Kırmızı pul biberlerde aflatoksin ve okratoksin a varlığının incelenmesi
Natural occurence of aflatoxin and ochratoxin a in red peppers
Yazar: SEBAHAT ÖZAKÇA
Danışman: YRD. DOÇ. HATİCE FUNDA KARBANCIOĞLU GÜLER
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2014
Konu: Gıda Mühendisliği = Food Engineering
Dizin: Aflatoksinler = Aflatoxins ; Kırmızıbiber = Paprika ; Kırmızıbiber = Paprika ; Mikotoksinler = Mycotoxins ; Okratoksin-A = Ochratoxin-A ; Okratoksinler = Ochratoxins

Özet:
Mikotoksinler; baharatlarda ve diğer gıdalarda başlıca Aspergillus, Penicillum ve Fusarium gibi küf cinslerinin oluşturduğu toksik metabolitlerdir. Baharatlar genellikle sıcaklık, yağış ve nem dalgalanmalarının olduğu tropikal iklime sahip ülkelerde üretilmektedir. Hasat edilen baharatlar, geleneksel kurutma yöntemleri ile açık havada yere serilerek kurutulduğundan küf gelişmesine ve mikotoksin kontaminasyonuna açık hale gelirler. Kırmızıbiber, patlıcangiller (Solanaceae) familyasının Capsicum cinsine bağlı Capsicum annum L.'nin kurutulmuş bir formu olup ılıman iklimlerde yıllık yetişen bir kültür bitkisidir. Kırmızıbiber üretiminde kurutma, taşıma ve depolama sırasında yetersiz hijyen koşulları mikotoksin oluşumuna neden olabilir. 
Yapılan çalışmada Güneydoğu Anadolu Bölgesi ve İstanbul'daki market ve pazarlardan toplanan 54 adet kırmızıbiber ve isot örneklerinde aflatoksin ve okratoksin A varlığı çoklu immunoaffinite kolonla temizleme sonrasında floresans dedektör ile HPLC'de ters faz kolonda araştırılmıştır. 
Örneklerin %93'ünde  (52/54)  AFB1 (0,20-38,69 g/kg), %74'ünde (14/54) AFB2 (0,04-2,14 g/kg), %17'sinde (9/45)  AFG1  (0,13-0,88 g/kg) ve  %94'ünde (51/54) OTA (0,18-52,19 g/kg) bulunmuştur. Örneklerin %93'ünde toplam aflatoksin miktarı 0,20-40,59 g/kg arasında değişmektedir. Örneklerin hiçbirinde AFG2 varlığına rastlanmamıştır. 
Geleneksel yöntem ve işletme ile kurutulan biber örneklerinde bulunan AFB1 kontaminasyon düzeyleri kırmızı pul biber için sırasıyla 9,18 µg/kg ve 1,17 µg/kg; isot biberi için ise sadece geleneksel yöntemde 4,30 µg/kg düzeyinde tespit edilmiştir. Geleneksel yöntem ve işletme ile kurutulan kırmızı pul biber ve isot örneklerinde bulunan toplam aflatoksin kontaminasyon düzeyleri kırmızı pul biber için sırasıyla 9,75 µg/kg ve 1,28 µg/kg; isot biberi için ise sadece geleneksel yöntemde 4,60 µg/kg düzeyinde tespit edilmiştir. 
16 adet kırmızı pul biber (%30) ve 1 adet isot biberi (%2) örneği AFB1; 14 adet kırmızı pul biber  (%26 ) örneğinin ise toplam aflatoksin açısından sınrın üzerinde olduğu belirlenmiştir. Buna karşın hiçbir isot biberi toplam aflatoksin açısından yasal limitlerin üzerinde bulunmamıştır. 4 adet kırmızı pul biber (%7,4) ve 1 adet isot (%2) örneklerinin Avrupa Birliği limitlerini aştığı tespit edilmiştir.  
AFB1, AFB2 ve OTA mikotoksinlerinin eş zamanlı olarak kırmızı pul biberlerin %53,7'sinde bulunması GAP (İyi tarım uygulamaları) ve GMP'nin (İyi üretim uygulamaları) önemle uygulanması gerektiğini işaret etmektedir. Bunun yanında, yaralı ve hastalıklı kırmızı pul biberler ortamdan uzaklaştırılmalı, kurutma sırasında toprakla temastan kaçınılarak biberler hijyenik tesislerde kurutulmalı ve sonrasında mikotoksin kontaminasyonunu önlemek için güvenli ambalaj kullanılmalıdır. Bunların yanında Türkiye'de Gıda, Tarım ve Hayvancılık Bakanlığı tarafından  çiftçilere, gıda üreticilerine ve tüccarlara GMP ve GHP hakkında rutin eğitim programları düzenlenmelidir. 
