International Journal of Food Microbiology 161 (2013) 53–59
Contents lists available at SciVerse ScienceDirect
International Journal of Food Microbiology
journal homepage: www.elsevier.com/locate/ijfoodmicro
Nanodispersed eugenol has improved antimicrobial activity against Escherichia coli
O157:H7 and Listeria monocytogenes in bovine milk
Bhavini Shah 1, P. Michael Davidson, Qixin Zhong ⁎
Department of Food Science and Technology, The University of Tennessee, Knoxville, United States
a r t i c l e
i n f o
Article history:
Received 31 August 2012
Received in revised form 19 November 2012
Accepted 25 November 2012
Available online 30 November 2012
Keywords:
Eugenol
Nanodispersion
Escherichia coli O157:H7
Listeria monocytogenes
Milk
Tryptic soy broth
a b s t r a c t
There has been great interest in intervention strategies based on plant essential oils to control pathogens
such as Escherichia coli O157:H7 and Listeria monocytogenes (Lm). However, the poor solubility of essential
oils in water makes it difficult to disperse evenly in food matrices, impacting food quality and antimicrobial
efficacy. In the present study, eugenol was dispersed in nanocapsules prepared with conjugates of whey
protein isolate (WPI) and maltodextrin (MD, of various chain lengths). When eugenol was encapsulated in
the conjugate made with MD40 at a WPI:MD mass ratio of 1:2, the nanodispersion was transparent and
was characterized for antimicrobial efficacy against E. coli O157:H7 strains ATCC 43889 and 43894, and Lm
strains Scott A and 101 in tryptic soy broth (TSB) and milk with different fat levels (whole, 2% reduced fat,
and skim) at 35 or 32 °C, with comparison to the same levels of free eugenol. In TSB, antimicrobial efficacy
of nanodispersed eugenol against E. coli O157:H7 and Lm was not improved, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values being 0.25 g/L higher than those of
free eugenol. Free eugenol performed better in TSB because there was no interfering compound and the MIC
and MBC were below the solubility of eugenol. In milk, nanodispersed eugenol was consistently observed to
be more effective than free eugenol, with MIC and MBC values above the solubility limit of eugenol. The
nanodispersed eugenol was speculated to be evenly distributed in food matrices at concentrations above
the solubility limit and supplied the antimicrobial locally when the binding caused eugenol level below the
inhibition requirement. Nanodispersed eugenol thus provides a novel approach for incorporation in foods
to improve antimicrobial efficacy without changing turbidity.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Recent estimates indicate that in the US, there is a mean annual cost
of approximately $2.04 billion on foodborne illness caused by Listeria
monocytogenes (Lm) and $635 million for STEC Escherichia coli O157:
H7 (Scharff, 2012). E. coli O157:H7 has been known to cause outbreaks
most commonly associated with the consumption of undercooked
ground beef (Amalaradjou et al., 2010), unpasteurized apple cider
(Baskaran et al., 2010) and fresh produce (Boyer et al., 2011; Viazis et
al., 2011). In May 2011, a newly emerged mutant strain E. coli O104:
H4 that contaminated sprouts caused a severe outbreak in Germany,
and smaller outbreaks in France, Spain and UK (CDC, 2011b). Listeriosis
outbreaks have been associated with raw or unpasteurized milk, dairy
products, and cantaloupe (CDC, 2011a).
Thus, the food industry is constantly exploring efficient and costeffective intervention strategies to control the growth of pathogenic
⁎ Corresponding author at: Department of Food Science and Technology, The University
of Tennessee, 2605 River Drive, Knoxville, TN 37996-4591, United States. Tel.: +865 974
6196; fax: +865 974 7332.
E-mail address: [email protected] (Q. Zhong).
1
Current address: Mead Johnson & Company, LLC, Evansville, IN.
