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Occurrence of ochratoxin A in Turkish wines
Microchemical Journal 86 (2007) 241 – 247 www.elsevier.com/locate/microc
Occurrence of ochratoxin A in Turkish wines I. Var, B. Kabak ⁎ Cukurova University, Agricultural Faculty, Department of Food Engineering, TR-01330, Adana, Turkey Received 22 March 2007; accepted 6 April 2007 Available online 20 April 2007
Abstract A total of 95 wine samples including 34 white, 10 rosé and 51 red wines originating from four different Turkish areas were analysed for ochratoxin A (OTA). An analytical method based on immunoaffinity column (IAC) for clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD) was used to determine OTA in wines. The limit of detection (LOD) was estimated as 0.006 ng ml− 1 for white wine and 0.010 ng ml− 1 for rosé and red wines. The limit of quantification (LOQ) was estimated as 0.020 ng ml− 1 in white wine and 0.030 ng ml− 1 in rosé and red wines. Recovery experiments were carried out with spiked samples in the range 0.1–1 ng ml− 1 of OTA. The average OTA recoveries from spiked white wine samples varied from 79.43% to 85.07%; while the mean recoveries for rosé and red wine samples were in the range of 77.48–83.96% and 76.61–83.55%, respectively. OTA was detected in 82 (86%) wine samples at levels of b 0.006–0.815 ng ml− 1, which were below the maximum allowable limit established by the European Community. The mean OTA concentration in red wines was slightly higher than in white and rosé wines. Furthermore, our data indicate that the geographic region of origin has strong influence on OTA level for white, rosé and red wines: wines originating from Thrace (n = 44, mean = 0.158 ng ml− 1) and Aegean (n = 28, mean = 0.060 ng ml− 1) regions of Turkey were more contaminated with OTA compared with wines originating from central (n = 15, mean = 0.027 ng ml−1) and east Anatolia (n = 8, mean = 0.027 ng ml− 1) areas. This study showed that the occurrence of OTA in Turkish wines is high, but at levels that probably leads to a nonsignificant human exposure to OTA by consumption of wines. © 2007 Elsevier B.V. All rights reserved. Keywords: Ochratoxin A; Wine; Occurrence; HPLC-FD; Immunoaffinity column
1. Introduction Ochratoxin A (OTA) is the most common naturally occurring mycotoxin produced by mainly Aspergillus ochraceus, A. carbonarius and Penicillium verrucosum, and is receiving increasing attention [1]. While P. verrucosum is the main producer in cereals for OTA in temperate climates [2], A. ochraceus is typically associated with coffee, grapes and spices in warm and tropical regions [3]. OTA commonly occurs in sub-tropical and temperate climates [4], and can be found in a number of food products, including cereals, beer, coffee beans, cacao, spices, nuts, dried fruit, grape juice, as well as in human blood and
Abbreviations: OTA, ochratoxin A; HPLC, high performance liquid chromatography; IAC, immunoaffinity column; LOD, limit of detection; LOQ, limit of quantification. ⁎ Corresponding author. Tel.: +90 322 3386173; fax: +90 322 3386614. E-mail address: [email protected] (B. Kabak). 0026-265X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2007.04.002
animal derived products [5]. Since OTA is relatively stable like other mycotoxins within the range of conventional food processing temperatures, and partially degraded under fermenting process, it can also be detected in various manufactured food products [6]. Recent studies have shown that wines contain a considerable level of OTA. The occurrence of OTA in wine was reported in Switzerland for the first time [7]. The data on OTA in wines reported by several investigators from the past decades originated mainly from European countries, especially from Scandinavia, Mediterranean Sea Countries and the Balkans. The concentrations of OTA in wines were highly variable. In the European diet, wine has been identified as the second major source of human exposure to OTA, following cereals, corresponding to 15% [8]. OTA is a well-known nephrotoxic agent and has been associated with fatal human kidney disease, referred to as Balkan Endemic Nephropathy and with an increased incidence of tumours of the upper urinary effect [1]. The toxin has also a number of toxic effects against various experimental animals:
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carcinogenic, teratogenic, immunotoxic, genotoxic and possibly neurotoxic [5,9]. The Canadian authorities proposed a tolerable daily intake (TDI) of 1.2–5.7 ng kg− 1 body weight (b.w.) day− 1, based on carcinogenic properties of OTA [10]. The toxin was also evaluated by The Scientific Committee on Food, who proposed that exposure should be below 5 ng kg− 1 b.w. day− 1 [11]. In 2001, JECFA (The Joint FAO/WHO Expert Committee on Food Additives) set a provisional tolerable weekly intake (PTWI) of 100 ng kg− 1 b.w., corresponding to approximately 14 ng kg− 1 b.w. per day [1]. It was also classified by the International Agency for Research on Cancer (IARC) of WHO as a group 2B (possible human carcinogen) agent in 1993 [12], based on sufficient evidence for carcinogenicity in animal studies and inadequate evidence in humans. The Scientific Commission of the European Community have regulated the maximum allowable level of 2 ng ml − 1 OTA for wine (including sparkling wine, excluding liqueur wine and wine with an alcoholic strength of not less than 15% vol) and fruit wine as well as for aromatised wine, aromatised wine-based drinks and aromatised wine-product cocktails [13]. There is yet no maximum permissible level established for OTA in wine in Turkey, but there is a tendency to adopt the maximum EU limit. It requires each country to establish their maximum allowable limit for OTA so as to minimize the health hazard risk. To our knowledge, there is only one work on the occurrence of OTA in Turkish wines, but more available data are need to establish the maximum limits of OTA from the public health point of view. Therefore, the primary aim of the present study was to obtain further information concerning OTA occurrence in white, rosé and red wines produced in different regions of Turkey by high performance liquid chromatography with fluorescence detection (HPLC-FD). 2. Materials and methods 2.1. Wine samples Samples, including 34 white, 10 rosé and 37 red wines were purchased from supermarkets and local retail stores in Adana, Turkey during 2005 and 2006 and 14 red wines were obtained from manufacturers' stocks in Tekirdag. All information on the samples, e.g. the year of vintage and its origin, were taken from the labels. The prices of the wines (white, rosé and red) ranged from 2.5 to 12.5 Euro per bottle (750 ml). Samples were stored at room temperature and opened just before analysis. 2.2. Reagents, standard and materials 2.2.1. Reagents All solvents used for the analysis of OTA were obtained from Merck (Darmstadt, Germany) and were of HPLC grade. 2.2.2. Water In all analytical steps, highly purified water generated by a Millipore Synergy 18S Ultra-Pure Water System from Millipore (France) was used.
