Efficacy of polyethylene-based antimicrobial films containing principal constituents of basil

Abstract

The feasibility of low-density polyethylene (LDPE)-based films containing linalool or methylchavicol as antimicrobial (AM) packages to retard microbial growth on food surfaces was investigated. The AM LDPE-based films were tested for inhibition against selected microorganisms. Both compounds retained their AM activity, after an extrusion film-blowing process, against Escherichia coli in solid medium. Cheddar cheese was wrapped with the AM films and the packaged cheese samples were stored at 4 °C. The changes in the mesophilic aerobic bacteria and coliform, as well as yeast and mould counts were monitored. In addition, cheese samples inoculated with E. coli or Listeria innocua were wrapped with the AM films, stored at refrigerated (4 °C) or at abuse (12 °C) temperatures and the count of these microorganisms was monitored as a function of time. The results showed an inhibitory effect of these AM films against microbial growth in naturally contaminated cheese and in inoculated samples. The effect on suppression of E. coli and L. innocua growth was more pronounced at the abuse temperature. Methylchavicol-LDPE-based film exhibited a higher efficacy of inhibition than that of linalool-LDPE-based film. In addition, a sensory evaluation was performed with regards to possible taint in the flavour of the cheese. Taint in flavour as affected by linalool or methylchavicol was not significantly detectable by the panelists at the end of the storage period of 6 weeks. This study shows the potential use of polymeric films containing the principal constituents of basil as the AM components for enhancing quality and safety of cheeses.

Full text

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LWT 41 (2008) 779–788
Efficacy of polyethylene-based antimicrobial films containing
principal constituents of basil
Panuwat Suppakula, Kees Sonneveldb, Stephen W. Biggerb, Joseph Miltzc,?
aDepartment of Packaging Technology, Faculty of Agro-Industry, Kasetsart University, 50 Phaholoyothin Road, Chatuchak, Bangkok 10900, Thailand
bPackaging and Polymer Research Unit, School of Molecular Sciences, Victoria University, P.O. Box 14428, Melbourne 8001, Australia
cDepartment of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel
Received 12 March 2007; received in revised form 2 June 2007; accepted 14 June 2007
Abstract
The feasibility of low-density polyethylene (LDPE)-based films containing linalool or methylchavicol as antimicrobial (AM) packages
to retard microbial growth on food surfaces was investigated. The AM LDPE-based films were tested for inhibition against selected
microorganisms. Both compounds retained their AM activity, after an extrusion film-blowing process, against Escherichia coli in solid
medium. Cheddar cheese was wrapped with the AM films and the packaged cheese samples were stored at 41C. The changes in the
mesophilic aerobic bacteria and coliform, as well as yeast and mould counts were monitored. In addition, cheese samples inoculated with
E. coli or Listeria innocua were wrapped with the AM films, stored at refrigerated (41C) or at abuse (121C) temperatures and the count of
these microorganisms was monitored as a function of time. The results showed an inhibitory effect of these AM films against microbial
growth in naturally contaminated cheese and in inoculated samples. The effect on suppression of E. coli and L. innocua growth was more
pronounced at the abuse temperature. Methylchavicol-LDPE-based film exhibited a higher efficacy of inhibition than that of linalool-
LDPE-based film. In addition, a sensory evaluation was performed with regards to possible taint in the flavour of the cheese. Taint in
flavour as affected by linalool or methylchavicol was not significantly detectable by the panelists at the end of the storage period of
6 weeks. This study shows the potential use of polymeric films containing the principal constituents of basil as the AM components for
enhancing quality and safety of cheeses.
r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial packaging; Antimicrobial film; Basil; Linalool; Methylchavicol; Cheddar cheese
1. Introduction
Antimicrobial (AM) packaging, an innovative concept,
can be defined as a version of active packaging (AP) in
which the package, the product and the environment
interact to extend the lag phase and/or reduce the growth
rate of microorganisms. By this action, the shelf life of the
product is prolonged and its quality and safety are better
preserved (Suppakul, Miltz, Sonneveld, & Bigger, 2003a).
Floros, Dock, and Han (1997) reviewed the products and
patents in the area of AP and identified AM packaging as
one of the most promising versions of AP. Appendini and
Hotchkiss (2002) and Suppakul et al. (2003a) reviewed
recently the principles of AP and AM systems. Due to the
perceived lower risk to the consumer, the use of naturally
derived AM additives (e.g. bacteriocins, enzymes, peptides,
plant extracts and plant-volatile components) is of an
increasing interest (Nicholson, 1998). Examples of AM
materials containing plant extracts include clove extract-
impregnated low-density polyethylene (LDPE), grapefruit
seed extract-incorporated LDPE, huanglian extract or
rhubarb extract-embedded LDPE. However, there is no
published information in the scientific/technical literature
regarding packages containing basil extracts. As no single
AM additive can cover all the needs for food preservation,
it is important to investigate different kinds of potential
compounds in order for this technology to become
successful.
ARTICLE IN PRESS
www.elsevier.com/locate/lwt
0023-6438/$34.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2007.06.006
?Corresponding author. Tel.: +97248292451; fax: +97248293603
(direct) or +97248293399 (Dept.).
E-mail address: jmiltz@tx.technion.ac.il (J. Miltz).

