Ultrasound-assisted extraction and flavor quality assessment of in vitro biomimetically fermented Kopi Luwak.

Kopi Luwak, renowned for its distinctive flavor profile, has long been esteemed in specialty coffee circles; however, the conventional animal digestive process is fraught with significant ethical and sustainability controversies. Building upon these findings, the present study established a tightly controlled in vitro biomimetic fermentation system, complemented by ultrasound-assisted extraction and multi-omics analyses, to faithfully reconstruct and elevate the hallmark flavour of civet coffee. Leveraging metagenomic data from the civet gut, thirty core functional strains were selected from 1870 isolates to create a synthetic symbiotic consortium. Fermentation parameters were optimised in three stages-single-factor experiments, Box-Behnken response-surface design, and a genetic-algorithm-artificial-neural-network (GA-ANN) model. Fermentation products were recovered by ultrasound-assisted extraction (40 kHz, 400 W, 10 min); volatile and non-volatile metabolites were quantified in both targeted and untargeted modes via GC-MS and UHPLC-MS/MS, and their temporal dynamics were deciphered through time-series clustering and metabolic-network analysis. The optimal conditions-16.5 % inoculum, initial pH 6.25, 33 ℃, 135 h-yielded an average SCA cupping score of 82.92, with a maximum of 85.25, significantly higher than those of natural fermentation and conventional civet coffee (P < 0.05). Total acidity increased to 0.78 g L^-1, total polyphenol content reached 225.3 mg L^-1, and key bioactive compounds remained stable. GC-MS quantification showed 1.9-2.3-fold increases in 2,3-dimethoxyphenol, phenylethanol, and 5-methylfurfural, alongside 63 % and 41 % reductions in 2-methylpyrazine and caffeine, respectively. Multi-omics evidence indicated that lipid β-oxidation, the amino-acid Ehrlich pathway, and esterification jointly enriched fruity, nutty, and floral notes; the elevated copy numbers of ndmA/B and pyoA/B genes underpinned the attenuation of bitter compounds. Relative to conventional solvent and Soxhlet extraction, ultrasound-assisted extraction improved volatile recovery by approximately 28 %, reduced energy consumption by ≥66 %, and halved solvent usage. Overall, this work achieves a high-fidelity in vitro replication of the civet gut microbiome and its metabolic functions, enables precision flavour modulation through intelligent optimisation and green extraction, and demonstrates industrial feasibility-the processing cost per kilogram of raw beans is 76.7 % lower than that of the traditional animal-derived method.