Letters in Applied Microbiology 1998, 26, 118–122

Antimicrobial properties of plant essential oils and essences

against five important food-borne pathogens
A. Smith-Palmer1, J. Stewart2 and L. Fyfe1,3
1
Department of Dietetics and Nutrition, Queen Margaret College, 2Department of Medical Microbiology, University
of Edinburgh Medical School, and 3Centre for Food Research, Queen Margaret College, Edinburgh, UK
1617/97: received 23 July 1997 and accepted 19 August 1997

The antimicrobial properties of 21 plant

A . S MI T H- PA L ME R, J . S TE W AR T A N D L . F Y FE . 1998.
essential oils and two essences were investigated against five important food-borne pathogens,
Campylobacter jejuni, Salmonella enteritidis, Escherichia coli, Staphylococcus aureus and
Listeria monocytogenes. The oils of bay, cinnamon, clove and thyme were the most inhibitory,
each having a bacteriostatic concentration of 0·075% or less against all five pathogens.
In general, Gram-positive bacteria were more sensitive to inhibition by plant
essential oils than the Gram-negative bacteria. Campylobacter jejuni was the most
resistant of the bacteria investigated to plant essential oils, with only the oils of bay and thyme
having a bacteriocidal concentration of less than 1%. At 35 °C, L. monocytogenes was
extremely sensitive to the oil of nutmeg. A concentration of less than 0·01% was bacteriostatic
and 0·05% was bacteriocidal, but when the temperature was reduced to 4 °C, the
bacteriostatic concentration was increased to 0·5% and the bacteriocidal
concentration to greater than 1%.

INTRODUCTION only basic screening techniques, whereas this paper takes the
work a stage further to establish precise bacteriostatic and
In recent years there has been a dramatic increase in the
bacteriocidal concentrations; it also investigates the effect
number of reported cases of food-borne illness, with 82 041
of refrigeration temperature on these concentrations for L.
reported cases in 1995 (Monitor 1996). Consequently, there
monocytogenes. This is the first account of plant essential oils
is considerable interest in ways to stop this upward trend and
against Camp. jejuni which, although comparatively newly
reduce the incidence of food poisoning. One area of research
recognized as a food pathogen, is the most common cause of
is the development of new and improved methods of food
infectious intestinal disease (CDSC 1994).
preservation. Due to negative consumer perceptions of arti-
ficial preservatives, attention is shifting towards alternatives
that the consumers perceive as natural and in particular, plant MATERIALS AND METHODS
extracts, including their essential oils and essences. It is well
established that these extracts have antimicrobial properties Maintenance and preparation of cultures
against bacteria, moulds and yeast (Farag et al. 1989; Paster
et al. 1990; Aureli et al. 1992; Pai and Platt 1995). However, Cultures of Escherichia coli 8007, Staphylococcus aureus 10657,
it is only relatively recently that much attention has been Listeria monocytogenes 11994, Salmonella enteritidis 4444 and
given to their potential application as food preservatives. Campylobacter jejuni 11626 from the UK National Collection
This study examines the antimicrobial properties of 21 of Type Culture (61 Colindale Avenue, London) were main-
plant essential oils and two essences against five predominant tained on Tryptone Soya Agar (TSA; Oxoid, Basingstoke,
food-borne pathogens; Campylobacter jejuni, Salmonella enter- UK) at 4 °C.
itidis, Listeria monocytogenes, Staphylococcus aureus and Escher-
ichia coli. Most of the data published on the antimicrobial
properties of plant essential oils are fragmented and employ Agar well technique