2.2.3. Phosphate-buffered saline (PBS), pH 7.4 PBS was prepared by dissolving 0.2 g KCl, 0.2 g KH2PO4, 1.16 g anhydrous Na2HPO4, and 8.0 g NaCl in 900 ml distilled water. The pH was adjusted to 7.4 with 0.1 M HCl or NaOH and diluted to 1000 ml. 2.2.4. Standard OTA obtained from A. ochraceus was purchased from Sigma-Aldrich (USA) as a crystalline powder form. A stock solution (approximately 500 μg ml− 1) was prepared by solving 1 mg of OTA in 2 ml of toluene/acetic acid (99/1, v/v). The solution was left overnight at room temperature to ensure complete dissolution of the crystalline OTA. A 50 μl of OTA standard stock solution was transferred to a 25 ml brown volumetric flask and was quickly diluted with toluene/acetic acid to obtain a working solution, at 1 μg ml− 1. Standard working solution was stored at − 18 °C in the dark; the working solution was freshly prepared and held for less than 3 months. The working solution was brought to room temperature before use. 2.2.5. Immunoaffinity columns (IAC) OCHRAPREP® IAC containing specific monoclonal antibodies bound to a solid support material for OTA clean-up were obtained from R-Biopharm Rhone diagnostic (product code: P14B, Glasgow, Scotland). 2.3. IAC clean-up The method developed by Otteneder and Majerus [14] was used with some modifications for the present analysis. Briefly, 25 ml of wine samples was diluted with 25 ml PBS. The mixture was shaken vigorously and the pH adjusted with 10 M NaOH to 7.0–7.5. A 25 ml of the diluted solution (equivalent to 12.5 ml of the wine sample) was passed through IAC at a flow rate of about 2–3 ml per minute. The column was washed with 20 ml water (2 × 10 ml) and dried under nitrogen. OTA was then eluted by passing 1600 μl (2 × 800 μl) methanol/acetic acid (99/1, v/v) through the column at a flow rate 2–3 ml min− 1. The eluate was carefully evaporated in the vial to dryness under a stream of nitrogen. The residue was diluted in 400 μl mobile phase and stored at 4 °C until to the HPLC analysis. Finally, 20 μl of the aliquot was injected onto the HPLC column. 2.4. HPLC analysis The HPLC system (Agilent 1100) consisted of the CSI-6150 online vacuum degasser (Cambridge Scientific Instruments, England), isocratic pump (G 1310 A9, Agilent) and fluorescence detector. The separation was performed using a Silica 5 μm ACE 5 C18, 100 Å, 25 × 4.6 mm column supplied by Advanced Chromatography Technologies (Scotland). A Rheodyne 7725i stainless steel manual injector (Agilent, USA) with 20 μl loop was used. Two injections were performed for each sample. The mobile phase was acetonitrile/water/acetic acid (47/51/2, v/v/v) eluted at a flow rate of 1 ml min− 1. The fluorescence detector (Agilent 1100) was set at excitation and emission wavelengths of 337 and 470 nm, respectively, and the quantification OTA were
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Table 1 Recovery experiments of OTA in white, rosé and red wines Spiked Number of Spiking level OTA detected Recovery samples analysis (ng ml− 1) (mean ± S.D., ng ml− 1) (%, mean ± S.D.) White 10 wine 4 4 Rosé 10 wine 4 4 Red 10 wine 4 4
0.1 0.5 1 0.1 0.5 1 0.1 0.5 1
0.079 ± 0.002 0.421 ± 0.029 0.851 ± 0.036 0.077 ± 0.003 0.419 ± 0.028 0.839 ± 0.039 0.077 ± 0.003 0.500 ± 0.038 0.836 ± 0.053
79.43 ± 2.03 84.08 ± 4.41 85.07 ± 3.62 77.48 ± 3.23 83.86 ± 5.66 83.96 ± 3.97 76.61 ± 3.25 81.91 ± 7.63 83.55 ± 5.28
determined using concentration/fluorescence curves of OTA. Chemstation software (Agilent) was used to control the chromatograph and process the signals. The calibration curve was established by injecting four OTA standard solutions. The correlation coefficient was linear (r2 = 0.99850). The retention time was about 13 min for OTA. 2.5. Recovery experiments To evaluate the test sensitivity, OTA-free white, rosé and red wine samples were spiked with OTA at 0.1, 0.5 and 1 ng ml− 1. Four replicates for levels of 0.5 and 1 ng ml− 1 and 10 replicates for level of 0.1 ng ml− 1 were performed. A recovery percentage was calculated for white, rosé and red wines individually. OTA concentrations were determined using the protocol previously described. 3. Results and discussion The results of recovery studies for OTA are summarised in Table 1. Mean recoveries for white, rosé and red wines were in the range of 79.43–85.07%, 77.48–83.96%, and 76.61–83.55%,
Fig. 1. HPLC chromatogram from a red wine sample with 0.815 ng OTA ml−1.
respectively. As can be seen in Table 1, the highest recovery rate of OTAwas obtained at a level of 1 ng ml− 1. Similarly, the highest recovery values depending on the spiking levels were obtained from white wine. These results may show that the spiked levels and sample matrix have an important role in the recovery of OTA. Recovery values obtained in the present study are lower than those reported by Markaki et al. [15], Pietri et al. [16], Lopez de Cerain et al. [17], Stefanaki et al. [18], Blesa et al. [19], Ratola et al. [20], but are higher than that found by Czerwiecki et al. [21], and are similar to that revealed by Ng et al. [9]. The mean recovery was 79.43% for white wine spiked with OTA at 0.1 ng ml− 1, with a limit of detection (LOD) and limit of quantification (LOQ) of 0.006 and 0.020 ng ml− 1. The LOD and LOQ for rosé and red wines were 0.010 and 0.030 ng ml− 1, respectively. The LOD obtained from this study was lower than those reported by Lopez de Cerain et al. [17], Stefanaki et al. [18], Blesa et al. [19], Rosa et al. [22] and Brera et al. [23], but are higher than those found by Ng et al. [9], Markaki et al. [15], Pietri et al. [16], and Czerwiecki et al. [21].
Table 2 Occurrence and concentrations of OTA in red, rosé and white wines from four different regions of Turkey Origin of samples
n
White wines Thrace region Aegean region Central Anatolia East Anatolia
34 9 14 11 –
Rosé wines Thrace region Aegean region Central Anatolia East Anatolia Red wines Thrace region Aegean region Central Anatolia East Anatolia a b
Positive n (%)
Incidence of OTA contamination (ng ml− 1)
OTA concentration (ng ml− 1)
LOD a–0.1
0.1–0.5
N0.5
Mean
Range b
29 (85) 9 (100) 14 (100) 6 (55) –
19 2 11 6 –
9 6 3 0 –
1 1 0 0 –
0.108 0.249 0.051 0.029 –
bLOD–0.618 0.044–0.618 0.006–0.192 bLOD–0.072 –
10 2 5 1 2
9 2 5 1 1
8 1 5 1 1
1 1 0 0 0
0 0 0 0 0
0.052 0.103 0.041 0.020 0.035
bLOD–0.161 0.045–0.161 0.020–0.086 0.020 bLOD–0.035
51 33 9 3 6
44 (86) 30 (91) 8 (89) 1 (33) 5 (83)
32 20 6 1 5
10 8 2 0 0
2 2 0 0 0
0.110 0.134 0.088 0.023 0.026
bLOD–0.815 bLOD–0.815 bLOD–0.252 bLOD–0.023 bLOD–0.026
(90) (100) (100) (100) (50)
LOD, limit of detection: for white wine = 6 ng l− 1; for red wine = 10 ng l− 1. Minor and major level of OTA.
