Integrating physicochemical and microbial characterization of red rice broth fermented over an 18-hour period augmented with metagenomic and metabolomic approaches

Abstract

Fermentation enhances the nutritional properties of foods. Fermented water of Kerala red rice (Oryza sativa L. subsp. indica), traditionally consumed in South India remains underexplored scientifically. This study characterizes the nutritional, microbial, and metabolite profiles of Kerala red rice water (broth) after 18 hours of natural fermentation using biochemical assays, shotgun whole-genome metagenomic sequencing (Illumina NovaSeq X Plus), untargeted gas chromatography-mass spectrometry (GC-MS) metabolomics, and a phytase-mediated mineral release assay. Fermentation enhanced nutritional quality with increase in carbohydrates by 22.7%, protein by 163.52%, and free amino acids by 35.47% compared to unfermented controls. Phytase activity rose from negligible levels to 0.12 U mL⁻¹. Metagenomics identified 50 taxa, dominated by Proteobacteria (59.63%) and Firmicutes (40.12%), with ∼34% of the community carrying phytase-encoding genes. Dominant genera included Pantoea, Saccharibacillus, and Bacillus. Fermentation also enhanced mineral release, with calcium, iron, and zinc in the fermented rice water showing increases of approximately 1190%, 566%, and 93%, respectively, relative to unfermented controls over a 360 min in vitro digestion period. These findings provide the first integrated insight bridging traditional dietary practice with modern analytical science.