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Mode of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua

Authors:
  • Smith & Nephew, R&D Hull, UK
  • Unilever R&D Vlaardingen, The Netherlands

Abstract and Figures

To investigate the mode of action of vanillin, the principle flavour component of vanilla, with regard to its antimicrobial activity against Escherichia coli, Lactobacillus plantarum and Listeria innocua. In laboratory media, MICs of 15, 75 and 35 mmol l(-1) vanillin were established for E. coli, Lact. plantarum and L. innocua, respectively. The observed inhibition was found to be bacteriostatic. Exposure to 10-40 mmol l(-1) vanillin inhibited respiration of E. coli and L. innocua. Addition of 50-70 mmol l(-1) vanillin to bacterial cell suspensions of the three organisms led to an increase in the uptake of the nucleic acid stain propidium iodide; however a significant proportion of cells still remained unstained indicating their cytoplasmic membranes were largely intact. Exposure to 50 mmol l(-1) vanillin completely dissipated potassium ion gradients in cultures of Lact. plantarum within 40 min, while partial potassium gradients remained in cultures of E. coli and L. innocua. Furthermore, the addition of 100 mmol l(-1) vanillin to cultures of Lact. plantarum resulted in the loss of pH homeostasis. However, intracellular ATP pools were largely unaffected in E. coli and L. innocua cultures upon exposure to 50 mmol l(-1) vanillin, while ATP production was stimulated in Lact. plantarum cultures. In contrast to the more potent activity of carvacrol, a well studied phenolic flavour compound, the extent of membrane damage caused by vanillin is less severe. Vanillin is primarily a membrane-active compound, resulting in the dissipation of ion gradients and the inhibition of respiration, the extent to which is species-specific. These effects initially do not halt the production of ATP. Understanding the mode of action of natural antimicrobials may facilitate their application as natural food preservatives, particularly for their potential use in preservation systems employing multiple hurdles.
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Mode of antimicrobial action of vanillin against Escherichia
coli,Lactobacillus plantarum and Listeria innocua
D.J. Fitzgerald
1
, M. Stratford
2
, M.J. Gasson
1
, J. Ueckert
3
, A. Bos
3
and A. Narbad
1
1
Food Safety Science Division, Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, Norfolk, UK,
2
Unilever R&D,
Colworth House, Sharnbrook, Bedford, UK, and
3
Unilever R&D, Vlaardingen, Olivier Van Noortlaan, Vlaardingen, the Netherlands
2003/1072: received 24 November 2003, revised 1 March 2004 and accepted 1 March 2004
ABSTRACT
D . J . F I T Z G E R A L D , M . S T R A T F O R D , M . J . G A S S O N , J . U E C K E R T , A . B O S A N D A . N A R B A D . 2004.
Aims: To investigate the mode of action of vanillin, the principle flavour component of vanilla, with regard to its
antimicrobial activity against Escherichia coli,Lactobacillus plantarum and Listeria innocua.
Methods and Results: In laboratory media, MICs of 15, 75 and 35 mmol l
)1
vanillin were established for E. coli,
Lact. plantarum and L. innocua, respectively. The observed inhibition was found to be bacteriostatic. Exposure to
10–40 mmol l
)1
vanillin inhibited respiration of E. coli and L. innocua. Addition of 50–70 mmol l
)1
vanillin to
bacterial cell suspensions of the three organisms led to an increase in the uptake of the nucleic acid stain propidium
iodide; however a significant proportion of cells still remained unstained indicating their cytoplasmic membranes
were largely intact. Exposure to 50 mmol l
)1
vanillin completely dissipated potassium ion gradients in cultures of
Lact. plantarum within 40 min, while partial potassium gradients remained in cultures of E. coli and L. innocua.
Furthermore, the addition of 100 mmol l
)1
vanillin to cultures of Lact. plantarum resulted in the loss of pH
homeostasis. However, intracellular ATP pools were largely unaffected in E. coli and L. innocua cultures upon
exposure to 50 mmol l
)1
vanillin, while ATP production was stimulated in Lact. plantarum cultures. In contrast to
the more potent activity of carvacrol, a well studied phenolic flavour compound, the extent of membrane damage
caused by vanillin is less severe.
Conclusions: Vanillin is primarily a membrane-active compound, resulting in the dissipation of ion gradients and
the inhibition of respiration, the extent to which is species-specific. These effects initially do not halt the production
of ATP.
Significance and Impact of the Study: Understanding the mode of action of natural antimicrobials may
facilitate their application as natural food preservatives, particularly for their potential use in preservation systems
employing multiple hurdles.
Keywords: carvacrol, cytoplasmic membrane, Escherichia coli,Lactobacillus plantarum,Listeria innocua, mode of
action, natural antimicrobial, vanillin.
INTRODUCTION
Many herbs and plant extracts possess antimicrobial activ-
ities against a wide range of bacteria, yeasts and moulds (Kim
et al. 1995; Aziz et al. 1998; Ultee et al. 1998; Friedman
et al. 2002); thus they provide a potentially rich source of
novel biocides and preservatives. The antimicrobial activity
of these plants can sometimes be attributed to the low-
molecular weight phenolic compounds that are present
within them (Gould 1996; Davidson and Naidu 2000).
