Study On Preparation Of Difructose Anhydride Ⅲ By Inulin Fructotransferase-Membrane Coupled With Reactor

Difructose anhydride III (DFA III) is a novel low-caloric sweetener substitute which can promotemineral absorption and diuretic action, inhibit cancer and prevent tooth decay. It can be used in thefields of bakery, candy, beverage, medicine and other related industries. The traditional method of itspreparation used to hydrolyze inulin with inulin fructotransferase (IFTase) in enzyme reactor (ER). Theinulin hydrolyzate was directly concentrated, decolorized for DFA III preparation. The above-mentionedprocess was time-consuming and labor intensity. Moreover, because IFTase was not able to separatefrom the inulin hydrolyzate, IFTase was not recycled and wasted. Enzyme reaction could be inhibited bythe product. The yield of DFA III was lower as well. It required high temperatures or regulated pH toinactivate enzyme at the end of reaction. The traditional process made operations complicated and muchenergy consumed. In order to solve these problems, IFTase was obtained by Arthrobacter aurescensSK8.001through fermentation, concentration, salt precipitation and freeze drying in our previous work.On the basis of the previous work, we firstly explored to design IFTase-ultrafiltration (UF) membranecoupled with reactor (enzyme membrane reactor i.e. EMR), which was used to hydrolyze inulin forDFA III preparation. Secondly, DFA III inhibition effects of enzyme reaction were studied. Lastly,enzyme membrane reactor coupled with nanofiltration (NF) membrane was designed, which wasdeveloped to separate enzyme and to concentrate DFA III solution. The main contents are as follows:Arthrobacter aurescens SK8.001was cultured in medium fermentation tank (30L) for72h. IFTaseactivity was17.5U/mL in the enzyme solution. IFTase was concentrated by UF, which the yield was85-93%and the loss rate was10%. IFTase activity was increased from17.5U/mL to154.6U/mL,which was raised8.5times. IFTase specific activity was increased from4.4U/mg to15.6U/mg.Permeat flux was reduced from11.2L/m2h to3.2L/m2h with the increase of permeate volume. Throughregeneration, membrane flux could be recovered to over95%of original level.126.4g wet IFTaseprecipitations were obtained by concentration, precipitation and centrifugation.10.2g enzyme powderswere obtained by freeze drying. In order to overcome the defects of DFA III preparation with ER, EMR can be used as thesubstituted method for the conventional one. Because the molecular weights were much differentbetween IFTase and DFA III, UF membrane was selected and used to design EMR. The batch operationsof EMR were studied to hydrolyze inulin for DFA III production. The results showed that the molecularweight cut-off (MWCO) of5kDa UF membrane was selected, inulin concentration as the reactionsubstrate was100g/L, and DFA III concentration of product was78.4g/L. Compared with thetraditional ER, when the initial total enzyme activity was340U, IFTase could be recycled for6times.DFA III productivity was raised5.5times, and the purity was increased from80%to92%. Permeateflux was decreased from14.4L/m2h to4.8L/m2h with the permeate volume increasing. Throughregeneration, UF membrane flux could be recovered to over95%of original level.According to the batch operation results of EMR, the continuous operations of EMR were carriedout to study inulin hydrolyzate for DFA III production. The optimal continuous process conditions ofEMR were obtained: reaction volume for100mL, inulin concentration for100g/L, enzyme to substrateratio (E/S) for30U/g, temperature for60-70°C, pH5.0-7.0, stirring rate for200r/pm and hydrolysistime1h; TMP for20Psi, temperature for25°C, stirring rate for300r/pm. Compared with the ER,IFTase could be recycled, and enzyme retention rate was84%. DFA III productivity was raised for5times and purity was increased from80%to92%. The operation stability experiment showed that theEMR system could maintain the steady production of DFA III for over15h.Enzyme and product were separated from reaction liquid by EMR, which could reduce or eliminate the product inhibition effects of the enzyme reaction. DFA III inhibition type and inhibition constant ofIFTase were studied, as well as the continuous operation stability of EMR. The results were Km=3.04g/L, Vmax=0.82g/L/min, KI=1.02g/L; it indicated that DFA III was competitive inhibiter of IFTase.Inulin conversion was about71.6%at the initial operation period of1.5day, which was about47.8%in7day. The half-life (t1/2) of EMR was about8.2day. It showed that EMR had long operation stability.This study attempted to amplify EMR and determine the optimal process conditions resulting fromthe above-mentioned EMR experiments. DFA III concentration obtained by EMR was low, so weattempted to design EMR coupled with NF membrane for inulin hydrolyzate to prepare high DFA IIIconcentration. Firstly, the suitable NF membrane was selected and the NF operation conditions weredetermined. Secondly, the process of EMR coupled with NFmembrane was developed to separateenzyme and to concentrate product. The optimal operation conditions of EMR coupled with NFmembrane were obtained: reaction solution volume for5L, E/S for22.8U/g, inulin solution for100g/L,pH5.5, temperature for60oC, stirring rate for300r/pm, reaction time2h; feed volume from reactor for5L, UF membrane MWCO for5kDa, TMP for10Psi, stirring rate for400r/pm, temperature for25oC;feed volume from the permeate of UF process for5L, NF membrane MWCO for150Da, TMP for15Psi, stirring rate for300r/pm, temperature for25oC. The results showed that DFA III concentration wasincreased from76g/L to400g/L, DFA III yield was73%. Compared with the conventional method, thisapproach could simultaneously recycle enzyme and water, obtain the high concentration product, saveenergy and eliminate pollution. The outcomes could be useful in EMR coupled with NF membrane forcommercial utilization of high concentration of DFA III preparation.