Sonuçlar incelendiğinde, işletmede kurutulan biberlerdeki mikotoksin kontaminasyonunun geleneksel yöntemlerle kurutulan biberlere kıyasla daha az olduğu bulunmuştur.Biber tarımının ve kurutmasının yoğun olarak yapıldığı Güneydoğu Anadolu illerinde kurutma kooperatiflerini kullanmayı aktifleştirmek için çalışmalar yapılabilir.

Abstract:
In today's changing world, safety and security have generally remained basic human needs. Ensuring the safety of food has been a major focus of international and national action over the last few years. The problem of food and feed contamination with aflatoxins is currently a hot issue and has received a great deal of attention during the last three decades. 
Mycotoxins are toxic metabolites produced by some species of mould genera such as Aspergillus, Penicillium and Fusarium. Moulds cause a decrease in quality of the food on which they grow, and also create a risk for human health because of some toxic metabolites they produce, which are called mycotoxins. The food contaminated with mycotoxins, when consumed by human and animals may cause diseases called mycotoxicosis and poisons even resulting in death.
Moulds are widely distributed as environmental contaminants. Raw food materials, vegetables and spices may be contaminated before processing anywhere in the food chain. Spices are produced in countries with tropical climates that have high ranges of temperature, humidity and rainfall.
Chilli also called red pepper belongs to the genus Capsicum, under the nightshade family, Solanaceae. Chilli (the dried form of Capsicum annum L.) is the second largest consumed spice throughout the world, after black pepper. Turkey is the third largest producer of chilli and pepper with a production of 1.84 million metric tonnes, followed by China and Mexico.
The presence of mycotoxigenic moulds in Capsicum products is extremely common, this does not necessarily infer the presence of mycotoxins, since mycotoxin pro- duction can be influenced by the toxigenic capacity of the strain, intrinsic factors (such as substrate composition) and extrinsic factors (temperature and incubation time).
Because of poor hygienic conditions and deficient water activity (aw) control during processing of peppers can lead to fungal growth, which can result in mycotoxin accumulation in peppers. Transportation, packaging and storage affect the microbial load of red peppers.
The incidence of AFs in foods and feeds is relatively high in tropical and subtropical regions, where climatic conditions provide an optimal environment for the growth of moulds. Most spices are produced in tropical and subtropical regions. The hot and humid climate, production conditions with extended drying times, and often inadequate instruc- tions to the farmers, may cause considerable quality problems. It has been reported that 5–10 %of agricultural products in the world are being spoiled by mould to the extent that they cannot be consumed by human and animals.
Mould growth in agricultural products may cause an important hazard to human health by the formation of toxic metabolites called "mycotoxin". Aflatoxins (AF) belong to the group of mycotoxins. AFs are a group of highly toxic secondary metabolic products of Aspergillus flavus, Aspergillus parasiticus have carcinogenic, teratogenic effect to livestock and humans.
Most of the mycotoxins are produced by mould species belonging to genus Aspergillus, Penicillum and Fusarium. Aspergillus and Penicillum, which are called store mould, contaminate and grow especially following harvest, during drying and storage period. Insufficient drying and unsuitable storage conditions favor their growth. Red pepper is a very sensitive product for aflatoxinformation depending on unsuitable processing conditions. Many survey showed the presence of xerophilic mould species, especially Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus ochraceus, in most pepper samples.
Drying on bare ground is a wrong application that results in toxic mould contamination and mycotoxin production. Since large drying periods will enhance mould growth, drying time should be kept as short as possible. In southern Anatolia, climatic conditions are suitable to produce mycotoxins in chilli producing areas, therefore there is a potential risk for red peppers. GAP (Good Agricultural Process) and  HACCP (Hazard Analyses of Critical Control Points) should be applied to prevent mycotoxins throughout the entire red pepper production and processing chain. 
