From invisible to quantifiable: unmasking non-extractable HT2/T2 glycosides

Fusarium mycotoxins occur in cereals in both free and (poly)saccharide-bound forms; the latter is often underestimated due to analytical limitations. An enzymatic hydrolysis-based method was developed and optimised to release matrix-associated T-2 and HT-2 toxins from cereal polysaccharides. The optimised protocol combined boiling pretreatment, sequential addition of glycosidase, and 44-h hydrolysis under a temperature gradient. Application to malting barley and oat products revealed substantial increases in total detectable T-2 and HT-2 toxin levels compared to conventional extraction, with median increases of 94% in barley and 168% in oats, and maximum increases of 760% and 466%, respectively. The method represents an analytical worst-case release approach for estimating the maximum potentially releasable fraction of matrix-associated mycotoxins. Although not intended to simulate gastrointestinal digestion, it provides relevant data to support exposure assessment and further research on bound mycotoxin forms.