Periodic Reporting for period 1 - RADOV (RADiation harvesting of bioactive peptides from egg prOteins and their integration in adVanced functional products)
Reporting period: 2022-09-01 to 2024-02-29
Antimicrobial resistance (AMR) kills 700,000 people each year worldwide. Experts predict that this number will reach 10 million by 2050 if great efforts will not be made to revert this trend. The major cause of antimicrobial resistance is the excessive and inappropriate use of antibiotics. Preventing and controlling antibiotic resistance are therefore complex issues requiring comprehensive approaches. Such initiatives entail the reduction of the need for antibiotics by preventing and controlling infections and using antimicrobial agents that do not contribute or contribute much less to the development of multi-drug resistance.
Antimicrobial peptides (AMPs) can provide a defence against microbial infections. Electron-beam (e-beam) accelerators can induce chemical modifications in materials without requiring added catalysts and solvents. The production of bioactive peptides from egg proteins through e-beam irradiation is paramount. The process could preferably use the abundant waste stream of the egg industry, such as the eggshell membrane. That presents an important opportunity for the radiation processing industry to strengthen and expand its position in biomedical devices and packaging films. It will also reinforce e-beam’s important role in the emerging field of antimicrobial peptide therapeutics production.
In RADOV, the high-energy radiation from electron accelerators is used to produce peptides. The collection of the results acquired regarding the peptide structure, irradiation conditions, and related bioactivity properties will be one vital output of the project. To expand the application of ionizing radiation and to demonstrate the broad scope of its applications, new products containing bioactive proteins/ peptides will be designed and developed using e-beam irradiation. In particular, two target products will be developed as demonstrators of egg-derived AMPs by radiation-induced fragmentation: peptide-laden antimicrobial/antioxidant hydrogel wound dressings and peptide-grafted active food packaging film.
Antimicrobial peptides (AMPs) can provide a defence against microbial infections. Electron-beam (e-beam) accelerators can induce chemical modifications in materials without requiring added catalysts and solvents. The production of bioactive peptides from egg proteins through e-beam irradiation is paramount. The process could preferably use the abundant waste stream of the egg industry, such as the eggshell membrane. That presents an important opportunity for the radiation processing industry to strengthen and expand its position in biomedical devices and packaging films. It will also reinforce e-beam’s important role in the emerging field of antimicrobial peptide therapeutics production.
In RADOV, the high-energy radiation from electron accelerators is used to produce peptides. The collection of the results acquired regarding the peptide structure, irradiation conditions, and related bioactivity properties will be one vital output of the project. To expand the application of ionizing radiation and to demonstrate the broad scope of its applications, new products containing bioactive proteins/ peptides will be designed and developed using e-beam irradiation. In particular, two target products will be developed as demonstrators of egg-derived AMPs by radiation-induced fragmentation: peptide-laden antimicrobial/antioxidant hydrogel wound dressings and peptide-grafted active food packaging film.
RADOV started with the development of laboratory-scale production of irradiated egg proteins and peptides. Irradiation of egg proteins was conducted under different conditions using gamma sources and e-beam accelerators. After irradiation, the products' bioactivity was evaluated with a particular focus on their antibacterial- and antioxidant properties. Two different irradiation conditions resulted in the production of materials with anti-oxidant properties. Fragmentation to peptides was confirmed for one of such cases. Concerning this, around 700 samples have already been tested and the information about each irradiation (i.e. all irradiation conditions) and the following bioactivity (or lack of bioactivity) together with structural data will be collected in a database that is under construction. The procedures and protocols for bioactivity assessment, structural determination, bulk fractionation, peptide isolation, and quantification of peptides are being developed and optimized.
Simultaneously, the egg protein and peptide-containing products are being developed initially based on model proteins used for fragmentation. The first product is a hydrogel wound dressing containing egg protein/peptide with antimicrobial/antioxidant properties. Preliminary screening was conducted using the commercial hydrogel dressing. Hydrogel film's visual appearance, water solubility, and manageability were used as qualitative criteria for a preliminary screening. A mechanical characterisation by small angle oscillatory rheometry was performed on the most promising formulations. More in-depth analysis of the gelation behaviour and microstructure of the hydrogels on selected systems was also conducted. A novel hydrogel formulation that can be loaded with three different egg white proteins, lysozyme, ovotransferrin, and ovalbumin, was identified. Gelation can be induced by e-beam irradiation at the typical sterilisation dose, freeze-thawing cycles, or a combination of the two processes. The composition, mechanical properties, swelling behaviour, and microstructure of the hydrogel films are suitable for the envisaged application as wound dressings.
