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R.Lo Curto, F. Vilasi, T. M. Pellicanò, P.Munafò, G. Dugo PRESENCE OF OCHRATOXIN A IN EXPERIMENTAL WINES RELATED TO CHEMICAL AND NATURAL TREATMENTS EMPLOYED.

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Presentation on theme: "R.Lo Curto, F. Vilasi, T. M. Pellicanò, P.Munafò, G. Dugo PRESENCE OF OCHRATOXIN A IN EXPERIMENTAL WINES RELATED TO CHEMICAL AND NATURAL TREATMENTS EMPLOYED."— Presentation transcript:

1 R.Lo Curto, F. Vilasi, T. M. Pellicanò, P.Munafò, G. Dugo PRESENCE OF OCHRATOXIN A IN EXPERIMENTAL WINES RELATED TO CHEMICAL AND NATURAL TREATMENTS EMPLOYED ON GRAPES Milano ottobre 2002

2 Their presence is depending upon factors such as fungal strain, climate and geographical conditions, cultivation technique and foodstuff conservation. Milano ottobre 2002 MYCOTOXINS Class of highly toxic chemical compounds produced, under particular environmental conditions, by several moulds developing in many foodstuffs. May occur in various vegetal products as cereals, dried fruits, coffee bends, cocoa and common beverages as beer and wine. Among mycotoxins very important are the ochratoxins which include a group of metabolites, showing similar chemical structures, produced by strains of the genus Aspergillus (A. ochraceus) and Penicillium (P. ferrucosum)

3 Term indicates a group of metabolites, having similar chemical structure, produced by fungi of the genus Aspergillus ochraceus and Penicillium ferrucosum. Milano ottobre 2002 OCHRATOXIN The most studied compound, both for its diffusion and toxicological importance, is Ochratoxin A, chemically R-N-[(5-chloro-3,4-dihydro-8-hydroxy- 3-methyl-1-oxo-1H-2benzopyran-7-yl)carbonyl]-phenylalanine.

4 Highly toxic, it caused severe animal and human intoxications (Porcine nephropathy and Balcan Endemic nephropathy) Milano ottobre 2002 OCHRATOXIN A OTA exhibits nephrotoxic and teratogen activites and moreover suppressive actions on immune system, causing a diminution of immune globulin level and of other humoral factors both in mice and chicken and a reduction of cell immune responses Its presence at levels ranging 0,1- 40 ng\ml in biologic fluids demonstrates that about 50% of European countries, enclosed Italy, are exposed to this contamination

5 WHO/FAO/JECFA proposes that Provisional Tolerable Weekly Intake (PTWI) value, on renal damage basis, is 100 ng\Kg body weight Milano ottobre 2002 LEGISLATION Regulation 466/2001 EEC sets limits in food products Regulation 472/2002 EEC (modification of Regulation 466/2001 EEC) European Commission 2001 defines acceptable sampling and analysis methods

6 Milano ottobre 2002 TOLERABLE LIMITS ( g/Kg) OF OCHRATOXIN A IN SOME FOOD (472/2002 EEC) Childhood food0.5 No toasted coffee8 Toasted coffee and instant coffee4 Cocoa and derivate products0.5 Beer0.2 Pork and derivate products1 Cereals and derivate products3 Dried grape fruits (sultanas, currants, raisins)10

7 Milano ottobre 2002 OCHRATOXIN A AND WINE Wine is a product widely consumed and it may represents, after cereals, a major source of daily OTA intake for people. Codex Alimentarius Commission suggest that 15% of the total intake of this toxin is due to wine consumption Some Authors report a high toxin concentration (up to 7.0 ng\ml) with considerable level of contamination (incidence up to 92%) in red wine produced in southern regions of Europe and in North Africa. Other Authors report OTA contamination levels ranging 3,9% for white wines to 16,6% for red wines Generally, red wines contain a greater amount of OTA than the white or rosè ones. These differences can be attributed to climatic factors, grape cultivation and storage conditions apart from wine-making

