Letters in Applied Microbiology 1998, 26, 209–214

The synergistic preservative effects of the essential oils of

sweet basil (Ocimum basilicum L.) against acid-tolerant
food microflora
K.J. Lachowicz1, G.P. Jones1, D.R. Briggs1, F.E. Bienvenu2, J. Wan3, A. Wilcock3 and
M.J. Coventry3
1
School of Nutrition and Public Health, Deakin University, Geelong, 2Ovens Research Station, Department of Natural
Resources and Environment, Myrtleford, and 3Australian Food Industry Science Centre, Werribee, Victoria, Australia
1618/97: received 30 July 1997 and accepted 30 October 1997

K .J . L A CH OW I CZ , G . P. JO N ES , D . R. BR I GG S, F .E . B I EN VE N U, J. W AN , A . WI LC O CK AN D M. J.
C OV EN T RY . 1998. Essential oils extracted by hydrodistillation from five different varieties of
Ocimum basilicum L. plants (Anise, Bush, Cinnamon, Dark Opal and a commercial
sample of dried basil) were examined for antimicrobial activity against a wide range of
foodborne Gram-positive and -negative bacteria, yeasts and moulds by an agar well
diffusion method. All five essential oils of basil showed antimicrobial activity against most of
the organisms tested with the exception of Flavimonas oryzihabitans and Pseudomonas
species. The inhibitory effect of Anise oil, in comparison with mixtures of the predominant
components of pure linalool and methyl chavicol, against the acid-tolerant organisms,
Lactobacillus curvatus and Saccharomyces cerevisiae, was examined in broth by an indirect
impedance method. Synergistic effects between Anise oil, low pH (pH 4·2) and salt
(5% NaCl) were determined. The antimicrobial effect of Anise oil was also assessed in a
tomato juice medium by direct viable count, showing that the growth of Lact. curvatus
and S. cerevisiae was completely inhibited by 0·1% and 1% Anise oil, respectively. The
results of the current study indicate the need for further investigations to understand
the antimicrobial effects of basil oils in the presence of other food ingredients and preservation
parameters.