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The occurrence and levels of OTA in 95 white, rosé and red wines from four different regions of Turkey are summarised in Table 2. OTA was detected in 82 samples of wine, corresponding to 86% of the samples analysed ranged from b 0.006 to 0.815 ng ml− 1. Only three samples contained over 0.5 ng ml− 1 OTA, while it was not detected in 14% (13 out of 95) of the samples. Neither of the positive samples showed levels higher than the maximum acceptable level set by EU (2 ng ml− 1). The highest OTA concentration was found in a red wine from Thrace region of Turkey. With respect to white wines, 85% had OTA ranging from b 0.006 to 0.618 ng ml− 1 with an overall mean of 0.108 ng ml− 1. Even though fewer samples of rosé wine were analysed, these wines contained
OTA at levels ranging from b0.010 to 0.161 ng ml− 1. On the other hand, OTA concentration in red wines from Turkey ranged from b 0.010 to 0.815 ng ml− 1. An example of a HPLC chromatogram obtained from highest contaminated red wine sample (0.815 ng ml− 1) is shown in Fig. 1. During the last decade, the occurrence of OTA in different wines originating from various countries has been reported in the scientific literature. The results of these reports are summarised in Table 3. Most of the available data have been obtained from Mediterranean countries and both the occurrence of wine contamination and OTA levels were high in red wines, with respect to the white or rosé wines. As can be seen in Table 3, there were large differences in OTA levels, ranging
Table 3 OTA contents of wines originating from various countries Origin
No. of samples
Positive (%)
Mean (ng ml− 1)
Range (ng ml− 1)
Reference
Wines from the Swiss retail market
24 white 15 rosé 79 red 60 white 55 rosé 305 red 31 red 41 white 37 red 7 white 3 rosé 20 red 9 white 8 rosé 38 red 96 red 21 white 9 rosé 159 red 12 white 28 red 21 white 21 rosé 61 red 13 dessert 40 white 120 red 13 white 1 rosé 14 red 118 white 20 rosé 104 red 340 wines 30 white 2 rosé 27 red 67 wines 8 white 4 rosé 35 red 43 white 36 red 53 red 15 white 5 rosé 22 red
(27) (92) (78) (25) (40) (54) (100) 21 (51) 25 (68) 7 (100) 3 (100) 20 (100) 4 (44) 7 (88) 37 (97) 82 (85) 4 (19) 5 (55) 124 (78) 7 (58) 13 (46) 1 (5) 12 (57) 21 (34) 8 (62) 4 (10) 22 (18) 7 (54) – 9 (64) 63 (53) 13 (65) 71 (68) 69 (20) – – – – 8 (100) 4 (100) 35 (100) 10 (23) 5 (14) 49 (92) 2 (13) 1 (20) 7 (32)
0.011 0.025 0.039 0.108 0.119 0.201 – 0.151 0.269 – – – – – – 0.419 – – – – – – – – – 0.30 0.18 0.27 b0.02 0.68 0.25 0.17 0.34 – b0.01 b0.01 b0.01 b0.024 – – – 0.009 0.017 0.477 0.028 0.035 0.039
b0.003–0.178 b0.003–0.123 b0.003–0.388 b0.01–1.36 b0.01–2.38 b0.01–3.31 b0.002–3.40 b0.004–3.720 b0.008–2.320 0.028–0.18 0.04–0.54 0.10–3.24 0.01–0.97 0.01–1.15 0.01–7.63 b0.001–3.177 b0.01–0.21 b0.01–1.04 b0.01–4.00 0.154–0.208 0.056–0.316 b0.01–0.09 b0.01–0.46 b0.01–0.53 b0.01–0.40 b0.05–1.13 b0.05–3.19 b0.02–0.87 b0.02 b0.02–2.51 b0.05–1.72 b0.05–1.16 b0.05–2.69 b0.084–2.1 b0.01 b0.01 b0.01 b0.024 0.03–0.34 0.03–2.23 0.04–1.92 b0.004–0.156 b0.008–0.393 0.002–6.710 0.028 0.035 0.028–0.071
[7]
Wines of worldwide origin
Mediterranean Sea countries Mediterranean wines Moroccan wines
Italian wines
Italian wines Italian wines
Spanish wines Spanish wines
Spanish wines Greek wines
Greek wines
Portuguese wines Hungarian wines
Hungarian wines Turkish wines
Canadian wines Wines from EU countries and Argentina Wines from south American countries
[14]
[15] [9] [25]
[24]
[16] [23]
[17] [19]
[28] [6]
[18]
[20] [23]
[32] [26]
[9] [21] [22]
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Fig. 2. Turkish wine regions (number of analysis samples and mean (ng ml− 1) in parentheses).
from b0.002 to 7.630 ng ml− 1, depending on the origin. The highest OTA concentration reported was in a red wine from Italy [24]. According to our data, mean for red wine is slightly higher than white and rosé wines. This result confirms the preliminary observation by Otteneder and Majerus [14], Filali et al. [25], Soufleros et al. [6], Stefanaki et al. [18], Blesa et al. [19], Rosa et al. [22], Anlı et al. [26], Brera et al. [23], but it contrast to that reported by Lopez de Cerain [17], who observed that red wines showed lower level of OTA than white wines conducted with limited samples in a 2-year study. These differences can be explained by different wine-making methods, storage conditions, climatic factors and grape cultivation [27]. The winemaking procedure is one of the most critical factors in the occurrence and level of OTA in wines. In white wine, the grapes are crushed and pressed rapidly, while red grapes are left to macerate for an extended period of time (several days) which obviously permits higher amount of the toxin to be transferred to the juice in making a red wine [6,14,28,29]. In addition, this process provides good conditions for mould growth and OTA accumulation [14]. This might be also due to the fact that wines clarified with fining agents such as activated carbon, bentonite, gelatine and casein which might adsorb OTA (22). Activated carbon was found to be an efficiently agent that could be used to remove OTA in wines. In our previous study showed that treatment with activated carbon caused 87% of the available toxin decreases in the OTA levels (5 ng ml− 1) present in artificially contaminated in white wine (unpublished data). Similarly, potassium caseinate has been shown to remove 82% of OTA from wine when used at 150 g hL− 1 [30]. The second critical factor influencing presence of OTA is the geographic region of the grapes used in the wine-making. With respect to red wines, whose number in the survey is rather high and whose distribution among the regions is not homogeneous. The incidence and concentrations of OTA in red wines decreased in order Thrace region N Aegean region N east Anatolia N central Anatolia. OTA was detected over 0.1 ng ml− 1 in 10 out of 33 wines originating from Thrace region and 2 out of 9 samples from Aegean region, whereas red wines originating from central Anatolia and east Anatolia contained only small quantities of OTA (ranges b 0.010–0.023 ng ml− 1 and b 0.010– 0.026 ng ml− 1, respectively). Similar results were also found for the rosé wines, even though only 10 samples were analysed. All
rose wines from Aegean region, central and east Anatolia contained OTA lower than 0.1 ng ml− 1, whereas higher amounts of OTA were found in rosé wines from Thrace region. The same trend can be observed for the white wines. All white wines originating from Thrace and Aegean regions contaminated with OTA levels ranging from 0.044 to 0.618 ng ml− 1 and from 0.006 to 0.192 ng ml− 1, respectively, while OTA was detected in 6 out of 11 samples ranging from b 0.006 to 0.072 ng ml− 1 in central Anatolia. There was however no available data with white wine originating from east Anatolia. Our data indicate that, the geographic origin of wines has a strong influence on OTA contamination. The regions of wine samples analysed are represented in Fig. 2. Turkey's diverse regions have different climates with the weather system on the coasts contrasting with that prevailing in the interior. In the Thrace region, the climate is moderate and quite humid, with temperatures range from −16 to 40 °C (average 4 °C in winter and 27 °C in summer). The average humidity is 73%. The region of Aegean has Mediterranean influences, and the average temperatures in the winter are 9 °C and in summer 29 °C, with average humidity of 69%. In the central Anatolia, there is a steppe climate. Summers are hot and dry, in winter however the temperatures can drop below zero. The temperatures drop to − 25 °C in deep