Recent trends and analytical challenges in plant bioactive peptide separation, identification and validation.

Interest in research into bioactive peptides (BPs) is growing because of their health-promoting ability. Several bioactivities have been ascribed to peptides, including antioxidant, antihypertensive and antimicrobial properties. As they can be produced from precursor proteins, the investigation of BPs in foods is becoming increasingly popular. For the same reason, production of BPs from by-products has also emerged as a possible means of reducing waste and recovering value-added compounds suitable for functional food production and supplements. Milk, meat and fish are the most investigated sources of BPs, but vegetable-derived peptides are also of interest. Vegetables are commonly consumed, and agro-industrial wastes constitute a cheap, large and lower environmental impact source of proteins. The use of advanced analytical techniques for separation and identification of peptides would greatly benefit the discovery of new BPs. In this context, this review provides an overview of the most recent applications in BP investigations for vegetable food and by-products. The most important issues regarding peptide isolation and separation, by single or multiple chromatographic techniques, are discussed. Additionally, problems connected with peptide identification in plants and non-model plants are discussed regarding the particular case of BP identification. Finally, the issue of peptide validation to confirm sequence and bioactivity is presented. Graphical representation of the analytical workflow needed for investigation of bioactive peptides and applied to vegetables and vegetable wastes Graphical Abstract.