1. Eleonora Winkelhausen 1 Robert Pospiech 2 and Günther Laufenberg 2 1 Faculty of Technology and Metallurgy, University “Sts. Cyril and Methodius”, Rudjer Boskovich 16, 1000 Skopje, Republic of Macedonia; eleonora@ereb1.mf.ukim.edu.mk 2 Department of Food Technology, University Bonn, Römerstr.164, D-53117 Bonn, Germany; g.laufenberg@uni-bonn.de 18th congress of chemists and technologists of Macedonia 23.-25.09.04 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. 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. Micro organisms used Alternaria solani (tomatoes, potatoes, red peppers) Botrytis cinerea (grapes, berry-fruits, some vegetable) Fusarium culmorum (cereal grains, production of mycotoxins) Media Malt extract, 30 g/L, meatpeptone extract, 3 g/L with or without agar, 20 g/L Olive oil residue Olive press cake, collected during 2002/2003 harvest season from Kalamata organic cultivars in Greece was dried at 60 o C, packed in vacuumed plastic bags and stored at 4 o C until used. 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 1:4 w/v). The polyphenols were then extracted using a mixture of water and ethanol (1:1, v/v). Solid-liquid ratio was 1:6 (w/v). The extract was filtered (0.45  m) and concentrated by a rotary-evaporator at 30 o C. Phenols were determined spectrophotometrically at 720 nm using Folin- Ciocalteu reagent. 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 25°C. 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. Table 1. Influence of the phenolic compounds and Euparen MW G on the mycelial growth rate of the fungi after 180 h. 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. 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. 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) (  ) A. solani B. cinerea F. culmorum Fig.2. Growth of A. solani on medium with 0, 0.1 and 0.2 % (w/v) phenolic compounds 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). a b c RESULTS MATERIALS AND METHODS