Kuru üzümde okratoksin A ve fumonisin B2 varlığının incelenmesi
Determination of ochratoxin A and fumonisin B2 in dried vine fruits
Yazar: ZEYNEP ASLANOĞLU
Danışman: PROF. DR. ZEYNEP DİLEK HEPERKAN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2014
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Üzüm (Vitis vinifera), iklim ve toprak istekleri yönünden çok seçici olmayışı, çoğalma yöntemlerinin kolay oluşu ve çok çeşitli şekillerde tüketilebilmesi gibi sebeplerden dolayı dünyadaki en yaygın kültür bitkilerinden birisidir. Yüksek besinsel değeri ve yüksek mikro besin içeriği sayesinde milattan önce 1490'dan beri sevilen bir gıda maddesi olmuştur. Kuru üzüm, yüksek şeker içeriğinden dolayı, kalori değeri yüksek bir besin maddesidir. Ayrıca mineral maddelerden kalsiyum, potasyum, sodyum ve demir yönünden zengin olduğu gibi bazı vitaminler (A, B1, B2, Niasin ve C vitaminleri) yönünden de önemli bir kaynak olarak kabul edilmektedir. Dünyada üretilen üzümlerin her yıl yaklaşık 700-1,200 bin ton arasındaki bir miktarı kurutularak değerlendirilmektedir. 

Türkiye, ABD, Şili, İran, Güney Afrika Cumhuriyeti ve Yunanistan dünyanın en önemli çekirdeksiz kuru üzüm üreticisi ülkelerdir.Türkiye Çin, İtalya, Amerika, İspanya, Fransa'dan sonra altıncı yaş üzüm üreticisi ülke olmasının yanı sıra net üzüm ihracatçısıdır.Türkiye kuru üzüm ihracatı yönünden dünyada ilk sıralarda yer almaktadır. Türkiye açısından önemli bir ihraç ürünü olan kuru üzüm, 1996-1997 yılı ürünlerinde İngiltere'de yapılan analizlerde ortaya çıkan OTA varlığından dolayı önemli bir problemle karşı karşıya kalmış ve 1997 yılında ihraç edilen çekirdeksiz kuru üzümlerde büyük bir OTA krizi yaşanmıştır.

Üzüm fungal gelişime hassas olan meyvelerden biridir ve bu zamana kadar yapılan çalışmalar üzüm ve üzüm ürünlerinde en sık rastlanan mikotoksinin OTA olduğunu ortaya koymuştur. OTA  Aspergillus ve Penicillium türlerinin belli suşları  tarafından üretilen ve çeşitli gıda matrislerinde tespit edilen düşük ağırlıklı bir mikotoksindir. OTA pişirme, kavurma, fermentasyon gibi gıda proseslerine karşı dayanıklıdır. Yani  hububat, kavrulmuş kahve, şarap, bira ve hayvan yemi gibi  işlenmiş ürünler kontamine olabilmektedir. Yakın zamana kadar sadece mısır, buğday, arpa, çavdar, yulaf, pirinç gibi tahıl ürünlerinde bulunduğu bilinmekteyken günümüzde kuru meyve, yer fıstığı, kahve, kakao, baharatta, şarap, bira ve bazı hayvansal ürünlerde de OTA'ya rastlanmaktadır. Tarımsal ürünlerde OTA oluşumu uygun koşullar bulunduğu takdirde hasattan tüketime kadar hemen her aşamada meydana gelebilmektedir. OTA immünotoksik,  teratojenik, nefrotoksik, nörotoksik ve kanserojenik etkilerinden dolayı önemli halk sağlığı sorunu oluşturmaktadır.

