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İstanbul Teknik Üniversitesi (2013)

Determination Of Ochratoxin A And Aflatoxins In Pistachio Nuts

Sedefoğlu, Cansu

Titre : Determination Of Ochratoxin A And Aflatoxins In Pistachio Nuts

Antep Fıstıklarında Okratoksin A Ve Aflatoksin Varlığının İncelenmesi

Auteur : Sedefoğlu, Cansu

Université de soutenance : İstanbul Teknik Üniversitesi

Grade : Master of Science (MS) 2013

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 μg/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 μg/l. The detected levels of 2 samples, 23,542 and 11,504 μg/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.

Mots clés : Antep fıstığı Okratoksin A Aflatoksin HPLC Pistachio Nuts Ochratoxin A Aflatoxin HPLC


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