The relationship between diet, human health, and disease prevention is well-established, with food bioactive compounds (FBs) widely recognized for their beneficial effects. Metabolism is key in transforming precursor FBs molecules and facilitating their circulation in the human body. Urine has proven to be a valuable biofluid for monitoring dietary exposure. However, the low concentrations of FBs metabolites, their chemical variability, and the lack of appropriate reference standards present challenges in metabolite identification. To address these challenges, developing urine preparation methods for scalable metabolite isolation and unambiguous structure elucidation could significantly improve the coverage and accurate annotation of urine metabolites. Urine samples were collected from a healthy volunteer after hydroxytyrosol (HT) supplementation. Traditional urine pretreatment protocols, such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE), were tested alongside enrichment methods using resins (XAD-4, XAD-7, ion-exchange). Extracts were analyzed in parallel using HPLC-DAD/ELSD, UPLC-HRMS, and NMR to assess profiles and annotate metabolites. Methods were evaluated based on extraction yield, metabolite chemical and biochemical diversity, metabolite coverage, selectivity, as well as cost, ease and time. The most promising protocols were further tested on a larger scale. Among the methods evaluated, XAD-7 resin and LLE (Urine/EtOAc 1:3) showed the best performance. Furthermore, detailed identification of metabolites (endogenous and exogenous) per protocol was performed using LC-HRMS/MS and NMR. Additionally, investigation of each protocol performance in respect to the biochemical pathway in which metabolites are implicated was assessed. The suggested workflow is compatible with both profiling and isolation set-ups and could provide essential insights into urine metabolome and FBs biotransformation. It ensures confident identification and high coverage of metabolites, providing more complete and accurate interpretation of metabolism studies' results and, therefore, valuable input in profiling approaches towards the role of diet on human health.

Ceratonia siliqua L. (Fabaceae) is an evergreen sclerophyllous species that successfully overcomes the challenges of the Mediterranean climate. Commonly, biosynthesis of secondary metabolites is a major reaction of the plants thriving in the Mediterranean formations against temperature stress. Due to concerns about the climate crisis, we studied the impact of 6-day low (5 °C) and high (40 °C) temperature stress on young carob seedlings. In stressed plants, mainly the heat-treated, the leaves appear xeromorphic. Parameters of the physiology of the plants such as chlorophyll-a and -b, total phenolic content, and oxidative stress were measured and presented via Principal Component Analysis. Chlorophyll-a and -b contents are inferior in cold-stressed leaves while heat-stressed leaves accumulate more phenolics and experience higher oxidative stress as compared to their cold-stressed counterparts. The phytochemical profile of different extracts obtained from stressed carob leaves was identified so as to gain insight into metabolites produced under stress. Moreover, LC-HRMS/MS metabolomic workflow was utilized for the discovery of biomarkers, over- or under-regulated in stressed conditions. The antimicrobial activity of carob leaf extract fractions was assessed against six human pathogen strains and three phytopathogen bacterial strains. MeOH-H₂O and dichloromethane (DCM) extracts presented notable activity against Candida albicans and Saccharomyces cerevisiae, while DCM extracts inhibited the growth of Erwinia amylovora. We may conclude that carob tree exposure to temperature stress does not have a significant influence on secondary metabolic pathways.

Microbial production of bioactive molecules provides a promising alternative to traditional isolation or chemical synthesis approaches, enabling high-yield production of pure compounds without the need for heavy-metal catalysts, harsh conditions, or complex plant-based processes. Hydroxytyrosol (HT), a potent antioxidant with significant pharmacological properties, has attracted considerable interest due to its broad applications in pharmaceuticals, cosmetics, and food industries. While Escherichia coli systems have been effective for HT production, optimization of extraction yield and recovery process remain a challenge. Furthermore, isolation of HT and its metabolites continues to suffer from inefficiencies even when biosynthetic pathways are successful. Centrifugal techniques such as annular centrifugal extraction and partition chromatography methods e.g., Annular Centrifugal Extraction - ACE, Countercurrent chromatography - CCC, Centrifugal Partition Chromatography - CPC offer advantages over traditional methods, including the absence of solid stationary phases, reduced organic solvent use, speed, and scalability. However, these techniques have been underutilized in biotechnology workflows. Additionally, existing procedures often employ simplistic analytical methods, which lead to loss of valuable data for pathway monitoring, validation and metabolite discovery. This study presents a comprehensive workflow for the production, extraction, isolation, and identification of HT and derivatives from metabolically engineered E. coli biofactories. The workflow combines ACE for media extraction with CPC for high yield and purity HT isolation. It also emphasizes the analysis and structural elucidation of HT metabolites by means of HRMS/MS and 1 & 2D NMR, facilitating biosynthetic pathway monitoring and discovery of novel HT metabolites with potential enhanced biological activity. Using this approach, 23 compounds were isolated and identified contributing to the completion of the complex biosynthetic route of HT synthesis from l-tyrosine. This is first reported of direct isolation of pure HT metabolites from E. coli biofactories and to our knowledge the first application of liquid-liquid centrifugal techniques in the treatment of biotechnological materials.