Correspondence to: Dr Lorna Fyfe, Department of Dietetics and Nutrition, The agar well technique used was based on the well estab-
Queen Margaret College, Clerwood Terrace, Edinburgh EH12 8TS, UK. lished method of Deans and Ritchie (1987). Thus, 20 ml of
© 1998 The Society for Applied Microbiology

an 18 h bacterial culture were used to seed 500 ml of molten
TSA. Four, 4 mm diameter wells were punched in each TSA
plate and to each well was added 25 ml of neat essential oil or
essence. (Essences of onion and garlic were kindly supplied
by the Regency Mowbray Company and plant essential oils
by F. D. Copeland & Sons.) Plates seeded with Camp. jejuni
were incubated at 42 °C in 2·5 l gas jars with CampyGen
microaerophilic generating gas packs (Oxoid). The remaining
plates were incubated at 35 °C and the zones of inhibition
measured after 24 h. Triplicate sets of plates were prepared
on each occasion and each experiment repeated three times.
Bacteriostatic and bacteriocidal concentrations
Bacteriostatic and bacteriocidal concentrations were deter-
mined for the 15 oils and two essences showing the greatest
zones of inhibition. The appropriate volume of oil was added
to 9·5 ml TSB to give a final concentration of 0·005–1% after
the TSB was inoculated with 0·5 ml of an 18 h bacterial
culture. (1% was chosen as the maximum concentration
because higher levels would probably be unacceptable in
food.) Tubes inoculated with Camp. jejuni were incubated at
42 °C under microaerophilic conditions and the remaining
tubes at 35 °C with regular shaking. After 24 h, growth was
compared with the control both visually and through the
measurement of absorbance at 630 nm using a Dynatech MR
500 plate reader (Dynatech laboratories, Billinghurst, UK).
Samples (100 ml) from those tubes showing no growth were
plated onto TSA and incubated for 24 h under the same
conditions as used for the TSB.
The bacteriostatic concentration was the lowest con-
centration at which bacteria failed to grow in TSB, but were
cultured when 100 ml samples were plated on TSA.
The bacteriocidal concentration was the lowest con-
centration at which bacteria failed to grow in TSB and when
bacteria were not cultured after 100 ml samples were plated
on TSA (Colome et al. 1986.)
The bacteriocidal and bacteriostatic concentrations were
determined at 4 °C for L. monocytogenes with the five oils
that were the most inhibitory at 35 °C. This was achieved as
previously described, with the modifications that the inocu-
lum was grown at 4 °C for 10 d rather than using an overnight
culture, and the inoculated test tubes were also incubated for
10 d at 4 °C.
RESULTS
The results show the wide variation in the antimicrobial
properties of plant essential oils. The oils of almond, aniseed,
garlic, grapefruit, mandarin and orange showed no or very
slight inhibition, even when used neat in the agar well tech-
nique; by contrast, other oils produced zones of 10 and
11 mm. More precise data on the antimicrobial properties
© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 118–122
A NT IM I CR OB I AL PL A NT ES S EN TI A L O IL S 119
were obtained through the determination of bacteriostatic
and bacteriocidal concentrations.
The bacteriostatic and bacteriocidal concentrations showed
the oils of bay, cinnamon, clove and thyme to be the most
inhibitory oils examined. Table 2 shows that these four oils
had a bacteriostatic concentration of 0·075% or less against
all five pathogens, and Table 3 shows that with the exception
of Camp. jejuni, the bacteriocidal concentrations were 0·1%
or less. Tables 2 and 3 also show that in general, the bac-
teriostatic and bacteriocidal concentrations were lower for the
Gram-positive bacteria than for the Gram-negatives. Cam-
pylobacter jejuni appeared to be the most resistant of the
pathogens to the essential oils, with only the oils of bay and
thyme having a cidal effect at a concentration of less than
1%; however, when neat oils were used (Table 1), it was as
susceptible to inhibition as the other four pathogens.
Oil of nutmeg proved to be very specific in its inhibitory
properties. Thus, it was highly inhibitory against L. mono-
cytogenes at 35 °C, with a bacteriostatic concentration of less
than 0·01% and a bacteriocidal concentration of 0·05%. In
contrast, concentrations in excess of 1% would be required
to achieve stasis or death against the other four bacteria,
although zones of up to 10 mm were produced when used
neat in the agar well technique.
From Table 4 it can be seen that oil of nutmeg was less
inhibitory against L. monocytogenes at 4 °C, with a dramatic
increase in the bacteriocidal concentration from 0·05% at
35 °C to greater than 1% at 4 °C. Oil of bay was also shown
to be less inhibitory against L. monocytogenes at 4 °C, with an
increase in the bacteriocidal concentration to 0·1% compared
to 0·04% at 35 °C. For oils of clove, cinnamon and thyme,
comparable results were shown for bacteriostatic and bac-
teriocidal concentrations at the two temperatures, with any
difference being no more than 0·01%.
DISCUSSION
The determination of bacteriocidal and bacteriostatic con-
centrations is a more sensitive technique than the agar well
technique, which was used purely as a screening tool, to
eliminate those oils with no or very slight inhibitory proper-
ties against the five pathogens. The results of the static and
cidal concentrations showed that the two Gram-positive bac-
teria, Staph. aureus and L. monocytogenes, were more sensitive
to inhibition by plant essential oils than the three Gram-
negative bacteria, E. coli, Salm. enteritidis and Camp. jejuni.
Thus, generally lower bacteriostatic and bacteriocidal con-
centrations were required for the four most inhibitory oils,
bay, cinnamon, clove and thyme, against Staph. aureus and
L. monocytogenes. This is also illustrated by the sensitivity of
Staph. aureus and L. monocytogenes to the oils of eucalyptus,
marjoram, peppermint, rosemary and sage, with bac-
teriostatic concentrations of 0·02–0·075% and bacteriocidal
120 A . S MI T H- PA L ME R E T AL .

