From Waste to Functional Feed Ingredient: Biochemical and SHK-1 Cell Line Evaluation of Black Soldier Fly Larvae for Aquaculture Nutrition

Black Soldier Fly Larvae Meal (BSFLM) has gained attention as a sustainable feed ingredient in aquaculture, yet its functional properties at the cellular level remain underexplored. This study evaluated the antioxidative and proliferative effects of BSFLM derived from larvae fed different waste-based substrates (Kitchen Waste (KW); Agricultural Waste (AW); Aquaculture Sludge (AS); Aquaculture Offal (AO); Mix (MX)), using the Atlantic salmon (<i>Salmo salar</i>) SHK-1 cell line as an in vitro model. BSFLM treatments were assessed through four assays: oxidative stress mitigation under H₂O₂ exposure, baseline cellular proliferation, proliferation under protein-standardized conditions, and recovery from serum starvation. Each assay was carried out in three independent experiments with three replicates per treatment, and changes in coloration were quantified using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The results showed that BSFLM from plant-based substrates, particularly MX and KW diets, significantly (<i>p</i> < 0.05) improved cell viability across all assays. Under oxidative stress, MX (121.1% ± 5.9) and AW (119.9% ± 6.1) treatments maintained viability levels comparable to Vitamin C (119.3% ± 3.8) (250 ppm of DSM Stay-C) and the control (137.5% ± 11.6). In proliferation assays, MX (207.6% ± 16.3) and KW (196.3% ± 11.1) outperformed animal-based treatments AO (122.6% ± 4.4) and AS (113.1% ± 3.7), and these effects persisted under protein-standardized conditions, although the statistical significance was reduced. In the recovery from serum starvation assay, cells treated with MX (45.5% ± 1.9) and KW (42.0% ± 0.4) exhibited markedly higher viability than AS (15.5% ± 1.9) and AO (14.8% ± 2.2). The biochemical composition of BSFL reared on different substrates, including proximate, amino acid, fatty acid, and polyphenol profiles, was analyzed to contextualize the observed cellular responses. These findings highlight the superior functional properties of BSFLM derived from plant-based substrates and support its potential use as a targeted functional feed ingredient in aquaculture feed formulations.