Preparation and Application of a Solubilizing α-1,6 / α-1,4 Chimeric Oligosaccharide

Abstract

The present invention provides a method for enzymatically synthesizing and purifying α-1,6 / α-1,4 chimeric oligosaccharides, comprising: synthesizing isomaltooligosaccharides using sucrose as a substrate, generating α-1,6 / α-1,4 chimeric oligosaccharides in a glycosylation reaction with γ-cyclodextrin glucanotransferase using γ-cyclodextrin as a donor, and then obtaining α-1,6 / α-1,4 chimeric oligosaccharide products by means of cyclododecanone complexation and low-temperature precipitation, exo-dextranase hydrolysis, dialysis, and freeze-drying. Further disclosed is a method for rapidly determining the degree of polymerization of α-1,6 / α-1,4 chimeric oligosaccharides by hydrolyzing with β-amylase. The chimeric oligosaccharides obtained by the present invention can be used as host molecules to increase the solubility of resveratrol in water.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation, and relates to a method for preparing and purifying an α-1,6 / α-1,4 chimeric macrosaccharide, a method for simply measuring the average chain length, and applications thereof. Background Art

[0002] Resveratrol, a representative stilbene, has a variety of physiological functions. However, due to its poor water solubility, these active functions are poorly exerted in humans or animals. A single-molecule dispersion system composed of a drug carrier represented by cyclodextrin can improve the bioavailability of hydrophobic guest molecules. However, the size of the guest molecules that cyclodextrin can encapsulate is limited by the size of the rigid cavity of cyclodextrin.

[0003] α-Glucans with a degree of polymerization between 11 and 100 are defined as macrosaccharides. Isomaltooligosaccharide obtained by the action of dextran dextrinase or 4,6-α-glycosyltransferase on maltodextrin is a linear non-branched chimeric sugar composed of a non-reducing end α-1,6 sugar chain and a reducing end α-1,4 sugar chain, and shows application prospects in aspects such as solubilization, reduction of the penetration of pro-inflammatory substances, and immune response. However, the shorter α-1,4 sugar chain at the reducing end loses its original advantage of having an amphiphilic helical cavity formed by continuous α-1,4 bonds in neutral and acidic aqueous solutions due to the special bond angle.

[0004] Therefore, constructing a linear non-branched chimeric sugar composed of a non-reducing end α-1,4 sugar chain and a reducing end α-1,6 sugar chain can retain the amphiphilic helical cavity of the α-1,4 sugar chain while providing good water solubility by continuous α-1,6 glycosidic bonds. Using such a chimeric sugar as a carrier can achieve the single-molecule dispersion of hydrophobic guest molecules in water.

[0005] Enzymatic synthesis of dextran has the advantages of low energy efficiency and being controllable. The glucosucrase mutant L940W from Lactobacillus reuteri 180 can use sucrose as a substrate to synthesize oligosaccharides with only α-1,6 glycosidic bonds. γ-Cyclodextrin glucosyltransferase can use cyclodextrin as a donor to transfer glucose to the receptor substrate with an α-1,4 glycosidic bond to achieve the extension of maltooligosaccharide chains on the receptor substrate.

[0006] How to obtain α-1,6 / α-1,4 chimeric macrosaccharides is of great significance for the development of polysaccharides and the design of single-molecule dispersion carriers. Summary of the Invention

[0007] The inventors constructed a method for preparing α-1,6 / α-1,4 chimeric macrosaccharides by a multi-enzyme cascade reaction, provided a separation and purification method for removing the remaining cyclodextrin and isomaltooligosaccharides after the reaction, provided a method for simply measuring the average chain length of the chimeric macrosaccharides, and also verified the application of the chimeric macrosaccharides in solubilizing hydrophobic guest molecules.

[0008] The first object of the present invention is to provide a method for enzymatically preparing α-1,6 / α-1,4 chimeric oligosaccharides.

[0009] The second object of the present invention is to provide a method for separating and purifying α-1,6 / α-1,4 chimeric oligosaccharides.

[0010] The third object of the present invention is to provide a method for rapidly determining the average chain length by enzymatically degrading α-1,6 / α-1,4 chimeric oligosaccharides.

[0011] The fourth object of the present invention is to provide a single-molecule dispersion system carrier capable of solubilizing hydrophobic guest molecules.