Table 1 Antimicrobial properties of plant essential oils and essences against five food-borne pathogens, using the agar well technique.
The value is the distance (mm) across the zone of inhibition and the agar well (diameter 4 mm). Values are the mean values of three separate
experiments
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
Oil Escherichia coli Staphylococcus aureus Listeria monocytogenes Salmonella enteritidis Campylobacter jejuni
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
Almond 4 4 4 4 6·3
Aniseed 4·5 4 4 4·5 4·5
Basil 6·9 7·3 5·7 7·1 7·6
Bay 10·1 10·9 9·2 11·0 10·1
Cinnamon 10·1 7·5 6·8 10·9 8·9
Clove 9·7 8·0 8·4 11·1 9·0
Eucalyptus 10·3 8·5 5·4 7·5 8·3
Fennel 4·9 4·1 4·8 5·5 4·9
Garlic 4 4 4 4 4·4
Ginger 4·3 4·5 4·3 4·3 5·1
Grapefruit 4 4·5 4 4 4
Lemon 4 6·0 5·3 4 4·6
Lime 6·7 5·3 4·2 7·2 5·2
Marjoram 6·5 5·8 4·8 5·0 7·1
Mandarin 4 4·1 4·2 4 4
Nutmeg 6·9 6·5 7·7 7·6 10·1
Onion 5·3 6·7 5·6 6·7 5·5
Orange 4 4 4 4 4
Peppermint 6·8 6·4 5·3 6·3 7·1
Rosemary 8·7 5·9 7·1 9·3 9·3
Sage 7·8 7·4 4·8 7·2 8·1
Spearmint 9·3 9·8 6·1 7·5 11·1
Thyme 8·3 8·5 10·0 11·1 10·4
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—

Table 2 Bacteriostatic concentrations (%) for a range of plant essential oils and essences. This is the lowest concentration at which
bacteria failed to grow in broth, but were cultured when 100 ml samples were plated onto agar
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
Oil Escherichia coli Staphylococcus aureus Listeria monocytogenes Salmonella enteritidis Campylobacter jejuni
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
Aniseed ×1 ×1 ×1 ×1 ×1
Basil 0·25 0·1 0·05 0·1 0·25
Bay 0·05 0·05 0·02 0·05 0·075
Clove 0·04 0·04 0·03 0·04 0·05
Cinnamon 0·05 0·04 0·03 0·05 0·05
Eucalyptus ×1 0·1 0·075 ×1 ×1
Fennel ×1 ×1 ×1 ×1 ×1
Garlic ×1 ×1 ×1 ×1 ×1
Ginger ×1 ×1 ×1 ×1 ×1
Marjoram ×1 0·05 0·02 ×1 0·25
Nutmeg ×1 ×1 ³0·01 ×1 ×1
Onion ×1 ×1 ×1 ×1 ×1
Peppermint ×1 0·04 0·03 ×1 0·1
Rosemary ×1 0.04 0.02 ×1 0·5
Sage ×1 0·075 0·02 ×1 ×1
Spearmint 0·25 0·075 0·04 0·25 0·25
Thyme 0·05 0·02 0·02 0·04 0·04
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—

© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 118–122
 A NT IM I CR OB I AL PL A NT ES S EN TI A L O IL S 121

Table 3 Bacteriocidal concentrations (%) for a range of plant essential oils and essences. This is the lowest concentration at which
bacteria failed to grow in broth and when bacteria were not cultured after 100 ml samples were plated onto agar
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
Oil Escherichia coli Staphylococcus aureus Listeria monocytogenes Salmonella enteritidis Campylobacter jejuni
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
Aniseed ×1 ×1 ×1 ×1 ×1
Basil 0·5 0·5 0·25 0·5 ×1
Bay 0·1 0·075 0·04 0·075 0·5
Clove 0·1 0·04 0·04 0·075 1
Cinnamon 0·1 0·04 0·075 0·1 ×1
Eucalyptus ×1 0·25 0·25 ×1 ×1
Fennel ×1 ×1 ×1 ×1 ×1
Garlic ×1 ×1 ×1 ×1 ×1
Ginger ×1 ×1 ×1 ×1 ×1
Marjoram ×1 0·25 0·25 ×1 ×1
Nutmeg ×1 ×1 0·05 ×1 ×1
Onion ×1 ×1 ×1 ×1 ×1
Peppermint ×1 0·25 0·5 ×1 ×1
Rosemary ×1 0·1 0·1 ×1 ×1
Sage ×1 1 0·25 ×1 ×1
Spearmint 0·5 ×1 0·25 0·5 ×1
Thyme 0·1 0·03 0·03 0·04 0·05
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—