8 Milano ottobre 2002 A reversed-phase HPLC method was utilized for Ocrhratoxin A (OTA) determination in 23 white and red wine samples produced in the year 2000 in three experimental vineyards, situated in three different Italian regions and treated with different pesticides Analytical methods utilized to detect OTA include commercial immunoaffinity columns (Visconti et al.) and a HPLC system equipped with a RF detector. The method sensibility was 0.01 ng /ml -1

9 Milano ottobre wine samples (16 white and 7 red), collected in the year 2000 from three experimental vineyards situated in the countries Campania (Avellino), Sicily (Catania) and Tuscany (Grosseto), were analyzed. WINE SAMPLES

10 Milano ottobre 2002 White wines were produced in Sicily and Campania. Sicilian wines were produced from years old plants, grown up on Etna (300 m asl, S. Venerina, Catania, Italy), in a vulcanic soil. Vines were grafted with Inzolia and Carricante varieties in 1:1 ratio. Wines from Campania were produced from 25 years old plants grown up on Montefredane hills (700 m asl, Avellino, Italy), in a clayey soil. Vines were grafted with Fiano dAvellino variety. Red wines were produced in Tuscany from 25 years old plants, cultivated in Maremma Toscana coast (100 m asl, Grosseto, Italy), on a calcareous soil. Vines were grafted with Sangiovese variety, Morellino clone. Vinification process, started within 24 hours after grape harvesting. WINE CHARACTERISTICS AND PESTICIDE TREATMENTS

11 Milano ottobre 2002 Wine samplesPesticide and treatments utilized during grape ripening Sicily 11 Sulphur 80 Pb6 Quinoxyfen250 SC Sicily 31 Sulphur 80 Pb6 Fenarimol 12 SC Sicily 41 Sulphur 80 Pb6 Azoxystrobin 250 SC Sicily 51 Dinocap 350 EC6 Penconazole 100 EC Sicily 81 Sulphur 80 Pb6 Sulphur 80 Pb Sicily 91 Sulphur 80 Pb6 Dinocap350EC Sicily 101 Sulphur Powder6 Sulphur Powder Sicily 111 Water6 Water Pesticide treatments on Inzolia and Carricante(Sicily) variety grapes

12 Milano ottobre 2002 Wine samplesPesticide and treatments utilized during grape ripening Campania 12 Sulphur 80 Pb6 Quinoxyfen 250 SC Campania 22 Sulphur 80 Pb6 Fenarimol 12 SC Campania 32 Sulphur 80 Pb6 Azoxystrobin 250 SC Campania 42 Sulphur 50 PS6 Sulphur 50 PS Campania 52 Dinocap350EC6 Penconazole 100 EC Campania 62 Sulphur 80 Pb6 Dinocap 350 EC Campania 72 Water6 Water Campania 82 Sulphur 80 Pb6 Sulphur 80 Pb Pesticide treatments on Fiano di Avellino(Campania) variety grapes

13 Milano ottobre 2002 Wine samplesPesticide and treatments utilized during grape ripening Tuscany 1 2 Sulphur 80 Pb9 Quinoxyfen 250 SC Tuscany 2 2 Sulphur 80 Pb9 Fenarimol 12 SC Tuscany 3 2 Sulphur 80 Pb9 Azoxystrobin 250 SC Tuscany 4 2 Dinocap 350 EC9 Penconazole 100 EC Tuscany 5 2 Sulphur Powder9 Sulphur Powder Tuscany 6 2 Sulphur 80 BWG9 Sulphur 80 PBWG Tuscany 7 2 Dinocap 350 EC6 Sulphur + 3 Quinoxyfen 250 SC Pesticide treatments on Sangiovese(Tuscany) variety grapes

