a sensitive indicator molecule for the presence of biologically active

or

th

p

th

ca

FIpmadw

rganisms an

hey have diff

atent on 197

he antimicro

arbenicillin,

Figure 17. Apswich, MA

measured usinddition of A

with a R2=0.9

d is a rapid t

fferent mecha

77 was devel

bial drug su

cephalothin

TP Standar, United Statng a Zylux (

ATP swab rea9966

tool for the a

anisms of ac

loped with th

sceptibility o

n, penicillin G

rd Curve. Otes) was dilu(Femtomasteagent. Result

analysis of e

ction (Loima

he objective

of different a

G, and clind

Original ATPuted to createer FB14, D-7ts were analy

effectiveness

aranta et al. 1

of using the

antibiotics, s

damycin (Cha

P Standard (1e a standard 75173, Pforzyzed against

s of antimicr

1998). In fac

e luciferase a

such as: amp

appelle et al

10mM, (Biolcurve. Biolu

zheim, Germt the resultin

robials even

ct, a NASA

assay to mea

picillin,

l. 1977).

labs, P0756Suminescence

many) after ng standard c

69

if

asure

S, e was

curve

Fcad(dev2th

Figure 18. Aarvacrol-loaifferent concdarker bars) valuated. A)% carvacrolhe treatment

TP values oaded emulsicentrations ohave on Esc

) Palm Steari. C) Palm Sts can be foun

of Escherichions. The effof carvacrol cherichia colin, Lecithin,tearin, Lecithnd in the ann

hia coli O157ffect on ATP(lighter barsli O157:H7 l and 1% of chin, and 2.5%nex section.

7:H7 in preP levels (nM)s) and emulslux after tencarvacrol. B% carvacrol

esence and a) of emulsionions with no

n min of contB) Palm Stear

. T-test <0.0

absence of ns containin

o carvacrol tact time warin, Lecithin05. The rest o

70

ng

s n, and of

71

Previous studies have reported that the mechanism of action of carvacrol might be

due to the accumulation of the antimicrobial in the cytoplasmic membrane causing

expansion, and having repercussions in both the lipid ordering and the bilayer stability

hence damaging membrane integrity and increasing the proton passive flux across the

membrane (Ultee et al. 1999, Cox et al. 2000, Ultee et al. 2002). Earlier studies suggested

that the mode of action of carvacrol might be due to the impairment of various enzyme

systems, such as those involved in the production of cellular energy and synthesis of

structural components (Conner and Beuchat 1984). However, this work suggests that

carvacrol acts as an oxidative phosphorylation uncoupler blocking the electron transport

chain and therefore, ATP production. This hypothesis can be supported by Ultee et al.

(2002). They conducted several experiments using carvacrol against Bacillus cereus and

concluded that although carvacrol caused destabilization on the membrane and a decrease

on the membrane potential, it is more probable that the antimicrobial activity of carvacrol

might be due to the decrease in the ΔpH as a result of its hydroxyl group and a system of

delocalized electrons. With this being said, the action of carvacrol will result in the

absence of a proton motive force, and as consequence, a disablement on ATP synthesis

which leads to a weakening of vital processes in the cell and finally to cell death.

72

CHAPTER 4. CONCLUSIONS AND FUTURE WORK

In this research, the antimicrobial effect of nanoemulsions containing carvacrol was

investigated and a mechanism of action of this antimicrobial was proposed. The

bioluminescent response of Escherichia coli O157:H7 lux was monitored in presence of

each of the different nanoemulsions and the resulting sample was plated for cell viability

after ten minutes of contact time. Contrary to expected, a discrepancy between

bioluminescence and cell viability was found whereas the first one decreased abruptly in

presence of carvacrol regardless of the antimicrobial concentration, and unexpectedly,

bacterial cells survived when the emulsions applied had 1% (with concentrations of

500,750, and 1000 ppm) or 2% carvacrol (with concentrations of 500 ppm). The same

bioluminescent response was found when Escherichia coli O157:H7 lux was monitored

in presence of 2,4-Dinitrophenol, a well-known oxidative phosphorylation uncoupler.

The comparison of both scenarios lead to the hypothesis of the similarity of modes of

action. This finding was confirmed when bioluminescence was monitored in presence of

both carvacrol containing nanoemulsions and n-decanal. Spikes of light were observed

immediately after addition of n-decanal regardless of the presence of carvacrol. ATP

concentrations were then investigated in presence and absence of the antimicrobial. In

agreement with the previous results, the presence of carvacrol, which is proposed to act

by uncoupling oxidative phosphorylation, caused a decrease in ATP.