The second product integrated with bioactive peptide/peptide fraction is an active food packaging film grafted with antioxidant/antimicrobial egg peptides. Two types of commercial films underwent radiation grafting meant to introduce carboxyl or amine groups on the surface. UV-vis 2D fluorescence maps were generated by testing the functionalized films coupled to proteins previously labelled with fluorescent probes. Although screening has not yet been completed, data collected so far indicate that functionalized films can be successfully coated by selected hen egg proteins.
Simultaneously, the egg protein and peptide-containing products are being developed initially based on model proteins used for fragmentation. The first product is a hydrogel wound dressing containing egg protein/peptide with antimicrobial/antioxidant properties. Preliminary screening was conducted using the commercial hydrogel dressing. Hydrogel film's visual appearance, water solubility, and manageability were used as qualitative criteria for a preliminary screening. A mechanical characterisation by small angle oscillatory rheometry was performed on the most promising formulations. More in-depth analysis of the gelation behaviour and microstructure of the hydrogels on selected systems was also conducted. A novel hydrogel formulation that can be loaded with three different egg white proteins, lysozyme, ovotransferrin, and ovalbumin, was identified. Gelation can be induced by e-beam irradiation at the typical sterilisation dose, freeze-thawing cycles, or a combination of the two processes. The composition, mechanical properties, swelling behaviour, and microstructure of the hydrogel films are suitable for the envisaged application as wound dressings.
The second product integrated with bioactive peptide/peptide fraction is an active food packaging film grafted with antioxidant/antimicrobial egg peptides. Two types of commercial films underwent radiation grafting meant to introduce carboxyl or amine groups on the surface. UV-vis 2D fluorescence maps were generated by testing the functionalized films coupled to proteins previously labelled with fluorescent probes. Although screening has not yet been completed, data collected so far indicate that functionalized films can be successfully coated by selected hen egg proteins.
Ionizing radiation (IR) from particle accelerators and radio-isotopes has never been evaluated as a synthetic route to produce peptides from proteins. This is despite the fact that radiation processing is a well-established technology for food decontamination, including decontamination of protein-rich foods. Additionally, the interaction between ionizing radiation and proteins has been investigated for decades due to its relevance in understanding the biological effects of exposure to radiation on living organisms. It is well known that the interaction between gamma-photons or high-energy electrons and proteins can induce protein fragmentation. Therefore, e-beam irradiation has the potential to become an economical route for AMP synthesis by protein fragmentation. Testing of different irradiation conditions will assist in the construction of a public database including egg-peptide structure-bioactivity relationships for different irradiation conditions. Such storage can be used as a library for potentially useful bioactive compounds that can be integrated into several existing products to improve their functionality or become the core components of novel nutraceutical or pharmaceutical products.
The project will support the climate action of the European Union and UN SDG n. 13 (Climate action) through the reduction of by-product waste from the egg product industries. By demonstrating the potentials of IR processes, it can be proven that they are cost-effective approaches for the recovery of highly valuable peptides from the eggshell membrane currently considered as waste. Also, off-grade eggs regarded as by-products can be used in this manner contributing to a circular economy within the industrial chains. Moreover, the project will use IR-driven methods, leading to a decline in the application of enzymes and chemicals in industrial processes for the production of antimicrobial peptides. The approach will simultaneously reduce the environmental footprints related to such processes.
The project will support the climate action of the European Union and UN SDG n. 13 (Climate action) through the reduction of by-product waste from the egg product industries. By demonstrating the potentials of IR processes, it can be proven that they are cost-effective approaches for the recovery of highly valuable peptides from the eggshell membrane currently considered as waste. Also, off-grade eggs regarded as by-products can be used in this manner contributing to a circular economy within the industrial chains. Moreover, the project will use IR-driven methods, leading to a decline in the application of enzymes and chemicals in industrial processes for the production of antimicrobial peptides. The approach will simultaneously reduce the environmental footprints related to such processes.