Antep fıstıklarında Okratoksin a ve Aflatoksin varlığının incelenmesi
Determination of Ochratoxin a and Aflatoxins in pistachio nuts
Yazar: CANSU SEDEFOĞLU
Danışman: PROF. DR. DİLEK HEPERKAN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2013
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Antep fıstığı (Pistacia vera L.), Anacardiaceae ailesinin bir üyesi olup, kökeni Orta ve Batı Asya kurak bölgeleridir ve Akdeniz havzası genelinde dağılmıştır. Besin değeri yüksek, lezzetli bu ürün tarih boyunca birçok ülkeye yayılmış ve farklı bölgelerde yetiştirilmeye başlanmıştır. Sert kabuklu meyvelerden olan Antep fıstığı, ülkemiz için yüksek ekonomik değere sahip bir ihraç ürünüdür. Sert kabuklu meyveler sahip oldukları besin bileşenleriyle; steroller, vitaminler, mineraller, yağ asitleri, fenolik bileşikler, antioksidan ve antiproliferatif özellikleriyle tercih edilen bir üründür.
2011 yılı verilerine göre Türkiye dünya genelinde sahip olduğu %13?lük payla Antep fıstığı üretiminde üçüncü sırada yer almaktadır. 2011 yılı Antep fıstığı üretim kapasitesi 112.000 tona erişmiştir. Türkiye dünya Antep fıstığı ihracatında ise 2010 yılı verilerine göre 12. sırada yer almaktadır. Antep fıstığı ihracat miktarı 717 tondur. Dünya antep fıstığı ithalat verileri incelendiğinde ise Türkiye ilk 20 ülke arasında yer almamaktadır. Türkiye?nin ihracat yaptığı ülkelerle ilgili verilere bakıldığında ise 2010 ve 2011 yılında İtalya, Almanya ve İsrail ilk 3 sırada yer alan ülkelerdir. İtalya %47,9?luk paya, Almanya %9,8 ve İsrail %9,0?luk paya sahiptir. 
Ticari değere sahip, lezzetli ve besin ögelerince zengin Antep fıstığı, üretim yöntemi ve diğer çevresel faktörler nedeniyle küf kontaminasyonu ve gelişimine maruz kalmaktadır. Bu küfler, doğada ve gıdalarda yaygın olarak bulunan, mikotoksin olarak adlandırılan ikincil metabolitleri oluşturmaktadırlar. Okratoksin A (OTA), Aspergillus ve Penicillium cinsi küfler tarafından üretilen mikotoksinlerdendir ve kahve, şarap, tahıllar, üzüm, baharat ve kakao ürünlerinde sıklıkla rastlanmaktadır. Toksin; berrak, renksiz, mavi floresans veren kristal bir bileşiktir ve asidik karaktere sahiptir. Zayıf organik asit gibi davranır ve göreceli kararlıdır; pişirme, kavurma ve fermantasyon gibi gıda işlemelerine dayanıklıdır. Üretimi karanlık evrelerde daha yüksek, ışık altında ise daha düşüktür. Hayvanlar ve insanlar üzerinde nefrotoksik, immunosupresif, teratojenik, genotoksik, karsinojenik, hepatotoksik özellik göstermektedir. Uluslararası Kanser Araştırma Merkezi (IARC) tarafından insanlar için olası kanserojenik gruba dahil edilmiştir (grup 2B). Hedef organ böbreklerdir ve Balkan Endemik Nefropatisi?ne (BEN) yol açtığı belirlenmiştir. 
Aflatoksin (AF); kristal yapıda, kloroform ve metanol gibi polar çözücülerde kolaylıkla çözünebilen, saf halde ultraviyole (UV) radyosyon altında parçalanan, düşük (<3) ve yüksek (>10) pH ve oksitleyici maddeler varlığında stabil olmayan bir birleşiktir. Gıda ve yemlerde ise bu mikotoksin oldukça stabildir. Işık ve UV radyasyona maruz kalmadıklarında, kristal halde yüksek sıcaklıklara dayanıklıdırlar. Tüm omurgalı canlılar üzerinde karsinojen, mutajen, teratojen, hepatotoksik ve bağışıklık sistemini baskılayıcı etkiye sahiptir. IARC tarafından aflatoksin B1 (AFB1) öncelikle ciğerleri hedef alan, grup 1 kanserojen madde olarak sınıflandırılmaktadır. 