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the major
constituent of vanilla beans and is produced naturally via
a multi-step curing process, however 90% of vanillin
Correspondence to: Arjan Narbad, Food Safety Science Division, Institute of Food
Research, Norwich Research Park, Colney Lane, Norwich, Norfolk, NR4 7UA, UK
(e-mail: arjan.narbad@bbsrc.ac.uk).
ª2004 The Society for Applied Microbiology
Journal of Applied Microbiology 2004, 97, 104–113 doi:10.1111/j.1365-2672.2004.02275.x
currently in use is synthetically produced (nature identical)
from lignin, eugenol or guaiacol (Hocking 1997; Ramachan-
dra Roa and Ravishankar 2000). Vanillin has generally
recognized as safe (GRAS) status and is used as a flavouring/
aroma compound in foods and fragrance industries. Syn-
thetic vanillin is also used as an intermediate in the chemical
and pharmaceutical industries for the synthesis of herbicides
and drugs (Walton et al. 2003). Recent reports have shown
that vanillin can act as an antioxidant improving the keeping
quality of precooked dried cereal flakes (Burri et al. 1989)
and afforded significant protection against protein oxidation
and lipid peroxidation in rat liver mitochondria (Kamat et al.
2000). Moreover, vanillin exhibits strong antimicrobial
properties with activity demonstrated against a number of
yeast and mould strains in laboratory media, fruit-based agar
systems, fruit purees and fruit juices (Lo
´pez-Malo et al.
1995; Cerrutti and Alzamora 1996; Fitzgerald et al. 2004).
However, to our knowledge no reports have yet detailed the
mode of action of vanillin inhibition.
Understanding of how antimicrobial compounds such as
vanillin function is desirable if they are to find commercial
application as natural preservatives. The mode of action of
nearly all antimicrobials can be classed into one or more of
the following groups: (a) reaction with the cell membrane,
(b) inactivation of essential enzymes, or (c) destruction or
inactivation of genetic material (Davidson 1993). Phenolic
compounds are hydrophobic in nature and are therefore
regarded as membrane active (Sikkema et al. 1994, 1995).
Recent mode of action studies using plant essential oils
(oregano, thyme and tea tree) or some of their phenolic
constituents (carvacrol, eugenol and thymol) against several
pathogenic bacteria and yeasts have shown that their activity
resides in their ability to perturb the cell membrane
resulting in the loss of chemiosmotic control leading to cell
death (Ultee et al. 1999; Cox et al. 2000; Lambert et al.
2001; Carson et al. 2002; Burt and Reinders 2003; Walsh
et al. 2003).
Our objective was to determine the mode of action of
vanillin inhibition of several food-related bacteria, namely E.
coli,Lactobacillus plantarum and Listeria innocua. The effect
of vanillin addition on respiration, membrane integrity, the
potassium gradient, pH homeostasis and ATP pools was
investigated.
MATERIALS AND METHODS
Bacterial strains and culture conditions
E. coli MC1022 and L. innocua (ATCC 33090) were
obtained from IFR stock cultures (Norwich, UK). Lact.
plantarum was obtained from Unilever R&D stock cultures
(Bedford, UK). E. coli cultures were grown in L-broth (10 g
bacteriological tryptone (Becton Dickinson, Oxford, UK),
5 g yeast extract (Becton Dickinson), 5 g NaCl and 1 g
glucose) at 37C with shaking (200 rev min
)1
). Lact.
plantarum cultures were grown statically at 30C in M17
broth (Oxoid, Basingstoke, UK) supplemented with 0Æ5%
(w/v) glucose (GM17). L. innocua cultures were grown in
BHI broth (Oxoid) at 35C with shaking (200 rev min
)1
).
For solid media 1Æ5% (w/v) agar (Difco) was added.
Chemical preparation
Stock solutions of 2Æ5 mol l
)1
vanillin (Sigma, Poole,
Dorset, UK) and 1 mol l
)1
carvacrol (Sigma) were prepared
in 99Æ7–100% (v/v) ethanol. These stocks were stored at
)20C in the dark until required.
Fluorescent probes propidium iodide (PI) and carboxy-
fluorescein diacetate succinimidyl ester (CFDA-SE) were
employed in this study to analyse the membrane integrity
and intracellular pH, respectively (Molecular Probes,
Leiden, The Netherlands). PI has an excitation maximum
(k
ex
) of 536 nm and an emission maximum (k
em
) of 617 nm
with a fluorescence enhancement of 20- to 30-fold upon
binding to DNA. A stock solution of 1 mg ml
)1
was
prepared in Milli Q water and stored at 2C. CFDA-SE is
cleaved by esterases upon entering the cytosol to yield
fluorescent carboxyfluorescein (CF) that has a k
ex
of 494 nm
and a k
em
of 518 nm. A stock solution of 1 mg ml
)1
was
prepared in dimethyl sulphoxide (DMSO; Sigma) and
stored at 2C.
A 200 lmol l
)1
nigericin (Sigma) stock was prepared in
99Æ7–100% (v/v) ethanol and stored at 2C.
