Phytochemical and metabolomics analysis of Quercus ilex acorns reveals substantial intraspecific variation, high nutritional and nutraceutical potential and rich associated microbiome

There is a currently renewed interest in the use of Q. ilex acorns for alimentary purposes, including nuts, flour, and various derivatives. This aligns with the UN Sustainable Development Goals and is supported by their traditional use since prehistoric times. To support their safe and sustainable use, we conducted a comprehensive phytochemical and metabolomic analysis of acorns from 14 trees across different regions in Spain. Acorns were classified by size and bitterness. Multiple complementary techniques were used, including Near Infrared Spectroscopy (NIRS), High Performance Liquid Chromatography (HPLC), colorimetric assays, macro- and micronutrient analysis, and untargeted Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS). The analysis annotated 1189 metabolic features, along with detailed profiles of carbohydrates, amino acids, fats, phenolics, and minerals. High variability was found among trees, with significant differences independent of geographic origin. Two main groups of acorns emerged, corresponding to a low degree of bitterness and large size vs. a high degree of bitterness and medium size. Notable features included high starch content, predominance of unsaturated fatty acids, and elevated calcium and sodium levels. Metabolomic profiles were highly tree-specific, and several newly reported compounds with potential bioactivity were annotated. Furthermore, a substantial number of metabolites were of microbial origin, revealing a diverse seed-associated microbiome. These findings highlight the nutritional and functional potential of Q. ilex acorns and support their valorization in sustainable food systems. The metabolomic signatures also offer promising markers for tree genotype identification and acorn quality assessment.