Ticari değeri yüksek bu ürünün, halk sağlığı açısından mikotoksin düzeyi önem taşımaktadır. Birçok ülkede, mikotoksinlerin insanlar ve hayvanlar üzerinde zararlı etkilerine karşı bilinçlenme başlamış ve gıdalarda bulunabilecek maksimum tolere edilebilir seviyeleri belirlenmiştir. 
Bu projede, Türkiye?de yetiştirilen Antep fıstıklarında okratoksin A ve aflatoksin varlığının incelenmesi ve miktarlarının belirlenmesi amaçlanmaktadır. Çalışma, Güneydoğu Anadolu Bölgesi?nde yer alan bir işletmeden temin edilen ve İstanbul marketten toplanan Antep fıstıkları ile yürütülmüştür. Gaziantep?ten temin edilen numunlerin her biri 2-3 tonu temsil etmektedir. İstanbul?da ise market, pazar ve küçük işletmelerden toplanan ürünler paketli ve paketsiz, kabuklu ve kabuksuz olarak farklı markalardan ve semtlerden temin edilmiştir. Analitik metot olarak immunoafinite kolon temizlemesini takip eden, floresans dedektörle birleştirilmiş yüksek performans sıvı kromatografisi kullanılmıştır. 
Antep fıstığı yüksek yağ içeriğine sahip bir üründür. Okratoksin A ve aflatoksin analizlerinde, ekstraksiyon aşamasında yağın ve diğer bileşenlerin uzaklaştırılması için organik çözgen ve su karışımı kullanılmıştır. Vicam marka immunoafinite kolonlarda toksinin saflaştırılması gerçekleştirilmiştir. Ardından HPLC ile analizlenen örneklerde toksin tayini ve miktar belirlenmesi gerçekleştirilmiştir. Aflatoksin analizinde KoBra-cell kimyasal türevlendirilmesi uygulanmıştır.  
73 örnekte okratoksin A ve aflatoksin taraması yapılan çalışmada, 54 örnek (%74) pozitif bulunmuştur. 19 örnekte ise toksin tespit edilmemiştir.
Tüm Antep fıstığı örneklerinin %6?sında 1,238 - 3,720 ?g/l arasında değişen düzeylerde OTA varlığı belirlenmiştir. OTA kontaminasyonu yalnızca Antep kırmızı örneklerinde (%7 oranında) tespit edilmiş, Antep yeşil ve Siirt çeşitlerinde OTA varlığına rastlanmamıştır. OTA tespit edilen örneklerin tümünde aflatoksin varlığına da rastlanmıştır.
Aflatoksin analizi sonucunda 73 adet örnekte 54 adet toplam aflatoksin (AFT) kontaminasyonu tespit edilmiştir. AFT seviyeleri, 0,001 ile 23,542 ?g/l arasında değişiklik göstermiştir. 23,542 ve 11,504 ?g/l?lik toksin içerikleri ile yasal limitlerin (doğrudan tüketim ve hammadde olarak kullanım için; 10 ?g/l) üzerinde 2 adet örnek tespit edilmiştir. 58 Antep kırmızı örneğinde 44 pozitif, 4 Antep yeşil örneğinde 4 pozitif ve 11 adet Siirt çeşidi örnekte 6 pozitif numune gözlenmiştir.
73 örnekte yalnızca AFB1 kontaminasyonu saptanan 20 örnek (%27) bulunmaktadır. Bu 20 örneğin 15 tanesi Antep kırmızı, 2 tanesi Antep yeşil ve 3 tanesi Siirt fıstığıdır. Tüm örnekler içinde 6 örnekte (%8), aflatoksin B1 ve aflatoksin B2 (AFB1 + AFB2) birlikte tespit edilmiştir. Bu 6 örneğin 4 tanesi Antep kırmızı ve 2 tanesi Antep yeşil çeşidine aittir. Antep kırmızı çeşidinde ait 1 örnekte (%1) aflatoksin B1 ve aflatoksin G1 (AFB1 + AFG1)  birlikte tespit edilmiştir. Siirt çeşidinde ait 1 örnekte (%1) aflatoksin B1 ve aflatoksin G2 (AFB1 + AFG2) birlikte tespit edilmiştir. 5 adet örnekte (%7) ise AFB1 + AFB2 + AFG2 belirlenmiştir. Bunların 3 tanesi Antep kırmızı diğer 2 tanesi de Siirt çeşidine aittir. Örneklerin 21?inde (%29) 4 aflatoksin türü birarada tespit edilmiştir. 4 aflatoksini bir arada bulunduran örneklerin tümünün Antep kırmızı olduğu gözlenmiştir.