[0012] The present invention provides a method for enzymatically synthesizing α-1,6 / α-1,4 chimeric oligosaccharides, which consists of the following steps:

[0013] (1) The reaction system uses a sodium citrate or sodium acetate solution as a buffer, sucrose as a substrate (0.1 - 5.0 M), adds dextransucrase for reaction, and inactivates the enzyme by boiling water bath after the reaction;

[0014] (2) Centrifuge to remove denatured proteins, dialyze the supernatant, freeze-dry, grind into powder, and sieve to obtain isomaltooligosaccharides;

[0015] (3) The reaction system uses a phosphate or glycine solution as a buffer, isomaltooligosaccharides as the acceptor substrate, γ-cyclodextrin as the donor substrate (the donor-acceptor ratio is 0.5 - 5:1), adds cyclodextrin glucosyltransferase for reaction,

[0016] Inactivate the enzyme by boiling water bath to prepare α-1,6 / α-1,4 chimeric oligosaccharides.

[0017] In one embodiment of the present invention, the concentration of the sucrose is preferably 2 M.

[0018] In one embodiment of the present invention, the dextransucrase is a mutant GTF180-ΔN L940W from Lactobacillus reuteri 180, the enzyme addition amount is 0.5 - 2 U / mL, preferably 1 U / mL; the reaction temperature is 30 - 40 °C, preferably 37 °C; the reaction time is 8 - 24 h, preferably 12 h.

[0019] In one embodiment of the present invention, the concentration of sodium citrate or sodium acetate in the dextransucrase buffer is 20 - 100 mM, the pH is 4.5 - 6.0, preferably a 50 mM sodium acetate solution with a pH of 5.0 containing 1 mM calcium chloride.

[0020] In one embodiment of the present invention, the pore size of the dialysis bag is 1000 Da, and the dialysis time is 36 - 50 h, preferably 48 h.

[0021] In one embodiment of the present invention, the concentration of the receptor substrate isomaltooligosaccharide is 5-20 mg / mL, preferably 10 mg / mL.

[0022] In one embodiment of the present invention, the cyclodextrin glucosyltransferase is γ-cyclodextrin glucosyltransferase derived from Bacillus FJAT-44876, the enzyme addition amount is 0.05-0.30 U / mg isomaltooligosaccharide, preferably 0.15 U / mg isomaltooligosaccharide; the reaction temperature is 40-55 °C, preferably 50 °C; the reaction time is 0.5-6 h, preferably 2 h.

[0023] In one embodiment of the present invention, the concentration of phosphoric acid or glycine in the cyclodextrin glucosyltransferase buffer is 20-100 mM, the pH is 6.0-10.0, preferably 25 mM glycine-sodium hydroxide buffer at pH 10.0.

[0024] In one embodiment of the present invention, the donor-receptor ratio is preferably 3:1 (w / w).

[0025] The present invention provides a method for purifying α-1,6 / α-1,4 chimeric oligosaccharides, which consists of the following steps:

[0026] (1) The pH of the above reaction system is adjusted to 5.0-7.0 with an acid solution, and after cooling, the denatured protein is removed by centrifugation;

[0027] (2) The supernatant is added with exo-dextranase and reacted for 10-20 h, inactivated by boiling water bath, and after cooling, the denatured protein is removed by centrifugation;

[0028] (3) The supernatant is heated to 70-85 °C, then 2-6% (w / v) cyclododecanone is added, shaken for 0.5-2 h, and cooled to 60-70 °C

[0029] Then continue to shake for 8-12 h, cool to 4-8 °C and continue for 10-20 h.

[0030] (4) The supernatant is aspirated and passed through a 0.45 μm organic filter membrane to separate from the insoluble matter in the solution.

[0031] (5) The filtrate is dialyzed and then freeze-dried and ground into powder to obtain pure α-1,6 / α-1,4 chimeric oligosaccharides.

[0032] In one embodiment of the present invention, the acid solution is hydrochloric acid or glycine solution, and preferably the pH end point is adjusted to 6.0.