Susceptibility testing
Susceptibility to vanillin is expressed as minimal inhib-
itory concentration (MIC). Experiments were conducted
in 10 ml volumes of media in 30 ml screw-capped bottles
supplemented with vanillin in duplicate. Cultures grown
in the presence of maximum ethanol levels only, acted as
controls. Cultures were incubated under optimal condi-
tions for a period of 96 h, after which the bottles were
examined for growth or absence of growth. MIC was
defined as the lowest concentration of vanillin required to
completely inhibit growth and was determined visually.
To establish the nature of the inhibitory activity of
vanillin, 100 ll samples were taken and surface-plated
onto agar and incubated under optimal conditions for up
to 48 h.
Determination of the effect of vanillin
on respiration
Overnight (16 h) cell cultures were subcultured (200 ll)
into 10 ml fresh media and incubated for a further 3 h
MODE OF ANTIMICROBIAL ACTION OF VANILLIN 105
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,97, 104–113, doi:10.1111/j.1365-2672.2004.02275.x
(E. coli)or5h(L. innocua). Cells were harvested by
centrifugation at 4C (1580 gfor 10 min) and washed once
with 50 mmol l
)1
potassium phosphate buffer [pH 7Æ0/
0Æ1% (w/v) glucose for E. coli or pH 7Æ4/0Æ2% (w/v)
glucose for L. innocua]. Cell suspensions were adjusted to a
density of 0Æ2atanO.D.
600nm
in the same buffers and kept
at 2C throughout the experiments. Oxygen consumption of
cell suspensions was measured using a calibrated Clark-type
oxygen electrode (Digital model 10, Rank Brothers Ltd,
Cambridge, UK) linked to Model 1325 Econo chart recorder
(Bio-Rad, Hercules, CA, USA). The chamber temperature
was maintained via a circulating water bath and cell
suspensions were preheated (5 min), then an initial respir-
ation rate was quantified before the addition of any
components. Vanillin was added to final concentrations of
0–40 mmol l
)1
and the addition of 2% (v/v) ethanol alone
acted as the control. All values were recorded from triplicate
measurements and suitable controls were included to ensure
that none of the test elements interfered with the assay.
Flow cytometry analysis
Fluorescent measurements were carried out using a calib-
rated Coulter EPICS ELITE flow cytometer (Beckman-
Coulter, High Wycombe, Bucks, UK) with 488 nm
excitation from an argon-ion laser. The sample analysis rate
was kept below 1000 events per second and ended after
10 000 events had been acquired. Red fluorescence (PI,
membrane integrity analysis) was detected through a
610 nm filter, green and orange fluorescence (CFDA-SE,
pH
i
analysis) were detected through 525 and 575 nm filters,
respectively. In membrane integrity experiments, vanillin
was added to a final concentration of 50 mmol l
)1
(E. coli
and L. innocua), or 70 mmol l
)1
(Lact. plantarum) to cells in
exponential phase of growth (ca 10
6
cells ml
)1
) and incuba-
ted under optimal conditions. Samples (20 ll) were taken
after 15 min or 1 h and added to 1 ml of 50 mmol l
)1
potassium phosphate buffer (pH 6Æ0) containing PI at a
working concentration of 5 lgml
)1
, mixed and incubated at
room temperature for 5 min prior to flow cytometry
analysis. A final sample was also taken following overnight
incubation (18 h). Cells grown in the absence of vanillin
acted as negative controls and heat-treated (80C for 4 min)
cells were used as positive controls. The effect of ethanol
only addition was also examined. Intracellular pH measure-
ments were performed with Lact. plantarum using a
modified method from Breeuwer et al. (1996). Cells (ca
10
6
cells ml
)1
) were exposed to 20 or 100 mmol l
)1
vanillin,
2lmol l
)1
nigericin or 4% (v/v) ethanol plus CFDA-SE
(20 lgml
)1
) and incubated for 10 min at room temperature.
Samples (10 ll) were then added to 1 ml potassium
phosphate buffer (50 mmol l
)1
) varying in pH from 4Æ5to
7Æ5 and then introduced into the flow cytometer. Flow
cytometry data was analysed using WinMDI v.2Æ8, available
as freeware from http://facs.scripps.edu/software.html.
Determination of intra- and extracellular
potassium levels
Intra- and extracellular potassium levels were determined
using a modified method from Ultee et al. (1999). Overnight
(16 h) cell cultures were centrifuged at 4C (1580 gfor
10 min) and the cell pellets washed twice in either
50 mmol l
)1
sodium HEPES buffer (pH 7Æ0 for E. coli,
pH 7Æ4 for L. innocua) or 50 mmol l
)1
sodium phosphate
buffer (pH 6Æ5) for Lact. plantarum. Cell suspensions were
adjusted to a density of 1Æ0 at an O.D.