Abstract:
Pistachio nut (Pistacia vera L.) is a member of Anacardiaceae family and its origin is arid regions of Central and West Asia and scattered throughout the Mediterranean basin. It has high nutritional value and delicious taste which resulted in spreading to many countries throughout the history and starting to be grown in different regions. Pistachio is one of the tree nuts which is an important export product for our country with its high economic value. Tree nuts have sterols, vitamins, minerals, fatty acids, phenolic compounds. Moreover, they show antioxidant and antiproliferative properties and they have been preferred with their rich nutritional components. According to data from the year 2011, Turkey took third place in globe pistachio production with 13% ratio. The production of Turkish pistachios in 2011 year has reached 112.000 tonnes. The world?s pistachio export data which belong to 2010 year shows that Turkey took twelfth place and the amount of export was 717 tons. According to the world?s pistachio import data which belong to same year shows that Turkey was not among the top 20 countries. In terms of exporting countries of Turkey in 2010 and 2011, Italy, Germany and Israel ranks the top three ones. Italy 47.9%, Germany 9.8% and 9.0% of Israel have a share.
Commercially valuable, tasty and rich in nutrients pistachio nuts are exposed to the mold growth due to the production method and other environmental factors. Fungi species are commonly found in nature as well as can occur jointly in a wide range of food commodities. Mycotoxins are secondary metabolites produced by some fungal species. 
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. Ochratoxin A (OTA) is a mycotoxin which is produced by Aspergillus and Penicillium genus. OTA is a pentaketide secondary metabolite that contaminates different plant products, cereals, coffee beans, nuts, cocoa, pulses, beer, wine, spices and dried vine fruits. It is mainly produced by Penicillium verrucosum and P. nordicum, and by a range of Aspergillus species assigned to sections Circumdati, Nigri and Flavi which Aspergillus ochraceus, Aspergillus niger and Aspergillus carbonarius species are the main source of ochratoxin contamination in foods and feeds. 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 its production is higher than under the light. OTA exhibits nephrotoxic, immunosuppressive, teratogenic, genotoxic, hepatotoxic properties which make it one of the most important mycotoxins of concern for human health. It has been grouped as possible cancerogen for human (group 2B) by International Cancer Research Center (IARC). Kidney has thought as target organ and it is associated with Balkan Endemic Nephropathy and urinary tract tumours. OTA contamination can also be transferred from feed into animal products, especially in organ meat (kidney, liver and blood).
OTA contamination is a great concern for tropical countries where the elevated temperature and water activity favor growth of mycotoxigenic fungi in agricultural products, especially in post-harvest activities. The European Commission Regulation has not established legal limits for OTA in pistachio nuts. Aspergillus species are more commonly associated with warmer and tropical climates from stored foods mostly. Growth and OTA production by members of the Aspergillus spp. Can be influenced by agricultural practices such as the use of pesticides, the time and conditions of harvesting, drying and handling the crop.
Aflatoxins (AFs) are the most toxic and the strongest natural carcinogens that are produced primarily by two species of Aspergilli, Aspergillus flavus and Aspergillus parasiticus. The AFs consist of about 20 similar compounds belonging to a group called the difuranocoumarins, but only four aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 (AFB1, AFB2, AFG1 and AFG2) are naturally found in foodstuffs. AFB1 is the most toxic and amongst the most commonly found in foods. These secondary metabolites can be found in a variety of food commodities, including cereals, spices and dried fruits (figs, raisins). Moreover, AFs have also been detected in oilseeds, peanuts and tree nuts (pistachios, almonds, pecans, walnuts etc.). These mycotoxins are crystalline, easily soluble in polar solvents such as chloroform and methanol. Pure form of AFs are degradable under ultraviolet (UV) radiation  and at low (<3) and high (>10) pH. Besides under the presence of oxidising agents it is unstable compound. These mycotoxins are very stable in food and feed compounds. They are resistant to temperatures above 100 °C in the crystalline form, unless exposed to light and UV radiation. When feed products are contaminated with mycotoxins, there might be a health hazard for animals. Furthermore, myctoxin residues in feed to be consumed by animals may cause a threat to humans. IARC has classified AFB1 as a group 1 carcinogen which means it is carcinogenic for human, mainly affecting the liver. AFB2, AFG1 and AFG2 are also classified as possible carcinogen to humans. They have carcinogenic, mutagenic, teratogenic, hepatotoxic and  immune-suppressing effects on human and animal health. Aflatoxicosis causes acute liver damage, liver cirrhosis, induction of tumors, impaired central nervous system, skin disorders, hormonal defects and decreased bone strength. This product has high commercial value and in terms of public health its presence in food and feed products has great importance. In many countries, the awareness against the harmful effects of mycotoxins become more of an issue. Those countries have established the maximum tolerable levels of mycotoxins to protect health of animals and human and prevent economical losses. According to European Commission Regulation (2010), pistachio nuts are not permitted to exceed 8,0 ng/g for AFB1 and 10 ng/g for total aflatoxins for direct human consumption or use as an ingredient in foodstuffs; 12,0 ng/g for AFB1 and 15,0 for total aflatoxins in pistachios to be subjected to sorting or other physical treatment, before human consumption or use as an ingredient in foodstuffs. 
