IJFS#1826_bozza Ital. J. Food Sci., vol. 32, 2020 - 712 SHORT COMMUNICATION OCCURRENCE OF DEOXYNIVALENOL IN BEERS COMMERCIALISED IN ITALY M. GRUMI, A. KUNOVA, M. ISOTTI, A. BARBIROLI and M. PASQUALI* DeFENS, Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy *Corresponding author: matias.pasquali@unimi.it ABSTRACT Deoxynivalenol (DON) is the most frequently detected mycotoxin in beer. This study represents a comprehensive assessment of DON occurrence in beers from the Italian market. Seventy-two craft and industrial beer samples were tested using the RIDASCREEN DON® ELISA method. DON was found in all samples. The average DON contamination was 34.3 μg/L (range: 6.1 - 111.2 μg/L). The highest contamination level was found in a wheat-based sample. Our study determined that wheat-based beers have a higher DON contamination than barley-based beers. Further studies are needed to verify the role of single ingredients on the risk of DON accumulation in beers. Keywords: consumption level, ELISA, fermented beverage, food safety, mycotoxins, wheat Ital. J. Food Sci., vol. 32, 2020 - 713 1. INTRODUCTION Deoxynivalenol (DON) is one of the most frequently detected mycotoxins in cereals and cereal-based products (PEDROSO PEREIRA et al., 2019). DON belongs to type-B trichothecenes mycotoxins and it is mainly produced by Fusarium graminearum and Fusarium culmorum (PASQUALI et al., 2016). Type-B trichothecenes involve DON, its acetylated derivatives (3-Acetyl-DON and 15-Acetyl-DON) nivalenol, and deoxynivalenol-3-glucoside (D3G). D3G might be the result of plant metabolism of DON or of some food processing operations (BERTHILLER et al., 2013). Although DON and its derivatives are not classified as being carcinogenic to humans, an exposure to this type of mycotoxins may be associated with a wide range of adverse health complications (EFSA CONTAM PANEL, 2017). The main effect of deoxynivalenol is the inhibition of protein synthesis. This leads to acute gastro-intestinal symptoms (e.g. emesis and diarrhoea) as well as, in case of long-term exposure, to immune system diseases or disorders (PEDROSO PEREIRA et al., 2019). Considering its potential to cause serious health issues, the European Commission established a range of maximum limits for DON in cereals and cereal-based foods (FERRIGO et al., 2016). In 2006, the European Commission proposed the maximum level for DON in cereals intended for direct human consumption of 750 μg/kg (EFSA CONTAM PANEL, 2017). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has also established a provisional maximum tolerable daily intake (PMTDI) of 1 μg/kg body weight for the sum of DON and its derivatives (PEDROSO PEREIRA et al., 2019). Since DON is largely detected in cereals and malt, which represent the key ingredients of beer, a DON contamination of this type of alcoholic beverages seems unavoidable (RODRÍGUEZ-CARRASCO et al., 2015). It is worth specifying that maximum levels for DON in beer have not been set so far. Monitoring DON in beer is important considering that this widely popular fermented beverage may significantly contribute to the intake of mycotoxins and even exceed the safety levels when following a regular diet (PAPADOPOULOU-BOURAOUI et al., 2004; RODRÍGUEZ-CARRASCO et al., 2015). Therefore, exposure of consumers to DON and its derivatives through beer consumption should not be underestimated, especially in case of “heavy drinkers” (PASCARI et al., 2018). The occurrence of deoxynivalenol in beer has been studied in various surveys all around the world: in Belgium (PAPADOPOULOU-BOURAOUI et al., 2004), in Poland (KUZDRALIŃSKI et al., 2013), in Austria, Hungary, Croatia