| Summary: | Fermented wheatgrass juice was prepared using a two-stage fermentation process by employing <i>Saccharomyces cerevisiae</i> and recombinant <i>Pediococcus acidilactici</i> BD16 (<i>alaD</i><sup>+</sup>). During fermentation, a reddish-brown hue appeared in wheatgrass juice due to production of different types of red pigments. The fermented wheatgrass juice has considerably higher content of anthocyanins, total phenols and beta-carotenes as compared to unfermented wheatgrass juice. It has low ethanol content, which might be ascribed to the presence of certain phytolignans in wheatgrass juice. Several yeast-mediated phenolic transformations (such as bioconversion of coumaric acid, hydroxybenzoic acid, hydroxycinnamic acid and quinic acid into respective derivatives; glycosylation and prenylation of flavonoids; glycosylation of lignans; sulphonation of phenols; synthesis of carotenoids, diarylnonanoids, flavanones, stilbenes, steroids, quinolones, di- and tri-terpenoids and tannin) were identified in fermented wheatgrass juice using an untargeted liquid chromatography (LC)-mass spectrometry (MS)-matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)/time-of-flight (TOF) technique. The recombinant <i>P</i>. <i>acidilactici</i> BD16 (<i>alaD</i><sup>+</sup>) also supported flavonoid and lignin glycosylation; benzoic acid, hydroxycoumaric acid and quinic acid derivatization; and synthesis of anthraquinones, sterols and triterpenes with therapeutic benefits. The information presented in this manuscript may be utilized to elucidate the importance of <i>Saccharomyces cerevisiae</i> and <i>P</i>. <i>acidilactici</i> BD16 (<i>alaD</i><sup>+</sup>) mediated phenolic biotransformations in developing functional food supplements such as fermented wheatgrass juice.
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