Fractionation of broccoli leaf extracts: Biostimulant activity on pak choi and induction of bacterial biofilms

Broccoli extracts have been discovered as plant biostimulant by improving crop vigour. However, their intrinsic chemical complexity makes it difficult to identify the specific mechanisms and bioactive components responsible for these effects. In this study, a broccoli by-product extract was subjected to physicochemical fractionation, including differential centrifugation, dialysis, size-exclusion chromatography, and Bio-Beads adsorption, to identify the components underlying its biological effects. The resulting fractions were characterized for mineral composition, metabolite content, and antioxidant capacity, and evaluated for their biostimulant potential in pak choi seedlings and their ability to induce biofilm formation in the rhizobacterium Bacillus velezensis . Fractionation revealed a clear functional specialization among fractions. The Bio-Beads filtrate (BBF) most consistently reproduced the growth-promoting effect of the crude extract, particularly by stimulating root elongation, whereas SepP3 (fraction 3 from the Sephadex column) was mainly associated with biomass accumulation. Global analyses indicated that ionic balance was the variable most strongly associated with seedling biomass, while antioxidant-related properties appeared to contribute secondarily. Furthermore, the crude extract, BBF, and the differential centrifugation supernatant (DCS) significantly promoted bacterial biofilm formation, suggesting that these fractions may also influence rhizosphere-associated microbial dynamics. These findings show that fractionation can isolate functionally specialized bioactive pools from complex plant-derived matrices. By identifying specific mineral balances and metabolite groups associated with plant performance, this work provides a mechanistic framework for developing targeted, high-efficiency biostimulants from agricultural residues.