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Basil (Ocimum basilicum L.) is one of the oldest identified
spices and its essential oils have been used extensively for
many years in food flavouring and perfumery. Numerous
investigations on basil essential oils have been reported
including taxonomy, chemistry and AM activity. Recently,
Suppakul, Miltz, Sonneveld, and Bigger (2003b) reviewed
the topic of basil essential oils with regards to their
chemical composition, their effect on microorganisms, and
their possible future use in food preservation or as an AM
additive in packaging materials. The principal constituents
of basil namely, linalool and methylchavicol, exhibit AM
properties against a wide spectrum of microorganisms
(Suppakul et al., 2003b). These compounds possess GRAS
status (Suppakul et al., 2003a). They are stable at relatively
high temperatures and may therefore have the potential to
be incorporated into polymers and used in AM packaging
applications.
Cheddar is a hard natural cheese. It varies in colour from
pale to deep yellow, and the flavour ranges from mild and
creamy (mild Cheddar) to strong and biting (mature
Cheddar) (Robinson, 1995). Cheddar cheese is produced
essentially by a microbial fermentation process of cow-milk
by selected lactic acid bacteria. Mesophilic cultures, with
an optimum temperature of ?301C, are used in the
production of Cheddar. The safety aspect is the driving
force for making cheese from pasteurized milk, as the most
common pretreatment. Some pathogens like Salmonella,
Escherichia coli O157:H7 and Listeria monocytogenes and
spoilage microorganisms are of high concern in the cheese
industry. Previous work indicated that L. monocytogenes
survives for more than a year in Cheddar cheese (Ryser &
Marth, 1987). Moreover, in all cheeses, E. coli is considered
to be the organism of primary concern (Hasell & Salter,
2003).
The current study was aimed at manufacturing, by
extrusion film-blowing, AM LDPE-based films containing
each of the two main constituents of basil, at determining
the AM activity of these films against selected microorgan-
isms and at assessing their feasibility to be used in cheese
packaging applications.
2. Materials and methods
2.1. Polymers
The polymers used in this study were: linear low-density
polyethylene (LLDPE) (Dowlex 2045 E, Dow Chemical,
Australia), LDPE (Alkathene XJF 143, Qenos Pty. Ltd.,
Australia), and an ethylene-based copolymer.
2.2. AM additives
The AM additives were linalool (L260-2, Aldrich
Chemical Company, Inc., USA) and methylchavicol
(AUSTL 21320, Aurora Pty. Ltd., Australia) with a purity
of 97% and 98%, respectively.
2.3. Chemicals
The chemicals: ethanol supplied by CSR Ltd., Australia;
iso-octane (Unichrom 2516-2.5L GL), purchased from
APS Chemicals, Australia; sodium dihydrogen orthopho-
sphate (NaH2PO4?2H2O) (30132), di-sodium hydrogen
orthophosphate (Na2HPO4) (30158.5000) and sodium
chloride (NaCl) (10241.AP) were purchased from BDH
Chemical Pty. Ltd., Australia.
2.4. Media
The media were nutrient broth (CM 1), nutrient agar
(CM 3), and potato dextrose agar (CM 139) purchased
from Oxoid, USA. Bacteriological agar (RM 250), plate
count agar (AM 144), tryptone soya broth (AM 185) and
YM broth (RM 229) were obtained from Amyl, Australia.
2.5. Count plates
The count plates were 3M PetrifilmTMaerobic count
plates, 3M PetrifilmTME. coli and coliform count plates
and 3M PetrifilmTMyeast and mould count plates. The
count plates were obtained from 3M Microbiology
Products, St. Paul, Minnesota.
2.6. Microorganisms
The microorganisms were Staphylococcus aureus (FSA
3601), Listeria innocua (FSA 2305), Escherichia coli (FSA
1301) and Saccharomyces cerevisiae (FSA 3301). All of the
test strains were obtained from the culture collection of
Food Science Australia, Werribee, Vic., Australia.
2.8. Preparation of AM LDPE-based films
LDPE-based films of 45–50mm in thickness, with and
without linalool or methylchavicol, were prepared from
LDPE pellets. A pre-blended master batch of an ethylene-
based copolymer (ethylene vinyl acetate—EVA) powder
containing linalool or methylchavicol was mixed with
virgin LDPE pellets and manufactured into films using the
same extruder. The purpose of using this copolymer was to
enhance the solubility and partial anchoring of the AM
additives in the polymer matrix. Films without linalool or
methylchavicol were used as controls and were prepared
under similar conditions as the films containing the active
agents.
2.9. Film thickness measurement
A hand-held micrometer (Hahn & Kolb, Stuttgart,
Germany) was used for measuring the film thickness. Five
readings were taken for each sample, one at the sample
centre and four measurements were taken around the
perimeter.
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2.10. Additive quantification
The amount of linalool or methylchavicol in the samples
was determined by gas chromatography (GC). The proce-
dure was as follows: a 5g of film sample was extracted for
18h by Soxhlet extraction using 150mL of iso-octane. An
aliquot (100mL) of the extract was then taken for GC
analysis. A Varian Star 3400-CX GC equipped with a fused
silica capillary column DB-5 (30m?0.25mm i.d., film
thickness 0.25mm, J & W Scientific, USA) was used. The
following conditions were applied: sample volume, 1.0mL,
initial column temperature, 801C, heating rate of 51Cmin–1
to 1801C, maintaining the column at this temperature for an
additional 5min; injector temperature, 2501C, split ratio,
1:100; FID detector temperature, 3001C; carrier gas,
nitrogen. The linalool and methylchavicol contents of
the samples were calculated from pre-prepared standard
calibration curves.
