Eleonora Winkelhausen1 Robert Pospiech2 and G

1
Eleonora Winkelhausen1 Robert Pospiech2 and
Günther Laufenberg21 Faculty of Technology and
Metallurgy, University Sts. Cyril and
Methodius, Rudjer Boskovich 16, 1000 Skopje,
Republic of Macedonia eleonora_at_ereb1.mf.ukim.edu.
mk2 Department of Food Technology, University
Bonn, Römerstr.164, D-53117 Bonn, Germany
g.laufenberg_at_uni-bonn.de
Growth inhibition of moulds by natural pesticides
derived from olive oil residues
INTRODUCTION Synthetic crop protection agents
play an essential role in the protection of
plants or harvested fruits against
microorganisms. They are effective and
economically advantageous but, by modern
standards, they lack selectivity and applied at
high rates they are threat to human health and
environment. Complying with the growing public
awareness of these hazards, increasing emphasis
is placed upon the search for novel, natural
products with pesticidal activity. Phenolic
compounds, present in the olive fruit, definitely
belong to this group.
Olive press cake, representing 40 of the
original olive weight used for oil production,
contain 0.3 phenolics, representing an abundant
and cheap source of natural antimicrobial
compounds.
The major phenolic compound in unripe olive
fruits Olea europaea is the secoiridoid
Oleuropein being responsible for their
bitterness. Therefore olives are often pickled
debittering the flavor.
MATERIALS AND METHODS
Micro organisms used Alternaria solani
(tomatoes, potatoes, red peppers) Botrytis
cinerea (grapes, berry-fruits, some vegetable)
Fusarium culmorum (cereal grains, production of
mycotoxins)
Extraction of the phenolic compounds The
extraction of the olive press cake was performed
in a stirred-tank batch extractor at 750 rev
min-1. The residual oil and pigments were removed
with hexane (ratio 14 w/v). The polyphenols were
then extracted using a mixture of water and
ethanol (11, v/v). Solid-liquid ratio was 16
(w/v). The extract was filtered (0.45 ?m) and
concentrated by a rotary-evaporator at 30oC.
Phenols were determined spectrophotometrically at
720 nm using Folin- Ciocalteu reagent.
Media Malt extract, 30 g/L, meatpeptone extract,
3 g/L with or without agar, 20 g/L
Assay of the antifungal activity The fungi
were grown on media containing 0, 0.1 and 0.2
(w/v) phenolic extract and 0.2 (w/v) Euparen MW
G (Bayer), a commercial agrochemical. The
cultivation was carried out in Erlenmeyer flasks
on a rotary shaker (125 rev min-1) at 25C. At
defined time intervals, the content of the entire
flask was vacuum filtered (0.45 ?m). The filtrate
was used for pH and redox potential measurements,
while the cell residue was washed and dried for
cell mass determination.
Olive oil residue Olive press cake, collected
during 2002/2003 harvest season from Kalamata
organic cultivars in Greece was dried at 60oC,
packed in vacuumed plastic bags and stored at 4oC
until used.
RESULTS
Fig. 1. Effect of the natural and commercial
antifungal compounds. Phenolic compounds 0 (?),
0.1 (?), 0.2 (w/v) (?), Euparen MW G 0.2
(w/v) (?)
Fig .2. Growth of A. solani on medium with 0,
0.1 and 0.2 (w/v) phenolic compounds
Table 1. Influence of the phenolic compounds and
Euparen MW G on the mycelial growth rate of the
fungi after 180 h.
Fig. 3. Morphology of the F. culmorum mycelium
observed on a light microscope. Growth on a
medium without (a) and with phenolic extract (b,
c).
CONCLUSIONS The presence of phenolic extract in
the medium that normally supports the growth of
the fungi, inhibited the growth of all three
fungi, with higher phenol concentration (0.2
w/v) being more effective. The medium with
Euparen influenced the growth of all three fungi
in the same manner. Although with reduced rate,
they grew till fifth day when their growth
slightly declined. In media containing phenolic
compounds, the growth was delayed and slow, but
did not decline. The results indicated the
presence of antifungal activity in the phenolic
extract and hence the possibility of its
application as an antimicrobial agent.
Further studies should focus on optimizing the
inhibitory concentration and conditions.
Attention should be paid to the mechanism of the
antifungal action.
18th congress of chemists and technologists of
Macedonia 23.-25.09.04