INTRODUCTION trum of 50 plant essential oils (including basil) against 25

Lactobacillus, yeasts and moulds are predominant spoilage
microflora of acidic foods and where vacuum or anaerobic
modified atmosphere packaging is utilized, Lactobacillus are
invariably the major spoilage genera (Fierheller 1991; Stiles
1991). Sweet basil (Ocimum basilicum L.) is a popular culinary
herb and its essential oil has been used for many years to
flavour foods, as an ingredient of dental and oral health care
products and in fragrances (Guenther 1952). Basil and its
essential oils are often used as a flavourant in tomato-based
products that also have high acidity and that are prone to
spoilage by acid-tolerant microflora (Dziezak 1989; Frier-
heller 1991).
Deans and Ritchie (1987) screened the antimicrobial spec-
Correspondence to: Dr M.J. Coventry, Australian Food Industry Science
Centre, Private Bag 16, Werribee, Victoria 3030, Australia.
© 1998 The Society for Applied Microbiology
genera of bacteria and all bacteria showed a reasonably broad
sensitivity to the oils tested. Food spoilage yeasts (Conner
and Beuchat 1984) and toxic moulds (Ray and Bullerman
1982) also showed substantial sensitivity to a wide variety of
plant essential oils. The antimicrobial and antifungal proper-
ties of essential oils of different species of Ocimum have been
predominantly associated with the main constituents, linalool
and methyl chavicol (Reuveni et al. 1984; Sinha and Gulati
1990) and there have been synergistic effects attributed to
these two components against Rhizopus nigrans (Reuveni et
al. 1984). Also, other food flavourants such as the essential
oils of mint (Mentha spp.) demonstrate synergistic inhibitory
effects with low pH and sodium chloride (Tassou et al. 1995)
against Listeria. However, there have been no previous inves-
tigations reported using basil essential oils to study such
effects.
210 K .J . L A CH OW I CZ ET A L.
The chemical characteristics of the essential oils of some
varieties of basil (Ocimum basilicum L.) grown in Australia
has been described by Lachowicz et al. (1996, 1997). The
purpose of the current investigation was to compare the
antimicrobial spectrum of these essential oils against food
microflora and further investigate preservative interactions
against acid-tolerant spoilage microflora.
MATERIALS AND METHODS
Essential oils
Five samples of essential oil from sweet basil (Ocimum basili-
cum L.) were used in this study. These five basil oils were
produced by hydrodistillation (Lachowicz et al. 1996) of dried
leaves from a commercial basil (supplied by Waters Trading
Pty Ltd, Lower Templestowe, Victoria, Australia) and four
varieties (Anise, Bush, Cinnamon and Dark Opal) of basil
grown at the Ovens Research Station (Myrtleford, Victoria,
Australia). After distillation, the oil was collected, dried using
anhydrous sodium sulphate and stored at 4 °C under N2 in
the dark until required. The chemical compositions of the
oils used in the current study have been previously reported
by Lachowicz et al. (1996, 1997). Linalool and methyl chav-
icol were purchased from Sigma Chemical Company (St
Louis, MO, USA).
Cultures and media
Cultures used in this investigation were obtained from the
culture collection of the Australian Food Industry Science
Centre (AFISC), Werribee, Victoria, Australia, and media
were obtained from Oxoid (West Heidelberg, Victoria, Aus-
tralia). Stock cultures were maintained at −80 °C in Nutrient
broth containing 16% (v/v) glycerol. Bacterial cultures were
propagated as described by Coventry et al. (1996). In addition,
Flavimonas and Yersinia were propagated for 16 h in Tryp-
tone Soy Broth with added 0·6% yeast extract (TSYE) at
30 °C. Yeasts were grown in Oxytetracycline Glucose Yeast
Extract (OGYE) broth for 16 h at 25 °C. Moulds were grown
on the surface of OGYE agar for 5 d at 25 °C and spore
suspensions of mould cultures were prepared by the method
of Batish et al. (1989) and held at 4 °C.
Agar well diffusion method
Five essential oils of basil and pure authentic linalool and
methyl chavicol were screened for antimicrobial activity
against a wide range of micro-organisms by an agar well
diffusion method (Wan et al. 1998). Results were quoted as
the radius of the zone of inhibition around the well (sub-
tracting the radius of the well).
© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 209–214
Assessment of antimicrobial activity of Anise oil by
indirect impedance method
The antimicrobial activity of Anise basil oil and mixtures of
pure linalool and methyl chavicol (of v/v ratios of 0:1, 0·2:0·8,
0·4:0·6, 0·6:0·4, 0·8:0·2 and 1:0) at 0·01, 0·1 and 1% (v/v)
against Lactobacillus curvatus (AFISC 2103) in MRS broth at
30 °C and Saccharomyces cerevisiae (AFISC 3301) in OGYE
broth at 25 °C were examined by the indirect impedance
method (RABIT impedance instrument, Don Whitley Scien-
tific Limited, Shipley, UK) as described previously (Wan et
al. 1998). Conductance changes were recorded at 6 min inter-
vals for 99 h. The time to detection of growth (TDG) was
indicated by three consecutive changes in conductivity
greater than 10 mS 6 min−1 (detection criterion value based
on manufacturer’s recommendation and the noise level of the
conductivity variation of a control broth medium).
To determine the effect of Anise oil (0·01, 0·1 and 1%, v/v)
and low pH and salt against Lact. curvatus and S. cerevisiae by
this method, the MRS broth and OGYE broth used were
modified by adjusting pH (6·2 or 4·2) or adding 5% (w/v)
NaCl. The water activity of the growth media was measured
using CX-1 Water Activity System (Decagon Devices, Inc.,
Pullam, Washington, USA).
Effect of basil essential oil on growth of
Lactobacillus curvatus and Saccharomyces cerevisiae
in tomato juice medium
Canned tomato juice obtained from a local supermarket was
supplemented with 0·5% w/v glucose and autoclaved for
30 min. Anise basil oil was added at 0·1 and 1% (v/v) by
vortexing for 1 min; medium without added Anise oil was
tested as a control. Triplicate portions (20 ml) of the tomato
juice containing Anise oil were inoculated with either Lact.
curvatus at 6·7 × 103 CFU ml−1 or S. cerevisiae at 2·1 × 102
CFU ml−1 and incubated at 15 °C. At weekly intervals, ali-
quots (1 ml) were removed for viable count determination of
Lact. curvatus and S. cerevisiae on pour plates of MRS agar
(incubated for 48 h at 30 °C) and OGYE agar (incubated for
3 d at 25 °C), respectively.
RESULTS
Antimicrobial activity of basil oils by agar well
diffusion
The antimicrobial activity of five basil essential oils (from
different varieties of basil: Anise, Bush, Cinnamon, Dark
Opal and commercial) and authentic linalool and methyl
chavicol was tested against a range of Gram-positive (13) and
Gram-negative (11) bacteria, yeasts (six) and moulds (three)
by an agar well diffusion method (Table 1). The majority (29
 A NT IM I CR OB I AL AC T IV IT Y OF BA S IL OI L 211

