effect of carvacrol-loaded nanoemulsions on a
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Open Access Theses Theses and Dissertations
Spring 2014
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
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NTENTS
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ntimicrobial
and spices ..
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vehicles ......
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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)
....................
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ons...............
....................
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....................
ence product
....................
of emulsion .
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LSIONS ON
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ty .................
) ..................
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tion ..............
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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)
TcoS
E
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2.3.1 N
paration
mulsions, gr
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decreases an
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ny years. Wit
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12
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McClements
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ffects of ther
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2011, McCl
plet aggrega
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ntually, an in
lements and
tion
ons are const
, gravity and
rces between
ts 1999, Horn
ether to form
he process w
plet.
droplets is p
o separation
lectrostatic)
ith decreasin
e solubility o
n of solubili
an in a larger
es to move ar
ncrease in m
Rao 2011).
tantly collidi
d applied me
n droplets the
n and Riege
m an aggrega
where two or
prevented by
(Tadros et a
and attractiv
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of the encaps
ized oil mole
r one.This co
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mean droplet
ing among e
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r 2001). Flo
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more particl
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al. 2004). Th
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sulated oil in
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each other’s
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ain aggregate
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ons (e.g. Van
t al. 2009).
ncreases.
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s et al. 2004
due to the
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ed or separat
when two o
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f repulsive
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13
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The ma
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anoencapsul
Nanoencapsu
nteractions w
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Nanoe
ncreasing the
izes, nanoem
rotecting the
Donsì et al. 2
o ensure safe
tutzenberger
ntimicrobial
Donsì et al. 2
Nanoe
nd pear juice
was achieved
active solubi
ajor issue wh
need to be a
duct’s sensor
lation of thes
ulation also in
with food ing
passive mec
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encapsulatio
eir solubility
mulsions can
em from inte
2011). In add
ety and quali
r 2008, Don
ls are more e
2011, Liang
encapsulatio
es inoculated
d with 1g/L t
ility
hen using ant
added due to
ry characteri
se phytophen
ncreases the
gredients as w
chanisms of c
Nanoemulsio
on is an effic
y and keepin
increase ph
eractions wit
dition, nanoe
ity of foods
sì et al. 2011
effective aga
et al. 2012)
on of terpene
d with Lacto
erpenes and
timicrobial e
their low w
stics (Gaysin
nols is an ef
eir physical s
well as incre
cell absorpti
ons as natura
ient alternat
g them stabl
hysical stabil
th food ingre
emulsions h
(Parris et al.
1). Previous
ainst food-bo
.
es from Mela
obacillus del
bacterial ina
essential oils
water solubili
nsky et al. 2
fficient way
stability by p
easing their b
ion (Weiss e
al antimicrob
tive to delive
le over time.
lity of the bio
edients and i
have been use
. 2005, Jang
studies have
orne pathoge
aleuca altern
lbrueckii; de
activation w
s in foods is
ity which can
008). Theref
of increasing
protecting th
bioactivity t
et al. 2009).
bial vehicles
er bioactive c
. Due to the
oactive com
improving b
ed to deliver
and Lee 200
e shown that
ens than pure
nifolia were
elay of antim
was successfu
the large
n interfere w
fore, the
g their solub
hem from the
through the
s
compounds
small partic
mpounds,
ioavailabilit
r antimicrob
08, Luo and
t encapsulate
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
th
by
p
(B
pr
(U
pr
T
(M
2
Fm1
Carva
986) (Figure
hyme (45%)
y the FDA a
art 172: Foo
Burdock 201
roducts are:
Ultee et al. 1
reservative i
Therefore, the
MIC) in diff
001).
Figure 5. Chmonoterpenic
.25g/L at 20
acrol is a mo
e 5). Carvacr
. This antim
and its regula
od additives p
10).The max
baked good
1999, Burdoc
in food prod
ere is a urge
ferent food sy
hemical struc phenols an0°C (Chen et
2.
noterpenic p
rol is found m
microbial has
ations are un
permitted fo
ximum levels
ds (15.75 ppm
ck 2010). Ho
ducts is some
to find an a
ystems with
ucture of card are isomert al. 2014)
4.1 Carva
phenol isome
mainly in or
been grante
nder the Cod
or direct addi
s that carvac
m), chewing
owever, the u
etimes limite
accurate/effec
out comprom
rvacrol and rs. Their wat
acrol
eric with thy
regano essen
ed GRAS (G
de of Federal
ition to food
crol can be ad
gum (8.42 p
use of this p
ed due to flav
ctive minim
mising the se
thymol. Boter solubility
ymol (Laurin
ntial oil (60 t
Generally Rec
l Regulation
d for human c
dded to certa
ppm), condim
phenolic com
vor consider
mal inhibitory
ensory aspec
oth compouny have been r
n and Stupar
to 74%) and
cognized As
ns Chapter 21
consumption
ain food
ments (18 pp
mpound as
rations.
y concentrati
cts (Lambert
nds are reported to b
21
r
in
s Safe)
1,
n
pm)
ion
t et al.
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
ns are
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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
re it is constae system is cwith black cl
ier Tube (Programmablantly stirred connected to loth to avoid
PMT) Systeme Logic Conusing a stir pa PC for dat
d penetration
m for real-tintroller (A) iplate (C) An
ata acquisition of environm
ime biolumiis connected n inlet vial (Don (E). The smental light
inescence d to the sampD) is for samsystem is clo(F).
37
ple mple osed
3
in
co
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th
b
B
v
w
F
em
ce
re
th
lu
Dose.3.4.1
Bacter
nactivate 50%
ontaining 2.5
pm. The per
he average c
acteria) were
BA50, the hig
Bacter
ials were pre
was monitore
B14, D-751
mission was
entrifuge 54
esulting pelle
he carbon so
uminometer.
e-response a
ricidal activi
% of the bac
5% of carva
rcentage of k
ontrol value
e used to det
her the antim
3.3.
rial dilutions
epared with
ed every min
73, Pforzhei
s reversible, t
15D for 10 m
et was resus
urce. Light w
and bacterici
ities are defi
cteria under t
crol were tes
killed bacteri
. Dose-respo
termine BA5
microbial act
5 Effect o
s were prepa
0.9 mL of w
nute for five
im, Germany
the antimicr
min at 16,10
pended with
was monitor
idal activitie
ined as the p
test conditio
sted at two a
ia was evalu
onse graphic
50 values by
tivity (Friedm
f carvacrol o
ared as previ
washed bacte
minutes usin
y). To under
robial was re
00 g) after fiv
h one mL of
red for anoth
s (BA50 valu
percentage of
ons (Friedma
additional co
uated by com
cs (concentra
linear regres
dman et al. 20
on biolumine
iously descri
eria and 0.1 m
ng a luminom
rstand if the
emoved by c
ve min of co
MSM conta
her five addit
ues)
f a test comp
an et al. 2004
oncentration
mparing each
ation of carv
ssion models
004).
escence
ibed (section
mL of nanoe
meter (Zylux
effect of car
entrifugation
ontact time w
aining glucos
tional minut
pound neede
4). Emulsion
s: 250 and 3
h dilution wit
vacrol vs % k
s. The lower
n 3.3.4). Gla
emulsion. Li
x Femtomas
rvacrol on ph
n (Eppfendo
with bacteria
se (1000 ppm
tes using a
38
ed to
ns
50
th
killed
r the
ass
ight
ster
hoton
orf
a. The
m) as
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