600nm
. After an initial
incubation period of 10 min under optimal conditions, the
experiments were started by energizing the cells with
glucose to final concentrations of 0Æ1% (w/v) for E. coli,
0Æ2% (w/v) for L. innocua and 0Æ5% (w/v) for Lact.
plantarum. At 10 min intervals, samples (1 ml) were
removed and added to sterile Eppendorf tubes and centri-
fuged immediately (2300 gfor 2 min). The extracellular
supernatants were collected (900 ll) and immediately frozen
using dry ice. The remaining supernatant was carefully
removed and discarded. To the remaining cell pellet 1 ml
TCA-EDTA buffer was added [10% (v/v) trichloroacetic
acid and 2 mmol l
)1
EDTA]. The cells were resuspended
and then centrifuged immediately (13 400 gfor 2 min); the
resulting intracellular supernatants were collected (900 ll)
and immediately frozen using dry ice. Vanillin, carvacrol or
ethanol (control) were added after the 20 min sampling
point. The potassium concentrations were measured using a
Flame Photometer (Atomic Absorption Spectrometer 3300,
Perkin-Elmer Ltd, Bucks, UK) set to read absorbance at
766Æ5 nm with a slit width of 0Æ1 mm. Values were
extrapolated against a standard calibration curve of KCl
after dilution (1 : 10) in HCl [final concentration 5% (v/v)
in Milli Q water]. All values were recorded from triplicate
measurements and suitable controls were included to ensure
that none of the test elements interfered with the assay.
Determination of intra- and extracellular ATP
levels
Cell preparation and sampling procedure were as described
above with the following amendments: 50 mmol l
)1
potas-
sium phosphate buffers were used and the extracellular
supernatants collected (500 ll) were diluted 1 : 2 with
2 mmol l
)1
EDTA before being frozen immediately using
dry ice. ATP levels were determined using an Enliten
rLuciferin/Luciferase ATP assay kit (Promega, Southamp-
ton, UK) with 10 ll sample and 100 ll reagent volumes per
assay. Luminescence (560 nm) was recorded with a Lumat
LB 6501 luminometer (Berthold UK Ltd, St Albans, Herts,
106 D.J. FITZGERALD ET AL.
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,97, 104–113, doi:10.1111/j.1365-2672.2004.02275.x
UK). All values were recorded from triplicate measurements
and suitable controls were included to ensure that none of
the test elements interfered with the assay.
Viability assays
Cells were prepared as described for the potassium deter-
mination assay in duplicate. Samples (20 ll) were taken at 0
and 1 h and a series of decimal dilutions were made in 1/4
strength Ringers solution pH 7Æ0 (Oxoid). The dilutions
were surface plated in triplicate onto the appropriate agar,
and the viable cell counts were enumerated after incubation
under optimal conditions for 24–48 h.
RESULTS
Antimicrobial activity of vanillin
The antimicrobial activity of vanillin was investigated
against E. coli,Lact. plantarum,andL. innocua in laboratory
media. After incubation for 96 h, MICs of 15, 75 and
35 mmol l
)1
were established for the three strains respec-
tively. Colony formation on agar plates inoculated with cells
from cultures exposed to MIC levels of vanillin indicated
that the inhibitory action of vanillin was bacteriostatic rather
than bactericidal.
Effect of vanillin on respiration
The addition of 2% (v/v) ethanol stimulated respiration in
glucose-energized cells of E. coli and L. innocua to 114 and
79% above the normal respiration rate of untreated cells,
respectively (Fig. 1). The addition of increasing concentra-
tions of vanillin resulted in the inhibitionof respiration;
however the addition of a minimum concentration of
30 mmol l
)1
was required before the inhibitory effect
overcame the stimulatory effect of ethanol in all cases.
Ethanol was added due to the requirement for a solvent in
the preparation of vanillin solutions. Maximum inhibition
levels of 19 and 52% were achieved with the addition of
40 mmol l
)1
vanillin to E. coli and L. innocua cell suspen-
sions, respectively. A linear relationship existed between the
inhibition of respiration and vanillin concentration in E. coli
cell suspensions with an R
2
value of 0Æ97. A weaker linear
relationship existed for cell suspensions of L. innocua with
an R
2
value of 0Æ83.
Effect of vanillin on membrane integrity
Flow cytometry was used in tandem with the nucleic acid
stain propedium iodide (PI) to measure the effect of vanillin
on membrane integrity. PI cannot enter cells with intact
membranes resulting in low fluorescence intensity in the red
spectrum, as shown by untreated E. coli cells (Fig. 2a). Cells
that have damaged membranes allow the stain to enter the
cell and bind to the DNA resulting in a 20–30-fold increase
in fluorescence intensity (Fig. 2b). A strong shift in intensity
was also observed with Lact. plantarum and L. innocua
cultures treated similarly (data not shown), however heat
–60·0
–40·0
–20·0
0·0
20·0
40·0
60·0
80·0
100·0
120·0
140·0
0 5 10 15 20 25 30 35 40
Vanillin (mmol l–1)
Relative change in respiration rate (%)
(a)
(b)
(a)
R
2 = 0·834
R
2 = 0·970
(b)
Fig. 1 Relative change* in the respiration rates of cell suspensions of
Listeria innocua (d) and Escherichia coli (m) in the presence of vanillin.
