Art15_Özcakmak.indd Journal of Applied Botany and Food Quality 85, 97 - 99 (2012) 1 Department of Food Processing, Terme Vocational School, Ondokuz Mayis University, Terme, Samsun, Turkey The effects of Heracleum platytaenium Boiss essential oil on the growth of ochratoxigenic Penicillium verrucosum (D-99756) isolated from Kashar Cheese Sibel Özçakmak (Received July 13, 2011) Summary The antifungal effects of essential oil isolated from Heracleum platytaenium Boiss on the growth of ochratoxigenic Penicillium verrucosum (D-99756) isolated from Kashar cheese were in- vestigated. Minimal inhibitory concentration (MIC) and Minimal lethal concentration (MLC) tests were performed by using broth dilution method. Controls were prepared with three groups; with pure essential oils (Eo) (positive control), methanol and without Eos. The serial doubling dilution of oil was prepared in methanol with concentrations ranging from 500 to 31µL/mL. Applied incu- bation periods were 25 oC for 10 days for MIC and 48 hours at the same temperature for MLC. After the incubation period, the tubes were controlled for either mycelia growth or turbidity and sediments. The maximal inhibitory dilution (the minimal inhibitory concentration), exhibiting any growth, was regarded as MIC. Tubes containing Penicillium verrucosum which did not display any growth were subcultured to determine if the inhibition was reversible or permanent. The Eo dilution, which is the not visible growth of typical P. verrucosum colonies on the plates, was accepted as lethal effect. MLC was the lowest concentration of antifungal to completely inhibit fungal growth. The MIC and MFC values were determined as 31 and 125 µL/mL, respectively. Introduction The presence and growth of fungi in food may cause spoilage and result in a reduction in quality and quantity. Growth of commonly occurring fi lamentous fungi in foods may result in production of toxins known as mycototoxins, which can cause a variety of ill effects which has a range of biological activities including acute toxicity, teratogenicity, mutagenicity and carcinogenicity in humans, beginning from allergic responses to immunosuppression and cancer. The most important mycotoxins are afl atoxins, ochratoxin-A, fumonisins, trichothecenes and zearalenone. Ochratoxin-A produced by Aspergillus ochraceus, A. niger, A. carbonarius, Penicillium verrucosum and P. viridicatum (PITT, 1987; ABARCA et al., 1994; VARGA et al., 1996) is probably carcinogenic and may cause urinary tract cancer and kidney disease (KROGH et al., 1974; PITT, 2000). Penicillium species are frequent contaminants of different food products and known as producers of a variety of secondary metabolites (FRISVAD and FITENBORG, 1983). P. verucosum is pri- marily encountered on cereals and cheeses (LARSEN et al., 2001). Various species of Heracleum have been reported to possess anti- bacterial and antifungal properties (CIESLA et al., 2008). Heracleum sp. is a member of Umbelliferae family whose essential oils (Eos) have antibacterial and antifungal effects (İŞCAN et al., 2004). The fruits and the leaves of this genus are used as fl avouring agent and spice for food and also antiseptic, carminative, digestive and analgesic effect (FIRUZI et al., 2010). Chemical composition of the Eos of some Heracleum species was investigated by some researches. The major compounds of different Heracleum species were reported as octyl acetate by IŞCAN et al. (2004), 1-octanol, octylbutyrate, octanol by JANSSEN et al. (1986) and KÜRKÇÜOĞLU et al. (1995) and myristicin, E-anethole, octyl isobutanoate, hexyl