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CN-122012652-A - Beta-cyclodextrin and preparation method and application thereof taking sucrose as substrate

CN122012652ACN 122012652 ACN122012652 ACN 122012652ACN-122012652-A

Abstract

The invention discloses a preparation method of beta-cyclodextrin and application thereof taking sucrose as a substrate, belonging to the technical field of cyclodextrin production, wherein sucrose is taken as a raw material, firstly sodium citrate buffer solution is added to prepare sucrose solution, the temperature and pH value of feed liquid are regulated and controlled, a sucrose glucosyltransferase mutant and a cyclodextrin glucosyltransferase mutant are added to react, after the reaction is finished, the feed liquid is transferred and heated, and then high-temperature resistant amylase is added to continuously stir, then active carbon is used for decoloring and filtering the feed liquid, n-hexane is added to the filtrate and crystallized under the condition of room temperature, finally n-hexane is removed by evaporation, oligosaccharide is removed by filtration, and the beta-cyclodextrin is prepared by vacuum drying.

Inventors

  • LIN BO
  • LIN QIQI
  • WU WEIBIN

Assignees

  • 广东筑美生物医疗科技有限公司

Dates

Publication Date
20260512
Application Date
20241108

Claims (10)

  1. 1. A method for preparing beta-cyclodextrin by taking sucrose as a substrate, which is characterized by comprising the following steps: (1) Taking sucrose as a raw material, adding sodium citrate buffer solution for modulation to obtain sucrose solution, and regulating and controlling the temperature and the pH value of feed liquid after stirring; (2) Adding sucrose glucosyltransferase mutant and cyclodextrin glucosyltransferase mutant into the sucrose solution prepared in the step (1) for reaction, transferring and heating the feed liquid after the reaction is finished, and adding the lycan-resistant high-temperature amylase in a volume ratio of 300:1 for continuous stirring; (3) Adding active carbon into the feed liquid, fully stirring, decoloring and filtering, adding normal hexane into the filtrate, and crystallizing at room temperature; (4) Removing n-hexane by evaporation, filtering to remove oligosaccharide, and vacuum drying to obtain the beta-cyclodextrin.
  2. 2. The method for preparing beta-cyclodextrin by using sucrose as a substrate according to claim 1, wherein the concentration of the sodium citrate buffer solution in the step (1) is 0.1M, the final concentration of the prepared sucrose solution is 250g/L, and the stirring condition is that stirring is carried out at 350rpm until the temperature of the feed liquid is 45 ℃.
  3. 3. The method for preparing beta-cyclodextrin by using sucrose as a substrate according to claim 1, wherein the temperature of the material liquid is controlled to be maintained at 45 ℃ in the step (1), and the pH value is adjusted to be 6.5-7.5.
  4. 4. The method for preparing beta-cyclodextrin by using sucrose as a substrate according to claim 3, wherein the adding volume ratio of the sucrose solution to the sucrose glucosyltransferase mutant and the cyclodextrin glucosyltransferase mutant in the step (2) is 15:1-25:1, the reaction time is 2-3h, the heating condition is heating at 90 ℃ for 15min, and the stirring condition is stirring at 90 ℃ for 12-15h.
  5. 5. The method for preparing beta-cyclodextrin by using sucrose as a substrate according to claim 4, wherein the mutant sucrose glucosyltransferase is obtained by mutating alanine at 179 th position of wild type sucrose glucosyltransferase into lysine, the amino acid sequence is shown as SEQ ID No. 1, and the nucleotide sequence is shown as SEQ ID No. 2.
  6. 6. The method for preparing beta-cyclodextrin by using sucrose as a substrate according to claim 4, wherein the mutant cyclodextrin glucosyltransferase is obtained by mutating tyrosine at 328 rd position of wild cyclodextrin glucosyltransferase into tryptophan, the amino acid sequence is shown as SEQ ID No. 3, and the nucleotide sequence is shown as SEQ ID No. 4.
  7. 7. The method for preparing beta-cyclodextrin by using sucrose as a substrate according to claim 3, wherein in the step (3), the mass of the activated carbon is 0.2% of the mass of the liquid material, the stirring condition is stirring for 1h at 60 ℃, and the volume ratio of the liquid material to the n-hexane is 1:1.5.
  8. 8. The method for preparing beta-cyclodextrin by using sucrose as a substrate according to claim 3, wherein the evaporation condition in the step (4) is that the evaporation is carried out for 2-6 hours at a temperature of 60 ℃ at a rotation speed of 100rpm and a vacuum degree of 335mbar, and the vacuum drying condition is that the evaporation is carried out for 24 hours at a temperature of 50 ℃.
  9. 9. A beta-cyclodextrin prepared according to the method of any one of claims 1-8.
  10. 10. Use of a beta-cyclodextrin prepared according to the method of any one of claims 1-8 for preparing a food, pharmaceutical or cosmetic product.