The values represent the mean of triplicate measurements. In the
absence of vanillin 2% (v/v) ethanol acted as a control. Linear
trendlines and R
2
values for L. innocua (a) and E. coli (b) are indicated
*Compared with the respiration rates of untreated cell suspensions (0Æ0)
75
0
75
0
100101102103104
Log red
ID
EventsEvents
(a)
(b)
Fig. 2 Flow cytometry histograms of red fluorescence (610 nm) of
untreated (a) or heat-treated (b) Escherichia coli cultures stained with PI
(5 lgml
)1
). Gated zones show intact cells (I) and damaged cells (D)
MODE OF ANTIMICROBIAL ACTION OF VANILLIN 107
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,97, 104–113, doi:10.1111/j.1365-2672.2004.02275.x
treatment of these cultures proved to be less effective
(Table 1). Escherichia coli cultures exposed to 50 mmol l
)1
vanillin showed a 29% increase in the number of cells with
damaged membranes within 60 min. After a longer incuba-
tion (overnight), some cells appeared to recover and this
level was reduced to 13%. Lactobacillus plantarum cultures
exposed to 70 mmol l
)1
vanillin had only 10% of cells with
damaged membranes after 60 min, this level increased to
55% after overnight incubation. Similarly L. innocua
cultures exposed to 50 mmol l
)1
vanillin had only a small
proportion of cells with damaged membranes (4% after
60 min), however unlike Lact. plantarum cultures this level
did not increase overnight. The addition of maximum
ethanol concentrations was found not to affect the mem-
brane integrity of any of the strains studied (data not
shown).
Effect of vanillin on intra- and extracellular
potassium levels
Disruption of the cytoplasmic membrane by vanillin would
also be indicated by an effect on ion gradients between the
cell and the external environment. Therefore intra (Kþ
in)-
and extra (Kþ
ext)-cellular potassium levels were measured.
Carvacrol, a membrane-active phenolic compound (Ultee
et al. 1999; Lambert et al. 2001), was used as a positive
control. Addition of 50 mmol l
)1
vanillin to E. coli cell
suspensions resulted in a rapid decrease of Kþ
in (6Æ3–
2Æ1 ppm ml
)1
) within 10 min (Fig. 3a). A corresponding
increase of Kþ
ext from 0Æ04 to 3Æ4 ppm ml
)1
was recorded.
Over the next 30 min only a slight reduction of Kþ
in was
observed indicating that a K
+
gradient was still being
maintained although to a lesser degree. Addition of
3Æ3 mmol l
)1
carvacrol resulted in the sudden and complete
Table 1 Flow cytometry measurements using propidium iodide for
identification of damaged cell membranes of Escherichia coli,Lactoba-
cillus plantarum and Listeria innocua treated with vanillin. Data from
control and heat-treated cells are also shown
Bacterial strain Treatment Sampling
time (min)
Membrane
damage (%)
E. coli Control* – 0
15 16
50 mmol l
)1
vanillin 60 29
Overnight 13
Heat–88
Lact. plantarum Control* – 3
15 8
70 mmol l
)1
vanillin 60 10
Overnight 55
Heat–53
L. innocua Control* – 1
15 4
50 mmol l
)1
vanillin 60 4
Overnight 2
Heat–46
*Ethanol was added to 2 or 2Æ8% (v/v) as required for the addition of
50 or 70 mmol l
)1
vanillin, respectively.
80C for 4 min.
0·00
2·00
4·00
6·00
8·00
10·00
12·00
14·00
0·00
1·00
2·00
3·00
4·00
5·00
6·00
7·00
8·00
6050403020100
6050403020100
K+ (ppm ml1)K+ (ppm ml1)K+ (ppm ml1)
0·00
2·00
4·00
6·00
8·00
10·00
12·00
14·00
6050403020100
Time (min)
(a)
(b)
(c)
Fig. 3 Intracellular (closed symbols, solid lines) and extracellular
(open symbols, dotted lines) potassium levels of glucose-energized
Escherichia coli (a), Lactobacillus plantarum (b) and Listeria innocua (c)
exposed to 50 mmol l
)1
vanillin (j()or3Æ3 mmol l
)1
carvacrol
(mn). Exposure to 2% (v/v) ethanol (r)) acted as a control.
Compounds were added at 20 min (indicated by arrow). The values
represent the mean of triplicate measurements
108 D.J. FITZGERALD ET AL.
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,97, 104–113, doi:10.1111/j.1365-2672.2004.02275.x
collapse of the K
+
gradient within 10 min. Viable cell counts
showed that the addition of vanillin resulted in a reduction
in cell numbers from 6Æ3·10
8
to 5Æ3·10
8
CFU ml
)1
,
while the addition of carvacrol resulted in the complete loss
of viability (Table 2). The addition of either vanillin or
carvacrol had a similar effect on K
+
ion gradients in Lact.
plantarum cell suspensions (Fig. 3b). At 50 mmol l
)1
vanil-
lin a continuous decrease in Kþ
in to near 0 after 40 min was
measured compared with the initial decrease of Kþ
in in
control cells which eventually stabilized, again a corres-
ponding increase of Kþ
ext was observed. No loss of cell
viability was observed with vanillin, while carvacrol caused a
complete loss of cell viability (Table 2). K
+
ion gradients
were still present in L. innocua cultures after 60 min
although Kþ
in levels were reduced from 12Æ3to5Æ5 ppm ml
)1
or 6Æ6 ppm ml
)1
after 40 min following the additions of
50 mmol l
)1
vanillin or 3Æ3 mmol l
)1
carvacrol, respectively
(Fig. 3c). A corresponding increase of Kþ
ext was again
observed. However, the viable cell counts only slightly
decreased from 1Æ2·10
9
to 9Æ0·10
8
CFU ml
)1
following
exposure to vanillin, but the addition of carvacrol was totally
bactericidal (Table 2).
