Structure And Characterization Of Branching Enzyme-Modified Waxy Rice Starch

The modified starch,which has a wide range of applications in food industry,can be prepared by physical,chemical and enzymatic treatment.After modification,the disadvantages of native starch such as insolubility in cold water,high tendency towards retrogradation after cooking and condensation at low temperature can be overcome.Compared with different modification methods,enzymatic treatment has many advantages,it has high reaction efficiency,mild reaction conditions and the enviromental friendly product,which plays a key role in starch modification process.Branching enzyme as the glucan transferases,can be used in above modification,which is responsible for catalyzing transglycosylation reaction to break?-1,4-glucoside and form?-1,6 branch points.This study focused on enzymatic modification of waxy rice starch with branching enzyme,then structural analysis,properties evaluation and its application in nutrient encapsulation were carried out.Firstly,the gene segment of branching enzyme from Bacillus stearothermophilus was acquired by genetic method,then the segment was sub-cloned into pET-22b?+?vector to obtain the plasmid.The recombinant plasmid is transformed into E.coli BL21?DE3?and branching enzyme was expressed by IPTG induction.The relative molecular weight of enzyme protein is 70 kDa,the maximum activity of the recombinant enzyme was at 75? and pH 7.0.It was stable in a high temperature as well as at pH 6.0⁹.5.Secondly,the waxy rice starch was modified by branching enzyme and structural characteristics of the modified starch were investigated.After enzymatic modification,the weight-average molecular weight?M_w?and dispersibility of waxy rice starch were both decreased.Moreover,it showed that the number of A chains?DP?13?in substrate increased,and B chains?DP>13?content decreased during the process of modification.It can be speculated that branching enzyme has a preference to cleave the long chains,and released chains with DP?13 for subsequent reaction.Furthermore,the?-1,6-linkage ratio increased from 5.0%to 6.6%and cyclic structure with DP 19³4 was formed after reactiont.The solubility,viscosity and digestibility of the modified starch were analyzed,it turned out that the enzyme-modified product has a high solubility with 83.3 g/100 mL at 25?,and the viscosity is becoming much lower than waxy rice starch.As far as the extent of digestibility detected by the in vitro Englyst assay,the slow digested starch?SDS?content changed from 8.5% to 21.0% and resistant starch?RS?increased from 6.9%to 18.5%due to the branched cyclic structure formed by branching enzyme.Thirdly,the detail conformation of the branched cyclic feature structure was investigated.It showed that the branched cyclic structure was consisted of twenty one glucose units linked by?-1,4-and?-1,6-glucosidic linkage.One point sixe?-1,6-linkages were directly involved in the formation of the cycilc structure with several non-cyclic side glucose units attached to the ring.The property of the branched cyclic structure by using Discovery Studio 3.5?DS 3.5?,UV-Vis spectra and Isothermal titration calorimetry?ITC?,the results indicated that the branched cyclic strucure contain partially hydrophobic cavity,which can be encapsulated with hydrophobic substances.Finally,the enzyme-modified starch was utilized for?-carotene inclusion complex preparation.The property study of inclusion complex showed that the solubility of?-carotene reached 179.1?g/mL,the corresponding load rate and amount of?-carotene was 1.3%and8.9?g/mg respectively in the inclusion complex with a mass ratio of 1:5.Fourier transform infrared spectrometer?FT-IR?,X-ray diffraction?XRD?,Thermogravimetric analyzer?TGA?and Differential scanning calorimeter?DSC?were used for inclusion complex characterization.Compared with crystal?-carotene,the stability of the encapsulated?-carotene increased greatly.After being lighted at room temperature for sixe weeks,the retention rate of?-carotene in complex is more than twice than crystal?-carotene.Meanwhile,after 20minutes of oxidation by FeCl₃ solution,the degradation in inclusion complex dispersion was about 20%and remained constant afterward,whereas the control showed a continuous degradation during the oxidation period.Therefore the enzyme-modified starch plays a key role in nutrients protection.