butanoate, octyl acetate and elemicin by FIRUZI et al. (2010). Materials and Methods Materials The essential oil of the plant Heracleum platytaenium Boiss was obtained from Biological Science Department, Faculty of Science and Literature in Samsun-Turkey. Preparation of the essential oil dilutions The serial doubling dilution of each oil (PINTO et al., 2007) was prepared in methanol (RASOOLI and ABYANEH, 2004) with concentrations ranging from 500 to 31 µL mL-1. Fungal strain and preparation of spore suspension The fungal strain used in this study was Penicillium verrucosum Dierck (D-99756), obtained from Faculty of Food Engineering in Istanbul Technical University, Istanbul-Turkey. The strain was cultured from frozen stocks and maintained on Malt Extracted Agar (MEA, 2% malt, 0.1% peptone, 1.5% agar) slants at 25 oC in the dark for 7-10 days. The strain was cultivated on MEA slants at 25 oC in the dark for 7-10 days (CABANAS et al., 2009). Spores were harvested by adding 10 ml of sterile saline solution (NaCl, 0.85%) containing Tween 80 (0.1% v/v). The spore suspension was adjusted to approximately 106 spores mL-1 using haemocytometer (ELIAS et al., 2006). Test organism was enumerated in growing broth media by serial dilution method (ÖZKAN et al., 2010). The viability and inoculums size of the strains were checked using quantitive colony counts. The plates were incubated for 72 h at 25 oC in the dark (TAVARES et al., 2008). Growth media and chemicals Yeast Extract Sucrose (YES) agar, YES broth, methanol, Tween-80, Sodium chlorid (NaCl) were obtained from Merck, Germany. YES culture media was used to determine inhibitory concentrations and YES agar was used to determine if the inhibition was reversible or permanent (BRAGULAT et al., 2001). 5 mL sterile YES broth tubes containing 106 spores mL-1 were dispensed into sterile tubes using for broth dilution method. Petri dishes (90 mm) were fi lled with 15- 20 mL of YES agar for fungicidal effect. Controls with and without methanol (negative controls) and using pure Eos (positive controls) incubated under the same conditions. Antifungal activity of essential oils with macrodilution method A macrodilution broth method was used to determine the minimal inhibitory concentrations (MIC) and minimal lethal concentrations 98 S. Özçakmak (MLC) (LASS-FLÖRL et al., 2003; TAVARES et al., 2008). Fifty micro liters from four Eos dilutions at various concentrations (500, 250, 125, 62, 31, µL mL-1) were added in 5 mL sterile YES broth tubes containing 106 spores mL-1 and these solutions mixed gently. The tubes were incubated at 25 oC for 7-10 days on an incubator shaker as to evenly disperse the oils throughout the broth in tubes. The fungal growth was indicated by the turbidity. The highest dilution (lowest concentration), showing no visible growth compared with Eo-free controls, was regarded as MIC. The oil dilutions supplying inhibitory effect were tested for lethal effect as well. The inoculations of 1 mL for pour plate and 30 µL for three point inoculation methods from the tubes were subcultured on YES agar plates and than incubated at 25 oC for 72 hours. The essential oil dilution, which is not visible growth of typical P. verrucosum colonies on the plates, was accepted as lethal effect. The MLC was defi ned as the lowest Eo concentration at which 99% of the inoculum was killed. All the experiments were performed in duplicate and three independent experiments were run with concordant results. Results The results of experimental design in this research are presented in Tab. 1, Fig. 1 and Fig. 2a - 2b. The mycelial