Fumonisinler birçok Fusarium türü tarafından üretilen bir mikotoksin olmakla birlikte genel olarak Fusarium verticillioides ve F. proliferatum tarafından üretilen ve dünya çapında sıklıkla mısır ve mısır ürünlerini kontamine eden mikotoksinlerdir. Yapılan çalışmalarda Aspergillus niger' in fumonisin B2 (FB2) ve B4 (FB4) üretme kapasitesinde olduğu bildirilmiştir. Hububat ve hububat kaynaklı gıda ürünleri için fumonisin limiti belirlenmiş olmasına rağmen, kuru üzüm için bu limit henüz belirlenmemiştir. Deney hayvanları üzerinde yapılan araştırmalar ve epidemiyolojik çalışma sonuçları incelendiğinde OTA ve FB2, Uluslararası Kanser Araştırma Ajansı (IARC) tarafından potansiyel kanserojen (2B) olarak değerlendirilmiştir. 

Bu çalışmada,  Anadolu'nun çeşitli bölgelerinden İzmir, Gaziantep, Besni, Kahramanmaraş, Konya, Bingöl illerinden 60 adet 7 farklı türde; Sultana (çekirdeksiz üzüm), Besni (kırmızı çekirdekli iri), Antep (çekirdekli siyah iri), Maraş (çekirdekli siyah küçük), Karaman (çekirdekli iri siyah),Urumu (çekirdekli sarı) , Dımışkı (çekirdekli siyah iri) kuru üzüm temin edilmiş. Üzümlerde OTA ve FB2 varlığı incelenmiştir. Elde edilen sonuçlar AB ülkelerinde ve Türk Gıda Kodeksi'nde kabul edilen maksimum limitlere göre değerlendirilmiştir. 
Kuru üzüm örneklerinde OTA ve FB2 varlığı yüksek performans sıvı kromatografisi (HPLC) yöntemi ile araştırılmıştır. OTA ve FB2 analizi, hazırlanan örneklerin ekstraksiyonu sonrasında immunoaffinite (IA) kolonla saflaştırılan ekstraktların HPLC yöntemi ile  analiziyle gerçekleştirilmiştir. Ters faz kolonda OTA ve FB2 ayrımı sonrasında fluoresans dedektörle tespit ve tayin gerçekleştirilmiştir. 
Kuru üzüm örneklerinin %18,3'ünde 0,22 -5,2639 µg/kg arasında değişen düzeylerde OTA belirlenmiştir. Toplanan 16 adet Besni üzümlerinden 5 (%31,3) tanesinde sırayla 1.09-1.43-2.67-1.75-0.39 µg/kg,  17 adet Antep üzümlerinden 5 (%29,4) tanesinde sırasıyla 0.34-0.26-2.63-0.22-1.65 µg/kg, 19 adet Sultana çekirdeksiz üzümlerinden 1 (%5,3) tanesinde 5.26 µg/kg OTA tespit edilmiştir. Bunun yanı sıra  farklı bölgelerden alınan kuru üzümlerden Antep üzümlerinin 1 tanesinde (%5.8) yani toplam üzümlerin %1,6'sında 8.8 µg/kg FB2 kontaminasyonu tespit edilmiştir. 
Kuru üzümlerde OTA ve FB2 oluşumunun önlenmesi amacıyla, bağ alanlarında, hasat sırasında ve sonrasında İyi Tarım Uygulamaları (GAP), İyi Üretim Uygulamaları (GMP) ve İyi Hijyen Uygulamaları (GHP)  oldukça önemlidir. Üretim esnasında her bir basamağın titizlikle kontrol edilmesi, son ürünün depolama koşullarının uygun olması, mikotoksin analizlerini düzenli olarak uygun yöntemlerle yapılması kontaminasyonların önlenmesinde etkilidir. Kuru üzümde OTA ve FB2 oluşumunun önüne geçilmesiyle hem halk sağlığı korunur hem de ekonomik kayıplar önlenmiş olur.

