Mycotoxins in horse feed: Incidence of deoxynivalenol in oat samples from stud farms

Reports concerning mycotoxins in horse feed are very rare and are typically restricted to fumonisins. As a non-ruminant monogastric species, horses may be more sensitive to adverse effects of mycotoxins, but the most severe effect of fumonisin B1 (FB1) in equines is that it causes fatal leucoencephalomalacia. In recent years, the European Food Safety Authority (EFSA) has evaluated several mycotoxins as “undesirable substances in animal feed” with the aim of establishing guidance values for the feed industry. In its evaluation of deoxynivalenol (DON), EFSA concluded that this toxin exhibited toxic effects in all species, but that horses were more tolerant towards this toxin than pigs. According to the available data, a systematic survey on mycotoxins in horse feed in Serbia has not been published. Therefore, the aim of this study was to investigate the incidence of mycotoxins in horse feed in Vojvodina. Samples of oats for horse consumption, collected in 2010, were analyzed by enzyme immunoassays (ELISA) for deoxynivalenol contamination. Twelve samples of oats were taken from twelve horse studs, with sport, school and hobby horses

Elisa and HPLC analyses of deoxynivalenol in maize and wheat

Deoxynivalenol (DON) is a part of the family of mycotoxins called trichothecenes which are produced by a number of different Fusarium mold species. The presence of DON in 25 wheat and 25 maize samples was examined by Enzyme Linked Immunosorbent Assay (ELISA) and High Performance Liquid Chromatography (HPLC) methods. The presence of DON was detected and determined in 5 (20%) maize and 6 (25%) wheat samples by both of the methods. Correlation between ELISA and HPLC results was established, with the correlation coefficients (r) of 0.9691 and 0.9735 for wheat and maize samples, respectively. The results obtained by ELISA method were significantly higher than those obtained by HPLC method. This fact can be explained by the presence of conjugated or masked mycotoxins in the samples, especially DON-3-glucoside (DON-3-Glc), which could not be determined by HPLC method due to the lack of external standards. Contrary to this, being insufficiently selective towards masked DON, ELISA method measures total DON content of a sample. According to the obtained results, ELISA can be used as a reliable screening method, but the confirmation of positive results must be done by HPLC method

Fumonisins and co-occurring mycotoxins in north Serbian corn

The presence of fumonisin has not been regulated in the legislation of the Republic of Serbia. Therefore, the data on contamination of cereals, especially corn, which is highly susceptible to contamination by this toxin, are not sufficient. This paper presents the results of testing the corn samples collected in the autumn 2009 on the territory of Bačka. Samples were analyzed for the contents of fumonisins and it was determined whether there is a correlation between the moisture content, total number and class of fungi, as well as the content of aflatoxin, ochratoxin and zearalenone. Using enzymatic immunoaffinity method it was discovered that the highest percentage of samples were contaminated with fumonisins, which was probably due to the presence of Fusarium molds as the most abundant ones. The positive samples contained fumonisin in the concentrations from 0.030 to 1.52 mg kg−1. The influence of the climate and moisture content of grain on fungal contamination and mycotoxin production was analyzed in order to investigate the predictability of the presence of mycotoxins