0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijfoodmicro.2012.11.020
and spoilage microorganisms to ensure microbial quality and safety
of consumer foods (Lambert et al., 2001). For minimally-processed
foods, naturally occurring antimicrobial compounds are a viable
option (Tserennadmid et al., 2011). Essential oils from aromatic
plants, generally herbs or spices, are one group of naturally occurring
antimicrobials. Traditionally used as flavoring agents, they are
gaining popularity due to their preservative effects such as antifungal, antibacterial and antioxidant properties (Burt, 2004). Thymol,
carvacrol and eugenol are some of the most potent compounds in
essential oils extracted from commonly used culinary ingredients
thyme, oregano and clove, respectively (Ananda et al., 2009). Their
antimicrobial activity is attributed to the presence of phenolic
groups in their chemical structures (Michiels et al., 2007). They are
effective against a broad spectrum of gram-negative and grampositive pathogenic bacteria such as E. coli, Lm, Staphylococcus
aureus, Salmonella typhimurium and many fungi (Bajpai et al., 2012;
De Martino et al., 2009; Oussalah et al., 2007). However, the
poor water-solubility of essential oils makes it difficult to incorporate into foods, and their tendency to bind with hydrophobic food
constituents reduces availability for antimicrobial action (Friedman
et al., 2004; Gaysinsky et al., 2007; Juven et al., 1994). Thus, their
application in complex food systems is used at a concentration
54
B. Shah et al. / International Journal of Food Microbiology 161 (2013) 53–59
much higher than that in model growth media to achieve the same
level of efficacy. This may cause the rejection by consumers, due to
the strong herbal aroma.
In order to overcome these challenges, essential oils may be
encapsulated to enhance solubility and dispersibility in aqueous media,
reduce losses due to binding with food constituents, and increase antimicrobial efficacy by promoting contact with bacterial cell components
(Weiss et al., 2009). Several studies have demonstrated the potential
of microencapsulating essential oils in proteins and polysaccharides
(Baranauskiene et al., 2006; Beristain et al., 2001). When prepared as
emulsions, surface properties of oil droplets such as hydrophobicity
and surface charge are not only important to the physical stability of
emulsions but also to antimicrobial effectiveness (Weiss et al., 2009).
Furthermore, interactions between bacteria and food components such
as proteins and lipids affect the localization of oil droplets in a food
matrix and thus overall antimicrobial efficacy (Pérez-Conesa et al.,
2011). Therefore, there is growing interest in designing structured
delivery systems to improve the dispersion stability and antimicrobial
activity, with examples being nano-emulsions, microemulsions, and
liposomes (Donsì et al., 2011; Donsì et al., 2012; Gaysinsky et al., 2007;
Pérez-Conesa et al., 2011; Weiss et al., 2009). However, each of these
examples has certain limitations, e.g., high energy used to prepare
nano-emulsions and large quantity of surfactant required for formulating
microemulsions. Much work is needed to advance delivery systems of
antimicrobials.
Previously, we reported an emulsion–evaporation process to
encapsulate eugenol in nanocapsules of whey protein isolate (WPI)–
maltodextrin (MD) conjugates (Shah et al., 2012). WPI–MD conjugates were produced via the Maillard reaction by dry-heating a mixture of WPI and MD. The conjugates were used to prepare emulsions
with the oil phase of eugenol dissolved in hexane. The emulsion was
spray-dried to remove hexane, yielding hollow microparticles. After
hydration of spray-dried powder, transparent dispersions were
observed for some preparations at a eugenol concentration well
above its solubility limit. Our earlier work presented a food
biopolymer-based system to disperse essential oils in aqueous
food systems without causing phase separation and turbidity. The
objective of the present study was to characterize the antimicrobial
efficacy of this promising nanodelivery system against E. coli O157:
H7 and Lm in growth medium (tryptic soy broth; TSB) and in a
model food system (whole, 2% reduced fat and skim milk).