Table 4 The bacteriostatic and bacteriocidal concentrations (%) Listeria monocytogenes is a pathogen of great concern to
of five plant essential oils against Listeria monocytogenes at the food industry, especially in foods normally stored under
4 °C and 35 °C refrigeration conditions where, unlike most food-borne
—
––––––––––––––––––––––––––––––––––––––––––––––––––––– pathogens, it is able to multiply (Junttila et al. 1988). Conse-
Bacteriostatic Bacteriocidal
quently, refrigeration should not be relied upon as the sole
—–––––––––––––––––– —––––––––––––––––––
35 °C 4 °C 35 °C 4 °C method for the control of L. monocytogenes, but should be
—
––––––––––––––––––––––––––––––––––––––––––––––––––––– incorporated with another means of preservation. One poss-
Bay 0·02 0·03 0·04 0·1 ible option is the use of plant essential oils. From Table 4,
Cinnamon 0·03 0·02 0·075 0·075 the most applicable would be clove, cinnamon and thyme, as
Clove 0·03 0·02 0·04 0·05 these retain their low bacteriostatic and bacteriocidal con-
Nutmeg ³0·01 0·5 0·05 ×1 centrations at 4 °C, unlike oils of bay, and especially nutmeg,
Thyme 0·02 0·02 0·03 0·03 which were less inhibitory at 4 °C than at 35 °C. This may be
—
––––––––––––––––––––––––––––––––––––––––––––––––––––– a result of changes to the site of action of these oils at the
lower temperature, or alterations in the bacterial membrane,
so reducing the penetration of the oils to the interior of the
cell. It is also possible that the effectiveness of oil of nutmeg
concentrations of equal to or less than 1%. In contrast, con- against L. monocytogenes at 35 °C is due to the release of
centrations of these oils in excess of 1% would be required inhibitory volatiles at this temperature. The influence of tem-
to achieve inhibition against the Gram-negative bacteria. The perature on the activity of plant essential oils has also been
difference in sensitivity between the Gram-negative and reported by Ting and Deibel (1991) who showed that refriger-
Gram-positive bacteria to inhibition by plant essential oils is ation temperature could enhance the inhibitory effect of sage,
supported by other researchers including Shelef (1983) and but not clove or oregano, against L. monocytogenes.
Farbood et al. (1976). It is not known exactly why Gram- It is widely accepted that higher concentrations of plant
negative bacteria should be less susceptible, but it may be essential oils are required in foods than in laboratory media
related to the outer membrane of Gram-negative bacteria (Farbood et al. 1976). Due to changes in the organoleptic
which endows the bacterial surface with strong hydrophilicity properties of food caused by high levels of plant essential oils,
and acts as a strong permeability barrier (Nikaido and Vaara it may not be possible to add concentrations high enough to
1985). cause bacterial cell death. However, in many cases, a con-
© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 118–122
122 A . S MI T H- PA L ME R E T AL .
centration sufficient to result in the stasis of growth may be
all that is required to achieve a safe product, provided that
the initial pathogen load is low. Also, as shown by the results
in Tables 2 and 3, the concentrations required for stasis can
be considerably lower than those for killing.
The low bacteriostatic and bacteriocidal concentrations of
some plant essential oils against some of the most important
causes of bacterial food poisoning provides an exciting poten-
tial for the future, especially in the light of the shift away from
artificial preservatives and the move towards more natural
alternatives.
ACKNOWLEDGEMENTS
The authors acknowledge F. D. Copeland & Sons, Colanol
House, 5 Westfield Street, London, and the Regency Mow-
bray Company, Regency House, Hixon Industrial Estate,
Hixon, Staffordshire, for the supply of plant essential oils
and essences.
REFERENCES
Aureli, P., Costantini, A. and Zolea, S. (1992) Antimicrobial activity
of some plant essential oils against Listeria monocytogenes. Journal
of Food Protection 55, 334–348.
CDSC (1994) Communicate Disease Surveillance Centre Report. Food-
© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 118–122
borne Disease Surveillance in England and Wales. Government
Statistical Service. London: Office for National Statistics.
Colome, J.S., Cano, R.J., Kubinski, A. and Grandy, D.V. (1986)
Laboratory Exercises in Microbiology. pp. 95. West Publishing
Company.
Deans, S. and Ritchie, G. (1987) Antimicrobial properties of plant
essential oils. International Journal of Food Microbiology 5, 165–
180.
Farag, R.S., Daw, Z.Y., Hewedi, F.M. and Baroty, G.S.A. (1989)
Antimicrobial activity of some Egyptian spice essential oils. Jour-
nal of Food Protection 52, 665–667.
Farbood, M.I., MacNeil, J.H. and Ostovar, K. (1976) Effect of
rosemary spice extractive on growth of micro-organisms in meats.
Journal of Milk and Food Technology 39, 675–679.
Junttila, J.R., Niemala, S.I. and Hirn, J. (1988) Minimum growth
temperature of Listeria monocytogenes and non-haemolytic
Listeria. Journal of Applied Bacteriology 65, 321–327.
Monitor (1996) Population and Health. National Statistics. Govern-
ment Statistical Service. London: Office for National Statistics.
Nikaido, H. and Vaara, M. (1985) Molecular basis of bacterial outer
membrane permeability. Microbiology Reviews 49, 1–32.
Pai, S.T. and Platt, M.W. (1995) Antifungal effects of Allium sativum
(garlic) extract against the Aspergillus species involved in oto-
mycosis. Letters in Applied Microbiology 20, 14–18.
Paster, N., Juven, B.J., Shaaya, E. et al. (1990) Inhibitory effect of
oregano and thyme essential oils on moulds and food borne
bacteria. Letters in Applied Microbiology 11, 33–37.
Shelef, L.A. (1983) Antimicrobial effects of spices. Journal of Food
Safety 6, 24–29.
Ting, W.T.E. and Deibel, K.E. (1991) Sensitivity of Listeria mono-
cytogenes to spices at two temperatures. Journal of Food Safety 12,
129–137.