Prisustvo zearalenona u najčešće uzgajanim sortama pšenice u Srbiji

A total of 45 samples of wheat from three different locations in Vojvodina were analyzed for the presence of zearalenone. Analytical methods based on clean-up by solid-phase extraction (SPE) columns and detection by liquid chromatography were used after validation. Limit of detection for ZEA in wheat was 18.6 μg/kg and the limit of quantification was 56.5 μg/kg. Recovery values ranged between 86% and 97%. The occurrence of ZEA in wheat was rather high with 53.3% of positive samples with the average value of 330 μg/kg. Incidences were found from 68 μg/kg to 1079 μg/kg. Contamination levels were above the established maximum limit for unprocessed cereals, other than maize, in as many as seventeen samples. These results were compared to the results of investigation of deoxynivalenol and fumonisin content, established in our previous work on the same samples. The results obtained were also compared to those of the neighboring countries where the relevant data existed and to the data of previous studies in our country.Na prisustvo zearalenona analizirano je ukupno 45 uzoraka pšenice sa tri različite lokacije u Vojvodini. Korišćene su analitičke metode zasnovane na prečišćavanju ekstrakcijom na čvrstoj fazi, te kvantifikacija tečnom hromatografijom, nakon validacije metode. Granica detekcije za zearalenon u pšenici je iznosila 18,6 μg/kg, a granica određivanja 56,5 μg/kg. Efikasnost metode je bila u opsegu od 86% do 97%. Zearalenon je bio prisutan u 53,3% ispitivanih uzoraka, sa prosečnim sadržajem od 330 μg/kg. Dobijene vrednosti sadržaja zearalenona su bile u opsegu od 68 μg/kg do 1079 μg/kg. U čak sedamnaest uzoraka je pronađena koncentracija ovog toksina koja prevazilazi maksimalni dozvoljeni sadržaj zearalenona u netretiranim žitaricama. Ovi rezultati su upoređeni sa vrednostima sadržaja deoksinivalenola i fumonizina u istim uzorcima dobijenim u našim prethodnim istraživanjima. Rezultati su takođe upoređeni sa dostupnim rezultatima dobijenim u našoj i susednim zemljama tokom prethodnih godina

Prisustvo deoksinivalenola u uzorcima strnih žita u žetvenoj 2009/10. godini

Fusarium head blight (FHB) is present in all growing regions of small grains and causes significant losses in yield and grain quality. In our environmental conditions, dominant species is Fusarium graminearum Group 2. During 2009/10 there was a significant Fusarium infestation on wheat, barley and triticale. The aim of this study was to examine the contents of deoxynivalenol (DON) in cereal samples taken after 2009/10 harvest season. We analyzed 22 NS varieties of small grains from Rimski Šančevi, including 16 varieties of winter wheat, one facultative wheat variety, four varieties of winter barley and one variety of triticale. Analytical methods based on clean-up by solid-phase extraction (SPE) columns and detection by liquid chromatography were used. Fifteen out the 22 analyzed samples were positive for the presence of DON at a mean level of 0.537 mg/kg. The highest concentration was 1.952 mg/kg. These findings were in correlation with percentage of the Fusarium damaged kernels.Patogeni iz roda Fusarium su prisutni u svim rejonima gajenja strnih žita i nanose značajne gubitke u prinosu i kvalitetu zrna. Fuzariozu klasa pšenice prouzrokuje veći broj vrsta iz roda Fusarium. U našim uslovima gajenja dominantna je vrsta Fusarium graminearum Grupa 2. U toku 2009/10. godine došlo je do značajne pojave fuzarioza na pšenici, ječmu i tritikaleu. Cilj rada je bio da se ispita sadržaj DON-a na uzorcima strnih žita uzetih posle žetve iz uslova prirodne zaraze. Analizirane su 22 novosadske sorte strnih žita iz lokaliteta Rimski Šančevi, od čega: 16 sorti ozime pšenice, jedna fakultativna sorta pšenice, četiri sorte ozimog ječma i jedna sorta tritikalea. Uzorci su uzeti sa parcela različitih površina u zavisnosti od značaja i rasprostranjenosti gajene sorte. Analitičko određivanje je zasnovano na prečišćavanju sirovog ekstrakta analiziranih uzoraka pomoću tzv. Mucosep kolona, a zatim je sadržaj DON-a kvantitativno određen tečnom hromatografijom. Od 22 analizirana uzorka strnih žita čak 15 (68,2%) je bilo pozitivno na prisustvo DON-a. Još veći procenat zaraženosti DON-om je utvrđen kada je u pitanju samo pšenica (82,4%). Prosečan sadržaj DON-a je iznosio 0,537 mg/kg a najveća koncentracija je utvrđena u uzorku tritikalea i iznosila je visokih 1,952 mg/kg. Od svih uzoraka koji su bili pozitivni na prisustvo ovog mikotoksina, 2 su prevazilazila koncentracije koje su propisane od strane Evropske komisije. Sve ovo ukazuje na visoku zaraženost strnih žita sa naših polja iz žetve 2010. Procenat fuzarioznih zrna kod 16 ispitivanih sorti pšenice kretao se od 1 do 11,5%, a gubici u masi 1000 zrna od 1,2 do 5,7%. Između jačine zaraze u polju i sadržaja mikotoksina DON ustanovljena je potpuna pozitivna korelacija kod pojedinih sorti. Fakultativna sorta pšenice Nataša je imala visoku koncentraciju DON od 1,572 mg/kg, pri stepenu zaraze u polju od 33,3% zaraženih klasova po 1 m2. Sorta Zvezdana imala je najniži procenat zaraze u polju od 1% i kod nje nije determinisano prisustvo mikotoksina DON

