Aspergillus flavus ve Aspergillus tamarii suşlarının siklopiazonik asit üretme potansiyelerinin belirlenmesi ve siklopiazonik asit üretimine sıcaklık ve süre etkisinin incelenmesi
Determination of cyclopiazonic acid production ability of Aspergillus flavus and Aspergillus tamarii and investigetion of heat and time effect on cyclopiazonic acid production by Aspergillus flavus and Aspergillus tamarii
Yazar: DENİZ DİNÇER
Danışman: PROF. DR. Z.DİLEK HEPERKAN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2012
Konu: Gıda Mühendisliği = Food Engineering ; Mikrobiyoloji = Microbiology
Dizin: Mikotoksinler = Mycotoxins

Özet:
Mikotoksinler, küfler tarafından üretilen, ikincil metabolit olarak adlandırılan toksik bileşiklerdir. Bu bileşikler insan ve hayvanlar için toksik özellik taşırken; tarım ürünlerinde verim kayıpları oluşturarak büyük ekonomik kayıplara sebep olmaktadırlar. Aspergillus ailesine ait olan Aspergillus flavus, sıklıkla rastlanan küftür. Ürettiği metabolitler içerisinde başta aflatoksin olmak üzere siklopiazonik asit de yer almaktadır. Aflatoksin, gıda ürünlerinde ve hayvan yemlerinde sıklıkla kontamine olarak bulunmaktadır. Fındık, mısır ve pamuk ürünlerinde gelişmesinin ardından bu ürünler tüketilemez hale gelmektedir. Aspergillus tamarii; siklopiazonik asit (CPA), tenuazonik asit ve kojik asit üreten Aspergillus ailesine ait olan bir küftür. A.flavus ve A.tamarii aynı aileden gelmesinin yanı sıra siklopiazonik asit üretme özelliği, ortak özellikleridir.Siklopiazonik asit indole-tetramik asit olan; Penicillium cyclopium Westling, P. patulum, P. viridicatum, P. puberulum, P. crustosum, P. camemberti, Aspergillus flavus, A. versicolor, A. Oryzae ve A.tamarii gibi küfler tarafından üretilen ikincil bir metabolittir. Karaciğer ve sindirim sistemi dokularında nekroza; iskelet kaslarında ve böbreklerde lezyon oluşumuna sebep olur. Siklopiazonik asit doğal kontaminasyon sonucunda bulunduğu gıdalara örnek olarak yerfıstığı, mısır, peynir, süt, domates ve domates püresi verilmektedir.Çalışmanın ilk aşamasında A.flavus ve A.tamarii suşlarının hangi sıcaklıkta maksimumum miktarda CPA ürettiğini bulmak amacıyla 5, 10, 15, 20, 25, 30, 35, 40 ve 45 °C sıcaklıklarda 7 gün süre ile Czapek Yeast Agar (CYA) besi yerinde inkübasyona bırakılmıştır. Petrilerden ekstrakte edilen CPA, HPLC-DAD kullanılarak miktar tayini yapılmış ve istatistiksel açıdan sonuçlar değerlendirilmiştir. Bu sonuçlardan, 30 °C'nin her iki suş içinde maksimum miktarda CPA üretimi sağladığı bulunmuştur. Bu sonuca ek olarak; her iki suş için de 5 ve 45 °C'de gelişim gözlenmediği belirlenmiştir.Çalışmanın ikinci aşaması olarak 2 saat farklı sıcaklıkta bekletilen petrilerin, 30 °C'de 7 gün inkübasyonlarını tamamlamasının ardından, ürettikleri CPA miktarları belirlenmiştir. Bu çalışmanın amacı CPA üretimine sıcaklığın etkisinin belirlenmesidir. Bu amaç ile A.flavus ve A.tamarii suşları ayrı ayrı olmak üzere 5, 10, 15, 20, 25, 30, 35, 40 ve 45 °C'de sıcaklıkta 2 saat bekletildikten sonra (optimum sıcaklık) 30 C°'de sıcaklıkta 7 gün inkübasyona bırakılmıştır. Petrilerden ekstrakte edilen CPA, HPLC-DAD kullanılarak miktar tayini yapılmış; sonuçlar istatistiki açıdan değerlendirilmiştir. Elde edilen sonuçlar; kontrol grubu olan 30 °C'de 7 gün süre ile inkübe edilen suşların ürettiği CPA miktarları ile karşılaştırılmıştır. A.flavus suşu için en az CPA ürettiği deney tasarısı; 35 °C'de 2 saat bekletilip 30 °C'de inkübasyonunun tamamlanmasının ardından elde edilmiştir. İstatistiki açıdan % 95xivgüven aralığında değerlendirilen sonuca göre 2 saat 25 veya 35 °C'de bekletilen petrilerin ürettiği CPA miktarları arasında fark bulunamamıştır. A.tamarii suşu için en az CPA ürettiği deney tasarısı; 25 °C'de 2 saat bekletilip 30 °C'de inkübasyonunun tamamlanmasının ardından elde edilmiştir. İstatistiki açıdan % 95 güven aralığında değerlendirilen sonuca göre 2 saat 25 veya 35 °C'de bekletilen petrilerin ürettiği CPA miktarları arasında fark bulunamamıştır. Her iki suş için kontrol grubu ile yapılan ikili karşılaştırma sonucunda; A.flavus suşu için 10 °C ve A.tamarii suşu için 15 °C'de dışında ki deneyde kullanılan her sıcaklık derecesi için; ?=0,05 olmak üzere, önemli derecede kontrol grubu ile arasında fark bulunmuştur.Bu çalışmalara ek olarak 30 °C'de 1, 3, 5 ve 7. günlerde üretilen CPA miktarları A.flavus ve A.tamarii suşları için incelenmiş, günlere göre CPA üretim miktarları belirlenmiştir. Her iki suşunda ilk günden itibaren CPA üretime başladığı ve 7. güne kadar artış gösterdiği bulunmuştur. Belirli sıcaklıkta farklı günlerde CPA miktarının belirlenmesine yönelik başka bir çalışma da 25 °C için yapılmıştır. 25 °C'de 1, 3 ve 7. günlerde üretilen CPA miktarları A.flavus ve A.tamarii suşları için incelenmiş, günlere göre CPA üretim miktarları belirlenmiştir. 30 °C'de olduğu gibi, 25 °C'de de ilk günden itibaren CPA üretimi her iki suş içinde tespit edilmiştir.