in winter, and rise to 40 °C in summer, with the average − 2 °C in winter and 23 °C in summer. Rainfall is low and it usually in form of snow. There is a great temperature difference between day and night. The overall humidity is 62.6%. The eastern Anatolia areas have long, bitterly cold with frequent, heavy snowfall winters and mild summers. The eastern Anatolia areas receive the greatest amount of snow from November until to the end of April. It has the lowest sun levels of all the regions studied. In the eastern Anatolia, the average temperature is − 13 °C in winter and 17 °C in summer [26]. The reason for the higher OTA content in the wines originating from Thrace and Aegean regions, located in the northwest and west of Turkey, respectively, relative to central and east Anatolia can be explained by their climates. Looking at the meteorological data, there are high temperatures in summer and high humidity when compared to central and east Anatolia. Similarly, several authors reported that the wines from the Mediterranean region contain high concentrations of OTA due to their climates being characterised by high humidity and high temperature [19,25]. Black Aspergilli are known to be very
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resistant to sun exposure and to hot and dry environments [8]. In a previous work conducted by Pardo et al. [31] has been showed that high relative humidity and temperature levels favoured ochratoxigenic A. ochraceus isolates growth on grapes and subsequently OTA production. In addition, it has been revealed that the vicinity of a sea also benefits the growth of OTA producing fungi by the increased degree of humidity of the air [23]. Moreover, both rates of occurrence and OTA levels might be varied from one year to the other depending on the meteorological conditions [15,19]. The present study agrees with Anlı et al. [26] who reported that wines originating from Thrace and Aegean regions much more contained OTA than central and east Anatolia. The mean value of OTA contamination have been reported to be 0.929, 1.372, 0.053 and 0.526 ng ml− 1 in red wines originating from Thrace, Aegean, central Anatolia and east Anatolia regions, respectively. Our results are also consistent with pervious surveys conducted in other countries that the geographic origin had a strong influence on OTA levels of wines. Soufleros et al. [6] revealed that wines from islands of Greece contained more OTA than north, south and central areas of Greece. Numerous investigators have concluded from their results that wines from southern countries of the Northern Hemisphere are more contaminated than those of wines from the northern areas [7,14,24]. Similarly, it has been reported that the frequency of occurrence and concentration of OTA in red wines increase from northern to southern Greece [18], from northern to southern Italy [16], and from northern to southern Hungary [32]. Recently, Czerwiecki et al. [21] have concluded that wines from France and Italy were found to contain more frequency and higher levels of OTA than Hungary and Bulgaria. A concentration gradient from north to south also leads to the assumption that OTA-producing fungi can be found more often on grapes growing in the south [14]. 4. Conclusions The results showed that most of the samples contained only small proportions of OTA, although the occurrence of contamination was rather high. Only three of them had values above 0.5 ng ml − 1 . The results support the general tendency that red wines contain more OTA than rosé and white wines. It must be noted that in comparison with wines from central and east Anatolia showed a high level of OTA, and the frequency of the mycotoxin occurrence. Data have also demonstrated that OTA levels in Turkish wines were substantially lower than published results from EU Member States. According to these results, it can be concluded that the contamination of Turkish wines with OTA does not exceed the maximum EU limit (2 ng ml − 1 ). Although more studies must be systematically conducted on the occurrence of OTA risk, it is not implying a high risk of human exposure to OTA through consumption of wine. The best way to protect consumers against OTA health hazards is to implement good agricultural practices (GAPs), good manufacturing practices (GMPs) and the hazard analysis and critical control point (HACCP) systems.
Acknowledgements This study was supported financially by the Scientific and Technical Research Council of Turkey (TUBITAK, project no. TOGTAG-3374) and Cukurova University Research Council. References [1] FAO/WHO (Food and Agricultural Organization/World Health Organization) (2001), Ochratoxin A, In “Safety evaluations of specific mycotoxins”. Prepared by the fifty-sixth meeting of the Joint FAO/WHO Expert Committee on Food Additives, 6–15 February, Geneva. [2] F. Lund, J.C. Frisvad, J. Appl. Microbiol. 95 (2003) 1117. [3] Codex Alimentarius Commission (2002), Proposed draft code of practice for the prevention (reduction) of mycotoxin contamination in cereals, including annexes on ochratoxin A, zearalenone, fumonisins and trichothecenes, CX/FAC 02/21, Joint FAO/WHO Food Standards Programme, Rotterdam, The Netherlands. [4] EC report (European Commission report) (1999), Opinion on the relationship between the use of plant protection products on food plants and the occurrence of mycotoxins in foods, European Commission SCP/ RESI063, Belgium. [5] Commission Regulation EC No. 472/2002 of 12 March 2002 amending Regulation (EC) No 466/2001 setting maximum levels for certain contaminants in foodstuffs, Official Journal of the European Union, L75, 18–20, 16.03.2002. [6] E.H. Soufleros, C. Tricard, E.C. Bouloumpasi, J. Sci. Food Agric. 83 (2003) 173. [7] B. Zimmerli, R. Dick, Food Addit. Contam. 13 (1996) 655. [8] R. Serra, L. Abrunhosa, Z. Kozakiewicz, A. Venâncio, Int. J. Food Microbiol. 2736 (2003) 1. [9] W. Ng, M. Mankotia, P. Pantazopoulos, R.J. Neil, P.M. Scott, Food Addit. Contam. 21 (2004) 971. [10] T. Kuiper-Goodman, Food Addit. Contam. 13 (1996) 53. [11] EC report (1998), Opinion on ochratoxin A, expressed on 17 September 1998. Scientific Committee on Food. [12] IARC (International Agency for Research on Cancer), Some Naturally Occurring Substances, Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins, 56, World Health Organization, Lyon, France, 1993, p. 489. [13] Commission Regulation (EC) No 1881/2006 of 19 December 2006, setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union, L 364, 5–24, 20.12.2006. [14] H. Otteneder, P. Majerus, Food Addit. Contam. 17 (2000) 793. [15] P. Markaki, C. Delpont-Binet, F. Grosso, S. Dragacci, J. Food Prot. 64 (2001) 533. [16] A. Pietri, T. Bertuzzi, L. Pallaroni, G. Piva, Food Addit. Contam. 18 (2001) 647. [17] A. Lopez de Cerain, E. González-Peñas, A.M. Jiménez, J. Bello, Food Addit. Contam. 19 (2002) 1058. [18] I. Stefanaki, E. Foufa, A. Tsatsou-Dritsa, P. Dais, Food Addit. Contam. 20 (2003) 74. [19] J. Blesa, J.M. Soriano, J.C. Moltó, J. Mañes, J. Chromatogr., A 1054 (2004) 397. [20] N. Ratola, L. Martins, A. Alves, Anal. Chim. Acta 513 (2004) 319. [21] L. Czerwiecki, G. Wilcznyñska, A. Kwiecieñ, Food Addit. Contam. 22 (2005) 158. [22] C.A.R. Rosa, C.E. Magnoli, M.E. Fraga, A.M. Dalcero, D.M.N. Santana, Food Addit. Contam. 21 (2004) 358. [23] C. Brera, M. Soriano, F. Debegnach, M. Miraglia, Microchem. J. 79 (2005) 109. [24] A. Visconti, M. Pascale, G. Centonze, J. Chromatogr., A 864 (1999) 89. [25] A. Filali, L. Ouammi, A.M. Betbeder, I. Baudrimont, R. Soulaymani, A. Benayada, E.E. Creppy, Food Addit. Contam. 18 (2001) 565. [26] E. Anlı, B. Çabuk, N. Vural, E. Baspınar, J. Food Biochem. 29 (2005) 611.