Effect of vanillin on pH homeostasis
The pH homeostasis of Lact. plantarum in the presence of
vanillin was investigated further by flow cytometry analysis
utilizing the pH-sensitive stain CFDA-SE. Cells with an
intact membrane would be able to maintain their internal pH
via ion channels and pumps in the presence of a moderate
change of pH. Therefore the mean ratio of green fluores-
cence (525 nm) that is pH sensitive and orange fluorescence
(575 nm) that is pH insensitive would also remain constant.
This ratio would change depending on the external pH if the
cell membrane integrity was disrupted and H
+
ion gradients
were no longer controlled. The mean ratio of cells treated
with either 4% (v/v) ethanol (control) or 20 mmol l
)1
vanillin remained fairly stable irrespective of the external pH
(Fig. 4). The slight change in mean ratio may be due to the
amount of stain that accumulates in the cell wall and is
dependent on external pH values. Addition of the H
+
ionophore nigericin (2 lmol l
)1
) resulted in a large shift in
the mean ratio as the external pH increased. The mean ratio
shifted from a maximum value of 333 at an external pH
(pH
ext
)4Æ5 to a minimum value of 220 at a pH
ext
7Æ5
indicating membrane damage and loss of pH homeostasis.
This shift was more obvious with the addition of
100 mmol l
)1
vanillin resulting in a maximum mean ratio
value of 378 at a pH
ext
4Æ5 and a minimum value of 243 at a
pH
ext
7Æ5.
Effect of vanillin on cellular energy (ATP)
Membrane perturbation, decrease in respiration and loss of
K
+
and H
+
ion gradients would suggest that energy
generation within the cells would be detrimentally affected.
The effect of vanillin on ATP concentration was therefore
investigated. Carvacrol was again used as a positive control.
Intracellular ATP (ATP
in
) levels continued to increase in E.
coli cell suspensions even after the addition of 50 mmol l
)1
vanillin although at a slightly reduced rate when compared
with control cells (Fig. 5a). Small quantities of external
ATP (ATP
ext
) were observed but these levels did not
increase significantly in either the control or vanillin treated
cells. Addition of 3Æ3mmoll
)1
carvacrol resulted in the
rapid decrease of ATP
in
to undetectable levels within
10 min. This coincided with an observed increase of ATP
ext
Table 2 Viable counts (CFU ml
)1
)ofEscherichia coli,Lactobacillus
plantarum and Listeria innocua cells after the addition of either
50 mmol l
)1
vanillin or 3Æ3 mmol l
)1
carvacrol. Exposure to 2% (v/v)
ethanol acted as a control. Values represent the mean of duplicate
measurements
Bacterial strain Treatment
Sampling point (h)
01
E. coli Control 9Æ33 ·10
8
4Æ00 ·10
8
50 mmol l
)1
vanillin 6Æ33 ·10
8
5Æ33 ·10
8
3Æ3 mmol l
)1
carvacrol 7Æ17 ·10
8
0
Lact. plantarum Control 4Æ10 ·10
8
4Æ53 ·10
8
50 mmol l
)1
vanillin 3Æ49 ·10
8
3Æ50 ·10
8
3Æ3 mmol l
)1
carvacrol 4Æ18 ·10
8
7Æ67 ·10
6
L. innocua Control 1Æ07 ·10
9
1Æ20 ·10
9
50 mmol l
)1
vanillin 1Æ24 ·10
9
9Æ00 ·10
8
3Æ3 mmol l
)1
carvacrol 9Æ92 ·10
8
0
200
225
250
275
300
325
350
375
400
4·55 5·56·567·57
pH (50 mmol l1 potassium phosphate buffer)
Mean ratio (575/525 nm)
Fig. 4 Fluorescence intensity ratios (575/525 nm) of Lactobacillus
plantarum cell suspensions stained with CFDA-SE (20 lgml
)1
) after
exposure to 2 lmol l
)1
nigericin (m), 20 mmol l
)1
(d)or
100 mmol l
)1
(j) vanillin. Exposure to 4% (v/v) ethanol (r) acted as
a control
MODE OF ANTIMICROBIAL ACTION OF VANILLIN 109
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,97, 104–113, doi:10.1111/j.1365-2672.2004.02275.x
level. Addition of 50 mmol l
)1
vanillin to Lact. plantarum
cell suspensions resulted in the stimulation of ATP
in
levels
compared with levels recorded in the control cell suspen-
sions (Fig. 5b). The addition of carvacrol decreased ATP
in
levels although this time only a small increase in ATP
ext
levels was observed. Reduced levels of ATP
in
were recorded
in cell suspensions of L. innocua treated with vanillin when
compared with control levels (Fig. 5c). However, significant
levels of ATP
in
still remained and no ATP
ext
were detected.