2.11. AM activity by agar plate test
The films were tested for their inhibition against
the target microorganisms: L. innocua, S. aureus (Gram-
positive bacteria), E. coli (Gram-negative bacterium)
and S. cerevisiae (yeast) by using an agar disc diffusion
method.
All test cultures used in the microbiological assay were
‘‘twice-passaged’’ 15h cultures grown in tryptone soya
broth, nutrient broth or YM broth. Cell densities of
106cfumL?1were calculated and prepared from cultures of
approximately 7.50?108cfumL?1for L. innocua and
E. coli, 4.85?108cfumL?1for S. aureus, and 4.75?
108cfumL?1for S. cerevisiae. Cell densities were also
confirmed by the ‘‘pour plate’’ method on plate count agar
for bacteria and on potato dextrose agar for yeast. Each
film sample was cut into a circle of 4mm in diameter and
sterilized with UV light for 2min (Cooksey, 2000), prior to
being placed on an agar plate surface seeded with 1mL of
test culture. The plates were incubated for 1–2 days at the
appropriate temperature for each culture. The clear zone
formed around the film disc in the medium was recorded as
an indication of inhibition of the microbial species. The
evaluation of the inhibitory activity was carried out in
quadruplicate by measuring inhibition zones with a Vernier
caliper (Mitutoyo, Japan). An average of four measure-
ments, taken 451 apart from each other, was used as the
result of each test.
2.12. Application of AM films on Cheddar cheese
2.12.1. Inhibition of microbial growth on cheese
2.12.1.1. Cheese preparation and storage.
was purchased from a local retail outlet. For the AM
packaging experiments, cubes (3cm?4cm?2cm) of the
cheese weighing ca. 20g each were used. During the storage
test, the cheese samples were divided randomly into three
lots for different packaging treatments (control film,
Cheddar cheese
linalool-LDPE-based
based film) and packaged by wrapping tightly the film
(8cm?12cm), which was previously sterilized with UV
light for 2min (Cooksey, 2000), around the cheese. The
packaged cheese samples were stored at 41C for 21 days
and periodically analysed for their microbiological quality
during storage.
filmandmethylchavicol-LDPE-
2.12.1.2. Microbiological analysis.
cheese were examined for levels of mesophilic aerobic
bacteria, coliform bacteria and yeasts and moulds. Three
packages from each treatment were aseptically opened just
before sampling. A cheese cube was aseptically transferred
to a sterile stomacher bag and 180mL of 0.1M sterile
sodium phosphate buffer solution (pH 7.0) was added. The
sample was then homogenized for 1min in a laboratory
blender (Seward Stomachers400, Seward Medical, UK).
A series of decimal dilutions were carried out according
to recommended microbiological protocols (Spencer &
Ragout de Spencer, 2001). In order to determine the
mesophilic aerobic and coliform bacterial counts and yeast
and mould counts, 1mL aliquots of serially diluted samples
were plated in duplicate on 3M PetrifilmTMaerobic total
count plates, 3M PetrifilmTME. coli and coliform count
plates, and 3M PetrifilmTMyeast and mould count plates,
respectively. Aerobic total count plates and E. coli and
coliform count plates were incubated aerobically for 24h at
371C, whereas yeast and mould count plates were
incubated for 5 days at 301C. The colonies were counted
and the results were expressed as cfug?1.
Cubes of Cheddar
2.12.2. Inhibition of bacterial growth on cheese
2.12.2.1. Inoculation of bacteria on cheese and storage at
refrigerated temperature.
Commercial, sliced Cheddar
cheese was purchased from a local supermarket. For the
packaging experiments, the slices (9cm?9cm?0.2cm)
weighing ca. 20g each were treated by UV light for 1h on
each side in a laminar flow cabinet (Taniwaki, Hocking,
Pitt, & Fleet, 2001). During the storage test, the cheese was
randomly divided into two sets for different bacterial
inoculations (E. coli and L. innocua). Each set of the cheese
samples was further divided randomly into three lots for
different packaging samples (LDPE as control film,
linalool-LDPE-basedand
films). The slices of Cheddar cheese were then surface
inoculated with E. coli or L. innocua at a level of 104cfug?1
and were packaged by wrapping the film (12cm?24cm),
that was previously sterilized with UV light for 2min
(Cooksey, 2000), tightly around the cheese and heat-sealing
the three open sides. The packaged cheese samples were
stored at 41C for 35 days and periodically collected for
analysis of bacteriological quality during storage.
methylchavicol-LDPE-based
2.12.2.2. Inoculation of bacteria on cheese and storage
whilst subjected to temperature abuse.
experiments was performed in a similar way as described
above. The packaged cheese samples were stored at 41C for
The second set of
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1 day and then subjected to a temperature abuse at 121C
(to represent the worst possible conditions during distribu-
tion and storage) for 15 days, performing periodically
bacteriological analyses.
2.12.2.3. Bacterial enumeration.
were tested, at appropriate intervals, for numbers of E. coli
and L. innocua. Two packages from each treatment were
opened aseptically just before sampling and the cheese
samples were aseptically transferred to a sterile stomacher
bag. This enumeration procedure was the same as the
microbiological analysis described in Section 2.12.1.