2. Materials and methods
2.1. Materials
Eugenol (99%) was purchased from Acros Organics (part of Thermo
Fisher Scientific, Morris Plains, NJ). WPI was a gift from Hilmar Cheese
Company (Hilmar, CA). MD of various chain lengths (MD40, MD100
and MD180) corresponding to an average dextrose equivalent of 4, 10
and 18, was obtained from Grain Processing Corporation (Muscatine,
IA). TSB, peptone, and agar (chemical grade) were products of Becton,
Dickinson and Company (Sparks, MD). Other chemicals were purchased
from Fisher Scientific (Pittsburgh, PA). Ultra-high temperature (UHT)pasteurized organic milk (whole, 2% reduced fat and skim) was
obtained from Private Selection, a brand of the Kroger Company
(Cincinnati, OH).
prepared with an oil phase (with 20%w/v eugenol in hexane) at 10%v/v
volume and an aqueous phase with conjugates dissolved at a net protein
concentration of 3.7%w/v by using a high-speed homogenizer (Cyclone
I.Q.2, The VirTis Company, Inc., Gardiner, NY) at 15,000 rpm for 3 min.
Emulsions were then spray dried. All spray drying experiments were
performed using a model B-290 mini spray-dryer (BÜCHI Labortechnik
AG, Flawil, Switzerland) at an inlet temperature of 150 °C, 35 m3/h air
flow rate, 600 kPa compressed air pressure, feed rate of 6.67 mL/min
and the recorded outlet temperature of 80–90 °C. The spray dried
capsules were collected and stored in a freezer at −18 °C. The content
of eugenol in spray-dried powder was quantified using high performance liquid chromatography. To prepare nanodispersions for microbial
tests, spray dried capsules were hydrated at a desired concentration of
eugenol in deionized water for 14 h at room temperature (21 °C). The
dispersions were characterized for particle size distributions using a
Delsa Nano-Zeta Potential and Submicron Particle Size Analyzer
(Beckman Coulter, Inc. Brea, CA).
2.3. Culture preparation
E. coli O157:H7 strains ATCC 43889 and ATCC 43894 and Lm strains
Scott A and 101 were stock cultures obtained from the Department of
Food Science and Technology at the University of Tennessee, Knoxville.
All cultures were grown in TSB and stored at −20 °C in glycerol as
stocks. Working cultures were obtained by inoculating 50 mL TSB
with 100 μL stock cultures and incubating for 24 h at 35 °C for E. coli
or 32 °C for Lm.
2.4. Antimicrobial susceptibility test in TSB
Bacterial inhibition in growth medium was studied using time-kill
assays (Davidson and Parish, 1989). The bacterial cultures were harvested
at late logarithmic phase (overnight) and diluted to approximately 5.0–
6.0 log CFU/mL. A total volume of 25 mL consisting of 12.5 mL of TSB,
10 mL of nanodispersed eugenol (or deionized water as control) and
2.5 mL of inoculum was used. The nanodispersions were prepared
such that the overall eugenol concentrations in the mixture were 1.0,
1.25, 1.5, 1.75, 2.0 and 2.25 g/L. Free eugenol was prepared similar to
nanodispersions, except that it was directly added into deionized water.
The mixture was adjusted to pH 6.8 and incubated at 35 °C for E. coli
treatments and 32 °C for Lm treatments. At regular intervals (0, 3, 6, 12,
24, and 48 h), a bacterial suspension sample (0.1 or 1.0 mL) was serially
diluted in 0.1% peptone, plated in duplicate (0.1 or 1.0 mL) using tryptic
soy agar (TSA), incubated for 24 h at the optimum growth temperature,
and then enumerated for viable count as CFU/mL. The lowest concentration inhibiting growth of the test microorganisms above the original
inocula, approximately 4.0–5.0 log CFU/mL (inhibition) after 48 h was
determined as the minimum inhibitory concentration (MIC), while the
lowest concentration tested where bacterial death (inactivation) was
observed after 48 h was determined as the minimum bactericidal concentration (MBC). The growth media, with and without bacterial culture
and no eugenol, were used as positive and negative controls, respectively,
in all treatments. The growth curves of positive controls with and without
WPI–MD conjugates were similar in preliminary trials.
