CN101570770A

CN101570770A - Enzymatic-process preparation method for maltotriose glycosyl-beta-cyclodextrin

Info

Publication number: CN101570770A
Application number: CNA200910033820XA
Authority: CN
Inventors: 于博; 金征宇; 徐学明; 谢正军
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2009-05-31
Filing date: 2009-05-31
Publication date: 2009-11-04
Also published as: WO2010139100A1

Abstract

The invention provides an enzymatic-process preparation method for maltotriose glycosyl-beta-cyclodextrin, which belongs to the technical field of cyclodextrin. The method comprises the steps: taking pulullan polysaccharide and beta-cyclodextrin as substrates, utilizing the hydrolysis property of pullulanase to transform the pulullan polysaccharide into high-purity maltotriose, ensuring that the maltotriose and the beta-cyclodextrin are appropriately proportioned, utilizing the reverse synthesis capability of the pullulanase to connect the maltotriose with the beta-cyclodextrin through alpha-(1,6) glycosidic bonds and obtaining a maltotriose glycosyl-beta-cyclodextrin product. The method has the advantages of simplifying the preparation process of the maltotriose glycosyl-beta-cyclodextrin, along with mild controllable reaction conditions, high production security and low cost. The obtained product has the characteristics of high solubility and high security, and is applicable in food, medicaments, cosmetics, additives, flavors and fragrances, environmental protection, analytical detection and other fields.

Description

A kind of enzymatic-process preparation method of G 3-

Technical field

A kind of enzymatic-process preparation method of G 3-, relating to a kind of is raw material with pulullan polysaccharide and beta-cyclodextrin, by the hydrolysis properties and the reverse synthesis capability of Pullulanase, prepares the method for branching cd, belongs to the cyclodextrin technical field.

Background technology

Cyclodextrin (Cyclodextrin) is a class cyclic oligosaccharide that is formed by connecting with α-(1,4)-glycosidic link by D-glucopyranose unit.Hydrophobic outer hydrophilic tubular structure has determined it to have the characteristic of molecular capsule in the cyclodextrin, can generate inclusion compound with the chemical compound lot effect, thereby shows advantages such as slowly-releasing, solubilising, anti-oxidant, anti-illumination, thermostability increase and covering smell.More and more hot to the research of cyclodextrin in recent years, obtained suitable achievement in medicine, food, environmental protection, daily use chemicals and supramolecular chemistry field.

According to the difference of glucose unit number, cyclodextrin can be divided into α-, β-, γ-, δ-, ε-etc. cyclodextrin.More common cyclodextrin be the α that forms by 6,7 and 8 glucosyl residues respectively-, β-and γ-Huan Hujing.The beta-cyclodextrin production technique that wherein contains 7 glucosyl residues is simple, and cost is lower, is at present industrial unique energy mass production and the wider cyclodextrin product of application.But beta-cyclodextrin has the problem of water-soluble relatively poor, hemolytic and non-digestive tract security, has restricted its further application in every respect.Branching cd is by introducing a kind of strategy that glycosyl improves the beta-cyclodextrin water solubility, glycosyl modification by beta-cyclodextrin can reach the water miscible purpose of increase on the one hand, then can reduce the haemolysis effect and the non-digestive tract security of mother body cyclodextrin on the other hand.Generally by chemical method and two kinds of methods of enzyme process, with respect to the chemical method modification, it is strong that enzyme modification then has specificity, advantages such as security height in the glycosyl modification of beta-cyclodextrin.

Summary of the invention

The preparation method who the purpose of this invention is to provide a kind of branching cd, be substrate promptly with pulullan polysaccharide and beta-cyclodextrin, utilize the hydrolysis properties and the reverse Synthesis of Pullulanase, earlier pulullan polysaccharide is converted into the trisaccharide maltose of higher degree, again trisaccharide maltose is grafted on the parent beta-cyclodextrin, thereby obtains G 3-.

