Quantitative PCR (qPCR) for estimating the presence of Fusarium and its mycotoxins in barley grains.

Members within the Fusarium sambucinum species complex (FSAMSC) are able to produce mycotoxins, such as deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN) and enniatins (ENNs) in food products. Consequently, alternative methods for assessing the levels of these mycotoxins are relevant for quick decision-making. In this context, qPCR based on key mycotoxin biosynthetic genes could aid in determining the toxigenic fungal biomass, and could therefore infer mycotoxin content. The aim of this study was to verify the use of qPCR as a technique for estimating DON, NIV, ENNs and ZEN, as well as Fusarium graminearum sensu lato (s.l.) and F. poae in barley grains. For this purpose, 53 barley samples were selected for mycobiota, mycotoxin and qPCR analyses. ENNs were the most frequent mycotoxins, followed by DON, ZEN and NIV. 83% of the samples were contaminated by F. graminearum s.l. and 51% by F. poae. Pearson correlation analysis showed significant correlations for TRI12/15-ADON with DON, ESYN1 with ENNs, TRI12/15-ADON and ZEB1 with F. graminearum s.l., as well as ESYN1 and TRI12/NIV with F. poae. Based on the results, qPCR could be useful for the assessment of Fusarium presence, and therefore, provide an estimation of its mycotoxins' levels from the same sample.

Determination of mycotoxins and their dietary exposure assessment in pale lager beers using immunoaffinity columns and UPLC-MS/MS.

The use of contaminated raw materials can lead to the transfer of mycotoxins into the final product, including beer. This study describes the use of the commercially available immunoaffinity column 11+Myco MS-PREP® and UPLC-MS/MS for the determination of mycotoxins in pale lager-type beers brewed in Czech Republic and other European countries. The additional aim of the work was to develop, optimize and validate this analytical method. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy were tested. The calibration curves were linear with correlation coefficients (R2 > 0.99) for all mycotoxins under investigation. The LOD ranged from 0.1 to 50 ng/L and LOQ from 0.4 to 167 ng/L. Recoveries of the selected analytes ranged from 72.2 to 101.1%, and the relative standard deviation under conditions repeatability (RSDr) did not exceed 16.3% for any mycotoxin. The validated procedure was successfully applied for the analysis of mycotoxins in a total of 89 beers from the retail network. The results were also processed using advanced chemometric techniques and compared with similar published studies. The toxicological impact was taken into account.

Determination of T-2 and HT-2 Toxins in Seed of Milk Thistle [Silybum marianum (L.) Gaertn.] Using Immunoaffinity Column by UPLC-MS/MS.

Milk thistle [Silybum marianum (L.) Gaertn.] achieved a significant increase in interest over the past few years from local and foreign pharmaceutical corporations. The silymarin complex of constituents extracted from milk thistle achenes provides compelling health benefits primarily thanks to antioxidant activities and hepatoprotective effects. However, consuming mycotoxin-contaminated plant material can cause immunosuppression and hepatotoxic problems. The aim of this study was to develop and validate a method for the determination of mycotoxin content in milk thistle. Fusarium toxins as T-2 and HT-2 toxins in grown milk thistle harvested from a breeding station in the Czech Republic during 2020-2021 were studied. The analysis of T-2 and HT-2 toxins was performed by UPLC-MS/MS after immunoaffinity columns EASI-EXTRACT® T-2 & HT-2 clean up. All analysed samples of milk thistle were contaminated with T-2 toxin and HT-2 toxin. The content of T-2 toxin in the samples from 2020 was in the range of 122.7-290.2 µg/kg and HT-2 toxin 157.0-319.0 µg/kg. In 2021, the content of T-2 toxin was in the range of 28.8-69.9 µg/kg and HT-2 toxin was 24.2-75.4 µg/kg. The results show that the climatic conditions of the year of harvesting have a highly statistically significant effect on the content of T-2 and HT-2 toxins in milk thistle.

Levels of T-2 toxin and its metabolites, and the occurrence of Fusarium fungi in spring barley in the Czech Republic.