2.5. Antimicrobial susceptibility test in milk
2.2. Preparation of nanodispersions
Procedures of preparing nanodispersions of eugenol were detailed
previously (Shah et al., 2012) and are summarized as follows. To
prepare conjugates, solutions containing WPI and MD at a mass ratio of
1:2, 1:1 and 2:1 were spray-dried, and the collected powder was
heated in an oven at 90 °C for 2 h for conjugation (via Maillard reaction).
To encapsulate eugenol by emulsion–evaporation, emulsions were
The above method was used for inhibition tests in milk with three
fat levels, by replacing TSB with milk, except the final overall eugenol
concentrations which were 3.5, 4.5, 5.5 and 6.5 g/L. These levels
were chosen after conducting a preliminary screening at 1.0, 1.5 and
2.0 g/L which yielded no or negligible pathogen inhibition. The milk
was used directly without sterilization. Portions of the milk were
incubated to ensure that there were no surviving microorganisms.
B. Shah et al. / International Journal of Food Microbiology 161 (2013) 53–59
Duplicates of spray dried samples were prepared for each conjugate.
Each spray dried sample was tested in duplicate against same bacterial
culture. Data from four replicate experiments (n = 4) were pooled to
calculate the statistical mean and standard deviation of the mean for
each condition tested. Data were analyzed by mixed model analysis of
variance (ANOVA, p b 0.05) using SAS 9.2 (SAS Institute, NC).
3. Results and discussion
(A)
3.2. Antimicrobial activity of nanodispersed eugenol in tryptic soy broth
Antimicrobial properties of nanodispersed eugenol against E. coli
O157:H7 ATCC 43889 and Lm strain Scott A in TSB were compared to
free eugenol, shown in Fig. 1 at levels of 1.0, 1.25 and 1.75 g/L. Against
E. coli O157:H7, an initial reduction of population was observed at
two lower eugenol levels, followed by recovery to ~5 log CFU/mL at
1.0 g/L and 1.6 log CFU/mL at 1.25 g/L. At a level of 1.75 g/L, E. coli
O157:H7 was completely inhibited by both forms of eugenol. Overall,
there was no difference between nanodispersed and free eugenol.
Both free eugenol and nanodispersed eugenol were observed to be
less efficacious against Lm than E. coli O157:H7. At 1.0 and 1.25 g/L
eugenol, the inhibition was similar for both forms of eugenol and Lm
recovered to a population similar to the initial population after 48-h
incubation. At 1.75 g/L, significant inhibition was observed, and free
eugenol was more efficacious than nanodispersed eugenol (Fig. 1B).
The trend that eugenol is more effective against E. coli O157:H7 ATCC
43889 than Lm strain Scott A and the overall antimicrobial effectiveness
generally agreed with the literature (Pérez-Conesa et al., 2011).
