Fractionation of Nondialyzable Melanoidin into Components by Electrofocusing Electrophoresis

Nondialyzable melanoidin formed in a model system of glucose and glycine was applied on thin layer gel electrofocusing in a polyacrylamide gel. The electrofocusing profile differed according to the reaction time: the pI of the melanoidin formed in the early stage of the reaction was less than 2.9 and, as the reaction progressed, the pI of the melanoidin formed gradually shifted to a less acidic value, from 2.9 to 3.3. Those from the xylose and glycine model system gave a similar profile to that of the glucose system.

Preparative separation of the nondialyzable melanoidin, which was formed by heating the glucose system for 7hr, was performed by flat-bed electrofocusing in Sephadex gel. At least 14 melanoidin bands were clearly electrofocused at a pH range of 2.7-3.3 and about 59% of the melanoidin applied remained unaffected by electrophoresis at the starting position. The major component of electrofocused melanoidin was pI 3.00, this being made up of 12.5% of the total amount of electrofocused melanoidin. The molecular weight of melanoidin affected by electrophoresis was about 25,000, regardless of the pI value of the melanoidin components. The reducing activity, estimated by the potassium ferricyanide method, showed that the lower the pI of melanoidin, the higher was the reducing activity.

The addition of hydrochloric acid to the melanoidin solution caused it to gradually become viscous and the melanoidin was precipitated below pH 3.0-3.5, corresponding approximately to ampholite. This feature can be used as a method to prepare nondialyzable melanoidin in a short time.