Enterohemorrhagic Escherichia coli (EHEC) are zoonotic bacterial pathogens responsible for serious human infections characterized by a broad spectrum of symptoms, from acute diarrhea to hemorrhagic colitis and the hemolytic uremic syndrome, particularly in children. Following asymptomatic colonization of the gastrointestinal tract of ruminants, EHEC are shed in animal feces, accidentally contaminating certain food products intended for human consumption. Contaminated raw milk and dairy products are a main source of human infections by EHEC strains, especially those belonging to the O26:H11 serotype. To control the flow of EHEC O26:H11 in the dairy sector and thus reduce human infections, we propose a “One Health” approach that combines distinct anti EHEC strategies, in dairy farms and dairy plants. At the farm level, we aim at reducing EHEC O26:H11 carriage by ruminants by combining two approaches based on (i) the use of probiotic or commensal E. coli strains naturally producing antimicrobial peptides, named microcins, that exert potent activity against EHEC while maintaining the gut microbiota equilibrium and on (ii) vaccines derived from bacterial outer membrane vesicles (OMVs) expressing specific EHEC antigens and optimized for efficient immunization of ruminants. We hypothesize that the administration to ruminants, soon after their birth, of commensal E. coli strains that occupy the same ecological niche as EHEC O26:H11 and produce microcins with anti-EHEC O26:H11 activity, will protect the ruminants from being colonized by EHEC O26:H11. We also hypothesize that the adjuvant properties of OMVs coupled to their immunogenicity resulting from the presentation of specific EHEC O26:H11 antigens will trigger an efficient immune response and make the intestinal environment of vaccinated ruminants unfavourable to colonization by EHEC O26:H11. At the milk processing level, we hypothesize that microcin-producing E. coli will inhibit the growth of EHEC O26:H11 during cheese manufacture, cooperatively or synergistically to the anti-EHEC activity of protective microbial consortia inoculated into milk along with lactic acid starters. These innovative anti-EHEC O26:H11 approaches will be first developed, optimized and evaluated using in vitro assays and in vivo experiments in mice. The capacity of microcin-producing E. coli to reduce the growth of EHEC O26:H11 during cheese manufacture will be studied using challenge tests in uncooked pressed cheeses and soft cheeses, and the absence of impact on the balance of cheese microbial communities will be verified. Finally, after having demonstrated the protective effects of microcin-producing E. coli and OMV-based vaccines against EHEC O26:H11 in mice and/or in cheeses, a pilot study will be performed where the efficacy of both strategies will be evaluated with dairy small ruminants (sheep).

PASPACK 4.0 aims to produce an innovative and sustainable bio-based food packaging from Mediterranean agri-food by-products and waste, especially pomegranate (peel and seeds) and dates (date pits, molasses), to contribute to addressing the dilemma of food waste and the overuse of plastic in food packaging. This will be achieved by harnessing innovative technologies, mainly those of fourth industrial revolution (I4.0) innovations, such as smart sensors, nanotechnology, artificial intelligence, blockchain, 3D printing, and radio-frequency identification (RFID), to develop a sustainable alternative smart food packaging with the following features: to be able to reduce food waste and plastic food packaging material; to be active to maintain food quality and improve shelf-life; to be sustainable and cost-effective, obtained from agri-food by-products and waste, using emerging green extraction and processing technologies; to be flexible and suitable for application to different food categories; and to be intelligent to monitor food quality and safety and tell the consumer about freshness of the packaged food. Our innovative approach will be based on polysaccharide-cellulose nanocrystals matrix, designed from date and pomegranate by-products and waste, enriched by bioactive compounds to enhance the antimicrobial and antioxidant capacity of the developed packaging film. An anthocyanin-based pH-sensitive indicator will be developed and RFID tags will be implemented to monitor food freshness. Moreover, blockchain applications will be used to provide relevant information about the traceability of food along the supply chain, constituting the originality and novelty of the proposed packaging solution. The smart packaging will be tailored and adopted to improve the shelf-life of highly perishable products, such as fish, meat, and cheese. The project is subdivided into seven work packages with well-defined goals and tasks, which falls perfectly under the multi-actor approach given the strong and diverse set of stakeholders (17 partners from 9 Mediterranean countries) involved in this project.