Chiral amino acid analysis of Japanese traditional Kurozu and the developmental changes during earthenware jar fermentation processes

• Enantiomers of all proteinogenic amino acids plus allo forms in the Japanese traditional Kurozu vinegar were determined.
• In addition to the widely observed d-Ala, d-Asp, d-Glu and d-Ser, considerable amounts of d-Leu and d-allo-Ile were found.
• Developmental changes of these d-amino acids during fermentation and aging processes have also been clarified.
• The present results are useful to design a highly functional natural beverage.

Enantioselective amino acid metabolome analysis of the Japanese traditional black vinegars (amber rice vinegar, Kurozu) was performed using two-dimensional high-performance liquid chromatography combining a microbore-monolithic ODS column and narrowbore-enantioselective columns. d-Amino acids, the enantiomers of widely observed l-amino acids, are currently paid attention as novel physiologically active substances, and the foodstuffs and beverages containing high amounts of d-amino acids are the subjects of interest. In the present study, the amino acid enantiomers were determined by two-dimensional HPLC techniques after pre-column fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole. In the first dimension, the amino acid enantiomers are separated as their d plus l mixtures by the reversed-phase mode, then the d-amino acids and their l-counterparts are separately determined in the second dimension by the enantioselective columns. As a result, large amounts of d-Ala (800–4000 nmol/mL), d-Asp (200–400 nmol/mL) and d-Glu (150–500 nmol/mL) were observed in some of the traditionally produced Kurozu vinegars. Relatively large or small amounts of d-Ser (50–100 nmol/mL), d-Leu (10–50 nmol/mL) and d-allo-Ile (less than 20 nmol/mL) were also present in these samples. Developmental changes in the d-amino acid amounts during the fermentation and aging processes have also been investigated