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[30] M. Castellari, A. Versari, A. Fabiani, G.P. Parpinello, S. Galassi, J. Agric. Food Chem. 49 (2001) 3917. [31] E. Pardo, S. Marin, V. Sanchis, A.J. Ramos, Food Microbiol. 22 (2005) 383. [32] B. Berente, Á. Móricz, K.H. Otta, G. Záray, L. Lékó, L. Rácz, Microchem. J. 79 (2005) 103.
Occurrence of ochratoxin A in Turkish wines I. Var, B. Kabak ⁎ Cukurova University, Agricultural Faculty, Department of Food Engineering, TR-01330, Adana, Turkey Received 22 March 2007; accepted 6 April 2007 Available online 20 April 2007
Abstract A total of 95 wine samples including 34 white, 10 rosé and 51 red wines originating from four different Turkish areas were analysed for ochratoxin A (OTA). An analytical method based on immunoaffinity column (IAC) for clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD) was used to determine OTA in wines. The limit of detection (LOD) was estimated as 0.006 ng ml− 1 for white wine and 0.010 ng ml− 1 for rosé and red wines. The limit of quantification (LOQ) was estimated as 0.020 ng ml− 1 in white wine and 0.030 ng ml− 1 in rosé and red wines. Recovery experiments were carried out with spiked samples in the range 0.1–1 ng ml− 1 of OTA. The average OTA recoveries from spiked white wine samples varied from 79.43% to 85.07%; while the mean recoveries for rosé and red wine samples were in the range of 77.48–83.96% and 76.61–83.55%, respectively. OTA was detected in 82 (86%) wine samples at levels of b 0.006–0.815 ng ml− 1, which were below the maximum allowable limit established by the European Community. The mean OTA concentration in red wines was slightly higher than in white and rosé wines. Furthermore, our data indicate that the geographic region of origin has strong influence on OTA level for white, rosé and red wines: wines originating from Thrace (n = 44, mean = 0.158 ng ml− 1) and Aegean (n = 28, mean = 0.060 ng ml− 1) regions of Turkey were more contaminated with OTA compared with wines originating from central (n = 15, mean = 0.027 ng ml−1) and east Anatolia (n = 8, mean = 0.027 ng ml− 1) areas. This study showed that the occurrence of OTA in Turkish wines is high, but at levels that probably leads to a nonsignificant human exposure to OTA by consumption of wines. © 2007 Elsevier B.V. All rights reserved. Keywords: Ochratoxin A; Wine; Occurrence; HPLC-FD; Immunoaffinity column
1. Introduction Ochratoxin A (OTA) is the most common naturally occurring mycotoxin produced by mainly Aspergillus ochraceus, A. carbonarius and Penicillium verrucosum, and is receiving increasing attention [1]. While P. verrucosum is the main producer in cereals for OTA in temperate climates [2], A. ochraceus is typically associated with coffee, grapes and spices in warm and tropical regions [3]. OTA commonly occurs in sub-tropical and temperate climates [4], and can be found in a number of food products, including cereals, beer, coffee beans, cacao, spices, nuts, dried fruit, grape juice, as well as in human blood and
Abbreviations: OTA, ochratoxin A; HPLC, high performance liquid chromatography; IAC, immunoaffinity column; LOD, limit of detection; LOQ, limit of quantification. ⁎ Corresponding author. Tel.: +90 322 3386173; fax: +90 322 3386614. E-mail address: [email protected] (B. Kabak). 0026-265X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2007.04.002
animal derived products [5]. Since OTA is relatively stable like other mycotoxins within the range of conventional food processing temperatures, and partially degraded under fermenting process, it can also be detected in various manufactured food products [6]. Recent studies have shown that wines contain a considerable level of OTA. The occurrence of OTA in wine was reported in Switzerland for the first time [7]. The data on OTA in wines reported by several investigators from the past decades originated mainly from European countries, especially from Scandinavia, Mediterranean Sea Countries and the Balkans. The concentrations of OTA in wines were highly variable. In the European diet, wine has been identified as the second major source of human exposure to OTA, following cereals, corresponding to 15% [8]. OTA is a well-known nephrotoxic agent and has been associated with fatal human kidney disease, referred to as Balkan Endemic Nephropathy and with an increased incidence of tumours of the upper urinary effect [1]. The toxin has also a number of toxic effects against various experimental animals:
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carcinogenic, teratogenic, immunotoxic, genotoxic and possibly neurotoxic [5,9]. The Canadian authorities proposed a tolerable daily intake (TDI) of 1.2–5.7 ng kg− 1 body weight (b.w.) day− 1, based on carcinogenic properties of OTA [10]. The toxin was also evaluated by The Scientific Committee on Food, who proposed that exposure should be below 5 ng kg− 1 b.w. day− 1 [11]. In 2001, JECFA (The Joint FAO/WHO Expert Committee on Food Additives) set a provisional tolerable weekly intake (PTWI) of 100 ng kg− 1 b.w., corresponding to approximately 14 ng kg− 1 b.w. per day [1]. It was also classified by the International Agency for Research on Cancer (IARC) of WHO as a group 2B (possible human carcinogen) agent in 1993 [12], based on sufficient evidence for carcinogenicity in animal studies and inadequate evidence in humans. The Scientific Commission of the European Community have regulated the maximum allowable level of 2 ng ml − 1 OTA for wine (including sparkling wine, excluding liqueur wine and wine with an alcoholic strength of not less than 15% vol) and fruit wine as well as for aromatised wine, aromatised wine-based drinks and aromatised wine-product cocktails [13]. There is yet no maximum permissible level established for OTA in wine in Turkey, but there is a tendency to adopt the maximum EU limit. It requires each country to establish their maximum allowable limit for OTA so as to minimize the health hazard risk. To our knowledge, there is only one work on the occurrence of OTA in Turkish wines, but more available data are need to establish the maximum limits of OTA from the public health point of view. Therefore, the primary aim of the present study was to obtain further information concerning OTA occurrence in white, rosé and red wines produced in different regions of Turkey by high performance liquid chromatography with fluorescence detection (HPLC-FD). 2. Materials and methods 2.1. Wine samples Samples, including 34 white, 10 rosé and 37 red wines were purchased from supermarkets and local retail stores in Adana, Turkey during 2005 and 2006 and 14 red wines were obtained from manufacturers' stocks in Tekirdag. All information on the samples, e.g. the year of vintage and its origin, were taken from the labels. The prices of the wines (white, rosé and red) ranged from 2.5 to 12.5 Euro per bottle (750 ml). Samples were stored at room temperature and opened just before analysis. 2.2. Reagents, standard and materials 2.2.1. Reagents All solvents used for the analysis of OTA were obtained from Merck (Darmstadt, Germany) and were of HPLC grade. 2.2.2. Water In all analytical steps, highly purified water generated by a Millipore Synergy 18S Ultra-Pure Water System from Millipore (France) was used.
