Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars

Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, {"type":"entrez-protein","attrs":{"text":"P81367","term_id":"3913899","term_text":"P81367"}}P81367 and {"type":"entrez-protein","attrs":{"text":"P81368","term_id":"3913900","term_text":"P81368"}}P81368; both reviewed and a targeted LC–MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR ({"type":"entrez-protein","attrs":{"text":"P81367","term_id":"3913899","term_text":"P81367"}}P81367) and TYMVR ({"type":"entrez-protein","attrs":{"text":"P81368","term_id":"3913900","term_text":"P81368"}}P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.     

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.     

The effect of tannins on Mediterranean ruminant ingestive behavior: the role of the oral cavity

Sheep, cattle and goat are domestic ruminants of significant economic interest in the Mediterranean region. Although sharing the same pasture ranges, they ingest different plants and plant parts and, consequently different levels of tannins. This suggests an ability to detect and adapt ingestion according to animal physiological limits of tolerance for plant secondary metabolites. This review will detail the effects of dietary tannins on feeding behavior, and the role of the oral cavity in this process, with focus on such ruminant species. The role of salivary protein profile in tannin perception in the oral cavity, and as a defense mechanism, will be discussed.