| Cycloamyloses or large-ring cyclodextrins (CA/LR-CDs) are cyclic α-1,4-glucanscomposed of more than nine glucopyranose units, which are prepared mainly from amylosewith the action of4-α-glucanotransferase. Cycloamyloses have many unique properties,which are very different from that of so-called small-ring cyclodextrins, due to their largercavities. In this paper, the4αGTase was prepared by separation and purification from clonedE.coliDH5α containing the gene of thermos scotoductus4αGTase, and then generated CAwith the extractive amylose as substrate. Further, the inclusion interaction between CA andgenistin, natamycin and the inhibition of CA against starch retrogration were studied in orderto provide theory and experiment gist for development and application of CA.The main content and results were as follow:(1) Optimization of the preparation and identification of CA. The recombinant E.coliDH5α was selected in LB medium containing ampicillin, finally the4αGTase was obtained byliquid fermentation, separation and purification. Plackett-Burman design was used to selectreaction conditions for CA preparation by the action of4αGTase on extractive amylose andthe optimal factors were conducted by a response surface. SDS-PAGE results showed that the4αGTase had one band with the molecular weight of57KDa, the enzymatic activity reached21.58U/mL. Enzyme, reaction temperature and time were three significant factors. Theoptimal conditions were enzyme100U/g, reaction temperature75℃for9.0h. On theseconditions, the yield of CA was35.2%by validation test. Identification results showed thatthe degree of polymerization was ranged from18to43，the ratio of α-1,4-linked cyclicglucans component was78.5%.(2) The inclusion interaction of genistin with CA was studied. The enhancing effect ofCA on the solubility of genistin is investigated by the phase-solubility diagram. Isothermaltitration calorimetry was used to study their interactions, the stability constant andthermodynamic parameters in the complexation. Moreover, the properties of the inclusioncompound were investigated by using the UV-Visible spectra, fourier transform infraredspectroscopy (FTIR) absorption spectra and differential scanning calorimeter. The resultsshowed that the solubility of genistin increase in aqueous solutions with added CA and the phase solubility diagram exhibited positive deviation from linearity with the increase in CAconcentration, showing type Ap diagram. The curve obtained for the titration was analyzed bya model assuming1:2complex formation with sequential binding sites. The binding constantsK1and K2were721L·mol-1,35.2×103L·mol-1, respectively. Analysis of the thermodynamicdates predicted the interaction power of inclusion complexation. The formation of theinclusion complex was confirmed by UV-Visible, FTIR spectroscopy and DSC.(3) The inclusion interaction of natamycin with CA was studied. The enhancing effect ofCA on the solubility of natamycin is investigated by the phase-solubility diagram.Thermodynamic parameters in the complexation were obtained. The formation of theinclusion compound was confirmed by UV-Visible spectra, FTIR absorption spectra and DSC.The results showed that the solubility of natamycin increase in aqueous solutions with addedCA and the phase solubility diagram exhibited negative deviation from linearity with theincrease in CA concentration, showing type ANdiagram. Analysis of the thermodynamic datespreliminarily inquired into the main driving force of the inclusion complexation. Theformation of the inclusion complex was confirmed by UV-Visible, FTIR spectroscopy andDSC.(4) The inhibition effect of CA on retrogradation of corn starch was studied. The impactof different concentrations of CA on transmittance of starch solution, freeze-thaw stability andretrogradation were investigated. The influence of CA on retrogradation of corn starch wasinvestigated by texture profile analysis, DSC, and X-ray diffraction (XRD). Results showedthat the CA could reduce the transparency of starch paste, significantly increase thefreeze-thaw stability and inhibit retrogradation of starch paste. The presence of CA at the levelof2.0%improved the textural properties. Further anaysis indicated the addition of CAtransformed the nucleation type due to a potential amylose-lipid-CA complex formation.Further, CA changed the crystalline type and reduced the relative crystallinity value ofretrogration. |
PDF Full Text Request