From lemon peels to bioactive peptides: protein recovery by pressurized liquid extraction and hydrolysates characterization by UHPLC-ESI-QTOF-MS/MS

Abstract

This study aimed to develop a sustainable and efficient method for protein recovery from lemon peels using Pressurized Liquid Extraction (PLE), optimized through experimental design and response surface methodology. The final protocol, employing 18 % (v/v) ethanol, 110 ◦C, and a 7-min extraction time, demonstrated high extraction efficiency (66 %) and environmental compatibility, achieving a greenness score of 0.59 (AGREEprep). The optimized protein extract was subsequently hydrolyzed using alcalase and thermolysin, and the resulting peptide fractions were characterized by spectrophotometric methods and by Ultra High-Performance Liquid Chromatography coupled to Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-ESIQTOF-MS/MS). A total of 58 peptides were identified, 39 of which originated from lemon proteins, along with 19 polyphenols, such as vicenin-2, narirutin or subaphylin, and other compounds (e.g., organic acids, amino acids, and purine nucleosides), revealing a diverse and multifunctional bioactive profile. Bioactivity assays demonstrated that the thermolysin hydrolysate exhibited the highest antioxidant (97 ± 1 % inhibition of hydroxyl radicals), antimicrobial (MIC = 0.75 mg/mL against Staphylococcus aureus), and antihypertensive activities (49 ± 3 % angiotensin-converting enzyme inhibition), likely due to the presence of peptides enriched in aromatic amino acids such as phenylalanine and tyrosine.