Metabolite Analysis using 1H NMR Spectroscopy
We used 1H NMR spectroscopyto analyze doenjang metabolites
across the entire fermentation period. Metabolites included
monosaccharides, organic acids, and nitrogen compounds such
as amino acids and BAs.To minimize quantification errors
due to large particles, 10 g of doenjang samples were dried
in an oven at 80 C for 1 h and ground into a fine
power using a pestle and mortar. For sufficient metabolite
extraction, 0.2 g of doenjang powder was resuspended in
1.5 mL of 99.9% deuterium oxide (D2O; Sigma–Aldrich,
USA) containing 5 mM sodium 2,2-dimethyl-2-silapentane-5-
sulfonate (DSS,97%; Sigma–Aldrich) and incubated on ice with
occasional shaking for1 h.The doenjang powder solutions
were centrifuged at 12,000 rpm and 4 C for 10 min,the
600 mL of the supernatant were transferred into NMR tubes.
We obtained 1H NMR spectra with a Varian Inova 600-
MHz NMR spectrometer (Varian,USA); doenjang metabolites
were identified and quantified using the Chenomx NMR
Suite program (version6.1;Chenomx,Canada). Metabolite
concentrations were calculated as m mol per g-dry weight
doenjang.