Technical scheme of the present invention: a kind of enzymatic-process preparation method of G 3-, with pulullan polysaccharide and beta-cyclodextrin is substrate, utilize the hydrolysis properties and the reverse synthesis capability of Pullulanase, at first pulullan polysaccharide is hydrolyzed to trisaccharide maltose, then it is grafted on the beta-cyclodextrin; Step is:

(1) with the pulullan polysaccharide is substrate, enzymatic hydrolysis α-(1,6)-glycosidic link makes its fracture, the trisaccharide maltose of preparation higher degree, control pulullan polysaccharide mass concentration is 3%～5%, the Pullulanase concentration that adds is controlled at the 100U/g pulullan polysaccharide, when 45 ℃ of pH values about 5.0, temperature of reaction, hydrolysis 8h, too high or too low pH, hydrolysis temperature and concentration of substrate can reduce the DE value of hydrolyzed solution; The boiling water bath enzyme that goes out, centrifugal remove zymoprotein after rotary evaporation be concentrated into certain volume.

(2) the adjusting trisaccharide maltose is 5: 1～6: 1 with the ratio of The quality of ss-cyclodextrin concentration, control trisaccharide maltose concentration is 1-1.2g/mL, can select the 20mL liquid glucose for use as experiment, regulating wherein, the amount of trisaccharide maltose is 20-24g, add the 4g beta-cyclodextrin again, because beta-cyclodextrin is water-soluble relatively poor, the beta-cyclodextrin in the mixed system can not dissolve fully, presents hypersaturated state.Regulating liquid glucose pH is 5.0, adds Pullulanase 200U/g beta-cyclodextrin, then at 50 ℃ of insulation reaction 48h; The boiling water bath enzyme that goes out, centrifugal remove zymoprotein after, use the 1000Da membrane filtration, to remove small molecules carbohydrate in the reaction solution such as glucose, maltose, trisaccharide maltose etc., in the solution of G 3-, add the dehydrated alcohol precipitating, the centrifugal supernatant liquor that discards, 50 ℃ of vacuum-dryings of precipitating thing obtain white foam shape solid, i.e. G 3-product.

Beneficial effect of the present invention: the branching cd that the inventive method prepares has better water solubility and medicament solubilization, and the haemolysis effect is lower than the parent beta-cyclodextrin.This method is the feedstock production G 3-with the pulullan polysaccharide, has save treating process and expense during trisaccharide maltose is produced, and the transformation efficiency of cyclodextrin reaches more than 30%, thereby greatly reduces production cost.The present invention has realized the simplification of G 3-preparation process, and the reaction conditions gentleness is controlled, production security height, lower-cost advantage; The product that it obtained has solubleness height, safe characteristics, is applicable to fields such as food, medicine, makeup, additive, essence and flavoring agent, environmental protection and analyzing and testing.

Embodiment

Embodiment 1

In the reaction system, select 3% pulullan polysaccharide solution for use, regulate pH value about 5.0, adding Pullulanase is the 100U/g pulullan polysaccharide, and the thermostatic water-circulator bath control reaction temperature is at 45 ℃, hydrolysis 8h, the boiling water bath enzyme that goes out, centrifugal remove zymoprotein after rotary evaporation be concentrated into certain volume, make high purity trisaccharide maltose slurry, be 85% through high-performance liquid chromatogram determination trisaccharide maltose content.Add beta-cyclodextrin, the adjusting trisaccharide maltose is 5: 1 with the ratio of The quality of ss-cyclodextrin concentration, regulates pH of mixed value about 5.0, and adding Pullulanase is the 200U/g beta-cyclodextrin, then at 50 ℃ of insulation reaction 48h.The boiling water bath enzyme 15min that goes out, centrifugal remove zymoprotein after, use the 1000Da membrane filtration, to remove small molecules carbohydrate in the reaction solution such as glucose, maltose, trisaccharide maltose etc.In the solution of G 3-, add the dehydrated alcohol precipitating, the centrifugal supernatant liquor that discards, behind the collecting precipitation, it is G 3-that 50 ℃ of vacuum-dryings obtain white foam shape solid.25 ℃ of its solubleness are 0.945mol/L, and 40 ℃ of its solubleness are 0.951mol/L; And 25 ℃ of beta-cyclodextrin solubleness are 0.016mol/L, and 40 ℃ of solubleness are 0.032mol/L.With the Myodigin is example, and the water solubility of G 3-and its formation mixture is 15.3 μ g/mL, and the water solubility of beta-cyclodextrin and its formation mixture is 0.94 μ g/mL.In the time of 37 ℃, G 3-causes that people's red blood corpuscle 50% hemolytic concentration is 10.52mmol/L, and the parent beta-cyclodextrin causes that people's red blood corpuscle 50% hemolytic concentration is 5.43mmol/L.