and Serbia (VARGA et al., 2013), in Spain (RODRÍGUEZ-CARRASCO et al., 2015), in Brazil (PIACENTINI et al., 2015), in Germany (BAUER et al., 2016), in Paraguay (ARRÚA et al., 2019) and in Mexico (WALL- MARTÍNEZ et al., 2019 b). Occurrence of DON in beers from the Italian market was assessed in two surveys with limited number of samples: by PIETRI et al. (2003) and by PETERS et al., (2017). The surveys showed that the small number of Italian samples analysed, compared with samples produced in other countries, had the lowest DON contamination levels (below than 10 μg/L). In 2018 the Italian beer consumption increased by 3.2%. Therefore, per capita consumption reached its historical peak of 33.6 L a year (ASSOBIRRA, 2018). Considering both the limited number of surveys focusing on the occurrence of DON in beers purchased from the Italian market and the significant raise of Italian craft breweries the aims of this work were: 1. to assess the level of deoxynivalenol in beer samples sold on the Italian market from May 2018 to December 2018 in order to update and enrich the available data; Ital. J. Food Sci., vol. 32, 2020 - 714 2. to compare the contamination of industrial beers to craft beers, taking into account the findings of PETERS et al., 2017 who identified higher DON incidence in craft beers collected all over Europe; 3. to define whether wheat-based beers had higher DON contamination compared to barley based beers. 2. MATERIALS AND METHODS Seventy-two beer samples were purchased from May to December 2018 in pubs, supermarkets and beer shops located in the North of Italy. Some of them were home brewed from semi-processed products. Most samples (53) were produced by Italian companies whereas nineteen samples came from different European countries. The foreign beers were produced in Germany (8), Belgium (5), Austria (1), Czech Republic (1), France (1), Netherlands (1), Sweden (1) and United Kingdom (1). None of the beers exceeded their expiration date. 1.5 mL of each sample were placed in separate test tubes and stored at -80°C for at least 24 hours, in order to reach a complete degassed condition. 50 μL of CO2-free samples were subjected to the analysis. The commercial competitive ELISA RIDASCREEN DON® (R-Biopharm AG, Germany) was used. The declared detection limit of RIDASCREEN DON® is 3.7 ppb for beer samples, with a cross-reactivity to DON (100%), 3-Acetyldeoxynivalenol (>100%), 15-Acetyldeoxynivalenol (approximatively 19%), Nivalenol (approximatively 4%), Fusarenon-X (<1%) and T-2 Toxin (<1%). All reagents required for the analysis – including standards – were contained in the kit. The PBS-Tween washing buffer was prepared by dissolving the provided salt in Milli-Q® ultra-pure water. The test procedure issued by the producer was strictly followed. Results were obtained by reading sample or standard absorbances at 450 nm using a Synergy (H1) microplate reader (BioTek®, US) spectrophotometer. The absorbance was inversely proportional to the DON concentration in the samples. Absorbance was expressed as a percentage value with respect to the zero standard (100 × (absorbance sample or standard)/(absorbance zero standard)). The values calculated for the standards were entered in a system of coordinates on a semilogarithmic graph against the DON concentrations (expressed in μg/L) using the online editor Line of Best Fit Generator “plot.ly” (PLOTLY TECHNOLOGIES, 2015). This allowed obtaining a calibration curve from which the DON concentration, actually contained in all the samples, was defined. All samples were measured at least twice in each analysis. Samples resulted off the chart were diluted using