Revestimentos bifuncionais edíveis para conservação de alimentos: probióticos produtores de péptidos antimicrobianos como uma estratégia para melhorar a segurança alimentar e a saúde humana

The main objective of Pep2Coat is to lead the way in developing a dual-purpose edible coating that will both i) preserve food products from spoilage, thereby reducing food waste, and ii) enhance human gut’s health by counteracting infectious diseases agents offering an alternative solution to the ongoing global crisis of bacterial and viral infections. To achieve this goal, the first step is to identify, produce, and characterize AMP from NGP candidates, with potential novel bioactivities, new targets and modes of action. These peptides are intended to be active preferably against food spoilage agents and, ideally, against human pathogens as well. To achieve that, 6 specific objectives were outlined: 1) To identify putative AMP gene clusters within NGP metabolic potential using culture-independent methods; 2) To develop a heterologous expression system for the identified AMP; 3) To produce and purify the putative AMP; 4) To characterize AMP bioactivity (mainly antimicrobial, but also eventual antiretroviral, antitumor, and/or anti-inflammatory activities) and safety for human consumption; 5) To develop edible bifunctional coatings containing the AMP or AMP-producing strain(s); 6) To assess and improve the properties of the coating in a food model system – cheese products as proof-of-concept. To achieve this, 6 scientific tasks are proposed, each with specific key deliverables. These will be described in detail in the relevant section. Task 1 will use culture-independent methods to assess the potential of gut bacteria, focusing on next-generation probiotics, in producing AMP. A single cell RNA-sequencing approach will be used on stool samples from healthy individuals and patients recovering from pathogen-induced inflammatory intestinal processes to identify variations in activity. Once the putative AMP(s) are identified, Task 2, will involve developing an heterologous expression system to allow its production using E. coli Nissle 1917 as the host due to its probiotic nature. The structural genes responsible for the final products will be cloned in frame with an His6-tag to facilitate the purification steps in Task 3. An optimized extraction and purification protocol will be developed in this phase to isolate the mature AMP and assess its structure, bioactivity, stability, and toxicity, envisaging the application for human consumption (Task 4). Task 5 will focus on creating an edible coating that includes the selected AMP or AMP-producing probiotic strain. The edible coatings will be tested in Task 6 in cheese models system. Task 7 related with coordination and project management will ensure the successful functioning of all Tasks and in Task 8 the project results will be widely disseminated to all stakeholders involved. At the end of Pep2Coat, AMP from gut microbiota in general, and particularly next generation probiotics, will have been investigated and characterized using innovative methodologies, contributing with new candidate molecules with new and diverse bioactivities, including against Gram-positive and Gram-negative bacteria, in particular food spoilage agents and human pathogens. Bifunctional edible coatings with AMPs and/or probiotics will be developed to improve food preservation, reduce food waste, and promote human gut health. These findings will have a true value-added application in the food industry, as well as in biomedical and nutraceuticals fields, aligning with the 2030 Agenda for Sustainable Development goals.