73

This study contributes to the knowledge of carvacrol and its antimicrobial

mechanism of action and opens opportunities for further directions. More studies are

required to determine the effects of environmental factors such as pH, temperature, and

media nutrients, in the carvacrol antimicrobial efficacy. There is a need to understand the

effect of carvacrol on not only Gram positives, and other foodborne pathogens, but on

eukaryotes such as yeast and amoeba as well. The results of this work could be compared

to other antimicrobials found in essential oils, such as eugenol, p-cymene, menthol,

geraniol, limonene, and specially thymol which is an isomer of carvacrol. Furthermore, it

is important to complement the results of this study with the evaluation of the membrane

potential and proton motive force in the presence of carvacrol and the bioluminescence

response from other bacterial strains expressing the luxCDABE genes used in this study.

74

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84

APPENDICES

F

App

Figure 19. E

pendix A

Effect of nanof carvacr

Nanoemuls

oemulsions rol at differe

sions effect o

containing Cent dilutions:

on bacterial

Coconut oil : 500, 750 an

luminescenc

(CO), Lecithnd 1000 ppm

ce productio

hin (Lu) andm.

84

on

d 1%

Fo

igure 20. Eff carvacrol a

ffect of nanoat different d

emulsions cdilutions: 500

ontaining Co0, 750 and 1

oconut oil (C000 ppm.

CO), Lecithi

in (Lu) and 2

85

2%

Fo

igure 21. Eff carvacrol a

ffect of nanoat different d

emulsions cdilutions: 500

ontaining Co0, 750 and 1

oconut oil (C000 ppm.

CO), Lecithi

in (Lu) and 2

86

2.5%

Fo

igure 22. Eff carvacrol a

ffect of nanoat different d

oemulsions cdilutions: 500

containing P0, 750 and 1

Palm Stearin000 ppm.

n (PS), Twee

en 20 (Tw) a

87

and 1%

Fo

igure 23. Eff carvacrol a

ffect of nanoat different d

oemulsions cdilutions: 500

containing P0, 750 and 1

Palm Stearin000 ppm.

n (PS), Twee

en 20 (Tw) a

88

and 2%

Fo

igure 24. Eff carvacrol a

ffect of nanoat different d

emulsions cdilutions: 500

ontaining Pa0, 750 and 1

alm Stearin (000 ppm.

(PS), Tween

n 20 (Tw) an

89

nd 2.5%

Fo

igure 25. Eff carvacrol a

ffect of nanoat different d

emulsions cdilutions: 500

ontaining Co0, 750 and 1

oconut oil (C000 ppm.

CO), Tween

20 (Tw) and

90

d 1%

Fo

igure 26. Eff carvacrol a

ffect of nanoat different d

emulsions cdilutions: 500

ontaining Co0, 750 and 1

oconut oil (C000 ppm.

CO), Tween

20 (Tw) and

91

d 2%

Fo

igure 27. Eff carvacrol a

ffect of nanoat different d

oemulsions cdilutions: 500

containing C0, 750 and 1

Coconut oil (C000 ppm.

CO), Tween

n 20 (Tw) an

92

nd 2.5%

93

Appendix B Non-normalized bioluminescent results

Bioluminescence was monitored every second for ten minutes. Tables SS shows

the non-normalized results for the 600th second. A noteworthy difference is observed

between the bioluminescence emitted by E. coli O157:H7 lux in presence and absence of

carvacrol.

Table 12. Non-normalized bioluminescent values of CO|Lu|C%. The last bioluminescent value of emulsions containing Coconut oil (CO), Lecithin (Lu) and carvacrol at 1, 2, and 2.5% are compared to the same emulsion lacking of carvacrol.

