Headspace SPME-GC-MS characterization of mushrooms and agroindustrial by-products for sustainable food protein development

The BIO.FUNGI.PRO project (PRIN 2022YXLFYH) aims to develop an integrated bio-based strategy for the sustainable production of fungal proteins and fiber-rich ingredients through the valorization of agri-food by-products, specifically cocoa bean shells (CBS) and rice husks (RH). (1) The project combines circular economy principles with advanced green technologies to optimize mushroom cultivation and transform fungal biomass into functional ingredients for innovative high-protein foods, culminating in the development of a sustainable “mushburger.” This study focuses on the characterization of volatile organic compounds (VOCs) emitted from raw matrices, including standard mushroom culture media, CBS- and RH-enriched substrates, and two commercial mushroom species, Agaricus bisporus and Pleurotus ostreatus. (2) Volatiles profiling was performed using Headspace Solid-Phase Microextraction coupled with Gas Chromatography–Mass Spectrometry (HS-SPME-GC-MS). employing an Agilent 8890 GC System coupled to an Agilent 5977C MSD and an HP-5ms Ultra Inert capillary column (30 m x 0.25 mm × 0.25 um). Five commercial SPME coatings (DVB/CAR/PDMS, DVB/PDMS, CAR/PDMS, PDMS, and Polyacrylate) were comparatively evaluated to optimize extraction performance. Preparatory tests assessed matrix stability under different storage treatments (fresh at 4–5°C, oven-dried at 40°C overnight, and freeze-dried). Oven-drying provided the most reproducible and representative VOC profiles, enabling the identification of more than 50 compounds in A. bisporus and over 40 in P. ostreatus. Carboxen-based fibers showed enhanced affinity for small polar compounds (aldehydes, ketones, alcohols), which are key contributors to mushroom aroma. Cooking simulations were also performed to monitor volatilome changes and potential formation of neo-formed compounds along processing. These preliminary results provide a robust analytical framework to monitor volatiles changes along the fungal production chain, supporting the development of safe, stable and potential sensory-acceptable mushroom-based protein foods within a circular bio-economy model. Aknowledgement This study is part of the BIO.FUNGI.PRO research project funded by the Italian Ministero dell’Università e della Ricerca (MUR) PRIN PNRR project no. 2022YXLFYH. The authors thank all the project’s partners.