Integrated recovery of calcium salts and collagen-derived fractions from eggshell waste using ultrasound-assisted acid treatment

Eggshells (ES) from the poultry industry are an abundant biowaste rich in calcium carbonate (CaCO 3 ) and collagen-containing eggshell membrane (ESM), yet integrated valorization of both fractions remains limited. This study presents a biorefinery approach combining ultrasound-assisted acetic acid (ACA-US) treatment for controlled demineralization and efficient separation of ESM from ES, followed by pepsin-assisted hydrolysis and stepwise ultrafiltration for recovery of collagen-derived fractions. Separated fractions were characterized using Raman spectroscopy, SEM-EDS, XRD, LC-MS, and amino acid (AA) profiling. ACA-US ensured effective removal of CaCO 3 while preserving collagen-related spectral features in ESM. Raman spectra showed intact Amide I, II, and III bands and a characteristic proline/hydroxyproline doublet, confirming preservation of native collagen structure prior to hydrolysis. SEM-EDS and XRD analyses verified CaCO 3 removal and its conversion into crystalline Ca salts with high Ca content. Following pepsin-assisted hydrolysis of isolated ESM, AA analysis revealed glycine, proline, and hydroxyproline as dominant residues. Ultrafiltration showed that the unsalted 50 kDa retentate contained the highest collagen and total AA content, followed by the 10 kDa retentate. Collagen-derived material was also detected in permeates, indicating distribution within ≥ 50 kDa and ≤ 50 kDa fractions due to partial fragmentation. Collagen recovery ranged from 1.24 to 2.87 g per 100 g ESM, while salted fractions showed reduced yields due to aggregation of non-collagenous proteins. This approach demonstrates a cleaner pathway for simultaneous recovery of mineral and protein fractions from ES waste, supporting circular bioeconomy valorization, although further optimization and safety assessment are required for practical industrial application and scale-up considerations.