Abstract:
Grape (Vitis vinifera) consist in the world's most widely cultivated plants because the lack of very selective in terms of climate and soil, the proliferation of methods is easy to create and can be consumed in a variety of ways.  Since ancient times, raisins have been valued as a form of preserving grapes so they would last through the winter months and could be more easily stored and transported. Grapes were considered to be a nutritious and healthful food and raisins a good source of energy because of their high sugar content. Thanks to it has got nutritional value and high micro-nutrient content, it has been a nourishment which has liked since 1940 BCE. Grape is a high calorific value nutrient due to high sugar content. Also it has  lots of mineral such as calcium, potassium, sodium and iron rich as well as some vitamins such asA, B1 , B2 , niacin and vitamin C. Grapes produced in the world each year in an amount of about 700 to 1.200 tons are evaluated by dried.Sun drying was seen as a natural way to produce raisins that not only gave them a particular intense flavor, but also maintained the delicate balance of nutrients of the original fruit. Some saw this age-old process as an extension of the natural ripening cycle of the grape. Indeed, it is almost certain that raisins and grapes occurred naturally before men intended to cultivate them.
Mycotoxins are toxic secondary metabolites produced by many phytopathogenic and food spoilage fungi, which can cause a variety of adverse effects in humans, from allergic responses to cancer and death. They 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. Turkey, USA, Chile, Iran, South Africa and Greece are the most important countries, producing seedless raisins. Turkey is the sixth  grape producers after China, Italy, USA, Spain, France as well as net exporters of grapes. Turkey is world leader for raisin. In 1996-1997 the raisins  from Turkey to Britain had been  analyzed and the analysing had uncovered OTA in raisins so that led to OTA  crisis in raisins occured which was exported in 1997.
Grape is one of the most perishable fruits for fungal growing. Studies that has been done up to this time presents the mycotoxin is seen in grape is OTA.  OTA was originally described as a metabolite of Aspergillus ochraceus but, currently, several fungal species belonging to the genera Aspergillus and Penicillium are known to biosynthesize it . With regard to the first genus, the ochratoxigenic species are included in the Circumndati and Nigri sections such as A. ochraceus, Aspergillus melleus, Aspergillus carbonarius and Aspergillus niger aggregate. OTA has been reported to be produced by  A. ochraceus, A. carbonarius and A. niger as the main producers commonly found in warm climates such as southern Europe and by Penicillium verrucosum and Penicillium nordicum in more temperate climates. OTA  is a mycotoxin mainly low is detected in various food matrices. This toxin is colorless, blue fluorescent crystalline compound and has acidic character; acting as a weak organic acid, and relatively stable to baking, roasting and fermentation, as well as in food products. In dark phase the production is higher than under the light. OTA  is resistant to food processes such as baking, roasting and processed products like cereals, roasted coffee, wine, beer and animal feed can be contaminated. Until recently  it was known that OTA  had only present in corn, wheat, barley, rye, oats, rice, cereal products but now it is known OTA is being also in dried fruit, peanuts, coffee, cocoa, spices, the wine, beer and animal products. In the case that suitable conditions OTA  can  occure in agricultural products in each step from harvesting to the consumption. OTA  constitutes a major public health problem due to immunotoxic, teratogenic, nephrotoxic, neurotoxic and carcinogenic effects. 
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).
Fumonisins are carcinogenic mycotoxins that were originally identified in Fusarium verticillioides. Fusarium proliferatum, F. subglutinans, F.oxysporum and F. globosum were found to be able to produce these toxins. A putative gene cluster for fumonisin biosynthesis in the phylogenetically very distantly related fungus Aspergillus niger, and fumonisin production has also been proved for several  A. niger isolates that came from culture collections, coffee beans and grapes. Fumonisins can be divided into structurally distinct groups, four of which have been designated A, B, C and P fumonisins. These fumonisin groups differ in structure by differences in the nitrogen function and by the length of the carbon backbone. In fumonisins A, B and P analogues (FAs, FBs and FPs) the backbone is 20 carbon atoms long, whereas in C type fumonisins (FCs) it is 19 carbon atoms long.  100 different isomers and stereomers of fumonisins have been identified. These fumonisins contaminate maize and maize products in the worldwide.  Fumonisin B1 is the most commonly found, in corn (maize) and corn-based foods, beer, milk, rice, sorghum, triticale, cowpea seeds, garlic (powder), garlic bulbs, onion powder, black radish, figs, black tea, beans, soybeans and asparagus. Some industrially important strains of Aspergillus niger were reported to produce, in addition to ochratoxin A, the mycotoxin fumonisin B2 . It has also been shown that strains of A. niger from coffee beans and raisins produced both fumonisin B2 and fumonisin B4. A. niger is produced fumonisin B2 from grape. A. niger strains isolated from raisins are capable of producing both fumonisin B2 and fumonisin B4 when cultured on either grapes or raisins. Although the fumonisin limit is determined for cereals and cereal products, this limit has not been established for the raisins. When studies on experimental animals and epidemiological study results have been analyzed, International Agency for Research on Cancer  evaluated that OTA and FB2 are potential carcinogen (2B). Fumonisins may be involved in esophageal cancer in South Africa, and have been shown to be involved in leukoencephalomalacia in horses, pulmonary edema in pigs, and cancer and neural tube defects in experimental rodents.