Emulsion  C%  Dilution (ppm)  Final light value (photons/s) 

Standard Deviation 

CO|Lu|C  0  500  108138  992.78 

CO|Lu|C  1  500  12020  22.63 

CO|Lu|C  0  750  986694  200651.45 

CO|Lu|C  1  750  17782  6151.83 

CO|Lu|C  0  1000  563098  119882.88 

CO|Lu|C  1  1000  10304  989.95 

CO|Lu|C  0  500  710476  0.00 

CO|Lu|C  2  500  32076  29059.26 

CO|Lu|C  0  750  765596  0.00 

CO|Lu|C  2  750  6968  1708.37 

CO|Lu|C  0  1000  4680898  38197.91 

CO|Lu|C  2  1000  8520  4830.95 

CO|Lu|C  0  500  1269550  76138.43 

CO|Lu|C  2.5  500  8426  726.91 

CO|Lu|C  0  750  1101834  49822.74 

CO|Lu|C  2.5  750  5248  1538.66 

CO|Lu|C  0  1000  978752  12942.88 

CO|Lu|C  2.5  1000  3696  452.55 

94

Table 13. Non-normalized bioluminescent values of CO|Tw|C%. The last bioluminescent value of emulsions containing Coconut oil (CO), Tween 20 (Tw) and carvacrol at 1, 2, and 2.5% are compared to the same emulsion lacking of carvacrol.

Emulsion  C%  Dilution (ppm) 

Final light value (photons/s) 

Standard deviation 

CO|Tw|C  0  500  256064  109149.00 

CO|Tw|C  1  500  23374  6287.59 

CO|Tw|C  0  750  225720  109397.90 

CO|Tw|C  1  750  12332  2522.96 

CO|Tw|C  0  1000  315610  3606.24 

CO|Tw|C  1  1000  6684  4038.99 

CO|Tw|C  0  500  5201999  121055.27 

CO|Tw|C  2  500  686666  892365.93 

CO|Tw|C  0  750  686666  892365.93 

CO|Tw|C  2  750  902570  1236104.68 

CO|Tw|C  0  1000  1090175  3667.06 

CO|Tw|C  2  1000  11080  2059.09 

CO|Tw|C  0  500  6469138  1278830.90 

CO|Tw|C  2.5  500  19318  9246.13 

CO|Tw|C  0  750  4400588  349050.53 

CO|Tw|C  2.5  750  7258  2231.63 

CO|Tw|C  0  1000  3773414  221853.34 

CO|Tw|C  2.5  1000  4344  610.94 

95

Table 14. Non-normalized bioluminescent values of PS|Tw|C%. The last bioluminescent value of emulsions containing Palm Stearin (PS), Tween 20 (Tw) and carvacrol at 1, 2, and 2.5% are compared to the same emulsion lacking of carvacrol.

Emulsion  C%  Dilution (ppm)  Final light value (photons/s) 

Standard Deviation 

PS|Tw|C  0  500 1952082  927036.79

PS|Tw|C  1  500 46610  10343.56

PS|Tw|C  0  750 1769052  1196503.87

PS|Tw|C  1  750 23282  3238.55

PS|Tw|C  0  1000 1858904  1064122.17

PS|Tw|C  1  1000 16926  3187.64

PS|Tw|C  0  500 1927568  0.00

PS|Tw|C  2  500 7984  1736.65

PS|Tw|C  0  750 1529052  0.00

PS|Tw|C  2  750 7344  379.01

PS|Tw|C  0  1000 1126988  0.00

PS|Tw|C  2  1000 6260  588.31

PS|Tw|C  0  500 1645170  128322.91

PS|Tw|C  2.5  500 120974  150181.00

PS|Tw|C  0  750 1416366  85964.39

PS|Tw|C  2.5  750 5240  248.90

PS|Tw|C  0  1000 1341966  99687.91

PS|Tw|C  2.5  1000 2304  2098.69

Fotote

A

Figure 28. Slf nanoemulsotal nanoemuen minutes. T

Appendix C

low additionsion (CO|Lu|ulsion volumThe red arro

Biolumin

n of nanoem|C2%) was a

me added waws represent

nescence res

mulsion to Eadded to E. cas 1 mL. Biot the slow ad

sponse to slo

Escherichia ccoli O157:Holuminescencddition of th

ow addition o

coli O157:H7 lux every ce was moni

he emulsion

of emulsion

H7 lux. One 30 seconds. itored in vivo

96

drop The

o for

Fcad(devcoS

Figure 29. Aarvacrol-loaifferent concdarker bars) valuated. Thomparison wtearin, Twee

TP values oaded emulsicentrations ohave on Esc

he presence owith the conten 20, and 2%

Appendix

of Escherichions. The effof carvacrol cherichia colof carvacrol trol. A) Palm% carvacrol

x D ATP

hia coli O15ffect on ATP(lighter barsli O157:H7 lreduces con

m Stearin, Tw. C) Palm St

Swab Hygie

7:H7 in preP levels (nM)s) and emulslux after ten

nsiderably thween 20, andtearin, Twee

ene test

esence and a) that emulsiions with no

n min of conthe ATP amoud 1% of carven, and 2.5%

absence of ions containio carvacrol tact time waunt in vacrol. B) Pa

% carvacrol

97

ing

s

alm

Fcad(devcoo

Figure 30. Aarvacrol-loaifferent concdarker bars) valuated. Thomparison wil, Lecithin,

