食品添加物ステビア甘味料の迅速分析法の開発と腸内細菌による成分変換解析へのアプローチ

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Stevia extract (Ste) and α-glucosyltransferase-treated stevia (Gts) are included in the List of Existing Food Additives in Japan. A rapid and simple method for simultaneous determination of the stevia sweeteners by HPLC coupled with UV and MS was developed in the present study. Both Ste and Gts contain steviol glycosides as major sweetening components. It was possible to distinguish the HPLC peaks of not only stevioside and rebaudioside A available as purified standard compounds, but also 10 other coexisting glycosides with the aid of LC/MS, owing to the characteristic structural profiles of the glycosides. Analysis with UV-HPLC using ODS column as the stationary phase and MeCN-water (30:70) as mobile phase and detecting at 210nm was commonly profitable for separation of many components of stevia. Further separation of overlapped peaks was achieved by hydroxyapatite (HA) column for Ste (MeCN-water 78:22) and for Gts (gradient from 78:22 to 70:30). Calibration curves drawn by total areas of the peaks of glycosides of Ste and Gts were almost rectilinear. The limits of detection for Ste were 2.0 μg/g (ODS) and 5.0 μg/g (HA), while those for Gts were 2.5 μg/g (ODS) and 25 μg/g (HA), respectively. The recoveries of the constituents of Ste added to several foods were 83.4 〜 95.0 % by ODS, and 80.7 〜 100.9 % by HA, and those of Gts were 84.7 〜 98.5 % by ODS, and 87.8 〜 95.0 % by HA, respectively. Along with the TLC study, the present UV-HPLC method was applied to determine the kind and amount of the sweetener in commercial foods. Pickles contained 23 μg/g of Ste, on the other hand, soft drink and rice cracker contained 269 μg/g, and 24 μg/g of Gts, respectively. Finally, constituents were measured by the present UV-HPLC method in experiments incubating the stevia sweeteners with extract of normal rat's feces. Major glucosides of both Ste and Gts were found to be converted to steviolbioside, possibly through hydrolyses by intestinal microbes. Thus, the present method was shown to be useful to examine the biotransformation of stevia sweeteners.
日本食品化学学会の論文
2001-08-31

食品添加物ステビア甘味料の迅速分析法の開発と腸内細菌による成分変換解析へのアプローチ

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