[0033] In one embodiment of the present invention, the exo-dextranase is glucan1,6-alpha-glucosidase DexB from Streptococcus mutans, and the enzyme dosage is 0.5 - 2 U / mL, preferably 1 U / mL; the reaction temperature is 30 - 37 °C, preferably 35 °C; the reaction time is 8 - 16 h, preferably 12 h.

[0034] In one embodiment of the present invention, the addition amount of cyclododecanone is preferably 5% (w / v), the temperature for heating the supernatant is preferably 75 °C, the temperature for maintaining is preferably 65 °C, the temperature for cooling and standing is preferably 4 °C, and the high-temperature oscillation frequency is 400 - 800 rpm, preferably 600 rpm.

[0035] In one embodiment of the present invention, the pore size of the dialysis bag is 1000 Da, and the dialysis time is 36 - 50 h, preferably 48 h.

[0036] The present invention provides a method for rapidly determining the average chain length by enzymatic degradation of α-1,6 / α-1,4 chimeric oligosaccharides. The method is as follows: The α-1,6 / α-1,4 chimeric oligosaccharides and sodium acetate buffer are configured into a reaction solution, the total sugar content z is measured by the phenol-sulfuric acid method, β-amylase is added for reaction, and the reducing sugar content h sam 、h con , the average chain length of the α-1,6 / α-1,4 chimeric oligosaccharides is DP = (DP 异麦芽低聚糖 + 1.5) / (1 - 2×(h sam - h con ) / z).

[0037] In one embodiment of the present invention, the concentration of the α-1,6 / α-1,4 chimeric oligosaccharides is 1 - 5 mg / mL, preferably 2 mg / mL.

[0038] In one embodiment of the present invention, the concentration of sodium acetate is 20 - 100 mM, preferably 50 mM; the pH is 5.0 - 7.0, preferably 6.0.

[0039] In one embodiment of the present invention, the enzyme dosage of β-amylase is 0.1 - 0.2 U / mL, preferably 0.16 U / mL; the reaction temperature is 30 - 40 °C, preferably 37 °C; the reaction time is 2 - 6 h, preferably 4 h.

[0040] The α-1,6 / α-1,4 chimeric oligosaccharides prepared by the present invention can preferably improve the solubility and dispersity of stilbene hydrophobic guest molecules represented by resveratrol in water, and have good application potential in the fields of food and medicine, etc.

[0041] Beneficial effects: The present invention designs a carrier for solubilizing hydrophobic guest molecules in a single-molecule dispersed form, an α-1,6 / α-1,4 chimeric large sugar. And a method for preparing and purifying the α-1,6 / α-1,4 chimeric large sugar is provided, and the average degree of polymerization can be rapidly determined by hydrolyzing the large sugar with β-amylase on the premise of knowing the average degree of polymerization of the known receptor isomaltooligosaccharide. A complete route from sugar preparation, separation, identification to application is established, which is of great significance for the development of sugars and the research and application of solubilizing single-molecule dispersed carriers. Description of the Drawings

[0042] Figure 1 It is a statistical chart of the degree of polymerization distribution of the intermediate product isomaltooligosaccharide of the present invention.

[0043] Figure 2 It is a one-dimensional proton nuclear magnetic resonance spectrum of the intermediate product isomaltooligosaccharide of the present invention.

[0044] Figure 3 They are high performance liquid chromatography diagrams before and after the reaction of different cyclodextrin glucanotransferases catalyzing different substrate compositions. a is the treatment of α- / γ-cyclodextrin with the commercial cyclodextrin glucanotransferase Toruzyme 3.0L, b is the treatment of α- / γ-cyclodextrin with γ-cyclodextrin glucanotransferase, c is the treatment of the mixed substrate of α- / γ-cyclodextrin and isomaltooligosaccharide with the commercial cyclodextrin glucanotransferase Toruzyme 3.0L, and d is the treatment of the mixed substrate of α- / γ-cyclodextrin and isomaltooligosaccharide with γ-cyclodextrin glucanotransferase.

[0045] Figure 4 It is a one-dimensional proton nuclear magnetic resonance spectrum of the product α-1,6 / α-1,4 chimeric large sugar of the present invention.

[0046] Figure 5 It is a schematic flow chart for preparing and purifying the α-1,6 / α-1,4 chimeric large sugar of the present invention.

[0047] Figure 6 It is a schematic structural diagram of the product α-1,6 / α-1,4 chimeric large sugar of the present invention.