Slices of Cheddar cheese
2.12.3. Sensory analysis
2.12.3.1. Cheese wrapping and storage.
was purchased from a local retail outlet. For the packaging
experiments, cubes (3cm?2cm?2cm) weighing ca. 10g
each were cut and then surface treated by exposure to UV
light for 1h in a laminar flow cabinet (Taniwaki et al.,
2001). During the storage test, the cheese was randomly
divided into three lots for different packaging material
samples (LDPE as control film, linalool-LDPE-based and
methylchavicol-LDPE-based films) and packaged by wrap-
ping tightly the film (6cm?8cm), that was previously
sterilized with UV light for 2min (Cooksey, 2000), around
the cheese. The packaged cheese samples were stored at
41C for 42 days and subjected to periodic sensory
evaluation.
Cheddar cheese
2.12.3.2. Triangle test.
engaged for evaluating the presence of the constituents of
basil (or basil tainted flavour) in the Cheddar cheese
wrapped in the AM LDPE-based films. The standard
triangle test method was used. In this study, each panelist
was presented with samples that had been subjected to two
sets of treatments: (i) six cube samples of Cheddar cheese,
in two sets: one set comprising of two cubes wrapped in the
control film, and one cube wrapped in the linalool-LDPE-
based film. In the second set: one cube wrapped in the
control film and two cubes wrapped in the linalool-LDPE-
based film, and (ii) six cube samples of Cheddar cheese in
two sets: one set comprising of two cubes wrapped in the
A panel of 10 members was
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Table 1
AM activity of LDPE-based films against E. coli as observed by the agar
disc diffusion assay
TreatmentLevel of
addition (g/
100g)
Level of
retention (g/
100g)
Zone of
inhibition
(mm)
LDPE
Linalool-LDPE-
copolymer
Methylchavicol-
LDPE-copolymer
–
1.00
–
0.338
–a
11.5270.19b
1.000.34510.0970.18
a(–) No inhibition.
bValues for zone of inhibition are represented as mean7SEM.
Fig. 1. Growth inhibition of E. coli by LDPE-based film containing
linalool. A similar result was obtained using methylchavicol as the AM
agent.
Fig. 2. Growth inhibition of E. coli by LDPE-based films containing
linalool: (a) film cutting at 0/901 of extrusion direction and (b) film cutting
at 45/451 of extrusion direction. Similar results were obtained using
methylchavicol as the AM agent.
P. Suppakul et al. / LWT 41 (2008) 779–788
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control film and one cube wrapped with methylchavicol-
LDPE-based film, and in the other set: one cube wrapped
in the control film and two cubes wrapped in the
methylchavicol-LDPE-based film. Panelists were asked to
choose the odd sample in each set of the three and to
indicate whether linalool or methylchavicol (or an off
flavour) could be detected. Cheddar cheese samples and
controls were tested in this manner after 1, 2, 3, 4 and 6
weeks of storage for detection of a possible tainted flavour.
Probability tables were employed to determine levels of
significant difference between samples and controls as
described by Roessler, Pangborn, Sidel, and Stone (1978).
2.13. Data analysis
Data points were represented by the mean. Data sets
were subjected to analysis of variance (ANOVA) and the
Tukey test at the 0.05 level of significance using KyPlot 2.0
for Windows (Kyence Inc., Japan).
3. Results and discussion
During the preparation of the LDPE-based films, the
additive could be properly incorporated in the polymer
melt, leading to a film with a uniformly dispersed AM
agent. This result was observed and confirmed by scanning
electron microscopy (Suppakul, Miltz, Sonneveld, &
Bigger, 2006). This finding is consistent with the study by
Hong, Park, and Kim (2000) on AM films in which clove
extract had been incorporated.
The transparency of the AM LDPE-based films in the
present study decreased slightly compared to the control
LDPE film. Methylchavicol had a larger effect on the
transparency than linalool. These findings are in agreement
with the results of Han and Floros (1997) studying the
optical properties of K-sorbate incorporated-LDPE and
LDPE films. The transparency of the AM films in the
present study was in the acceptable range for transparent
films and no difficulties in commercialization of these films
is envisioned. The temperature profile of 90–951C pre-
viously used by Han and Floros (1997) for the production
of their films could not be used in our study since our
polymers had higher melting temperatures (the vast
majority of commercial polyethylene grades have a melting
temperature above 1001C and therefore it is not clear how
the above mentioned temperature range could be applied).
The temperature profile of 160–1901C, previously used by
Ha, Kim, and Lee (2001), was used for manufacturing of
the AM LDPE-based films by extrusion film blowing and
resulted in a high loss of the AM agent. Lower
manufacturing temperatures are preferable in order to
minimize loss of the active agent by evaporation. The
limitations of the single screw extruder available for our
experiments further affected the expected results. In
preliminary experiments, linear low density polyethylene
(LLDPE) was used. However, because of the higher
melting temperature of this polymer compared to LDPE,
higher processing temperatures had to be applied resulting
in a much greater loss of the AM agents during processing.
Thus, the residual AM concentration in the polymer was
about 0.05g/100g only (Suppakul, Miltz, Sonneveld, &
Bigger, 2002). At a later stage, a blend of LDPE and an
ethylene-based copolymer was used. This combination
increased the retention of the residual active agent in the
extruded films to 0.34g/100g (initial concentration was
1.0g/100g in the blend) (Table 1). The increased AM agent
concentration in this polymer mixture is attributed to the
lower processing temperature and to the interaction
between the AM agents and the copolymer enabling the
‘‘anchoring/solubilizing’’ of the AM molecules within the
polymeric matrix.