ultra-pure water, in ratio 1:2 and 1:5 and then reanalysed. The pH of each sample was also measured using an XS Instruments® benchtop pHmeter supplied with an automatic temperature compensation and a microelectrode that was fit for the low-volume samples. The statistical analysis was carried out using the statistical software JASP Version 0.11.1 (JASP TEAM, 2019). A linear regression was performed to assess relationship among DON, pH and alcohol content values. Furthermore, analysis of variance (ANOVA) followed by Bonferroni post hoc test was performed in order to determine the significance of fixed factors “wheat”, “type of brewing process” and “type of fermentation” on the DON concentration. All tests were executed at a significance level of P<0.05. Ital. J. Food Sci., vol. 32, 2020 - 715 3. RESULTS With reference to the whole collection of beer samples, 46 samples were classified as “craft beer” whereas 26 samples were classified as “industrial produced beer”. Wheat was one of the ingredients in 21 samples. Three of the selected samples were “gluten free” beers. The results of the analysis are summarised in Table 1. Table 1. Samples description and results of the analysis. Sample Country of origin 1%ABV Type of fermentation Type of brewing process Wheat Special features pH 2DON (𝝁𝒈/𝑳) B1 Germany 7.5 bottom industrial 4.66 46.9±7.2 B2 Germany 5.3 top industrial 4.36 71.5±15.7 B3 Germany 5.4 top industrial 4.40 35.6±3.3 B4 Germany 5 bottom industrial 4.48 25.7±8.4 B5 Germany 4.9 bottom industrial 4.49 43.3±9.6 B6 Germany 5 top industrial yes 4.47 32.1±7.7 B7 Germany 5 bottom industrial 4.50 57.2±19.5 B8 Italy 4.7 top industrial 4.58 17.2±0.5 B9 Italy 6.5 top craft yes 4.51 45.0±2.9 B10 Italy 5.5 top craft 4.57 55.9±14.6 B11 Italy 4.5 top craft yes 4.31 54.3±13.5 B12 Italy 5 top craft yes 3.82 61.4±5.4 B13 Italy 6.9 top craft yes 4.50 49.3±12.9 B14 Italy 5.2 top craft yes 4.64 25.8±0.8 B15 Italy 7.8 top craft 4.96 54.8±7.1 B16 Italy 5.6 top craft 4.58 50.5±15.6 B17 Italy 3.9 top craft 4.17 18.1±2.7 B18 Italy 5 bottom industrial 4.93 18.6±10.2 B19 Italy 4.5 bottom craft 4.43 19.9±7.3 B20 Italy 5 bottom craft yes 4.64 44.5±17.8 B21 Italy 6 top craft 4.64 18.4±2.1 B22 Italy 4.5 top craft gluten free 4.50 17.8±8.8 B23 Italy 5.6 top craft yes 4.46 20.7±0.6 B24 Italy 5.6 top craft 4.36 13.1±2.1 B25 Italy 4.7 bottom industrial 4.39 10.8±2.0 B26 Italy 4.7 bottom industrial gluten free 4.66 6.1±0.1 B27 Italy 5.5 bottom industrial 4.60 20.0±0.8 B28 Italy 4.5 bottom industrial 4.59 16.1±1.2 B29 Italy 4.7 bottom industrial 4.44 15.7±1.4 B30 Italy 5 bottom industrial 4.72 22.7±0.7 B31 Italy 5.5 top craft gluten free 4.53 13.1±1.7 B32 Italy 7 top craft 4.31 95.8±5.7 B33 Italy 5 top craft 4.41 31.9±1.0 B34 Italy 0.49 n.d. industrial alcohol-free 4.83 9.5±0.4 Ital. J. Food Sci., vol. 32, 2020 - 716 B35 Italy 5 top craft yes 4.05 65.9±12.2 B36 Belgium 4.9 top industrial yes 4.46 60.8±6.8 B37 Italy 5 top industrial yes 4.19 76.3±9.6 B38 Italy 5.1 bottom industrial 4.22 12.0±0.8 B39 Belgium 8 top craft yes 4.40 27.8±4.0 B40 Italy 5.2 bottom craft 4.80 10.7±0.5 B41 Italy 4.6 bottom craft 4.55 22.3±3.2 B42 Belgium 7.5 top industrial yes 4.77 50.7±7.1 B43 Germany 12 top industrial yes 4.73 56.3±9.6 B44 Italy 8 top industrial 4.62 36.4±7.9 B45 Italy 8 bottom craft 4.70 80.3±2.1 B46 Italy 7.5 bottom industrial 4.92 19.1±0.8 B47 Nether-lands 6.5 top craft yes 4.35 37.6±15.3 B48 United Kingdom 4.6 bottom craft 4.55 9.3±0.6 B49 Italy 5.4 bottom craft 4.56 54.5±9.8 B50 Italy 5 bottom craft 4.73 18.9±1.8 B51 Belgium 9.5 top craft 4.36 24.3±8.4 B52 Italy 8 top craft 4.80 24.1±14.1 B53 France 5.5 bottom industrial 4.43 12.7±3.1 