INTEGRATED AND SUSTAINABLE STRATEGY FOR THE INVESTIGATION OF NOVEL FOOD SAFETY,FOCUSING ON BIOACTIVE PEPTIDES AND PHYTOCOMPLEXES

In recent years, there has been increasing attention within the European food sector toward foods that are expected to provide health benefits in addition to basic nutrition. This shift is actually reflected in the growing interest in functional foods and in the search for new ingredients that may contribute to health maintenance or disease prevention. In this context, bioactive compounds have received particular attention, especially concerning food-derived bioactive peptides (BAPs) and natural extracts obtained from different food sources. Notably, BAPs can be released from both animal and plant proteins, and, over the years, a plethora of studies have reported and demonstrated their multifunctional bioactivities, although their effectiveness could actually depend on several factors that need to be thoroughly analyzed. Many researchers have explored alternative protein sources as a sustainable reservoir of potentially bioactive peptides, while, at the same time, sustainability concerns increasingly influence food science research, since a large amount of by-products is generated during food processing, although many of these materials could be valorized, being still rich in proteins and other compounds with potential biological activity. Despite the promising properties of BAPs, their development as nutraceutical ingredients faces significant challenges, including potential degradation during gastrointestinal digestion, variable intestinal absorption, and uncertain biological activity in systemic circulation. A comprehensive evaluation requires, therefore, integrated approaches, alongside careful consideration of production methods, green technologies, and regulatory compliance to ensure the safety and efficacy of the final product. Within this scenario, my PhD project was conducted as part of Spoke 3 of the PNRR - funded PE10 project on Sustainable Food Systems, entitled “ON Foods - Research and Innovation Network on Food and Nutrition Sustainability, Safety and Security (Working ON Foods)", addressing the safety evaluation of novel ingredients and traditional food products. Specifically, this thesis aims to develop an integrated methodological framework for the production and characterization of bioactive peptides obtained from sustainable and novel sources, built around the need to evaluate bioactive food-derived compounds in a realistic and biologically meaningful way, taking into account not only their bioactive potential but also their safety, metabolic stability, and actual relevance after digestion and intestinal absorption. For this reason, the work followed a stepwise experimental strategy in which food processing, in vitro and cellular models, and in vivo studies were closely interconnected. Different protein-rich matrices were selected, including both animal and plant sources, as well as by-products from the food production chain. Proteins were extracted and hydrolyzed using food-grade enzymes in both standard and immobilized conditions for some peculiar matrices to target applicability within the food and nutraceutical context. In detail, enzymatic hydrolysis was carried out either through conventional enzymatic protocols or in combination with simulated gastrointestinal digestion of the resulting peptides, depending on the specific objective of each case study. For selected raw matrices, specifically soybean okara and herring side streams, ultrasound-assisted processing was explored as a green pretreatment strategy, with the aim of altering the structural organization of the starting materials and improving the release of bioactive compounds and protein accessibility prior to extraction and enzymatic hydrolysis. High-resolution mass spectrometry (HRMS) approaches, namely untargeted peptidomic and metabolomic techniques, were exploited to define the specific composition of peptides or secondary metabolites present in the final products, providing a molecular-level understanding of the functional potential of these matrices, besides evaluating the composition of their bioaccessible and bioavailable fractions, respectively. Overall, this methodological strategy reflects an integrated approach, combining complementary models and international expertise to address the complexity of developing safe and biologically relevant food-derived ingredients for nutraceutical applications.