In a recent study (Terjung et al., 2012), eugenol was mixed with a
medium chain triacylglyceride and emulsified with Tween 80 to
different droplet sizes (80–3000 nm). When tested against E. coli C 600
and L. innocua in a non-selective medium at 37 °C using eugenol up to
0.8 g/L, no inhibition was observed for L. innocua, while emulsions with
smaller droplets were less effective in inhibiting E. coli C 600. By using
high speed centrifugation, it was observed that eugenol concentration
in the aqueous phase was lower in emulsions with smaller particles,
which was hypothesized to be caused by localization of eugenol at
the oil/water interface. In contrast, nanoemulsions were observed to
improve antimicrobial activities of terpene mixture and D-limonene in
growth media and fruit juices against Saccharomyces cerevisiae (ATCC
16664), E. coli (ATCC 26), and Lactobacillus delbrueckii sp. lactis (Donsì
et al., 2011). A follow-up investigation using carvacrol, limonene and
cinnamaldehyde at 1 g/L revealed that the improved antimicrobial
efficacy in growth media after encapsulation in nanoemulsions was
caused by increased solubility of these antimicrobials (Donsì et al.,
6
4
2
3.1. Nanodispersion properties
0
0
10
20
30
40
50
40
50
Time (h)
(B)
10
8
Log CFU/ml
Physical properties of nanodispersions were detailed previously
(Shah et al., 2012). Five samples were chosen for antimicrobial tests
in the present study. These included three treatments using MD40
at WPI:MD mass ratios of 1:2, 1:1, and 2:1 that had a mean diameter
of 127, 314, and 255 nm at pH 7.0, respectively, which corresponded
to eugenol loading levels of 7.3%, 7.8%, and 7.2% in spray-dried
powder, respectively. The other two samples were prepared at a
WPI:MD mass ratio of 1:1 using MD100 and MD180, with eugenol
loading levels of 7.5% and 7.9% in spray-dried powder, respectively,
that corresponded to a mean diameter of 357 and 237 nm at pH 7.0,
respectively. The dispersion with the smallest diameter, using
MD40 at a WPI:MD mass ratio of 1:2, was transparent and was thus
characterized more extensively for antimicrobial experiments, while
other samples had varying turbidity. Except the last section studying
eugenol dispersed by different conjugates, antimicrobial experiments
were carried out using the transparent dispersion.
10
8
Log CFU/ml
2.6. Data and statistical analysis
55
6
4
2
0
0
10
20
30
Time (h)
Fig. 1. Inhibition of Escherichia coli O157:H7 ATCC 43889 at 35 °C (A) and Listeria
monocytogenes Scott A at 32 °C (B) by 1.0 (triangles), 1.25 (squares) and 1.75 (circles)
g/L eugenol in tryptic soy broth adjusted to pH 6.8. Filled and open symbols represent
free and nanodispersed eugenol treatments, respectively. Nanodispersions were prepared
using WPI–MD40 conjugate with a mass ratio of 1:2. Control of bacterial culture without
eugenol is shown in diamonds. Error bars represent standard deviation of mean from four
measurements (n=4), two from each of two nanodispersion replicates.
2012). Unfortunately, we did not quantify the amount of dissolved
eugenol, because the dispersion was transparent and the separation of
dissolved versus nanodispersed eugenol was a challenge. Nevertheless,
concentrations in Fig. 1 and those tested in growth media in the literature
are below the solubility of eugenol, which is 2.46 g/L at 25 °C (HSDB,
2012). The possible binding between eugenol and WPI–MD conjugates,
in comparison to all available free eugenol, likely caused the lowered
antimicrobial activity of nanodispersions in TSB (Fig. 1).
3.3. Antimicrobial activity of nanodispersed eugenol in milk
Because binding between eugenol and food components like proteins and lipids reduces the antimicrobial activity (Baranauskiene
et al., 2006; Gutierrez et al., 2008; Gutierrez et al., 2009), milk with
three fat levels (full fat (~ 4%), reduced fat (2%) and skim (b0.5%))
were utilized in this research. Fig. 2 shows the comparison of free
and nanodispersed eugenol against E. coli O157:H7 ATCC 43889 at
concentrations of 3.5, 4.5, and 5.5 g/L. The treatments with 6.5 g/L
eugenol were not plotted because complete inhibition was observed
in all experiments. In skim milk (Fig. 2A), inhibition was observed at
3.5 g/L eugenol, with nanodispersion showing better activity, while
inactivation was observed at two higher eugenol levels. As fat levels
increased (Fig. 2B and C), a higher eugenol level was needed to
inhibit the growth of E. coli O157:H7. The treatment with 4.5 g/L
was ineffective in inhibiting E. coli O157:H7 in full fat milk. At
56
B. Shah et al. / International Journal of Food Microbiology 161 (2013) 53–59
8
8
Log CFU/ml
(A) 10
Log CFU/ml
(A) 10
6
4
6
4
2
2
0
0
0
10
20
30
40
0
50
10
(B)
(B)
10
Log CFU/ml
Log CFU/ml
30
40
50
40
50
40
50
10
8
8
6
4
6
4
2
2
0
0
0
10
20
30
40
0
50
10
20
30
Time (h)
Time (h)
(C) 10
(C) 10
8
Log CFU/ml
8
Log CFU/ml
20
Time (h)
Time (h)
6
4
6
4
2
2
0
0
0
10
20
30
40
50
Time (h)
Fig. 2. Inhibition of Escherichia coli O157:H7 ATCC 43889 in skim (A), 2% reduced fat
(B) and full-fat milk (C) by 3.5 (triangles), 4.5 (squares) and 5.5 (circles) g/L eugenol at
35 °C. Filled and open symbols represent free and nanodisdispersed eugenol treatments,
respectively. Nanodispersions were prepared using WPI–MD40 conjugate with a mass
ratio of 1:2. Control of bacterial culture without eugenol is shown in diamonds. Error
bars represent standard deviation of mean from four measurements (n=4), two from
each of two nanodispersion replicates.