B54 Italy 9 top craft 4.67 10.1±0.2 B55 Czech Republic 4.4 bottom industrial 4.69 15.0±0.7 B56 Belgium 6.5 spontaneous craft yes 3.45 111.2±14.1 B57 Italy 6 top craft 4.66 10.3±1.3 B58 Sweden 5 bottom craft yes 4.55 23.3±3.2 B59 Italy 9 top craft 4.53 45.0±6.2 B60 Italy 4.9 top craft 4.56 50.5±15.7 B61 Italy 4.9 bottom craft 4.79 48.5±8.2 B62 Italy 9.7 top craft 4.80 82.6±13.8 B63 Italy 8.7 top craft 4.60 35.2±5.1 B64 Italy 4.7 top craft 4.78 7.5±1.2 B65 Italy 5.2 top craft yes 4.38 17.2±1.0 B66 Italy 4.5 bottom craft 4.57 37.8±1.0 B67 Italy 8.5 top craft 4.49 46.4±4.8 B68 Italy 4.6 top craft yes 4.31 25.5±2.2 B69 Italy 4.6 top craft 4.39 28.9±2.8 B70 Italy 6.5 top craft 3.98 35.0±6.9 B71 Italy 6.5 top craft 4.77 19.1±4.9 B72 Italy 5.5 bottom craft 4.51 20.5±5.6 1percentage alcohol by volume; 2 mean value±standard deviation. Ital. J. Food Sci., vol. 32, 2020 - 717 DON contamination levels obtained by RIDASCREEN® DON ELISA DON was found in all samples with a contamination incidence of 100%. The contamination ranged from 6.1 μg/L to 111.2 μg/L. The average contamination level was 34.3 μg/L, with a median of 25.8 μg/L. Only in 43.06% of samples (31) the DON contamination was greater than the average contamination level (34.3 μg/L). The highest contamination level (111.2 μg/L) was found in a sample that included wheat among the ingredients (Table 1). pH values ranged from 3.45 to 4.96 with an average value of 4.51 and a median of 4.53. The percentage alcohol by volume (%ABV) ranged from a minimum value below 0.5% (non-alcoholic beer sample) to a maximum of 12%. The average alcohol content was 5.8% with a median of 5.2%. In order to assess the influence of the two variables pH and %ABV on DON contamination levels, a linear regression model was computed. Both %ABV and pH were partially correlated with samples DON content (P>0.001) but R2 were negligible (R2 = 0.109, R2 = 0.123). Analysis of Variance (ANOVA) was performed in order to determine the potential impact of both wheat (as ingredient) and the type of brewing process (industrial or craft beer) on DON content. The ANOVA revealed a positive effect of wheat as ingredient on DON contamination of the samples (P value = 0.001), with an average DON contamination of wheat-based beers of 46.6 μg/L (±23.1) and an average DON contamination of beers without wheat of 29.9 μg/L (±20.8). On the contrary, there was no difference between the types of brewing process on the final DON content (P value = 0.966). 4. CONCLUSIONS This study represents the first comprehensive assessment of the DON level in beers sold on the Italian market. Moreover, it identifies wheat-based beers as potentially contributing to higher level of DON accumulation in consumers. The screening results showed a weak correlation between the alcohol content (%ABV) and the DON contamination levels. Higher alcohol levels were related to significantly higher DON levels in beers by other researchers such as PAPADOPOULOU-BOURAOUI et al., 2004; KOSTELANSKA et al., 2009; PETERS et al., 2017; KSIENIEWICZ-WOŹNIAK et al., 2019; WALL-MARTÍNEZ et al., 2019 b. The requirement of a higher input of fermentable sugars in malt wort, in order to reach higher alcohol levels, seems to be a possible explanation. Indeed, the further supplement of grains may be associated with a higher risk of mycotoxins contamination (KOSTELANSKA et al., 2009; PETERS et al., 2017; PASCARI et al., 2018; WALL-MARTÍNEZ et al., 2019 b). A previous study (WALL-MARTÍNEZ et al., 2019 a) did not find any correlation between pH and DON contamination values. However, in our study we observed a slightly negative correlation that suggests that higher