Table 1 Effect of basil essential oils on growth of microorganisms on agar plate*

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
Radius† (mm) of zone of inhibition by basil essential oils
—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
AFISC Dark Methyl
No. Indicator culture Anise Bush Cinnamon Opal Commercial Linalool chavicol
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
0110 Aeromonas hydrophila 3 3 3 3 3 3 0
0114 Aer. hydrophila 2 2 2 3 2 6 1
0303 Bacillus cereus 5 4 3 6 3 9 0
0320 B. subtilis 2 2 2 2 2 5 0
0605 Brochothrix thermosphacta 1 1 1 2 1 4 2
1105 Debaryomyces hansenii 1 1 1 1 1 3 0
1201 Enterobacter aerogenes NCTC 10006 1 1 1 1 1 2 0
1204 Ent. agglomerans 1 1 1 1 1 6 0
1301 Escherichia coli NCTC 8196 2 2 2 2 2 5 0
1406 Flavimonas oryzihabitans 0 0 0 0 0 3 0
2102 Lactobacillus plantarum 1 2 1 2 1 4 2
2103 Lact. curvatus 2 2 2 2 2 2 2
2206 Leuconostoc cremoris 2 2 1 2 2 3 0
2305 Listeria innocua 1 1 1 1 1 2 1
2310 L. monocytogenes 1 1 1 1 1 2 1
2612 Mucor piriformis 10 23 15 26 9 11 5
2802 Penicillium candidum 9 15 11 10 13 13 6
2804 P. expansium 3 5 7 4 5 6 5
3020 Proteus vulgaris 2 2 2 3 2 4 0
3101 Pseudomonas aeruginosa 0 0 0 0 0 0 0
3105 Ps. fluorescens 0 0 0 0 0 0 0
3108 Ps. putida 0 0 0 0 0 0 0
3301 Saccharomyces cerevisiae 3 3 3 4 4 4 5
3412 Salmonella typhimurium 4 4 5 4 4 5 2
3501 Serratia marcescens 3 3 3 3 3 6 0
3601 Staphylococcus aureus NCTC 6571 2 2 2 2 2 5 0
3801 Yersinia enterocolitica 2 2 2 2 2 7 0
3901 Enterococcus faecalis 1 1 0 1 1 3 0
4202 Candida colliculosa 5 6 4 5 6 6 2
4206 C. formata 5 11 8 9 5 6 4
4208 C. humicola 4 4 5 4 4 7 4
4704 Rhodotorula sp. 4 3 5 6 6 4 3
5001 Zygosaccharomyces bailii 2 4 2 6 4 10 8
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
*Cultures were inoculated at 106 cfu ml−1 to agar medium and 4 mm wells were filled with 10 ml of essential oil.
†Radius of zone of inhibition around the well (subtracting the radius of well).

