Physical, Chemical, and Enzymatic Pretreatment of Spent Hops and Its Impact on Xanthohumol Extraction Yield

Spent hops from the supercritical extraction process represent a valuable source of xanthohumol (XN), a prenylated flavonoid with demonstrated anticancer, antidiabetic, antibacterial, and anti-inflammatory properties. However, XN is thermally sensitive and readily isomerizes into the less bioactive iso-XN at elevated temperatures, necessitating mild extraction conditions. Previous studies have shown that the pretreatment of plant biomass can enhance the extraction efficiency of bioactive compounds. In this study, various pretreatment methods-including physical (freeze-thaw, ultrasound, and microwave), chemical (acid and base hydrolysis), and enzymatic approaches-were applied to spent hops prior to extraction, and XN yields were compared to those obtained from untreated samples. The experiments, performed in triplicate, yielded meaningful results which helped understand the raw material's behavior in applied conditions. Due to the compound's high thermal sensitivity, ultrasound and microwave pretreatments require precise control to prevent excessive temperature increases, making low-temperature methods more suitable. Additionally, exposure to elevated pH adversely affected XN extraction efficiency, limiting the applicability of strong alkaline pretreatments. Among the evaluated methods, freeze-thaw pretreatment proved to be a simple and effective strategy, enhancing XN extraction yields by up to 10.7 ± 0.7% through the optimization of soaking time, the solid-to-liquid ratio, and the thawing temperature. Identifying an inexpensive and efficient pretreatment method could reduce extraction time while improving yield, contributing to the sustainable utilization of spent hops as an XN source.