5.5 g/L, E. coli O157:H7 was inactivated in all milk treatments. The
overall trend, i.e., improved activity with the nanodispersion, was
also observed when tested against Lm strain Scott A (Fig. 3), although
the overall degree of inhibition was not as high as the E. coli treatments (Fig. 2). The best improvement in antilisterial properties
after nanodispersion was observed at 5.5 g/L eugenol in full fat
milk, with Lm strain Scott A at ca. 2.6 log CFU/mL after 48-h incubation which contrasted with the recovery to 5.5 log CFU/mL after 48 h
for free eugenol.
The eugenol concentrations required to inhibit bacteria in milk were
much higher than those in TSB (Fig. 1), verifying the interference of
0
10
20
30
Time (h)
Fig. 3. Inhibition of Listeria monocytogenes strain Scott A in skim (A), 2% reduced fat
(B) and full-fat milk (C) by 3.5 (triangles), 4.5 (squares) and 5.5 (circles) g/L eugenol at
32 °C. Filled and open symbols represent free and nanodispersed eugenol treatments,
respectively. Nanodispersion was prepared using WPI–MD40 conjugate with a mass
ratio of 1:2. Control of bacterial culture without eugenol is shown in diamonds. Error
bars represent standard deviation of mean from four measurements (n=4), two from
each of two nanodispersion replicates.
protein and fat on eugenol activity. The concentrations in Figs. 2 and 3
are all above the solubility of eugenol, 2.46 g/L at 25 °C (HSDB, 2012).
The improved antimicrobial properties likely resulted from improved
dispersibility of eugenol. Eugenol has a density of 1.06 g/mL (HSDB,
2012) and the insoluble fraction precipitates during incubation,
unavailable to supply the depleted eugenol (due to binding with
proteins and lipids) in the bulk phase when test tubes were incubated.
In contrast, nanodispersed eugenol is evenly distributed in the liquid
dispersion and can be released locally to keep the concentration of
dissolved eugenol sufficiently high to inhibit the bacteria. Because
binding between eugenol dissolved in the continuous phase and fat
B. Shah et al. / International Journal of Food Microbiology 161 (2013) 53–59
57
Table 1
Minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of eugenol (g/L) required for inhibition and inactivation of Escherichia coli O157:H7 and Listeria
monocytogenes in tryptic soy broth (TSB) and whole, 2% reduced fat and skim milk, at pH 6.8.