3.1. AM activity by agar plate test
All prepared LDPE-based films containing linalool or
methylchavicol exhibited a significantly positive AM
activity against E. coli in the agar disc diffusion test
(Table 1 and Fig. 1). Colonies of E. coli could not be
viewed in the clear zone directly above the film samples
containing the constituents of basil, whereas such colonies
were formed all over the control plates. The microbial
inhibition indicates that a portion of either linalool or
methylchavicol was released from the extruded film sample
and diffused into the agar layer, retarding the development
of microbial cells in the agar. Although linalool and
methylchavicol are almost insoluble in pure water, they are
slightly soluble in the water held by the agar due to the
presence of some hydrophobic substances. Fig. 1 suggests
an eccentric characteristic in the AM activity of the film in
the extrusion and cross directions consistent with an
anisotropic material. Film cutting at 0/901 to the extrusion
direction (Fig. 2a) showed a zone of inhibition in
agreement with the study of Cutter, Willett, and Siragusa
(2001). However, these authors provided no explanation
for this eccentric characteristic. A statistically significant
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05 1520
0
1
2
3
4
5
6
Microbial count/log (cfu g-1)
25 10
Time/days
Fig. 3. Inhibition of mesophilic aerobic bacteria on Cheddar cheese using
LDPE-based AM films refrigerated at 41C, LDPE (~), linalool-LDPE-
copolymer (K), methylchavicol-LDPE-copolymer (’).
P. Suppakul et al. / LWT 41 (2008) 779–788
783

Page 6

difference (pp0.05) was found between the films with
different active agents. According to Elgayyar, Draughon,
Golden, and Mount (2001), the present results show that
the LDPE-based films possess ‘‘moderately inhibitory’’
characteristics against E. coli. Linalool showed a higher
level of inhibition than methylchavicol in the AM films
prepared by extrusion or coating, in spite of the fact that
methylchavicol possesses a greater extent of AM activity
than linalool. The reason may stem from the faster
diffusion of linalool and its greater solubility in water
(and subsequently a more pronounced presence in the
aqueous-based agar media), compared to methylchavicol
(Suppakul et al., 2003b). Linalool and methylchavicol are
known to possess a broad spectrum of AM activity against
a variety of microorganism such as Aeromonas hydrophila,
Bacillus cereus, E. coli, L. monocytogenes, S. aureus,
S. cerevisiae, Aspergillus sp., and Penicillium sp. (Suppakul
et al., 2003b).
No inhibitory action of linalool or methylchavicol
against L. innocua, S. aureus and S. cerevisiae of the two
AM agents in the LLDPE was found (Suppakul et al.,
2002). This lack of action stems probably from the fact that
the concentration of the agents in the films was below the
values required for demonstrating inhibitory effects against
these organisms. Additionally, a lower concentration of
basil extract in a film matrix will result in a decrease in
either linalool or methylchavicol in the aqueous phase of
the agar layer where microbial proliferation takes place.
In this study, LLDPE was replaced by a blend of LDPE
and the EVA copolymer resulting in a higher level of
residual AM agent in the film. However, still no inhibition
of linalool or methylchavicol against L. innocua, S. aureus
and S. cerevisiae was observed. This might be due to a
limitation of the agar disc diffusion method when it is used
with a lipophilic compound. To overcome this problem,
Sanla-Ead, Jangchud, Chonhenchob, and Suppakul (2006)
have recently proposed an alternative ‘‘Headspace diffu-
sion method’’, for antimicrobial activity testing of lipophi-
lic compounds. The LDPE-based films containing the
relatively low concentration (0.34g/100g) of linalool or
methylchavicol inhibited the activity of E. coli, a Gram-
negativebacterium.Other
Ag-zirconium (An, Hwang, Cho, & Lee, 1998), clove
extract (Hong et al., 2000), lacticin and nisin (An, Kim,
AM additives, including
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0520 253040
3.60
3.80
4.00
4.20
4.40
Bacterial count/log (cfu g-1)
35 1510
Time/days
2.50
3.00
3.50
4.00
4.50
Bacterial count/log (cfu g-1)
0520 25 3040351510
Time/days
Fig. 4. Bacterial inhibition on Cheddar cheese using LDPE-based AM
films refrigerated at 41C, (a) E. coli, (b) L. innocua, LDPE (~), linalool-
LDPE-copolymer (K), methylchavicol-LDPE-copolymer (’).
02468
2.50
3.00
3.50
4.00
4.50
Bacterial count/log (cfu g-1)
Time/days
16141210
3.25
3.50
3.75
4.00
4.25
4.50
4.75
02468
Time/days
161412 10
Bacterial count/log (cfu g-1)
Fig. 5. Bacterial inhibition on Cheddar cheese, using LDPE-based AM
films where the samples were subjected to temperature abuse conditions.
Conditions: 1 day at 41C and the remainder of time at 121C, (a) E. coli,
(b) L. innocua, LDPE (~), linalool-LDPE-copolymer (K), methylchavi-
col-LDPE-copolymer (’).
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Lee, Paik, & Lee, 2000) failed to retard the growth of E.
coli, even at much higher concentrations. The reason might
be that Gram-negative bacteria are generally more resistant
to the growth inhibition and killing effects of various
antibiotics and AM agents (Salton, 1994) due to the strong
hydrophilicity of their surface that acts as a strong
permeability barrier (Nikaido & Vaara, 1985). The surface
also possesses divalent cations that stabilize the lipopoly-
saccharide association within the membrane, and may
prevent active compounds from reaching the cytoplasmic
membrane (Russel, 1991).
