Valorization of Expired Milk into Protein Hydrolysate as a Plant Biostimulant: Characterization and Application on Hydroponically Grown Cos Lettuce

To meet global food demand, agricultural systems must enhance crop performance, productivity, and sustainability. Biostimulants have emerged as a promising strategy, particularly in vegetable production, due to their ability to enhance plant growth and resilience. This study characterized milk-derived protein hydrolysates (MPH) produced from expired milk and evaluated their potential as biostimulants for hydroponic cos lettuce. Hydrolysis of expired milk with hydrochloric acid achieved 94.55% hydrolysis and yielded 80.77% free amino acids. MPH was applied at volumes of 0, 1, 3, and 5 mL L−1 in combination with Hoagland and Arnon nutrient solution. The 1 mL L−1 (MPH1) treatment significantly increased shoot and root biomass and canopy size while reducing nitrate accumulation and enhancing total flavonoid and ascorbic acid content, as well as antioxidant capacity. HPLC analysis showed that MPH1 treatment promoted the accumulation of key metabolites, including vanillic acid, para-coumaric acid, salicylic acid, ferulic acid, gallic acid, syringic acid, quercetin, myricetin, and naringenin. MPH1 improved uptake of phosphorus, potassium, calcium, magnesium, and iron, contributing to mineral biofortification and nutritional quality. These results demonstrate that MPH at 1 mL L−1 is an effective biostimulant, improving yield and quality while reducing nitrate levels in hydroponically grown cos lettuce, offering a sustainable solution for food waste valorization.