Bacteria
Escherichia coli O157:H7
Strain
ATCC 43889
MIC (g/L)
MBC (g/L)
MIC (g/L)
MBC (g/L)
MIC (g/L)
MBC (g/L)
MIC (g/L)
MBC (g/L)
Nanodispersed eugenola in TSB
Free eugenol in TSB
Nanodispersed eugenola in skim milk
Free eugenol in skim milk
Nanodispersed eugenola in 2% milk
Free eugenol in 2% milk
Nanodispersed eugenola in whole milk
Free eugenol in whole milk
1.25
1.0
3.5
3.5
4.5
4.5
4.5
5.5
1.75
1.5
4.5
4.5
5.5
5.5
5.5
6.5
1.25
1.0
3.5
3.5
4.5
4.5
4.5
5.5
1.75
1.5
4.5
4.5
5.5
5.5
5.5
6.5
1.75
1.5
3.5
3.5
4.5
4.5
5.5
6.5
2.0
1.75
6.5
6.5
6.5
6.5
6.5
6.5
1.75
1.5
3.5
3.5
4.5
4.5
5.5
6.5
2.0
1.75
6.5
6.5
6.5
6.5
6.5
6.5
a
Listeria monocytogenes
ATCC 43894
Scott A
101
Nanodispersions were prepared using conjugates made with MD at a WPI:MD40 mass ratio of 1:2.
globules is stronger at a higher fat content, a higher level of eugenol is
needed to inhibit the growth of bacteria and nanodispersed eugenol
shows better improvement in antimicrobial activity than free eugenol.
3.4. MIC and MBC
Table 1 summarizes MIC and MBC of free and nanodispersed
eugenol against two strains each of E. coli and Lm in TSB and milk,
according to the criteria described previously. At the tested conditions, results were similar for the two strains of each pathogen. The
discussion hereafter thus is focused on E. coli O157:H7 ATCC 43889
and Lm Scott A. In TSB adjusted to pH 6.8, nanodispersed eugenol
demonstrated MIC of 1.25 g/L for E. coli and 1.75 g/L for Lm, and
(B)
14
MD 40, 1:2
MD 40, 2:1
MD 40, 1:1
Free eugenol, 1.25 g/l
Control
12
Log CFU/ml
10
14
MD 40, 1:1
MD 100, 1:1
MD 180, 1:1
Free eugenol, 1.25 g/l
Control
12
10
Log CFU/ml
(A)
MBC of 1.75 g/L for E. coli and 2.0 g/L for Lm, all of which was
0.25 g/L higher than the comparable conditions for free eugenol.
The present findings are similar to the MIC of approximately 1.5 g/L
for both bacteria when eugenol was solubilized by surfactant micelles
(Gaysinsky et al., 2005). The MIC of free eugenol was lower than the
1.6 g/L for E. coli in a study (Pei et al., 2009), possibly due to differences in the methodology. Nevertheless, our results showed that
nanodispersed eugenol was not more effective than free eugenol
(p > 0.05) in TSB.
MIC and MBC of nanodispersed eugenol against E. coli and Lm
were similar to those of free eugenol in skim and 2% reduced fat
milk but were overall significantly lower than those of free eugenol
in full fat milk (p b 0.05). The MIC results were in the range of a
8
6
8
6
4
4
2
2
0
0
0
10
20
30
40
50
0
10
Time (h)
(D)
14
MD 40, 1:2
MD 40, 2:1
MD 40, 1:1
Free eugenol, 1.75 g/l
Control
12
Log CFU/ml
10
30
40
50
40
50
Time (h)
14
MD 40, 1:1
MD 100, 1:1
MD 180, 1:1
Free eugenol, 1.75 g/l
Control
12
10
Log CFU/ml
(C)
20
8
6
8
6
4
4
2
2
0
0
0
10
20
30
Time (h)
40
50
0
10
20
30
Time (h)
Fig. 4. Inhibition of Escherichia coli O157:H7 ATCC 43889 by 1.25 g/L eugenol (A and B) at 35 °C and Listeria monocytogenes Scott A by 1.75 g/L eugenol (C and D) at 32 °C in tryptic
soy broth adjusted to pH 6.8, using nanodispersions produced with different chain lengths of MD and mass ratios of WPI:MD, as labeled in the legend. Error bars represent standard
deviation of mean from four measurements (n = 4), two from each of two nanodispersion replicates.
