Synthesis Of Shallow Porous Carriers Based On Glass Beads Cross-linking Chitosan And Preparation Of Fructooligosaccharide By Immobilized Inulinase

Compared with free enzyme,immobilized enzyme can be recycled and reused,which can meet the requirements of continuous and stable industrial production.Whether the enzyme can be successfully immobilized and the activity of the immobilized enzyme depends on the material and properties of the carriers.Nevertheless,there are three bottlenecks in the current immobilized enzyme carriers:(a)most of the microspheres with pore structure are deep pore carriers with high mass transfer resistance,making the immobilized enzymes difficult to play catalytic function and maintain high half-life and activity recovery;(b)even if the enzymes are immobilized on the surface,it is easy to fall off due to agitation,collision or friction,and the space for action is limited because of the lack of flexible arms;(c)the carriers,with low density and mechanical strength,are commonly friable under the action of stirring force,which seriously affects the life of immobilized enzyme.In this paper,firstly,micron glass beads were used as the core layer for surface chemical modification.Then chitosan,as the film-forming agent,was covered on the surface of the glass beads with glutaraldehyde as the cross-linker and nano-scale stearic acid powder as the pore-forming agent,so as to prepare shallow porous microsphere carriers based on core-shell structure formed by glass beads cross-linked chitosan for immobilizing enzymes.Secondly,lysine,as a flexible arm,was grafted on the shallow porous microsphere carriers to multiply the number of active groups of the carriers,so that the optimized synthesis conditions for preparing immobilized endo-inulinase and the enzymatic properties before and after immobilization were systematically studied.Finally,a complete set of technological process was established by using fresh Jerusalem artichoke as raw material,extracting inulin with hot water leaching,preparing fructan with sucrase,and preparing fructooligosaccharide with flexible immobilized inulinase.Furthermore,the flexible immobilized inulinase was assembled into a column reactor to explore the application of continuous hydrolysis of fructan to prepare fructooligosaccharides.The research conclusions obtained in this paper are summarized as follows:(1)The shallow porous microsphere carriers based on core-shell structure were prepared by using glass beads as the core layer,chitosan as the shell layer,glutaraldehyde as the cross-linker and nano-scale stearic acid powder as the porogen.The results of chemical composition analysis showed that the grafting rate of chitosan was about 18.30%.The results of statistical analysis showed that the thickness of chitosan coating was about 2.16 μm,and the average size of the surface shallow pores was about 0.12 μm.(2)Lysine was successfully grafted onto the surface of the shallow porous microsphere carriers as a flexible arm by using N,N-diisopropylcarbodiimide as the condensing agent.The method and optimal synthesis conditions for immobilization of inulinase on the shallow porous microsphere carriers grafted flexible arm were established.Under the optimal conditions,the activity,coupling rate and activity recovery rate of flexible immobilized inulinase were 46.29 U / mg,87.50% and 86.10%,respectively.The enzymatic properties of by direct immobilized inulinase and flexible immobilized inulinase were compared.The overall efficiency of flexible immobilized inulinase was higher than that of direct immobilized inulinase.In particular,the repeated half-life of the two is 47 times and 61 times,which is significantly improved compared to some other immobilized carriers for inulinase,showing potential practical value.(3)It was proved that invertase can specifically hydrolyze glucose residues at the reducing end of inulin,and cleave it into fructan and glucose.Under optimal conditions,the production rate of fructan is 87.72%.The method and optimal process conditions for preparing fructooligosaccharides with flexible immobilized inulinase as catalysts were established.Under the optimal conditions,the production rate of fructooligosaccharides was 92.39%,and the total yield was 10.90% of fresh Jerusalem artichoke.The degree of polymerization of fructooligosaccharides products is mainly 3-5,of which trimeric fructose accounts for 51.29%,tetrameric fructose accounts for 28.56%,and pentafructose accounts for 20.15%.In the form of a flexible immobilized inulinase column reactor,a process for the continuous hydrolysis of fructan to prepare fructooligosaccharides was developed.The continuous use half-life of the immobilized inulinase column is 73.5 h,and its efficiency is increased to 7.43 times of free inulinase.