Sıcaklığın Aspergillus section Nigri üyelerinin gelişme ve okratoksin A oluşturması üzerine etkisinin incelenmesi
Determination the effect of temperature on growth and ochratoxin A production properties of Aspergillus section Nigri members
Yazar: AYŞEGÜL MUTLU
Danışman: YRD. DOÇ. DR. FUNDA KARBANCIOĞLU GÜLER
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2012
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Mikotoksinler çesitli bitkisel ve hayvansal orijinli gıdalarda yaygın olarak bulunan küflerin ürettiği düşük molekül ağırlıklı ikincil metabolitledir. Miktoksijenik küflerin dağılımı ve son üründe ki miktarları iklimsel faktörlerden ve gıda işleme koşullarına bağlı olarak değişkenlik göstermektedir.Aspergillus section Nigri üyeleri OTA kontaminasyonundan başlıca sorumlu küflerdir. Okratoksin A, Aspergillus ochraceus, A. carbonairus, A. niger ve Penicillium verrocosum'un da dahil olduğu bazı Aspergillus ve Penicillium türleri tarafından üretilmektedir. OTA, Uluslararası Kanser Araştırma Ajansı (IARC) tarafından?Grup IIB? muhtemel karsinojen olarak sınıflandırılmıştır.Bu çalışmanın ilk aşamasında Ege Bölgesi'nden toplanan kuru incirlerden izole edilen 30 adet Aspergillus section Nigri izolatının (10 adet A.carbonarius, 20 adet A. niger aggregate) OTA oluşturma özellikleri 25°C ve 30°C'de 7 ve 10 günlük inkübasyon süresi sonunda belirlenmiştir. A. carbonarius izolatlarının CYA besiyerinde %90 oranında OTA üreticisi olduğu belirlenmiştir. A. niger ise YES besiyerinde %45 oranında OTA üretimiştir. İzolatların 25 ve 30°C'de okratoksin A üretme özellikleri dikkate alınarak seçilen 3 adet Aspergillus carbonarius ve 1 adet Aspergillus niger agg.'in gelişmesine ve OTA üretimine sıcaklığın etkisi 8 farklı sıcaklıkta (5, 10, 15, 20, 25, 30, 35, 40°C) incelenmiştir. Okratoksin A üretimleri A. carbonarius izolatları için CYA besiyerinde, A . niger agg. izolatı için ise YES besiyerinde yüksek basınçlı sıvı kromatografisi ile belirlenmiştir.İzolatların tümünün en hızlı geliştiği sıcaklık 30°C, en yavaş geliştiği sıcaklık ise 15°C'dir. 30°C'de en hızlı gelişen izolatın, günde 25,968 mm gelişen 6Y1 nolu Aspergillus niger agg., en yavaş gelişen izolatın ise günde 16,152 mm gelişen 46D2 nolu Aspergillus carbonarius olduğu saptanmıştır. Bütün izolatlar için de en uzun lag fazı süresi 15°C'dedir.8-04D2 nolu Aspergillus carbonarius izolatının en hızlı geliştiği sıcaklık 20°C, optimum OTA ise üretimi 15°C'de gerçekleşmiştir. 21-04D2 nolu A. carbonarius izolatı ise en hızlı 25°C'de gelişirken maksimum düzeyde OTA üretimi 20°C'de gerçekleşmektedir. 46D2 nolu A. carbonarius'un optimum gelişme sıcaklığı 25°C iken OTA üretimi en fazla 20°C'de belirlenmiştir. 6Y1 nolu Aspergillus niger agg. izolatı ise en hızlı gelişmeyi 30°C'de gösterirken, optimum OTA üretimini ise A. carbonarius izolatlarına oranla çok düşük miktarda olmakla birlikte 25°C'de gerçekleştirmiştir.

Abstract:
Mycotoxins are low molecular weight secondary metabolites that have adverse effects on humans, animals, and crops that result in illnesses and economic losses., commonly found in various plant and animal origin foods. Distribution of mycotoxin producer fungi quantities in the final product varies depending on climatic factors and food processing conditions. General interest in mycotoxins rose in 1960 when a feed-related mycotoxicosis called turkey X disease, which was later proved to be caused by aflatoxins, appeared in farm animals in England. After this importance of mycotoxins increased all over the word.Ochratoxin A is a toxic secondary metobolite, naturally produced by some Aspergillus and Penicillium species. Aspergillus ochraceus, Penicillium verrucosum and Aspergillus section Nigri members are the main producers of ochratoxin A. These mould species are capable of growing on several substrates under different environmental conditions; so contamination of ochratoxin A in food products can be seen all over the world. Its occurrence has been reported in a wide variety of foods and beverages such as cereals and cereal products, pulses, dried vine fruits (currants, raisins and sultanas), coffee, cocoa products, nuts, spices, black table olive, olive oil, wine, grape juice and beer. Ochratoxin A has also been detected in food products from non-ruminant animals exposed to ochratoxin A in animal feeding stuffs.Ochratoxin A has been shown to be mutagenic, to cause cancer and kidney damage in a number of animal species. Teratogenic, immunotoxic and hepatotoxic effects of ochratoxin A have been determined. Ochratoxin A has been associated with Balkan Endemic Nephropathy. Balkan Endemic Nephropathy is a chronic progressive kidney disease, predominantly affect women, which may lead to death. OchratoxinA has been also linked with the development of urothelial cancer in some human populations. With regard to the animal studies and epidemiological studies on humans, Ochratoxin A has been classified as possible human carcinogen (Group IIB) by International Agency for Research on Cancer (IARC).Exposure to ochratoxin A mainly occurs in the food chain by the consumption of the cereals and cereal products. Because of the adverse effects, maximum ochratoxin A levels in foods such as cereals, dried wine fruits, coffee and foods for infants and young children are regulated by the European Union and Turkish Food Codex.Aspergillus section Nigri members especially Aspergillus carbonarius primarily responsible for OTA contamination in dried fruits as well as dried