Cheese Powder Extraction Protocols

To enable accurate chemical compound extractions, powders of the cheese samples were prepared. Therefore, cheese core and rind samples were frozen using liquid nitrogen (Air Liquide, Paris, France) and subsequently milled into a fine powder with a coffee grinder (De’Longhi KG49, Treviso, Italy). These cheese powders were subjected to two types of extractions. To assess organic acids, free amino acids, and biogenic amines, an aqueous extraction was performed, as described previously (Le Boucher et al., 2016 (link); Zhang et al., 2019 (link)). Briefly, 1.0 g of cheese powder was mixed with 9.0 mL of ultrapure water (Milli-Q; Merck) on a rotating wheel at 30 rpm for 30 min at room temperature, followed by centrifugation at 1,000 × g for 5 min. Extracts were stored at −25°C until further analysis. To assess volatile organic compounds, ethyl acetate extracts were prepared by mixing 0.5 g of cheese powder with 9.5 mL of ethyl acetate (SupraSolv^® grade; Merck) and supplemented with 100 μg/L of toluene-D8 (Sigma-Aldrich) as internal standard (IS). Ethyl acetate extracts were filtered with a Millex Syringe Driven Filter Unit (Millex; Merck) and immediately used for further analysis.