Advancing Aquafeed: Prebiotic, Antimicrobial, Antioxidant and Immunomodulatory Benefits of Innovative Ingredients from Farming Systems

Mariana GirãoDiogo AmaralGabriel CamposRalph UrbatzkaHelena PeresRodrigo Ozorio^*Maria Fátima Carvalho

• Interdisciplinary Center for Marine and Environmental Research, University of Porto, Matosinhos, Portugal

Aquaculture plays a vital role in meeting the increasing global food demand driven by population growth. Sustainable development in the sector depends on optimizing fish nutrition and reducing antibiotic use, with bioactive ingredients offering a promising means to enhance animal health and productivity. Applying circular economy principles, specifically valorising underutilized biomasses as functional feed ingredients, provides a synergistic strategy to boost both environmental sustainability and economic efficiency by minimizing waste and input costs. This study investigated waste-derived biomasses from fungi (Pleurotus ostreatus), invertebrates (Tenebrio molitor and Eisenia fetida), microalgae (Phaeodactylum tricornutum and Scenedesmus spp.) and aquatic plants (Lemna minor and Nasturtium officinale) sourced from European freshwater aquaculture systems as novel feed ingredients with functional properties. These were screened for their prebiotic potential, as well as for their antimicrobial, antioxidant and immunomodulatory properties. Prebiotic effects were assessed by measuring the ability of the biomasses to promote the growth of seven probiotic bacterial strains relevant to fish and human health. All strains except Bifidobacterium longum ATCC15708 showed enhanced growth in the presence of at least one biomass, with strain-specific responses indicating selective prebiotic effects. Organic and aqueous biomass-derived extracts were tested for antimicrobial activity against 13 human and fish pathogens using the disc diffusion assay. Extracts from P. ostreatus, T. molitor, E. fetida, and L. minor inhibited growth of Aeromonas hydrophila, A. sobria, A. salmonicida, Lactococcus garvieae, Listonella anguillarum, Tenacibaculum maritimum, and Staphylococcus aureus, with inhibition zones ranging from 9-16 mm at 1 mg·mL⁻¹ of extract. When tested for their pro- and anti-inflammatory properties in RAW 264.7 cells, several fungal, microalgal, and aquatic plant extracts exhibited significant anti-inflammatory activity by reducing nitric oxide production without affecting cell viability. Antioxidant assessments revealed marked differences among biomasses, with aqueous extracts of aquatic plants showing the highest DPPH radical scavenging capacity, microalgal activity varying with solvent polarity, and fungi and invertebrates exhibiting comparatively low antioxidant potential. Our findings highlight the beneficial properties of low-value, circular-economy biomasses, supporting their development as sustainable, cost-effective aquafeed ingredients. Further research is underway to characterize the in vivo effects of these ingredients when incorporated into experimental fish diets.