Potencijal za proizvodnju fumonizina kod izolata Fusarium verticillioides sa zrna kukuruza i pšenice iz Srbije

The production of fumonisins by potentially toxigenic Fusarium verticillioides isolates originating from Serbian maize and wheat kernels was tested in vitro. A total of six F. verticillioides isolates were incubated on yeast extract sucrose medium (YESA) for 4 weeks at 25 °C in the dark. Their toxin production potential was tested by applying a modified HPLC method for determination of fumonisins in cereals, since the TLC method gave no results. Analyses were performed on a HPLC-FLD system after sample extraction from YESA and extract clean-up on a SPE column. Although the isolates were tested for fumonisin B1, B2 and B3, only fumonisin B1 was detected. The results showed that all tested isolates had toxigenic potential for fumonisin B1 production. The average fumonisin B1 production of the isolates ranged from 7 to 289 µg/kg, thus indicating a highly variable toxigenic potential among the isolates. Isolate 1282 expressed the highest toxigenic potential for fumonisin B1 production (289 µg/kg), while isolate 2533/A showed a questionable potential for fumonisin production (7 µg/kg).Proizvodnja fumonizina kod potencijalno toksikogenih Fusarium verticillioides izolata sa zrna kukuruza i pšenice poreklom iz Srbije testiran je in vitro. Ukupno šest izolata F. verticillioides inkubirano je na agarizovanoj podlozi sa ekstraktom kvasca i saharozom (YESA) tokom četiri nedelje, u mraku na 25 °C. Kapaciteti odabranih izolata za proizvodnju toksina detektovani su primenom modifikovane HPLC metode za određivanje fumonizina u zrnu žitarica, jer brza trijažna TLC metoda nije dala pozitivne rezultate. Analiza je izvedena na HPLC-FLD sistemu nakon ekstrakcije uzorka iz YESA kultura izolata i prečišćavanja ekstrakta na SPE koloni. Sve analize su urađene u tri ponavljanja. Iako su izolati F. verticillioides testirani na prisustvo fumonizina B1, B2 i B3, samo je fumonizin B1 bio detektovan. Rezultati su pokazali da svi testirani izolati imaju potencijal u sintezi fumonizina B1. Prosečna vrednost proizvedenog fumonizina B1 kod izolata F. verticillioides kretala se od 7 do 289 µg/kg, što ukazuje na izuzetno varijabilan toksigeni potencijal istih izolata. Izolat označen sa 1282 pokazao je najveći potencijal za biosintezu fumonizina B1 (289 µg/kg), dok je izolat 2533/A ispoljio diskutabilan potencijal za proizvodnju istog fumonizina (7 µg/kg)

Investigations of the capability of Fusarium isolates from corn for biosynthesis of fusariotoxins

The aim of this paper was to investigate the potential of zearalenone (ZEN) and type A trichothecenes (T-2 toxin and diacetoxyscirpenole - DAS) for biosynthesis by Fusarium spp. isolated from corn kernels contaminated by mycotoxins. The samples of corn kernels (2004 and 2005 harvest) originating in different regions of Bačka (Vojvodina, Serbia) were tested. Mycotoxicological investigations showed in most cases a significant contamination with deoxynivalenol (DON 200 - 2,460 μg/kg) and ZEA (520 1,680 μg/kg). Isolations and identifications of fusaria established only the presence of species F. verticillioides, after one month storage in freezer conditions, in fusariotoxin positive samples. The control cultures known as ZEA producers - F. graminearum GZ-LES, i.e. T-2 toxin and DAS producer - F. sporotrichioides KF-38/1/R were also tested. In vitro toxicological investigations of isolated fusaria were performed in liquid semisynthetic media (GPK or SPK), and on wet sterilized corn kernels, respectively. Under testing conditions, analyzed F. verticillioide and F. sporotrishioides isolates were not ZEA producers. Contrary to them, F. graminearum GZ-LES pure culture was very good producer of fusariotoxins; it biosynthesized max. 465,900 μg/kg DON, and 4,416 μg/kg ZEA, respectively. Cultivation conditions influenced a great deal of T-2 toxin production under laboratory conditions. In most cases, higher yields were obtained during the cultivation of F. verticillioides in liquid glucose medium (80-240 μg/L). Contrary to the control strain F. sporotrichioides KF-38/1/R that under the same conditions synthesized, besides T-2 toxin (4.000 μg/L) and DAS (240 μg/L), isolates of F. verticillioides from corn grain did not show that ability

