Global lemon production reached 23.6 M tons in 2023, with major uses including fresh consumption, juice production, and food flavorings. Around 50% of the fruit becomes by-products (peels, pulp, and seeds), which are not only rich in fibers and polysaccharides that make up the cell walls but also organic acids and bioactive flavonoids with health benefits. To extract these valuable compounds sustainably, enzyme-assisted extraction (EAE) is a promising green technology that facilitates the release of those cell wall-bound molecules, enhancing their yield. This study focused on the solid–liquid extraction of lemon peel by-products using a 1:100 ratio with 1% aqueous solutions of four enzymes (glucanase, cellulase, pectin lyase, and pectinmethylesterase). As a control (CTRL), no enzyme was used. The major flavonoids in each sample were quantified via HPLC-MS (ESI-QqQ) in multiple reaction monitoring mode and total phenolic content (TPC) was analyzed via UV-vis spectroscopy to evaluate extraction efficiency across enzymatic treatments.

All enzymes yielded higher TPC in the extracts compared to the CTRL sample. Glucanase demonstrated the greatest extraction yield of TPC (~32 %), though the difference from pectin lyase was not significant. However, pectinmethylesterase yielded higher concentrations of the main analyzed metabolites with ~36% more citric acid, ~7% more hesperidin, and ~22% more eriocitrin compared to the CTRL. In contrast, pectin lyase extracted the highest concentration of diosmetin-6,8-di-C-glucoside, exceeding the CTRL by ~81%. Interestingly, glucanase-treated extracts showed undetectable levels of hesperidin and eriocitrin, yet elevated concentrations of diosmetin-6,8-di-C-glucoside. This suggests that glucanase selectively cleaves O-glycosidic bonds, yielding aglycones, while preserving C-glycosidic linkages. In conclusion, the application of EAE enhances the extraction yield of lemon peel metabolites using water as a green solvent, without requiring organic solvents. For further research, incubation times and enzyme selection are critical for process optimization.