Extraction, Purification And Partial Characterization Of The Sweet Modifying Glycoprotein, Miraculin From Richardella Dulcifica

Abstract

In the present study miraculin, a non-nutritive taste modifier from the tropical plant, Richardella dulcifica has been purified to homogeneity. It was extracted from the pulp of the fruits in 0.5M NaCl and purified at ordinary temperatures by a three step chromatographic process namely; Hydrophobic interaction chromatography, (HIC), Ion exchange chromatography, (IEC), and Gel filtration chromatography (GFC) respectively. The relative molecular weight (Mr) of purified miraculin was about 43, 000 and 100 fig/ml of the lyophilized product provoked sweetness with lemon. N-terminal amino acid analysis revealed two forms of miraculin, the major form being more than 70 % of the total miraculin population. Protease activity was detected and measured in the pulp of fresh and aged fruits. The activity .and specific protease activity in the pulp of freshly picked berries were 0.0756 units/mg pulp and 0.2414 units/mg protein respectively. Corresponding values for the aged (at least six months old) berries were 0.0369 unit/mg pulp and 0.1342 unit/mg protein. The difference was significant (p s 0.05) . Total protein content measured for the fresh and aged berries were 31.3% + 1.9% and 27.4% + 0.3% (w/w) respectively. The difference was not significant (p <; 0.05), in addition only approximately 10 % (w/w) of the fruits contained the active principle, miraculin. A predictive model: Z = 0.027348 - 0.015177^ - 0.003364/tpH + 0.000128pH + 0.029960/i2 where Z is the solubility, was developed to explain the effect of ionic strength (/x) and pH on the solubility of miraculin. The model had an R2 Of 66.12%. Analysis of variance (ANOVA) showed ionic strength to be the most influential variable in the model. The protein had the maximum solubility at low pH and low ionic strength, with the minimum solubility recorded at high pH and high ionic strength. A thermal denaturation investigation, established that water-glycerol mixtures substantially increased the melting temperature of the purified product.