Metabolomic profiling of bioactive compounds in traditional indian red rice beer using high-resolution mass spectrometry (HR-MS)

• Fermentation parameters of traditional Indian rice beer were optimized using RSM. • RRB (3.05g starter, 104 h fermentation) exhibited high sensory acceptability. • Metabolomics identified 40 BACs such as FAs, esters, sphingolipids, and terpenoids. • Ceramides and oxylipins can reduce oxidation, inflammation, and microbial cells. • RRB is both a culturally significant and bioactive-rich beverage for consumers. Globally, the brewing of rice beer is a traditional practice by different ethnic groups through spontaneous fermentation by locally grown starter cultures. These traditional rice beers produced across India remain underexplored, despite their cultural importance and potential functional value. Most of these beverages are prepared through spontaneous fermentation using indigenous starter cultures, yet less is known about how controlled process optimization influences their quality and bioactive profile. This study delineates optimizing fermentation parameters of a traditional red rice beer (RRB) which would lead to a beverage with targeted pH, acidity, TSS, and alcohol content, thereby enhancing its quality and potentially its bioactive metabolite diversity. Using response surface methodology, optimal conditions were identified at 3.05 g starter culture and 104 h fermentation has produced beverage with pH 4.38, TSS 24.82 °Brix, acidity 0.59 g/L, and 8.01% ethanol content. The optimized RRB also showed favourable colour attributes and high sensory acceptability. Untargeted LC-MS metabolomics of RRB has revealed 1,188 differential metabolites, out of which 40 major bioactive compounds were annotated. Fatty acid ethyl esters, PUFA derivatives, monoacylglycerols, sphingolipids, phenolics, terpenoids, ceramides, and oxylipins were covered in the RRB metabolome in the RT range from 25.01 to 28.43 min. These metabolites are known for their anti-inflammatory, anti-CVD, anti-cancer, anti-oxidant and skin health-promoting actions. This study has demonstrated that controlled optimization not only standardizes product quality but also enhances the functional potential of traditional beer, as evidenced by the diverse profile of health-relevant metabolites.