Mycotoxins have been widely studied by many research groups but further multidisciplinary research is needed to better understand and clarify many issues. This study describes the use of high-performance liquid chromatography coupled with ion trap mass spectrometry (HPLC-MS) to measure T-2 toxin and its metabolites, such as HT-2 toxin, neosolaniol (NEO) and diacetoxyscirpenol (DAS), as well as masked glucosylated mycotoxins in Fusarium-infected Czech spring barley. In total, 152 spring barley samples from the 2018 harvest were analyzed by the ELISA screening method for the presence of T-2 toxin. The most contaminated samples (15), which exceeded the recommended maximum level set by the EU for the sum of T-2 and HT-2 toxin in unprocessed cereals (200 µg/kg), were analyzed by HPLC-MS/MS and microbiological testing. Isolated fungi were evaluated microscopically and identified by polymerase chain reaction (PCR) assays. The prevalence of Fusarium species in spring barley across the Czech Republic in 2018 showed a predominance of F. poae (12 barley samples) and F. tricinctum (9 barley samples). Other strains (F. sporotrichioides and F. langsethiae) were present at a lower frequency, in 1 and 2 samples, respectively. The average concentration of T-2 plus HT-2 toxin was 107.7 µg/kg, while NEO and DAS were found in a few samples at values close to their limit of quantification. HT-2 glucoside was identified in all samples.

Fate and Behavior of Field-Applied Pesticides during Malting and Mashing Processes.

The present work aimed to study the fate of field-applied pesticides during malting and mashing processes. Twenty-four field-collected barley samples were subject to micromalting followed by lab-scale mashing to investigate the carryover of residual pesticides from barley to malt and then from malt to sweet wort. The citrate-buffered QuEChERS sample preparation method was adapted for simultaneous determination of 57 pesticide residues in grain, malt, spent grains, and sweet wort samples using ultra-performance liquid chromatography coupled with tandem mass spectroscopy (UPLC-MS/MS). Residues of four fungicides (fenpropimorph, pyraclostrobin, tebuconazole, and trifloxystrobin) and two insecticides (chlorpyrifos and pirimiphos-methyl), frequently found in the barley samples, were investigated in detail in this study. The carryover percentages of these pesticides to malt, against the concentration of residues in barley grain, ranged from 22% for pirimiphos-methyl up to 78% for fenpropimorph. The results confirm a general rule that residues of pesticides with log P values >2 remain on the malt, but it was found that their transfer potential is more related to its individual physical-chemical properties but does not much correlate to their log P values. In the second part of the study, a noticeable carryover from malt to sweet wort was observed for pyraclostrobin, fenpropimorph, and tebuconazole residues, and these values ranged from 2 to 15%. Moreover, the analysis of pesticide residues in spent grain after mashing revealed that the spent grain samples contain on average once as much pyraclostrobin and tebuconazole residues as the original malt. It was concluded that (1) pyraclostrobin and tebuconazole residues could be incorporated into or associated with macromolecules in barley grain to form "hidden" (bound) forms, and (2) the parent compounds are subsequently released from their hidden forms during mashing.

Characterization of the Fusarium sambucinum species complex and detection of multiple mycotoxins in Brazilian barley samples.

This study investigated the fungal diversity in Brazilian barley samples, focusing on the Fusarium sambucinum species complex and the presence of multiple mycotoxins: aflatoxins B1, B2, G1, G2 beauvericin (BEA), enniatins (ENNs) A, A1, B, and B1, deoxynivalenol (DON), fumonisins (FB) B1 and B2, HT-2 and T-2 toxins, nivalenol (NIV) and ochratoxin A (OTA) from two different regions, São Paulo (SP) and Rio Grande do Sul (RS). The majority of the isolates belonged to the Fusarium sambucinum species complex (FSAMSC), with F. graminearum s.s. characterized as the major contaminant. F. meridionale and F. poae were the second most frequent fungi isolated from SP and RS, respectively. All of the F. graminearum s.s. isolates demonstrated 15-ADON genotype, whereas F. poae and F. meridionale were all NIV. The majority of the F. cortaderiae isolates were NIV, with only one 3-ADON genotype. Mycotoxin analysis revealed that none of the samples were contaminated by aflatoxins, OTA, FB2 and type A trichothecenes, however, all of the samples were contaminated with at least one Fusarium toxin. Contamination by DON, ZEA, ENNB and ENNB1 levels were significantly higher in RS. Co-contamination of BEA, DON, ENNs, NIV and ZEA in 18.5% and 24.2% of the analyzed samples was observed, from SP and RS respectively. More than 20% of the samples from RS presented DON and ZEA levels above the regulations established by Europe and Brazil. The results provide further information on the FSAMSC from South America and detected multiple Fusarium toxins in barley samples. This highlights the importance for further studies on the possible interactions of these mycotoxins in order to determine potential risks to animal health.

