Antimicrobial properties of herbs and spices have been recognized and used

since ancient times for food preservation and in medicine (Zaika

1988; Conner 1993). Scientific reports on natural antimicrobial agents also
date to back more than a century, e.g. Chamberlain reported in 1887 the action
of essential oil vapours on anthrax spores, as cited by Maruzzella and
Sicurella (1960). A renewed interest in natural preservation appears to be
stimulated by present food safety concerns, growing problems with microbial
resistance, and a rise in production of minimal processed food joined with
green image policies of food industries.
Numerous studies have documented the antifungal (Thompson 1989; Mishra
and Dubey 1994; Cox et al. 1998; zcan
1998; Cosentino et al. 1999; Aligiannis et al. 2001; Elgayyar et al. 2001
) and anti-bacterial (Lachowicz et al. 1998; Wan et al. 1998; Canillac and
Mourey 2001) effect of plant essential oils. Examinations of indigenous local
herbs and plant material have also been reported from around the world e.g.
India (Ahmad and Beg 2001), Australia (Cox et al. 1998), Argentina
(Penna et al. 2001) and Finland (Rauha et al. 2000). Screening experiments
with 1352 essential oils and major active components against five to 25 microorganisms (Conner and Beuchat 1984; Deans and Ritchie 1987; SmithPalmer et al. 1998; Hammer et al. 1999; Dorman and Deans 2000) have
reported thyme, clove, cinnamon, bay, oregano, garlic and lemongrass to be
some of the best broad spectrum candidates for inhibition of food-borne
pathogens and spoilage organisms. However, comparisons of studies that have
used different methodologies are difficult, especially regarding minimal
inhibitory concentrations (MIC), and the need for uniform and reliable
procedures when testing activity has been emphasized (Davidson and Parish
1989; Smith and Navilliat 1997; Mann and Markham 1998). The
extensively used method is diffusion (from discs, drops, wells or double plate)
(Zaika 1988; Sivropoulou et al. 1996; Mangena and Muyima 1999) or
agar and broth dilution (mixing the agent into the growth media)
(Paster et al. 1990; Cosentino et al. 1999; zcan and Boyraz 2000).
Unfortunately, results obtained by the different methods using the same oils do
not always compare (Hili et al. 1997). Solubility and diffusion rates of the oil
compounds in the aqueous agar media are of paramount importance, and can
lead to misinterpretations in diffusion tests. Emulsifying agents (Tween-20 or
Tween-80) are often added to ensure uniform mixing in dilution tests (Smith
and Navilliat 1997), but emulsifiers and solvents have been shown to
decrease the activity (Remmal et al. 1993; Hili et al. 1997; Mann and
Markham 1998). A 02% agar solution has subsequently been recommended
as the least disturbing detergent (Remmal et al. 1993).
A third method for testing essential oils is the microatmosphere method
(Maruzzella and Sicurella 1960) or cup method (Zaika 1988) and
fumigation (Paster et al. 1995; Arras and Usai 2001). This method brings
the vapours of essential oils into contact with growth media and microorganisms. Thus, the problem of inadequate mixing is circumvented, and
instead distribution in the air-phase of the active compounds is important.

Evaporation inside Petri plates (ca. 8 mm distance between agar surface and
oil) can generate inhibition zones (Maruzzella and Sicurella 1960),
indicating a gradient and non-uniform air-phase distribution of vapour
compounds. The concentration of compounds in the head-space has also been
reported to be different from the composition of the liquid oil, reflecting
differences in volatility of the compounds (Benjilali et al. 1984). Thus, the
dynamics of the volatile system are very important and conflicting results
between volatile methods and diffusion or dilution methods could be expected.
To make the most appropriate assessments of potential antimicrobials, the
future application should be considered when choosing a screening method. For
active packaging (Nielsen and Rios 2000) and fumigation of storage crops
this would be a volatile screening method, whereas substitutes for synthetic
preservatives like sorbates, propionates and benzoates should be assayed by
dilution methods.
Composition of media, exposure time and environmental factors (temperature,
pH, aW) are important for all test methods. The lack of congruence between
agar media assays and real food studies (Arras and Usai 2001; Ultee and
Smid 2001) emphasizes the role that fat and protein content of the food play
in solubility and chemical reactions with the active compound
(Juven et al. 1994; Smith-Palmer et al. 2001), and encourages the use of
food product analogues instead of standard laboratory media. This study was
performed in a model system for bread, because bread is a staple diet
worldwide that spoils fast when not preserved. In Denmark, rye bread is a very
important bread type. The main spoilers of rye bread are Penicillium
roqueforti, P. corylophilum and Eurotium ssp. (Lund et al. 1996). Also yeasts
e.g. Endomyces fibuliger (Legan and Voysey 1991) and Aspergillus flavus are
commonly found.