[0048] Figure 7 It is a schematic diagram of the principle for rapidly determining the average degree of polymerization of the large sugar by hydrolyzing the large sugar with β-amylase in the present invention.

[0049] Figure 8 It is the phase solubility curve of the α-1,6 / α-1,4 chimeric large sugar and γ-cyclodextrin of the present invention for resveratrol. Detailed Embodiments

[0050] The present invention will be further described below in conjunction with embodiments.

[0051] Example 1

[0052] Add 2 M sucrose to 50 mM sodium acetate buffer at pH 5.0 containing 1 mM CaCl₂. After incubating at 37 °C for 20 min, add 1 U / mL dextransucrase GTF180-ΔN L940W and react with shaking at 300 rpm at 37 °C for 12 h. After the reaction is completed, terminate the reaction by heating in a boiling water bath at 100 °C for 20 min. Cool the reaction solution to room temperature and centrifuge at 12,000×g for 15 min. Take the supernatant and dialyze it in a 1 kDa dialysis bag at room temperature for 48 h, then lyophilize it, grind it into powder and sieve it to obtain a powdery isomaltooligosaccharide sample.

[0053] Dissolve isomaltooligosaccharide (2 mg / mL) in ultrapure water and centrifuge at 7000×g for 10 min at 25 °C. No precipitation is observed. For high performance anion exchange chromatograph-pulsed amperometric detection (HPAEC-PAD) analysis, equipped with a CarboPacTM PA1 chromatographic column (4×250 mm), the injection volume is 20 μL. The two mobile phases are (A) 1 mol / L NaAc solution and (B) 0.25 mol / L NaOH solution, and the flow rate is 1 mL / min. Elution gradient: 0–35 min 0%–21% A, 35–36 min 21%–0% A, 36–42 min 0% A, and 0–42 min 60% B. Using the undialyzed 0.1 M sucrose substrate dextransucrase reaction product as a reference sample, the degree of polymerization distribution of the isomaltooligosaccharide in Example 1 is obtained by peak area normalization method as Figure 1 shown.

[0054] Disperse isomaltooligosaccharide (2%, w / v) in D₂O in a boiling water bath for 1 h, then lyophilize it. Repeat the above operations twice and then dissolve it in D₂O containing sodium 3-(trimethylsilyl)propionate-d₁₀ and incubate at 60 °C. Determine the linkage composition of the isomaltooligosaccharide in Example 1 by ¹H nuclear magnetic resonance (¹H NMR) as Figure 2 shown.

[0055] Example 2

[0056] Add different concentrations of sucrose to 50 mM sodium acetate buffer at pH 5.0 containing 1 mM CaCl₂. After incubating at 37 °C for 20 min, add 1 U / mL dextransucrase GTF180-ΔN L940W and react with shaking at 300 rpm at 37 °C for 12 h. After the reaction, terminate the reaction by heating in a boiling water bath at 100 °C for 20 min. Cool the reaction solution to room temperature and centrifuge at 12000×g for 15 min. Take the supernatant, dialyze it in a 1 kDa dialysis bag at room temperature for 48 h, then lyophilize, grind it into powder and pass it through a sieve to obtain a powdery isomaltooligosaccharide sample. Dissolve isomaltooligosaccharide (5 mg / mL) in ultrapure water and centrifuge at 7000×g for 10 min at 25 °C. No precipitation is observed. Filter the sample solution through a 0.45 μm water membrane for analysis by high performance gel permeation chromatograph refractive index detector (HPGPC-RID). The chromatographic column is an UltrahydrogelTM column (7.8×300 mm), the injection volume is 30 μL, the mobile phase is 0.1 mol / L NaNO₃, and the flow rate is 0.5 mL / min. The results are shown in Table 1.

[0057] Table 1 Molecular sizes of isomaltooligosaccharides prepared at different sucrose concentrations.

[0058] Sucrose concentration <![CDATA[Mn a > <![CDATA[Mw b > <![CDATA[PDI c > 0.05M 1668 2559 1.53 0.10M 1795 2625 1.46 0.20M 2098 3063 1.46 0.50M 2033 2958 1.45 1.0M 2102 2982 1.42 2.0M 1412 1827 1.28 5.0M 1447 1878 1.30

[0059] a Number-average molecular weight

[0060] b Weight-average molecular weight

[0061] c The value of Mw / Mn characterizes the dispersity of the substance. The smaller the value, the more uniform the dispersion of the substance.

