Ochratoxin A and 2'R-Ochratoxin A in Selected Foodstuffs and Dietary Risk Assessment.

The aim of this study was to estimate the contamination of grain coffee, roasted coffee, instant coffee, and cocoa purchased in local markets with ochratoxin A (OTA) and its isomerization product 2'R-ochratoxin A (2'R-OTA), and to assess risk of dietary exposure to the mycotoxins. OTA and 2'R-OTA content was determined using the HPLC chromatography with immunoaffinity columns dedicated to OTA. OTA levels found in all the tested samples were below the maximum limits specified in the European Commission Regulation EC 1881/2006. Average OTA concentrations calculated for positive samples of grain coffee/roasted coffee/instant coffee/cocoa were 0.94/0.79/3.00/0.95 µg/kg, with the concentration ranges: 0.57-1.97/0.44-2.29/0.40-5.15/0.48-1.97 µg/kg, respectively. Average 2'R-OTA concentrations calculated for positive samples of roasted coffee/instant coffee were 0.90/1.48 µg/kg, with concentration ranges: 0.40-1.26/1.00-2.12 µg/kg, respectively. In turn, diastereomer was not found in any of the tested cocoa samples. Daily intake of both mycotoxins with coffee/cocoa would be below the TDI value even if the consumed coffee/cocoa were contaminated with OTA/2'R-OTA at the highest levels found in this study. Up to now only a few papers on both OTA and 2'R-OTA in roasted food products are available in the literature, and this is the first study in Poland.

Modified Fusarium Mycotoxins in Cereals and Their Products-Metabolism, Occurrence, and Toxicity: An Updated Review.

Mycotoxins are secondary fungal metabolites, toxic to humans, animals and plants. Under the influence of various factors, mycotoxins may undergo modifications of their chemical structure. One of the methods of mycotoxin modification is a transformation occurring in plant cells or under the influence of fungal enzymes. This paper reviews the current knowledge on the natural occurrence of the most important trichothecenes and zearalenone in cereals/cereal products, their metabolism, and the potential toxicity of the metabolites. Only very limited data are available for the majority of the identified mycotoxins. Most studies concern biologically modified trichothecenes, mainly deoxynivalenol-3-glucoside, which is less toxic than its parent compound (deoxynivalenol). It is resistant to the digestion processes within the gastrointestinal tract and is not absorbed by the intestinal epithelium; however, it may be hydrolysed to free deoxynivalenol or deepoxy-deoxynivalenol by the intestinal microflora. Only one zearalenone derivative, zearalenone-14-glucoside, has been extensively studied. It appears to be more reactive than deoxynivalenol-3-glucoside. It may be readily hydrolysed to free zearalenone, and the carbonyl group in its molecule may be easily reduced to α/ß-zearalenol and/or other unspecified metabolites. Other derivatives of deoxynivalenol and zearalenone are poorly characterised. Moreover, other derivatives such as glycosides of T-2 and HT-2 toxins have only recently been investigated; thus, the data related to their toxicological profile and occurrence are sporadic. The topics described in this study are crucial to ensure food and feed safety, which will be assisted by the provision of widespread access to such studies and obtained results.

Recent Research on Fusarium Mycotoxins in Maize-A Review.

Maize (Zea mays L.) is one of the most susceptible crops to pathogenic fungal infections, and in particular to the Fusarium species. Secondary metabolites of Fusarium spp.-mycotoxins are not only phytotoxic, but also harmful to humans and animals. They can cause acute or chronic diseases with various toxic effects. The European Union member states apply standards and legal regulations on the permissible levels of mycotoxins in food and feed. This review summarises the most recent knowledge on the occurrence of toxic secondary metabolites of Fusarium in maize, taking into account modified forms of mycotoxins, the progress in research related to the health effects of consuming food or feed contaminated with mycotoxins, and also the development of biological methods for limiting and/or eliminating the presence of the same in the food chain and in compound feed.

Dynamics of Deoxynivalenol and Nivalenol Glucosylation in Wheat Cultivars Infected with Fusarium culmorum in Field Conditions─A 3 Year Study (2018-2020).

Fusarium head blight (FHB) caused by pathogenic species of Fusarium fungi is one of the most important diseases of cereal plants and a factor contributing to losses in plant production. The growth of FHB-associated species is often accompanied by biosynthesis of secondary metabolites─mycotoxins, which serve as a virulence factor. The aim of the study was to evaluate the ratios between deoxynivalenol (DON) and nivalenol (NIV) and their derivatives in the ears of six cultivars of winter wheat with varying resistance to FHB, taking into account a range of factors (weather conditions, location, cultivar, and year) after inoculation with Fusarium culmorum, during a 3 year field experiment, 2018-2020. The presence of toxins in the ears was measured within 21 days of inoculation. The toxins were found in the ears as soon as on the third day from the start of the experiment, whereas relative humidity higher than 80% was a decisive factor for FHB incidence. All wheat cultivars showed the ability to biotransform DON and NIV present in the ears to glucosides, that is, deoxynivalenol-3-glucoside (DON-3G) and nivalenol-3-glucoside (NIV-3G). The levels of these metabolites showed significant correlation with the levels of their basic analogues. In most cases, higher levels of DON and NIV in wheat ears and higher levels of their metabolites were observed, but the relative levels of DON-3G/DON and NIV-3G/NIV at relatively high levels of toxins were lower compared to the ear samples with relatively low toxin levels. The presented results are the first studies, which systematically correlate a variety of wheat cultivars with their extent to glucosylate trichothecenes.