figs. Mould growth and mycotoxin production are affected by different environmental factors such as water activity (aw), tempature and pH.In this study 30 isolates of Aspergillus section Nigri members (10 Aspergillus carbonarius, 20 Aspergillus niger agg.), isolated from dried figs, investigated for their ochratoxin a production capacity on agar at 25 and 30°C. Ochratoxin A producer 3 isolates of Aspergillus carbonarius and 1 isolates of Aspergillus niger agg. search for growth and and OTA production capacities at eight different temprature (5, 10, 15, 20, 25, 30, 35 and 40°C). Also the effect of incubation time on ochratoxin A production is aimed to evaluate changes. Last stage, it is purposed to determine the optimum growth and ochratoxin A production temprature of Aspergillus section Nigri members.The first stage of this study was to investigate the ochratoxin producing abilities of 30 isolates of Aspergillus section Nigri members (10 Aspergillus carbonarius, 20 Aspergillus niger agg.) isolated from dried figs. Ochratoxin A production properties of these isolates were determined at 25 ° C and 30 ° C for 7 and 10 day incubation. Incidence of ochratoxin A production by Aspergillus carbonarius isolates were 90% on Czapek yeast exrtact agar. Forty five percent of Aspergillus niger agg. was ochratoxin A producer on yeast extract sucrose. Aspergillus carbonarius isolates were produced ochratoxin A within the range of 0,006-26,92 µg/g. Aspergillus niger agg. isolates were produced ochratoxin A lower levels than Aspergillus carbonarius.In the second stage of the study, depending on the ochratoxin A production characteristics of isolates at 25 and 30 °C, 3 Aspergillus carbonarius and 1 Aspergillus niger agg. were selected and effect of temperature on growth and ochratoxin A production was investigated at eight different temperatures (5, 10, 15, 20, 25, 30, 35, 40 °C).Growth rate, lag phase time and ochratoxin A production properties of A. carbonarius and Aspergillus niger agg. isolates were determined on CYA and YES, respectively. The growth rate was determined by periodical measurement of two right-angled diameters of the colonies. Colony diameters vs. time was plotted and growth rates (mm/ day) were evaluated from the slope by linear regression. Lag phase for growth was determined as the time (days) to reach 5 mm of diameter The determination of OTA in culture medium was carried out by using high performanceliquid chromatography (HPLC) with fluorescence detection after extraction with methanol.None of the four isolates of Aspergillus section Nigri grown at 5 and 10°C. Aspergillus carbonarius isolates growth was observed between 15 and 40°C for 8-04D2, between 15 and 30°C for 21-04D1 and 46D2. A. niger agg. isolate grown between 15 and 40°C with an optimum at 30°C. On the other hand, there is no statistical difference between 30 and 35°C. Optimum growth rate for all isolates were determined at 30°C, and minimum growth rate at 15°C. The highest growth rate at 30°C was calculated for Aspergillus niger agg. 6Y1 as 25.968 mm /day. The lowest growth rate at 30°C was determined for Aspergillus carbonarius 46D2 as 16.152 mm/day. Statistical analysis of variance (ANOVA) showed that temperature were significantly effect the growth rate of Aspergillus section Nigri members.For all strains, the longest lag phase period was observed at 15° C. Aspergillus section Nigri 46D2 isolate have the longest lag phase period over 8 days at 15°C. The shortest lag phase period for all Aspergillus carbonarius isolates was determined at 25°C. On the contrary, A. niger agg. 6Y1 isolate had the shortest lag phase period at 40°C. Statistical analysis of variance (ANOVA) showed that temperature had significant effect on the extension of lag phase of A. carbonarius and A. niger agg.A. carbonarius isolate produced OTA at each temperature and incubation time which growth was observed. On the other hand, temperature levels in which OTA production occurred were narrower than that for fungal growth for Aspergillus niger agg 6Y1. OTA amounts accumulated by the Aspergillus carbonarius isolates 8-04D2, 21-04D1 and 46D2 and Aspergillus niger agg. 6Y1 ranged from 0,01 to 26,92 µg/g. Maximum OTA production was found at 15°C and 10 day for the 8-04D2 Aspergillus carbonarius isolate. Optimum ochratoxin A production temperature was different from each other. A. carbonarius 8-04D2 produced optimum amount of OTA at 15°C, Aspergillus carbonarius 21-04D1 and 46D2 produced optimum amount of OTA at 20°C. Aspergillus niger agg. 6Y1isolate produced optimum amount of ochratoxin A at 25°C at very low levels compared to Aspergillus carbonarius isolates.A. carbonarius 8-04D2 isolate grew fastest at 20°C, and its optimum ochratoxin A production was at 15°C. Optimum growth of A. carbonarius 21-04D1 isolate was at 25°C and maximum OTA production was determined at 20°C. A. carbonarius 46D2 was grown optimally at 25°C while maximum OTA production was observed at 20°C. Aspergillus niger agg. 6Y1 showed optimum growth at 30°C, optimum OTA production was at 25°C.According the results of this study, Aspergillus carbonarius is more toxigenic than A. niger agg. because higher percentage of ochratoxin production capacity. For Aspergillus section Nigri members, 20-30°C is optimum for growth and 15-20°C is the optimum temperature for ochratoxin A production. There was no correlation between optimum growth temperature and ochratoxin A production in Aspergillus section Nigri members.