[0062] Example 3

[0063] Add 30 mg / mL γ-cyclodextrin, 10 mg / mL isomaltooligosaccharide to 25 mM glycine-sodium hydroxide buffer at pH 10.0. After incubating at 50 °C for 30 min, add 1.5 U / mL γ-cyclodextrin glucosyltransferase and react with shaking at 300 rpm at 50 °C for 2 h. After the reaction, terminate the reaction by heating in a boiling water bath at 100 °C for 40 min. After the reaction solution is cooled to room temperature, adjust the pH to 6.0 with 0.2 M glycine, transfer it to 4 °C for storage for 12 h, and then centrifuge at 12000×g at 4 °C for 15 min. After the supernatant is incubated at 35 °C for 20 min, add 1.0 U / mL exo-dextranase and react with shaking at 300 rpm at 35 °C for 12 h. After the reaction, terminate the reaction by heating in a boiling water bath at 100 °C for 20 min, and centrifuge the reaction solution at 12000×g at room temperature for 15 min. After the supernatant is heated to 80 °C, slowly add 5% (w / v) cyclododecanone and continuously shake at 600 rpm for 1 h. After cooling to 65 °C, continue to shake for 11 h. Transfer the mixed liquid to 4 °C and let it stand for 12 h, then aspirate the supernatant and filter it through a 0.45 μm organic filter membrane. The filtrate is dialyzed in a 1 kDa dialysis bag at room temperature for 48 h and then freeze-dried, ground and sieved to obtain the pure product α-1,6 / α-1,4 chimeric oligosaccharide.

[0064] The sample (2%, w / v) was fully dispersed in D2O by boiling water bath for 1 h, freeze-dried, and the above operations were repeated twice, then dissolved in D2O containing sodium 3-(trimethylsilyl)propionate-d4 and incubated at 60 °C. The bond type composition of α-1,6 / α-1,4 chimeric oligosaccharide was determined by one-dimensional nuclear magnetic resonance hydrogen spectrum (1H Nuclear magnetic resonance, 1H NMR) as Figure 4 shown.

[0065] Comparative Example 1

[0066] 30 mg / mL of γ-cyclodextrin, 30 mg / mL of α-cyclodextrin, 30 mg / mL of γ-cyclodextrin and 10 mg / mL of isomaltooligosaccharide, and 30 mg / mL of α-cyclodextrin and 10 mg / mL of isomaltooligosaccharide were respectively added to 25 mM glycine-sodium hydroxide buffer at pH 10.0. After incubation at 50 °C for 30 min, 1.5 U / mL of γ-cyclodextrin glucosyltransferase was added, and the reaction was carried out with shaking at 300 rpm at 50 °C for 2 h. After the reaction was completed, the reaction was terminated by heating in a boiling water bath at 100 °C for 40 min. 30 mg / mL of γ-cyclodextrin, 30 mg / mL of α-cyclodextrin, 30 mg / mL of γ-cyclodextrin and 10 mg / mL of isomaltooligosaccharide, and 30 mg / mL of α-cyclodextrin and 10 mg / mL of isomaltooligosaccharide were respectively added to 50 mM phosphate buffer at pH 6.0. After incubation at 60 °C for 30 min, 1.5 U / mL of commercial β-cyclodextrin glucosyltransferase (Toruzyme 3.0L) was added, and the reaction was carried out with shaking at 300 rpm at 60 °C for 2 h. The composition of cyclodextrin and malt oligosaccharide before and after the reaction in different groups was monitored by high performance liquid chromatography, and the results are as Figure 3 shown. The reaction conditions are statistically shown in Table 2 below.

[0067] Table 2 Statistics of the reaction of different cyclodextrin glucosyltransferases catalyzing different substrates

[0068]

[0069] γ-Cyclodextrin glucosyltransferase can open the ring of γ-cyclodextrin and carry out transglycosylation reaction when isomaltooligosaccharide is used as the acceptor, while commercial β-cyclodextrin glucosyltransferase (Toruzyme 3.0L) can consume cyclodextrin substrates in the presence or absence of isomaltooligosaccharide to generate two other cyclodextrins and malt oligosaccharides, which is not conducive to the monitoring of the reaction process and the separation of products.