TP values oaded emulsicentrations ohave on Esc

he presence owith the contand 2% carv

of Escherichions. The effof carvacrol cherichia colof carvacrol trol. A) Cocovacrol. C) C

hia coli O15ffect on ATP(lighter barsli O157:H7 lreduces con

onut oil, Lecoconut oil, L

7:H7 in preP levels (nM)s) and emulslux after ten

nsiderably thcithin, and 1%Lecithin, and

esence and a) that emulsiions with no

n min of conthe ATP amou% of carvacrd 2.5% carva

absence of ions containio carvacrol tact time waunt in rol. B) Cocoacrol

98

ing

s

onut

99

Appendix E Minimal Inhibitory Concentration

The minimal Inhibitory Concentration (MIC) of nanoemulsions against Escherichia

coli O157:H7 was determined by comparing two methods: Well Diffusion Agar Mueller

Hinton Agar (WDA) and Disk Diffusion Agar (DDA).

Well Diffusion Agar

The method of Puupponen‐Pimiä et al. (2001) was slightly modified. A sterile

cotton swab was immersed into 10mL Escherichia coli O157:H7 lux. The excess was

removed and LB Kan plates were inoculated by spreading. The plate was kept at room

temperature for about 10 minutes until dry. A 4 mm well was opened using a sterile cork

borer. Emulsions were diluted in sterile and distilled water to the desired carvacrol

concentrations based on the carvacrol content (500, 750, and 1000 ppm). 50 µL of

emulsion diluted to the desired concentration was added to each well.

Plates were stored at 4°C overnight and then were moved to an incubator at 37°C

overnight 912-18hr). The diameter of inactivation zone was recorded using an Electronic

Digital Caliper VWR.

Disk Diffusion Agar

Bacteria were spread in LB Kan plates as explained in the Well Diffusion Agar

method. 50 µL of the emulsion at different concentration (500, 750, 1000 ppm) were

applied to sterile filter paper disks (7 mm). Disks were placed at the top of the plate and

the same incubation process used for WDA was conducted (Klančnik et al. 2010)

100

Table 15. MIC values emulsions diluted to 500 ppm. Two different methods were used: Well Diffusion Agar (WDA) and Disk Diffusion Agar (DDA) 500 ppm 

Nanoemulsion  WDA  DDA 

CO|Lu|C1%  1.41±0.76  0.39±0.00 

CO|Lu|C2%  2.00±0.17  1.61±0.46 

CO|Lu|C2.5%  3.93±0.50  3.09±0.65 

CO|Tw|C1%  0.00±0.33  2.48±0.25 

CO|Tw|C2%  2.98±1.65  2.96±0.89 

CO|Tw|C2.5%  2.54±0.28  3.68±0.69 

PS|Lu|C1%  1.44±0.01  0.00±0.00 

PS|Lu|C2%  2.03±0.10  0.85±0.33 

PS|Lu|C2.5%  9.96±1.75  3.14±1.24 

PS|Tw|C1%  2.92±0.16  0.34±0.47 

PS|Tw|C2%  6.01±2.67  2.89±1.73 

PS|Tw|C2.5%  12.50±5.78  4.48±0.94 

Table 16. MIC values emulsions diluted to 750 ppm. Two different methods were used: Well Diffusion Agar (WDA) and Disk Diffusion Agar (DDA)

750 ppm 

Nanoemulsion  WDA  DDA 

CO|Lu|C1%  1.91±0.11  0.95±0.11 

CO|Lu|C2%  5.32±2.09  1.98±0.04 

CO|Lu|C2.5%  5.51±0.55  4.15±0.16 

CO|Tw|C1%  1.18±0.21  2.63±0.00 

CO|Tw|C2%  4.74±0.01  3.98±0.02 

CO|Tw|C2.5%  3.61±1.08  3.35±0.39 

PS|Lu|C1%  3.53±0.32  0.67±0.29 

PS|Lu|C2%  3.96±1.16  1.19±0.45 

PS|Lu|C2.5%  5.49±0.22  1.60±0.45 

PS|Tw|C1%  5.20±0.82  1.47±0.42 

PS|Tw|C2%  7.76±2.46  3.40±2.02 

PS|Tw|C2.5%  13.37±5.93  4.92±1.36 

    