58
B. Shah et al. / International Journal of Food Microbiology 161 (2013) 53–59
(A) 14
10
MD 40, 1:1
MD 100, 1:1
MD 180, 1:1
Free eugenol, 4.5g/l
Control
12
10
Log CFU/ml
12
Log CFU/ml
(B) 14
MD 40, 1:2
MD 40, 1:1
MD 40, 2:1
Free eugenol, 4.5 g/l
Control
8
6
8
6
4
4
2
2
0
0
0
10
20
30
40
50
0
10
Time (h)
(C) 14
(D) 14
40
50
40
50
MD 40, 1:1
MD 100, 1:1
MD 180, 1:1
Free eugenol, 4.5g/l
Control
12
10
Log CFU/ml
Log CFU/ml
10
30
Time (h)
MD 40, 1:2
MD 40, 2:1
MD 40, 1:1
Free eugenol, 4.5 g/l
Control
12
20
8
6
8
6
4
4
2
2
0
0
0
10
20
30
40
50
0
10
20
30
Time (h)
Time (h)
Fig. 5. Inhibition of Escherichia coli O157:H7 ATCC 43889 (A and B) at 35 °C and Listeria monocytogenes Scott A at 32 °C (C and D) by 4.5 g/L eugenol in 2% reduced fat milk adjusted
to pH 6.8, using nanodispersions produced with different chain lengths of MD and mass ratios of WPI:MD, as labeled in the legend. Error bars represent standard deviation of mean
from four measurements (n = 4), two from each of two nanodispersion replicates.
previously reported MIC of 5 g/L in semi-skimmed milk (Gaysinsky
et al., 2007). Nanodispersions are expected to have increased surface
area, increased eugenol solubility, and reduced surface tension
(Weiss et al., 2009), all of which can theoretically enhance antimicrobial activity. As for eugenol, it has been suggested that it is
capable of contacting and passing through hydrophilic sections of
bacterial cell envelope (García-García et al., 2011; Karapinar and
Esen Aktug, 1987). However, the exact molecular mechanism of
antimicrobial activity as impacted by nanodispersion and surface
chemistry of nanocapsules remains unknown.
3.5. Antimicrobial activity of nanodispersed eugenol as impacted by
conjugate structure
The impacts of conjugate structure on antimicrobial properties
of nanodispersed eugenol were studied for 5 conjugates: MD40
at WPI:MD40 mass ratios of 1:2, 1:1, and 2:1 and MD100 and
MD180 at a WPI:MD mass ratio of 1:1. Tests were conducted at
eugenol concentrations corresponding to MIC of nanodispersed
eugenol (Table 1), i.e., 1.25 g/L for E. coli O157:H7 ATCC 43889 and
1.75 g/L for Lm Scott A in TSB (Fig. 4) and 4.5 g/L for both bacteria
in 2% reduced fat milk (Fig. 5). The results showed that the inhibition
patterns were not significantly impacted by the length of MD and the
diameter of dispersions, possibly because the variations of these
treatments were not significant enough to render differences at the
studied conditions.
The present study demonstrated that antimicrobial efficacy of
nanodispersed eugenol against E. coli O157:H7 and Lm was not
improved in TSB. This was because the MIC and MBC were all below
the solubility of eugenol and the minimized binding enabled the better
availability of free eugenol than that dispersed by WPI–MD conjugate.
Nanodispersion did not change the antimicrobial characteristics of
eugenol that is more effective against gram negative E. coli O157:H7
than gram positive Lm. However, nanodispersion enabled the even distribution of eugenol in milk at concentrations above the solubility limit
of the antimicrobial, which improved antimicrobial efficacy in milk with
interfering components. Nanodelivery systems thus have the promise
to reduce the amount of antimicrobials without changing turbidity of
food products. We are planning to further study if the level of eugenol
can be reduced further by combination with other antimicrobials and
if nanodispersed eugenol has satisfactory sensory characteristics.
Acknowledgments
This work was supported by the University of Tennessee and the
USDA National Institute of Food and Agriculture under the Project
Number TEN02010-03476.
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