out of 33 strains) of the tested organisms were sensitive to all
five basil oils, with Mucor piriformis showing greatest sensi-
tivity. However, Flavimonas oryzihabitans, Pseudomonas aeru-
ginosa, Ps. fluorescens and Ps. putida were resistant to all the
oils tested. The spectrum of antimicrobial activity did not
vary greatly between oils from different varieties of basil
except that Enterococcus faecalis was resistant to Cinnamon
basil oil but sensitive to the other four basil oils. Authentic
pure linalool showed a similar antimicrobial spectrum to those
of basil oils. However, pure methyl chavicol showed a much
© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 209–214
narrower antimicrobial spectrum with activity against only
16 of the 33 strains of organisms tested. The paraffin oil
control did not produce any detectable zone of growth inhi-
bition in any of the indicator culture lawns.
Anise basil oil was chosen for further investigation against
Lact. curvatus and S. cerevisiae, two acid tolerant spoilage
organisms that can grow in the low pH of tomato based food
products. This oil contained 44% (v/v) linalool and 27%
(v/v) methyl chavicol (Lachowicz et al. 1997) and exhibited
an antimicrobial spectrum similar to the other varieties tested.
212 K .J . L A CH OW I CZ ET A L.

Antimicrobial activity of Anise oil against Lact. antimicrobial effect was observed with the combination of
curvatus and S. cerevisiae in combination with 5% of NaCl and 0·1% Anise oil in MRS (pH 6·2), which
low pH and salt completely inhibited the growth of Lact. curvatus for the
duration of the experiment (TDG × 99 h) compared with
The combined effect of Anise oil (0, 0·01, 0·1 and 1%,
the Anise oil (51·4 h) or 5% NaCl (28·3 h) alone. The decrease
v/v) with a low pH (pH 4·2) and/or addition of 5% NaCl
in pH of the MRS broth (pH 4·2, TDG of 28·5 h) in com-
(reflecting a decreased water activity of MRS and OGYE
bination with 0·1% Anise oil also demonstrated a similar
broth from 0·983 to 0·958 and from 0·985 to 0·960, respec-
synergistic effect against Lact. curvatus (TDG × 99 h).
tively) on growth of Lact. curvatus in MRS broth at 30 °C
In contrast, the addition of 5% NaCl to OGYE broth
and S. cerevisiae in OGYE broth at 25 °C was determined by
(pH 6·2) resulted in complete inhibition of S. cerevisiae
the indirect impedance method (Table 2). Without added
(TDG × 99 h) even without the inclusion of Anise oil
essential oil, the time to detection of growth (TDG) of Lact.
(Table 2). No synergistic effect was observed with the com-
curvatus and S. cerevisiae was 12·3 h and 20·3 h, respectively.
binations of low pH (pH 4·2) and Anise oil against this organ-
The addition of 0·01% Anise oil in broth did not have any
ism.
inhibitory effect on either of the organisms tested. The
addition of 0·1% Anise oil increased the TDG for both
organisms to approximately 51 h and at 1% Anise oil, no
Antimicrobial activity of combinations of linalool
substantial growth of either organism was detected during
and methyl chavicol in comparison with Anise oil
the 99 h test period. The addition of 5% NaCl to MRS
(pH 6·2) broth without Anise oil increased the TDG of Lact. The antimicrobial activity of various combinations of pure
curvatus from 12·3 h to approximately 28 h. A synergistic linalool and methyl chavicol (at v/v ratios of 1:0, 0·8:0·2,