2.2.3. Phosphate-buffered saline (PBS), pH 7.4 PBS was prepared by dissolving 0.2 g KCl, 0.2 g KH2PO4, 1.16 g anhydrous Na2HPO4, and 8.0 g NaCl in 900 ml distilled water. The pH was adjusted to 7.4 with 0.1 M HCl or NaOH and diluted to 1000 ml. 2.2.4. Standard OTA obtained from A. ochraceus was purchased from Sigma-Aldrich (USA) as a crystalline powder form. A stock solution (approximately 500 μg ml− 1) was prepared by solving 1 mg of OTA in 2 ml of toluene/acetic acid (99/1, v/v). The solution was left overnight at room temperature to ensure complete dissolution of the crystalline OTA. A 50 μl of OTA standard stock solution was transferred to a 25 ml brown volumetric flask and was quickly diluted with toluene/acetic acid to obtain a working solution, at 1 μg ml− 1. Standard working solution was stored at − 18 °C in the dark; the working solution was freshly prepared and held for less than 3 months. The working solution was brought to room temperature before use. 2.2.5. Immunoaffinity columns (IAC) OCHRAPREP® IAC containing specific monoclonal antibodies bound to a solid support material for OTA clean-up were obtained from R-Biopharm Rhone diagnostic (product code: P14B, Glasgow, Scotland). 2.3. IAC clean-up The method developed by Otteneder and Majerus [14] was used with some modifications for the present analysis. Briefly, 25 ml of wine samples was diluted with 25 ml PBS. The mixture was shaken vigorously and the pH adjusted with 10 M NaOH to 7.0–7.5. A 25 ml of the diluted solution (equivalent to 12.5 ml of the wine sample) was passed through IAC at a flow rate of about 2–3 ml per minute. The column was washed with 20 ml water (2 × 10 ml) and dried under nitrogen. OTA was then eluted by passing 1600 μl (2 × 800 μl) methanol/acetic acid (99/1, v/v) through the column at a flow rate 2–3 ml min− 1. The eluate was carefully evaporated in the vial to dryness under a stream of nitrogen. The residue was diluted in 400 μl mobile phase and stored at 4 °C until to the HPLC analysis. Finally, 20 μl of the aliquot was injected onto the HPLC column. 2.4. HPLC analysis The HPLC system (Agilent 1100) consisted of the CSI-6150 online vacuum degasser (Cambridge Scientific Instruments, England), isocratic pump (G 1310 A9, Agilent) and fluorescence detector. The separation was performed using a Silica 5 μm ACE 5 C18, 100 Å, 25 × 4.6 mm column supplied by Advanced Chromatography Technologies (Scotland). A Rheodyne 7725i stainless steel manual injector (Agilent, USA) with 20 μl loop was used. Two injections were performed for each sample. The mobile phase was acetonitrile/water/acetic acid (47/51/2, v/v/v) eluted at a flow rate of 1 ml min− 1. The fluorescence detector (Agilent 1100) was set at excitation and emission wavelengths of 337 and 470 nm, respectively, and the quantification OTA were
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Table 1 Recovery experiments of OTA in white, rosé and red wines Spiked Number of Spiking level OTA detected Recovery samples analysis (ng ml− 1) (mean ± S.D., ng ml− 1) (%, mean ± S.D.) White 10 wine 4 4 Rosé 10 wine 4 4 Red 10 wine 4 4
0.1 0.5 1 0.1 0.5 1 0.1 0.5 1
0.079 ± 0.002 0.421 ± 0.029 0.851 ± 0.036 0.077 ± 0.003 0.419 ± 0.028 0.839 ± 0.039 0.077 ± 0.003 0.500 ± 0.038 0.836 ± 0.053
79.43 ± 2.03 84.08 ± 4.41 85.07 ± 3.62 77.48 ± 3.23 83.86 ± 5.66 83.96 ± 3.97 76.61 ± 3.25 81.91 ± 7.63 83.55 ± 5.28
determined using concentration/fluorescence curves of OTA. Chemstation software (Agilent) was used to control the chromatograph and process the signals. The calibration curve was established by injecting four OTA standard solutions. The correlation coefficient was linear (r2 = 0.99850). The retention time was about 13 min for OTA. 2.5. Recovery experiments To evaluate the test sensitivity, OTA-free white, rosé and red wine samples were spiked with OTA at 0.1, 0.5 and 1 ng ml− 1. Four replicates for levels of 0.5 and 1 ng ml− 1 and 10 replicates for level of 0.1 ng ml− 1 were performed. A recovery percentage was calculated for white, rosé and red wines individually. OTA concentrations were determined using the protocol previously described. 3. Results and discussion The results of recovery studies for OTA are summarised in Table 1. Mean recoveries for white, rosé and red wines were in the range of 79.43–85.07%, 77.48–83.96%, and 76.61–83.55%,
Fig. 1. HPLC chromatogram from a red wine sample with 0.815 ng OTA ml−1.
respectively. As can be seen in Table 1, the highest recovery rate of OTAwas obtained at a level of 1 ng ml− 1. Similarly, the highest recovery values depending on the spiking levels were obtained from white wine. These results may show that the spiked levels and sample matrix have an important role in the recovery of OTA. Recovery values obtained in the present study are lower than those reported by Markaki et al. [15], Pietri et al. [16], Lopez de Cerain et al. [17], Stefanaki et al. [18], Blesa et al. [19], Ratola et al. [20], but are higher than that found by Czerwiecki et al. [21], and are similar to that revealed by Ng et al. [9]. The mean recovery was 79.43% for white wine spiked with OTA at 0.1 ng ml− 1, with a limit of detection (LOD) and limit of quantification (LOQ) of 0.006 and 0.020 ng ml− 1. The LOD and LOQ for rosé and red wines were 0.010 and 0.030 ng ml− 1, respectively. The LOD obtained from this study was lower than those reported by Lopez de Cerain et al. [17], Stefanaki et al. [18], Blesa et al. [19], Rosa et al. [22] and Brera et al. [23], but are higher than those found by Ng et al. [9], Markaki et al. [15], Pietri et al. [16], and Czerwiecki et al. [21].