Description

Beta-cyclodextrin and preparation method and application thereof taking sucrose as substrate Technical Field The invention belongs to the technical field of cyclodextrin production, and particularly relates to beta-cyclodextrin and a preparation method and application thereof taking sucrose as a substrate. Background Cyclodextrin (CD) is a cyclic oligosaccharide formed by the linkage of D-glucopyranose through an alpha-1, 4-glycosidic bond, and generally contains 6-12 glucosyl units. Common cyclodextrins include α -cyclodextrin, β -cyclodextrin and γ -cyclodextrin, consisting of 6, 7 and 8 glucose units, respectively. The glucose unit in the cyclodextrin molecular structure is in a chair-shaped conformation and cannot rotate at will, so that the cyclodextrin molecular structure is in a conical annular molecular shape and is a hollow cylinder. The cyclodextrin cavity is lipophilic and the external structure is hydrophilic, which enables CDs to contain guest hydrophobic substances or groups that are suitable for their size and shape, thereby altering the physical and chemical properties of the guest molecule, enhancing its solubility and increasing its stability, to enhance its quality of use. Beta-cyclodextrin (beta-cyclodextrin, beta-CD) is a cyclic polymer formed by connecting 7 glucose units through alpha-1, 4-glycosidic bonds, has a hollow cylindrical three-dimensional structure with a hydrophobic inner cavity and a hydrophilic outer part, can accommodate hydrophobic guest molecules with the shape and the size, has the characteristics of no toxicity or extremely low toxicity, safety and innocuity, is a substance allowed to be added by a food additive application standard (GB 2760-2014), can be used for improving the defects of easy volatilization and poor stability of hydrophobic guest molecules such as organic acid, fatty acid, aromatic, polar compound and the like, is widely applied in the fields of foods, materials, medicines and the like, and has high economic value. Currently, the industrial production of cyclodextrin is all prepared by an enzymatic method, namely starch is used as a raw material, and cyclodextrin is generated under the catalysis of cyclodextrin glycosyltransferase (Cyclomaltodextrin Glucanotransferase, CGTase). Since the solubility of β -CD in water is minimal compared to the other two cyclodextrins, it can be easily obtained by stepwise crystallization. However, α -CD and γ -CD are currently industrially difficult to separate and purify, so β -CD is the most widely used, accounting for more than 90% of cyclodextrin production. Industrial production of beta-cyclodextrin is generally achieved using beta-cyclodextrin glucosyltransferase (beta-Cyclodextringlycosyltransferase, beta-cgtase) to act on starch. beta-CGTase is a member of the alpha-amylase 13 family (GH 13) and can catalyze starch to generate various products, such as cyclodextrin, oligosaccharide and various dextrin substances by utilizing hydrolysis, disproportionation, cyclization and coupling functions. However, CGTase is an exoenzyme which starts to degrade from the non-reducing end of starch molecules and cannot cross the branching point (alpha-1, 6 glycosidic bond), and starch substrates usually contain 75% -80% of amylopectin, which greatly reduces the utilization rate of starch substrates and the production efficiency of beta-cyclodextrin in the cyclodextrin production process. The Chinese patent with publication number of CN104762345A and application number of 2015 and 3 month and 19 discloses a preparation process of beta-cyclodextrin, pullulanase or isoamylase is used for carrying out synergistic reaction with CGTase to produce beta-cyclodextrin, but pH is required to be regulated, pretreatment and enzyme reaction process are higher in temperature, enzyme reaction time is longer, energy consumption and improvement efficiency of beta-cyclodextrin production are not reduced, the Chinese patent with publication number of CN116656759A and application number of 2023 and 5 month and 25 provides a method for preparing beta-cyclodextrin, dextrin debranching enzyme is used for hydrolyzing alpha-1, 6 glycosidic bonds in the cyclization reaction process, limitation of branch points on beta-CGTase is relieved, beta-cyclodextrin preparation efficiency is improved, but starch still needs to be pretreated firstly to reduce the molecular weight of the starch due to larger molecular weight, and the method increases production cost and process complexity. When combining the current preparation of beta-cyclodextrin using starch as a substrate, there are disadvantages in that starch as a polysaccharide has a variable degree of polymerization, a wide molecular weight distribution, may affect the uniformity of the final product, and necessary pretreatment (e.g., enzymolysis) may increase costs, etc. There is a need to develop a method for preparing beta-cyclodextrin with relatively small molecules, definite and consistent molecular s