Purification and identification of the promoter of sediment formation from raw soy sauce by heating

Soy sauce contains promoters of sediment formation at 60°C. These promoters were extracted with 1-butanol from raw soy sauce, followed by acetonitrile extraction. The acetonitrile-soluble fraction was used for further separation of the promoter of sediment formation, as the specific activity of the acetonitrile-soluble fraction was higher than that of the insoluble fraction. The promoter of sediment formation could be separated as a single peak from the main low-molecular weight peak on a Sephadex G-10 column by means of HPLC on a LiChrospher (RP-18) column. The active fraction for sediment forming-activity separated by three columns, namely, a LiChrospher (RP-18) column, an Alltech 700CH carbohydrate column and a Shodex OHpak Q-801 column, was observed as a single peak. 0.08 g of promoter was obtained from 1l of raw soy sauce. The ¹³C, ¹H NMR and ¹H-¹H COSY spectral data identified the purified promoter as l-glutamic acid 5-n-butyl ester. l-Glutamic acid 5-n-butyl ester was synthesized and identified as a promoter by HPLC and ¹H NMR analysis. In addition, the existence of l-glutamic acid 5-n-butyl ester in raw soy sauce was confirmed by HPLC analysis of the extract without 1-butanol extraction from raw soy sauce.