Introduction Valorizing Agro-Industrial Waste: Exploring Antimicrobial Peptides Prepared from Feather Keratin Justine Horner,a Amira Ben Mansour,a Sutida Jansod,a Claire Gay,a Roger Martia* a University of Applied Sciences Western Switzerland (HES-SO), Haute Ecole d’Ingénierie et d’Architecture de Fribourg (HEIA-FR), Boulevard de Pérolles 80, CH-1700 Fribourg, Switzerland Results and outlooks Microbial colonization on surfaces contributes to infections, imposing significant health and economic burdens. Biobased alternatives to commercial antimicrobial coatings are increasingly sought after for sustainable solutions. Antimicrobial peptides (AMPs), biomolecules found in various natural sources, are known for effectively inhibiting microbial growth. Bioactivity of peptides are associated with their structural characteristics. Therefore, our strategy was to generate a large pool of antimicrobial candidates, by a large screening of various preparation methods, combining sometimes several hydrolysis approches The RELIANCE consortium consists of 15 partners spanning 8 EU and 2 non-EU countries. Partners include research institutions, universities, SMEs, and large industries. Acknowledgements Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them. This work is part of a project that has received funding from the European Union’s Horizon Europe research and innovation program under grant agreement No. 101058570 (RELIANCE). This work has received funding from the Swiss State Secretariat for Education, Research and Innovation (SERI). The goal of this project is to explore keratin- based AMPs from feather waste, a sustainable and abundant source, to produce contact-killing coatings for self- disinfecting surfaces. Various extraction and purification strategies were employed to generate a wide range of AMP candidates with optimal bioactivity. Peptides extraction Peptides size also matters. That’s why ultrafiltration has been used not only as a purification methods, but also in a way of separating peptides mixtures into fractions of several mass intervals. From feathers to antimicrobials Feather Waste Contact killing antimicrobial peptides Keratin hydrolysates Peptides mixtures Surface functionalization The project now advances to the next phase: coupling keratin-based antimicrobial peptides with nanoparticles, which will be applied to surfaces via cold atmospheric plasma deposition to create self-disinfecting materials. Keratin structure Feathers are mainly composed of keratin, a mechanically strong structural protein, insoluble in water, and resistant to degradation. To break down this protein into a mixture of peptides, different types of bonds must be broken : • Disulfide (S-S) bonds that contribute to the strength of keratin • Some peptide bonds that constitute the backbone of this molecule, but not all of them. Screening of peptides preparation methods In accordance with the process flow delineated on the left, more than 50 distinct samples encompassing various keratin peptide fractions were produced, in quantities ranging from a few milligrams to over 1 gram. Antimicrobial activity Preliminary analyses have shown promising antimicrobial activity of keratin-derived peptides in solution, particularly against viruses and Gram-negative bacteria. Process scale-up Production has been scaled-up 20 x for one of the most bioactive peptides samples, revealing different technical challenges, in particular for the work-up stages, but generating yields very close to the equivalent small scale experiments (around 40% of purified peptides regarding the mass of feather feed). cys-S-S-cys CAP DEPOSITION CHEMICAL COUPLING Nanoparticles AMP Functionalized nanoparticles + Self disinfectant surfaces t = 0 t