From Function to Metabolome: Metabolomic Analysis Reveals the Effect of Probiotic Fermentation on the Chemical Compositions and Biological Activities of Leaves.

This study aimed to investigate the impact of probiotic fermentation on the active components and functions of leaves (PFL). PFL was fermented for 7 days using six probiotics ( SWFU D16, ATCC 8014, ATCC 53013, CICC 6038, ATCC 334, and CICC 6045). The total phenol and flavonoid contents, antioxidant abilities, as well as α-glucosidase and acetylcholinesterase inhibition abilities of PFL during the fermentation process were evaluated, and its bioactive compounds were further quantified by high-performance liquid chromatography (HPLC). Finally, non-targeted ultra-HPLC-tandem mass spectroscopy was used to identify the metabolites affected by fermentation and explore the possible mechanisms of the action of fermentation. The results showed that most of the active component contents and functional activities of PFL exhibited that it first increased and then decreased, and different probiotics had clearly distinguishable effects from each other, of which fermentation with ATCC 53013 for 1 day showed the highest enhancement effect. The same trend was also confirmed by the result of the changes in the contents of 12 phenolic acids and flavonoids by HPLC analysis. Further metabolomic analysis revealed significant metabolite changes under the best fermentation condition, which involved primarily the generation of fatty acids and their conjugates, flavonoids. A total of 574 and 387 metabolites were identified in positive ion and negative ion modes, respectively. Results of Spearman's analysis indicated that some primary metabolites and secondary metabolites such as flavonoids, phenols, and fatty acids might play an important role in the functional activity of PFL. Differential metabolites were subjected to the KEGG database and 97 metabolites pathways were obtained, of which biosyntheses of unsaturated fatty acids, flavonoid, and isoflavonoid were the most enriched pathways. The above results revealed the potential reason for the differences in metabolic and functional levels of PFL after fermentation. This study could provide a scientific basis for the further study of PFL, as well as novel insights into the action mechanism of probiotic fermentation on the chemical composition and biological activity of food/drug.