Ultrasound pretreatment modifies the physicochemical properties and enhances the antioxidant activity of coffee leaf kombucha

This study investigated the effects of ultrasound pretreatment on the physicochemical properties, bioactive composition, and antioxidant potential of coffee leaf kombucha, along with its protective effects against oxidative stress in H2O2-induced HepG2 cells. Ultrasound pretreatment under optimized conditions (40 kHz, 8 W/L, 20 min) significantly improved the antioxidant activity (36.74 %) and α-glucosidase inhibitory activity (3.59 %) of the resulting coffee leaf kombucha (UCK2) compared with the untreated control (CK) (P < 0.05). In addition, ultrasound pretreatment also enhanced the levels of lactic acid (116.22 %), vitamin C (44.87 %), 4-caffeoylquinic acid (4-CQA) (2.91 %), epicatechin (EC) (9.98 %), mangiferin (8.03 %), and isoquercitrin (18.14 %) (P < 0.05). Both CK and UCK2 mitigated oxidative stress in H2O2-induced HepG2 cells by reducing reactive oxygen species (ROS) and malondialdehyde (MDA) levels while enhancing catalase (CAT) and superoxide dismutase (SOD) activities. Molecular analysis further showed that both samples activated Keap1/Nrf2 pathway through up-regulating Nrf2 and Nqo1 and down-regulating Keap1, and suppressed the TLR4/NF-κB inflammatory pathway by down-regulating TLR4, NF-κB, iNOS, and COX-2 gene expression (P < 0.05). UCK2 showed significantly stronger inhibition of NF-κB, iNOS, and COX-2 gene expression than the CK group (P < 0.05). These in vitro findings indicate that ultrasound pretreatment is an effective approach for enhancing the functional and bioactive qualities of coffee leaf kombucha, and confers cytoprotection through coordinated Keap1/Nrf2 and TLR4/NF-κB modulation. However, further in vivo studies are needed to confirm these findings.