In contrast to the results of the present study, LDPE film
containing chitosan polymer or oligomer investigated by
Hong et al. (2000) and a cast corn zein film containing
lysozyme studied by Padgett, Han, and Dawson (1998) as
well as a LDPE film coated with nisin tested by An et al.
(2000) exhibited no reduction of E. coli cells. For chitosan,
lysozyme and nisin, penetration through the outer mem-
brane can be accomplished by the use of a chelating agent
such as EDTA or by an osmotic shock. Removal of the
stabilizing effect of the cations by chelating agents could
increase cell permeability to AM additives (Hancock, 1984;
Padgett et al., 1998). Due to the low water solubility of
lipophilic molecules, emulsifiers such as Tween 20 (poly-
oxyehtylene-2-sorbitan monolaurate), Tween 80 (polysor-
bate 80) and Triton X-100, or solvents like ethanol, are
often used to enhance the solubility of hydrophobic
compounds in both solid and liquid media. These lipophilic
molecules may become soluble within the micelles formed
by non-ionic surfactants such as Tween 20 and 80, and
thereby be partitioned out from the aqueous phase of the
suspension (Schmolka, 1973). Kazmi and Mitchell (1978)
claimed that AM agents solubilized within micelles do not
contribute to the AM activity, as they do not come in direct
contact with the target microorganisms. Unlike chitosan,
lysozyme and nisin, a chelating agent (e.g. EDTA) is not
required for linalool and methylchavicol in order to
enhance outer membrane penetration. This is an advantage
of the latter compounds. It was also found in the present
study that non-ionic surfactants such as Tween 20 or 80 are
not required in order to improve the distribution of linalool
or methylchavicol within an aqueous media even though
they have limited solubility.
3.2. Application of AM films on Cheddar cheese
3.2.1. Microbial growth
Changes in the mesophilic aerobic bacterial counts, in
Cheddar cheese samples wrapped with the additive-free
LDPE and the AM films stored under refrigeration, are
presented in Fig. 3. The mesophilic aerobic plate counts
gradually increased from 4.18 to 4.78log(cfug?1) at the
end of the storage period (21 days) in the control samples.
Samples wrapped with linalool-LDPE-based and methyl-
chavicol-LDPE-based films showed a dramatic reduction
in bacterial populations by 2.14 and 1.90 log units,
respectively, during the first 2 days. Then, bacterial growth
in these samples increased progressively up to 4.35 and
4.29log(cfug?1). A similar pattern of bacterial growth was
observed during storage at 41C for 10 days of ground beef
samples irradiated with 1kGy of gamma-ray and coated
with protein-based film, as reported by Ouattara et al.
(2002).
In the present study, Cheddar cheese packaged in LDPE-
based film with 0.34g/100g linalool or methylchavicol
showed significantly lower growth of total aerobic bacteria
for the period of 15 and 9 days, respectively, compared to
the control (Fig. 3). There were insufficient data to evaluate
the shelf life of Cheddar cheese wrapped with the AM films
compared to the control on the basis of 107cfug?1as a
criterion for quality limit at an acceptable microbial level.
There were no significant differences between LDPE-
based AM films and additive-free LDPE film in coliform
bacteria and yeast and mould counts throughout the
studied storage period (21 days); the counts were less than
0.5cfug?1. There was also no visual colony of moulds on
cheese surfaces at the end of the storage. Mould growth
was visible a week later after the end of the storage period
(i.e. at 28 days) in the sample wrapped with the unmodified
LDPE film. The delay in mould growth on cheese wrapped
with this additive-free LDPE film suggested that a certain
time period was required for mould to access oxygen and to
develop mycelium reaching out beyond the surface area
covered by the LDPE film. No visual colony of moulds was
observed up to 2 months when the AM LDPE-based film
(containing 0.34g/100g linalool or methylchavicol) was
used. Weng and Hotchkiss (1992) reported that no growth
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Table 2
Triangle test results of Cheddar cheese wrapped with linalool-LDPE-
based film refrigerated at 41C
Storage time (weeks) Correct responseSignificance level
1
2
3
4
6
2
3
3
3
5
NSa
NS
NS
NS
NS
The test was carried out with 10 panelists.
aNS: non-significant difference.
Table 3
Triangle test results of Cheddar cheese wrapped with methylchavicol-
LDPE-based film refrigerated at 41C
Storage time (weeks) Correct responseSignificance level
1
2
3
4
6
8
4
3
8
5
99%
NSa
NS
99%
NS
The test was carried out with 10 panelists.
aNS: non-significant difference.
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of Aspergillus toxicarius and Penicillium sp. was observed
visually up to 10 days, when LDPE film containing
1000mgkg?1imazalil, an antimycotic agent, was used to
wrap Cheddar cheese.
The results of the present study show that LDPE-based
films containing linalool or methylchavicol have an
inhibitory effect for microbial growth in Cheddar cheese
containing a natural level of microbial contamination. The
complexity of the microbial flora in Cheddar cheese makes
it difficult to explain well the reduced microbial growth
obtained by the linalool-LDPE-based and methylchavicol-
LDPE-based films. Ha et al. (2001) found that grapefruit
seed extract-coated film showed an AM activity against
B. cereus, Bacillus subtilis, E. coli, Leuconostoc mesenter-
oides, Micrococcus flavus, S. aureus, and S. cerevisiae on the
agar disc diffusion assay. However, the level of grapefruit
seed extract that was added (0.5 and 1.0g/100g) did not
produce any noticeable difference in microbial growth on
packaged ground beef.