growth was visible in negative control tube (Fig. 1b) while any fungal growth wasn’t seen in positive control tubes (Fig. 1a). The highest dilution concentration (31 µL mL-1) was MIC value (Fig. 1c). The growth of P. verrucosum was inhibited 100% at MIC value. Heracleum species has just been investigated for anticandidal activity by NAEINI et al. (2009), İŞCAN et al. (2003 and 2004) and KULJANABHAGAVAD et al. (2010). The researches reported that different Heracleum spp. had notable anticandidal effect. In this study, it was determined that the mycelial formation of P. verrucosum could be prevented using H. platytaenium essential oil. Tab. 1: MIC/MFC values of Heracleum platytaenium Eo against ochratoxigenic P. verrucosum strain. The inhibitory and lethal effects of H. platytaenium Eo dilutions (µL mL-1) on P. verrucosum. Positive controls Negative controls 500 250 125 62 31 MIC + - + + + + + MLC + - + + + - - +: inhibition positive -: inhibition negative Fig. 1: The inhibitory effects of H. platytaenium Eo dilutions on P. verrucosum after 9 days of incubation at 25°C. The left tube represents complete inhibition at pure form of Eo, the right tube represents complete inhibition at 31 µL mL-1 and the middle tube represents negative control tube. 2a 2b Fig. 2: MFC results after 72 hours of incubation at 25 °C using three point inoculation (Fig. 2a) and pour plate mehods (Fig. 2b). The left petri dishes for non-additive and the right petri dishes for additive with H. platytaenium Eo dilutions represent subcultured inoculations from MIC tubes. 1a 1b 1c The lethal effect was assayed using two methods. The results both were the same. Oil required for MLC value is more than MIC. The inoculations subcultured on YES agar plates could be killed at 125 µL mL-1 oil dilutions (Fig. 2a - 2b). Effects of Heracleum platytaenium Boiss essential oil on the growth of ochratoxigenic Penicillium verrucosum 99 Discussion In this study, it was demonstrated that H. platytaenium had inhibitory and toxic effects against P. verrucosum. In general, the cytotoxic activity of Eos is mostly due to the presence of aldehydes, alcohols, methylene dioxy compounds and phenols (KULJANABHAGAVAD et al., 2010). FIRUZI et al. (2010) reported that Heracleum species had the cytotoxic activity mostly due to the presence of myristicin, elemicin and (E)-anethole. Heracleum species has just been investigated for anticandidal activity by NAEINI et al. (2009), İŞCAN et al. (2003 and 2004) and KULJANABHAGAVAD et al. (2010). This survey is the fi rst report that the inhibitory effect of H. platytaenium against ochratoxigenic P. verrucosum. Acknowledgements The present research was funded by the Scientifi c Research Programme of Ondokuz Mayis University. The authors thank also Terme Vocational School for their support. References ABARCA, M.L., BRAGULAT, M.R., CASTELLA, G., CABANES, F.J., 1994: Ochratoxin A production by strains of Aspergillus niger var. niger. Appl. Environ. Microbiol. 60, 2650-2652. BRAGULAT, M.R., ABARCA, M.L., CABANES, F.J., 2001: An easy screening method for fungi producing ochratoxin A in pure culture. Int. J. Food Microbiol. 71, (2-3), 139-144. CABANAS, R., ABARCA, M.L., BRAGULAT, M.R., CABANES, F.J., 2009: In vitro activity of imazalil against Penicillium expansum: Comparison of the CLSI M38-A broth microdilution method with traditional techniques. Int. J. Food Microbiol. 