14 Milano ottobre 2002 Standard of OTA, sodium chloride, polyethylene glycol (Peg 8000), sodium hydrogencarbonate, glacial acetic acid and toluene were obtained from Fluka (Milano-Italy) Immunoaffinity columns were purchased from Vicam ( Waterton, MA, USA ) Acetonitrile, methanol and water (HPLC grade) were supplied from Carlo Erba Reagents (Milano-Italy) STANDARDS

15 Milano ottobre 2002 HPLC/RF ANALYSIS HPLC Shimadzu: Controller System SCL-10A 1 Pump LC-10A Detector RF-10AXL: ( ex =330 nm, em =460 nm) Injector Rheodyne: loop da 20 l Reversed-phase Supelco Column C 18 (5 m 150 x 4.6 mm)

16 Milano ottobre 2002 Analyses were run at room temperature in isocratic conditions with a mobile phase compose of water / acetonitrile / acetic acid 99:99:2 v/v/v at a flow rate of 1 ml/min. OTA quantification is made by measuring peak areas at OTA retention time and by comparing them with calibration curve. A wine sample was added with OTA to confirm the OTA identification standard. The sensibility test of method was of 0,01 ng/ml of mycotoxin. HPLC/RF ANALYSIS

17 Milano ottobre 2002 HPLC chromatogram of standard OTA

18 Milano ottobre 2002 Wine SamplesOchratoxin A (mg L -1 ) Sicily 1n. d. Sicily Sicily Sicily Sicily Sicily Sicily Sicily 11traces Tuscany Tuscany Tuscany Tuscany Tuscany Tuscany Tuscany Concentration of OTA in wine samples

19 Milano ottobre 2002 No OTA traces were found in white wine sample from Campania In six samples from Sicily OTA traces were found; (0.03 ng\ml for sample treated with dinocap). The sample treated with quinoxifen and the sample treated with water were found not contaminated with OTA Values of OTA found in red wines show that all samples are contaminated The wine produced with grapes treated with sulphur 80 PBWG only, showed a Ochratoxin A content (2.00 ng\ml) higher than the other samples, followed by the sample obtained from grapes treated with powdered sulphur (0.71 ng\ml). The concentration of OTA in the other samples spanned from 0.07 ng\mg in sample treated with azoxystrobin to 0.24 ng\mg in sample treated with dinocap and penconazole. The values of OTA found, are comparable with those reported in literature on micotoxins presence in red wines. Results

20 Milano ottobre 2002 Values of OTA found in wine samples show that mycotoxins more frequently detected in red than in white wines The OTA levels reported in this work are comparable with those reported in literature on mycotoxin presence in red wine Moreover the content of Ochratoxin A in the wines can be considered an index of pesticides effectiveness on parasitic fungi. The experimental vineyards located in Campania seem not to be subjected to pollution from OTA-producing fungi. The experimental Sicilian cultivars seem, on the contrary, more easily subjected to OTA contamination since, of eight wine samples, six were found contaminated. Values of OTA found in red wines show that experimental vineyards located in Tuscany are strongly subject to infection from OTA-producing fungi. Synthetic pesticides can reduce OTA concentration from 96.5% in the sample treated with azoxystrobin, to 88% in the sample treated with dinocap and penconazole. Since OTA is strictly related to the growth of some toxigenic fungi on grapes, the different Ochratoxin A content in wine samples can be also considered an efficiency test of the pesticides used. CONCLUSION

21 Milano ottobre 2002 The experimental vineyards located in Campania seem not to be subjected to pollution from OTA-producing fungi. The experimental Sicilian cultivars seem, on the contrary, more easily subjected to OTA contamination since, of eight wine samples, six were found contaminated. Values of OTA found in red wines show that experimental vineyards located in Tuscany are strongly subject to infection from OTA-producing fungi. Synthetic pesticides can reduce OTA concentration from 96.5% in the sample treated with azoxystrobin, to 88% in the sample treated with dinocap and penconazole. Since OTA is strictly related to the growth of some toxigenic fungi on grapes, the different Ochratoxin A content in wine samples can be also considered an efficiency test of the pesticides used. CONCLUSION


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