| The structure of cyclodextrin(CD)molecule is a truncated cone-shape with a hydrophile exterior and hydrophobic cavity,which could form inclusion complexes with the hydrophobic molecule,and the properties of inclusion complex formed have changed in physical,chemical and biological activity.Based on this,in this paper,three kinds of stevioside hydrolysate inclusion complexes were prepared and characterized,and the properties of inclusion complexes were further explored.Firstly,the steviolbioside(Sbio),steviol and isosteviol(Ist)were prepared with stevioside(Stv)as raw material from stevia leaf by enzymatic method and chemical hydrolysis.Secondly,a series of inclusion complexes made by CD and stevioside derivatives were characterized,and the possible binding mode of stevioside derivatives with ?-CD was identified using Auto Dock 4.2 software.Finally,the properties of inclusion complexes were studied,such as,antioxidant activity,stability and so on.The specific contents and main conclusions are as follows:1 The stevioside(Stv)was hydrolyzed by Na OH,Fe3+ and ?-glucosidase from Penicillium multicolor,respectively,and effects of temperature,dosage of catalyst,concentration of substrate and time on the reaction were investigated.When the experiment about steviolbioside prepared with Stv hydrolyzed by Na OH at the experimental conditions,the Stv conversion rate reached 97.8% and the yield of steviolbioside was 91.8%.Under the optimum conditions of Stv hydrolyzed by Fe3+ to prepare isosteviol for 8h,the Stv conversion reached 99.9% and the yield of isosteviol was 83.2%.the experiment of Stv hydrolyzed by the ?-glucosidase from Penicillium multicolor to prepare steviol reacted for 8 h at the optimum conditions,the Stv conversion reached 95.2% and the yield of steviol and steviolbioside was 56.4% and 36.8%,respectively.The hydrolysis pathway was explored,the ester keys of C19 position of Stv was superior to break compared with glycosidic linkage at the position of C13,and did not break the ?-1,2 glycosidic linkage of sophorose at the position of the C13.2 Under the experimental conditions,the inclusion effect of ?-CD is the best,which makes the solubility of steviolbioside,steviol and isosteviol increased by 24 times,95 times and 75 times,respectively.The phase solubility curves of the stevioside hydrolysate and ?-CD belong to the typical AL type(inclusion ratio is 1:1).Thermodynamic study of inclusion process showed that,the process of inclusion between steviolbioside and ?-CD is exoergic,spontaneous and entropy was increased.,While the process of steviol and ?-CD is endothermic,spontaneous and positive entropy,and inclusion of isosteviol and ?-CD is the same as steviol.Inclusion rate of inclusion complexes between ?-CD and steviolbioside,steviol,isosteviol is 90.5%,94.6% and 97.8%,respectively.The formation of inclusion complexes were confirmed by HPLC,IR,TGA,XRD,SEM and NMR.3 Doing molecular docking between cyclodextrin and stevioside derivatives(steviol,isosteviol and steviolbioside)by Auto Dock 4.2 Software,and the lowest binding energy conformation was obtained.The guest molecule wrapped inside of the ?-CD cavity.The carboxyl group at the C19 position of the steviol was close to the narrow opening of the ?-CD cavity,and the hydroxyl group at the C13 position of the steviol was close to the wide opening of ?-CD cavity.The carboxyl group at the C19-position of the isosteviol is near the narrow opening of the ?-CD cavity,while the two hydrophilic glucosyl group at the C13 position of the isosteviol is near the wide opening of the ?-CD cavity.The hydrophobic aglycone of the steviolbioside is completely encapsulated inside the cavity of the ?-CD and the hydrophilic sophorosyl at the C13-position are outside the ?-CD cavity and near the wide opening of the ?-CD cavity.The carboxyl group at the C19-position of steviolbioside is close to the narrow opening of the ?-CD cavity.4 Finally,the related properties and influencing factors of the inclusion complex were investigated.The results of stability experiment showed that,the stability of the inclusion complex at a concentration of 5-25 mg/m L at room temperature: Steviol/?-CD(1:3.5)> Steviol /?-CD(1:2.5),Ist/?-CD(1:3)> Ist/?-CD(1:2),Sbio/?-CD(1:2)> Sbio/?-CD(1:1.5).Sbio/ y-CD(1:1.5)was unstable with the increase of concentration and the elongation of time and Sbio / ?-CD(1: 1.5)at 25 mg/m L precipitated 19.8% after 30 days.The effect of p H on the stability of inclusion complex at concentration of 10 mg/m L and 20 mg/m L was further investigated.The results showed that,the stability of Steviol/?-CD(1:2.5),Steviol/?-CD(1:3.5),Ist/?-CD(1:3)and Sbio/?-CD(1:2)was good,and Ist/?-CD(1:2)and Sbio/?-CD(1:1.5)was stable at p H 6.86 and p H 9.18,but the stability was relatively poor in the solution of p H 4.01.The results of antioxidant experiments showed that,the ability of the inclusion complex to remove superoxide anion radical(O2-)and hydroxyl radical(·OH)is close to that of ?-cyclodextrin,which is stronger than that of the guest molecule.The Addition of cyclodextrin is beneficial to improve the ability of guest molecules to remove superoxide anion radicals(O2-)and hydroxyl radical(·OH).In addition,the results showed that the rate of hydrolysis of stevioside with ?-galactosidase enzymatic form Aspergillus sp was increased after ?-CD inclusion,The time of reaction is 10 h under the optimal reaction conditions,and the conversion rate of Sbio was 99.0%,and the yield of Smono was 65.1%. |
PDF Full Text Request