Short Communications: An improved automated colorimetric analysis of fructose in fermentation media

There is a widespread interest in the commercial application of D-glucose-isomerase for the large-scale conversion of D-glucose into fructose-containing syrups[ ]. As such, there is a need for a rapid and reliable method for the routine analysis of glucose-isomerising activity in fermentation broths. The analytical methods currently in use include the incubation of the enzyme preparation with a solution of D-glucose followed by colorimetric analysis of the fructose produced [2,3]. Of the many proposed colorimetric methods such as the reaction of anthrone with sulphuric acid [4-6], resorcinol with hydrochloric acid [7], thiobarbituric with hydrochloric acid [8], and the cysteine-carbazole-sulphuric acid procedure , the latter commonly known as the "Dische Reaction" [9,10] stands out as being particularly advantageous for the selective and sensitive analysis of fructose in the presence of other carbohydrates. Cadmus and Strandburg reported an automated procedure using only cysteine and sulphuric acid reagents for the quantitative assay of D-fructose in the presence of a large excess of aldoses and other ketoses. Lloyd et al 12] described a semi-automated procedure using the Dische reaction for the determination of D-fruct0se in biological materials. This note describes a simplified and rapid automated procedure which, retains the accuracy of the cysteine-carbazole-sulphuric acid procedure while using milder reaction conditions than those formerly prescribed 12-14]. There are also fewer analytical modules included than previously described. The method is capable of analysing fructose in the range of 0.01-0.1 g/1 solution. Twenty samples per hour are analysed. Materials and method All chemicals should be of reagent grade. Distilled water is used throughout to make up the solutions to volume. Weigh 300 g Renex-30 (available from J.T. Baker Chemical Company, Phillipsburg, New Jersey 08865 USA) into a 100 ml. volumetric flask. Make up to volume with water. Store the reagent in a plastic bottle. L-eystein hydroehloride solution, 2% (w/v) Weigh 2.0 g L-cysteine hydrochloride into a 100 ml volumetric flask. Make up to volume with water. Add 0.5 ml Renex-30 solution and mix the contents thoroughly. Keep the reagent in a brown bottle and prepare fresh daily. into a 100 ml volumetric flask. Add 50 ml ethylene glycol monomethyl ether and make up to volume with water. Shake the contents to dissolve and add 0.5 ml Renex-30 solution. Keep the reagent in a brown bottle and store in refrigerator. This reagent is stable for four days. Sulphuric acid solution, 80% (v/v) Cautiously, add 1600 ml concentrated sulphuric …