8 Oleochemistry Products
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the main components. EOs are complex mixtures of numerous molecules, and their
biological effects are the result of a synergism of all components. Several studies
have concluded, in fact, that essential oils resulted to be more effective in terms, of
antimicrobial activity, with respect to an artificial mixture of the major components
[73]. Essential oils and their components, commonly used as flavouring in the food
industry, also present interesting antibacterial, antifungal and antioxidant properties
[34].
8.3.3.1 Methods for Antimicrobial Assessment
The task of assessment of EOs, for their antibacterial and antifungal properties, is
generally pursued through different conventional and widely used techniques initially
developed for antibiotic testing that proceeds through a visual or an instrumental
evaluation. Two fundamental basic techniques, briefly described in this paragraph,
are widely used for the assessment of both antibacterial and antifungal activities of
essential oils:
1. The agar diffusion method (paper disc or well),
2. The dilution method (agar or liquid broth).
Anyway, some specific properties of EOs, like volatility, high viscosity and
hydrophobicity, require that some modifications must be applied to the mentioned
standard assessment methods as they were initially developed for aqueous-phase
antimicrobial substances. In the application of these tests on EOs, even if a proper
solubilizing agent or emulsifier must be used, unequal distribution of the oil through
the medium can occur affecting the results. It must be checked also if the dispersing
agent can affect the growth or the differentiation of the microorganisms under assessment. Another critical issue, in the assessment test, could be the possible selective
evaporation of EOs components during a long incubation period so the test condition
must be carefully controlled. Usually, microorganisms derived from internationally
certified collections are employed but can also be isolated from the natural environment [20, 24, 32]. All parameters which may affect the experimental results have to
be strictly defined and the growth experiments must be repeated from two to seven
times to ascertain reproducibility [36].
The agar diffusion method is the most widely used test for antimicrobial/antifungal
activity assessment. The method is recognized by the most authors [122] as precise
and reliable, even if in some cases it furnishes semi-quantitative results or, according
to some authors, only qualitative [11] and with low repeatability [51]. It can, however,
estimate the degree of microorganisms’ growth inhibition and their morphological
changes in a very simple way.
The method consists [53] in filling Petri dishes with agar broth and inoculated
with microorganisms. The EO to be tested is incorporated in the system by means of
a paper disc or through a hole directly in the agar medium. Critical parameters are:
the diameter of the disc, the amount of EO added, the type of dispersing agent.
223
the main components. EOs are complex mixtures of numerous molecules, and their
biological effects are the result of a synergism of all components. Several studies
have concluded, in fact, that essential oils resulted to be more effective in terms, of
antimicrobial activity, with respect to an artificial mixture of the major components
[73]. Essential oils and their components, commonly used as flavouring in the food
industry, also present interesting antibacterial, antifungal and antioxidant properties
[34].
8.3.3.1 Methods for Antimicrobial Assessment
The task of assessment of EOs, for their antibacterial and antifungal properties, is
generally pursued through different conventional and widely used techniques initially
developed for antibiotic testing that proceeds through a visual or an instrumental
evaluation. Two fundamental basic techniques, briefly described in this paragraph,
are widely used for the assessment of both antibacterial and antifungal activities of
essential oils:
1. The agar diffusion method (paper disc or well),
2. The dilution method (agar or liquid broth).
Anyway, some specific properties of EOs, like volatility, high viscosity and
hydrophobicity, require that some modifications must be applied to the mentioned
standard assessment methods as they were initially developed for aqueous-phase
antimicrobial substances. In the application of these tests on EOs, even if a proper
solubilizing agent or emulsifier must be used, unequal distribution of the oil through
the medium can occur affecting the results. It must be checked also if the dispersing
agent can affect the growth or the differentiation of the microorganisms under assessment. Another critical issue, in the assessment test, could be the possible selective
evaporation of EOs components during a long incubation period so the test condition
must be carefully controlled. Usually, microorganisms derived from internationally
certified collections are employed but can also be isolated from the natural environment [20, 24, 32]. All parameters which may affect the experimental results have to
be strictly defined and the growth experiments must be repeated from two to seven
times to ascertain reproducibility [36].
The agar diffusion method is the most widely used test for antimicrobial/antifungal
activity assessment. The method is recognized by the most authors [122] as precise
and reliable, even if in some cases it furnishes semi-quantitative results or, according
to some authors, only qualitative [11] and with low repeatability [51]. It can, however,
estimate the degree of microorganisms’ growth inhibition and their morphological
changes in a very simple way.
The method consists [53] in filling Petri dishes with agar broth and inoculated
with microorganisms. The EO to be tested is incorporated in the system by means of
a paper disc or through a hole directly in the agar medium. Critical parameters are:
the diameter of the disc, the amount of EO added, the type of dispersing agent.