The addition of carvacrol resulted in a gradual decrease in
ATP
in
with a corresponding increase of ATP
ext
levels.
DISCUSSION
Many natural compounds, including phenolic compounds
derived from plants, exhibit strong antimicrobial properties
and therefore have the potential to be applied to food
products as novel preservatives (Beuchat and Golden 1989;
Gould 1996; Friedman et al. 2002). However, there is
relatively little knowledge about the specific mode of action
of these compounds. In this study, we investigated the effect
of vanillin on the cytoplasmic membrane of the food-related
bacteria E. coli,Lact. plantarum, and L. innocua. The
antimicrobial activity of vanillin was found to be dependent
on the time of exposure, concentration and the target
organism. Jay and Rivers (1984) reported that the inhibitory
activity of vanillin was more effective against nonlactic
Gram-positives than Gram-negative bacteria. The high
MIC reported here for Lact. plantarum is in agreement with
these findings, however the E. coli strain used here had the
lowest MIC of the three organisms studied indicating that
certain Gram-negative bacteria can be equally susceptible to
the antimicrobial activity of vanillin. The inhibitory action
of vanillin at MIC was found to be bacteriostatic in contrast
to the more potent phenolic antimicrobials such as carvacrol
and thymol that are bactericidal (Ultee et al. 1998; Friedman
et al. 2002). Furthermore, it is recognized that if vanillin is
to be used as a preservative at the MIC values established
here it will impart the characteristic flavour and hence
organoleptic quality in the individual food application needs
to be examined.
Initial experiments revealed that glucose-dependent res-
piration was inhibited with increasing levels of vanillin.
These observations could indicate that vanillin affected
membrane integrity; however the direct inhibition of
respiratory enzymes or processes cannot be excluded using
the method employed here. Cox et al. (2000) observed that
tea tree oil, where the antimicrobial activity is attributed to
terpinen-4-ol also inhibited respiration in E. coli. Phenolic
compounds primarily target the cytoplasmic membrane due
to their hydrophobic nature and will therefore preferentially
partition into the lipid bilayer (Sikkema et al. 1994, 1995;
Weber and de Bont 1996). Interactions between both lipids
and membrane embedded proteins with the phenolic
compound results in the destabilizing of the membrane
and loss of integrity. The observed increase in the uptake of
the nucleic acid stain PI upon exposure to vanillin suggested
the integrity of the cell membrane was compromised;
however a significant proportion of the cell populations still
had functional membranes. Similar observations have been
0·00
1·00
2·00
3·00
4·00
5·00
6·00
7·00
8·00
9·00
0 102030405060
0102030405060
Time (min)
0102030405060
ATP (nmol l1 ml1)ATP (nmol l1 ml1)ATP (nmol l1 ml1)
0·00
1·00
2·00
3·00
4·00
5·00
6·00
7·00
8·00
9·00
10·00
0·00
2·00
4·00
6·00
8·00
10·00
12·00
14·00
16·00
18·00
(a)
(b)
(c)
Fig. 5 Intracellular (closed symbols, solid lines) and extracellular
(open symbols, dotted lines) ATP levels of glucose-energized Escheri-
chia coli (a), Lactobacillus plantarum (b) and Listeria innocua (c) exposed
to 50 mmol l
)1
vanillin (j()or3Æ3 mmol l
)1
carvacrol (mn).
Exposure to 2% (v/v) ethanol (r)) acted as a control. Compounds
were added at 20 min (indicated by arrow). The values represent the
mean of triplicate measurements
110 D.J. FITZGERALD ET AL.
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,97, 104–113, doi:10.1111/j.1365-2672.2004.02275.x
reported in cultures of E. coli and Staph. aureus when
exposed to tea tree oil (Cox et al. 2000). This would indicate
that the destabilizing effect of vanillin on the membrane is at
a sublethal level for the majority of the microbial population
and may provide an explanation for the bacteriostatic
activity of vanillin observed here.
One function of the cytoplasmic membrane is to act as a
selectively permeable barrier for small ions such as H
+
,K
+
,
Na
+
and Ca
2+
. The gradients of these ions between the
intra- and extracellular environments play an important role
for the cell in the regulation of cytoplasmic pH, control of
turgor pressure and generation of cellular energy (Kroll and
Booth 1981; Booth 1985; Sikkema et al. 1995). This study
has shown that vanillin caused a rapid leakage of intracel-
lular K
+
ions and a resulting increase in extracellular K
+
.