No evidence of chemical interaction between polymers
and linalool or methylchavicol has been found in the
literature. Ouattara, Sabato, and Lacroix (2001) hypothe-
sized that impregnation of active compounds in a
polymeric matrix would lower their diffusion and lead to
higher concentrations of the compounds on the surfaces of
foods for a longer period. It was reported that incorpora-
tion of hydrophobic compounds into hydrophilic polymers
caused structural modifications in the polymer matrix,
leading to an increased network tortuosity resulting in an
impeding transport of molecules through the network and
to a reduced water uptake. Further research is ongoing to
investigate the AM effects of these agents in Cheddar
cheese with food spoilage and/or pathogenic culture
inoculation.
3.2.2. Bacterial growth
3.2.2.1. E. coli.
cheese wrapped with LDPE, linalool-LDPE-based, and
methylchavicol-LDPE-based films decreased by a factor of
24, 28 and 47, respectively, after 35 days of storage at 41C
(Fig. 4). A similar pattern of inhibition of E. coli was found
on Cheddar cheese stored under temperature abuse
conditions (121C) as shown in Fig. 5. The LDPE,
linalool-LDPE-based and
films reduced the E. coli populations by a factor of 14, 27
and 34, respectively, after 15 days.
The numbers of E. coli on Cheddar
methylchavicol-LDPE-based
3.2.2.2. L. innocua.
genes, L. innocua is often selected for inhibition studies
because it is not pathogenic, but closely related to
L. monocytogenes, as shown by results of DNA–DNA
hybridization, multilocus enzyme analysis, and 16S rRNA
sequencing experiments (Swaminathan, 2001). Films con-
taining linalool or methylchavicol reduced L. innocua
populations by a factor of 2.7 and 3.4, respectively, after
35 days of storage at 41C (Fig. 4). In the control samples,
the Listeria population was also reduced after 35 days
An identical strain of L. monocyto-
under refrigeration storage conditions, but by a factor of
2.2. The inhibition of L. innocua on Cheddar cheese
subjected to the temperature abuse condition is depicted in
Fig. 5. The values of L. innocua on Cheddar cheese packed
withlinalool-LDPE-based
based films were reduced by a factor of 4.0 and 5.6,
respectively, after 15 days of the abuse simulation. For the
additive-free LDPE film, the Listeria population initially
remained constant and decreased slightly (by a factor of
1.4) during the following 7 days of the temperature abuse
condition. Regardless of the storage temperature and
sample treatments, the growth of Listeria on Cheddar
cheese may have occurred during the first day due to the
availability of nutrients leached from the cheese into the
moisture, due to the presence of oxygen on the surface of
the product and due to previous exposure to the relatively
high moisture and oxygen environment as reported by
Holliday, Adler, and Beuchat (2003).
Populations of E. coli in the Cheddar cheese decreased
also during storage in the control samples. The decrease
was faster in samples stored at the temperature abuse
conditions, compared to 41C. This is consistent with the
results of Holliday et al. (2003) on the viability of E. coli
O157:H7 in butter, ‘‘yellow fat spread’’ and margarine
stored at 4.4 and 211C. It is also consistent with the results
of Cagri, Ustunol, and Ryser (2002) on the survival of
E. coli O157:H7 in Bologna and fermented summer sausage
stored at 41C. Similarly to the observation with E. coli, the
population of L. innocua was reduced faster in cheese
samples stored at 41C. However, in the abuse condition,
the population of L. innocua remained almost constant.
Holliday et al. (2003) also showed that the population of
L. monocytogenes decreased continuously in salted butter
stored at 4.41C, whilst it remained almost unchanged in
salted butter stored at 211C for 7 days. The effect in our
results is thus similar also to that obtained by Dawson,
Carl, Acton, and Han (2002) who claimed that the growth
rate of L. monocytogenes is expected to be lower at 41C
than at elevated temperatures. Limjaroen, Ryser, Lock-
hart, and Harte (2005) reported that polyvinylidene
chloride (Saran F-310) films containing 1.5 and 3.0g/
100g sorbic acid decreased L. monocytogenes populations
on Cheddar cheese by a factor of 6.3 and 21.4, respectively,
after 35 days of storage at 41C. Cutter (1999) investigated
the effectiveness of triclosan-incorporated plastic (TIP)
against bacteria on beef surfaces. A small decrease in the
number of E. coli was observed during 5 days of storage
under simulated temperature abuse conditions of vacuum-
packaged beef wrapped with TIP containing 1500ppm of
triclosan. However, after an additional storage period of
up to 14 days under these conditions, the number of E. coli
increased.
It seems that the agar disc diffusion assay on packaging
films with typical test strains of microorganisms provides
limited information on the effectiveness of the film in
actual food packaging situations. Actual packaging trials
need to be performed in order to evaluate the efficacy of
and methylchavicol-LDPE-
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the AM packaging film (Ha et al., 2001). In the previous
section, it was stated that no AM activity against
L. innocua was found in the agar disc diffusion test. A
later study was therefore carried out to challenge the
efficacy of these AM films against L. innocua in Cheddar
cheese. In this study, linalool-LDPE-based and methylcha-
vicol-LDPE-based films showed significant inhibitory
effects against L. innocua when this organism was
inoculated on Cheddar cheese. In these tests, the film was
tightly contacted with the surface of the Cheddar cheese.
