| In the paper, the method of mensurating extracellular-trehalose, the technology of synthesizing extracellular-trehalose, as well as the extraction and purification of trehalose were studied.A new method with less samples and shorter time was studied to determine extracellular-trehalose accurately.The method of high-temperature passivation was determined to synthesize extracellular-trehalose. The optimized conditions to stimulate yeasts is: culture yeasts under the temperature of 39 C for 22h, add 1.5% NaCl and culture to the 23rd hour, increase the temperature to 39 C and culture to the 24th hour, regulate the pH5.0 and culture to the 25th hour, then stop the reaction, centrifugating and collecting the yeast. The synthesizing technology of extracellular-trehalose is: the initial glucose concentration 3%, pH6.5-7.0, culture yeasts for 1h under the temperature of 39 C, then increase the temperature to 50 C, culture yeasts for 5h, then stop the reaction.The fed-batch reaction in which the extracellular-trehalose was synthesized was tested in the 5L bioreactor. It is found in the experimentation that the suitable oxygen concentration of 10% is needed to accumulate extracellular-trehalose. With the increase of seed volume, the concentration of trehalose in the reaction liquor increases too.Extraction and purification technology of trehalose from yeast was studied in the experiment. The extraction recovery of trehalose is above 98.81% under the condition: temperature 80 C, the concentration of yeasts 70g/L as well as the extraction time 60 min. The extraction liquor was purified by anion and cation exchange chromatography under the condition: flow rate 3~6mL/min, temperature 40 C.The anion and cation exchange chromatography is used to purify extracellular-trehalose. By calculating the volume of extracellular-trehalose reaction liquor, the trehalose was separated from the glucose completely under the condition: flow rate 1 .0mL/min, temperature 40 C. The suitable crystaling conditions are the alcohol volume: 4 times of crystaling liquor Vol. and crystaling temperature 40 C. The purity of trehalose produced by the technology is about 99% with the recover rate of 89%. |