129, 26-29. CIESLA, Ł., BOGUCKA-KOCKAB, A., HAJNOSC, M., PETRUCZYNIKA, A., WAKSMUNDZKA-HAJNOSA, M., 2008: Two-dimensional thin-layer chro- matography with adsorbent gradient as a method of chromatographic fi ngerprinting of furanocoumarins for distinguishing selected varieties and forms of Heracleum spp. J. Chromatogr. A. 1207, (1-2), 160 168. ELIAS, B.C., SAID, S., ALBUQUERQUE, S., PUPO, M.T., 2006: The influence of culture conditions on the biosynthesis of secondary metabolites by Penicillium verrucosum Dierck. Microbiol. Res. 161, 273-280. FIRUZI, O., ASADOLLAHI, M., GHOLAMI, M., JAVIDNIA, K., 2010: Compo- sition and biological activities of essential oils from four Heracleum species. Food Chem. 122, 117-122. FRISVAD, FILTENBORG, O., 1983: Classification of terverticillate Penicillia based on profi les of mycotoxins and other secondary meta-bolites. Appl. Environ. Microbiol. 46, 1301-1310. İŞCAN, G., DEMIRCI, F., KURKCUOGLU, M., KIVANC, M., BASER, K.H.C., 2003: The bioactive essential oils of Heracleum sphondylium L. subsp. ternatum (Velen.). Brummitt. Z. Naturforsch. C. 58, 195-200. İŞCAN, G., OZEK, T., OZEK, G., DURAN, A., BASER, K.H.C., 2004: Essential oils of three species of Heracleum anticandidal activity. Chem. Nat Compd. 40, 544-547. JANSSEN, A.M., CHIN, N.L.J., SCHEFFER, J.J.C., BAERHEIM, S.A., 1986: Sci. Pharm. Weekbl. 8, 289-292. KROGH, P., HALD, B., ENGLUND, P., RUTQVIST, L., SWAHN, O., 1974: Contamination of Swedish cereals with ochratoxin A. Acta Pathol. Microbiol. Scand. [B] 82, 301-302. KULJANABHAGAVAD, T., SRIUBOLMAS, N., RUANGRUNGSI, N., 2010: Che- mical composition and antımicrobial activity of the essentıal oil from Heracleum sıamıcum. J. Health Res. 24, 55-60. KÜRKCÜOGLU, M., OZEK, T., BASER, K.H.C., MALYER, H., 1995: Compo- sition of the essential oil of Heracleum platytaenium Boiss. from Turkey. J. Ess. Oil Res. 7, 69-70. LASS-FLÖRL, C., SPETH, C., KOFLER, G., DIERCH, M.P., GUNSILIUS, E., WÜRZNER, R., 2003: Effect of increasing inoculum sizes of Aspergillus hypae on MICs and MFCs of antifungal agents by broth microdilution method. Int. J. Antimicrob. Agents 21, 229-233. LARSEN, T.O., SVENDSEN, A., SMEDSGAARD, J., 2001: Biochemical cha- racterization of ochratoxin A producing strains of the genus Penicil- lium. Appl. Environ. Microbiol. 67, 3630-3635. NAEINI, A., KHOSRAVI, A.R., CHITSAZ, M., SHOKRI, H., KAMLNEJAD, M., 2009: Anti-Candida albicansactivity of some Iranian plants used in traditional medicine. J. Mycol. Med. 19, 168-172. ÖZKAN, G., SAGDIC, O., GOKTURK, R.S., UNAL, O., ALBAYRAK, S., 2010: Study on chemical composition and biological activities of essential oil and extract from Salvia pisidica. LWT - Food Sci. Technol. 43, 186- 190. PINTO, E., SALGUEIRO, R., CAVALEIRO, C., PALMEIRA, A., GONÇALVES, M.J., 2007: In vitro susceptibility of some species of yeasts and filamentous fungi to essential oils of Salvia offi cinalis. Ind. Crop Prod. 26, 135-141. PITT, J.I., 1987: Penicillium vindicatum, Pemcillium verrucosum and production of ochratoxin A. Appl. Environ. Microbiol. 53, 266-269. PITT, J.I., 2000: Toxigenic fungi and mycotoxins. Brit. Med. Bull. 56, 184- 192. RASOOLI, I., ABYANEH, M.R., 2004: Inhibitory effects of thyme oils on growth and afl atoxin production by Aspergillus parasiticus. Food Control. 15, 479-483. TAVARES, A.C., GONÇALVES, M.J., CAVALEIRO, C., CRUZ, M.T., LOPEZ, M.C., CAHOTO, J., SALGUEIR, L.R., 2008: Essential oil of Daucus carota subsp. halophilus: Composition, antifungal and cytotoxicity. J. Ethnopharmacol. 119, 129-134. VARGA, J., KEVEII, E., RINYU, E., TEREN, J., KOZAKIEWICZ, Z., 1996: Ochratoxin production by Aspergillus species. Appl. Environ. Microbiol. 62, 4461-4464. Address of the author: Ph. Dr. Sibel Özçakmak, Tel.: +90 362 8761318 / 114, Fax: +90 362 8761317, E-mail: sibelo@omu.edu.tr