Study On Technology Of Fructo-oligosaccharides Production By Immobilized Fructosyltransferase And Glucose Isomerase From Sucrose

Fructooligosaccharides(FOS) is one of functionnal oligosaccharides to human body.Fructooligosaccharides also have increasingly become important because of their desirable physiological activities, such as being low in calories, anti-cariogenicity and acting as a growth factor for bifidobacteria in the intestinal flora. FOS are not susceptible to decomposition by human or animal digestive enzymes. Their prebiotic properties favor an adequate function of the human digestive system. For the production of this functional oligosaccharides from sucrose, the enzymes of fructosyltransferase(FTase) derived from bacteria and fungi have been mainly used. However, glucose is a by-product result from chemical reaction in the production process which can inhibit the reaction. The yield of oligosaccharides is not high, usually around 50%. It is of great significance to find a way to eliminate the inhibition of glucose in the industrial production of oligosaccharides.Fructosyltransferase immobilized in porous anion-exchange resin were used to produce fructooligosaccharides(FOS). In this study, the preparation of free fructosyltransferase, the selection of carrier, the optimization of immobilization, the properties of immobilized fructosyltransferase and glucose isomerase have been investigated. The production of FOS with immobilized fructosyltransferase and glucose isomerase in the different manner of cascade reaction, batch reaction and feeding batch reaction in column have been compared in this article.Fructosyltransferase was extracted from the cells of Aspergillus oryzae with rubbing treatment, and the ratio of extraction was up to about 64.26%. D102 p anion-exchange resin was selected as a carrier to immobilize fructosyltransferase through absorption and cross-linking. The microstructure of immobilized enzyme was observed by SEM.The optimal conditions of enzyme immobilization were investigated by the single factor experiment. 62.85 U enzyme per g wet resin was immobilized for 8 h at 25℃ and p H 7.0,then crosslinked with 0.01~0.05%(final concentration) glutaraldehyde for 6 h at 4℃The optimum temperature and p H of the immobilized fructosyltransferase were 50℃ and6.5. Thermal and p H stability were both improved by immobilization comparing with the free enzyme. The Km value was 1.28 M(for sucrose), which was 5 times higher than that(0.29 M)of the free enzyme. This indicated that the affinity between enzyme and substrate decreased.The optimum temperature and p H of the immobilized glucose isomerase were 80℃ and 7.0~8.0. The Km value was as low as 0.325 M(for glucose), which indecated that the immobilized glucose isomeras could be used to mitigate the inhibition caused by glucose.Both in the step reaction and cascade reaction,the contents of fructose significantly increased. But the amount of fruto-oligosaccharides was still about 50%.However, 8g sucrose was added into the mixed sugar liquid which had treated with immobilized TFase and GI for4 h successively.The fruto-oligosaccharides concentration reached 56.51%. The yield of fruto-oligosaccharides increased by 5.21%.After continuous operation of the column packed with immobilized enzyme over 28days(more than 8 h per day), 83.27% of initial enzyme activity was kept. The half-life of the immobilized enzyme at 60℃ was estimated to be 42 days. Mainly because of the higher concentration of substrate(50%) and operation temperature(60℃), no microbial contamination was found during production of FOS. In addition, the mechanical strength of the immobilized enzyme was unchanged.