Table 2 Effect of Anise basil oil and combinations of linalool and methyl chavicol on growth of Lactobacillus curvatus in MRS broth
at 30 °C and Saccharomyces cerevisiae in OGYE broth at 25 °C
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
TDG* (h)
Essential oil/ Linalool to Concentration (%) of basil oil
component methyl chavicol Media ––––––––––––––––––––––––––––––––
—
Culture combination ratio (v/v) condition 0 0·01 0·1 1·0
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
Lactobacillus curvatus Anise oil 0·44 : 0·27 pH 6·2 12·3 12·8 51·4 ×99
pH 6·2¦5% NaCl 28·3 28·4 ×99 ×99
pH 4·2 28·5 29·5 ×99 ×99
pH 4·2¦5% NaCl 34·3 37 ×99 ×99
Mixtures of 1:0 pH 6·2 nd† nd 13·5 ×99
linalool and methyl 0·8 : 0·2 pH 6·2 nd nd 13·5 ×99
chavicol 0·6 : 0·4 pH 6·2 nd nd 13·6 ×99
0·4 : 0·6 pH 6·2 nd nd 13·6 ×99
0·2 : 0·8 pH 6·2 nd nd 14·3 ×99
0:1 pH 6·2 nd nd 14·5 ×99

Saccharomyces cerevisiae Anise oil 0·44 : 0·27

pH 6·2 20·3 20·1 51 ×99
pH 6·2¦5% NaCl ×99 ×99 ×99 ×99
pH 4·2 20·8 21·1 44·2 ×99
pH 4·2¦5% NaCl ×99 ×99 ×99 ×99
Mixtures of 1:0 pH 6·2 nd nd 24·1 ×99
linalool and methyl 0·8 : 0·2 pH 6·2 nd nd 24·8 ×99
chavicol 0·6 : 0·4 pH 6·2 nd nd 25·5 ×99
0·4 : 0·6 pH 6·2 nd nd 25·5 ×99
0·2 : 0·8 pH 6·2 nd nd 30·6 ×99
0:1 pH 6·2 nd nd 27·5 ×99
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
*Time to detection of growth.
†Not determined.

© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 209–214
 A NT IM I CR OB I AL AC T IV IT Y OF BA S IL OI L 213

0·6:0·4, 0·4:0·6, 0·2:0·8 and 0:1) was also determined in broth
systems against Lact. curvatus (in MRS broth at 30 °C) and S.
cerevisiae (in OGYE broth at 25 °C) by an indirect impedance
method (Table 2). Substitution of Anise oil with various mix-
tures of the two major components (linalool and methyl chavi-
col) at a combined concentration of 1% (v/v) also resulted in
complete inhibition of both Lact. curvatus and S. cerevisiae
(TDG × 99 h), independent of the ratio of linalool and
methyl chavicol (1:0–0:1). At a combined concentration of
0·1% (v/v), however, decreased antimicrobial activity (com-
pared with Anise oil) was observed with all combination ratios
with a TDG range from 13·5 to 14·5 h for Lact. curvatus and
24·1–30·6 h for S. cerevisiae. Only a slight increase in TDG
(24·1–30·6 h) for S. cerevisiae was observed with the increase
of methyl chavicol proportion from 0 to 80%.
Effect of Anise oil on growth of Lact. curvatus and
S. cerevisiae in tomato juice medium
The antimicrobial activity of Anise oil was also verified in
tomato juice medium (pH 4·1) challenged with Lact. curvatus
(104 cfu ml−1) and S. cerevisiae (102 cfu ml−1) and incubated
at 15 °C (Fig. 1). In tomato juice medium without added
Anise oil, Lact. curvatus grew to approximately 108 cfu ml−1
10
a to all of the basil essential oils tested. In contrast, Flavimonas
Viable count (log cfu ml–1)
within 1 week. The addition of 0·1% (v/v) Anise oil decreased
the viable count of Lact. curvatus from 104 cfu ml−1 to below
the detection limit (1 cfu ml−1) at week 1 and maintained
the complete inhibition of growth for the duration of the
experiment (4 weeks) (Fig. 1a). The addition of 1% (v/v)
Anise oil produced immediate reduction in the viability of
Lact. curvatus in tomato juice medium. The addition of 1%
(v/v) Anise oil to medium also completely inhibited the
growth of S. cerevisiae (Fig. 1b). However, at 0·1% (v/v) of
Anise oil, the growth of this organism was only inhibited for
1 week while the viable count increased to 106 and 107 cfu
ml−1 at weeks 2 and 3, respectively.
DISCUSSION
Essential oils obtained from Ocimum basilicum L. and other
Ocimum species were generally more effective against Gram-
positive (Bacillus sp., Staphylococcus sp., Micrococcus sp., Sar-
cina sp., Lactobacillus sp.) than Gram-negative bacteria
(Enterobacter sp., Pseudomonas sp., Escherichia coli, Salmonella
sp.) (Prasad et al. 1986; Farag et al. 1989). Comparisons such
as this depend on the methodology used to determine the
antimicrobial activity and the diversity of micro-organisms
tested. In the current study, the spectrum of activity obtained
by an agar well diffusion method showed that Aeromonas
hydophila, Bacillus cereus, Salmonella typhimurium, Serratia
marcescens and particularly yeasts and moulds were sensitive

