Nisa OZSOY, HİLAL ÖZKILINÇ, Cigdem Uysal PALA

Molecular Characterization of Natural Fungal Flora in Black Olives: From Field to Table

In this study, molecular markers were used to determine fungal flora in black olive fruits from field surveys to the table, following the fermentation process. Field samples were collected from different locations of Canakkale province, including Gokceada (Imbros), where organic farming is employed. Some of the fruits from field samples were used for black table olive production and then fungal flora was tracked during the fermentation process. Fungal isolation was also conducted on some commercial samples. Fifty seven isolates from field samples, 56 isolates from the fermentation process and 17 isolates from commercial products were obtained. Among these isolates, 41 Alternaria, 43 Penicillium, 19 Aspergillus, 8 Monascus and 19 other genera were determined using amplified sizes of the Beta-tubulin gene region. Species level identification was carried out based on sequences of Beta-tubulin amplicons, which provided accurate identification, especially where the genera were morphologically highly similar. The occurrence and prevalence of fungal species changed in fungal collections from the field to the fermentation process. While Alternaria alternata was common in field samples, they were absent during fermentation. Many of these identified species, such as Penicillium expansum, Aspergillus niger and Monascus pilosus, which are known as potential toxin producers such as aflatoxin, ochratoxin A and citrinin, were found both in natural and fermented samples, even at the end of the fermentation process. These results showed that some fungal species which survive on olives from the field to the table are potential toxin producers and can be successfully characterized by amplification and sequencing of Beta-tubulin gene.

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Adlouni CE, Tozlovanu M, Naman F, Faid M, Pfohl-Leszkowicz A. 2006. Preminary data on the presence of mycotoxins (ochratoxin A, citrinin and aflatoxin B1) in black table olives "Greek style" of Moraccon origin. Mol. Nutr. Food Res. 50, 507-512.Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J. Mol. Biol., 215: 403-410.

Aran N. 2012. Gıda Biyoteknolojisi, Geliştirilmiş 2. Baskı, Nobel Yayınevi. İstanbul Teknik Üniversitesi. pp. 49.

Arroyo-López FN, Dura´n-Quintana MC, Ruiz-Barba JL, Querol A, Garrido-Fernandez A. 2006. Use of molecular methods for the identification of yeast associated with table olives. Food Microbiol. 23:791–796. Baffi MA, Romo-Sanchez S, Ubeda-Iranzo J, Briones-Perez AI. 2012. Fungi isolated from olive ecosystems and screening of their potential biotechnological use. New Biotechnology. 29: 451-456.Baskou D. 2015. Olive and olive oil bioactive contituents. Olive oil chemistry and technology, 2nd edition, AOCS press. Carbone I, Kohn LM. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91: 553-556.

Carlucci A, Raımondo ML, Cibelli F, Phillips AJL, Lops F. 2013. Pleurostomophora richardsiae, Neofusicoccum parvum and Phaeoacremonium aleophilum associated with a decline of olives in Southern Italy, Phytopathologia Mediterranea. 52(3): 517−527.

Chliyeh M, Rhimini Y, Selmaoui1 K, Touhami1 AO, Filali-Maltouf A, El Modafar C, Moukhli, A, Oukabli, A, Benkirane R, Douira A. 2014. Survey of the fungal species associated to olive-tree (Olea europaea L.) in Morocco. Int. J. Rec. Biotech. 2: 15-32. Ghitakou S, Koutras K, Kanellou E, Markaki. 2006. Study of aflatoxin B1 and ochratoxin A production by natural microflora and Aspergillus parasiticus in black and green olives of Greek origin. Food Microbiol 23: 612-621.

Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microb. Apr., 1323– 1330.

Gonvances JS, Ferracin LM, Vieira MLC, Iamanaka BT, Taniwaki MH, Pelegrinelli Fungaro MH. 2012. Molecular analysis of Aspergillus section Flavi isolated from Brazil nuts. World J Microbiol Biotechnol. 28: 1817–1825.

Feng W, Zheng X. 2006. Control of Alternaria alternata by cassia oil in combination with potassium chloride or sodium chloride. Journal of Applied Microbiology. 101: 1317–1322.

Hall TA. 1999. Bioedit: A user-friendly biological sequence alignment editor and analysis program for Windows95/98/NT. Nucleic Acids Symposium Series. 41: 95–98.

Heperkan D, Meric BE, Sismanoglu G, Dalkılıç G, Güler FK. 2006. Mycobiota, mycotoxigenic fungi and citrinin production in black olives. Advances in Food Microbiology,571, 203-210. Heperkan D. 2013. Microbiota of table olive fermentations and criteria of selection for their use as starters. Frontiers in Microbiology. 4: 1-11. Hong SY, Oh JH, Lee I. 2011. Simultaneous enrichment of deglycosylated ginsenosides and monacolin K in red ginseng by fermentation with Monascus pilosus. Bioscience, biotechnology, and biochemistry. 75: 1490-1495. Lumi Abe, C. A., Bertechini Faria, C., Fernandes de Castro, F., et al. 2015. Fungi Isolated from Maize (Zea mays L.) Grains and Production of Associated Enzyme Activities. International Journal of Molecular Sciences, 16(7): 15328–15346.

Magnani M, Fernandes T, Prete CEC, Homechim M, Ono EYS, Vilas-Boas LA, Satori D, Furlaneto MC, Fungaro MHP. 2005. Molecular identification of Aspergillus spp. isolated from coffee beans. Sci. Agric. (Piracicaba, Braz.). 62: 45-49.

Marsilio V, Campestre C, Lanza B, De Angelis M. 2001. "Sugar and polyol compositions of some European olive fruit varieties (Olea europaea L.) suitable or table olive purposes". Food Chem. 72: 485–490.

Oliveria I, Pereira JA, Lino-Neto T, Bento A, Baptista P. 2012. Fungal diversity associated to the olive moth, Prays Oleae bernard: A survey for potential entomopathogenic fungi. Microb Ecol. 63: 964-974.

Özkaya MT, Tunalıoğlu R, Eken Ş, Ulaş M, Tan M, Danacı A, İnan N, Tibet İ. 2010. “Türkiye zeytinciliğinin sorunları ve çözüm önerileri”. Türkiye Ziraat Mühendisliği V. Teknik Kongresi 11- 15 Ocak 2010.

Pereira AP, Ferreira ICFR, Marcelino F, Valentao P, Andrade PB, Seabra R, Estevinho L, Bento A, Pereira JA. 2007. Phenolic compounds and antimicrobial activity of olive (Olea europaea l. cv. cobrançosa) leaves. Molecules. 12: 1153-1162.

Piccolo SL, Mondello V, Giambra S, Conigliaro G, Torta L, Burruano S. 2014. Arthrinium phaeospermum, Phoma cladoniicola and Ulocladium consortiale, New Olive Pathogens in Italy. J Phytopathol 162: 258–263.