As a lipophilic compound, linalool and methylchavicol can
diffuse well into the surface of the cheese, which is a fat-
based food product and, in turn, the inhibitory effect on
the growth of L. innocua on the surface of the cheese was
positive. The inhibition of E. coli and L. innocua on
Cheddar cheese stored under the temperature abuse
conditions exhibited promising results when using the
AM films. It is known that an increase in temperature from
refrigerated conditions (41C) to 121C enhances or ex-
pedites the growth of microorganisms. In spite of that,
these films showed a significant reduction in the growth of
E. coli and a highly significant decrease in the growth of
L. innocua at the higher abuse temperature. This result
could be explained by the fact that the higher the
temperature, the higher the release rate of the active agent
from the film onto the food surface. Thus, there is a merit
in the use of these natural plant extracts over conventional
AM agents like sorbic acid, K-sorbate or triclosan.
Antimicrobial films containing the basil extracts yielded
encouraging protection against E. coli and L. innocua in
Cheddar cheese, especially under the temperature abuse
conditions. It should be noted, however, that for this
maximum inhibition to occur, there is a need for direct
contact between the food and the AM packaging material.
It is believed that AM films containing basil extracts would
contribute to the shelf life extension of Cheddar cheese and
probably also of other foods.
3.2.3. Sensory evaluation
The sensory evaluation results are summarized in
Tables 2 and 3. The panelists did not perceive a difference
in flavour between Cheddar cheese wrapped in linalool-
LDPE-based film and LDPE film (control) throughout the
storage period of 6 weeks at 41C.
A significant difference in flavour, at the 99% confidence
level, was found between Cheddar cheese wrapped in
methylchavicol-LDPE-based film and LDPE film after
1 week. At longer periods, the panelists found no
significant difference in flavour and 80% of the panelists
had correct answers at 4 weeks of storage. This might be a
result of ‘‘correct guessing’’ by the panelists with the
probability of1
3in the triangle test. Then, a non-significant
difference in flavour was detected at 6 weeks. It was
observed that the panelists spent less time for the sensory
evaluation in the first week in order to detect methylcha-
vicol in the cheese sample. After that week, the panelists
spent more time and were unable to detect differences in
the samples. The majority of panelists detected a methyl-
chavicol flavour but could not detect a linalool flavour at
1 week of storage. This might be a result of the more
distinct flavour of methylchavicol compared to linalool.
In summary, the present study shows that taint in
flavour from linalool was not significantly detectable by the
sensory evaluation panels. As far as taint in flavour from
methylchavicol is concerned, it may not present a problem
in the sensory properties of Cheddar cheese. Hence, low
doses of linalool or methylchavicol can possibly be used as
AM additives in AM films to extend the shelf life of
Cheddar cheese (which has its own unique flavour),
seemingly without any detrimental effect on flavour, unlike
the following cases. Ouattara et al. (2001) reported that low
scores were obtained in sensory evaluation tests for odour
and taste of pre-cooked shrimp (Penaeus sp.) coated with a
protein-based solution containing 0.9 or 1.8mL/100g of a
mixture of thyme oil and trans-cinnamaldehyde. They
claimed that these low scores resulted from the intrinsic
sensory attributes of thyme oil and trans-cinnamaldehyde.
Mejlholm and Dalgaard (2002) claimed that 0.05mL/100g
oregano oil yielded a distinct, although pleasant, flavour to
cod fillets, and the oil delayed spoilage reactions and
extended the shelf life of the fish. Consequently, the active
ingredients of volatile natural plant extracts at very low
concentration should be considered for food applications
in order to avoid some of the pitfalls in relation to
detrimental effects on flavour. Alternatively, flavour
matching between plant extract and food product could
be used. For instance, wasabi extract-incorporated sheet
(as an AM packaging material) is commercially used for
bento, a type of Japanese lunch box.
4. Conclusions
The findings of the present study demonstrate that the
natural AM components of basil (linalool and methylcha-
vicol) can be successfully incorporated into LDPE-based
polymers and retain their inhibitory effect against micro-
bial growth in model (i.e. solid medium) and real (Cheddar
cheese) systems. As far as taint in flavour is concerned,
linalool or methylchavicol may not present a problem in
sensory evaluation studies. Therefore, these additives may
be useful in AM packaging of some foods by enhancing
microbial stability and food safety. Additional studies are
required to investigate the loss of the AM additive and the
retained AM activity during film storage.
Acknowledgements
On behalf of the Royal Thai Government, P. Suppakul
gratefully thanks the Australia Agency for International
Development (AusAID) for providing financial support.
This work was partially supported by a fund for the
promotion of research at the Technion-Israel Institute of
Technology. J. Miltz expresses his thanks and appreciation
for this support. The authors thank Dr. Alison Duncan,
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Irawati Prasatya, and Joe Pelle, School of Molecular
Sciences, Victoria University; Juan Carrasset, Ku Heng Sai
and Dow Chemical (Australia) Ltd. as well as Qenos Pty.
Ltd., Australia, for their support towards the experiments.
Many thanks are also due to the panelists in sensory
evaluation for their valuable time and efforts.
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