8 oryzihabitans and Pseudomonas species showed no sensitivity

to any of the oils tested.
6 Sinha and Gulati (1990) and Simon et al. (1990) reported
that the antimicrobial activity of essential oils from Ocimum
4 species was predominantly associated with the main con-
stituents linalool and methyl chavicol. In the current study,
2
these two components were also the predominate constituents
0 in the basil essential oils examined, at considerably different
0 1 2 3 4 concentrations, however (Lachowicz et al. 1996, 1997). The
Time (week) linalool content varied form 17% in commercial oil to 57%
in Opal oil and the methyl chavicol content varied from 0%
10
b in Opal oil and Bush oil to 41% in Anise oil. Authentic
Viable count (log cfu ml–1)

8 linalool and methyl chavicol were combined in a range of

ratios for assessment of antimicrobial activity against Lact.
6 curvatus and S. cerevisiae in broth systems in comparison with
Anise oil. The lesser antimicrobial activity of the linalool-
4 methyl chavicol combinations compared with Anise oil indi-
cates that other less predominant component(s) in Anise oil
2
most likely contribute to the antimicrobial activity.
0 Synergistic effects were reported with essential oils from
0 1 2 3 4 Ocimum basilicum and O. sanctum where a 1:1 combination of
Time (week) the oils was more effective than each oil alone against various
Fig. 1 Effect of Anise basil oil at 0 (), 0·1 (r) and 1·0% () Gram-positive and Gram-negative bacteria and some fungi
on growth of (a) Saccharomyces cerevisiae at 25 °C and (b) (Sinha and Gulati 1990). In addition, synergistic effects of
Lactobacillus curvatus at 30 °C in tomato juice medium other plant essential oils and low pH (Juven et al. 1994;
© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 209–214
214 K .J . L A CH OW I CZ ET A L.
Tassou and Nychas 1994; Tassou et al. 1995) and sodium
chloride (Kurita and Koike 1982) were observed. In the cur-
rent study, Anise oil combined with low pH and 5% sodium
chloride showed synergistic effects against Lact. curvatus
based on the comparison of TDG values obtained for the
combinations of 0·1% (v/v) Anise oil and low pH or NaCl
with those for these three preservation parameters acting
alone. However, no synergistic effect was observed against S.
cerevisiae and these results, together with those published by
previous researchers, highlight the need to examine anti-
microbial effects against particular microflora of interest.
Little is known of the mechanisms involved in creating
synergistic antimicrobial effects with plant essential oils
against specific microflora.
Basil essential oil is often used as a flavourant in tomato-
based products that have a low pH. The antimicrobial effect
of basil essential oil at 0·1% and 1% against Lact. curvatus
and S. cerevisiae in tomato juice media was consistent with
the results obtained by determining growth by impedance
measurements in broth. However, further application and
challenge assessment studies are required to more extensively
quantify the preservative effects of the various basil essential
oils and their constituents in low acid foods.
ACKNOWLEDGEMENT
This project was financially supported by the Agriculture
and Food Initiative, Department of Natural Resources and
Environment, Victoria, Australia and a Deakin University
postgraduate scholarship awarded to K.J. Lachowicz.
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