Deciphering Butea monosperma Kombucha Fermentation Through NMR and Live-Cell Imaging

This study employed live-cell imaging and nuclear magnetic resonance (NMR) spectroscopy to investigate the microbiological and metabolic changes occurring during the fermentation of B. monosperma (Palash) flower kombucha. Throughout the 14-day fermentation period, live-cell imaging enabled direct visualization of SCOBY development, showing the progressive formation of a dense and interconnected network of cellular and aggregate structures. Semi-quantitative ImageJ analysis showed marked increases in image-derived surface coverage, particle count, and aggregate size, consistent with active fermentation. Complementary ¹H and ¹³C NMR analyses revealed fermentation-associated changes in the chemical composition of both green tea kombucha (GTK) and Palash flower kombucha (PFK). These included alterations in carbohydrate- and glycoside-associated resonances and the appearance of additional signals consistent with microbial transformation of tea constituents. Fermentation also produced significant physicochemical and antioxidant changes. The pH decreased from 6.0-6.2 to 2.90 and 2.88, while titratable acidity increased to 1.87% and 1.73% in GTK and PFK, respectively. Total soluble solids decreased from 11.6-11.9 Brix to 5.0 Brix in GTK and 6.5 Brix in PFK. In PFK, DPPH radical-scavenging activity increased from 60.93% to 80.41%, total phenolic content increased from 128.99 to 263.24 mg GAE/L, and total flavonoid content reached 94.74 mg QE/L. Overall, fermentation improved the physicochemical properties, antioxidant capacity, and chemical composition of Palash flower kombucha, highlighting its potential as a functional fermented beverage.