Abstract:
Mycotoxins are the secondary metabolites produced by specific fungi. They are toxic to human and animals, at the same time they cause significant reductions in crop yield and cause economic losses. Aflatoxin, ohcrotoxin, patulin, fumonisin, cyclopiazonic acid, zearalenone, deoxynivalenol are example of the some mycotoxins. Althought having many researches and the introduction of good manufacturing practices in the food production, storage, and distribution chain, mycotoxins still continue to be a problem, hence their presence still important topic. Some mycotoxins can also be metabolized by animals fed contaminated grains and excreted into, for example, milk, or accumulated in eggs, meat, and/or other tissues, thus these food commodities are re-entering the food chain. In addition to that some mycotoxins? chemical structures are changed, then conjugated forms are occurred. So, they can not detect due to differences in polarity between the native precursors and their metabolites. These unexpected conjugated forms of mycotoxins are called ?masked mycotoxins?. Masked or modified mycotoxins are usually bounded mycotoxins with a more polar substance such as glucose, amino acid and sulfates. The most well-known substances within the group of fungal conjugates are 3-acetyl deoxynivalenol (3ADON) and 15-acetyl deoxynivalenol (15ADON), and also zearalenone 4-sulfate (Z4S). In plant conjugates; DON is converted to DON-3-glucopyranoside (D3G) and ZON can be altered to ZON-4-glucopyranoside (Z4G).Aspergillus flavus is the member of Aspergillus section flavi. Some type of A.flavus can produce aflatoxin (AF), and cyclopiazonic acid. When these fungi grow in commodities such as peanuts, corn and cottonseed, the resulting contamination with aflatoxins often makes the commodities unsuitable for consumption. Aspergillus tamarii is the member of Aspergillus section, too. Cyclopiazonik acid, tenuazonic acid and kojic acid is produced by A.tamarii. Besides A.flavus and A.tamarii come from same family, the ability of production of CPA is common behavior of them.Cyclopiazonic acid is a mycotoxin wtih an indole tetramic acid structure produced by Penicillium cyclopium Westling, P. patulum, P. viridicatum, P. puberulum, P. crustosum, P. camemberti, Aspergillus flavus, A. versicolor, A. Oryzae ve A.tamarii. It causes necrosis of liver or gastrointestinal tissue and necrotic changes in skeletal muscle and kidney. In addition to that, CPA causes weight loss, diarrhea, degeneration and necrosis of the muscles and viscera, and convulsion and death in rodents, birds, dogs and swine. CPA causes two mycotoxicoses, first one is ?Kodua posining? caused by consuming Kodua millet drink infected by high concentration of CPA, and the second one is ?Turkey X? disease together with AF. ?Kodua poisoning? is characterized by vomiting and nausea in human. And also, generally it can be seen on man. The ?Turkey X? disease was concluded by killing of 100.000 poultry. In the beginning of the search, AF was found only responsibility of this disease. However, finding the neurological semptoms on the poultry, CPA found the second responsibility of it.CPA is produced by different kind of fungies that is the reason of it can be seen different food products. CPA has been found to occur naturally in several products such as peanuts, corn, cheese, milk, tomato paste, puree and figs.When human consume a large range of contamined food commodities, the risk of exposure to CPA is arising, hence the determination of CPA in food samples represents an important aspect of food safety. However, still there is no regulation of CPA in Europe and USA.To detect and evaluate level of CPA in food commodities several different methods are used. As an example, immunoassay, capillary electrophoresis, and chromatography have been reported. There are some problems can affect the results. The problem is to removing the interference from the sample matrix. In addition to that CPA has a low pKa (3.5), that it can be reason of some acidic dissolvations in the extract, needs be