Embodiment 2

In the reaction system, select 5% pulullan polysaccharide solution for use, regulate pH value about 5.0, adding Pullulanase is the 100U/g pulullan polysaccharide, and the thermostatic water-circulator bath control reaction temperature is at 45 ℃, hydrolysis 8h, the boiling water bath enzyme that goes out, centrifugal remove zymoprotein after rotary evaporation be concentrated into certain volume, make high purity trisaccharide maltose slurry, be 83% through high-performance liquid chromatogram determination trisaccharide maltose content.Add beta-cyclodextrin, the adjusting trisaccharide maltose is 6: 1 with the ratio of The quality of ss-cyclodextrin concentration, regulates pH of mixed value about 5.0, and adding Pullulanase is the 200U/g beta-cyclodextrin, then at 50 ℃ of insulation reaction 48h.The boiling water bath enzyme 15min that goes out, centrifugal remove zymoprotein after, use the 1000Da membrane filtration, to remove small molecules carbohydrate in the reaction solution such as glucose, maltose, trisaccharide maltose etc.In the solution of G 3-, add the dehydrated alcohol precipitating, the centrifugal supernatant liquor that discards, behind the collecting precipitation, it is G 3-that 50 ℃ of vacuum-dryings obtain white foam shape solid.25 ℃ of its solubleness are 0.941mol/L, and 40 ℃ of its solubleness are 0.949mol/L.And 25 ℃ of beta-cyclodextrin solubleness are 0.016mol/L, and 40 ℃ of solubleness are 0.032mol/L.With the Myodigin is example, and the water solubility of G 3-and its formation mixture is 15.2 μ g/mL, and the water solubility of beta-cyclodextrin and its formation mixture is 0.94 μ g/mL.In the time of 37 ℃, G 3-causes that people's red blood corpuscle 50% hemolytic concentration is 10.61mmol/L, and the parent beta-cyclodextrin causes that people's red blood corpuscle 50% hemolytic concentration is 5.43mmol/L.

Claims

1, a kind of enzymatic-process preparation method of G 3-, it is characterized in that with pulullan polysaccharide and beta-cyclodextrin be substrate, utilize the hydrolysis properties and the reverse synthesis capability of Pullulanase, at first pulullan polysaccharide is hydrolyzed to trisaccharide maltose, then it is grafted on the beta-cyclodextrin; Step is:

(1) with the pulullan polysaccharide is substrate, enzymatic hydrolysis α-(1,6)-glycosidic link makes its fracture, the trisaccharide maltose of preparation higher degree, control pulullan polysaccharide mass concentration is 3%～5%, the Pullulanase concentration that adds is controlled at the 100U/g pulullan polysaccharide, when 45 ℃ of pH values 5.0, temperature of reaction, and hydrolysis 8h, the boiling water bath enzyme that goes out, centrifugal remove zymoprotein after rotary evaporation be concentrated into certain volume;

(2) the adjusting trisaccharide maltose is 5: 1～6: 1 with the ratio of The quality of ss-cyclodextrin concentration, and control trisaccharide maltose concentration is 1-1.2g/mL, and regulating liquid glucose pH is 5.0, adds Pullulanase 200U/g beta-cyclodextrin, then at 50 ℃ of insulation reaction 48h; The boiling water bath enzyme that goes out, centrifugal remove zymoprotein after, use the 1000Da membrane filtration, to remove the small molecules carbohydrate in the reaction solution, in the solution of G 3-, add the dehydrated alcohol precipitating, the centrifugal supernatant liquor that discards, 50 ℃ of vacuum-dryings of precipitating thing obtain white foam shape solid, i.e. G 3-product.