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

ABSTRACT

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 ¹ H and ¹³ C 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.