Casein-based film enriched with lignin as a biodegradable substrate for enzyme immobilization

In the last decades, the negative impact of petroleum derived materials on the environment is more and more evident; beyond the unavoidable reduction in the use of classical plastic, another promising approach is the development of alternative materials prepared starting from natural, biodegradable, and more sustainable biomolecules, particularly undervalued or discarded ones. Caseins are the most abundant proteins in milk, with important nutritional value but also interesting film-forming properties. Lignin is a polyphenolic polymer found in wood and derived from a by-product of the cellulose extraction processes; it is well known for its antibacterial, antioxidant, and UV-protecting properties. In the present work, casein was isolated from UHT skimmed bovine milk through acidification and used alone or in combination with lignin to produce films that are biodegradable and environmentally friendly. Casein and casein-lignin films presented a thickness in the range of 180-260 μm and a compact, non-porous texture. The presence of lignin did not affect the morphology of the films but influenced their mechanical properties. For casein and casein-lignin films covalently crosslinked with transglutaminase (TGM), the solubility decreased to 40-50% and the samples retained their shape. The results show that TGM-containing films are suitable as substrates for the immobilization of enzymes; herein, the FAD-dependent glucose oxidase from Aspergillus niger was added to the film and the enzyme remained stable and active against glucose for weeks, as demonstrated by the colorimetric detection of the H₂O₂ produced in the catalysed reaction. This study opens up the possibility of combining two products of natural origin for the production of films through processes with low environmental impact, thus offering interesting scenarios in the immobilization of macromolecules for the detection of target molecules.