In this study, 60 samples in five different types of dried vine fruits, Antep, Sultana, Besni , Maraş, Karaman,Urumu, Dımışkı  were supplied from the regions of Anatolia such as İzmir, Gaziantep, Besni, Kahramanmaraş,Konya, Bingöl. The presence of OTA in grapes and FB2 were examined. The results obtained in the EU and the Turkish Food Codex  had been evaluated according to the accepted maximum limit.
An analytical method based on immunoaffinity column clean-up and high performance liquid-chromatography with fluorimetric detection was used to determine the occurrence of OTA and FB2 in dried vine fruits and sultanas. The limit of detection (LOD) and limit of quantification (LOQ) for OTA were 0,2 μg/kg, 0,26 μg/kg  respectively and the recovery value was %86,3. The limit of detection (LOD) and limit of quantification (LOQ) for FB2 were 8 μg/kg, 8,63 μg/kg  respectively and the recovery value was %75,5. In order to minimize public health risk, the European Commission and Turkish Food Codex established a maximum level of 10 μg/kg for OTA in dried vine fruits.
The presence of OTA and FB2 in the samples was explored by high performance liquid chromatography method. Following extraction of the samples prepared immunoaffinity (IA) of the extract purified by column, The analysis of OTA and FB2 was performed by HPLC method. After the seperation of OTA and FB2 are detected by reversed phase column assays were performed by detectors.OTA was determined at levels from  0.22-5.2639 0.22 µg/kg  in 18,3%  of the samples. OTA was detected in 5 (31.3%) of gathered 1 Besni, 1.09-1.43-2.67-1.75-0.39 µg/kg, in 5(29.4%) of 17 Antep, 0.34-0.26-2.63-0.22-1.65 µg/kg, in 1(5,3%) of 19 Sultana, 5.26 µg/kg. In addition, FB2 was found in 1 sample  (1,6%) sample in Antep raisin (5.5%)  in the content of 8.8 µg/kg. 
The percentage of contamination in the sample from Southeastern Anatolia Region was remarkably high when comparing to the other regions such as Aegean, Central Anatolia and The Mediterranean. Taking precautions in Southeastern Anatolia region is very significant for public health.
In order to prevent the formation of OTA and FB2 in raisins, during and after harvest of Good Agricultural Practices (GAP), Good Manufacturing Practices (GMP) and Good Hygiene Practices (GHP) are very important. Preventing OTA and FB2 formation in dried vine fruits, both public health is protected and economic losses can be avoided. Raw material control, good agricultural practices (GAP), good manifacturing practices (GMP), good hygene practices (GHP) are necessary in vineyards, during harvesting and post harvesting phases , in order to prevent OTA and fumonisin B2. While producing raisins, it is vitally important to prevent contamination in raw material to prevent OTA and fumonisin B2. Contamination being detected indicates that good agricultural practices haven't been used or contaminated grapes have been used. Good agricultural practices contain setting up vineyards, pruning, soil cultivation, watering, applying hormones, plant protection and harvesting process. Each of these stages should be controlled carefully. In vineyards, during harvesting and post harvesting, occurrence of OTA and fumonisin B2 can be prevented using good manufacturing practices. During production, every step should be controlled meticulously and storage conditions should be proper to regular micotoxine analysis work in order to prevent contamination. Preventing OTA and fumonisin B2 is necessary in terms of protecting health and preventing economic losses.