Highly efficient method for synthesizing difructose anhydride III

Abstract

The invention discloses a high-efficiency synthesis method of difructose anhydride III. The method comprises the following steps: firstly converting sucrose into inulin by using inulosucrase without separating polysaccharide, and then converting inulin by using inulin fructotransferase to synthesize the functional disaccharide difructose anhydride III. The method has the advantages of simple process and high efficiency, and a conversion rate of synthesizing inulin into difructose anhydride III can reach 40%-54%. In order to obtain purer difructose anhydride III, yeast is utilized to remove small molecule monosaccharides in a reaction solution, and the finally obtained purer difructose anhydride III can be easily separated and purified. Thus, the method has broad market application prospects.

Claims

1. A method of synthesizing with high efficiency difructose anhydride III, comprising firstly converting sucrose into inulin by adding inulosucrase without separating polysaccharide, and then converting inulin by adding inulin fructotransferase to synthesize the functional disaccharide difructose anhydride III; wherein the amino acid sequence of the inulosucrase is set forth in SEQ ID NO.1; wherein the process for converting sucrose into inulin comprises: completely dissolving the sucrose in water, wherein the concentration of the sucrose is at 400-500 g/l, regulating pH to be at 5.0-6.0, controlling temperature to be at 20-30 C., adding inulosucrase with an amount of 1-15 U/g of sucrose, and performing thermostatic reaction for 25-60 minutes to obtain a sucrose conversion solution; wherein the process for converting inulin into difructose anhydride III comprises: heating the sucrose conversion solution to 50-60 C., and adding inulin fructotransferase into the sucrose conversion solution, wherein the concentration of the inulin fructotransferase is at 1-20 U/g of the sucrose conversion solution, controlling the pH to be at 5.0-6.0, and performing thermostatic reaction for 2-12 hours.

2. The method of claim 1, wherein the the inulosucrase is controlled to be at 5-15 U/g of sucrose, pH is controlled to be at 5.0-6.0, the temperature is controlled to be at 20-30 DEG C., and the thermostatic reaction lasts for 30-60 minutes.

3. The method of claim 1, wherein the concentration of the inulin fructotransferase is 5-10 U/g of the sucrose conversion solution, the pH value is controlled to be at 5.0-6.0, the temperature is controlled to be at 55-60 C., and the thermostatic reaction lasts for 6-12 hours.

4. The method of claim 1, further comprising a step of eliminating residual saccharides to remove sucrose, glucose, fructose and part of fructooligosaccharides therein.

5. The method of claim 4, further comprising removing residual saccharides by adding yeast.

6. The method of claim 1, wherein the amino acid sequence of the inulin fructotransferase is set forth in SEQ ID NO.2.

7. The method of claim 4, further comprising cooling the reaction solution, adding yeast into the reaction solution to absorb and remove the sucrose, the glucose, the fructose and part of the fructooligosaccharides in the reaction solution, controlling pH to be at 5.0-6.0, controlling temperature to be at 28 C., and reacting in a 200 rpm shaking table for 24-36 hours.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1: Synthesis of inulin (diluted solution) from substrate sucrose by using inulosucrase.

(2) FIG. 2: Conversion of synthesized inulin into difructose anhydride III (diluted solution) by using inulin fructotransferase.

(3) FIG. 3: Treatment of the synthesized difructose anhydride III solution by using yeast.

DETAILED DESCRIPTION

(4) The examples of converting sucrose into difructose anhydride III by using inulosucrase and inulin fructotransferase are as follows. The method of the invention is illustrated by the examples, but is not limited to the examples listed.

Example 1

(5) Completely dissolve substrate sucrose in water, control concentration to be 400 g/l, regulate a pH value to be 5.5, control temperature to be 20 C., add inulosucrase (addition amount being 10 U/g of sucrose), and perform thermostatic reaction for 30 minutes;

(6) Increase the temperature of a reaction system to 55 C., add inulin fructotransferase into the reaction system (the concentration of the added inulin fructotransferase being 5 U/g), control the pH value to be still 5.5, and perform thermostatic reaction for 12 hours.

(7) Take a solution after reaction, centrifuge, filter supernate through a microporous filter membrane (0.22), and analyze the filtrate by HPLC equipped with a differential refraction display. HPLC conditions are as follows: a Sugarpak1, 6.5 mm id300 mm calcium cation exchange column is used, pure water is used as a mobile phase, column temperature is 85 C., and flow velocity is 0.4 ml/min, and the sample size is 10 l. The concentration of a difructose anhydride III standard sample is 0.5%. The concentration of the difructose anhydride III synthesized in the reaction solution is calculated by utilizing a ratio of the peak area of the difructose anhydride III in the reaction solution to the peak area of the 0.5% difructose anhydride III standard sample. A conversion rate of the obtained difructose anhydride III is up to 54%.

Example 2

(8) Completely dissolve substrate sucrose in water, control concentration to be 300 g/l, regulate a pH value to be 5.0, control temperature to be 22 C., add inulosucrase (addition amount being 15 U/g of sucrose), and perform thermostatic reaction for 60 minutes;

(9) Increase the temperature of a reaction system to 60 C., adding inulin fructotransferase into the reaction system (the concentration of the added inulin fructotransferase being 10 U/g), control the pH value to be still 5.0, and perform thermostatic reaction for 6 hours. A conversion rate of the obtained difructose anhydride III is up to 48%.

Example 3

(10) Completely dissolve substrate sucrose in water, control concentration to be 500 g/l, regulate a pH value to be 6.0, control temperature to be 30 C., adding inulosucrase (addition amount being 5 U/g of sucrose), and perform thermostatic reaction for 60 minutes;

(11) Increase the temperature of a reaction system to 50 C., add inulin fructotransferase into the reaction system (the concentration of the added inulin fructotransferase being 20 U/g), control the pH value to be still 6.0, and perform thermostatic reaction for 20 hours. A conversion rate of the obtained difructose anhydride III is up to 43%.

Example 4

(12) Produce difructose anhydride III according to reaction and detection conditions in Embodiment 1, naturally cool a reaction system to 28 C., add yeast into the reaction system, control pH to be 5.0-6.0. and react in a 28 C. 200 rpm shaking table for 24-36 hours. The present embodiment is mainly implemented by removing sucrose, glucose, fructose and part of fructooligosaccharides in a reaction solution by yeast fermentation to obtain the purer difructose anhydride III, thereby facilitating the subsequent detection and separation purification in industrial implementation. Finally, a conversion rate of difructose anhydride III is close to that in Embodiment 1, but the solution after reaction does not contain sucrose, glucose, fructose and part of fructooligosaccharides any more, thereby improving the purity of the difructose anhydride.