Synthesis, separation, and purification of glucosyl-β-cyclodextrin by one-pot method

Glucosyl-β-cyclodextrin (G₁ -β-CD) has wide application prospects in the food, pharmaceutical, agriculture, and chemical industry as its better solubility and biocompatibility than parent β-cyclodextrin (β-CD). However, researches on G₁ -β-CD were few due to difficulties in its preparation and separation. In the present study, a one-pot method was as follows: first, maltosyl-β-cyclodextrin (G₂ -β-CD) was prepared through the reverse synthesis capability of pullulanase. Then, G₁ -β-CD was obtained through hydrolysis by glucoamylase in the same reaction system and further purified by gel filtration chromatography. The results revealed that the optimal conditions for the reverse synthesis were as follows: n_G2 :n_β-CD , 8:1; enzyme concentration, 60 U/ml; reaction temperature, 60°C; pH, 4.5; reaction time, 60 hr. The optimal conditions for G₂ -β-CD hydrolysis were as follows: reaction time, 8 hr; enzyme concentration, 40 U/ml. Finally, the pure G₁ -β-CD samples were obtained by Sephadex G-25 gel column separation and identified by HPLC, LC-MS/MS, and NMR chromatography. PRACTICAL APPLICATIONS: Commercially available modified CDs are almost synthesized using the chemical method, such as methyl-, sulfonylbutyl-, and hydroxypropyl-CD. However, the chemical synthesis of modified CDs has some security issue, such as organic solvent residue, which limits its application in foods. Based on the relevance references, G1-β-CD not only has the inclusion properties of the parent β-CD but also improves solubility and reduces the hemolysis effect and nephrotoxicity. However, the synthesis and separation of G1-β-CD is an obstacle to its expansion in production and application. In addition, there are few studies on G1-β-CD and few companies selling the product on the market. In this manuscript, the G1-β-CD was successfully synthesized by one-pot method and the optimal preparation and separation conditions of G1-β-CD were investigated.