Ion gradients completely collapsed in cell suspensions of
Lact. plantarum, however partial gradients still remained in
cell suspensions of E. coli and L. innocua. Carvacrol and tea
tree oil have also been shown to cause leakage of K
+
ions
(Ultee et al. 1999; Cox et al. 2000). In this study carvacrol
was used as a positive control and the results obtained here
were consistent with those of Ultee et al. (1999). However,
unlike vanillin, the addition of carvacrol resulted in the
complete loss of viability. If membrane permeability was
increased for K
+
ions then it would be assumed that H
+
ion
gradients would also be affected. The addition of vanillin
(100 mmol l
)1
) or the proton ionophore nigericin
(2 lmol l
)1
)toLact. plantarum cultures resulted in the loss
of pH homeostasis. However, the addition of 20 mmol l
)1
vanillin did not affect pH homeostasis and further demon-
strates the concentration dependence of vanillin activity.
Proton ion gradients generate the two components of the
proton motive force, the pH gradient (DpH) and the
electrical potential (Dw) that are used to generate ATP via
the membrane-located ATPase (Sikkema et al. 1995; David-
son 1997). The loss of ion gradients, particularly the H
+
,
could be expected to be detrimental for the generation of
cellular energy (ATP), while it may affect the cell indirectly
through decreased enzyme activity, although this inhibition
would be dependent on both the external pH and the
optimal pH at which any particular enzyme functions. In
this study addition of vanillin resulted in either the
stimulation of ATP (Lact. plantarum) or slightly reduced
levels of ATP production (E. coli and L. innocua) when
compared with levels produced in control cell suspensions.
These results indicate an apparent contradiction. Although
the generation of ATP, at least for a short period of time
could be possible through the passive influx of H
+
via the
ATPase enzyme, a partial K
+
gradient still existed in E. coli
and L. innocua cell suspensions that may have been sufficient
to drive ATP generation. Lactobacillus plantarum are oxy-
gen-tolerant organisms that do not produce ATP via an
oxidative phosphorylation system, instead producing ATP
via substrate level phosphorylation. This energy-generating
system could potentially remain unaffected by vanillin in the
short term i.e. glucose as a substrate was present initially,
while the same hypothesis may also hold true for the
generation of ATP via glycolysis only in the other two
strains. The addition of carvacrol resulted in the rapid
depletion of intracellular ATP levels and a corresponding
increase in extracellular ATP levels indicating leakage of
ATP in E. coli and L. innocua. Helander et al. (1998) also
observed a similar change in ATP levels when E. coli cells
were exposed to either carvacrol or thymol. Ultee et al.
(2002) reported a novel mode of action for carvacrol
inhibition of B. cereus. It was proposed that carvacrol acts
as a trans-membrane carrier of monovalent cations by
exchanging its hydroxyl H
+
for another ion such as K
+
. The
observations made in our study showing K
+
leakage and also
the loss of pH homeostasis (Lact. plantarum only) would
suggest that vanillin does not work in the same manner.
This is further substantiated by earlier work showing the
importance of the aldehyde group rather than the hydroxyl
group of the vanillin structure in the inhibitory activity of
vanillin against Saccharomyces cerevisiae (Fitzgerald et al.
2003). The extent of membrane damage induced by any
given compound can be related to its intrinsic hydrophobi-
city that can be determined experimentally by its partition
coefficient in octanol-water (P
o/w
), compounds with a higher
P
o/w
will partition further into the cell membrane (Weber
and de Bont 1996). Vanillin has a log P
o/w
of 1Æ09 while
carvacrol has a log P
o/w
of 3Æ64 (Ultee et al. 2002) which
could account for the relatively weak membrane perturba-
tion observed upon exposure to vanillin and the severe
membrane damage caused by exposure to carvacrol reported
here and elsewhere (Kim et al. 1995; Helander et al. 1998;
Ultee et al. 1998).
Phenolic compounds have been shown to inhibit DNA,
RNA and protein synthesis (Nes and Eklund 1983), glucose
uptake (Evans and Martin 2000) and enzyme activities
(Rico-Munoz et al. 1987; Wendakoon and Sakaguchi 1995;
Kreydiyyeh et al. 2000). Furthermore, several reports have
detailed the binding interactions between vanillin and
proteins including bovine serum albumin, soy, fababean
and milk proteins (Ng et al. 1989; Li et al. 2000; Chobpat-
tana et al. 2002). Therefore we cannot rule out the
possibility that vanillin could also inhibit key membrane
proteins or other cellular functions.
In summary our observations using the food-related
bacteria E. coli,Lact. plantarum,andL. innocua have shown
that the inhibitory activity of vanillin resides primarily in its
ability to detrimentally affect the integrity of the cytoplasmic
membrane, with the resultant loss of ion gradients, pH
homeostasis and inhibition of respiratory activity. Energy
generation remains largely unaffected or can indeed be
stimulated for at least a short period of time (1 h). The
MODE OF ANTIMICROBIAL ACTION OF VANILLIN 111
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,97, 104–113, doi:10.1111/j.1365-2672.2004.02275.x
extent of the membrane damage appears to be sublethal in
the majority of cells within an inhibited microbial popula-
tion, exhibited as a bacteriostatic action of inhibition at
MIC.
ACKNOWLEDGEMENTS
We thank Dave Hart (IFR) for his assistance with the
potassium measurements. This work was supported at the
Institute of Food Research by a Unilever studentship
awarded to D. Fitzgerald.
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MODE OF ANTIMICROBIAL ACTION OF VANILLIN 113
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