Carbon Stable Isotope Analysis for Glucose in Sake: Simple Freeze-Dried Sake Can Substitute for Glucose Following HPLC Isolation

Abstract

Carbon stable isotope analysis of ethanol is an established method for determining the authenticity of alcoholic beverages, but there is no accepted method for measurements of glucose carbon stable isotopic composition (δ 13C). This study aims to establish two methods to determine accurately the δ 13C value of glucose isolated from sake by high-performance liquid chromatography (HPLC) after three clean-up steps using ion exchange chromatography and in simple freeze-dried sake (total nonvolatile matter) and to examine two methods that yield different δ 13C values. In the glucose isolation procedure, the δ 13C values of glucose were not significantly affected by the glucose concentration, solvent (water or aqueous acetonitrile), drying method (freeze-dry or nitrogen-spraying), the clean-up step using ion exchange chromatography (weakly acidic cation-exchange, strongly acidic cation-exchange, and weakly basic anion-exchange resins), or HPLC isolation. Glucose nitrogen spray-dried after isolation by ion exchange chromatography and HPLC showed a carbon isotope discrimination value of <0.1 ‰. The glucose δ 13C values ranged from −26.8 to −25.5 ‰ in authentic sake and from −27.0 to −11.0 ‰ in commercial sake samples. This HPLC isolation approach may provide a first step toward visualizing the brewing process via isotopic carbon flow during fermentation. Additionally, there was no significant difference between the δ 13C values of glucose and total nonvolatile matter from sake. The δ 13C measurement of simple freeze-dried sake can substitute for glucose δ 13C measurement using HPLC isolation, thus aiding high-throughput detection of carbon derived from C4 plants in sake.