Table 2 Occurrence and concentrations of OTA in red, rosé and white wines from four different regions of Turkey Origin of samples
n
White wines Thrace region Aegean region Central Anatolia East Anatolia
34 9 14 11 –
Rosé wines Thrace region Aegean region Central Anatolia East Anatolia Red wines Thrace region Aegean region Central Anatolia East Anatolia a b
Positive n (%)
Incidence of OTA contamination (ng ml− 1)
OTA concentration (ng ml− 1)
LOD a–0.1
0.1–0.5
N0.5
Mean
Range b
29 (85) 9 (100) 14 (100) 6 (55) –
19 2 11 6 –
9 6 3 0 –
1 1 0 0 –
0.108 0.249 0.051 0.029 –
bLOD–0.618 0.044–0.618 0.006–0.192 bLOD–0.072 –
10 2 5 1 2
9 2 5 1 1
8 1 5 1 1
1 1 0 0 0
0 0 0 0 0
0.052 0.103 0.041 0.020 0.035
bLOD–0.161 0.045–0.161 0.020–0.086 0.020 bLOD–0.035
51 33 9 3 6
44 (86) 30 (91) 8 (89) 1 (33) 5 (83)
32 20 6 1 5
10 8 2 0 0
2 2 0 0 0
0.110 0.134 0.088 0.023 0.026
bLOD–0.815 bLOD–0.815 bLOD–0.252 bLOD–0.023 bLOD–0.026
(90) (100) (100) (100) (50)
LOD, limit of detection: for white wine = 6 ng l− 1; for red wine = 10 ng l− 1. Minor and major level of OTA.
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The occurrence and levels of OTA in 95 white, rosé and red wines from four different regions of Turkey are summarised in Table 2. OTA was detected in 82 samples of wine, corresponding to 86% of the samples analysed ranged from b 0.006 to 0.815 ng ml− 1. Only three samples contained over 0.5 ng ml− 1 OTA, while it was not detected in 14% (13 out of 95) of the samples. Neither of the positive samples showed levels higher than the maximum acceptable level set by EU (2 ng ml− 1). The highest OTA concentration was found in a red wine from Thrace region of Turkey. With respect to white wines, 85% had OTA ranging from b 0.006 to 0.618 ng ml− 1 with an overall mean of 0.108 ng ml− 1. Even though fewer samples of rosé wine were analysed, these wines contained
OTA at levels ranging from b0.010 to 0.161 ng ml− 1. On the other hand, OTA concentration in red wines from Turkey ranged from b 0.010 to 0.815 ng ml− 1. An example of a HPLC chromatogram obtained from highest contaminated red wine sample (0.815 ng ml− 1) is shown in Fig. 1. During the last decade, the occurrence of OTA in different wines originating from various countries has been reported in the scientific literature. The results of these reports are summarised in Table 3. Most of the available data have been obtained from Mediterranean countries and both the occurrence of wine contamination and OTA levels were high in red wines, with respect to the white or rosé wines. As can be seen in Table 3, there were large differences in OTA levels, ranging
Table 3 OTA contents of wines originating from various countries Origin
No. of samples
Positive (%)
Mean (ng ml− 1)
Range (ng ml− 1)
Reference
Wines from the Swiss retail market
24 white 15 rosé 79 red 60 white 55 rosé 305 red 31 red 41 white 37 red 7 white 3 rosé 20 red 9 white 8 rosé 38 red 96 red 21 white 9 rosé 159 red 12 white 28 red 21 white 21 rosé 61 red 13 dessert 40 white 120 red 13 white 1 rosé 14 red 118 white 20 rosé 104 red 340 wines 30 white 2 rosé 27 red 67 wines 8 white 4 rosé 35 red 43 white 36 red 53 red 15 white 5 rosé 22 red
(27) (92) (78) (25) (40) (54) (100) 21 (51) 25 (68) 7 (100) 3 (100) 20 (100) 4 (44) 7 (88) 37 (97) 82 (85) 4 (19) 5 (55) 124 (78) 7 (58) 13 (46) 1 (5) 12 (57) 21 (34) 8 (62) 4 (10) 22 (18) 7 (54) – 9 (64) 63 (53) 13 (65) 71 (68) 69 (20) – – – – 8 (100) 4 (100) 35 (100) 10 (23) 5 (14) 49 (92) 2 (13) 1 (20) 7 (32)
0.011 0.025 0.039 0.108 0.119 0.201 – 0.151 0.269 – – – – – – 0.419 – – – – – – – – – 0.30 0.18 0.27 b0.02 0.68 0.25 0.17 0.34 – b0.01 b0.01 b0.01 b0.024 – – – 0.009 0.017 0.477 0.028 0.035 0.039
b0.003–0.178 b0.003–0.123 b0.003–0.388 b0.01–1.36 b0.01–2.38 b0.01–3.31 b0.002–3.40 b0.004–3.720 b0.008–2.320 0.028–0.18 0.04–0.54 0.10–3.24 0.01–0.97 0.01–1.15 0.01–7.63 b0.001–3.177 b0.01–0.21 b0.01–1.04 b0.01–4.00 0.154–0.208 0.056–0.316 b0.01–0.09 b0.01–0.46 b0.01–0.53 b0.01–0.40 b0.05–1.13 b0.05–3.19 b0.02–0.87 b0.02 b0.02–2.51 b0.05–1.72 b0.05–1.16 b0.05–2.69 b0.084–2.1 b0.01 b0.01 b0.01 b0.024 0.03–0.34 0.03–2.23 0.04–1.92 b0.004–0.156 b0.008–0.393 0.002–6.710 0.028 0.035 0.028–0.071
[7]
Wines of worldwide origin
Mediterranean Sea countries Mediterranean wines Moroccan wines
Italian wines
Italian wines Italian wines
Spanish wines Spanish wines
Spanish wines Greek wines
Greek wines
Portuguese wines Hungarian wines
Hungarian wines Turkish wines
Canadian wines Wines from EU countries and Argentina Wines from south American countries
[14]
[15] [9] [25]
[24]
[16] [23]
[17] [19]
[28] [6]
[18]
[20] [23]
[32] [26]
[9] [21] [22]
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245
Fig. 2. Turkish wine regions (number of analysis samples and mean (ng ml− 1) in parentheses).