Deoxynivalenol occurrence in Serbian maize under different weather conditions

The aim of this paper was to investigate deoxynivalenol (DON) occurrence in maize samples originating from two harvest seasons in Serbia. The key differences between harvest seasons were weather conditions, specifically the humidity. The samples were analyzed using high performance liquid chromatography with DAD detection, after clean-up on SPE columns. In samples from 2014, DON was found in 82 (100.0%) samples with the average content of 2.517 mg/kg (ranged from 0.368 to 11.343 mg/kg). Two samples exceeded maximum level permitted by EU regulations. However, analyzing larger number of samples (163) from 2015 harvest season, DON was present in 51 (31.3%) samples in significantly lower concentrations (average of 0.662 mg/kg, ranged from 0.106 to 2.628 mg/kg). None of the samples from 2015 exceeded maximum level permitted by EU regulations. The data on DON presence in Serbian maize were in relation to the different weather conditions that prevailed during the two harvest seasons. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 172042

Application of ATR-FTIR analysis for determination of fumonisins in corn

In order to develop rapid, inexpensive and, at the same time, reliable method for the analysis of molds of the genus Fusarium as an indicator of the presence of fumonisins in corn samples, possible application of Fourier transform infrared spectroscopy (FTIR) with attenuated total reflection (ATR) technique was examined. The content of fumonisins in contaminated corn samples had previously been quantified by ELISA method. At the spectrum of the sample contaminated with a high concentration of mycotoxins, there was a lack of the peak at 1,743 cm-1, but the peak was observed at 1709 cm-1. To the purpose of result classification the principal component analysis (PCA) and cluster analysis were applied. Conclusions of the two methods were similar both when applying ATR technique in the whole region of the spectrum (1,150-1,770 cm-1) and when the whole spectrum was divided into two regions: 1,150-1,450 and 1,450-1,770 cm-1. However, classification of samples was somewhat better in the ranges 1,150-1,770 and 1,450-1,770 cm-1 . Of the 16 analyzed corn samples, only very contaminated corn sample with 190 mg/kg was correctly classified as compared to the other samples with the content of less than 10 mg/kg. Also, it was found-passing recording of spectra of contaminated corn of the same genotype in order to avoid the possible impact of different hybrids on the spectrum. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. OI 172042

Validation and application of FTIR spectroscopy in raw milk analysis

The aim of this study was to investigate whether FTIR spectroscopy is an accurate and valid technique for the assessment of quality parameters in raw cow's milk: fat, protein, lactose, and total solids. The assessment was based on calibration series and comparison with reference material. Furthermore, it takes into account the results obtained in the inter-laboratory comparisons (proficiency testing). The calibration samples were purchased from the accredited regional reference laboratories. The validation parameters included linearity, accuracy, repeatability, reproducibility, and robustness. The linearity ratio was 0.95%. The biases calculated for the fat, protein, lactose and dry matter were -0.33, 0.31, -0.25, and 0.06 respectively. The F value from the F-test was used to determine the significant differences between two independent sets of the results. The obtained results were as follows: 1.469 for fat, 1.634 for protein, 1.192 for lactose, and 0.528 for dry matter. The intra-laboratory reproducibility calculated as the Horwitz Ratios for all parameters were within the criterion limits (0.5 to 0.8). The data obtained for carry-over were 0.27% for fat, 0.52% for protein, 0.47% for lactose, and 0.47% for dry matter. Based on the obtained results it can be concluded that the FTIR spectroscopy is a reliable instrumental technique for the determination of fat, protein, lactose and total solids in raw cow's milk