The purpose of  this study to investigate the ochratoxin A and aflatoxins presences and detect their amounts in pistachio nuts cultivated in Turkey. This study was carried out with pistachio nuts that some of the samples were obtained from a company which is located in Southeastern Anatolia Region and the remaining samples were collected from Istanbul market. Each of the samples gathered from Southeastern Anatolia Region represents 2-3 tonnes. In Istanbul, the pistachios are collected from the markets, bazaars and small businesses with packaged and unpackaged, shelled and unshelled product properties that were supplied in different districts with different brands. High performance liquid chromatography (HPLC) is now the most commonly used chromatographic technique for the detection of wide diversity of mycotoxins. In this study, an analytical method based on immunoaffinity column clean-up and followed by the high performance liquid chromatography equipped with fluorescence detection were performed for both ochratoxin A and aflatoxins analysis. 73 pistachio samples were analysed and the results were evaluated with respect to European Commission Regulation and Turkish Food Codex.
At the beginning of the analysis the samples were subjected to extraction and subsequently clean-up stage. The tree nuts have high amount of lipid content. Thus, its important to performance effective extraction. By the purpose of removal of the oil phase and the other components, organic solvent and water mixture were used. In order to purification of toxin, samples were subjected to immunoaffinity columns with the brand of Vicam. Afterwards, samples were analysed by HPLC to detect the presences of toxin and determine their amounts in pistachio samples. In aflatoxins analysis the koBra-cell derivatization was applied which differ from ochratoxin A analysis.
73 pistachio samples were analysed and the results obtained from the study are 54 of them (74%) were positive with mycotoxin contamination. The screening 19 pistachio samples were toxin-free. OTA was identified in 4 samples (6%) from the levels ranging from 1,238 to 3,720 ?g/l levels. 
In all red kernel pistachios the contamination ratio was 7%. OTA was not detected in green kernel pistachios and Siirt cultivars. In all OTA found samples, the presence of aflatoxins were also detected.
The analysis of aflatoxins was performed and results showed that 54 of 73 samples were contaminated by these detrimental toxins. The total aflatoxin (AFT) levels of positive samples are ranging from 0,001 to 23,542 ?g/l. The detected levels of 2 samples, 23,542 and 11,504 ?g/l, were above the legal limits. 44 of 58 red kernel pistachios, 4 of 4 green kernel pistachios and 6 of 11 Siirt cultivars were positive with AFT. 
AFB1 contaminate 20 pistachio nuts of total 73 samples (27%). These 20 positive samples include 14 red kernel pistachios, 2 green kernel pistachios and 3 Siirt cultivars. AFB1 and AFB2 was found together in 6 samples of total 73 samples (8%). These 6 samples include 4 red kernel pistachios and 2 green kernel pistachios. Just 1 red kernel pistachio sample was found to be contaminated by AFB1 and AFG1. Moreover, 1 sample of Siirt cultivar type was positive with AFB1 and AFG2 types. AFB1, AFB2 and AFG2 were detected together in 5 samples of total 73 samples (7%). 2 samples of them belong to the type of red kernel pistachio and the remaining 2 samples belong to the type of Siirt cultivar. Four types of aflatoxins, AFB1, AFB2, AFG1 and AFG2 were found in 20 samples of total 73 samples. All positive samples which contaminated by them belong to the red kernel pistachio samples.
Raw material control, good agricultural practices (GAP), good manifacturing practices (GMP), good hygene practices (GHP) are necessary during harvesting and post harvesting phases, in order to prevent OTA and AFs. It is vitally important to prevent contamination in raw material to prevent to mycotoxin producing. As well as controlling raw material, the place where it is kept being under control is significant. During harvesting and post harvesting, occurrence of OTA and AFs can be prevented using good manufacturing practices, proper storing conditions and using high quality raw material. Preventing OTA and AFs is necessary in terms of protecting health and preventing economic losses.