Activity-guided fractionation and taste dilution analysis (TDA), followed by LC-MS/MS, LC-TOF-MS, and 1D/2D-NMR spectroscopy, led to the identification of the cyclic diarylheptanoid asadanin exhibiting a human bitter recognition threshold of 13 μmol/kg, as the major inducer of the sporadic bitter off-taste of hazelnut kernels (Corylus avellana L.). Sensory analysis of hazelnut samples from two origins (Ordu/2013 and Akçakoca/2014) and from Cimiciato-infected hazelnut kernels, followed by LC-MS/MS quantitation of 1 and calculation of dose-over-threshold (DoT) factors, showed established evidence for the Cimiciato infection as the major inductor of asadanin biosynthesis in hazelnut kernels and, as a consequence, as the reason for bitter off-taste development.

Activity-guided fractionation in combination with sensory analytics, LC-TOF-MS, and 1D/2D-NMR spectroscopy enabled the identification of the bitter tasting diarylheptanoids asadanin, giffonin P, and the previously not reported ( E)-7,9,10,13-tetrahydroxy-1,7-bis(2-hydroxyphenyl)hept-9-en-11-one and 4,12,16-trihydroxy-2-oxatricyclo[13.3.1.1]-nonadeca-1(18),3,5,7(20),8,15,17-heptaen as well as the yet unknown astringent compounds 2-(3-hydroxy-2-oxoindolin-3-yl) acetic acid 3- O-6'-galactopyranosyl-2″-(2″oxoindolin-3″yl) acetate and 3-( O-β-d-glycosyl) dioxindole-3-acetic acid in Cimiciato-infected hazelnuts exhibiting a bitter off-taste. Quantitative LC-MS/MS studies, followed by dose/activity considerations confirmed for the first time asadanin to be the key contributor to the bitter taste of Cimiciato-infected hazelnuts. Furthermore, quantitative studies demonstrated that neither the physical damage alone nor a general microbial infection is able to initiate a stress-induced asadanin generation, but most likely either specific Cimiciato-specific microorganisms associated with the bugs or specific chemical stimulants in the bugs' saliva is the cause triggering asadanin biosynthesis. Finally, also germination was found for the first time to activate diarylheptanoid biosynthesis, resulting in higher contents of bitter tasting phytochemicals and development of the bitter off-taste.