Fusarium Mycotoxins Stability during the Malting and Brewing Processes.

Mycotoxins are widely studied by many research groups in all aspects, but the stability of these compounds needs further research for clarification. The objective of this study is to evaluate deoxynivalenol and zearalenone stability during all steps of the malting and brewing processes. The levels of these compounds decreased significantly during the production process (barley to beer). During the malting process, the DON levels decreased significantly in the steeping, germination, and malting steps (62%, 51.5%, and 68%, respectively). Considering ZEN, when the levels were compared between barley and the last step of the process, a significant decrease was observed. Most of the mycotoxins produced were transferred to the rootlets and spent grains, which is advantageous considering the final product. Furthermore, the mycotoxin dietary intake estimation was included in this study. The results proved that if the concentrations of target mycotoxins in raw material are under the limits established by the regulations, the levels decrease during the malting and brewing processes and make the beer secure for consumers. The quality of the five commodities involved in the beer process plays a decisive role in the creation of a safe final product.

Development and optimization of ultra-high performance supercritical fluid chromatography mass spectrometry method for high-throughput determination of tocopherols and tocotrienols in human serum.

The goal of this study was to develop an effective supercritical fluid chromatography method using single quadrupole MS for analysis of all isomeric forms of vitamin E. Finally, two fast and effective methods, the high resolution one and the high speed one, for the determination of 8 vitamin E isomers in human serum were developed. Rapid high-throughput liquid-liquid extraction was selected as a sample preparation step. Sample pretreatment of 100 µL human serum was consisted of protein precipitation with 200 µL ethanol and liquid-liquid extraction by 400 µL hexane/dichloromethane (80/20, v/v). The separation was performed on BEH 2-EP (3.0 × 100 mm, 1.7 µm) stationary phase, using isocratic elution with carbon dioxide and 10 mM ammonium formate in methanol in the ratio 98:2 for high resolution method with run time 4.5 min and in the ratio 95:5 for high speed method, where the run time was 2.5 min. The method development included optimization of key parameters: the choice of the suitable stationary phase and the composition of mobile phase, where an influence of various modifiers, their ratio and additives were tested, and optimization of fine tunning parameters including BPR pressure, flow-rate and column temperature. Quantification of all isomeric forms was performed using SIM (single ion monitoring) experiments in ESI positive ion mode. Both high speed and high resolution chromatographic methods were validated in terms of precision, accuracy, range, linearity, LOD, LOQ and matrix effects using the same LLE procedure. The high resolution method provided more sensitive results (LOD: 0.017-0.083 µg mL(-1)) and better linearity (r(2) > 0.9930) than the high speed one (LOD: 0.083-0.25 µg mL(-1), r(2) > 0.9877) at the cost of double time of analysis.

Antioxidant vitamins in barley green biomass.

Two malting hulled varieties (Sebastian, Malz) and one nonmalting hull-less variety (AF Lucius) were used to assess vitamins C and E in the green biomass of young plants of spring barley ( Hordeum vulgare L.) in three stages of growth and development (BBCH 29, 31, 32-33). The samples from sampling I (BBCH 29) had statistically significantly higher vitamin C content and vitamin E activity compared to sampling I (BBCH 31). The highest average vitamin content was determined in the malting variety Sebastian (vitamin C, 520 mg 100 g(-1) DM; vitamin E, 73.06 mg kg(-1) DM) compared to the varieties Malz (501 mg 100 g(-1) DM; 61.84 mg kg(-1) DM) and AF Lucius (508 mg 100 g(-1) DM; 67.81 mg 100 g(-1) DM). The locality Kromeríz (Czech Republic, CR), with vitamin C and E contents of 524 mg 100 g(-1) DM and 68.74 mg kg(-1) DM, respectively, proved to be more suitable for growing green biomass compared to the locality Zabcice (CR) (content of vitamins C and E, 477 mg 100 g(-1) DM and 66.39 mg kg(-1) DM, respectively). During the research period (2005-2007), it was determined that the green mass of young plants of spring barley was a significant source of vitamins C and E in the growth stage BBCH 29; in later samplings (BBCH 32-33) the vitamin levels dropped (by as much as 48%). These vitamins are important antioxidants for human health. Therefore, "green barley" can be recommended for the preparation of natural dietary supplements and is preferred to synthetic vitamin preparations.