beer pH is correlated to lower DON content. Further studies are needed to decipher this phenomenon. Given the minor pH diversity it is not possible to postulate that alkaline pH are the cause of decreased DON stability as it was found for baking products (YOUNG et al., 1984). As it is known that the brewing process can include a pH correction before selling, our observations may not be associated to any specific processing of the beer. According to our surveillance, wheat-based beers represent higher risk for consumers. As stated by a recent report of the U.S. Department of Agriculture Economic Research Ital. J. Food Sci., vol. 32, 2020 - 718 Service, wheat may represent 5-10% of the whole malts used by U.S. breweries. The increased use of wheat may be related to the significant growth of craft beer production along with the increased popularity of several wheat beers produced by leading international brewers (JIN et al., 2018). The scientific report of the European Food Safety Authority (EFSA, 2013) states that wheat has an average DON contamination about three times higher (434.4 μg/kg) than barley (176.1 μg/kg). That might explain the greater DON contamination values of wheat-based beers when compared to other beer types. Similar results were reported by KSIENIEWICZ-WOŹNIAK et al. (2019), who found a very high percentage of wheat-based beer samples positive to DON. Previously published studies (PETERS et al., 2017; WALL-MARTÍNEZ et al., 2019 b) found a higher risk of mycotoxin contamination in craft beers. Diversely, our analysis did not reveal any significant differences between industrial and craft beers for what concerns DON contamination, which is in accordance with the study conducted by ARRÚA et al., 2019. EFSA estimates that the contribution of DON deriving from beer, in adult population, is approximatively 0.5-5.3% (EFSA, 2013). The average contamination value obtained in this study (34.3 μg/L) is three times higher than the average value (13.5 μg/L) taken into consideration by EFSA, 2013. Based on the average DON value found in our study, the consumption of a heavy drinker of 70 kg of body weight, consuming 0.5 L of beer per day, will determine a DON daily intake of 24.5 %. Considering the 2018 beer per capita consumption of 33.6 L (0.092 L of beer per day) of the italian consumers 4.5% of the TDI will be reached. These numbers are substantially higher than those found by PIETRI et al. (2003). Annual climate and weather variability may contribute to modify levels of mycotoxins in field crops (BEYER et al., 2014), which will eventually lead to modified levels of DON in beer. For this reason, annual monitoring of grains for DON contamination would be essential to investigate the variability of malts contamination. For the year 2018, our data suggests that through the consumption of beer, the Italian population received a higher percentage of DON from beer consumption compared to the average estimation from EFSA. Hence, our study suggests that the intake of DON through beer consumption should be updated for each nation, possibly on a yearly based manner. To verify if this can be simply attributed to a “year effect” or to the changes in the ingredients used for beer production further monitoring is needed. Future studies should also focus on the impact of other grains on final DON levels in beer. Specifically, since maize is used in beer production and it has an average DON contamination (1041.9 μg/kg) significantly higher than barley (176.1 μg/kg) (EFSA, 2013), an investigation of maize impact on DON contamination would be of interest. 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