[0070] Control Example 2

[0071] The specific implementation method refers to Example 2, the difference is that the initial content of γ-cyclodextrin and the reaction time of γ-cyclodextrin glucosyltransferase are respectively modified to 10 mg / mL and 1 h, and the other conditions remain unchanged.

[0072] Control Example 3

[0073] The specific implementation method refers to Example 2, the difference is that the initial content of γ-cyclodextrin is modified to 20 mg / mL, and the other conditions remain unchanged.

[0074] Control Example 4

[0075] For the specific implementation method, refer to Example 2, with the difference that the reaction time of γ-cyclodextrin glucosyltransferase is modified to 104 h, and the other conditions remain unchanged.

[0076] Control Example 5

[0077] For the specific implementation method, refer to Control Example 3, with the difference that the content of initial γ-cyclodextrin is modified to 40 mg / mL, and the other conditions remain unchanged.

[0078] Control Example 6

[0079] For the specific implementation method, refer to Example 2, with the difference that the content of initial γ-cyclodextrin and the reaction time are respectively modified to 50 mg / mL and 6 h, and the other conditions remain unchanged.

[0080] Example 4

[0081] The α-1,6 / α-1,4 chimeric oligosaccharide (2 mg / mL) and 50 mM sodium acetate buffer at pH 6.0 are prepared into a reaction solution as the blank group. After preheating at 37 °C for 20 min, 0.16 U / mL of β-amylase is added, and the reaction is carried out with shaking at 37 °C and 300 rpm for 4 h as the experimental group. The total sugar content z is determined by the phenol-sulfuric acid method, and the reducing sugar contents hsam and hcon of the experimental group and the blank group are determined by the DNS reducing sugar detection method. The average chain length of the α-1,6 / α-1,4 chimeric oligosaccharide is DP = (DP 异麦芽低聚糖 + 1.5) / (1 - 2×(hsam - hcon) / z).

[0082] The average degree of polymerization data of the α-1,6 / α-1,4 chimeric oligosaccharide obtained from the examples and different control examples are shown in Table 3.

[0083] Table 3 Calculation and data statistics of the average degree of polymerization of the α-1,6 / α-1,4 chimeric oligosaccharide obtained from different treatment groups

[0084] Item 2×(hsam - hcon) / z / % Average degree of polymerization of α-1,4 chain segments Average degree of polymerization of chimeric oligosaccharides Example 3 40.6 9.9 20.7 Comparative Example 2 34.6 8.0 18.8 Comparative Example 3 38.2 9.1 19.9 Comparative Example 4 38.8 9.3 20.1 Comparative Example 5 41.7 10.3 21.1 Comparative Example 6 43.6 11.0 21.8

[0085] Example 5

[0086] The α-1,6 / α-1,4 chimeric oligosaccharide obtained from Example 3 and commercial γ-cyclodextrin are prepared into aqueous solutions with different concentrations (M). An excessive amount of solid resveratrol powder is added thereto, and the mixture is shaken at 300 rpm at 25 °C for 72 h and then allowed to stand at room temperature for 6 h. The supernatant is taken and passed through a 0.45 μm water membrane, and the resveratrol content in the filtrate is measured at 300 nm. The different carrier concentrations and the corresponding resveratrol contents are shown in Table 4 below, and a solubility phase diagram of resveratrol is made as Figure 8 shown. The solubilization effect of the α-1,6 / α-1,4 chimeric oligosaccharide on resveratrol is better than that of γ-cyclodextrin.