Transformation of Selected Fusarium Toxins and Their Masked Forms during Malting of Various Cultivars of Wheat.

This study investigated the impact of malting of six wheat cultivars inoculated with Fusarium culmorum on the dynamics of content changes of selected Fusarium toxins. The grains of all the tested cultivars showed a high content of deoxynivalenol (DON), zearalenone (ZEN), and their derivatives, whereas nivalenol (NIV) and its glucoside were found only in the Legenda cultivar. Our experiments confirmed that the malting process of wheat grain enables the secondary growth of Fusarium, and mycotoxin biosynthesis. The levels of toxins in malt were few-fold higher than those in grain; an especially high increase was noted in the case of ZEN and its sulfate as the optimal temperature and pH conditions for the biosynthesis of these toxins by the pathogen are similar to those used in the grain malting process. This is the first paper reporting that during the malting process, biosynthesis of ZEN sulfate occurs, instead of glycosylation, which is a typical modification of mycotoxins by plant detoxication enzymes.

Transformation of Selected Trichothecenes during the Wheat Malting Production.

The transformation of deoxynivalenol (DON), nivalenol (NIV), and their glucosides (DON3G and NIV3G) during the malting of grains of two wheat varieties was studied. The concentration of DON3G and NIV3G started to increase significantly before the concentration of DON and NIV increased. This may reflect the transformation of the parent mycotoxin forms into their glucosides due to xenobiotic detoxification reactions. After a sharp rise during the last 2 days of the process (day 6 and 7), the DON concentration reached 3010 ± 338 µg/kg in the Legenda wheat-based malt and 4678 ± 963 µg/kg in the Pokusa wheat-based malt. The NIV concentration, at 691 ± 65 µg/kg, remained the same as that in the dry grain. The concentration of DON3G in the Legenda and Pokusa wheat-based malt was five and three times higher, respectively, than that in the steeped grain. The concentration of NIV3G in the Legenda wheat-based malt was more than twice as high as that in the steeped grain. The sharp increase in the concentration of DON at the end of the malting process reflected the high pathogen activity. We set aside some samples to study a batch that was left undisturbed without turning and aeration, for the entire period of malting. The concentration of DON in the malt produced from the latter batch was 135% and 337% higher, for Legenda and Pokusa, respectively, than that in the malt produced from the batch that was turned and aerated. The NIV concentration was 22% higher in the latter batch.

Transformations of Selected Fusarium Toxins and Their Modified Forms During Malt Loaf Production.

An increasing number of studies have found that modified mycotoxins, such as free mycotoxins, naturally occur in food, and severely impact food safety. The present study investigated concentrations of trichothecenes nivalenol (NIV), deoxynivalenol (DON), and zearalenone (ZEN), together with their modified forms, nivalenol-3-glucoside (NIV-3G), deoxynivalenol-3-glucoside (DON-3G), and zearalenone-14-glucoside (ZEN-14G) and zearalenone-14-sulfate (ZEN-14S), respectively, at successive stages of malt loaf production (flour, dough kneading/fermentation, loaf baking). Toxins in bakery products originate in flour produced from wheat grain that is naturally contaminated with Fusarium culmorum. Mycotoxin concentrations were determined using high-performance liquid chromatography-high resolution mass spectrometry, and did not significantly change during the successive stages of bread production. After the dough kneading/fermentation stage, concentrations of NIV-3G and DON-3G were slightly increased, whereas those of ZEN and ZEN-14S were slightly decreased. The largest average decrease (21%) was found in ZEN-14G. After the baking stage, the average concentrations of NIV-3G, DON-3G, ZEN-14S, and ZEN-14G in the loaf crumb and crust decreased by 23%, 28%, 27%, and 20%, respectively, compared with those in the dough. During this technical process, the concentration of ZEN-14G in loaf crumb significantly decreased by an average of 48%, and those of ZEN, ZEN-14S, and ZEN-14G in loaf crust decreased by an average of 29%, 42%, and 48%, respectively. Considering the possibility of modified mycotoxins degradation to free forms, as well as the ability to synthesize them from free forms during technological processes, it would be prudent to consider them together during analysis.

Selected Trichothecenes in Barley Malt and Beer from Poland and an Assessment of Dietary Risks Associated with their Consumption.

