UHPLC-Q-Orbitrap-HRMS metabolomics and molecular networking to assess non-thermal debittering of orange juice: Insights into compositional quality preservation

Industrial debittering of orange juice often affects its nutritional and sensory quality due to heating or clarification steps. As a non-thermal alternative, this study explores for the first time the application of an expanded bed vortex flow reactor (EB-VFR) to selectively remove bitter limonin, avoiding heating and clarification steps that could compromise the chemical integrity of orange juice. The process efficiency and its further compositional impact were assessed using ultra-high-performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS), integrating a comprehensive threefold analytical strategy: (i) monitoring of limonin reduction, (ii) untargeted metabolomics to assess global changes and for marker discovery, and (iii) suspect screening to evaluate specific changes in key juice compounds. Multivariate and univariate analyses highlighted 35 discriminant metabolites differentiating EB-VFR-treated and control juices (variable importance in projection, VIP ≥ 1.0, p -values <0.05, and fold change threshold >1.1). Limonin and bitter glycosylated flavonoids (naringin, neohesperidin) markedly decreased, achieving up to >99% limonin removal below the sensory bitterness threshold (6 mg/L). In contrast, metabolites associated with flavor and freshness properties of fruit juices, such as amino acids ( L -phenylalanine), organic acids (DL-malic acid), and oxidized sugars, significantly increased. Pathway and molecular networking analyses indicated selective changes in metabolites linked to the tricarboxylic acid (TCA), alanine-glutamate, and butanoate. Notably, ascorbic acid (vitamin C) remained unaltered, underscoring the nutritional preservation achieved by this novel non-thermal treatment. Overall, the EB-VFR demonstrates strong potential as a promising non-thermal and energy-efficient debittering approach, supporting its potential feasibility for bitterness-free orange juice production. • Non-thermal EB-VFR efficiently removes limonin bitterness from orange juice. • It achieves up to 99.4% limonin reduction while preserving flavor quality. • UHPLC-HRMS metabolomics reveals 35 key post-debittering metabolic shifts. • Amino and organic acids, and sugars increased, enhancing flavor and freshness. • Vitamin C is fully preserved under sustainable EB-VFR processing.