consider carefully when deciding the method. As generally used in other mycotoxins analyses, chloroform and dichloromethane, which are toxic to human) are used. Recent studies show that they are searching less toxic solvents, easy and quick extraction methods and high recovery results.In the first part of study, to find the optimum temperature where the maximum CPA production was occurred, different temperatures were evaluated. These temperatures were 5, 10, 15, 20, 25, 30, 30, 35, 40 and 45 °C. The fungal strains were used in this study were A.flavus and A.tamarii, obtained from Istanbul Technical University, Food Microbiology Laboratory. All cultures were maintained on malt extract agar (MEA). Spore concentration was determined by thome lame. The concentration of spores was 1-5x106 cfu/ml was used by single point cultivation. These strains were incubated on Czpek Yeast Agar (CYA) for 7 days at different temperatures mentioned before. After the incubation CPA was extracted by using methanol. The quantification of CPA was determined by HPLC-DAD by reverse-phase NH2 columns with ammonia in the mobile phase. Data analyzes was run using the statistical package MiniTab 15.30 C° was chosen from the result for optimum temperature to obtain the maximum production level of CPA for A.flavus and A.tamarii. In addition to that, there were not any growing at 5 C° and 45 C° for all strains.In the second part of the study to find out heat effects on CPA production, 2 hours waiting in the different temperatures in the beginning of incubation was used. After 2 hour waiting in 5, 10, 15, 20, 25, 30, 35, 40 or 45 C°, A.flavus and A.tamari were continued to incubation at 30 C° until end of 7th days. CPA was extracted and then was quantified by HPLC-DAD. Data analyzes was run using the statistical package MiniTab 15. The result was compared with control group (optimum condition: incubation at 30 C° for 7 days).The minimum CPA production of A.flavus was obtained by 2 hours waiting at 35 C°. If the result with compare 95 % confidence limit, there was no differences between waiting 25 and 35 C°. In addition to that, the minimum CPA production of A.tamarii was obtained by 2 hours waiting at 25 C°. If the result with compare 95 % confidence limit, there was no differences between waiting 25 and 35 C°. Except 10 C° for A.flavus and 15 C° for A.tamarii, all 2 hours waiting temperatures had important differences from control group.In addition to these studies, the effects of incubation days (1.3.5 and 7) at 30 C° on CPA production by A.flavus and A.tamarii was investigated. . The strains were incubated on CYA at 30 C° for 1, 3, 5 and 7 days. As it mentioned before CPA was extracted by methanol, and then analyzed by HPLC-DAD.CPA production was determined in the beginning of the incubation (1st day) and continued by increasing rate to end of the 7th day. The results of CPA production by A.flavus were 1.83, 30.56, 66.95 and 133.01µg/ml by following incubation days. The results of CPA production by A.tamarii were 0.82, 3.07, 4.09 and 9.29 µg/ml by following incubation days.Another study at the constant temperature with different incubation days was examined. 25 C° was used for constant temperature and the incubation days were 1, 3 and 7. A.flavus and A.tamarii were incubated on CYA, and then CPA was extracted by methanol.The result of HPLC-DAD showed that CPA production was started in the beginning of the incubation (1st day) and increased to end of the incubation period. The results of CPA production by A.flavus were 2.13, 53.443 and 76.43 µg/ml by following incubation days. The results of CPA production by A.tamarii were 0.28, 1.09 and 5.9 µg/ml by following incubation days.This research is the first that shows different temperatures? effect on production of CPA by A.flavus and A.tamarii. Our results show that A.flavus and A.tamarii start to produces CPA after the 1st incubation at 25 C° and 30 C°. In addition to that, incubating 2 hours at different temperatures and put 30 C° for 7 days incubation can be a protection method, and also can be clue of optimization of harvest hours from field. For example, our result show that if there is small changing in the temperature (20-25-30-35 C°), the negative effect of it on the CPA production will be higher. So, if the working conditions are considered, there is a chance to reduce of production of CPA. As a conclusion this research has a lots of important outgoings makes its first research in their topic.