Sake and that added with tryptophan (Try) were both heated and fractionated by cellulose-column chromatography as shown in Figs. 1 and 2, from which the formation of peak Y containing a yellowgreen fluorescent substance (represented as YS below) was found to be favored by heating the mixture of sake and Try at acidic media.
Ether-soluble basic fraction was obtained from sake, fractionated by cellulose-column chromatography, and the fractions of peak Y were collected, concentrated after neutralization, and subjected to thinlayer chromatography (TLC). The yellow-green fluorescent spots of Rf O. 03 on TLC were dissolved in IN HC1, from which a yellow viscous substance was obtained after concentration. The UV-ray absorption spectrum of this substance was compared with that of the substance obtained from sake heated with Try by the same method as above, by which these two substances were found to be identical with each other as shown in Fig. 3.
The YS' were also obtained from several kinds of materials in Part 3 by the same methods as described above, and they were proved to be identical with each other from the results of paperpartition chromatography and TLC as seen in Figs. 4 and 5.
Isolation and purification of YS were carried out by the method schematically shown in Fig. 6, and results of fractionation were shown in Figs. 7 and 8. The YS, mp. 183 deg;C, C
16H
14N
20
3, was obtained as a plane crystal with some shade of yellow, of which UV-ray and infra-red ray absorption spectra were shown in Figs. 10 and 11, respectively. Fluorescent intensities were compared with those of other authentic fluorescent substances as shown in the Table. Thus, YS was found to be a new fluorescent substance in sake, which increased its amount by heating or aging of sake.
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