Eighty-seven samples of malt from several Polish malting plants and 157 beer samples from the beer available on the Polish market (in 2018) were tested for Fusarium mycotoxins (deoxynivalenol (DON), nivalenol (NIV)), and their modified forms ((deoxynivalenol-3-glucoside (DON-3G), nivalenol-3-glucoside (NIV-3G), 3-acetyldeoxynivalenol (3-AcDON)). DON and its metabolite, DON-3G, were found the most, among the samples analyzed; DON and DON-3G were present in 90% and 91% of malt samples, and in 97% and 99% of beer samples, respectively. NIV was found in 24% of malt samples and in 64% of beer samples, and NIV-3G was found in 48% of malt samples and 39% of beer samples. In the malt samples, the mean concentration of DON was 52.9 µg/kg (range: 5.3-347.6 µg/kg) and that of DON-3G was 74.1 µg/kg (range: 4.4-410.3 µg/kg). In the beer samples, the mean concentration of DON was 12.3 µg/L (range: 1.2-156.5 µg/L) and that of DON-3G was 7.1 µg/L (range: 0.6-58.4 µg/L). The concentrations of other tested mycotoxins in the samples of malt and beer were several times lower. The risk of exposure to the tested mycotoxins, following the consumption of beer in Poland, was assessed. The corresponding probable daily intakes (PDIs) remained a small fraction of the tolerable daily intake (TDI). However, in the improbable worst-case scenario, in which every beer bottle consumed would be contaminated with mycotoxins present at the highest level observed among the analyzed beer samples, the PDI would exceed the TDI for DON and its metabolite after the consumption of a single bottle (0.5 L) of beer.

Contamination of Wheat Cultivated in Various Regions of Poland during 2017 and 2018 Agricultural Seasons with Selected Trichothecenes and Their Modified Forms.

Cross-interaction of antibodies within the immunoaffinity columns used in this study facilitated the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), their glucoside derivatives (NIV-3G, DON-3G), and 3-acetyl-deoxynivalenol (3-AcDON) in wheat grain harvested in various regions of Poland. In Poland, 2018 was a warm, dry agricultural season, and hence, was relatively less favourable for cereal cultivation than 2017. Data on the natural occurrence of NIV-3G in wheat grain are among the first published in the literature. DON was the most frequently found mycotoxin in the tested samples; the percentage occurrence of DON-positive samples was 92% in 2017 and 61% in 2018. Moreover, DON concentrations were generally higher in 2017 samples (5.2⁻1670.7 µg/kg) than those in 2018 samples (range 5.0⁻461.7 µg/kg). A similar pattern was found for DON-3G. However, no statistically significant differences between the samples from the two agricultural seasons were observed for the other three mycotoxins that were analysed, and their concentrations were generally considerably lower. DON was strongly correlated with DON-3G (correlation coefficient r = 0.9558), while NIV was strongly correlated with NIV-3G (r = 0.9442). The percentage occurrence of NIV-3G- and DON-3G-positive samples was 14% in 2017 and 49% in 2018. The NIV-3G/NIV ratio was 5.9⁻35.7%, while the DON-3G/DON ratio range was 3.2⁻53.6%. In 2018, wheat samples from Southern Poland exhibited statistically significantly higher levels of DON than those from Northern Poland. The dry and hot summer of 2018 not only reduced wheat yields, but also limited development of Fusarium spp. Therefore, grain harvested that year was generally contaminated with relatively low levels of mycotoxins. Lower levels of DON were also accompanied by lesser amounts of DON-derivatives.

Natural Occurrence of Nivalenol, Deoxynivalenol, and Deoxynivalenol-3-Glucoside in Polish Winter Wheat.

The presence of mycotoxins in cereal grain is a very important food safety factor. The occurrence of "masked" mycotoxins has been intensively investigated in recent years. In this study, the occurrence of nivalenol, deoxynivalenol-3-glucoside, and deoxynivalenol in 92 samples of winter wheat from Polish cultivars was determined. The frequency of the occurrence of deoxynivalenol and nivalenol in the samples was 83% and 70%, respectively. The average content of the analytes was: for deoxynivalenol 140.2 µg/kg (10.5-1265.4 µg/kg), for nivalenol 35.0 µg/kg (5.1-372.5 µg/kg). Deoxynivalenol-3-glucoside, the formation of which is connected with the biotransformation pathway in plants, was present in 27% of tested wheat samples; its average content was 41.9 µg/kg (15.8-137.5 µg/kg). The relative content of deoxynivalenol-3-glucoside (DON-3G) compared to deoxynivalenol (DON) in positive samples was 4-37%. Despite the high frequency of occurrence of these mycotoxins, the quality of wheat from the 2016 season was good. The maximum content of DON, as defined in EU regulations (1250 µg/kg), was exceeded in only one sample. Nevertheless, the presence of a glycosidic derivative of deoxynivalenol can increase the risk to food safety, as it can be hydrolyzed by intestinal microflora.