| Backgroung:Corn stigma is the dry style and stigma of corn which belongs to gramineouscrop. The resources of stigma in China are very rich while the vast majority ofthem are di--scarded, without being made full use of. The pharmacologyexperiment and clinical application shows that corn stigma containspolysaccharide composition which has great curative effect on diabetes.Therewill be a broad prospect in development and application of corn stigma asmedicinal resource if we can clear its hypoglycemic mechanism. Suppressingthe enzymes activity of alpha amylase and alpha glycosidas--e can lower theblood sugar levels in human body. So the impact to polysaccharide of alphaamylase and alpha glycosidase enzymes by corn stigma can be studied. However, the vitality of the mixture of free alpha amylase and alpha glycosidase enzymesand polysaccharide solution can not be measured. If the free enzymes areimmobilized into the immobilized enzymes by magnetic nanoparticles, theenzymes will be sucked out, through additional magnetic field, and the dynamicchanging vitality of the mixed corn stigma polysaccharide solution can bemeasured completely. There is a very wide range of applications of immobilizedenzyme in the domain of food science and it is important to strive for maximumuse of immobilized enzymes because of the high price of it.Magnetic nanoparticles have become a good carrier of immobilized enzymedue to many characteristics, including their controllable morphology and particlesize, ea--sy surface functionalization, and separation of target substances underthe action of external magnetic field, etc. Among them, non-toxic Fe3O4magnetic nanoparticle is applied most widely, which can be used for biologicaldetection, biological separation, targeted drug delivery, and so forth. Formagnetic nanoparticle immobilized enzyme, on the one hand, it can greatlyimprove the stability and repeated utilization rate of enzyme and decrease theuse-cost of enzyme; On the other hand, it can separate the substance that reactswith immobilized enzyme via external magnetic field, and can keep the enzymeas well as its substrate not to be contaminated.The α-amylase and α-glucosidase mainly act on food starch to break it downinto glucose, causing the rise of postprandial blood glucose level, so blood sugarconcen--tration can be reduced through inhibiting the activity of these twoenzymes, therein, α-glucosidase inhibitor has been applied to clinicalcommercialization as hypoglyce--mic agent. In the research on plantcompositions for decreasing blood sugar, polysa--ccharide may be one ofthe α-glucosidase inhibitors. In order to discuss the hypogly--cemic mechanism of polysaccharide in corn stigma, we chose Fe3O4magnetic nan--oparticles ascarrier, α-amylase and α-glucosidase as immobilized enzymes, to inves--tigatethe role of polysaccharide in corn stigma and the two kinds of immobilized en--zymes.The main research work was as follows:(1) to explore the optimal method of the preparation of magneticnanoparticles, and select the best immobilized enzyme particles. Two kinds ofmagnetic nanoparti--cles were prepared:(i) Fe3O4(MNP) naked magneticbeads were prepared by che--mical co-precipitation method, then they werecoated with easily modified SiO2lay--er to get SMNP, afterwards, APTS wasadded for functional modification to obtain AMNP;(ii) amine-functionalizedFe3O4magnetic nanoparticles were prepared by one-step synthesis under hightemperature and high pressure, which were then coup--led with high polymersPAA and NHS and connected with carboxyl functional grou--ps, after that,carboxyl was activated by EDC, so as to get carboxylic--functionalize--dmagnetic nanoparticles. Particle activity was studied through the combinedquanti--ty with BSA. The particle morphology and particle size werecharacterized with TE--M, and functional groups were tested with FTIR.Results showed that, two kinds of particles had uniform morphology, gooddispersity as well as superparamagnetism, and could quickly aggregate anddisperse; Amino functional groups had been conect--ed to AMNP successfully;Amino functional groups and carboxyl functional groups had been connected tocarboxylic magnetic nanoparticles. By comprehensive comp--arison, it wasfound that, the dispersity and the combined quantity with BSA, etc. ofamino-functionalized and carboxylic-functionalized magnetic nanoparticlesprepared by one-step method were superior to those of AMNP synthesized by chemical copre--cipitation method. Finally we choose the step prepared aminomagnetic nanoparticle--s and carboxyl magnetic nanoparticles as a carrier of theimmobilized enzyme.(2) With the preparation of magnetic nanoparticles immobilized alphaamylase and alpha glycosidase enzymes immobilization, choosing the bestimmobilized enzy--me to effect the stigma of polysaccharide solution.Weselected amine-functionalized magnetic nanoparticles andcarboxyl-functionalized magnetic nanoparticles to imm--obilize α-amylaseand α-glucosidase of different sources and different specific activity（represents the purity of enzyme）,and discussed the influence of the purity offree enzymes on immobilization, as well as optimum pH and operationalstability of immobilized enzymes. Results displayed that, neither of unpurifiedand low specific activity α-amylase and α-glucosidase were immobilized on thetwo kinds of particles, while both purified and high specific activity α-amylaseand α-glucosidase were im--mobilized on the two kinds of particles successfully;The pH stability and operatio--nal stability (repeated utilization rate) ofimmobilized enzymes were superior to th--ose of free enzymes; And carboxylic magnetic nanoparticles had better affect.So we think that the purity of enzyme is one of the factors, which affects the rateof immobi--lized enzyme, ultimately we chose carboxyl-functionalizedmagnetic nanoparticles immobilized enzyme to immobilize the corn stigmapolysaccharide solution.(3) To make sure the immobilized enzyme can be sucked out after the effectof corn stigma polysaccharide solution, and to determine the change of enzymeactivity in order to speculate the hypoglycemic mechanism of corn stigmapolysaccharide, whether including the alpha amylase and alpha glycosidase enzyme inhibition or not. Carboxyl-functionalized magnetic nanoparticleimmobilized enzymes were selected to react with solution of polysaccharidefrom corn stigma for a certain time,with dei--onized water as contrast.Then the activities of enzymes over time were checkedrespectively, and results indicated that the activity of α-amylase presented atrend of gradual decline as action time extended, and disappeared after8hours;The activity of α-glucosidase also had a trend of gradual decline, and alldisappeared after12ho--urs; However, the activity of immobilized enzymesthat reacted with deionized wa--ter did not change basically, indicating thatthere were inhibitors lowering enzyme activity in polysaccharide from cornstigma, and further prompting that polysacchar--ide from corn stigma mayregulate blood sugar level by inhibiting α-amylase and α-glucosidase. |
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