Tu1

N

C

C

C

C

C

C

Fcoh

Table 17. MIsed: Well D1000 ppm 

Nanoemulsio

CO|Lu|C1% 

CO|Lu|C2% 

CO|Lu|C2.5%

CO|Tw|C1% 

CO|Tw|C2% 

CO|Tw|C2.5%

PS|Lu|C1% 

PS|Lu|C2% 

PS|Lu|C2.5%

PS|Tw|C1% 

PS|Tw|C2% 

PS|Tw|C2.5%

Figure 31. Zooli O157:H7igher the inh

A

IC values emiffusion Aga

on 

% 

% 

% 

% 

ones of bact7. A) Well Dhibition zone

mulsions dilar (WDA) an

terial inhibiDifussion Age, the higher

luted to 100nd Disk Diff

WDA 

2.56±0.71

8.29±1.53

9.14±1.97

1.81±0.16

6.52±0.49

6.77±1.80

4.06±0.10

5.49±0.22

11.49±0.9

6.63±0.51

8.88±2.24

13.93±6.2

ition of carvgar (WDA). B

the antimicr.

00 ppm. Twofusion Agar

1 

3 

7 

6 

9 

0 

0 

2 

94 

1 

4 

23 

vacrol-loadeB) Disk Diffrobial activi

B

o different m(DDA) 

DDA 

1.07±0

3.01±0

4.48±0

3.29±0

4.30±0

4.27±0

1.17±0

1.60±0

4.76±1

1.22±0

3.95±0

5.70±1

ed nanoemuffusion Agar ity.

methods wer

0.28 

0.17 

0.28 

0.78 

0.41 

0.93 

0.42 

0.45 

1.11 

0.18 

0.66 

1.27 

ulsions on E(DDA). The

101

e

E. e

102

Appendix F Statistical output

Table 18. One-way ANOVA (p<0.05). Independent variables: Emulsifier, Oil, Carvacrol Dilution and interactions. Dependent variable: Bacterial Enumeration.

Source DF Sum of Squares Mean Square F Value Pr > F

Model 19 1.4729828E14 7.7525408E12 4.49 <.0001

Error 52 8.9768951E13 1.726326E12

Corrected Total 71 2.3706723E14

R-Square Coeff Var Root MSE Bacterial Mean

0.621335 161.1430 1313897 815361.1

Source DF Type I SS Mean Square F Value Pr > F

Emulsifier 1 2.4125624E12 2.4125624E12 1.40 0.2425

Oil 1 2.0818541E12 2.0818541E12 1.21 0.2772

Carvacrol 2 2.6766922E13 1.3383461E13 7.75 0.0011

Dilution 2 2.9918883E13 1.4959441E13 8.67 0.0006

Emulsifier*Oil 1 4.7448428E12 4.7448428E12 2.75 0.1034

Emulsifier*Carvacrol 2 3.0226784E13 1.5113392E13 8.75 0.0005

Emulsifier*Dilution 2 573971103611 286985551806 0.17 0.8473

Oil*Carvacrol 2 2.0497385E13 1.0248693E13 5.94 0.0048

Oil*Dilution 2 7.5593397E12 3.7796698E12 2.19 0.1222

Carvacrol*Dilution 4 2.2515731E13 5.6289327E12 3.26 0.0185

103

Table 18 continued

Source DF Type III SS Mean Square F Value Pr > F

Emulsifier 1 2.4125624E12 2.4125624E12 1.40 0.2425

Oil 1 2.0818541E12 2.0818541E12 1.21 0.2772

Carvacrol 2 2.6766922E13 1.3383461E13 7.75 0.0011

Dilution 2 2.9918883E13 1.4959441E13 8.67 0.0006

Emulsifier*Oil 1 4.7448428E12 4.7448428E12 2.75 0.1034

Emulsifier*Carvacrol 2 3.0226784E13 1.5113392E13 8.75 0.0005

Emulsifier*Dilution 2 573971103611 286985551806 0.17 0.8473

Oil*Carvacrol 2 2.0497385E13 1.0248693E13 5.94 0.0048

Oil*Dilution 2 7.5593397E12 3.7796698E12 2.19 0.1222

Carvacrol*Dilution 4 2.2515731E13 5.6289327E12 3.26 0.0185