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Wheat is one of the most consumed foods in the world and unfortunately causes allergic reactions which have important health effects. The α-amylase/trypsin inhibitors (ATIs) have been identified as potentially allergen components of wheat. Due to a lack of data on optimization of ATI extraction, a new wheat ATIs extraction approach combining solvent extraction and selective precipitation is proposed in this work. Two types of wheat cultivars (Triticum aestivum L.), Julius and Ponticus were used and parameters such as solvent type, extraction time, temperature, stirring speed, salt type, salt concentration, buffer pH and centrifugation speed were analyzed using the Plackett-Burman design. Salt concentration, extraction time and pH appeared to have significant effects on the recovery of ATIs (p < 0.01). In both wheat cultivars, Julius and Ponticus, ammonium sulfate substantially reduced protein concentration and inhibition of amylase activity (IAA) compared to sodium chloride. The optimal conditions with desirability levels of 0.94 and 0.91 according to the Doehlert design were: salt concentrations of 1.67 and 1.22 M, extraction times of 53 and 118 min, and pHs of 7.1 and 7.9 for Julius and Ponticus, respectively. The corresponding responses were: protein concentrations of 0.31 and 0.35 mg and IAAs of 91.6 and 83.3%. Electrophoresis and MALDI-TOF/MS analysis showed that the extracted ATIs masses were between 10 and 20 kDa. Based on the initial LC-MS/MS analysis, up to 10 individual ATIs were identified in the extracted proteins under the optimal conditions. The positive implication of the present study lies in the quick assessment of their content in different varieties especially while considering their allergenic potential.
Necrotrophic as well as saprophytic small-spored Altemaria (A.) species are annually responsible for major losses of agricultural products, such as cereal crops, associated with the contamination of food and feedstuff with potential health-endangering Altemaria toxins. Knowledge of the metabolic capabilities of different species-groups to form mycotoxins is of importance for a reliable risk assessment. 93 Altemaria strains belonging to the four species groups Alternaria tenuissima, A. arborescens, A. altemata, and A. infectoria were isolated from winter wheat kernels harvested from fields in Germany and Russia and incubated under equal conditions. Chemical analysis by means of an HPLC-MS/MS multi-Alternaria-toxin-method showed that 95% of all strains were able to form at least one of the targeted 17 non-host specific Altemaria toxins. Simultaneous production of up to 15 (modified) Altemaria toxins by members of the A. tenuissima, A. arborescens, A. altemata species-groups and up to seven toxins by A. infectoria strains was demonstrated. Overall tenuazonic acid was the most extensively formed mycotoxin followed by alternariol and alternariol mono methylether, whereas altertoxin I was the most frequently detected toxin. Sulfoconjugated modifications of alternariol, alternariol mono methylether, altenuisol and altenuene were frequently determined. Unknown perylene quinone derivatives were additionally detected. Strains of the species-group A. infectoria could be segregated from strains of the other three species-groups due to significantly lower toxin levels and the specific production of infectopyrone. Apart from infectopyrone, alterperylenol was also frequently produced by 95% of the A. infectoria strains. Neither by the concentration nor by the composition of the targeted Altemaria toxins a differentiation between the species-groups A. altemata, A. tenuissima and A. arborescens was possible.
Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding their functions in such a disorder is still unclear and for further study, the need for pure ATI molecules is one of the limiting problems. In this work, a simplified approach based on the successive fractionation of ATI extracts by reverse phase and ion exchange chromatography was developed. ATIs were first extracted from wheat flour using a combination of Tris buffer and chloroform/methanol methods. The separation of the extracts on a C18 column generated two main fractions of interest F1 and F2. The response surface methodology with the Doehlert design allowed optimizing the operating parameters of the strong anion exchange chromatography. Finally, the seven major wheat ATIs namely P01083, P17314, P16850, P01085, P16851, P16159, and P83207 were recovered with purity levels (according to the targeted LC-MS/MS analysis) of 98.2 ± 0.7; 98.1 ± 0.8; 97.9 ± 0.5; 95.1 ± 0.8; 98.3 ± 0.4; 96.9 ± 0.5, and 96.2 ± 0.4%, respectively. MALDI-TOF-MS analysis revealed single peaks in each of the pure fractions and the mass analysis yielded deviations of 0.4, 1.9, 0.1, 0.2, 0.2, 0.9, and 0.1% between the theoretical and the determined masses of P01083, P17314, P16850, P01085, P16851, P16159, and P83207, respectively. Overall, the study allowed establishing an efficient purification process of the most important wheat ATIs. This paves the way for further in-depth investigation of the ATIs to gain more knowledge related to their involvement in NCWS disease and to allow the absolute quantification in wheat samples.
Spotlight on the underdogs
(2017)
Alternaria (A.) is a genus of widespread fungi capable of producing numerous, possibly health-endangering Alternaria toxins (ATs), which are usually not the focus of attention. The formation of ATs depends on the species and complex interactions of various environmental factors and is not fully understood. In this study the influence of temperature (7 °C, 25 °C), substrate (rice, wheat kernels) and incubation time (4, 7, and 14 days) on the production of thirteen ATs and three sulfoconjugated ATs by three different Alternaria isolates from the species groups A. tenuissima and A. infectoria was determined. High-performance liquid chromatography coupled with tandem mass spectrometry was used for quantification. Under nearly all conditions, tenuazonic acid was the most extensively produced toxin. At 25 °C and with increasing incubation time all toxins were formed in high amounts by the two A. tenuissima strains on both substrates with comparable mycotoxin profiles. However, for some of the toxins, stagnation or a decrease in production was observed from day 7 to 14. As opposed to the A. tenuissima strains, the A. infectoria strain only produced low amounts of ATs, but high concentrations of stemphyltoxin III. The results provide an essential insight into the quantitative in vitro AT formation under different environmental conditions, potentially transferable to different field and storage conditions
Semi-natural habitats (SNHs) are becoming increasingly scarce in modern agricultural landscapes. This may reduce natural ecosystem services such as pest control with its putatively positive effect on crop production. In agreement with other studies, we recently reported wheat yield reductions at field borders which were linked to the type of SNH and the distance to the border. In this experimental landscape-wide study, we asked whether these yield losses have a biotic origin while analyzing fungal seed and fungal leaf pathogens, herbivory of cereal leaf beetles, and weed cover as hypothesized mediators between SNHs and yield. We established experimental winter wheat plots of a single variety within conventionally managed wheat fields at fixed distances either to a hedgerow or to an in-field kettle hole. For each plot, we recorded the fungal infection rate on seeds, fungal infection and herbivory rates on leaves, and weed cover. Using several generalized linear mixed-effects models as well as a structural equation model, we tested the effects of SNHs at a field scale (SNH type and distance to SNH) and at a landscape scale (percentage and diversity of SNHs within a 1000-m radius). In the dry year of 2016, we detected one putative biotic culprit: Weed cover was negatively associated with yield values at a 1-m and 5-m distance from the field border with a SNH. None of the fungal and insect pests, however, significantly affected yield, neither solely nor depending on type of or distance to a SNH. However, the pest groups themselves responded differently to SNH at the field scale and at the landscape scale. Our findings highlight that crop losses at field borders may be caused by biotic culprits; however, their negative impact seems weak and is putatively reduced by conventional farming practices.