from b0.002 to 7.630 ng ml− 1, depending on the origin. The highest OTA concentration reported was in a red wine from Italy [24]. According to our data, mean for red wine is slightly higher than white and rosé wines. This result confirms the preliminary observation by Otteneder and Majerus [14], Filali et al. [25], Soufleros et al. [6], Stefanaki et al. [18], Blesa et al. [19], Rosa et al. [22], Anlı et al. [26], Brera et al. [23], but it contrast to that reported by Lopez de Cerain [17], who observed that red wines showed lower level of OTA than white wines conducted with limited samples in a 2-year study. These differences can be explained by different wine-making methods, storage conditions, climatic factors and grape cultivation [27]. The winemaking procedure is one of the most critical factors in the occurrence and level of OTA in wines. In white wine, the grapes are crushed and pressed rapidly, while red grapes are left to macerate for an extended period of time (several days) which obviously permits higher amount of the toxin to be transferred to the juice in making a red wine [6,14,28,29]. In addition, this process provides good conditions for mould growth and OTA accumulation [14]. This might be also due to the fact that wines clarified with fining agents such as activated carbon, bentonite, gelatine and casein which might adsorb OTA (22). Activated carbon was found to be an efficiently agent that could be used to remove OTA in wines. In our previous study showed that treatment with activated carbon caused 87% of the available toxin decreases in the OTA levels (5 ng ml− 1) present in artificially contaminated in white wine (unpublished data). Similarly, potassium caseinate has been shown to remove 82% of OTA from wine when used at 150 g hL− 1 [30]. The second critical factor influencing presence of OTA is the geographic region of the grapes used in the wine-making. With respect to red wines, whose number in the survey is rather high and whose distribution among the regions is not homogeneous. The incidence and concentrations of OTA in red wines decreased in order Thrace region N Aegean region N east Anatolia N central Anatolia. OTA was detected over 0.1 ng ml− 1 in 10 out of 33 wines originating from Thrace region and 2 out of 9 samples from Aegean region, whereas red wines originating from central Anatolia and east Anatolia contained only small quantities of OTA (ranges b 0.010–0.023 ng ml− 1 and b 0.010– 0.026 ng ml− 1, respectively). Similar results were also found for the rosé wines, even though only 10 samples were analysed. All
rose wines from Aegean region, central and east Anatolia contained OTA lower than 0.1 ng ml− 1, whereas higher amounts of OTA were found in rosé wines from Thrace region. The same trend can be observed for the white wines. All white wines originating from Thrace and Aegean regions contaminated with OTA levels ranging from 0.044 to 0.618 ng ml− 1 and from 0.006 to 0.192 ng ml− 1, respectively, while OTA was detected in 6 out of 11 samples ranging from b 0.006 to 0.072 ng ml− 1 in central Anatolia. There was however no available data with white wine originating from east Anatolia. Our data indicate that, the geographic origin of wines has a strong influence on OTA contamination. The regions of wine samples analysed are represented in Fig. 2. Turkey's diverse regions have different climates with the weather system on the coasts contrasting with that prevailing in the interior. In the Thrace region, the climate is moderate and quite humid, with temperatures range from −16 to 40 °C (average 4 °C in winter and 27 °C in summer). The average humidity is 73%. The region of Aegean has Mediterranean influences, and the average temperatures in the winter are 9 °C and in summer 29 °C, with average humidity of 69%. In the central Anatolia, there is a steppe climate. Summers are hot and dry, in winter however the temperatures can drop below zero. The temperatures drop to − 25 °C in deep in winter, and rise to 40 °C in summer, with the average − 2 °C in winter and 23 °C in summer. Rainfall is low and it usually in form of snow. There is a great temperature difference between day and night. The overall humidity is 62.6%. The eastern Anatolia areas have long, bitterly cold with frequent, heavy snowfall winters and mild summers. The eastern Anatolia areas receive the greatest amount of snow from November until to the end of April. It has the lowest sun levels of all the regions studied. In the eastern Anatolia, the average temperature is − 13 °C in winter and 17 °C in summer [26]. The reason for the higher OTA content in the wines originating from Thrace and Aegean regions, located in the northwest and west of Turkey, respectively, relative to central and east Anatolia can be explained by their climates. Looking at the meteorological data, there are high temperatures in summer and high humidity when compared to central and east Anatolia. Similarly, several authors reported that the wines from the Mediterranean region contain high concentrations of OTA due to their climates being characterised by high humidity and high temperature [19,25]. Black Aspergilli are known to be very
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resistant to sun exposure and to hot and dry environments [8]. In a previous work conducted by Pardo et al. [31] has been showed that high relative humidity and temperature levels favoured ochratoxigenic A. ochraceus isolates growth on grapes and subsequently OTA production. In addition, it has been revealed that the vicinity of a sea also benefits the growth of OTA producing fungi by the increased degree of humidity of the air [23]. Moreover, both rates of occurrence and OTA levels might be varied from one year to the other depending on the meteorological conditions [15,19]. The present study agrees with Anlı et al. [26] who reported that wines originating from Thrace and Aegean regions much more contained OTA than central and east Anatolia. The mean value of OTA contamination have been reported to be 0.929, 1.372, 0.053 and 0.526 ng ml− 1 in red wines originating from Thrace, Aegean, central Anatolia and east Anatolia regions, respectively. Our results are also consistent with pervious surveys conducted in other countries that the geographic origin had a strong influence on OTA levels of wines. Soufleros et al. [6] revealed that wines from islands of Greece contained more OTA than north, south and central areas of Greece. Numerous investigators have concluded from their results that wines from southern countries of the Northern Hemisphere are more contaminated than those of wines from the northern areas [7,14,24]. Similarly, it has been reported that the frequency of occurrence and concentration of OTA in red wines increase from northern to southern Greece [18], from northern to southern Italy [16], and from northern to southern Hungary [32]. Recently, Czerwiecki et al. [21] have concluded that wines from France and Italy were found to contain more frequency and higher levels of OTA than Hungary and Bulgaria. A concentration gradient from north to south also leads to the assumption that OTA-producing fungi can be found more often on grapes growing in the south [14]. 4. Conclusions The results showed that most of the samples contained only small proportions of OTA, although the occurrence of contamination was rather high. Only three of them had values above 0.5 ng ml − 1 . The results support the general tendency that red wines contain more OTA than rosé and white wines. It must be noted that in comparison with wines from central and east Anatolia showed a high level of OTA, and the frequency of the mycotoxin occurrence. Data have also demonstrated that OTA levels in Turkish wines were substantially lower than published results from EU Member States. According to these results, it can be concluded that the contamination of Turkish wines with OTA does not exceed the maximum EU limit (2 ng ml − 1 ). Although more studies must be systematically conducted on the occurrence of OTA risk, it is not implying a high risk of human exposure to OTA through consumption of wine. The best way to protect consumers against OTA health hazards is to implement good agricultural practices (GAPs), good manufacturing practices (GMPs) and the hazard analysis and critical control point (HACCP) systems.
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