Brown juice processed from alfalfa green biomass as a source of phytohormones and saponins

The valorization of by-products from green biorefineries is critical for environmental sustainability and economic viability. During the extraction of leaf proteins from alfalfa green biomass, significant volumes of brown juice (BJ) are generated. This study assessed the phytohormonal profile and anti-nutritional elements in BJ post-lacto-fermentation, examining its potential as a plant biostimulant. Fresh BJ was lacto-fermented using Pediococcus acidilactici, Lactobacillus paracasei, and Lactobacillus plantarum at 35 °C for 48 h. Phytochemical analysis via HPLC-MS/MS identified 18 saponins in BJ, with only medicagenic acid remaining post-fermentation, whose concentration increased 39-fold. Additionally, 23 plant hormone derivatives were quantified, with significant increases observed in indole-3-acetic acid, gibberellin GA1, jasmonic acid, and abscisic acid, while others like p-coumaric acid, caffeic acid, and ferulic acid decreased. These results demonstrate lacto-fermentation's ability to stabilize BJ, reduce haemolytic saponins, and enhance phytohormone concentrations, supporting its utility as a sustainable plant growth enhancer.