Green extraction of hyaluronic acid from eggshell membranes using ultra-mild acetic acid: Process optimization, structural validation and waste valorization

• Ultra-mild (0.1 M) acetic acid enables HA recovery from eggshell waste. • Acid concentration is the only significant factor affecting yield. • 80-fold reduction in acid strength vs. strong-acid protocols. • Room temperature extraction preserves HA functional groups. • Eggshell membranes are valorized into a high-value biopolymer. Eggshell membranes represent an abundant agro-industrial waste with potential for the recovery of high-value biomolecules such as hyaluronic acid (HA). In this study, a low-impact extraction approach using ultra-mild acetic acid under ambient conditions was evaluated as an alternative to conventional methods based on strong acids or enzymatic treatments. A sequential experimental design strategy was implemented to assess the influence of acetic acid concentration, extraction time, and solvent volume on HA recovery. Quantitative analysis was performed using a modified carbazole method, while Fourier Transform Infrared Spectroscopy (FTIR) was used for structural assessment. Results indicate that acetic acid concentration was the only significant factor affecting HA recovery, with lower concentrations (0.1 M) yielding higher amounts of HA compared to 1 M. In contrast, extraction time and solvent volume showed no statistically significant influence within the evaluated ranges. FTIR analysis revealed the presence of functional groups consistent with HA, although the coexistence of other biomolecules cannot be excluded. Although the yield obtained (1.85 mg HA/g membrane) is lower than that reported for enzymatic or strong acid-based methods, the proposed approach significantly reduces chemical consumption and operates under mild conditions without the need for additional reagents or energy input. These findings highlight the potential of eggshell membrane waste as a feedstock for sustainable biomolecule recovery and support the development of low-impact extraction strategies aligned with circular bioeconomy principles.