[0087] Statistics of Resveratrol Content Corresponding to Different Carrier Concentrations in Table 4

[0088]

[0089]

[0090] The present invention discloses a method for the preparation, purification, rapid determination of the degree of polymerization of α-1,6 / α-1,4 chimeric oligosaccharides and their application in solubilizing hydrophobic guest molecules. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The products of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the products described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Claims

1. A method for enzymatic synthesis of α-1,6 / α-1,4 chimeric oligosaccharides, characterized in that, It consists of the following steps: (1) The reaction system uses sodium citrate or sodium acetate solution as a buffer, sucrose as a substrate, and dextransucrase is added for reaction. After the reaction, the enzyme is inactivated by boiling water bath. (2) The denatured proteins are removed by centrifugation. The supernatant is dialyzed, freeze-dried, ground into powder and sieved to obtain isomaltooligosaccharide. (3) The reaction system uses phosphate or glycine solution as a buffer, isomaltooligosaccharide as the acceptor substrate, γ-cyclodextrin as the donor substrate, and cyclodextrin glucosyltransferase is added for reaction. The enzyme is inactivated by boiling water bath to prepare α-1,6 / α-1,4 chimeric oligosaccharide; the cyclodextrin glucosyltransferase is the γ-cyclodextrin glucosyltransferase derived from Bacillus sp. FJAT-44876. Wherein the donor-acceptor ratio is 0.5-5:

1.

2. The method according to claim 1, wherein The concentration of the sucrose is 0.1-5.0 M.

3. The method according to claim 1, wherein The dextransucrase is the mutant GTF180-ΔN L940W from Lactobacillus reuteri 180. The enzyme addition amount is 0.5-2 U / mL; the reaction temperature is 30-40 °C; the reaction time is 8-24 h.

4. The method according to claim 1, wherein The addition amount of the γ-cyclodextrin glucosyltransferase is 0.05-0.30 U / mg of isomaltooligosaccharide; the reaction temperature is 40-55 °C; the reaction time is 0.5-6 h.

5. A method for purifying an α-1,6 / α-1,4 chimeric oligosaccharide, characterized in that, It consists of the following steps: (1) The pH of the reaction system in step (3) of claim 1 is adjusted to 5.0-7.0 with an acid solution, and after cooling, the denatured proteins are removed by centrifugation. (2) The supernatant is added with exo-dextranase for reaction for 10-20 h, the enzyme is inactivated by boiling water bath, and after cooling, the denatured proteins are removed by centrifugation. (3) The supernatant is heated to 70-85 °C and then 2-6% (w / v) cyclododecanone is added, shaken for 0.5-2 h, cooled to 60-70 °C and continuously shaken for 8-12 h, and cooled to 4-8 °C and continuously shaken for 10-20 h. (4) The supernatant is aspirated and filtered through a 0.45 μm organic filter membrane to separate from the insoluble substances in the solution. (5) The filtrate is dialyzed, freeze-dried and ground into powder to obtain pure α-1,6 / α-1,4 chimeric oligosaccharide. The acid solution is hydrochloric acid or glycine solution, and the pH adjustment end point is 6.

0.

6. The method according to claim 5, characterized in that, The exo-dextranase is glucan 1,6-alpha-glucosidase DexB from Streptococcus mutans. The enzyme addition amount is 0.5-2 U / mL; the reaction temperature is 30-37 °C; the reaction time is 8-16 h.

7. The method according to claim 5, wherein The addition amount of cyclododecanone is 5% (w / v), the temperature to which the supernatant is heated is 75 °C, the maintained temperature is 65 °C, the cooling temperature is 4 °C, and the shaking frequency is 400-800 rpm.

8. A method for rapidly determining the average chain length by enzymatic degradation of α-1,6 / α-1,4 chimeric oligosaccharides, characterized in that, The method is as follows: The α-1,6 / α-1,4 chimeric oligosaccharide prepared by the method described in claim 5 is formulated into a reaction solution with sodium acetate buffer, and the total sugar content z is determined by the phenol-sulfuric acid method. Then, β-amylase is added for reaction, and the reducing sugar content h before and after the reaction is determined by the DNS reducing sugar detection method sam 、h con , the average chain length of the α-1,6 / α-1,4 chimeric oligosaccharide is DP = (DP 异麦芽低聚糖 + 1.5) / (1 - 2×(h sam - h con ) / z); DP 异麦芽低聚糖 is the degree of polymerization of the isomaltooligosaccharide obtained in step (2) of claim 1 9. The method according to claim 8, wherein The concentration of the α-1,6 / α-1,4 chimeric oligosaccharide is 1-5 mg / mL.

10. The method according to claim 8, wherein The addition amount of β-amylase is 0.1-0.2 U / mL; the reaction temperature is 30-40 °C; the reaction time is 2-6 h.

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