This study aims to determine the co-occurrence of aflatoxin (AF) and ochratoxin (OTA) in 54 red chilli flakes and isot pepper samples which are collected from especially southeastern Anatolia of Turkey (Adana, Ankara, Gaziantep, Kahramanmaraş, Şanlıurfa)  and İstanbul markets. The mycotoxins were determined by high performance liquid chromatography coupled with fluorescence detection (HPLC-FD) after multiple immunoaffinity (IAC) clean-up. The LODs and LOQs were calculated for each mycotoxin; AFB1 (0.11 µg/kg; 0.18 µg/kg), AFB2 (0.04 µg/kg ; 0.08 µg/kg), AFG1 (0.13 µg/kg ; 0.18 µg/kg), AFG2 (0.04 µg/kg; 0.11 µg/kg), OTA (0.1 µg/kg; 0.21 µg/kg)
Aflatoxin B1 was detected in 52 of 54 of red chilli flakes (93 %) concentrations ranged between 0.20-38.69 g/kg; Aflatoxin B2 was found in 14 of 54 red chilli flakes (74 %) with a range 0.04-2.14 g/kg; Aflatoxin G1 was found in 9 of 54 of red chilli flakes (17%) with levels ranges from 0.13 g/kg to 0.88 g/kg; OTA was determined in 51 of 54 of red chilli flakes (94%) with a range between 0.18-52.19 g/kg. None of the red chilli flake and red chilli powder samples contain AFG2. The total aflatoxin contamination levels in positive red chilli flake and red chilli powder samples varied from 0.20 to 40.59 µg/kg.
The mean AFB1 contamination levels in red chilli flake and isot pepper samples according to their traditional and mechanical drying productions are; 9.18 and  1.17 µg/kg;  4.30 µg/kg- not detected, respectively. The mean total AF contamination levels in red chilli flake and isot pepper samples according to their traditional and mechanical drying productions are; 9.75 and 1.28 µg/kg;  4.60 µg/kg- not detected, respectively. 
AFB1 levels in 16 red chilli flake (30 %) and 1 isot samples (2 %)  were above the EU limit of 5 µg/kg, while 14 red chilli flakes (26 %) exceeded legal limit of 10 µg/kg for total AFs while none of the isot samples exceed the limits. OTA levels in 4 red chilli flake (7.4 %) and 1 isot samples (2 %)  were above the EU limit of 10 µg/kg.
AFB1 and AFB2 were detected more frequently than AFG1 and AFG2 in both red chilli flake and red chilli powder samples. This could be explained by both occurrence and invasion of red chilli samples by A. flavus rather than A. parasiticus. In a similar work, red-scaled pepper samples from southeastern Anatolia of Turkey were contaminated with A. niger, A. flavus, A. versicolor, A. ochraceus and Penicillium spp. 
With regard to OTA, 51 of 54 red chilli flakes (94 %) showed OTA contamination. Four of them were contaminated with OTA at levels greater than regulatory limits establised in EU legislation (10 µg/kg). 
The ranges of OTA in positive red chilli flakes were 0.18 – 52.19 µg/kg, with a mean concentration of 4.62 µg/kg. For red chilli flakes OTA was detected in 40 of  41 samples (97.5 %). For isot type pepper, OTA was detected in 11 of 13 samples (85 %)  respectively. Besides AFB1, AFB2 and OTA was simultaneously detected in 29 red scaled peppers (53.7 %).  
Red chilli flake is highly susceptible to fungal contamination and subsequently mycotoxin formation due to their environmental (high humidity and high temperature) and processing conditions. Red chilli can be contaminated with toxigenic fungi in the field during crop production, but also during drying process and in storage. For this reason, fresh fruits must be washed after harvesting and injured and diseased fruits must be removed. The moisture content of red chillies (65-80%) on harvesting should be immediately reduced to about 10%by drying process to avoid mould growth and mycotoxin accumulation.
The simultaneous occurrence of AFs and OTA in a half part of red chillies indicates the importance of good agricultural practicing (GAP) and good manufacturing practising (GMP), including removing injured and diseased fruits, avoiding the contact with soil during drying, improving the drying method and storage conditions, hygienic aspects and safe packaging to avoid mycotoxin contamination. Moreover, training programs should be presented routinely for farmers, food manufacturers and traders about GAP and GMP by Ministry of Food, Agriculture and Livestock.
Our data also showed that red chilli flakes and isot peppers were simultaneously contaminated by AFs and OTA, while co-occurrence of AFB1, AFB2 and OTA was observed in 53.7 % (29/54 samples).
A combined intake of different type of mycotoxins may lead to a possible higher risk for adverse health effects than the intake of one of these mycotoxins alone. 
This is the first study concerning the simultaneous occurrence of aflatoxin and ochratoxin in red chilli flake and isot peppers with using multiple immunoaffinity coloumn from Turkey. Although there is little information about interaction between mycotoxins and the issue of combined toxicity is quite complex, it is expected that mycotoxins with similar mode of action would have at least an additive effect.The results will shed light on the cause of the co-occurence of AFs and OTA in red scaled peppers.